Revista Română de Medicină de Laborator Vol. 10, Nr.

1, Martie 2008 13

Bioanalytical method validation

Silvia Imre1, Laurian Vlase2, Daniela Lucia Muntean1
1. Department of Drugs Analysis, University of Medicine and Pharmacy from Targu-Mures, Faculty of
Pharmacy, Gheorghe Marinescu 38, 540139 Targu-Mures, Romania
2. Department of Biopharmacy and Pharmacokinetics, University of Medicine and Pharmacy „Iuliu
Hatieganu”, Faculty of Pharmacy, Victor Babes 41, 400012 Cluj-Napoca, Romania

Abstract

A syntetic discussion on bioanalytical methods validation is presented from the point of view of regula-
tory documents, scientific articles and books. The validation parameters are described, together with an example
of validation methodology applied in the case of chromatographic methods used in bioanalysis, taking in account
to the recent Food and Drug Administration (FDA) guidelines and documents of the International Conference on
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH).

Introduction aged method fulfills a number of performance

criteria after following a method validation
The reliability of analytical method is a protocol.
matter of great importance in analysis and is, of
course, a prerequisite for correct interpretation
Publications on bioanalytical methods
of data.
As it is known, analytical method vali-
validation
dation is an experimental procedure which
The scientific literature about analytical
demonstrates that a specific analytical method
and bionalytical methods validation is reach
generates reliable, accurate and precise infor-
and includes different categories: guidelines of
mation about a sample. Bioanalytical methods
the European and US committees, review arti-
are used for the quantitation of drugs and their
cles, books, documents published by interna-
metabolites in biological matrices. Should we
tional conferences or congresses etc.
validate a bioanalytical method is not a question
Since the publications of the European
anymore, validation proving the quality of the
and US committees at the beginning of ’90
analyst’s work, users of bioanalytical data get
years, many laboratories have started to re-
confidence in the results and it is required by
design their processes by involving analysts and
the regulatory agencies. Before using a bioana-
statisticians, in order to define strategies that
lytical method for quantitative determinations
will allow the fulfillment of the regulatory re-
of drugs and their metabolites, an applicant lab-
quirements, while being practicable and scien-
oratory must first demonstrate that the envis-
14 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008

tifically consistent. The Romanian National ulatory agencies of the European Union, the
Agency of Drugs harmonized their require- United States of America and Japan: the first
ments under the latest international regulations document, approved in 1994, concentrated on
which provide assistance in developing bioana- the theoretical background and definitions in
lytical method validation used in human clinical validation13, the second, approved in 1996, on
pharmacology, bioavailability and bioequiva- methodology and practical issues14. The recent
lence studies requiring pharmacokinetic evalua- book edited by Ermer J. and Miller J.H. (2005)7
tion. is the latest document about these topics. De-
Frank T. Peters and Hans H. Maurer19 spite the fact, that these were focussed on ana-
made in 2002 an excelent summary of the most lytical methods for pharmaceutical products
important documents published since 1991: rather than bioanalysis, they still contain helpful
• The review on validation of bioanalyti- guidance on some principal questions and defi-
cal methods published by Karnes et al. (1991) - nitions in the field of analytical method valida-
intended to provide guidance for bioanalytical tion.
chemists15. Compliance with the 2001 FDA guid-
• The Shah et al. report (1992) on the ance can be considered today a minimum re-
conference on "Analytical Methods Validation: quirement to test the performance of a bioana-
Bioavailability, Bioequivalence and Pharma- lytical method. At the beginning of this docu-
cokinetic Studies" held in Washington in 199022 ment the FDA states very clearly that its guid-
- guidance for bioanalysts for the next years; ance for bioanalytical method validation repre-
contains the parameters of bioanalytical meth- sents its current thinking on this topic and that
ods which should be evaluated, and some ac- an alternative approach may be used if such an
ceptance criteria were established but no specif- approach satisfies the requirements of applica-
ic recommendations on practical issues like ex- ble statutes and regulations. This statement al-
perimental designs or statistical evaluation had lows bioanalytical laboratories to adjust or
been made. modify the FDA recommendations, depending
• Hartmann et al. (1994) analyzed the on the specific type of bioanalytical method
1990 Conference Report performing statistical used.
experiments on the established acceptance crite- In addition to such important docu-
ria for accuracy and precision9. Based on their ments, different scientific journal published
results they questioned the suitability of these their opinion on these aspects. Journals like
criteria for practical application. Journal of Chromatography B17 or Clinical
• Hartmann et al. (1998) review on vali- Chemistry have established their own criteria
dation of bioanalytical chromatographic meth- for validation. Other perspectives are included
ods - theoretical and practical issues were dis- in a recent valuable book edited by Chan C.C.
cussed in detail10. et al. (2004)3.
• The Shah et al. (2000) report on an up- It is also necessary to present the guide
date conference of the 1990 Washington con- published in 1997 by La Société Française des
ference23 - template for the guidelines (2001) of Sciences et Techniques Pharmaceutiques (SF-
the U.S. Food and Drug Administration STP)5 that provided the bioanalyst, on the one
(FDA)25. hand, with a better understanding on the way to
• The documents of the International proceed and on the other hand, real data for
Conference on Harmonisation of Technical Re- qualifying his own computations that he could
quirements for Registration of Pharmaceuticals perform using a commercial spreadsheet4, 6, 11. It
for Human Use (ICH) and approved by the reg- should be noted that this guide was published
 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008 15

