Selected Reaction Monitoring Mode Research Articles

Wide-Targeted Semi-Quantitative Analysis of Acidic Glycosphingolipids in Cell Lines and Urine to Develop Potential Screening Biomarkers for Renal Cell Carcinoma.

Glycosphingolipids (GSLs), mainly located in the cell membrane, play various roles in cancer cell function. GSLs have potential as renal cell carcinoma (RCC) biomarkers; however, their analysis in body fluids is challenging because of the complexity of numerous glycans and ceramides. Therefore, we applied wide-targeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM) based on theoretical mass to perform a comprehensive measurement of GSLs and evaluate their potency as urinary biomarkers. In semi-quantitative lipidomics, 240 SRM transitions were set based on the reported/speculated structures. We verified the feasibility of measuring GSLs in cells and medium and found that disialosyl globopentaosylceramide (DSGb5 (d18:1/16:0)) increased GSL in the ACHN medium. LC-MS/MS analysis of urine samples from clear cell RCC (ccRCC) patients and healthy controls showed a significant increase in the peak intensity of urinary DSGb5 (d18:1/16:0) in the ccRCC group compared with that in the control group. Receiver operating characteristic analysis indicated that urinary DSGb5 could serve as a sensitive and specific marker for RCC screening, with an AUC of 0.89. This study demonstrated the possibility of urinary screening using DSGb5 (d18:1/16:0). In conclusion, urinary DSGb5 (d18:1/16:0) was a potential biomarker for cancer screening, which could contribute to the treatment of RCC patients.

Detailed profiling of polysorbate 80 oxidative degradation products and hydrolysates using liquid chromatography-tandem mass spectrometry analysis.

Polysorbate 80 (PS80) is an amphipathic, nonionic surfactant that is commonly used to stabilize proteins in biopharmaceutical formulations. PS80 undergoes oxidative and/or enzymatic degradation. However, because PS80 is a complex mixture consisting of many constituents, comprehensive evaluations of its oxidative degradation products are difficult and insufficient. Our previously reported comprehensive liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based method for PS80 effectively provides an overall profile of PS80 components under simple LC conditions. In this study, we attempted to shorten the analysis time. Furthermore, PS80 was oxidatively degraded in a solution containing histidine and iron, and the oxidative degradation products were evaluated using a modified LC/MS/MS method. In addition, enzymatically hydrolyzed PS80 samples were analyzed. We succeeded in shortening the analysis time from 70 to 20 min while maintaining the resolution of the PS80 components of the same selected reaction monitoring transition. Both the previously reported oxidative degradation products and the newly discovered products were successfully detected, and their composition ratios and changes over time were observed. Changes in the hydrolysates over time are shown in the analysis of the hydrolyzed PS80 samples. This study clearly showed the presence of changes in PS80 oxidative and/or enzymatic degradation products, including those previously unreported. These results demonstrate that a detailed profiling of PS80 degradation products can be performed using LC/MS/MS, which is less expensive and more generally adopted than high-resolution MS.

Determination of Acrylamide in Coffee, Cereals, Baby Food, Cocoa, Dry Pet Food, Potato Products, Vegetable Crisps, Biscuits, Tea, Nuts, and Spices by LC-MS/MS in a Single-Laboratory Validation: First Action 2023.01.

