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• EditBioanalytical
Master text stylesDevelopment and Validation
Method
• Second level
• Third level
Sameer Kumar Singdevsachan, M.Sc., Ph.D.
Business Manager-Biopharma
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Therapeutic DrugsMaster title style

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• Third level

• The terms drug, medicine and pharmaceutical product are commonly used interchageably.
An Active Pharmaceutical Ingredient (API) is the chemical substance contained in a
pharmaceutical, which is responsible for its therapeutic effect. Some pharmaceuticals
contain more than one active ingredient (combination product).
 Complexity of Therapeutic Drugs

Small Molecule Biologics

Acetylsalicylic acid1
~ 180 daltons
Insulin2
~ 5,700 daltons

Growth hormone3
191 amino acids
~ 22,000 daltons

Monoclonal antibody4
~ 1,300 amino acids
~ 150,000 daltons

Increasing Complexity

1. Aspirin (acetylsalicylic acid) prescribing information, Bayer.

2. Insulin product information, Sigma-Aldrich. www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/i6634pis.pdf. Accessed February 6, 2017.
3. Online Mendelian Inheritance in Man. www.omim.org/entry/139250?search=human%20growth%20hormone&highlight=hormone%20growth%20human. Accessed February 6, 2017
4. Chennamsetty N, et al. mAbs. 2009;1:580-582
Complexity
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Produced by chemical synthesis
• Second level
Chemical Low molecular weight
Well defined structure
• Third level Mostly process independent
drugs Completely characterized
Stable
Mostly non-immunogenic

Produced by living cells

High molecular weight
Biologics Complex & heterogeneous
structure
Strongly process dependent
Impossible to fully characterize
Unstable
Immunogenic
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Process edit Master
of Therapeutic Drug title style
Development

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• Third level
Bio-analytical
Method
Development
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Bio-analytical Master
Method title style
Development

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• Bioanalytical method development is the process of creating a procedure to enable a
compound of interest to be identified and quantified in a biological matrix.
• Second level
• Third
As per•the level a bioanalytical method is used for “quantitative determination of drugs
FDA guideline,
and / or metabolites in biological matrices such as blood, serum, plasma, or urine… tissue and
skin samples.” Such methods are primarily applied in pharmacological, pharmacokinetic, and
toxicological studies and are mainly performed in animals and humans during preclinical and
clinical trials.

• Analytical methods, on the contrary, are intended for demonstrating quality parameters
(like identity, purity or content) of a biopharmaceutical product. Over the years, analytical
methods have proven to be the backbone of biopharmaceutical industry beside respecting good
manufacturing practices (GMP) during production. Analytical methods ensure the safety, purity,
potency and consistency of drug substances (DS) or drug products (DP).
Clickofto
Need edit Master
bioanalytical title
method style and validation
development

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• Sponsors text
are applying styles
for investigational new drug application (IND), new drug application (NDA),
Abbreviated new drug application (ANDA) to FDA.
• Second level
To fulfill Third
• the levelthey have to submit human clinical pharmacology, bioavailability (BA), and
formalities,
bioequivalence (BE) studies, requiring pharmacokinetic (PK) evaluation including non-human
pharmacology and toxicology studies and preclinical studies, for this purpose there is a need to
develop and validate bioanalytical method.
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Instrumentation Master title style

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• Second level
• Third level

LCMSMS

HPLC UHPLC

GC GCMSMS
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High to edit Master title style (HPLC)
Liquid Chromatography

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• HPLC is a form of liquid chromatography used to separate compounds that are
dissolved in solution.
• Second level
Third level
• HPLC is• characterized by the use of high pressure to push a mobile phase
solution through a column of stationary phase allowing separation of complex
mixtures with high resolution.

• HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a

separation column, and a detector.

• Compounds are separated by injecting a sample mixture onto the column.

