Isocratic analysis assay method

The principles of green chemistry by adopting the most efficient and environmentally friendly processes should be practiced whenever possible in the HPLC laboratory. One obvious approach is reduction of solvent consumption by using solvent recycling for isocratic analysis and narrowbore LC columns. Another area is to find ways to reduce sample size and the number of sample preparation steps without sacrificing method performance.2 A case study to illustrate this principle for a tablet assay is shown in Table 11.1. Another example is an environmental analysis of soil/sediment sample3 is shown in Table 7.7. 

In the case of amino-/3-lactarns, an additional reaction step with acetic anhydride was necessary to protect the amino groups of AMO and AMP (82). This step was also incorporated into the assay for determination of five penicillins (AMO, AMP, PenG, PenV, CLO) (83). The previously described methods were modified by replacing the acylating agent, acetic anhydride, with benzoic anhydride this permitted the elution of all derivatives under isocratic conditions within a very short period of time. This modification reduced analysis time per sample from over 1 hour to about 22 min without loss in detection sensitivity (84). 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a Cl 8 column. Bile samples do not require any sample dean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL... 

New, comprehensive methods such as the one developed by Kimura et al. will eventually render HPLC methods for the determination of PLP redundant. The latter methods had the advantage of simplicity and a high turnover potential, but yielded information only on PLP and sometimes also PL. Now it has become possible to use isocratic HPLC and obtain information on all the Bg vitamers without the inconvenience of unacceptably long analysis times. It has already been pointed out that the simultaneous analysis of PLP and PL sometimes yielded explanations for depressed PLP levels observed under certain circumstances. It is expected that a more comprehensive vitamin Bg assay, which includes at least 4-PA together with PLP and PL, will be even more informative when applied in studies of vitamin Bg metabolism in various clinical conditions. Perhaps it will be possible to explain certain observations of a low PLP status by increased formation of 4-PA Perhaps the new HPLC methods wiU enable us to find an explanation for ethnic differences in PLP levels which are not dietary induced (161) ... 

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