Liquid Chromatography-tandem Mass Spectrometric Method Research Articles

Development of a rapid LC-MS/MS method for the simultaneous quantification of various flavonoids, isoflavonoids, and phytohormones extracted from Medicago truncatula leaves

Flavonoids, isoflavonoids, and phytohormones are small metabolites of plants, which are involved in the regulation of plant development and/or defense against pathogens. Quantitation of these metabolites in plants is important to basic research on crop protection and in the food and herbal industry. Hence, a robust, reliable, and sensitive method is required for the analysis of these compounds in plant samples. In the present study, a high-performance liquid chromatographic-tandem mass spectrometric (HPLC–ESI-MS/MS) method was developed and validated for the determination of isoflavonoids including genistein, daidzein, formononetin, biochanin A, 2′-hydroxyformononetin, 2′-methoxyformononetin, medicarpin, flavonoids; liquiritigenin, naringenin, and phytohormones; salicylic acid and jasmonic acid in Medicago truncatula. The analytes were separated by means of C-18, reversed-phase chromatography, and detected using QTRAP mass spectrometer. The on-column limit of detection of all the analytes was as low as 0.03 pg, whereas the limit of quantification of all the compounds was observed up to 0.1 pg levels. Further the method was also validated in terms of selectivity, linearity (r 2 > 0.99), recovery (90.6–110.3%), accuracy (RE% ≤ 3%) and precision (RSD% ≤ 3%). As a proof of concept, the developed method was successfully used for the quantitation of these metabolites from leaf extract of M. truncatula.

Liquid chromatography-tandem mass spectrometric method for trace quantification of ethyl methanesulfonate: a genotoxic impurity in dapoxetine hydrochloride

BackgroundDapoxetine hydrochloride is a selective serotonin reuptake inhibitor drug for treating premature ejaculation. This study was designed to develop and validate a sensitive and selective LC–MS/MS method for trace analysis of genotoxic impurity ethyl methanesulfonate in Dapoxetine hydrochloride.ResultsChromatographic separation was achieved on the Shodex RSpak DS-413 column, 150 × 4.6 mm, 3.0 µm using eluent containing a equal volumes of acetonitrile and 0.1% v/v formic acid in water was used in the isocratic elution mode at a pump flow of 1.0 mL/min. No interference was observed at the retention time of ethyl methanesulfonate, indicating that the developed method is specific and selective for trace level quantification.The developed method was found to be linear in the concentration range of 1–50 ppm with coefficient of regression of 0.9997. Detection limit and quantification limit were determined to be 0.6 ppm and 1.0 ppm respectively. Acceptable RSD values (< 10.0%) and recovery results (> 90%) obtained from the accuracy and precison experiments indicate that the developed method is precise and accurate in the concentration range of 1–50 ppm. Ethyl methanesulfonate solutions were stable for two days when stored at room and refrigerated temperatures.ConclusionThe developed method has the ability to quantify ethyl methanesulfonate in dapoxetine hydrochloride. Thus, the anticipated method has high probability to adopt in the quality testing laboratories of pharmaceutical industry.

A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Telmisartan and Chlorthalidone in Rat Plasma and its Application to a Pharmacokinetic Study

A selective, sensitive, rapid, and stable liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed for the simultaneous quantification of chlorthalidone (CLT) and telmisartan (TEL) in rat plasma. Sample preparation involved liquid-liquid extraction by ethyl acetate. Analytes and internal standard (IS) were separated on Gemini C18 (50 x 4.6 mm, 5 μm) using a mixture of methanol-2 mM ammonium acetate (80:20 v/v) as the mobile phase at a flow rate of 0.4 mL/min. Hydrochlorothiazide (HCTZ) was used as an internal standard. Detection was carried out using Electrospray positive and negative ionization mass spectrometry using multiple reaction monitoring of the transition at m/z 515.90→276 for TEL and m/z 337.1→190.05 for CLT, m/z 296.1→205.3 for HCTZ respectively. For TEL and CLT, the method was linear (R2 ≥ 0.995) within the range of 6-1200 ng/mL and 3-600 ng/mL respectively. The retention time for TEL, CLT, and IS was found to be 3.65±0.03 min, 1.83±0.02 min, and 1.76±0.01 min. The mean recovery for both analytes and IS was greater than 70 %. The developed method was validated according to the USFDA guideline. The quantitative method was validated for specificity, linearity, accuracy, precision, recovery, dilution integrity, matrix effect, and analyte stability. The method was successfully applied to a pharmacokinetic study in rat plasma following oral administration.

An ultra-performance liquid chromatography-tandem mass spectrometric method for the identification and quantification of selected natural antioxidants in prickly pear samples

The aim of the present study was to develop a rapid, simple and reliable method for the identification and quantification of ten natural antioxidants in prickly pears using liquid chromatography with electrospray ionization tandem mass spectrometry. The analytical method was fully-validated, and calibration curves were established with correlation coefficients >0.99. A direct extraction method of analytes (ascorbic acid, catechin, gallic acid, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, rutin, and taurine) with aqueous methanol resulted in a short analysis time and a good extraction efficiency. LOD values were from 0.17 to 15.0 μg kg−1 and LOQ values were from 0.50 to 45.4 μg kg-1 with recoveries ranging from 62.2 to 104.0 %. The expanded uncertainty was 9.7–19.0 % expressed at a coverage level of k = 2 at a confidence level of approximately 95 %. The method was successfully applied to yellow prickly pear samples from Cyprus. All above antioxidant compounds were present in all prickly pear samples analyzed except for Myricetin and Luteolin which were not detected in any of the samples.

Development and validation of a LC-MS/MS method for quantitation of 3-hydroxypentanoic acid and 3-oxopentanoic acid in human plasma and its application to a clinical study of glucose transporter type I deficiency (G1D) syndrome

Interest in human and experimental animal metabolism of substrates containing an odd number of carbons capable of fueling the tricarboxylic acid cycle such as heptanoic acid has motivated us to develop and validate a selective and specific liquid chromatographytandem mass spectrometric method for the simultaneous, quantitative determination of the ketone body byproducts 3-hydroxypentanoic acid and 3-oxopentanoic acid in plasma. Human plasma samples were protein-precipitated with methanol containing 0.2% formic acid. Chromatographic resolution was achieved on a Phenomenex Luna C18 column using gradient elution with mobile phases of water containing 0.1% formic acid and methanol containing 0.1% formic acid at 0.3 mL/min flow rate. The retention times of 3-hydroxypentanoic acid, 3-oxopentanoic acid and sulbactam (internal standard) were 3.85, 4.23, and 5.11 min, respectively. Validation was conducted in accordance with United States Food and Drug Administration guidance. The validated range of 3-hydroxypentanoic acid was 0.078–5 µg/mL and 0.156–10 µg/mL for 3-oxopentanoic acid. The method was accurate and precise over this range and exhibited 10-fold dilution integrity in human plasma. Recovery> 88% was achieved for analytes and internal standard. There was no matrix effect observed in human plasma. Both 3-hydroxypentanoic acid and 3-oxopentanoic acid were stable across conditions including autosampler, benchtop and freeze-thaw, as well as demonstrated long-term stability at −80 °C. The method was applied to the measurement of 3-hydroxypentanoic acid and 3-oxopentanoic acid concentrations in plasma from subjects receiving the triglyceride triheptanoin (as a source of heptanoate) for the experimental treatment of glucose transporter type I deficiency (G1D) syndrome.

Presence of Alternaria toxins in maize from Republic of Serbia during 2016–2017

The aim of this study was to apply modern analytical tools, liquid chromatography-tandem mass spectrometric method (LC-MS/MS), for identification and quantitation of Alternaria metabolites in maize samples. Maize samples were collected from the main maize-producing regions (Bačka, Banat, and Srem) of the Republic of Serbia during two harvest seasons (2016 and 2017). The most commonly detected Alternaria toxin in maize samples from both years was tenuazonic acid. On the other hand, determined mean concentrations of quantified tenuazonic acid were significantly higher in maize samples from Banat from both maize growing seasons in comparison to its concentrations in maize samples from Bačka and Srem. As a consequence of influence of microclimate conditions in investigated regions, the highest percentages of contaminated maize samples by Alternaria toxins, in both the years 2016 and 2017, were 48% and 47%, respectively from Srem, while the lowest percentages of 29% and 23%, respectively were found in Bačka. Novelty impact statement The main novelty of this study is achieved through increased knowledge and creation of one of the few database from this part of Europe regarding the frequencies of occurrence of “emerging” Alternaria toxins in maize, depending on weather conditions and some of the applied agrotechnical measures during a two-year period.

Effect of environmental factors on polyketide synthase gene expression, usnic acid biosynthesis, and ground cover of a lichen-fungus, Cladonia uncialis

Lichens are composed of symbiotic slow-growing organisms and are often exposed to extreme microenvironmental conditions, resulting in the production of unique secondary metabolites. One of the most commonly produced secondary metabolites is usnic acid, which is thought to be produced by two genes. The objectives of the present study were to compare polyketide synthase (PKS) gene expression and usnic acid concentration in Cladonia uncialis (L.) Weber ex F.H. Wigg., with two environmental factors. Seventy-five lichen samples were collected from three locations in Newfoundland, Canada, using a strip transect method (×5 transects, ×5 quadrats). Usnic acid concentration was measured using the liquid chromatography tandem mass-spectrometric method and gene expression of two PKS genes (methylphloracetophenone oxidase (MPAO) and methylphloracetophenone synthase (MPAS)) was examined using quantitative real-time PCR. The results showed that percent ground cover of C. uncialis was affected by soil pH level but not soil moisture, and usnic acid concentration was not affected by either soil pH or soil moisture. MPAO gene expression level was significantly affected by soil pH level but not soil moisture, and MPAS gene expression level was not affected by either soil pH level or soil moisture. There was no significant relationship between MPAS and MPAO gene expression levels and usnic acid concentration. These findings suggest that soil pH may be important for the production of usnic acid by C. uncialis but the genes involved require further study.

Multi-Class UHPLC-MS/MS Method for Plant Toxins and Cyanotoxins in Food Supplements and Application for Belgian Market Samples.

The presence of plant toxins and/or cyanotoxins in food supplements implies consumer health risks. Therefore, a targeted ultra-high performance liquid chromatographic-tandem mass spectrometric method to detect/quantify 25 toxins simultaneously in food supplement formulations was developed and validated. Full validation for tablets/powders and secondary validation for a liquid and soft gel capsule indicated that most compounds were efficiently extracted (≥ 75%), while others were only partly extracted (18 - 61%). Trueness was fulfilled (70 - 120%), with some exceptions (mostly at the lowest validation level). Intralaboratory repeatability, intra- and interlaboratory reproducibility values of ≤ 20%, ≤ 25%, and ≤ 25% were obtained for most, respectively. Matrix effects were found to be significant for most compounds. Good sensitivity (µg/kg level) was observed for galegin(e), lycopsamine, lycorine, rubiadin, skimmiamine, and vascin(e), in contrast to helveticoside, lucidin, lucidin-3-primveroside, plumbagin(e), and thujone, which were detected at the mg/kg level. The other compounds were characterized by a sensitivity between 10to 1000 µg/kg. The validated methodology was applied for 52 food supplements (tablets, capsules, liquids/syrup, etc.) purchased from the Belgian market. In more than 25% of the samples, one or more toxins were detected (concentrations determined using standard addition). Lycopsamine, microcystin LR, solamargine, thujone, and vasicin(e) were the most frequently detected toxins. A clear link between the toxins detected and the plant species on the food supplement ingredient list could not always be established. This generic "dilute-and-shoot" procedure can be used for further research on toxins in food supplements and by extension other plant/algae-based food/feed commodities (herbs, edible flowers, etc.).

Simultaneous Determination of Seven Bioactive Constituents from Salvia miltiorrhiza in Rat Plasma by HPLC-MS/MS: Application to a Comparative Pharmacokinetic Study

The roots of Salvia miltiorrhiza (Danshen) is a precious herbal medicine used to treat cardiovascular diseases. This study establishes a high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method to quantify seven bioactive constituents from Danshen in rat plasma simultaneously. Chromatographic separation is performed on an Agilent Eclipse Plus C18 column (150 mm × 2.1 mm, 5 μm), utilizing a gradient of acetonitrile and 0.2% formic acid aqueous solution as the mobile phase, at a flow rate of 0.6 mL/min. We conduct a tandem mass spectrometric detection with electrospray ionization (ESI) interface via multiple reaction monitoring (MRM) in both positive and negative ionization mode. Our results show that a linear relationship is established for each analyte of interest over the concentration range of 0.5–300 ng/mL with r ≥ 0.9976. The validated method is successfully used to compare the pharmacokinetic properties of crude and wine-processed Danshen extract orally administered to rats. Cmax of tanshinone IIA, Cmax, and AUC0-t of dihydrotanshinone I decrease significantly (p &lt; 0.05) in the wine-processed group. No significant changes for other compounds are observed. These results might provide meaningful information for the further application of wine-processed Danshen and understanding of wine-processing mechanisms.

Simultaneous determination of 3-n-butylphthalide and its metabolite 10-hydroxy-butylphthalide in rat plasma using liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study.

3-n-Butylphthalide (NBP) is a potent drug for the treatment of ischemic stroke. The aim of this study was to develop a simple and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method for the simultaneous determination of NBP and its circulating metabolite 10-hydroxy-NBP in rat plasma using senkyunolide I as the internal standard (IS). The analytes and IS were extracted from the plasma by ethyl acetate-ethyl ether (1:5, v/v) and then separated on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 μm). The mobile phase consisted of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, which was delivered at a flow rate of 0.3 mL/min with gradient elution. MS detection was achieved under selective reaction monitoring mode with precursor-to-product transitions at m/z 191.1 > 145.1 for NBP, m/z 207.1 > 171.1 for 10-hydroxy-NBP and m/z 207.1 > 161.1 for IS, respectively. The assay showed excellent linearity over the concentration range of 0.5-1000 ng/mL for both analytes, with correlation coefficient greater than 0.998. The other validation parameters were all within the required limits. The validated UPLC-MS/MS method has been further applied to the pharmacokinetic study of NBP and 10-hydroxy-NBP in rats after they were orally administered with NBP (30 mg/kg).

Pharmacokinetic Analysis of Rosmarinic Acid and its Analog in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry.

The anticancer effects of rosmarinic acid (RA) are a hotspot of current research. In order to enhance its pharmacological activity, N-substituted RA was prepared, and it has been shown to exhibit notable antitumor effects. In order to elucidate the underlying mechanisms of action, pharmacokinetic analysis is necessary. In the present study, liquid chromatography-tandem mass spectrometric method, was used to determine the concentrations of RA and its analog, (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A2) in plasma from rats. The analyses were divided into a C18 column (1.9μm, 2.1mm × 100mm) with a security guard C18 column (5μm, 2.1mm × 10 mm) and a triple-quadrupole mass spectrometry with an electrospray ionization ion-source generates ions. The sample pretreatment is relevant to the one-step protein precipitation with isopropanol:ethyl acetate (v/v, 1:1) This method presented a linear association within ranges at the concentration of 5-2000 ng/mL for A2 and RA. Relative standard deviations in daily courses were <15% and the relative errors registered within 15%. The methods used in the present study make the unambiguous quantification and identification of RA and A2 possible in vivo. The present study is the first to focus on determining A2 and RA in rat plasma following oral administration. The results may provide a meaningful basis for the evaluation of the application of RA and its analog in clinical practice and also provide a reference method for the pharmacokinetic analysis of RA analogs.

Quality by design optimization of a liquid chromatographic-tandem mass spectrometric method for the simultaneous analysis of structurally heterogeneous pharmaceutical compounds and its application to the rapid screening in wastewater and surface water samples by large volume direct injection

This study focused on the Analytical Quality by Design (AQbD) optimization of the chromatographic separation and mass spectrometric detection of a wide group of structurally heterogeneous model pharmaceutical compounds (PhCs) and transformation products (TPs), chosen to cover the challenging issues of the co-presence of compounds characterized by (i) a wide range of physicochemical properties, (ii) the same mass transitions, and (iii) different ionisation modes. Italian consumption of PhCs were also considered as election criteria of target analytes. Octadecyl and pentafluorophenyl stationary phases, acetonitrile/methanol ratios and acidity of the eluents, column temperature, initial organic phase percentage, and elution gradient were investigated by AQbD, aiming at optimizing critical resolutions, sensitivities, and analysis time. Statistically significant models were obtained in most cases with fitting and cross-validation coefficients in the ranges of 0.681-0.998 and 0.514-0.967, respectively. After optimization, the analysis of target analytes was performed in a single chromatographic run, adopting a mixed acquisition mode based on scheduled acquisition windows comprising both single polarity and continuous polarity switching. For most investigated analytes the method provided detection limits in the sub-ng/L to low ng/L range, meeting for macrolides the sensitivity requested by the “Watch List” 2018/840/EU. The optimized method was applied to the direct injection analysis of PhCs and TPs in four wastewater treatment plant (WWTP) effluents and surface water (SW) samples collected in the receiving water bodies. Absolute values of matrix effect were found to be far higher than 20% for most target analytes in most samples. Seventeen PhCs and two TPs were quantified in at least one sample, at the wide concentration range of about 1-3200 ng/L. The most occurring PhCs in both WWTP effluents and SWs were levofloxacin (202-1239 and 100-830 ng/L), furosemide (865-3234 and 230-880 ng/L), ketoprofen (295-1104 and 270-490 ng/L), and ibuprofen (886-3232 and 690-1440 ng/L).

Meconium and maternal hair analysis vs. medical records to monitor antidepressants and benzodiazepines exposure during pregnancy

Psychiatric disorders and drugs used for their treatment, can have negative consequences for the pregnancy and the development of the newborn. Therefore, meconium, maternal hair and maternal medical records were compared for the detection of antidepressants and benzodiazepines exposure during pregnancy. Moreover, correlations between neonatal outcomes and meconium or maternal hair results were evaluated. Two liquid chromatography-tandem mass spectrometric methods for the determination of the most common benzodiazepines and antidepressants in meconium and maternal hair were applied to the analysis of 145 matched samples. Statistical analyses were performed with IBM SPSS Statistics software. According to the medical records, 5.5% of the mothers used prescribed antidepressants during pregnancy and 23.1% benzodiazepines (10.5% at the time of delivery). For antidepressants, 5.5% of meconium and 24.8% of maternal hair specimens were positive; for benzodiazepines, 16.6 and 15.9% of meconium and hair specimens, respectively, were positive. Overall agreement meconium/hair, and medical records/hair, for positive cases, was very poor (28.1 and 18.9%, respectively), while improved between medical records/meconium (60%). Regarding the evaluated neonatal parameters, we only found a significant negative correlation between male weight and meconium-positive results for benzodiazepines. Maternal hair was more sensitive to detect antidepressants use, while meconium analysis can provide detection of neonatal benzodiazepines exposure at the time of delivery. Therefore, combination of both matrices is recommended, since it shows a more complete picture of prenatal exposure to these drugs. No clear influence of prenatal exposure to antidepressants or benzodiazepines on neonatal outcomes was noticed.

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2–2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7–111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.

Application of an ultra-performance liquid chromatography-tandem mass spectrometric method for the detection and quantification of cannabis in cerumen samples

In this study, cerumen, a non-conventional biological secretion, was examined as an alternative matrix for forensic analyzis. A fully validated analytical UPLC-MS/MS method was developed for the detection and quantification of the most prevalent psychoactive illicit drug globe wide, Δ9-tethrahydrocannabinol, commonly known as THC, and four major cannabinoids found in cannabis Sativa. The method was validated, and standard external calibration curves were established with correlation coefficients > 0.99. A validated experimental procedure, along with a direct extraction of cannabinoids with acidified acetonitrile resulted in a short total analyzis time and a good extraction efficiency for all the analytes under study. LOD and LOQ values were determined to be 0.01–0.08 pg/mg and 0.04–0.23 pg/mg, respectively. To prove applicability of the proposed assay, volunteers were selected, and cerumen samples were examined for cannabis. The analyzis by use of UPLC-MS/MS indicated that all samples were positive, reporting recent cannabis abuse. Surprisingly, both THC and Cannabinol (CBN) were detected, and quantification was possible in 75% of the cases.

A validated UHPLC-MS/MS method for determination of TQ-B3203 in human plasma and its application to a pharmacokinetic study in Chinese patients with advanced solid tumor.

TQ-B3203 is a new topoisomerase I inhibitor derived from camptothecin. In this paper, a simple and reliable ultra high-performance liquid chromatography-tandem mass spectrometric method was developed and validated for determination of TQ-B3203 in human plasma with TQ-B3203-d8 used as the internal standard. Bis(p-nitrophenyl)phosphate (2mol/L) was added to ensure the stability of TQ-B3203 in human plasma. Plasma samples were protein precipitated by methanol and processed samples were chromatographed on an AQUITY BEH C8 column (50 × 2.1mm, id 1.7 μm) with acetonitrile and water (0.1% formic acid) as the mobile phase. The calibration curves showed good linearity (R≥0.99) over the concentration range of 0.5-500ng/mL. Within- and between-run precision were ≤5.8% and the accuracy was within the range of -8.3 to 14.0%. This method was further successfully applied to a pharmacokinetic study of TQ-B3203 in Chinese advanced solid cancer patients after administration of TQ-B3203 liposome injection.

Isoprostanes in wastewater as biomarkers of oxidative stress during COVID-19 pandemic

Isoprostanes are the potential biomarkers of endogenous human metabolism and proven clinically to provide the quantitative measure of systematic oxidative injury. An ultra-performance liquid chromatography-tandem mass spectrometric analytical method capable of determining four biomarkers of oxidative stress (8-iso-PGF2α, 2,3-dinor-iPF2α-III, PGE2, and 5-iPF2α-VI) in wastewater was developed and validated. Isoprostanes were quantified in the range of 31.1–1270 ng/L in raw wastewater samples in two communities in western Kentucky and Tennessee during the first four months of the COVID-19 pandemic. Consistent detection of PGE2 and 5-iPF2α-VI in wastewater suggested that PGE2 and 5-iPF2α-VI can be a reliable biomarker of community oxidative anxiety. The higher 4-month average mass load of isoprostanes [(ranged from 22.9 mg/d/1000 people to 807 mg/d/1000 people] may be attributed to the elevated community level oxidative anxiety owing COVID-19 uncertainties. The average mass loads of PGE2 and 5-iPF2α-VI in a community were significantly increased (two-tailed p < 0.001) from the first month of COVID-19 pandemic to the second month; however, significantly decreased (two-tailed p < 0.001) in the third month. Wastewater-based-epidemiological determination of isoprostanes can be a near-real-time and cost-effective approach of a trend in community depression. This is the first report of the quantification of PGE2 and 5-iPF2α-VI in wastewater and estimation of the community level oxidative anxiety.

A validated liquid chromatography-tandem mass spectrometric method for the determination of co-administered ranitidine and metronidazole in plasma of human volunteers

This work describes the development, optimization and validation of a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for the simultaneous analysis of co-administered ranitidine (RAN) and metronidazole (MET) in plasma of healthy human volunteers. A simple protein precipitation procedure using 1 mL acetonitrile was applied to extract both drugs and metoclopramide as an internal standard (IS) from plasma. The chromatographic separation was achieved on a C18 column with isocratic elution of the mobile phase consisting of acetonitrile and 0.1% formic acid (90 : 10, v/v) at a flow rate of 0.35 mL min-1. The positive ionization mode was used for detecting the ions, by observing the pairs of transition m/z 314.82 < 176.06 for RAN, m/z 172.03 < 127.95 for MET and m/z 299.86 < 277.10 for IS. The linearity range was from 5-600 ng mL-1 for RAN and 2-40 μg mL-1 for MET. The method was found to be sensitive and accurate with good simple extraction recovery and matrix effect, according to FDA guidelines for bioanalytical methods. The developed method could be applied for further bioavailability studies that could be useful in therapeutic drug monitoring.

Ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitation of twelve phenolic components in different parts of sarcandra glabra

Objective: The study objective was to determine phenolic components for the quality control (QC) of cultivated Sarcandra glabra (Thunb.) Makino ( S. glabra). Materials and Methods: A sensitive, ultra-high-performance liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 12 phenolic components has been developed. Six caffeoylquinic acids, two caffeoylshikimic acids, and four flavanonol glucosides were selected for the comprehensive analysis of distribution in different parts (root, stem, and leaf). Results: Twelve phenolic components were linear in the concentration range of 0.005–5.0 μg/mL ( R2 > 0.995). The relative standard deviation of intra-day and inter-day precision across three validation runs over the entire concentration range was <5%. The recovery determined was within 5% in terms of relative error. Our results showed that the 12 phenolic compounds were mainly distributed in leaves and stems far more than those in roots. Conclusions: This study provided an ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitative method of 12 phenolic components for the QC of cultivated S. glabra. It was found that the phenolic components were significantly accumulated in the aerial parts (stems and leaves) of cultivated S. glabra.

Simultaneous determination and pharmacokinetics of tetrandrine, fangchinoline, and cyclanoline in rat plasma by ultra-high performance liquid chromatography-mass spectrometry after oral administration of stephaniae tetrandrae radix extract

Objective: The objective of the study was to develop a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of tetrandrine, fangchinoline, and cyclanoline in rat plasma and to investigate their pharmacokinetics after oral administration of Stephaniae Tetrandrae Radix extracts. Methods: Sample pretreatment involved methanol pretreatment and liquid–liquid extraction of ethyl acetate from plasma with methanol. Tramadol was used as the internal standard. The analysis was performed using an high strength silica T3 column (100 mm × 2.1 mm, 1.8 μm) and a gradient elution method consisting of mobile phase solution A (0.1% formic acid in water) and B (acetonitrile) at a flow rate of 0.4 mL/min. The detection was performed using a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode and using an electrospray ionization source in the positive ionization mode. Results: High efficiency was achieved with an analysis time of 4 min/sample. The calibration curve linear in the concentration range of 1250 ng/ml ( R2 ≥ 0.9900) and the lower limit of quantification is 1 ng/ml. The intraday and interday precision (relative standard deviation) values were lower than 9.4. Accuracy (relative error) was within 10.3% at all three quality control levels. Conclusions: This method was successfully applied in pharmacokinetics of tetrandrine, fangchinoline, and cyclanoline in rats after oral administration of Stephaniae Tetrandrae Radix extracts. The maximum plasma concentration ( Cmax) of tetrandrine, fangchinoline, and cyclanoline was 124.71 ± 16.08, 84.56 ± 3.28, and 57.61 ± 6.26 ng/mL, respectively. The time to reach Cmax was 10.39 ± 3.04 for tetrandrine, 10.17 ± 3.04 for fangchinoline, and 6.40 ± 3.16 for cyclanoline. The pharmacokinetic results might help further guide the clinical application of Stephaniae Tetrandrae Radix.