• The different component in the mixture pass through the column at

differentiates due to differences in their partition behaviour between the mobile
phase and the stationary phase.
 TYPES OF HPLC TECHNIQUES:

C. Based on elution technique

A. Based on modes of chromatography
– Isocratic separation
– Normal phase mode
– Gradient separation
– Reverse phase mode

D. Based on the scale of operation

B. Based on principle of separation
– Analytical HPLC
– Adsorption chromatography
– Preparative HPLC
– Ion exchange chromatography
– Ion pair chromatography
E. Based on the type of analysis
– Size exclusion(or) Gel permeation chromatography
– Qualitative analysis
– Affinity chromatography
– Quantitative analysis
– Chiral phase chromatography

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 Flow Diagram showing components of HPLC instrument

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 Basic procedure of HPLC analysis
isocratic
elution

Isocratic

Mobile Phase
Separation

40 CV
20%

0%
Column volumes (CV)

Gradient
Separation
Mobile Phase

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Gas to edit Master
Chromatography (GC) title style

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• Gas chromatography (GC) is an analytical
technique applicable to gas, liquid, and solid
• Second level
samples (components that are vaporized by
heat). If a mixture of compounds is analyzed
using •GC Third level
system, each compound can be
separated and quantified.

Gas Chromatography (GC)

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GC to Configuration
System edit Master title style

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• Second level
• The configuration of a GC system is very
simple.• Third level
There are three main GC system
components: the sample injection unit,
which heats the liquid sample and vaporizes
it; the column, which is used to separate
each compound; and the detector, which
detects the compounds and outputs their
concentrations as electrical signals.
 Mass Spectrometry
• Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge
ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a
function of the mass-to-charge ratio.

• The analytes are detected based on their MW. The obtained information is especially useful for
compound structure identification. However, its use is not limited to structure identification and
can be used to quantify very low detection limit of elemental and molecular components
 Working principle of Mass Spectrometer

• In a typical MS procedure, a sample, which may be solid, liquid, or gaseous, is ionized, for example by bombarding
it with a beam of electrons. This may cause some of the sample's molecules to break up into positively charged
fragments or simply become positively charged without fragmenting.

• These ions (fragments) are then separated according to their mass-to-charge ratio, for example by accelerating
them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the
same amount of deflection.

• The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier.
Results are displayed as spectra of the signal intensity of detected ions as a function of the mass-to-charge ratio.

• The atoms or molecules in the sample can be identified by correlating known masses (e.g. an entire molecule) to
the identified masses or through a characteristic fragmentation pattern.

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 Mass Spectrometer

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Bioanlytical Master title style
Workflow

The Master
• Edit bioanalytical
textworkflow
styles contains many steps from sample collection
to sample analysis and data reporting.
• Second level
• Third level

Sample Analysis & Data

Pre-planing Sampling Reporting
Preparation Calibration Evaluation
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Steps to edit Master
in method title style
development

•1. Edit Master text styles

Method selection and information of sample
• Second level
2. Selection of initial method conditions
• Third level
3. Checking the analytical method in aqueous standards

4. Development and optimization of sample processing method

5. Checking the analytical method in biological matrix

6. Pre-validation
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1. Method Master
selection title style
and information of sample

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• Literature survey shall be conducted to have first-hand
information on drug profile and its pharmacokinetic
• Second level
Monograp
properties. h

• Third
• Collection level properties of the analytes
of physicochemical
and the related compounds are essential for the
development of the analytical method. Research
Paper

• Based on the drug’s physicochemical properties such as

molecular size, shape, structure, functional groups,
polarity, partition coefficient, solubility, dissociation
constant etc., choose the internal standard having Book
comparable molecular structure and physicochemical
properties with respect to the analytes.
Click to edit
2. Selection Master
of initial methodtitle style& optimisation
conditions

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• Second
• Setting level
the initial method conditions include diluent selection based on the solubility of the
drug, drug metabolites and internal standard and compatibility with analytical method.
• Third level
• The lowest concentration to be quantified shall be assessed using aqueous solutions during
this phase. Run time and resolution between the peaks should be taken care during this phase.

