A multi‐analyte approach to help in assessing the severity of acute poisonings – Development and validation of a fast LC–MS/MS quantification approach for 45 drugs and their relevant metabolites with one‐point calibration

Diagnosis, monitoring of the efficiency of detoxification, and estimating the prognosis of acute poisonings are important tasks in emergency toxicology. Comprehensive screening and quantification of relevant substances by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) help in assessing the severity of most acute poisonings. Turnaround time for such analyses must be short enough to impact on clinical decisions. Therefore, a multi‐analyte LC–MS/MS approach with a 5‐minute gradient was developed and validated for 45 drugs and their active metabolites as a complement to an existing GC–MS approach using the same liquid–liquid extraction. The determination ranges were defined by quality control samples of low and high, representing concentrations from low therapeutic to highly toxic levels. To shorten the turnaround time, one‐point calibration was used. Validation showed low matrix effects and ionization effects of co‐eluting analytes thanks to APCI source as well as sufficient recoveries, precisions, and selectivities. For accuracy, 32 of the 45 compounds fulfilled the criteria for quantification in lower therapeutic and 41 in overdosed and toxic concentrations, considering limits of ±30% deviation. The reuse of the processed calibrator for a period of 30 days was possible for 32 compounds, showing sufficient stability at 8°C. In addition, analysis of authentic blood samples showed the applicability and yielded drug levels, which were comparable to those determined by fully validated therapeutic drug monitoring methods. In conclusion, the present approach in combination with the GC–MS approach should provide sufficient support for clinical assessment of the severity of poisonings with 68 compounds in an acceptable turnaround time.     

Development and validation of a fast and simple multi‐analyte procedure for quantification of 40 drugs relevant to emergency toxicology using GC‐MS and one‐point calibration

Diagnosis and prognosis of poisonings should be confirmed by comprehensive screening and reliable quantification of xenobiotics, for example by gas chromatography–mass spectrometry (GC‐MS) or liquid chromatography‐mass spectrometry (LC‐MS). The turnaround time should be short enough to have an impact on clinical decisions. In emergency toxicology, quantification using full‐scan acquisition is preferable because this allows screening and quantification of expected and unexpected drugs in one run. Therefore, a multi‐analyte full‐scan GC‐MS approach was developed and validated with liquid‐liquid extraction and one‐point calibration for quantification of 40 drugs relevant to emergency toxicology. Validation showed that 36 drugs could be determined quickly, accurately, and reliably in the range of upper therapeutic to toxic concentrations. Daily one‐point calibration with calibrators stored for up to four weeks reduced workload and turn‐around time to less than 1 h. In summary, the multi‐analyte approach with simple liquid‐liquid extraction, GC‐MS identification, and quantification over fast one‐point calibration could successfully be applied to proficiency tests and real case samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Power of Orbitrap‐based LC‐high resolution‐MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC–HR–MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSⁿ or GC–MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on‐spot cleavage followed by liquid extraction (DUSglucE), and one worked‐up by acid hydrolysis, liquid–liquid extraction, and acetylation for GC–MS analysis. The 3 series of LC–HR–MS/MS results were compared among themselves, to corresponding published LC–MSⁿ data, and to screening results obtained by conventional GC–MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC–HR–MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one‐third more hits in comparison to the corresponding published LC–MSⁿ results with ≤49% detected hits. The GC–MS approach identified 56% of all detected hits. In conclusion, LC–HR–MS/MS provided the best screening results after conjugate cleavage and precipitation.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.     

Evaluation of UV–HPLC and mass spectrometry methods for specific activity determination

The specific activity (SA) values determined using two different methods were compared for a set of tritium‐labeled and carbon‐14‐labeled compounds. The methods employed were as follows: (a) liquid chromatography/mass spectrometry (LC/MS) isotopic peak intensity distribution, and (b) determination of the tracer mass concentration using ultraviolet–high‐performance liquid chromatography analysis coupled with the radioactive solution concentration measured by liquid scintillation counting. In general, at lower SA, the accuracy and or precision of the LC/MS‐determined SA value decreased significantly. Because of this decrease in accuracy, a rough guideline of ~10% of the theoretical maximum SA is recommended as the lower cutoff for MS‐based SA measurements. If the tracer contains heteroatoms that possess significant percentages of heavy isotopes at natural abundance (e.g. Cl and Br), then the MS‐based SA cutoff recommendation is approximately 25–30% of the fully labeled compound in the tracer mixture. Additionally, IsoPat² was found to be the preferred calculation method for LC/MS‐based SA determination because SA values via this program were more consistent with those obtained by ultraviolet concentration calibration with solution count.     

