Drug Monitoring by HPLC: Recent Developments, Nova Science Publishers, Incorporated, 2010. Proquest Ebook Central

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.
Drug Monitoring by Hplc : Recent Developments, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,
PHARMACOLOGY – RESEARCH, SAFETY TESTING
AND REGULATION SERIES
DRUG MONITORING BY HPLC:

RECENT DEVELOPMENTS
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DRUG MONITORING BY HPLC:

RECENT DEVELOPMENTS
VICTORIA SAMANIDOU
AND
EFTICHIA KARAGEORGOU
Nova Science Publishers, Inc.

New York
Antibiotic Resistance: Causes and Risk Factors,

Mechanisms and Alternatives
Adriel R. Bonilla and Kaden P. Muniz (Editors)
2009. ISBN: 978-1-60741-623-4
Antibiotic Resistance: Causes and Risk Factors,

Mechanisms and Alternatives
Adriel R. Bonilla and Kaden P. Muniz (Editors)
2009. ISBN: 978-1-61668-162-3
Poisons: Physiologically Active Substances

S. B. Zotov and O. I. Tuzhikov
2009. ISBN: 978-1-60741-973-0
Drug Monitoring by HPLC: Recent Developments

Victoria Samanidou and Eftichia Karageorgou
2010. ISBN: 978-1-60876-183-8
Copyright © 2010 by Nova Science Publishers, Inc.
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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Samanidou, Victoria.
Drug monitoring by HPLC : recent developments / Victoria Samanidou and Eftichia
Karageorgou.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-61324-472-2 (eBook)
1. High performance liquid chromatography. 2. Drugs--Analysis. I. Karageorgou, Eftichia. II.
Title.
[DNLM: 1. Drug Monitoring--methods. 2. Chromatography, High Pressure Liquid. WB 330
S187d 2009]
RS189.5.H54S26 2009
615'.1901--dc22
2009034548
Published by Nova Science Publishers, Inc.  New York
CONTENTS
Preface vii
Chapter 1 Introduction 1
Chapter 2 Brief Introduction to HPLC 5
Chapter 3 Sample Preparation 11
Chapter 4 Anti-Cancer Drugs 21
Chapter 5 Bronchodilators 29
Chapter 6 Cardiovascular Drugs 33

Chapter 7 Antipsychotics 41
Chapter 8 Antibiotics 49
Chapter 9 Antiepileptic Drugs 53
Chapter 10 Antidepressants 83
Chapter 11 Immunosuppressants 105
Chapter 12 Conclusions 111
Index 115
PREFACE
Drug monitoring in clinical chemistry refers to the measurement of drug

concentration in blood serum/plasma so that an optimal concentration is obtained
to benefit patient with minimal toxic adverse effects. Drug monitoring addresses
drugs with narrow effective range or a narrow therapeutic/toxic index. It is
required to individualise and optimise drug therapy. Moreover it supports
pharmacokinetic and drug metabolism studies.
Drugs that usually require monitoring include: cardioactive medications,
antiepileptic drugs, antibiotics (e.g. aminoglycosides), anti-cancer drugs,
immunosuppressants, antidepressants (e.g. tricyclic antidepressants),
bronchodilators (Theophylline), antipsychotics etc. Monitoring of medication is

also important to detect poisoning with above drugs in forensics.
In pharmacology, many drugs are used without monitoring of blood levels, as
their dosage can generally be varied according to the clinical response of the
patient. In some drugs insufficient levels will lead to undertreatment or resistance,
and excessive levels can lead to toxicity and tissue damage.
The available analytical methods for monitoring drug levels in patient
specimens in human blood or serum/plasma include: immunoassays, such as
microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay
technique (EMIT), fluorescent polarization immunoassay (FPIA),
radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) and
most frequently fluorescence polarisation immunoassay (FPIA) and liquid
chromatography-based methods. Chromatography-based methods include high
performance liquid chromatography (HPLC) with ultraviolet detection, HPLC-
mass spectrometry (HPLC-MS), and HPLC-tandem mass spectrometry (HPLC-
MS/MS). In clinical practice, immunochemical assays are applied to screen the
drugs in biological fluids. These assays while they are sensitive, they are not
viii Victoria Samanidou and Eftichia Karageorgou
sufficiently selective and can only be used to eliminate negative samples.

Confirmation of the initial screening should always be implemented by a
separation assay usually a chromatographic one, which should have sufficient
sensitivity and selectivity to confidently identify the analyte down to the cut-off
level.
Currently HPLC-MS/MS has gained increasing popularity in clinical
laboratories due to the advantages of the technology over other methods, while the
capital cost for instruments has been decreased. HPLC-MS/MS provides high
specificity and sensitivity for the simultaneous measurement of several drugs
and/or their major metabolites in one single analytical run.
In this book chromatographic methods published in the last decade are
reviewed regarding their applicability to drug monitoring.
VICTORIA SAMANIDOU
Assistant Professor of Analytical Chemistry, Laboratory of Analytical
Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, R-
54124 Thessaloniki, Greece,Tel. +302310-997698, Fax. +302310-997719, e-mail:
samanidu@chem.auth.gr, http://users.auth.gr/samanidu.
EFTICHIA KARAGEORGOU
MSc in Analytical Chemistry, Laboratory of Analytical Chemistry
Department of Chemistry Aristotle University of Thessaloniki GR-54124
Thessaloniki,Greece.
Chapter 1
INTRODUCTION
Incessant discovery of new drugs is a worldwide demand. New drugs may be
derived from natural products or be produced synthetically. New compounds have
to be comparatively tested with their predecessors. Toxicity, biological half-life,
ease of administration are important factors to be checked when a new drug is
under development. Research on absorption, distribution, metabolism and
excretion of the new drugs must proceed at the same time with development
process. Therapeutic Drug Monitoring (TDM) is the measurement of specific
drugs at different time intervals in order to maintain a relatively constant
concentration of the medication in the bloodstream. Drugs that require monitoring
tend to have a narrow ―therapeutic range‖, which means that the necessary
quantity to be effective is not far from the quantity that causes significant side
effects and/or signs of toxicity.
Maintaining this steady state is mandatory and not as simple as compared
with a standard dose of medication. Personalization of dosage is a significant
issue, since absorption, metabolism, utilization, and elimination of drugs will be
different in each person, based upon their age, general state of health, genetic
makeup, and the interference of other medications that they are taking. This rate
may change over time and vary from day to day. [1]
Luckily not all medications require therapeutic monitoring. Most drugs have a
wide therapeutic range and can be prescribed based upon pre-established dosing
schedules. The effectiveness of these treatments is evaluated, while it is not
usually necessary to determine the concentration of the drug in the bloodstream.
Many of the drugs that are monitored therapeutically are taken for a lifetime.
They must be maintained at steady concentrations year after year, while the
patient ages and life events such as pregnancies, temporary illnesses, infections,
emotional and physical stress, accidents, and surgeries may occur. Over time other
2 Victoria Samanidou and Eftichia Karageorgou
chronic conditions such as cardiovascular disease, kidney disease, thyroid disease,

liver disease, and HIV/AIDS may arise that also require lifetime medication and
that may affect the processing of their monitored drugs. Therapeutic drug
monitoring put up with these changes. Patient non-compliance is assessed by
identifying the effect of drug interactions caused by drug higher or lower
concentrations. This helps to individualize dosages to fit the current needs of the
specific patient helping tolp identify decreases in the efficiency of and
dysfunctions in the body in metabolizing and eliminating therapeutic drugs. [1]
It has become clear by now that therapeutic drug monitoring is of great value
for specific classes of drugs. The following criteria should be fulfilled so that
TDM measurements are clinically worthwhile:
Relationship between plasma drug concentration and therapeutic

response and/or toxicity.
Relationship between plasma concentration and drug dosage.
Clinical indication for no response to treatment; suspected non-
compliance; signs of toxicity. [2]
In vivo–factors affecting therapeutic drug levels include: Patient compliance,

Bioavailability (access to circulation, interaction with related receptors; ionized
and free, or bound to a carrier molecule e.g. albumin), and Pharmacokinetics.
Drug concentrations are affected by:
Interaction with diet or other medication.

Absorption on large molecule size components, e.g. proteins.
Lipid solubility, which affects the volume of distribution as highly lipid-
soluble substances have high affinity for adipose tissue and a low
tendency to remain in the vascular compartment.
Biotransformation, by hepatic metabolism, in which polar groups are
introduced into relatively insoluble molecules by oxidation, reduction or
hydrolysis.
Physiological factors like age affect drug levels. Lower doses are
necessary in both infants and the elderly, in the former because the
metabolism mechanism is not fully operational. In elderly people the
organism may suffer from decrease in cardiac and/or renal function,
enzyme activity etc.
Enzyme induction may reduce the drug's activity,
Introduction 3
Enzyme inhibition may result in the increase of drug activity, prolonging

the action of various drugs e.g. metronidazole.
Genetic factors.
Associated diseases may also affect drug distribution or metabolism, e.g.
renal disease with decrease in clearance and increase of drug levels, or
hepatic disease, in which there is a decrease in albumin production and
enzyme activity resulting in a functional increase in drug levels. [3]
As it has been already mentioned not all medications require therapeutic

monitoring. For example many anti-infective agents (e.g. aminoglycosides
antibiotics) have a wide therapeutic range, which means that standard doses can
be used. However, with some antibiotics, toxicity is associated with persistently
high concentrations whereas with others, treatment failure can result from
persistently low concentrations. Antibiotics are ―time-dependent‖. The aim when
this medication is prescribed is to maintain concentrations above the Minimum
Inhibitory Concentration (MIC) for most of the dosage interval or ―concentration-
dependent‖. In the latter situation the aim is to achieve high peak concentrations,
while allowing the concentration to fall to low levels between doses.
A field most commonly requiring TDM is psychiatry. The routine analysis of
drugs used in psychiatry is popular in many European countries. Monitoring
concentrations of antiepileptic drugs became popular during the 1980‘s, when it
was recognized that it could help to reduce variability in response and toxicity.
Most of the older anticonvulsants are eliminated by hepatic metabolism, leading
to a wide range of dose requirements and a high incidence of drug interactions.
Target ranges have been identified for a number of antidepressants and
antipsychotics, many of which are metabolized by cytochrome P450 2D6.
Significant differences in dosage requirements have been demonstrated between
poor or extensive metabolisers.
Although anticancer drugs have narrow therapeutic ranges, concentrations are
not routinely monitored because of a lack of data on concentration-effect
relationships. One exception, however, is methotrexate, where folic acid rescue
therapy is based on monitoring the methotrexate concentration 24-48 hours after
high dose therapy and continuing until concentrations are below 0.05 μmol/L. For
other anticancer drugs, monitoring the enzyme responsible for their metabolism
has been used to help adjust doses and reduce toxicity.
Bronchodilators belong also to drugs requiring TDM. Although theophylline
has largely been replaced by other drugs with less potential for adverse effects, it
is still used in some patients. It is principally cleared by hepatic metabolism, thus
dosage requirements vary widely and drug interactions remain a major concern.
Bronchodilator effects have been demonstrated within the normal target range of
10-20 mg/L, but lower concentrations are associated with anti-inflammatory and
steroid sparing effects. Consequently, there is some support for reducing the target
range to 5-15 mg/L, which might reduce the incidence of toxic effects. [4]
It can be concluded by now that the appropriate use of therapeutic drug
monitoring requires measuring the concentration of a drug in the patient‘s blood.
In the following paragraphs drugs requiring TDM are classified according to
their activity and target and methods are summarized to provide information on
their application. Figure 1 illustrates the chemical structures of most common
analytes requiring drug monitoring.
REFERENCES
[1] www.labtestonline.org (Accessed January 2009)
[2] www.toxlab.co.uk (Accessed January 2009)
[3] www.thefreedictionary.com (Accessed January 2009)
[4] www.pjonline.com (The Pharmaceutical Journal Vol.273 2004) (Accessed
January 2009)
SUGGESTED FURTHER READING

[1] Dasgupta, A. Handbook of Drug Monitoring Methods, Therapeutics and
Drugs of Abuse.p.446, 44 2008.
[2] Hempel, G. Drug Monitoring and Clinical Chemistry, 5. Institut fur
Pharmazeutische und Medizinische Chemie Westfalische Wilhelms-
Universitat Munster. Hardbound, 2004.
[3] Dasgupta, A. Advances in Chromatographic Techniques for Therapeutic
Drug Monitoring CRC Press, 2009.
[4] Evans, W.E.; Schentag, J.J.; Jusko, W.J. Applied Pharmacokinetics:
Principles of Therapeutic Drug Monitoring. Lippincott Williams & Wilkins
[5] Bauer, L.A. Applied Clinical Pharmacokinetics. McGraw-Hill, 2001.
Chapter 2
BRIEF INTRODUCTION TO HPLC

2.1. Introduction
Chromatography is a science that counts more than one century, since its
introduction, while in the past twenty years has expanded exponentially because
of the availability to be applied for the analysis of various analytes in a very wide
variety of matrices.
In principle, chromatographic techniques belong to separation science, where
separation is achieved by regulating the magnitude of the distribution coefficient
between two distinguished phases: one stationary and one mobile. Mixture
components are separated while they migrate with different rates in the stationary
phase by means of the mobile phase. Depending on the type of these phases there
are several chromatographic techniques. When the mobile phase is a liquid, the
chromatography is called liquid chromatography. Liquid chromatography in its
various forms, where HPLC is the most important and dominant, is of major
importance in all areas related to chemistry.
The most sophisticated type of liquid chromatography is HPLC where the
mobile phase runs through the stationary by means of a pump at elevated
pressures.
HPLC has been used in an extremely wide range of analytical methods and it
is impossible to give a comprehensive set of examples that would illustrate its
wide applicability in a variety of matrices.
HPLC is probably the most universal type of analytical procedure; it has
achieved this position as a result of the constant evolution of the equipment used
to provide higher efficiencies at faster analysis time with a constant incorporation
of new column packings.
Five different mechanisms are responsible for separation in HPLC:
1. Adsorption of the analytes on the sorbent used as a stationary phase.

2. Partitioning of the solutes between two liquid phases. These two
mechanisms can be applied in normal or reversed phase mode, where
analytes are retained due to their polarity. In normal phase, most non-
polar compounds elute first and the most polar compounds elute last,
because the mobile phase is less polar than the stationary phase. The
opposite stands for reversed phase, which covers the vast majority of
applications. In this mode the mobile phase is more polar than the
stationary phase, so that more polar compounds elute earlier.
3. Ion exchange, where separation is based upon electrostatic interactions
between electrically charged ions or ionizable compounds.
4. Size exclusion, where biomolecules can be separated based on their size,
rather than on their charge or polarity, by passing, or filtering, them
through a controlled-porosity packing material.
5. Affinity, where molecules are separated due to a highly specific
interaction between targeted analytes and the packing material e.g. the
isolation of antibodies in a serum sample specific for a particular
antigenic determinant.
2.2. HPLC Instrumentation
The main components of an HPLC system are shown in Figure 2.1. These
include:
1. Mobile phase reservoir: Solvent Reservoirs are used to store Mobile-

Phase.
2. Pump: The HPLC pump is used to deliver the mobile phase at constant
flow so that the separation of the components of the mixture occurs in a
reasonable time. There are two types of pumping systems Isocratic and
Gradient.
Brief Introduction to HPLC 7
Pump Chromatographic Detector

Column
Injector
Mobile Phase
Reservoir
Chromatogram
Waste
A typical HPLC apparatus
Figure 2.1. Main components of an HPLC system.

Figure 2.1. Main components of an HPLC system.
Isocratic: In this mode the mobile phase composition is kept constant
throughout the chromatographic run.
Gradient: In this mode the mobile phase composition can be changed during
chromatographic run to achieve a better or/and faster separation. There are two
types of gradient systems: Low-pressure mixing and High-pressure mixing. In the
second mode two pumps are required.
1. Injection Port: Sample introduction unit. Usually this is a six port valve
injection with a loop which can be partially or fully filled.
2. Analytical column: This contains the stationary phase for separating the
components of the sample. It is regarded as the ―heart‖ of the
chromatographic system responsible for the efficiency of
chromatographic separation.
3. Detector: The device used to detect the separated compounds.
There are several detectors available. However UV-VIS Detector, Photo-

Diode Array Detector, fluorescence Detector, are more commonly used. The
Photo Diode Array Detector has the advantage of identification by means of UV-
vis spectrum comparison. It can give a three dimensional view of chromatogram
(Intensity Vs Time) and Spectra (Intensity Vs Wavelength) simultaneously. It also
gives information on peak purity. The new ELSD detector is proving to be an
important detector especially in polymer analysis; while the MS as detector is
outstanding. Figure 2.2 cites the most common detection techniques used in
HPLC.
DETECTION TECHNIQUES
Identification
FTIR
Electrochemical Spectroscopic NMR
MS
Molecular Atomic
•Conductivity Spectroscopic Spectroscopic
•Potentiometry techniques techniques
•Amperometry
•UV/vis •Atomic Absorption
•Coulometry
•Photodiode array Spectrometry
(PDA) •Atomic Emission
•Fluorescence Spectrometry
Figure 2.2. Most
common detection •Refractive index
techniques in HPLC.
6. Data Acquisition System: to process the detector output and integrate it to

form the chromatogram. Personal computers with sophisticated software are used
to process the chromatogram for quantitative analysis, identification, peak purity
test etc. The same software may also be used to control various parameters of the
system.
Beside these main parts there may also be optional units such as
1. Precolumn or guard column to protect the analytical column.

2. Autosampler.
3. Column thermostat to maintain the column at an elevated temperature.
4. Fraction collector to obtain the separated analytes for preparative
chromatography.
Brief Introduction to HPLC 9
2.3. Liquid Chromatography-Mass Spectrometry
Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an

analytical technique that combines the separation capabilities of HPLC with the
mass detection capabilities of mass spectrometry.
Ion Sources: used for the ionization of sample molecules include Electrospray
Ionisation (ESI), EI (Electron Ionisation), Matrix-Assisted Laser
desorption/Ionisation (MALDI), Chemical Ionisation (CI) Fast Atom
Bombardment (FAB), Atmospheric Pressure Chemical Ionization (APCI),
Atmospheric Pressure Photoionization (APPI), Thermospray Ionization.
Mass analysers: used to Sort Ions by Mass (m/z) include: Quadrupole, Ion
Trap, Magnetic Sector Field, Electric Sector Field, Time-Of-Flight (TOF).
Detectors: Detection of ions is achieved by a Microchannel Plate, an Electron
Multiplier, a Faraday cup, a photomultiplier etc.
Tandem mass spectrometry, known as MS/MS, involves multiple steps of
mass spectrometry selection, with some form of fragmentation during different
stages of mass analysis. This hyphenated to HPLC is a powerful technique which
can also provide structural information.
It is beyond the scope of this book to give more details upon this well known
technique, since it has been comprehensively covered by textbooks dedicated to
the analytical technique theory, instrumentation and applications. The aim of this
chapter is to give supportive fundamental information to the clinical chemist.
SUGGESTED FURTHER READING-REFERENCES

[1] www.chromatography-online.org/HPLC.html
[2] Papadoyannis, I.N. ΗPLC in Clinical Chemistry. Editor J. Cazes, Marcel
Dekker Inc. New York & Basel, 1990.
[3] Snyder, L.R.; Glajch, J.L.; Kirkland, J.J. Practical HPLC Method
Development. Wiley John & Sons, 1997.
[4] Neue, U.D. HPLC Columns: Theory, Technology, and Practice. Wiley-
VCH Inc. Milford, MA USA, 1997.
[5] Dong, M.W. Modern HPLC for Practicing Scientists. John Wiley & Sons
Inc. New Jersey, 2006.
[6] Kazakevich, Y.V. & LoBrutto, R. HPLC for Pharmaceutical Scientists.
Wiley-Interscience, New Jersey USA, 2007.
[7] Snyder, L.R. & Dolan, J.W. High-Performance Gradient Elution: The
Practical Application of the Linear-Solvent-Strength Model. John Wiley &
Sons Inc. New Jersey, 2007.
[8] Samanidou, V.F. & Papadoyannis, I.N. HPLC: the dominant separation
technique with a wide range of applications, edited collection:
"Chromatography: Types, Techniques and Methods." Nova Publishers,
2009.
Chapter 3
SAMPLE PREPARATION
3.1. Introduction
Sample preparation is widely accepted to be the most important part of any

analytical practice. It is usually time consuming and since it requires many steps it
arises to be the most error-prone phase in any sample processing.
The objectives of any sample preparation technique include:
1) Modification or elimination of sample matrix to prepare it for

introduction (injection) on to the chromatographic column.
2) Impurities removal and sample purification.

3) Analytes‘ enrichment by pre-concentration when being in not detectable
amounts. The latter should be done with precautions because impurities
and matrix interferences if present are inevitably concentrated as well.
[1,2].
Therapeutic drug monitoring (TDM) may be used as a tool for the

improvement of drug therapy. The ideal sample preparation technique for
biological samples prior to HPLC or LC/MS analysis should be ―no preparation‖.
A similar approach is to avoid the preparation step by using a technique known as
"dilute and shoot". This technique could only be efficient when levels of targeted
analytes are relatively high, and the matrix components do not co-elute.
When HPLC is the analytical technique of choice then sample pre-treartment
is especially necessary:
to prolong instrument‘s and above all column life time.

to improve method detectability providing lower LOD and LOQ values
to change the sample solvent and make it suitable for introduction to the
chromatographic system.
Sample pretreatment may include various steps after sampling such as:
1. Preservation during storage.

2. Dilution, freeze drying, filtration, centrifugation, pH adjustment etc.
3. Extraction-Isolation of analytes of interest.
4. Derivatization prior to analysis to improve either chromatographic
separation or detection capability of the method.
There is no ideal or universal sample pretreatment technique. The analytical

chemist should take into consideration all requirements in order to choose the
most efficient sample preparation protocol, which should:
be simple and fast

provide high extraction efficiency
be accurate, precise and reproducible
be specific for the targeted analytes
provide sample free from interference and impurities
be easily automated in order to be readily applied in routine analysis
provide high sample throughput

involve the minimum manipulation to reduce analytes‘ loss
be solvent-free or use as less solvent amount as possible, so as to be
friendly to the environment
be of low cost.
Final decision upon the optimal sample preparation technique depends also on
sample volume and required quantities for measurement. [3-6]
3.2. Sample Preparation in Drug Monitoring
In drug monitoring sample preparation aims to eliminate endogenous

inteference from biological samples. Biological fluids may need dilution, protein
precipitation and an extraction step for the isolation of analytes of interest.
Sample dilution can only be applied in a few cases if the concentrations of the
analytes are sufficiently high that they can still be detectable.
Sample Preparation 13
Blood serum and plasma are routinely used for drug monitoring. Plasma
samples contain a significant amount of salt and proteins that can be precipitated
or adsorbed on reversed phase packings. The adsorbed protein can easily foul the
column resulting in changes in the separation and ultimately clogging the column.
Serum is the straw-colored liquid that separates from the clot that forms in whole
blood. Plasma is prepared from whole blood that has treated with an anti-clotting
substance such as heparin. It is the supernatant that results when the cellular
components of blood are removed by centrifugation. A method developed for
plasma can normally be applied without modification or slightly modified to
serum as well.
Urine is one of the most commonly studied for drug analysis, particularly
because of its relative ease of collection, and because it is nearly universal means
of excretion of parent drug compounds metabolites or both. As a matrix, it has
moderate complexity, and typically contains both organic and inorganic
constituents, as well as a relatively high variability.
A protein - free sample can be obtained by protein precipitation, for
example by addition of acetonitrile or an acidic solution such as perchloric acid,
trichloroacetic acid to the sample. After centrifugation the supernatant can be
directly injected into the chromatographic system, if it is free from endogenous
compounds. However, in most cases matrix components still interfere.
Therefore biological samples prior to HPLC analysis should be further purified in
an organic solvent. This is accomplished mainly by liquid-liquid extraction (LLE)
and solid-phase extraction (SPE). In the extraction method the compound of

interest is removed from the biological matrix (plasma, serum, urine, etc.) under
suitable conditions that selectively isolate (extract) the desired components and
leave behind unwanted materials. A typical sample preparation protocol is shown
in Figure 3.1. [7-10]
Nearly all analytical problems can be solved by LLE and SPE. Therefore,
these methods can be characterized as universal from a scientific and technical
view.
Liquid-liquid extraction (LLE) is the traditional extraction technique by
which analytes of interest are extracted from the sample matrix with an organic
solvent with a large affinity for the analyte. This technique has some drwbacks
such as: 1. The use of large volumes of extraction solvent, 2. The formation of
emulsions during the mixing procedure. 3. Time-consuming evaporation
procedures. 4. Co-extraction of proteins and other matrix components. 5. It is not
suitable for very polar analytes. Since many samples also have very polar
metabolites this method has limitations.
Sample
SamplePreparation
Preparation
Generic
GenericProtocol
Protocol
Blood plasma/serum
Steps
Steps
Protein precipitation
1. Deproteinization e.g. Acetonitrile, TFA etc
Centrifugation
2. Cleanup-isolation
Solid Phase Extraction
(e.g. RP-18, HLB,
mixed mode)
1. Wash to remove interference
2. Elute anaytes of interest
3. Analysis HPLC, HPLC-MS/MS Figure 3.1 Typical sample preparation

protocol for blood plasma/serum
samples in drug monitoring.
So called modern extraction techniqes are preferred nowadays such as solid

pahse extraction (SPE) or solid phase microextarction (SPME).
Comparing solid-phase extraction with classical liquid- liquid extraction, it

displays the following advantages:
- An almost quantitative recovery can be obtained, provided that the

optimal conditions regarding sorbent, wash and elution solvents have
been chosen.
- The extracts obtained with solid-phase extraction are cleaner than
those obtained with liquid-liquid extraction due to the better selectivity
of solid-phase extraction.
- Lower volumes of both samples and solvents are required.
- Problems such as the formation of emulsions are absent in solid-phase
extraction.
- Solid-phase extraction methods are not time consuming and have proven
to be applicable with large numbers of samples. Since 10, 12, 24 or
even more samples can be processed simultaneously, solid-phase
extraction is considerably faster than liquid-liquid extraction.
Solid - phase extraction (SPE) is an extraction process that comprises a solid

and a liquid phase. In SPE the sorbent is packed between two fritted disks in a
polypropylene cartridge. The basic principle of SPE is that intercepts the
components of interest, mainly organic molecules, by a special sorbent placed in a
disposable extraction minicolumn.
Sorbents used in SPE involve: Reversed phase, high - hydrophobic octadecyl
(C18), octadecyl (C18), octyl (C8), ethyl (C2), cyclohexyl, phenyl. Wide-pore
reversed phase, butyl (C4 ). Normal phase, silica modified by cyano (-CN), amino
(-NH2), diols (-COHCOH). Adsorption, silica gel (-SiOH), florisil (Mg2SiO3),
alumina (Al2O3). Ion-exchangers, amino (-NH2), quaternary amine (N+),
carboxylic acid (-COOH), aromatic sulfonic acid (ArSO2OH). Wide-pore ion
exchangers, carboxylic acid (-COOH), polyethyleneimine [-(CH2CH2NH)n - ].
The particle size allows the use of small pressure to force the sample and
wash solutions through the column.
Separation mechanisms involved in SPE are similar to those encontered in
liquid chromatography: adsorption (silica), bonded-phase partition (normal-
phase= sample solvent less polar than the adsorbent, reversed phase with or
without ion-painting= sample solvent more polar than the adsorbent) ion-
exchange, size exclusion.
SPE consists of four steps as shown in Figure 3.2:

1. Sorbent conditioning which is the preparation of the sorbent. This is also

called solvation (activation of functional groups). It aims to facilitate interactions
between analytes of interest and sorbent. After conditioning removal of the excess
of solvation solvent then washing with a solvent suitable for analyte retention of
the sorbent is required; however drying of sorbent should be prevented.
2. Sample Loading: The sample solution is forced through the sorbent of the
cartridge. Analyte are retained in this step. Some undesired compounds may also
be adsorbed by the sorbent.
3. Washing with an appropriate solvent for undesired matrix components
removal.
4. Elution: Selective desorbing the compounds of interest from the sorbent
with a suitable elution solvent, and collecting the cartridge effluent. The extract is
directly injected or evaporated and reconstituted in the mobile phase or after
addition of internal standard.
SOLID PHASE EXTRACTION STEPS
interferences
Analyte of interest
1. Conditioning 2. Sample loading 3. Washing 4. Elution
Figure 3.2. Steps in Solid Phase Extraction
Figure 3.2. Steps in Solid Phase Extraction.
Optimization assays are required in each of these steps to reassure high

recoveries and purification efficiency.
The suitable sorbent type and mass as well as washing and elution solvents
are critical for high recovery rates without loss in recovery.

Some polymeric sorbents such as Hydrophilic-lipophilic polymers used in
OASIS by WATERS and Nexus Abselut by Varian are designed to extract
extensive spectrum of analytes: lipophilic, hypophilic, acidic, basic and neutral,
where no conditioning is required. These sorbents significantly simplify
extraction protocols. [11]
Two different approaches can be chosen for SPE: either the analyte is retained
on the sorbent whilst components pass through the waste. The analyte will be
eluted later from the sorbent with a suitable solvent to be analysed. Or the matrix
components are adsorbed, whilst the analyte is evacuated.
The first approach is generally prefered as less sorbent is required and isolate
preconcentration is possible.
Advantages of this principle should be greater than those of other extraction
methods with only a very low quantity (micro) of the extraction agent, for
example, SPE with disc technology. In contrast to conventional SPE with packed-
bed columns, micro or non micro columns, this arrangement allows the
combination of all steps of sample preparation in one step as described above. [12,
13]
SPE cartridges can be used either with centrifugation, positive pressure or

under vacuum manually or in a manifold as shown in Figure 3.3.
Vacuum Manifold Centrifugation

Positive Pressure
(Manually)
Figure 3.3. Solid Phase Extraction modes (From Biotage Sweden AB, with Permission).
Some promising approaches in SPE are based on special packings such as

restricted access
Figure materials
3.3. Solid (RAMs),
Phase Extraction and(From
modes molecular imprinting
Biotage Sweden AB, with materials
Permission) (MIPs).
On column sample preparation with column-switching techniques and on-line
SPE in LC using RAMs, LC-GC coupling and membrane-based sample
preparation dialysis, electrodialysis, ultrafiltration are also introduced as a useful
tool for sample pretreatment and sampling for complicated matrix samples. On
line dialysis has been used in on-line bioprocess monitoring. It has the advantages
of easy operation rapid isolation of components of interest from complicate and

dirty matrix and free or less use of organic solvents. Serum, plasma, muscle
tissue can be analyzed after sampling by means of on line dialysis. [14-17]
Although these methods have their own merits, most of them are only found
in isolate applications, oftentimes, they do not achieve sensitivity and selectivity
of LLE and SPE and, finally, some methods need expensive equipment. Other
problems are fouling of membranes in membrane-based sample preparation and
irreversible in on-line SPE.
Solid Phase Microextraction SPME is a modern solvent-free
adsorption/desorption technique used to analyze volatile and nonvolatile
compounds in both liquid and gaseous samples. An SPME unit consists of a fused
silica fiber coated with a cross linked polymeric organic liquid such as
polydimehylsiloxane phase and bonded to a stainless steel plunger and a holder.
The fiber assembly consists of an outer protective septum piercing needle and an
inner fiber attachment needle to which the fiber is epoxied.
Organic compounds can be directly introduced into any gas chromatograph or
GC/MS system, as well as in an HPLC system with the proper interface consisting
of a six port injection valve and a desorption chamber that replaces the injection
loop in the HPLC system.
Analytes can be removed in a moving stream of mobile phase (dynamic
desorption) or when analytes are more strongly adsorbed to the fiber the fiber can
be soaked in mobile phase or another stronger solvent for a specific period of time
(e.g. 1 minute) before the material is injected onto the column (static desorption).
SPME is an alternative to the most common sample preparation techniques
used in any laboratory such as liquid-liquid extraction or SPE for semivolatile
compounds.
As no solvent is used, SPME saves preparation time and cost and often
improves the limits of detection in an analysis.
However SPME can display these advantages only in some areas of
biomedical analysis, i.e., the matrix and the volatility of target analyte have to be
taken into account. First of all, the combination of low volatility of analyte and a
complex matrix with polymer components, e.g. proteins in plasma or cell cultures,
considerably limits the application of SPME. The extraction is very slow in
contrast to LLE and SPE with packed bed columns.
The advantages of SPME can be used for both the assay of low volatile and
highly volatile analytes in urine or other body fluids, with no or only a low
concentration of polymer biomolecules. The problems of sensitivity and delayed
time are considerably decreased in comparison to plasma. A number of methods
with good precision, accuracy, sensitivity and selectivity were demonstrated
which were also simple and fast.

