Multiple Reaction Monitoring Method Research Articles

Targeted lipidomics-based study of radiation-induced metabolite profiles changes in plasma of total body irradiation cases

Purpose Lipidomics is an important tool for triaging exposed individuals, and helps early adoption of prevention and control strategies. The purpose of this study was to screen significantly perturbed lipids between pre- and post-irradiation of human plasma samples after total body irradiation (TBI) and explore potential radiation biomarkers for early radiation classification. Methods Plasma samples were collected before and after irradiation from 22 hospitalized cases of acute myeloid leukemia (AML) prepared for bone marrow transplantation. Acute total-body γ irradiation was performed at doses of 0, 4, 8, and 12 Gy. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM) method was utilized. Self-paired studies before and after irradiation were performed to screen potential lipid categorization markers and markers of dose-response relationships for radiation perturbation in humans. Based on the screened potential markers, a human TBI dose estimation model was developed. Results In total, 426 individual lipids from 14 major classes were quantified and 152 potential biomarkers with categorical characteristics were screened. A total of 80 lipids (32 TGs, 29 SMs, 9 FAs, 5 CEs, 5 PIs) were upregulated at 4 Gy, and a total of 91 lipids (39 SMs, 18 TGs, 15 HexCers, 7 CEs, 6 Cers, 3 LacCers, 2 LPEs, 1 PI) were upregulated at 12 Gy. Comparison of the ROC curves between the non-exposed and exposed groups at different doses showed AUC values ranging from 0.807 to 0.876. The metabolic pathways of potential lipid markers are mainly sphingolipid and glycerolipid metabolism, unsaturated fatty acid biosynthesis, fatty acid degradation and biosynthesis. Among the 13 dose-dependent radiosensitive lipids, CE (20:5), CE (18:1) and PI (18:2/18:2) were gradually incorporated into the TBI dose estimation model. Conclusion This study suggested that it was feasible to acquire quantitative lipid biomarker panels using targeted lipidomics platforms for rapid, high-throughput triage. Lipidomics strategies for radiation biodosimetry in humans were established with lipid biomarkers with good dose-response relationship.

A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring.

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.

Lipidomics analysis of bone marrow in a mouse model of postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a major public health problem worldwide, afflicting many postmenopausal women. Although many studies have focused on the biological role of individual lipids in osteoporosis, no studies have systematically elucidated the lipid profile of osteoporosis. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology based on multiple reaction monitoring (MRM) method was used to compare the levels of lipid molecules in bone marrow cells of osteoporotic mice (OVX) group and sham-operation (Sham) group. Principal component analysis (PCA) was used for multivariate statistics. Differential lipids were obtained by bar graph, heatmap and volcano map. A total of 400 lipid molecules were identified. A total of 199 lipid molecules were identified to be associated with PMOP, including 6 phospholipids and 3 sphingolipids. These differential lipid molecules provide a systematic lipid profile for osteoporosis, which helps to discover new candidate osteoporosis biomarkers, and their changes at the molecular level can be used as new targets for diagnosis or prevention.

Integrated identification-quantification (ID-Quant) workflow utilizing UPLC-QTOF-MS for the therapeutic drug monitoring of multi-component antibiotics without pure standards: Validation using teicoplanin

The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MSE full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.

The bone marrow lipidomics of mice reveal sex-related differences.

Osteoporosis is a common skeletal disorder characterized by an imbalance between bone resorption and formation, exhibiting a higher prevalence in women compared with men. While previous studies have primarily focused on genomics and genetics in osteoporosis susceptibility, there is a lack of systematic exploration of sex-specific differences in lipid levels in mouse bone marrow. Multiple reaction monitoring-based liquid chromatography-trandem mass spectrometry(LC-MS/MS) was used to quantify lipidomic profiles in bone marrow samples from three female mice and three male mice. The LC-MS/MS technique based on the multiple reaction monitoring method identified and quantified 184 lipids from 15 lipid classes. The contents of most lipids in the bone marrow cells of female mice were higher than those in male mice, including four polyunsaturated fatty acids, three phospholipids and four sphingolipids. Among all the lipid molecules, lactosylceramide (d18:0/16:0) showed the highest fold change in female mice, while its precursor lipid, glucosylceramide, was the most up-regulated in male mice. This study, focusing on bone marrow lipidomics, elucidates significant sexual dimorphism in lipid levels within bone marrow cells. It provides novel evidence supporting the higher prevalence of osteoporosis in women and enhances our understanding of the connection between sex-specific lipid levels and the risk of osteoporosis.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Co-stimulatory pathway competitive assay development using Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Discovery and identification of interspecies peptide biomarkers in the seahorse species using liquid chromatography tandem mass spectrometry and chemometrics

