Liquid Chromatography-high Resolution Mass Spectrometry Method Research Articles

7Method for Detection of Naturally Occurring Toxins in Human Urine Using Liquid Chromatography High Resolution Mass Spectrometry.

Natural toxins present an ongoing risk for human exposure that requires a rapid, accurate diagnosis for proper response. In this study, a qualitative liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed and validated for the detection of a large, diverse selection of natural toxins. Data-dependent acquisition was performed to identify compounds with an in-house mass spectral library of 129 hazardous toxins that originate from plants, animals, and fungi. All 129 compounds were spiked into human urine, extracted, and evaluated for spectral library matching. Of these, 92 toxins met the quality criteria and underwent validation in urine matrix based on American National Standards Institute (ANSI) guidelines. A generalized workflow for method expansion was developed and enables the rapid addition of relevant compounds to the established method. This LC-HRMS method achieves efficient detection of natural toxins in urine, and the created workflow can rapidly increase compound coverage via method expansion.

Identification of organic contaminants in water and related matrices using untargeted liquid chromatography high-resolution mass spectrometry screening with MS/MS libraries

Nontargeted and suspect screening with liquid chromatography-high resolution mass spectrometry (LC-HRMS) has become an indispensable tool for quality assessment in the aquatic environment – complementary to targeted analysis of organic (micro)contaminants. An LC-HRMS method is presented, suitable for the analysis of a wide variety of water related matrices: surface water, groundwater, wastewater, sediment and sludge, including extracts from passive samplers and on-site solid phase enrichment, while focusing on the data processing aspect of the method. A field study is included to demonstrate the practical application and versatility of the whole process. HRMS/MS data were recorded following LC separation in both (ESI) positive and negative ionization modes using data dependent as well as data independent acquisition. Two vendor (Agilent's Personal Compound Database and Library and from National Institute of Standards and Technology) and one open (MassBank/EU) tandem mass spectral libraries were utilized for the identification of compounds via mass spectral match. The development of a novel software tool for parsing, grouping and reduction of MS/MS features in data files converted to mascot generic format (MGF) helped to substantially decrease the amount of time and effort needed for MS library search. While applying the method, in the course of the entire field study, 18771 detections (from 870 individual compounds) in total were recorded in 275 samples, resulting in 68.3 identified compounds per sample, on average. Among the top ten most frequently detected contaminants across all samples and sample types were pharmaceutical compounds carbamazepine, 4-acetamidoantipyrine, 4-formylaminoantipyrine, tramadol, lamotrigine and phenazone and industrial contaminants toluene-2-sulfonamide, tolytriazole, tris(2-butoxyethyl) phosphate and benzotriazole. Exploratory data analysis methods and tools enabled us to discover organic pollutant occurrence patterns within the comprehensive sets of qualitative data collected from various projects between the years 2018–2023. The results may be used as valuable inputs for future water quality monitoring programs.

Enhanced Surveillance of >1100 Pesticides and Natural Toxins in Food: Harnessing the Capabilities of LC-HRMS for Reliable Identification and Quantification

The consequences of climate change along with diverse food regulations and agricultural practices worldwide are complexifying the occurrence and management of chemical contaminants in food. In this context, we present an ultra-high-performance liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach for the simultaneous identification and quantitation of over 1100 pesticide residues, mycotoxins, and plant toxins in cereals and fruits and vegetables. Analytical conditions were optimized to maximize the scope of the targeted molecules, the reliability of compound identification, and quantification performance within a single method. The method was further transferred and validated in another laboratory to assess its ruggedness. Validation according to the SANTE 11312/2021v2 guidelines showed that 92% and 98% of the molecules fulfill the quantification criteria at the lowest validated level in the cereals and fruits and vegetables groups, respectively. Analysis of fifteen certified reference materials led to a 96% satisfactory rate of z-scores confirming method’s competitiveness. Furthermore, the occurrence of these contaminants was studied in 205 cereals and grains samples collected worldwide. The low µg/kg quantification limits make this LC-HRMS method a valuable tool to ensure compliance toward regulations and to screen for non-regulated substances for which occurrence data are crucial for an appropriate risk evaluation.

