aThe rules are in fact more detailed: Highest atomic number > highest atomic mass > cis- prior to trans- > like pairs (RR) or (SS) prior to unlike > lone pairs are considered an atom of atomic number 0
Quantitative methods must have good precision (reproducibility) and accuracy (the results must reflect the true concentration of the analyte). Selectivity (freedom from interference, specificity) is important when a single species is to be measured, but broad specificity may be useful when screening for the presence of a particular class of compounds as discussed above. The recovery of the analyte, i.e. the proportion of the compound of interest that is recovered from the sample matrix during an extraction, for example, is important if sensitivity is limiting, but need not be an issue if the LLoD, accuracy, and precision of the assay are acceptable.
Table 1.5 Some terms used in stereochemistry
| Absolute stereochemistry | The absolute spatial configuration of atoms of a molecule |
| Chiral | Hand-like, i.e. left- and right-handed mirror images |
| Enantiomer | One mirror image from of a pair of non-superimposable optically active compounds |
| Epimers | Optically active molecules with more than two chiral centres differing at only one chiral centre |
| Epimerization | Partial racemization of one chiral centre in a molecule with two or more chiral centres |
| Diastereomers | Stereoisomers that are not mirror images of each other |
| Inversion | Conversion of one enantiomer to the other |
| Meso | Optically inactive isomer in which the activity of chiral centres are balanced |
| Racemate | Equimolar mixture of both enantiomers of an optically active compound |
| Racemization | Conversion of a single enantiomer to a racemate |
Ideally, whatever the methodology employed, quantitative assay calibration should be by analysis of standard solutions of each analyte (normally 6–8 concentrations across the calibration range) prepared in the same matrix and analyzed as a batch along with the test samples. A graph of response against analyte concentration should be prepared and used to calculate the analyte concentration in the sample (so-called ‘external standard’ method). Use of a full calibration sequence may not be possible in emergency toxicology, for example, and in such circumstances single-point calibration is often acceptable if properly validated (Section 3.2.4.10).
Any quantitative analysis is a measurement and, in common with all measurements, has associated errors, both random and systematic. In chromatographic and other separation methods the ‘internal standard’ method is often used to reduce the impact of systematic errors such as variations in injection volume or evaporation of extraction solvent during the analysis. Thus, a known amount of a second compound that behaves similarly to the analyte during the analysis, but elutes at a different place on the chromatogram or is otherwise detected independently of the analyte (the internal standard, ISTD) is added at an appropriate stage in the analysis. Subsequently, the detector response of the analyte relative to the response of the ISTD is plotted against analyte concentration when constructing a calibration graph (Section 3.3). In the case of MS methods, stable isotope-labelled analogues of the analyte should be used if available. The use of a marketed medicament should be avoided otherwise a significant bias may result if the patient has taken this drug.
1.3.4 Quality control and quality assessment
Once an analytical method has been validated and implemented it is important to be able to show that the method continues to perform as intended. In qualitative work, known positive and negative specimens should normally be analyzed in the same analytical sequence as the test sample. A negative control (‘blank’) helps to ensure that false positives (owing to, for example, contaminated reagents or glassware, or carry-over from a previous analysis) are not obtained. Equally, inclusion of a true positive serves to check that any reagents have been prepared properly and have remained stable.
Table 1.6 Terms used when reporting method validation
| Term | Notes |
| Accuracy | The difference between the measured value and the accepted (‘true’) value |
| Calibration range | The range of concentrations between the highest and lowest calibration standards. This should encompass the range of concentrations found in the test samples |
| Carry over | Signal from the last sample analysis enhancing response in the next analysis |
| Carry under | Signal from the last sample analysis reducing response in the next analysis |
| Coefficient of variation (CV) | An alternative term for relative standard deviation (RSD) |
| ‘Cut-off’ | An analyte concentration above or below which positive results are not reported (see Section 18.1.2) |
| Higher or upper limit of quantification (HLoQ/ULoQ) | The highest concentration that can be quantified. Not always quoted, but important in assays with a clear upper limit to a calibration graph, for example immunoassays and fluorescence assays |
| Internal quality control (IQC) | A sample of known analyte concentration that is analyzed as if it were a real sample |
| Internal standard (ISTD) |
A second compound, not
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