Mass Spectrometry Method Research Articles

Selective quantification of estrogens in serum with aptamer-affinity and LC-MS/MS analysis

In the previous study, we developed an accurate and automated immunologic mass spectrometry (iMS) method for the simultaneous quantification of multiple steroid hormones by target capturing with monoclonal antibodies and LC-MS/MS analysis. However, antibodies are less accessible to a wide variety of low-molecular-weight analytes, which limited the application of iMS method. At the same time, multiple monoclonal antibodies needed also increased the complexity and cost of the iMS assay. In the present study, we developed an aptamer-affinity and LC-MS/MS analysis method with a class-specific aptamer for the selective enrichment and simultaneous determination of estrogens (estrone (E1) and estradiol (E2)) in human serum. The DNA aptamer was coupled to magnetic beads to enrich the target estrogens from serum sample after protein precipitation. The magnetic beads were then washed and eluted with acetonitrile solution, and targets were analyzed with LC-MS/MS. The sample preparation process of enrichment, washing and elution works in an automatic manner. Matrix effect was negligible as the high specificity of target recognition and enrichment by the aptamer. The aptamer-affinity assay for estrogen quantification was validated for sensitivity, linearity, accuracy and precision. The limit of detection (LOD) and quantification (LOQ) were0.01 ng/mLand0.02 ng/mL for E1 and E2, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch CVs of5.5 %-7.3 % and 5.2 %-6.0 % for E1 and E2, respectively, and inter-batch CVs of 3.8 %-10.0 % and 5.2 %-6.3 % for E1 and E2, respectively. Recoveries were in the range of99.1 %-107.0 % and 94.9 %-107.7 % for E1 and E2, respectively. Quantification results of 40 serum samples by the aptamer-affinity and solid-phase extraction (SPE) LC-MS/MS method showed good consistency, with the correlation coefficient of 0.93 and 0.98 for E1 and E2, respectively. Clinical application of the aptamer-affinity and LC-MS/MS method was further demonstrated for quantifying serum estrogen levels of 170 pregnant women at different gestational weeks.

Ultrafast metaproteomics for quantitative assessment of strain isolates and microbiomes

BackgroundMicrobial communities are essential in human health and environmental regulation, but present a challenge for the analytical science due to their diversity and dynamic range. Tandem mass spectrometry provides functional insights on microbial life cycle, but is time-consuming. MALDI TOF excels in rapid species identification, but not functional assessment. To address critical challenges in human health and environmental sustainability, microbiology needs advanced mass spectrometry methods and bioinformatic tools enabling both rapid identification and accurate assessment of functional activity of microbial communities. ResultsWe show for the first time that both identity and functional activity of microorganisms and their communities can be accurately determined in experiments as short as 7 min per sample, using the basic Orbitrap MS configuration without peptide fragmentation. The approach was validated using strain isolates, mock microbiomes composed of bacteria spiked at known concentrations and human fecal microbiomes. Our new bioinformatic algorithm identifies the bacterial species with an accuracy of 95 %, when no prior information on the sample is available. Microbiome composition was resolved at the genus level with the mean difference between the actual and identified components of 12 %. For mock microbiomes, Pearson coefficient of up to 0.97 was achieved in estimates of strain biomass change. By the example of Rhodococcus biodegradation of n-alkanes, phenols and its derivatives, we showed the accurate assessment of functional activity of strain isolates, compared with the standard label-free and label-based approaches. SignificanceOur approach makes microbial proteomics fast, functional and insightful using the Orbitrap instruments even without employing peptide fragmentation technology. The approach can be applied to any microorganisms and can take a niche in routine functional assessment of microbial pathogens and consortiums in clinical diagnostics together with MALDI TOF MS and 16S rRNA gene sequencing.

