Method development and validation of

pharmaceuticals according to ICH guidelines

by LC-MS

Presented by
JNV Indira Devi
Research Scholar
Pharmaceutical Analysis
Andhra University
 History
 Francis William Aston was awarded the nobel prize for establishing the
isotopic constitution, using a new device called a mass spectrograph in 1922.
 Martin and Synge, received nobel in 1952 demonstrated partition
chromatography by separating some quite tricky bio-compounds.
 V. L. Tal'roze and collaborators started the development of LC-MS in the
early 1970s, when they first used capillaries to connect LC columns and MS
ion sources.
 McLafferty and collaborators in 1973 developed the first and most obvious
way of coupling LC with MS, and was known as the capillary inlet interface.
 Fenn, 2002 was awarded nobel prize for his ground breaking work in using
electrospray ionization (ESI) for enabling macro biomolecule analysis with
MS. 
 Tanaka, 2002 was awarded nobel prize for his contributions in the
development of soft desorption ionization for mass spectrometric methods.
 LC-MS System Components
Principle:
• LC/MS is a technique that combines physical separation
capabilities of liquid chromatography with mass analysis
capability of Mass spectrometry.

• Mass spectrometers work by ionizing molecules and then sorting and

identifying the ions according to their mass-to-charge (m/z) ratios.
 TYPES OF INTERFACES
• It is difficult to interface a liquid chromatography to a mass- spectrometer cause
of the necessity to remove the solvent.

• The commonly used interfaces are:-

(1) Electrospray ionization (ESI)
(2) Thermospray ionization (TSI)
(3) Atmospheric pressure chemical ionization (APCI)
(4) Atmospheric pressure photoionization(APPI)
 Electro Spray Ionization (ESI)
• ESI draws sample solutions to the tip of a capillary tube, where it applies a high
voltage of about 3 to 5 kV.
• ESI provides the softest ionization method available, which means it can be used for
highly polar, least volatile, or thermally unstable compounds.
 Atmospheric pressure chemical ionization (APCI)

• APCI vaporizes solvent and sample molecules by spraying the sample solution
into a heater (heated to about 400ºC) using a gas, such as N2.
• Solvent molecules are ionized by corona discharge to generate stable reaction
ions.
 Atmospheric pressure photoionization(APPI)

• The LC eluent is vaporized using a heater at atmospheric pressure. The resulting

gas is made to pass through a beam of photons generated by a discharge lamp
(UV lamp) which ionizes the gas molecules.
 Mass Analyser
• They deflect ions down a curved tubes in a magnetic fields based on
their kinetic energy determined by the mass, charge and velocity.
• The magnetic field is scanned to measure different ions.
 Types of mass analyzer:-
(1) Quadrapole mass filter.
(2) Time of flight
(3) Ion trap
(4) Fourier transform ion cyclotron resonance (FT-ICR)
 Quadrupole Mass Analyzer
• A Quadrupole mass filter consists of four parallel metal rods with
different charges
• Two opposite rods have an applied + potential and the other two
rods have a - potential
• The applied voltages affect the trajectory of ions traveling down
the flight path
• For given DC and AC voltages, only ions of a certain mass-to-
charge ratio pass through the quadrupole filter and all other ions are
thrown out of their original path
 TOF (Time of Flight) Mass Analyzer

• TOF Analyzers separate ions by time without the use of an electric or

magnetic field.
• In a crude sense, TOF is similar to chromatography, except there is no
stationary/ mobile phase, instead the separation is based on the kinetic energy
and velocity of the ions.
 Method development
Table 1: Things to know about the sample
Sample property Details

Matrix What compounds other than the analyte are in the sample?

Concentration • How much of the compound is present in the sample?

• What is the concentration range (low or high)?

Quantity How many compounds are present in the sample?

Chemical/physical properties • pKa values

• molecular size and weight
• electrical charge
• sample solubility
• sample volatility
• stability and toxicity
• hydrophobicity/polarity
• chemical/biological reactivity
• UV spectra
Table 2: Method goals

Method Details
goal
Detect Is the compound present?

Quantitate How much of the compound is present?

Identify What is the compound?

Characterize What are the compound properties?

Purify/isolate Do you want to collect the compound for further use?

 Table 3: Analysis requirements

Method Analysis requirement

goal
Detection • What detection technique can be used to ana- lyze the sample? Is it UV absorbing, can you ionize it,
does it have observable thermal characteristics, etc.

Quantitation • How will you quantify (e.g., internal standard,externalstandard,absolute detection)?

• Whatisyourconcentrationrangeor sampleamount(lowor high)?
• Howmanysamplesdoyou need?
• Whatlevelsofaccuracyandprecision arerequired?

Identification • How will you identify the compound (e.g., what detection technique will you use)?
• How will you determine purity (e.g., UV spec- tral purity, percent area)?

Characterization What properties or property levels do you need to determine?

Purification/isolation • Do you want to isolate purified material?

• Do you need to recover 100% of your sample?
Sample matrix Is there more than one sample matrix before analysis? Will the sample matrix interfere
with your analysis?