Phenethylamine‐derived new psychoactive substances 2C‐E‐FLY, 2C‐EF‐FLY,and 2C‐T‐7‐FLY: Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings

Psychoactive substances of the 2C‐series are phenethylamine‐based designer drugs that can induce psychostimulant and hallucinogenic effects. The so‐called 2C‐FLY series contains rigidified methoxy groups integrated in a 2,3,6,7‐tetrahydrobenzo[1,2‐b:4,5‐b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C‐E‐FLY, 2C‐EF‐FLY, and 2C‐T‐7‐FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 fraction (pS9) incubations were analyzed by liquid chromatography?high‐resolution tandem mass spectrometry (LC?HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)?MS, LC?MSⁿ, and LC?HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N‐acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N‐acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO‐A and B. Interindividual differences in the metabolism of the 2C‐FLY drugs could be caused by polymorphisms of enzymes involved or drug–drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C‐E‐FLY and 2C‐EF‐FLY have to be considered during interpretation of analytical findings.     

Identification of the anabolic steroid 6β‐chlorotestosterone in a dietary supplement

Capsules that were labeled to be performance‐enhancing dietary supplements obtained during an investigation were found to contain an unrecognized steroid‐like substance. This compound was isolated by liquid chromatography (LC) fraction collection and characterized using several qualitative analytical techniques, including ultraviolet (UV) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐high resolution accurate mass‐mass spectrometry (LC–HRAM–MS), as well as ¹H, ¹³C, and two‐dimensional nuclear magnetic resonance (NMR) spectrometry. This multi‐technique analytical approach was used to identify the designer steroid as 6β‐chloro‐4‐androsten‐17β‐ol‐3‐one (6β‐chlorotestosterone), an analog of testosterone about which little has been published.     

Gas chromatography−mass spectrometry analysis of non‐hydrolyzed sulfated steroids by degradation product formation

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non‐hydrolyzed sulfated steroids with gas chromatography?mass spectrometry (GC–MS) instead of the conventionally applied liquid chromatography?mass spectrometry (LC–MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization?GC?triple quadrupole MS (CI?GC‐TQMS) and low energy?electron ionization?GC?quadrupole time‐of‐flight?MS (LE?EI?GC?QTOF?MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC–MS, forming two isomers. In CI, the dominant species (ie, [MH – H₂SO₄]⁺ or [MH – H₄S₂O₈]⁺ for bis‐sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE?EI, [M – H₂SO₄]^.+ and/or [M – H₂SO₄ – CH₃]^.+ are the dominant species in most cases. Based on the common GC–MS behavior of non‐hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC–MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC–MS and LC–MS analysis of non‐hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC–MS after hydrolysis. For non‐hydrolyzed sulfated steroids, GC–MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC–MS.     

Identification of the designer steroid Androsta‐3,5‐diene‐7,17‐dione in a dietary supplement

A liquid chromatography‐mass spectrometry (LC–MS) screen for known anabolic‐androgenic steroids in a dietary supplement product marketed for “performance enhancement” detected an unknown compound having steroid‐like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC–UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC–FT–IR–MS), liquid chromatography–high resolution accurate mass–mass spectrometry (LC–HRAM–MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error?0.57 ppm) for the protonated species [M + H]⁺, corresponding to a molecular formula of C₁₉H₂₄O₂. Based on the GC–FT–IR–MS data, NMR data, and accurate mass, the compound was identified as androsta‐3,5‐diene‐7,17‐dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.     

Retrospective analysis for valproate screening targets with liquid chromatography–high resolution mass spectrometry with positive electrospray ionization: An omics‐based approach

Liquid chromatography coupled with high‐resolution mass spectrometry (LC–HRMS) is an important analytical tool in the systematic toxicological analysis performed in forensic toxicology. However, some important compounds, such as the antiepileptic drug valproate (valproic acid; VPA), cannot be directly detected with positive electrospray ionization (ESI⁺) due to poor ionization. Here we demonstrate an omics‐based retrospective analysis for the identification of indirect screening targets for VPA in whole blood with LC–ESI⁺–HRMS. Analysis was performed utilizing data acquired across four years from LC–ESI⁺–HRMS, with VPA results from a quantitative LC–MS/MS method. The combined data with VPA results were split into an exploration set (n = 68; 28% positive) and a test set (n = 37; 32% positive). Eight indirect targets for VPA were identified in the exploration set. The evaluation of these targets was confirmed with retrospective target analysis of the test set. Using a combination of two out of the eight indirect targets, we attained a sensitivity of 92% (n = 12; VPA concentration range: 4.4–29.7 mg/kg) and 100% specificity (n = 25) for VPA with LC–ESI⁺–HRMS. VPA screening targets were identified with retrospective data analysis and could be appended to the existing screening procedure. A sensitive and specific screening with LC–ESI⁺–HRMS was achieved with targets corresponding to the sodium adducts of C₇H₁₄O₃ and C₈H₁₄O₃. Three chromatographic resolved isomer peaks were observed for the latter, and the consistently most intense peak was tentatively identified as 3‐hydroxy‐4‐en‐VPA.     

Diphenidine,a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC‐MS,LC‐MSn,and LC‐HR‐MSn

Diphenidine is a new psychoactive substance (NPS) sold as a ‘legal high’ since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐(high resolution)‐mass spectrometry (LC‐(HR)‐MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono‐ and bis‐hydroxylation at different positions, partly followed by dehydrogenation, N,N‐bis‐dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis‐hydroxy‐aryl groups. Mono‐ and bis‐hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome‐P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy‐aryl, hydroxy‐piperidine, and bis‐hydroxy‐piperidine. In incubations with CYP2D6 hydroxy‐aryl and hydroxy‐piperidine metabolites were detected. After application of a common users’ dose, diphenidine metabolites could be detected in rat urine by the authors’ GC‐MS as well as LC‐MSⁿ SUSA. Copyright © 2016 John Wiley & Sons, Ltd.     

Urinary detection of conjugated and unconjugated anabolic steroids by dilute‐and‐shoot liquid chromatography‐high resolution mass spectrometry

Anabolic androgenic steroids (AAS) are an important class of doping agents. The metabolism of these substances is generally very extensive and includes phase‐I and phase‐II pathways. In this work, a comprehensive detection of these metabolites is described using a 2‐fold dilution of urine and subsequent analysis by liquid chromatography‐high resolution mass spectrometry (LC‐HRMS). The method was applied to study 32 different metabolites, excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of at least 21 anabolic steroids. The method has been fully validated for 21 target compounds (8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection limits in the range of 1–10 ng mL^?1 in urine. For the conjugated compounds, for which no reference standards are available, metabolites were synthesized in vitro or excretion studies were investigated. The detection limits for these compounds ranged between 0.5 and 18 ng mL^?1 in urine. The simple and straightforward methodology complements the traditional methods based on hydrolysis, liquid‐liquid extraction, derivatization and analysis by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS). Copyright © 2014 John Wiley & Sons, Ltd.     

Profiling and Characterization of Sialylated N-glycans by 2D-HPLC (HIAX/PGC) with Online Orbitrap MS/MS and Offline MSn

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor–Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization–sequential mass spectrometry (NSI–MSⁿ). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (−) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(−) NSI–MSⁿ investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.     

Increasing the linear dynamic range in LC–MS: is it valid to use a less abundant isotopologue?

Liquid chromatography‐mass spectrometry (LC–MS) has quickly become the analytical method of choice in forensic toxicology laboratories due to its ability to detect a very wide range of compounds in a single analysis. One of the major limitations of LC–MS however, is a relatively limited linear dynamic range for quantitation. A new approach to combating this problem is to use the [⁺¹M + H]⁺ isotope mass peak for quantitation, thereby reducing saturation at the detector and extending the linear range. This is particularly useful in full‐scan applications, such as quadrupole‐time‐of‐flight (QTOF) mass spectrometry, where the isotopic mass peaks are acquired as a matter of course. Due to the variation in abundance of naturally occurring isotopes for common elements, especially ¹³C, this technique has the potential to lead to additional quantitative error. Through a review of published isotope ratio mass spectrometry data, we have assessed this potential for error and found that it is likely to be less than 2% and unlikely to be more than 4%, although this may not apply to compounds containing high numbers of nitrogen or sulphur atoms. This additional potential error must be considered before using this technique as it may not be appropriate for all applications. We have deemed it fit for purpose for our application and demonstrate the applicability of this technique to a quantitative LC‐TOF method. © 2017 Commonwealth of Australia. Drug Testing and Analysis © 2017 John Wiley & Sons, Ltd.     

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam,norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 ‘designer benzodiazepines’ monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4′‐chlorodiazepam (Ro5–4864)] offered as ‘research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography?mass spectrometry (GC–MS), liquid chromatography?tandem mass spectrometry (LC MS/MS), liquid chromatography?quadrupole time of flight?mass spectrometry (LC?QTOF?MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4’‐Chlorodiazepam biotransformation consisted of N‐dealkylation and hydroxylation. It has to be noted that 4′‐chlorodiazepam and its metabolites show almost identical LC–MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.     

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC‐positive samples for these compounds was performed by liquid chromatography–quadrupole time of flight–mass spectrometry (LC–QTOF–MS). Once identified, mass transitions for o‐, p‐ and m‐isomers of hydroxy COC as well as p‐ and m‐isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography–tandem mass spectrometry (LC–MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC‐positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p‐OH‐BE, m‐OH‐BE as well as p‐OH‐NCOC and m‐OH‐NCOC have been identified for the first time in COC‐positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC‐positive hair samples.     

A study of the origin of chloramphenicol isomers in honey

Due to the unexpected detection of chloramphenicol isomer residues in honey, we have studied the hypothesis of unauthorized or unintended use of unregistered veterinary drug preparations. First, we have investigated honey samples in which a discrepancy was observed between the results of the immunological screening methods and the confirmatory liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In all samples, previously identified to be contaminated with the banned antibiotic chloramphenicol according to LC–MS/MS only, the presence of dextramycin (SS‐para isomer of chloramphenicol) was detected by chiral LC–MS/MS. The source of dextramycin in honey was investigated by studying the preparations utilized in apiaries from which the above non‐compliant honey samples have been received. In all these preparations (beehive strips applied against the mite Varroa destructor) chloramphenicol was detected in the concentrations ranging from 33 to 34,400 μg kg⁻¹. Chiral LC–MS/MS demonstrated the presence of chloramphenicol and dextramycin in different ratios, and it was concluded that these preparations can be the source of chloramphenicol and dextramycin residues in honey. These preparations were of foreign production and are not officially registered in accordance with current legislation.     

Differentiation of structural isomers in a target drug database by LC/Q‐TOFMS using fragmentation prediction

Isomers cannot be differentiated from each other solely based on accurate mass measurement of the compound. A liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) method was used to systematically fragment a large group of different isomers. Two software programs were used to characterize in silico mass fragmentation of compounds in order to identify characteristic fragments. The software programs employed were ACD/MS Fragmenter (ACD Labs Toronto, Canada), which uses general fragmentation rules to generate fragments based on the structure of a compound, and SmartFormula3D (Bruker Daltonics), which assigns fragments from a mass spectra and calculates the molecular formulae for the ions using accurate mass data. From an in‐house toxicology database of 874 drug substances, 48 isomer groups comprising 111 compounds, for which a reference standard was available, were found. The product ion spectra were processed with the two software programs and 1–3 fragments were identified for each compound. In 82% of the cases, the fragment could be identified with both software programs. Only 10 isomer pairs could not be differentiated from each other based on their fragments. These compounds were either diastereomers or position isomers undergoing identical fragmentation. Accurate mass data could be utilized with both software programs for structural elucidation of the fragments. Mean mass accuracy and isotopic pattern match values (SigmaFit; Bruker Daltonics Bremen, Germany) were 0.9 mDa and 24.6 mSigma, respectively. The study introduces a practical approach for preliminary compound identification in a large target database by LC/Q‐TOFMS without necessarily possessing reference standards. Copyright © 2010 John Wiley & Sons, Ltd.     

液相色谱 质谱联用法鉴定9种皮质激素药物

目的:建立皮质激素的液相色谱 质谱分析方法,为限制药物滥用提供检测手段。方法:采用RP-HPLC-UV-MS联用法,同时对9 种皮质激素进行色谱分离及质谱鉴定,利用质谱解析软件研究了该类化合物的质谱裂解规律,并应用本法鉴定了药物制剂、送检物及尿样中的皮质激素。结果:在正离子检测方式下,9 种皮质激素的质谱断裂方式存在共性,即对于含有氟的分子,二级质谱优先脱去HF;含有醋酸酯的分子,二级质谱易产生脱CH₃COOH的特征碎片离子。每种化合物的检测限约为6 ng。结论:本法可用于皮质激素的体外、体内定性分析。