SPME is an encouraging development for sample preparation in biomedical
analysis. The most striking attribute of SPME is a low recovery as reported for
many methods. This is not unexpected because SPME is an equilibrium extraction
but not an exhaustive extraction.
A wider application of SPME in TDM is expected for the near future.
However, more studies are necessary, for example, the potential of SPME to
analyze directly the free concentration of drugs in plasma. [18-22]
REFERENCES
[1] Papadoyannis, I.N. & Samanidou, V.F. Sample Preparation Prior to HPLC.
Encyclopedia of Chromatography. p. 1499 – 1515. Marcel Dekker. Jack
Cazes Ed. Second Edition, 2003
[2] Hassan, E.Y.; Enein. A.; Papadoyannis, I.N.; Samanidou,V. F. Sample
Pretreatment in Clinical Chemistry. Chapter 1: Separation Techniques in
Clinical Chemistry. Marcel Dekker. NewYork,USA; 2003.
[3] Henion, J.; Brewer, E.G. LC-MS Sample Preparation. Today’s Chemist at
Work p.36-42 February 1999.
[4] Majors, R. E. New Approaches to Sample Preparation. LC-GC
International 1995 8, 128-133.
[5] Pearce, J.C.; McDowall, R. D.; El Sayed, A.; Pichon, B. Automated analysis
of drugs in plasma using liquid-solid extraction and HPLC. International
Laboratory November/December 34-40, 1990.
[6] Rossi, D. T. ; Zhang, N. Automating solid-phase extraction: current aspects
and future prospects. Journal of Chromatography A 2000 885, 97-113.
[7] Wells, M. Principles of extraction and the extraction of semivolatile
organics from liquids in Sample Preparation Techniques in Analytical
Chemistry. John Wiley & Sons, Inc.; 2003.
[8] Snow, N.H. Solid phase extraction of drugs from biological matrices.
Journal of Chromatography A 2000 885, 445-455.

[9] Hennion, M.C. Solid-phase extraction: method development, sorbent, and
coupling with liquid chromatography. Journal of Chromatography A 1996
85, 63-54.
[10] Majors, R.E. Liquid Extraction Techniques for Sample Preparation. LC-GC
[11] Huck, W.; Bonn, G.K. Recent developments in polymer-based sorbents for
solid-phase extraction. Journal of Chromatography A 2000 885, 51-72.
[12] Fritz, J.S.; Masso, J.J. Miniaturized solid-phase extraction with resin disks.
Journal of Chromatography A 2000 909, 79-85.
[13] Bert Ooms, J.A.; Van Gils, G.J.M.; Duinkerken, A.R.; Halmingh, O.
Development and validation of protocols for solid-phase extraction coupled
to LC and LC-MS. International Laboratory 2000 11, 18-23.
[14] Johnsson, J.A.; Mathiasson, L. Membrane based techniques for sample
enrichment. Journal of Chromatography A 2000 902, 205-225.
[15] Majors, R. E. The use of membranes in sample preparation. LC-GC
[16] Delaunay-Bertoncini, N.; Pichon, V.; Hennion, M.-C. Immunoextraction: A

highly selective method for sample preparation. LC/GC Europe 2001 14,
164-172.
[17] Boos, K.S.; Rudolphi, A. The Use of restricted-Access media in HPLC, Part
I- Classification and Review. LC-GC International 1998 11, 84-95.
[18] Queiroz, M.E.C.; Lanças, F.M. Practical Tips on Preparing Plasma Samples
for Drug Analysis Using SPME. LC-GC North America 2004 22, 970-980.
[19] Lord, H.; Pawliszyn, J. Microextraction of drugs. Journal of
Chromatography A 2000 902, 17-63.
[20] Lord, H. ; Pawliszyn, J. Review Evolution of solid-phase microextraction
technology. Journal of Chromatography A 2000 885, 153-193.
[21] Lord, H.L.; Pawliszyn, J.; Recent Advances in Solid-Phase Microextraction.
LC-GC International 1998 11, 776-785.
[22] Ulrich, S. Solid-phase microextraction in biomedical analysis. Journal of
Chromatography A 2000 902, 167-194.
Chapter 4
ANTI-CANCER DRUGS
The available anticancer drugs have discrete mechanisms of action, which
may vary in their effects on different types of normal and cancer cells which
demonstrate very few biochemical differences. There is no single cure for cancer,
since there is not a single type of cancer. On the contrary there are more than a
hundred different types of cancer. The effectiveness of many anticancer drugs is
limited by their toxicity to normal rapidly growing cells in the intestinal and bone
marrow areas. A final problem is that cancerous cells, which are initially
suppressed by a specific drug, may develop a resistance to that drug. For this
reason cancer chemotherapy may consist of using several drugs in combination
for varying lengths of time.

Besides their beneficial effect, chemotherapy drugs may sometimes cause
toxic effects.
The common approach in chemotherapy is to decrease the growth rate (cell
division) of the cancer cells. Side effects may be seen in systems that naturally
have a rapid turnover of cells including skin, hair, gastrointestinal, and bone
marrow. These healthy, normal cells also end up damaged by the chemotherapy
program.
The most commonly-used anticancer agents can be divided into three main
categories based on their mechanism of action.
a. Those which stop the synthesis of pre DNA molecule building blocks:
These agents work by blocking some step in the formation of nucleotides or
deoxyribonucleotides (necessary for making DNA).
b. Those which directly damage the DNA in the nucleus of the cell: These
agents chemically damage DNA and RNA. They disrupt replication of the DNA
and either totally prevent replication or cause the manufacture of false DNA or
RNA.
c. Those which affect the synthesis or breakdown of the mitotic spindles:
These drugs disrupt the formation of mitotic spindles and therefore interrupt cell
division. [1]
Resveratrol is a phytoalexin produced naturally by several plants, when under

attack by pathogens such as bacteria or fungi. In mouse and rat experiments, anti-
cancer, anti-inflammatory, blood-sugar-lowering, chelating and other beneficial
cardiovascular effects of resveratrol have been reported.
Since 1997 when Jang reported that topical resveratrol applications prevented
the skin cancer development in mice treated with a carcinogen, dozens of studies
of the anti-cancer activity of resveratrol in animal models have been reported. In
vitro resveratrol interacts with multiple molecular targets, and has positive effects
on the cells of breast, skin, gastric, colon, esophageal, prostate, and pancreatic
cancer, and leukemia. The study of pharmacokinetics of resveratrol in humans
concluded that even high doses of resveratrol might be insufficient to achieve
resveratrol concentrations required for the systemic prevention of cancer due to its
poor systemic bioavailability. [2, 3]
Table 1 summarises the chromatographic methods for therapeutic drug
monitoring of anti-cancer drugs. In the following paragraphs an overview of

published methods on the analysis of anti-cancer drugs is provided.
4. 1. Analytical Methods
Monitoring of drug substances and metabolism products is routinely

accomplished using HPLC.
(E)-3,5,4Ά-trimethoxystilbene (BTM-0512) (trans-3,5,4Ά-trihydroxystilbene)
is a resveratrol analog with a variety of pharmacological action, including anti-
cancer properties, anti-allergic activity, estrogenic activity, antiangiogenic
activity, and vascular-targeting activity against microtubule-destabilization.
Pharmacokinetic data and suitable methods for determination of the compound in
plasma are mandatory. A rapid and sensitive liquid chromatographic–mass
spectrometric method for determination of (E)-3,5,4Ά-trimethoxystilbene in rat
plasma, using, has been developed and validated. Plasma samples were treated
with acetonitrile to precipitate proteins. Samples were then analyzed by HPLC on
Anti-Cancer Drugs 23
a 250mm x 4.6 mm, 5-μm, C18 column with methanol–water, 80:20 (v/v),
containing 10 mM ammonium acetate and 0.2% formic acid (pH 3.0), as mobile
phase, delivered at 0.85 mL/min. Carbamazepine was used as internal standard A
single-quadrupole mass spectrometer with an electrospray interface operated in
selected-ion monitoring mode was used to detect [M + H]+ ions at m/z 271.3 for
(E)-3,5,4Ά- trimethoxystilbene and m/z 237.5 for the internal standard. Linearity
was observed at concentrations ranging from 0.01 to 5.0 μg/mL. Intra-day and
inter-day precision provided RSD values lower than 12.9%, while accuracy was in
the range 94.8–104.7%. The limit of detection in plasma was 0.005 μg/mL. The
method was successfully applied to determine the concentration of (E)-3,5,4Ά-
trimethoxystilbene after oral administration of 86 mg/kg of the drug to rats and
can be used to investigate the pharmacokinetics of the compound. [4]
In vitro and in vivo biotransformation studies play an important role during
the drug development process. A major goal of studying the biotransformation of
a drug in both in vitro and in vivo models is to ensure toxicology studies expose
animals to the same therapeutic agent and metabolites as those observed in
humans. Wabnitz et al in their study, investigated the metabolism of CI-1040
using in vitro and in vivo models compared with metabolism observed in a human
bile sample. Samples sample were obtained from a cancer patient receiving a
MAP-Erk Kinase (MEK) inhibitor CI-1040 during an efficacy study against
tumor growth. MAP-Erk Kinase inhibitors are atypical in selectively inhibiting
various signals in the mitogenic cascade. MEK is involved in the transmission of
oncogenic and proto-oncogenic signals. A substance that blocks these key-

signaling molecules that are involved in tumor growth, progression and
metastasis, would be of significance in the treatment of cancer, and inflammatory
diseases. CI-1040 (2-(2-chloro-4-iodo-phenylamino)-Ncyclopropylmethoxy-3, 4-
difluoro-benzamide, is a potent and highly selective inhibitor of MEK. It directly
inhibits purified MEK with a 50% inhibitory concentration (IC50) of 17 nM (23).
CI-1040 reduces gene over-expression, when an abnormality is present in the
MEK pathway leading to increased cell growth and tumor production. Pre-clinical
data from various animal models has indicated significant tumor growth
inhibition. CI-1040 recently underwent phase II clinical trials.
Human bile and plasma samples were obtained immediately after
administration of CI-1040 to a patient with advanced colon cancer. A combination
of HPLC-radiochromatography (HPLC-RAM), LC/MS and LC/MS/MS
experiments were used to analyze all resulting metabolites.
Table 4.1. Overview of HPLC methods for therapeutic drug monitoring of anti-cancer drugs.
Analytes Sample type Sample Preparation Chromatography Recovery % Detection Reference

Resveratrol (trans-3,5,4Ά- Rat plasma Deproteinization with ACN C18 250mm x4.6 mm, 5 μm, 94.8–104.7% Single-quadrupole mass 4
trihydroxystilbene) MP: MeOH–water, 80:20 Linearity: 0.01 to 5.0 spectrometer ESI in
(v/v), 10 mM ammonium μg/mL SIM mode [M + H]+ at
acetate and 0.2% formic acid m/z 271.3 for (E)-
(pH 3.0), FR: 0.85 mL/min). 3,5,4Ά-
IS: carbamazepine trimethoxystilbene and
m/z 237.5 for IS.
Dup 941 and its Standard Zorbax SB-C8 4.6 mm x 15 UV diode array 210– 5
impurities solutions of cm, MP: ACN– 600 nm.
drug water–TFA (10:90:0.1,
v:v:v) gradient to ACN–
water–TFA (40:60:0.1, v:v:v).
6-thioguanine (6-TGN) Human red Deproteinization by HClO4 with Purospher RP 18-e, 5 μm. 73.1% and 84.0% for 6-TG at 341 nm and 7
and methyl 6- blood cell dithiothreitol by heating the sample Gradient. MP: 0.02 mol/L 6-TGN and Me6- Me6-MP at 304 nm
mercaptopurine for 45 min at 100 °C. potassium phosphate (pH 3.5) MPN derivatives,
nucleotides (Me6-MPNs) :MeOH (40:60 v/v). FR: 1.2 respectively
mL/min.
6-Thioguanine, 6- Human red Acid hydrolysis C18 (25 x 0.46 cm) UV: 6-TG at 342 nm, 8
mercaptopurine, and 6- blood cell Me6-MP at 303 nm
methylmercaptopurine
Dithiothreitol
6-MP, 6-MMP, Blood Incubation C18 5 μm (125x4 mm) Recovery: 70-89.9% UV: 290 nm 9
dithiothreitol, allopurinol, samples LiChrocart. MP: acetic acid Linear range: 6-MP
S-adenosyl-L-methionine 0.1% and ACN 100% in linear (0.15–8 mM)
gradient. FR: 1 mL /min. SAM (1–12 mM)
Methotrexate (MTX), Human Oxidation using 10-3M KMnO4 MP: Tris–NaCl buffer 89-122% Photometric and 10
urine (pH 5.0) and 35 min of oxidation solution with 15 mM Tris and Fluorimetric detectors,
time 1mM NaCl , pH 6.8 with HCl. in series, at 230 nm and
FR: 1.0 mL/min 444 nm (λex=280 nm)
5-fluorouracil (5FU), Εnvironmen Synergi 4u Max-RP Recovery: 92.4-99.9% MS/MS 11
methotrexate (MTX) and tal samples capillary column (0.5 x 50 Linearity: 1.1
cyclophosphamide (CP) of large mm, 5 μm, 80 A° ). MP: 20 and 3333.3 μg/L for
general mM ammonium acetate, pH 4 MTX and CP and
hospital with acetic acid and MeOH. between 33.3 and
Gradient. FR: 10 μL/min. 3333.3 for 5FU.
Unlabeled CI-1040 was administered (100 mg/day, QD) for 15 days to a

patient suffering from colon cancer. Bile was collected by the insertion of a T-
tube directly into the bile duct over a 14-h period. Metabolites were also
monitored in the patient‘s plasma. [5]
A reversed-phase high-performance liquid chromatographic assay was
developed to quantify intracellular metabolites of the cytotoxic drug 6-
mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-
thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a
single sample. A similar assay measures the parent thiopurine compounds. The
limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 · 108 red blood
cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-
methylmercaptopurine metabolites, respectively. [6]
6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6-
MPNs) are the two major metabolites found in erythrocytes after administration of
azathioprine. In order to understand the role of these metabolites in the
pharmacologic and toxic activity of thiopurines, an HPLC method was developed
for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes
after deproteinization by perchloric acid with dithiothreitol by Dervieux and
Boulieu. The nucleotides were hydrolyzed to their bases by heating the sample for
45 min at 100 °C. During acid hydrolysis Me6-MP was converted into a
compound analyzed on a Merck Purospher RP 18-e column, 5-μm, protected by a
Purospher RP 18-e guard column with a linear gradient elution mode with 0.02
mol/L potassium phosphate (pH 3.5) and 0.02 mol/L potassium phosphate (pH
3.5):methanol (40:60 v/v). The flow rate was 1.2 mL/min. Detection of 6-TG and
Me6-MP derivative was performed at 341 nm and 304 nm respectively. With this
procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN
derivatives, respectively, were found. [7]
Stefan et al in their paper presented in-depth methodological analysis and
optimization of the two previously described HPLC procedures [10 and 11] to
improve precision, turn-around time, ruggedness, and cost effectiveness.
Reversed-phase chromatography with UV detection was performed on a Waters
HPLC system. The two protocols were improved with regards to chromatographic
conditions, as well as reagent preparation, storage, and use. 6-Thioguanine
nucleotides (6-TGNs) were analyzed by optimized techniques in samples from
patients on thiopurine therapy (n = 24) and the results were compared. 6-
Mercaptopurine (6-MP) was used as internal standard in both procedures.
Isocratic elution with 5% acetonitrile (ACN) in 20 mmol/L phosphate buffer pH
2.5 allowed for minimal background interference in both protocols. 6-
Thioguanine, 6-mercaptopurine, and 6- methylmercaptopurine (6-MMP) eluted at
around 4, 5, and 6 min, respectively. Dithiothreitol (DTT) was critical only during
the acid hydrolysis step. With these optimized protocols recovery of 6-TGNs was
on average 1.38-fold higher in the Dervieux–Boulieu method and no interfering
peaks hindered analysis. [8]
Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the
metabolism of thiopurine drugs. A genetic polymorphism is responsible for large
inter-individual differences observed in TPMT activity. An HPLC method was
reported avoiding an extraction step and the use of radioactive reagents, based on
the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP)
using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates
(RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and
between-run variations showed high precision. The formation of 6-MMP was
linear with respect to the lysate concentration and time. The results of HPLC
method correlated with those of the radiochemical method. Because of the
absence of extraction step, this method of TPMT activity determination reduces
analysis variation and is time-saving and suitable for routine monitoring of TPMT
activity and for fundamental studies. [9]
An HPLC method, using UV and fluorimetric serial detection, for the
simultaneous determination of methotrexate (MTX), five disease marker
pteridines, and the reference metabolic subproduct creatinine (CREA) in human
urine was established after oxidation process using 10-3 M KMnO4 (pH 5.0) and
35 min of oxidation time to transform the analytes in the highly fluorescent
pteridinic rings. Chromatographic separation was achieved on a C18 Nova-Pack

column (150 × 3.9mm, 5 μm, Waters, Millipore Iberica, Barcelona, Spain). The
mobile phase consisted of Tris–NaCl buffer solution containing 15 mM Tris and 1
mM NaCl and adjusted to pH 6.8 with HCl delivered at a flow rate of 1.0 mL/min.
MTX and the assayed pteridines were monitored by Fluorescence at λem=444 nm
and λex=280 nm and creatinine was monitored by absorption measurements at 230
nm. All components were well resolved in approximately 7 min. Detection limits
were 10 ng/mL for MTX, less than 1 ng/mL for all of the pteridines, and 4 ng/mL
for CREA. [10]
A high-performance liquid chromatographic/electrospray ionization tandem
mass spectrometric
(HPLC/ESI-MS/MS) method was developed for the simultaneous
quantification of 5-fluorouracil (5FU), methotrexate (MTX) and
cyclophosphamide (CP) in environmental samples. Micro-HPLC analysis was
performed at a flow-rate of 10 μL/min using 20 mM ammonium acetate, adjusted
to pH 4 with acetic acid (solvent A), and methanol (solvent B). Separation was
accomplished on a Synergi 4u Max-RP capillary column (0.5 × 50 mm, 5 μm, 80
A° ), (Phenomenex, Torrance, CA, USA), by gradient elution as follows: after 1

min of isocratic elution, solvent B was increased from 15 to 50% in 0.5 min along
a linear gradient curve, then increased to 65% in 15 min. Finally, solvent B was
decreased from 65 to 15% in 0.5 min and the column equilibration was conducted
isocratically for 8 min. The total run time was 25 min. Transitions of the
protonated and deprotonated molecular ions ([M- H]- for 5FU, [M+ H]+ for the
other analytes and IS were monitored for quantitative analysis in the multiple
reaction monitoring (MRM) mode. Transitions m/z 129 42, 455  308, 261
140 and 323 154 were selected for 5FU, MTX, CP and TP (IS), respectively.
The present method offers high sensitivity, with detection limits of 1.1 μg/L for
MTX and CP and 33.3 μg/L for 5FU, avoiding any sample preconcentration
procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and
99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the
method suitable for the routine determination of these three antineoplastic drugs.
[11]
REFERENCES
[1] www.elmhurst.edu (February 2009)
[2] Farina, A.; Ferranti, C.; Marra, C. An improved synthesis of resveratrol.
Nat. Prod. Res. 2006 20, 247–52.

[3] Elliott, P.J.; Jirousek, M. Sirtuins: Novel targets for metabolic disease.
Current Opion in Investigational Drugs 2008 9, 1472–4472.
[4] Ma, N.; Liu, W.-Y.; Li, H.-D.; Jiang, X.-Y.; Zhang, B.-K.; Zhu, R.-H.;
Wang, F.; Liu, W.; Liu, X.; Xiang, D.-X. Determination of (E)-3,5,4 A-
Trimethoxystilbene in Rat Plasma by LC with ESI-MS. Chromatographia
2007 66, 251–255.
[5] Wabnitz, P.A.; Mitchell, D.; Wabnitz, D.A.M. In Vitro and in Vivo
Metabolism of the Anti-Cancer Agent CI-1040, a MEK Inhibitor, in Rat,
Monkey, and Human. Pharmaceutical Research 2004 21, 9.
[6] Lennard. L.; Singleton. H. High-performance liquid chromatographic assay
of the methyl and nucleotide metabolites of 6 mercaptopurine:
quantification of red blood cell 6 thioguanine nucleotide, 6 thioinosinic acid
and methylmercaptopurine metabolites in a single sample. Journal of
Chromatography 1992 58, 383–390.
[7] Dervieux, T.; Boulieu, R. Simultaneous determination of 6-thioguanine and
methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by
HPLC. Clinical Chemistry 1998 44, 551-555.
[8] Stefan, C.; Walsh, W.; Banka, T.; Adeli, K.; Verjee, Z.; Improved HPLC
methodology for monitoring thiopurine metabolites in patients on thiopurine
therapy. Clinical Biochemistry 2004 37, 764– 771.
[9] Anglicheau , D.; Sanquer , S.; Loriot , M.-A.; Beaune , P.; Thervet, E.
Thiopurine methyltransferase activity: new conditions for reversed-phase
high-performance liquid chromatographic assay without extraction and
genotypic–phenotypic correlation. Journal of Chromatography B 2002 773,
119–127.
[10] Duran Meras, I.; Espinosa Mansilla, A.; Rodriguez Gomez, M. J.
Determination of methotrexate, several pteridines, and creatinine in human
urine, previous oxidation with potassium permanganate, using HPLC with
photometric and Xuorimetric serial detection. Analytical Biochemistry 2005
346, 201–209.
[11] Sabatini, L.; Barbieri, A.; Tosi, M.; Saverio Violante, F. A new high-
performance liquid chromatographic/ electrospray ionization tandem mass
spectrometric method for the simultaneous determination of
cyclophosphamide, methotrexate and 5-fluorouracil as markers of surface
contamination for occupational exposure monitoring. Journal of Mass
Spectometry 2005 40, 669–674.
Chapter 5
BRONCHODILATORS
Bronchodilators are medicines that help open the bronchial airways of the
lungs, so that more air can flow through them.
People with asthma have problematic breathing, since their airways are
inflamed and become narrowed. Air moves smoothly through the airways and into
the lungs during inhaling. Exhaling happens automatically, when the person stops
breathing in. In a person with asthma incoming air can slide around the blockage,
because the act of breathing in makes the airways expand. The problem in people
with asthma comes during exhaling. The air remains trapped in the lungs so that
the patient can then only take shallow breaths. Bronchodilators work by relaxing
the smooth muscles lining the airways. This results in opening of the airways
wider and allows air to leave the lungs. These drugs also are used to relieve
breathing problems associated with emphysema, chronic bronchitis, and other
lung diseases.
Some bronchodilators are inhaled, using a nebulizer or an inhalation aerosol.
Others are taken as injections or orally. Dosage depends on the type of
bronchodilator and may be different for different patients. [1]
Theophylline, also known as 1,3 dimethylxanthine, is a methylxanthine drug
used in therapy for respiratory diseases. Due to its numerous side-effects, is are
now less administered for clinical use. The mechanism of action of theophylline
involves relaxing of bronchial smooth muscle, increasing of heart muscle
contractility and efficiency, increasing heart rate, increasing blood pressure,
increasing renal blood flow, anti-inflammatory effects and central nervous system
stimulatory effect. Its bioavailability is quantitative. Theophylline is distributed in
the extracellular fluid, in the placenta, in the mother's milk and in the central
nervous system. The protein binding is 40%. The volume of distribution is 0.5
L/kg and may increase in neonates and those suffering from cirrhosis or
malnutrition, whereas the volume of distribution may decrease in those suffering

from obesity.
Theophylline is metabolized extensively (up to 70%) in the liver, by N-
demethylation via cytochrome P450 1A2. It is metabolized by parallel first order
and Michaelis-Menten pathways. Metabolism may become saturated (non-linear),
even within the therapeutic range. Small dose increases may result in large
increases in serum concentration. Clearance of the drug is influenced by age, so
that it is increased in children 1 to 12, teenagers 12 to 16, adult smokers, elderly
smokers or decreased in elderly. Other dieseases such as cystic fibrosis,
hyperthyroidism, acute congestive heart failure, cirrhosis, hypothyroidism and
febrile viral illness may also affect theophylline‘s clearance positively or
negatively. [2,3]
Table 5.1. summarises the chromatographic methods for therapeutic drug
monitoring of bronchodilators.
Table 5.1. Overview of HPLC methods for therapeutic drug

monitoring of bronchodilators.
Analytes Sample Sample Preparation Chromatography Detection Reference

type
Theophylline Rat Blood LLE with isopropyl Novapak C18 4
Ciprofloxacin samples alcohol. Vortex Mix. (3.9x150 mm) MP:

Centrif. Evap. organic ACN, MeOH, TCA
Layer and pH=3 with 1M NaOH.
reconstitution of IS: isopropyl analog of
residue with MP. ciprofloxacin.
Theophylline Human LLE with chloroform/ Waters RP C18, 39x150 UV: 267 5
serum isopropanol. Vortex mm.MP:ACN: nm
Mix and Centrif. phosphate buffer (94:6
Organic phase dried v:v). FR: 1.6 mL /min.
under N2. Extract IS: (b-OH ethyl-
dissolved with MP. theophylline 40 mg /L)
Theophylline Intestinal LLE with chloroform– Waters Novapak C-18 UV: 271nm 6
fluid isopropyl alcohol 1:1. (4 μm). MP: 10mM
Dilution in KH2PO4. KH2PO4–ACN–
Extraction vortex and MeOH (900:25:90 v/v).
centrif. The organic FR: 2mL/min.
layer evap. to dryness. IS: diprophylline
The residue dissolved
in MP.
Bronchodilators 31
5.1. Analytical Methods
Theophylline requires frequent control because of its narrow therapeutic

index.
Theophylline and ciprofloxacin were determined in blood samples of
cadmium-exposed and control rats. Cadmium has been associated with a number
of adverse health effects but the impact of those effects on the pharmacokinetics
of different drugs has not been investigated. The pharmacokinetics of theophylline
and ciprofloxacin were studied in cadmium-exposed and control rats (72 rats)
following i.p. (6.5 mg/kg) and p.o. (10 mg/kg) administration, respectively. The
third-generation off springs of rats exposed to 100 mg/mL of cadmium chloride in
drinking water were used in this study. Following eight weeks of exposure,
animals received the drugs as a single dose.
Blood samples were withdrawn at different time-points and the plasma
concentrations of both drugs were analyzed by HPLC after LLE extraction with
2mL of chloroform. The pharmacokinetic parameters of theophylline and
ciprofloxacin were altered significantly in the cadmium exposed animals.
HPLC analysis was performed within 8 min using a Novapak C18 column
(3.9x150 mm, 5 μm).
A significant effect of chronic exposure to cadmium on the pharmacokinetics
of the two selected drugs was demonstrated in this study. Theophylline Cl/F was
found to be reduced by 41% due to the effect of Cd exposure on the liver, which
could be hazardous to humans, since theophylline has a narrow therapeutic
window. The pharmacokinetics of ciprofloxacin on exposure to cadmium was not
altered as drastically as that of theophylline. [4]
Theophyline was determined in human serum. Samples were extracted by
chloroform and isopropanol mixture. After centrifugation, the organic phase was
dried under nitrogen. Finally the extract was dissolved in 100 mL of mobile
phase. HPLC–UV measurements were performed using a Waters RP C18, 39 ×
150 mm column. The mobile phase consisted of acetonitrile: phosphate buffer
(94:6 v:v) delivered at a flow rate of 1.6 mL/min. The detector wavelength was set
at 267 nm and the temperature of the column maintained at 50°C. [5]
Theophylline was determined in intestinal fluid after LLE with 2 mL
chloroform–isopropyl alcohol 1:1, vortex mixing for 30 s and centrifugation, the
water layer was discarded and the organic layer evaporated to dryness under a
gentle stream of air. The residue was dissolved in 200 μL mobile phase, of which
100 μL was injected into the HPLC system. The column used was a Waters
Novapak C-18 column (4 μm) and the mobile phase was 10 mM KH2PO4–
acetonitrile– methanol (900:25:90 v/v). The flow was maintained at 2 mL/min.
Spectrophotometric detection at 271 nm was applied. Diprophylline theophylline

was used as internal standard. Less than 20% of the total dose was dissolved from
the slow-release pellets after 120min, while dissolution from the immediate-
release formulation was found to be complete after 45–90 min in the different
media. [6]
REFERENCES
[1] www.healthatoz.com (Accessed February 2009)
[2] www.theophyllineatpriory.com (Accessed February 2009)
[3] www.rxlist.com (Accessed February 2009)
[4] Zaghloul, I. Y.; Radwan, M.A.; Aly, Z.H. The effect of chronic cadmium
exposure on the pharmacokinetics of theophylline and ciprofloxacin in rats.
Journal of Trace Elements in Medicine and Biology 2007 21, 132–137.
[5] Jourquin, G.; Kauffmann, J.-M. Fluorimetric determination of theophylline
in serum by inhibition of bovine alkaline phosphatase in AOT based
water/in oil microemulsion. Journal of Pharmaceutical and Biomedical
Analysis 1998 18, 585–596.
[6] Brouwers, J.; Ingels, F.; Tack, J.; Augustijns, P. Determination of
intraluminal theophylline concentrations after oral intake of an immediate
and a slow-release dosage form. Journal of Pharmacy and Pharmacology

