A sensitive GC-EIMS method for simultaneous detection and quantification of JWH-018 and JWH-073 carboxylic acid and hydroxy metabolites in urine.

Synthetic cannabinoids, including JWH-018 and JWH-073, belong to a class of aminoalkylindoles (AAIs) that are smoked to produce an effect similar to tetrahydrocannabinol. Compounds in this class are often collectively known as 'Spice'. After ingestion, these compounds are extensively metabolized to their hydroxy and carboxylic acid metabolites. During forensic analysis, detection of these metabolites in urine is an indication of past exposure to the parent compounds. The analytical process involved hydrolysis of conjugated metabolites by glucuronidase, solvent extraction, derivatization by trifluoroacetic anhydride and hexafluoroisopropanol and GC-EIMS detection. Identification of the unknown was based on the criteria of GC retention time within ±2% and mass spectral ion ratio within ±20% of that of a standard. Deuterated internal standards of the carboxylic acid metabolites were used for quantification. The acid (JWH-018-COOH, JWH-073-COOH) and hydroxy (JWH-018-OH, JWH-073-OH) metabolites were linear over the concentration range of 0.1-10 and 0.2-10 ng/mL, respectively, with a correlation coefficient-square, R(2) > 0.999 (N = 5). Extraction recoveries of the metabolites were within 79 and 87%. The method was applied to 17 urine specimens collected as part of a military law enforcement investigation. Nine of the specimens tested positive for one or more of the metabolites. When the procedure was extended to screen other AAI compounds, two of the specimens were found to contain JWH-210, JWH-250 (JWH-302 or JWH-201) and JWH-250 (C4 isomers). The GC-EIMS method presented here was found to be suitable for detecting JWH-018 and JWH-073 metabolites and other AAI compounds in urine.

Urinary excretion of ecgonine and five other cocaine metabolites following controlled oral, intravenous, intranasal, and smoked administration of cocaine.

Urinary excretion of ecgonine (EC) was compared to that of cocaine, benzoylecgonine, ecgonine methyl ester and minor metabolites, meta-hydroxybenzoylecgonine, para-hydroxybenzoylecgonine, and norbenzoylecgonine, following controlled administration of oral, intravenous, intranasal, and smoked cocaine. Urine EC concentrations peaked later than all other analytes and had longer detection times than the other minor metabolites. With a 50 ng/mL cutoff concentration and following low doses of 10 to 45 mg cocaine by multiple routes, detection times extended up to 98 h. Maximum concentrations (Cmax) were 6-14 mole % of those for benzoylecgonine, Cmax increased with dose, time to maximum concentration (Tmax) was independent of dose, and route of administration did not have a significant impact on Cmax or Tmax for metabolites. EC is an analyte to consider for identifying cocaine use due to its stability in urine and long detection times.

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.

Stable isotopes as valid components for identification of drugs in biological specimens.

Selected ion monitoring in mass spectrometric methods (SIM-MS) is generally used to confirm the presence of a drug in biological samples. Criteria for identification of a compound by MS are based on some specific guidelines. However, some disparities exist between the guidelines as to how many and what type of ions to monitor. Although European guidelines allow the monitoring of isotopic ions, such monitoring is not valid by SOFT/AAFS guidelines. The feasibility of monitoring a stable isotopic ion as an alternate to the fragment ion was examined in our study. Area ratios of stable isotopic ion m/z 275 and its parent ion m/z 274 of optical isomers of methamphetamine as (R)-(-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl derivative were found to be within +/- 4% of theoretical value (14.969) calculated from fragment formula C(13)H(15)F(3)NO(2) and isotopic abundances (C(13) = 1.1%, H(2) = 0.015%, F = 0%, N(15) = 0.37%, and O(17) = 0.037%). In another example, the area ratios of a stable isotopic ion m/z 283 and the parent ion m/z 282 of 6-pentafluoropropionyl codeine was also within +/- 4% of theoretical value (20.542) calculated from fragment formula C(18)H(20)NO(2). This relationship between the isotopic abundance and fragment composition was also useful in assigning structures to many fragment ions of methamphetamine, LSD, morphine, and 6-acetylmorphine derivatives, whereas structural assignment to the ions based on mass alone was difficult. The predictability of the relative abundance of the examined isotopic ions has proven reliable in our studies. The use of an isotope was found to be an important additional tool for compound identification by MS.