Continue..
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Analytical edit Master title
parameters style
to be optimized

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The various parameters to be optimized during method development includes

• Second level
✓ Mode of separation
• Third level
✓ Selection of stationary phase

✓ Selection of mobile phase

✓ Analytical Condition
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a. Mode Master title style
Separation

It’s important to choose suitable mode of separation based on physico-chemical properties molecule.
• Edit Master text styles
Second
•functional level
▪ Reverse Phase Mode, the stationary phase is non polar hydrophobic packing with octyl or octa decyl
group bonded to silica gel and the mobile phase is polar solvent.
• Third level
▪ This is suitable mode of separation for polar and moderately polar compounds/molecules.

▪ The polar compound gets eluted first in this mode and non polar compounds are retained for longer
time. As most of the drugs and biopharmaceuticals are polar in nature, they are not retained for
longer times and hence elute faster.

▪ The different columns used are Octa Decyl Silane (ODS) or C18, C8,C4, (in the order of increasing
polarity of the stationary phase).

▪ An aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as ionization
control, ion suppression, ion pairing and complexation) to control retention and selectivity.
b. Selection of Stationary Phase
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• Selection of the column is the first and the most important step in method development,
because the column is the heart of separation process.
• Second level
• The appropriate choice of separation column includes different approaches
• Third level
Column dimensions
Nature of packing material
Shape of the particles
Size of the particles
Surface area
Pore volume
End capping
c. Selection of Mobile Phase
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• The primary objective in selection and optimization of mobile phase is to achieve optimum
separation of all the individual impurities and degradants from each other and analyte peak.
• Second level
• The following are the parameters to be considered during selection and optimization of mobile
phase.• Third level

Buffer
pH of the buffer
Mobile phase composition
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• EditandMaster
Buffer its role: text styles
Buffer and its strength play an important role in deciding the peak symmetries
•
• Second
and separations.level
The retention time depends on molar strength of buffer. Molar
• Third
strength level
is proportional to retention time. In order to achieve better separation
the strength of the buffer can be increased.

• Commonly used buffers are

✓ Acetic buffers includes ammonium acetate, sodium acetate.
Click to edit Master title style
• Another important component is the influence of the pH since this can change the hydrophobicity of
• Edit Master text styles
the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to
control the pH.
• Second level
• Third level
• The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed
silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte.

• Ammonium formate is commonly added in mass spectrometry to improve detection of certain

analytes by the formation of ammonium adducts.

• A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the
mobile phase if mass spectrometry is used to analyze the column eluent.

• Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in
the detector and solvent delivery system, but can be effective in improving retention of analytes
such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic
acids.
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• Edit
pH Master
of buffer: text styles
• • Second
pH level role in achieving the chromatographic separation as it
plays an important
controls the elution properties by controlling the ionization characteristics.
• Third level
• A different concentration of buffer was chosen to achieve required separations.

• It is important to maintain the pH of mobile phase in the range of 2.0 to 8.0 as

most of the columns does not withstand out of this range as Siloxane linkages
are cleaved below pH 2 and at above pH 8 silica dissolves.
d. Analytical
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Master title style

•• Edit
SeveralMaster
instrumenttext
separation of analytes.
styles
parameters are play important role for detection and

• Second level
• It’s also impact the peak shape which is very important for high accuracy
• Third level
results.

• Flow rate
• Column oven temperature
• Detector wavelength
• Data acquisition rate
Click to edit
3. Checking Mastermethod
the analytical title style
in aqueous standards

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• Before going to analyze a method in biological matrix,
• Second
first level
check the analytical method in aqueous standards.

• Third level
• Prepare aqueous calibration curve standards, at least with
five concentrations, including the highest and lowest.

• Concentration of the highest standard shall be based on

Cmax and lowest standard shall be tentatively fixed based
on the preliminary studies. Make injections of each
calibration curve standard and find the correlation
coefficient. Correlation co-efficient (r) should not be less
than 0.99.
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4. Development Master title of
optimization style
sample processing

••Edit
WhenMaster text styles
the instrumental method is concluded with aqueous standards,
Second
•prepare level
matrix sample.

