Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 4 pp.

473ñ479, 2011 ISSN 0001-6837

Polish Pharmaceutical Society

HPLC-UV DETERMINATION OF MORPHINE IN HUMAN PLASMA AND ITS

APPLICATION TO THE CLINICAL STUDY

DANUTA SZKUTNIK-FIEDLER1*, EDMUND GRZEåKOWIAK1, MICHA£ GACA2,

and MAGDALENA BOROWICZ1

1
Department of Clinical Pharmacy and Biopharmacy, Poznan University of Medical Sciences, 14 åw. Marii
Magdaleny St., 61-861 PoznaÒ, Poland
2
Clinics of Obstetrical and Gynecological Anaesthesiology, Poznan University of Medical Sciences,
33 Polna St., 60-535 PoznaÒ, Poland

Abstract: A sensitive and specific high-performance liquid chromatography method with ultraviolet detection
(HPLC-UV) has been developed for the quantification of morphine sulfate [(5α,6α)-7,8-didehydro-4,5-epoxy-
17-methylmorphinan-3,6-diol], (CAS: 52-26-6) in human plasma. The analyte was extracted from plasma sam-
ples with chloroform ñ isopropyl alcohol (90:10, v/v) and analyzed on a Bondapak C18 column. The calibration
curves were linear within the range of 10ñ150 ng/mL. The lower limit of quantitation was 10 ng/mL with 0.5
mL plasma sample. The mean recovery of the drug from plasma samples was 83.39%. The results from analy-
sis of quality-control samples at concentrations of 30, 75, and 150 ng/mL were indicative of good accuracy and
precision. This method was successfully used to analyze morphine in plasma samples of patients after abdom-
inal hysterectomy.

Keywords: high performance liquid chromatography, UV detector, plasma samples, morphine, hysterectomy,
pain

Morphine [(5α,6α)-7,8-didehydro-4,5-epoxy- Many different chromatographic assays for the

17-methylmorphinan-3,6-diol] (Fig. 1) is a phen-
antrene opioid receptor agonist and its main effect is
binding to and activating the µ-opioid receptors in
the central nervous system. Morphine is also a κ-opi-
oid and δ-opioid receptor agonist, κ-opioidís action
is associated with spinal analgesia, miosis (pinpoint
pupils) and psychomimetic effects, whereas δ-opioid
is believed to play a role in analgesia. Morphine is
the most frequently used as an analgesic drug for
both postoperative and cancer pain (1).
Figure 1. Chemical structure of morphine
determination of morphine in biological fluids can
be found in the literature (1ñ40).
It is a well-known fact that morphine measure-
ments can be best performed by using mass spec-
trometry (HPLC-MS) (2ñ12), tandem mass spec-
trometry (HPLC-MS/MS) (11ñ24) and GC-MS (4,
25ñ27) detection. In many cases these techniques
have achieved both the desired sensitivity and fast
run time, which is important in drug analysis.
Unfortunately, these combinations are very expen-
sive and therefore not routinely used for monitoring
of morphine in laboratories.
Chromatographic procedures, described previ-
ously, were based also on fluorescence (1, 4, 10,
28ñ30), electrochemical (ECD) (10, 31ñ37) or diode
array (DAD) detection (10, 38ñ40).
The combined ECD detection with UV was
also described by some authors (4, 10).
Opiate analgesics may also be monitored by
immunoassay or radioimmunoassay (RIA), which
are very sensitive tests, but cross-reactions of ana-
lytes with the antisera complicate very often the
assay and provide false results (10).

* Corresponding author: e-mail: d.szkutnik@wp.pl; phone: +48 61 6687853; fax: +48 61 6687858

473
474 DANUTA SZKUTNIK-FIEDLER et al.