Seahorses have important edible and medicinal values including strengthening the body, tonifying the liver and kidneys, and reducing swelling. And there are abundant seahorse species on Earth. Many seahorses have large price differences due to the scarcity of resources, and some seahorses with similar appearances appear to be confused for use. While in market trading, Hippocampus is susceptible to loss of specialized morphology characteristics, making it difficult to distinguish between specific species. Here we report an effective method based on peptide biomarkers for the identification of seahorse species. Peptide biomarkers for each species were predicted using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with chemometrics software. One unique biomarker peptide for each species was synthesized and verified, and finally developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring method. The results indicate that the method has great potential for species-specific identification of seahorses and their preparations, among others.

Optimization of therapeutic drug monitoring of vancomycin in newborns using “Dried Blood Spot” method

Therapeutic drug monitoring (TDM) is used to increase the individualization of pharmacotherapy, especially in patient groups with a high interindividual variability in pharmacokinetic (PK) parameters. One of these groups of patients is newborn children, for whom drug therapy, especially drugs with a narrow therapeutic range, causes a few difficulties or cannot be used in principle.The aim of the work was to develop and validate quantitative HPLC-MS/MS methods for the determination of vancomycin in “dried blood spot” samples using new protocols and comparison of the results obtained with the results in plasma samples using standard sample preparation methods.Materials and methods. To prepare stock and standard solutions of vancomycin and norvancomycin as an internal standard, dry portions of the corresponding certified standards of vancomycin (Servier, France) and norvancomycin (Augsburg, Germany, purity grade >95.0%) were used. A chromatographic separation of the components was carried out on a Poroshell 120 C18 column (4.6×50 mm, 2.7 µm). When developing conditions for a mass spectrometric detection of the desired substances using the multiple reaction monitoring (MRM) method, precursor ions and their corresponding product ions were determined.Results. A quantitative HPLC-MS/MS method for the determination of vancomycin in «dried blood spot» samples was developed and validated. A comparison was made between vancomycin concentrations in «dried blood spot» samples and plasma samples. Moreover, more than 95% of the calculated average concentrations are within the limits of d-2s and d+2s, which correspond to the values of –10.2 and 12.2. That confirms the suitability of the developed method for the analysis of patient samples.Conclusion. The results obtained make it possible for us to recommend the “dried blood spot” method for therapeutic monitoring of vancomycin, additional studies of PK in this group of patients with subsequent use of this drug in newborns and pediatric patients.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Rapid detection of heat stress biomarkers in Atlantic salmon (Salmo salar) liver using targeted proteomics

AbstractMost fish are ectothermic; therefore, their physiology is significantly affected by temperature. Aquaculture fish have limited ability to avoid elevated water temperatures, with impacts increasing as a result of climate change. To date, quantifying gene expression has been proposed to monitor heat stress in salmon liver. This study aimed to establish a faster multiplexed proteomics method to measure the abundance of thermal stress biomarkers in liver of salmon reared at 15°C or 20°C. Moreover, this study aimed to determine the effects that sample pooling, and data normalisation using housekeeping (HK) protein peptides would exert over the statistical significance of these thermal stress markers. A multiple reaction monitoring (MRM) mass spectrometry method, comprised 45 peptides derived from thermal stress markers and 10 peptides from HK proteins, was applied to measure these markers in liver of salmon reared at 15°C or 20°C. When samples were processed individually, 34 peptides were significant between salmon livers at 15°C or 20°C. In pooled samples, this decreased to five significant peptides. Peptides hprt1_HYADDLDR (hypoxanthine phosphoribosyl transferase) and gapdh_VPTPNVSVVDLTVR (glyceraldehyde‐3‐phosphate dehydrogenase) were the most stable and unstable HK protein peptides, respectively. When data was normalised with hprt1_HYADDLDR, 16 peptides were significant in individual samples and 13 in pooled samples. Significant peptides serpinh1a_ADLSNISGK, SerpinH1_TNSILFIGR, ela2_VVGGEDVR and gapdh_VPTPNVSVVDLTVR were common regardless of data strategy. A fast and reliable MRM method was established to validate thermal stress markers in salmon liver, where individual samples yielded better results than pooled samples. Sample pooling was only better when combined with normalisation as it validated twice the number of markers than sample pooling alone. This method could be applied to monitoring stress response in experiments involving feeding additives designed to mitigate thermal stress or in selective breeding programs to help understanding family variance in thermal tolerance.

