Rapid Liquid Chromatography-tandem Mass Research Articles

Development and validation of a rapid HPLC-MS/MS method for simultaneous determination of cyclosporine A and tacrolimus in whole blood for routine therapeutic drug monitoring in organ transplantation.

Therapeutic drug monitoring is an integral part of organ transplantation. A rapid, simple, economical, and robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the immunosuppressants cyclosporine A and tacrolimus might increase detection efficiency. In this study, we developed and validated a rapid HPLC-MS/MS method. Whole blood samples of 100 μL were prepared by protein precipitation with acetonitrile and 0.5mol. L-1 ZnSO4. Chromatography was performed on a pre-column using a gradient elution with 20mmol. L-1 ammonium formate and 0.1% (v/v) formic acid in water (mobile phase A) and 0.1% (v/v) formic acid in methanol (mobile phase B) at a flow rate of 1.5mL.min-1. The analysis time was 2.2min. Electrospray ionization and multiple reaction monitoring were performed. The lower limit of quantification was set at 1ng. L-1 for tacrolimus and 50 ng. L-1 for cyclosporine A. The method showed adequate accuracy and precision with a sufficient linear range. The calibration curve range of tacrolimus and cyclosporine A was 1-30 and 50-1500 ng·mL-1, respectively. All correlation coefficients were >0.99. The developed HPLC-MS/MS is rapid and can be used for simultaneous monitoring of tacrolimus and cyclosporine A.

Simultaneous Quantification of Serum Symmetric Dimethylarginine, Asymmetric Dimethylarginine and Creatinine for Use in a Routine Clinical Laboratory.

Background Symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) are naturally occurring amino acids classed as uremic toxins by the EUTOX working group. SDMA is principally excreted through the kidneys and is a well-known renal function marker, ADMA is a potent inhibitor of nitric oxide production. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous measurement of SDMA, ADMA and creatinine. Method Serum samples were prepared by protein precipitation and dilution with acetonitrile prior to injection onto a Waters TQS-Micro. Multiple reaction monitoring was used to detect SDMA, ADMA, creatinine, and their corresponding internal standard transitions after separation with a HILIC analytical column. Sample stability and intra-individual variation studies were also assessed following ethical approval. Results The retention time for creatinine was 0.43, SDMA 1.10 and ADMA 1.14 minutes. Mean recovery for creatinine was 103%, SDMA was 100% and ADMA was 103%, matrix effects were minimal (<6%). Lower limit of quantitation for creatinine and SDMA/ADMA was 17.5 µmol/L and 0.1 µmol/L respectively. Analytical imprecision showed a coefficient of variation <10% for all analytes across the working range of the assays. Intra-individual variation for creatinine was 4.7%, SDMA 7.5% and ADMA 7.6%. Discussion We have developed a rapid assay for LC-MS/MS measurement of SDMA, ADMA and creatinine in a routine clinical laboratory. It is simple, reproducible, and easy to perform. The stability of SDMA and ADMA pre- and post-centrifugation allows for their routine use without any special sample handling requirements.

Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Total and Free Testosterone in Human Serum and Its Application to Monitoring Biomarker Response of Elite Athletes.

Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid-liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02-100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage.

Development of a rapid and robust hydrop interaction liquid chromatography tandem mass spectrometry method for the detection of 13 endogenous amino acids as well as trimethylamine oxide in serum and tissues of the mice.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0mm, 3μM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from -12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients

ObjectiveTo establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole. MethodsA triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC18 RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards. ResultsThe calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation. ConclusionThis newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice.

Stability Evaluation and Pharmacokinetic Profiling of Vepdegestrant in Rodents Using Liquid Chromatography-Tandem Mass Spectrometry.