Cocaine and metabolites urinary excretion after controlled smoked administration.

Understanding cocaine and metabolites urinary excretion following smoking is important for interpretation of urine test results in judicial, workplace and treatment settings. In National Institute on Drug Abuse approved studies on a secure research unit, six subjects smoked placebo, 10, 20, and 40 mg cocaine with a precise dose delivery device and six different subjects smoked 42 mg cocaine in a glass pipe. Urine specimens (n = 700) were collected for up to seven days and analyzed for cocaine (COC), benzoylecgonine (BE), ecgonine methylester (EME), m-hydroxybenzoylecgonine (mOHBE), p-hydroxybenzoylecgonine (pOHBE), norbenzoylecgonine (NBE), and ecgonine (EC) by gas chromatography-mass spectrometry. Results (mean +/- SE) for the 40-mg precise delivery doses are as follows: (Table can not be represented) Mean C(max) for all analytes linearly increased with increasing dose. T(max) was not dose-dependent. All metabolites were detected in some subjects within 2 h. EC concentrations were significantly higher after smoked cocaine in a precise delivery coil compared to a glass "crack" pipe.

Cyanide and thiocyanate in human saliva by gas chromatography-mass spectrometry.

A method is described for simultaneous determination of cyanide (CN) and thiocyanate (SCN) in human saliva, or oral fluid. SCN concentrations in body fluids appeared to be important in classifying patients as smokers or nonsmokers, in determining some clinical conditions, and in specimen validity testing in forensic drug testing. The human saliva samples were diluted and the anions were separated by an extractive alkylation technique. Tetrabutylammonium sulfate was used as phase-transfer catalyst and pentafluorobenzyl bromide as the derivatizing agent. The products were analyzed by a gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring method. 2,5-Dibromotoluene was used as internal standard for quantitation of CN and SCN in saliva. The calibration plot was linear over the concentration range from 1 to 100 micromol/L (0.026-2.60 microg/mL) for CN (R=0.9978) and 5 to 200 micromol/L (0.29-11.6 microg/mL) for SCN (R=0.9996). The method was used to examine 10 saliva specimens. The concentration ranged from 4.8 to 29 micromol/L (0.13-0.75 microg/mL) for CN and 293 to 1029 micromol/L (17-59.7 microg/mL) for SCN. The SCN results were similar to those obtained from a method using oxidation of SCN to CN with colorimetric detection (R=0.9882). The proposed GC-MS confirmatory method was found useful when the concentrations of CN and SCN in saliva needed to be accurately determined.

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry.

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.

Spectrophotometric detection of iodide and chromic (III) in urine after oxidation to iodine and chromate (VI).