Exploring peptides from toad venom for source identification by LC-MS/MS using MRM method

Toad venom is a traditional Chinese medicine (TCM) with various sources and wide-ranging preparations. Previous quality assessment studies primarily concentrated on small molecular compounds like toad dienolactones and indole alkaloids, studies on macromolecular peptides and proteins as quality assessment standards remained at the qualitative stage, lacking the development of practical and convenient quantitative methods. In this study, to explore the peptides from toad venom as a new method for identifying and evaluating its source, a complete scan of the water extract of peptides from toad venom was conducted using HPLC-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF) 5600, leading to the identification of peptides based on mass spectrometry data. Subsequently, HPLC- Quadrupole-Linear Ion Trap Mass Spectrometer (Q-Trap) 5500 employing Multiple Reaction Monitoring (MRM) mode was utilized to quantitatively analyze peptides in various sources of toad venom, followed by Partial Least Squares Discriminant Analysis (PLS-DA) to further analyze the data and evaluate the effectiveness. This study highlights the importance of exploring macromolecular substance in natural products research and provides a foundation for further studies on toad venom.

Comprehensive analysis of different types of ginsenosides in the different parts of American ginseng by targeted and nontargeted MS/MS scanning.

To comprehensively study the ginsenosides distribution in the various tissues of American ginseng, the qualitative and quantitative-targeted and nontargeted mass spectroscopic methods were established using the high-performance liquid chromatography coupled with Qtrap triple quadrupole mass spectrometry (HPLC-QtrapQQQ-MS). The total ginsenosides of the root, stem, and leaf of American ginseng were determined by a colorimetric method, and the contents showed the order from high to low root, stem, and leaf. Eighty-two kinds of ginsenosides were detected in the different parts of American ginseng by enhanced mass scan-information-dependent data acquisition (IDA)-enhanced product ion (EPI) scan mode, including 69 from the root, 62 from the stem, and 48 from the leaf. An HPLC-multiple reaction monitoring (MRM) method was established, and 28 representative ginsenosides were further quantified in the three parts. Nearly all ginsenosides had the highest contents in the root and the lowest content in the leaf. Three types of ginsenosides (protopanaxadiol [PPD]-, protopanaxatiol [PPT]-, and oleanolic acid [OA]-types) were analyzed by precursor ion-IDA-EPI and MRM-IDA-EPI scan modes. Root had the most abundant ginsenosides in PPD- and PPT-type ginsenosides. Meanwhile, the OA-type ginsenosides are significantly enriched in the stem and leaf of American ginseng. The results provided a supplement to the quality assessment of American ginseng. PRACTICAL APPLICATION: The distribution profile of ginsenosides in the parts of American ginseng is different. Except for the root, the stem, and leaf of American ginseng have the most abundant ginsenosides in oleanolic acid type. The results reported herein can help the manufacturers choose appropriate materials to extract the ginsenosides.

Effect of oxyresveratrol on the quality and membrane lipid metabolism of shiitake mushroom (Lentinus edodes) during storage.

The effect of oxyresveratrol on postharvest quality and membrane lipid metabolism of shiitake mushroom was investigated. The result exhibited that oxyresveratrol retarded browning, maintained firmness and alleviated occurrence of decay of shiitake mushroom. The oxidation and hydrolysis of membrane phospholipids were suppressed by oxyresveratrol treatment, which was associated with reduced LOX and PLD activities and increased SOD and CAT activities. The membrane lipidomics of shiitake mushroom was determined by LC-MS. 385 lipid species and 13 fatty acids in membrane lipids were identified by multiple reaction monitoring method. Compared with control group, the phospholipic acid and lysophospholipid reduced by 29.24% and 21.29% in oxyresveratrol-treated group, respectively, which alleviated hydrolysis of phospholipid. Meanwhile, oxyresveratrol maintained the unsaturation of fatty acids and alleviated oxidation of phospholipid. These results demonstrated that oxyresveratrol could play a dual role of inhibiting the oxidation and hydrolysis of phospholipids to mitigate cellular damage of shiitake mushroom.

Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry for Simultaneous Analysis of 22 Analytes of Oncheong-Eum, a Traditional Korean Herbal Formula

Oncheong-eum (OCE) is a traditional Korean herbal formula comprising eight medicinal herbs for treating skin disorders, including eczema and skin rashes. Here, we sought to simultaneously analyze 22 analytes of OCE using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). All analytes were separated on a Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) maintained at 45 °C by gradient elution with a mobile phase of 0.1% (v/v) aqueous formic acid–acetonitrile. By applying a multiple reaction monitoring method, we rapidly determined the various analytes simultaneously. The coefficient of determination of the regression equation prepared in the tested concentration range of each authentic reference standard was ≥0.9950 and showed good linearity. The accuracy ranged from 84.23% to 115.47%, and the relative standard deviation values for intra- and interday precisions ranged from 0.84% to 9.57%, respectively. Analysis of OCE samples using this method showed that they contained up to 27.10 mg/g of active ingredients. The method can provide data to improve the consistency and, thus, the future quality of OCE preparations and other traditional herbal formulas.