Vepdegestrant (formerly ARV-471), a novel proteolysis-targeting chimera (PROTAC), targets estrogen receptor alpha (ERα) for degradation, offering a promising option to treat advanced ER-positive breast cancer. We developed and validated a sensitive and rapid liquid chromatography-tandem mass spectrometry method to quantify vepdegestrant in rodent plasma using bavdegalutamide (formerly ARV-110) as an internal standard. Plasma samples were prepared with protein precipitation using acetonitrile and analyzed using reverse-phase C18 columns and a mobile phase of 10 mM ammonium formate in distilled water and acetonitrile. The method demonstrated linearity from 1 to 1000 ng/mL in mouse and rat plasma, meeting all validation criteria, and successfully applied to in vivo and in vitro studies. Pharmacokinetic analysis revealed low-to-moderate clearance (313.3, 1053 mL/h/kg) and oral bioavailability (17.91, 24.12%) of vepdegestrant in mice and rats, respectively. It was unstable in buffer solutions across pH 2-10 and in phosphate-buffered saline (pH 7.4), likely due to adsorption, but remained stable in mouse and rat plasma at varying temperatures. In liver microsomes, vepdegestrant exhibited moderate stability in rats but was stable in mice, dogs, and humans. These findings enhance the understanding of pharmacokinetic properties of vepdegestrant supporting further development of PROTAC drugs.

Simultaneous determination of three tyrosine kinase inhibitors and three triazoles in human plasma by LC-MS/MS: applications to therapeutic drug monitoring and drug-drug interaction studies

Tyrosine kinase inhibitors (TKIs) and triazole antifungals are the first-line drugs for treating chronic myeloid leukemia (CML) and fungal infections, respectively, but both suffer from large exposure differences and narrow therapeutic windows. Moreover, these two types of drugs are commonly used together in CML patients with fungal infections. Multiple studies and guidelines have suggested the importance of therapeutic drug monitoring (TDM) of TKIs and triazoles. Currently, methods for the simultaneous determination of both types of drugs are limited. We developed a simple, rapid, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of three commonly used TKIs (imatinib, dasatinib, and nilotinib) and three commonly used triazoles (voriconazole, itraconazole, and posaconazole) in human plasma. The analytes were eluted on a Welch XB-C18 analytical column (50 × 2.1 mm, 5 µm) at 0.7 mL/min, using a gradient elution of 10 mM ammonium formate (A) and methanol–acetonitrile-isopropanol (80:10:10, v/v/v) containing 0.2 % formic acid (B) with a total analysis time of 3.5 min. The calibration curves were linear over the range from 20 to 4000 ng/mL for imatinib and nilotinib, from 2 to 400 ng/mL for dasatinib, and from 50 to 10,000 ng/mL for voriconazole, itraconazole, and posaconazole. Selectivity, accuracy, precision, recovery, matrix effect, and stability all met the validation requirements. The method was successfully used for TDM in CML patients who co-treated with both TKIs and triazoles. Drug-drug interaction analysis between TKIs and triazoles showed that a significant positive correlation was observed between imatinib and voriconazole, as well as dasatinib and voriconazole. Therefore, this method can be well applied in clinical TDM for patients receiving TKIs, triazoles, or both simultaneously.

A quantitative method for the analysis of total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma with liquid chromatography-tandem mass spectrometry assay.

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC-MS/MS was performed within a run time of 7.5min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1-50 and 0.05-100 mg/l, respectively) and quadratic for other analytes (0.1-50 mg/l, except for ampicillin: 0.1-20 mg/l; R2 > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.

Determination of doxorubicin in plasma and tissues of mice by UPLC-MS/MS and its application to pharmacokinetic study

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous determination of doxorubicin (DOX) in mouse plasma and tissues, including the heart, liver, spleen, lung, kidney and tumor, and to investigate the pharmacokinetics and distribution in mice. In this study, daunorubicin (DNR) was used as an internal standard, and the mobile phase consisted of ammonium formate 2 mM containing 0.1 % formic acid (A) and acetonitrile (B), the chromatographic column was ACQUITY UPLC BEHTM C18 with a gradient elution at a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in positive ion pattern was utilized for the ion separation of DOX, with the ions used for quantitative analysis being DOX m/z 544.28 → 397.10 and DNR m/z 528.35 → 321.08, respectively. The results showed that a good linear relationship in the calibration curve range of 1–800 ng/mL in mouse plasma and 1–2500 ng/g in tissues (R2 > 0.99) with the limits of quantification of 1 ng/mL in plasma and tissues. The method exhibited good matrix effect and extraction recovery, with the intra-day and inter-day precision of plasma and tissue were less than 10.3 % and 15.4 %, and the relative error (RE) were both less than ±14.8 % and ±18.9 %, respectively. The stability results under different conditions were found to be accurate. It also revealed the distribution of DOX in various tissues of mice, with the concentration ranking as liver > heart > kidney > spleen > lung > tumor. This method was successfully used to the study for the pharmacokinetics in plasma and drug distribution in tissues of BALB/c mice.