LC-HRMS analysis of phospholipids bearing oxylipins

BackgroundSeveral oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed. ResultsOptimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC. SignificanceThe developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs. The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism. The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity). A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS) Fingerprinting and Chemometrics for Coffee Classification and Authentication.

Nowadays, the quality of natural products is an issue of great interest in our society due to the increase in adulteration cases in recent decades. Coffee, one of the most popular beverages worldwide, is a food product that is easily adulterated. To prevent fraudulent practices, it is necessary to develop feasible methodologies to authenticate and guarantee not only the coffee's origin but also its variety, as well as its roasting degree. In the present study, a C18 reversed-phase liquid chromatography (LC) technique coupled to high-resolution mass spectrometry (HRMS) was applied to address the characterization and classification of Arabica and Robusta coffee samples from different production regions using chemometrics. The proposed non-targeted LC-HRMS method using electrospray ionization in negative mode was applied to the analysis of 306 coffee samples belonging to different groups depending on the variety (Arabica and Robusta), the growing region (e.g., Ethiopia, Colombia, Nicaragua, Indonesia, India, Uganda, Brazil, Cambodia and Vietnam), and the roasting degree. Analytes were recovered with hot water as the extracting solvent (coffee brewing). The data obtained were considered the source of potential descriptors to be exploited for the characterization and classification of the samples using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). In addition, different adulteration cases, involving nearby production regions and different varieties, were evaluated by pairs (e.g., Vietnam Arabica-Vietnam Robusta, Vietnam Arabica-Cambodia and Vietnam Robusta-Cambodia). The coffee adulteration studies carried out with partial least squares (PLS) regression demonstrated the good capability of the proposed methodology to quantify adulterant levels down to 15%, accomplishing calibration and prediction errors below 2.7% and 11.6%, respectively.

Carbon-doped Co3O4-MgO catalyzed peroxymonosulfate activation via an enhanced Co(III)/Co(II) cycle for rapid chloramphenicol degradation

Advanced oxidation processes based on heterogeneous peroxymonosulfate activation (PMS) have been a feasible strategy for removing organic pollutants. In this study, the waste filter membranes in the laboratory were used as a carbon material, which combines with cobalt and magnesium to obtain the carbon-doped cobalt-magnesium binary oxides (C/Co3O4–MgO) after calcination to activate PMS for chloramphenicol (CAP) degradation. The results revealed that the degradation efficiency of CAP in the C/Co3O4–MgO/PMS system reached 98% within 15 min, much higher than those in C/Co3O4/PMS (62%) and Co3O4–MgO/PMS (68%) systems. The excellent catalytic performance of the C/Co3O4–MgO material was attributable to a good synergistic interaction between C, Co, and Mg. Electron paramagnetic resonance test and quenching experiments confirmed that •OH, SO4•−, and 1O2 were reactive oxygen species involved in CAP degradation. The intermediates of CAP degradation were analyzed via a high-performance liquid chromatography–high-resolution mass spectrometry method, and the possible degradation pathways of CAP were proposed. C/Co3O4–MgO exhibited good stability and reusability. The concentration of leached Co ions was lower than 0.53 mg L−1, and over 95% of CAP was still decomposed in the fifth cycle. Furthermore, the degradation of rhodamine B, sulfamethazine, and phenacetin via PMS activation by C/Co3O4–MgO was investigated. These three pollutants are almost completely degraded within 10 min. Therefore, C/Co3O4–MgO is an excellent heterogeneous catalyst used for activating PMS to degrade organic pollutants.

Targeted and Suspect Fatty Acid Profiling of Royal Jelly by Liquid Chromatography-High Resolution Mass Spectrometry.