Figure 2. UV spectrum of morphine (Photodiode Array Detector, Waters 474)

A B
Figure 3. Chromatograms of morphine in human plasma. (A) plasma sample at 1 h after i.v. administration of morphine in patient no. 5
(retention time of morphine 5.037 min). (B) Blank plasma

Capillary zone electrophoresis (CZE) does not
have sufficient sensitivity for pharmacokinetic stud-
ies (10).
For these reasons, high performance liquid
chromatography is the most popular choice for the
determination of morphine and its metabolites in
biological fluids (4, 10).
In the present study, a sensitive and inexpen-
sive HPLC-UV assay for the determination of mor-
phine in human plasma is described. During devel-
opment of a modified HPLC method with fluores-
cence detection, adequate parameters for the extrac-
tion and UV detection of morphine were found. It
offers an alternative to the previously described pro-
cedures available for pharmacokinetic and monitor-
ing studies of morphine.
Using this HPLC-UV assay, plasma concentra-
tions of morphine in patients after hysterectomy
were analyzed.
MATERIALS AND METHODS
Materials
Morphine sulfate, CAS: 52-26-6 (pure for
analysis) was obtained from Sigma-Aldrich
(Steinheim, Germany). Morphine sulfate ñ solution
 HPLC-UV determination of morphine in human plasma and its application...
for injections, 20 mg/mL, batch no: 05BDO107 was
supplied by Polfa Warszawa, Poland. Acetonitrile,
n-hexane, methanol (HPLC grade) and sodium
hydroxide (analytical grade, pure for analysis) were
purchased from Merck (Darmstadt, Germany). All
other chemicals (chloroform, isopropyl alcohol, eth-
ylene diamine tetraacetic acid sodium salt, boric
acid, sodium borate, monopotassium phosphate,
hydrochloric acid ) were of analytical reagent grade
(Merck, Germany). Human plasma was obtained
from the Laboratory of the Blood Donation Centre
(PoznaÒ, Poland).
Standard solutions
Standard stock solutions of morphine sulfate
were prepared by dissolving accurately weighed
substances (100 mg) in bidistilled water (100 mL) to
obtain final concentration of 1 mg/mL. Stock solu-
tions were stored at 4OC and remained stable for at
least 1 month.
Working solutions (used with the purpose of
validation and calibration) were prepared ex tempore
by further dilution of stock solution with bidistilled
water to provide final concentrations of 10, 20, 30,
50, 75, 100, and 150 ng/mL for morphine sulfate in
plasma.
For preparing calibration curves, a volume of
10 mL of an adequate working solutions were
spiked to 0.5 mL of blank heparinized plasma, and
then extracted, according to procedure described
below.
Extraction of morphine sulfate from plasma sam-
ples:
To 0.5 mL of plasma sample with morphine,
0.5 mL of borate buffer solution (pH 8.9) and 6 mL
of chloroform ñ isopropyl alcohol solution (90:10,
v/v) were added. The mixture was vortexed for 20
min and centrifuged (2000 ◊ g, 10 min). The organ-
ic layer was transfered to another tube, to which 2
mL of 5 mM ammonium phosphate buffer pH 9.3
was added and the mixture was vortexed for 1 min
and centrifuged (2000 ◊ g, 5 min). The supernatant
was discarded and the organic layer containing mor-
phine was transfered into a glass vial, 150 mL of 0,1
M hydrochloric acid was added and the mixture was
vortexed for 2 min and centrifuged (2000 ◊ g, 5
min). The separated acid layer was transferred to
another glass tube, 15 mL of 1 M sodium hydroxide
and 1 mL of hexane were added and the mixture was
vortexed for 20 s and centrifuged (1500 ◊ g, 2 min).
The organic layer was transfered to 1.5 mL
polypropylene Eppendorf tube and 50 µL was
injected into the chromatographic system.
475
HPLC analysis
First of all, in order to find optimum wave-
length for ultraviolet detection of morphine, the UV
spectrum of this drug was determined (Fig. 2).
Using Photodiode Array Detector (Waters 474) the
wavelength of morphine was set at 226 nm.
This method was based on the HPLC methods
with fluorescence detection (1, 10, 29, 30).
The HPLC system was constructed from the
following components: HPLC Waters 2695
Separations Module with autosampler, Waters 2487
Dual l Absorbance Detector. Analytes were separat-
ed by an analytical column Bondapak C18 (150 mm
◊ 4 mm, 10 µm) from Waters. Temperature of col-
umn was maintained at 25OC. Total analysis time
was 7 min and the flow rate was set at 0.8 mL/min.
Samples were eluted with a mobile phase: acetoni-
trile : bidistilled water : methanol (60:840:100,
v/v/v) with addition of 0.012 M monopotassium
phosphate, and 0.5 mM ethylenediaminetetraacetic
acid disodium salt.
Data collection and processing was carried out
by Empower Pro software.
Clinical study
Consent for the study
The study was approved by the Ethics
Committee at the Poznan University of Medical
Sciences. A day before the surgery, patients
received written information with a consent for par-
ticipation in the studies.
Characterization of the investigated population
Subjected were 15 patients (women, 54.3 ± 4.6
years of age, weight 64.5 ± 9.4 kg) after hysterecto-
my, whom applied intravenous bolus (i.v.) of mor-
phine (Morphini sulfas ñ solution for injections, 20
mg/mL) with dose titration to achieve the effective
morphine dose (2 mg of morphine given every 2ñ3
min, until the pain was eliminated or lessened ñ VAS
< 4), then constant infusion was maintained after cal-
culating the infusion rate based on the biological
morphine half-life (t0.5 = 3 h); breakthrough pains
were treated with administration of an additional
bolus dose of 2 mg i.v. After the surgery, the patients
additionally received 100 mg of ketoprofen (Ketonal
ñ solution for injections, 100 mg/2 mL, i.v.).
Patients were qualified to ASA risk groups I (n
= 12) and II (n = 3), without history of liver and kid-
ney diseases, with normal laboratory results, accord-
ing to standards of premedication care, not taking
analgesics on a chronic basis and without con-
traindications to morphine analgesia. Surgeries were
carried out under general anesthesia with intratra-
476 DANUTA SZKUTNIK-FIEDLER et al.

