Monday, 8 July 2019

Validation of analytical procedures

Validation of analytical procedures

Validation of analytical procedures

The International Conference on Harmonisation (ICH) has adopted the following terms for defining how the quality of an assay is controlled.

The analytical procedure

The analytical procedure provides an exact description of how the analysis is carried out. It should describe in detail the steps necessary to perform each analytical test. The full method should describe:
(i) the quality and source of the reference standard for the compound being analyzed
(ii) the procedures used for preparing solutions of the reference standard
(iii) the quality of any reagents or solvents used in the assay and their method of preparation
(iv) the procedures and settings used for the operation of any equipment required in
the assay
(v) the methodology used for calibration of the assay and methodology used for the processing of the sample prior to analysis. In fact, it is difficult to be comprehensive in this short account, since the description of a fully validated method is a lengthy document.

Levels of precision

The ICH guidelines define precision as follows:
“the precision of an analytical procedure expresses the closeness of agreement (degree of scattering) between a series of measurements obtained from multiple a sampling of the same homogeneous sample under the prescribed conditions... The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.”

Thisisbroadlywhatwasdescribedinmoredetailabovefortheassayofparacetamol tablets. There is no absolute guideline for how good precision should be for the active ingredient in a formulation but, in general, a precision of <? 1.0% is desirable. The precision achievable depends on the nature of the sample being analyzed. The RSDs achievable in the analysis of trace impurities in bulk drug or drugs in biological fluids may be considerably greater than? 1.0%because of the increased likelihood of losses when very low concentrations of analyte are being extracted and analyzed. The precision of the assay of a particular sample, in the first instance, is generally obtained by repeating the assay procedure a minimum of five times starting from five separate aliquots of the sample(e.g.five weight soft tablet powder or five volume of elixir)giving a total of 25 measurements. Repetition of the sample extraction gives a measure of any variation in recovery during extraction from the formulation matrix. One difficulty in defining the precision of an assay is in indicating which steps inthe assayshouldbeexamined. Initially, an assay will be characterized in detail by thereafter, in re-determining precision (e.g. in order to establish repeatability and intermediate precision), the certainelementsinthe assay may be taken for granted. For example, the thesame standard calibration solution may be used for several days provide the dataset ability to storage has been established. Similar there needs to be a limited number of samples extracted for assay, provided it has been established that the recovery of the sample upon extraction does not vary greatly. According to the ICH guidelines, precision may be considered at three levels: repeatability, intermediate precision, and reproducibility.


Repeatability expresses the precision obtained under the same operating conditions over a short interval of time. Repeatability can also be termed intra-assay precision. It is like that the assay would be repeated by the same person using a single instrument. Within repeatability, it is convenient to separate the sample preparation method from the instrument performance. 

Figure 1.3 shows the levels of precision including some of the parameters which govern the system precision of a high-pressure liquid chromatography(HPLC)instrument. It would be expected that they stem precision of a well-maintained instrument would be better than the overall repeatability where sample extraction and dilution steps are prone to greater variation than the instrumental analysis step.

An excellent detailed summary of levels of precision is provided by Omar. For example, Figure 1.4 

shows the results obtained from five repeat injections of a mixture of the steroid speeds one(P)and hydrocortisone(H)intoaHPLCusingamanualloop injector. The mixture was prepared by pipetting 5 ml of a 1 mg/ml stock solution of each steroid into a 100 ml volumetric flask and making up to volume with water. The precision obtained for the areas of the hydrocortisone peak is? 0.3%; the injection process in HPLC is generally very precise and one might expect even better precision from an automated injection system, thus this aspect of system precision is working well. The precision obtained for the prednisone peak is? 0.6%; not quite as good, but this is not to do with the inject but is due to a small impurity peak(I)which runs closely after the prednisone causing some slight variation in the way the prednisone peak is integrated. The integration aspect of the system precision is not working quite as well when challenged with a difficulty in the integration method but the effect is really only minor.

For the repeat, analysis is carried out on a subsequent day on a solution freshly prepared by the same method. The injection precision for hydrocortisonewas? 0.2%.The variation for the means of the areas of hydrocortisone peaks obtained on the 2 days was? 0.8%; this indicates that there was a small variation in the sample preparation (repeatability) between the 2 days since the variation in injection precision is? 0.2%–? 0.3%. Usually, itis expected that instrument precision is better than the precision of sample preparation if a robust type of instrument is used in order to carry out the analysis. Table 1.2 shows the results for the repeat absorbance measurement of the same sample with a UV spectrophotometer in comparison with the results obtained from the measurement of five samples by a two-stage dilution from a stock solution. In both cases the precision is good but, as would be expected, the better precision is obtained where it is only instrumental precision that is being assessed. Of course, instruments can malfunction and produce poor precision, for example, a spectrophotometer might be nearing the end of the useful lifetime of its lamp and this would result in poor precision due to unstable readings.

A target level for system precision is generally accepted to be <? 1.0%. This should be easily achievable in a correctly functioning HPLC system but might be more difficult in, for instance, a gas chromatography assay where the injection process is less well controlled. The tolerance levels may be set at much higher RSD in trace analyses where instruments are operated at higher levels of sensitivity and achieving 100% recovery of the analyte may be difficult.

