Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists

In France during a famous bicycle race, the newspapers documented the degree in which doping seemed to be supervised in some teams by managers and doctors. Use of anabolic steroids and other substances was officially banned in the mid-seventies by sports authorities. This policy has been enforced through urine testing before competition. It is well known, however, that a latency period is all that is necessary to defeat these tests. Nevertheless, hair analysis could be a promising tool when testing for periods that are not accessible to urinalysis any more. We have developed different sensitive methods for testing hair for amphetamines, anabolic steroids and their esters and corticosteroids. For amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate, derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an ultrasonic bath and subsequently purified using a C18 solid phase extraction column. Analysis was realized by high performance liquid chromatography coupled to electrospray-ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue was derivatized with MSTFA prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair (out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times (triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30 analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30 analyses).     

Supercritical fluid chromatography in forensic science: a critical appraisal

The application of supercritical fluid chromatography (SFC) in forensic science is reviewed. The applications centre on the analysis of explosives and of drugs of abuse. The systems employed are discussed in the context of comparison with gas chromatography and high-performance liquid chromatography methods which are traditionally used for such analyses. The advantages and disadvantages of SFC over these methods are discussed. Recommendations are made for the developments which are required in SFC technology if it is to find greater application in forensic science.     

New materials used for the synthesis of 2-chlorophenyl cyclopentyl ketone seized from an illicit ketamine manufacturing unit

Ketamine deemed as a psychoactive substance has gained popularity for recreational use owing to its hallucinogenic and dissociative effects. Understanding the synthetic processes of ketamine can provide essential clues for law enforcement officers against illicit ketamine manufacturing. In this case report, a chemical company was being monitored by law enforcement officers due to its importation of precursors and materials that could be used for the synthesis of illicit drugs. After materials and products seized from this chemical company were employed for analyses using gas chromatography–mass spectrometry, liquid chromatography–high-resolution mass spectrometry, and nuclear magnetic resonance analyses, ketamine, hydroxylamine, 2-chlorophenyl cyclopentyl ketone, and cyclopentanone p-toluenesulfonylhydrazone were identified. In addition, a novel process for the synthesis of ketamine precursor 2-chlorophenyl cyclopentyl ketone from cyclopentanone p-toluenesulfonylhydrazone and 2-chlorobenzaldehyde was validated. This is the first report to uncover this novel process for the synthesis of 2-chlorophenyl cyclopentyl ketone and can be used to increase awareness among law enforcement officers and forensic practitioners about these novel starting materials for the synthesis of ketamine.     

Sample Mining and Data Mining: Combined Real-Time and Retrospective Approaches for the Identification of Emerging Novel Psychoactive Substances

Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns. A multitude of NPS have been identified in the United States, often implicated in forensic investigations. The most common and effective manner for identifying NPS is by use of mass spectrometry and the true utility lies within nontargeted acquisition techniques. During this study, a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology testing. A SCIEX TripleTOF™ 5600 + with SWATH^® acquisition was used. Resulting data were compared against an extensive library database containing more than 800 compounds. The LC-QTOF-MS assay was applied to the reanalysis of biological sample extracts to discover emergent NPS. More than 3,000 sample extracts were analyzed, and more than 20 emerging NPS were detected for the first time. Among these were isopropyl-U-47700, 3,4-methylenedioxy-U-47700, fluorofuranylfentanyl, N-methyl norfentanyl, 2F-deschloroketamine, 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone.     

A Polydrug Intoxication Involving Methoxetamine in a Drugs and Driving Case

Methoxetamine ((RS)2‐(3‐methoxyphenyl)‐2‐(ethylamino)cyclohexanone)) is becoming a drug of interest among practitioners of forensic toxicology. In this case report, we describe the case background, standard field sobriety tests, sampling, and analysis of this drug in a whole blood sample as well as screening methods and analysis from a driver operating under the influence of intoxicating substances. Methoxetamine was isolated from the blood sample using mixed mode solid phase extraction. After elution and evaporation, the residue was dissolved in mobile phase (consisting of acetonitrile and aqueous formic acid) for analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS). The case sample was found to contain clonazepam, 7‐aminoclonazepam, carboxy‐THC, Ddphenhydramine, and MDMA. The case sample was found to contain 10 ng/mL of the drug (methoxetamine) in whole blood. The results of this drug analysis and previous analyses are discussed in terms of this driver operating under the influence of drugs.     

