Validation of a method to detect cocaine and its metabolites in nails by gas chromatography-mass spectrometry

The objective of the present work was to compare previously published methods and provide validation data to detect simultaneously cocaine (COC), benzoylecgonine (BE) and norcocaine (NCOC) in nail. Finger and toenail samples (5mg) were cut in very small pieces and submitted to an initial procedure for external decontamination. Methanol (3 ml) was used to release analytes from the matrix. A cleanup step was performed simultaneously by solid-phase extraction (SPE) and the residue was derivatized with pentafluoropropionic anhydride/pentafluoropropanol (PFPA/PFP). Gas chromatography-mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). Confidence parameters of validation of the method were: recovery, intra- and inter-assay precision, as well as limit of detection (LOD) of the analytes. The limits of detection were: 3.5 ng/mg for NCOC and 3.0 ng/mg for COC and BE. Good intra-assay precision was observed for all detected substances (coefficient of variation (CV)<11%). The inter-assay precision for norcocaine and benzoylecgonine were <4%. For intra- and inter-assay precision deuterated internal standards were used. Toenail and fingernail samples from eight declared cocaine users were submitted to the validated method.     

Postmortem Liver and Kidney Tissue Concentrations of Heroin Biomarkers and Their Metabolites in Heroin-Related Fatalities*†

A method was developed and validated for analyzing 6-monoacetylmorphine, morphine, 6-acetylcodeine, and codeine in routine postmortem liver and kidney specimens using liquid chromatography–tandem mass spectrometry. Samples were prepared with a Stomacher instrument followed by solid-phase extraction. All calibration curves [0.5–1000 ng/g] were linear with coefficients of determination greater than 0.99 and limits of quantification of 1.0 ng/g. Within-run precision ranged between 2.0% and 8.0%, between-run precision ranged between 1.0% and 9.0%, and accuracy ranged between −5.0% and +3.0%. Matrix effects ranged from −18% to +9%. After matrix effects were excluded, analytical recoveries ranged from 76% to 94%. The distributions of 6-monoacetylmorphine, morphine, 6-acetylcodeine, and codeine were investigated in 31 postmortem cases in which heroin was the primary cause of death. In the current study, the median free morphine ratios were calculated for liver to blood and kidney to blood, which were 2.2 and 4.0, respectively. The current report highlights the importance of testing multiple specimens, including liver and kidney, in heroin-related deaths, especially if no blood samples are available. Furthermore, this work presents new information regarding the distribution of heroin metabolites in liver and kidney.     

Development and Validation of a Method Using Dried Oral Fluid Spot to Determine Drugs of Abuse

A liquid chromatography–mass spectrometry method using dried oral fluid spots was developed and validated for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, amphetamine, and 3,4‐methylenedioxymethamphetamine. The oral fluid was applied to a Whatman 903 grade paper and submitted to a drying time of 2.5 h. The extraction procedure was optimized by chemometric approach using simplex centroid design. Spots were extracted with a mixture of acetonitrile, buffer, and methanol. Calibration curves covered a linear concentration range of 40–500 ng/mL. Validation parameters of linearity, precision, accuracy, selectivity, carryover, matrix effects, and stability were evaluated and showed satisfactory results. Spot homogeneity was also satisfactory, with less than 15% of deviation from nominal concentration. Spot volume did not influence accuracy when less than 100 μL of the sample was applied to the spot. The validation of the proposed method suggests a potential application in different scenarios in toxicology.     

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial

A qualitative and quantitative analytical method was developed and validated for the determination of 49 licit and illicit drugs in oral fluid. Small oral fluid samples, volume 1mL, were collected from volunteers using a modified Omni-Sal device and the analytes were extracted from an oral fluid/buffer mixture using a single Bond Elut Certify solid phase extraction cartridge. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-repetitive full scan mass spectrometry (GC-MS) were used in parallel to analyze the extracts for the targeted drugs. Extracts were analyzed by GC-MS in their underivatized form and as their pentafluoropropionyl derivatives. Deuterated internal standards were used for quantification of drugs of abuse by LC-MS-MS to minimize matrix effects. Methadone-d(9) and tumoxetine were used as the internal standards for quantification of non-derivatized and derivatized analytes respectively by GC-MS. Linearity was demonstrated over the range 5-200 ng/mL and limits of detection were less than 4 ng/mL for each drug analyzed. The method demonstrated acceptable recoveries for most of the analytes and good intra- and inter-day precision. Acquisition of data by repetitive full scan GC-MS allows the addition of further analytes to the target menu.     