before the recent FDA’s guide and introduces analysis.

new concepts in three different areas: stages of • Analytical method validation - a proce-
the validation, test of acceptability of a method dure employed to demonstrate that an analytical
and design of experiments to perform. The method used for quantification of analytes in a
main authors of SFSTP guide recently pub- biological matrix quantifies the analyte with a
lished (2003) an article which objectives were degree of accuracy and precision appropriate to
to identify and explain the progress permitted the task.
by the SFSTP guide, point out some of the limi- o Full validation: establishment of
tations and suggest ways to overcome them2. all validation parameters to apply to sample
An interesting thing is that no references about analysis for the bioanalytical method for
the recently published FDA guide was made in each analyte.
this article, just the first FDA guide (1992) is o Partial validation: modification of
cited, even if the FDA document published in validated bioanalytical methods that do not
2001 provides more detailed aspect regarding necessarily call for full revalidation.
experimental procedures. o Cross-validation: comparison of
Many other scientific articles try to add validation parameters of two bioanalytical
a practical point of view on bioanalytical and methods.
analytical methods validation1, 8, 12, 16, 18, 20, 24, 26. In order to understand the validation
process it is necessary to define the analytical
Current validation practice on terms used, including the validation parameters
bioanalytical methods validation (Figure 1):
• Specificity/selectivity – the ability of
In today’s drug development environ- the method to measure and differenciate the an-
ment, highly sensitive and selective methods alyte signal in the presence of components that
are required to quantify drugs in matrices such may be expected to be present. There has been
as blood, plasma, serum, or urine. Chromato- some controversial discussion about the termi-
graphic methods are the most commonly used nology for this validation characteristic. In con-
technology for the bioanalysis of small trast to the ICH, most other analytical organisa-
molecules and the general terms presented be- tions define this as selectivity, whereas speci-
low take in account to this type of analytical ficity is regarded in an absolute sense, as the
method. “ultimate degree of selectivity” (IUPAC). Se-
It is well accepted the FDA Guidance lectivity is the ability of the bioanalytical
for Industry, Bioanalytical Methods Validation method to measure and differentiate the ana-
(2001) as a reference for current validation lytes in the presence of components that may be
practice and a briefly description of it is given expected to be present. Specificity is the ability
here. to assess unequivocally the analyte in the pres-
ence of components that may be expected to be
a. Glossary present. For example, in high-performance liq-
uid chromatography with UV detection (HPLC-
The general concepts could be ex-
UV), a classic chromatographic method, the
pressed as follows:
method is specific if the assigned peak at a giv-
• Validation - the process of checking if en retention time belongs only to one chemical
something satisfies a certain criterion. entity; in liquid chromatography with mass
• Analytical method - a comprehensive spectrometry detection (LC-MS) the detector
description of all procedures used in sample could measure selective an analyte, even if this
16 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008