Tandem mass spectrometry-based assay for heparan-N-sulphatase in paediatric CSF: A potential pharmacodynamic biomarker for mucopolysaccharidosis type IIIA therapy

Mucopolysaccharidosis type IIIA is a lysosomal storage disorder caused by mutations in the gene coding for heparan-N-sulphatase, a crucial enzyme in the degradation of heparan sulfate. In mucopolysaccharidosis type IIIA, heparan sulfate accumulates in the lysosomes, predominantly affecting the central nervous system. It is the most common and most severe form of mucopolysaccharidosis type III, with onset typically before the age of ten years. There is an ongoing effort to develop therapies that aim at restoring enzyme function in the brain. This study introduces a novel tandem mass spectrometry method for assessing heparan-N-sulphatase activity in pediatric cerebrospinal fluid from healthy and disease individuals.Analysis of cerebrospinal fluid samples revealed marked differences in enzyme activity, with mucopolysaccharidosis type IIIA individuals exhibiting significantly reduced levels. This new method could serve as a valuable tool for evaluating the efficacy of future therapeutic interventions targeting sulphatase activity restoration in the brain.

B-171 Iohexol Mass Spectrometry Method Validation and Comparison in Clinical Pediatric Laboratory

Abstract Background Accurate assessment of kidney function is important yet challenging in pediatric patients due to changing muscle mass affecting serum creatinine. There are several key methods to assess kidney function in this population, including estimated glomerular filtration rate (eGFR) from serum creatinine, urine creatinine clearance, and eGFR from serum iohexol clearance, which often provides a more reliable estimate. Iohexol is a non-ionic iodinated contrast agent filtered freely through the glomeruli without tubular secretion, making it an ideal GFR marker.In this study, we validated the LC-MS/MS method to quantitatively measure iohexol in clinical pediatric plasma and serum samples. The clinical performance was compared with the novel Verispray method. Methods A series of iohexol standards (Caymen Chemical) were prepared at 10, 100, 500, 1000 µg/mL in serum (UTAK). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and Verispray method were developed to quantitatively measure iohexol concentrations in spiked pediatric specimens. The internal standard (IS) solution was prepared with Iohexol-D5 (Cayman Chemical) at 9 µg/mL in UPLC grade methanol. Specimen or calibration/QC (50 µL) were mixed with IS solution (450 µL), and then centrifuged at 14,000xg for 3 minutes. The supernatants were analyzed. For LC-MS/MS, 3 µL supernatants were chromatographed on a AccucoreTM Vanquish C18 50 x 2.1mm (Thermo Scientific) using a gradient of water with 0.1% formic acid (FA) against methanol with 0.1% FA. Iohexol and IS were measured by multiple reaction monitoring using a TSQ Quantis Plus Mass Spectrometer (ThermoFisher). For Verispray, 10 µL supernatants were loaded on the Verispray sample plate (ThermoFisher) without LC portion. The samples were extracted from the plate paper using 95% acetonitrile with 5% water and 0.1% FA before directly injected into the mass spectrometer. The LC-MS/MS method was evaluated for linearity, patient comparison, carryover, matrix effect, and stability. 40 patients were analyzed between methods. Results Linearity was assessed over 5 days at 4 calibrator levels, the coefficient of variation (CV) ranged from 3.46 - 5.99%. The patient comparison of 40, 20 serum and plasma respectively, was conducted with Mayo Clinics, and good concordance was observed. Matrix effects were evaluated with spiked samples prepared with 50% mobile phase A and B, the ion enhancement was at 10.7%, within the allowable limits. No significant carryover was seen. The samples were stable at room temperature for 48 hours and 4oC for 7 days. Freezing is not recommended. The comparison between Verispray and LC-MS/MS showed significant bias between and within the performing personnel, the deviation can be up to 40% comparing to LC-MS/MS method. Conclusions The LC-MS/MS method demonstrated stable and consistent analytical performance for iohexol measurement. However, the Verispray method showed significant inter- and intra-user variabilities. As the Verispray sample spot can only be used once, respotting is required for any additional measurement, reducing reproductivity. It is highly recommended to duplicate all samples when using Verispray, to limit preparation to one user per patient run. Overall, our validation indicates the LC-MS/MS method provides reliable performance as a useful alternative to other complex gold standard pediatric tests of kidney function.