Standardization of method for the analysis of dextromethorphan in urine

Dextromethorphan (DMP), an antitussive, is one of the most popular drugs among the younger generation in Korea. It usually is taken for its hallucinogenic properties and overdoses have been responsible for the fatalities that have been reported frequently. To control the abuse of DMP, the authorities restricted its use through classifying it as a controlled drug on October 2003. The purpose of this study is to provide a standard method for the analysis of DMP and its main metabolite, dextrorphan (DTP) in biological specimens. At first we established a standard operating procedure (SOP) for DMP/DTP in urine, and a method validation was performed. We also quantified DMP from 16 drug abuser's urine samples all of which were positive in the screening test for DMP. For the detection of DMP/DTP, urine samples were adjusted with 6N NaOH (pH 11) and extracted with ethylacetate. Thin layer chromatography was used as the screening test, and the final identification for DMP/DTP was used by GC/MS. The ions (m/z 271 for DMP, m/z 257 for DTP and m/z 86 for lidocaine as internal standard) were extracted from the full scan mass spectrum and were used for quantification. The selectivity, linearity of calibration, accuracy, within- and between day precision, limit of detection and quantification, recovery and stability were examined as parts of the method validation. Extracted calibration curves were linear from 100 to 2000 ng/mL for DMP and DTP with correlation coefficients better than 0.999. Limit detection was 50 ng/mL for DMP and DTP. Within-run precision (%CV) for DMP and DTP at three different concentrations (100, 500 and 1000 ng/mL) was 6.10-18.85%, and between-run precision was 1.70-7.86% for DMP and DTP. Absolute recovery for DMP and DTP was 57-74%, and relative recovery (extraction efficiency) was 80-89%. For 16 drug abuser's urine samples, the concentrations of DMP and DTP were 0.16-52.63 and 0.41-23.75 microg/mL, respectively. Method validation is an important requirement in the practice of chemical analysis, and it will be particularly useful in verifying the reliability of analytical results in the field of forensic science.     

Screening for Corticosteroid Adulterants in Korean Herbal Medicines

The objective of this study was to determine the presence of corticosteroids in illegal herbal medicines using ultra‐high‐performance liquid chromatography–tandem mass spectrometry. We collected 212 herbal medicine samples that were advertised as being effective for treatment of joint pain and bone aches. Samples were from the Korean commercial market during a span of four years (2010–2013), and the method was validated. The limits of quantification ranged from 0.47 to 15.0 ng/mL, and recoveries ranged from 80.6% to 119.5%. The intra‐ and interday precision ranged from 0.18% to 8.82% and from 0.09% to 8.96%, respectively. Among the samples, three samples (1.4%) were identified as adulterants. Dexamethasone was the only compound detected in the adulterated products. As the corticosteroid–adulteration of herbal medicines may become a major problem and lead to side effects, the continued development of screening procedures for herbal medicines is critical.     

Quantification of psilocin in human whole blood using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC–MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7–200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification.     

Simultaneous Determination of 13 Anticoagulant Rodenticidesin Human Blood by Liquid Chromatography–Tandem Mass Spectrometry and its Application in Three Poisoning Cases

Anticoagulant rodenticides are widely used for rodent control around the world. A rapid and sensitive method was developed and validated for the simultaneous determination of 13 anticoagulant rodenticides (coumafuryl, pindone, valone, warfarin, coumatetralyl, coumachlor, diphacinone, dicumarol, chlorophacinone, bromadiolone, difenacoum, flocoumafen, and brodifacoum) in human blood by liquid chromatography–tandem mass spectrometry. After liquid–liquid extraction, the anticoagulant rodenticides were separated on an Eclipse Plus C18 column. Linearities were observed for each analyte in blood ranging from 0.5 to 50 ng/mL, with correlation coefficients over 0.99. The limits of detection ranged from 0.01 to 0.2 ng/mL, and the limits of quantification were 0.5 ng/mL for all analytes. The intraday and interday precisions were <15%, and accuracies ranged from 80.3% to 111.0%. This validated method with high sensitivity has been applied in three anticoagulant rodenticide poisoning cases and has been used successfully in monitoring blood concentrations for months.     

Detection and validated quantification of 21 benzodiazepines and 3 "z-drugs" in human hair by LC-MS/MS

A method for detection and quantification of 21 benzodiazepines and the pharmacologically related "z-drugs" in human hair samples was developed and fully validated using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After methanolic and methanolic/aqueous extraction, the analytes were separated using two different LC-MS systems (AB Sciex 3200 QTRAP and AB Sciex 5500 QTRAP). Separation columns, mobile phases and MS modes for both systems were: Phenomenex Kinetex, 2.6 μm, 50/2.1; 5mM ammonium formate buffer pH 3.5/methanol, total flow 0.75 mL/min; electrospray ionization (ESI), multiple reaction monitoring (MRM), information dependent acquisition (IDA), enhanced product ion scan (EPI). The assays were found to be selective for the tested compounds (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxymidazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem and zopiclone), all validation criteria were in the required ranges according to international guidelines, except for bromazepam. Matrix effects, and process efficiencies were in the acceptable ranges evaluated using the post-extraction addition approach. Lower limits of quantification were between 0.6 and 16 pg/mg of hair. The LC-MS/MS assay has proven to be applicable for determination of the studied analytes in human hair in numerous authentic cases (n=175).     