• Third
• Based on thelevel
literature survey data on analyte and internal standard’s
physicochemical properties like structure, functional groups, pH, partition co-
efficient, dissociation constant, polarity and solubility, set and optimize the
sample preparation technique like

✓ protein precipitation,
✓ liquid-liquid extraction and
✓ solid phase extraction.
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•• Edit
Prefer Master text styles
protein precipitation when sensitivity of the drug is more and check
for recovery, precision and interferences.
• Second level
• • Third
Prefer level
liquid-liquid extraction when sensitivity of the drug is less, and check
for recovery, precision and interferences.

• When the recovery and reproducibility is less in liquid-liquid extraction,

prefer solid phase extraction for better sensitivity, recovery, precision and low
interferences.
Click to edit
5. Checking Mastermethod
the analytical title style
in biological matrix

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• Checking the developed bioanalytical method with matrix samples for accuracy,
precision and recovery is essential before finalizing the method for pre-validation.
• Second level
• Third
• Minimum level
three aliquots each of Higher Quality Control (HQC) and Lower Quality
Control (LQC) and Lower Limit of Quantification (LLOQ) matrix samples are analysed
with one set of extracted calibration curve standards including matrix blank and zero
standard (blank with only internal standard) and the results shall be compared for
recovery with aqueous quality control samples of equivalent concentration.

• The method is accepted if it meets the criteria of accuracy, precision and recovery. If
needed, the method shall be considered for modification.
Click to edit Master title style
6. Pre-validation

•• Edit Master text styles

When the method is evaluated to be reliable, prepare a brief procedure with
• Second
the details of level
sample preparation, instrumental conditions and method
conditions, to proceed for pre-validation.
• Third level
• Selectivity, Accuracy, Precision, Recovery parameters should be evaluated in
Pre-validation stage.
Click to edit Master title style
Background

• Method development involves optimizing the procedures and conditions involved with
• Edit Master
extracting text
and detecting styles
the analyte.
• Second
• Method level
development includes the optimization of the following bioanalytical parameters (which
• Third
are discussed) level
to ensure that the method is suitable for validation:

✓Reference standards ✓Critical reagents

✓Calibration curve ✓Quality control samples (QCs)
✓Selectivity and specificity ✓Sensitivity
✓Accuracy ✓Precision
✓Recovery ✓Stability of the analyte in the matrix

• Bioanalytical method validation proves that the optimized method is suited to the analysis of the
study samples
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Types of edit Master
Validation title style

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• Second level
• Third level Method Validation

Full Validation Partial Validation Cross Validation

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1. Full Validation

•• Edit Master
A full validation text styles
is required
–•IfSecond
the method level
is developed and implemented for the first time,
Third
– If a•new levelis analyzed, or
drug entity
– If metabolites are added to an existing assay for quantification.
Click toValidation
2. Partial edit Master title style

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• A partial validation can be performed if validated bioanalytical methods have been
modified. It can range from the determination of a within-day accuracy and precision
• Second level
to a nearly full validation.
• Typical situations for a partial validation are
• Third level
– Method transfers between laboratories and analysts,
– Instrument and/or software platform changes,
– Changes in species within the same matrix,
– Changes in matrix within the same species,
– Change in analytical methodology, and
– Change in sample processing procedures.
Click
3. Crossto edit Master title style
Validation

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• In a cross validation two bioanalytical methods for the same analyte are compared.
• Cross validations are necessary when two or more bioanalytical methods are used to
Second
•generate level
data within the same study. They should be conducted with spiked matrix
standards and subject samples.
• Third level
• A cross validation should be also considered when
– Sample analyses within a single study are conducted in more than one laboratory or
– Data generated using different analytical techniques in different studies are included
in a regulatory submission.
Click to Parameters
Validation edit Master title style

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• Analytical method validation is only one kind of validation among many other validations (e.g.
process validation, cleaning validation, transport validation etc.) required during drug
• Second level
development and especially for manufacturing.

• Third
• For validation levelmethods, there exists a guideline [ICH Q2(R1)] that determines the
of analytical
parameters of the validation procedure required by the pharmaceutical companies to follow.
As per this guideline, analytical methods are categorized based on their application in purity,
identity, content, and potency testing.