Royal jelly (RJ) is a bee product produced by the mandibular and hypopharyngeal glands of worker honeybees which has attracted special attention because of its numerous pharmacological activities and its applications to dermatology and cosmetics. In 2020, we demonstrated a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the determination of seven medium-chain FFAs in RJ samples. The aim of the present work was to extend our studies on FA profiling of RJ, exploring the presence of common long-chain saturated, mono-unsaturated and poly-unsaturated free FAs in RJ samples using this LC-HRMS method. Among twenty common FAs studied by a targeted approach, palmitic acid, stearic acid and oleic acid were found at concentrations higher than the rest of the FAs (the concentrations of these three acids ranged from 37.4 to 48.0, from 17.7 to 24.0 and from 9.4 to 11.1 mg/100 g of fresh RJ, respectively). The high mass accuracy of LC-HRMS allowed the application of a suspect approach, which enabled the exploration of various C9 and C11 FAs, as well as hydroxylated C12 FAs. Nonenoic acid was indicated as the most abundant among these acids. In addition, for the first time, the presence of a variety of regio-isomers of hydroxymyristic, hydroxypalmitic and hydroxystearic acids was demonstrated in RJ samples.

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.

Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools.

The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs. SIGNIFICANCE STATEMENT: Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

Fluoro-cotton assisted non-targeted screening of organic fluorine compounds from rice (Oryza sativa L.) grown in perfluoroalkyl substance polluted soil

The toxicity and environmental persistence of perfluorooctanoic acid (PFOA), and perfluorooctane sulfonate (PFOS) are of great concern for food intake in humans. However, PFASs conversion or conjugation to other substances in rice grown on PFASs polluted soil has not been explored clearly. These unknown transformed or conjugated products of PFOA and PFOS could be harmful to human health. The restriction factor in evaluating the possible transformation of PFOA and PFOS is mainly attributed to the lack of an efficient method for screening PFOA and PFOS and their related metabolites. To circumvent this challenge, we established a non-targeted screening method by combining a fluoro-cotton fiber-based solid phase extraction (FC-SPE) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) to monitor the formation of possible organic fluorine compounds from rice (Oryza sativa L.) grown on PFASs. We synthesized fluoro-cotton fibers to serve as the FC-SPE packing material and characterized by field-emission scanning electron-microscope, Fourier transform infrared, and X-ray photoelectron spectroscopy measurements. The optimal extraction conditions for the prepared FC-SPE were investigated. The performance of FC-SPE in LC-MS analysis was validated by linearity, precision, recovery, and matrix effect. Then the FC-SPE combined with LC-HRMS was used to specifically capture organic fluorine compounds from complex matrices via F–F interaction, including rice seedlings grown in PFOA and PFOS polluted soil and soil samples. By the established FC-SPE LC-HRMS method, in total 429 features were found as the possible organic fluorine compounds from rice seedlings grown in PFOA polluted soil among the 1781 features from the rice seedlings. Finally, we employed a13C metabolic tracing analysis of organic fluorine compounds in combination with the FC-SPE LC-HRMS method to further identify the features that detected from rice seedlings grown in PFOA polluted soil. The final result indicated that there were not any new organic fluorine metabolites screened out from rice grown in PFOA or PFOS polluted soil.

Development and application of a High-Resolution mass spectrometry method for the detection of fentanyl analogs in urine and serum

IntroductionThe use of illicitly manufactured synthetic opioids, specifically fentanyl and its analogs, has escalated exponentially in the United States over the last decade. Due to the targeted nature of drug detection methods in clinical laboratories and the ever-evolving list of synthetic opioids of concern, alternative analytical approaches are needed. MethodsUsing the fentanyl analog screening (FAS) kit produced by the Centers for Disease Control and Prevention (CDC), we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) synthetic opioid spectral library and data acquisition method using information dependent acquisition of product ion spectra. Chromatographic retention times, limits of detection and matrix effects, in urine and serum, for the synthetic opioids in the FAS kit (n = 150) were established. All urine and serum specimens sent to a clinical toxicology laboratory for comprehensive drug testing in 2019 (n = 856) and 2021 (n = 878) were analyzed with the FAS LC-HRMS library to determine the prevalence of fentanyl analogs and other synthetic opioids, retrospectively (2019) and prospectively (2021). ResultsThe limit of detection (LOD) of each opioid ranged from 1 to 10 ng/mL (median, 2.5 ng/mL) in urine and 0.25–2.5 ng/mL (median, 0.5 ng/mL) in serum. Matrix effects ranged from −79 % to 86 % (median, −37 %) for urine, following dilution and direct analysis, and −80 % to 400 % (median, 0 %) for serum, following protein precipitation. The prevalence of fentanyl/fentanyl analogs in serum samples increased slightly from 2019 to 2021 while it remained the same in urine. There were only 2 samples identified that contained a fentanyl analog without the co-occurrence of fentanyl or fentanyl metabolites. Analysis of the established MS/MS spectral library revealed characteristic fragmentation patterns in most fentanyl analogs, which can be used for structure elucidation and drug identification of future analogs. ConclusionsThe LC-HRMS method was capable of detecting fentanyl analogs in routine samples sent for comprehensive drug testing. The method can be adapted to accommodate testing needs for the evolving opioid epidemic.