Tests for oxidizing adulterants in urine are a continuing challenge to the drug-testing program. Iodine was found to destroy morphine and 6-acetylmorphine almost immediately. The effects were less evident on 11 -nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-acid). When the urine solution was tested for iodine by a chromogenic substrate, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), no iodine was detected. Masking drug and adulterant simultaneously made iodine a preferred oxidizing adulterant for drug abusers. In this study, the reduced iodide was oxidized by sodium nitrite to iodine. The excess nitrite was decomposed by sulfamic acid and the iodine was detected by ABTS. Linearity was 12.7 to 635 mg/L (0.1 to 5 mmol/L, y = 0.9966x + 0.0016, R2 = 1.0000). Precisions (coefficient of variation) were within +/- 4.1% and quantitative accuracies were within 97% of expected values (n=5). Chromate, iodate, periodate, and persulfate interfered with the method. To alleviate the problem, the positive specimens were tested again by an iodine-specific method. After oxidation, the samples were treated with sodium azide and ammonium thiocyanate. In presence of thiocyanate, the azide reduced iodine to iodide almost immediately, and the solutions showed negative response to ABTS. The results were compared with that of a control group tested without thiocyanate. When iodine was present, the ratios of thiocyanate to control were less than 6%. Chromate was also found to destroy THC-acid in urine, and during storage most of the chromate changed to chromic (III). In this study, chromic was oxidized to chromate by hydrogen peroxide and sodium hydroxide and detected by 1,5-diphenylcarbazide. Linearity was 5.2 to 156 mg/L (0.1 to 3.0 mmol/L, y = 1.0285x - 0.0034, R2 = 0.9998). Precisions were within +/- 8.5% and quantitative accuracies were within 92% of expected values (n=5). The test was not interfered by other oxidizing agents. Both iodide and chromic oxidation methods showed urine backgrounds less than 1.27 and 0.52 mg/L, respectively (< 0.01 mmol/L). It indicated that a response more than 10 times of the background could be considered as oxidant contamination or adulteration of urine specimens.

Enantiomeric separation and quantitation of (+/-)-amphetamine, (+/-)-methamphetamine, (+/-)-MDA, (+/-)-MDMA, and (+/-)-MDEA in urine specimens by GC-EI-MS after derivatization with (R)-(-)- or (S)-(+)-alpha-methoxy-alpha-(trifluoromethy)phenylacetyl chloride (MTPA).

In drug testing, the presence of methamphetamine in urine is generally confirmed by a gas chromatography-mass spectrometry (GC-MS) method. Derivatization of the compound to a perfluoroalkylamide, prior to confirmation, typically yields better chromatographic separation. Once methamphetamine is detected, a second GC-MS test is necessary to distinguish positive results from the use of over-the-counter medication, Vicks inhaler, or from use of a prescription drug, selegiline (Deprenyl). R-(-)-Methamphetamine is the urinary product from legitimate use of these medications. The second GC-MS test is to confirm illicit use of (S)-(+)-methamphetamine. In the procedure, the two methamphetamine isomers are changed to the chromatographically separable diastereomers by a chiral derivatizing agent, (S)-(-)-trifluoroacetylprolyl chloride (TPC). But the method has inherent limitations. Racemization of the reagent produces mixed diastereomers even from pure (S)-(+)-methamphetamine. Instead of using TPC, we utilized (R)-(-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride (MTPA) to prepare the amides of diastereomers of methamphetamine. No racemization was observed with this reagent. The method was extended to resolve GC peaks of (R)-(-)- and (S)-(+)-isomers of amphetamine, 3,4-methylenedioxyamphetamine (MDA), N-methyl-MDA (MDMA), and N-ethyl-MDA (MDEA). Three ions from the drug and two ions from the deuterated internal standard were monitored to characterize and quantitate the drugs. For MDEA, only one ion was used. The quantitation was linear over 25 to 5000 ng/mL for MDEA and 25 to 10,000 ng/mL for all other drugs. Correlation coefficients were > 0.996. Precision calculated as the coefficient of variation at the calibrator concentration of 500 ng/mL was within +/- 11% for all drugs. The method was applied to test 43 urine specimens. In 91% of the methamphetamine-positive specimens, only the (S)-(+)-isomer was detected. In all MDMA-positive specimens, the concentrations of (R)-(-)-isomer were greater than the (S)-(+)-isomer indicating longer retention of (R)-(-)-isomer in the human body. The specimen concentrations (R + S) compared well with that of a non-chiral method that used 4-carboethoxyhexafluorobutyryl chloride as derivatizing agent. But the MTPA method has some advantage. It alone can replace the two GC-MS methods needed to confirm the presence of (S)-(+)-isomers of amphetamine and methamphetamine.

Six spectroscopic methods for detection of oxidants in urine: implication in differentiation of normal and adulterated urine.