A-099 Case Study of a Patient With Heterophile Antibodies Interfering with Both Competitive and Immunometric Assays

Abstract Background Immunoassay results may be affected by the presence of interfering substances including heterophile antibodies. These heterophile antibodies can cause either an increase or decrease in the result based on assay design and antibody target. We report a case study wherein we identified several analytes whose results were either increased or decreased by heterophile antibodies in a patient. Methods 74 year male presented to Endocrinology with history of hypocalcemia and his previous lab tests were suggestive for idiopathic hypoparathyroidism, Hashimoto’s disease and Addison’s disease raising physician concern for a possible Type 1 autoimmune endocrinopathy. Cortisol was ordered (for an ACTH stimulation test for adrenal insufficiency); upon testing the post-stimulation cortisol result was higher than the analytical measurement range (AMR), and so the sample was diluted 1:2 and repeated. The repeat cortisol result after correction for the dilution factor was well within the AMR and prompted an investigation for an interfering substance. Serial dilution of analytes was performed and send-out testing for the same with another immunoassay from a different manufacturer and/or mass spectrometry method where available were done. Results The results for most analytes did not match with the reference lab results. Serial dilution showed non-parallelism in the results. For most analytes, after dilution of the sample by 1:4 or 1:8 times, the interfering substance was diluted enough to obtain a result matching the reference lab result. Conclusions Based on non-parallelism in serial dilution results, discordant results with other methods, and loss of interference after dilution it was determined that the most likely cause was a heterophile antibody. Formal testing using blocking antibodies was not done as it seemed that the reference lab used did not run our method and their assays were not affected by this particular heterophile antibody. Analysis of the assay designs and analyte results showed that competitive assays showed falsely high results, while immunometric assays showed falsely low results. The physician was contacted and the case discussed in detail and it was recommended that further testing on this individual be sent out to a reference lab using the alternate method or to use mass spectrometry based assays for further testing if needed. Today, the patient continues to have low calcium, high phosphate and an undetectable PTH suggestive of idiopathic hypoparathyroidism but his other endocrine functions are within normal limits.

Advances in Chiral Metabolomic Profiling and Biomarker Discovery.

Chiral metabolomics entails the enantioselective measurement of the metabolome present in a biological system. Over recent years, it has garnered significant interest for its potential in discovering disease biomarkers and aiding clinical diagnostics. D-Amino acids and D-hydroxy acids, traditionally overlooked as unnatural, are now emerging as novel signaling molecules and potential biomarkers for a range of metabolic disorders, brain diseases, kidney disease, diabetes, and cancer. Despite their significance, simultaneous measurements of multiple classes of chiral metabolites in a biological system remain challenging. Hence, limited information is available regarding the metabolic pathways responsible for synthesizing D-amino/hydroxy acid and their associated pathophysiological mechanisms in various diseases. Capitalizing on recent advancements in sensitive analytical techniques, researchers have developed various targeted chiral metabolomic methods for the analysis of chiral biomarkers. Here, we highlight the pivotal role of chiral metabolic profiling studies in disease diagnosis, prognosis, and therapeutic interventions. Furthermore, we describe cutting-edge chromatographic and mass spectrometry methods that enable enantioselective analysis of chiral metabolites. These advanced techniques are instrumental in unraveling the complexities of disease biomarkers, contributing to the ongoing efforts in disease biomarker discovery.

A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

B-282 Cost and Benefits of Utilization of Ion Mobility Mass Spectrometry Testing for Drugs of Abuse Compared to Immunoassay Screening in a Large Urban Hospital

