Journal of Clinical Pharmacy and Therapeutics (1989) 14,465-473.

THE VALIDATION CRITERIA FOR ANALYTICAL METHODS

USED IN PHARMACY PRACTICE RESEARCH
A. C. Mehta
Department of Pharmacy, The General Infirmary, Leeds, Yorkshire, U.K.

SUMMARY

An integral part of analytical method development is validation, i.e. once the

method has been devised it is necessary to evaluate it under the conditions
expected for real samples before being used for a specific purpose. Although
the validation stage is crucial in method development, the importance of this
step is often overlooked. This paper attempts to clarify the nomenclature of
method validation and describes the validation procedure for analytical
methods used for the determination of drugs in biological fluids and
formulations.
INTRODUCTION
An ability to develop new assays for drugs and metabolites is of obvious importance to
hospital pharmacy laboratories involved in practice research. In hospital pharmacy the
research is mainly of an applied and collaborative nature and the results are of direct
practical importance. Many of the investigations therein require reliable and
thoroughly validated analytical methods in order to measure drugs in complex media,
such as biofluids or formulations. Table I shows some typical investigations that require
involvement by the hospital pharmacy in analytical method devetopment.
ANALYTICAL T E C H N I Q U E S
Most hospital workers prefer chromatographic methods [gas chromatography (GC) or
high performance liquid chromatography (HPLC)] for drug assays due to their selectivity (i.e. their ability to measure several compounds simultaneously), sensitivity, and
overall versatility. HPLC has a further advantage over GC in that it can be used for
polar, thermaliy labile drugs, particularly those that are not easily analysed by G C (e.g.
antibiotics). Immunochemical methods are also used in hospital pharmacy laboratories;
they are relatively expensive (because of the cost of reagent kits) but are less timeconsuming and easier to use than the chromatographic techniques. They are well suited
to projects that involve therapeutic monitoring in patients, provided that the assay kit
for the drug in question is available. Homogeneous enzyme immunoassays, such as
Syvas EMIT system, can be used on biofluids without clean-up steps. Spectroscopy
(ultraviolet, infrared, atomic absorption) and other analytical techniques are also used
in hospital pharmacy laboratories but are mainly used for routine quality control (QC)
465

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A . C.Mehta
Table 1. R&Dactivities in hospital pharmacy which require some analytical input

1. Method development for quality control of finished products and special formulations where official
methods are not available
2. Testing of raw materials (devisingof limit tests)
3. Testing of containers and closures
4. Medical gas testing
5. Monitoring of medicinal products purchased on regional contracts, e.g. dissolution and bioavailability

testing of generic tablets

6 . Stability testing of special formulations (ointments, creams, intravenous mixtures)
7. Clinical trials and phase I and 11 pharmacokinetic studies
8. Therapeutic drug monitoring and monitoring of patient compliance
9. Evaluation of a new analytical equipment or a software

work. A more detailed discussion of the analytical techniques used in hospital pharmaceutical work is not appropriate in this paper; the interested reader should consult
reviews published elsewhere (1-4).
VALIDATION PARAMETERS
Due to a variation in sample composition or instrumental response, the existing (i.e.
published) methods for drug analysis often need modifying to suit the requirements of
the laboratory that performs the assay. Thus, in addition to the newly developed
methods, modified methods also require validation to assess their performance after
modification. In general, validation of an analytical procedure with respect to linearity,
accuracy, precision, sensitivity and limit of detection, specificity, and recovery is
required (5-9). In addition to the scientificcriteria, the developed method will also need
to be evaluated further on the basis of cost and ease and speed of operation.
The extent (level) of validation usually depends on the specific application. For
pharmacokinetic studies the assay must be selective and sensitive enough to follow
absorption, distribution, and elimination of the drug. Furthermore, it must be accurate
and precise in order to reveal differences between drugs, formulations or subjects. For
therapeutic drug monitoring (TDM), the emphasis is on a fast, simple and reliable
method. As determinations for TDM are usually performed at comparatively high
concentrations (steady-state levels), extreme sensitivity i s not often needed. An exception to this is the monitoring of free (unbound) drugs in plasma or serum. For this the
assay needs to be very sensitive especially to highly protein-bound drugs (e.g. phenytoin) because the percentage of free drug is very low. Free level assays are done after
removing protein from biological samples by separation methods such as ultrafiltration
or equilibrium dialysis (10).
In formulation studies if the chosen method is intended for QC of a major constituent
in a dosage form, then a highly selective and sensitive assay is not usually required
provided that the excipients do not interfere with the assay. On the other hand, an assay
for stability study demands a different approach, i.e. it must be sufficiently sensitive and
selective to detect and separate low levels of degradation products. Moreover, throughout the study, the assay must be sufficiently precise especially if the extent of decomposition measured is small. The individual validation criteria for method development are
discussed in the following sections.

