Absolute quantitation assay

A plethora of chemical reactions that are intimately associated with the quantitative analysis essentially belong to the class of reversible reactions. These reactions under certain prevailing experimental parameters are made to proceed to completion, whereas in certain other conditions they may even attain equilibrium before completion. In the latter instance, erroneous results may creep in with regard to the pharmaceutical substance under estimation. Hence, it has become absolutely necessary first to establish the appropriate conditions whereby the reactions must move forward to attain completion so as to achieve the ultimate objective in all quantitative assays. 

Definitive quantitative assay uses calibrators fitted to a known model to provide absolute quantitative values for unknown samples. Typically, such assays are only possible where the analyte is not endogenous, for example, a small-molecule xenobiotic drug. 

Demonstration of selectivity and specificity of an assay for a biomarker must be considered in the context of whether the assay is a definitive quantitative assay or a relative quantitative assay (see Section 1.2). It can be difficult to show absolute specificity and selectivity for a relative assay due to the uncertainty of the exact species being measured and unknown components of the matrix. At a minimum, reproducible measurements of endogenous samples with and without spiking of the reference analyte should be demonstrated. On the other hand, likely truncated forms of a well-characterized biomarker measured by a definitive quantitative assay can be tested for specificity, as in the following example. 

It should be understood that in most cases, the calibration material provided may not be 100% pure (most are not supplied with a Certificate of Analysis). However, notwithstanding the recommendations below, this is often not a major issue since the majority of biomarker assays will, by definition, be relative quantitative assays that do not measure absolute concentration. In addition, in many assay kits, particularly more recent ones, the calibration material may not be well characterized or purified, or it may not be fully representative of the endogenous analyte to be measured. Therefore, we would recommend procuring material from at least one other third-party manufacturer against which concentrations can be checked to ensure consensus. If there is disparity, another source may be required. It is also a good idea to check with the kit manufacturer as to where they have sourced their calibration material, since they often do not manufacture it themselves. On more than one occasion with certain assays we... 

Since we accept that the majority of biomarker assays will be relative (as opposed to absolute) quantitative methods, the main concern is one of reproducibility of the assay over the time course of the relevant study for which it is being used. Therefore, it is often common at the validation stage to set no accuracy acceptance criteria for the QC samples analyzed in the validation batches but to evaluate the intra- and interassay performance of the method using the QC sample results obtained therein. Thereafter, it can be assessed as to whether the assay s performance meets the requirement of the study and if so, acceptance criteria for the sample analysis batches can be set a priori. 

Figure 11.35 Diagram illustrating a method for providing absolute quantitation of metabolites in incurred serum samples when no analytical standard is available. The method exploits the trend towards equimolar responses for a drug candidate and its metabolite at low flowrates ( 10 nL.min ) that permits measurement of the concentration ratio of metabolite to parent compound via the observed ratio of signals for the two using nanospray ionization (1 nano/Ip,nano)- However, use of a fast nonselective extraction procedure like protein precipitation for the LC-ESl-MS/MS assays is not suitable for the nanospray experiment since the extracts are too complex, so a more selective procedure, liquid-liquid extraction (LLE) in this case, must be used to prepare these extracts. The connection between the two procedures is achieved by analyzing the LLE extracts by both nanospray and the LC-ESI-MS method used for the assays of the incurred samples, yielding an LC-ESI-MS peak area ratio (A] lc/ms/- p,lc/ms) comparison with (iM.mmo/Ip.nano) that is interpreted as the concentration ratio. This comparison yields a calibration factor k ji = (lM,nano/Ip,nano)/(AM.LC/Ms/Ap Lc/Ms) that enables quantitation of the metabolite relative to that of the parent compound subsequently, absolute quantitation of the latter by conventional LC-MS using an analytical standard to prepare a calibration curve permits absolute quantitation of the metabolite also. Only a limited number of nanospray analyses (one in the example shown) is required to derive a k j value applicable to a complete set of study samples. Reproduced from Valaskovic, Rapid Commun. Mass Spectrom. 20, 1087 (2006), with permission of John Wiley Sons Ltd.

Fusari and coworkers3 have developed a similar method for the assay of phenytoin and phenobarbital in dosage forms. They have used Silanized Celite 545 as the support material, 25% amyl alcohol in chloroform as the stationary phase and 0.05M borate buffer of pH 9.5 as the mobile phase. Phenobarbital was eluted with the mobile phase and phenytoin eluted with absolute ethanol. Ultraviolet absorption was used to quantitate the substances. 

Spectrofluorimetry differs from absorption spectrophotometry in not yielding an absolute scale of values. For this reason it is essential to employ a reference standard for quantitative measurements. For example, some pharmacopoeial tests, such as the test for uniformity of content for digitoxin tablets, employ a spectrofluorimefric assay and comparison wi an ofticial reference standard. Quantitative Spectrofluorimetry has been proposed for a munber of naturally fluorescent compoimds, including ergometrine, riboflavine, tiie catechol-amines, phenothiazines, the barbiturates (at pH 13), and certain antibiotics such as chlortetracycline and oxytetracycline. 

The most serious problem encountered with the assay system is a quantitative variability that is, a particular set of extracts assayed under identical conditions in separate assays may give figures which are quantitatively different. The reason for this is unknown. The relative cyclic AMP activity in the extracts is the same that is, when sample B contains twice as much cyclic AMP as sample A on day 1, it will also contain twice as much on day 2. However, the absolute values of cyclic AMP may vary. 

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