Liquid chromatography tandem mass spectrometry method for the quantification of sarpogrelate, a selective 5-HT(₂A) receptor antagonist, in plasma: application to a pre-clinical pharmacokinetic study

A simple LC‐MS/MS method was developed and validated for the estimation of sarpogrelate in 50 µL of rat plasma. The analyte and internal standard (IS) were extracted from rat plasma by acetonitrile precipitation and they were separated on a reversed‐phase C₈ column with gradient program. The MS acquisition was performed with multiple reaction monitoring mode using m/z 430.2 to m/z 135.0 for analyte and m/z 448.2 to m/z 285.3 for IS. The calibration curves were linear over the range of 1–1000 ng/mL with the correlation coefficient greater than 0.999. With dilution integrity up to 20‐fold, the upper limit of quantification was extendable up to 15,000 ng/mL. The method was successfully applied to the analysis of rat plasma samples after single dose oral administration of sarpogrelate at 5 mg/kg to rats for the determination of its pharmacokinetics. Following oral administration the maximum mean concentration in plasma (C_max, 11514 ng/mL) was achieved at 0.25 h (T_max) and the area under curve (AUC_0–24) was 11051 ± 3315 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.9 ± 1.1 h and 490 ± 171 mL/h/kg, respectively. We believe that development of a method in rodent plasma would facilitate the ease of adaptability of sarpogrelate in human plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Caffeine and paraxanthine HPLC assay for CYP1A2 phenotype assessment using saliva and plasma

Caffeine has been extensively used as a probe to measure CYP1A2 activity in humans with caffeine clearance or the paraxanthine (major metabolite of caffeine) to caffeine concentration ratio being regarded as the preferred metric. A simple reverse‐phased C₁₈ HPLC assay using ethyl acetate liquid–liquid extraction was developed to quantitate caffeine and paraxanthine concentrations in saliva and plasma. The mobile phase consisted of acetonitrile–acetic acid–H₂O (100:1:899) and analytes were quantitated with UV detection at 280 nm. The extraction recovery for paraxanthine and caffeine was approximately 70% in both saliva and plasma. The assay was linear over the concentration ranges 0.05–2.50 and 0.05–5.00 µg/mL, for paraxanthine and caffeine, respectively, in saliva. In plasma the assay was linear over the ranges 0.025–2.50 and 0.025–5.00 µg/mL for paraxanthine and caffeine, respectively. Intra‐ and inter‐assay precision and accuracy were less than 15%. Detection limits were 0.015 µg/mL for paraxanthine and caffeine in saliva, while it was 0.005 µg/mL for paraxanthine and caffeine in plasma. Utility was established in samples collected from two healthy volunteers who abstained from caffeine for 24 h and received a single 100 mg oral dose of caffeine. The assay developed is a robust, simple and precise technique to measure caffeine and paraxanthine in saliva and plasma of healthy volunteers after a single oral dose of caffeine. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolite profiles of epimedin C in rat plasma and bile by ultra‐performance liquid chromatography coupled with quadrupole‐TOF‐MS

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C₁₈ column and detected by ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative study on effects of single and multiple oral administration of mungbean (Phaseolus radiatus L.) seed extract on the pharmacokinetics of aconitine by UHPLC‐MS

The study was aimed to investigate the effects of single and multiple oral administration of mungbean (Phaseolus radiatus L.) seed extract (ME) on the pharmacokinetics of aconitine in rats. The Sprague–Dawley rats were randomly divided into three groups (six rats each group). In group 1, rats were orally administered 500 µg/kg aconitine after receiving a single oral dose of 1 g/kg ME. In group 2, rats were orally administered with 500 µg/kg aconitine at day 7 of treatment with 1 g/kg/day ME. In group 3, rats were orally administered with 500 µg/kg aconitine. Blood samples were collected at different time points (0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 h). The concentration of aconitine in rats plasma was determined by a fully validated ultra‐high‐performance liquid chromatography coupled with mass spectrometry method. The results showed that single and multiple oral co‐administration of ME significantly altered the pharmacokinetic parameters of aconitine. Copyright © 2014 John Wiley & Sons, Ltd.     

A simple high‐performance liquid chromatography for the determination of linezolid in human plasma and saliva

Linezolid is an antimicrobial agent for the treatment of multiresistant Gram‐positive infections. A practical high‐performance liquid chromatography method was developed for the determination of linezolid in human plasma and saliva. Linezolid and an internal standard (o‐ethoxybenzamide) were extracted from plasma and saliva with ethyl acetate and analyzed on a Capcell Pak C₁₈ MG column with UV detection at 254 nm. The calibration curve was linear through the range 0.5–50 µg/mL using a 200 μL sample volume. The intra‐ and interday precisions were all <6.44% for plasma and 5.60% for saliva. The accuracies ranged from 98.8 to 110% for both matrices. The mean recoveries of linezolid were 80.8% for plasma and 79.0% for saliva. This method was used to determine the plasma and saliva concentrations of linezolid in healthy volunteers who were orally administered a 600 mg dose of linezolid. Our liquid–liquid extraction procedure is easy and requires a small volume of plasma or saliva (200 μL). This small volume can be advantageous in clinical pharmacokinetic studies, especially if children participate. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane in rat plasma. II. Pharmacokinetic profile in male and female Sprague-Dawley rats evaluated by capillary electrophoresis

This paper describes a pharmacokinetic study performed in Sprague-Dawley rats after i.v. administration of a single 6-mg/kg dose of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane (Altropane). Plasma samples were collected from the retro-orbital sinus at times up to 3 h after drug administration, extracted by solid-phase extraction, and the drug levels determined by capillary electrophoresis (CE). Pharmacokinetic parameters were determined by a standard noncompartmental model using WinNonlin version 1.5. The maximum plasma concentrations, clearances of the drug, and areas under the curve for male and female rats were 5.74 and 7.26 microg/ml, 135.7 and 98.5 ml/kg x min, and 44.23 and 60.92 microg x min/ml, respectively. The drug was cleared very rapidly from the systemic circulation, with a terminal t(1/2) of 7 to 10 min and a mean residence time of about 11 min for both sexes. The volume of distribution was approximately 1 l/kg. No metabolites were detected when the samples were analyzed individually. However, after samples were pooled and concentrated, traces of two unknown peaks that may represent metabolites were detected in concentrates from the last two timepoints. Part I of this work [J. Chromatogr. A, 895 (2000) 87] describes validation of CE methods for the analysis of aqueous and plasma samples of Altropane, including its solid-phase extraction from rat plasma.     

Determination of Mercury and Thallium in Seawater by Electrothermal Vaporization Inductively Coupled Plasma Mass Spectrometry

Electrothermal vaporization inductively coupled plasma mass spectrometry (ETV‐ICP‐MS) has been applied to the determination of Hg and Tl in seawater samples. Various modifiers were tested for the best signal of these elements. After preliminary studies, 0.3% EDTA, 0.1%m/vTAC and 1% v/v HCl were added to the sample solution to work as the modifier. Since the sensitivities of Hg and Tl in various seawater matrices and aqueous standard solutions were quite different, standard addition method and isotope dilution method were used for the determination of Hg and Tl in these seawater samples. This method was applied to the determination of Hg and Tl in NASS‐4 and CASS‐3 reference seawater samples and seawater samples collected from the Kaohsiung area. Results obtained by isotope dilution method and method of standard additions agreed satisfactorily. Detection limits were in the range of 5‐15 and 0.4‐0.5 ng l^?1 for Hg and Tl in seawater, respectively, with the ETV‐ICP‐MS method. The precision between sample replicates was better than 18%) for all the determinations.     

Quantification of urinary AICAR concentrations as a matter of doping controls

Influencing the endurance in elite sports is one of the key points in modern sports science. Recently, a new class of prohibited substances reached in the focus of doping control laboratories and their misuse was classified as gene doping. The adenosine monophosphate activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance even in sedentary mice after treatment. Due to endogenous production of AICAR in healthy humans, considerable amounts were present in the circulation and, thus, were excreted into urine. Considering these facts, the present study was initiated to fix reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 499 athletes were analysed, and AICAR concentrations in urine were determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of sport and type of sample collection (in competition/out of competition). The method was fully validated for quantitative purposes considering the parameters linearity, inter- (12%, 7% and 10%) and intraday precision (14%, 9% and 12%) at low, mid and high concentration, robustness, accuracy (approx. 100%), limit of quantification (100 ng/mL), stability and ion suppression effects, employing an in-house synthesised ¹³C₅-labelled AICAR as internal standard.     

