High-resolution Mass Spectrometry Method Research Articles

Correlation analysis between cocaine samples seized in China by the rapid detection of organic impurities using direct analysis in real time coupled with high-resolution mass spectrometry

A direct analysis in real time (DART) coupled with high-resolution mass spectrometry (HRMS) method was established to detect organic impurities in cocaine samples seized in China. This approach allowed discrimination between linked and unlinked seizures with quick sample preparation and high throughput. The responsivities of the mass spectra of 20 organic impurities found in 47 cocaine samples were explored as factors in multivariate analysis. Based on the data from 20 organic impurities in 47 linked/unlinked cocaine samples, 25 combinations of data pretreatment methods and distance/correlation measurements were compared to choose the combination with the best discriminating power for cocaine profiling. Data pre-treatment by calculating the logarithm (L) followed by Squared Euclidean distance (Squ Dij) was chosen as the best combination for the optimal discrimination of linked and unlinked samples. Hierarchical cluster analysis (HCA) was used to analyse another 46 cocaine samples, and 38 of the 46 cocaine samples were divided into 9 linked groups. Relevant information was provided for forensic science intelligence and case analysis.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

A sensitive method detecting trace levels of levonorgestrel using LC-HRMS

ObjectiveTo develop a high resolution mass spectrometry (HRMS) method to quantify levonorgestrel (LNG) in serum. Study designLevonorgestrel was extracted using solid phase extraction and measured using liquid chromatography (LC) HRMS. ResultsLow limit of quantification (LLOQ) was 25 pg/mL and low limit of detection (LLOD) was 12.5 pg/mL. Precision and accuracy bias were <10%. LNG in serum samples from Mirena® users ranged between 37 to 219 pg/mL (n=12). In eight out of 22 patients with suspected intrauterine device (IUD) expulsion LNG was detected (26–1272 pg/mL). ConclusionA sensitive, fast and simple LC-HRMS method was developed to detect trace levels of LNG.

Qualitative and quantitative analysis of sesquiterpene lactones in Centipeda minima by UPLC–Orbitrap–MS & UPLC-QQQ-MS

An ultra-high performance liquid chromatography coupled with high-resolution Orbitrap mass spectrometry (UPLC–Orbitrap–MS) method has been used to identify sesquiterpene lactones in the methanolic extract of Centipeda minima. Fifteen sesquiterpene lactones were tentatively identified based on retention time and accurate mass of external standards or exact accurate mass searching (within 2 ppm) by comparison of some previous isolated sesquiterpene lactones. Meanwhile, a rapid, sensitive, precise, and reliable ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QQQ-MS) method has been developed to evaluate the quality of Centipeda minima through a simultaneous determination of five sesquiterpene lactones, namely brevilin A, arnicolide C, arnicolide D, microhelenin C, minimolide F. Chromatographic separation was achieved on a Waters Acquilty UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with a mobile phase consisting of a) 0.1% formic acid and b) a mixture of acetonitrile and methanol 50:50 v/v under an isocratic elution (42:58) manner. Positive electrospray ionization mode with multiple reaction monitoring was applied for the detection of the five sesquiterpene lactones. Method validation for linearity, accuracy and precision was also carried out. Finally, the method was successfully used for the analysis of 10 batches of Centipeda minima samples collected in China. Brevilin A and arnicolide D were the dominant sesquiterpene lactones in Centipeda minima and could be proposed as suitable markers for the quality control of Centipeda minima.

