Tandem Mass Spectrometry Detection Research Articles

Multi-immunoaffinity column for the simultaneous analysis of bisphenol A and its analogues in Chinese foods by liquid chromatography tandem mass spectrometry.

Bisphenol A (BPA) and its four analogues have been receiving considerable attention owing to their potential endocrine disrupting effects. The European Food Safety Authority has proposed 0.04ng/kg·body weight/day of thetemporary tolerable daily intake for BPA. Therefore, a more sensitive analytical method was urgently needed for the necessity of the risk reassessment of bisphenols (BPs). The matrix effect of Chinese foods is a challenge for the analysis of ultra-trace analytes due to the presence of various spices. A multi-immunoaffinity column (mIAC) was prepared for the purification of BPA, BPB, BPF, BPS, and BPAF in Chinese foods following ultra-high-performance liquid chromatography tandem mass spectrometry detection (UHLPC-MS/MS). The recoveries of each of BPs were ranged from 84.6% to 116.7%, and the intra-day precision and inter-day precision were ranged from 1.6% to 12.4%, and from 4.1% to 14.0%, respectively. This is the first report on the mIACs for simultaneous clean-up and analysis of BPs in complex Chinese foods.

Application of a multi-toxin detect method to analyze mycotoxins occurrence in plant-based beverages

In recent years, plant-based beverages have gained popularity on the market due to environmental and ethical concerns, as well as milk intolerances and allergies. However, raw materials employed in the manufacture of these products are susceptible to mycotoxin contamination. For this reason, a new method based on a QuEChERS extraction procedure followed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) detection was developed for the analysis of 29 mycotoxins in oat, rice, soy, and almond beverages. The method was validated in terms of linearity, detection and quantification limits, matrix effect, recoveries, accuracy and precision. Satisfactory performance characteristics were achieved, with recoveries above 70% for most mycotoxins. Several commercial samples were analyzed, aflatoxins were frequently detected in rice and almond beverages, while T-2 and HT-2 toxins were identified in oat-based products. In addition, emerging mycotoxins such as enniatins and beauvericin were detected in the four types of beverages.

Determination of underivatized amino acids in human plasma using ion pair liquid chromatography/tandem mass spectrometry

Accurate quantification of amino acids (AA) is essential for several applications, including clinical research, food analysis, and pharmaceutical studies. In this study, we developed an analytical method based on liquid chromatography with electrospray ionization coupled to tandem mass spectrometry detection (LC-ESI-MS/MS). This method was devised to accurately quantify a spectrum of amino acids, notably taurine, creatinine, glutathione (GSH), and sulfur-containing amino acids (SAAs) such as methionine, cysteine, and homocysteine, using only 10 μL of human plasma. A stable isotope derivative of each AA is used as an internal standard (IS) for accurate quantification. For retention and separation on a C18 column, heptafluorobutyric acid (HFBA) was employed as an ion pair agent. Multiple reaction monitoring (MRM) in positive mode with the precursor-to-product ion transitions at m/z is used for quantification. The method showed excellent linearity for all AA with a high correlation coefficient (r > 0.9927). The linear fit indicates that the detector response is linear over the tested range of standard concentrations. The accuracy and precision of the method were within the acceptable range of 92–110% and < 15%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were in the range of 0.001–1.80 µM and 0.004–6.0 µM, respectively. No significant ion suppression or carry over was observed. In conclusion, the assay was validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The assay has been successfully applied to the analysis of human plasma.

Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry

Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (μSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the μSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN–H2O (15:85, v/v)–1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the μSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed μSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 μg mL−1 in plasma. LOQ values were 2 μg mL−1 for INDS and 7 μg mL−1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

Determination of Aflatoxins in Peanut Oil by One-Step Solid-Phase Extraction with Metal-Organic Framework MIL-101 (Cr) Filled Syringe Filter and HPLC-MS/MS

A simple, rapid and sensitive method for the determination of aflatoxins in peanut oil was developed using MIL-101(Cr) material filled syringe filter for one-step solid-phase extraction (SPE) with high-performance liquid chromatography—tandem mass spectrometry (HPLC-MS/MS) detection. The MIL-101(Cr) sorbent was characterized and its mass, the loading solution, and the eluent were optimized; under these conditions, a matrix matching internal standard method was developed. The linear range was from 0.1 to 20 ng/mL with correlation coefficient (r) exceeding 0.9998. The recoveries were between 94.7% and 105.9% with relative standard deviations from 2.02% 6.88%. The recoveries of inter-day and intra-day measurements were from 93.0% to 98.5% and 97.6% to 100.7%. The precision values for inter-day and intra-day analyses were from 6.09% to 7.68% and 2.56% to 4.30%, respectively. The pretreatment efficiency of the developed method was superior to literature procedures and used to determine aflatoxins in 126 peanut oil samples from local markets. 38 samples had positive results and 1 sample exceeded the allowable limit. Hence, the developed protocol was suitable for the determination of aflatoxins in peanut oil.

