María Jesús Tabernero

Determination of ketamine and amphetamines in hair by LC/MS/MS

A liquid chromatography-tandem mass spectrometry method was developed for the determination of ketamine (with its metabolite norketamine) and some amphetamines (amphetamine, methamphetamine, methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine). This method was developed to determine these compounds in hair and is able to simultaneously quantify all of them in human hair. Hair samples (20xa0mg) were washed and pulverized, and an extraction with formic acid (0.01%) and ultrasonication for 4xa0h was used. Deuterated analogs of the analytes were used as internal standards for quantification. Linearity from 0.5 to 25xa0ng/mg was obtained for both ketamine (and norketamine) and amphetamines with correlation coefficients exceeding 0.99. The limit of detection and the limit of quantification obtained were 0.1 and 0.5xa0ng/mg, respectively, for ketamine and amphetamines. A total of 25 hair samples from known drug abusers (relating to designer drug consumption or consumption of amphetamines) were examined by this validated method. The results show that the proposed method is suitable for testing these drugs in a single sample of hair. In addition, it is simpler and faster than analysis by conventional methods such as gas chromatography-mass spectrometry, which usually require a more laborious extraction procedure and, in most of cases, an additional derivatization process.

Analysis of ethyl glucuronide in hair samples by liquid chromatography‐electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS)

Many different biomarkers can be used to evaluate ethanol intake. Ethyl glucuronide (EtG) is a direct phase II and minor metabolite of ethanol formed through the UDP‐glucuronosyl transferase‐catalyzed conjugation of ethanol with glucuronic acid. Its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. A sensitive LC‐MS/MS procedure has been developed and fully validated according to the guidelines of forensic toxicology for the analysis of EtG in hair. Sample preparation and chromatographic separation were thoroughly optimized. The analysis was performed in the multiple reaction monitoring mode using the transitions m/z 221u2009→u2009203 (for the quantification) and 221u2009→u200985 or 75 (for the qualification) for EtG, and m/z 226u2009→u2009208 (for quantification) and 226u2009→u200975 or 85 (for qualification) for EtG‐D5, used as the internal standard. Analyses were carried out using an Inertsil ODS‐3 column (100u2009×u20093u2009mm i.d., 3u2009µm particle size) and a mobile phase composed of formic acid and acetonitrile. Various SPE cartridges and solvents were tested in order to obtain the highest recoveries and cleanest extracts. The assay linearity of EtG was confirmed over the range from 20 to 2500u2009pgu2009mg−1, with a coefficient of determination (R2) above 0.99. The lower limit of quantitation (LLOQ) was 20u2009pgu2009mg−1 and the limit of detection was 10u2009pgu2009mg−1. Intra‐ and inter‐day assays were less than 15% except at the LLOQ (20%). The analytical method was applied to 72 post‐mortem hair samples. EtG concentration in the hair ranged from 0 to 653u2009pgu2009mg−1 hair. Copyright

Hair testing for cocaine and metabolites by GC/MS: criteria to quantitatively assess cocaine use

A simple, rapid and sensitive method has been developed and validated for the determination of cocaine and its main metabolites (benzoylecgonine and cocaethylene) in human hair. The method involved solid‐phase extraction with an Oasis HLB extraction cartridge and subsequent analysis by GC/MS. The limit of detection was 0.01u2009ngu2009mg−1 for cocaine, 0.04 for benzoylecgonine and 0.03 for cocaethylene. The method validation included linearity (with a correlation coefficient >0.99 over the range 0.2–50u2009ngu2009mg−1), intra‐ and inter‐day precision (always lower than 12%) and accuracy (mean relative error always below 17%) to meet the bioanalytical acceptance criteria. The procedure was further applied to 40 hair samples from self‐reported cocaine users arrested by the police who provided a positive urine‐analysis for cocaine, and was demonstrated to be suitable for its application in forensic toxicology. New approaches were raised to detect false‐negative results that allow a better interpretation of hair testing results. Copyright

Determination of Cannabinoids in Human Hair by GC/MS

Abstract An analytical method for the determination of the three most important components of cannabis [viz., Δ9 tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN)] and the main metabolite of THC (11‐nor‐Δ9 tetrahydrocannabinol‐9‐carboxylic acid [THC‐COOH]) in human hair, using gas chromatography/mass spectrometry (GC/MS), is proposed. Hair samples were washed and hydrolyzed with 2.5 M NaOH to complete dissolution. Extraction was done with 9:1 (v/v) n‐hexane/ethyl acetate and separated basic and neutral cannabinoids (viz. CBD, THC, and CBN) first, and acid cannabinoids (THC‐COOH) after addition of 6 N HCl to pH 2–3. The first extract was analyzed using THC‐d3 as internal standard. The second extract was derivatized with bistrimethylsilyltrifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) and analyzed using THC‐COOH‐d3 as internal standard. Calibration curves run over the concentration range 0.1–5.0 ng/mg for each compound exhibited good correlation coefficients and excellent sensitivity. The coefficients of variation were less than 8% and the relative errors laid in the range ±7%. Thirty‐five hair samples were studied, obtaining concentrations in the ranges 0.01–15.26 ng/mg for CBD, 0.04–0.29 ng/mg for THC, 0.03–6.66 ng/mg for CBN, and 0.05–0.80 ng/mg for THC‐COOH.