Designer drugs in herbal aphrodisiacs

In the presented case three herbal aphrodisiacs (Libidfit, Satibo and Viamax) were investigated for the presence of regular pharmaceuticals against erectile dysfunction. However, high-performance liquid chromatography with diode array detection and mass spectrometry (HPLC-DAD-MS) and nuclear magnetic resonance (NMR) analyses revealed the presence of ingredients, having a molecular structure strongly resembling those of sildenafil (Viagra) and vardenafil (Levitra). The health risk posed by these analogous substances is high because they were found to be potent phosphodiesterase 5 (PDE5) inhibitors used in pharmacologically relevant quantities having no known safety profile. Based on structural and functional analogy these analogs represent a new class of designer drugs and should be taken off the market for being unapproved drug substances. In the Libidfit court case this was done successfully, setting a precedent.     

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

Quantification of Methamphetamine in Mouse Thighbones Buried in Soil

Bone samples are used for analysis of drugs in decomposed or skeletonized bodies. Toxicological analyses of buried bones are important for determining the causes and circumstances of death. In this study, methamphetamine and amphetamine concentrations in heart blood, thigh muscles, and thighbones were analyzed using solid‐phase extraction with liquid chromatography–tandem mass spectrometry. Methamphetamine concentrations in heart blood, thigh muscle, and thighbone ranged from 0.041 to 0.873 μg/mL, 0.649 to 2.623 μg/g, and 56.543 to 643.371 μg/g, respectively. Thighbone concentrations were significantly higher than those in heart blood or thigh muscles were. Methamphetamine concentrations in buried thighbone (4.010–45.785 μg/g) were significantly lower than those of unburied thighbones were (56.543–643.371 μg/g). Methamphetamine and amphetamine were detected in thighbones buried for 7–180 days. These findings indicate that the methamphetamine concentrations in bone are higher and decrease after burial in soil.     

Analysis of pain management drugs, specifically fentanyl, in hair: application to forensic specimens

This article discusses the immunoassay screening of pain management drugs, and the mass spectrometric confirmation of fentanyl in human hair. Hair specimens were screened for fentanyl, opiates (including oxycodone), tramadol, propoxyphene, carisoprodol, methadone, and benzodiazepines and any positive results were confirmed using gas chromatography or liquid chromatography with mass spectral detection. The specific focus of the work was the determination of fentanyl in hair, since autopsy specimens were also available for comparison with hair concentrations. Using two-dimensional gas chromatography with electron impact mass spectrometric detection, fentanyl was confirmed in four of nine hair specimens collected at autopsy. The accuracy of the assay at 10 pg/mg was 95.17% and the inter-day and intra-day precision was 5.04 and 13.24%, respectively (n=5). The assay was linear over the range 5-200 pg/mg with a correlation of r(2)>0.99. The equation of the calibration curve forced through the origin was y=0.0053x and the limit of quantitation of the assay was 5 pg/mg. The fentanyl concentrations detected were 12, 17, 490, and 1930 pg/mg and the results were compared with toxicology from routine post-mortem analysis. The screening of pain management drugs in hair is useful in cases where other matrices may not be available, and in routine testing of hair for abused drugs.     

Identification of synthetic cannabinoids in herbal incense blends in the United States

Abstract: Synthetic cannabinoid agonists are chemically diverse with multiple analogs gaining popularity as drugs of abuse. We report on the use of thin layer chromatography, gas chromatography mass spectrometry, high‐performance liquid chromatography, and liquid chromatography time of flight mass spectrometry for the identification and quantitation of these pharmacologically active chemicals in street drug dosage forms. Using these approaches, we have identified the synthetic cannabinoids JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐200, JWH‐210, JWH‐250, CP47,497 (C=8) (cannabicyclohexanol), RCS‐4, RCS‐8, AM‐2201, and AM‐694 in various commercially available products. Other noncannabinoid drugs including mitragynine have also been detected. Typical concentrations of drug in the materials are in the range 5–20 mg/g, or 0.5–2% by weight for each compound, although many products contained more than one drug.     