Assessment of the presence of UV filters and UV stabilizers in stranded dolphin blubber

The presence of ultraviolet filters (UVFs) and stabilizers (UVSs) was evaluated for the first time in the common bottlenose dolphin (Tursiops truncatus). UVFs and UVSs are compounds of growing concern because their effects on the environment are not completely known. UVFs and UVSs are added to personal care products (PCPs), such as cosmetics and products related to sun care and once released to the aquatic ecosystem, marine organisms can bioaccumulate these substances.This work aimed to determine the presence of 12 UVFs and UVSs in cetacean blubber samples to assess the pollution to which these animals of the highest trophic chain levels are exposed due to human activity. Analytical determinations were carried out using a method based on microwave-assisted extraction combined with ultrahigh-performance liquid chromatography and tandem mass spectrometry detection. The developed method was successfully applied to determine the target compounds in the blubber tissues of five necropsied common bottlenose dolphins.Three of the 12 studied compounds, namely 2-ethylhexyl 2-cyano-3,3-diphenylprop-2-enoate (octocrilene, OC), 2-hydroxy-4-methoxybenzophenone (benzophenone 3, BP3) and 3-methylbutyl (E)-3-(4methoxyphenyl) prop-2-enoate (IMC), were detected in several samples. Of the identified compounds, OC was present in all the samples and at the highest concentration within the range from 52.61 ± 18.59 to 108.0 ± 11.32 ng·g−1.

Analytical method for the determination of usually prescribed antibiotics in human nails using UHPLC-MS/MS. Comparison of the efficiency of two extraction techniques

Antibiotics are a group of drugs used for the treatment of bacterial diseases. They are used in both human and veterinary medicine and, although they are not permitted, they are sometimes used as growth promoters. The present research compares two extraction techniques: ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) in order to evaluate their efficiency in the determination of 17 usually prescribed antibiotics in human nails. The extraction parameters were optimized using multivariate techniques. Once both techniques were compared, MAE was selected as optimal due to its greater experimental practicability together with the better extraction efficiencies it provides. Target analytes were detected and quantified by ultra-high performance liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). The run time was 20 min. The methodology was then successfully validated, obtaining acceptable analytical parameters according to the guide used. Limits of detection were between 0.3 and 3 ng g−1 and limits of quantification were in the range from 1.0 to 4.0 ng g−1. Recovery percentages ranged from 87.5% to 114.2%, and precision (in terms of standard deviation) was less than 15% in all cases. Finally, the optimized method was applied to nails taken from 10 volunteers and the results revealed the presence of one or more antibiotics in all the samples examined. The most commonly found antibiotic was sulfamethoxazole, followed by danofloxacin and levofloxacin. The results demonstrated, on the one hand, the presence of these compounds in the human body and, on the other hand, the suitability of nails as a non-invasive biomarker of exposure.

Hidden threat lurking in extensive hand hygiene during the Covid-19 pandemic: investigation of sensitizing molecules in gel products by hyphenated chromatography techniques

During the Covid-19 pandemic, health agencies worldwide have recommended frequent handwashing and sanitizing. A variety of hand gel products were made available on the market, often with fragrances added to curtail the strong smell of alcohol. Commonly used Citrus fragrances contain volatile aroma constituents and non-volatile oxygen heterocyclic compounds (OHCs), consisting mostly of polymethoxyflavones, coumarins, and furocoumarins. The latter have long been investigated for their phototoxic properties, and their safety as cosmetic product ingredients has been debated recurrently. To this concern, twelve commercial Citrus-scented products were investigated in this study. An extraction method was optimized for thirty-seven OHC compounds, obtaining absolute mean recovery values in the 73.5–116% range with only few milliliters of solvent consumption. Analysis by ultra-high-pressure liquid chromatography with tandem mass spectrometry detection evidenced that three samples did not conform to the labeling requirements for fragrance allergens (coumarin) laid down by the European Union Regulation on Cosmetic Products. The total furocoumarin (FC) content of the samples investigated was in the 0.003–3.7ppm range, with some noteworthy exceptions. Specifically, in two samples, the total FCs were quantified as 89 and 219 ppm, thus exceeding the safe limits recommended up to a factor of 15. Finally, the consistency of the volatile fingerprint attained by gas chromatography allowed drawing conclusions on the authenticity of the Citrus fragrances labeled, and several products did not conform to the information reported on the label concerning the presence of essential oils. Besides the issue of product authenticity, analytical tools and regulatory actions for widespread testing of hand hygiene products are urgent, to protect consumers’ health and safety.Graphical