Acrylamide (AA) is a process contaminant naturally formed during the cooking of starchy food at high temperatures. Considering existing risks of misquantification inherent to the analysis of AA, an AOAC initiative raised the need for a consensus standard to determine AA in a broad variety of food. A quantitative LC-MS/MS method for AA determination in food was validated in a single-laboratory study. Targeted performance requirements in terms of target matrixes, limit of quantification, recovery, and precision were as defined per Standard Method Performance Requirement (SMPR®) 2022.006. The proposed method derives from EN 16618:2015 standard pending modifications brought to the (1) sample preparation (simplified, potentially automated); (2) scope of application (significantly extended); and (3) LC conditions (improved selectivity). Confirmatory detection of AA is conducted by LC-MS/MS in the Selected Reaction Monitoring mode (SRM), and isotopic dilution was applied for quantification approach using either 2,3,3-d3-acrylamide (d3-AA), or 13C3-2,3,3-d3-acrylamide (13C3-d3-AA) as labeled internal standard. A total of 16 laboratory samples from nine matrix categories were included in the validation process. A full validation was conducted on coffee (instant, roast), infant cereal, cocoa powder, pet food (croquettes), tea (green tea), spices (black pepper), and nuts (roasted almonds) with satisfactory performances both in terms of recovery (97-108%) and precision (RSDr and RSDiR <12%). The method applicability was further demonstrated through the analysis of quality control materials and reference materials including French fries, potato crisps, vegetable crisps, instant coffee, infant food, and biscuits (cookies), with accuracy values determined within a 94-107% range. The performances of the presented method are in agreement with the acceptance criteria stipulated in SMPR 2022.006. The Expert Review Panel for acrylamide approved the present method as AOAC Official First Action 2023.01.

Gas chromatography-mass spectrometry selected ion monitoring and gas chromatography-tandem mass spectrometry selected reaction monitoring analyses of mono-, di- and tri-unsaturated C25 highly branched isoprenoid alkene biomarkers in sea ice and sediment samples: A comparative study.

The efficiency of selected ion monitoring (SIM) and selected reaction monitoring (SRM) analyses for the quantification of three mono-, di- and tri-unsaturated highly branched isoprenoid (HBI) alkenes (IP25 , IPSO25 and HBI III, respectively), often used as proxies for the occurrence of Arctic and Antarctic sea ice or the adjacent open waters, was compared. Gas chromatography (GC)-mass spectrometry (MS)/SIM and GC/MS/MS/SRM analyses were carried out on dilute solutions made from purified standards of these three HBIs, and then on hydrocarbon fractions of several sediment and sea ice sample extracts. More efficient and specific SRM transitions were selected after collision-induced dissociation of each precursor ion at different collision energies. SRM analysis avoided any overestimation of IP25 resulting from the contribution of the coeluting 13 C mass isotopomer of IPSO25 (M+ ˙ + 2) to the SIM target ion. In contrast, SRM analysis is less reliable for IPSO25 quantification in cases where several regio-isomers are present, likely due to intense double bond migrations following electron impact. In the case of HBI III, SRM analysis constitutes a potentially suitable alternative to SIM analysis, especially in terms of improving limit of detection. Despite the intense migrations of HBI double bonds under electron ionization, the selected SRM transitions should be more suitable than SIM target ions for IP25 and HBI III quantification in complex hydrocarbon fractions of natural samples. However, the advantage is less evident for IPSO25 due to the presence of numerous regio-isomers.

Quantification of Vanillin, Ethyl Vanillin and Methyl Vanillin Among Thai Rice Cultivars by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

A method was developed and validated for vanillin, ethyl vanillin, and methyl vanillin determination in Thai rice cultivars by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) under the selected reaction monitoring mode. In rice samples, the limit of detection was 13.3, 11.2 and 5.0 mg/kg for vanillin, ethyl vanillin, and methyl vanillin. A high correlation coefficient was obtained (R2 = 0.9999, 0.9999 and 0.9998) within the linear range (0 - 300 mg/L) in the MS method. The recoveries ranging from 90.63 % ± 6.25 to 108.7 % ± 2.35 were obtained in different spiking concentrations. The average RSD% of intra-day for vanillin, ethyl vanillin, and methyl vanillin was 6.90, 5.13 and 2.16 %, and the inter-day were 6.72, 5.30 and 5.64 %, respectively. The developed method was applied to 24 commercial rice samples. Vanillin, were found in 24 samples ranging from 95.2 to 773.2 mg/kg. However, ethyl vanillin and methyl vanillin were not detected under LOD. The developed UPLC-MS/MS method for vanillin, ethyl vanillin and methyl vanillin determination include the merits of simple preparation steps and high sensitivity. HIGHLIGHTS Method development for analyzing vanillin, ethyl vanillin, and methyl vanillin in rice grain by UPLC-MS/MS. High accuracy and precision method for determining vanillin and its derivatives. Quantitative analysis of vanillin, ethyl vanillin, and methyl vanillin in Thai rice cultivars. GRAPHICAL ABSTRACT