2005 57, 987–995
Chapter 6
CARDIOVASCULAR DRUGS
The angiotensin converting enzyme (ACE) inhibitor captopril, which was
developed around 1975 was the first drug designed to block a particular target
protein, and has subsequently become the preferred therapy for hypertension and
congestive heart failure. [1]
The statins (or HMG-CoA reductase inhibitors) are a class of drugs that lower
cholesterol levels in people with or at risk of cardiovascular disease. They lower
cholesterol by inhibiting the enzyme HMG-CoA reductase, which is the rate-
limiting enzyme of the mevalonate pathway of cholesterol synthesis. Inhibition of
this enzyme in the liver stimulates low-density lipoprotein (LDL) receptors,
resulting in an increased clearance of LDL from the bloodstream and a decrease in

blood cholesterol levels. The first results can be seen after one week of use and
the effect is maximal after four to six weeks.
Based on clinical trials and the increasing focus on aggressively lowering
LDL-cholesterol, the statins continue playing an important role in the primary as
well as in secondary prevention of coronary heart disease, myocardial infarction,
stroke and peripheral artery disease. Statins also appear to have a favorable effect
on inflammation, cancer etc. [2,3]
Table 6.1 summarises the chromatographic methods for therapeutic drug
monitoring of cardiovascular drugs. In the following paragraphs an overview of
published methods on the analysis of these drugs is provided.
Table 6.1. Overview of HPLC methods for therapeutic drug monitoring of cardiovascular drugs.
Table 6.1. Overview of HPLC methods for therapeutic drug monitoring of cardiovascular drugs.
Analytes Sample Sample Preparation Chromatography Recovery % Detection Reference
type
Eprosartan Plasma SPE Waters Atlantis dC18, 93.4-102.8%. UV: 232 nm 5
Samples 100Χ3.9mm, 3μm, 100 °A, Linearity:
35±0.2 ◦C, MP: 0.031% TFA 150–4000
and ACN 0.026% TFA, ng/mL
gradient. FR=1.25 mL/min.
IS=irbesartan
XUESETONG XUESETO Filtration through a 0.45 A ZORBAXSB-C18 95.5 - 100.0 DAD detector: 203 6
injection: saponins NG μm membrane. (2.1x150mm, 5μm) Gradient. % nm
made injection MP: ammonium acetate Lineariry: ESI-MS/MS:
from Panax and ACN, FR: 0.4 ml/min, at 0.0002-
notoginseng 25 ◦C. 1.0900
(μg/μl)
Pholedrine (40- Blood SPE (SPEC C18) LC- MS/MS: RP-18 gradient 67% MS/MS 8
hydroxymethamphet plasma and 50 to 70% of B MeOH/ACN linearity: 1– positive ionization,

amine) urine 3/1 (v/v), 0.02% acetic acid) in 100 ng/mL (MRM) mode
A (5 mM ammonium
acetate/ACN 95/5 (v/v), 0.02%
acetic acid. IS: D11-
methamphetamine, D5-
methylenedioxymethampheta
mine
Homocysteine Total Derivatization with Discovery C18, MP: ACN– 95% Fluorimetric 9
plasma ammonium-7- potassium λ (Ex): 385 and
fluorobenzo-2-oxa-1,3- dihydrogenphosphate λ(Em): 515 nm
diazole-4-sulphonate after buffer 0.1 M (8:92, v:v)
reduction with tri-n- (pH=2.1).
butylphosphine
Captopril and its Human LLE C-18. MP: MeOH-ACN- Linearity: 5 Fluorescence 11
disulfide metabolites plasma phosphate buffer (0.02 ng/mL detection at ex 235
(total captopril) molΒ·L-1, pH 6.4) (30:30:135, (LOQ) and nm and em 440 nm
v/v), FR: 1 mL/min 300 ng/mL
Cardiovascular Drugs 35
The 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase

inhibitors, more commonly known as ‗statins‘, are a novel class of drugs widely
used for the treatment of hypercholesterolaemia in patients with established
cardiovascular disease as well as those at high risk of developing atherosclerosis.
High performance liquid chromatography (HPLC) in combination with tandem
mass spectrometry (MS/MS) is the dominant analytical technique for the
quantification of statins in biological samples. An excellent and comprehensive
review has be written on the most of the methods used for quantification of statins
are in plasma and they are suitable for therapeutic drug monitoring of these drugs.
[4]
A solid phase extraction-reversed phase high performance liquid
chromatographic (SPE-RP-HPLC) method with photometric detection for
monitoring the antihypertensive drug eprosartan has been validated in order to
assure good quantitation of eprosartan in plasma samples obtained from patients
under cardiovascular treatment. No interferences were observed from endogenous
compounds of plasma and other drugs which are commonly co-administered in
elderly patients. The recoveries of eprosartan from plasma samples, measured at
three levels of the linear concentration range (150–4000 ng/mL) were found to be
between 93.4 and 102.8%. The intra-day and inter-day precision and accuracy
were always lower than 13% with regards to RSD and 4% with regards to RE.
The analytical column was a Waters Atlantis dC18, 100 × 3.9mm, 3 μm,
thermostated at 35±0.2 ◦C, protected with a guard column Waters Bondapak C18
10 μm. The mobile phase consisted of a mixture of water 0.031% TFA and
acetonitrile 0.026% TFA, low pressure mixed, and delivered in gradient mode at a
flow rate of 1.25 mL/min. Irbesartan was used as internal standard. Photometric
detection was performed at 232 nm.
The use of this method can save effort when monitoring patients who take
several medications, especially when polar drugs are mixed. The validity, LOQ
and the linearity range of the method make it acceptable for eprosartan monitoring
during 24 h after dose intake which is necessary to assure that antihypertensive
drug plasma levels are included in the therapeutic range during all the interdose
range to decrease the incidence of cardiovascular events. [5]
An interested paper has been reported on the development of an HPLC–ESI-
MS/MS method for the qualitative and quantitative determination of gensinosides
and notoginsenosides, which is helpful to improve the quality control of Panax
notoginseng and its pharmaceutical preparations such as Xuesetong injection.
The latter is one of the most widely used proprietary medicines in traditional
Chinese medicine, which consists of total saponins made from Panax

notoginseng, used to treat cardiovascular diseases. Analysis was performed within
25 min, on a ZORBAXSB-C18 column (2.1 × 150 mm, 5μm) and a ZORBAX
ODS C18 guard column (4.6 × 12.5mm, 5 μm). Gradient elution was applied with
mobile phase consisted of aqueous ammonium acetate and acetonitrile, delivered
at a flow-rate of 0.4 mL/min and the system operated at 25 ◦C. Full scan and time
programmed selected reaction monitoring (SRM) were used for qualitative and
quantitative analysis of saponins, respectively in negative mode. Twenty-seven
saponins were identified and nine of them were quantified. Ten XUESETONG
injections were analyzed and compared. The results showed that there is a great
variation among different samples. The developed method is rapid, accurate and
sensitive for qualitative and quantitative analysis of saponins in Xuesetong
injection. Moreover, it also can be used for the quality control of Panax
notoginseng raw material and its preparations. The method can be modified to
include the analysis of biofluids. [6]
An interesting study investigated the interaction of exercise training and
chronic nitroglycerin treatment on blood pressure and alterations in nitric oxide
(NO), glutathione (GSH), antioxidant enzyme activities and lipid peroxidation in
rats. Fisher rats were divided into four groups: (1) sedentary control, (2) exercise
training for 8 weeks, (3) nitroglycerin (15 mg/kg, s.c. for 8 weeks) and (4) training
+ nitroglycerin for 8 weeks. Blood pressure, heart rate and respiratory exchange
ratio were monitored weekly for 8 weeks using tail-cuff method and
oxygen/carbon dioxide analyzer, respectively. The animals were sacrificed 24 h

after last treatment and plasma was isolated and analyzed using HPLC, ELISA
and UV-VIS spectrophotometric techniques.
Biochemical changes were accompanied by a significant increase in
respiratory exchange ratio (p < 0.001) without a significant change in blood
pressure and heart rate. Chronic nitroglycerin administration significantly
increased nitric oxide levels (p < 0.05), glutathione levels (p < 0.001), superoxide
dismutase activity (p < 0.05), Glutathione S-transferase activity (p < 0.05), and
decreased malon-dialdehyde levels (p < 0.05). These biochemical changes were
accompanied by a significant decrease in blood pressure (p < 0.05) and without
any significant changes in heart rate and respiratory exchange ratio. Interaction of
exercise training and chronic nitroglycerin treatment resulted in normalization of
plasma nitric oxide, malon- dialdehyde, lactate levels, and catalase activity. The
combination of exercise and nitroglycerin significantly enhanced glutathione
levels (p < 0.05), and the up-regulation of superoxide dismutase (p < 0.001),
glutathione peroxidase (p < 0.05), glutathione reductase (p < 0.05) and
Glutathione S-transferase (p < 0.001) activities. These biochemical changes were
Recent Developments in Drug monitoring by HPLC 37
accompanied by normalization of blood pressure and a significant increased in

respiratory exchange ratio (p < 0.001). The data suggest that the interaction of
physical training and chronic nitroglycerin treatment resulted in the maintenance
of blood pressure and the up-regulation of plasma antioxidant enzyme activities
and glutathione levels in the rat. [7]
Pholedrine (40-hydroxymethamphetamine) is a cardiovascular agent exerting
hypertensive and adrenergic effects. High doses may cause a drop in the
peripheral circulation blood flow and increase blood pressure, heart rate and body
temperature up to a state of central respiratory paralysis. A 15-year-old girl who
suffered from heavy agitation and hallucinations was admitted to the intensive
care unit in a comatose state. The toxicological analysis using GC/MS revealed a
considerable amount of pholedrine in blood and urine. A method for determining
pholedrine in human body fluids utilizing high-performance liquid
chromatography (HPLC)/tandem mass spectrometry (LC-MS/MS) with a turbo
ion-spray source was developed, using D11-methamphetamine and D5-
methylenedioxymethamphetamine as internal standards.
Samples were prepared by SPE extraction using SPEC-C18AR columns.
Chromatographic separation was achieved on an RP-18 stationary phase applying
gradient elution from 50 to 70% of B (methanol/acetonitrile 3/1 (v/v), 0.02%
acetic acid) in A (5 mM ammonium acetate/acetonitrile 95/5 (v/v), 0.02% acetic
acid, at pH 5). Supra-pure acetic acid was added to the post-column effluent with
a flow rate of 0.2 mL/min to optimize ionization.
Detection was carried out in the positive ionization, multiple reaction

monitoring (MRM) mode. The chromatograms showed no interference from other
substances. The limit of detection (LOD, S/N=3) of pholedrine was 0.8 ng/mL.
The intra-day R.S.D. between 5 and 80 ng/mL were 3.8–8.7% and the inter-day
R.S.D. between 5 and 100 ng/mL were 6.7–10.7%. The pholedrine concentrations
in blood and urine collected when the girl was still alive were 16.1 mg/mL
(R.S.D. 10.5%) and 1120 mg/mL (R.S.D. 8%), respectively. In post-mortem
samples, they were 23.0 mg/mL (R.S.D. 5.1%) in heart blood and 27.3 mg/g
(R.S.D. 6.6%) in the liver.
Interactions with endogenous or exogenous substances, such as metabolites,
were not observed. Calibration was linear in a range from 1 to 100 ng/mL, and
hence was suited for determining therapeutic concentrations. Concentrations of
toxicological relevance need dilution of the matrix and appropriate calibration. [8]
Total plasma homocysteine (tHcy) in newborn children may be a useful
biochemical marker for genetic risk of premature cardiovascular disease. A rapid,
isocratic HPLC method able to process very small amount of newborn plasma
samples was developed by Bartesaghi et al. Plasma sample from 1 to 10 mL was
derivatized with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate after

reduction with tri-n-butylphosphine and analyzed on Discovery C18 column, with
a solution of acetonitrile–dihydrogenphosphate 0.1 M (8:92 v:v at pH=2.1).
Fluorimetric detector with excitation (Ex) and emission (Em) wavelengths set at
385 and 515 nm respectively was used. The method is suitable for routine analysis
of tHcy and other aminothiols (Cys, Cys-Gly, Glutathione) assessed for clinical
and research purposes. The method was applied for monitoring tHcy levels in
1400 apparently healthy newborn babies. This HPLC method allows measurement
of tHcy in newborn during the routinary capillary blood collection in the fourth
living day without any other invasive procedure. [9]
Metoprolol and captopril were determined in human plasma of volunteers.
[10] Captopril and its disulfide metabolites (total captopril) human plasma liquid-
liquid extraction HPLC using a C-18 reversed phase column. The mobile phase
consisted of a methanol-acetonitrile-phosphate buffer (0.02 molΒ·L-1, pH 6.4)
mixture (30:30:135, v/v), and was set at a flow rate of 1 mL/min. Linearity was
observed with 5 ng/mL (lower limit of quantitation) and 300 ng/mL for total
captopril in plasma fluorescence detection at the excitation and emission
wavelengths of 235 nm and 440 nm. [11]
REFERENCES
[1] www.bmb.leeds.ac.uk (Accessed January 2009)

[2] www.jlr.org (Accessed January 2009)
[3] www.bmj.com (Accessed January 2009)
[4] Nirogi, R.; Mudigonda, K.; Kandikere, V. Chromatography–mass
spectrometry methods for the quantitation of statins in biological samples.
Journal of Pharmaceutical and Biomedical Analysis 2007 44, 379–387.
[5] Ferreiros, N.; Iriarte, G.; Alonso, R.M.; Jimenez, R.M.; Ortız, E. Validation
of a solid phase extraction-high performance liquid chromatographic
method for the determination of eprosartan in human plasma. Journal of
Chromatography A 2006 1119, 309–314.
[6] Lai, C.M.; Li, S.P.; Yu, H.; Wan, J.B.; Kan, K.W.; Wang, Y.T. A rapid
HPLC–ESI-MS/MS for qualitative and quantitative analysis of saponins in
―XUESETONG‖ injection. Journal of Pharmaceutical and Biomedical
Analysis 2006 40, 669–678.
[7] Husain, K.; Somani, S.M.; Boley T.M.; Hazelrigg, S.R. Interaction of
physical training and chronic nitroglycerin treatment on blood pressure and
Recent Developments in Drug monitoring by HPLC 39
plasma oxidant/antioxidant systems in rats. Molecular and Cellular

Biochemistry 2003 247, 37–44.
[8] Romhild, W.; Krause, D.; Bartels, H.; Ghanem, A.; Schoning, R.; Wittig, H.
LC-MS/MS analysis of pholedrine in a fatal intoxication case. Forensic
Science International 2003 133, 101–106.
[9] Bartesaghi, S.; Accinni, R.; De Leo, G.; Cursano, C.F.; Campolo, J.;
Galluzzo, C.; Vegezzi, P.G.; Parodi, O. A new HPLC micromethod to
measure total plasma homocysteine in newborn. Journal of Pharmaceutical
and Biomedical Analysis 2001 24, 1137–1141.
[10] Qu, F.-J.; Zhang, X.-J.; Song, L.; Xu, K.-J. Studies on the pharmacokinetic
interaction between metoprolol and captopril in healthy volunteers. Chinese
Pharmaceutical Journal 2001 36, 111-113.
[11] Zhong, D.; Li, X.; Wang, A.; Chen, X. Determination of captopril plus its
disulfide metabolites in human plasma. Yaoxue Xuebao 1998 33, 605-609.
Chapter 7
ANTIPSYCHOTICS
Antipsychotics are a group of psychoactive drugs commonly used to treat
psychosis typified by schizophrenia. A wide range of antipsychotics have been
developed till now. The first generation of antipsychotics, known as typical
antipsychotics, was discovered in the 1950s. The second generation is known as
atypical antipsychotics. Both classes of medication act by blocking receptors in
the brain's dopamine pathways. Excess release of dopamine in the mesolimbic
pathway has been linked to psychotic experiences. Besides beneficial effects a
number of side effects have been observed in relation to specific medications. The
development of new antipsychotics, and the relative efficacy of different ones, is
an important ongoing field of research. The decision upon the most appropriate
drug for an individual patient requires careful consideration. Antipsychotics are
used in the treatment of schizophrenia, mania, and delusional disorder. They
might be also used to counter psychosis associated with a wide range of other
diagnoses, such as psychotic depression, as well as to treat non-psychotic
disorders e.g. Tourette syndrome, or Asperger's syndrome.
Typical antipsychotics are not particularly selective and also block dopamine
receptors in other pathways, leading to unwanted side effects. They are classified
on a spectrum of low potency to high potency.Potency refers to the ability of the
drug to bind to dopamine receptors. High-potency antipsychotics such as
haloperidol, in general, have doses of a few milligrams and cause less sleepiness
and calming effects than low-potency antipsychotics, such as chlorpromazine and
thioridazine, which have dosages of several hundred milligrams. The latter have a
greater degree of anticholinergic and antihistaminergic activity, which can
counteract dopamine-related side effects.
Atypical antipsychotic drugs have a similar blocking effect on D2 receptors.
Some also block or partially block serotonin receptors (particularly 5HT2A, C and
5HT1A receptors): ranging from risperidone, which acts on serotonin receptors, to

amisulpride. The latter has no serotonergic activity. The additional effects on
serotonin receptors may be why some of them can benefit the 'negative symptoms'
of schizophrenia. [1,2]
Aripiprazole has been approved by the Food and Drug Administration (FDA)
in 2002 for the treatment of schizophrenia. More recently it received FDA
approval for the treatment of acute manic and mixed episodes associated with
bipolar disorder, and as an adjunct for the treatment of depression. Its mechanism
of action is different from other atypical antipsychotics (e.g., clozapine or
risperidone). Aripiprazole appears to be a D2 partial agonist and selective agonist
instead of antagonizing the D2 receptor. It acts also as a partial agonist at the 5-
HT1A receptor, and like the other atypical antipsychotics displays an antagonist
profile at the 5-HT2A receptor. It has moderate affinity for histamine and α-
adrenergic receptors and for the serotonin transporter, and no appreciable affinity
for cholinergic muscarinic receptors. [3]
Chlorpromazine, a phenothiazine antipsychotic, is the oldest of the
antipsychotic drugs, synthesized in 1950. It is a typical antipsychotic, mainly used
in the treatment of schizophrenia, though it has also been used to treat severe
manic episodes in people with bipolar disorder. Its use has been characterised as
the single biggest advance in psychiatric treatment. Currently its use has been
largely supplanted by the newer atypical antipsychotics.
Chlorpromazine works on a variety of receptors in the central nervous system
including anticholinergic, antidopaminergic and antihistamine effects as well as

some antagonism of adrenergic receptors. It has minimal effect on the
serotonergic pathways. It also has anxiolytic (alleviation of anxiety) properties.
[4,5]
Clozapine is an antipsychotic medication used in the treatment of
schizophrenia. The first of the atypical antipsychotics to be developed, it was first
introduced in Europe in 1971. It was voluntarily withdrawn by the manufacturer
in 1975, after it was shown to cause agranulocytosis that led to death in some
clozapine-treated patients. In 1989, the FDA approved clozapine's use only for
treatment-resistant schizophrenia. In 2002 it was approved by the FDA that
clozapine for patients with schizophrenia, reduces the risk of suicidal behavior.
Due to its potential to cause many severe side effects, it is relegated to third-line
use. It is only used in patients when all other anti-psychotics have failed. Safer use
of clozapine requires weekly blood monitoring for around five months followed
by four weekly testing thereafter. [6, 7]
.
Antipsychotics 43
Table 7.1. Overview of HPLC methods for therapeutic drug monitoring of antipsychotics.

Chlorpromazine, haloperidol, Human Plasma with EDTA as anticoagulant. Chromsep C8 (150×4.6 mm, 5 μm) ≥93% UV: 238 nm 9
Loxapine, clotiapine), plasma Centrif. SPE with cyanopropyl at RT. MP: ACN (30%, v/v) and a
(clozapine, quetiapine and cartridges. pH 3.0, 30 mM phosphate buffer
risperidone) and their active with 0.5% triethylamine (70%,
metabolites (N- v/v). FR: 1.0 mL/min. IS:
desmethylclozapine, clozapine amitriptyline
N-oxide and
9-hydroxyrisperidone
48 antidepressants and Human Protein precipitation Chromolith Speed ROD C18, 50 92-111% MS/MS in 10
antipsychotics serum mm x 4.6mm , 5 μm. MP:MeOH Linearity: positive MRM
and 5mM acetic acid, pH 3.9 and 0.5–2000 mode
ammonia solution, gradient. FR: ng/mL
1.0 mL/min.
18 antidepressants, four Human SPE Nucleosil (250x4.6 mm ) 75-99% UV: 230 nm 11
atypical antipsychotics serum containing 100-5-Protect 1
and active metabolites (endcapped), MP: 25 mM KH2PO4

(pH 7.0)–ACN (60:40). FR: 1
mL/min. IS: melperone
Aripiprazole and dehydro- Human LLE with heptane and isopropanol 98:2 C18 X Bridge® C18 3.5-μm 100 mm 98 to 113% DAD: 200 to 12
aripiprazole plasma (v/v) x 4.6mm. MP: ACN: ammonium linear :2–1000 350 nm
buffer (10mM; pH 8.35) (60:40, ng/mL
v/v) FR: 1mL/min. IS:
chlorohaloperidol
Quetiapine Human SPE on Oasis HLB. Elution with MeOH. C8 (150x4.6 mm , 5 92% UV: 254 nm 13
plasma mm). MP: ACN, MeOH and pH Linearity: 4-
1.9 phosphate buffer. IS: /400 ng/ml
triprolidine.
Risperidone was approved by FDA in 1993 for the treatment of

schizophrenia. In 2007 risperidone was approved as the only drug agent available
for treatment of schizophrenia in ages from 13 to 18 years old; it was also
approved for treatment of bipolar disorder in youth and children ages 10–18,
joining lithium. In 2003 the FDA approved risperidone for the short-term
treatment of the mixed and manic states associated with bipolar disorder. Like
other atypical antipsychotics, risperidone has also been used off-label for the
treatment of anxiety disorders, such as obsessive-compulsive disorder; severe,
treatment-resistant depression with or without psychotic features; Tourette
syndrome; disruptive behavior disorders in children; and eating disorders. [8]
monitoring of antipsychotics. In the following paragraphs an overview of
published methods on the analysis of antipsychotics is provided
Antipsychotics neuroleptics (chlorpromazine, haloperidol, loxapine and

clotiapine), atypical antipsychotics (clozapine, quetiapine and risperidone) and
their active metabolites (N-desmethylclozapine, clozapine N-oxide and 9-
hydroxyrisperidone were determined in human plasma.
The blood samples were collected from patients subjected to therapy with two
or more of the drugs of interest for at least 2 weeks at constant daily doses. Blood
samples were usually drawn 12 h after the last drug administration. Blood was
stored in glass tubes containing EDTA as the anticoagulant, and then centrifuged
(within 2 h from collection) at 1,400×g for 15 min; the supernatant (plasma) was
then transferred to polypropylene tubes and stored at −20 °C until HPLC analysis.
Control plasma was obtained in the same way from blood drawn from healthy
volunteers not subjected to any pharmacological treatment.
The solid-phase extraction procedure was carried out on IST Isolute
cyanopropyl (CN) cartridges (50 mg/1 mL). Separation was obtained using a C8
Chromsep column (150 × 4.6-mm, 5 μm) at room temperature. The mobile phase
consisted of a mixture of acetonitrile (30%, v/v) and a pH 3.0, 30 mM phosphate
buffer containing 0.5% triethylamine (70%, v/v), delivered at a flow rate of 1.0
mL/min. Amitriptyline was used as the internal standard. Column effluent was
monitored at 238 nm. The limits of quantitation (LOQ) were lower than 2.6
ng/mL, while the limits of detection (LOD) were lower than 0.9 ng/mL, for all
analytes. The method was applied successfully to plasma samples from
Antipsychotics 45
schizophrenic patients undergoing polypharmacy with two or more different

antipsychotics. [9]
An interested HPLC method was developed for the simultaneous
determination of 48 antidepressants and antipsychotics in human serum. This
method describes the simultaneous determination of amisulpride, amitriptyline,
aripiprazole, benperidol, chlorpromazine, chlorprothixene, citalopram,
clomipramine, clozapine, desipramine, doxepin, fluoxetine, flupentixol,
fluphenazine, fluvoxamine, haloperidol, hydroxyrisperidone, imipramine,
levomepromazine, maprotiline, mianserine, mirtazapine, moclobemide,
norclomipramine, nordoxepin, norfluoxetine, nortriptyline, O-
desmethylvenlafaxine, olanzapine, opipramol, paroxetine, perazine, perphenazine,
pimozide, pipamperone, quetiapine, reboxetine, risperidone, sertraline, sulpiride,
thioridazine, trazodone, trimipramine, venlafaxine, viloxazine, ziprasidone,
zotepine and zuclopenthixol with a single sample/ triple injection approach. Drugs
were assigned to subgroups covering low, medium and high concentrations
(overall range of therapeutic levels to be considered: 0.5–2000 ng/mL) by further
dilution of the supernatant obtained after the first protein precipitation.
Chromatographic separation was necessary for isobaric mass fragments and
performed on a monolithic column Chromolith Speed ROD C18, 50 mm x 4.6
mm, 5 μm.
The mobile phase was a mixture of methanol and 5mM acetic acid, pH 3.9
and ammonia solution delivered by a gradient elution program. The flow rate was
1.0 mL/min. The injection interval was 8 min. A set of three internal standards
was used for quantification of drugs, which are characterised by widely different
hydrophobicity. After electrospray ionization positive ion fragments were
detected in the multiple reaction monitoring (MRM) mode. Regression parameters
of calibration curves and limits of quantification showed good covering of
therapeutic and subtherapeutic ranges, within the range 0.5–2000 ng/mL for all
analytes. Average coefficients of variation were 6.1% for intra-assay and 7.4% for
inter-assay comparisons, while average deviations from spiked concentrations
were 4.8% for intra-assay and 4.2% for inter-assay comparisons, respectively.
Recovery rates, measured as the percent recoveries of spiked serum samples
against standard solutions without serum matrix, varied between 92 and 111%,
with an average of 101%, except for olanzapine for which response was much
higher (185%) in serum matrix than in matrix-free controls. The method allows a
general view on the individual intake of psychoactive drugs and its accurate
quantification as well. [10]
Therapeutic drug monitoring of antidepressants necessitates efficient, fast and
reliable analytical methods validated by external quality control. An isocratic
reversed-phase HPLC method with ultraviolet detection was developed and

optimised to quantify eighteen antidepressants, four atypical antipsychotics and
active metabolites mirtazapine, reboxetine, moclobemide, venlafaxine, O-
desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine,
sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine,
doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine,
maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine,
risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of
the drugs and metabolites, the chromatographic separation was achieved on a
Nucleosil 100-Protect 1 column. The mobile phase consisted of 25 mM potassium
dihydrogenphosphate (pH 7.0)–acetonitrile (60:40) delivered at a flow-rate of 1
mL/min. Melperone was used as internal standard 75-99%. UV detection was
applied at 230 nm. The method was validated for therapeutic and toxic serum
ranges. A linear relationship (r=0.998) was obtained between the concentration
and the detector signal. Recoveries were between 75 and 99% for the drugs and
metabolites. The accuracy of the quality control samples, expressed as percent
recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard
deviations were 0.9–10.2% and 0.9–9.7%, respectively. This method is applicable
to rapidly and effectively analyze serum or plasma samples for therapeutic drug
monitoring of about 30 antidepressants and atypical antipsychotics, since it allows
an efficient and rapid analysis of serum concentrations within 24 h with a single
system. [11]
A high-performance liquid chromatographic method with diode array

detection (HPLC-DAD) was developed for quantification of aripiprazole and
dehydro-aripiprazole, in human plasma after LLE with heptane and isopropanol in
a ratio of 98:2 (v/v) as extracting solvents. The HPLC chromatographic separation
of compounds was carried out on a C18 column X Bridge® 3.5-μm, 100mm ×
4.6mm (Waters). Compounds were eluted isocratically using a mobile phase
consisting of acetonitrile: ammonium buffer (10mM; pH 8.35) (60:40, v/v) with a
flow rate of 1 mL/min. Column effluent was monitored using a diode array
detector in the range 200-350 nm. Chlorohaloperidol was used as internal
standard. Recovery obtained ranged from 98 to 113%. The total run time was 7
min at a flow-rate of 1.0 mL/min. The precision values were less than 12% and
the accuracy values were ranging from 98 to 113% and the lower limit of
quantification was 2 ng/mL for both compounds. Calibration curves were linear
over a range of 2–1000 ng/mL. The mean plasma concentrations in patients
treated with aripiprazole were 157 and 29 ng/mL for aripiprazole and dehydro-
aripiprazole, respectively. This method is a rapid, reproducible and accurate
method to quantify both aripiprazole and dehydroaripiprazole. Due to the fact that
Antipsychotics 47
is is easy and less expensive than those based on mass spectrometry detection,
which is not available in many laboratories, it represents therefore an alternative
procedure for routine therapeutic drug monitoring of patients treated with
aripiprazole. [12]
A precise and feasible high-performance liquid chromatographic (HPLC)
method for the analysis of the novel antipsychotic drug quetiapine in plasma has
been developed. The analysis was carried out on a C8 (150 × 4.6 mm, 5 μm)
column, using a mixture of acetonitrile, methanol and pH 1.9 phosphate buffer as
the mobile phase. Triprolidine was used as the internal standard. Mean recovery
was 92%. Linearity ranged from 4 to 400 ng/mL. UV detection was performed at
254 nm. Careful pretreatment of the biological samples was implemented by
means of solid-phase extraction (SPE) Oasis HLB (Hydrophilic/Lipophilic
Balance) cartridges (30 mg/1 mL). The application to some plasma samples of
patients treated with quetiapine containing tablets gave satisfactory results. The
accuracy was good (quetiapine mean recovery 92%), as well as the precision
(mean RSD 3.3%). The method seems to be suitable for the clinical monitoring of
patients treated with quetiapine. The proposed method for the determination of
quetiapine in human plasma based on the use of liquid chromatography with
spectrophotometric detection resulted to be simple, accurate and precise, fast and
feasible. Furthermore, it has good selectivity; in fact, no interference was found
upon examining 21 different CNS drugs. The method is suitable for the analysis
of quetiapine in human plasma, applicable for the TDM of patients undergoing
chronic treatment with quetiapine. [13]
REFERENCES
[1] Swainston, H.T.; Perry, C.M. Aripiprazole: a review of its use in
schizophrenia and schizoaffective disorder. Drugs 2004 64, 1715–1736.
[2] Zuardi, A.W; Crippa, J.A.S.; Hallak, J.E.C.; Moreira, F.A.; Guimarães, F.S.
Cannabidiol as an antipsychotic drug. Brazilian Journal of Medical and
Biological Research 2006 39, 421–429.
[3] Lawler, C.P. et al. Interactions of the novel antipsychotic aripiprazole
(OPC-14597) with dopamine and serotonin receptor subtypes.
Neuropsychopharmacology 1999 20, 612–27.
[4] www.curedisease.com (Accessed January 2009)
[5] www.cochrane.org (Accessed January 2009)
[6] Benkert, H. Kompendium der Psychiatrischen Pharmakotherapie (German),
Springer Verlag, 4th. ed.
[7] Bandelow, B.; Bleich, S.; Kropp, S. Handbuch Psychopharmaka (German),

Hogrefe, 2nd. ed.
[8] www.fda.gov (Accessed January 2009)
[9] Mercolini, L.; Grillo, M.; Bartoletti, C.; Boncompagni, G.; Raggi, M.A.
Simultaneous analysis of classical neuroleptics, atypical antipsychotics and
their metabolites in human plasma. Anaytical Bioanaytical Chemistry 2007
388, 235–243.
[10] Kirchherr, H.; Kuhn-Velten, W.N. Quantitative determination of forty-eight
antidepressants and antipsychotics in human serum by HPLC tandem mass
spectrometry: A multi-level, single-sample approach. Journal of
Chromatography B 2006 843, 100–113.
[11] Frahnert, C.; Rao, M.L.; Grasmader, K. A nalysis of eighteen
antidepressants, four atypical antipsychotics and active metabolites in serum
by liquid chromatography: a simple tool for therapeutic drug monitoring.
Journal of Chromatography B 2003 794, 35–47.
[12] Lancelin, F.; Djebrani, K.; Tabaouti, K.; Kraoul, L.; Brovedani, S.; Paubel,
P.; Piketty, M.-L.; Development and validation of a high-performance liquid
chromatography method using diode array detection for the simultaneous
quantification of aripiprazole and dehydro-aripiprazole in human plasma.
[13] Mandrioli, R.; Fanali, S.; Ferranti, A.; Raggi, M.A. HPLC analysis of the
novel antipsychotic drug quetiapine in human plasma. Journal of
Pharmaceutical and Biomedical Analysis 2002 30, 969-977.
Chapter 8
ANTIBIOTICS
Antibiotics are chemical substances produced by microorganisms that either
destroy (bactericidal) or inhibit the growth of other microorganisms
(bacteriostatic). Antibiotics can be either broad spectrum, which means that they
are active against a wide range of microorganisms. Narrow spectrum drugs target
a specific group of microorganisms and are able to interfere with a metabolic
process specific to those organisms. In general antibiotics work by: (i) preventing
the synthesis of bacterial cell wall components (e.g. penicillins); (ii) damaging the
bacterial cytoplasmic membrane; (iii) interfering with protein or nucleic acid
synthesis.
Not all antibiotic classes require therapeutic drug monitroring.