MetaboMSDIA: A tool for implementing data-independent acquisition in metabolomic-based mass spectrometry analysis

Data-dependent acquisition (DDA) is the most widely used mode in untargeted metabolomic analysis despite its limited tandem mass spectrometry (MS2) detection coverage. We present MetaboMSDIA for complete processing of data-independent acquisition (DIA) files by the extraction of multiplexed MS2 spectra and further identification of metabolites in open libraries. In the analysis of polar extracts from lemon and olive fruits, DIA allows one to obtain multiplexed MS2 spectra for 100% of precursor ions compared to 64% of precursor ions from average MS2 acquisition in DDA. MetaboMSDIA is compatible with MS2 repositories and homemade libraries prepared by analysis of standards. An additional option is based on filtering molecular entities by searching for selective fragmentation patterns according to selective neutral losses or product ions to target the annotation of families of metabolites. Combining both options, the applicability of MetaboMSDIA was tested by annotating 50 and 35 metabolites in polar extracts from lemon and olive fruit, respectively. MetaboMSDIA is particularly proposed to increase the acquisition coverage in untargeted metabolomics and to improve spectral quality, which are two critical pillars for the tentative annotation of metabolites. The R script used in MetaboMSDIA workflow is available at github repository (https://github.com/MonicaCalSan/MetaboMSDIA).

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Quantitative determination of polychlorinated biphenyls in chicken based on QuEChERS extraction and GC-MS/MS detection

Polychlorinated biphenyls (PCBs), a class of synthetic organic compounds consisting of chlorine atoms on two coupled biphenyl rings, have been declared class A carcinogens by the World Health Organization. Here, we report a new method for the determination of PCBs content in chicken. The method employs QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction, followed by gas chromatography and tandem mass spectrometry (GC-MS/MS) detection for the identification and quantification of seven indicative PCBs (PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, PCB180). The PCBs were extracted by acetone with an Association of Official Analytical Chemists (AOAC) extraction kit, followed by a clean-up step using primary secondary amine, octadecyl silica, florisil, and anhydrous magnesium sulfate. PCBs in the extracts were subsequently quantified by GC-MS/MS. The method was validated, and the correlation coefficients of the curves were found to be higher than 0.99. The limits of detection for the seven indicative PCBs ranged from 0.1 to 0.5 ng/g, and the limits of quantification were 0.3–1.5 ng/g. Satisfactory recovery rates ranged from 96% to 115%, with a maximum relative standard deviation of 9%. For simple pretreatment and accurate detection, this method is quick and efficient in detecting batches of seven indicative PCBs in chicken.

Validation of an isotope dilution mass spectrometry (IDMS) measurement procedure for the reliable quantification of steroid hormones in waters.

Reliable data are compulsory to efficiently monitor pollutants in aquatic environments, particularly steroid hormones that can exert harmful effects at challenging analytical levels below the ng L-1. An isotope dilution two-step solid-phase extraction followed by an ultra-performance liquid chromatography separation coupled to tandem mass spectrometry (UPLC-MS/MS) detection method was validated for the quantification of 21 steroid hormones (androgens, estrogens, glucocorticoids, and progestogens) in whole waters. To achieve a realistic and robust assessment of the performances of this method, the validation procedure was conducted using several water samples representative of its intended application. These samples were characterized in terms of concentration of ionic constituents, suspended particulate matter (SPM), and dissolved organic carbon contents (DOC). For estrogens that are part of the European Water Framework Directive Watchlist (17beta-estradiol and estrone), the performances met the European requirements (decision 2015/495/EU) in terms of limit of quantification (LQ) and measurement uncertainty. For 17alpha-ethinylestradiol, the challenging LQ of 0.035ng L-1 was reached. More generally, for 15 compounds out of 21, the accuracy, evaluated in intermediate precision conditions at concentrations ranging between 0.1 and 10ng L-1, was found to be within a 35% tolerance. The evaluation of the measurement uncertainty was realized following the Guide to the expression of Uncertainty in Measurement. Finally, a water monitoring survey demonstrated the suitability of the method and pointed out the contamination of Belgium rivers by five estrogens (17alpha-ethinylestradiol, estriol, 17alpha-estradiol, 17beta-estradiol, and estrone) and three glucocorticoids (betamethasone, cortisol, and cortisone) which have been up to now poorly documented in European rivers.