The use of high-resolution mass spectrometry (HRMS) for the analysis of DNA and other macromolecules: A how-to guide for forensic chemistry

High-resolution mass spectrometry (HRMS) is a highly effective technique for the analysis of DNA and other biological macromolecules and has the potential to be a valuable tool for forensic chemists. In this work, we introduce concepts of HRMS, discuss benefits of the technique, and outline the steps involved in developing an application utilizing this platform. We examine the importance of optimizing instrumental conditions, selecting an appropriate solvent, and interpreting mass spectra with considerations to determining charge state, mass accuracy and resolution. Specifically, we present a method for the analysis of short single stranded oligonucleotides (26- and 28-base) using Fourier-transform ion cyclotron mass spectrometry (FT-ICR MS), while exploring the importance of sample matrix. Results of this work indicate that analysis of small oligonucleotides with 50:50 water:acteonitrile + 20 mM imidazole (C3H4N2) produced a mass spectrum with the −5 charge state as the base peak. Charge states of −4 up to −15 were also observed, although at much lower ion intensities. Analysis of the oligonucleotides using water and 20 mM imidazole produced a charge state distribution from −5 to −11 over the mass range m/z 200–2500. These two solvents, paired with the optimized HRMS method, produced multiple charge states, high resolution mass spectra, and high ion signal intensity that allowed for unambiguous peak identification. This work serves as a “how-to guide” for forensics scientists who require efficient and accurate HRMS protocols. Our research supports using nucleic acids for diagnostics to analyze oligonucleotides, short DNA fragments and other biological macromolecules using HRMS.

UHPLC-HRMS study of anti-Alzheimer’s drug candidates: metabolism of 7-MEOTA-tryptophan hybrids hampers their passage into brain

Being among the top five causes of death in the developed world, Alzheimer’s disease represents a major socio-economic issue. We administered a single intramuscular dose of two new hybrid anti-Alzheimer’s compounds, with 7-methoxytacrine (7-MEOTA; acetylcholinesterase inhibitor) and tryptophan (inhibitor of amyloid accumulation) in their structure, to rats.Using validated ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) methods, we uncovered their inability to enter the site of action – the brain. We discuss four possible explanations: i) physico-chemical properties, ii) lack of active/facilitated transport, iii) effective efflux and/or iv) extensive metabolism.High-resolution mass spectrometric analyses proved that the compounds are easily hydrolysed at amide bond between tryptophan and the linker both in vitro and in vivo. Contrary to the parent compounds these metabolites – analogues of 7-MEOTA – can enter the brain in significant amounts.

Rapid removal of acesulfame potassium by acid-activated ferrate(VI) under mild alkaline conditions

Acesulfame potassium (ACE) is a widely used artificial sweetener that has consistently been detected in wastewater and surface waters. The high-valent iron-based green oxidant known as ferrate(VI) (potassium ferrate(VI); Fe(VI)) had low reactivity with ACE (i.e. 4 h (or 240 min) contact time removed only ∼ 67% ACE) at a molar ratio of 6.0 ([Fe(VI)]:[ACE]). Comparatively, it took 60 s (or 1 min) to remove ∼94% ACE when HCl (786 μM) was added to a mixture of Fe(VI)-ACE at the same molar ratio of 6.0 (or acid-activated Fe(VI)). Significantly, the final pH (i.e. 7.6–8.1) was similar for Fe(VI) and acid-activated Fe(VI). An empirical model using response surface methodology was developed that could describe reasonably well the removal efficiency of ACE. Inorganic constituents of wastewater (Cl−, Na+, Ca2+, and Mg2+) had no significant effect on the oxidation of ACE by acid-activated Fe(VI). The degradation efficiency of ACE decreased in the presence of 10 mg/L of natural organic matter (NOM) but remained unchanged at 5 mg NOM/L. Sulfamic acid as the oxidized product of ACE was identified by liquid chromatography high resolution mass spectrometry method. Reaction pathways include ring opening of ACE through hydrolytic transformation. Acid-activated Fe(VI) has advantage of rapid removal of ACE under mild alkaline conditions of wastewater treatment plants compared to other oxidation processes such as chlorination, ozonation, and light-based processes.