Abstract Background Urine drugs of abuse testing (DOAU) in US hospitals are mainly orders from the emergency department performed by immunoassay screening. Testing can be relatively expensive, and results may suffer from non-idealities in sensitivity, specificity, and from panel limitations. A potential approach to these issues for immunoassays is instead to employ mass spectrometry (MS), where a large and flexible analyte list can be evaluated providing needed specificity and reducing send out costs for confirmatory testing. We compared results and cost impacts of a DOAU assay on a prototype, ion mobility mass spectrometry system to those obtained from an array of immunoassays offered as routine tests at a large urban hospital. Methods Immunoassay screen-positive and screen-negative urine samples were isolated from among those submitted for a DOAU panel (amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, opiates, and methadone) or for additional single drug tests (fentanyl, oxycodone, phencyclidine, buprenorphine, propoxyphene). Sample aliquots (3 mL) were retrieved after not more than 5 days of refrigerated storage of the original sample and were subsequently frozen. These samples were then subjected to MS analysis using the prototype system where identification of 62 compounds was based on mass and ion mobility-derived Collision Cross Section (CCS). An established library of m/z, SLIMCCSHe, peak intensity, and peak width for each analyte was used, with acceptable method performance characteristics as based on applicable CLIA validation guidelines. The MS list included compounds covered by the immunoassay classes as well as additional drugs not expected to be positive among the immunoassays test results but of value for routine toxicological assessments. Results comparisons, utility of results, sample-to-result turnaround times, and overall financial implications were compared between the two workflows. Results Among immunoassay screen-negative samples, MS analysis showed additional positive results for metabolites of parent drugs as well as novel psychoactive substances and known adulterants in >10% of cases. Among the immunoassay screen-positive results, MS analysis confirmed immunoassay results (>90%) but picked up an additional positive component in over 15% of cases. Cost per complete patient result for the immunoassay was approximately $2.80 in comparison to an estimated cost of $5 for the MS method. Confirmation testing for positive immunoassay screen results (16% of all samples) would cost an additional $70 per sample, whereas the need for confirmation testing is eliminated for the MS method. Thus, use of the MS testing workflow would be superior to an immunoassay with reflex testing workflow while at the same time providing a clinically superior service of more diverse positive test results and elimination of often overlooked false negatives. Turnaround time between immunoassay (1h) and MS (50m) were judged to be similar. Conclusions The MS approach confirmed >90% of all immunoassay positive screen results. In addition, the MS method identified additional positive results not detected by the immunoassay despite an overall similar estimated time for sample to patient. Need for development of a user friendly, MS analyzer for use in hospitals is supported based on cost-effective and analytically superior tests for DOAU compared to screens performed using immunoassay panels.

B-170 Age-Specific reference intervals for ethanolamine plasmalogen species in red blood cells using liquid chromatography tandem mass spectrometry

Abstract Background Plasmalogens are critical membrane structural components that are mainly generated by de novo synthesis starting in peroxisomes. Hence, patients with defects in peroxisome biogenesis (PBD) exhibit markedly reduced plasmalogen levels. Plasmalogen ratios are traditionally measured by gas chromatography-mass spectrometry (GC-MS); however, this method entails a lengthy sample extraction and derivatization and does not report concentrations of individual plasmalogen species. We have developed a robust and easy-to-implement liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the 18 most abundant ethanolamine plasmalogen (PlsEtn) species in packed red blood cells (RBCs). However, no reference intervals have been published for individual PlsEtn. Here, we describe the establishment of age-specific reference intervals for individual PlsEtn species and their total values (16:0, 18:0, 18:1 species, and total plasmalogens). Methods Plasmalogens were extracted using methanol containing two labeled internal standards, shaking for one hour at room temperature. Chromatographic separation was performed using an Acquity Premier BEH C18 UPLC column with a binary gradient of 5 mM ammonium acetate in water:methanol (15:85) and 5 mM ammonium acetate in methanol. Analysis was performed using a XEVO TQ-XS Mass Spectrometer with Ultra-High Performance Liquid Chromatography (Waters) in multiple reaction monitoring mode. Eighteen PlsEtn species were quantified using four commercially available standards; additionally, totals were calculated for 16:0, 18:0 or 18:1 species, and for total plasmalogens. Reference intervals were established using 376 RBCs from self-reported healthy volunteers and de-identified clinical samples referred for unrelated testing (182 females and 194 males; range 0 to 88 years). Data was analyzed using the R programming language. The study was approved by the Institutional Review Board of the University of Utah. Results Initial age groups were identified using a model-based clustering algorithm followed by iterative Harris-Boyd analysis. Finally, the adjacent groups were merged if their means differed by less than 10%. Once the final age groups were partitioned, data in each individual age group were analyzed using parametric or non-parametric statistics to determine reference intervals (95%, with 90%confidence intervals). PlsEtn species displayed the lowest concentration in the first few months of life, which increased in childhood until adolescence or adulthood (depending on PlsEtn). For most species, the concentrations increased over time reaching a plateau between 18 and 48 years of age, and then starting to decrease. The total values followed the same trend, with neonates showing significantly lower values compared to other age groups. Conclusions We applied a novel statistical approach to identify age groups and determine age-specific reference intervals for 18 individual PlsEtn in RBC and their totals. Lacking previously published data, this study is critical for supporting test implementation in clinical laboratories.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

