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Because the internal standard method eliminates some of the errors found in the external standard method it does not automatically follow that the internal standard method should always be used. The precision of many LC external standard methods is very good (e.g. <0.4% RSD for a purity determination) given that (i) the repeatability of injection volumes in modern injectors is much better than it used to be, especially if the injection is automated and (b) there are many methods for which sample
Figure 7.6 Internal standard choice in reversed-phased LC. (a) Analyte, (b) Suitable internal standard if readily available; similar physicochemical properties to analyte but probably enough difference in hydrophobicity to allow separation, (c) Unsuitable, different pÊë from analyte, therefore large difference in k' at some pH values, (d) Unsuitable, not the best match for >.max and likely significantly larger k' values, (e) Highly unsuitable, very different molar absorbance, (f) Highly unsuitable, different behaviour relative to analyte with changes in pH (i.e. neutral of basic analyte).
preparation is simple, involving no loss of the analyte. For these cases there is no advantage in using an internal standard method especially since it involves an extra step and extra measurements. Moreover there are cases where the use of an internal standard would actually lead to poorer precision. This would be the case if the LC peaks were tailed and
the major source of error was in the estimation of peak area. The external standard method involves one peak area measurement per chromatogram while the internal standard method involves two. Hence there is greater error in the internal standard method (a2totai = cr2i + a22 + ñò2ç + • • ¦)•
7.4 Method validation
Having developed a separation of analyte(s) from all possible other components that are likely to arise in the samples being analysed and decided how the quantitation will be carried out, the proposed analytical method must be validated before it can be used. In other words, it must be demonstrated that it is suitable for its intended purpose by carrying out a series of tests. The reason for carrying out these tests is often given as being to satisfy the demands of regulatory bodies. However while these demands were the catalysts for the trend in recent years for analytical methods to be thoroughly validated, they have merely served to raise awareness. It is a simple fact that the results of any analytical method are worthless if the method is not suitable for its intended purpose.
The validation process begins in method development in that the documentation reporting the validation data must include a record of the method development process, giving details of the conditions explored and the rationale of the progression of the process. The validation proper consists of a series of tests for which there are acceptance criteria which vary depending on the type of assay being carried out. Literal definitions of the parameters for which tests must be carried out are discussed elsewhere in this book but are repeated here as a reminder.
Since achieving specificity is often the most difficult aspect of developing an assay, establishing specificity is inextricably involved in the method development process. Therefore when it comes to validation, it should be simply a case of demonstrating specificity. It may be the case that when more thorough checks are made as in the validation specificity tests, the method is not as specific as was first thought.
An assay is specific if the ‘analytical response’ (i.e. that which is measured) arises from the analyte of interest and cannot arise from any other compound likely to be present in the sample. Therefore for an HPLC method, unless a selective detector is being used, it is necessary to demonstrate the peaks arising from all other compounds likely to be present in the sample are at least baseline-resolved from the main peak.
For all such likely other compounds for which specimen samples were available, k' would be determined under the conditions of the HPLC method to demonstrate that it was different from k! of the compound being determined. Specimen samples of likely interferences are not commonly available. Then it is necessary to obtain a chromatogram of a typical sample or better still a chromatogram of a sample which is thought to be enriched in likely interferences. For example, for a method to determine purity, a chromatogram of a highly impure sample would be obtained. For acceptable specificity there should be no peaks that are only partially resolved from the main peak. Also the main peak should be ‘homogeneous’; i.e. there should be no peaks underlying the main peak.
Establishing peak homogeneity is no easy matter. It may involve studying the sample using a variety of different HPLC conditions and/or using techniques such as thin-layer chromatography (TLC) and proton nuclear magnetic resonance spectroscopy ('H NMR) to search for additional impurities. There is some assurance of specificity if all the impurities found by the alternative methods are accounted for in the chromatogram obtained using the conditions of the method that is being validated. In other words, if they can be observed in the chromatogram then they cannot be hidden under the main peak. UV diode-array detection may also be useful in checking peak homogeneity. The UV spectra taken at various points on the peak should be a good match (there are various ways of testing the degree of match) for the UV spectrum of the pure compound. If the UV spectrum of a pure reference standard is not available, then at least the UV spectrum across the peak should not change. If there is partial resolution under the main peak then the UV spectrum or absorbance ratio of two or more wavelengths would change from the front edge to the apex to the tailing edge of the peak. This is quite a discriminating test if there is a co-eluting compound which has a significantly different UV spectrum from the compound being determined. However, if the UV spectrum of the interference is similar to that of the compound being determined, as is frequently the case with co-eluting structurally related interferences, then quite large amounts (up to 10-20% by weight) of interference may be ‘missed’. In time, with the advent of low cost LC-MS (HPLC coupled directly on-line with a mass spectromonitor), peak homogeneity testing will be much less problematic. Mass spectra of even closely related compounds are distinctly different and accordingly it should be possible using LC-MS to detect very low levels (<0.1% by weight) of interferences under the main peak.