Spectrometric-based assay methods

However, these titration methods can be used in early development when a reference standard is not available. Also, the spectrometric-based assay methods such as ultraviolet (UV) may be nonspecific because most of the drug substance impurities contain a similar chromophore as the parent molecule. If UV is used, UV absorption is measured at one or more wavelengths and the absorbance value is recorded for a particular concentration. Sandor Gorog has critically evaluated the difference between specific and nonspecific assay methods in the European and US Pharmacopoeias [23]. The difference between the mean and the accepted true value with a defined confidence interval should be reported in the acceptance criteria. 

Solubility screens using LC/MS detection do not require an ultra-pure sample of the test compound due to the selective detection of the mass spectrometer. Mass spectrometric detection offers high selectivity and low detection limits, which eliminates the need to develop complex chromatographic methods. The LC/MS-based solubility screen surpasses the traditional HPLC/UV-based equilibrium solubility assay with increased throughput, minimal manual intervention, and high sensitivity and selectivity. 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

A problem which arises after cleavage not only from polystyrene-based polymeric carriers is the presence of impurities derived presumably from linker or resin components [57]. The occurrence of these impurities hampers routine product analysis and may have considerable influence also on the screening assay. In the mass spectrometric analysis, they can compete with poorly ionizable products, thus inhibiting a reliable analysis. At worst, they may even preclude detection of the expected compound, leading to a misinterpretation of the synthesis results. It has been found that the extraneous signals in the mass spectra are primarily dependent upon the type of resin used. Differences between analogous resins from different suppliers, or even different charges are also observed. As yet, it has not been possible to characterize these impurities with the methods of mass spectrometry and NMR. 

Sample Preparation Because large amounts of proteins are present in biological samples (except urine), conventional HPLC columns will not tolerate the direct introduction of these samples for quantitative analysis. Most bioanalytical assays have a sample preparation step to remove the bulk proteins from the samples [2], In addition, there are other important reasons for a sample preparation step when developing LC-MS/MS methods. These include the reduction of matrix components from the samples and minimization of ion suppression (also called matrix effects ) in the mass spectrometric detection [18]. Once a bioanalytical method has been developed, the method performance must remain consistent over the duration of the study. The results generated based on a validated method procedure should be free from systematic error and any other characterized errors and meet the predefined acceptance criteria. Sample preparation is used to... 

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