Development of a detection method for 10 non-steroidal anti-inflammatory drugs residues in four swine tissues by ultra-performance liquid chromatography with tandem mass spectrometry

The ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) detection method was developed for the residues of 10 NSAIDs (salicylic acid, acetylsalicylic acid, acetaminophen, diclofenac, tolfenamic acid, antipyrine, flunixin meglumine, aminophenazone, meloxicam, metamizole sodium) in swine muscle, liver, kidney, and fat. Swine tissue samples were extracted by phosphorylated acetonitrile with the addition of an appropriate amount of internal standard working solution, defatted with acetonitrile-saturated n-hexane, and purified by Hydrophile-Lipophile Balance (HLB) solid-phase extraction column, then separated by UPLC BEH shield RP18 column with 0.1% formic acid in water/0.1% formic acid in acetonitrile with gradient elution, which was detected in the multiple reaction monitoring (MRM) modes. The correlation coefficient of the standard curve equation is greater than 0.99, and the coefficient of variation within and between batches is less than 14.4%. We evaluated the analytical method using two green assessment tools. The method established in this study met the requirements of NSAID residue analysis and provides analytical tools for determining and confirming NSAIDs in swine tissue samples. This is the first report on the simultaneous determination of 10 NSAIDs in four swine tissues by the UPLC-MS/MS method and accurate quantification using deuterated internal standards.

In the case of extensively putrefied bodies, the analysis of entomological samples may support and complement the toxicological results obtained with other alternative matrices

Entomotoxicological analysis is not part of routine toxicological analysis. This work aims to present two cases to illustrate the potential of entomological samples as complementary matrices to identify substances in cases of advanced putrefaction. (Case#1) A woman wasexhumed after 14 months to ascertain the exact cause of death. She died after six weeks of hospitalization because of intestinal ischemia followed by multiorgan failure. (Case#2) The corpse of a woman, known to have a psychiatric disorder, was discovered in her apartment. The state of decomposition of the body was consistent with a post-mortem period of several weeks (approximately 6 weeks). Toxicological investigations were performed in the biological and entomological samples of case#1 (hair, adipocere, brain, and pupae) and of case#2 (hair, bone, flies, and pupae) using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, several drugs and metabolites were detected. In particular, the pupae analyses allowed the objectification of morphine administration, whereas morphine was only found in adipocere, but not in hair nor in brain. In case#2, the pupae analyses allowed the detection of three metabolites of quetiapine, and the flies analyses allowed the detection of valpromide, which was only detected in hair. In conclusion, the pupae and flies analyses in these two cases complemented the results obtained in the other alternative biological samples, which may guide hypotheses about the possible causes of death. Nevertheless, additional data and case reports would be of benefit to assess the value of entomotoxicology in routine forensic investigations.

Efficacy of Alternative Dose Regimens of Exemestane in Postmenopausal Women With Stage 0 to II Estrogen Receptor–Positive Breast Cancer

