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[Display omitted] • A fast and sensitive method was established for determination of 12 analytes. • The pharmacokinetics of Zingiberis Rhizoma − Jujubae Fructus herb pair were profiled. • The absorption of the analytes was decreased in the stomach cold syndrome rats. • The compatibility of the herb pair promoted the absorption of analytes in model rats. The Zingiberis Rhizoma − Jujubae Fructus herb pair (ZJHP) is a classic herb pair in traditional Chinese medicine. The herb pair shows the effect of dispelling cold, harmonizing the middle and improving gastrointestinal function, and is widely used for patients with stomach cold syndrome (SCS), stomachache and anemofrigid cold. The gingerols, shogaols, flavonoids and triterpenic acids are the important bioactive ingredients of ZJHP. However, few pharmacokinetic studies have been investigated in vivo for the above compounds. To comprehend the kinetics of active components and promote their curative application, a fast and sensitive ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) method was established for simultaneous determination of 12 analytes in normal and SCS rats in this study. The results showed that the pharmacokinetic parameters (C max , T max , t 1/2z , MRT 0-t , AUC 0-t and AUC 0-∞) in SCS model were significantly different from those in normal rats. In addition, the pharmacokinetics of rats given ZJHP were also varied from single herb oral administration, especially in model condition. These results indicated that the in vivo processes of the above analytes changed under pathological conditions and the compatibility of the herb pair could significantly influence the absorption of active components, which might provide an insight and further supports for the clinical application of ZJHP. [ABSTRACT FROM AUTHOR]

Abstract Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (K m) value of genistein was obvious increased while its maximal velocity (V max) and intrinsic clearance (CL int) values were significantly decreased by diabetic RLMs, and the V max and CL int values of kaempferol and isorhamnetin were notably enhanced while their K m values were markedly reduced. For the half-time (t 1/2) values of four target compounds and the K m , V max and CL int values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE. Highlights • The biotransformation of GBE in diabetic rat liver microsomes (RLMs) was first investigated. • An off-line 2D LC-DAD-q/TOF-MS was first developed and optimized for the analysis. • 6 mono-glucuronide conjugation metabolites were tentatively characterized. • The metabolic stability of quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs was obtained. • The enzyme kinetics of quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs was first reported. [ABSTRACT FROM AUTHOR]

Abstract Identification of metabolic alterations is useful for elucidating the pathophysiology and mass spectrometry-aided metabolomic, which provides small molecule metabolites to understand underlying mechanisms of complex diseases. In this work, we developed a novel liquid chromatography coupled with tandem mass spectrometry-based metabolomics screen coupled with multivariate statistical analysis workflow to obtaining the metabolite annotation, visualization and rapid characterization of small molecular metabolites, then revealed the altered metabolic pathway in a rat model of alcohol-induced liver damage rats as case study. The ions were detected in both positive and negative mode, and the key biomarkers were selected through multivariate statistical screening analysis. We employed the enhanced mass spectrometry coverage method to comprehensive analysis of the metabolite data. Importantly, we further find these small molecule metabolites were associated with multiple metabolic pathways. The elucidation of these altered metabolic pathways should help identify new and specific therapeutic targets. In addition, this metabolomics workflow coupled to LC–MS/MS has been successfully applied to the identification of small molecule metabolites in this complex disease. We strongly believe that metabolomics studies will benefit from incorporating liquid chromatography coupled with tandem mass spectrometry strategies. Highlights • LC-MS has been successfully applied to the identification of small molecule metabolites. • Identification of metabolic alterations is useful for elucidating the pathophysiology. • Metabolomics provides tool to understand mechanisms of alcohol liver damage. • Biomarkers were selected through multivariate statistical screening analysis. • The altered metabolic pathways should help identify specific therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract (+)-Borneol, a bicyclic monoterpene, has been shown to possess valuable biological properties and potential as a pharmaceutical agent due to anti-inflammatory, anti-oxidant and GABA receptor-enhancing functions; it also enhances the permeability of the blood brain barrier to improve the efficacy of CNS drugs. In this study, we have developed a simple, selective, and rapid liquid chromatography-tandem mass spectrometry method for the assay of (+)-borneol in rat plasma. Verapamil was used as an internal standard. Plasma samples were deproteinized using methanol. The analyte was detected by a mass spectrometer with positive atmospheric pressure chemical ionization by multiple reaction monitoring mode for transitions at m / z [M + H]+ 137.2 → 81.0 for (+)-borneol and 455.2 → 165.1 for verapamil. The method has been fully validated to ensure good selectivity, a satisfactory lower limit of quantification at 10.0 ng/mL, acceptable intra- and inter-day accuracy, and high precision. The method was used for the pharmacokinetic evaluation of (+)-borneol in Sprague-Dawley rats after intravenous, oral, and sublingual administration. The results indicate that oral bioavailability of (+)-borneol was extremely low but sublingual administration yielded rapid absorption and favorable bioavailability of (+)-borneol. Graphical abstract Unlabelled Image Highlights • A simple, selective, and rapid LC-MS/MS method was validated for quantify (+)-borneol in rat plasma. • The sensitive, robust and high throughput method has been sucessfully applied for a pharmacokinetic study. • The pharmacokinetic profile of (+)-borneol as a stereoisomer was first reported. [ABSTRACT FROM AUTHOR]

Abstract A selective, sensitive and fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the determination of dihydromyricetin, dihydroquercetin, myricetin and quercetin in rat plasma after intragastric administration of Hoveniae Semen total flavonoids. Baicalin was selected as the internal standard. Analytes were extracted from the rat plasma by protein precipitation with acetonitrile and separated on a C18 chromatographic column (Agilent ZORBAX Eclipse Plus, 4.6 mm × 100 mm, 3.5 μm) using the mobile phase containing acetonitrile (A) and 0.1% formic acid-water (B) by gradient elution at 0.5 mL/min flow rate. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to detect analytes. The analytes were measured by multiple reaction monitoring (MRM) in the negative ionization mode. The lower limit of quantification of dihydromyricetin, dihydroquercetin, myricetin and quercetin were 0.70, 8.16, 1.62 and 0.56 ng/mL, respectively. The accuracy, intra-day and inter-day precision and recovery were all satisfactory and the compounds were stable in rat plasma under all tested conditions. The approach was successfully applied to study pharmacokinetic characteristics of the four bioactive flavonoids in plasma after administering Hoveniae Semen total flavonoids intragastrically to rat. Further investigation was carried out to assess pharmacokinetic comparability of the four bioactive flavonoids after intragastric administration of Hoveniae Semen total flavonoids to mixture of flavonoids. Highlights • Pharmacokinetics of 4 main flavonoids in Hoveniae Semen were determined. • Only 6.5 min was in need for one analytical run. • The pharmacokinetic of Hoveniae Semen was firstly studied. • Comparative pharmacokinetics of Hoveniae Semen total flavonoids and a mixture of flavonoids were firstly investigated. [ABSTRACT FROM AUTHOR]

Abstract Methylisothiazolinone (MI) is a preservative used in consumer products to control bacterial and fungal growth. MI can be toxic, act as a skin sensitizer and irritant, and initiate lung diseases; therefore, it is important to understand the mechanisms underlying the toxicity of MI in the body. In this study, we developed a method to analyze plasma MI by using an LC-MS/MS-coupled multiple reaction monitoring (MRM) technique, which follows the fragments of a target metabolite in rat plasma. The MRM transition of MI was m / z 116 ➔ 101, and the lower limit of quantification (LLOQ) was set at 10 ng/mL. Including the concentration of LLOQ, a seven-point calibration curve explained much of the variation in the response, and it was strongly linear (R2 = 0.9998); its intra- and inter-day accuracy and precision values were within 15% of the standard deviation (SD%) and along with the FDA and Korea Ministry of Food and Drug Safety guidelines. For intravenous (iv) pharmacokinetic studies of MI using rats, we developed an analytical method that was useful in detecting the profile of MI in the plasma. We also determined half-life, and area under the curve (AUC) of MI using a non-compartment model, and these might be useful for the study of toxic mechanisms of MI in the body. [ABSTRACT FROM AUTHOR]