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467

~ A L I B R A T I O NAND L I N E A R I T Y
At least six calibration points (excluding the blank), which cover the expected range of
concentrations of the drug in the samples, are needed for calibration, i.e. the graph of
instrumental response versus analyte concentration. In biofluid analysis, drug-free
specimens of biofluids (blank) spiked with appropriate amounts of the drug should
always be employed as calibration standards. Aqueous or methanolic solutions are
unacceptable as standards because they differ markedly from biological matrices. Blank
samples should be included in each calibration run in order to detect interference from
substances other than the analyte, which are present in the sample, e.g. endogenous
substances and co-administered drugs in biological fluids or excipients in formulations.
If necessary, calibrations (including blank) should be duplicated to improve the precision of the calibration line. Variation in the blank (particularly in biological fluids) or
instrumental response makes it imperative to run a calibration with every batch of
samples. The calibration data should always be plotted as well as subjected to linear
regression analysis (if a linear response is obtained) (1 1). Any dubious points on the
calibration line should be checked and no results that fall outside the calibration range
should be reported.
The majority of calibration graphs produced routinely are linear; however, they need
not always be so provided that they are reproducible and reasons for the non-linearity
are understood. Non-linear curves may indicate variation in extraction yields over a
concentration range, and in chromatography, overloading of a column or detector.
Non-linear calibration can be linearized, for example, by taking logy andjor log x
vaiues, instead ofy and x values, while plotting calibration curves. Such conversions are
routinely applied to the data obtained using certain analytical techniques such as
radioimmunoassay.
ACCURACY AND PRECISION
Accuracy (or bias) refers to the difference between the observed value and the true or
known value and is expressed in terms of error. Precision (or reproducibility) describes
the variation or scattering of the data around the mean. Accuracy and precision are not
absolute characteristics of the method but depend on the expertise and equipment
available in a particular laboratory. Because of the complexity of the media used and the
presence of trace quantities of drugs, the accuracy and precision of drug analysis in
biofluids are not usually as good as those of bulk drug or formulation analysis.
Accuracy can be determined by replicate analysis of samples containing known
amounts of the drug. The deviation from the true values serves as a record of the
methods accuracy. Usually three concentrations (a low, an intermediate and a high
value within the calibration range) are tested to determine whether or not accuracy is
concentration dependent. It should be noted that outside the linear calibration range the
accuracy is unknown. Accuracy usually decreases rapidly as the limit of detection (see
below) is approached.
The within-day precision (repeatability) can be determined by duplicate measurement of drug levels at three different concentrations, preferably in at least 10 samples for
each concentration. Similarly, between-day precision (reproducibility) is measured by
analysis preferably on at least 10 separate days. The results are expressed as the standard
deviation or the coefficient of variation (CV) at each level. The more meaningful figure

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A. C . Mehta

for precision would be for between-day precision as this is more appropriate to the real
situation. Furthermore, to test the methods ruggedness, between-day precision experiments can be carried out under slightly varying conditions which may be encountered
during daily application of the method. For example, different workers may apply the
method on different instruments using the same sample.
Although a CV value of 10% should be acceptable as the minimum acceptable precision, higher precision (CV < lOyA) should be aimed at particularly in the middle of the
calibration range. It must be emphasized that good precision does not necessarily imply
good accuracy: a systematic error may lead to precise but inaccurate results. Systematic
errors can be eliminated by careful checks of the method or equipment.
The accuracy of a newly developed (or modified) method can be assessed by comparing the results obtained by it with those found with a comparison (or reference) method
of known accuracy and precision. Correlation of the two methods against each other by
linear regression analysis is the preferred statistical approach for comparing the accuracy of two analytical methods ( 7 , l i ) . The idea behind this is to identify any systematic
(positive or negative) errors in a new procedure compared to a reference method. For
example, in a newly developed immunoassay, some antibodies may cross-react with
drug metabolites to give higher results than those obtained with the reference method.
GC and HPLC are often used as reference methods because they are less susceptible to
interferences from other substances. Sometimes, however, it is not possible to conduct
method comparison studies simply because either a widely accepted reference method is
not available or is costly and requires special facilities.
Once the assay has been established for routine use its accuracy and precision should
be regularly monitored to ensure that it continues to work satisfactorily. For this a
number of separately prepared QC samples are analysed along with actual samples at
intervals depending on the total number of actual samples. As a rough guide one control
for every 10 samples or two per small batch should suffice. QC samples are prepared by
adding known amounts of drugs to blank specimens which are frozen in small aliquots
(1-2 ml) for later use. Controls should be run in duplicate, preferably at three concentrations corresponding to levels below, within, and above the experimental range or at
least at two concentrations, one at the high end and one at the low end of the range. The
sample results are acceptable if the QC results are within l O g b of the known values. QC
materials based on serum or urine are commercially available for many drugs which are
assayed routinely for therapeutic or toxic purposes.
S E N S I T I V I T Y AND T H E L I M I T O F D E T E C T I O N
Sensitivity and the limit of detection (LOD) are two related but distinct parameters
which are used to pinpoint the ability of an analytical technique to detect and quantify a
trace substance (concentration mg/l or below) in a complex chemical or biological
medium (12, 13). They provide important i n f o ~ a t i o nwhen attempting to compare
analytical methods, instruments, or to decide which method to use to tackle a particular
problem.
There is a certain degree of confusion in the terminology which has been used to
describe sensitivity and LOD. Sometimes one term is misused to denote the other. A
method is said to be sensitive if a small change in concentration (c) causes a large change
in instrumental response ( x ) , i.e. when the derivative dx/dc is large. The sensitivity is