Intermediate precision

Intermediate precision expresses within-laboratory variation of precision when the analysis is carried out by different analysts, on different days and with different equipment. Obviously, a laboratory will want to cut down the possibility for such variations being large and thus it will standardize on particular items of equipment, particular methods of data handling, and make sure that all their analysts are trained to the same standard.


Reproducibility expresses the precision between laboratories. Such a trial would be carried out when a method was being transferred from one part of a company to another. The data obtained during such a method transfer does not usually form part of the marketing dossier submitted in order to obtain a product license. For new methodologies, a popular method for surveying the perform microfame methods to carry out a round robin trial, where many laboratories are asked to carry out a qualitative and quantitative analysis of a sample where the composition is only known to those organizing the trial.


As described above, methods may be precise without being accurate. The determination of accuracy in the assay of an unformulated drug substance is relatively straightforward. The simplest method is to compare the substance being analyzed with a reference standard analyzed by the same procedure. The reference standard is a highly characterized form of the drug which has been subjected to extensive analysis including a test for elemental composition. The methods for determining the accuracy of an assay of a formulated drug are less straightforward. The analytical procedure may be applied to a drug formulation prepared on a small scale so that the amount of drug in the formulation is more precisely controlled than in a bulk process; a placebo formulation spiked with a known amount of drug or the formulated drug spiked with a known amount of drug. The accuracy of the method may also be assessed by comparison of the method with a previously established reference method such as a pharmacopoeial method.

Accuracy should be reported as percent recovery in relation to the known amount of analyte added to the sample or as the difference between the known amount and the amount determined by analysis. In general, at least five determinations, at 80, 100 and 120% of the label claim for the drug in the formulated product, should be carried out in order to determine accuracy.

Standard operating procedure (SOP) for the assay
of paracetamol tablets

The terms defined above are perhaps best illustrated by using the example of the simpleassaythatwementionedbefore.TheassayinBox1.4islaidoutinthestyleofa standardoperatingprocedure(SOP).Thisparticularsectionoftheoperatingprocedure describes the assay itself, but there would also be other sections in the procedure dealing with safety issues, the preparation, and storage of the solutions used for extraction and dilution, the glassware required and a specification of the instrumentation to be used.

The assay described in Box 1.4 assesses the precision of some of the operations within the assay. If a single analyst were to assess the repeatability of the assay, instructions might be issued to the effect that the assay as described was to be repeated five times in sequence, i.e. completing one assay before commencing another. If between-day repeatability were to be assessed, the process used for determining the repeatability would be repeated on two separate days. If the within-laboratory reproducibility were to be assessed two or more analysts would be assigned to carry out the repeatability procedure. In arriving at an SOP such as the one described in Box 1.4, there should be some justification in leaving out certain steps in the complete assay. For instance, weighing is often the most precise step in the process and thus repeat weighings of samples of tablet powder would not be necessary to guarantee precision; the precision of the extraction might be more open to question.

Each of the sections within an assay would have other SOPs associated with them, governing, for instance, the correct use and care of balances, as listed in Box 1.5.

Compound random errors

Systematic errors in analysis can usually be eliminated, but true random errors are due to operations in an assay which are not completely controlled. A common type of random error arises from the acceptance of manufacturers’ tolerances for glassware. Table 1.3 gives the RSD values specified for certain items of grades A and B glassware.

Anestimateofcompoundrandomerrorsisobtainedfromthesquarerootofthesum of the squares of the RSDs attributed to each component or operation in the analysis. If the analysis of paracetamol described in Box 1.4 is considered, then, assuming the items of glassware are used correctly, the errors involved in the dilution steps can be simply estimated from the tolerances given for the pipette and volumetric flasks. Thus it can be seen that the compound error from the glassware differs little from the largesterrorintheprocess. Of course the glassware error scan is eliminated by calibration of the glassware prior to use, but, in general, analysts will accept manufacturers’ tolerances. The tolerated random error from glass war could be readily eliminated; other random errors, such as variation in the extraction efficiency, are more difficult to control.

Reporting of results

In calculating an answer from the data obtained in an analysis it is important to not indicate a higher level of precision than was actually possible in the assay. As mentioned in the previous section, when considering the accuracy of glassware used with the assumption that it complied with the BS grade A standard, it was obvious that there was some uncertainty in any figure < 1%. It might be possible toimproveonthis degree of precision by calibrating glassware; however, any improvement in precision in the real world would take time and hence have cost implications. Thus for the purposes of most analyses, and for the purposes of the calculations in this book, it would seem sensible to report four significant figures, i.e. to 0.1%. In the process of carrying out calculations, five figures can be retained and rounded up to four figures at the end of the calculation. Since in pharmaceutical analyses the percentage of the stated content of a drug in a formulation may be reported as being between 90 and 99.9%, if the first significant figure is 9, then at the end of the calculation a more realistic estimate of precision is given by rounding the answer up to three significant figures. The SD or RSD reported with the answer should reflect the number of significant figures given; since there is usually uncertainty in figures < 1% of the answer,theRSDshouldnotbereportedbelow0.1%.Taking this into consideration the correct and incorrect ways of reporting some answers are given in Table 1.4.

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