Simultaneous Determination of Two Metabolites of Gelsemium elegans by LC-MS/MS in Rat Urine and its Application to Pharmacokinetic Study

Background: G. elegans is a highly useful medicinal plant with broad and superior clinical pharmacological effects. However, it also exhibits high toxicity to humans, so it must be used with extreme caution in clinical practice. Previous studies on G. elegans have mainly focused on its main ingredients from perspectives of pharmaceutical analysis, pharmacokinetics, and pharmacodynamics. The kinetic behavior of G. elegans' main metabolites in vivo has not yet been reported, which is also crucial for studying the herb's toxification and detoxification process. Aims: This study aimed to establish a quantitative method to describe the metabolic profile of the two main metabolites of koumine after oral administration of G. elegans to rats. Objective: The objective of this study was to test two major metabolites of G. elegans in rat urine after administration using a rapid and sensitive high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) method developed in advance. Methods: A Kromasil C18 column was used as the stationary phase for chromatographic separation, with an isocratic mobile phase consisting of mixture of water (2 mM ammonium formate), formic acid, and methanol (25:0.05:75, v/v/v) at a flow rate of 0.40 mL/min. The mass spectrometer was equipped with an electrospray ionization (ESI) source operating in positive ionization mode, and detection was performed using a selective reaction monitoring (SRM) mode. Detection was performed in selective reaction monitoring (SRM) mode. Protein precipitation was used to pretreat the rat urine samples before analysis, with acetonitrile as the precipitation solvent. In this study, a "relative quantification" strategy was employed, which avoided the expensive and time-consuming separation of standard substances. The assay was validated in terms of specificity, linearity, precision, stability, recovery, and other aspects. Results: The intra- and inter-day precision values were less than 12.7%. The recoveries at three different concentrations were all above 73.1%. The stability study showed that the analytes were stable during the experiment. The method was then used to study the kinetic profiles of N-demethylkoumine and N-oxidatekoumine in rat urine for the first time. Conclusion: In this study, the established LC/MS method was fully validated and proven to be sensitive and accurate for the simultaneous determination of the two main metabolites of G. elegans in rats' urine samples. These results could provide references for the safe use of koumine and G. elegans in clinical applications.

Quantitative analysis of residual aurintricarboxylic acid in biotherapeutic process streams using liquid chromatography triple quadrupole mass spectrometry

Aurintricarboxylic acid (ATA) is an excipient that can be added to the therapeutic protein manufacturing process as a component of the Chinese hamster ovary (CHO) cell culture media. ATA inhibits cell apoptosis and promotes cell growth in both serum-free and protein-free media. The addition of ATA is beneficial to the manufacturing process at the cell growth stage, however, residual ATA not consumed by cells can have toxicological effects on patients and its removal is required during downstream processing. To ensure manufacturing control and patient safety, the determination of residual ATA during downstream processing of biotherapeutics is required. The aim of this study was to develop and validate a liquid chromatography triple quadrupole mass spectrometry method for monitoring process clearance of ATA during downstream processing. Chromatographic separation of ATA was achieved using a 50 × 3 mm, 3 µm Imtakt Cadenza HS-C18 column and a triple quadrupole mass spectrometer operated in selected reaction monitoring mode was used for sensitive and specific detection. Linearity was assessed over a range of 62.5 ng/mL to 2000 ng/mL. Accuracy and precision were within 20% of the theoretical spike levels across the three concentration levels evaluated utilising two different antibodies, an IgG1 and an IgG4. Analyte specificity and selectivity were deemed acceptable based on no extraneous peaks detected. The method was successfully applied to monitoring process clearance of ATA in a tangential flow filtration (TFF) unit operation for downstream processing of monoclonal antibodies.