Standard-Free Quantification of Dicarboxylic Acids: Case Studies with Salt-Rich Effluents and Serum.

The present study evaluates the ionization efficiency (IE) of linear and branched C2-C14 dicarboxylic acids (DCAs) by electrospray ionization (ESI) under different conditions. The influence of the concentration of organic modifier (MeOH); mobile phase additive; and its concentration, pH, and DCA structure on IE values is studied using flow injection analysis. The IE values of DCAs increase with the increase of MeOH concentration but also decrease with an increase of pH. The former is due to the increase in solvent evaporation rates; the latter is caused by an ion-pairing between the diacid and the cation (ammonium), which is confirmed by the study with different amines. The investigation of DCA ionization in the presence of different acidic mobile phase additives showed that a significant improvement in the (-)ESI responses of analytes was achieved in the presence of weak hydrophobic carboxylic acids, such as butyric or propanoic acid. Conversely, the use of strong carboxylic acids, such as trichloroacetic acid, was found to cause signal suppression. The results of the IE studies were used to develop the liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method that provided instrumental limits of detection in the range from 6 to 180 pg. Furthermore, upon applying the nonparametric Gaussian process, a model for the prediction of IE values was developed, which contains the number of carbons in the molecule and MeOH concentration as model parameters. As a case study, dicarboxylic acids are quantified in salt-rich effluent and blood serum samples using the developed LC-HRMS method.

Prevalence of Licit and Illicit Drugs Use during Pregnancy in Mexican Women.

For the first time, the present study employed hair testing to investigate the prevalence of classical drugs of abuse and new psychoactive substances use during gestation in a cohort of 300 Mexican pregnant women. An interview was conducted to collect data on sociodemographic aspects of the patients, and a 9 cm-long hair strand was taken from the back of the head of each mother one month after delivery. A validated ultra-high-performance liquid chromatography–high-resolution mass spectrometry method was used for the screening of classic drugs, new psychoactive substances, and medications in maternal hair. Out of 300 examined hair samples from pregnant women, 127 (42.3%) resulted positive for psychoactive substances: 45 (35.4%) for cannabis only, 24 (18.9%) for methamphetamine only, 13 (10.2%) for cocaine only, 1 (0.3%) for heroin, 1 for N-N-dimethyltryptamine (0.3%), 1 for ketamine (0.8%), and 35 (16.3%) for more than one psychoactive substance. Furthermore, seven samples (2.3%) resulted positive for new psychoactive substances (NPS): two samples for synthetic cannabinoids, two for synthetic cathinones, and three for nor-fentanyl, and 3.3% of women hair resulted positive for anticonvulsant, antidepressant, and antipsychotic medications. Finally, 83 women hair samples (27.7%) tested positive for nicotine. Nonsteroidal anti-inflammatory drugs (NSAIDs) and other painkillers (60.0%), medications for the treatment of nausea and vomiting (12.3%), antihistamines (8.7%) and nasal/sinus decongestants (6.7%), cough suppressants (5.0%), and bronchodilator agents (5.0%) were also detected in pregnant women hair. The gestational use of psychoactive substances and exposure to tobacco smoke, assessed by hair testing, were associated with a significantly younger age and with a low education grade of the mothers (p < 0.005). This study provides a significant preliminary indication of the under-reported gestational consumption of licit and illicit psychoactive and pharmacologically active drugs in a Mexican environment, showing the value of toxicological and forensic analyses in the global effort to determine the health risks caused by classic drugs and new psychoactive substances during pregnancy.