B-325 Retrospective Study Comparing Immunoassay Screen and Mass Spectrometry Confirmation Results in an Opioid Dependant Population

Abstract Background Retrospective analysis of clinical laboratory data is employed to monitor the performance of instruments, evaluate test utilization and support operational changes. Urine drug testing in the context of opioid use disorder is used to assist in the management of patients. This testing is typically performed by an immunoassay screen followed by mass spectrometry confirmation. Immunoassay screens identify drug class and can cross-react with off-target substances to produce false-positive or false-negative results. Mass spectrometry confirmations provide more sensitive and specific results. The objective of this study was to utilize historical data to determine the concordance between immunoassay and mass spectrometry results from patients in an opioid dependency treatment program. Methods Urine drug screening results spanning a 3-year period (2020–2023) were mined to identify samples that screened positive for amphetamines, benzodiazepines, opiates, fentanyl/carfentanil, oxycodone/oxymorphone, and cocaine. These results were compared to the mass spectrometry confirmation results. The immunoassay results were collected on an Olympus AU480 instrument and the mass spectrometry results were collected using an in-house developed dynamic multiple reaction monitoring method on both Agilent 6470 and 6460 triple quadrupole instruments. Results Of the 15 850 urine drug records reviewed, 28.8% screened positive for amphetamines, 23.5% screened positive for benzodiazepines, 14.2% screened positive for opiates, 23.8% screened positive for fentanyl/carfentanil, 4.2% screened positive for oxycodone/oxymorphone, and 11.2% screened positive for cocaine. Of the amphetamine class samples that screened positive, 95.3% confirmed positive and 4.7% confirmed negative. Of the benzodiazepine class samples that screened positive, 50.8% confirmed positive and 49.2% confirmed negative. Of the opiate class samples that screened positive, 79.8% confirmed positive and 20.2% confirmed negative. Of the fentanyl/carfentanil samples that screened positive, 97.2% confirmed positive and 2.8% confirmed negative. Of the oxycodone/oxymorphone samples that screened positive, 58.5% were confirmed positive and 41.5% confirmed negative. Of the cocaine samples that screened positive, 99.8% confirmed positive and 0.2% confirmed negative. Conclusion Our findings determined there were discrepancies among all classes when comparing immunoassay vs mass spectrometry results. There was good agreement between immunoassay and mass spectrometry results in the detection of fentanyl/carfentanil, cocaine and amphetamines. The differences between methods were most significant in the benzodiazepine, opiate and semi-synthetic opioid classes. This may be due to different cut-off levels between methods, specificity limitations of immunoassays and/or changing drug use patterns resulting in mass spectrometry multiple reaction monitoring false negative results.

Metabolite Profiling for Typization of "Rucola della Piana del Sele" (PGI), Eruca sativa, through UHPLC-Q-Exactive-Orbitrap-MS/MS Analysis.

In August 2020, the Eruca sativa cultivar "Rucola della Piana del Sele" obtained from the European Union the prestigious PGI (protected geographical indication) label, which certifies the uniqueness of its characteristics and increases its prestige both nationally and, above all, internationally. This plant is recognized as a product of excellence, with a unique flavor and unmistakable aroma. Therefore, since there are no methods to characterize the PGI product, a metabolomic approach was applied to characterize E. sativa grown in the Piana del Sele and different geographical areas. As E. sativa has very wide cultivation, this study sought to compare the metabolite profiles of rocket grown in Piana del Sele, Bergamo, and Brescia, as well as in Switzerland, making a comparison also with the metabolite profile of E. sativa grown spontaneously. To determine the best procedure to distinguish "Rucola della Piana del Sele" from the others, different extraction procedures were carried out using different solvents and fresh or freeze-dried plant matrices. The different extracts were analyzed by liquid chromatography coupled with high-resolution mass spectrometry experiments, using chemometric analyses to identify biomarker metabolites that characterize the PGI product. The LC-ESI-Q-Exactive-MS/MS profiles of methanol and hydroalcoholic extracts of different cultivars of E. sativa were found to be rich in bioactive compounds such as glucosinolates, glycosylated flavonoids, fatty acids, and lipids. The LCMS data were analyzed by principal component analysis (PCA); the score scatter plot shows significant separation among Eruca samples grown in different geographical areas. In detail, loading the scatter plot revealed Eruca grown in Piana del Sele to be richer than other cultivars in glycosylated quercetin 3,3',4'-O-triglucoside (7), quercetin-3,4'-O-diglucoside-3'-O-(6-sinapoyl-glucoside) (10), and quercetin diglucoside (30). Finally, considering the biological interest in erucin, the myrosinase product of glucoerucin, the latter was quantified in the extracts by LC-ESI/QTrap/MS/MS using the multiple reaction monitoring (MRM) method; E. sativa from Piana del Sele showed the highest content of glucoerucin.