A-001 The Postprandial Levels of the Metabolites in the Metaorganismal Trimethylamine N-Oxide (TMAO) Pathway are Altered in A Diet-, Microbe-, and Sex-Specific Manner

Abstract Background Cardiovascular disease (CVD) remains the leading cause of death in developed countries. Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis, using samples obtained from fasting patients. Although the strong association between TMAO levels and CVD risk has been established in fasting blood samples, here we are investigating the potential for a single meal to dynamically alter TMAO-related metabolites based on sex, diverse dietary substrates, and microbial influences. Methods 35 healthy participants were randomized to one of four study groups with approximately 8-9 subjects in each arm. Half of the participants were randomly assigned to receive a three-day broad spectrum antibiotic regimen (ciprofloxacin, metronidazole, and vancomycin) while the others received no antibiotics. These two groups were further subdivided into a group consuming a highly processed meal (originating from local fast food restaurants) or alternatively a whole food meal (containing a variety of fruits, vegetables, and healthy fiber). Blood samples were taken at baseline (after an overnight fast), and then postprandially at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after meal ingestion. Targeted plasma metabolites were quantified using a stable isotope dilution, liquid chromatography - tandem mass spectrometry (LC-MS/MS) method Results While plasma TMAO levels between food groups did not change significantly within the total cohort, there were clear individualized responses to either highly processed or whole food meals. Separation based on sex showed that TMAO levels were significantly reduced in females at 6 hours but remained steady in males for both food groups. Particularly, in the processed food group, TMAO levels were significantly lower in females than males at the 6-hour time point. Neither of these observations held true for TMAO precursors choline, carnitine, or betaine. However, plasma levels of a dietary precursor for TMAO, γ-butyrobetaine, showed clear diet-microbe-host interactions. For males in the processed food group, plasma γ-butyrobetaine levels were significantly increased in subjects on broad spectrum antibiotics. Conclusions Our results show the postprandial levels of TMAO, and its nutrient precursors dynamically change in a diet-, microbe-, and sex-dependent manner. These findings provide new insights into the postprandial levels of TMAO-related metabolites and may inform precision nutritional approaches in those who could benefit from TMAO-lowering strategies.

A-316 A quantitative gas chromatography tandem mass spectrometry method for metabolic profiling of urine organic acids and acylglycines

Abstract Background The analysis of urine organic acids (UOA) and acylglycines (UAG) are an essential investigation for the diagnosis inborn errors of metabolism (IEMs). The vast majority of methods for these analytes use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity and enables the combination of qualitative screening with quantitative analysis. We describe the development and validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA and UAG to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. Results The optimal temperature gradient for high-resolution UOA and UAG separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N=36) derivatized analytes. Total analysis time was 33.8 min per injection. This method permits the measurement of 11 samples in one batch and is highly linear for all analytes (R^2 >0.99 and average slope 1.04). The limit of quantification (LOQ) was established and ranged from <0.05 to 2.1 µmol/mmol creatinine. Intra- and inter-day imprecision (CV%) was determined using matrix-matched abnormal and normal QC. Intra-day imprecision (N=10) ranged from 1.3% (2-ketoglutaric acid) to 9.7% (3-hydroxybutyric acid). Inter-day imprecision (N=30 over 15 days) ranged from 2.2% (ethylmalonic acid and methylmalonic acid) to 20.5% (3-hydroxy-3-methylglutaric acid). The method had an average accuracy of 113±14% for analytes included in the ERNDiM quantitative organic acids (urine) external quality assurance program. For analytes not included in this proficiency testing, the method showed good agreement when compared to a national reference laboratory, with average Deming correlation coefficients and regression slopes being 0.9944 and 1.1, respectively. No interferences were detected when monitoring the analyte quantifier and qualifier ion ratios in patient urine. Conclusions The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA and UAG used to investigate IEMs. The method has acceptable sensitivity, specificity, precision and accuracy for clinical routine clinical testing.

