Search

Acotiamide is a new prokinetic drug that is used to treat functional dyspepsia (FD). A sensitive and specific LC-MS-MS method has been developed and validated for the analysis of acotiamide in rat plasma. The assay involved a simple protein precipitation (PPT) step with methanol-acetonitrile (50:50, v/v) and a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid. The analytes were chromatographed on a reversephase Agilent Zorbax XDB C18 column (2.1 mm× 50 mm, 3.5 μm) with a flowrate of 0.50 mL/min. The analytes were monitored by tandem-mass spectrometry with positive electrospray ionization. The precursor-product transitions (m/z) in the positive ion mode were 451.4→271.3 and 386.2→122.2 for acotiamide and buspirone (internal standard, IS), respectively. The assay was shown to be linear over the range of 0.10-200 ng/mL, with a lower limit of quantification of 0.10 ng/mL. The method was shown to be reproducible and reliable with the inter- and intra-batch accuracy and precision were within ±15%. The assay has been successfully used for pharmacokinetic evaluation of acotiamide after intravenous and oral administration of 10 mg/kg acotiamide in rats. The oral absolute bioavailability (F) of acotiamide in rats was estimated to be 38.4 ± 13.5% with an elimination half-life (t1/2) value of 9.11 ± 0.40 h. [ABSTRACT FROM AUTHOR]

This studywas designed to develop a sensitive, simple and rapid method for the quantitation of morroniside in rat urine using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) and to investigate the excretion of morroniside in rat urine. The mobile phase consisted of water-acetonitrile (gradient elution) at a flow rate of 0.4 mL/min. Detection was performed using positive-ion electrospray ionization in multiple reaction monitoring (MRM) modes. And the detection of morroniside in rat urine by the LC-MS-MS was accurate and precise from 1.0 to 2,500 ng/mL (a correlation coefficient of 0.9953). The recoveries and matrix effects were all in line with the biological sample measurement requirements. The intraday accuracy was 88.68-105.78% with precision of 6.50-11.19% and the interday accuracy was 95.77-102.43% with precision of 7.08-10.40%. Excretion data of morroniside in rat urine indicated that 21.43? (i.g.) and 100.35% (i.v.) of the dose administered was excreted as unconverted form, respectively. And the maximal excretion ratewas 27.57 and 482.42 µg/h after oral and intravenous administration, respectively. These results indicated that the developed method has satisfactory sensitivity, accuracy and precision for the quantification of morroniside in rat urine. [ABSTRACT FROM AUTHOR]

AM-2201 is a popular synthetic cannabinoid first synthesized in 2000. AM-2201 pharmacokinetic and pharmacodynamic data are scarce, requiring further investigation. We developed a sensitive method for quantifying AM-2201 and 13 metabolites in plasma to provide a tool to further metabolic, pharmacokinetic and pharmacodynamic studies. Analysis was performed by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed by gradient elution on a biphenyl column with 0.1% formic acid in water/0.1% formic acid in acetonitrile:methanol 50:50 ( v/v ) mobile phase. Sample preparation (75 μL) consisted of an enzymatic hydrolysis and a supported liquid extraction. The method was validated with human plasma with a 0.025 or 0.050–50 μg/L working range, and cross-validated for rat plasma. Analytical recovery was 88.8–110.1% of target concentration, and intra- (n = 30) and inter-day (n = 30) imprecision < 11.9% coefficient of variation. Method recoveries and matrix effects ranged from 58.4–84.4% and −62.1 to −15.6%, respectively. AM-2201 and metabolites were stable (±20%) at room temperature for 24 h, at 4 °C for 72 h, and after three freeze-thaw cycles, and for 72 h in the autosampler after extraction. The method was developed for pharmacodynamic and pharmacokinetic studies with controlled administration in rats but is applicable for pre-clinical and clinical research and forensic investigations. Rat plasma specimen analysis following subcutaneous AM-2201 administration demonstrated the suitability of the method. AM-2201, JWH-018 N -(5-hydroxypentyl), and JWH-018 N -pentanoic acid concentrations were 4.8 ± 1.0, 0.15 ± 0.03, and 0.34 ± 0.07 μg/L, respectively, 8 h after AM-2201 administration at 0.3 mg/kg (n = 5). [ABSTRACT FROM AUTHOR]