Abstract 2-ethyl-6-methyl-3-hydroxypyridine (EMHP) succinate is the original antioxidant and antihypoxic drug commonly prescribed in Russia. The objective of this study was to develop a rapid, simple and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for EMHP quantitation in rat brain tissue with the use of a bead beating homogenizer. The comparison between two approaches to brain tissue preparation was performed, when spiking the blank brain tissue with EMHP reference standard and internal standard (IS) before and after homogenization step. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 column (1.8 μm, 2.1 × 50 mm) and elution was performed with the mobile phase, consisting of 10 mM of ammonium formate aqueous solution with 0.1% formic acid as solvent A and 0.1% formic acid in methanol as solvent B [44%(А):56%(В), v/v]. Flow rate was 0.4 mL/min and the total run time for each sample analysis was 2.0 min. EMHP and amantadine, IS of this study, were analyzed in positive ionization mode. Ion transitions of m / z 138.0 → 123.0 for EMHP and m/z 152.0 → 135.0 for amantadine were selected in multiple reaction monitoring mode. The developed method for EMHP determination in rat brain samples was validated for selectivity, linearity, accuracy, precision, matrix effects, and stability. The lower and upper limits of quantification were determined to be 1 and 1500 ng/g, respectively. The developed and validated HPLC-MS/MS method was successfully applied to determine EMHP concentrations in rat brain tissue following the intraperitoneal administration at a dose of 3.4 mg/kg. Highlights • HPLC-MS/MS method was firstly developed to quantify EMHP succinate in rat brain tissue. • EMHP standard and IS were added before bead beating homogenization. • The method was validated and applied to a pharmacokinetic study in rats. [ABSTRACT FROM AUTHOR]

Abstract Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d 3 -DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan- O -demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes. [ABSTRACT FROM AUTHOR]

Abstract Disopyramide as an antiarrhythmic agent has been used for treating ventricular tachycardia and metabolized into its major metabolite, mono-isopropyl-disopyramide, by CYP3A4. We developed a novel, selective, highly sensitive, accurate, rapid method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of disopyramide and mono-isopropyl-disopyramide in rat plasma. This study is the first report for the assay validation using LC-MS/MS in biological fluids after simple protein-precipitation method. The most sensitive signals by multiple reaction monitoring (MRM) showed at m/z 340.2 → 239.2 and 298.2 → 239.2 with same fragment ion for disopyramide and mono-isopropyl-disopyramide, respectively. The lower limit of quantification (LLOQ) was determined at 2 ng/mL for both analytes and the linear concentration ranges were found to be 2–2000 ng/mL for disopyramide and 2–1000 ng/mL for mono-isopropyl-disopyramide. Finally, this assay was successfully applied to pharmacokinetic analysis of disopyramide and mono-isopropyl-disopyramide after oral and intravenous administration of disopyramide. [ABSTRACT FROM AUTHOR]

Abstract A rapid, simple and selective approach based on ultra-high performance liquid chromatography tandem with mass spectrometry was established and validated for simultaneous quantification of six compounds in rat plasma, including astragaloside I, astragaloside II, astragaloside IV, calycosin‑7‑ O ‑β‑ d ‑glucoside, ononin and formononetin. The approach was applied to a comparative pharmacokinetic investigation of routine aqueous extract and ultrafine powder of Astragalus propinquus. Six compounds were extracted from plasma by using one-step protein precipitation. Chromatographic separation was conducted with a Waters BEH C 18 UHPLC column using mobile phases of acetonitrile/0.1% formic acid-water. Multiple reaction monitoring (MRM) was optimized for mass quantification in a triple quadrupole mass spectrometer. The approach elicited good linearity for the six compounds (r2 > 0.9991) in the concentration ranges. The lower limits of quantification (LLOQ) of astragaloside IV, astragaloside II, astragaloside I, calycosin‑7‑ O ‑β‑ d ‑glucoside, ononin and formononetin were determined as 6.0, 9.6, 11.8, 5.8, 6.9, and 9.4 ng/mL, respectively. Intra- and inter-day precision, accuracy, selectivity and stability of the method met the required limits. The extraction recoveries of the six compounds were from 91.1% to 107.5% and the matrix effects ranged from 92.1% to 106.7%. The normalized C max and AUC 0–t values of calycosin‑7‑ O ‑β‑ d ‑glucoside, and formononetin of the ultrafine powder group was much higher than that of the routine aqueous extract. Administration of Astragalus propinquus in the form of ultrafine powder showed enhanced bioavailability than that of routine aqueous extract. Highlights • A highly sensitive and effective UHPLC–MS/MS approach was developed and validated. • Only 6.0 min was in need for one analytical run. • Six components were simultaneously quantitated in rat plasma. • Comparison of PK between routine extract and ultrafine powder of Astragalus propinquus Schischkin for the first time [ABSTRACT FROM AUTHOR]

Abstract A dispersed solid phase extraction method for high performance liquid chromatography-tandem mass spectrometry analysis of lysophosphatidylcholines and underivatized neurotransmitters in rat brain tissues has been developed. Lysophosphatidylcholines (16:0, 18:0, 18:1, 20:4, 22:6), dopamine, 5-hydroxytryptamine, glutamine, dihydroxy-phenyl acetic acid, 5-hydroxyindole acetic acid and γ-aminobutyric acid were included. Dispersed solid phase extraction method was developed with the aiming to minimize the matrix effects. The cleanup procedure has been followed by rat brain homogenate, nitrogen blow drying, reconstitution, dispersive solid phase extraction and separation. Type and size of sorbent, the re-dissolving solution of the tissues, dilute solution, elution solution and the appropriate acidic or alkaline environment of the system were optimized for the purpose of obtaining a minimized matrix effect. Reversed-phase liquid chromatography was used for separation and mass spectrometry with multiple reaction monitoring was used for quantification. Electrospray ionization in negative ion mode was suitable for dihydroxy-phenyl acetic acid and 5-hydroxyindole acetic acid, while the other substances were detected in positive ion mode. The method was validated with linear range, method detection limit, limit of quantitation, matrix effects, relative recovery and the intra and inter-day precision. Linear range was at 0.1–1000 ng/mL for γ-aminobutyric acid and glutamine, 1.0–1000 ng/mL for dopamine, 10–1000 ng/mL for 5-hydroxytryptamine, 50–1000 ng/mL for 5-hydroxyindole acetic acid, dihydroxy-phenyl acetic acid and lysophosphatidylcholines. The mean matrix effect was between 52.48% and 123.12% and the CVs ranged from 0.41% to 10.96%. Mean relative recovery was from 65.17% to 114.89%, and the CVs ranged from 0.14% to 8.68% at 3 concentration levels for all compounds. Intra-day precision was with coefficients of variation between 0.20% and 14.7%. Inter-day precision values varied <9.21%, with coefficients of variation between 0.18% and 9.21%. The developed method was used to quantify lysophosphatidylcholines and underivatized neurotransmitters in healthy rat brain tissues in this study. [ABSTRACT FROM AUTHOR]

Capillary scale (100 mm × 150 μm id) UPLC/MS/MS, performed using reversed-phase gradient chromatography on sub 2 μm particles, has been successfully employed for the characterization of the metabolites of the drug tienilic acid (TA) excreted via the urine following oral administration to the rat. The capillary LC system provided a significant increase (range ca. 11–33-fold) in sensitivity compared with a conventional 150 mm × 2.1 mm id UPLC system. An investigation of the effect of the injection volume and sample mass loading on the capillary column on the results obtained for both endogenous metabolites and TA was performed. This demonstrated that the injection of up to 2 μL of rat urine onto the system was permitted whilst still providing excellent chromatographic results and robustness. Qualitative analysis of the urine revealed the presence of TA itself and a total of 15 metabolites of the drug, including those resulting from biotransformations such as hydroxylation or conjugation. The capillary chromatography system was shown to be robust, and capable of providing comprehensive drug metabolite profiles from small format urine samples such as those obtained from preclinical studies in rodents. [ABSTRACT FROM AUTHOR]