Influence of concomitant drugs on methadone elimination half‐life in patients under a maintenance treatment

Methadone elimination half‐life was determined in 18 patients under maintenance treatments and found to range from 2.05 to 49.6 h. A study of potential correlations between this parameter and the presence of concomitant drugs, inhibitors or inducers of cytochrome P450, revealed an increase or decrease of methadone elimination half‐life, respectively. Twenty‐four‐hour urinary excretion of methadone was determined in three patients, who were found to release 22.3–49.8% of the dose taken. The amount excreted varied with the chemical form (unaltered drug or its main metabolite) in the three cases. No statistically significant correlation was found between urine pH and the elimination half‐life (p < 0.50).

Use of High Performance Liquid Chromatography for the Determination of Cocaine and Benzoylecgonine in Human Hair

Abstract A high performance liquid chromatographic method involving diode array detection (HPLC–DAD) is proposed for the determination of cocaine and benzoylecgonine in human hair. Samples were washed, dried, and subjected to enzyme hydrolysis and liquid–liquid extraction in Toxitubes A® prior to analysis on a Nova‐Pak C18 (150 × 3.9 mm) column with a 70:30 (v/v) methanol–0.02 M phosphate buffer pH 7, at a constant flow‐rate of 0.4 mL/min as the mobile phase, and articaine as reference compound. The detector response was linear over the concentration range 2–200 ng/mg hair for both drugs. The detection and quantitation limits were 1 ng/mg and 2 ng/mg, respectively. The precision was quite acceptable, with coefficients of variation less than 5%. An overall 52 specimens from cocaine users were analysed with the proposed method and found to contain cocaine and benzoylecgonine concentrations over the ranges 4.2–554.7 ng/mg (average 57.3 ng/mg) and 0–1167.6 ng/mg (average 159.7 ng/mg), respectively.

Rapid determination of quetiapine in blood by gas chromatography–mass spectrometry. Application to post-mortem cases

A simple, fast and sensitive method for the determination of quetiapine in human blood has been developed and validated. The method involved a basic liquid–liquid extraction procedure and subsequent analysis by gas chromatography–mass spectrometry, previous derivatization with bis(trimethylsilyl)‐trifluoro‐acetamide and chorotrimethylsilane (99 : 1). The methods of validation included linearity with a correlation coefficientu2009>u20090.99 over the range 0.02–1u2009µgu2009ml–1, intra‐ and interday precision (alwaysu2009<u200912%) and accuracy (mean relative error alwaysu2009<u200912%) to meet the bioanalytical acceptance criteria. The limit of detection was 0.005u2009µgu2009ml–1. The procedure was further applied to post mortems from the Institute of Legal Medicine, University of Santiago de Compostela. Copyright

Analysis of opiates and cocaine by RIA and GC‐MS: distribution of their metabolites in urine and hair from drug addicts

Two analytical techniques (RIA and GC‐MS) were used for the simultaneous identification and determination of heroin, cocaine and their metabolites in the urine and hair of 200 drug addicts. Opiates tests were positive in 182 hair samples and 145 urine samples, whereas cocaine tests were positive in 173 hair samples and in 63 urine samples. Drug content of hair, as determined by RIA, varied over the ranges of 0–30 ng/mg (opiates) and 0–924 ng/mg (cocaine). Metabolite distribution was studied by GC‐MS in samples taken from 50 individuals. Tests revealed 6‐monoacetylmorphine to be ubiquitous in hair and morphine to be the major component in urine. Cocaine was found to invariably occur at higher concentrations than its metabolites in hair with the opposite result in urine.

GC-MS determination of amphetamines in human urine

Abstract An analytical method for the simultaneous determination of amphetamine (AP), methamphetamine (MA), 3,4‐methylenedioxyamphetamine (MDA), 3,4‐methylenedioxy‐methamphetamine (MDMA), and 3,4‐methylenedioxy‐ethamphetamine (MDEA) in urine, using gas chromatography–mass spectrometry (GC–MS), is proposed. The analytes were subjected to liquid–liquid extraction with 4∶1 (v/v) methylene chloride/isopropanol at pH 12, and derivatized with pentafluoropropionic anhydride (PFPA) and ethyl acetate. Calibration curves for the five analytes in methanol and urine were constructed over the range 0.5–5.0 µg·ml−1, using their respective pentadeuterated analogues as internal standards. The average extraction recoveries from the urine were higher than 91% for the five amphetamines. The limits of detection and quantitation achieved were 2.02 and 6.74 ng·ml−1 for AP; 0.56 and 1.88 ng·ml−1 for MA; 2.74 and 9.13 ng·ml−1 for MDA; 2.95 and 9.85 ng·ml−1 for MDMA; and 5.41 and 18.03 ng·ml−1 for MDEA. The accuracy was in the range ±4% and the coefficients of variation were less than 9% for all analytes. The analysis of real urine samples with the proposed method provided 24 positives for amphetamines, with the following average concentrations: 0.56 µg·ml−1 for AP, 6.78 µg·ml−1 for MA, 0.90 µg·ml−1 for MDA, 8.78 µg·ml−1 for MDMA, and 1.13 µg·ml−1 for MDEA.

Bile Analysis for Cocaine and Benzoylecgonine in Overdose Cases

Abstract A method for determining cocaine and benzoylecgonine (BEG) in human bile using high performance liquid chromatography (HPLC) with ultraviolet detection at 235 nm is proposed. The method uses a Lichrospher RP18 column and methanol-phosphate buffer as mobile phase. Following solid phase extraction with Bond Elut Certify cartridges, the linearity of the method was examined over the analyte concentration range 0.125–5 µg/mL in bile. The precision of the method was acceptable, with coefficients of variation less than 5%. The average extraction yield was 82% for cocaine and 76% for BEG. The proposed method was applied to 30 bile samples from individuals fatally poisoned with cocaine.