Successful therapeutic cancer prevention requires definition of the minimal effective dose. Aromatase inhibitors decrease breast cancer incidence in high-risk women, but use in prevention and compliance in adjuvant settings are hampered by adverse events. To compare the noninferiority percentage change of estradiol in postmenopausal women with estrogen receptor-positive breast cancer given exemestane, 25 mg, 3 times weekly or once weekly vs a standard daily dose with a noninferiority margin of -6%. This multicenter, presurgical, double-blind phase 2b randomized clinical trial evaluated 2 alternative dosing schedules of exemestane. Postmenopausal women with estrogen receptor-positive breast cancer who were candidates for breast surgery were screened from February 1, 2017, to August 31, 2019. Blood samples were collected at baseline and final visit; tissue biomarker changes were assessed from diagnostic biopsy and surgical specimen. Biomarkers were measured in different laboratories between April 2020 and December 2021. Exemestane, 25 mg, once daily, 3 times weekly, or once weekly for 4 to 6 weeks before surgery. Serum estradiol concentrations were measured by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry detection. Toxic effects were evaluated using the National Cancer Institute terminology criteria, and Ki-67 was assessed by immunohistochemistry. A total of 180 women were randomized into 1 of the 3 arms; median (IQR) age was 66 (60-71) years, 63 (60-69) years, and 65 (61-70) years in the once-daily, 3-times-weekly, and once-weekly arms, respectively. In the intention-to-treat population (n = 171), the least square mean percentage change of serum estradiol was -89%, -85%, and -60% for exemestane once daily (n = 55), 3 times weekly (n = 56), and once weekly (n = 60), respectively. The difference in estradiol percentage change between the once-daily and 3-times-weekly arms was -3.6% (P for noninferiority = .37), whereas in compliant participants (n = 153), it was 2.0% (97.5% lower confidence limit, -5.6%; P for noninferiority = .02). Among secondary end points, Ki-67 and progesterone receptor were reduced in all arms, with median absolute percentage changes of -7.5%, -5.0%, and -4.0% for Ki-67 in the once-daily, 3-times-weekly, and once-weekly arms, respectively (once daily vs 3 times weekly, P = .31; once daily vs once weekly, P = .06), and -17.0%, -9.0%, and -7.0% for progesterone receptor, respectively. Sex hormone-binding globulin and high-density lipoprotein cholesterol had a better profile among participants in the 3-times-weekly arm compared with once-daily arm. Adverse events were similar in all arms. In this randomized clinical trial, exemestane, 25 mg, given 3 times weekly in compliant patients was noninferior to the once-daily dosage in decreasing serum estradiol. This new schedule should be further studied in prevention studies and in women who do not tolerate the daily dose in the adjuvant setting. ClinicalTrials.gov Identifier: NCT02598557; EudraCT: 2015-005063-16.

Development of the thin film solid phase microextraction (TF-SPME) method for metabolomics profiling of steroidal hormones from urine samples using LC-QTOF/MS.

In the present study, the development and optimization of a thin film solid phase microextraction method (TF-SPME) was conducted for metabolomics profiling of eight steroid compounds (androsterone, dihydrotestosterone, dihydroepiandrosterone, estradiol, hydroxyprogesterone, pregnenolone, progesterone and testosterone) from urine samples. For optimization of extraction method, two extraction sorbents (PAN-C18 and PS-DVB) were used as they are known to be effective for isolation of low-polarity analytes. The stages of sample extraction and analyte desorption were considered as the most crucial steps in the process. Regarding the selection of the most suitable desorption solution, six different mixtures were analyzed. As a result, the mixture of ACN: MeOH (1:1, v/v) was chosen in terms of the highest analytes' abundances that were achieved using the chosen solvent. Besides other factors were examined such as the volume of desorption solvent and the time of both extraction and desorption processes. The analytical determination was carried out using the ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry detection in electrospray ionization and positive polarity in a scan mode (UHPLC-ESI-QTOF/MS). The developed and optimized TF-SPME method was validated in terms of such parameters as extraction efficiency, recovery as well as matrix effect. As a result, the extraction efficiency and recovery were in a range from 79.3% to 99.2% and from 88.8% to 111.8%, respectively. Matrix effect, calculated as coefficient of variation was less than 15% and was in a range from 1.4% to 11.1%. The values of both validation parameters (recovery and matrix effect) were acceptable in terms of EMA criteria. The proposed TF-SPME method was used successfully for isolation of steroids hormones from pooled urine samples before and after enzymatic hydrolysis of analytes.

Validation of the quantification of phosphatidylethanol 16:0/18:1 concentrations in TASSO-M20 devices.

Phosphatidylethanol 16:0/18:1 (PEth), found in whole blood, is a biomarker for alcohol consumption with high sensitivity, specificity, and a long detection window. The TASSO-M20 device is used to self-collect capillary blood from the upper arm and has advantages over finger stick methods. The purpose of this study was to (1) validate PEth measurement using the TASSO-M20 device, (2) describe the TASSO-M20 for blood self-collection during a virtual intervention, and (3) characterize PEth, urinary ethyl glucuronide (uEtG) and self-reported alcohol in a single participant over time. PEth levels in blood samples dried on TASSO-M20 plugs were compared to those in (1) liquid whole blood (N=14) and (2) dried blood spot cards (DBS; N=23). Additionally, the self-reported drinking, positive or negative uEtG results (dip card cutoff ≥300 ng/mL), and observed self-collection of blood with TASSO-M20 devices for PEth levels were obtained over time during virtual interviews of a single contingency management participant. High-performance liquid chromatography with tandem mass spectrometry detection was used to measure PEth levels for both preparations. PEth concentrations from dried blood on TASSO-M20 plugs and liquid whole blood were correlated (0 to 1700 ng/mL; N=14; r2 =0.988; slope=0.951) and in a subgroup of samples with lower concentrations (N=7; 0 to 200 ng/mL; r2 =0.944, slope=0.816). PEth concentrations from dried blood on TASSO-M20 plugs and DBS were correlated (0 to 2200 ng/mL; N=23; r2 =0.927; slope=0.667) and in a subgroup of samples with lower concentrations (N=16; 0 to 180 ng/mL; r2 =0.978, slope=0.749). Results of the contingency management participant indicate that changes in PEth levels (TASSO-M20) and uEtG concentrations were consistent with each other and with changes in self-reported alcohol use. Our data support the utility, accuracy, and feasibility of using the TASSO-M20 device for blood self-collection during a virtual study. The TASSO-M20 device had multiple advantages over the typical finger stick method, including consistent blood collection, participant acceptability, and less discomfort as indicated by acceptability interviews.