A sensitive, selective, and reliable LC-MS/MS method was developed and validated for simultaneous quantification of venlafaxine (VEN) and its 5 metabolites (ODV, NDV, NNDDV, OHV and NODDV) in rat plasma. The calibration ranges are 15.0 to 6000 ng/mL for VEN, 1.00 to 400 ng/mL for ODV, 5.00 to 2000 ng/mL for NDV, 1.00 to 400 ng/mL for NNDDV, 10.0 to 4000 ng/mL for OHV, and 0.200 to 20.0 ng/mL for NODDV. Briefly, 50 μL of rat plasma was extracted using liquid-liquid extraction (LLE) with methyl tert -butyl ether (MTBE). The analytes were separated on an Agilent SB-Phenyl (50 mm × 4.6 mm, 3.5 μm) column using a binary gradient of 0.1% formic acid in water versus 0.1% formic acid in acetonitrile at a flow rate of 0.8 mL/min. The method was validated following FDA guidance for bioanalytical method validation. Validated method was successfully applied to a pharmacokinetic study of VEN orally administered to rats. [ABSTRACT FROM AUTHOR]

Only focusing on the circulating levels is insufficient for the comprehensive understanding of the physiological disposition of herbal medicine in vivo . Therefore, we conducted the comprehensive investigation on the in vivo dynamic process of orally administrated Gouteng - Baitouweng (GB), a classical herb pair with anti-Parkinson potentials. Serving as the technical base, a sensitive and selective liquid chromatography–tandem mass spectrometry method was established and validated in the plasma, liver and brain, for simultaneous determination of five alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine and geissoschizine methyl ether) and three saponins (anemoside B4, anemoside A3 and 23-hydroxybetulinic acid). Following liquid-liquid extraction, favorable chromatographic behaviors of eight analytes were obtained on Waters Xbrigde C18 column within 13 min. This method elicited good linearity for the analytes at the concentration range of 0.3–1000 or 1.8–6000 ng/mL with favorable precision, accuracy and stability. Following oral administration of GB (25 g/kg) in rats, this method was applied to the quantitative analysis in the portal vein plasma, liver, systemic plasma, and brain. Consequently, anemoside B4 was of the highest exposure, followed by 23-hydroxybetulinic acid, anemoside A3, rhynchophylline and isocorynoxeine in vivo . Notably, three saponins were all observed with certain exposure in the brain, along with rhynchophylline at low levels. Besides, five alkaloids and 23-hydroxybetulinic acid underwent serious liver first-pass effect. Hence, the pharmacokinetics, liver first-pass effect, liver and brain distribution of ingredients in GB were clarified, which laid a solid foundation for interpreting its efficacy and safety. [ABSTRACT FROM AUTHOR]

The fruit of Ziziphus jujuba ( Jujubae Fructus ) has been used as food and crude drug for thousands of years. Although several chemical and biological studies have revealed triterpenic acid as the main bioactive constituent of Jujubae Fructus responsible for immune-regulatory activity, only few pharmacokinetic studies have been conducted. To comprehend the kinetics of triterpenic acids and promote their curative application, a sensitive and efficient ultra-performance liquid chromatography coupled with mass spectrometry method (UPLC-MS/MS) was established. UPLC-MS/MS was applied for the simultaneous determination of ceanothic acid, epiceanothic acid, pomonic acid, alphitolic acid, maslinic acid, betulinic acid, and betulonic acid in normal and immunosuppressed rat plasma samples. After sample preparation, chromatographic separation was performed on an Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 μm) with acetonitrile: methanol (1:1, v/v) and 0.5% ammonium acetate in water as mobile phase. The established method was validated and found to be specific, accurate, and precise for the seven triterpenic acids, and was successfully applied for the pharmacokinetic study of rat plasma samples. The results showed that the pharmacokinetic parameters (C max , T max , AUC 0–t , AUC 0–∞, and CLz/F) in the plasma samples of immunosuppressed rats were significantly different from those in normal rats, and might provide an insight for the clinical usage of triterpenic acids from Jujubae Fructus . [ABSTRACT FROM AUTHOR]

A HILIC-UHPLC–MS/MS untargeted urinary metabonomic method combined with quantitative analysis of five potential polar biomarkers in rat urine was developed and validated, to further understand the anti-osteoporosis effect of Gushudan(GSD) and its mechanism on prednisolone-induced osteoporosis(OP) rats in this study. The metabolites were separated and identified on Waters BEH HILIC (2.1 mm × 100 mm, 1.7 μm) column using the Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, USA) coupled with a Micromass Quattro Micro™ API mass spectrometer (Waters Corp, Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential polar biomarkers including creatinine, taurine, betaine, hypoxanthine and cytosine, which were related to energy metabolism, lipid metabolism and amino acid metabolism, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode, using tinidazole as internal standard (I.S.). Good linearities (r > 0.99) were obtained for all the analytes with the low limit of quantification from 1.00 to 12.8 μg/mL. The relative standard deviation (RSD) of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from −14.3% to 13.5%. The recovery was more than 85.0%. And the validated method was successfully applied to investigate the urine samples of the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group in rats. Compared to the control group, the level of creatinine, taurine, betaine, hypoxanthine and cytosine in the model group revealed a significant decrease trend (p < 0.05), while the Gushudan-treatment group showed no statistically differences by an independent sample t -test. This paper provided a better understanding of the therapeutic effect and mechanism of GSD on prednisolone-induced osteoporosis rats. [ABSTRACT FROM AUTHOR]

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5 v/v) at an isocratic flow rate of 300 μL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1–1000 ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45 min in rats after oral treatment of 150 mg/kg arecoline. [ABSTRACT FROM AUTHOR]

DA-9805 is a new botanical antiparkinson drug candidate formulated using an ethanolic extract of the root of Bupleurum falcatum , the root cortex of Paeonia suffruticosa , and the root of Angelica dahurica . In this study, a sensitive and rapid LC–MS/MS method was developed to simultaneously determine, saikosaponin a, paeonol, and imperatorin, three active/representative ingredients of DA-9805, in rat plasma. Plasma was extracted by mixture of ethyl acetate and methyl tertiary butyl ether. Chromatographic separation was carried out using a C 18 column and a gradient elution of mobile phases consisting of 5 mM formic acid in water and acetonitrile. Total chromatographic run time was 10.5 min. Multiple reaction monitoring mode was used for mass spectrometry; the transitions were m / z 779.5 → 617.2 for saikosaponin a in negative-ion mode, m / z 167 → 149 for paeonol and m / z 271.1 → 203 for imperatorin in positive-ion mode. Calibration curves were constructed in the range of 0.5–1000 ng/mL for saikosaponin a, 20–10000 ng/mL for paeonol, and 0.2–1000 ng/mL for imperatorin. All the validation data, including the selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability satisfied the acceptance requirements. The method was successfully applied in a pharmacokinetic study of saikosaponin a, paeonol, and imperatorin following oral administration of DA-9805. [ABSTRACT FROM AUTHOR]