Evaluation of vapor profiles of explosives over time using ATASS (Automated Training Aid Simulation using SPME)

Despite numerous instrumental achievements, canines are still considered the most effective field method for explosive detection. However, due to strict explosive regulations and safety requirements, it can be a challenge for agencies with "bomb dogs" to train using neat explosive materials. This establishes a need for non-explosive canine training aids with the same volatile component profiles as the explosives that they represent. In order to compare mimic materials to their explosive counterparts, a technique must be established that not only allows for identification of volatile compounds but also can monitor changes in the headspace profile over time with respect to time and temperature. The Automated Training Aid Simulation using SPME (or ATASS) was developed for that purpose. As described, ATASS was used to observe changes in the volatile profile of three explosives (Composition C-4, 2,4-dinitrotoluene (DNT), and triacetone triperoxide (TATP)) and respective prototype training materials (0.1% by mass C-4, 1% by mass 2,4-DNT, and 1% by mass TATP). Samples were prepared in vials and metal tins within a gallon (≈ 3785 mL) paint can to simulate common field techniques for canine training. Monitoring these materials in real time provides a better understanding of the major volatile components present and how the relative abundances of these components can change over time. The results presented indicate that ATASS successfully allows for a sufficient comparison between explosive and non-explosive training materials.     

A novel method for cyanide quantification in human whole blood using ion chromatography with amperometric detection and its application to cyanide intoxication cases

Cyanide is a highly toxic agent that has been frequently used for suicide in South Korea. It is also used in various industrial fields, such as metal plating, in which many accidental cyanide intoxications have occurred. To overcome the disadvantages of conventional cyanide analysis methods, a simple and fast method for the analysis of cyanide in whole blood using ion chromatography (IC) with amperometric detection was developed in this study. Whole blood samples were deproteinized, diluted, and analyzed using an IC–amperometric detection system. The limits of detection and quantitation were 0.1 and 0.2 mg/L, respectively. The method showed good linearity in the range of 0.2 to 50 mg/L with R² > 0.99. The intra- and inter-assay precision and accuracy values were <10%. The established method was successfully applied to analyze whole blood samples from three cyanide intoxication cases.     

Fast quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) using microwave-accelerated derivatisation and gas chromatography–triple quadrupole mass spectrometry

A rapid and sensitive determination of cannabinoids in urine is important in many fields, from workplace drug testing over toxicology to the fight against doping. The detection of cannabis abuse is normally based on the quantification of the most important metabolite 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine. In most fields THCA needs to be present at a concentration of exceeding 15 ng/mL before a positive result can be reported.The method described in this paper, combines a 4 min GC–MS/MS method with a fast sample preparation procedure using microwave assisted derivatisation in order to complete the quantification of THCA in urine in 30 min, using only 1 mL of urine.The method is selective, linear over the range 5–100 ng/mL and shows excellent precision and trueness and hence, the estimated measurement uncertainty at the threshold level is small. The method also complies with applicable criteria for mass spectrometry and chromatography. Therefore the method can be used for rapid screening and confirmatory purposes.     

Quantitation of Synthetic Cannabinoids in Plant Materials Using High Performance Liquid Chromatography with UV Detection (Validated Method)

Plant based products laced with synthetic cannabinoids have become popular substances of abuse over the last decade. Quantitative analysis for synthetic cannabinoid content in the laced materials is necessary for health hazard assessments addressing overall exposure and toxicity when the products are smoked. A validated, broadly applicable HPLC‐UV method for the determination of synthetic cannabinoids in plant materials is presented, using acetonitrile extraction and separation on a commercial phenylhexyl stationary phase. UV detection provides excellent sensitivity with limits of quantitation (LOQs) less than 10 μg/g for many cannabinoids. The method was validated for several structural classes (dibenzopyrans, cyclohexylphenols, naphthoylindoles, benzoylindoles, phenylacetylindoles, tetramethylcyclopropylindoles) based on spike recovery experiments in multiple plant materials over a wide cannabinoid contents range (0.1–81 mg/g). Average recovery across 32 cannabinoids was 94% for marshmallow leaf, 95% for damiana leaf, and 92% for mullein leaf. The method was applied to a series of case‐related products with determined amounts ranging from 0.2 to >100 mg/g.     

