Ray H. Liu

Detection of Cocaine Metabolite in Perspiration Stain, Menstrual Bloodstain, and Hair

Low nanogram and picogram quantities of cocaine metabolite equivalents were detected in extracts from perspiration stains, menstrual bloodstains, and hair using radioimmunoassay. The theory of drug inclusion in hair and its significance are discussed.

Simultaneous analysis of amphetamine, methamphetamine, and 3, 4-methylenedioxymethamphetamine (MDMA) in urine samples by solid-phase extraction, derivatization, and gas chromatography/mass spectrometry

A rapid and effective solid-phase extraction procedure using Bond Elute Certify bonded silica sorbent cartridges was adopted to extract amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) from urine samples. The extract was derivatized with trichloroacetic anhydride prior to gas chromatography/mass spectrometry (GC/MS) analysis with selected ion monitoring of the following ions: 190, 91, 188; 204, 91, 202; 162, 135, 202; 194, 123; and 211, 209 for the derivatized amphetamine, methamphetamine, MDMA, d5-amphetamine, and d9-methamphetamine, respectively. The first of the ions listed for each compound was used for quantitation. The compound d5-amphetamine was used as the internal standard for amphetamine, and d9-methamphetamine was used for methamphetamine and MDMA. Results showed a higher than 65% recovery and a reproducibility with less than a 5% coefficient of variation. When a sample size of 2 mL was used, the lowest detectable concentration was about 50 ng/mL, and a near-perfect fit can be obtained (within the 250 to 4000-ng/mL concentration range studied) using a second-order polynomial model.

Improved Gas Chromatography/Mass Spectrometry Analysis of Barbiturates in Urine Using Centrifuge-Based Solid-Phase Extraction, Methylation, with d5-Pentobarbital as Internal Standard

Effective solid-phase extraction, derivatization, and GC/MS procedures are developed for the simultaneous determinations of butalbital, amobarbital, pentobarbital, and secobarbital, using a deuterated pentobarbital (d5-pentobarbital) as the internal standard. Buffered (pH 7) urine samples were extracted with Bond Elute Certify II cartridge. Iodomethane/tetramethylammonium hydroxide in dimethylsulfoxide was used for methylation, while a HP 5970 MSD equipped with a 13 m J & W DB-5 column (5% phenyl polysiloxane phase) and the Thru-Put Target software package were used for GC/MS analysis and data processing. This protocol was found to be superior, in both chromatographic performance characteristics and quantitation results, over a liquid-liquid extraction procedure without derivatization using hexobarbital as the internal standard. Extraction recoveries observed from control samples containing four barbiturates range from 80% to 90%. Good one-point calibration data are obtained for all four barbiturates in the 50 to 3200 ng/mL range. Interestingly, the one-point calibration data for pentobarbital are inferior to the other three barbiturates--due to interference from the internal standard (d5-pentobarbital). The calibration data of pentobarbital are best described by a hyperbolic curve regression model. Precision data (% CV) for GC/MS analysis, over-all procedure, and day-to-day performance are approximately 2.0%, 6.0%, and 8.0%, respectively. With the use of a 2 mL sample size, the attainable detection limit is approximately 20 ng/mL.

Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse--pentobarbital example.

Internal standards are commonly used for the quantitative determination of drugs of abuse and their metabolites (drug/metabolite) in biological fluids and tissues by the selective ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS) procedure. Analogs of drugs/metabolites that are labeled with three or more deuterium atoms (isotopic analog) at appropriate positions are considered to be the most effective internal standards for these applications. Before a specific deuterated analog can be adopted as an internal standard in a GC/MS assay, the mass spectrum of the compound or its derivative must be evaluated along with the corresponding spectrum from the parent drug/metabolite. There should be an adequate number of sufficiently high-mass ions (typically three for the drug/metabolite and two for the isotopic analog) that can be attributed to each analyte, and these ions should be sufficiently free of interference from the other analyte of the pair (cross-contribution). Interferences may be caused by the presence of an isotopic impurity in the deuterated analog (extrinsic factor) or may be due to the ion fragmentation characteristics of the compound (intrinsic factor). The extrinsic factor may be corrected by the manufacturer with different synthetic methods and purification procedures, while the intrinsic factor may be partially or wholly corrected through the use of different chemical derivatives (sample preparation stage) or different ionization (GC/MS assay stage) procedures. In this study, pentobarbital/d5-pentobarbital is used as the exemplar analyte/deuterated analog pair to illustrate the ion selection and evaluation procedures. Full-scan mass spectra were employed for preliminary ion selection. SIM data were then used to calculate the extent, if any, of cross-contributions.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectra interference between diquat and paraquat by second derivative spectrophotometry