Differentiation of aminohydroxypropanesulfonic acid structural isomers using tandem mass spectrometry-based methods

d-Cysteinolic acid (D-CA) is an important metabolite within the biosulfur cycle. A structural isomer, (R)-3-amino-2-hydroxypropanesulfonate ((R)-AHPS), is less common in nature but potentially can be misidentified as D-CA due to their many shared physical properties. To support confident assignment of these two isomers by use of mass spectrometry alone, this study explores the fragmentation reactions of their [M+H]+ and [M−H]− ions using collision-induced dissociation (CID). Electrospray ionization mass spectrometry (ESI-MS) experiments were conducted on authentic standards using an ion trap mass spectrometer, while a triple-quadrupole (QqQ) mass spectrometer was used in the selective reaction monitoring (SRM) mode to record energy-resolved CID. Density-functional theory (DFT) calculations were carried out at the M06/6-31+G* level of theory to study gas-phase fragmentation mechanisms. The data generated revealed kinetically-controlled fragmentations involving participation of neighboring amino groups in the positive ion mode. Negative ion mode MS analysis could distinguish the structural isomers through different collision energy-resolved results for m/z 95 product ions, CH3SO3−. DFT calculations revealed an enthalpy (ΔH) (Gibbs energy (ΔG)) gap of 31.8 (31.4) kJ/mol between transition state barriers of a concerted mechanism for D-CA, and a more preferred stepwise mechanism for (R)-AHPS.

Development and validation of an HPLC-MS/MS method to quantify the KRAS inhibitor adagrasib in mouse plasma and tissue-related matrices.

We developed and validated an assay utilizing a liquid chromatography-tandem mass spectrometry technique to quantify the KRAS inhibitor adagrasib in mouse plasma and seven tissue-related matrices. The straightforward protein precipitation technique was selected to extract adagrasib and the internal standard salinomycin from the matrices. Gradient elution of acetonitrile and water modified with 0.5% (v/v) ammonium hydroxide and 0.02% (v/v) acetic acid on a C18 column at a flow rate of 0.6ml/min was applied to separate the analytes. Both adagrasib and salinomycin were detected with a triple quadrupole mass spectrometer with positive electrospray ionization in a selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of adagrasib was demonstrated during the validation. In addition, the reported precision values (intra- and inter-day) were between 3.5 and 14.9%, while the accuracy values were 85.5-111.0% for all tested levels in all investigated matrices. Adagrasib in mouse plasma was reported to have good stability at room temperature, while adagrasib in tissue-related matrices was stable on ice for up to 4h (matrix dependent). Finally, this method was successfully applied to determine the pharmacokinetic profile and tissue distribution of adagrasib in wild-type mice.

Development and application of a supercritical fluid chromatography-tandem mass spectrometry method for the simultaneous determination of pantoprazole enantiomers in rat plasma

The study aimed to develop a supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for the enantioselective separation and quantification of pantoprazole in rat plasma. The method utilized protein precipitation with acetonitrile to prepare plasma samples. SFC was performed on an Acquity ultra-performance convergence chromatography (UPCC) system. An Acquity UPCC Trefoil™ CEL2 column was employed for enantioseparation, and isocratic elution was achieved using a mobile phase consisting of CO2-methanol (81:19, v/v). The detection of pantoprazole enantiomers and the internal standard phenacetin was carried out using a Xevo TQD triple quadrupole mass spectrometer in selected reaction monitoring mode with positive electrospray ionization. The developed method successfully achieved full separation of S- and R-pantoprazole enantiomers. Calibration curves were linear in the concentration range of 10–5000 ng/mL for both S- and R-pantoprazole. The method met the acceptance criteria for carry-over, accuracy, precision, extraction recovery, matrix effect, stability, and dilution integrity, demonstrating its stereoselectivity, sensitivity, accuracy, precision, and reliability. The validated method was then applied to investigate the toxicokinetics of racemic pantoprazole and evaluate the potential of chiral inversion between S- and R-pantoprazole in rats. Following oral administration of 200 mg/kg racemic pantoprazole once daily for 90 consecutive days, there was no significant difference in toxicokinetic parameters between the two enantiomers on both day 1 (single-dose experiment) and day 90 (multiple-dose experiment). This suggests that there is no enantioselectivity in the toxicokinetic behaviors of racemic pantoprazole in rat plasma. Additionally, bidirectional chiral inversion between S- and R-pantoprazole in plasma was observed after single-dose and multiple-dose oral administrations of S-pantoprazole (20, 100, and 200 mg/kg) or R-pantoprazole (200 mg/kg) to rats. Compared to single and low doses, there was no accumulation of each enantiomer in rat plasma following repeated and escalated dosing. Overall, this study provided the first report on the toxicokinetics of pantoprazole in rat plasma and presented the first SFC-MS/MS method for the simultaneous quantification of pantoprazole enantiomers in rat plasma.