Specificity
and
selectivity

Dilution effect Accuracy

Repeatability,
intermediate
Recovery precision and
reproducibility

Validation
parameters

Analyte
stability Robustness

Lower limit of
quantification
LLOQ and
upper limit of
quantification
ULOQ
Limit of
Linearity
detection
LOD

Fig. 1. Validation parameters of a bioanalytical method

is not fully separated from endogenous com- assay, inter-assay).
pounds etc. Despite this controversy, there is a o Reproducibility, i.e., the precision
broad agreement that specificity/selectivity is between laboratories (collaborative or inter-
the critical basis of each analytical procedure. laboratory studies), is not required for sub-
• Precision - the closeness of agreement mission, but can be taken into account for
(degree of scatter) between a series of measure- standardisation of analytical procedures.
ments obtained from multiple sampling of the • Accuracy: the degree of closeness of
same homogeneous sample under the pre- the determined value to the nominal or known
scribed conditions. Precision may be considered true value under prescribed conditions. This is
at three levels: repeatability, intermediate preci- sometimes termed trueness. It is expressed as
sion and reproducibility. As parameters, the bias% or relative error%.
standard deviation, the relative standard devia- • Robustness: a measure of its capacity
tion (coefficient of variation) should be calcu- to remain unaffected by small, but deliberate
lated for each level of precision. variations in method parameters and provides
o Repeatability expresses the analyti- an indication of its reliability during normal us-
cal variability under the same operating age
conditions over a short interval of time • Limit of detection (LOD): the lowest
(within-assay, intra-assay). concentration of an analyte that the bioanalyti-
o Intermediate precision includes the cal procedure can reliably differentiate from
influence of additional random effects with- background noise.
in laboratories, according to the intended • Lower limit of quantification (LLOQ):
use of the procedure, for example, different the lowest amount of an analyte in a sample that
days, analysts or equipment, etc. (between- can be determined quantitatively with suitable
 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008 17

precision and accuracy. methanol or acetonitrile and kept refrigerated at

• Upper limit of quantification (ULOQ): −20ºC if there are no problems of stability or
the highest amount of an analyte in a sample solubility.
that can be determined quantitatively with pre- • Working solutions: solutions prepared
cision and accuracy. from the stock solution through dilution in the
• Standard curve: the relationship be- appropriate solvent at the concentration request-
tween the experimental response value and the ed for spiking the biological matrix.
analytical concentration (also called a calibra- • Calibration standard: a biological ma-
tion curve); usually this relationship is linear. trix to which a known amount of analyte has
• Quantification range: The range of been added or spiked. Calibration standards are
concentration, including the LLOQ and ULOQ used to construct calibration curves from which
that can be reliably and reproducibly quantifi ed the concentrations of analytes in QCs and in un-
with suitable accuracy and precision through known study samples are determined.
the use of a concentration response relationship. • Internal standard: test compound(s)
• Recovery: the extraction efficiency of (e.g., structurally similar analog, stable labeled
an analytical process, reported as a percentage compound) added to both calibration standards
of the known amount of an analyte carried and samples at known and constant concentra-
through the sample extraction and processing tion to facilitate quantification of the target ana-
steps of the method. lyte(s).
• Dilution effect: The ability to dilute • Sample: a generic term encompassing:
samples originally above the upper limit of the o Blank: a sample of a biological ma-
standard curve should be demonstrated by accu- trix to which no analytes have been added
racy and precision parameters in the validation. that is used to assess the specificity of the
• System suitability: determination of in- bioanalytical method.
strument performance (e.g., sensitivity and o Quality control sample (QC): A
chromatographic retention) by analysis of a ref- spiked sample used to monitor the perfor-
erence standard prior to running the analytical mance of a bioanalytical method and to as-
batch. sess the integrity and validity of the results
• Reinjection reproducibility: It is neces- of the unknown samples analyzed in an in-
sary to be determined if an analytical run has to dividual batch. They are also used to calcu-
be reanalyzed in the case of instrument failure. late the accuracy and precision of the
• Stability: the chemical stability of an method.
analyte in a given matrix under specific condi- o Unknown sample: a biological
tions for given time intervals. sample that is the subject of the analysis.
Other definitions should be given:
b. Validation methodology of the
• Biological matrix: a discrete material of
chromatographic methods applied for drugs
biological origin that can be sampled and pro-
determination in human plasma
cessed in a reproducible manner. Examples are
blood, serum, plasma, urine, feces, saliva, spu- Includes two phases:
tum, and various discrete tissues. • pre-study method validation – method
• Stock solutions: the original solutions developing; method validation; it is performed
prepared directly by weighing the reference before the unknown samples analysis
standard of the analyte and dissolving it in the • routine-run method validation – during
appropriate solvent. Usually, stock solutions are unknown samples analysis
prepared at a concentration of 1-3 mg/mL in
18 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008