Simultaneous measurement of 17 endogenous steroid hormones in human serum by liquid chromatography-tandem mass spectrometry without derivatization

Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180 µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11β-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11β-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11β-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05 ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1 ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5 ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.

Quantification of treprostinil concentration in rat and human using a novel validated and rapid liquid chromatography-tandem mass spectrometry method: Experimental and clinical applications in ischemia–reperfusion injury

Treprostinil (Remodulin®) is a Food and Drug Administration (FDA) approved prostacyclin analog to treat pulmonary arterial hypertension. Recently, treprostinil has been investigated to reduce ischemia–reperfusion injury (IRI) during transplantation, which currently has no approved treatment. A validated analytical method is necessary to measure treprostinil concentrations in biological specimens. Here, a novel, sensitive, and specific method to measure treprostinil concentrations in rat serum, human serum, and human plasma has been developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Biological samples were processed by protein precipitation before chromatography and 6-keto Prostaglandin F1α-d4 was used as an internal standard. A gradient method was established with a total run time of 4 min. The assay was linear over the range of 0.25–75.0 ng/ml with accuracy (92.97–107.87 %), intra-assay precision (1.16–3.34 %), and inter-assay precision (1.11–4.58 %) in all biological matrices, which are within FDA acceptance criteria. No significant variation in treprostinil or 6-keto Prostaglandin F1α-d4 concentrations were observed under the investigated storage conditions. This novel, sensitive, and specific LC/MS-MS method is cost-effective and suitable for measuring treprostinil concentrations in animal studies and human biological samples for clinical applications.

Quantitation of thiorphan in human plasma using LC-MS/MS and its application to a bioequivalence study

Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1–200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

Rapid and reliable quantification of urinary malondialdehyde by HILIC-MS/MS: A derivatization-free breakthrough approach

BackgroundThe development of fast analytical methods is crucial for the research, discovery, and confirmation of crucial biomarkers. Furthermore, the implementation of fast analytical strategies contributes to efficient and time-effective procedures. In this sense, analysis of malondialdehyde (MDA) has become an important tool for understanding the role of oxidative stress in various diseases and for evaluating the efficacy of therapeutic interventions. ResultsA rapid and robust liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) has been developed to determine endogenous amounts of malondialdehyde (MDA) in human urine without any associated derivatization reaction. MDA was separated in 4 min through a Urea-HILIC column and was analyzed using a triple quadrupole mass spectrometer in negative electrospray ionization mode. With a 50-fold dilution as the only sample pretreatment after alkaline hydrolysis, no matrix effect was present, which allowed for a fast and simple quantification by means of an external standard calibration with a limit of detection of 0.20 ng mL−1. The whole methodology was validated by analyzing unspiked and spiked urine samples from ten healthy individuals and comparing with the results obtained by the standard addition method. MDA was detected in all cases, with natural concentrations varying from 0.11 ± 0.03 to 0.31 ± 0.03 mg g−1 creatinine. Accuracies were found to be satisfactory, ranging from 95 % to 101 %. The proposed method also exhibited good repeatability and reproducibility (RSD<15 %) for four quality control levels. SignificanceThe main significance of this method is the avoidance of a derivatization reaction for the determination of urinary MDA, this constituting a step forward when compared with previous literature. This breakthrough not only streamlines time analysis to less than 5 min per sample but also results in a more robust procedure. Consequently, the method here developed could be applied to subsequent future research involving the determination of MDA as a lipid peroxidation biomarker, where simple, rapid, and reliable methods could represent a significant improvement.

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC-MS/MS.