Chiral LC-MS/MS method for the discrimination of triiodothyronine enantiomers on a crown ether-based chiral stationary phase.

Considering the greater pharmaceutical and clinical interest of triiodothyronine (T3 ) thyroid hormone, an effective D/L-T3 enantiomer separation was performed on a crown ether-based chiral stationary phase by LC-MS/MS. In optimal analytical condition and selected reaction monitoring mode, the two enantiomers of T3 were baseline separated within 10min. The limit of detection and limit of quantitation were found to be 0.05 and 0.10ng/μl; 0.20 and 0.50 ng/μl for D- and L-T3 , respectively. During validation, this method proved to be feasible, accurate as well as enantioselective and sensitive for the resolution of T3 enantiomers. For commercial D- and L-T3 chemicals, the enantiomeric impurities as the other enantiomer were 0.11% and 4.61%. On the other hand, the impurity as D-T3 for commercial pharmaceutical products (liothyronine sodium tablets, two suppliers) was 0.68% and 6.57%.

Comprehensive analysis of oxylipins using reverse phase liquid chromatography and data dependent acquisition workflow on LTQ-Orbitrap® Velos Pro

Oxylipins – involved in inflammatory processes – are reported in several diseases, in biological, pharmacological, and physiological fields. To face the structural complexity of oxylipins, the study of isomers and isobars species relied on Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) in tandem mass spectrometry such as triple quadrupole, quadrupole-Time of Flight (TOF). Unfortunately, false positive signals in cellular matrix could occur using MRM or SRM mode since the MS/MS spectrum of each molecule is not acquired with the previous mode to help molecule confirmation. Using the versatile ability of LTQ-Orbitrap® Velos Pro mass spectrometer, we developed a novel method based on data dependent acquisition (DDA) workflow for oxylipins analysis. To reach sufficient data points per peak and a better sensitivity to quantify oxylipins traces, an optimization of the acquisition frequency was carried out both on linear trap and Orbitrap analyzers. A segmentation of the chromatographic profile and an optimization of the collision energies by HCD (higher energy collision dissociation) for each eicosanoid increased the acquisition frequency significantly and the detection threshold: around 2 pg for some prostanoids and 0.02–2 pg for some leukotrienes and oxidized species. We validated our method in terms of specificity (RSD <10%), sensitivity, accuracy and precision. The intra and inter-day accuracy were between 86.56% and 114.93%. Besides, a relative standard deviation less than 15% as intra- and inter-day precision were obtained for almost all molecules. A linear range between 2.5 and 12,500 pg was reached. DDA approach on LTQ-Orbitrap® constitutes an alternative to MRM mode on triple quadrupole for eicosanoids quantification in complex matrices. Finally, this method helped us to compare for the first time the amount of prostanoids released by J774 and THP-1 macrophages under lipopolysaccharide (LPS) stimulation.

Ultrahigh-performance liquid chromatography-tandem mass spectrometry quantitation of o-cresol in hydrolyzed human urine to assess toluene exposure.