Amino acids promote black carbon aggregation and microbial colonization in coastal waters off Vietnam

The combustion of fossil fuels and biomass produces pyrogenic organic matter usually known as ‘black carbon’ (BC), which are transported across the atmosphere as particulate aerosol, eventually deposited on land and oceans. Soil studies have investigated the potential microbial colonization and remineralization of BC particles, but this process has been seldom studied in marine waters. BC provides a significant input of organic carbon to the oceans, yet its fate and role in biogeochemical cycling remains unknown. Here we explored the microbial colonization of BC particles in coastal seawater samples collected in Halong Bay (northern Vietnam). Using high-resolution mass spectrometry and microscopy methods, we observed an increasing colonization of BC particles by marine microbes in the presence of amino acids. Our results suggest that natural organic matter (NOM) present in seawater may promote the microbial colonization and eventual remineralization of BC particles. Future experiments should explore the potential microbial remineralization of BC particles to unveil the role of this massive source of carbon to marine ecosystems.

Development of two complementary LC-HRMS methods for analyzing sotatercept in dried blood spots for doping controls.

Aim: sotatercept is a therapeutic Fc-fusion protein with erythropoiesis-stimulating activity. Due to a potential abuse of the drug by athletes in professional sports, a sensitive detection method is required. In sports drug testing, alternative matrices such as dried blood spots (DBS) are gaining increasing attention as they can provide several advantages over conventional matrices. Materials & methods: Herein, two complementary LC-high-resolution mass spectrometry (HRMS) detection methods for sotatercept from DBS, an initial testing procedure (ITP) and a confirmation procedure (CP)were developed and validated for the first time.Both methods comprise an ultrasonication-assisted extraction, affinity enrichment, proteolytic digestion and HRMS detection. Results & conclusion:For the multianalyte ITP, artificial samples fortified with sotatercept, luspatercept and bimagrumab, and authentic specimens containing bimagrumab were successfully analyzed as proof-of-concept. The validated detection methods for sotatercept are fit for purpose and the ITP was shown to be suitable for the detection of novel IgG-based pharmaceuticals in doping control DBS samples.

Development and validation of an ultra-high performance liquid chromatography-high resolution mass spectrometry method for simultaneous quantification of cyanogenic glycosides and secoisolariciresinol diglucoside in flaxseed (Linum usitatissimum L.)

An ultra performance liquid chromatography electrospray ionization high-resolution mass spectrometry (UPLC/ESI-HRMS) method was developed and validated for simultaneous quantification of cyanogenic glycosides (CGs), [linustatin (LIS) and neolinustatin (NLIS)], and the main lignan, secoisolariciresinol diglucoside (SDG) in Linoforce® (LF) [flaxseed (Linum usitatissimum L.) coated with two herbal extracts (Senna alexandrina mill and Frangula alnus)]. CGs and SDG were extracted from defatted ground LF by a new procedure consisting of an aqueous methanol ultrasound-assisted extraction followed by an aqueous alkaline ultrasound-assisted extraction of the residue. The combined extracted solutions were then hydrolyzed by 0.02 M NaOH to release SDG from its hydroxymethyl glutaryl ester-linked complex (SDG-HMG). After hydrolysis, the sample was acidified and analyzed directly, without the need of any additional clean-up steps, by UPLC/ESI-HRMS in positive mode. The identification of CGs and SDG was confirmed by the similar retention time and similar MS spectra to the corresponding authentic standards. The quantification was performed using the corresponding extracted ion chromatograms and amygdalin as internal standard. The overall method was validated in terms of linearity, stability, selectivity, precision and accuracy. The developed method was successfully applied to the quantification of CGs and SDG in LF and also in non-coated flaxseed. This is the first report on the simultaneous quantification of CGs and SDG in LF and flaxseed.

The Metabolome: a Key Measure for Exposome Research in Epidemiology

Application of omics to study human health has created a new era of opportunities for epidemiology research. However, approaches to characterize exogenous health triggers have largely not leveraged advances in analytical platforms and big data. In this review, we highlight the exposome, which is defined as the cumulative measure of exposure and biological responses across a lifetime as a cornerstone for new epidemiology approaches to study complex and preventable human diseases. While no universal approach exists to measure the entirety of the exposome, use of high-resolution mass spectrometry methods provide distinct advantages over traditional biomonitoring and have provided key advances necessary for exposome research. Application to different study designs and recommendations for combining exposome data with novel data analytic frameworks to study complex interactions of multiple stressors are also discussed. Even though challenges still need to be addressed, advances in methods to characterize the exposome provide exciting new opportunities for epidemiology to support fundamental discoveries to improve public health.

