Buddha D. Paul

Validity Testing of Commercial Urine Cocaine Metabolite Assays: I. Assay Detection Times, Individual Excretion Patterns, and Kinetics After Cocaine Administration to Humans

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.

Electron ionization mass fragmentometric detection of urinary ecgonidine, a hydrolytic product of methylecgonidine, as an indicator of smoking cocaine†

When cocaine is smoked, methylecgonidine (anhydroecgonine methyl ester) is also consumed as a pyrolytic product. Methylecgonidine, on incubation with human liver homogenate, was metabolized to a stable compound, ecgonidine. The compound was also formed when methylecgonidine was exposed to a urine pH > or = 8.0. Ecgonidine is a zwitterion and highly water soluble. A method was developed to identify ecgonidine quantitatively in urine. After removal of cocaine, benzoylecgonine and methylecgonidine from urine at pH 5.5 +/- 0.5 using a solid-phase extraction (SPE) technique, the pH of the solution was readjusted to 2.0-3.0. The acidic solution reduced the dissociation of the carboxylic acid and improved the lipophilic and cationic character of ecgonidine. The compound was extracted from the solution with the SPE technique with an 89-99% yield. Ecgonidine was then detected as a tert-butyldimethylsilyl derivative by a gas chromatographic/electron ionization mass spectrometric method. Quantitation was linear over the concentration range 7-2000 ng ml-1. Concentrations as low as 7 ng ml-1 can be detected by this procedure. Ecgonidine was detected in > 95% of benzoylecgonine-positive urine specimens from a random drug testing program, indicating smoking as the major route of cocaine administration.

Gas chromatographic-electron-impact mass fragmentometric determination of lysergic acid diethylamide in urine☆

A sensitive method for the detection and quantitation of lysergic acid diethylamide (LSD) in urine was developed. After initial solvent extraction, the compound was further purified by liquid-liquid extraction or by solid-phase extraction. The trimethylsilyl derivative of LSD was detected by gas chromatography-mass spectrometry (GC-MS) operated in the electron-impact mode with selected-ion monitoring. The presence of LSD was confirmed by comparing retention times and relative abundances of ions of unknowns with that of a standard. The recovery of this procedure was greater than 89%. The intra-run and inter-run coefficients of variation were less than 5% and less than 7%, respectively. This procedure allows detection of LSD concentrations as low as 29 pg/ml. Quantitation of LSD was linear over the concentration range 50-2000 pg/ml.

Isomerization of delta‐9‐THC to delta‐8‐THC when tested as trifluoroacetyl‐, pentafluoropropionyl‐, or heptafluorobutyryl‐ derivatives

For GC-MS analysis of delta-9-tetrahydrocannabinol (delta-9-THC), perfluoroacid anhydrides in combination with perfluoroalcohols are commonly used for derivatization. This reagent mixture is preferred because it allows simultaneous derivatization of delta-9-THC and its acid metabolite, 11-nor-delta-9-THC-9-carboxylic acid present in biological samples. When delta-9-THC was derivatized by trifluoroacetic anhydride/hexafluoroisopropanol (TFAA/HFIPOH) and analyzed by GC-MS using full scan mode (50-550 amu), two peaks (P1 and P2) with an identical molecular mass of 410 amu were observed. On the basis of the total ion chromatogram (TIC), P1 with a shorter retention time (RT) was the major peak (TIC 84%). To identify the peaks, delta-8-THC was also tested under the same conditions. The RT and spectra of the major peak (TIC 95%) were identical with that of P1 for delta-9-THC. A minor peak (5%) present also correlated well with the latter peak (P2) for the delta-9-THC derivative. The fragmentation pathway of P1 was primarily demethylation followed by retro Diels-Alder fragmentation (M - 15-68, base peak 100%) indicating P1 as a delta-8-THC-trifluoroacetyl compound. This indicated that delta-9-THC isomerized to delta-8-THC during derivatization with TFAA/HFIPOH. Similar results were also observed when delta-9-THC was derivatized with pentafluoropropionic anhydride/pentafluoropropanol or heptafluorobutyric anhydride/heptafluorobutanol. No isomerization was observed when chloroform was used in derivatization with TFAA. In this reaction, the peaks of delta-8-THC-TFA and delta-9-THC-TFA had retention times and mass spectra matching with P1 and P2, respectively. Because of isomerization, perfluoroacid anhydrides/perfluoroalcohols are not suitable derivatizing agents for analysis of delta-9-THC; whereas the TFAA in chloroform is suitable for the analysis.