Puerarin, a bioactive natural C -glycoside isoflavonoid isolated from Pueraria lobata (Willd.) Ohwi, possesses many potential health benefits. However, the in vivo metabolic fates of puerarin have not been comprehensively clarified yet. In this study, an efficient strategy based on UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes was developed to profile and characterize puerarin metabolites in rat urine and plasma. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filtering (MMDF) were utilized to screen potential puerarin metabolites from HR-ESI–MS 1 stage. The mass fragmentation behaviors of five reference standards, including puerarin, daidzin, daidzein, genistin, and genistein, were comprehensively studied for construction of diagnostic product ions (DPIs), which could be employed to implement rapid identification of puerarin metabolites. Finally, a total of 66 metabolites (prototype compound included) were tentatively or positively identified based on standard substances, chromatographic retention times, accurate mass measurement, and corresponding Clog P values. Our results demonstrated that puerarin underwent multiple in vivo metabolic reactions including methylation, hydroxylation, dehydroxylation, hydrogenation, deglycosylation, glycosylation, sulfonation, glucuronidation, and their complicated reactions. In conclusion, the newly discovered puerarin metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method was developed and fully validated for simultaneous determination of puerarin, 3′-hydroxy puerarin, 6″- O -xylosyl puerarin, 3′-methoxy puerarin, mirificin, puerarin-7- O -glucoside, daidzin, daidzein, daidzein-7- O -glucoside, ginsenoside-Rd, notoginsenoside-R1, ginsenoside-Re, ginsenoside-Rg1, ginsenoside-Rb1 in rat plasma after intravenous injection of 5 mL/kg Gegen-Sanqi compatibility solution (containing Pueraria flavonid 10.8 mg/mL and Panax notoginsenosidum 5.4 mg/mL). After addition of internal standard (IS) baicalin, the analytes and IS were recovered from rat plasma by protein precipitation using acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on a Capcell pak MG C18 column (3.0 mm × 75 mm, 3.0 μm) at 35 °C with a flow rate of 0.75 mL/min. Mass spectrometry was conducted using multiple reaction monitoring in positive mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The precision and accuracy of the LC–MS/MS assay based on the three analytical quality control (QC) levels were well within the acceptance criteria from FDA guidance for bioanalytical method validation. Method recoveries were higher than 75% and the matrix effects were minimal. All analytes were stable under tested conditions. It was the first time to study the pharmacokinetics of daidzein-7- O -glucoside in rat plasma. To the best of our knowledge, this is the first study for simultaneous determination of so many analytes in rat plasma after intravenous injection of Gegen-Sanqi compatibility solution. It was expected that the present work would provide some helpful references for the apprehension of the mechanism of action and further clinical efficacy studies of Gegen-Sanqi herb-pair. [ABSTRACT FROM AUTHOR]

Orientin and vitexin, 4’-hydroxyl-2-phenylchromen-4-one, are both major flavones derivatives found in Trollius ledebourii possessing definite pharmacological activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on Male Sprague Dawley rats of orientin and vitexin were tested, respectively. A systematic method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to characterize metabolites by means of electrospray ionization (ESI) mass spectrometry in positive ion mode. An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to observe probable relevant metabolites. By comparison of chromatographic behaviors with reference substances, exact protonated ions, MS/MS fragment ions and relevant literature, a total of 12 metabolites of orientin and 23 metabolites of vitexin were detected, respectively, which suggested that orientin is more metabolically stable than vitexin. Oxidation, methylation, acetylation, reduction, loss of C 6 H 10 O 5 and glucuronide conjugation were the major biotransformation routes of both of them in rats. More significant, glutamine conjugation, loss of CO and loss of CH 2 O were the unique metabolic pathways of vitexin compared with that of orientin for the first time. Besides, most metabolites were observed in rat urine and feces, implying that urine and feces were the active metabolic places for flavones. This is the first study on metabolisms of orientin and vitexin in vitro and in vivo simultaneously and the proposed metabolic pathways of them might provide further understanding of their pharmacological mechanisms and later study on their excretion. [ABSTRACT FROM AUTHOR]

Brigatinib is the second-generation anaplastic lymphoma kinase – inhibitor in non-small cell lung cancer and it can overcome the crizotinib-resistance. Chromatographic separation was carried out on an Acquity Ultra Performance Liquid Chromatography (UPLC) unit with a BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase was composed of acetonitrile and 0.1% formic acid in water. No endogenous interfering compounds was discovered at retention time of brigatinib (0.56 min) and imatinib (IS, 1.41 min). MS/MS detection was performed in positive mode. And the MRM transitions were m / z 584.09 → 484.08 and m / z 494.3 → 394.2 for brigatinib and IS, respectively. This method was assessed to be stable, specificity, and no matrix effect in three concentrations (0.004, 0.4, 4 μg/mL). The intra-day and inter-day precisions were less than 11.09% and 6.43%. And intra-day and inter-day accuracies were ranged from −3.88% to 5.44%. The recovery of brigatinib was from 85.26% to 96.05%. Additionally, the method had a good linearity in the range of 0.002–5 μg/mL. The presented method was effectively implemented to determine the concentration of brigatinib in rat plasma. [ABSTRACT FROM AUTHOR]

Polymyxin B has resurfaced as a last-line treatment for multi-drug resistant Gram-negative bacteria. Accurate characterization and quantification of polymyxin B components is necessary to optimize this therapy. We developed and validated a robust, straightforward LC–MS/MS method to quantify polymyxin B1 and B2, the primary polymyxin B components, in various matrices (cation-adjusted Mueller-Hinton broth (CAMHB), human and rat plasma). Of sample preparation approaches investigated, two protein precipitation/extraction methods were developed as part of an analytical strategy based upon reverse-phase LC–MS/MS using electrospray ionization in positive multiple-reaction monitoring mode. Both methods were validated over therapeutically and experimentally relevant concentration ranges (CAMHB: 0.1–8.0 μg/mL, rat and human plasma: 0.05–4.0 μg/mL). Quality control samples spanning a relevant concentration range were employed to assess intra- and inter-day accuracy (relative error (%RSD)) and precision (coefficient of variation (CV%)). For polymyxin B1 and B2 in CAMHB, inter-day standard deviations were 1.18–4.59% and 0.777–1.23%, respectively, and accuracies were 94.2–99.3% and 94.4–99.1%. For rat plasma, inter-day standard deviations were 1.53%–5.64% and 4.07%–8.26%. Accuracies were 100.6–108.9% and 96.1–108.1%. For human plasma, inter-day standard deviations were 2.77–7.32% and 1.55–7.29%. Accuracies were 89.6–96.4% and 92.9–102.0%. Extraction recoveries for all matrices were >93.5%. Adsorption, storage, and long-term stability were assessed and were acceptable. Accuracy, precision, and cost-efficiency make this an ideal approach for quantifying polymyxin B in in vitro and in vivo samples including those from rat and human subjects. [ABSTRACT FROM AUTHOR]

Gentiopicroside is a natural secoiridoid glycoside that may require metabolic activation to exert pharmacological effects. In this study, two active metabolites of gentiopicroside (M1 and M2) were isolated from rat urines and identified with our previous method. Most importantly, a fast, sensitive and selective ultra high-performance liquid chromatography–tandem mass spectrometry method was developed to simultaneously determine gentiopicroside and its two metabolites in rat plasma. The analytes and internal standard (swertiamarin) were separated on an ACQUITY UPLC ® BEH C18 column (2.1 × 50 mm, 1.7 μm) using gradient elution by acetonitrile and 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometry detector was operated in the multiple reaction monitoring with positive ionization mode. The method had a good linearity over the concentration range of 0.2–10,000 ng/mL for gentiopicroside and 0.1–5000 ng/mL for the two metabolites. The validated method was successfully applied to the pharmacokinetic study of gentiopicroside and its metabolites after single oral administration of gentiopicroside (150 mg/kg) to rats (n = 8). The pharmacokinetic differences between gentiopicroside and its two metabolites were identified.Results provided the evidence for in vivo metabolism-based activation of gentiopicroside. [ABSTRACT FROM AUTHOR]

Cucurbitacin B (CuB) and cucurbitacin E (CuE) are tetracyclic triterpene compounds from Cucurbitaceae , and the main bioactive compounds of cucurbitacins tablets that used to treatment of chronic hepatitis. Pharmacological research has been very comprehensive, and there are few studies on pharmacokinetics, especially about CuE. An Ultra High Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-MS/MS) method with high selectivity, simplicity and sensitivity has been used for quantitative analysis of Cucurbitacin B (CuB) and cucurbitacin E (CuE). Plasma samples were pretreatment by Liquid-liquid extraction (LLE) method with dichloromethane. The chromatographic separation was achieved on a C 18 column (Agilent Eclipse Plus, 1.8 μm, 50 × 2.1 mm) using gradient elution with water – methanol at a flow rate of 0.3 mL/min and the column temperature was set at 30 °C. The method was validated according to FDA guidelines. Lower limit of quantification (LLOQ) was 1.60 ng/mL for CuB and 1.58 ng/mL for CuE. Correlation coefficients of CuB and CuE were more than 0.99 in rat plasma. All values of intra-day and inter-day precision (RSD%) were not exceeded 15%, the accuracy (RE%) were within −5.57 to 5.20% for CuB and −3.33 to 7.37% for CuE. The mean extraction recoveries were more than 80%. Pharmacokinetic parameters were also evaluated by UHPLC-MS/MS method. The results suggestion that this method was successfully applied to pharmacokinetic study of CuB and CuE in rat plasma after oral administration cucurbitacin tablets. [ABSTRACT FROM AUTHOR]