Occurrence and Degradation Potential of Fluoroalkylsilane Substances as Precursors of Perfluoroalkyl Carboxylic Acids.

Polyfluoroalkylsilanes (PFASis) are a class of artificial chemicals with wide applications in surface coating, which arouse attention due to their hydrophobic/oleophobic properties and potential biological effects. In this study, a robust high-resolution mass spectrometry method through direct injection into a Fourier transform ion cyclotron resonance instrument was established, with the aid of CF2-scaled Kendrick mass defect analysis and isotope fine structure elucidation. The occurrence of 8:2 polyfluoroalkyl trimethoxysilane (8:2 PTrMeOSi) and 8:2 polyfluoroalkyl triethoxysilane (8:2 PTrEtOSi), as well as their cationic adducts, solvent substitutions, and other compound analogues, were identified in commercial antifingerprint liquid products. In the hydroxyl radical-based total oxidizable precursor assay, differential molar yields of products were observed with regard to varied PFASi carbon-chain lengths and terminal groups. The yields of perfluoroalkyl carboxylic acids (PFCAs) from 8:2 PTrMeOSi conversion were the highest (92 ± 9%, n = 3), with the C ( n - 1) perfluoroheptanoic acid (PFHpA, 49 ± 11%, n = 3) as the dominating product. Distinct conversion of 8:2 PTrMeOSi in the simulated solar exposure experiments found that C ( n) perfluorooctanoic acid (PFOA, 0.6 ± 0.04 ‰, n = 3) was predominant, and 8:2 fluorotelomer carboxylic acid (8:2 FTCA, 0.59 ± 0.08‰, n = 3), 8:2 fluorotelomer unsaturated carboxylic acid (8:2 FTUCA, 0.09 ± 0.00‰, n = 3) intermediates were also observed. To our knowledge, this is the first report regarding the occurrence and degradation potential of several fluoroalkylsilane substances as PFCA precursors.

Application of multifold characteristic ion filtering combined with statistical analysis for comprehensive profiling of chemical constituents in anti-renal interstitial fibrosis I decoction by ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high resolution mass spectrometry

A Chinese herbal formula of Anti-Renal Interstitial Fibrosis I Decoction (ARIF I) is composed of Eucommia ulmoides Oliv., Scutellaria baicalensis Georgi, and Rheum. officinale Baill., and it has been used for the treatment of chronic kidney disease for many years. However, the comprehensive chemical analysis of the formulated ARIF I has yet to be clarified. In this study, an ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC/Q-orbitrap HRMS) method executed in full scan/data-dependent MS2 (full MS/dd-MS2) mode has been established in negative ion mode to analyze the major chemical constituents in ARIF I as well as in its three constituent herbs. To rapidly and efficient identify these compounds, we employed a combination of statistical analysis of the MS2 data and fragmentation pathway method to screen for elementary characteristic ions and sub-characteristic ions. Then, we applied multifold characteristic ion filtering to comprehensively profile ARIF I extract chemical constituents with diverse structures. A total of 360 compounds, including anthraquinones, sennosides, galloyl glucoside derivatives, catechin and its derivatives, flavonoids, iridoids, phenylpropanoids, lignan, organic acid, and other compounds were rapidly identified or tentatively characterized by multifold characteristic ion filtering based on their accurate mass spectra (within 5 ppm error) and structure types or by comparison with reference standards. All the ARIF I compounds were also detected in the individual constituent herbs. This study provides a basis for further pharmacokinetic studies of ARIF I and may help in the discovery of new chemical drugs to treat chronic kidney disease. This study also provides a more efficient approach for the screening of appropriate characteristic ions, and a practical strategy for the detection of the types of compounds identified in this study in other herbal medicines.

Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry Method for the Simultaneous Determination of Mycotoxins and Phytoestrogens in Plant-Based Fish Feed and Exposed Fish.

New protein sources in fish feed require the assessment of the carry-over potential of contaminants and anti-nutrients from feed ingredients into the fish, and the assessment of possible health risks for consumers. Presently, plant materials including wheat and legumes make up the largest part of aquafeeds, so evaluation of the transfer capabilities of typical toxic metabolites from plant-infesting fungi and of vegetable phytoestrogens into fish products is of great importance. With the aim of facilitating surveillance of relevant mycotoxins and isoflavones, we have developed and validated a multi-analyte LC-HRMS/MS method that can be used to ensure compliance to set maximum levels in feed and fish. The method performance characteristics were determined, showing high specificity for all 25 targeted analytes, which included 19 mycotoxins and three isoflavones and their corresponding aglycons with sufficient to excellent sensitivities and uniform analytical linearity in different matrices. Depending on the availability of matching stable isotope-labelled derivates or similar-structure homologues, calibration curves were generated either by using internal standards or by matrix-matched external standards. Precision and recovery data were in the accepted range, although they varied between the different analytes. This new method was considered as fit-for-purpose and applied for the analysis of customised fish feed containing wheat gluten, soy, or pea protein concentrate as well as salmon and zebrafish fed on diets with these ingredients for a period of up to eight weeks. Only mycotoxin enniatin B, at a level near the limit of detection, and low levels of isoflavones were detected in the feed, demonstrating the effectiveness of maximum level recommendations and modern feed processing technologies in the Norwegian aquaculture industry. Consequently, carry-over into fish muscle was not observed, confirming that fillets from plant-fed salmon were safe for human consumption.

Improved oil spill dispersant monitoring in seawater using dual tracers: Dioctyl and monoctyl sulfosuccinates sourced from corexit EC9500A

A high resolution mass spectrometry method was developed for the environmental impact monitoring of oil spill dispersants. Previously reported instability of dioctyl sulfosuccinate (DOSS) dispersant tracer was addressed by the new procedure. The method monitors both DOSS and its degradation product, monooctyl sulfosuccinate (MOSS), by liquid chromatography time-of-flight mass spectrometry. The related isomer, 4-(2-ethylhexyl) 2-sulfobutanedioate, was chromatographically resolved from MOSS but was not a product of DOSS degradation. Using this direct injection method (10 μL), the practical lower limit of quantitation was 0.5 nM for each analyte, a concentration equivalent to 0.22 ng mL−1, or 0.30 ng mL−1 including initial dilution factor with acetonitrile. The method was shown applicable to analysis of the dispersants Corexit® EC9500 A, Finasol OSR 52, Slickgone NS, and Slickgone EW for which DOSS is an active ingredient. A marine microcosm study of Corexit EC9500A, together with diluted bitumen (dilbit), at 15 ± 1 °C, provided evidence of the stoichiometric conversion of DOSS to MOSS under conditions reflecting a western Canadian marine environment. The advantage of the developed method is in its ability to extend environmental seawater sample collection time from 4 days for DOSS alone, to 14 days when both DOSS and MOSS are simultaneously analysed and results combined. The collection time is likely extended beyond the 14 day period with cooler temperatures. Preservation of collected seawater samples using sodium hydroxide, converting DOSS into MOSS in situ, was rejected due to stability issues. Addition of disodium ethylenediaminetetraacetic acid did not improve hold times, thus eliminating the theory of cation induced micelle effects causing DOSS loss.