Monitoring of Levamisole Concentration in Serum of Traffic Participants after Cocaine Consumption

This study highlights the problem of levamisole‐adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC‐MS/MS and solid‐phase extraction was applied. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole‐positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.     

Fast gas chromatography of explosive compounds using a pulsed-discharge electron capture detector

The detection of a mixture of nine explosive compounds, including nitrate esters, nitroaromatics, and a nitramine in less than 140 sec is described. The new method employs a commercially available pulsed-discharge electron capture detector (PDECD) coupled with a microbore capillary gas chromatography (GC) column in a standard GC oven to achieve on-column detection limits between 5 and 72 fg for the nine explosives studied. The PDECD has the benefit that it uses a pulsed plasma to generate the standing electron current instead of a radioactive source. The fast separation time limits on-column degradation of the thermally labile compounds and decreases the peak widths, which results in larger peak intensities and a concomitant improvement in detection limits. The combination of short analysis time and low detection limits make this method a potential candidate for screening large numbers of samples that have been prepared using techniques such as liquid-liquid extraction or solid-phase microextraction.     

Evaluation of a method for simultaneous quantification of codeine, ethylmorphine and morphine in blood.

Codeine, ethylmorphine and morphine are the most commonly detected opiates in forensic blood samples in Norway. A method for the simultaneous quantification of these opiates utilizing solid phase extraction and gas chromatography-mass spectrometry has been evaluated. The detection limits were 0.026 mumol/l for codeine, 0.025 mumol/l for ethylmorphine and 0.032 mumol/l for morphine (corresponding to 7.8, 7.8 and 9.1 micrograms/l, respectively). The analytical variations at concentrations of 1.0 mumol/l codeine, 1.0 mumol/l ethylmorphine and 0.5 mumol/l morphine were less than 5%.     

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06–5.30 ng/mL and a limit of quantification of 0.18–16.50 ng/mL, high linearity (R² > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.     

Simple and simultaneous quantification of cyanide,ethanol, and 1-propanol in blood by headspace GC–MS/NPD with Deans switch dual detector system

Cyanide is a powerful and rapidly acting poison. In Japan, cyanide poisoning is rare, and regular cyanide testing can be costly and time consuming. In contrast, alcohol analysis is routinely performed in most forensic laboratories. In this study, we attempted to develop a method for the simultaneous quantification of cyanide and alcohols in blood using headspace gas chromatography (HS–GC). As nitrogen-phosphorus detection (NPD) is more sensitive to hydrogen cyanide than mass spectrometry (MS), a Deans switch was used to switch the detectors during a single run. The separation provided by three analytical columns, PoraBOND Q, CP-Sil 5 CB, and HP-INNOWax, was investigated, and PoraBOND Q was selected. The use of HS–GC–MS/NPD with a Deans switch enabled the simple and simultaneous quantification of cyanide, ethanol, and 1-propanol. Eighteen other volatile compounds were detected in the SIM/scan mode of the MS.     

Rapid GC–MS confirmation of amphetamines in urine by extractive acylation

Amphetamine and related derivatives are widely abused central- and psychostimulants. Detection of certain derivatives, such as methcathinone, by commonly available immunoassay screening techniques is insufficient. Multi-analyte confirmations for amphetamine type stimulants are therefore required, but traditional gas chromatography–mass spectrometry methods necessitate lengthy analytical procedures with prolonged sample turn-around times. A validated rapid GC–MS assay for urinary confirmation of amphetamine, methamphetamine, methcathinone, ephedrine, norephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine and N-methyl-1-(3,4 methylenedioxyphenyl)-2-butanamine is reported. The method entailed in situ derivatization of urine specimens by extractive acylation with pentafluoropropionic anhydride, followed by rapid chromatography on a microbore capillary column. Analytes were separated in less than 3 min and quantified simultaneously by selected-ion monitoring using stable isotope substituted internal standards. The total instrument cycle-time was 6 min per sample. The limits of detection were between 1.5 ng/mL and 6.25 ng/mL for the various analytes. Intermediate precision and accuracy were in the range of 6.3–13.8% and 90.5–107.3% for the respective analytes at the lower limit of quantitation, and between 5.8–12.6% and 95.4–103.1% for the high control. Long-term storage of methcathinone positive specimens at ?20 °C proved insufficient stability of this analyte. The proposed assay is precise and accurate for confirmation of amphetamine and derivatives in urine. The complementary approach of extractive-derivatization and fast GC–MS analysis is especially applicable in routine clinical settings where reduced sample turn-around times are required. Further investigation of cathinone as a possible metabolite of methcathinone is warranted, based on results from analyzed authentic urine samples.