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In recent years, a number of new methods have been reported that make use of immobilized enzymes either on microarrays or in bioaffinity columns for high-throughput screening of compound libraries. A key question that arises in such methods is whether immobilization may alter the intrinsic catalytic and inhibition constants of the enzyme. Herein, we examine how immobilization within sol-gel-derived materials affects the catalytic constant (kcat), Michaelis constant (KM), and inhibition constant (KI) of the clinically relevant enzymes Factor Xa, dihydrofolate reductase, cyclooxygenase-2, and gamma-glutamyl transpeptidase. These enzymes were encapsulated into sol-gel-derived glasses produced from either tetraethyl orthosilicate (TEOS) or the newly developed silica precursor diglyceryl silane (DGS). It was found that the catalytic efficiency and long-term stability of all enzymes were improved upon entrapment into DGS-derived materials relative to entrapment in TEOS-based glasses, likely owing to the liberation of the biocompatible reagent glycerol from DGS. The KM values of enzymes entrapped in DGS-derived materials were typically higher than those in solution, whereas upon entrapment, kcat values were generally lowered by a factor of 1.5-7 relative to the value in solution, indicating that substrate turnover was limited by partitioning effects or diffusion through the silica matrix. Nonetheless, the apparent KI value for the entrapped enzyme was in most cases within error of the value in solution, and even in the worst case, the values differed by no more than a factor of 3. The implications of these findings for high-throughput screening are discussed.

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A sensitive and selective method was developed to quantitate allopregnanolone and its 5β isomer pregnanolone in human plasma using liquid chromatography-differential mobility separation combined with MS/MS detection. The method employed a simple liquid-liquid extraction of 100 μL plasma with hexane/ethyl acetate. After extraction, the sample was derivatized using a quaternary aminooxy reagent. Separation of allopregnanolone, pregnanolone and their 3β epimers (epiallopregnanolone and epipregnanolone) was achieved using a Phenomenex Kinetex C18 2.1×100 mm 2.6 μm column. A linear calibration curve was obtained over the concentration range from 10 pg/mL to 25,000 pg/mL and the inter- and intra-day accuracy of the QC samples were between 90-110% with the inter- and intra-day precision less than 10%. The lower limit of quantitation is 50 fg (157 amol) on column for both allopregnanolone and pregnanolone which is 100 fold less than the underivatized compounds. The recovery is above 95% and the extracted samples are stable for at least 6 days when stored at 4°C. Plasma samples from normal, pregnant and postpartum women were analyzed using this method.

A multiresidue, multiclass semiquantitative screening analysis of 39 drug residues covering 8 drug classes, including aminoglycosides in veal muscle, based on a single multiresidue extraction routine and using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), is presented. Sample preparation involves extraction of a 5 g diced tissue sample with 10 mL of acetonitrile/ water (86:14), incubated at 60 degrees C for 1 h, and then cooled for 10 min in ice. Formic acid is added to the suspension, then mixed, and centrifuged. The supernatant is retained, and the pellet is extracted with 10 mL of water for aminoglycosides and again centrifuged. Approximately 9.5 mL of each of the supernatants from both extracts is combined and diluted with water to 25 mL. The final solution is then defatted with 20 mL of hexane prior to analysis. Liquid chromatography for the aminoglycosides is carried out with ZIC-HILIC and for the remainder of the compounds with an Atlantis dC18 minicolumn. LC-ESI-MS/MS in positive and negative ionization modes (three injections total) is carried out, and two ion transitions per analyte are monitored. The method provides semiquantitative analysis to identify incurred positive drug classes in a rapid and cost-effective manner. Of particular interest is the detection of numerous compounds in the low nanograms per gram concentration range, which are not typically detected using receptor-based screening methods. All identified drugs were confirmed using internationally recognized regulatory methods, with no apparent false positives.

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