Detection of Methylphenidate in Equine Hair Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography–high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid–liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.

Mycotoxins Exposure of French Grain Elevator Workers: Biomonitoring and Airborne Measurements.

It is now recognized that additional exposure to mycotoxins may occur through inhalation of contaminated dust at a workplace. The aim of this study was to characterize the multi-mycotoxin exposure of French grain elevator workers using biomonitoring and airborne measurements. Eighteen workers participated in the study. Personal airborne dust samples were analyzed for their mycotoxin concentrations. Workers provided multiple urine samples including pre-shift, post-shift and first morning urine samples or 24 h urine samples. Mycotoxin urinary biomarkers (aflatoxin B1, aflatoxin M1, ochratoxin A, ochratoxin α, deoxynivalenol, zearalenone, α-zearalenol, β-zearalenol, fumonisin B1, HT-2 toxin and T-2 toxin) were measured using a liquid chromatography–high-resolution mass spectrometry method. Grain elevator workers were highly exposed to organic airborne dust (median 4.92 mg.m−3). DON, ZEN and FB1 were frequent contaminants in 54, 76 and 72% of air samples, respectively. The mycotoxin biomarkers quantified were DON (98%), ZEN (99%), α-ZEL (52%), β-ZEL (33%), OTA (76%), T-2 (4%) and HT-2 (4%). DON elimination profiles showed highest concentrations in samples collected after the end of the work shift and the urinary DON concentrations were significantly higher in post-shift than in pre-shift-samples (9.9 and 22.1 µg/L, respectively). ZEN and its metabolites concentrations did not vary according to the sampling time. However, the levels of α-/β-ZEL were consistent with an additional occupational exposure. These data provide valuable information on grain worker exposure to mycotoxins. They also highlight the usefulness of multi-mycotoxin methods in assessing external and internal exposures, which shed light on the extent and pathways of exposure occurring in occupational settings.

Development and validation of a LC-HRMS method for the quantification of cannabinoids and their metabolites in human plasma

The resurgence of Cannabis therapeutic discoveries have led to the need for sensitive and selective analytical methods for the detection of cannabinoids and their metabolites in biological matrices. High resolution mass spectrometry (HRMS) enables good sensitivity and provides more selectivity due to its accurate mass measurement of the targeted compounds. The aim of this study was to develop and validate a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantitative analysis of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (Δ9-THC) and its major metabolites 11-Hydroxy-Δ9-THC and 11-Nor-9-carboxy-Δ9-THC in human plasma. The method utilized a simple liquid-liquid extraction of the cannabinoids from plasma samples followed by an isocratic chromatographic separation and detection by ESI-HRMS Q-Exactive plus platform. The lower limit of quantification (LLOQ) was 0.2 ng/ mL for the targeted cannabinoids and its metabolites with sample volume of 0.5 mL plasma. The method was linear from 0.2 to 100.0 ng/mL with an average correlation coefficient of >0.995 using weighted (1/x) linear least-squares regression. No significant carry-over was noticed for all analytes and the extraction recovery ranged from 60.4 % to 85.4 %. Dilution results indicated no influence on the accuracy of analysis. The method's intra-day and inter-day precision (CV %) ranged from 2.90 to 10.80 % and accuracy within -0.9 to 7.0 from nominal. Matrix effect ranged from 1.1 % to 49.8 %. The analytes were stable in the autosampler for 6 and 12 h, respectively. This method was sensitive and can be applicable to cannabinoids pharmacokinetics study.

