No abstract

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research.

The pharmacokinetic interaction between indinavir and ritonavir was evaluated in five groups of healthy adult volunteers to explore the potential for twice-daily (b.i.d.) dosing of this combination. All subjects received 800 mg of indinavir every 8 h (q8h) on day 2. In addition, subjects in group I received one dose of 800 mg of indinavir on day 1 and 800 mg of indinavir q8h on day 17. Subjects in Groups II and IV each received one dose of 600 mg of indinavir on days 1 and 17, and subjects in groups III and V each received one dose of 400 mg of indinavir on days 1 and 17. During days 3 to 17, ritonavir placebo or ritonavir at 200, 300, 300, or 400 mg q12h was given to groups I, II, III, IV, and V, respectively. Ritonavir at steady state probably inhibited the cytochrome P-450 3A metabolism of indinavir and substantially increased plasma indinavir concentrations, with the area under the plasma concentration-time curve (AUC) increasing up to 475% and the peak concentration in serum (C max) increasing up to 110%. The C max/trough concentration ratio decreased from 50 in standard q8h regimens to less than 14 when indinavir was administered with ritonavir. For a constant indinavir dose, an increase in the ritonavir dose yielded similar indinavir AUCs, C maxs, and concentrations at 12 h (C 12s). For a constant ritonavir dose, an increase in the indinavir dose resulted in approximately proportional increases in the indinavir AUC, less than proportional increases in C max, and slightly more than proportional increases inC 12. Ritonavir reduced between-subject variability in the indinavir AUC and trough concentrations and did not affect indinavir renal clearance. With the altered pharmacokinetic profile, indinavir likely could be given as a b.i.d. combination regimen with ritonavir. This could potentially improve patient compliance and thereby reduce treatment failures.

Sample preparation is a major task in a regulated bioanalytical laboratory. The sample preparation procedure significantly impacts assay throughput, data quality, analysis cost, and employee satisfaction. Therefore, selecting and optimizing an appropriate sample preparation method is essential for successful method development. Because of our recent expertise, this article is focused on sample preparation for high-performance liquid chromatography with mass spectrometric detection. Liquid chromatography with mass spectrometric detection (LC-MS) is the most common detection technique for small molecules used in regulated bioanalytical laboratories. The sample preparation technologies discussed are pre-extraction and post-extraction sample processing, protein precipitation (PPT), liquid-liquid extraction (LLE), offline solid-phase extraction (SPE), and online solid-phase extraction. Since all these techniques were in use for more than two decades, numerous applications and variations exist for each technique. We will not attempt to categorize each variation. Rather, the development history, a brief theoretical background, and selected references are presented. The strengths and the limitations of each method are discussed, including the throughput improvement potential. If available, illustrations from presentations at various meetings by our laboratory are used to clarify our opinion. Drug Dev Res 68: 2007 r2007 Wiley-Liss, Inc.

Acetonitrile, an organic solvent miscible with aqueous phase, has seen thousands of publications in the literature as an efficient deproteinization reagent. The use of acetonitrile for liquid-liquid extraction (LLE), however, has seen very limited application due to its miscibility with aqueous phase. The interest in LLE with acetonitrile has been pursued and reported in the literature by significantly lowering the temperature of the mixture or increasing the salt concentration in the mixture of acetonitrile and aqueous phase, resulting in the separation of the acetonitrile phase from aqueous phase, as observed in conventional LLE. However, very limited application of these methods has been reported. The throughput was limited. In this report, we report a new sample preparation technique, salting-out assisted liquid-liquid extraction with acetonitrile, for high-throughput good laboratory practice sample analysis using LCMS, Two compounds from an approved drug, Kaletra, were used to demonstrate the extractability of drugs from human plasma matrix. Magnesium sulfate was used as the salting-out reagent. Extracts were diluted and then injected into a reversed phase LC-MS/MS system directly. One 96-well plate was extracted with this new approach to evaluate multiple parameters of a good laboratory practice analytical method. Results indicate that the method is rapid, reliable and suitable for regulated bioanalysis. With minimal modification, this approach has been used for high-throughput good laboratory practice analysis of a number of compounds under development at Abbott.