• These methods are tested for multiple parameters such specificity, linearity, limit of detection
and quantitation, trueness, precision, robustness, and range, depending on the category of the
method.
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1. Selectivity

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• Selectivity is the ability of an analytical method to differentiate and quantify in a sample
the analyte in the presence of other components.
• Second level
• Third
obtained level
• For selectivity, analyses of blank samples of the respective biological matrix should be
from at least 6 sources.

• Each blank sample should be tested for interference which could originate from
endogenous matrix components, metabolites, decomposition products and concomitant
medication.
Click to edit Master title style
2. Sensitivity

Lower Limit of Quantification (LLOQ)

•• Edit
The LLOQMaster text
is the lowest styles
concentration of an analyte that can be measured with acceptable
Second level
accuracy and precision.
•
• The lowest standard on the calibration curve should be accepted as LLOQ if the following
• Third
conditions are met:level

– The analyte response should be at least 5 times the response compared to blank response.
– The analyte response should be reproducible with an imprecision of maximum 20% and an
accuracy of 80–120%.

Limit of Detection (LOD)

• Limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected but
not necessarily quantified. LOD is the injected amount that results in a peak with a height at
least three times as high as the baseline noise level.
Click to edit Master title style
3. Linearity

• Linearity evaluates the facility of the bioanalytical method to acquire test results that are
• Edit Master text styles
directly proportionate to the concentration of the analyte in the sample.

• Second
• According to FDAlevel
guidelines, the linear range of the method (standard curve) should contain
• Third level
at least five standard points matrix-based as studied samples, using single or replicate
aliquots.

• In all bioanalytical trends needs to have an appropriate operational range but not necessarily
be linear. If linear regression doesn’t work, and the dynamic range is large, quadratic
regression may be used. In some cases standard curve is weighting by 1/x or 1/x2 to obtain
accurate back-calculated values for standards and quality control (QC) samples.

• In addition the standard points during each run must be freshly prepared for each assay. The
variation in back-calculated values of standards should not exceed ± 15% except LLOQ (±
20%). The acceptance criterion for the standard curve is that at least 75% of standards
including the LLOQ while 67% of QC samples (low, medium, high) should meet this criteria.
Click to edit Master title style
• The aim of consideration of quality control (QC)
• Edit Master text styles
samples is to demonstrate the accuracy and precision
of an analyte in biological matrix with known
• Second level
concentrations.

• Third
• This process can belevel
completed by analysis of replicate
sets of desired analyte of known concentrations (QC
samples) in the same matrix. At least, three
concentration levels should be used except lower limit
of quantification (LLOQ); quality control high (QCH),
quality control medium (QCM) and quality control low
(QCL).
➢ LLOQ (lowest concentration of the standard curve)
➢ ULOQ (upper limit, highest concentration of the standard curve)
➢ QC-Low (3 × LLOQ)
• The standards and QC samples should be matrix ➢ QC-Mid (linear midpoint)
based as the studied samples and the minimum ➢ QC-High (80% of ULOQ)
number of QCs should be at least 5% of the number
of unknown samples. At least six sources of blank
matrix should be used for preparing of QC samples.
Click to edit Master title style
4. Accuracy

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• The accuracy of an analytical method describes the closeness of mean test results obtained by
the method to the nominal value (concentration) of the analyte.
• Second level
• Third
should belevel
• Accuracy is determined by replicate analysis of samples containing known amounts of analyte.
Accuracy measured using a minimum of 3 concentrations and 5 determinations per
concentration.

• The mean value should be within 15% of the nominal value except at lower limit of
quantification (LLOQ, see below), where it should not deviate by more than 20%.
Click to edit Master title style
5. Precision

• The precision of an analytical method describes the closeness of individual measures of an

• Edit Master text styles
analyte when the procedure is applied repeatedly to multiple aliquots of a single
homogeneous volume of biological matrix.
• Second level
• Precision should be measured using a minimum of 3 concentrations and 5 determinations per
• Third level
concentration.