Toluene is a volatile organic compound used in domestic and industrial applications. The main routes of workplace exposure to toluene are inhalation and dermal contact. As toluene exposure can cause severe nervous system damage, its quantification is crucial to prevent occupational illness. Toluene is metabolized mainly as hippuric acid, S-benzylmercapturic acid and epoxides. These are rapidly converted to o-/p-cresol, which is then excreted in the urine as conjugated glucuronides and sulfates. o-Cresol and its conjugates can be chemically hydrolyzed to form free o-cresol, which can then serve as a urinary biomarker of toluene exposure. Current analytical methods for quantifying o-cresol in hydrolyzed urine are, however, either weakened by interference, are not sensitive enough or require water-sensitive sample preparation. Development of a liquid chromatography-tandem mass spectrometry method for assessing exposure to toluene is thus required. Urine samples were acidified and heated to form free o-cresol and then derivatized with dansyl chloride and diluted. Extracts were separated by reverse-phase chromatography on a BEH phenyl column and then analyzed using a triple quadrupole instrument in selected reaction monitoring mode. The dansyl chloride derivatization step was optimized to produce the derivative within a reaction time of 3min. Hydrolysis efficiency in forming free o-cresol from conjugated metabolites was evaluated using o-cresol-β-d-glucuronidespiked human urine: complete hydrolysis occurred in 45 min. Dynamic range was 0.4 to 40 μM, and the method was useful for toluene monitoring in non-occupational (0.1μmol/mmol creatinine) as well as occupational (0.3μmol/mmol creatinine) exposure. The calculated limit of detection and limit of quantitation of the method were 0.06 and 0.21 μM, respectively. Intraday and interday precisions were 3.2% and 4.4%, respectively. Method accuracy was established as 99% using ClinChek® urine controls. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for analysis of o-cresol was developed for biological monitoring of toluene exposure in human urine. This is the method of choice used by occupational health and safety practitioners in the province of Québec, Canada.

Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples

The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry – Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15–100% MeOH and ACN), flow rate (8–100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO2/MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS.

Rapid and efficient LC-MS/MS diagnosis of inherited metabolic disorders: a semi-automated workflow for analysis of organic acids, acylglycines, and acylcarnitines in urine.

The analysis of organic acids in urine is an important part of the diagnosis of inherited metabolic disorders (IMDs), for which gas chromatography coupled with mass spectrometry is still predominantly used. Ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for urinary organic acids, acylcarnitines and acylglycines was developed and validated. Sample preparation consists only of dilution and the addition of internal standards. Raw data processing is quick and easy using selective scheduled multiple reaction monitoring mode. A robust standardised value calculation as a data transformation together with advanced automatic visualisation tools are applied for easy evaluation of complex data. The developed method covers 146 biomarkers consisting of organic acids (n=99), acylglycines (n=15) and acylcarnitines (n=32) including all clinically important isomeric compounds present. Linearity with r2>0.98 for 118 analytes, inter-day accuracy between 80 and 120 % and imprecision under 15 % for 120 analytes were achieved. Over 2 years, more than 800 urine samples from children tested for IMDs were analysed. The workflow was evaluated on 93 patient samples and ERNDIM External Quality Assurance samples involving a total of 34 different IMDs. The established LC-MS/MS workflow offers a comprehensive analysis of a wide range of organic acids, acylcarnitines and acylglycines in urine to perform effective, rapid and sensitive semi-automated diagnosis of more than 80 IMDs.

Analysis of Phenolic Acids and Flavonoids in Rabbiteye Blueberry Leaves by UPLC-MS/MS and Preparation of Nanoemulsions and Extracts for Improving Antiaging Effects in Mice.