Determination of Macrolides in Water Using Solid-Phase Extraction and Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

Macrolides are a group of compounds used to treat bacterial infections in humans and animals. Their widespread use results in the contamination of the water environment, which, on the one hand, has a detrimental effect on aquatic organisms and, on the other hand, can lead to the emergence of resistant strains of microorganisms. All of the above determines the need for monitoring of these compounds in the environment, particularly, in water objects. Usually, the high-performance liquid chromatography combined with tandem mass spectrometry method is used to solve this problem, however, this work shows the possibility of using the supercritical fluid chromatography-tandem mass spectrometry method. An approach for the determination of four common macrolides, namely erythromycin, clarithromycin, midecamycin, and josamycin, was developed. The use of solid-phase extraction allowed to achieve limits of quantification at 0.57-6.8ng/L. The presented approach was validated and tested on a real object-a sample of municipal wastewater.

Development and Validation of an Eco-Friendly Liquid Chromatography–Tandem Mass Spectrometry Method for Rapid Quantification of Γ-Aminobutyric Acid in Cordyceps Sinensis and Its Related Species

Introduction: Cordyceps sinensis, a renowned traditional Chinese medicinal substance, contains pharmacologically beneficial compounds, including γ-aminobutyric acid (GABA), necessitating efficient analytical methods for quality assessment. This study aims to develop a rapid, cost-effective, and eco-friendly liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for GABA quantification in Cordyceps sinensis and its related species. The objective is to enhance the quality control standards within the Cordyceps sinensis industry and for those of its related species. Methods: The study innovated a swift analytical procedure. Optimal conditions were determined through systematic evaluations of extraction solvents, durations, and chromatographic settings, prioritizing speed and solvent minimization. Results: The proposed LC–MS/MS method achieved precise quantification of GABA in a reduced time frame and significantly lowered solvent consumption, enhancing method efficiency by 22-fold compared to reported LC methods. The method exhibited robustness and scored the highest blue applicability grade index score, underscoring its suitability for academic and industrial applicaition. Conclusion: The validated LC–MS/MS approach offers a sustainable avenue for the rapid and accurate measurement of GABA in Cordyceps sinensis and its related species, improving the quality control process and ensuring product authenticity. This method stands out as a model for green analytical chemistry, propelling forward the eco-friendly analytical evaluation of health-related compounds.

The evolution of amino acids under asteroidal aqueous alteration

Carbonaceous chondrites contain amino acids, with variable abundances and isotope compositions between and within carbonaceous chondrites. The parent body processes, and the presence of clay minerals may explain those differences. Here, we experimentally investigate the evolution of 6 amino acids (glycine, β-alanine, α-alanine, 2-aminoisobutyric acid, γ-aminobutyric acid, and isovaline) exposed to hydrothermal conditions in the presence or absence of silicates. We determined the chemical nature and isotopic composition of the organic compounds of the soluble and solid fractions of the residues using X-ray diffraction, spectroscopy, and mass-spectrometry methods. Glycine and α-alanine exhibit a rather high stability, which is consistent with the measured abundances of α-alanine and glycine in chondrites having experienced various degrees of aqueous alteration. In the meantime, the evolution of β-alanine under hydrothermal conditions leads to the formation of a new compound, which likely results from the decarboxylation and deamination of β-alanine followed by recombination. More than 95 % of γ-ABA was transformed into 2-pyrrolidione though self-cyclization during the aqueous alteration. The solid residues of the experiments conducted in the presence of clay minerals contain organic material, with abundances varying depending on the amino acid used for the experiments (TOC isovaline > 2-aminoisobutyric acid > γ-aminobutyric acid > glycine > α-alanine > β-alanine). Clay minerals thus preferentially trap branched amino acids over chained amino acids, likely within their interlayer spaces as suggested by XRD data. The δ13C values of amino acids have not changed significantly during the experiments, even with the presence of silicates. Thus, the δ13C values of amino acids reported in CR and CM chondrites likely relate to synthetic conditions or the origin of their precursors (i.e. inherited from the pre-accretion processes).

