Current applications of high-resolution mass spectrometry for the analysis of new psychoactive substances: a critical review.

The proliferation of new psychoactive substances (NPS) in recent years has resulted in the development of numerous analytical methods for the detection and identification of known and unknown NPS derivatives. High-resolution mass spectrometry (HRMS) has been identified as the method of choice for broad screening of NPS in a wide range of analytical contexts because of its ability to measure accurate masses using data-independent acquisition (DIA) techniques. Additionally, it has shown promise for non-targeted screening strategies that have been developed in order to detect and identify novel analogues without the need for certified reference materials (CRMs) or comprehensive mass spectral libraries. This paper reviews the applications of HRMS for the analysis of NPS in forensic drug chemistry and analytical toxicology. It provides an overview of the sample preparation procedures in addition to data acquisition, instrumental analysis, and data processing techniques. Furthermore, it gives an overview of the current state of non-targeted screening strategies with discussion on future directions and perspectives of this technique. Graphical Abstract Missing the bullseye - a graphical respresentation of non-targeted screening. Image courtesy of Christian Alonzo.

Characterization of hallucinogenic phenethylamines using high-resolution mass spectrometry for non-targeted screening purposes.

Hallucinogenic phenethylamines such as 2,5-dimethoxyphenethylamines (2C-X) and their N-(2-methoxybenzyl) derivatives (25X-NBOMe) have seen an increase in novel analogues in recent years. These rapidly changing analogues make it difficult for laboratories to rely on traditional targeted screening methods to detect unknown new psychoactive substances (NPS). In this study, twelve 2C-X, six 2,5-dimethoxyamphetamines (DOX), and fourteen 25X-NBOMe derivatives, including two deuterated derivatives (2C-B-d6 and 25I-NBOMe-d9 ), were analyzed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Collision-induced dissociation (CID) experiments were performed using collision energies set at 10, 20, and 40 eV. For 2C-X and DOX derivatives, common losses were observed including neutral and radical losses such as NH3 (17.0265 Da), •CH6 N (32.0500 Da), C2 H7 N (45.0578 Da) and C2 H9 N (47.0735 Da). 2C-X derivatives displayed common product ions at m/z 164.0837 ([C10 H12 O2 ]+• ), 149.0603 ([C9 H9 O2 ]+ ), and 134.0732 ([C9 H10 O]+• ) while DOX derivatives had common product ions at m/z 178.0994 ([C11 H14 O2 ]+• ), 163.0754 ([C10 H11 O2 ]+ ), 147.0804 ([C10 H11 O]+ ), and 135.0810 ([C9 H11 O]+ ). 25X-NBOMe had characteristic product ions at m/z 121.0654 ([C8 H9 O]+ ) and 91.0548 ([C7 H7 ]+ ) with minor common losses corresponding to 2-methylanisole (C8 H10 O, 122.0732 Da), 2-methoxybenzylamine (C8 H11 NO, 137.0847 Da), and •C9 H14 NO (152.1074 Da). Novel analogues of the selected classes can be detected by applying neutral loss filters (NLFs) and extracting the common product ions. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous Screening and Quantification of Basic, Neutral and Acidic Drugs in Blood Using UPLC-QTOF-MS.

An analytical method using ultra performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) was developed and validated for the targeted toxicological screening and quantification of commonly used pharmaceuticals and drugs of abuse in postmortem blood using 100 µL sample. It screens for more than 185 drugs and metabolites and quantifies more than 90 drugs. The selected compounds include classes of pharmaceuticals and drugs of abuse such as: antidepressants, antipsychotics, analgesics (including narcotic analgesics), anti-inflammatory drugs, benzodiazepines, beta-blockers, amphetamines, new psychoactive substances (NPS), cocaine and metabolites. Compounds were extracted into acetonitrile using a salting-out assisted liquid-liquid extraction (SALLE) procedure. The extracts were analyzed using a Waters ACQUITY UPLC coupled with a XEVO QTOF mass spectrometer. Separation of the analytes was achieved by gradient elution using Waters ACQUITY HSS C18 column (2.1 mm x 150 mm, 1.8 µm). The mass spectrometer was operated in both positive and negative electrospray ionization modes. The high-resolution mass spectrometry (HRMS) data was acquired using a patented Waters MSE acquisition mode which collected low and high energy spectra alternatively during the same acquisition. Positive identification of target analytes was based on accurate mass measurements of the molecular ion, product ion, peak area ratio and retention times. Calibration curves were linear over the concentration range 0.05-2 mg/L for basic and neutral analytes and 0.1-6 mg/L for acidic analytes with the correlation coefficients (r2) > 0.96 for most analytes. The limits of detection (LOD) were between 0.001-0.05 mg/L for all analytes. Good recoveries were achieved ranging from 80% to 100% for most analytes using the SALLE method. The method was validated for sensitivity, selectivity, accuracy, precision, stability, carryover and matrix effects. The developed method was tested on a number of authentic forensic samples producing consistent results that correlated with results obtained from other validated methods.