CKD-712 is a potential treatment for sepsis, as it exhibits protective effects against lipopolysaccharide-mediated platelet aggregation, inducible nitric oxide synthase expression, and cecum-ligation puncture-induced septic mortality in mice. In this study, we develop a rapid and sensitive LC–MS/MS method for determining CKD-712 in rat plasma. CKD-712 and papaverine hydrochloride (an internal standard) were analyzed using an LC–MS/MS system consisting of an Agilent HPLC system (HP-1100) equipped with an Atlantis HILIC Silica (2.1 × 50 mm, 3 μm) column and a API 4000 (Applied Biosystems/MDS Sciex, USA) in a positive ESI mode. We utilized multiple reaction monitoring (MRM) at m / z transitions of 306.2–164.0 to analyze CKD-712, and 340.3–202.1 m / z for IS, with a mobile phase of acetonitrile (0.025% trifluoroacetic acid):20 mM ammonium acetate (94:6, v/v) at a flow rate of 0.25 mL/min. The lower limit of quantification (LLOQ) was 5 ng/mL, with a linearity ranging from 5 to 1000 ng/mL ( r > 0.999). Validation parameters including specificity, precision, accuracy, matrix effect, recovery, dilution effect and stability results were well within acceptance criteria, and applied successfully on a pharmacokinetic study in rats. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA), a chronic systemic inflammatory disorder affects many adults. Sinomenine, a natural product, has been clinically available for the treatment of RA in China. SND-117, a sinomenine derivative with much more potent activity, might serve as a candidate for anti-arthritis. The aim of the present study was to develop a sensitive and rapid high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method for quantification of SND-117 in rat plasma and to understand its absolute bioavailability. The HPLC–MS/MS method was developed and fully validated for determination of SND-117 in rat plasma, and the pharmacokinetic differences were investigated after different administration routes. The pharmacokinetics parameters were calculated by non-compartment model with DAS 3.0 software. After the oral or intravenous administration of different doses of SND-117, the time to peak is 1.5 h, half-life time is 8–10 h. The absolute oral bioavailability of SND-117 in rats was 9.60%. The results showed that SND-117 in rats was quickly absorbed, slowly eliminate, and the kinetics were linear. This method was suitable for pharmacokinetic studies of SNA-117 in rats. [ABSTRACT FROM AUTHOR]

Till date, no analytical method published to detect Cisplatin has been validated according to the U.S. Food and Drug Administration (FDA) guidance using liquid chromatography mass spectrometry (LC–MS/MS). We report, a validated LC–MS/MS method for quantitative determination of cisplatin in rat plasma and urine according to FDA guidlines. Cisplatin is a platinum containing compound used for the treatment of different types of cancers. Quantitative determination of cisplatin has been carried out using atomic absorption spectroscopy, high pressure liquid chromatography with phosphorescence, ultra-violet detection, or with inductively coupled plasma mass spectrometry. Few LC–MS/MS methods have been reported for the analysis of cisplatin either for direct quantification or indirect by derivatizing with organic compounds but none of the reported methods have validated the method. The developed and validated assay presented here is a highly sensitive LC–MS/MS method developed and validated for the quantitative determination of cisplatin following derivatization with diethyldithiocarbamate (DDTC) in order to detect platinum (Pt) of cisplatin, suitable for pharmacokinetic studies in rats and to further use it to study human toxicology. Chromatographic separation was achieved using a Poroshell 120 EC-C 18 column (3 × 50 mm, 2.7 μm) with a binary gradient mobile phase. Quantification was performed on a triple quadruple with electrospray ionization and detection was performed using multiple reaction monitoring. The method has a limit of detection of 1 ng/mL, and the quantifiable range was 3–3000 ng/mL in rat plasma and urine. The method was accurate and precise with an accuracy and precision for intra-day and inter-day of ±20% for lower limit of quantitation and of ±15% for low, mid and high quality control samples. This method was successfully applied to study the pharmacokinetic profile of cisplatin in rat plasma and urine given a range of doses from 0.5 to 3.5 mg/kg. [ABSTRACT FROM AUTHOR]

Glimepiride (GLI) is prescribed for the management of type-2 diabetes where as pitavastatin (PIT) for the treatment of diabetes associated dyslipidemia. Both the drugs are metabolized by CYP2C9 and have the potential of altering the enzyme through either inhibition or induction. In this respect, we present a simple, fast and validated bioanalytical LC–MS/MS method for the simultaneous estimation of GLI and PIT from rat plasma. Waters XTerra RP HPLC column (4.6 × 100 mm, 5 μm) with mobile phase consisting of acetonitrile and 10 mM ammonium acetate (pH-6.0) in the ratio 85:15 ( v/v ) at a flow rate of 1 mL/min was used for the chromatographic separation. The negative ionization mode with MRM transitions: m / z 420.17 → 288.13 for PIT, m / z 489.59 → 350.12 for GLI and m / z 380.08 → 316.31for celecoxib as internal standard (IS). A total run time of 3 min and LLOQ was found to be 5 ng/mL for both PIT and GLI. The method was applied to study the drug interaction between GLI and PIT in rat liver microsomes. In vivo rat pharmacokinetics study showed there was a 1.29-fold increase in AUC 0-∞ and 1.2-fold decrease in the clearance of PIT in presence of GLI. No notable difference in the pharmacokinetic profile of GLI was observed upon the intravenous co-administration of PIT. [ABSTRACT FROM AUTHOR]

Crocin and crocetin in rat plasma were simultaneously analysed using ultra-performance liquid chromatography tandem mass spectroscopy (UPLC–MS/MS), and method was fully validated. For the first time, levels of both crocin and crocetin in plasma were profiled after oral administration of crocin, and this UPLC–MS/MS approach was applied to evaluate pharmacokinetics and relative bioavailability of crocin and crocetin in rats. It was shown that crocin transformed into crocetin quickly in the gastrointestinal tract, and crocetin was 56–81 fold higher exposed in rat plasma than crocin after oral administration of crocin. A comparison study revealed that an oral administration of equal molar crocin achieved higher exposure of crocetin in rat plasma than that of crocetin. It was suggested that oral administration of crocin has the advantages over crocetin, and crocetin may be the active component potentially responsible for the pharmacological effect of crocin. [ABSTRACT FROM AUTHOR]

A liquid chromatography coupled with electrospray ionization mass spectrometry method was developed and validated for simultaneous determination of seven bioactive constituents including astragaloside IV, calycosin, glycyrrhizic acid, enoxolone, saikosaponin D, ferulic acid and hesperiden in rats’ various tissues using diclofenac as the internal standard (IS). Biological samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid and 4 mM ammonium acetate). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. The mass transitions were as follows: m / z 829.7 → 783.3 for astragaloside IV, m / z 283.3 → 267.7 for calycosin, m / z 821.6 → 350.0 for glycyrrhizic acid, m / z 469.9 → 425.2 for enoxolone, m / z 825.7 → 779.6 for saikosaponin D, m / z 192.5 → 133.9 for ferulic acid, m / z 609.1 → 301.0 for hesperiden and m / z 293.6 → 249.9 for the IS, respectively. The lower limits of quantification for the seven analytes in different rat tissues were 0.2–20 ng/mL. Bu-zhong-yi-qi-tang (Hochuekkito in Japan, Bojungikki-tang in Korea) is one of the most frequently prescribed traditional herbal formulas used in Korea, Japan, and China to treat gastrointestinal diseases, cancer and chronic fatigue syndrome. The validated method was successfully applied to a tissue distribution study of the seven components in rat tissue after oral administration of Bu-zhong-yi-qi-tang concentrated granule. The results of the tissue distribution study showed that the high concentration of seven components were mainly in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Genipin-1- o -glucuronic acid and genipin-monosulfate are two major metabolites from geniposide and genipin. Based on diabetic rat model, we developed a simultaneous quantification method to investigate their comparative pharmacokinetics by online mircrodialysis-ultra performance liquid chromatography–mass spectrometry (MD-UPLC–MS/MS) without their standard compounds. Online microdialysis sampling could avoid unexpected contamination or degradation of the analytes during the storage and transfer steps. Combined with good sensitivity, selectivity and selectivity of UPLC–MS/MS, online MD-UPLC–MS/MS method could real-timely monitor metabolites in rat blood for quantitative analysis. Our research found that AUC 0→t of genipin-1- o -glucuronic acid and genipin-monosulfate in blood of diabetic group were 17.68 and 7.58 times than those in normal group, respectively, and AUC 0→t of genipin-1- o -glucuronic acid was 2.28 times than that of genipin-monosulfate in blood of diabetic group, which revealed the effect of diabetes on the pharmacokinetic properties of the two metabolites. This study not only provides an approach for pharmacokinetic studies for various metabolites from herb medicines, but also can predict druggability of their bioactive metabolites. The insight obtained should facilitate drug development and toxicity research. [ABSTRACT FROM AUTHOR]

Identification of metabolic profile of traditional Chinese medicine in vivo is always a challenge task. Usually, screening out and identifying the exogenous compounds manually from total ion chromatograms (TICs) of biologic samples is time-consuming and strenuous. In this study, a systematic identification strategy based on LC–MS was adopted to clarify the metabolic profiling of Zhi-Zi-Hou-Po decoction ( ZZHPD ) in rat. Bile, urine and feces samples of rat were obtained after oral administration and then analyzed by LC–MS after proper preparation. The xenobiotics were screened out from TICs globally and rapidly by untargeted metabolomics-driven strategy (UMDS) based on the combined of XCMS online (a web-based platform to process LC–MS data), MetAlign (a software to process LC–MS data) and SIMCA-P (a software for data analysis). Most of the xenobiotics were identified by means of series product ions filtering (sPIF), which was based on the database-hit of ZZHPD (including prototype and metabolites). Then the unmatched constituents were identified tentatively and their source and metabolic pathway were clarified by using diagnostic fragment ions strategy (DFIS). As a result, a total of 83 compounds including 44 prototype compounds and 39 metabolites were rapidly identified or tentatively characterized from the biologic samples, and among them, four of which were found for the first time. Further research on the correlations of these prototype compounds and metabolites revealed that glucuronidation is the main metabolic pathways of ZZHPD in rat bile and urine, while prototype compounds were the abundant ingredients detected in rat feces. [ABSTRACT FROM AUTHOR]

A fast, selective and sensitive procedure for quantitation of the camphor-based anti-influenza agent camphecene in whole rat blood was developed and validated using dried blood spots and LC–MS/MS. The method was validated according to recommendations of the FDA and EMA in terms of selectivity, linearity, accuracy, precision, recovery, matrix factor, stability, and carry-over. Sample preparation included spotting 20 μL of whole blood taken from the tail vein onto the paper, drying and extracting the analyte, followed by evaporation of the solvent and analysis of the residue. HPLC separations were run on a reversed-phase microcolumn; the time of analysis was less than 2 min. MS/MS detection was performed on a triple quadrupole mass-spectrometer using multiple reaction monitoring (MRM) mode. Transitions 196.4 → 122.2/153.3 and 152.2 → 93.1/107.2 were monitored for camphecene and 2-adamantylamine hydrochloride (internal standard), respectively. The intra- and inter-day precisions and accuracies, matrix factor, carry-over and recovery were within acceptable limits. Despite low extraction recovery (less than 2%), the sensitivity of the method was enough to detect the analyte in the concentration range 50–2500 ng/mL. The application of the method was shown in pharmacokinetic studies of camphecene in rats at a dose of 10 mg/kg. [ABSTRACT FROM AUTHOR]

It has been proved that the purine metabolic pathway has been implicated in various biological disorders including gout, diabetes, coronary heart diseases, and neurodegenerative diseases. The analysis of the purine metabolic pathway in organisms reveals important alterations under different physiological and pathological conditions, which contributes to the pathological study, diagnosis, and therapy of related diseases. In the present study, an ultra-high performance liquid chromatography with ultraviolet and tandem mass spectrometry (UHPLC-UV–MS/MS) method was developed for conducting the comprehensive analysis of the metabolite profiles of the purine pathway in rat plasma through a single analysis. The purine metabolites including adenosine-5′-monophosphate, guanosine-5′-monophosphate, adenosine, inosine, guanosine, inosine-5′-monophosphate, deoxyadenosine, deoxyguanosine, deoxyinosine, xanthine, hypoxanthine, and uric acid, were separated and quantified in the short running time of 10 min. After rapid chromatographic separation achieved by an Agilent Zorbax SB-Aq column, high concentration of uric acid and the remaining purine metabolites at lower levels were respectively detected by ultraviolet detector and triple quadruple mass spectrometry within a single analysis. The proposed method was validated by applying charcoal-stripped plasma as a matrix and it was proved to be linear ( R 2 > 0.982), accurate (with a relative error for accuracy < ± 15% and the relative standard deviation for intra- and inter-run precision <11%) and reproducible (with a matrix effect ranging between 86.49% and 111.44% with a maximum RSD of 8.69%). As a result, the method was successfully applied to the quantification of the endogenous purine metabolites in rat plasma. It was found that the concentration levels of the purine metabolites may keep a physiological balance as an integrated system in normal individuals and the concentration level of uric acid in rat plasma was 64 μM which was more than 200 times greater than the other purine metabolites. The established method and the measurement of the concentration of these purines in normal rat plasma may help the investigation of the action mechanisms between purine disorders and related diseases. [ABSTRACT FROM AUTHOR]

We established and validated a specific and sensitive analytical method for the determination of 1-vinyl-2-pyrrolidone (VP) as 1-vinyl-2-pyrrolidone-mercapturic acid (VPMA) in urine using an electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) column switching method. An online solid phase extraction (SPE) for sample cleanup was performed by column switching to a restricted access material and back-flushing to the analytical column. A Phenomenex Luna C8 column was used for sample separation (150 mm; ID 4,6 mm; 3 μm). D4-VPMA served as an isotope labeled internal standard and was detected in negative multiple-reaction monitoring (MRM) mode. The Limit of quantification (LOQ) for VPMA was 1.5 μg/L, the intra-day precision of three concentrations (2 μg/L, 75 μg/L and 400 μg/L) of spiked urine samples ranged from 2.7 to 7.3%, the inter-day precision from 3.4 to 14.4%. The accuracy ranged from 6.2 to 9.0%, for the intra-day experiments and from 0.3 to 6.9% for the inter-day experiments. The method was applied to urines of Sprague-Dawley rats exposed to VP as a proof of principle of VPMA as a potential biomarker. [ABSTRACT FROM AUTHOR]

Phellodendrine, a quaternary ammonium alkaloid extracted from the dried bark of Phellodendrom chinensis Schneid and Phellodendrom amurense Rupr, has the effect of suppressing cellular immune response, reducing blood pressure and antinephritis. However, few investigations have been conducted for the pharmacokinetic study of phellodendrine. Thus, a rapid, simple and reliable ultra-high performance liquid chromatography–tandem quadrupole mass spectrometry (UHPLC–QQQ MS/MS) method has been established for quantification of phellodendrine in rat plasma and tissues by using magnoflorine as internal standard. The chromatographic separation was achieved on an Agilent ZORBAX SB-C18 column (4.6 mm × 50 mm, 1.8 μm) by gradient elution using 0.1% aqueous formic acid (A) and methanol (B). Triple quadrupole mass detection with multiple reaction monitoring mode was used to monitor the ion transitions, at m / z 342.20 → 192.20 for phellodendrine and m / z 342.20 → 58.20 for internal standard, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of phellodendrine after intravenous administration. The lower limits of quantification were 0.5 ng/mL for plasma samples, 2.5 ng/g for brain and 1 ng/g for other tested tissues. Precisions and accuracy values were within the Food and Drug Administration acceptance criteria, the recovery and matrix effects were between 87.8–113.5%. The area under the curve (AUC 0−t ) ranged from 15.58 to 57.41 mg/L min and Cmax were between 1.63–4.93 mg/L. The results showed that phellodendrine was eliminated in 120 min in plasma and most of tissues and the highest concentrations of phellodendrine were found in the kidney. This study may provide a basis for the further study of phellodendrine. [ABSTRACT FROM AUTHOR]

