Quantification of drugs and other toxins
Calibration methods
Choice of an appropriate calibration method is critical to obtaining reliable results. Single-point calibrations are generally unacceptable for post mortem toxicology, unless it can be shown that the calibration is stable, reproducible and linear over the desired range, and that appropriate controls are used to validate the calibrations when specimens are being analysed. Multipoint calibrations are preferred, unless the calibration is known to be very stable and linear (e.g. GC FID headspace ethanol analysis). A minimum of three calibration points is usually recommended, although five or more are preferred, depending on the linearity and precision of the method. An appropriate internal standard is considered almost essential for all GC- or HPLC-based methods to help minimise matrix effects and also to correct for other variables, such as slight differences in transfer volumes when using liquid–liquid extraction or SPE. An internal stand ard should ideally be similar in chemical structure to the target analyte (e.g. an alkyl analogue). Use of stable isotope analogues (e.g. deuterated) is preferred, where available, because these analogues are virtually identical chemically to their non-deuterated counterparts and hence behave in the same way through sample extraction and analysis. They can only be used where the detection method can differentiate between the deuterated and non-deuterated analogues, meaning that their use is generally linked with MS detection. A good internal standard can provide much greater accuracy and precision than would otherwise be possible with difficult postmortem specimens. Quantitative results are only acceptable if the analyte concentration lies within the validated calibration range (Fig. 1). If the concentration lies outside that range, it may be reported as greater than or less than the calibration range, as appropriate, or additional calibrators or controls could be run to validate the calibration in that range. Standard calibration lines should not be extrapolated (Fig. 7.4) and if an accurate result is required when the initial quantification lies outside the scope of the standard curve, the specimen should be diluted and re-analysed.
Figure 1 Illustration of a calibration line showing values for samples within and outside values for calibrators. Value B lies within the range of the calibrators and can be considered acceptable. Values for A and C are outside the range. Further calibrators should be prepared covering the range for samples A and C. In the case of sample A, it may be possible to dilute the sample and reanalyse using the same set of calibrators if quantification uses peak areas (if peak area ratios are used, then the ratio will not change even if the sample is diluted). Extrapolation of the line to include value A is not correct as the accuracy of the result cannot be guaranteed, although it may be permissible in some circumstances.
Ideally, an assay calibration should be linear and produce a good correlation coefficient (e.g. better than 0.98). Not infrequently the calibra tion line will be nonlinear and may require a quadratic fit. Some GC or HPLC assays are inher ently nonlinear. For example, at very high or low concentrations, ions common to both the analyte and internal standards may cause a devi ation from linearity. A large deviation from linearity usually indicates the use of an inappropriate internal standard, poor chromatography, poor analyte recovery during the extraction or that the dynamic range of the detector has been exceeded. The method of standard addition can be useful in quantitative postmortem analysis. In essence, the specimen being analysed is used as the matrix to prepare calibrators. Preparing standards in solvents is rarely acceptable in toxicological analysis because it does not take into account losses which will occur during extraction processes. Hence the norm is to spike a known mass of the target analyte into a portion of the same matrix as is being analysed. For post mortem toxicology, blood, liver, etc. sampled from a body which may have been recovered some time after death may be in a rather different condition from fresh blood and hence the analyte may behave differently in terms of extraction of the specimen compared with its behaviour in the matrix used for the standards, potentially giving rise to an inaccurate result. Also, interferences may be present in the specimen that are not present in the matrix used for standards, again potentially giving rise to errors. Because the method of standard addition utilises the specimen being analysed, it avoids these difficulties. Multiple calibrators are prepared by adding known amounts of the analyte to tubes that contain the target specimen. An internal standard should be used; if possible, these should be deuterated analogues in MS techniques. The calibration line can be back-extrapolated to the x-axis and the concentration determined (see Fig. 2). This method can be used with some success for difficult matrices. Ideally, another person in the laboratory should also independently prepare a control (in the same sample matrix).
Figure 2 Illustration of the graph arising from the stand ard addition method. The line of best fit, including the value for the sample with no calibrator added, is back extrapolated to the x-axis to determine the concentration of analyte in the sample.