Rhodamine B is an illegal and potentially carcinogenic food dye. The aim of this study was to develop a convenient, rapid, and sensitive UHPLC-MS/MS method for pharmacokinetic studies in rats. Rat plasma samples were deproteinized with acetonitrile and separated by UHPLC on a reverse-phase C18e column (100 mm × 2.1 mm, 2 μm) using a mobile phase consisting of methanol—5 mM ammonium acetate (90:10, v/v). Detection was performed using a triple quadrupole tandem mass spectrometer in the selected reaction monitoring mode at [M] + ion m / z 443.39 → 399.28 for rhodamine B and [M+H] + ion m / z 253.17 → 238.02 for 5-methoxyflavone as the internal standard. This method was specific and produced linear results over a concentration range of 0.5–100 ng/mL, with a lower limit of quantitation of 0.5 ng/mL. All validation parameters, including the inter-day, intra-day, matrix effect, recovery, and stability in rat plasma, were acceptable according to the biological method validation guidelines developed by the FDA (2001). This method was successfully applied to a pharmacokinetic study in rats; oral administration of 1 mg/kg of rhodamine B yielded a time to maximum concentration ( T max ) of 1.3 ± 0.4 h and an elimination half-life of 8.8 ± 1.4 h, with a clearance of 229.7 ± 19.4 mL/h/kg. These pharmacokinetic results provide a constructive contribution to our understanding of the absorption mechanism of rhodamine B and support additional food safety evaluations. [ABSTRACT FROM AUTHOR]

A simple, sensitive and accurate ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of AKI603 in rat plasma has been firstly developed and validated. After a simple liquid–liquid extraction (LLE) with ethyl acetate, the analytes were separated on C18 column (2.1 × 100 mm, 1.9 μm, Thermo) by gradient elution with mobile phase of water (A) (containing 5 mM ammonium acetate and 0.1% formic acid) and methanol (B) with a flow rate of 0.3 mL min −1 and then analyzed by mass spectrometry in the positive multiple reactions monitoring (MRM) mode. The mass transitions monitored were m / z 410.0 → 352.9, m / z 457.1 → 367.9 for AKI603 and internal standard (Ly-7z), respectively. The developed method was validated for specificity, linearity and lower limit of quantification, intra- and inter-day precision and accuracy, extraction recovery, matrix effect and stability whose values satisfied the acceptable limits. The calibration curves for AKI603 was linear in concentration ranges of 0.025–5000 ng mL −1 . The method has been successfully used to the bioavailability study of AKI603 administered to rats intravenously (2.5 mg/kg) or orally (25 mg/kg). The oral bioavailability of AKI603 in rats was calculated as 28.7 ± 9.7%. [ABSTRACT FROM AUTHOR]

Metal-organic framework MIL-101(Cr) was successfully used as an efficient sorbent in a vortex-assisted dispersive solid-phase extraction (VA-DSPE) and applied for the determination and the pharmacokinetic of imatinib mesylate in rat plasma by UPLC–MS/MS. In the enrichment of imatinib from rat plasma, the analyte was efficiently adsorbed on MIL-101(Cr) and simply recovered by using initial mobile phase (0.1% formic acid-methanol (6:4 v / v )) as elution solvent. Meanwhile, the protein in the plasma samples was excluded from the porous structure of MIL-101(Cr), leading to direct extraction of drug molecule from protein-rich biological samples without any other pretreatment procedure. After being removed, the supernatant was filtered and directly injected into the UPLC–MS/MS for the analysis of the target. The experimental parameters, including nature of MOFs, amount of MIL-101(Cr), pH value of aqueous solution, extraction time, type and volume of elution solvent, were systematically optimized. After VA-DSPE, chromatographic separation was performed on an ACQUITY UPLC ® BEH C 18 column (2.1 mm × 100 mm, 1.7 μm) with a 3 min gradient elution using 0.1% formic acid and methanol as mobile phase at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the positive ionization mode. The lower limit of quantification of 1 ng/mL was achieved and the mean recovery of the analyte was higher than 81.2%. Moreover, computational simulation was primarily applied to predict the adsorption behavior and revealed the molecular interactions and free binding energies between MIL-101(Cr) and imatinib with the molecular modeling method, providing certain explanation of the adsorption mechanism. The originally established pretreatment and detection method has some merits, such as less solvent consumption, easy operation, higher sensitivity and lower matrix effect. And the MIL-101(Cr) exhibited a potential as an efficient sorbent in the enrichment of the analyte from complex biosamples. [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]

