Detection of metabolites of lysergic acid diethylamide (LSD) in human urine specimens: 2-oxo-3-hydroxy-LSD, a prevalent metabolite of LSD.

Seventy-four urine specimens previously found to contain lysergic acid diethylamide (LSD) by gas chromatography-mass spectrometry (GC-MS) were analyzed by a new procedure for the LSD metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) using a Finnigan LC-MS-MS system. This procedure proved to be less complex, shorter to perform and provides cleaner chromatographic characteristics than the method currently utilized by the Navy Drug Screening Laboratories for the extraction of LSD from urine by GC-MS. All of the specimens used in the study screened positive for LSD by radioimmunoassay (Roche Abuscreen). Analysis by GC-MS revealed detectable amounts of LSD in all of the specimens. In addition, isolysergic diethylamide (iso-LSD), a byproduct of LSD synthesis, was quantitated in 64 of the specimens. Utilizing the new LC-MS-MS method, low levels of N-desmethyl-LSD (nor-LSD), another identified LSD metabolite, were detected in some of the specimens. However, all 74 specimens contained O-H-LSD at significantly higher concentrations than LSD, iso-LSD, or nor-LSD alone. The O-H-LSD concentration ranged from 732 to 112 831 pg/ml (mean, 16340 pg/ml) by quantification with an internal standard. The ratio of O-H-LSD to LSD ranged from 1.1 to 778.1 (mean, 42.9). The presence of O-H-LSD at substantially higher concentrations than LSD suggests that the analysis for O-H-LSD as the target analyte by employing LC-MS-MS will provide a much longer window of detection for the use of LSD than the analysis of the parent compound, LSD.

Urine concentrations of ecgonine from specimens with low benzoylecgonine levels using a new ecgonine assay.

A new approach to detecting drug positives for cocaine in urine having benzoylecgonine concentrations below the Department of Defense (DoD) cutoffs was examined by measuring the concentrations of the metabolite ecgonine. The DoD cutoff concentrations for determining a positive for cocaine are 150 and 100 ng/mL for radioimmunoassay and gas chromatography-mass spectrometry, respectively. To facilitate this approach, a new assay was developed for ecgonine using only 1 mL urine. The urine was passed through an anion-exchange cartridge, and the eluant was evaporated to dryness in a water bath under nitrogen. The residue was subjected to nonylation and a standard back extraction procedure before a second derivatization with propionic anhydride. A total of 139 urine specimens were analyzed in this manner, and 104 yielded ecgonine concentrations greater than 50 ng/mL. The average ecgonine concentration in the latter specimens was approximately 5 times the comparable benzoylecgonine concentration. By monitoring ecgonine alone or in conjunction with benzoylecgonine, the number of cocaine positives detected in urine could be dramatically increased.

Detection of a GC/MS artifact peak as methamphetamine.

Recently, certain laboratories reported the presence of methamphetamine in several negative urine specimens. We have demonstrated an artifact peak, by SIM GC/MS, in negative urine specimens, that frequently matches the retention time and identity ion ratio criteria for methamphetamine. This peak resulted from the presence of high levels of pseudoephedrine (PS) or ephedrine (EP) and was produced after derivatization with 4-carbethoxyhexafluorobutyryl chloride (CB), heptafluorobutyric anhydride (HFB), and N-trifluoroacetyl-l-prolyl chloride (TFAP). Use of N-methyl trideuterated PS and two deuterated EP compounds in spiked samples provided additional proof that PS and EP can produce the artifact peak. The above results were achieved using a GC injection port temperature of 300 degrees C and low split and isothermal conditions. Thermal transformation in the injector is involved because lowering and raising the injector temperature results in the disappearance and subsequent reappearance of the artifact peak. Proof that the artifact peak is actually methamphetamine was provided by full-scan GC/MS data of the artifact peak produced after use of all three derivatizing reagents. GC/MS analysis has demonstrated that urine (65 specimens) with concentrations of PS or EP ranging from 100,000 to 1,100,000 ng/mL will test negative for methamphetamine, while the same specimens will trigger positive results by radioimmunoassay and the Emit monoclonal assay. Full-scan GC/MS of the CB derivative for PS and EP reveals what appears to be both mono- and diderivatized products. SIM analysis can differentiate between PS and EP in urine specimens because the relative amounts of mono- and diderivatized products are very different for PS than for EP.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrospective analysis of some L-methamphetamine/L-amphetamine urine data.

A limited analysis is presented of data accumulated over about three years for specimens containing either nonracemic L-methamphetamine/L-amphetamine or racemic mixtures of the D and L stereoisomers. 55 data points for both nonracemic L-methamphetamine and racemic mixtures show that it is possible to report a specimen containing L-methamphetamine positive for illegal D-methamphetamine based on current guidelines unless chiral assays are performed on selected methamphetamine positives. Generation of only 45 racemic specimens from chiral analysis of about 5,000 urine methamphetamine positives demonstrates the overwhelming prevalence of nonracemic illegal D-methamphetamine in Southern California.

Quantitation of methamphetamine and amphetamine in urine by capillary GC/MS Part II. Derivatization with 4-carbethoxyhexafluorobutyryl chloride.

An analytical procedure for methamphetamine and amphetamine has been developed using 4-carbethoxyhexafluorobutyryl chloride. This derivatizing reagent has all the previously reported advantages of trichloroacetic anhydride (TCAA) in that it produces stable derivatives having both a higher mass fragmentation pattern and lesser volatility than derivatives of trifluoroacetic anhydride (TFAA) or heptafluorobutyric anhydride (HFBA). In addition, the assay uses deuterated internal standards for both methamphetamine and amphetamine. The procedure employs a liquid-liquid extraction and back extraction followed by acylation of the amines to their respective 4-carbethoxyhexafluorobutyramides. Excess derivatizing reagent is converted to the diethyl ester with anhydrous ethanol. At a column temperature of approximately 180 degrees C, the retention times for amphetamine and methamphetamine are 4 min and 5.5 min respectively. The monitored positive ions are: 248, 266, 294 (amphetamine); 270, 298 (amphetamine-d6); 262, 280, 308 (methamphetamine); 287, 315 (methamphetamine-d9). As a result of this high mass fragmentation pattern, which eliminates the 91 and 118 ions as criteria for both identification and quantitation, potential interferences from phentermine and phenethylamine are totally eliminated.

Quantitation of methamphetamine and amphetamine in urine by capillary GC/MS. Part I. Advantages of trichloroacetyl derivatization.

A urine assay for methamphetamine and amphetamine that is compatible with our existing high-volume GC/MS assays for other drugs of abuse has been developed. Trichloroacetic anhydride is used for derivatization and its derivative is substantially less volatile than other commonly used derivatives. The internal standard is the primary amine 2-methylphenethylamine. The procedure utilizes an initial liquid-liquid extraction, a liquid-liquid back extraction for specimen cleanup, and derivatization for removal of excess trichloroacetic anhydride and acid by-product. A GC temperature of about 180 degrees C results in retention times of approximately 2.8, 3.2, and 4.3 min for amphetamine, the internal standard, and methamphetamine, respectively. Five ions are monitored: 91+, 118+, 188+ for amphetamine; 105+, 118+ for 2-methylphenethylamine; and 91+, 118+, 202+ for methamphetamine. Full scan GC/MS data from a variety of other derivatives are examined and used to illustrate the advantages of derivatizing molecules with strongly electronegative atoms near the reaction site. This situation forces fragmentation patterns in which positive charges are located on larger and structurally acceptable identifying mass fragments of the original methamphetamine or amphetamine molecule.