Properties Does the analysis technique allow you to deter- mine sample properties?
Methodology

 The main sample preparation methods include: – Ultrafiltration

 – Solvent extraction/desalting
 – Liquid-liquid extraction
 – Solid phase extraction (SPE)
 – Immunoaffinity
 – On-column concentration
 – Column switching (LC/LC)
 Considerations Prior to Method
Validation
 Suitability of Instrument
 Status of Qualification and Calibration
 Suitability of Materials
 Status of Reference Standards, Reagents, Placebo

 Suitability of Analyst
 Status of Training and Qualification Records
 Suitability of Documentation
 Written analytical procedure and proper approved protocol
 with pre-established acceptance criteria
 Purpose of Method Validation

 Identification of Sources and Quantitation of Potential errors

 Determination if Method is Acceptable for Intended Use

 Establish Proof that a Method Can be Used for Decision

Making

 Satisfy Requirements
 Published Validation Guidelines
 1978 Current Good Manufacturing Practices (cGMPs)
 1987 FDA Validation Guideline
 1989 Supplement 9 to USP XXI

 1994 CDER Reviewer Guidance: Validation of

Chromatographic Method
 1995 ICH Validation Definitions:
Q2A, Text on Validation of Analytical
procedures
 1997 ICH Validation Methodology:
Q2B, Validation of Analytical Procedures:
Methodology
 1999 Supplement 10 to USP 23 <1225>: Validation
of Compendial Methods
 1999 CDER “Bioanalytical Method Validation
for Human Studies”
 Requirements & Parameters

USP ICH
 Specificity
 Specificity  Linearity
 Linearity and Range  Range
 Accuracy  Accuracy
 Precision  Precision
 Limit of Detection  Repeatability
 Limit of Quantitation  Intermediate
 Ruggedness Precision
 Robustness  Reproducibility
 Limit of Detection
 Limit of Quantitation
 ICH GUIDELINES:
VALIDATION OF ANALYTICAL
PROCEDURES:
S.N Parameter Solution TEXT Procedure
AND METHODOLOGY-Acceptance createria
o used Q2(R1)
1 System suitability USP Tailing <2.0

Standard Injecting Standard solution USP Platecount >2000

 
USP Resolution >2.0

2 Specificity Diluent, Injecting Blank and Placebo No Interference on STD

Placebo
3 Linearity Correlation Coefficient: 0.999

API Std Conc. in differ from 25%-150%

4 Precision:      

4.1 Method precession Sample Injecting Samples solution % Assay:95-105%; %RSD<2.0%

(Intra-day)
(With in
the day)
like
Precision
4.2 System precession Standard Solution

Injecting Standard solution one vials into 6 times (Same Solution) %RSD<2.0

4.3 Intermesiate precession Sample Injecting Samples solution (Different Day, Different Analayst, Different Instrument ) % Assay:95-
(Inter-day/Reggudness) (Differ from Method Precision) 105%;
Solution %RSD<2.0%

5 Accuracy API+Placebo % Recovery:95-

105%;
Preparing API and Placebo in Samle concentration (50%, 100% 150% Level) %RSD<2.0%

6 LOD

API One injection S/N 3-10

7 LOQ

API One injection S/N 20-30

8 Robustness Blank, STD & % Assay:95-

Sample 105%;
Injecting Blank , Standard (3-Injections), Sample (3-preparations) %RSD<2.0%
9 Forced degredation Sample Applying Stress on Sample Solution (Acid, Alkali, Peroxide, Reduction, Thermal, Photo and % Degradation:
studies Hydrolysis) 10-20%

% Assay: 80-90%
9.1 Acid % Degradation:
10-20%
Sample Sample Solution having (0.1-5 N) HCl to Neutralize with (0.1-5 N) NaOH

% Assay: 80-90%
9.2 Base % Degradation:
10-20%
Sample Sample Solution having (0.1-5 N) NaOH to Neutralize with (0.1-5 N) HCl

% Assay: 80-90%
9.3 Peroxide % Degradation:
10-20%
  Sample Solution having (5-30%) Hydrogen Peroxide

% Assay: 80-90%
9.4 Thermal % Degradation:
10-20%
  Sample was exposed 105°C for 3Hr

% Assay: 80-90%
9.5 Photo % Degradation:
10-20%
  Sample was exposed UV light for 72Hrs

% Assay: 80-90%
9.6 Hydrolysis % Degradation:
10-20%
  Sample solution having water

% Assay: 80-90%
10 Assay % % Assay: 98-
102%
Sample One batch sample was prepared as sample procedure for 2-Preparations
 Performance Calculations:
Note: Where terms W and t both appear in same equation they must be
expressed in same units.
Relative retention (Selectivity)  = (t2 - ta) / (t1 - ta)
Theoretical plates n = 16 (t / W) 2
Capacity factor K' = (t2 / ta) - 1
Resolution R = 2 (t2 - t1) / (W2 + W1)
Peak asymmetry T = W0.05 / 2f
Plates per meter N=n/L
Height Equivalent to Theoretical Plates L/n
  where,  = Relative retention.

 t2 = Retention time of the second peak measured from point of injection.

 t1 = Retention time of the first peak measured from point of injection.

 ta = Retention time of an inert peak not retained by the column, measured from point of injection.

 n = Theoretical plates.

 t = Retention time of the component.

 W = Width of the base of the component peak using tangent method.

 K' = Capacity factor.

 R = Resolution between a peak of interest and the peak preceding it

 W2 = Width of the base of component peak 2.

 W1 = Width of the base of component peak 1.

 T = Peak asymmetry, or tailing factor.

 W0.05= Distance from the leading edge of the peak measured at a point

 W0.05 = Distance from the leading edge to the tailing edge of the peak measured at a point 5 % of the peak height from the baseline.

 f = Distance from the peak maximum to the leading edge of the peak.

 N = Plates per meter and L = Length of column, in meters.

 LOD
  = 3.3 × σ/S
 LOQ = 10 × σ/S
 Accuracy:
% error = (accepted - experimental) / accepted *100%
 Precision:
deviation = (average - actual)

 %Assay Calculation:

 Standard deviation:

Where x = sample

xi = mean value of samples

n = number of samples
 Relative standard deviation: