Tandem Mass Spectrometric Method Research Articles

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The method attended method validation criteria. The limits of detection were equal or lower than 2.0ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0ng/mL, from 0.1 to 5.0ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24°C for 24h, all analytes were stable at -20°C for 14 days and at -80°C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Investigation of Rosa species by an optimized LC-QTOF-MS/MS method using targeted and non-targeted screening strategies combined with multivariate chemometrics.

Plants of the Rosa genus are renowned for their pronounced and pleasant aroma and colors. The aim of this work was to develop a novel liquid chromatographic triple quadrupole time-of-flight tandem mass spectrometric (LC-QTOF-MS/MS) method for the investigation of the bioactive fingerprint of petals of different genotypes belonging to Rosa damascena and Rosa centifolia species. Central composite design (CCD) of response surface methodology (RSM) was used for the optimization of the LC-QTOF-MS/MS method. The method was validated and target, suspect, and non-target screening workflows were applied. Statistical analysis and chemometric tools were utilized to explore the metabolic fingerprint of the Rosa species. RSM revealed that the optimal extraction parameters involved mixing 11 mg of sample with 1mL of MeOH:H2 O (70:30, v/v). Target analysis confirmed the presence of 11 analytes, all of which demonstrated low limits of quantification (LOQs; as low as 0.048 ng mg-1 ) and sufficient recoveries (RE: 85%-107%). In total, 28 compounds were tentatively identified through suspect analysis. Non-target analysis enabled the generation of robust OPLS-DA and HCA models that classified the samples according to their species with 100% accuracy. A novel LC-QTOF-MS/MS method was developed and applied in the analysis of 47 R.centifolia and R.damascena flowers belonging to different genotypes.

Simultaneous determination of ethyl glucuronide, cocaine, cocaethylene, and benzoylecgonine in hair by using LC–MS/MS

Alcohol and cocaine (COC) are commonly co-used drugs that cause addiction and have harmful effects. Their abuse may threaten the health of the abuser and public safety by causing serious accidents or crimes. The recidivism rate of drug-related crimes closely correlates with alcoholism. Several incidences of alcohol consumption in combination with drug abuse have been reported. Here, liquid chromatography tandem mass spectrometric method was developed to simultaneously analyze ethyl glucuronide (EtG), a metabolite of ethanol; COC; cocaethylene (CE), an alcohol-derived metabolite of COC; and benzoylecgonine (BZE), a major metabolite of COC, to determine the concurrent use of alcohol with COC. For pre-treatment, ultracentrifugation (5 min, 50,000 g) and mixed-mode anion exchange solid-phase extraction were used to increase the recovery of target compounds and minimize the matrix effect of hair. The lower limits of quantification were: 7 pg/mg (EtG), 2 pg/mg (COC), 10 pg/mg (CE), and 1 pg/mg (BZE). The correlation coefficient (r) of the calibration curve within the quantified range of target compounds was ≥ 0.9978. The intra- and inter-day accuracies were − 6.1–9.7% and − 9.3–8.3%, and intra- and inter-day precisions were 0.5–10.3% and 0.6–14.4%, respectively. The recovery, matrix effect, process efficiency, and autosampler stability were 89.2–104.8%, 81.6–105.4%, 81.5–107.1%, and 96.6–109.7%, respectively. The novel analytical method was validated with hair samples from individuals suspected of alcohol and COC use, and the method could distinguish between independent and concurrent use. Based on the findings, the analytical approach developed in this study is anticipated to be valuable in drug and alcohol dependence tests that require the simultaneous detection of alcohol and COC abuse.

Liquid Chromatography Tandem Mass Spectrometric Method for Quantification of Margetuximab in Rat Plasma and Application to a Pharmacokinetic Study.

Margetuximab was approved for the treatment of advanced HER2+ breast cancer. A feasible analytical technique that can measure this drug was obligatory. In light of this, a novel and thoroughly validated liquid chromatographic (LC)-tandem mass spectrometric (MS/MS) approach was developed for the quantification of margetuximab in rat plasma. The liquid-liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile and formic acid buffer (30:70) as a mobile phase on Waters, alliance e-2695 model HPLC having Symmetry C18 column, 150 mm × 4.6 mm, 3.5-µm column. The overall runtime was 6 min at a flow rate of 1.0 ml/min. The method showed significant sensitivity and acceptable linearity over the concentration range of 6-120 ng/ml. Accuracy was within 98.51-99.92%. The intraday precision ranged between 0.41 and 8.98% CV. Also, the findings of pharmacokinetic parameters such as Cmax, tmax, AUC0-∞, AUC0-t, and half-life results of margetuximab showed that the technique was helpful for accurately measuring drug concentrations in rat plasma. The method that was developed was useful and effective for quantifying margetuximab.

LC-HRMS methods for the quantification of two artemisinin drugs and their metabolites in rat blood and plasma.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2nm) and its metabolite D-ART (18.8-751.9nm), and the linear range was 40.0-4,000.0nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

Quantification of asciminib by LC-MS/MS method in human plasma: Validation and stability studies

An accurate, sensitive, and linear liquid chromatographic tandem mass spectrometric method was developed for the quantification of asciminib in human plasma. Voriconazole, internal standard (IS), and asciminib were isolated from the plasma samples by C18-cartridges. Chromatographic isolation was processed with Atlantis/dC18 (2.1 × 100 mm, 3 μm) stationary phase and a mobile solvent system of formic acid (0.1%) and methyl alcohol (20:80, v:v), conveyed at a 0.7 ml/minute flowing rate. Analytes were quantified by ionization in a positive approach with electrospray ionization at the mass transitions of (m/z): asciminib, 450.11/239.09, and voriconazole (IS), 350.3/281.1. No interference by constituents of plasma blank or other components was detected. The association between asciminib concentration levels and their respective peak response fractions to voriconazole was rectilinear between 39.0 and 1,586.0 ng/ml. Intraday and interday precisions were ≤4.79% for asciminib. The interday bias was between −4.28% and 5.76%, and the intraday bias was −3.75% and 4.53%. The mean measured extraction recovery of asciminib was 99.06%. The recovery of IS was 98.34%. Asciminib was subjected to long-term, bench-top, freeze-thaw, short-term stability, dry extract, auto-sampling, and stock solution stability at Low quality control and High quality control levels, and it was stable at all these conditions. The established technique can be utilized for the regular quantification of asciminib in plasma samples in industries, forensic laboratories, and clinical research organizations.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Greenness assessment of UPLC/MS/MS method for determination of two antihypertensive agents and their harmful impurities with ADME/TOX profile study

Hypertension is described by the world health organization (WHO) as a serious medical problem that significantly affects the heart, brain and kidneys. It is a major cause of premature death worldwide. The present study aims to quantify the combination of captopril (CPL), hydrochlorothiazide (HCZ) and their harmful impurities; captopril disulphide (CDS), chlorothiaizde (CTZ) and salamide (SMD). In-silico study was conducted for estimation of pharmacokinetic parameters (ADMET) as well as toxicity profile of the proposed impurities. The results showed that the three impurities under investigation had poor permeability to CNS and cannot pass the blood–brain barrier (BBB), reducing the likelihood of causing side effects in the brain. On the other hand, all studied impurities were found to be hepatotoxic. In consequence, a highly sensitive and green ultra-performance liquid chromatography- tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for separation of the cited drugs in the presence of their harmful impurities; methanol and 0.1% formic acid (90:10, v/v) mixture was used as a mobile phase, eluted at a constant flow rate of 0.7 mL/min at room temperature. Detection was adopted using a tandem mass spectrometer in a positive mode only for CPL and negative mode for HCZ, CDS, CTZ and SMD. Separation was performed within 1 min. Calibration graphs were found to be linear in the ranges of (50.0–500.0 ng mL−1), (20.0–500.0 ng mL−1), (10.0–250.0 ng mL−1), (5.0–250.0 ng mL−1) and (20.0–400.0 ng mL−1) corresponding to CPL, HCZ, CDS, CTZ and SMD, respectively. Additionally, comparative study of greenness profile was established for the proposed and reported methods using five green metric tools. The proposed method was found to be greener than the reported HPLC method. The developed (UPLC/MS/MS) method was validated according to (ICH) guidelines and it was found to has greater sensitivity, shorter analysis time and lower environmental impact compared to the reported methods.

Liquid Chromatography Tandem Mass Spectrometric Method Development and Validation for the Quantification of Orlistat in Biological Matrices

A specific, linear and precise liquid chromatographic-tandem mass spectrometric method was established and validated for the quantitation of orlistat in sample plasma. Zorbax C18 (4.6 mm i.d.× 50.0 mm; 5.0 μm) stationary phase was utilized to achieve chromatography elution, through a flowing rate of 0.90 mL/min. Isocratic elution was done using methanol, acetonitrile and 0.10% v/v HCOOH in a fraction of 80:10: 10 v/v/v as the mobile phasic system. For drug and internal standard separation, the precipitation extraction technique used acetonitrile as solvent. A triple quadrupole mass detector was employed for the quantification of ions. Electrospray ionization in a positive ionizing method, which was executed in multiple reaction monitorings (MRM) with parent/product ion transitions of m/z 496.4→337.31 for orlistat and 506.23→57.07 for amprenavir internal standard. The calibration graph was executed between the concentrations of 4.75–190.0 ng/mL and the resulting equation was y = 0.0058x + 0.0022 with r2 value of more than 0.99. Orlistat recovery values were found to be more than 93.65%, and its accuracy, measured in relative error, was in the range of -4.48 to 3.49%. Accuracy findings, sensitivity and recovery values of orlistat in the sample plasma for the established technique evidences its importance in pharmacokinetic and bioequivalence study.

Stability Indicating LC-MS/MS Method Development and Validation for the Quantification of Cabotegravir in Biological Samples

The major goal of current research study was to create a sensitive tandem mass spectrometric method using electrospray ionisation and liquid chromatography for quantifying cabotegravir in biological matrices. A stationary Phenomenex C18 column with dimensions of 50 × 4.6 mm and 5.0 μm particle size of was used to achieve chromatographic elution. With the flowing rate of 0.80 mL/min, isocratic separation was done using methanol and 0.10% V/V HCOOH in a fraction of 85:15 V/V as the mobile phasic system. For drug and internal standard separation, liquid-liquid extraction was carried out using methanol and ethyl acetate (1:4) solvent solution. On repeated reaction monitoring, fragment and product ionic values were seen at m/z 406.12→142.04 for cabotegravir and 450.12→160.03 for bictegravir internal standard. Drug’s linearity graph had a r2 value of 0.9998 and was rectilinear at concentrations between 400 and 16000 ng/mL. The inter- and intra-batch accuracy %relative standard deviation values ranged from 2.54 to 5.21. The percent recovery results of the lower quality control (LQC), median quality control (MQC), and higher quality control (HQC) sample solutions were 102.85, 97.84, and 94.27%, respectively. This approach has excellent recoveries. Studies on stability were processed under various circumstances, and stability values ranged from 92.93 to 103.89%. When exposed to various stability conditions, cabotegravir is more steady for a longer time, and the approach was successfully applicable to routine examination of cabotegravir in biological samples.

Rapid identification of chemical composition and quality markers of Gynostemma pentaphylla by ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry connected with high-performance liquid chromatography

Gynostemma pentaphylla, one of the traditional Chinese medicinal materials, which has gradually become a research hotspot due to its rich pharmacological activities such as hypolipidemic properties, anti-inflammatory, anti-cancer and etc. The main active ingredients of Gynostemma pentaphyllum are saponins (gypenoside), flavonoids and polysaccharides. The pharmacologically active dose depends on the content of the active ingredient, so it is necessary to establish a reliable quality evaluation standard for Gynostemma pentaphyllum. However, the existing Gynostemma pentaphyllum quality control methods are not perfect, Ginsenoside Rb1 was regarded as a signature ingredient of Gynostemma pentaphylla in local standards. Surprisingly, the high-performance liquid chromatogram results displayed that the content of Ginsenoside Rb1 was not suitable as a quality marker. Therefore, a triple quadrupole time-of-flight tandem mass spectrometer method was developed to explore other potential quality markers in Gynostemma pentaphylla. As a result, a total of 24 compounds was identified tentatively. While another stable and reliable component Gypenoside XLVI was found and the content was carried out. It was expected to replace Ginsenoside Rb1 to establish a more reliable and precise quality standard of Gynostemma pentaphylla.

A High-Throughput Absolute Quantification of Protein-Bound Tryptophan from Model and Crop Seeds.

This protocol describes a high-throughput absolute quantification protocol for the aromatic essential amino acid, tryptophan (Trp). This procedure consists of a milligram-scale alkaline hydrolysis followed by an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common alkaline hydrolysis protocol, followed by water extraction that includes L-Trp-d5 as an internal standard to enable the quantification of the absolute level of the bound Trp with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples for Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound tryptophan from seeds.

Bifunctional Ti4+-modified paper for selective extraction or removal of phospholipids and paper spray mass spectrometry for bioanalysis in urine and plasma

BackgroundPhospholipids (PLs) are major constituents of cell membranes, play important roles in cell proliferation and death, as well as in signal transduction, and therefore are relevant biomarkers for different pathologies. On the other hand, when the analysis of small compounds, such as therapeutics in blood is desired, then phospholipids are part of the matrix and cause serious interference during analysis. Currently, both the analysis and removal of PLs from biological samples are limited by extensive sample preparation and instrumental separation. ResultsA fast and simple quantitative Ti4+-modified paper spray tandem mass spectrometric (TiPS-MS/MS) method was established in urine, where the enrichment of phospholipids was achieved, as well as reduction of matrix effects (primarily caused by high salt content) that ultimately led to improved sensitivity and selectivity. The method could achieve a physiologically relevant limit of detection (0.01–0.03 μg mL−1). Also, the usefulness of the Ti4+-modified paper was investigated in the opposite mode, namely for the selective removal of phospholipids from matrices such as plasma. Clonidine is used as model compound, as the detection of this compound is known to suffer from ion suppression by phospholipids. Compared with blank paper spray tandem mass spectrometry, the limit of detection could be improved from 0.3 μg mL−1 to 0.03 μg mL−1 by employing a Ti4+-modified paper on top of the spray tip to capture phospholipids from the sample. Significance and noveltyA novel Ti4+-modified paper was developed to allow for rapid solid-phase extraction of phospholipids from urine or selective removal from plasma, followed by direct paper spray mass spectrometric detection as a fast and convenient sample preparation and analysis combination. The paper properties are based on the Ti4+ metal ion, which can selectively bind phosphate-containing compounds under acidic conditions, and its applicability was demonstrated in relevant biological matrices.

Simultaneous quantitation of multiple myeloma related dietary metabolites in serum using HILIC-LC-MS/MS.

Recent studies from targeted and untargeted metabolomics have consistently revealed that diet-related metabolites, including carnitine (C0), several species of acylcarnitines (AcyCNs), amino acids, ceramides, and lysophosphatidylcholines (LPCs) may serve as potential multiple myeloma (MM) biomarkers. However, most of these approaches had some intrinsic limitations, namely low reproducibility and compromising the accuracy of the results. This study developed and validated a precise, efficient, and reliable liquid chromatography tandem mass spectrometric (LC-MS/MS) method for measuring these 28 metabolic risk factors in human serum. This method employed isopropanol to extract the metabolites from serum, gradient elution on a hydrophilic interaction liquid chromatographic column (HILIC) for chromatographic separation, and multiple reaction monitor (MRM) mode with positive electrospray ionization (ESI) for mass spectrometric detection. The correlation coefficients of linear response for this method were more than 0.9984. Analytical recoveries ranged from 91.3 to 106.3%, averaging 99.5%. The intra-run and total coefficients of variation were 1.1-5.9% and 2.0-9.6%, respectively. We have simultaneously determined the serological levels of C0, several subclasses of AcyCNs, amino acids, ceramides, and LPCs within 15 min for the first time. The established LC-MS/MS method was accurate, sensitive, efficient, and could be valuable in providing insights into the association between diet patterns and MM disease and added value in further clinical research.

Preclinical pharmacokinetics and metabolism study of SCO-267, a GPR40 full agonist, in beagle dogs using ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

SCO-267 is a GPR40 full agonist that has been developed for the treatment of diabetes. To support its preclinical and clinical development, in this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometric method for the determination of SCO-267 in dog plasma using cabozantinib as internal standard (IS). The chromatographic separation was obtained on a Waters acquity BEH C18 column (50×2.1 mm, I.D., 1.7 μm) and the detection was performed using Thermo TSQ triple quadrupole mass spectrometer with multiple reactions monitoring (MRM) positive mode at m/z 615.3>230.1 for SCO-267 and m/z 502.5>323.3 for IS. The method was validated over the concentration range of 1-2000 ng/mL with the lower limit of quantification of 1 ng/mL. The selectivity, linearity, precision and accuracy over this range were acceptable. The extraction recovery was more than 88.73% and no matrix effect was observed. SCO-267 was demonstrated stable during the storage and processing period. The new method was successfully applied to the pharmacokinetic study in beagle dogs following a single oral and intravenous administration. The oral bioavailability was 64.34%. In addition, the metabolites from dog liver microsomal incubation and plasma collected after an oral administration were identified by a UHPLC-HRMS method. The biotransformation pathways of SCO-267 involved oxygenation, O-demethylation, N-dealkylation and acyl glucuronidation.

A fast ultra performance supercritical fluid chromatography-tandem mass spectrometric method for profiling of targeted phytosterols

Phytosterols are essential structural components of plant cell membranes and possess health-related benefits, including lowering blood cholesterol levels in humans. Numerous analytical methods are being used to profile plant and animal sterols. Chromatography hyphenated to tandem mass spectrometry, is a better option due to its specificity, selectivity, and sensitivity. An ultra-performance supercritical fluid chromatography hyphenated with atmospheric pressure chemical ionization (APCI) tandem mass spectrometric method was developed and evaluated for fingerprint analysis of seven phytosterols. Mass spectrometry fragmentation behavior was used for phytosterol identification, and multiple reaction monitoring scanning was utilized for phytosterol confirmation, where APCI outperformed superiority in terms of ion intensity, particularly in the production of [M + H-H2O]+ ions rather than [M + H]+ ions. The chromatographic conditions were thoroughly evaluated, and the ionization parameters were optimized as well. In a 3 min. run, the seven phytosterols were separated concurrently. The calibration and repeatability tests were conducted to check the instrument's performance, and the results indicated that all of the phytosterols tested had correlation coefficients (r2) greater than 0.9911 over the concentration range of 5–5000 ng/mL. The limit of quantification was below 20 ng/mL for all the tested analytes except for stigmasterol and campesterol. The partially validated method was applied for the evaluation of phytosterols in pure coconut oil and palm oil in order to demonstrate its applicability. Total sterols in coconut and palm oils were 126.77 ng/mL and 101.73 ng/mL, respectively. In comparison to earlier methods of phytosterol analysis, the novel method offers a far faster, more sensitive, and more selective analytical process.

Introducing direct probe electrospray ionization tandem mass spectrometry in apiculture: Comparison with other mass spectrometric methods for the determination of antibiotics in bees and honey

Amphenicols and tetracyclines (TCs) antibiotics use in apiculture is mainly focused on the treatment of bee brood diseases. Despite their efficiency, the serious genotoxic effects that some elicit led to their banning for use in apiculture and for chloramphenicol (CAP) in all food-producing animals. In this work, three tandem mass spectrometric methods were developed and validated for the analysis and quantification of CAP, thiamphenicol (TF), florfenicol (FF), tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) in honey and honeybees. With the first, we have demonstrated the first use of probe electrospray ionization (PESI) in conjunction with tandem mass spectrometry (MS/MS) for antibiotics determination in honey and bees entailing a minimum sample preparation for bees, resulting in rapid chemical analysis for both commodities. The second was a reversed-phase HPLC-ESI-MS/MS method sharing the same scope. With the third chiral HPLC-ESI-MS/MS method, it was attempted to separate CAP stereoisomers in both commodities since reports have shown that only the D-threo isomer displays antimicrobial properties. The analytical methods fulfilled validation criteria demonstrating recoveries in the range of 74–92%, managing to address the minimum required performance limit (MRPL) for CAP, with the exception of PESI-MS/MS. The latter displayed a detection capability (CCβ) for all compounds in proximity to 1 ng/g, and an overall runtime of 1.5 min. In total, 68 samples of honeybees and honey were evaluated for amphenicols and TCs residues. CAP was detected only in two honeybee samples, while one honey sample was positive to TC and OTC was detected in traces in another sample. PESI-MS/MS managed to quantify both TC in honey and CAP in bees in proximity to the values assigned by the conventional HPLC-ESI-MS/MS methods. With regard to the separation of CAP's isomers, the chiral method managed to resolute all isomers and quantify CAP in one bee sample in the form of its L-threo stereoisomer. Overall, ABCs detection in honey and related matrices still constitutes a challenge, considering their prevalence and potential applications in major honey-producing countries.

A Sensitive Ultra-performance Liquid Chromatography-Tandem Mass Spectrometric Method for Determination of Secalonic Acid F in Rat Plasma and Its Application to Pharmacokinetic Study.

Secalonic acid F (SAF) is a fungal secondary metabolite exhibited interesting pharmacological effect. In this study, a simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the determination of SAF in rat plasma. Emodin was selected as the internal standard (IS), and plasma samples were prepared by liquid-liquid extraction with ethyl acetate. Chromatographic separation was operated on an Agilent SB-C18 column, and the mobile phase was a mixture of 0.5% formic acid in water and methanol (V:V =20:80) at a flow rate of 0.3mL/min. Detection was carried out with a 6460 triple-quadrupole mass spectrometer using electrospray ionization in the multiple-reaction monitoring mode. The MS/MS ion transitions monitored were m/z 639.3→415.4 and 269.0→225.1 for SAF and IS, respectively. Results showed the calibration curve of SAF was linear in the range of 2-500ng·mL-1 with the correlation coefficient > 0.99. The matrix effect, extraction recovery, dilution effect, intraday and inter-day precision and accuracy were all in acceptable limits. The analytes were also stable under different conditions. The validated method was successfully applied to a pharmacokinetic study after oral administration in Sprague-Dawleyrats at a dose of 10mg/kg.

Measurement of kynurenine pathway metabolites by tandem mass spectrometry

ObjectivesRecent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites. MethodsA tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples. ResultsThe developed method was linear at the concentrations of 48.8 – 25,000, 0.98 – 500, 1.2–5000, 1.2–5000, and 0.98–250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively. ConclusionsA fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients.

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' urine samples. This method could be a supplement to the plasma pharmacokinetics of Semen Strychni.