Development of a high resolution mass spectrometry method for the determination of danshensu and salvianolic acid B

In this work, a highly sensitive ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method has been developed and validated by the quantifications of danshensu (DSS) and salvianolic acid B (SAB) in Danhong injection (DHI) in human plasma, urine and feces for the pharmacokinetic and excretion studies. The DSS and SAB were extracted from biological matrix by direct protein precipitation with fivefold volume of methanol and separated by a Waters CORTECS C18+ chromatographic column (100 × 2.1 mm, 2.7 μm) with gradient elution (mobile phase: methanol–H2O, both containing 0.1% formic acid). The detection of analytes was achieved using negative ion ESI in Full MS/dd-MS2 (data-dependent MS2) scan mode. The human subjects received either a single intravenous injection (i.v.) dose (containing 0.331 mg/kg DSS and 0.117 mg/kg SAB) or a single intramuscular injection (i.m.) dose (containing 0.663 mg/kg DSS and 0.234 mg/kg SAB) of DHI. For i.v., the Cmax, half-life (t1/2) and AUC0−∞ were 906.2 ± 31.82 μg/L, 0.604 ± 0.103 h and 242.19 ± 18.24 μg/L × h for DSS and 1118.5 ± 35.64 μg/L, 0.893 ± 0.159 h and 277.63 ± 11.24 μg/L × h for SAB, respectively. For i.m., the Cmax, Tmax, t1/2 and AUC0−∞ were 243.0 ± 29.1 μg/L, 0.67 h, 0.71 ± 0.03 h and 318.98 ± 7.62 μg/L × h for DSS and 119.4 ± 6.51 μg/L, 0.50 h, 1.08 ± 0.18 h and 185.63 ± 4.23 μg/L × h for SAB, respectively. Based on the AUC0−∞ obtained from i.m. and i.v. administrations, the absolute bioavailabilities (F) were estimated to be 65.9% for DSS and 33.4% for SAB.

New psychoactive substances in oral fluid of drivers around a music festival in south-west France in 2017

Driving under the influence of drugs (DUID) is a worldwide problem with potentially major forensic and life-threatening consequences. Although it is obvious that new psychoactive substances (NPS) could lead to impaireddriving, the prevalence of NPS use in a DUID context is unknown as the applied roadside screening tests for drugs of abuse (DOA) are not adapted for NPS detection. This works aims to tested oral fluid (OF) specimens for NPS in French drivers circulating around two music festivals (Artsenik 2017 and Garorock 2017) in order to assess the prevalence of consumption and the kind of used NPS in this particular population. OF samples consisted in dried saliva spots obtained from used Drugwipe-5S® tests (after a positive or negative roadside screening test for DOA). These OF were analyzed using a liquid chromatography coupled with tandem mass spectrometry or high-resolution mass spectrometry method. NPS were detected in 17 out of the 229 OF collected specimens (7.4%). Eleven various NPS were identified (number of identification): 5F-AKB48 (2), MAM2201 (1), JWH122 (1), 4F-PVP (1), 3- or 4-MMC (2), fluoromethamphetamine (1), ketamine (3), MXE (3), methoxyketamine (1), 6-APB (2) and 25C-NBOMe (1). There is an apparent effect of the music festival proximity on the prevalence of NPS in OF from this controlled driver population compared to that of 140 controlled drivers from Northern France analyzed in the same period (7.4% versus 3%). The variety of used NPS appears to be increasing (e.g. large proportion of cyclohexanones). In addition, 5% of drivers initially roadside-tested negative for DOA were in fact driving after NPS use in this specific population. From a forensic perspective, these results confirm the reality of driving after NPS use in French drivers, notably in those driving to or from a music festival.