Rabbiteye blueberry leaves, a waste produced after harvest of blueberry, are rich in polyphenols. This study aims to analyze phenolic acids and flavonoids in blueberry leaves by UPLC-MS/MS and prepare nanoemulsions for determining anti-aging activity in mice. Overall, 30% ethanol was the most suitable extraction solvent for total phenolic acids and total flavonoids. A total of four phenolic acids and four flavonoids were separated within seven minutes for further identification and quantitation by UPLC-MS/MS in selective reaction monitoring (SRM) mode, with 3-O-caffeoylquinic acid being present in the highest amount (6474.2 μg/g), followed by quercetin-3-O-galactoside (1943.9 μg/g), quercetin-3-O-rutinoside (1036.6 μg/g), quercetin-3-O-glucoside (867.2 μg/g), 5-O-caffeoylquinic acid (815.8 μg/g), kaempferol-3-O-glucoside (309.7 μg/g), 3,5-dicaffeoylquinic acid (195.3 μg/g), and 4,5-dicaffeoylquinic acid (60.8 μg/g). The blueberry nanoemulsion was prepared by using an appropriate ratio of soybean oil, Tween 80, glycerol, ethanol, and water at 1.2%, 8%, 2%, 2%, and 86.8%, respectively, and mixing with dried blueberry extract, with the mean particle size and zeta potential being 16 nm and -54 mV, respectively. A high stability was observed during storage of nanoemulsion for 90 days at 4 °C and heated at 100 °C for 2 h. An animal study revealed that this nanoemulsion could elevate dopamine content in mice brain as well as superoxide dismutase, glutathione peroxidase, and catalase activities in mice liver while reducing the contents of malondialdehyde and protein carbonyl in mice brains. Collectively, the high-dose nanoemulsion possessed the highest efficiency in improving mice aging with a promising potential for development into a health food.

Comparison of chromatographic conditions for the targeted tandem mass spectrometric determination of 354 mammalian metabolites

Metabolomics is becoming increasingly popular in livestock research, but no single analytical method can cover the entire metabolome. As such, we compared similar and complementary chromatographic methods with respect to analyte coverage and chromatographic properties of mammalian metabolites. We investigated 354 biologically relevant primary metabolites from 19 compound classes including amino acids, bile acids, biogenic amines, carboxylic acids, lipids, nucleotides and sugars. A total of 2063 selected reaction monitoring transitions were optimized on a triple quadrupole mass spectrometer. We then determined the retention profiles and peak parameters of our compounds using an anion exchange chromatography (AIC), three reversed-phase (RP) and three hydrophilic interaction liquid chromatography (HILIC) methods. On average, HILIC methods covered 54% of all metabolites with retention factors >1, while average RP coverage was 41%. In contrast to RP, HILIC methods could also retain polar metabolites such as amino acids and biogenic amines. Carboxylic acids, nucleotides, and sugar related compounds were best separated by AIC or zwitterionic pHILIC with alkaline eluents. Combining two complementary HILIC and RP methods increased the library coverage to 92%. By further including important short chain fatty acids, a combination of HILIC, RP and AIC methods achieved a coverage of 97%. The resulting dataset of LC and MS/MS parameters will facilitate the development of tailor-made quantitative targeted LC-MS/MS methods to investigate the mammalian metabolome.

Lack of pharmacokinetic interaction between derazantinib and naringin in rats

Context Derazantinib—an orally bioavailable, ATP competitive, multikinase inhibitor—has strong activity against fibroblast growth factor receptors (FGFR)2, FGFR1, and FGFR3 kinases. It has preliminary antitumor activity in patients with unresectable or metastatic FGFR2 fusion-positive intrahepatic cholangiocarcinoma (iCCA). Objective This experiment validates a novel sensitive and rapid method for the determination of derazantinib concentration in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and applies it to the study of drug-drug interaction between derazantinib and naringin in vivo. Materials and methods A Xevo TQ-S triple quadrupole tandem mass spectrometer was used for mass spectrometry monitoring in selective reaction monitoring (SRM) mode with transitions of m/z 468 96 → 382.00 for derazantinib and m/z 488.01 → 400.98 for pemigatinib, respectively. The pharmacokinetics of derazantinib (30 mg/kg) was investigated in Sprague-Dawley (SD) rats divided into two groups (with the oral pretreatment of 50 mg/kg naringin or not). Results The newly optimized UPLC-MS/MS method was suitable for the determination of derazantinib in rat plasma. It was also successfully employed to evaluate the effect of naringin on derazantinib metabolism in rats. After pretreatment with naringin, there was no significant difference in the pharmacokinetic parameters (AUC0→t, AUC0→∞, t1/2, CLz/F, and Cmax) of derazantinib when compared with derazantinib alone. Conclusion Co-administration of naringin with derazantinib was not associated with significant changes in pharmacokinetic parameters. Thus, this study suggests that the combination of derazantinib with naringin can safely be administered concomitantly without dose adjustment.