Six separate methods to detect oxidants in urine were developed. The presence of the oxidants was established by initial oxidation of ferrous to ferric ion and detecting the ferric by chromogenic oxidation or complex formation. The reagents for chromogenic oxidation were N,N-dimethylamoino-1,4-phenylenediamine (DMPDA), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS), and 2-amino-p-cresol (APC), and the reagents for the chromogenic complex were xylenol orange (XO), 8-hydroxy-7-iodo-5-quinolinesulfonic acid (HIQSA), and 4,5-dihydroxy-1,3-benzene-di-sulfonic acid (HBSA). All methods showed comparable results when tested for ferric, chromate, permanganate, oxychloride, hydrogen peroxide, oxone, tert-butylhydroperoxide, and cumenehydroperoxide at a concentration of 1.0 mmol/L in water (CV < 7%). The nitrite results are comparable only with DMPDA and APC. Periodate responded to the highest oxidation number (ON = 8) by chromogenic oxidation but lowest (ON = 2) by the chromogenic complex. The iodate responded only to the chromogenic oxidation with ON = 6. The linearity of the procedures was established by chromate in water. The linear concentrations were 0.09-12.00 mE/L for DMPDA, ABTS, APC, and HBSA and 0.09-6.00 mE/L for XO and HIQSA. In all methods, the correlation coefficients were > or = 0.9991 and precisions were within +/- 5.6%. The methods were used to test oxidants in 238 urine specimens. The chromate at 3.0 mE/L in water was used as standard. The correlation coefficients of 0.9600-0.9853 and the ANOVA test (F = 0.90, F(critical) = 2.22 at P(0.48)) indicated that the methods correlated well. The median concentration of oxidants in the specimens was 0.21 mE/L with an average and standard deviation of 0.62 +/- 1.19 (range 0.04-8.83 mE/L). When Grubbs' statistical test was applied to the specimen results, no specimen was found to be outlier or could be considered as adulterated. The Grubbs' test also revealed that the threshold concentration to identify urine adulteration was 29 mE/L at confidence level of 99%.

Effects of oxidizing adulterants on detection of 11-nor-delta9-THC-9-carboxylic acid in urine.

Bleach, nitrite, chromate, and hydrogen peroxide-peroxidase are effective urine adulterants used by the illicit drug users to conceal marijuana-positive results. Methods for detecting nitrite and chromate are available. Effects of other oxidizing agents that could possibly be used as adulterants and are difficult to detect or measure are presented in this report. Urine samples containing 40 ng/mL of 11-nor-delta9-THC-9-carboxylic acid (THC-acid) were treated with 10 mmol/L of commonly available oxidizing agents. Effects of horseradish peroxidase of activity 10 unit/mL and extracts from 2.5 g of red radish (Raphanus sativus, Radicula group), horseradish (Armoracia rusticana), Japanese radish (Raphanus sativus, Daikon group), and black mustard seeds (Brassica nigra), all with 10 mmol/L of hydrogen peroxide, were also examined. After 5 min, 16 h and 48 h of exposure at room temperature (23 degrees C) the specimens were tested by a gas chromatographic-mass spectrometric method for THC-acid. A control group treated with sodium hydrosulfite to reduce the oxidants, was also tested to investigate the effect of oxidizing agents on THC-acid in the extraction method. THC-acid was lost completely in the extraction method when treated with chromate, nitrite, oxone, and hydrogen peroxide/ferrous ammonium sulfate (Fenton's reagent). Some losses were also observed with persulfate and periodate (up to 25%). These oxidants, and other oxidizing agents like permanganate, periodate, peroxidase, and extracts from red radish, horseradish, Japanese radish and black mustard seeds destroyed most of the THC-acid (> 94%) within 48 h of exposure. Chlorate, perchlorate, iodate, and oxychloride under these conditions showed little or no effect. Complete loss was observed when THC-acid was exposed to 50 mmol/L of oxychloride for 48 h. Several oxidizing adulterants that are difficult to test by the present urine adulterant testing methods showed considerable effects on the destruction of THC-acid. The time and temperature for these effects were similar to those used by most laboratories to collect and test specimens. In several cases, the loss of THC-acid was > 94%.