Aminoglycosides are a group of antibiotics that are used to treat certain bacterial
infections. They can be used against certain Gram-positive bacteria, but are not
typically employed because other antibiotics are more effective and have fewer
side effects. They are primarily used to combat infections due to aerobic, Gram-
negative bacteria: Pseudomonas, Acinetobacter, and Enterobacter species, among
others. Aminoglycosides are also effective against mycobacteria, the bacteria
responsible for tuberculosis. They are not effective against anaerobic bacteria
(bacteria that cannot grow in the presence of oxygen), viruses, and fungi, only one
aminoglycoside, paromomycin, is used against parasitic infection.
This group of antibiotics includes at least eight drugs: amikacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin.
All of these drugs have the same basic chemical structure. [1]
Streptomycin was the first of aminoglycosides to be discovered, and was the
first antibiotic used to treat tuberculosis. It is is a bactericidal antibiotic derived
from the actinobacterium Streptomyces griseus. It kills sensitive microbes by
inhibiting protein synthesis; it binds to the 16S rRNA of the bacterial ribosome,
interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This

prevents initiation of protein synthesis thus leading to the death of microbial cells.
Humans have structurally different ribosomes from bacteria, thereby allowing the
selectivity of this antibiotic for bacteria. Streptomycin must be administered by
regular intramuscular injection. [2]
Spiramycin is a 16-membered ring macrolide (antibiotic). It was discovered in
1952 as a product of Streptomyces ambofaciens, used as preparations for oral
administration, since 1955, while in 1987 its parenteral form was introduced into
practice. The antibacterial action involves inhibition of protein synthesis in the
bacterial cell during translocation. Resistance to spiramycin can develop by
several mechanisms and its prevalence is to a considerable extent proportional to
the frequency of prescription in a given area. The antibacterial spectrum
comprises Gram-positive cocci and rods, Gram-negative cocci and also
Legionellae, mycoplasmas, chlamydiae, some types of spirochetes, Toxoplasma
gondii and Cryptosporidium sp., Enterobacteria, pseudomonads and pathogenic
moulds are resistant. Its action is mainly bacteriostatic, on highly sensitive strains
it exerts a bactericide action, with the significant advantage its great
gastrointestinal tolerance. [3]
Metronidazole is a nitroimidazole anti-infective medication used mainly in
the treatment of infections caused by susceptible organisms, mainly anaerobic
bacteria and protozoa. Metronidazole is also used in the treatment of the
dermatological conditions. It is a pro-drug, converted in anaerobic organisms by
the redox enzyme pyruvate-ferredoxin oxidoreductase. The nitro group of

metronidazole is chemically reduced by ferredoxin (or a ferredoxin-linked
metabolic process) and the products are responsible for disrupting the DNA
helical structure, thus inhibiting nucleic acid synthesis.
Metronidazole is selectively taken up by anaerobic bacteria and sensitive
protozoal organisms, because of the ability of these organisms to reduce
metronidazole to its active form intracellularly. [4,5]
Table 8.1. summarises the chromatographic methods for therapeutic drug
monitoring of antibiotics. In the following paragraphs an overview of published
methods on the analysis of antibiotics is provided.
Metronidazole and spiramycin I were determined in human plasma, saliva

and gingival crevicular fluid. For plasma, the samples were thawed at room
temperature and vortexed, then centrifuged for 5 min at 3500 rpm at
Antibiotics 51
approximately +4 ◦C. To 500 μL of plasma, 50 μL of 0.1 M sodium hydroxide

solution, and 0.1 M 1mL of pH 9 buffer solution were added, followed by 6 mL of
ethyl acetate after vortexing for a few seconds. A similar extraction procedure was
applied for saliva.
Table 8.1 Overview of HPLC methods for therapeutic drug

monitoring of antibiotics.

type
Metronidazole and Human LLE with ethyl acetate. Kromasil C18, 5μm Human plasma: MS/MS 6
spiramycin I plasma, (150mm×4.6mm,), 77, 76 and 92%
saliva and Gradient. MP: ACN, for metronidazole
gingival water and formic acid. and spiramycin I.
crevicular FR: 0.9 ml/min. IS: Human saliva:
fluid Ornidazole 67, 82 and 76%,.
Human GCF
approximately
100% for both.
Evernimicin Human Ultrafiltration PRP-1 5-mm at 40 oC 99.4-103% UV: 302 nm 7
plasma MP: 57% 0.2 M Linearity: 25 to
ammonium acetate 2500 ng/mL
(pH 8.75, with
triethylamine), 43%
ACN, FR: 1.0 mL/min.
For the human gingival crevicular fluid, the samples were thawed at room
temperature. Twenty microlitres internal standard at 0.4 ng/ μL was added,
followed by 50 μL of acetonitrile to precipitate proteins. Separation was achieved
by LC–MS/MS on a 5 μm Kromasil C18 column (150 mm×4.6 mm, 5 μm), with a

gradient using acetonitrile, water and formic acid at a flow rate of 0.9 mL/min.
Ornidazole was used as an internal standard. [6]
Evernimicin was determined in human plasma after ultrafiltration. An aliquot
of the ultrafiltrate mixed with 40 μL of acetonitrile was transferred into the HPLC
system. The analytical column was a 5-mm polymeric reversed-phase PRP-1
column maintained at 40oC. The mobile phase, consisting of 57% 0.2 M
ammonium acetate (pH 8.75, adjusted with triethylamine) and 43% acetonitrile,
was delivered at 1.0 mL/min. Linearity was observed in the range 25 to 2500
ng/mL. UV detection was used at 302 nm. This method has been used for the ex
vivo assessment of evernimicin protein binding in human plasma from safety and
tolerance as well as liver dysfunction and renal insufficiency studies.
The assay was successfully used to support single- and multiple-rising dose
safety and tolerance studies in the clinic as well as to assess the effect of liver
dysfunction or renal insufficiency on the binding of evernimicin to human plasma
proteins. [7]
REFERENCES
[1] www.healthatoz.com (Accessed January 2009)
[2] Kingston, W.; Waksman, S. Streptomycin and the Balance of Credit for
Discovery. Journal of the History of Medicine and Allied Sciences 2004 59,
441-462.
[3] www.drugs.com (Accessed January 2009)
[4] http://ntp.niehs.nih.gov (Accessed January 2009)
[5] http://cmr.asm.org (Accessed January 2009)
[6] Sagan, C.; Salvador, A.; Dubreuil, D.; Poulet, P.; Duffaut, D.; Brumpt, I.
Simultaneous determination of metronidazole and spiramycin I in human
plasma, saliva and gingival crevicular fluid by LC–MS/MS. Journal of
Pharmaceutical and Biomedical Analysis 2005 38, 298–306.
[7] Zhong, R.; Hernandez, A.; Alton, K.B.; Kishnani, N.S.; Patrick, J.E. High-
performance liquid chromatographic method for the quantification of
unbound evernimicin in human plasma ultrafiltrate. Journal of
Chapter 9
ANTIEPILEPTIC DRUGS
Modern treatment of seizures started in 1850 with the introduction of
bromides. In 1910, phenobarbital, which then was used to induce sleep, was found
to have antiseizure activity and became the drug of choice for many years. A
number of medications similar to phenobarbital were developed, including
primidone. In 1940, phenytoin (PHT) was found to be an effective drug for the
treatment of epilepsy, and since then it has become a major first-line antiepileptic
drug (AED) in the treatment of partial and secondarily generalized seizures.
In 1968, carbamazepine (CBZ) was approved, for the treatment of trigeminal
neuralgia; and in 1974, for partial seizures. Ethosuximide has been used since
1958 as a first-choice drug for the treatment of absence seizures without

generalized tonic-clonic seizures. Valproate was licensed in Europe in 1960 and in
the United States in 1978, and today is worldwide used for generalized epilepsies.
Novel antiepileptic drug with good efficacy, fewer toxic effects, better
tolerability, and no need for blood level monitoring were developed after 1990,
approved in the United States as add-on therapy only, with the exception of
topiramate and oxcarbazepine; lamotrigine is approved for conversion to
monotherapy. Understanding the mechanism of action and pharmacokinetics of
antiepileptic drugs is important in clinical practice, so that they can be used
effectively, especially in multi-drug treatment. Many structures and processes are
involved in the development of a seizure, including neurons, ion channels,
receptors, glia, and inhibitory and excitatory synapses. The antiepileptic drugs are
designed to modify these processes to favor inhibition over excitation in order to
stop or prevent seizure activity.
The antiepileptic drugs can be grouped according to their main mechanism of
action, although some of them have several actions and others have unknown
mechanisms of action. The main groups include sodium channel blockers, calcium
current inhibitors, gamma-aminobutyric acid (GABA) enhancers, glutamate

blockers, carbonic anhydrase inhibitors, hormones, and drugs with unknown
mechanisms of action. [1]
Carbamazepine was discovered in 1953. It was initially introduced as a drug
to treat trigeminal neuralgia in 1962. In 1971, Drs. Takezaki and Hanaoka first
used carbamazepine to control mania in patients refractory to antipsychotics. Dr.
Okuma, working independently, did the same successfully. Carbamazepine would
be studied for bipolar disorder throughout the 1970s. The mechanism of action of
carbamazepine is stabilization of the inactivated state of sodium channels,
meaning that fewer of these channels are available to open, making brain cells less
excitable.
Common side effects include drowsiness, headaches and migraines, motor
coordination impairment and/or upset stomach. With normal use, small reductions
in white cell count and serum sodium are common, however, in rare cases, the
loss of platelets may become life-threatening. Therefore frequent blood tests
during the first few months of use are mandatory, followed by three to four tests
per year for established patients. [2]
Oxcarbazepine is an anticonvulsant and mood stabilizing drug, used primarily
in the treatment of epilepsy and bipolar disorder. It is a derivative of
carbamazepine, adding an extra oxygen atom on the dibenzazepine ring. This
difference helps reducing the impact on the liver of metabolizing the drug, and
also prevents the serious forms of anemia or agranulocytosis occasionally
associated with carbamazepine. It has a similar mechanism as carbamazepine -

sodium channel inhibition and is generally used to treat the same conditions.
Oxcarbazepine has been recently found associated with a greater enhancement in
mood and reduction in anxiety symptoms than other drugs employed to treat
epilepsy. [3]
Lamotrigine is an anticonvulsant drug used in the treatment of epilepsy and
bipolar disorder. For epilepsy it is used to treat partial seizures, primary and
secondary tonic-clonic seizures, and seizures associated with Lennox-Gastaut
syndrome. The exact way lamotrigine works is unknown. One proposed
mechanism of action for lamotrigine involves an effect on sodium channels,
although this remains to be established in humans. In vitro pharmacological
studies suggest that lamotrigine inhibits voltage-sensitive sodium channels,
thereby stabilizing neuronal membranes and consequently modulating presynaptic
transmitter release of excitatory amino acids (e.g. glutamate and aspartate).
Lamotrigine shares very few side-effects with other, unrelated
anticonvulsants known to inhibit sodium channels, (e.g. Oxcarbazepine), which
Antiepileptic Drugs 55
may suggest that lamotrigine has a different mechanism of action. It is inactivated

by hepatic glucuronidation.
Lamotrigine has recently been reported to be a useful treatment for some
people with post-traumatic stress disorder and borderline personality disorder. A
recent study reported beneficial effects on individuals with schizoaffective
disorder, bipolar subtype with depression.
The pharmacokinetics of lamotrigine are quite complicated, with highly
varying half-life and blood plasma levels. Lamotrigine has fewer drug interactions
than many anticonvulsant drugs, although a pharmacokinetic interaction with
sodium valproate in particular is an indication for blood monitoring. [4, 5]
Phenobarbital is the most used anticonvulsant worldwide and the oldest still
commonly used. It has sedative and hypnotic properties but it has been replaced
by the benzodiazepines for these indications. In most countries it is no longer
recommended as a first-line medication, rather it is considered as an alternate
when a patient fails to respond to treatment with more modern anti-epileptic-
drugs. It is still commonly used around the world to treat neonatal seizures.
Phenobarbital is indicated in the treatment of all types of seizures except absence
seizures. Phenobarbital is no less effective at seizure control than more modern
drugs such as phenytoin and carbamazepine. It is, however, significantly less well
tolerated.
For treatment of status epilepticus benzodiazepines such as diazepam or
lorazepam are the first choice. Phenobarbital has an oral bioavailability of
approximately 90%. Peak plasma concentrations are reached 8 to 12 hours after

oral administration. It is one of the longest-acting barbiturates available – it
remains in the body for a very long time (half-life of 2 to 7 days) and has very low
protein binding (20 to 45%). Phenobarbital is metabolized by the liver, mainly
through hydroxylation and glucuronidation, and induces many isozymes of the
cytochrome P450 system. It is excreted primarily by the kidneys. [6]
Phenytoin sodium is a commonly used antiepileptic. Phenytoin acts to
decrease the unwanted, runaway brain activity seen in seizure by reducing
electrical conductance among brain cells by stabilizing the inactive state of
voltage gated sodium channels. Aside from seizures, it is an option in the
treatment of trigeminal neuralgia as well as certain cardiac arrhythmias. [7]
Primidone is an anticonvulsant of the pyrimidinedione class whose active
metabolites, phenobarbital (major) and phenylethylmalonamide (minor), are also
anticonvulsants. It is used mainly to treat complex partial, simple partials,
generalized tonic-clonic seizures, myoclonic, akinetic seizures. Unlike other
anticonvulsants such as carbamazepine and valproic acid, primidone is rarely used
in the treatment of bipolar disorder or any other psychiatric problem.
Primidone was once a mainstay anticonvulsant in the treatment of partial and

generalized seizures and was the treatment of choice for secondarily generalized
seizures originating in the temporal lobes, especially when combined with
phenytoin. Primidone works via interactions with voltage-gated sodium channels,
which inhibit high-frequency repetitive firing of action potentials. It is much less
toxic in overdose than phenobarbital. Along with carbamazepine, phenobarbital,
and phenytoin, primidone is an inducer of metabolic enzymes in the liver, as it
accelerates the metabolism of many other pharmaceuticals. [8]
monitoring of antiepileptics. In the following paragraphs an overview of methods
on the analysis of antiepileptics is provided.
The therapeutic efficacy of anti-epileptic drugs depends on the achievement

of well-defined plasma concentrations. A validated analytical method is essential
to yield results that satisfactorily allow the monitoring of patients during therapy.
In a routine laboratory, where a large number of samples have to be analyzed
every day, requirements such as short analysis times, simple instrumentation, and
chromatographic conditions, as well as an easily applicable technique, without
affecting the accuracy and reproducibility of the analytical methods are

mandatory.
A sensitive and reproducible stir bar-sorptive extraction and high-
performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for
therapeutic drug monitoring of carbamazepine, carbamazepine-10, 11-epoxide,
phenytoin and phenobarbital in plasma samples is described and compared with a
liquid:liquid extraction method. Important factors in the optimization of SBSE
efficiency such as pH, extraction time and desorption conditions (solvents, mode
magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries
ranging from 72 to 86%, except for phenytoin (62%).
Chromatographic separation was achieved at room temperature on a
LiChrospher 100 RP-18 column (125 mm × 4 mm, 5 μm, Merck, Damstadt,
Germany). The mobile phase consisted of water: acetonitrile (78:22, v/v)
delivered at a flow-rate of 1 mL/min. The ultraviolet detector was set at 220 nm.
Table 9.1. Overview of HPLC methods for therapeutic drug monitoring of antiepileptics.

type
Carbamazepine, Human Stir bar-sorptive SBSE/HPLC-UV vs 72 to 86%, except UV: 220 nm 9
carbamazepine- plasma extraction vs LLE LLE/HPLC-UV: for phenytoin
10,11-epoxide, Lichrospher 100 RP-18 (62%)
phenytoin and (125 mm × 4 mm, Linearity: 0.08–
phenobarbital 5μm). RT. MP: water: 40.0 μgmL−1 for
ACN (78:22, v/v). FR:1.0 carbamazepine,
mL/min. (IS): carbamazepine-
tolylbarbituric 10,11-epoxide
Acid and phenobarbital

and 0.125–40.0
μgmL−1 for
phenytoin
Lamotrigine (LAM), Human LLE with diethylether Glass column (3×150 Linearity: UV: 220 nm 10
primidone (PD), serum mm) with stationary 0.5–25 mg/Lfor
phenobarbital (PB), Phase Separon SGX C18, PEMA and LAM;
phenytoin 5 μm. 1.25–25 mg/Lfor
(DPH), carbamazepine MP: water/ACN/MeOH PD and CMZ;
(CMZ), and two active 72:23:5 (v/v/v) with TEA. 0.625 –12.5
metabolites 2-phenyl- pH: 3.5–7.0. mg/Lfor EPO;
2-ethyl-malonamide FR: 1 mL/min. 1.5–60 mg/Lfor
(PEMA) and 10,11- IS: 5-Ethyl-5-p- PB; and 1.25–50
dihydro-10,11- tolylbarbituric acid mg/Lfor DPH
epoxycarbamazepine
(EPO)
Table 9.1. Continued.

type
Primidone, phenobarbital, Human SPE. Elution with Alltima 3 C18 (15 cm × 98-103%. Linearity DAD: 215 and 11
Phenytoin, carbamazepine serum ACN/MeOH (7/3, v/v) 0.46 cm) at 45◦C. MP: (mg/L): PRM 0–21.8 275 nm
with its two major MeOH (14.5 vol.%), ACN ZNS 0–21.8
metabolites carbamazepine- (19.5 vol.%) and 25 mm CBZD 0–9.28
10,11-epoxide and Phosphate buffer with LTG 0–21.6
carbamazepine-10,11- 12.5mm of sodium HCB 0–38.8
(trans)-dihydrodiol chloride, pH 6.2 (66 PHB 0–50.4
lamotrigine, vol.%). FR: 0.9 mL/min. CBZE 0–6.26
hydroxycarbazepine (active IS:5-ethyl-5-para-tolyl PHT 0–31.0

metabolite of oxcarbazepine) barbituric acid CBZ 0–14.8
and zonisamide
Lamotrigine, oxcarbazepine Human Samples were cleaned Betasil C-6, 250 mm × 4.6 95–107% for UV: 215 nm 12
and its metabolite 10- serum from interfering mm, 5μm. 25◦C. At 0–5 lamotrigine, 101–
monohydroxycarbazepine proteins and lipids by min 103% for
(MHD) transfer onto a pre- MHD and 97 to
column, using a Perfect 120% for
bond C-8 material, oxcarbazepine.
With 8% ACN in water Linearity: 30–5000
as a pre-column eluent ng/mL for
lamotrigine,
60–10000 ng/mL for
10-mono-
hydroxycarbazepine
(MHD) and 90–1000
ng/mL
For oxcarbazepine

type
Drug vigabatrin and R-(−)- Human Deproteinize with ACN Chiralcel-ODR (250 mm 96±7% for S-(+)- UV: 340 nm 13
and S-(+)-enantiomers plasma and the supernatant is × 4.6 mm, 10 μm) at 25 vigabatrin and
derivatized with 2,4,6 ◦C. MP: potassium 94±3%
trinitrobenzene sulfonic hexafluorophosphate/AC For R-(−)-vigabatrin.
acid (TNBSA). The N/ethanol. Gradient. FR: Linearity:0.5–40
Trinitrobenzene 0.9 mL/min. IS: 1- μg/mL
derivatives were then aminomethyl-cycloheptyl-
concentrated on High acetic acid
Performance
Extraction Disk
Cartridges
Zonisamide Human Plasma samples 4.6 mm · 250 mm, 5 μm, 94.57 ± 6.33to UV: 215 nm 14
(ZNS), primidone (PRI), plasma Vortex-mixed Zorbax RX-C8 MP: 103.49 ± 5.78%. for PRI, LTG,
lamotrigine (LTG), For 30 s then MeOH–ACN– 0.1% TFA, Linearity (μg/mL): MHD, PB,
phenobarbital (PB), centrifuged at 12,000g 235: 120: 645 (v/v), Carbamazepine1–25, PHT, and
phenytoin (PHT), for 10 min at 4 oC. FR: 1.5 mL/min. 40oC Carbamazepine CBZE, and at
oxcarbazepine 10,11-epoxide 1–10, 235 nm for
(OXC), and carbamazepine Lamotrigine 1–25, ZNS, OXC,
(CBZ) and two of their Monohydroxycarbam and CBZ.
active metabolites, azepine 1–50,
monohydroxycarbamazepine Oxcarbazepine 0.5–
(MHD) and carbamazepine 25, Phenobarbital 5–
10,11-epoxide (CBZE) 100, Phenytoin 1–50,
Primidone 5–50,
Zonisamide 1–80.

type
Lamotrigine (LTG), Human Deproteinization by Synergi 4 μm Hydro-RP, 100-104% UV: 210 nm 15
oxcarbazepine‘s (OXC) main plasma ACN 150 mm × 4 mm, MP: Linearity: 1–20
active metabolite potassium dihydrogen μg/mL for
monohydroxycarbamazepine phosphate buffer (50 mm, lamotrigine, 2–40
and felbamate ph 4.5) and ACN/MeOH μg/mL for
(3/1) (65:35, v/v) FR: 1.0 monohydroxycarbam
mL/min. azepine and 10–120
I.S:4-methylprimidone. μg/mL for felbamate
Pregabalin (PGB), Human Deproteinized with Home-made Linearity: 63 mg/l Fluorescence 16

gabapentin (GBP) and serum TCA and precolumn Column (15 cm × 0.46 For PGB, 40 mg/Lfor detector: λex
vigabatrin (VGB) derivatization with o- cm) packed with Alltima GBP and 62 mg/Lfor =330 nm and
phtaldialdehyde 3 C18 kept at 30 ◦C. MP: VGB λem = 450 nm.
(OPA) and MeOH (8.0 vol. %), ACN
3-Mercaptopropionic (17.5 vol.%) and 20 mm
acid phosphate buffer ph 7.0
(74.5 vol.%). FR: 0.8
mL/min. pH 7.50
IS: Norvaline
Piracetam(2-oxo-1- Standard Hibar RT 250-4, ELSD 17
pyrrolidineacetamide, VPA- solutions Lichrosorb RP-8. 250 mm
Na (2-propylpentanoic acid, × 4 mm, 5μm, 25 ◦C. FR:
sodium salt, PRM (2- 0.5 mL/min. Gradient.
desoxyphenobarbital MP: (A) ammonium
And CBZ (5H-dibenz[b, acetate (B) ethanol and
f]azepine-5-carboxamide (C) isopropyl alcohol.

type
Oxcarbazepine Human SPE (Elution MeOH) Microsorb MV Rainin >94% UV: 237 nm 18
and metabolites plasma (C18 , 150 × 4.6 mm, 5 Linearity: 100–4000
mm) MP: (FR, 1 mL min ng mL for OXCBZ,
) 15 mM phosphate 1.0–40.0 mgmL for
buffer–MeOH–ACN– CBZ-10OH and 0.3–
triethylamine 12.0 mg mL for
(62.25:20.0:17.5:0.25, CBZ-dioh; 2.0 mg
v/v/v/v) pH= 3.5 with 1 M mL (constant) for the
HCl. IS: 10,11-dihydro- I.S.

10,11-
epoxycarbamazepine
Oxcarbazepine (OXC), 10- Human Deproteinization with Symmetry C18, 3.5 μm 60±4% for OXC, MS/MS 19
hydroxycarbazepine (MHD) serum acetone and SPE on 2.1mm × 100 mm, (40 88±1% for MHD and
and C8 cartridge. ◦C). MP: ACN 40% with 62±15% for
Trans-diol-carbazepine 0.02% formic acid. FR: DHD (mean±SD)
(DHD) 350 μL/min. Linearity: 0.78–50
IS: Cyheptamide (CYE) mg/L for MHD and
0.078–5.0 mg/Lfor
OXC and DHD
Carbamazepine (CBZ), Human LLE with Linearity: 1.0–15.0 UV: 190–350 20
carbamazepine-10,11- plasma dichloromethane and 0.5–12.0 μg/mL nm
Epoxide (CBZE) of CBZ and CBZE
respectively

type
Eslicarbazepine acetate, Human SPE on Oasis Lichrocart 250-4 Chiradex 94.00-102.23% UV: 225 nm 21
oxcarbazepine, S- plasma cartridges, (β-cyclodextrin, (5 μm) Linearity: 0.4–8
licarbazepine and R- conditioned with 30°C.isocratic MP: water– μg/mL for
licarbazepine MeOH, ACN and MeOH (88:12, v/v), eslicarbazepine
water–ACN (95:5, FR: 0.7 mL/min acetate and
v/v). Elution with oxcarbazepine, and
ethyl acetate 0.4 – 80 μg/ mL for
each licarbazepine
enantiomer.
Oxcarbazepine (OXC) and its Human LLE with diethyl Phenomenex® Luna C18 5 95.8% for OXC and MRM positive 22
active metabolite, 10,11- plasma ether– μm (150 mm × 4.6 mm) 86.7% for MHD Electrospray
dihydro-10- diclhoromethane isocratic MP: (ACN/water Linearity: 20–5250 ionization
hydroxycarbamazepine (60:40 v/v) using (50:50 v/v) + 20mm acetic ng/mL for OXC and (ESI+).
(MHD) deuterade acid). FR: 1.0 mL/min. 40–10,500 ng/mL for m/z 253 > 208
carbamazepine (d10- IS: d10-carbamazepine MHD for OXC, m/z
carbamazepine) as 255 > 194 for
IS. MHD and m/z
247 > 204 for
IS.

type
Lamotrigine, carbamazepine Human LLE with ethylacetate Μbondapak C18 22°C, 94%-98% UV:270 nm 23
and zonisamide plasma MP: aqueous 30 mm Linearity:1–30
and potassium phosphate μg/mL for
serum buffer (pH 3.7 with 5% lamotrigine, 2–
phosphoric acid) and 20 μg/mL for
ACN (65:35). FR: 1.2 carbamazepine, and
mL/min. IS: 1–40 μg/mL for
chloramphenicol zonisamide
Oxcarbazepine and its main Human LLE with methyl-tert- X-TERRA C18 (150mm x 80% for UV: 237 nm 26
metabolites 10-hydroxy-10, plasma butyl ether. 4.6 mm, 5μm at 40 ◦C. oxcarbazepine and
11- Dihydrocarbamazepine and MP: 20mm KH2PO4, 55% for metabolites.
and 10,11-dihydroxy-trans- cerebros ACN, Linearity: 25–1000
10,11-dihydrocarbamazepine pinal And n-octylamine ng/mL, 1000–25 000
fluid (76:24:0,05, v/v/v). ng/mL, and 100–
Isocratic. FR: 0.7 mL/min 4000 ng/mL for
IS: bromazepam Oxcarbazepine, 10-
hydroxy-10, 11-di-
hydrocarbamazepinea
nd 10,11-di-hydroxy-
trans-10,11-di-
hydrocarbamazepine
respectively.