Desorption atmospheric pressure photoionization-mass spectrometry in routine analysis of confiscated drugs

A comprehensive study was made, where desorption atmospheric pressure photoionization (DAPPI) was applied to the direct analysis of confiscated drugs and pharmaceuticals of various forms and matrices. The analyzed samples included herbal products [Catha edulis (khat), Psilocybe mushrooms, opium and Spice], designer drugs in tablet and powder form [e.g. meta-chlorophenylpiperazine (mCPP), 3-fluoromethamphetamine (3-FMA), methylenedioxypyrovalerone (MDPV) and methylone], and anabolic steroids in oil and tablets. The analyses were performed with ion trap mass spectrometer in MS and MS(2) modes and the obtained spectra were compared with GC-MS results. Contamination of the mass spectrometer was avoided by careful adjustment of the distance of the sample from the mass spectrometer inlet. DAPPI proved to be a fast and specific analysis technique, which does not require any sample preparation, and which therefore suits well to this type of forensic analysis.     

Prevalence of psychoactive substances in truck drivers in the Nord-Pas-de-Calais region (France)

A previous study conducted in 1995 showed that psychoactive drug use by workers was higher in safety/security workstations than in the rest of the labour force. In order to verify this finding, we conducted a new study in 2003-2004 in the Nord-Pas-de-Calais region, restricted to truck drivers. The aim of this study was to allow harmonizing the professional practice of the occupational physicians, proposing drug prevention and drug testing policies, validating the analytical methods and the guidelines in case of positive testing results. One thousand truck drivers were studied. Urines were tested for amphetamines, cannabinoids, cocaine, opiates, benzodiazepines, buprenorphine and methadone by immunoassay. Urine ethanol determinations were performed by an ADH method. Positive urines for drugs of abuse, methadone or buprenorphine were then tested by gas chromatography or liquid chromatography coupled to mass spectrometry. Out of the 1000 drivers, cannabinoids were detected in 85 cases, opiates in 41 cases, amphetamines in 3 cases and cocaine in only one case. Buprenorphine was detected in 18 cases, methadone in 5 cases and benzodiazepines in 4 cases. Urine ethanol was positive in 50 cases. We found only one case with 6-monoacetylmorphine. Other positive opiates were metabolites of antitussives. The relatively low number of benzodiazepine positive urines could be explained by the lack of sensitivity of the test we used. All these results confirm those of the previous study for cannabinoids and ethanol in safety/security workstations. Positive results for methadone and buprenorphine are eight times higher than in the general population. In conclusion, the authors think that it will be of a great interest to test urine of truck drivers for other classes of psychoactive drugs, using a liquid chromatography-mass spectrometry method.     

Validation of the Direct Analysis in Real Time Source for Use in Forensic Drug Screening

Abstract:  The Direct Analysis in Real Time (DART) ion source is a relatively new mass spectrometry technique that is seeing widespread use in chemical analyses world-wide. DART studies include such diverse topics as analysis of flavors and fragrances, melamine in contaminated dog food, differentiation of writing inks, characterization of solid counterfeit drugs, and as a detector for planar chromatography. Validation of this new technique for the rapid screening of forensic evidence for drugs of abuse, utilizing the DART source coupled to an accurate mass time-of-flight mass spectrometer, was conducted. The study consisted of the determination of the lower limit of detection for the method, determination of selectivity and a comparison of this technique to established analytical protocols. Examples of DART spectra are included. The results of this study have allowed the Virginia Department of Forensic Science to incorporate this new technique into their analysis scheme for the screening of solid dosage forms of drugs of abuse.     

Drugs and driving: a systematic analytical approach

To collect useful epidemiological data about drug involvement in highway safety, it is essential that sensitive and specific analytical procedures be used to establish the presence of and to determine the concentrations of drugs and metabolites in samples collected from drivers. This paper describes a comprehensive and systematic screening procedure requiring 6 mL of blood, which has been used for the analysis of samples collected from injured and fatally injured drivers. The procedure uses radioimmunoassay, gas chromatography with selective detectors, and high performance liquid chromatography. Drugs and metabolites presumptively identified are then confirmed primarily using gas chromatography--chemical ionization mass spectrometry.     