Comparison of two methods for ginsenosides quantitation.

The present study provides a comparison of two liquid chromatography-tandem mass spectrometry methods for ginsenosides analysis. The two methods have the same liquid chromatography separation procedure, and both use tandem mass spectrometry detection. However, one method uses multiple reaction monitoring transitions commonly recommended in the literature starting with [M + Na]+ as the molecular ions and with detection of specific fragment ions from the molecules M, while the other is an original method using [M + Cs]+ as molecular ions and Cs+ as fragment ion. The method using [M + Cs]+ as molecular ion has a very high sensitivity allowing the measurement of concentrations in the injecting solutions as low as 4ng/ml with peaks at this concentration showing signal to noise ratio of 20 or higher. The procedures were utilized for the measurement of eight ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf (S), Rg1, and Rg2), although the method using [M + Cs]+ has the potential for measuring other ginsenosides. As an application, the ginsenosides were measured in several types of ginseng root, several dietary supplements containing ginseng extracts, four energy drinks, and a sample of ashwagandha.

Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.

Defects in deoxyribonucleoside triphosphate (dNTP) metabolism are associated with a number of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders affect the muscles, liver, and brain, and the concentrations of dNTPs in these tissues are already normally low and are, therefore, difficult to measure. Thus, information about the concentrations of dNTPs in tissues of healthy animals and animals with MDS are important for mechanistic studies of mtDNA replication, analysis of disease progression, and the development of therapeutic interventions. Here, we present a sensitive method for the simultaneous analysis of all four dNTPs as well as all four ribonucleoside triphosphates (NTPs) in mouse muscles using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. The simultaneous detection of NTPs allows them to be used as internal standards for the normalization of dNTP concentrations. The method can be applied for measuring dNTP and NTP pools in other tissues and organisms.

Determination of vitamin D3 conjugated metabolites: a complementary view on hydroxylated metabolites.

Experts typically define vitamin D deficiency levels by the determination of a circulating 25-hydroxyvitamin D3-calcifediol prohormone. A large part of the population is characterized by deficient vitamin D levels (calcifediol < 20 ng mL-1) despite individuals not being affected by any disorder. Cholecalciferol (vitamin D3) and/or calcifediol supplementation is a common practice for vitamin D-deficient individuals as recommended by international scientific societies and official agencies. In the last few years, several studies have reported the presence of conjugated vitamin D3 metabolites, mainly glucuronidation and sulfation derivatives, although simultaneous quantitative measurements involving phase I and II vitamin D metabolites have not been carried out. A quantitative method based on tandem mass spectrometry detection is proposed here for the combined determination of phase I and phase II vitamin D3 metabolites in human serum. As phase I and phase II metabolites are preferentially ionized in different modes, a switching polarity mode was adopted to determine both groups of compounds in serum at high sensitivity levels (pg mL-1). The validation of this proposal was successfully accomplished by following the Center for Drug Evaluation and Research (CDER) guidelines. Its applicability was tested in a cohort of volunteers with mostly deficient baseline levels. Considering the sulfated form of calcifediol, the sum of its concentrations showed sufficient baseline vitamin D levels in all individuals, suggesting that this could be a novel strategy for vitamin D deficiency definition. Therefore, phase II metabolites are proposed to be included when evaluating the vitamin D status since they provide more information about the overall status of the vitamin D endocrine system. Nevertheless, further studies are required to confirm the biological activity of these conjugated metabolites and the suitability of this strategy for the description of vitamin D deficiency.