type
Fluvoxamine Rat LLE with n-hexane. C18 150 × 4.6 mm, 5 μm. 97.3–104.7% Fluorometric 27
plasma Pre-column MP: ACN- TFA (0.1% Linearity: 0.015–1.5 λex= 470 nm
derivatization with 4- v/v) 60:40 v/v. 25°C. FR: μg/mL and λem =540
fluoro-7- nitro-2,1,3- 1.0, mL/min.IS: nm.
benzoxadiazole (NBD- Propafenone
F). Hydrochloride
Levomepromazine, Blood SPE Lichrocart 125 mm × 3 62.0–94.4% and MS ion trap 28
Oxcarbazepine (OXCBZ) and hair mm., 5μm, filled with 39.0–77.1% equipped
and its two metabolites, 10- samples Purospher RP 18. Linearity: 0.05 to with an APCI
hydroxycarbazepine (CBZ- Gradient MP= 0.1% 20.00 μg/mL for
10OH) and trans-diol- formic acid and 95% OXCBZ and its
carbazepine (CBZ-dioh) ACN + 5% of the phase metabolites and
[A]. FR: 4 mL/min. IS: levomepromazine
CBZ epoxy and (blood) and 0.50–
levomepromazine. 100.00 ng/mg for
OXCBZ and its
metabolites (hair).
Carbamazepine Human LLE Average recovery: UV: 280–350 29
And Carbamazepine serum 96.56% Linearity: 0- nm
epoxide 14.0 μg/mL for CBZ
and 0-4.2 μg/mL for
CBZ-EP

type
Lamotrigine Human LLE with C18. Aqueous phosphoric 86% UV: 210 or 30
plasma and dichloromethane buffer, ACN and Linearity: 1.95–39.0 285 nm
Serum with 5% 3-methyl-2- diethylamine. FR: 0.9 mm
butanol (isoamyl mL/minIS:tyramine
alcohol). cloride
The commercial stir bar Twister for sorptive extraction was obtained from
Gerstel (Gerstel GmbH, Mulheim an der Ruhr, Germany), consisting of a 10 mm
long glass-encapsulated magnetic stir bar, externally coated with 22 μg of PDMS.
This layer is 0.5 mm thick, corresponding to a volume of 24 μL of PDMS. Prior to
the first use, the stir bars were conditioned for 24 h with an acetonitrile: methanol
solution (80:20, v/v). Among the successive extractions, the used stir bars were
cleaned in methanol for 30 min at 50 ◦C, under magnetic stirring rate of 1200 rpm,
followed by a drying step using a lint-free tissue.
For the desorption, the stir bars were rinsed lightly with Milli-Q water (1.0
mL), dried with lint-free tissue, and placed in a glass vial containing 1.0 mL of
solvent, ensuring total immersion. Desorption was performed by ultrasonic
treatment for 15 min at room temperature (25 ◦C) or by magnetic agitation for the
same period at the same temperature.
After the desorption process, the stir bars were removed by means of a
magnetic rod and the solvent was evaporated until dryness. The dry residues were
re-dissolved in 200 μL of the mobile phase, and 100 μL of this extract were
injected into the HPLC-UV system.
LLE was performed using 1 mL of sodium acetate buffer 0.75 M (pH 5.0) and
5mL dichloromethane to 1mL of plasma. The SBSE/HPLC-UV method was
linear over a working range of 0.08–40.0 μg/mL for carbamazepine,
carbamazepine-10, 11-epoxide and phenobarbital and 0.125–40.0 μg/mL for
phenytoin. The intra-assay and inter-assay precision and accuracy expressed as
coefficients of variation (CVs) for all compounds were less than 8.8% and all
inter-CVs were less than 10%. Limits of quantification were 0.08 μg/mL for
carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125
μg/mL for phenytoin. No interference of the drugs normally associated with
antiepileptic drugs was observed. The SBSE/HPLC-UV methodology developed
presents high sensitivity and enough reproducibility to permit the quantification of
carbamazepine, carbamazepine-10, 11-epoxide, phenytoin and phenobarbital in
human plasma. The method has been successfully applied to analysis of real
samples demonstrating that it works equally as well as the routine extraction
method for therapeutic drug monitoring of antiepileptic drugs. [9]
An HPLC procedure for the determination of lamotrigine (LAM)
simultaneously with other antiepileptic drugs, primidone (PD), phenobarbital
(PB), phenytoin (DPH), carbamazepine (CMZ), and two active metabolites 2-
phenyl-2-ethyl-malonamide (PEMA) and 10,11-dihydro-10,11-
epoxycarbamazepine (EPO) was developed and validated for their determination
in human serum for routine TDM in patients. The method involves an ordinary
RP system and a liquid –liquid extraction with diethylether. Α glass column (36
×150 mm) with stationary phase Separon SGX C18, 5 μm was used. Mobile phase
consisted of ternary mixtures of water/ACN/methanol (pH = 7) in ratio 72:23:5
(v/v/v) with addition of TEA as a modifier. UV detection was carried out at a
wavelength of 220 nm and the whole analysis took 15 min. The method was linear
in the range of 0.5–25 mg/L for PEMA and LAM; 1.25–25 mg/L for PD and
CMZ; 0.625 – 12.5 mg/L for EPO; 1.5–60 mg/L for PB; and 1.25–50 mg/L for
DPH, respectively. Within-day CV% and between-day CV% were within 10%.
The mixture consisting of water/ACN /methanol/TEA with was selected as the
universal mobile phase for both basic drugs and acid drugs, which were analyzed.
Preparation of patient samples is rapid, simple, accurate, and reproducible. A low
volume of a sample, only 50 μL of blood serum which is needed for the whole
analysis, is highly convenient namely for TDM in children. The developed HPLC
method allows simultaneous analysis of five AEDs with their two active
metabolites and now is used for the routine therapeutic drug monitoring of
epileptic patients both in children and adults. [10]
A rapid, simple and robust method is presented for the simultaneous
determination of seven antiepileptic drugs (AEDs), including primidone,
phenobarbital, phenytoin, carbamazepine with its two major metabolites
carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and
the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of
oxcarbazepine) and zonisamide in serum by HPLC-diode array detector (DAD) at
215-275 nm. After solid-phase extraction, separation is achieved on an Alltima
3C18 analytical column using isocratic elution with a mixture of acetonitrile,

methanol and phosphate buffer at 45 ◦C. The method was validated, including
experimental design in combination with statistical evaluation (ANOVA) to study
the robustness of chromatography and sample preparation. Commonly co-
administered antiepileptic drugs did not interfere with the method. Intra-day
precision (RSD < 1.9%), linearity, lower limit of quantitation (LOQ < 0.065
mg/L) and robustness make the method suitable for daily therapeutic drug
monitoring and pharmacokinetic studies.
Prior to SPE 0.1 mL of serum is diluted with 0.5 mL of the internal standard
working solution. After conditioning the extraction column with 1mL methanol
followed by 1mL water the sample mixture was poured into the column reservoir.
The sample was then drawn through the column with a maximum flow-rate of 1
mL/min. Consequently the column was washed with 2mL of water at maximum
speed. The drugs were then eluted with three times 0.1 mL of a mixture of
acetonitrile/methanol (7/3, v/v).
After diluting the eluate with 1 mL of water 50 μL was injected onto the
HPLC system.
The method is in daily use by the authors for routine therapeutic drug
monitoring for a considerable time without any problems. The experimental
results with respect to linearity, accuracy, precision, specificity and sensitivity
demonstrate the reliability of the procedure for its intended application. [11]
Using isocratic column-switching HPLC a group method for automated
quantitative analysis of the antiepileptic drugs lamotrigine, oxcarbazepine and its
metabolite 10-monohydroxycarbazepine (MHD) that are also used in psychiatry
as mood stabilizers was developed in human serum. Samples were cleaned from
interfering proteins and lipids by transfer onto a pre-column, using a
PerfectBond® C-8 material, with 8% acetonitrile in water as a pre-column eluent.
Separation was performed by isocratic elution onto the analytical column
(Betasil® C6 5 μm, 250 mm × 4.6 mm) at a flow rate of 1.0 mL/min with
potassium dihydrogenphosphate buffer (20 mmol/L, pH3.0)/acetonitrile (70/30;
v/v) as analytical eluent. UV- detector was set to 215 nm for all three compounds.
The process of switching from the pre-column to the analytical column was
executed by an electric 10-port valve incorporated in a thermostatted column
compartment, set to 25 ◦C. At 0–5 min, serum samples were delivered to the pre-
column by pre-column eluent (8% acetonitrile in water). At the same time the
analytical eluent flushed the analytical column for preparing separation of the
drug mixture. At 5–10 min, the switching valve was set to the analytical position
and analytical eluent delivered the matrix free drug mixture to the analytical
column in backflush mode.
The valve was set back to the starting position from 10 on to 18 min to
prepare the pre-column for the next sample injection. The analytical run was
finished within 18 min. Detection limit was 30 ng/mL for lamotrigine, 35 ng/mL
for oxcarbazepine and 25 ng/mL for 10-monohydroxycarbazepine. The method
was found to be suitable for automated analysis of serum samples of patients
treated with lamotrigine and oxcarbazepine.
The HPLC method described for the determination of lamotrigine,
oxcarbazepine and its active metabolite 10- monohydroxycarbazepine turned out
to be rapid, precise, accurate and specific over the entire therapeutic range, using
the advantages of an on-line sample preparation by column-switching procedure.
For separation of lamotrigine, oxcarbazepine and MHD in human serum, injection
of 100 μL serum turned out to be sufficient. Intraday variations within the
concentration levels specified were always below 2% for all analytes, indicating
high precision of the method. An imprecision of up to 2% for lamotrigine and
MHD and below 8% for oxcarbazepine among interday measurement experiments
is considered acceptable. The application of this HPLC method offers the
possibility of a simultaneous detection of various antidepressants and neuroleptics
within the same run that might be prescribed to patients suffering from bipolar
disorders. [12]
A rapid and simple HPLC method for the determination of the R-(−)- and S-
(+)-enantiomers of the antiepileptic drug vigabatrin in human plasma is described
by Franco et al. After adding the internal standard (1-aminomethyl-cycloheptyl-
acetic acid), plasma samples (200 μL) are deproteinized with acetonitrile and the
supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA).
Separation is achieved on a reversed-phase cellulose-based chiral column
(Chiralcel-ODR, 250 mm × 4.6 mm) using 0.05 M potassium
hexafluorophosphate (pH 4.5)/acetonitrile/ethanol (50:40:10 v/v/v) as mobile
phase at a flow-rate of 0.9 mL/min. Chromatographic selectivity is improved by
concentrating the derivatives on High Performance Extraction Disk Cartridges
prior to injection. Detection was performed at 340 nm. Calibration curves are
linear over the range of 0.5–40 μg/mL for each enantiomer, with a limit of
quantification of 0.5 μg/mL for both analytes. The assay is suitable for therapeutic
drug monitoring and for single-dose pharmacokinetic studies in man.
For sample preparation 200 μL aliquot of plasma was mixed with 200 μL of
internal standard working solution (12.5 μg/mL) and with 200 μL of acetonitrile.
After vortexing for 15 s and centrifuging for 10 min at 1400g, 200 μL of
supernatant were transferred into glass tubes. To each tube were added 70 μL of
saturated sodium borate solution and 5 μL of the derivatizing agent (5% TNBSA),
resulting in an amber-orange colored solution. The tubes were then tightly capped,
incubated at 50 ◦C for 10 min, and vortexed for 10 s. The caps were then removed
and the derivatization was stopped by adding 250 μL of 0.25 M acetic acid, which
resulted in a color change from amber-orange to yellow. The trinitrobenzene
derivatives were then concentrated on High Performance Extraction Disk
Cartridges and interfering substances were washed away. The method described
offers significant advantages in terms of simplicity and ease of use, and it has
sufficient sensitivity to allow quantification of the concentrations of each
enantiomer which are observed after administration of single oral doses of the
racemate in infants and in adults. Therefore, this method can provide a useful tool
not only for therapeutic drug monitoring purposes, but also for the conduction of
pharmacokinetic studies in a variety of clinical settings. Τhe assay is currently
being applied to the investigation of R-(−)- and S-(+)- vigabatrin
pharmacokinetics during pregnancy in women with epilepsy. [13]
A simple reversed-phase high-performance liquid chromatographic (HPLC)
method has been developed for the simultaneous determination of the antiepileptic
drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG),
phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine
.
(CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD)

and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL)
was pretreated by deproteinization with 300 μL methanol containing 20 μg/mL
propranolol hydrochloride as internal standard. HPLC was performed on a C8
column (4.6 mm × 250 mm; 5 μm) with methanol–acetonitrile–0.1%
trifluoroacetic acid, 235:120:645 (v/v), as mobile phase at a flow rate of 1.5
mL/min. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm, and
PRI, LTG, MHD, PB, PHT, and CBZE by UV detection at 215 nm. Relationships
between response and concentration were linear over the concentration ranges 1–
80 μg/mL for ZNS, 5–50 μg/mL for PRI, 1–25 μg/mL for LTG, 1–50 μg/mL for
MHD, 5–100 μg/mL for PB, 1–10 μg/mL for CBZE, 0.5–25 μg/mL for OXC, 1–
50 μg/mL for PHT, and 1–25 μg/mL for CBZ. Intra-day and inter-day
reproducibility were adequate (coefficients of variation were < 11.6%) and
absolute recovery ranged from 95.2 ± 6.13 to 107.7 ± 7.76% for all the analytes;
for the IS recovery was 98.69 ± 1.12%.
The main advantages of the assay are the rapid, single-step, protein
precipitation procedure, isocratic elution, and short analysis time, which enabled
the method to be cost-effective, and suitable for analysis of a large number of
samples in routine work for therapeutic monitoring of the nine analytes. [14]
A very simple and fast method has been developed and validated for the
simultaneous determination of the antiepileptic drugs: lamotrigine (LTG),
oxcarbazepine‘s (OXC) main active metabolite monohydroxycarbamazepine and
felbamate in plasma of patients with epilepsy using HPLC with

spectrophotometric detection. Plasma sample (500 μL) pre-treatment was based
on simple deproteinization by acetonitrile. Liquid chromatographic analysis was
carried out on a Synergi 4 μm Hydro-RP, 150 mm × 4 mm column, using a
mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and
acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0
mL/min. UV detection was performed at 210 nm. Calibration curves were linear
(mean correlation coefficient >0.999 for all the three analytes) over a range of 1–
20 μg/mL for lamotrigine, 2–40 μg/mL for monohydroxycarbamazepine and 10–
120 μg/mL for felbamate.
Both intra and interassay precision and accuracy were lower than 7.5% for all
three analytes. Absolute recoveries ranged between 100 and 104%.
The simple sample pre-treatment, combined with the fast chromatographic
run permit rapid processing of a large series of patient samples.
Venous blood samples (5 mL) were drawn from patients at 8 a.m., before
their first morning dose of AEDs. Τhe proposed method proved to possess
adequate specificity, sensitivity, accuracy and precision for a reliable
simultaneous determination of LTG, MHD and FBM in patients with epilepsy. By

minimizing plasma preparation steps and grouping different new AEDs in the
same assay the method allows a large series of patient samples to be processed in
a single analytical session, a task which can be very advantageous in a TDM
setting. [15]
A rapid, simple and robust method is developed for the simultaneous
determination of the γ-amino-n-butyric acid (GABA) derivatives pregabalin
(PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by HPLC.
Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant
are precolumn derivatized with o-phtaldialdehyde (OPA) and 3-
mercaptopropionic acid. Separation is achieved on an Alltima 3 C18 column using
isocratic elution; the drugs are monitored using fluorescence detection (excitation
wavelength 330 nm and emission wavelength 450 nm). Norvaline was used as an
internal standard. The method was linear up to at least 63 mg/L for PGB, 40 mg/L
for GBP and 62 mg/L for VGB. Lower limits of quantitation (LOQ) are 0.13
mg/L for PGB, 0.53 mg/L for GBP and 0.06 mg/L for VGB. No interferences
were found from commonly coadministered antiepileptic drugs and from
endogenous amino acids. The method is suitable for routine therapeutic drug
monitoring and for pharmacokinetic studies. The experimental results with respect
to linearity, accuracy, precision, specificity and sensitivity demonstrate the
reliability of the procedure for its intended application. [16]
A novel, rapid, accurate, sensitive, reproducible and robust HPLC–ELSD
method for the simultaneous separation and quantitation of four antiepileptic

drugs: Piracetam(2-oxo-1-pyrrolidineacetamide, VPA-Na (2-propylpentanoic
acid, sodium salt, PRM (2-desoxyphenobarbital and CBZ (5H-dibenz[b,
f]azepine-5-carboxamide has been developed and validated in terms of precision,
accuracy, linearity of detector response and robustness. Chromatographic
separation was performed on a C8 Hibar pre-packed column 250 mm × 4 mm, 5
μm, Lichrosorb RP-8 column maintained at 25 ◦C during analysis.
Mobile phase consisted of ammonium acetate, ethanol and isopropyl alcohol
delivered under gradient elution at a flow rate of 0.5 mL/min. Detection was
performed using evaporative light scattering detector (ELSD). Optimal
instrumental conditions were obtained by assessing the effect of various critical
experimental parameters such as evaporator tube temperature, carrier gas flow
rate, photomultiplier gain on separation efficiency, accuracy, reproducibility and
sensitivity of measurement on all four AEDs. This study illustrates the potential
for use of HPLC–ELSD in drug level monitoring of patients undergoing mono- or
polytherapy for epilepsy. [17]
A method based on high-performance liquid chromatography with UV

detection in combination with solid-phase extraction for sample pretreatment has
been developed for the simultaneous analysis of the antiepileptic drug
oxcarbazepine and its main metabolites in human plasma. The extraction of the
analytes from plasma samples was carried out by means of a selective solid-phase
extraction procedure using hydrophilic–lipophilic balance cartridges. The
separation was obtained on a Varian Microsorb MV Rainin reversed-phase
column (C18, 150 × 34.6 mm, 5 μm) with a Varian C18 precolumn (30 × 34.6 mm,
5 μm). The mobile phase was a 15 mM phosphate buffer–methanol–acetonitrile–
triethylamine (62.25:20.0:17.5:0.25, v/v/v/v) mixture, at pH= 3.5 with 1 M HCl
delivered at a flow-rate of 1 mL/min. UV detection was performed at 237 nm.
Under these chromatographic conditions, oxcarbazepine and its metabolites
10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxy-
carbamazepine and the internal standard are baseline separated in less than 9 min.
The extraction yield values were 94% for all analytes. The LOQs reached (15
ng/mL) are below the concentrations expected in patients. The SPE procedure
appropriately adopted for sample pre-treatment enabled avoidance of
chromatographic interference when analysing samples from patients treated with
multiple drugs. The method was applied to plasma samples from patients
undergoing chronic treatment with oxcarbazepine, both in monotherapy and in
polytherapy. Based on the analytical parameters precision, accuracy, limit of
quantitation and analysis time the method is suitable for routine application in
therapeutic drug monitoring. [18]

A fast, sensitive and specific LC/MS/MS method for the simultaneous
analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-
carbazepine (DHD), in human serum, has been developed and validated. Serum
drugs were extracted by C8 solid-phase cartridges (SPE) after sample pre-
treatment with acetone to precipitate the proteins and separated in less than 3 min
on column (Symmetry C18 3.5 μm 2.1 mm × 100mm, Waters, USA) at 40◦C. The
mobile phase consisted of acetonitrile 40% containing 0.02% formic acid
delivered isocratically. Analytical flow rate of 350 μL/min. A split was included
so that only approximately 1/3 of the column eluent entered into ESI probe. The
temperature of the autosampler was maintained at 8 ◦C and 10 μL were
automatically injected into the HPLC. A tandem mass spectrometer, as detector,
was used for quantitative analysis in positive mode by a multiple reaction
monitoring. Calibration curves, obtained on two ranges of concentration (0.78–50
mg/L for MHD and 0.078–5.0 mg/L for OXC and DHD). Within-day and
between-days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and
from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard.
Samples from 24 treated patients were analysed and drug serum concentrations
obtained by this method are in agreement with those of other methods and also are
well correlated (r = 0.88) in comparison to the routine HPLC-UV method. Based
on the analytical results and short run time, the method is suitable to support
routine analysis of therapeutic drugs monitoring from human serum of treated
patients or for pharmacokinetic studies. [19]
Carbamazepine (CBZ) undergoes enzyme biotransformation through
epoxidation with the formation of its metabolite, carbamazepine-10, 11- epoxide
(CBZE). Carbamazepine (CBZ) and carbamazepine-10, 11-epoxide (CBZE) were
determined in human plasma using a simple chemometrics-assisted
spectrophotometric method. A liquid extraction procedure using dichloromethane
was operated to separate the analytes from plasma, and the UV absorbance spectra
of the resultant solutions were subjected to partial least squares (PLS) regression.
An HPLC method was also employed for comparison. The respective mean
recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 %
and 103.00 % for PLS and 99.40 % and 102.20 %. Analysis of the CBZ and
CBZE in the real patients‘ plasma by the two methods indicated excellent
agreement between the results obtained by both methods. Thus, both can be used
to monitor the levels of CBZ and CBZE in plasma for pharmacokinetic studies.
[20]
Eslicarbazepine acetate (BIA 2-093) is a novel central nervous system drug
undergoing clinical phase III trials for epilepsy and phase II trials for bipolar
disorder. A simple and reliable chiral reversed-phase HPLC-UV method was

developed and validated for the simultaneous determination of eslicarbazepine
acetate, oxcarbazepine, S-licarbazepine and R-licarbazepine in human plasma.
The analytes and internal standard were extracted from plasma by a solid-phase
extraction using Waters Oasis® HLB cartridges. Chromatographic separation was
achieved by isocratic elution with water–methanol (88:12, v/v), at a flow rate of
0.7 mL/min, on a LichroCART 250-4 ChiraDex (β-cyclodextrin, 5 μm) column at
30°C. BIA 2-265 used as internal standard. All compounds were detected at 225
nm. Calibration curves were linear over the range 0.4–8 μg/mL for
eslicarbazepine acetate and oxcarbazepine, and 0.4 – 80 μg/ mL for each
licarbazepine enantiomer. The overall intra- and interday precision and accuracy
was less than 15%. Mean relative recoveries varied from 94.00 to 102.23% and
the limit of quantification of the assay was 0.4 μg/mL for all compounds. The
method seems to be a useful tool to individually assess the pharmacologically
active metabolites licarbazepine enantiomers and their prodrugs ESL and OXC. It
can be applied to clinical trials but also in the routine therapeutic drug monitoring
assays or in bioequivalence studies that involve the new antiepileptic drug ESL
and the already marketed OXC. [21]
A fast and sensitive method to quantify oxcarbazepine (OXC) and its active
metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (MHD) in human plasma
using HPLC–MS/MS has been developed. The method involved liquid–liquid
extraction (LLE), with diethyl ether–dichloromethane (60:40 v/v) using deuterade
carbamazepine (d10-carbamazepine) as internal standard (IS). The analytes and IS
were separated using an isocratic mobile phase (acetonitrile/water (50:50 v/v) +
20mM acetic acid) on the analytical column Phenomenex® Luna C18, 5 μm,
(150mm × 4.6 mm) at room temperature. Detection was performed by a
Micromass Quatro LC mass spectrometer in the reaction monitoring mode using
positive electrospray ionization (ESI+). The MS–MS ion transition monitored
were m/z 253 > 208 for OXC, m/z 255 > 194 for MHD and m/z 247 > 204 for IS.
Linearity was observed over the range 20–5250 ng/mL for OXC and 40–10,500
ng/mL for MHD. The lower limits of quantification obtained as a result of the
LLE procedure was 20 ng/mL for OXC and 40 ng/mL for MHD. The suitability
of the assay for pharmacokinetics studies was determined by measuring OXC and
MHD concentration after administration of a single 10 mL of OXC oral
suspension (6%) in plasma human of healthy volunteers.
The method has proved to be fast and reliable, with each sample requiring
less than 4 min of analysis time. Finally, the suitability of LC–MS/MS method to
identify and quantify OXC and MHD in human plasma has been successfully
demonstrated. [22]
An HPLC assay with ultraviolet detection was developed for the simultaneous
determination of the anti-epileptic drugs lamotrigine, carbamazepine and
zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide
and the internal standard chloramphenicol were extracted from serum or plasma
using liquid–liquid extraction under alkaline conditions using ethylacetate. The
method was linear in the range 1–30 μg/mL for lamotrigine, 2– 20 μg/mL for
carbamazepine, and 1–40 μg/mL for zonisamide. Within- and between-run
precision studies demonstrated coefficient of variation <10% at all tested
concentrations. Other anti-epileptic medications tested did not interfere with the
assay. The method is appropriate for determining lamotrigine, carbamazepine and
zonisamide serum or plasma concentrations for therapeutic monitoring.
To 250 μL of sample (calibrator, control or patient sample) in a 16 × 125 mm
glass culture tube, 100 μL of IS solution, 1.5 mL NaOH and 4.0 mL ethylacetate
were added. The separation was performed at 22°C with a μBondapak C18
column. The mobile phase was a mixture of aqueous 30 mM potassium phosphate
buffer (adjusted to pH 3.7 with 5% phosphoric acid) and acetonitrile (65:35) at a
flow rate of 1.2 mL/min. Chloramphenicol was used as internal standard.

Recovery rates were 94%-98%. Linearity was observed in the range 1–30 μg/mL
for lamotrigine, 2– 20 μg/mL for carbamazepine, and 1–40 μg/mL for zonisamide.
UV detection was performed at 270 nm. The HPLC method described here is
simple, sensitive and specific and allows for the simultaneous determination of
three commonly prescribed anti-epileptic drugs. [23]
A simple reversed-phase high-performance liquid chromatographic (HPLC)
method has been developed for the simultaneous determination of the antiepileptic
drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG),
phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine
(CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD)
and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL)
was pretreated by deproteinization with 300 μL methanol containing 20 μg/mL
propranolol hydrochloride as internal standard. HPLC was performed on a C8
column (4.6 mm x 250 mm, 5 μm) with methanol–acetonitrile–0.1%
trifluoroacetic acid, 235:120:645 (v/v), as mobile phase delivered at a flow rate of
1.5 mL/min. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm,
and PRI, LTG, MHD, PB, PHT, and CBZE at 215 nm. Linear relationships
between response and concentration were observed over the concentration ranges
1–80 μg/mL for ZNS, 5–50 μg/mL for PRI, 1–25 μg/mL for LTG, 1–50 μg/mL
for MHD, 5–100 μg/mL for PB, 1–10 μg/mL for CBZE, 0.5–25 μg/mL for OXC,
1–50 μg/mL for PHT, and 1–25 μg/mL for CBZ. Intra-day and inter-day
reproducibility were adequate (coefficients of variation were lower than 11.6%)

and absolute recovery ranged from 95.2 to 107.7 % for all the analytes. The
results showed the method was rapid, specific, precise, and accurate, and response
was a linear function of concentration over the therapeutic range of interest,
therefore the method is suitable for routine clinical use. [24]
An HPLC method with UV detection has been developed for the
simultaneous determination of levomepromazine, clozapine and their main
metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, O-
desmethyl-levomepromazine, N-desmethylclozapine and clozapine N-oxide. The
analytes were separated on a C8 reversed-phase column using a mobile phase
composed of acetonitrile and a pH 2.0, 34 mM phosphate buffer containing 0.3%
triethylamine (29:71, v/v). Loxapine was used as the internal standard. Baseline
separation was achieved in less than 20 min. Sample pre-treatment was performed
by means of solid-phase extraction on BondElut C1 cartridges (100 mg/1 mL)
yielding >91% recovery for all analytes and appropriate sample purification from
endogenous interference. The method was validated in terms of extraction yield,
precision and accuracy.
The method is suitable for the therapeutic drug monitoring (TDM) of patients
undergoing polypharmacy with levomepromazine and clozapine. It has been
successfully applied to the analysis of plasma samples from patients subjected to
treatment with LMP and CLZ. [25]
An isocratic reversed-phase HPLC-UV procedure for the determination of
oxcarbazepine and its main metabolites 10-hydroxy-10,11- dihydrocarbamazepine
and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine in human plasma and
cerebrospinal fluid has been developed and validated. After addition of
bromazepam as internal standard, the analytes were isolated from plasma and
cerebrospinal fluid by liquid–liquid extraction with 0.5 mL of 0.1M NaOH and
5mL of methyl-tert-butyl ether. Separation was achieved on a X-TERRA C18
column using a mobile phase composed of 20 mM KH2PO4, acetonitrile, and n-
octylamine (76:24:0.05, v/v/v) delivered isocratically at a flow rate of 0.7
mL/min, at 40 ◦C and detected at 237 nm. Bromazepam was used as internal
standard. The described assay was validated in terms of linearity, accuracy,
precision, recovery and lower limit of quantification according to the FDA
validation guidelines. Linearity was observed within the range: 25–1000 ng/mL,
1000–25 000 ng/mL, and 100–4000 ng/mL for oxcarbazepine, 10-hydroxy-10,11-
dihydrocarbamazepine, and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine,
respectively Accuracy ranged from 92.3% to 106.0% and precision was between
2.3% and 8.2%.
The method was simple, precise, accurate, selective and sufficiently sensitive
and seems suitable for the quantitative determination of oxcarbazepine, 10-

hydroxy-10,11-dihydrocarbamazepine and 10,11-dihydroxytrans- 10,11-
dihydrocarbamazepine in plasma and cerebrospinal fluid samples, obtained in the
conduct of clinical pharmacokinetic studies, after oral administration of
oxcarbazepine, both in monotherapy and polytherapy. [26]
A sensitive, simple and reliable method using HPLC was reported for the
determination of fluvoxamine (FLU), a selective serotonin reuptake inhibitor
(SSRI), in rat plasma after LLE with n-hexane and pre-column derivatization with
4-fluoro-7- nitro-2,1,3-benzoxadiazole (NBD-F). Extracted plasma samples were
mixed with NBD-F at 60°C for 5 min and injected into HPLC. The HPLC C18
column was 150 × 4.6 mm, 5μm. The mobile phase consisted of acetonitrile and
trifluoroacetic acid (0.1% v/v) in water, at a volume ratio 60:40. The samples
were eluted from the column at 25°C at a flow rate of 1.0 mL/min.
Retention times of FLU and an internal standard (propafenone) derivative
were 15.5 and 13.5 min, respectively. Linearity was observed in the range 0.015–
1.5 μg/mL. The lower limits of detection and quantification of FLU were 0.008
and 0.015 μg/mL, respectively. The coefficients of variation for intra-day and
inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Propafenone
hydrochloride solution in water was added as an internal standard. Fluorometer
detector was operating at an excitation wavelength of 470 nm and an emission
wavelength of 540 nm. No interference was observed by other SSRIs and
centrally acting drugs. Results indicate that the method is useful to determine the
FLU levels in rat plasma of volumes as small as 100 μL and can be applied to
pharmacokinetic studies and therapeutic drug monitoring of FLU in patients. [27]
Levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-
hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) were
determined in blood and hair samples after SPE.
A typical use of hair analysis in forensic toxicology is the documentation of
previous drug administration. This is illustrated in a suicidal death of a 58-year-
old epileptic patient who was treated with oxcarbazepine and probably with
levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS
included hair (6 cm length) besides postmortem blood. The method was validated
for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-
hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in
various biological matrices.
The analysis revealed differences between the concentration levels of
oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and
hair, suggesting different mechanisms of penetration of metabolites and their
precursors into this matrix. Samples were extracted by means of solid phase
extraction (SPE). The method provided good relative and absolute recoveries of
62.0–94.4% and 39.0–77.1%, respectively, for all analytes across the linear
dynamic range.
The chromatographic separation was performed with a LiChroCART column
125 mm×3mm, 5μm, filled with Purospher RP 18 and a LiChroCART precolumn
4 mm×4 mm, 5 μm, filled with LiChrospher 60 RP – select. The mobile phase
consisted of a gradient mixture of A: 0.1% formic acid in demineralised water and
B: 95% acetonitrile + 5% of A delivered at a flow rate of 0.4 mL/min. Linearity
was obsreved in the range: 0.05 to 20.00 μg/mL for OXCBZ and its metabolites
and levomepromazine (blood) and 0.50–100.00 ng/mg for OXCBZ and its
metabolites in hair. [28]
Carbamazepine and carbamazepine epoxide were determined in human serum
of real patients by a procedure assisted by chemometric tools. First, a response
surface methodology based on a mixture design was applied in order to select the
best conditions for the extraction step. Finally, partial least squares multivariate
calibration (PLS-1) was applied to second-derivative UV spectra, eliminating a
shift baseline effect that originated in the extraction procedure. The performance
assessment included: (a) a three-level precision study, (b) a recovery study

analyzing spiked samples, and (c) a method comparison with high-performance
liquid chromatography (HPLC) and fluorescence polarization immunoassay
(FPIA) applied on real patient samples. The obtained results show the potentiality
of the presently studied methodology for the monitoring of patients treated with
this anticonvulsant.
The combination of UV spectrophotometry coupled to both optimized-analyte
extraction and multivariate calibration (PLS-1) leads to a powerful tool to be
applied to drug monitoring. The results obtained by applying the developed
method on real patient serum samples and by comparing it with reference methods
show the enormous potentiality of this analytical strategy. Carbamazepine was
determined with high accuracy and precision by using a very simple, quick and
inexpensive method.
Average recovery was 96.56%. Linearity ranged from 0 to14.0 μg/mL for
CBZ and 0-4.2 μg/mL for CBZ-EP. [29]
Lamotrigine was determined in human plasma and serum. Aliquots of sample
(patient, calibrators and controls) were mixed with internal standard solution,
NaOH and dichloromethane containing 5% 3-methyl-2-butanol (isoamyl alcohol).
After centrifugation, the aqueous (upper) phase was discarded and the organic
phase was evaporated to dryness and reconstituted in 200 mL methanol.
Separation was performed on a reversed-phase C18 column. The mobile phase was
prepared by mixing aqueous phosphoric buffer, acetonitrile and diethylamine. The
flow-rate was 0.9 mL/min. Tyramine chloride was used as internal standard.
Linearity was observed in the range 1.95–39.0 mM. UV detection was performed
at 210 or 285 nm. [30]
Recently direct plasma injection LC/MS/MS technique has been increasingly
used in pharmaceutical research and development due to the demand for higher
throughput of sample analyses. Two on-line extraction methods including high
flow LC/MS/MS and high flow column switching LC/MS/MS were investigated.
The evaluations were conducted and focused on their performances with respect
to peak responses, separation efficiency, and signal to-noise ratio in a multiple-
component LC/MS/MS assay. Two HPLC pumps were used-with one for high
flow delivery and one for gradient elution. High flow LC was achieved by the use
of 4 mL/min flow rate on a 1 × 50 mm Waters Oasis column. A 2 × 100 mm YMC
column was coupled via a column-switching valve. The extracted analytes were
analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole
MS/MS.
The on-line extraction HFCS LC/MS/MS method has been successfully used
not only in the plasma assays of N-in-1 PK studies but also in urine and synovial
fluid assays where the biological fluids contain dirty matrices. In fact, good
sensitivity and separation efficiency were achieved from all biological fluids
including directly injected plasma, serum, whole blood, urine, and bile samples.
The resulting dynamic range, lower limit of quantification, QC accuracy and
precision were within the acceptable range for discovery research and
development. [31]
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Chapter 10
ANTIDEPRESSANTS
An antidepressant is a psychiatric medication used for treeatment of major
depression or dysthymia. Drug groups most commonly prescribed by psychiatrists
include monoamine oxidase inhibitors (MAOIs), tricyclics, and second-generation
antidepressants such as Selective serotonin reuptake inhibitors (SSRIs), and
Serotonin norepinephrine reuptake inhibitors (SNRI's). The effectiveness and
adverse effects are the subject of many studies.
Most antidepressants have a delayed onset of action and are usually
administered over a long-term period of weeks, months, or sometimes years.
Antidepressants are often used in the treatment of other conditions, including
anxiety disorders, bipolar disorder, obsessive compulsive disorder, eating