Isotope dilution LC-orbitrap-HRMS with automated sample preparation for the simultaneous quantification of 11 antimycotics in human serum

IntroductionThe aim of this project was to develop and validate an isotope-dilution liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantification of the 11 most widely used systemic antimycotics and to study whether HRMS is a feasible alternative for therapeutic drug monitoring (TDM) when compared to tandem MS (MS/MS) technology. MethodsAfter protein precipitation, followed by automated online sample clean-up the analytes were separated within 4 min on a C18 column using an acetonitrile-water gradient. Eleven antimycotics, namely 5-flucytosine, amphotericin B, anidulafungin, fluconazole, isavuconazole, itraconazole, ketoconazole, micafungin, OH-itraconazole, posaconazole and voriconazole were finally quantified in full MS scan mode using positive electrospray ionization (ESI +) with a mass range fromm/z 110–1300 using HRMS. The method was comprehensively validated on the basis of the European Medicines Agengy (EMA) method validation protocol using commercially available IVD kit components. ResultsGood linear relationship between peak area responses and drug concentrations (R2 > 0.995) and excellent selectivity were observed for all antimycotics in this study. Inaccuracy and imprecision of all quality controls were consistently below ± 12.6% and ± 8.1%, respectively. Quantification results were in agreement with an IVD LC–MS/MS method. ConclusionHRMS was shown to be suitable for TDM of small molecules when compared to tandem mass spectrometry. The novel HRMS method is quickly installed and may be a robust and reliable tool for routine TDM of antimycotics in clinical laboratories.

Application of Orbitrap high resolution mass spectrometry for the screening and identification of 15 adulterated weight loss compounds in dietary supplements

A liquid chromatography-Orbitrap high resolution mass spectrometry (LC-HRMS) method and a TraceFinder database were developed for the screening and identification of 15 adulterated weight loss compounds in dietary supplements. The samples were extracted with methanol and filtered through a 0.22 μm microfiltration membrane prior to LC-HRMS analysis. The Full MS/dd-MS2 mode was utilized in both positive and negative ion modes and the collected data were imported into the TraceFinder screening software. The established compound database and screening method were used for rapid, automatic, and high-precision screening to determine if the weight loss compounds were adulterated. The method validation results indicated that all of the analytes showed excellent linear relationships with regression coefficients (r) above 0.998. The recoveries were in the range of 79.7%-95.4% while the precisions ranged from 3.3% to 8.7%. The method and database were used to screen weight loss adulterants in 29 batches of dietary supplements; six batches of samples tested positive for adulterants with the identification of four compounds including sibutramine. This method enables the automatic high-precision screening and identification of adulterants, providing a novel and powerful tool for combating the increasingly rampant occurrence of adulteration in dietary supplements.

Development and validation of an ultra-high performance liquid chromatography – high resolution mass spectrometry method for the quantification of total and free teicoplanin in human plasma

ObjectivesThe antibiotic teicoplanin, used for the treatment of infections caused by Gram-positive bacteria, is highly bound to plasma proteins (percentage protein binding, %PB, around 90%) and therapeutic plasma levels of total teicoplanin are 10–100 mg/L. Because of the low free concentrations (i.e. <1–10 mg/L), current immunoassays are not able to quantify free teicoplanin concentrations, although they might be more relevant in therapeutic drug monitoring than total concentrations. Design and methodsIn this study, an ultra-high performance liquid chromatography – high resolution mass spectrometry (UHPLC-HRMS) method for the quantification of total and free teicoplanin in K2EDTA plasma samples was developed and validated. Furthermore, %PB obtained by ultrafiltration was compared with that obtained by equilibrium dialysis using spiked samples from healthy subjects. Analytes were separated using a phenylhexyl column, gradient mobile phase analysis was used, total run time was 4.5 min and teicoplanin was detected by orbitrap MS. ResultsThe precision and accuracy were below 15% and within ±15%, respectively and teicoplanin was found to be stable for at least 14 days in plasma at 4 °C. The %PB of teicoplanin in spiked plasma from healthy subjects as obtained by ultrafiltration (94.1 ± 1.3%) was in good agreement with that obtained by equilibrium dialysis (93.6 ± 0.4%), whereas mean %PB of teicoplanin in samples from infected patients who received the antibiotic was 87.7 ± 4.2% (range: 79.6–95.4%). ConclusionA novel highly sensitive UHPLC-HRMS method was developed and validated for the quantification of total and free teicoplanin in human K2EDTA plasma samples. Amongst others, this method is suitable for therapeutic drug monitoring.