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469

thus defined as the slope of the calibration line and provided that the plot is linear, the
slope can be measured at any point on it. On the other hand, LOT) determines the lowest
concentration or amount (mass) of analyte which can be distinguished from the blank
with reasonable confidence (7, 13). According to the IUPAC (International Union of
Pure and Applied Chemistry), LOD is defined as the mean blank value plus three times
the standard deviation (SD) of the blank (14). The blank is also called the background or
the noise and comprises the electronic noise coming from the instrument and the signal
coming from the blank sample, i.e. from all the constituents of a typical sample other
than the analyte. A multiplication factor of 3 for SD corresponds to a 990,, confidence
interval for a Gaussian (normal) distribution (13).
Many other definitions are given to LOD, e.g. the concentration corresponding to
half the bottom standard or three times the background signal, but they are qualitative
and do not reflect the inherent scatter in the calibration line. The half-the-bottomstandard approach would result in a much higher or lower LOD depending on the value
of the arbitrarily chosen lowest calibration standard. The three times the background
method gives a higher LOD than that obtained using the IUPAC definition and is very
popular in the area of drug bioanalysis. In this method, LOD is determined by testing
serially diluted samples until the signal to noise ( S / ) ratio reaches 3. The popularity of
the three times the background method in the bioanalytical field may be due to the fact
that the higher value of the resultant LOD provides a wider safety margin, which may be
preferable, given the unpredictable nature of the biological blank. If no blank exists,
LOD can be defined as the concentration or amount for which the estimated precision
becomes greater than a pre-set limit, say a CV value of 209, (5).
Meas~rementof LOD (IUPAC approach)
The measurements of the blank signal and its SD are at the centre of the estimation of
LOD if the IUPAC definition is adopted. They can be measured directly by performing
the blank experiments several times, but this is a time-consuming procedure; instead,
the value of the intercept of the calibration line on they-axis can be used as an estimate of
the blank signal itself. It takes into account all points on the calibration line and gives a
more accurate estimate of the blank than a single measured value does. Similarly the SD
of the regression line can be used as the SD of the blank. The regression line approach to
calculating the LOD is quite suitable in practice (1 1) and has been used to calculate the
LOD of an HPLC procedure for the determination of propranolol in plasma (15).
Variability of LOD
Because of the different approaches used to calculate the LOD, it is essential that the
method used should be specified in all publications. The volume of the sample needed to
achieve the stated LOD should also be given. The LOD is governed by the type and
condition of the instrument (light source, chromatographic column, detector, etc.) and
by procedural factors such as recovery of the compound. It also varies with the type of
samples (e.g. presence of compounds similar to analyte), and different batches of blank
control (e.g. urine). For thcse reasons the published LOD should be considered only as
a useful but approximate guide to analytical procedure. It is necessary to reassess it
whenever changes in conditions affecting it are suspected.