disorders, chronic pain, mood disorders, and dysmenorrheal and other hormone-
mediated conditions.
Selective serotonin reuptake inhibitors (SSRIs) are a family of
antidepressants acting by preventing the reuptake of serotonin (also known as 5-
hydroxytryptamine, or 5-HT) by the presynaptic neuron, thus maintaining higher
levels of 5-HT in the synapse. It is thought that one cause of depression may be an
inadequate amount of serotonin, a chemical used in the brain to transmit signals
between neurons.
Members of this family include fluoxetine, paroxetine, escitalopram,
citalopram, sertraline, and fluvoxamine. These antidepressants typically have
fewer adverse events and side effects than the tricyclics or the MAOIs. Some side
effects may decrease as a person adjusts to the drug, but other side effects may be
persistent. They are considered to be safer than the first generation
antidepressants.
Serotonin-norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine,
milnacipram and duloxetine are a newer form of antidepressant that work on both
norepinephrine and 5-HT. They typically have similar side effects to the SSRIs,
although there may be a withdrawal syndrome on discontinuation that may
necessitate dosage control.
Noradrenergic and specific serotonergic antidepressants (NASSAs) are a
newer class of antidepressants which increase norepinephrine (noradrenaline) and
serotonin neurotransmission by blocking presynaptic alpha-2 adrenergic receptors
while at the same time minimizing serotonin related side-effects by blocking
certain serotonin receptors. Mirtazapine is the only member of this class in
clinical use. Mianserin has also similar mechanism of action.
Norepinephrine (noradrenaline) reuptake inhibitors (NRIs) act via
norepinephrine (known also as noradrenaline). NRIs are thought to have a
positive effect on concentration and motivation.
Norepinephrine-dopamine reuptake inhibits the neuronal reuptake of
dopamine and norepinephrine (noradrenaline).
Tricyclic antidepressants (TCAs) are the oldest class of antidepressant drugs.
Representative members are amitriptyline and desipramine. They act by blocking
the reuptake of certain neurotransmitters such as norepinephrine (noradrenaline)
and serotonin. They are used less commonly now due to the development of more
selective and safer drugs. In overdoses they can be lethal, as they may cause a
fatal arrhythmia. However, tricyclic antidepressants are still used because of their
effectiveness, especially in severe cases of major depression.
Monoamine oxidase inhibitor (MAOIs) may be used if other antidepressant
medications are ineffective. Because there are potentially fatal interactions

between this class of medication and certain foods (particularly those containing
Tyramine), red wine, as well as certain drugs, classic MAOIs are currently rarely
prescribed. MAOIs work by blocking the enzyme monoamine oxidase which
breaks down the neurotransmitters dopamine, serotonin, and norepinephrine
(noradrenaline). MAOIs can be as effective as tricyclic antidepressants, although
they can have a higher incidence of dangerous side effects (as a result of
inhibition of cytochrome P450 in the liver).
Duloxetine is a serotonin-norepinephrine reuptake inhibitor (SNRI) used for
major depressive disorder, generalized anxiety disorder, pain related to diabetic
neuropathy and fibromyalgia. Preclinical studies demonstrate that duloxetine
potently inhibits neuronal serotonin and norepinephrine reuptake, and this
inhibition is balanced throughout the dosing range. It is also considered a less
potent inhibitor of dopamine reuptake. Since it has no significant affinity for
dopaminergic, adrenergic, cholinergic, histaminergic, opioid, glutamate, and
GABA receptors it can be considered as a selective reuptake inhibitor at the 5-HT
and NA receptors. Duloxetine undergoes extensive metabolism, but the major
Antidepressants 85
circulating metabolites do not contribute significantly to the pharmacologic

activity. Its pharmacokinetic is dose proportional over the therapeutic range.
After 3 days a steady-state is usually achieved. [1]
Mianserin is a tetracyclic antidepressant that has antihistaminic and
hypnosedative, but almost no anticholinergic, effect. Mianserin is a weak inhibitor
of norepinephrine reuptake and strongly stimulates the release of norepinephrine.
Interactions with serotonin receptors in the central nervous system have been also
found. Mianserin blocks inhibitory α2-autoreceptors on central noradrenergic
nerve endings, and so may increase the amount of noradrenaline in the synaptic
cleft. It may also cause agranulocytosis and aplastic anaemia. [2,3]
Fluoxetine hydrochloride is an antidepressant of the selective serotonin
reuptake inhibitor (SSRI) class. Fluoxetine is approved for the treatment of major
depression, obsessive-compulsive disorder, bulimia nervosa, anorexia nervosa,
panic disorder and premenstrual dysphoric disorder. Despite the availability of
newer agents, it remains extremely popular.
The bioavailability of fluoxetine is relatively high (72%), and peak plasma
concentrations are reached in 6 to 8 hours. It is highly bound to plasma proteins,
mostly albumin.
Fluoxetine is metabolized in the liver by isoenzymes of the cytochrome P450
system, including CYP2D6. Only one metabolite of fluoxetine, norfluoxetine
(demethylated fluoxetine), is biologically active. The concentration of the drug
and its active metabolite in the blood increases through the first few weeks of
treatment, and their steady concentration in the blood is achieved only after four
weeks. Complete excretion of the drug may take several weeks. [4]
Venlafaxine is an antidepressant of the serotonin-norepinephrine reuptake
inhibitor (SNRI). It is prescribed for the treatment of major depression and
anxiety disorders. Due to the side effects and suspicions that venlafaxine may
significantly increase the risk of suicide, it is not recommended as a first line
treatment of depression. However, it is often effective for depression not
responding to SSRIs.
Table 10.1. Overview of HPLC methods for therapeutic drug monitoring of antidepressants.
Analytes Sample Sample Preparation Chromatography Recovery % Detection Referenc

type e
Trazodone and its main active Human SPE: C8 cartridges (50 Genesis C8 >91% UV: 255nm 7
metabolite 3-(1- plasma mg, 1 mL). Elution (15 mm × 4.6 mm, Linearity: TRZ
Clorophenyl)piperazine (m-cpp) with MeOH. 5 μm) RT. MP: ACN 10–2000 ng/mL
(30%, v/v) and pH 3.5, m-CPP: 10–1000
50mM phosphate ng/mL
buffer with 0.3% (v/v)
triethylamine (70%,
v/v). FR: 1.2mL/min.
(IS): Loxapine
Mianserin hydrochloride Human LLE with N-hexane. Hypersil-Hypurity C18 81.3–84.1% ESI–MS: 8
plasma (150 mm × 2.1mm, Linearity: 1.0– (SIM) at m/z
5μm) at 45 ◦C. MP: 200.0 ng/mL 265 [M+H]+

10mM ammonium for mianserin
acetate (pH 3.4)– and m/z 369
MeOH–ACN (35:50:15, [M+H]+ for
v/v/v). cinnarizine.
FR: 0.22 mL/min (I.S.)
cinnarizine
Trazodone, fluoxetine, Urine SPE: laboratory made Eclipse X-DB-C8 (4.6 × 72.4-97% UV: 254 nm. 9
Mianserine samples 30 mg MWCNTs 150 mm). MP: NaH2PO4
Desipramine imipramine cartridges buffer (pH 3.0)–ACN–
nortryptiline amitryptiline MeOH 70:25:5 (v/v).
trimipramine clomipramine FR: 1.2 mL min−1

Amitriptyline, Urine, plasma (HF-LPME): Zorbax Extend C18 (100 Linearity: 5–500 UV: 215 nm 10
Imipramine and sertraline and tap water Extraction from 11.0 mm × 2.1 mm). MP: μg/L
mL of aqueous solution 0.02 M acetic acid (pH
with pH 12.0 into n- 4.0) and MeOH (54:46)
dodecane impregnated FR: 0.25mL/min.
in a hollow fiber and IS: chloropromazine.
back extracted into 24
μL of aqueous solution
located inside the
lumen of the hollow
fiber and adj. pH 2.1
using 0.1M of H3PO4
Amitriptyline doxepin Pharmaceutical Human plasma Kromasil C8 (250 × 4 91.0 -114.0% UV: 238 nm 11
Clomipramine formulations samples were mm), 5 μm, RT. MP: Linearity: 1–5
Imipramine and biological pretreated to remove CH3COONH4 0.05 M– μg/mL
Fluids proteins. Urine samples CH3CN 45:55 v/v,
were analysed both FR: 1.5 mL/min.
directly after twofold IS: bromazepam.
dilution and filtration
and after SPE.
Duloxetine Human plasma SPE: Waters Oasis C8. MP: 60% aqueous >90% UV: 230 nm 12
mixed-mode reversed phosphate buffer with Linearity: 2–200
phase—strong cation triethylamine, pH 3.0 ng/mL
exchange (MCX) and 40% CAN. FR: 1
cartridges (30 mg/ 1 mL/min. IS: Loxapine.
mL). Elution with
ammonia/
water/MeOH (5/15/80,
w/w/v).

type e
Imipramine, amitriptyline, Human LLE LiChrospher 60 RP- 72%-86%, except UV: 230 nm 13
clomipramine, fluoxetine, plasma select B for moclobemide
sertraline, paroxetine, citalopram, (250 mm × 4 mm, 5μm). (59%). Linearity:
mirtazapine, moclobemide and MP: 35% ACN: MeOH 2.5–2000 ngmL−1
duloxetine (92:8, v/v) and 65% of
sodium acetate buffer,
pH 4.5.
FR: 1.0mLmin−1.
IS: etidocaine.
Levomepromazine, clozapine Human SPE: BondElut C1 C8 (150 mm × 4.6 mm, >91% UV: 254 nm 14
and their main plasma cartridges (100 mg, 1 5 μm; Phenomenex. MP: Linearity: LMP
Metabolites: n-desmethyl- mL). ACN and a phosphate 9–200 ng/mL;

levomepromazine, Elution with MeOH. buffer (34 mM, pH 2.0) NDLM
levomepromazine sulphoxide, o- with 0.3% triethylamine 10–150 ng/mL;
desmethyl-levomepromazine, n- (29:71, v/v). FR: 1.0 LMSO 5–500
desmethylclozapine and mL/min. IS: Loxapine. ng/mL; ODLM 7–
clozapine 150 ng/mL; CLZ
N-oxide. 20–2500 ng/mL;
DMC 15–1000
ng/mL; NOX 10–
200 ng/mL; IS
200 ng/mL

type
Venlafaxine, fluoxetine, Human SPE apolar, polymeric, HPLC-DAD 70 and 109% for DAD: 210-380 15
viloxazine, fluvoxamine, plasma ion-exchange, mixed GC-MS all compounds, nm
mianserin, mirtazapine, modes combining ion- except for MS: the spectra
Melitracen, reboxetine, exchange properties trazodone (39%) obtained
citalopram, maprotiline, Elution with MeOH were measured
sertraline, paroxetine and and 3mL of buffer (pH in the SIM
trazodone) together with eight 6.5 or 2.5). mode
of their metabolites
(odesmethylvenlafaxine,
norfluoxetine,
desmethylmianserine,desmethy
lmirtazapine,desmethylcitalopr
am,didesmethylcitalopram,des
methylsertraline and m
chlorophenylpiperazine)
Amitriptyline and nortriptyline Serum Dilution (1:10) in 0.15 Kromasil C18 with 5 98.5-101.6% UV: 240 nm 16
M SDS-6% pentanol at μm, 250 mm × 4.6 mm. Electrochemical
pH 7, and filtration MP: a 0.15 M SDS-6% detection at 650
through 0.45 μm nylon (v/v) pentanol at pH 7. mV.
membranes. FR: 1.5 mL/min.
Three intermediates in the Standard DKTP as a 250mm × 4.6 mm, 5 μM 99.7- 101.5% UV: 241 nm 17
synthesis of S-duloxetine, the solutions hydrochloride salt was LichroCART RP-18 Linearity: AT 5–
antidepressant drug, viz., 2- added. Incubation at 30 500 (μg/mL)
acetyl thiophene (AT), N,N- ◦C in an orbital shaker DKTP &DHTP
dimethyl-3-keto- 200 rpm. LLE with 20–500 (μg/mL)
(2-thienyl)-propanamine ethyl acetate.
(DKTP) and (S)-N,N-dimethyl-
3-hydroxy-(2-thienyl)-
propanamine (DHTP)

type e
Fluoxetine, citalopram, Human SPE on Waters Oasis. C18 (250 mm × 4.6 mm, > 73.2% Linearity: (ESI-MS) in 18
paroxetine and venlafaxine plasma Elution with % HAc- 5 μm), MP: (formic acid 5.0–1000.0 ng/mL the selected
MeOH. 0.6‰, ammonium ion recording
acetate: 30 mmol/L)– (SIR) mode
ACN (35:65, v/v), FR:
0.85 mL/min. IS:
fluvoxamine
Eighteen antidepressants, four Human SPE Nucleosil 100-5- (250 75-99% UV: 230 nm 19
atypical antipsychotics serum ×4.6 mm), MP: 25 mM
And active metabolites potassium dihydrogen
phosphate (pH 7.0)–
ACN (60:40). FR: 1

mL/min. IS: melperone
Amitriptyline (AMI), Plasma and LLE Nova-Pak C18, (4.6 × 92-105% UV: 242 nm 20
nortriptyline (NORT), Serum using hexane at pH 11 150 mm). Linearity: 20 and
imipramine (IMI), desipramine MP: KH2PO4 buffer, 400 ng /mL
(DESI), clomipramine ACN and diethylamine,
(CLOMI), and norclomipramine pH= 8 with H3PO4. FR:
(NCLOMI) 0.9 mL/ min. RT.
IS: econazole.
Fluoxetine, norfluoxetine Human SPE on C8 cartridge. C8 (150 × 4.6 mm). MP: Linearity: 8–200 Fluorescence: 21
plasma Elution with MeOH. ACN and water with ng /mL λem =290
HClO4 and Nm, λexc = 230
tetramethylammonium nm.
perchlorate
FR: 1 mL/min.

type e
Modafinil (provigil) Human LLE with ethyl acetate 4.6 mm × 250mm 80.0- 98.9%. UV:220-233 22
plasma and and ethyl acetate– Symmetry C18, Linearity: 0.1–20.0 nm
urine acetic acid (100:1, v/v). MP: MeOH–water– μg/mL
acetic acid 500:500:1,
v/v) FR: 1.0 mL/min.
IS: Phenylthioacetic acid
Venlafaxine is a bicyclic antidepressant, and is usually categorized as a

serotonin-norepinephrine reuptake inhibitor (SNRI), but it has been referred to as
a serotonin-norepinephrine-dopamine reuptake inhibitor. It acts by blocking the
transporter "reuptake" proteins for key neurotransmitters affecting mood, thereby
leaving more active neurotransmitters in the synapse. In high doses it weakly
inhibits the reuptake of dopamine. Venlafaxine is well absorbed with at least 92%
of an oral dose being absorbed into systemic circulation. It is extensively
metabolized in the liver via the CYP2D6 isoenzyme to O-desmethylvenlafaxine,
which is just as potent a serotonin-norepinephrine reuptake inhibitor as the parent
compound, meaning that the differences in metabolism between extensive and
poor metabolizers are not clinically important in terms of efficacy. Steady-state
concentrations of venlafaxine and its metabolite are attained in the blood within 3
days. Therapeutic effects are usually achieved within 3 to 4 weeks. [5,6]
monitoring of antidepressants. In the following paragraphs an overview of
published methods on the analysis of antidepressants is provided. Figure 10.2
illustrates an example of a chromatographic separation of SNRI‘s and SSRI‘s in
blood plasma.
AU 0,16
0,14
0,12
0,1
0,08
0,06
0,04
0,02
0
0,5 2,5 4,5 6,5
t (min)
Figure 10. 2. HPLC chromatogram of SNRIs and SSRIs determination in human plasma in
the presence of BAM as the internal standard. Experimental data from author‘s laboratory.
VEN: venlafaxine, BAM: bamifylline (IS), PAR: paroxetine, DUL: duloxetine and FLU:
fluoxetine.
Antidepressants 93
Trazodone and its main active metabolite 3-(1-clorophenyl) piperazine (m-

CPP) were determined in human plasma 12 h after the last drug administration.
Trazodone is a second-generation antidepressant with serotonin antagonist
activity. The metabolite is considered to be involved in some side effects of
trazodone therapy, and for this reason its determination is very important during
therapeutic drug monitoring. Analysis was performed with a Jones
Chromatography Genesis C8 column (150mm × 4.6mm, 5 μm) operated at room
temperature using a mobile phase composed of aqueous phosphate buffer (70%),
containing triethylamine, at pH 3.5 and acetonitrile (30%). UV detection at 255
nm was applied. Loxapine was used as the internal standard. C8 reversed phase
cartridges IST (Mid Glamorgan, UK) Isolute (50 mg/1 mL) were applied for
sample preparation with extraction yields values better than 90%. The method was
successfully applied to plasma samples from depressed patients undergoing
therapy with trazodone; accuracy results were satisfactory (recovery >91%). The
method is very selective since no endogenous compounds or any of the tested
Central Nervous System drugs caused any interference in the analysis of TRZ and
m-CPP in depressed patients‘ plasma. The proposed method has high accuracy
and a wide response function range, which allows the determination of the
analytes not only at therapeutic doses of TRZ, but also in overdose cases and
whentakenatsub-therapeuticdoses.[7]
A rapid, convenient and selective method using high performance liquid
chromatography coupled with electrospray ionization mass spectrometry (HPLC–
ESI/MS) has been developed and validated to determine mianserin in human
plasma. Mianserin and the internal standard (I.S.), cinnarizine were extracted from
plasma by N-hexane:dimethylcarbinol (98:2, v/v) after addition of sodium
hydroxide. LC separation was performed on a Thermo Hypersil-Hypurity C18 (5
μm, 150mm × 2.1mm) with the mobile phase consisting of 10 mM ammonium
acetate (pH 3.4)–methanol–acetonitrile (35:50:15, v/v/v) at 0.22 mL/min. The
retention time of mianserin and cinnarizine was 3.4 and 2.1 min, respectively.
Quadrupole MS detection and quantitation was performed by monitoring at m/z
265 [M+H]+ for mianserin and m/z 369 [M+H]+ for cinnarizine (IS). The method
was validated over the concentration ranges of 1.0–200.0 ng/mL for mianserin.
The recovery was 81.3–84.1%. Limit of quantitation was 1.0 ng/mL for
mianserin. This method proved to be suitable for the bioequivalence study of
mianserin hydrochloride tablets. [8]
As it has already been obvious antidepressants are widely used for the
treatment of psychiatric disorders and therefore their monitoring in biological
fluids is quite important taking into account that they can produce dangerous
biochemical imbalances in toxic doses. A method for the determination of
antidepressants: Trazodone, fluoxetine, Mianserine, Desipramine, Imipramine,
Nortryptiline, Amitryptiline Trimipramine and Clomipramine in urine samples is
presented using solid-phase extraction (SPE) and high-performance liquid
chromatography (HPLC) with ultraviolet (UV) detection.
Home-made cartridges containing 30 mg multiwall carbon nanotubes are
employed for isolation of the analytes from the sample, allowing also the
preconcentration of the analytes prior to the HPLC analysis. Chromatographic
separation was carried out on an Eclipse X-DB-C8 column (4.6 × 150 mm) using
as mobile phase sodium dihydrogen phosphate buffer (pH 3.0)–acetonitrile–
methanol 70:25:5 (v/v). The ionic liquid 1-butyl-3-methylimidazolium
trifluoromethanesulfonate was added at a concentration of 20 mmol/L in order to
suppress the effect of the silanol groups on the chromatographic separation. The
flow rate of the isocratic separation was 1.2 mL/min and the injection volume was
20 μL. Analytes were detected at 254 nm. Limits of detection were 12.3 ng/mL
for trazodone and 90.1 ng/mL for fluoxetine. The method, validated for sensitivity
and precision, has been successfully applied to the determination of
antidepressants in urine samples from hospital patients after a course of treatment
with antidepressants and it proved suitable for the therapeutic monitoring of
antidepressants in urine samples. [9]
The applicability of hollow fiber-based liquid phase microextraction (HF-
LPME) was evaluated for the extraction and preconcentration of three

antidepressant drugs (amitriptyline, imipramine and sertraline) prior to their
determination by HPLC-UV in urine and plasma. The extraction was performed
due to pH gradient between the inside and outside of the hollow fiber membrane.
In order to obtain high extraction efficiency, the parameters affecting the HF-
LPME were studied and optimized. All the extractions were carried out using an
Accurel Q3/2 polypropylene hollow fiber membrane (Wuppertal, Germany) with
a 0.2 μm pore size, 600 μm internal diameter and 200 μm wall thickness.
Separations were carried out on a Zorbax Extend C18 column (100 mm × 2.1 mm,
with 3.5 μL particle size). A mixture of 0.02 M acetic acid solution (pH 4.0) and
methanol (54:46) at a flow rate of 0.25 mL/min was used as a mobile phase in
isocratic elution mode. Linearity was observed in the range 5–500 μg/L. UV
detection was applied at 215 nm. Chloropromazine was used as internal standard.
The limits of detection (LODs) ranged between 0.5 and 0.7 μg/L (based on S/N =
3). Finally, the applicability of the proposed method was evaluated by extraction
and determination of the drugs in urine, plasma and tap water samples. The results
indicated that hollow fiber microextraction method has excellent clean-up and
Antidepressants 95
high-preconcentration factor and can be served as a simple and sensitive method

for monitoring of antidepressant drugs in the biological samples. [10]
Α simple, rapid and sensitive HPLC method was developed and validated for
the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin,
clomipramine (CLO) and imipramine, in pharmaceutical formulations and
biological fluids. A Kromasil C8 analytical column (250 × 4 mm, 5 μm) was used
for the separation within 6 min, using a mobile phase consisting of 0.05 M
CH3COONH4 and CH3CN (45:55 v/v) delivered at 1.5 mL/min isocratically.
Quantification was performed at 238 nm, with bromazepam (1.5 ng/μL) as
internal standard. Linearity was observed within the range: 1–5 μg/mL The
determination of TCAs in blood plasma was performed after protein precipitation
with ACN. Urine analysis was performed by means of SPE using Lichrolut RP-18
Merck cartridges providing high absolute recoveries (higher than 94%). Direct
analysis of urine was also performed after two-fold dilution. The developed
method was fully validated in terms of selectivity, linearity, accuracy, precision,
stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5)
revealed RSD lower than 13%. Recoveries from biological samples ranged from
91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1–
0.6 ng in blood plasma and 0.2–0.5 ng in extracted urine or 0.4–0.7 in diluted
urine. The method was applied to real samples of plasma from a patient under
CLO treatment. The method is suitable for the pharmacokinetics and
bioavailability studies of TCAs. It is also a useful tool in human medicine for
estimating and personalising the effective drug dose in patients. [11]

Duloxetine is the most recent serotonin and norepinephrine reuptake inhibitor
(SNRI) drug introduced for the therapy of depression. Thus, it is evident that there
is a need for having on hand new reliable analytical methods for the determination
of duloxetine plasma levels in depressed patients. A method dealing with the
development of a rapid and sensitive high-performance liquid chromatographic
method for duloxetine analysis in human plasma has been presented. The assays
were carried out using a C8 reversed-phase column and a mobile phase composed
of 60% aqueous phosphate buffer containing triethylamine at pH 3.0 and 40%
acetonitrile. The UV detector was set at 230 nm and loxapine was used as the
internal standard. Pre-treatment of plasma samples was developed, based on solid-
phase extraction (SPE) Waters (Milford, MA, USA) Oasis mixed-mode reversed
phase—strong cation exchange (MCX) cartridges (30 mg/1 mL). The extraction
yields values were higher than 90%. Linearity was found in the 2–200 ng/mL
range; the limit of quantitation was 2.0 ng/mL. The method was applied to plasma
samples from depressed patients undergoing therapy with duloxetine. Precision
data and accuracy results were satisfactory and no interference from other drugs
was found.
The HPLC method presented here for the analysis of DLX is feasible and
rapid: a chromatographic run lasts less than five minutes. The SPE procedure
implemented for the sample pre-treatment, based on MCX cartridges, gives good
extraction yields (>90%) and satisfactory precision (RSD% < 5.1%). Thus, the
method seems to be suitable for the therapeutic drug monitoring of duloxetine in
depressed patients‘ plasma. [12]
A high-performance liquid chromatography method is presented for the
determination of 10 frequently prescribed tricyclic and nontricyclic
antidepressants: imipramine, amitriptyline, clomipramine, fluoxetine, sertraline,
paroxetine, citalopram, mirtazapine, moclobemide and duloxetine in human
plasma. The simple and accurate sample preparation step, consisted of
liquid:liquid extraction with recoveries ranging between 72% and 86%, except for
moclobemide (59%). The extraction consisted of the addition of 25 μL of
etidocaine (IS), 200 mg NaCl, 50μL of sodium hydroxide 1.5 M, and 5 mL
hexane-isoamyl alcohol (99:1, v/v) to 1mL of plasma.
Chromatographic separation was achieved isocratically, at room temperature,
on a LiChrospher 60 RP-select B column (250 mm × 4 mm, 5 μm). The mobile
phase consisted of 35% a mixture of acetonitrile: methanol (92:8, v/v) and 65% of
sodium acetate buffer, pH 4.5 delivered at a flow-rate of 1.0mL/min under
isocratic conditions with UV detection at 230 nm. Linearity was observed over a
working range of 2.5–1000 ng/mL for moclobemide, 5–2000 ng/mL for

citalopram, duloxetine, fluoxetine, 10–2000 ng/mL for sertraline, imipramine,
paroxetine, mirtazapine and clomipramine. The intra-assay coefficients of
variation (CVs) studied at three concentrations (50, 200, and 500 ng/mL) for all
compounds were less than 8.8%, and all inter-CVs were less than 10%. Limits of
quantification were 2.5 ng/mL for moclobemide, 5 ng/mL for citalopram,
duloxetine and amitriptyline, and 10 ng/mL for mirtazapine, paroxetine,
imipramine, fluoxetine, sertraline, and clomipramine. No interference of the drugs
normally associated with antidepressants was observed. The method has been
successfully applied to the analysis of real samples, for monitoring of ten
frequently prescribed tricyclic and non-tricyclic antidepressant drugs, so it can be
readily applied to the routine therapeutic drug monitoring. [13]
An HPLC method with UV detection has been developed for the
simultaneous determination of levomepromazine, clozapine and their main
metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, O-
desmethyl-levomepromazine, N-desmethylclozapine and clozapine
Antidepressants 97
N-oxide in human plasma. The analytes were separated on a C8 (150 mm ×

4.6 mm, 5 μm; Phenomenex) column supplemented with a C8 cartridge
precolumn, using a mobile phase composed of acetonitrile and a pH2.0, 34 mM
phosphate buffer containing 0.3% triethylamine (29:71, v/v). UV detection was
performed at 254 nm. Loxapine was used as the internal standard. Baseline
separation of the 7 analytes (and the IS) was achieved in less than 20 min. A
reliable biological sample pre-treatment procedure by means of solid-phase
extraction on BondElut C1 cartridges was implemented, which allows to obtain
good extraction yields (>91%) for all analytes and appropriate sample purification
from endogenous interference. The method was validated in terms of extraction
yield, precision and accuracy. Linearity was observed in the ranges: LMP 9–200
ng/mL; NDLM 10–150 ng/mL; LMSO 5–500 ng/mL; ODLM 7–150 ng/mL; CLZ
20–2500 ng/mL; DMC 15–1000 ng/mL; NOX 10–200 ng/mL.
These assays gave RSD% values for precision always lower than 4.9% and
mean accuracy values higher than 92%. The method is suitable for the therapeutic
drug monitoring (TDM) of patients undergoing polypharmacy with
levomepromazine and clozapine. [14]
A solid phase extraction procedure (SPE) for 13 ‗new‘ antidepressants
(venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine,
melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and
trazodone) together with eight of their metabolites (O desmethylvenlafaxine,
norfluoxetine, desmethylmianserine, desmethylmirtazapine, desmethylcitalopram,
didesmethylcitalopram, desmethylsertraline and m-chlorophenylpiperazine) from

plasma was optimized using HPLC-DAD at 210-380 nm as monitoring system. A
total number of 10 sorbents (apolar, polymeric, ion-exchange and mixed mode
combining ion-exchange properties with, respectively, C8 or a styrene–
divinylbenzene polymer) was evaluated. Based on recovery, reproducibility and
absence of interfering substances the strong cation exchanger gave the best
results. Recoveries were determined at low and high therapeutic and toxic levels
and ranged between 70 and 109% for all compounds, except for trazodone (39%).
[15]
Amitriptyline and nortriptyline are tricyclic antidepressants which act by
enhancing the actions of norepinephrine and serotonin caused by blocking the re-
uptake of various neurotransmitters at the neuronal membrane. A micellar liquid
chromatographic procedure was developed to determine these drugs in serum
samples for use in clinical monitoring. The chromatographic determination was
carried out using a 0.15 M SDS-6% (v/v) pentanol buffered at pH 7, in a Kromasil
5 C18 column, 5 μm, 250 mm × 4.6 mm from Scharlab (Barcelona, Spain). The
mobile phase was 0.15 M SDS-6% (v/v) pentanol at pH 7 delivered at a flow-rate
of 1.5 mL/min. Detection was performed at 240 nm and 650 mV. The analysis
time was 14 min. The limits of detection (ng/mL) in serum were 0.25 and 0.31 for
amitriptyline and nortriptyline, respectively. Repeatability and intermediate
precision were evaluated at three different concentrations in serum samples.
Untreated serum samples were injected directly into the HPLC system after
filtration, leading to be a simple procedure that can be applied in routine analyses
for Therapeutic Drug Monitoring. No interference was observed from endogenous
compounds and other drugs. Recoveries obtained were from 95 to 102%. The
applicability of the procedure developed to determine amitriptyline and
nortriptyline was verified by analysing them in spiked and real serum samples.
[16]
A simple reversed-phase high-performance liquid chromatographic method
employing C18 column has been developed for simultaneous analysis of three
intermediates in the synthesis of S-duloxetine, the antidepressant drug, viz., 2-
acetyl thiophene (AT), N,N-dimethyl-3-keto- (2-thienyl)-propanamine (DKTP)
and (S)-N,N-dimethyl-3-hydroxy-(2-thienyl)-propanamine (DHTP). Good
separations were achieved on a 250 mm × 4.6 mm, 5 μM particle LichroCART
RP-18 column (Merck, Germany) by employing an isocratic system using
acetonitrile and 0.05M phosphate buffer (pH 7.0) containing 0.02% diethylamine.
The detection was carried out at 241 nm. High recovery rates were obtained 99.7-
101.5%. The method was validated in terms of linearity, range, accuracy and
precision. Linearity was demonstrated within the range: AT 5–500 (μg/mL),
DKTP & DHTP 20–500 (μg/mL). The developed HPLC method is simple,
accurate and reproducible and can be used as a routine analytical tool during the
synthesis of S-duloxetine. [17]
The four most commonly prescribed non-tricyclic antidepressants:
Fluoxetine, citalopram, paroxetine and venlafaxine were simultaneously
determined by high performance liquid chromatography–electrospray ionization
mass spectrometry (HPLC–MS/ESI) in human plasma of patients undergoing
antidepressant treatment.
The analytes in plasma were extracted by solid-phase-extraction column on
(Waters Oasis) after samples had been alkalinized. The HPLC separation of the
analytes was performed on a MACHEREY-NAGEL C18 (250mm × 4.6 mm, 5
μm, Germany) column, using water (formic acid 0.6‰, ammonium acetate: 30
mmol/L)–acetonitrile (35:65, v/v) as mobile phase, with a flow-rate of 0.85
mL/min. Fluvoxamine was used as internal standard. The compounds were
ionized in the electrospray ionization (ESI) ion source of the mass spectrometer
and were detected in the selected ion recording (SIR) mode.
Antidepressants 99
The calibration curves were linear in the 5.0–1000.0 ng/mL range for all
compounds. The average extraction recoveries for all the four analytes were above
73.2%. The limits of detection (LODs) were 0.5, 0.3, 0.3 and 0.1 ng/mL for
fluoxetine, citalopram, paroxetine and venlafaxine, respectively. The intra- and
inter-day variation coefficients were less than 15.0%. The method is accurate,
sensitive and simple for routine therapeutic drug monitoring (TDM) as well as
toxicologic screening, and for the study of the pharmacokinetics and metabolism
of the four drugs. [18]
An isocratic reversed-phase HPLC method with ultraviolet detection was
developed and optimised to quantify eighteen antidepressants and four atypical
antipsychotics commonly prescribed psychotropic drugs including seven
metabolites. Among these 29 compounds are new psychotropic drugs for which
so far only single-component assays are described in the literature: mirtazapine,
reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine,
fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline,
nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine,
norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline,
amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-
risperidone in human serum. After solid-phase extraction on 3M-Empore high-
performance extraction disk cartridges (Varian, Darmstadt, Germany) and a Baker
spe-12G vacuum instrument of the drugs and metabolites, the chromatographic
separation was achieved on a Nucleosil 100-5-Protect 1 (endcapped) column with
acetonitrile–potassium dihydrogenphosphate buffer as mobile phase. Melperone