Development and validation of an LC-MS/MS method for the detection of sodium pentachlorophenolate residues on cutting boards.

The objective of this study was to develop and validate an automated solid-phase extraction (SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the detection of sodium pentachlorophenolate (PCP-Na) residues on cutting boards. Given the potential hazards and environmental persistence of PCP-Na, a sensitive and reliable method is crucial for monitoring its residues in food contact materials to ensure consumer safety. Wood shavings from cutting boards were extracted using 10% methanol in water, followed by purification using an automated SPE system. The eluent was concentrated, reconstituted, and analyzed by UPLC-MS/MS. An isotope-labeled internal standard was used to mitigate matrix effects, enhancing detection sensitivity. The method was validated by assessing linearity, limit of detection (LOD), limit of quantification (LOQ), recovery rates, and relative standard deviations (RSDs) across various concentration levels. The method demonstrated excellent linearity over a concentration range of 0 to 100 μg/L with a regression equation of Y = 1.035X-0.7771 and an R² of 0.9996. The LOD and LOQ were determined to be 0.4 and 1.0 μg/kg, respectively. Recovery rates ranged from 71.75% to 96.50% with RSDs between 5.19% and 16.66%. When applied to 30 market cutting board samples, PCP-Na residues were detected in 50% of the samples, with concentrations ranging from 0 to 83,990 µg/kg. This study presents a robust UPLC-MS/MS method for the detection of PCP-Na on cutting boards, offering improved sensitivity and simplified sample preparation. The high detection rate in commercial samples underscores the need for stringent monitoring and regulatory measures to mitigate the exposure risk to consumers.

The Increasing Trend of Triazole-Resistant Candida from Vulvovaginal Candidiasis.

Candida vaginitis is widely prevalent worldwide and is one of the common gynecological disorders. The aim of this study is to analyze the sensitivity of recurrent vulvovaginal (RVVC) candidiasis to antifungal drugs and its relationship with vaginal microbiota. We Isolated and cultured Candida from RVVC patients, mass spectrometry and broth microdilution method were used to identify and determine MIC values of antifungal drugs. Clinical medical records and vaginal microbiota of RVVC patients were also collected. The main pathogens causing RVVC are predominantly Candida albicans (70.26%), but in recent years, there has been an increasing proportion of Candida glabrata(24.46%). However, only 15.70% of Candida albicans were sensitive to Voriconazole, 35.84% to Fluconazole and 25.60% to Itraconazole. No fluconazole-resistant Candida glabrata was found. Most Candida krusei strains were sensitive to voriconazole (81.80%). More important MIC values of triazoles were increased in Candida species, when exposed to clotrimazole. In addition, we found that the vaginal microecology of candida vaginitis and bacterial vaginitis was significantly different. Triazoles resistant Candida species have emerged, leading to the failure of empirical anti-infective therapy. At the same time, the vaginal microecology of candida vaginitis and bacterial vaginitis was significantly different. In addition, a new breakpoint for Candida from RVVC needs to be established.

Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry

Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

The Impact of Family Relationship on Serum 25-Hydroxyvitamin D (25OHD) Concentrations in Individuals Residing in Pune, India.

