Measurement of benzoylecgonine in whole blood using the Abbott ADx analyzer.

An alternate procedure has been developed for the processing of whole blood for the estimation of benzoylecgonine with the use of the Abbott ADx reagents and analyzer. This procedure allows for handling of relatively large numbers of samples without the need to evaporate extraction solvent. Blood samples were diluted with an equal volume of phosphate buffer-methanol (80:20 v/v), and the proteins were removed by centrifugation through a membrane filter device. A comparison of the proposed method with an acetone solvent extraction procedure has been made, and results were shown to be equivalent. Recoveries of 94-105% benzoylecgonine were obtained for added concentrations of 25-500 ng/mL.

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.

The effects of pH and temperature on the in vitro bindings of delta-9-tetrahydrocannabinol and other cannabinoids to bovine serum albumin.

Albumin is a major carrier of drugs and fatty acids in biological fluids. These protein-drug complexes serve to solubilize, transport these compounds to sites of action, and have been associated with increased half-life for these compounds. The authors are interested in the pH and temperature effects of the binding of delta-9-tetrahydrocannabinol to albumin. Ultrafiltration techniques were used in the separation of free to bound compounds. Cannabinoids bind to bovine serum albumin rapidly. The cannabinoid binding sites are more sensitive to temperature changes (37-47 degrees C) than changes in pH with 37 degrees C and pH 7.4 resulting in optimal binding. These conditions would result in the greatest viability in the cells, while allowing for the use of a variety of compounds in in vitro studies for the administration of compounds to isolated cells and cell lines.

Confirmation of drugs of abuse in urine drug screens by direct exposure probe mass spectrometry.

A direct exposure probe (DEP) mass spectrometric method was developed to confirm the presence of cocaine (C), phencyclidine (P), benzoyl ecognine (BE) and 11-norcarboxy tetrahydrocannabinol (11-nor-c-THC) in previously screened urine specimens. Essentially, a urine sample is lyophilized overnight and reconstituted in 30 microliters of a 3:1 mixture of ethyl acetate:methanol. One microliter is placed on a rhenium filament and left to air dry. The specimen was analyzed by negative ion chemical ionization DEP using ammonia as reagent gas at 0.20 Torr with an ion source temperature of 100 degrees C. An electronics setting of 1700 V (EM), 0.30 mA filament current, and 100 eV with scanning at m/z 100-650 (1 sec/SCAN) resulted in simple spectra with easily identifiable molecular ions for C, BE, P and 11-nor-c-THC. The sensitivity of the assay was 1 ng for the drugs of abuse. The method was validated by analyzing 50 urine samples that have been previously screened and confirmed for drugs at the University of Illinois Toxicology Laboratory. The results showed that the DEP method confirmed 87%, 71%, 100%, and 85% of the BE, C, P and 11-nor-c-THC. In summary, a rapid and sensitive DEP method for the confirmation of drugs of abuse in urine has been developed which can serve as a useful adjunct to gas chromatographic/mass spectrometric confirmation.

Analysis of retinoids by direct exposure probe mass spectrometry.

Recently, our laboratory has investigated the depletion of Vitamin A and its metabolites in experimental animals. High Pressure Liquid Chromatography (HPLC) was used to measure retinal oxidase activity by monitoring the conversion of retinaldehyde (RALD) to retinoic acid (ROIC). In order to obtain more information about these compounds, a Direct Exposure Probe mass spectrometric method was developed to confirm the presence of ROIC and RALD in HPLC peaks. A rapid negative ion chemical ionization (NICI) method using ammonia as reagent gas was developed to detect the presence of ROIC and RALD with picogram sensitivity. The ROIC and RALD peaks were collected from HPLC, extracted with hexane, evaporated under nitrogen and reconstituted in ethanol before placing onto a rhenium filament with current programmed from 0-1.3 A at 50 mA/sec. The instrument employed was a Finnigan 4510 operated in the NICI mode and scanned from m/z 100-650 with a source temperature of 80 C. Other parameters were electron energy 140 eV and filament current at 0.25 mA. Prominent ions were generated at m/z 284 and m/z 300 for RALD and ROIC which were subsequently monitored in the selected ion monitoring mode. In summary, we have developed a rapid retinoid identification method (2.5 minutes) which is more sensitive (pg vs ng) than HPLC and does not require elaborate sample preparation or derivitization. This method can be used as an important adjunct to HPLC enzyme studies.