(3000 ng/mL) was used as internal standard. UV detector was used at 230 nm.
The method was validated for therapeutic and toxic serum ranges. Recoveries
were between 75 and 99% for the drugs and metabolites. The accuracy of the
quality control samples, expressed as percent recovery, ranged from 91 to 118%;
intra- and inter-assay-relative standard deviations were 0.9–10.2% and 0.9–9.7%,
respectively. This method is applicable to rapidly and effectively analyze serum or
plasma samples for therapeutic drug monitoring of about 30 antidepressants and
atypical antipsychotics, within 24 h with a single system, thus reducing the time
for apparatus preparation and system in stabilities linked to this process. [19]
A reversed-phase high performance liquid chromatography (HPLC) method
for the determination of plasma and serum levels of amitriptyline (AMI),
nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine
(CLOMI), and norclomipramine (NCLOMI) is described, based upon single step
liquid:liquid extraction with hexane of these compounds at pH 11 (recovery
between 92 and 105%). A a Nova-Pack C-18 HPLC cartridge column was used
for separation while the mobile phase composed of a phosphate buffer with 50%
(v:v) acetonitrile and about 0.2% (v:v) diethylamine (final pH: 8) and solute
detection at 242 nm. Using 1 mL of plasma or serum and econazole as internal
standard, drug levels between 20 and 400 ng/mL were found to provide linear
calibration graphs. For drug concentrations in the range of 70–120 ng/mL,
intraday and interday imprecisions (n=5) were determined to be <6.0, and <15%,
respectively. The assay was employed for therapeutic drug monitoring and
clinical toxicology. The described method is demonstrated to be suitable and
sufficiently reliable for diverse clinical investigations of compliance, drug
overdoses, drug-induced psychoses and substance abuse. [20]
An HPLC method with fluorescence detection for the monitoring of
fluoxetine which is an atypical antidepressant drug that selectively inhibits the
neuronal reuptake of serotonin, and is widely used in the treatment of depressive
disorders in plasma. After SPE on C8 cartridge the analytical separation was
performed on a reversed phase C8 column (150 × 4.6 mm), while the mobile phase
was a mixture of acetonitrile and water containing perchloric acid and
tetramethylammonium perchlorate delivered at a flow rate of 1 mL/min. The
fluorescence detector (exc at 230 nm, em at 290 nm) yielded very low levels of
analytes in plasma with a good selectivity. Several drugs, such as clozapine,
risperidone and diazepam, currently used in psychiatry, did not interfere with
chromatographic analysis of FLU.
Fluoxetine had a retention time of 9.7 min, while norfluoxetine 8.1 min.
Linearity was obtained over the concentration range 8–200 ng/mL for both
substances. The method seems suitable, in accuracy and precision, for the
determination of fluoxetine plasma levels of patients; furthermore, it is rapid and
sensitive. [21]
Modafinil was determined in human plasma and urine. Modafinil is a novel
antidepressant also used as wake-promoting drug that is being used for the
management of excessive sleepiness in patients with narcolepsy.
It has pharmacological properties similar to that of amphetamine, but without
some of the side effects associated with amphetamine-like stimulants. Since
modafinil has the potential to be abused, accurate drug-screening methods are
needed for its analysis. An HPLC method for the quantitative analysis of
modafinil in plasma and urine was developed. (Phenylthio) acetic acid was used
as an internal standard for the analysis of both plasma and urine. Modafinil was
extracted from urine and plasma with ethyl acetate and ethyl acetate–acetic acid
(100:1, v/v), respectively, and analyzed on a Symmetry C18 reverse phase column
(4.6mm×250mm) with methanol–water–acetic acid (500:500:1, v/v) as the mobile
phase delivered at a flow rate of 1.0 mL/min. Recoveries from urine and plasma
Antidepressants 101
were 80.0 and 98.9%, respectively and the limit of quantitation was 0.1 μg/mL at
233 nm. No interference was observed.
The HPLC method can be used for screening plasma and urine samples for
the presence of the modafinil and it can be used for therapeutic drug monitoring,
pharmacokinetic studies, and drug abuse screening. [22]
REFERENCES
[1] Wong, D.T.; Robertson, D.W.; Bymaster, FP.; Krushinski, J.H.; Reid, LR.
LY227942, an inhibitor of serotonin and norepinephrine uptake:
biochemical pharmacology of a potential antidepressant drug. Life Sciences
1988 43, 2049–2057.
[2] www.sciencedaily.com (Accessed January 2009)
[3] www.nature.com (Accessed January 2009)
[4] Wong, D.T.; Bymaster, F.P.; Engleman, E.A. Prozac, the first selective
serotonin uptake inhibitor and an antidepressant drug: twenty years since its
first publication. Life Sciences 1995 57, 411-441.
[5] Golden, R.N.; Nicholas, L. Antidepressant efficacy of venlafaxine.
Depression and anxiety 2000 12, 45–49.
[6] Bielski, R.J.; Ventura, D.; Chang, C.C. A double-blind comparison of
escitalopram and venlafaxine extended release in the treatment of major

depressive disorder. The Journal of clinical psychiatry 2004 65, 1190–1196.
[7] Mercolini, L.; Colliva, C.; Amore, M.; Fanali, S.; Raggi, M.A. HPLC
analysis of the antidepressant trazodone and its main metabolite m-CPP in
human plasma. Journal of Pharmaceutical and Biomedical Analysis 2008
47, 882–887.
[8] Xu, P.; Li, H.-D.; Chen, B.-M.; Ma, N.; Yan, M.; Zhu, Y.-G. Determination
of mianserin in human plasma by high performance liquid chromatography–
electrospray ionization mass spectrometry (HPLC–ESI/MS): Application to
a bioequivalence study in Chinese volunteers. Journal of Pharmaceutical
and Biomedical Analysis 2008 47, 994–999.
[9] Cruz-Vera, M.; Lucena, R.; Cárdenas, S.; Valcárcel, M. Combined use of
carbon nanotubes and ionic liquid to improve the determination of
antidepressants in urine samples by liquid chromatography. Analytical
Bioanalytical Chemistry 2008 391, 1139–1145.
[10] Esrafili, A.; Yamini, Y.; Shariati, S. Hollow fiber-based liquid phase
microextraction combined with high-performance liquid chromatography
for extraction and determination of some antidepressant drugs in biological

fluids. Analytica Chimica Acta 2007 604, 127–133.
[11] Samanidou, V.F.; Nika, M.K.; Papadoyannis, I.N. Development of an
HPLC method for the monitoring of tricyclic antidepressants in biofluids.
Journal of Separation Science 2007 30, 2391 – 2400.
[12] Mercolini, L.; Mandrioli, R.; Cazzolla, R.; Amore, M.; Raggi, M.A. HPLC
analysis of the novel antidepressant duloxetine in human plasma after an
original solid-phase extraction procedure. Journal of Chromatography B
2007 856, 81–87.
[13] Malfara, W.R.; Bertucci, C.; Costa Queiroz, M.E.; Dreossi Carvalho, S. A.;
Pires Bianchi, M.; Cesarino, E.J.; Crippa, J.A.; Costa Queiroz, R.H.
Reliable HPLC method for therapeutic drug monitoring of frequently
prescribed tricyclic and nontricyclic antidepressants. Journal of
[14] Mercolini, L.; Bugamelli, F.; Kenndler, E.; Boncompagni, G.; Franchini, L.;
Raggi, M.A. Simultaneous determination of the antipsychotic drugs
levomepromazine and clozapine and their main metabolites in human
plasma by a HPLC-UV method with solid-phase extraction. Journal of
[15] Wille, S.M.R.; Maudens, K. E.; Van Peteghem, C.H.; Lambert, W.E.E.
Development of a solid phase extraction for 13 ‗new‘ generation
antidepressants and their active metabolites for gas chromatographic–mass
spectrometric analysis. Journal of Chromatography A 2005 1098, 19–29.

[16] Bose, D.; Durgbanshi, A.; Martinavarro-Domınguez, A.; Capella-Peiro, M.-
E.; Carda-Broch, S.; Esteve-Romero, J.; Gil-Agust, M. Amitriptyline and
nortriptyline serum determination by micellar liquid chromatography.
Journal of Pharmacological and Toxicological Methods 2005 52, 323 –
329.
[17] Soni, P.; Mariappan, T.T.; Banerjee, U.C. High-performance liquid
chromatographic method for the simultaneous estimation of the key
intermediates of duloxetine. Talanta 2005 67, 975–978.
[18] Juan, H.; Zhiling, Z.; Huande, L. Simultaneous determination of fluoxetine,
citalopram, paroxetine, venlafaxine in plasma by high performance liquid
chromatography–electrospray ionization mass spectrometry (HPLC–
MS/ESI). Journal of Chromatography B 2005 820, 33–39.
[19] Frahnert, C.; Rao, M.L.; Grasmader, K. A nalysis of eighteen
antidepressants, four atypical antipsychotics and active metabolites in serum
by liquid chromatography: a simple tool for therapeutic drug monitoring.
Antidepressants 103
[20] Theurillat, R.; Thormann, W. Monitoring of tricyclic antidepressants in

human serum and plasma by HPLC: characterization of a simple, laboratory
developed method via external quality assessment. Journal of
Pharmaceutical and Biomedical Analysis 1998 18,751–760.
[21] Raggi, M.A.; Mandrioli, R.; Casamenti, G.; Bugamelli, F.; Volterra, V.
Determination of fluoxetine and norfluoxetine in human plasma by high-
pressure liquid chromatography with fluorescence detection. Journal of
[22] Schwertner, H.A.; Kong, S.B. Determination of modafinil in plasma and
urine by reversed phase high-performance liquid-chromatography. Journal
of Pharmaceutical and Biomedical Analysis 2005 37, 475–479.
Chapter 11
IMMUNOSUPPRESSANTS
Immunosuppressive drugs inhibit or prevent activity of the immune system.
They are used in immunosuppressive therapy to prevent the rejection of
transplanted organs and tissues, to treat autoimmune diseases or diseases that are
most likely of autoimmune origin as well as to treat some other non-autoimmune
inflammatory diseases. However these drugs may present side-effects, such as
hypertension, dyslipidemia, hyperglycemia, peptic ulcers, liver, and kidney injury.
The immunosuppressive drugs also interact with other medicines and affect their
metabolism and action.
monitoring of immunosupressants.
Cyclosporin which is potent immunosuppressive agent characterized by a

narrow therapeutic range blocks the replication of lymphocytes and inhibits the
release and production of interleukin-2. It plays an important role in a range of
clinical conditions, including organ transplantation. Low cyclosporin
concentrations can increase the chance of graft rejection, while high
concentrations are associated with nephrotoxicity. Therefore, therapeutic drug
monitoring is prudent. However, cyclosporin is difficult to be measured and early
immunoassays detected both parent drug and a range of metabolites, leading to
wide variability in proposed target ranges. Current assay methods are more
specific and the focus has changed from analytical issues to the relationships
between pharmacokinetic parameters and response.
Table 11.1. Overview of HPLC methods for therapeutic drug monitoring of immunosuppressants.

type
Cyclosporine A) Whole Blood sample acidified with HCl Normal-phase Sperisorb S5 31–1500 ng/mL UV: 212 nm 1
and its major, partly blood (0.18M) and LLE with diethyl CN, 250-4 Phasesep, 5 μm, for CyA and of
active metabolites ether. 25 cm × 4.6mm, at 60 ◦C, 31–1000 ng/mL
AM1, AM9, AM4N, MP: hexane-isopropanol for metabolites.
and AM19 s (93:7, v/v). IS: cyclosporine LOD: 15 ng/mL
D (CyD).
Εverolimus Ηuman SPE after protein precipitation Ultrasphere C8 with 3 μm; 75 1–200 ng/mL UV: 278 nm 3
whole using zinc sulfate solution and mm × 4.6 mm, at 60 ◦C. MP:
blood acetone. Bond-Elut cartridges C18. 56% ACN in water FR: 1
mL/min.
Tacrolimus, Blood Protein precipitation with ACN. AtlantisTM C18 (150 mm × Linearity: 1000 MS: SRM in 5
2.0 mm) 3 μm or a YMCTM
sirolimus and samples Vortex and centrifuge at 14000 g to 1 ng/mL positive ion
cyclosporin A for 10 min. CN (150mmx. 2.0mm) 3 μm. mode:
MP: ACN/water 0.1% acetic cyclosporine A
acid (negative ion mode) or 1225+/1114+,
0.1% formic acid (positive sirolimus
ion mode) 937+/409+ and
Response. FR: 0.45 mL/min tacrolimus
at 50 ◦C. IS: Ascomycin 827+/616+
Everolimus (RAD), Blood Deproteinization by aqueous zinc Ultrasphere C8 3 μm; 75 × Linearity: 1-160 UV: 278 & 214 6
cyclosporineA(CsA) samples sulfate and acetone. SPE Bond– 4.6 mm at 60oC. MP: 56% ng/mL for RAD nm
and cyclosporine Elut cartridge (C18) Wash with ACN in water. and 20-10,000
D (CsD) methanol / water (30:70) and FR: 1 mL/min. ng/mL for CsA.
hexane. Elution with ACN. Recovery:
76.8% for RAD,
65.5 % for
DMRP, 86.2 %
for CsA and
65.1 % for CsD.
Immunosuppressants 107
Cyclosporine A, is characterised by inter- and intra-individual variability and

a lack of correlation between drug dosage and blood levels. Blood levels of CyA
should be routinely monitored to assess organ rejection and toxicity. For this
reason a simple and efficient optimized high-performance liquid chromatograph
(HPLC) method for the simultaneous determination of cyclosporine A (CyA) and
its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole
blood from transplant patients was developed using cyclosporine D (CyD) as
internal standard. Blood sample was acidified with HCl (0.18 M) and extracted
with diethyl ether. The separation was performed on a 25 cm × 4.6 mm, normal-
phase column (Sperisorb S5 CN, 250-4 Phasesep, 5 μm), maintained at 60 ◦C,
with hexan-isopropanol (93:7, v/v) as mobile phase. The compounds were
quantified by UV detection at 212 nm. Linearity for all five compounds was tested
in the range of 31–1500 ng/mL for CyA and of 31–1000 ng/mL for metabolites.
The limit of detection was found to be 15 ng/mL for all compounds. This method
is also suitable for measuring cyclosporine A and metabolite concentrations in
routine monitoring of patients undergoing treatment with CyA. [1]
CyA as well as its metabolites (AM9, AM19, AMl, and AM4N) were
evaluated in whole blood samples from 117 patients using commercially available
immunological assays (AxSYM, EMIT, Dimension) and HPLC. [2]
In the clinical practice of organ transplantation, everolimus is used in
combination with cyclosporine (CsA), the most common antirejection agent. Both
drugs show a narrow therapeutic window, which requires strict monitoring of
their blood concentration. Simple methods for simultaneous measurement of

everolimus and CsA concentration are necessary.
A high-performance liquid chromatography–ultraviolet (HPLC–UV) method
for determining everolimus concentrations in human whole blood was developed
and validated. Sample preparation involved a solid-phase extraction after protein
precipitation using zinc sulfate solution and acetone. Bond-Elut cartridges (C18,
200 mg, 3 mL, Varian) were used for sample clean-up, on aVac Elut 20 Manifold
(Varian). The HPLC system used for separation of everolimus from internal
standard (IS) and endogenous components consisted of an analytical column
(Ultrasphere C8, 3 μm, 75 mm×4.6 mm, Beckman, Fullerton, CA) heated at 60 ◦C.
UV detector was set at 278 nm. The mobile phase consisted of 56% acetonitrile in
water and was delivered at a flow rate of 1 mL/min. The method showed a linear
relationship between peak height ratios and blood concentrations in the range of
1–200 ng/mL. The method was found to be precise, accurate, and sensible making
it useful for routine therapeutic monitoring of everolimus. [3]
The previously described method was further implemented to allow
concomitant measurement of everolimus and CsA. Blood samples were
deproteinized by addition of an aqueous zinc sulfate solution and acetone. After

centrifugation the clear supernatant was further purified on Bond–Elut cartridge
(C18, 200 mg/3 mL, Varian). The method was found accurate and precise and
useful for simultaneous therapeutic monitoring of the two drugs. [4]
The potential of a cyano HPLC column for the analysis of three
immunosuppressants was investigated by Hatsis and Volmer. Tacrolimus,
sirolimus and cyclosporine A, were used to investigate differences in the retention
and efficiency of a cyano column compared to the more widely used C18 column.
The cyano column showed comparable retention for all three compounds, whereas
the C18 column showed stronger retention, especially for cyclosporin A. HPLC
separations were carried out on either an AtlantisTM C18 column (150mm ×
2.0mm, 3 μm) (Waters, Milford, MA, USA) or a YMCTM CN column (150mm ×
2.0mm, 3 μm) (Waters). Separations were performed isocratically using
acetonitrile/water mobile phases at a flow rate of 0.45 mL/min. Column
temperature was maintained at 50 ◦C. Mobile phases contained 0.1% acetic acid
(in negative ion mode) or 0.1% formic acid (in positive ion mode) to enhance
electrospray response. The overall chromatographic run time was approximately 4
min, including 1 min for the injection. The efficiencies at 50 ◦C were up to 12
times higher on the cyano column. Baseline separation was achieved in less than
three minutes with the cyano column, using an isocratic mobile phase of 52/48
(v/v) acetonitrile/water at 0.45 mL/min. The analysis of immunosuppressant drugs
in human whole blood was performed with the cyano column using a selected
reaction monitoring (SRM) method for each analyte with negative ion mode
electrospray ionization on a triple quadrupole mass spectrometer. Detection limits
were 0.05 ng/mL for sirolimus, 0.1 ng/mL for cyclosporin A and 0.2 ng/mL for
tacrolimus. Good agreement was obtained with the actual levels of
immunosuppressant drugs in patient samples with an average error of less than
10%. [5,6]
REFERENCES
[1] Khoschsorur, G.; Erwa, W.; Fruehwirth, F.; Stettin, M.; Meinitzer, A.;
Hoebarth, G.; Zelzer S.; Halwachs-Baumann, G. High-performance liquid
chromatographic method for the simultaneous determination of
cyclosporine A and its four major metabolites in whole blood Talanta 2005
65, 638–643.
[2] Stettin, M.; Halwachs-Baumann, G.; Genser, B.; Fruhwirth, F.; Marz, W.;
Khoschsorur, G.A. Determination of cyclosporine A in whole blood:
Immunosuppressants 109
Comparison of a chromatographic method with three different

immunological methods. Talanta 2006 69, 1100–1105.
[3] Baldelli, S.; Murgia, S.; Merlini, S.; et al. High-performance liquid
chromatography with ultraviolet detection for therapeutic drug monitoring
of everolimus. Journal of Chromatography B 2005 816, 99– 105.
[4] Baldelli, S.; Zenoni, S.; Merlini, S.; Perico, N.; Cattaneo, D. Simultaneous
determination of everolimus and cyclosporine concentrations by HPLC with
ultraviolet detection. Clinica Chimica Acta 2006 364, 354 – 358.
[5] Hatsis, P.; Volmer, D.A. Evaluation of a cyano stationary phase for the
determination of tacrolimus, sirolimus and cyclosporin A in whole blood by
high-performance liquid chromatography–tandem mass spectrometry.
[6] Golden, R.N.; Nicholas, L. Antidepressant efficacy of venlafaxine.
Depression and anxiety 2000 12, 45–49.
Chapter 12
CONCLUSIONS
Therapeutic Drug Monitoring (TDM) is the measurement of specific drugs at
different time intervals in order to maintain a relatively constant concentration of
the medication in the bloodstream. Drugs that require monitoring are those which
tend to have a narrow ―therapeutic range‖. This means that the necessary quantity
to be effective is very close to the quantity that causes significant side effects
and/or signs of toxicity.
There are several cases in clinical trials, where drug monitoring is required to
individualise and optimise drug therapy. Moreover drug monitoring supports
pharmacokinetic and drug metabolism studies.
The usefulness of TDM to optimize drug management the pharmacotherapy

has been strongly supported by the significant variability of plasma drug
concentration among the patients under treatment with several classes of drugs:
cardioactive medications, antiepileptic drugs, antibiotics (e.g. aminoglycosides),
anti-cancer drugs, immunosuppressants, antidepressants (e.g. tricyclic
antidepressants), bronchodilators (theophylline), antipsychotics etc.
Therapeutic drug monitoring necessitates efficient, fast and reliable analytical
methods validated by external quality control. Chromatography-based methods
have sufficient sensitivity and selectivity to confidently identify the analytes of
interest down to the cut-off level. HPLC is usually the technique of choice for
confirmative determination of several classes of drugs. Nowadays HPLC-MS/MS
has gained increasing popularity in clinical laboratories due to the advantages of
the technology over other methods, while the investment cost for instruments has
been decreased. HPLC-MS/MS provides high specificity and sensitivity for the
most drugs. In addition, HPLC-MS/MS is able to simultaneously measure several
drugs and/or their major metabolites in one single analytical run.
Sample preparation is considered as the bottleneck in sample analysis.

Biological samples usually require sample pre-treatment involving more than one
steps. Prior to HPLC analysis biological samples may require extraction. In the
extraction method the compound of interest is removed from the biological matrix
(plasma, serum, urine, etc.) under suitable conditions that selectively isolate
(extract) the desired components and leave behind unwanted materials. The
sample as prepared for chromatograph should be concentrated and prepurified in
an organic solvent. This is accomplished mainly by liquid-liquid extraction (LLE)
and solid-phase extraction (SPE). Nearly all analytical problems can be solved by
LLE and SPE. Therefore, these methods can be characterized as universal from a
scientific and technical point of view.
The use of SPE poses several advantages with respect to the liquid–liquid
extraction procedures used as the SPE procedure is faster and requires lower
volumes of organic solvents. A protein precipitation step is usually implemented
prior to SPE using organic solvents such as acetonitrile or acids as trifluoroacetic
acid.
Moreover modern techniques such as SPME may act as a substitution and
improvement of classical methods. Besides that SPME may lead to new
approaches in biomedical analysis. For example, the potential of SPME to analyze
directly the free concentration of drugs in plasma should be more investigated in
future.
The following concluding remarks can be extracted from this thorough
literature search:
 The demand for rapid methods in the field of drug monitoring will
continue to increase in the future.
 Fully automated systems will be required for routine analysis to provide
the nevessary means to estimate therapeutic levels, monitor the levels of
the drugs and personalize dosages.
 Bioanalytical methods will continue to be developed for safer
manipulation of biological fluids.
 HPLC-MS/MS will play a predominant role in drug monitoring since
mass spectrometric detection will allow not only parent analytes, but their
metabolism products to be identified. This will allow metabolic paths to
be explored in the field of clinical chemistry.
In this book chromatographic methods published in the last decade are

presented regarding their applicability to drug monitoring of most commonly
Conclusions 113
researched analytes. Special emphasis has been given to sample preparation

modes.
APPENDIX
ACN: Acetonitrile
DAD: Diode Array Detection
EDTA: Ethylenediaminetetraacetic acid
FR: Flow Rate
IS: Internal Standard
LLE: Liquid –Liquid Extraction
MeOH: Methanol
MP: Mobile Phase
RP: Reversed Phase
SPE: Solid Phase Extraction
TCA: Trichloroacetic acid
TFA: Trifluoroacetic acid
INDEX
adjustment, 12
administration, 1, 23, 25, 31, 36, 44, 50, 55,
A 69, 74, 76, 77, 93
adsorption, 15, 17
absorption, 1, 26
adult, 30
accidents, 1
adults, 67, 69
accuracy, 18, 23, 27, 35, 46, 47, 56, 66, 68,
adverse event, 83
70, 71, 72, 73, 75, 76, 78, 79, 93, 95, 96,
aerobic, 49
97, 98, 99, 100
aerosol, 29
ACE, 33
age, 1, 2, 30
acetate, 23, 26, 36, 37, 51, 60, 62, 66, 71, 73,
agent, 16, 23, 37, 44, 69, 107

80, 86, 88, 89, 90, 91, 93, 96, 98, 100
agents, 3, 21, 22, 85
acetic acid, 26, 37, 45, 59, 62, 69, 74, 87, 91,
agonist, 42
94, 100, 106, 108
agranulocytosis, 42, 54, 85
acetone, 61, 72, 106, 107, 108
air, 21, 29, 31
acetonitrile, 13, 22, 25, 31, 35, 36, 37, 38, 44,
airways, 29
46, 47, 51, 56, 66, 67, 68, 69, 70, 72, 74,
albumin, 2, 3, 85
75, 76, 77, 78, 93, 94, 95, 96, 97, 98, 99,
alcohol, 31, 60, 65, 71, 78, 96
100, 107, 108, 112
alkaline, 32, 74
achievement, 56
alkaline phosphatase, 32
acid, 3, 13, 15, 23, 25, 26, 27, 37, 45, 49, 50,
alpha, 84
51, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64,
alternative, 18, 47
67, 69, 70, 71, 72, 74, 75, 76, 77, 87, 90,
amine, 15
91, 94, 98, 100, 106, 108, 112, 113
amino, 15, 54, 71
acidic, 13, 16
amino acid, 54, 71
Acinetobacter, 49
amino acids, 54, 71
action potential, 56
aminoglycosides, vii, 3, 49, 111
activation, 15
ammonia, 45, 87
acute, 30, 42
ammonium, 23, 26, 36, 37, 38, 46, 51, 60, 71,
adipose, 2
86, 90, 93, 98
adipose tissue, 2
amphetamine, 100
116 Index
anaemia, 85 asthma, 29
anaerobic, 49, 50 atherosclerosis, 35
anaerobic bacteria, 49, 50 autoimmune diseases, 105
analog, 22 availability, 5, 85
anemia, 54 avoidance, 72
angiotensin converting enzyme, 33
animal models, 22, 23
animals, 23, 31, 36 B
anorexia nervosa, 85
babies, 38
ANOVA, 67
bacteria, 22, 49, 50
antagonism, 42
bacterial, 49, 50
antagonist, 42, 93
bacterial infection, 49
antiangiogenic, 22
bacteriostatic, 49, 50
antibacterial, 50
barbiturates, 55
antibiotic, 49, 50
behavior, 44
antibiotics, vii, 3, 49, 50, 51, 111
beneficial effect, 21, 41, 55
anti-cancer, 3, 21, 22, 24, 111
benzodiazepines, 55
anticholinergic, 41, 42, 85
bile, 23, 25, 79
anticoagulant, 44
bile duct, 25
anticonvulsant, 54, 55, 56, 78
binding, 29, 50, 51, 55
anticonvulsants, 3, 54, 55
bioanalytical, 101, 112
antidepressant, 83, 84, 85, 89, 92, 93, 94, 98,
bioavailability, 22, 29, 55, 85, 95
100, 101, 102
biomolecules, 6, 18
antidepressant medication, 84
biotransformation, 2, 23, 73
antidepressants, vii, 3, 45, 48, 68, 83, 84, 86,
bipolar, 42, 44, 54, 55, 69, 73, 83

90, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102,
bipolar disorder, 42, 44, 54, 55, 69, 73, 83
103, 111
blocks, 23, 85, 105
antineoplastic, 27
blood, vii, 4, 13, 22, 25, 26, 27, 29, 31, 33, 36,
antioxidant, 36, 37, 39
37, 38, 42, 44, 53, 54, 55, 64, 67, 70, 77,
antipsychotic, 41, 42, 47, 48, 81, 82, 102
79, 81, 85, 92, 95, 106, 107, 108, 109
antipsychotic drugs, 41, 42, 81, 102
blood collection, 38
antipsychotics, vii, 3, 41, 42, 43, 44, 45, 46,
blood flow, 29, 37
48, 54, 90, 99, 102, 111
blood plasma, 55, 92, 95
anxiety, 42, 44, 54, 83, 85, 101, 109
blood pressure, 29, 36, 37, 38
anxiety disorder, 44, 83, 85
bloodstream, 1, 33, 111
anxiolytic, 42
body fluid, 18, 37
application, 4, 18, 47, 68, 71, 72, 81
body temperature, 37
aqueous solution, 87
bone marrow, 21
aripiprazole, 45, 46, 47, 48
borate, 69
Aristotle, viii
borderline, 55
arrhythmia, 84
borderline personality disorder, 55
arrhythmias, 55
Bose, 102
artery, 33
bottleneck, 112
aspartate, 54
bovine, 32
assessment, 51, 78, 103
brain, 41, 54, 55, 83
Index 117
brain activity, 55 cerebrospinal fluid, 63, 76, 81