A sensitive LC–MS/MS method for simultaneous determination of cabozantinib and its metabolite cabozantinib N‐oxide in rat plasma and its application in a pharmacokinetic study

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N‐oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC–MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C₁₈ column (2.1 × 150 mm, 3 μm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500–5000 ng/mL for CBZ and 0.525–2100 ng/mL for CBN. The extraction recoveries were constant and the intra‐ and inter‐batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague–Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose‐proportional increases in exposure.     

Application of tail vein serial microsampling for plasma or dried plasma spots in toxicokinetic assessment in rats using acetaminophen as the model compound

In the current study, two groups of rats (five per group) were administered a single oral dose of 500 mg/kg acetaminophen. For toxicokinetic assessment, the Group 1 animals were bled via conventional sparse (two animals/time point) sublingual vein bleeding (~0.5 ml) with anesthesia, while the Group 2 animals were bled via serial tail vein microsampling (~0.075 ml) without anesthesia. All collected blood was processed for plasma. Each Group 2 plasma sample (~30 μl) was divided into ‘wet’ and ‘dried’ (dried plasma spots). All plasma samples were analyzed by LC–MS/MS for acetaminophen and its major metabolites acetaminophen glucuronide and acetaminophen sulfate. In addition, plasma and urine samples were collected for analysis of corticosterone and creatinine to assess stress levels. Comparable plasma exposure to acetaminophen and its two metabolites was observed in the plasma obtained via conventional sparse sublingual vein bleeding and serial tail vein microsampling and between the ‘wet’ and ‘dried’ plasma obtained by the latter. Furthermore, comparable corticosterone levels or corticosterone/creatinine ratios between the two groups suggested that serial microsampling without anesthesia did not increase the levels of stress as compared with conventional sampling with anesthesia, confirming the utility of microsampling for plasma or dried plasma spots in rodent toxicokinetic assessment.     

Enantioselective pharmacokinetics of mabuterol in rats studied using sequential achiral and chiral HPLC

The enantioselective pharmacokinetics of mabuterol was studied in six rats after single oral dose administration of mabuterol racemate. Serial plasma samples were collected and the pharmacokinetic behavior of each enantiomer in rats was characterized using a sequential achiral and chiral liquid chromatographic method. This method involved the separation of mabuterol racemate from endogenous substances on an achiral ODS column and enantiomeric separation on a Chirobiotic V column. The plasma-concentration data were analyzed for individual mabuterol enantiomer using 3P97 software. After i.g. administration of mabuterol racemate at a dose of 10 mg/kg, both enantiomers were slowly absorbed, reaching mean C(max) of 266.8 and 277.9 ng/mL at t(max) of 5.3 and 5.7 h for R- and S-mabuterol, respectively. The AUC(0-infinity) (5,938.9 ng h/mL) of R-mabuterol was significantly higher than that (4,446.1 ng h/mL) of S-mabuterol, and the half-life (14.5 h) was longer than that (9.6 h) of S-mabuterol (p < 0.001 and p < 0.01, respectively), showing that enantioselective pharmacokinetics between mabuterol enantiomers occur during the metabolism phase.     

Determination of antazoline hydrochloride in rat plasma and excreta by reversed‐phase ion‐pair chromatography and its application to pharmacokinetics

A reversed‐phase ion pair chromatography method with liquid–liquid extraction analytical method was developed and validated for the determination of antazoline hydrochloride in plasma and excreta of rat. The aim of our study was to characterize the preclinical pharmacokinetics and excretion profiles of antazoline hydrochloride in rats after intravenous injection at the dose of 10 mg/kg. Plasma and excreta samples were extracted with ethyl acetate, and phenacetin was used as the internal standard. The result showed that the method is suitable for the quantification of antazoline hydrochloride in plasma and excreta samples. Analysis of accuracy (90.89–112.33%), imprecision (<7.1%) and recovery (>82.5%) showed adequate values. After a single intravenous administration at 10 mg/kg to rats, plasma concentration profile showed a relative fast elimination proceeding with a terminal elimination half‐life of 3.53 h. Approximately 61.8 and 14.2% of the administered dose were recovered in urine and bile after 72 and 24 h post‐dosing respectively; 5.9% of the administered dose was recovered in feces after 72 h post‐dosing. The above results show that the major elimination route is urinary excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetics and tissue distribution of coptisine in rats after oral administration by liquid chromatography–mass spectrometry

Coptisine, one of the main components isolated from Coptidis rhizoma, has been reported to have many beneficial pharmacological effects including anti‐inflammatory, anti‐hypercholesterolemia, neuroprotective and cardioprotective properties. However, to date the information related to the in vivo pharmacokinetics (PK) of coptisine is very limited. The purposes of our study are to establish a fast and sensitive quantification method of coptisine using liquid chromatography–mass spectrometry (LC–MS) and evaluate the PK profile of coptisine in rats. The calibration curve for coptisine was linear from 0.78 to 50 ng/mL. After single‐dose oral administration of coptisine, the mean peak plasma concentration values for groups treated with 30, 75 and 150 mg/kg doses ranged from 44.15 to 66.89 ng/mL, and the mean area under the concentration–time curve values ranged from 63.24 to 87.97 mg/L h. The absolute bioavailability was calculated to range from 1.87 to 0.52%. Coptisine remained in all analyzed samples at low concentrations after oral administration of 30 mg/kg.     

Study of human neutrophil peptides in saliva by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides – HNP 1, 2, and 3 – in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI‐TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva‐absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age‐dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging. Copyright © 2009 John Wiley & Sons, Ltd.     

Particle size analysis of perfluorocarbon emulsions in a complex whole blood matrix by sedimentation field-flow fractionation

The goal of this study was to show that sedimentation field-flow fractionation (SdFFF) could be used to study changes in the particle size distribution of perfluorocarbon (PFC) emulsion droplets in ex vivo whole blood samples. A PFC emulsion containing 40% w/v perfluorooctyl bromide (PFOB), 20% w/v perfluorodecyl bromide (PFDB), and 6% w/v egg yolk phospholipid (EYP) was manufactured by high pressure homogenization. The emulsion was infused intravenously to rats at a dose of 2.7 g PFC/kg body weight. Blood samples were collected at 0, 3, 6, and 24 h and analyzed (without additional sample manipulation) by SdFFF. Excellent chromatographic separation between the blood components and PFC emulsion droplets was achieved. After infusion, the particle size distribution broadened slightly. With time, the larger sized droplets were selectively removed by circulating monocytes and tissue resident macrophages of the reticuloendothelial system, causing the particle size distribution to shift to lower median diameters. SdFFF is an excellent technique for studying the in vivo stability of colloidal drug particles following intravenous administration.     

Pharmacokinetics,bioavailability and metabolism of scopoletin in dog by ultra‐high‐performance liquid chromatography combined with linear ion trap–Orbitrap tandem mass spectrometry

A highly sensitive and selective method based on ultra‐high‐performance liquid chromatography combined with linear ion trap–Orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS) has been developed and validated for the determination of scopoletin in dog plasma. The analyte was extracted from plasma samples using acetonitrile and separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) with 0.05% ammonium hydroxide and acetonitrile as mobile phase. The developed method was linear over the concentration range of 1–500 ng/mL, with a correlation coefficient >0.9988. The intra‐ and inter‐day precisions (RSD) were <8.93% while the accuracy (RE) ranged from ?6.50 to 8.12%. Extraction recovery, matrix effect and stability for dog plasma samples were within the required limits. The validated method has been successfully applied to investigate the pharmacokinetics and metabolism of scopoletin in dog plasma after intravenous (1 mg/kg) and oral (10, 25, 50 mg/kg) administration. The results revealed that (a) scopoletin showed short elimination half‐life in dog; (b) its oral bioavailability was low (within the range of 5.69–7.08%); (c) scopoletin showed dose‐independent pharmacokinetic profiles in dog plasma over the dose range of 10–50 mg/kg; and (d) glucuronidation was the predominant metabolic pathway in dog.     

A liquid chromatography–tandem mass spectrometry method for the quantitation of actarit in rabbit plasma: application to pharmacokinetics and metabolic stability

Actarit (ATR), 4‐acetylaminophenylacetic acid is an orally effective disease‐modifying anti‐rheumatic drug widely prescribed for the treatment of rheumatoid arthritis. The present study demonstrates the first report on a selective and sensitive liquid chromatography–tandem mass spectrometry method for the quantification of ATR in rabbit plasma using p‐coumaric acid as an internal standard (IS). Following liquid–liquid extraction, chromatographic separation of the reconstituted samples was achieved isocratically on a Syncronis‐C18 column with a mobile phase consisting of aqueous ammonium acetate (10 mM, pH 4)‐ methanol and acetonitrile mixture (8 : 92, v/v) at a flow rate of 0.6 ml/min. ATR and IS were detected using electrospray ionization operated in negative multiple reaction monitoring mode. The calibration curve was linear (r² ≥ 0.990) over the concentration range of 1–4000 ng/ml with a lower limit of quantitation of 1 ng/ml. The mean extraction recovery of ATR and IS from rabbit plasma was greater than 85%. The method complied well with US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, dilution integrity, carry‐over effect and stability. The method was successfully applied to in vitro metabolic stability (using rabbit liver microsomes) and in vivo pharmacokinetic study after oral administration of ATR at a dose of 10 mg/kg in New Zealand rabbits. Copyright © 2015 John Wiley & Sons, Ltd.     

Noninvasive biochemical monitoring of physiological stress by Fourier transform infrared saliva spectroscopy

Physical stress affects the immune system, activates the sympathetic (SNS) and parasympathetic (PNS) subsystems of autonomic nervous system (ANS), and increases the activity of the hypothalamic-pituitary-adrenal axis (HPA). The specific response of the major regulatory systems depends on the human functional state. Saliva is a unique diagnostic fluid, the composition of which immediately reflects the SNS, PNS, HPA and immune system response to stress. A new method of saliva biomarker determination by Attenuated Total Reflection Fourier-Transform Infrared (ATR FTIR) spectroscopy has been developed to monitor the exercise induced metabolic changes in saliva from male endurance athletes. The method has been tested using a group of professional athletes by analysing saliva samples collected before and after the exercise, and the saliva composition monitoring by ATR FTIR spectroscopy was shown to be suitable for real-time checking of response to stress.     

Determination of cadmium,mercury and lead in seawater by electrothermal vaporization isotope dilution inductively coupled plasma mass spectrometry

Electrothermal vaporization isotope dilution inductively coupled plasma mass spectrometry (ETV-ID-ICP-MS) has been applied to the determination of Cd, Hg and Pb in seawater samples. The isotope ratios of the elements studied in each analytical run were calculated from the peak areas of each isotope. Various modifiers were tested for the best signal of these elements. After preliminary studies, 0.15% m/v TAC and 4% v/v HCl were added to the sample solution to work as the modifier. The ETV-ID-ICP-MS method has been applied to the determination of Cd, Hg and Pb in NASS-4 and CASS-3 reference seawater samples and seawater samples collected from Kaohsiung area. The results for reference sample NASS-4 and CASS-3 agreed satisfactorily with the reference values. Results for other samples determined by isotope dilution and method of standard additions agreed satisfactorily. Detection limits were approximately 0.002, 0.005 and 0.001 ng ml⁻¹ for Cd, Hg and Pb in seawater, respectively, with the ETV-ICP-MS method. Precision between sample replicates was better than 20% for most of the determinations.     

Simultaneous Gas Chromatographic Determination of Diazapam and its Major Metabolites in Human Plasma,Urine, and Saliva

Abstract

A glc analysis method was developed for the simultaneous determination of diazepam (I), and its major metabolites N-desmethyldiazepam (II), oxazepam (III), and hydroxydiazepam (IV) in human plasma, urine, and saliva. Medazepam (V) was used as the internal standard to control extraction efficiency and permit precise and accurate determination of I-IV. Extraction and glc analysis of physiological fluid samples in triplicate required only 40 minutes using the developed method. Three human subjects were given either 20 or 5 mg of I via oral administration and serial specimens of blood, urine, and saliva collected. All samples were analyzed using the developed assay method. Results indicate that the method could be applied to pharmacokinetic studies of I where plasma, urine, and/or saliva is to be monitored.