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95-99.97 (norvancomycin), 725.90-100.04 (vancomycin), 384.16-67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5-50 μg/ml for serum and 0.5-100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.

Advanced UPLC-MS/MS Method for the Quantification of SIPI6398 in Rat Plasma and Its Pharmacokinetic Characterization.

SIPI6398 is a novel anti-schizophrenia agent with a new mechanism of action and demonstrates better target selectivity and safety compared to its competitors. However, few in vivo studies on the pharmacokinetics and bioavailability of SIPI6398 have been performed. A rapid and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was developed for accurate quantification of SIPI6398 in rat plasma. A simple protein precipitation of acetonitrile-methanol (9 : 1, v/v) was used to treat plasma. Chromatography was performed on a UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) at a flow rate of 0.4 ml/min. The mobile phase consisted of acetonitrile-water (with 0.1% formic acid) and gradient elution was used, and the elution time was 4 minutes. Quantitative analysis was performed using electrospray ionization (ESI) in positive ion detection mode with multiple reaction monitoring (MRM) mode. To evaluate the pharmacokinetics and bioavailability, SIPI6398 was administered to rats in two different ways: oral (4 mg/kg) and intravenous (2 mg/kg) administration. The calibration curve for the UPLC-MS/MS approach shows excellent linearity in the range of 1-2000 ng/mL with an r value above 0.99. The precision, accuracy, recovery, matrix effect, and stability results all meet the criteria established for biological analytical methods. The UPLC-MS/MS method was successfully applied it to pharmacokinetics study of SIPI6398. The bioavailability of SIPI6398 was calculated to be 13.2%. These studies have the potential to contribute towards a more comprehensive comprehension of the pharmacokinetics and bioavailability of SIPI6398.

Determination of nine components of fried Ziziphi Spinosae Semen extract in beagle dog plasma by UPLC-MS/MS and its application in pharmacokinetic research

This study established a convenient, rapid, and sensitive ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, swertisin, N-nornuciferine, 6-feruloylspinosin, and jujuboside B in beagle dog plasma after oral administration of fried Ziziphi Spinosae Semen(FZSS) extract. The Waters HSS-T3 C_(18) column(2.1 mm×100 mm, 1.8 μm) was used. The methanol-aqueous solution(containing 0.01% formic acid) was adopted as the mobile phase for gradient elution. The nine components and two internal standards were completely separated within 8 min. The mass spectrometry detection was performed in multiple reaction monitoring(MRM) mode by positive and negative ion switching of electrospray ionization. The analytical method was validated in terms of specificity, selectivity, linear range, accuracy, precision, recovery, matrix effect, and stability. It could meet the requirement of pharmacokinetic research after oral administration of FZSS extract to beagle dogs. The results showed that the time to reach the peak concentration(T_(max)) of magnoflorine,(R)-coclaurine, vicenin Ⅱ, isospinosin, spinosin, 6-feruloylspinosin, and jujuboside B was 2.40-3.20 h, and the elimination halflife(t_(1/2)) was 2.08-6.79 h after a single-dose oral administration of FZSS to beagle dogs. The exposure of magnoflorine and spinosin was high, with a peak concentration(C_(max)) of 76.7 and 31.5 ng·mL~(-1) and an area under the curve(AUC_(0-∞)) of 581 and 315 ng·h·mL~(-1), respectively. The exposure of the remaining five compounds was lower, with a C_(max) of 0.81-13.0 ng·mL~(-1) and an AUC_(0-∞) of 6.00-106 ng·h·mL~(-1). This study provides a reference for the follow-up research of FZSS.

Simultaneous determination of folic acid photolysis products and oxidized guanine derivatives in plasma by liquid chromatography-tandem mass spectrometry.