Tamoxifen (TAM) is a non-steroidal estrogen receptor antagonist that enhances erlotinib (ERL)-induced cytotoxicity in the treatment of NSCLC. ERL and TAM are metabolized by CYP3A4 enzymes. In addition, both drugs have the potential of altering the enzymatic activity through either inhibition (ERL) or induction (TAM). Thus it was expected that pharmacokinetics (PK) drug–drug interactions (DDIs) could be encountered following their co-administration. In this respect, a bioanalytical UPLC–MS/MS method has been developed and validated for the simultaneous determination of ERL and TAM in rat plasma samples, using ondansetron (OND) as an internal standard (IS). Plasma samples were prepared using mixed mode cationic solid phase extraction (SPE) STRATA™ −X-C 33 μm cartridges with good extraction recovery of both drugs from rat plasma (E r % from −13.92 to −3.32). The drugs were separated on a Waters BEH™ C18 column with an isocratic elution using a mobile phase composed of a mixture of acetonitrile and water, each with 0.15% formic acid, in the ratio of 80: 20, v/v. Quantitation was carried out using the positive ionization mode with multiple reaction monitoring (MRM) at m / z 394.20 > 278.04 (ERL), m / z 372.25 > 72.01 (TAM), and m / z 294.18 > 170.16 (OND). The method was fully validated as per the FDA guidelines over the concentration range of 0.2–50 ng/mL with very low lower limit of quantification (LLOQ) of 0.2 ng/mL for both ERL and TAM. The intra- and inter-day assay precision (in terms of relative standard deviation, RSD) and accuracy (in terms of percentage relative error, % E r ) were evaluated for both drugs and the calculated values evaluated at four different concentration levels were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ. The method was successfully applied to the study of possible PK-DDI following the oral administration of ERL and TAM in a combination, compared to their single administration. [ABSTRACT FROM AUTHOR]

Gout is a common metabolic disorder caused by the deposition of monosodium urate crystals within joints. A new kind of xanthine oxidase inhibitor, WSJ-537, was developed as a potential drug. In order to investigate the pharmacokinetic behavior in vivo , a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for determination the concentration of WSJ-537 in rat plasma was developed. After extraction by protein precipitation method with acetonitrile, the chromatographic separation was accomplished on a Venusil ASB C 18 column(2.1 mm × 50 mm, 3 mm)at a flow rate of 0.3 mL min −1 with the mobile phase consisting of acetonitrile-ammonium acetate (33:67, v/v ). An electrospray ionization (ESI) source was applied and operated in the positive ion mode. The plasma concentration was detected by multiple reactions monitoring (MRM) mode with the target fragment ions m / z 410.2 → m / z 368.1 for WSJ-537 and m / z 244.1 → m / z 185.0 for the IS. Good linearity was observed in the range of 20–800 ng mL −1 (r = 0.9947). The recovery of WSJ-537 in rats plasma was more than 85%. This method was suitable for pharmacokinetic studies after oral administration of 10 mg/kg WSJ-537 in rats. [ABSTRACT FROM AUTHOR]

Sarsasapogenin, a natural compound from Chinese medical herb Anemarrhena asphodeloides Bge., has recently received a great deal of attention due to its various bioactivities. In this study, an easy and applicable liquid chromatography tandem mass spectrometry method for the quantification of sarsasapogenin in rat plasma was developed. Sample preparation was accomplished through a simple one-step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m / z 417.4/273.2 for sarsasapogenin, and 415.2/271.4 for diosgenin (internal standard). The calibration curve was linear over the range of 0.5–500 ng/mL (r = 0.9994), with a lower limit of quantification at 0.5 ng/mL. The RSD of intra- and inter-day precision was below 6.41%, and accuracy ranged from 87.60% to 99.20%. The RSD of matrix effect and recovery yield was within ±15% of nominal concentrations and sarsasapogenin was stable during stability tests. This validated method had been successfully applied to the preclinical pharmacokinetic studies of sarsasapogenin in rats. The half-life (t 1/2 ) was (15.1 ± 2.3), (16.1 ± 3.0) and (15.4 ± 3.9) h after single intragastric administration of 25, 50 and 100 mg/kg sarsasapogenin, respectively. And it was found that, the area under the plasma concentration versus time curve (AUC 0-72h ) and the maximum plasma concentration (C max ) were linearly related to dose. [ABSTRACT FROM AUTHOR]

A liquid chromatography tandem mass spectrometry (LC–MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6- 2 H 2 ]glucose and [U- 13 C 6 ]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U- 13 C 6 ]glucose and an oral glucose load enriched with [6,6- 2 H 2 ]glucose. The sensitivity is sufficient for analysis of the equivalent to <5 μL of blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments. [ABSTRACT FROM AUTHOR]

In this study, we developed and validated a new, rapid, specific and sensitive ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to simultaneously determine parecoxib sodium (PX) and its active metabolite, valdecoxib (VX), in rat plasma. Plasma samples were prepared by plasma protein precipitation combined with a liquid-liquid extraction method. The separation was carried out on a Kinetex C 18 column (2.1 mm × 50 mm, 2.6 μm) with a gradient elution using methanol (A) and a 2 mM ammonium acetate aqueous solution (B). The analysis was performed in less than 3 min with a flow rate of 0.2 mL/min. Ketoprofen was used as an internal standard (IS). Mass spectrometric detection was conducted with a triple quadrupole detector equipped with electrospray ionization in the negative ion mode (ESI − ) using multiple reaction monitoring (MRM). The calibration curves were linear over the concentration ranges of 5–4000 ng/mL for PX and 5–2000 ng/mL for VX with all correlation coefficients greater than 0.998. The intra- and inter-day relative standard deviations (RSD) for both analytes were within 15% and the accuracy was within 85–115% at all quality control levels. The mean extraction recoveries for all analytes obtained from three concentrations of QC plasma samples were more than 89.0% efficient. Selectivity, matrix effect, dilution integrity and stability were also validated. The method was successfully used to investigate the pharmacokinetics of PX and VX in rat plasma after intravenous and intramuscular administration of PX. [ABSTRACT FROM AUTHOR]

7′( Z )-(8″ S , 8‴ S )-epi-Salvianolic acid E (compound 1 ) and (7 ′R , 8′ R , 8″ S , 8‴ S )-epi-salvianolic acid B (compound 2 ), two novel analogs of salvianolic acid B (Sal B), have been recently isolated from Salvianolic acid for injection. They both show powerful antioxidant effects, including inducing NQO1 activity and scavenging DPPH free radical, and potential protecting effects for cerebral ischemia. However, no reports have been described the pharmacokinetic study of them. In this study, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for the determination of compound 1 , compound 2 and Sal B in rat plasma, respectively. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters Acquity UPLC ® HSS T3 column (1.7 μm particles, 2.1 mm i.d. × 100 mm) with the mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B) (65:35, v/v). Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization (ESI) by multiple reaction monitoring (MRM) in the negative ion mode. Monitored transitions were set at m / z 717.0 → 519.0, 717.1 → 519.1, 717.2 → 518.9 and 320.9 → 152.1 for compound 1 , compound 2 , Sal B and chloramphenicol (internal standard, IS), respectively. Linear calibration curves were acquired over the concentration range of 2.0–1000 ng/mL for the three analytes in rat plasma. The extraction recoveries, matrix effects, intra- and inter-day precisions and accuracies of the three analytes were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of compound 1 , compound 2 and Sal B after intravenous administration of 6.0 mg/kg in rats, respectively. The results indicated that compound 1 and compound 2 were both eliminated more slowly than Sal B. Exposure levels of both compound 1 and Sal B were higher than compound 2 in the same dosage range. This study provided critical reference for the pharmacokinetic study of compound 1 and compound 2 . [ABSTRACT FROM AUTHOR]