Pre-study method validation the concentration. In HPLC-UV-based assays

Specificity the use of 1/x2 weighed linear regression analy-
As a first step of method validation, sis can significantly reduce the LLOQ obtain-
specificity is verified using six different plasma able, where the standard deviation of y varies
blanks obtained from healthy human volunteers with x. A comparison between the weighed
who had not previously taken any medication. least squares procedure and the conventional
A general approach to prove the selectivity least squares calibration shows improvements
(specificity) of the method is to verify that: the in accuracy at the lower end. The principal ad-
response of interfering peaks at the retention vantage in this case is for clinical pharmacology
time of the analyte is less than 20% of the re- and pharmacokinetic studies when concentra-
sponse of an LLOQ standard, or the response at tion values being measured by the method are
the LLOQ concentration is at least five times near LLOQ.
greater than any interference in blanks at the re- Calibration is performed using singli-
tention time of the analyte; the responses of in- cate, duplicate or triplicate (it depends on
terfering peaks at the retention time of the inter- method precision) calibration standards on five
nal standard are ≤5% of the response of the different occasions. The concentration range
concentration of the internal standard used in should cover the expected concentration in bio-
the studies. logical samples. A calibration curve should
Standard curve. Quantification range consist of a blank sample (matrix sample pro-
The relationship between the detector cessed without the IS), a zero standard (matrix
response and concentration should be demon- sample processed with internal standard), and 6
strated to be well defined and reproducible. The to 8 nonzero standards. The number of stan-
calibration curve model is determined usually dards can be increased for a complex curve or a
by the least squares analysis. In general, a poly- curve covering a very large range. The simplest
nomial function is considered: relationship that provides acceptable backcalcu-
y = b0 + b1x + b2x2 + b3x3 + ...; lated concentrations for the standards should be
for the linear model, the terms x2 and used first to fit the calibration curve. If a
larger are ignored and for the quadratic model, weighting factor is used, it should be defined
terms larger than x2 are not considered. Even if during validation. Distribution of the residuals
the use of the quadratic model is allowed and (% difference of the back-calculated concentra-
used extensively by some bioanalytical labora- tion from the nominal concentration) should be
tories, the use of linear regression models investigated. The calibration model is accepted,
should be attempted first. Usually, a deviation if the residuals were within ±20% at the lower
from the linear model should be investigated limit of quantification (LLOQ) and within ±
and avoided. Nonlinearity could be due to in- 15% at all other calibration levels and at least
jection techniques, sample holdup on glassware, 2/3 of the standards met this criterion, including
cross-talk in MS/MS, interferences, and too highest and lowest calibration levels. Calibra-
wide a concentration range. Weighing functions tion standards not meeting the acceptance crite-
reduce the influence of values obtained for ria should be eliminated from the calibration
higher concentrations on slope and intercept but curve calculations.
selection of weighing should be justified. From Lower limit of quantification
statistical considerations the most common The lower limit of quantification is es-
weighing function for LC-MS- and LC- tablished as the lowest calibration standard with
MS/MS-based assays should be 1/x, due to the an accuracy and precision less than 20%.
fact that variance in y increases in proportion to
 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008 19