A High-Resolution Liquid Chromatography-Mass Spectrometry Method for Identification of Toxic Natural Products in Clinical Cases

Abstract Introduction/Objective Many natural products have biological effects on humans and animals. Poisoning caused by natural products is often found in clinical toxicology cases. Liquid chromatography-high-resolution-mass spectrometry (LC-HRMS) has recently emerged as a powerful analytical tool for large-scale target screening, and the application of LC-HRMS can be expanded to solve the clinical cases of natural product poisoning. Methods The LC-HRMS method is based on a spectral library of 121 natural products. The spectral library was constructed by analyzing standards either in a Q-TOF mass spectrometer (only MS2 spectra acquired) or in an Orbitrap Tribrid mass spectrometer (MS2 and MS3 spectra acquired). Results The LC-HRMS method was verified for the limit of detection (LOD) and matrix effects in both serum and urine matrices. For each compound, the LOD was evaluated from 1.0 ng/ml to 1000 ng/ml for urine samples and from 0.50 ng/ml to 500 ng/ml for serum samples. The matrix effects were determined at three concentration levels andranged from 30.4% to 123.5% for urine samples and from 23.4% to 132.9% for serum samples. The LC-HRMS method was successfully applied to identify the culprits in three clinical cases. In addition, the combined use of MS2 and MS3 spectra enhanced the accuracy of compound identification, in library search reducing the importance of retention time that varies among instruments and consumable lots. In Case 1, the patient presented with paresthesias, arrhythmias, and stiffened arms and legs. The toxic alkaloid aconitine was identified in the serum sample and the extract of herbs that the patient ingested. In Case 2, the patients presented with weakness, dizziness, and vomiting. The symptoms were caused by mistakenly taking Nicotiana glauca leaves and the alkaloid anabasine was identified as the culprit. In Case 3, the patients were suspected of intoxicated by taking too much extract of lupini beans. The culprit alkaloids from lupini beans lupanine and sparteine were found in the serum samples. Conclusion The involvement of a toxicology laboratory with the capability to perform the LC-HRMS method and with experience in the investigation of undifferentiated cases provides a unique diagnostic advantage in cases where exposure to toxic substances is possible.

Measurement of hydroxychloroquine in blood from SLE patients using LC-HRMS\u2014evaluation of whole blood, plasma, and serum as sample matrices

BackgroundHydroxychloroquine (HCQ) is the standard of care in the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other inflammatory rheumatic diseases and potentially for the treatment in COVID-19 patients. Determination of HCQ for therapeutic drug monitoring (TDM) can be performed in whole blood (WB), serum, and plasma. Direct comparisons of WB, serum, and plasma levels of HCQ in patients with SLE have not previously been reported. We describe a method for the determination of HCQ in human blood using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices.MethodsA method for the determination of HCQ in human blood using LC-HRMS was developed, validated, and applied for the determination of HCQ levels in WB, serum, and plasma from 26 SLE patients. The reproducibility of the method, in the three matrices, was evaluated using quality control samples and repeated preparations and measurements of patient samples. The performance of the developed method for HCQ measurement in serum was further evaluated by comparison with two previously reported extraction methods.ResultsThe performance of the presented method demonstrated high accuracy and precision. A large range of HCQ concentrations was observed for the SLE patients in all three matrices (WB, serum, and plasma). The mean levels in WB were approximately two-fold the levels in serum and plasma (813 ng/mL compared to 436 ng/mL and 362 ng/mL, respectively). Spiked quality controls showed high reproducibility for all matrices (coefficient of variation, CV, approx. 5%), whereas in patient samples, equally high-precision was only found using WB as the matrix (CV 3%). The CV for serum and plasma was 14% and 39%, respectively. Two alternative methods applied to serum samples did not demonstrate improved precision.ConclusionsA LC-HRMS method for the measurement of HCQ in human blood was developed and validated. Whole blood was found to be the superior sample matrix in terms of sample reproducibility. Thus, whole blood samples should be used for HCQ analysis when patients are monitored for HCQ treatment effects. The assay is in clinical use to monitor levels of HCQ in patients.

Stability of prostaglandin E1 solutions stored in polypropylene syringes for continuous intravenous administration to newborns

ObjectiveWe aimed to monitor the physicochemical stability of prostaglandin E1 (PGE1) 1.5 and 15 µg/mL in 10% dextrose stored in polypropylene syringes.MethodsWe developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS)...