Database Creator for Mass Analysis of Peptides and Proteins, DC-MAPP: A Standalone Tool for Simplifying Manual Analysis of Mass Spectral Data to Identify Peptide/Protein Sequences.

Proteomic studies typically involve the use of different types of software for annotating experimental tandem mass spectrometric data (MS/MS) and thereby simplifying the process of peptide and protein identification. For such annotations, these softwares calculate the m/z values of the peptide/protein precursor and fragment ions, for which a database of protein sequences must be provided as an input file. The calculated m/z values are stored as another database, which the user usually cannot view. Database Creator for Mass Analysis of Peptides and Proteins (DC-MAPP) is a novel standalone software that can create custom databases for "viewing" the calculated m/z values of precursor and fragment ions, prior to the database search. It contains three modules. Peptide/Protein sequences as per user's choice can be entered as input to the first module for creating a custom database. In the second module, m/z values must be queried-in, which are searched within the custom database to identify protein/peptide sequences. The third module is suited for peptide mass fingerprinting, which can be used to analyze both ESI and MALDI mass spectral data. The feature of "viewing" the custom database can be helpful not only for better understanding the search engine processes, but also for designing multiple reaction monitoring (MRM) methods. Post-translational modifications and protein isoforms can also be analyzed. Since, DC-MAPP relies on the protein/peptide "sequences" for creating custom databases, it may not be applicable for the searches involving spectral libraries. Python language was used for implementation, and the graphical user interface was built with Page/Tcl, making this tool more user-friendly. It is freely available at https://vit.ac.in/DC-MAPP/.

Validation of the Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry Method for Simultaneous Analysis of Eighteen Compounds in the Traditional Herbal Prescription, Sanjoin-Tang

Sanjoin-tang (SJIT) is an ancient oriental medicine prescription listed in the Jinguiyaolue that is mainly used for the treatment of primary insomnia. This study was conducted to develop and validate an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) simultaneous analysis method for the quality control of SJIT using 18 target compounds. The 18 analytes were separated on an Acquity UPLC BEH C18 column maintained at 45 °C using a mobile phase composed of distilled water and acetonitrile. The MS system was used to simultaneously detect all analytes using the multiple reaction monitoring (MRM) method of Xevo TQ-XS coupled with an electrospray ionization source. The concentrations of the 18 analytes investigated in the SJIT samples ranged from below the limit of detection to 9.553 mg/g. In conclusion, the validated UPLC–MS/MS MRM analysis method can be used to obtain basic data to establish chemical-nonclinical linkage efficacy and for the clinical research and quality evaluation of SJIT.

Identification and Quantification of Resveratrol and Its Derivatives in Franconian Wines by Comprehensive Liquid Chromatography–Tandem Mass Spectrometry

Resveratrol and its derivatives are valued compounds in foods such as wine. To date, their occurrence and concentrations in Franconian wines are not known. Thus, the present study investigated resveratrol and its derivatives in Franconian wines. First, comprehensive ultrahigh-performance liquid chromatography (UHPLC)–tandem mass spectrometry methods were applied to identify cis-/trans-resveratrol, cis-/trans-piceid, deoxyrhapontigenin, trans-piceatannol, and cis-/trans-astringin. Second, a sensitive UHPLC–scheduled multiple reaction monitoring method for absolute quantification of the major resveratrol derivatives was developed, validated, and applied to 19 Franconian wine samples. Recovery rates ranged between 95.0 and 109.3% and reproducibility between 2.6 and 10.3%. The limits of detection/quantification were between 0.01 and 0.08 μg/mL. The samples contained 0.07–2.61 μg/mL cis-resveratrol, 0.05–3.82 μg/mL trans-resveratrol, 0.24–9.01 μg/mL cis-piceid, and 0.25–8.30 μg/mL trans-piceid. Additionally, the concentrations of deoxyrhapontigenin (0.02–0.53 μg/mL), trans-piceatannol (0.05–1.35 μg/mL), and trans-astringin (0.04–0.67 μg/mL) were quantified.