Brazilian, 47 channel blocker, 53
breakdown, 22 channels, 53, 54, 55, 56
breathing, 29 chemical structures, 4
broad spectrum, 49 chemometrics, 73
bronchial airways, 29 chemotherapy, 21
bronchitis, 29 children, 30, 37, 44, 67
bronchodilator, 29 Chinese medicine, 36
buffer, 25, 26, 31, 38, 44, 46, 47, 51, 58, 60, chiral, 69, 73
61, 63, 65, 66, 67, 68, 70, 72, 74, 75, 78, chloride, 31, 58, 78
86, 87, 88, 89, 90, 93, 94, 95, 96, 97, 98, 99 chloroform, 31
building blocks, 21 chlorpromazine, 41, 44, 45
bulimia, 85 cholesterol, 33
bulimia nervosa, 85 cholinergic, 42, 84
butyl ether, 63, 76 chromatograms, 37
butyric, 71 chromatographic technique, 5
chromatography, vii, 5, 8, 9, 25, 67, 103
chronic pain, 83
C ciprofloxacin, 31, 32
circulation, 2, 37, 92
cadmium, 31, 32
cirrhosis, 29, 30
calcium, 53
citalopram, 45, 46, 83, 88, 89, 90, 96, 97, 98,
calibration, 37, 45, 99, 100
99, 102
cancer, 21, 22, 23, 33
classes, 2, 41, 49, 111
cancer cells, 21
classical, 14, 48, 112
cancerous cells, 21
clinical trial, 23, 33, 73, 111
capillary, 26, 38, 81
clinical trials, 23, 33, 73, 111
capital cost, viii
CLO, 95
caps, 69
clozapine, 42, 44, 45, 46, 75, 76, 81, 88, 96,
carbon, 36, 94, 101
97, 99, 100, 102
carbon dioxide, 36
CNS, 47
carbon nanotubes, 94, 101
Co, 13
carbonic anhydrase inhibitors, 54
coefficient of variation, 74
carboxylic, 15
coenzyme, 35
carcinogen, 22
colon, 22, 23, 25
cardiac arrhythmia, 55
colon cancer, 23, 25
cardiovascular disease, 2, 33, 35, 36, 37
complexity, 13
carrier, 2, 71
compliance, 2, 100
catalase, 36
components, 2, 5, 6, 7, 11, 13, 15, 16, 17, 18,
cation, 87, 95, 97
26, 49, 107, 112
cell, 18, 21, 22, 23, 25, 26, 27, 49, 50, 54
composition, 7
cell culture, 18
compounds, 1, 6, 7, 13, 15, 17, 18, 25, 35, 46,
cell division, 21, 22
66, 68, 73, 89, 93, 96, 97, 98, 99, 107, 108
cell growth, 23
cellulose, 69
central nervous system, 29, 42, 73, 85
118 Index
concentration, vii, 1, 2, 3, 4, 11, 18, 23, 26, detection, vii, 7, 9, 12, 18, 23, 25, 26, 27, 28,
30, 35, 46, 68, 70, 72, 74, 75, 77, 84, 85, 32, 35, 37, 38, 44, 46, 47, 48, 51, 56, 67,
93, 94, 100, 107, 111, 112 68, 70, 71, 72, 74, 75, 76, 78, 80, 81, 89,
conditioning, 15, 16, 67 93, 94, 95, 96, 97, 98, 99, 100, 103, 107,
conductance, 55 109, 112
conduction, 69 detection techniques, 7
congestive heart failure, 30, 33 diabetic neuropathy, 84
Congress, iv dialysis, 17
contamination, 28 diet, 2
control, 8, 31, 36, 46, 54, 55, 74, 84 Discovery, 38, 52
conversion, 26, 53 diseases, 3, 105
coronary heart disease, 33 disorder, 41, 44, 47, 55, 83, 85
correlation, 28, 70, 107 distribution, 1, 2, 3, 5, 29
correlation coefficient, 70 disulfide, 38, 39
cost effectiveness, 25 division, 21, 22
cost-effective, 70 DNA, 21, 22, 50
coupling, 17, 19 donor, 26
covering, 45 dopamine, 41, 47, 84, 92
CRC, 4 dopaminergic, 84
creatinine, 26, 28 dosage, vii, 1, 2, 3, 32, 84, 107
culture, 74 dosing, 1, 84
cyclodextrin, 62, 73 drinking, 31
cyclohexyl, 15 drinking water, 31
cyclophosphamide, 26, 28 drowsiness, 54
Cyclosporin, 105 drug abuse, 101
cyclosporine, 106, 107, 108, 109 drug design, 33

Cyclosporine A, 106, 107 drug interaction, 2, 3, 55
cystic fibrosis, 30 drug metabolism, vii, 111
cytochrome, 3, 30, 55, 84, 85 drug therapy, vii, 11, 111
cytoplasmic membrane, 49 drug treatment, 53
cytosolic, 26 drug use, 29, 54
cytotoxic, 25 drug-induced, 100
drying, 12, 15, 66
dyslipidemia, 105
D dysthymia, 83
DAD, 46, 58, 67, 89, 97, 113

death, 42, 50, 77 E
delivery, 78
deoxyribonucleotides, 21 eating, 44, 83
depressed, 93, 95, 96 eating disorders, 44, 83
depression, 41, 42, 44, 55, 83, 85, 95 effluent, 15, 37, 44, 46
depressive disorder, 84, 100, 101 elderly, 2, 30, 35
derivatives, 25, 59, 69, 71 electrophoresis, 81
desipramine, 45, 46, 84, 90, 99 electrostatic interactions, 6
desorption, 9, 17, 18, 56, 66 ELISA, vii, 36
Index 119
emission, 38, 71, 77 fibromyalgia, 84

emotional, 1 fibrosis, 30
emphysema, 29 filtration, 12, 87, 89, 98
emulsions, 13, 14 first generation, 41, 83
enantiomer, 62, 69, 73 flow rate, 25, 26, 31, 35, 37, 38, 44, 45, 46,
enantiomers, 59, 69, 73, 80 51, 68, 70, 71, 72, 73, 75, 76, 77, 78, 94,
encapsulated, 66 100, 107, 108
environment, 12 fluid, 29, 31, 50, 51, 52, 63, 76, 79, 81
enzyme immunoassay, vii fluorescence, vii, 7, 38, 71, 78, 80, 81, 100,
enzyme-linked immunosorbent assay, vii 103
enzymes, 56 fluoxetine, 45, 46, 83, 85, 86, 88, 89, 92, 94,
epilepsy, 53, 54, 69, 70, 71, 73, 79, 80 96, 97, 99, 100, 102, 103
epoxy, 64 fluphenazine, 45
equilibrium, 18 fluvoxamine, 45, 46, 76, 81, 83, 89, 90, 97, 99
Erk, 23 folic acid, 3
erythrocytes, 25 Food and Drug Administration (FDA), FDA,
escitalopram, 83, 101 42, 44, 76
ESI, 9, 26, 27, 35, 38, 62, 72, 74, 86, 90, 93, forensic, 77
98, 101, 102 fouling, 17
ESL, 73 fragmentation, 9
estimating, 95 fungi, 22, 49
ethanol, 59, 60, 67, 69, 71
ethyl acetate, 51, 62, 89, 91, 100
evaporation, 13 G
evolution, 5
GABA, 54, 71, 84

excitation, 38, 53, 71, 77
gamma-aminobutyric acid, 54
excitatory synapses, 53
gas, 17, 71, 102
exclusion, 6, 15
gas chromatograph, 17, 102
excretion, 1, 13, 85
gastric, 22
exercise, 36
gastrointestinal, 21, 50
experimental design, 67
gel, 15
exposure, 28, 31, 32
gene, 23
extraction, 12, 13, 14, 15, 16, 18, 19, 26, 28,
generalized anxiety disorder, 84
31, 35, 37, 38, 44, 46, 47, 51, 56, 57, 66,
generalized seizures, 53, 56
67, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
generalized tonic-clonic seizure, 53, 55
93, 94, 95, 96, 97, 98, 99, 102, 107, 112
generation, 31, 83, 93, 102
extraction process, 15
gentamicin, 49
Germany, 56, 66, 94, 98, 99
F gingival, 50, 51, 52
glass, 44, 66, 69, 74
failure, 3 glia, 53
family, 83 glutamate, 54, 84
fatal arrhythmia, 84 glutathione, 36
FDA approval, 42 glutathione peroxidase, 36
fiber, 17, 18, 87, 94, 101 Gram-negative, 49, 50
120 Index
Gram-positive, 49, 50 hypertensive, 37

grouping, 71 hyperthyroidism, 30
groups, 2, 15, 36, 53, 83, 94 hypnotic, 55
growth, 21, 23, 49 hypothyroidism, 30
growth inhibition, 23
growth rate, 21
guidelines, 76 I
identification, 7, 8
H imbalances, 94
immersion, 66
half-life, 1, 55 immune system, 105
hallucinations, 37 immunoassays, vii, 105
haloperidol, 41, 44, 45 immunological, 107, 109
health, 1, 31 immunosuppressive agent, 105
health effects, 31 immunosuppressive drugs, 105
heart, 7, 29, 30, 33, 36, 37 imprinting, 17
heart rate, 29, 36, 37 impurities, 11, 12
heating, 25 in vitro, 23
height, 107 in vivo, 23
heptane, 46 inactive, 55
hexafluorophosphate, 59, 69 incidence, 3, 4, 35, 84
hexane, 64, 76, 86, 90, 93, 96, 99, 106 indication, 2, 55
high risk, 35 individual differences, 26
high-frequency, 56 inducer, 56
high-performance liquid chromatography, 37, induction, 2

48, 56, 72, 78, 79, 81, 94, 96, 101, 107, 109 infants, 2, 69
histamine, 42 infection, 49
HIV, 2 infections, 1, 49, 50
HIV/AIDS, 2 inflammation, 33
homocysteine, 37, 39 inflammatory, 4, 22, 23, 29, 105
hormone, 83 inflammatory disease, 23, 105
hormones, 54 inhalation, 29
hospital, 94 inhibition, 3, 23, 32, 50, 53, 54, 84
HPLC-MS/MS, vii, viii, 111, 112 inhibitor, 23, 33, 76, 84, 85, 92, 95, 101
humans, 22, 23, 31, 54 inhibitors, 23, 33, 35, 54, 83, 84
hydro, 72 inhibitory, 23, 53, 85
hydrolysis, 2, 25, 26 inhuman, 80
hydrolyzed, 25 initiation, 50
Hydrophilic, 16, 47 injection, 7, 11, 18, 35, 38, 45, 50, 68, 69, 78,
hydrophobic, 15 94, 108
hydrophobicity, 45 injections, 29, 36
hydroxide, 51, 93, 96 injury, 105
hydroxylation, 55 inorganic, 13
hyperglycemia, 105 insertion, 25
hypertension, 33, 105 instruments, viii, 111
Index 121
intensive care unit, 37 lipid peroxidation, 36

interaction, 2, 6, 36, 37, 39, 55 lipids, 58, 68
interactions, 2, 3, 6, 15, 55, 56, 84 lipophilic, 16, 72
interface, 17, 23 lipoprotein, 33
interference, 1, 12, 25, 37, 47, 66, 72, 75, 77, liquid chromatography, vii, 5, 15, 19, 35, 37,
93, 96, 97, 98, 101 47, 48, 56, 72, 78, 79, 80, 81, 93, 94, 96,
interleukin, 105 98, 99, 101, 102, 103, 107, 109
interleukin-2, 105 liquid phase, 6, 15, 94, 101
interval, 3, 45 liquids, 19
intoxication, 39 lithium, 44
intramuscular, 50 liver, 2, 30, 31, 33, 37, 51, 54, 55, 56, 84, 85,
intramuscular injection, 50 92, 105
invasive, 38 liver disease, 2
investment, 111 LOD, 11, 37, 44, 106
ion channels, 53 low-density, 33
ion exchangers, 15 low-density lipoprotein, 33
Ion-exchange, 15 lumen, 87
ionic, 94, 101 lung, 29
ionization, 9, 26, 28, 37, 45, 62, 74, 93, 98, lung disease, 29
101, 102, 108 lungs, 29
ions, 6, 9, 23, 27 lymphocytes, 105
isoenzymes, 85
isolation, 6, 12, 17, 94
isozymes, 55 M
M.O., 80
K M1, 106, 107
kidney, 2, 105 major depression, 83, 84, 85

kidneys, 55 major depressive disorder, 84, 101
Kinase, 23 malnutrition, 30
management, 100, 111
mania, 41, 54
L manic, 42, 44
manic episode, 42
lactate level, 36
manifold, 17
LDL, 33
manipulation, 12, 112
leukemia, 22
manufacturer, 42
life-threatening, 54
maprotiline, 45, 46, 89, 97, 99
lifetime, 1
mass spectrometry, vii, 9, 35, 37, 38, 47, 48,
light scattering, 71
81, 93, 98, 101, 102, 109
limitations, 13
matrix, 11, 13, 15, 16, 17, 18, 37, 45, 68, 77,
linear, 25, 26, 27, 30, 35, 37, 46, 66, 67, 69,
112
70, 71, 73, 74, 75, 77, 99, 100, 107
measurement, vii, viii, 1, 12, 38, 68, 71, 107,
linear function, 75
111
lipid, 2, 36
measures, 25
122 Index
media, 20, 32 monoamine, 83, 84

medication, vii, 1, 2, 3, 41, 42, 50, 55, 83, 84, monoamine oxidase, 83, 84
111 monoamine oxidase inhibitors, 83
medications, vii, 1, 3, 35, 41, 53, 74, 84, 111 monotherapy, 53, 72, 76
medicine, 36, 95 mood, 54, 68, 79, 83, 92
MEK, 23, 27 mood disorder, 83
membranes, 17, 19, 54, 89 morning, 70
Merck, 25, 56, 95, 98 motivation, 84
metabolic, 26, 27, 49, 50, 56, 112 motor coordination, 54
metabolism, vii, 1, 2, 3, 22, 23, 26, 56, 84, 92, mouse, 22
99, 105, 111, 112 multivariate, 77, 78
metabolite, 58, 60, 62, 67, 68, 70, 73, 74, 77, multivariate calibration, 77, 78
85, 86, 92, 93, 101, 107 muscarinic receptor, 42
metabolites, viii, 13, 23, 25, 27, 28, 37, 38, muscle, 17, 29
39, 44, 46, 48, 55, 57, 58, 59, 61, 63, 64, muscle tissue, 17
66, 67, 70, 72, 73, 75, 76, 77, 79, 80, 81, mycobacteria, 49
85, 89, 90, 96, 97, 99, 102, 105, 106, 107, myocardial infarction, 33
108, 111
metabolizing, 2, 54
metastasis, 23 N
methamphetamine, 37
NaCl, 26, 96
methanol, 23, 25, 26, 31, 37, 38, 45, 47, 66,
nanotubes, 94, 101
67, 70, 72, 73, 75, 78, 93, 94, 96, 100, 106
narcolepsy, 100
methionine, 26
natural, 1
MHD, 58, 59, 61, 62, 68, 70, 71, 72, 74, 75,
nebulizer, 29
79
neonatal, 55
mice, 22
neonates, 29
microbes, 49
nephrotoxicity, 105
microbial, 50
nerve, 85
microbial cells, 50
nervous system, 29
microemulsion, 32
neuroleptics, 44, 48, 68
microorganisms, 49
neurons, 53, 83
microtubule, 22
neurotransmission, 84
migraines, 54
neurotransmitters, 84, 92, 97
milk, 29
nitric oxide (NO), 36
milligrams, 41
nitrogen, 31
minicolumn, 15
noise, 78
mitogenic, 23
noradrenaline, 84, 85
mitotic, 22
norepinephrine, 83, 84, 85, 92, 95, 97, 101
mixing, 7, 13, 31, 78
normal, 4, 6, 15, 21, 54, 107
MMP, 25, 26
normalization, 36
moclobemide, 45, 46, 88, 96, 99
North America, 20
Modafinil, 91, 100
nucleic acid, 49, 50
models, 22, 23
nucleic acid synthesis, 49, 50
molecules, 2, 6, 9, 15, 23
nucleotides, 21, 25, 27
Index 123
nucleus, 22 PDMS, 66
nylon, 89 peak concentration, 3
peptic ulcer, 105
perchlorate, 90, 100
O permit, 66, 70
pH, 12, 23, 25, 26, 37, 38, 44, 45, 46, 47, 51,
obesity, 30
56, 57, 58, 60, 61, 63, 66, 67, 69, 70, 72,
obsessive-compulsive, 44, 85
74, 75, 86, 87, 88, 89, 90, 93, 94, 95, 96,
obsessive-compulsive disorder, 44, 85
97, 98, 99
occupational, 28
pharmaceutical, 35, 78, 95
ODS, 36
pharmaceuticals, 56
oil, 32
pharmacokinetic, vii, 31, 39, 55, 67, 69, 71,
olanzapine, 45
73, 76, 77, 81, 85, 101, 105, 111
on-line, 17, 68, 78
pharmacokinetic parameters, 31, 105
opioid, 84
pharmacokinetics, 22, 23, 31, 32, 53, 55, 69,
optimization, 25, 56
74, 95, 99
oral, 23, 32, 50, 55, 69, 74, 76, 92
pharmacological, 22, 44, 54, 100
organ, 105, 107
pharmacological treatment, 44
organic, 13, 15, 17, 31, 78, 112
pharmacology, vii, 101
organic solvent, 13, 17, 112
pharmacotherapy, 111
organic solvents, 17, 112
PHB, 58
organism, 2
phenotypic, 28
oxidation, 2, 26, 28
phenytoin, 53, 55, 56, 57, 59, 66, 67, 69, 75,
oxide, 36, 44, 75, 88, 97
79
oxygen, 36, 49, 54
phosphate, 25, 31, 44, 47, 58, 60, 61, 63, 67,
70, 72, 74, 75, 86, 87, 88, 90, 93, 94, 95,
P 97, 98, 99
placenta, 29
pain, 83, 84 plants, 22
pancreatic, 22 plasma levels, 35, 95, 100
pancreatic cancer, 22 plasma proteins, 51, 85
panic disorder, 85 platelets, 54
paralysis, 37 play, 23, 112
parasitic infection, 49 PLC, vii
parenteral, 50 PLS, 73, 77, 78, 80
paroxetine, 45, 46, 83, 88, 89, 90, 92, 96, 97, poisoning, vii
98, 99, 102 polar groups, 2
partial seizure, 53, 54 polarity, 6
partition, 15 polarization, vii, 78
pathogenic, 50 polyethyleneimine, 15
pathogens, 22 polymer, 7, 18, 19, 97
pathways, 30, 41, 42 polymer-based, 19
patients, 3, 25, 28, 29, 35, 42, 44, 45, 46, 47, polymers, 16
54, 56, 66, 68, 69, 70, 71, 72, 73, 76, 77, polymorphism, 26
79, 80, 93, 94, 95, 96, 97, 98, 100, 107, 111 polypropylene, 15, 44, 94
124 Index
poor, 3, 22, 92
pore, 15, 94
Q
porosity, 6
quadrupole, 23, 78, 108
postmortem, 77, 81
quality assurance, 81
post-traumatic stress disorder, 55
quality control, 35, 45, 72, 99, 111
potassium, 25, 28, 46, 59, 60, 63, 68, 69, 70,
quetiapine, 44, 45, 46, 47, 48, 82, 99
74, 90, 99
precipitation, 12, 13, 45, 70, 95, 106, 107, 112
pregnancy, 69 R
premenstrual dysphoric disorder, 85
pressure, 7, 15, 17, 29, 35, 36, 37, 38, 103 range, vii, 1, 3, 4, 5, 10, 23, 30, 35, 37, 41, 45,
presynaptic, 54, 83, 84 46, 49, 51, 66, 67, 68, 69, 70, 73, 74, 75,
prevention, 22, 33 76, 77, 78, 79, 84, 93, 94, 95, 96, 98, 99,
PRI, 59, 69, 75 100, 105, 107, 111
probe, 72 rat, 22, 37, 76, 77, 81
prodrugs, 73 rats, 23, 31, 32, 36, 39
production, 3, 23, 105 raw material, 36
program, 21, 45 reagent, 25
propranolol, 70, 75 reagents, 26
prostate, 22 receptors, 2, 33, 41, 42, 53, 84, 85
protein, 12, 13, 29, 33, 45, 49, 50, 51, 55, 70, recovery, 14, 16, 18, 26, 46, 47, 64, 70, 75,
95, 106, 107, 112 76, 78, 93, 97, 98, 99
protein binding, 29, 51, 55 red blood cells, 25, 27
protein synthesis, 49, 50 red wine, 84
proteins, 2, 13, 18, 22, 51, 58, 68, 72, 85, 87, redox, 50
92 refractory, 54
protocol, 12, 13 regression, 73
protocols, 16, 19, 25 regular, 50
protozoa, 50 regulation, 36
Prozac, 101 rejection, 105, 107
PRP, 51 relationship, 46, 107
Pseudomonas, 49 relationships, 3, 75, 105
psychiatric disorder, 93 relevance, 37
psychiatric disorders, 93 reliability, 68, 71
psychiatrists, 83 renal, 2, 3, 29, 51
psychoactive drug, 41, 45 renal disease, 3
psychoses, 100 renal function, 2
psychosis, 41 replication, 22, 105
psychotic, 41, 44 research and development, 78, 79
psychotropic drug, 99 reservoir, 6, 67
psychotropic drugs, 99 residues, 66
pumping, 6 resin, 19
pumps, 7, 78 resistance, vii, 21
purification, 11, 16, 75, 97 respiratory, 29, 36, 37
pyruvate, 50 resveratrol, 22
Index 125
retention, 15, 93, 100, 108 septum, 17

reversed-phase high performance liquid serotonergic, 42, 84
chromatography, 99 serotonin, 41, 42, 47, 76, 83, 84, 85, 92, 93,
ribosome, 49 95, 97, 100, 101
ribosomes, 50 sertraline, 45, 46, 83, 87, 88, 89, 94, 96, 97,
rings, 26 99
risk, 33, 35, 37, 42, 85 serum, vii, 6, 13, 30, 31, 32, 45, 46, 48, 54,
risperidone, 42, 44, 45, 46, 99, 100 57, 58, 60, 61, 63, 64, 66, 67, 68, 71, 72,
RNA, 22 74, 77, 78, 79, 80, 81, 90, 97, 99, 102, 103,
robustness, 67, 71 112
rods, 50 shares, 54
RP-HPLC, 35 shoot, 11
runaway, 55 short run, 73
short-term, 44
side effects, 1, 41, 42, 49, 54, 83, 84, 85, 93,
S 100, 111
signaling, 23
safety, 51
signals, 23, 83
saliva, 50, 52
signs, 1, 2, 111
salt, 13, 60, 71, 89
silanol groups, 94
sample, 6, 7, 9, 11, 12, 13, 15, 16, 17, 18, 19,
silica, 15, 17
20, 23, 25, 27, 37, 45, 48, 67, 68, 69, 70,
SIR, 90, 98
72, 74, 75, 78, 93, 94, 96, 97, 106, 107,
skin, 21, 22
112, 113
skin cancer, 22
sampling, 12, 17
sleep, 53
saponins, 36, 38
smokers, 30
scattering, 71
smooth muscle, 29
schizoaffective disorder, 47, 55
sodium, 51, 53, 54, 55, 56, 58, 60, 66, 69, 71,
schizophrenia, 41, 42, 44, 47
88, 93, 94, 96
schizophrenic patients, 45
sodium hydroxide, 51, 93, 96
SDS, 89, 97
software, 8
search, 112
solid phase, 14, 35, 38, 77, 97, 102
second generation, 41
solubility, 2
sedative, 55
solvation, 15
sedentary, 36
solvent, 12, 13, 15, 16, 17, 18, 26, 66
seizure, 53, 55
solvents, 14, 16, 46, 56, 112
seizures, 53, 54, 55, 56
sorbents, 16, 19, 97
selective serotonin reuptake inhibitor, 76, 85
Spain, 26, 97
selectivity, viii, 14, 17, 18, 47, 50, 69, 95,
species, 49
100, 111
specificity, viii, 27, 68, 70, 71, 111
sensitivity, viii, 17, 18, 27, 66, 68, 69, 70, 71,
spectrophotometric, 36, 47, 70, 73, 80
79, 94, 95, 111
spectrophotometric method, 73, 80
separation, viii, 5, 6, 7, 9, 10, 12, 13, 26, 37,
spectrophotometry, 78
45, 46, 56, 67, 68, 71, 72, 73, 74, 75, 77,
spectrum, 7, 16, 41, 49, 50
78, 79, 80, 92, 93, 94, 95, 96, 97, 98, 99,
speed, 67
100, 107, 108
126 Index
springs, 31
stability, 95
T
stabilization, 54
tacrolimus, 106, 108, 109
stabilizers, 68
tandem mass spectrometry, vii, 35, 37, 48, 81
stages, 9
targets, 22, 27
stainless steel, 17
teenagers, 30
standard deviation, 46, 99
temperature, 8, 31, 37, 66, 71, 72, 108
standards, 37, 45
temporal, 56
statins, 33, 35, 38
temporal lobe, 56
status epilepticus, 55
textbooks, 9
steady state, 1
therapy, 3, 25, 28, 29, 33, 44, 53, 56, 93, 95,
steel, 17
105
steroid, 4
Thessaloniki, viii
stomach, 54
thioridazine, 41, 45
storage, 12, 25
thyroid, 2
strains, 50
time consuming, 11, 14
Streptomyces, 49, 50
tissue, vii, 2, 17, 66
stress, 1
tolerance, 50, 51
stroke, 33
tonic, 54
styrene, 97
tonic-clonic seizures, 54
subgroups, 45
total plasma, 39
substance abuse, 100
toxic, vii, 4, 21, 25, 46, 53, 56, 94, 97, 99
substances, 2, 22, 37, 49, 69, 97, 100
toxic effect, 4, 21, 53
substitution, 112
toxicity, vii, 1, 2, 3, 21, 107, 111
suffering, 25, 29, 69
toxicological, 37, 77
sugar, 22
toxicology, 23, 77, 100
suicidal, 42, 77
toxicology studies, 23
suicidal behavior, 42
training, 36, 38
suicide, 85
transfer, 58, 68
sulfate, 106, 107, 108
transition, 74
supernatant, 13, 44, 45, 59, 69, 71, 108
translocation, 50
superoxide, 36
transmission, 23
superoxide dismutase, 36
transplant, 107
surgeries, 1
transplantation, 105, 107
switching, 17, 68, 78, 79
treatment-resistant, 42, 44
symptoms, 42, 54
trichloroacetic acid, 13, 71
synapse, 83, 92
tricyclic antidepressant, vii, 84, 95, 96, 97, 98,
synapses, 53
102, 103, 111
syndrome, 41, 44, 54, 84
tricyclic antidepressants, vii, 84, 95, 97, 98,
synovial fluid, 79
102, 103, 111
synthesis, 21, 22, 27, 33, 49, 50, 89, 98
trifluoroacetic acid, 70, 75, 76, 112
systemic circulation, 92
trigeminal, 53, 54, 55
trigeminal neuralgia, 53, 54, 55
tuberculosis, 49
tumor, 23
Index 127
tumor growth, 23 volatility, 18

turnover, 21 vortex, 31
tyramine, 65
W
U
water, 31, 32, 35, 51, 56, 57, 58, 62, 66, 67,
ultraviolet, vii, 46, 56, 74, 94, 99, 109 68, 73, 74, 76, 77, 87, 90, 91, 94, 98, 100,
United States, 53 106, 107, 108
urine, 13, 18, 26, 28, 37, 78, 91, 94, 95, 100, wavelengths, 38
101, 103, 112 withdrawal, 84
women, 69
V
Y
vacuum, 17, 99
validation, 19, 48, 76, 80, 81 yield, 56, 72, 75, 97
validity, 35
valproic acid, 55
values, 11, 23, 27, 46, 72, 93, 95, 97 Z
variability, 3, 13, 105, 107, 111
zinc, 106, 107, 108
variation, 26, 36, 45, 66, 70, 75, 76, 96, 99
zinc sulfate, 106, 107, 108
venlafaxine, 45, 46, 83, 85, 90, 92, 97, 98, 99,
ziprasidone, 45
101, 102, 109
Victoria, iii, viii
viruses, 49

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DRUG MONITORING BY HPLC:

DRUG MONITORING BY HPLC:

Nova Science Publishers, Inc.

Antibiotic Resistance: Causes and Risk Factors,

Antibiotic Resistance: Causes and Risk Factors,

Poisons: Physiologically Active Substances

Drug Monitoring by HPLC: Recent Developments

2010. ISBN: 978-1-60876-183-8

NOTICE TO THE READER

required, the services of a competent person should be sought. FROM A DECLARATION OF

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Published by Nova Science Publishers, Inc.  New York

Chapter 6 Cardiovascular Drugs 33

Drug monitoring in clinical chemistry refers to the measurement of drug

bronchodilators (Theophylline), antipsychotics etc. Monitoring of medication is

sufficiently selective and can only be used to eliminate negative samples.

chronic conditions such as cardiovascular disease, kidney disease, thyroid disease,

Relationship between plasma drug concentration and therapeutic

In vivo–factors affecting therapeutic drug levels include: Patient compliance,

Drug concentrations are affected by:

Interaction with diet or other medication.

Enzyme inhibition may result in the increase of drug activity, prolonging

As it has been already mentioned not all medications require therapeutic

SUGGESTED FURTHER READING

BRIEF INTRODUCTION TO HPLC

Five different mechanisms are responsible for separation in HPLC:

1. Adsorption of the analytes on the sorbent used as a stationary phase.

2.2. HPLC Instrumentation

1. Mobile phase reservoir: Solvent Reservoirs are used to store Mobile-

Pump Chromatographic Detector

A typical HPLC apparatus

Figure 2.1. Main components of an HPLC system.

There are several detectors available. However UV-VIS Detector, Photo-

6. Data Acquisition System: to process the detector output and integrate it to

1. Precolumn or guard column to protect the analytical column.

2.3. Liquid Chromatography-Mass Spectrometry

Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an

chapter is to give supportive fundamental information to the clinical chemist.

SUGGESTED FURTHER READING-REFERENCES

Sample preparation is widely accepted to be the most important part of any

1) Modification or elimination of sample matrix to prepare it for

2) Impurities removal and sample purification.

Therapeutic drug monitoring (TDM) may be used as a tool for the

to prolong instrument‘s and above all column life time.

1. Preservation during storage.

There is no ideal or universal sample pretreatment technique. The analytical

be simple and fast

provide high sample throughput

3.2. Sample Preparation in Drug Monitoring

In drug monitoring sample preparation aims to eliminate endogenous

and solid-phase extraction (SPE). In the extraction method the compound of

3. Analysis HPLC, HPLC-MS/MS Figure 3.1 Typical sample preparation

So called modern extraction techniqes are preferred nowadays such as solid

Comparing solid-phase extraction with classical liquid- liquid extraction, it

- An almost quantitative recovery can be obtained, provided that the

Solid - phase extraction (SPE) is an extraction process that comprises a solid

SPE consists of four steps as shown in Figure 3.2:

1. Sorbent conditioning which is the preparation of the sorbent. This is also

SOLID PHASE EXTRACTION STEPS

Optimization assays are required in each of these steps to reassure high

are critical for high recovery rates without loss in recovery.

SPE cartridges can be used either with centrifugation, positive pressure or