Analysis of new designer drugs in post-mortem blood using high-resolution mass spectrometry.

An analytical method was developed and validated for the purpose of detecting and quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines and piperazines. Using only 100 µL whole blood, a salting-out-assisted liquid-liquid extraction with acetonitrile was performed to isolate target compounds followed by chromatographic separation using a Waters ACQUITY ultra performance liquid chromatograph coupled to a Waters XEVO quadrupole time-of-flight mass spectrometer. Mephedrone-d3 was used as an internal standard. A gradient elution was used in combination with a Waters ACQUITY HSS C18 column (2.1 × 150 mm, 1.8 µm). Samples were analyzed using the detector in positive electrospray ionization mode with MS(E) acquisition. All compounds of interest were resolved in a 15 min run time and positively identified based on accurate mass of the molecular ion, two product ions and retention time. All analyte calibration curves were linear over the range of 0.05-2 mg/L with most correlation coefficient (r(2)) values >0.98. The limits of detection were within the range of 0.007-0.07 mg/L and limits of quantification within 0.05-0.1 mg/L. All analytes were stable 48 h after extraction and most were stable in blood after 1 week stored in a refrigerator and 3 freeze-thaw cycles. No carryover was observed up to 10 mg/L and no interferences from common therapeutic drugs or endogenous compounds. Recoveries ranged from 71 to 100% and matrix effects were assessed for blank, post-mortem and decomposed blood. All bias and % coefficient of variation values were within the acceptable values of ±15 and ≤15%, respectively (±20 and ≤20% at lower limit of quantification). The method was applied to several forensic cases where the subject exhibited behavior characteristic of designer drug intoxication and where routine screening for a panel of drugs was negative.

A validated LC-MS-MS method for simultaneous identification and quantitation of rodenticides in blood.

A rapid, highly sensitive and specific analytical method for the extraction, identification and quantification of nine rodenticides from whole blood has been developed and validated. Commercially available rodenticides in Australia include coumatetralyl, warfarin, brodifacoum, bromadiolone, difenacoum, flocoumafen, difethialone, diphacinone and chlorophacinone. A Waters ACQUITY UPLC TQD system operating in multiple reaction monitoring mode was used to conduct the analysis. Two different ionization techniques, ES+ and ES-, were examined to achieve optimal sensitivity and selectivity resulting in detection by MS-MS using electrospray ionization in positive mode for difenacoum and brodifacoum and in negative mode for all other analytes. All analytes were extracted from 200 µL of whole blood with ethylacetate and separated on a Waters ACQUITY UPLC BEH-C18 column using gradient elution. Ammonium acetate (10 mM, pH 7.5) and methanol were used as mobile phases with a total run time of 8 min. Recoveries were between 70 and 105% with limits of detection ranging from 0.5 to 1 ng/mL. The limit of quantitation was 2 ng/mL for all analytes. Calibration curves were linear within the range 2-200 ng/mL for all analytes with the coefficient of determination ≥0.98. The application of the proposed method using liquid-liquid extraction in a series of clinical investigations and forensic toxicological analyses was successful.

Management of intentional superwarfarin poisoning with long-term vitamin K and brodifacoum levels.

CONTEXT: Brodifacoum is a widely available superwarfarin used as a commercial rodenticide. Toxicity from long-acting anticoagulant rodenticides, primarily from uncontrolled bleeding, has been reported. Very little published toxicokinetic data are available for human brodifacoum poisoning. Management is also contentious with uncertainty over the dose, frequency, and duration of antidote treatment with vitamin K. The role of brodifacoum levels in guiding management is not entirely established. METHODS: A novel, highly sensitive method was developed for measuring all commercially available rodenticide-hydroxycoumarin anti-coagulants. Monthly brodifacoum levels were performed in two patients to determine half-life and expected time for levels to fall below 10 µg/L. RESULTS: We report two concurrent cases at our clinical toxicology service that required prolonged treatment with oral vitamin K to achieve normalisation of coagulation studies. Brodifacoum elimination appears to follow first-order kinetics. Case 1 had a brodifacoum elimination half-life of 33 days and was treated with vitamin K (100 mg) for 6 months. Case 2 was treated with vitamin K (100 mg) for 3 months with a half-life of 15 days. DISCUSSION: Our cases illustrate the positive experience in the utility of brodifacoum levels to confirm diagnosis and aid in directing antidote therapy. Large ingestions of brodifacoum-containing rodenticides are likely to require high-dose oral vitamin K administered daily. A brodifacoum level below 10 µg/L was associated with a normal coagulation profile following completion of vitamin K(1) therapy in our cases; this level may prove to be a safe treatment cessation threshold.