cheal intubation, after induction with propofol, mus-
cle relaxation with rocuronium.
Course of the study
The patients had 5 mL of venous blood sam-
pled 7 times, from a peripheral vein, in aseptic con-
ditions; the first sample to exclude the patientís tak-
ing morphine, second ñ after the surgery, but before
administration of morphine ñ sample 0, with subse-
quent samples 1, 2, 4, 8 and 12 h after administration
of morphine. Blood was sampled to syringes, trans-
ferred to test tubes with lithium heparin, and then
centrifuged at temperature of 20OC for 10 min and
frozen at temperature of ñ70OC. These samples were
used for determination of plasma concentrations of
morphine (HPLC-UV).
The patients were monitored for 12 h after the
surgery. Pain intensity was evaluated according to a
VAS scale before administration of morphine, and 1,
2, 4, 8 and 12 h after administration. During this peri-
od, arterial blood pressure, heart rate, diuresis, SpO2
and breath rate per minute were monitored. Depth of
sedation was also estimated with an Aldrete scoring.
All cases of undesirable effects were recorded.
RESULTS
The HPLC method was validated in accor-
dance with the published guidelines (41, 42).
Specificity
The specificity of the method was examined by
comparing the chromatogram of the drug-free plas-
ma sample with that of the plasma sample spiked
with morphine. Representative chromatograms of
morphine are shown in Fig. 3.
Retention time of morphine was approximate-
ly 5.037 min. The total run time for each sample
injection was about 7 min. There were no interfering
peaks in the blank plasma at the retention time of
morphine. No carry-over (memory effect) problem
was observed in this assay as 50 µL of mobile phase
was injected into the HPLC system after analyzing
plasma samples containing morphine.
Linearity
Calibration curves (n = 6) were prepared by
spiking 10 µL of the appropriate working solution to
0.5 mL of blank human plasma. Final concentrations
of morphine in plasma samples were: 10, 20, 30, 50,
75, 100 and 150 ng/mL. Three spiked plasma sam-
ples were analyzed at each concentration level.
Calibration curves for the plasma assay developed
with peak area of morphine (y) versus drug concen-
tration (x) were found to be linear in the examined
concentration range.
Coefficient of variation (CV) for slope (a =
274.04 ± 25.76; n = 6) and intercept (b = 82.17 ±
13.24; n = 6) were 9.4 and ñ16.11%, respectively.
Assay linearity in the range of the morphine con-
centrations to be expected was concluded from the
mean correlation coefficient of r = 0.994 ± 0.003 (n
= 6); CV was 0.34%.
Recovery
The recovery of morphine was assessed by
comparing the response of five replicates of extract-
ed QC samples (30, 75 and 150 ng/mL) to the
response of the pure standard at the same concentra-
tion level.
The standard specimens were dissolved in
water assigned for injection and injected directly
into the HPLC chromatograph. The recovery of
morphine was greater than 90% for all tested con-
centrations. The results are shown in Table 1.