• The imprecision determined as coefficient of variation (CV) at each concentration level should
not exceed 15% except for the LLOQ, where it should not exceed 20%.

• Precision is further subdivided into

– Within-day precision, which assesses precision during a single analytical run, and
– Between-day precision, which measures precision with time, and may involve different
analysts, equipment, reagents, and laboratories.
Click to edit Master title style
6. Recovery

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• The recovery of an analyte is the detector response obtained from an amount of the
analyte added to and extracted from the biological matrix, compared to the detector
• Second level
response obtained for the nominal concentration of the pure authentic standard.

• Third
• Recovery level
experiments should be performed by comparing the analytical results for
extracted samples at three concentrations with unextracted standards that represent
100% recovery.

• Recovery of the analyte need not be 100% but the extent of recovery of an analyte and
an internal standard should be consistent, precise, and reproducible.
Click to edit Master title style
7. Carryover

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• Carry-over is a main problem causing drop in accuracy and precision of LC-UV, LC-MS and
LCMS/MS analyses.
• Second level
• Third
due to thelevel
• Carry-over occurs by formerly injection of sample analyte that elutes upon following
analyses sample matrix or analytical system or both. It can be occurred due to
adsorption of analytes on the LC injector or LC connections.

• Carry-over is depending on analyte chemical properties such as lipophobicity /

hydrophobicity and the sample concentration. Carry-over effect is more pronounced at low
analyte concentration.

• Carry-over can be tested by injecting a blank (mobile phase) after highest concentration of
the standard curve. The response from the blank sample should be less than 20% of LLOQ
peak response.
Click toeffect
8. Matrix edit Master title style

• Edit Master text styles

• Matrix effect is the effect on bio analytical method caused by all other components of the
sample except the specific compound to be quantified. It happens due to ion
• Second level
suppression/enhancement by the others ions present in the biological matrix which might get
ionized during detection and will give false results.
• Third level
• Matrix effect studied by comparing the response of extracted samples spiked before extraction
with response of the blank matrix sample to which analyte has been added at the same nominal
concentration just before injection.

• Matrix effect is done in LCMS-MS to find out if there is any ion suppression or enhancement
effect by the matrix.
Click to edit Master title style
9. Stability

• The stability of the analyte in the sample matrix is a critical issue in bioanalytical method
• Edit Master text styles
validation.

Second
• •The analyte can level
decompose during the sample storage (in refrigerators, on lab bench or
• Third level
on autosampler).

• Stock solution stability should be assessed to know how long time it can be stored
without significant loos (<15%) short term (one day) and long term (a number of
months).

• The stability of analyte in standard samples (matrix-based) need to be considered in short

term (4, 8 and 24 hours) and long term (more than one day).

• Freeze and thaw analyte stability is other parameter that should be determined (three
freeze and thaw cycles) using at least three aliquots at two different concentrations (low
and high) in each stability test.
Click to edit Master title style
10. Robustness

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• It is defined as the measure of the ability of an analytical method to remain unaffected by
small but deliberate variations in method parameters (e.g. pH, mobile phase composition,
• Second level
temperature and instrumental settings) and provides an indication of its reliability during
normal usage.
• Third level
• Determination of robustness is a systematic process of varying a parameter and measuring
the effect on the method by monitoring system suitability and/or the analysis of samples.
Click to edit Master title style
Summary

• Edit Master
• The efficient text styles
development and validation of analytical methods are critical
elements in the development of pharmaceuticals/biopharmaceuticals. In
Second method
•bioanalytical level development should not be restricted to pure and
neat analyte solutions.
• Third level
• This is overview of the sample preparation of drug in biological matrix and
to provide practical approaches for determining selectivity, specificity,
lower limit of quantitation, linearity, accuracy, precision, recovery, stability,
ruggedness of chromatographic methods to support pharmacokinetic,
toxicokinetic, bioavailability, and bioequivalence studies.
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• Second level
• Third level
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• Second level
• Third level
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