A rapid and accurate method, combining solid-phase extraction and second-order derivative spectrophotomety approaches, is developed for the simultaneous determination of diquat (DQ) and paraquat (PQ) in blood, tissue and urine samples. Supernatant resulting from the precipitation of protein (with trichloroacetic acid) in plasma and tissue or Amberlite IRA-401 resin treated urine are passed through a mini-column packed with Wakogel gel (Silica gel). Analytes are then eluted with a non-organic solvent, 0.2mol/l HCl solution containing 2mol/l NH(4)Cl. UV spectrum of the eluent in 220-350nm range provides effective screen to detect the presence of DQ and/or PQ. In the presence of DQ or PQ alone, the analyte present is quantitated by conventional zero- or second-order derivative spectrophotometry. The calibration curve in the 0.1-5.0mg/l range for either analyte obeys Beers law. When both DQ and PQ are present, their concentrations are determined by the peak amplitudes of their respective second-derivative spectra after the addition of alkaline dithionite reagent. Interference is negligible when the DQ/PQ concentration ratio is within the 5.0-0.2 range. Using a 2-ml of sample size, the detection limits for DQ and PQ in plasma are 0.02 and 0.005mg/l. The corresponding detection limits for urine samples (10ml sample size) are 0.004 and 0.001mg/l. Recoveries of DQ and PQ in triplicate plasma and urine samples spiked with 0.5mg/l of analytes are 93 and 85%. The precision of the proposed method resulting from triplicate study of spiked urine samples varies from 3.2 to 4.6% at 0.5mg/l of DQ and PQ, respectively.

Enantiomeric composition of abused amine drugs: chromatographic methods of analysis and data interpretation

1. Introduction1.1. EnantiomersEnantiomersarepairsofnonsuperimposablemirror-imagecompounds,typicallyincludeone or more asymmetric (chiral) carbons (carbon bonded to four different groups). The twoindividual components of an enantiomer-pair have identical chemical and physical proper-ties, differing only in the way they react with other chiral compounds and the direction inwhich they rotate plane-polarized light, and therefore, cannot be resolved by conventionalchromatographic methodologies.The configuration of the groups bonded to the chiral atom is designated as S or R, while

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine specimens

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.

SEPARATION OF ENANTIOMERIC EPHEDRINE AND PSEUDOEPHEDRINE-HIGH PRESSURE LIQUID CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS

Ephedrine and pseudoephedrine (ψ-ephedrine), frequently found in packaged drugs of abuse, are common over-the-counter pharmaceuticals. Present in high concentrations, these compounds have reportedly caused false identification of methamphetamine in urine specimens. Furthermore, (−)-ephedrine and (+)-ψ-ephedrine are used for manufacturing (+)-methamphetamine. Thus, knowledge on the enantiomeric compositions of these compounds may help identify their sources, providing valuable information to the investigation process. High pressure liquid chromatography (HPLC) and capillary electrophoresis (CE) methods were evaluated and compared for their application in analyzing the enantiomeric compositions of these two compounds. A chiral column (Supelcosil LC-(S) Naphthyl Urea) was found effective in resolving the resulting four components when derivatized with both of the following two chiral derivatization reagents: N-trifluoroacetyl-l-prolyl chloride (l-TPC) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC). A C18 column, although less effective, can also adequately resolve these four components for identification purposes. With CE, inclusion of 30 mM hydroxypropyl-β-cyclodextrin in 50 mM phosphate buffer (pH 2.5) was very effective in resolving all four components using an uncoated fused silica capillary without prior derivatization.

Isotopic Analogue as the Internal Standard for Quantitative Determination of Benzoylecgonine: Concerns with Isotopic Purity and Concentration Level

Empirical data is used to demonstrate the observation and quantification of benzoylecgonine in negative test samples when high concentrations of deuterated benzoylecgonine are used as the internal standard in the assay process. On the quantitative determination of true positive samples, inaccuracy introduced by the isotopic impurity of the internal standard is calculated as a function of the impurity and the concentration levels of the internal standard used.

Simultaneous Gas Chromatography/Mass Spectrometry Assay of Methadone and 2-Ethyl-1, 5-Dimethyl-3,3-Diphenylpyrrolidine (EDDP) in Urine

An efficient extraction and gas chromatography/mass spectrometry (GC/MS) procedure has been developed for the simultaneous determination of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine in urine samples. The merits of this procedure include (1) effective high-volume sample processing; (2) excellent gas chromatography characteristics; (3) high precision for quantitative methadone determination--1.0% coefficient of variation (CV) for GC/MS injection replicates and 1.2% for extraction replicates; (4) excellent linearity within the range (0 to 1200 ng/mL) studied; and (5) adequate detection limits (50 ng/mL) for most practical purposes. The detection limit for methadone may be improved 40-fold by using a different internal standard.