An understanding of the influence of family relationship on vitamin D concentrations could be useful in designing community-based strategies to improve vitamin D status. Hence, the aim of this study was to explore if family relationship had an impact on total serum 25-hydroxyvitamin D (25OHD) concentrations of individuals living in Pune, India. This cross-sectional study included 104 families. Data collected included anthropometry (height and weight), body composition (bioelectric impedance), sunlight exposure (validated questionnaire), and blood samples for estimation of serum 25OHD2 and 25OHD3 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Total serum 25OHD was computed as the sum of the serum 25OHD2 and 25OHD3 concentrations. Linear mixed model analysis was employed to assess the impact of family on serum 25OHD2 and total 25OHD concentrations. The strength of the family-level component was assessed by calculating the intra-family correlation for each model separately for children and adults. More than half (63%) of the participants of the participants (235 adults, 118 children) had poor daily sunlight exposure (< 30 minutes). Around one-third, both adults and children, were found to be vitamin D deficient. Intra-family correlation was found to be higher in children than in adults for serum 25OHD2 (0.6 vs 0.4) and total serum 25OHD (0.5 vs 0.1) concentrations. Our findings indicate that the likelihood of having similar vitamin D concentrations is higher among children as compared to adults in a family. This suggests a role played by genetic factors or possibly shared habits among children in a family, thus resulting in stronger correlations. This can facilitate the adoption of early preventive measures for treatment or prevention of vitamin D deficiency, even if one child among siblings is vitamin D insufficient.

Simultaneous determination of 102 synthetic cannabinoids in electronic cigarette oil by liquid chromatography-tandem mass spectrometry

Synthetic cannabinoids (SCs), which are among the most widely abused new psychoactive substances, are much more potent and have greater efficacy than natural cannabis. SCs can be disguised in various ways and are commonly sold in the form of electronic cigarette oil. SCs belong to a large family with structures consisting of a core with substituents, linker, ring with substituents, and tail. New SCs can be developed by adding substituents, such as halogen, alkyl, and alkoxy groups, to the aromatic ring system or by changing the alkyl chain length. Since the emergence of so-called first-generation SCs, subsequent developments have led to eighth-generation indole/indazole amide-based SCs. As of July 1, 2021, the entire category of SCs was added to the list of controlled substances, but implementation requires urgent improvements in detection technologies. Typically, each method is limited to a few SCs. Owing to the vast number of chemically diverse SCs and their fast update speed, the determination and identification of various types of SCs using a single method is challenging. Therefore, rapid, sensitive, and accurate quantitative methods that includes various types of SCs must be developed to meet the demand for the qualitative and quantitative analysis of new SCs in seized electronic cigarette oil. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 102 SCs in electronic cigarette oil. The mass spectrometry and liquid-phase conditions influencing SC separation and determination were optimized. Using the external standard method, 102 SCs were successfully identified in electronic cigarette oil. The samples were extracted using methanol. Target analytes were separated on a Shimadzu Shim-pack GIST-HP C18 AQ column (100 mm×2.1 mm, 1.9 μm) at a column temperature of 40 ℃. The mobile phases consisted of (A) 0.1% formic acid aqueous solution and (B) methanol-acetonitrile (1∶1, v/v). The gradient elution conditions were as follows: 0-8 min, 55%A-15%A; 8-15 min, 15%A; 15-16 min, 15%A-55%A; 16-18 min, 55%A. The flow rate was 0.4 mL/min and the injection volume was 1 μL. Operating in the multiple reaction monitoring mode, the 102 SCs were identified within 18 min. Each SC exhibited a good linear relationship in the range of 1-100.0 μg/L with a correlation coefficient (r)≥0.9915. The limits of detection were 0.01-0.30 μg/L and the limits of quantification were 0.04-0.99 μg/L, which meet the requirements for analyzing SCs in actual samples. Precision was determined using standard solutions with 2, 10, and 50 μg/L of the SCs. The precisions (n=6) were 0.3%-6.0%. The recoveries of the 102 SCs, as evaluated by spiking electronic cigarette oil at low (2 μg/mL), medium (10 μg/mL), and high (50 μg/mL) levels, were 80.1%-119.8%. Good performance was observed for the analysis of real samples. The developed method is accurate, rapid, sensitive, and effective for the determination of the 102 SCs in electronic cigarette oil, satisfying the requirements for practical qualitative and quantitative analysis.