Accuracy and precision concentration should be within ±15% and CV%

The within- and between-run precision < 15%.
(expressed as coefficient of variation, CV%) Four plasma standards at each of the
and accuracy (expressed as relative difference two levels are prepared and let at room temper-
between obtained and theoretical concentration, ature four hours before processing (RTS study).
Bias%) of the assay procedure is determined by After that the extracted samples are analyzed
analysis on the same day of five different sam- with fresh standards.
ples at each of the lower (2 or 3 x LLOQ), Other four pairs are prepared, immedi-
medium (30-50% of the ULOQ) and higher ately processed and stored in the HPLC au-
(80% of the ULOQ) levels of the considered tosampler (PPS study). The samples are inject-
concentration range and one different sample of ed periodically over the expected longest stor-
each on five different occasions, respectively. age times of the samples in autosampler before
Sometimes, the selected concentrations could injection. The extracted samples (ready to in-
include values which are relevant in practice. ject) kept at autosampler temperature is finally
Recovery analyzed with fresh standards.
The recoveries at each of the previously For the freeze-thaw stability (FTS),
three levels of concentration and limit of quan- aliquots at the same low and high concentra-
tification are measured by comparing the re- tions are prepared. These samples are subjected
sponse of the treated plasma standards with the to three cycles of freeze-thaw operations in
response of standards in solution with the same three consecutive days. After the third cycle the
concentration of analytes as the prepared plas- samples are analyzed against calibration curve
ma sample. of the day. The mean concentration calculated
Stability for the samples subjected to the cycles and the
Stock solution stability: The stability of nominal ones are compared.
the stock solutions of drug and internal stan- For long-term stability (LTS), in the
dards should be evaluated at room temperature first validation day, there are injected and ana-
for at least 6 hours. If the stock solutions are lyzed four samples at each of low and high con-
kept refrigerated or frozen over a period of centrations, and values are calculated against
time, the stability over that period should be calibration curve of the day. Other two sets with
evaluated by comparing the response of the the same plasma concentrations were stored in
aged stock solution to that of a freshly prepared freezer and analyzed together with calibration
stock solution. Stock solution stability should samples after the expected storage period. The
be performed at one concentration in at least values are calculated against calibration curve
duplicate. of the day and the mean values for the stored
The stability of the analytes in human samples and nominal concentrations are com-
plasma: it is investigated in four ways, in order pared. The requirement for stable analytes is
to characterize each operation during the pro- that the difference between mean concentra-
cess of bioequivalence studies: room-tempera- tions of the tested samples in various conditions
ture stability (RTS), post-preparative stability and nominal concentrations had to be in ±15%
(PPS) in the autosampler, freeze-thaw stability range.
(FTS) and long-term stability (LTS) (at a freez- Dilution study
ing temperature at which it is known that the The ability to dilute samples with con-
analyte is stable). For all stability studies, plas- centrations above the upper limit of quantifica-
ma standards at low and high concentrations are tion is also investigated. Plasma standards (n =
used. The acceptance criterion: the mean found 5) with the concentration levels above the
20 Revista Română de Medicină de Laborator Vol. 10, Nr. 1, Martie 2008

ULOQ are diluted with blank plasma in order to No substitute for Good Science. The Workshop
get a concentration within the calibration range, Report and/or the Guiance can provide only the
then processed and analyzed, five samples in guiding principles.”
the same run and one sample on five different
occasions. The mean found concentration is
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