Simultaneous Determination of 16-Hydroxystrychnine and Demethylbrucine by LC-MS/MS in Rat Urine and Its Application to Pharmacokinetic Study.

This paper aimed to establish a method to help investigate the combination mechanism of traditional Chinese medicine from the metabolic perspective. Semen Strychni has been a frequently used herb in clinics for a long time. In traditional Chinese medical science, Semen Strychni always combinate with other herbs to modulate its nature of severe toxicity. However, the mechanism of the combination is still unclear. Previous research mostly focused on the components of the herbs. The metabolic processes of the main components of the Semen Strychni are also very important and have rarely been reported. This study tended to develop a rapid and sensitive high-performance liquid chromatographic- tandem mass spectrometric (HPLC-MS/MS) method for the determination of two major metabolites of Semen Strychni in rat urine. Chromatographic separation was carried out on a C18 column. Detection was performed by a selective reaction monitoring (SRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. Analysis of analytes from rat plasma was carried out by protein precipitation using acetonitrile. The assay was validated in terms of specificity, precision, recovery, etc. The intra- and inter-day precision values were less than 13.1%. The recoveries at three levels were more than 69.1%. The method was then used to study the kinetic profiles of the metabolites of Semen Strychni in rat urine for the first time. The results demonstrate that the established LC/MS method in this study is accurate and sensitive for the simultaneous determination of the two metabolites of Semen Strychni in rats&#039; urine samples. This method could be a supplement to the plasma pharmacokinetics of Semen Strychni.

LC-MS/MS analysis of elastin crosslinker desmosines and microscopic evaluation in clinical samples of patients with hypertrophy of ligamentum flavum

Ligamentum flavum (LF) pathologies often lead to severe myelopathy or radiculopathy characterized by reduced elasticity, obvious thickening, or worsened ossification. Elastin endows critical mechanical properties to tissues and organs such as vertebrae and ligaments. Desmosine (DES) and isodesmosine (IDES) are crosslinkers of elastin monomers called tropoelastin. These crosslinkers are potential biomarkers of chronic obstructive pulmonary disease. As a biological diagnostic tool that supplements existing symptomatic, magnetic resonance imaging scanning or radiological imaging diagnostic measures for LF hypertrophy and associated pathologies, an isotope-dilution liquid chromatography-tandem mass spectrometry method with selected reaction monitoring mode for the quantitation of DESs in human plasma, urine, cerebrospinal fluid (CSF), and yellow ligamentum was investigated. Isotopically labeled IDES-13C3,15N1 was used as an internal standard (ISTD) for DES quantitation for the first time. The samples plus ISTD were hydrolyzed with 6 N hydrochloric acid. Analytes and ISTD were extracted using a solid phase extraction cellulose cartridge column. The assays were repeatable, reproducible, and accurate with % CV ≤ 7.7, ISTD area % RSD of 7.6, and % AC ≤ (101.2 ± 3.90) of the calibrations. The ligamentum samples gave the highest average DES/IDES content (2.38 μg/mg) on a dry-weight basis. A high percentage of the CSF samples showed almost no DESs. Urine and plasma samples of patients showed no significant difference from the control (p-value = 0.0519 and 0.5707, respectively). Microscopy of the yellow ligamentum samples revealed dark or blue-colored zones of elastin fibers that retained the hematoxylin dye and highly red-colored zones of collagen after counterstaining with van Gieson solution. Thus, we successfully developed a method for DES/IDES quantitation in clinical samples.