Folic acid (FA) is easily photodegraded to yield 6-formylpterin and pterin-6-carboxylic acid, which can generate reactive oxygen species and result in the formation of oxidized guanine derivatives such as 8-hydroxy-2'-deoxyguanosine and 8-hydroxy-guanosine.In this study, we developed a simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry strategy for the simultaneous determination of FA photolysis products and oxidized guanine derivatives in plasma samples. Chromatographic separation was performed on a Waters HSS T3 column (2.1 × 100mm, 5.0μm) with gradient elution at a flow rate of 0.25mL/min. Plasma samples were first pretreated with 1% formic acid, followed by protein precipitation with methanol. The developed method showed good linear relationships between 1 and 2000ng/mL (r2>0.99). The intra- and inter-day precisions ranged from 2.6% to 7.5% and from 2.5% to 6.5%, respectively. Recoveries of the analytes were between 75.4% and 112.4% with the relative standard deviation<9.1%. Finally, the method was applied to quantify FA photolysis products and oxidized guanine derivatives in rats with light and non-light conditions.

Comparative pharmacokinetics of six components in normal and rheumatoid arthritis rats after intragastrical administration of Qianghuo Shengshi Decoction granules by LC-MS/MS

ObjectiveTo investigate the plasma pharmacokinetics of six representative components (nodakenin, osthole, 5-O-methylvisammioside, ferulic acid, liquiritigenin, and liquiritin), which were the ingredients of Qianghuo Shengshi Decoction (QSD) granules, in normal and rheumatoid arthritis (RA) rats administrated QSD granules intragastrically. MethodsA rapid and accurate ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of six components in plasma, and it showed a good specificity, linearity, intra-day and inter-day precision, intra-day and inter-day accuracy, extraction recovery, stability, and the less matrix effect. ResultsThe validated LC-MS/MS method was successfully used to compare the plasma pharmacokinetics of six ingredients between normal and RA rats after intragastrical administration of QSD granules and differences in the pharmacokinetics were found in two types of rats. The absorption rate in the RA rats was lower for nodakenin, osthole, 5-O-methylvisammioside, liquiritigenin and liquiritin than in the normal group, while the absorption rate of ferulic acid remained constant in two groups. Incomparisonwith the normal rats, the exposure concentration of nodakenin was higher and that of other five components except for nodakenin was lower under pathological conditions. Additionally, the absorptive amount of nodakenin, osthole, 5-O-methylvisammioside and liquiritin was increased and that of ferulic acid and liquiritigenin was reduced in the RA rats than in the normal rats. Compared with the normal rats, the retention time of nodakenin, ferulic acid and liquiritin was reduced in vivo, whereas the retention time of osthole, 5-O-methylvisammioside and liquiritigenin was raised in the body for the RA rats. In contrast to the normal rats, the data demonstrated an increase in the elimination velocity of nodakenin and a decrease in the elimination velocity of the other five components except for nodakenin in the pathological state. ConclusionThis study showed that the pharmacokinetic behavior of the six components, nodakenin, osthole, 5-O-methylvisammioside, ferulic acid, liquiritigenin, and liquiritin, is different in vivo between normal and pathological states of rats, and this research provided the necessary experimental data to explain the pharmacokinetics of QSD granules in both normal and pathological states and provide some references for its clinical application at some level.

Extensive analysis of monosaccharide diversity in fermented soybean paste using LC-MS/MS

Doenjang, a widely consumed fermented soybean paste in Korea, contains abundant carbohydrates. However, the composition of various monosaccharides present in fermented foods has not been thoroughly investigated. Thus, we used a rapid and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously analyze monosaccharides in the soybean paste. The method exhibited excellent linearity, sensitivity, and accuracy as confirmed by spiking experiments. In the soybean paste, we identified 11 monosaccharides, with galactose, glucose, and arabinose being predominant, alongside galacturonic acid, N-acetylglucosamine, and glucuronic acid. Analyzing 8 modified and 9 traditional soybean paste samples from different regions, we found a significant correlation between manufacturing method and monosaccharide profiles, with traditional soybean paste exhibiting greater monosaccharide diversity. This method is adaptable for rapid and efficient analysis of monosaccharides in various fermented foods.

Accurate redox state indication by in situ derivatization with N-ethylmaleimide - Profiling of transsulfuration and glutathione pathway metabolites by UPLC-MS/MS

BackgroundReduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies. MethodsHere we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined. ResultsTwelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state. ConclusionThe described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.