Table 1. Results of intra-day and inter-day validation of morphine in human plasma.

Concentration Concentration
added founded SD CV (%) Bias (%) Recovery (%) n
(ng/mL) (ng/mL)
Intra-day
30 31.02 3.31 10.66 3.40 110.13 5
75 75.47 2.17 2.88 0.63 100.10 5
150 148.12 9.25 6.24 -1.20 103.78 5
Inter-day
30 28.81 3.04 10.57 -3.97 98.62 15
75 74.18 2.09 0.03 -1.09 92.20 15
150 153.78 10.36 6.74 2.50 96.43 15
SD = standard deviation; CV = coefficient of variation
 HPLC-UV determination of morphine in human plasma and its application... 477

Figure 4. The mean plasma concentration vs. time profiles of morphine in women after hysterectomy (n = 15, i.v. administration of mor-
phine)

Table 2. Stability of morphine in human plasma samples.

Short-term stability (48 h)

in an autosampler
Concentration of
30 75 150
morphine (ng/mL)
Mean 33.04 75.08 159.96
SD 0.61 1.13 2.67
CV (%) 1.84 1.50 1.67
Long-term stability (30 days)
Concentration of
30 75 150
morphine (ng/mL)
Mean 31.67 76.18 148.12
SD 1.45 3.63 5.89
CV (%) 4.58 4.76 3.98
Long-term freezer stability ñ freeze-thaw
stability after three cycles
(ñ80OC, 30 days)
Concentration of
30 75 150
morphine (ng/mL)
% of the initial conc. of
98.20 95.15 97.45
morphine after 30 days
SD 2.14 1.58 2.34
CV (%) 0.02 0.02 0.02