Esculin, a coumarin derivative found in Fraxinus rhynchophylla , has been reported to possess multiple biological activities. This present study is designed to investigate the metabolic profile of esculin in vivo based on ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) for the first time. After oral administration of esculin (100 mg/kg) for rats, plasma, urine, feces and bile samples were collected to screen metabolites. As a result, a total of 19 metabolites (10 phase I metabolites and 9 phase II metabolites) were found and identified. Results showed that metabolic pathways of esculin included hydrolysis, dehydrogenation, hydroxylation, methylation, dehydrogenation, glucuronidation, sulfation, and glycine conjugation. It was also found that after oral administration of esculin, the esculin could be metabolized to esculetin in vivo via deglycosylation, and esculetin was found in all biological samples. This study also laid solid basis for in-depth development of esculin and provided important information for clarifying the biotransformation process of esculin in vivo . [ABSTRACT FROM AUTHOR]

There is a relationship between circadian rhythm and metabolic disorders. The active agent, SR8278, could competitively bind to and inhibit the nuclear receptor, Rev-erb (a major modulator of mammalian circadian clock system), to regulate the metabolism in organisms. However, we had limited knowledge of the pharmacokinetic (PK) characteristics of SR8278. Here, we describe a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify SR8278 in vivo . The linearity range and the limit of quantification (LOQ) for SR8278 were 30–3000 ng/mL and 6 ng/mL, respectively. The inter-day and intra-day variability were within 10%. This UPLC-MS/MS method was successfully used to characterize the PK behaviors of SR8278 in normal and diabetic rats after intravenous (i.v.) injection at a dosage of 2 mg/kg. No significant differences were observed in the PK parameters of SR8278 in normal and diabetic rats. Specifically, the values of areas under plasma concentration time curves (AUC), initial plasma concentrations (C 0 ), elimination half-lives (t 1/2 ), and clearances (CL) were 608.33 ± 295.25 vs . 598.59 ± 276.92 ng·h/mL, 2410.25 ± 202.36 vs . 3742.11 ± 1300.21 ng/mL, 0.17 ± 0.08 vs . 0.11 ± 0.04 h, 3330.83 ± 1609.48 vs . 3364.81 ± 1111.38 mL/kg·h for SR8278 in normal rats vs . diabetic rats, respectively. In conclusion, a UPLC-MS/MS method was successfully developed and validated for the first time, with a wide linearity range, low LOQ, small sample volume (10 μL), rapid analysis (4 min) and excellent recoveries (>80%). It was also used to clarify the PK characteristics of SR8378 in rats. The same PK behaviors of SR8278 in normal and diabetic rats showed that diabetes may have little or no effect on the disposition, metabolism and/or elimination in vivo , which may be of great importance for future clinical studies. [ABSTRACT FROM AUTHOR]

Semen Oroxyli, a Traditional Chinese Medicine, has many significant pharmacological activities such as analgesic, apoptotic, anti-inflammatory, anticancer, and immunostimulant activities. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous quantitation of four flavonoids (oroxin A, oroxin B, baicalin, and chrysin) of Semen Oroxyli in rat plasma. After the addition of internal standard, plasma samples were pretreated with acetonitrile via a single-step protein precipitation. Chromatographic separation was performed on a Capcell Pak C 18 column (100 mm × 2.0 mm, 5 μm particles) with isocratic elution using a mobile phase of methanol and 2 mM ammonium acetate buffer solution (75:25, v/v) at a flow rate of 0.45 mL/min. The analytes were detected without interference in the selection reaction monitoring mode with negative electrospray ionization. The validated method exhibited good linearity over a wide concentration range (r ≥ 0.9958), and the lower limits of quantification were 1.0–5.5 ng/mL for all the analytes. The mean extraction recoveries of the analytes from rat plasma exceeded 80.6%. The intra- and inter-day precisions at three QC levels were both less than 11.5%, and the accuracies ranged from −6.2% to 10.3%. The LC–MS/MS method was successfully applied to a pharmacokinetic study of the four flavonoids in rat plasma after oral administration of Semen Oroxyli extract. [ABSTRACT FROM AUTHOR]

A simple, rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated for the quantitative determination in rat plasma of a new candidate for AD treatment, namely PC 48 (a 7-MEOTA-donepezil like compound) in rat plasma. Sample preparation involved pH adjustment with sodium hydroxide followed by solvent extraction with ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on an Ascentis Express RP-Amide column (75 mm × 2.1 mm, 2.7 μm) with a gradient mobile phase consisting of 0.05 M aqueous formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry on an LTQ XL system using the MS/MS CID (collision-induced dissociation) mode. The method was linear in the range 0.1–1000 ng/ml (r 2 = 0.999) with a lower limit of quantitation of 0.1 ng/mL. Extraction recovery was in the range 63.5–72.1% for PC 48 and 70.5% for reserpine (internal standard, IS). Intra- and inter-day precisions measured as relative standard deviation were below 10.8% and accuracy was from −7.2% to 7.4%. The method was successfully applied to a pharmacokinetic study involving intramuscular application of 3.86 mg/kg PC 48 to rats for the first time. Pharmacokinetic parameters for PC 48 include C max 39.09 ± 4.45 ng/mL, T max 5.00 ± 3.08 min, AUC 0 − t 23374 ± 4045 min ng/mL and t 1/2 1065 ± 246 min. [ABSTRACT FROM AUTHOR]

A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method for the quantification of shanzhiside methyl ester, 8- O -acetylshanzhiside methyl ester and luteolin-7- O -β- d -glucopyranoside of Lamiophlomis rotata Pill in rat plasma was developed and validated. After liquid–liquid extraction with n -butyl alcohol/ethyl acetate (70:30, v/v ), analytes and paeoniflorin (internal standard, IS) were separated on an Acquity BEH UPLC C 18 column (100 × 2.1 mm, 1.7 μm) with gradient elution at a flow rate of 0.2 mL/min. All calibration curves had good linearity (r > 0.9929) over the concentration ranges of 1–1000 ng/mL for shanzhiside methyl ester and 8- O -acetylshanzhiside methyl ester, 0.3–150 ng/mL for luteolin-7- O -β- d -glucopyranoside. The intra- and inter-day precisions were all within 11.1% and the accuracy (relative error, RE%) all ranged from −13.6% to 5.3%. The method also guaranteed an acceptable selectivity, recovery and stability, which was successfully applied to a pharmacokinetic study of the three analytes in rats after oral administration of Lamiophlomis rotata Pill. [ABSTRACT FROM AUTHOR]

For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C 18 column (150 mm × 3.9 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8 mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m / z 368.9→ m / z 191.9 (fraxin), m / z 206.9→ m / z 191.8 (fraxetin) and m / z 339.0→ m / z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r ≥ 0.995) over a concentration range of 5.00–3000 ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from −5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50 mg/kg to rats. [ABSTRACT FROM AUTHOR]

Rapid, sensitive, selective and accurate ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of elbasvir (ELB) in rat plasma with deuterated elbasvir (ELB-D6) as internal standard (IS).Sample preparation was done by protein precipitation using acetonitrile containing 50 ng/mL IS. Chromatographic separation was achieved by an UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with a gradient mobile phase consisting of acetonitrile–water (containing 5.0 mM ammonium acetate with 0.01% acetic acid, pH 4.5) as mobile phase at a flow rate of 0.3 mL/min for 3 min. ELB was monitored using positive electrospray triple quadrupole mass spectrometer (Waters Xevo TQ-S) via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 882.51 → 656.42 and m / z 888.49 → 662.43 for ELB and ELB-D6, respectively. The achieved lower limit of quantification was 1.0 ng/mL. The validated method had an excellent linearity in the range of 1.0–2000 ng/mL (r 2 > 0.996). Recovery efficiency at three levels QC concentrations of 2.0 (low), 160 (medium) and 1600 (high) ng/mLwas in the range of 98.29–106.40% for ELB. Matrix effect was found to be minimal. The intra- and inter-day precisions were less than 7.01%. The intra- and inter-day accuracies were determined to be within ±6.23% for all accuracy measurements. The validated simple and rapid UPLC–MS/MS method was successfully used to the pharmacokinetics study of ELB in rats, providing its applicability in relevant preclinical studies. [ABSTRACT FROM AUTHOR]