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470

Approaches to improving sensitivity and LOD

It is seldom advisable to use an instrument constantly at its highest sensitivity setting

or to work near the LOD of a method. High sensitivity of the instrument cannot always
achieve better detection limits, because a concomitant increase in noise level would
result in only a small increase in the overall S / N ratio. A low noise instrument is
obviously advantageous in this respect. The LOD value and the sensitivity of the
procedure can be improved by a sample clean-up step prior to analysis (e.g. selective
extraction), the use of appropriate analytical techniques [e.g. chromatographic separation (GC, LC) coupled with sensitive detection] and the derivatization of the drug if
necessary. In addition, the LOD value can often be improved by judicious choice of
experimental conditions, for example, in chromatography, by adjusting the size of the
sample, the reconstitution solvent or the injection volume.

SPECIFICITY
Specificity (or selectivity) is the ability of an analytical method to determine solely the
compound(s) of interest in the presence of potentially interfering compounds such as
the constituents of the biofluids, the presence of other drugs, excipients, impurities, or
degradation products. Thus, the analytical method should be tested during the method
developmentphase for suspected interfering substances. For GC and HPLC this means
that there is no other peak but the drug peak at the retention time of the test drug.
In biofluid analysis there is a higher potential for interference from other drugs and
their metabolites when the analytical method is used in clinical trials or compliance
studies instead of volunteer studies. In these situations it is not possible to test in
advance for all possible interfering drugs and it is wiser to restrict the specificity tests to
a limited number of drugs which may be co-administered. However, specificity should
be reassessed when a drug not previously tested for interference has been co-prescribed.
Specificity can be incorporated into a work-up step (e.g. selective extraction), the
analytical technique (e.g. chromatographic separation and use of specific detectors) or
via a change in experimental parameters (e.g. change in mobile phase or wavelength of
detection in HPLC). Each laboratory should determine the specificity of its own assay
methods and any limitation of specificity in a method should be mentioned in its description. It would also be sensible to reinvestigate the specificity of a published method
before putting it to a new use, because a method which works in one situation may give
rise to problems, or even fail, in another (7).
E X T R A C T I O N EFFICIENCY AND RECOVERY
Recovery experiments are particularly useful to test the various stages of an analytical
method and to assess losses in sample processing, e.g. solvent extraction. Ideally several
recovery experiments should be performed by adding various amounts of authentic
standard to a typical blank specimen. Spiking should cover at least 3, i.e. low, middle,
and high, concentrations in the expected range, because recovery may vary with concentration. The analytical recovery is calculated as
Recovery

Amount found
=

Amount added

x 100q/,.

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47 1

Absolute recovery can be determined by comparing the peak height or area for extracted
samples at each standard concentration of drug, with that for unextracted samples of
identical concentrations made up in reconstitution fluid, preferably the mobile phase in
the case of HPLC. The absolute recovery of a procedure should be reproducible and
preferably > 7tiolO.If recovery is low (say 600/,) but reproducible, it may still be acceptable; if however, it is low, variable, and unpredictable, the reason for this should be
investigated and eliminated or an alternative approach to isolating the drug should be
sought (7).
Novel approach
Literature search
1
including official - ~ A ~ ; - ; e l e c + i o n
compendia and
text books

Reconmmda+iofa coienque

Method lalidation

linearity
Comparison with a
reference method

Spiked sampies

-day
Day- to -day

---m&*--

Collaborative ?esrs*

Be tween la bora toiies*

Crass-reactivity studies
silldies

(for irnrnunoassays)

*If the assay is to be performed in different laboratories.

Fig. 1. Various stages for the evolution and evaluation of a new analytical method.

In trace analysis of drugs, the sensitivity and LOD of the method are greatly
influenced by the recovery of the drug to be determined; therefore maximizing the
overall recovery of the assay is essential for achieving high sensitivity and low LOD.
Good recovery is aided by efficient solvent extraction and by preventing drug losses.
Adsorption of the drug to glassware, instability and/or volatility of the drug, co-precipitation, incomplete reaction during derivatization, and last but not least, negligence on
the part of the analyst, are some of the many causes of sample loss during analysis.
In recovery studies, as the weighed amount of the drug is taken as the true amount, it
is imperative that the spiked samples are prepared very carefully, in the expectation that
the variances of the sample preparation (weighing, preparation of solutions, etc.) will be
small compared to the total variance of the overall method (7).