Precision and accuracy The intra- and inter-day precision was

Blank plasma samples were spiked with mor-
phine at three concentration levels QC (30, 75 and
150 ng/mL). The intra-day accuracy and precision
were determined using five replicates of QC sam-
ples on the same day; the inter-day accuracy and
precision were determined by analyzing three cali-
bration curves with five replicates of QC samples on
three separate days.
expressed as the coefficient of variation value: CV =
[SD/C] ◊ 100%, where: SD is the standard deviation
of the mean concentration of morphine and C is the
mean concentration of morphine (Tab. 1).
The accuracy (bias) for morphine was
expressed as the percentage deviation of observed
concentration from theoretical concentration.
478 DANUTA SZKUTNIK-FIEDLER et al.
Bias = [(found concentration ñ added concentra-
tion)/added concentration] ◊ 100% (Tab. 1).
Limits of quantitation and detection
The limit of quantitation (LOQ) of morphine,
defined as the lowest concentration that could be
measured with accuracy and precision, i.e., within
±20% of the actual value, was 10 ng/mL.
The lowest amount of morphine in the sample,
which can be detected but not necessarily quantitat-
ed under stated experimental conditions (detection
limit, LOD), was based on SD of response and
slope: LOD = 3.3δ/S, where S is the slope of the cal-
ibration curve and δ is SD of blank response (34).
LOD of morphine in this method was 5.23
ng/mL.
Stability
Short-term stability of plasma samples was
examined by supplementing blank plasma with
appropriate amounts of working solutions of mor-
phine to yield quality-control (QC) samples contain-
ing 30, 75, and 150 ng/mL. Each sample was ana-
lyzed at room temperature for 48 h (every 4 h),
including their residence time in an autosampler. CV
values were: 1.84, 1.50 and 1.67% for morphine con-
centrations of 30, 75 and 150 ng/mL, respectively.
Long-term stability of plasma samples at room
temperature was assessed for 30 days. The concen-
tration of the stability QC samples (30, 75, and 150
ng/mL) were compared to the mean of back-calcu-
lated values for the standards at the appropriate con-
centration from the first day of long term stability
testing. CV values for analyzed QC samples: 30, 75,
and 150 ng/mL were 4.58, 4.76 and 3.98%, respec-
tively.
To determine long-term freezer stability of
morphine in plasma, QC samples (30, 75, and 150
ng/mL) were analyzed in triplicate after storage at
ñ 80OC for 1, 2, and 30 days. The drug was regarded
as stable if more than 90% was intact at the end of
the study period. The amount of the initial concen-
tration of morphine remaining after this time was:
98.20 ± 2.14%, 95.15 ± 1.58% and 97.45 ± 2.34%
for 30, 75 and 150 ng/mL, respectively.
The results of stability of morphine plasma
samples are presented in Table 2.
Clinical study
The mean plasma concentration versus time profiles
of morphine in women after hysterectomy are pre-
sented in Figure 4.
In research patient group no serious adverse
effects were observed. During the research, blood
pressure, pulse value, breathing frequency and seda-
tion level were not significantly different from val-
ues of these parameters before the morphine was
applied.
DISCUSSION
A specific and sensitive HPLC-UV method for
measurement of morphine plasma concentrations
was described. This method is in accordance with
guidelines for validation of analytical methods
employed in bioavailability and pharmacokinetic
studies in men and animals (41, 42).
The chromatograms of morphine, obtained
under this assay conditions, were sharp and sym-
metrical; total run time was less than 7 min (Fig. 3).
The sample volume required is only 0.5 mL, com-
pared with 1.0ñ2.0 mL for other methods (4, 10).
Extraction recoveries for morphine were consistent,
precise and reproducible throughout the validation
experiments and were within the acceptance criteria.
The intra- and inter-run precision was less than 11%,
and the accuracy was within ±3.97% (Tab. 1), which
proves the acceptable accuracy and precision of the
method developed.
Stability experiments showed that no signifi-
cant degradation occurred at ambient temperature
for 40 h, 30 days and during the three freeze-thaw
cycles for morphine plasma samples. The small
mean CV values for 30, 75 and 150 ng/mL indicat-
ed analyte stability.
Standard solutions of morphine prepared in bidis-
tilled water were stable for at least 1 month at 4OC.
The reagents used in the method are inexpen-
sive and readily available and the procedure does
not involve any critical experimental conditions.
This HPLC method may be suitable for quantifica-
tion of morphine sulfate in small samples of plasma.
The fact, that our assay of morphine is less sensitive
than that using, for example, mass spectrometry
detection is evident. However, HPLC-MS or HPLC-
MS-MS for quantification of morphine is far more
expensive than the HPLC-UV method.
Detection limit of the assay is sufficient for
drug monitoring in optimizing of postoperative
analgesia. The procedure is sensitive and selective
as well as suitable for determining the pharmacoki-
netics of morphine in clinical studies. It offers a suit-
able alternative to existing HPLC techniques.
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Received: 22. 07. 2010