Pancuronium bromide (Pavulon) isolation and identification in aged autopsy tissues and fluids

The isolation and detection of pancuronium bromide was developed for aged autopsy samples to identify and confirm this compound in questioned tissue samples. A novel protocol was optimized for the isolation of the target drug in highly decomposed tissues. Solid-phase extraction (SPE) cartridges containing styrene-divinylbenzene were investigated. This polymer retained quaternary drugs and facilitated sequential elution upon washing with commonly available solvents. The semi-purified SPE samples were prescreened by pyrolysis GC-MS. A candidate specimen was then confirmed by microbore high-performance liquid chromatography/electrospray-ionization/mass spectrometry (microHPLC-ESI-MS/MS) with a triple-quadrupole mass spectrometer. The developed procedures provided a qualitative or semiquantitative (at best) basis for the investigation of difficult cases involving overdoses of polar drugs.     

Occurrence of contamination by controlled substances in Euro banknotes from the Spanish archipelago of the Canary Islands

The social problems of drug abuse are a matter of increasing global problem. Nowadays, international agencies need fresh methods to monitor trends of the use of illicit drugs. In this sense, small amounts of drugs are transferred to banknotes and they could be detected and quantified. An analytical procedure based upon extraction with organic solvent, liquid chromatography separation, and mass spectrometric detection allowed the identification of 21 drugs and metabolites in 120 used Euro banknotes collected in the Canary Islands (Spain). Most of the banknotes analyzed showed detectable drug residues (92.5%). Cocaine was the most frequently detected drug, present in approximately 90% of the samples. In addition, 75%, 35%, and 15% of the banknotes showed residues of amphetamine derivatives, opiates, and benzodiazepines, respectively. An average of three drug residues per banknote was detected. In summary, the presence of drug residues in banknotes could be useful as tracer for drugs prevalence.     

Analysis of amphetamines by supercritical fluid chromatography,high-performance liquid chromatography,gas chromatography and capillary zone electrophoresis; a preliminary comparison

A supercritical fluid chromatographic method with UV detection (SFC–UV) for the quantitative separation of phenylisothiocyanate (PITC)-derivatised amphetamines is described and compared to high-performance liquid chromatography–diode array detection (HPLC–DAD), gas chromatography–flame ionisation detection (GC–FID) and capillary zone electrophoresis–diode array detection (CZE–DAD) analyses of amphetamine and related compounds. Difficulties in the analysis of common amphetamines by SFC are discussed. Of the methods described in this paper, the SFC method offered the greatest sensitivity at 0.01–0.02 μg of drug on column. Suggestions are made for the use of combinations of these techniques for the identification of amphetamines when gas chromatography–mass spectrometry is not available.     

Development of gas chromatography–mass spectrometry (GC–MS) and other rapid screening methods for the analysis of 16 ‘legal high’ cathinone derivatives

The prevalence of so-called ‘legal high’ drugs in forensic science drug casework has increased markedly in recent years. This has given rise to both legal and analytical challenges in the identification of these substances. The requirement for validated, reliable and rapid testing methodologies for these compounds is obvious. This work reports the analysis of sixteen synthesised cathinone derivatives encountered in casework using presumptive testing, thin layer chromatography and gas chromatography–mass spectrometry (GC–MS).     

The Characterization of 3,4-Dimethylmethcathinone (3,4-DMMC)

Analogs and derivatives of traditional illicit drugs are ever increasing in variety and creativity. Staying abreast of the new developments is a constant challenge for every forensic laboratory. Recently, a seizure from Australian Customs Service presented our laboratory with the designer cathinone 3,4-dimethylmethcathinone (3,4-DMMC). Gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, and ultraviolet (UV) spectrophotometry were employed to analyze the spectroscopic characteristics of this cathinone. As an analog, 3,4-DMMC exhibits similar if not identical IR and UV profiles to mephedrone (4-MMC) and methcathinone; however, the retention time from GC is unique as expected, and the electron impact fragmentation pattern is consistent with the fragmentation pattern of other cathinones. The chemical shifts of the carbons and hydrogens were assigned by both one- and two-dimensional NMR techniques, while the molecular weight was confirmed by LC/MS.