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A. C. Mehta
INTERNAL STANDARD

During sample preparation an accurately known amount of a known compound

[internal standard (IS)] is added to the sample at the earliest possible stage in the
expectation that any procedural loss of sample will be accompanied by an equivalent loss
of IS. T h e ratio of the detector response (peak height or area) for the drug and the I S is
then used in calibration and assay. T h e use of an IS, however, does not lessen the
importance of careful work.
Ideally an I S should be stable and easily available. I t should have physicochemical
and chromatographic properties close to those of the test drug. I t should elute separately
but near to the analyte. If a derivative of the analyte is to be prepared, then I S should be
capable of giving a derivative in the same way. For these reasons most workers prefer to
use an IS structurally similar to the drug to be assayed. One drug can be used as an I S for
another provided that the drug used as an I S is not already part of the patients therapeutic regime. Guidelines are available for the proper application of the I S technique in
the determination of drugs in biological material (7, 16).
CONCLUSIONS
T h e validation procedure described in this paper represents the minimum amount of
work which should be done to gain confidence in the suitability of an analytical method
to produce meaningful results. It is obvious from this paper that method development
and validation require considerable effort and time and can only be justified if the
method is to be used for sizeable samples and for a worthwhile project. It is true that in
smaller laboratories method validation increases the workload, but it does improve the
quality of results.
As mentioned earlier, the extent to which a method needs to be validated depends on
its application. Each investigator should decide for himself which parameters are more
relevant to the particular assay and design the validation procedure accordingly. Figure
1 summarizes the various stages for the validation of a new analytical method. Several
examples of method validation can be cited in the literature, for instance, the validation
of prednisolone assay for stability studies (17) and that of ranitidine assay for pharmacokinetic studies (18). If the validation procedure described in this paper is followed
during method development and application, then the analytical laboratory can be
satisfied that it has provided an efficient and professional contribution to the particular
study.
REFERENCES

1. Couper, I. & Driver, N. (1980) Drug analysis and quality control. In: Textbook of Hospital Pharmacy
(eds M. C. Allwood & J. T. Fell), pp. 199-228. Blackwell Scientific Publications, Oxford.
2. Glenn, A.L. (1980) Development of analytical methods. In: Textbook of Hospital Pharmacy (eds M. C.
Allwood & J. T. Fell), pp. 229-273. Blackwell Scientific Publications, Oxford.
3. Lesne, M. (1983) Modem drug analysis in biological fluids suitable for clinical pharmacokinetics.
Journal of Pharmaceutical and Biomedical Analysis, 1,415-434.
4. de Silva, J.A.F. (1985) Analytical strategies for therapeutic monitoring of drugs in biological fluids.
Journal of Chromatography, 340,3-30.
5. Chamberlain, J. (1985) Analysis of Drugs in Biological Fluids. CRC Press, Boca Raton.
6. Dadger, D. & Smyth, M.R. (1986) Validation of chromatographic methods of analysis of drugs in
biological samples. Trends in Analytical Chemistry, 5 , 115-1 17.

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7. Mehta, A.C. (1987) Drug determination in biological fluids: approaches to method validation. Talanta,
34,609-613.
8. Shoenmakers, P.J.& Mulholland, M. (1988) An overview of contemporary method development in
liquid chromatography. Chromatographia, 25,737-748.
9. Inman, E.L., Frischmann, J.K., Jimenez, PJ., Winkel, G.D., Persinger, M.L. & Rutherford,
B.S. (1987) General method validation guidelines for pharmaceutical samples. Journal of Chromatographic Science, 25,252-256.
10. Mehta, A.C. (1 989) Therapeutic monitoring of free (unbound) drug levels: analytical aspects. Trends in
Analytical Chemzstry, 8, 107-1 12.
11. Miller, J.C. & Miller, J.N. (1988) Statistics f o r Analytical Chemistry. Ellis Horwood, Chichester.
12. Miller, J.N. (1982) Matrix and sensitivity constraints in trace organic analysis. Analytical Proceedings,
19,114-117.
13. Mehta, A.C. (1989) Trace measurement. Laboratory Practice, 38,29-30.
14. IUPAC (1978) C # m ~ e n d of
~ Analytical
u~~
~omenclature.p. 117. Pergamon, Oxford.
15. Smith, K.A., Wood, S. & Crous, M. (1987) Rapid high performance liquid chromatographic method
for the determination of propranolol in plasma. Analyst, 112,407-409.
16. Haefelfinger, P. (1981) Limits of the internal standard technique in chromatography. Journal of
Chromatography, 218,73-81.
17. Stroud, N., Richardson, N.E., Davies, D.J.G. & Norton, D.A. (1980) Quality control of prednisolone sodium phosphate. Analyst, 105,455-461.
18. Salem, M.S., Gharaibeh, A.M., Alkaysi, H.N. & Badwan, A. (1988) High performance liquid
chromatographic analysis of ranitidine in plasma and urine. Journal of Clinical Pharmacy and Therapeutics, 13,351-357.