Deoxyglycychloxazol (TY501) is a glycyrrhetinic acid derivative which exhibits high anti-inflammatory activity and reduced pseudoaldosteronism compared to glycyrrhetinic acid. In this study, a sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the quantitation of TY501 in rat plasma. Plasma samples were treated by precipitating protein with methanol and supernatants were separated by a Symmetry C 8 column with the mobile phase consisting of methanol and 10 mM ammonium formate (containing 0.1% of formic acid) (90:10, v/v). The selected reaction monitoring (SRM) transitions were performed at m/z 647.4→191.2 for TY501 and m/z 473.3→143.3 for astragaloside aglycone (IS) in the positive ion mode with atmospheric pressure chemical ionization (APCI) source. Calibration curve was linear over the concentration range of 5–5000 ng/mL. The lower limit of quantification was 5 ng/mL. The mean recovery was over 88%. The intra- and inter-day precisions were lower than 6.0% and 12.8%, respectively, and the accuracy was within ±1.3%. TY501 was stable under usual storage conditions and handling procedure. The validated method has been successfully applied to a pharmacokinetic study after oral administration of TY501 to rats at a dosage of 10 mg/kg. [ABSTRACT FROM AUTHOR]

In this study, a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine mangiferin and neomangiferin in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a Xevo TQD triple quadruple mass spectrometer coupled with an electrospray ionization (ESI) source. The MRM transitions of m / z 423.2 → 303.1 and m / z 585.0 → 273.1 were used to quantify for mangiferin and neomangiferin, respectively. The linearity of this method was found to be within the concentration range of 5–2000 ng/mL for mangiferin, and 2–1000 ng/mL for neomangiferin in rat plasma, respectively. Only 3.0 min was needed for an analytical run. This assay was used to support a preclinical study to investigate the pharmacokinetics of mangiferin and neomangiferin in rats. [ABSTRACT FROM AUTHOR]

A sensitive UPLC–MS/MS method was developed and validated for the determination of brusatol in rat plasma. Chromatographic separation was carried out on a C 18 column using methanol and 10 mM ammonium acetate containing 0.1% (v/v) formic acid (55:45, v/v). The lower limit of quantification (LLOQ) was 1.0 ng/mL for brusatol in plasma. The intra- and inter-day precision for the analyte ranged from 3.2% to 9.2% and 1.3% to 7.8%, and the accuracy was between 97.3% and 108.5%. The method was successfully applied in a pharmacokinetic study of brusatol following intravenous injection (0.5, 1.0, and 2.0 mg/kg) of brusatol. [ABSTRACT FROM AUTHOR]

A rapid, sensitive, and accurate bioanalytical method was established for the quantitation and pharmacokinetic investigation of loratadine (LTD) in rat plasma by liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS/MS) using loratadine-d5 as internal standard (ISTD). The analyte and ISTD were extracted by solid-phase extraction and chromatographic separation was achieved on Gemini NX- Reverse Phase C18 (50 × 4.6 mm;5 μ) using mobile phase mixture of 5 mM ammonium formate buffer in water (pH 3.5 ± 0.1 with formic acid), and acetonitrile (20:80 v/v), at a flow rate of 0.400 mL/min with injection volume of 10 μL. LTD and ISTD were detected at m / z 383.3 → 337.4 and 388.4 → 337.3 with retention time of 2.62 and 2.59 min, respectively. High sensitivity (1.0 ng/mL) was achieved using small volume of rat plasma (20 μL) and the method was validated over a linearity range of 1.05–405.41 ng/mL with high correlation coefficient (r = 0.9998). The extraction method displayed a mean process efficiency of 63.25 and 65.47% for LTD and ISTD, respectively. The validated method when successfully applied for quantification of LTD in rat plasma revealed enhanced bioavailability of orally administered LTD-loaded self-nanoemulsifying drug delivery system (SNEDDS) (C max , 466.65 ± 18.94 ng/mL and AUC 0-t 633.00 ± 12.44 ng-h/mL) over LTD-suspension (C max , 104.75 ± 2.87 ng/mL and AUC 0-t 287.00 ± 9.11 ng-h/mL). The in vivo-in silico prediction by the GastroPlus™ software showed good prediction accuracy for LTD-SNEDDS (fold error < 2). The Loo-Reigelman method (2-compartment) presented best model-fitting indicating adequate in vitro–in vivo correlations. Conclusively, the developed sensitive analytical method displayed enhanced systemic availability of LTD-SNEDDS, and the in vivo in silico approach revealed sufficiently good GI simulation. [ABSTRACT FROM AUTHOR]

A rapid and sensitive ultra fast liquid chromatography tandem mass spectrometry method (UFLC–MS/MS) was developed and validated for the simultaneous determination of six Saikosaponins (SSs) (SSa, SSb 1 , SSb 2 , SSd, SSc, SSf) of Bupleurum Dropping Pills (BDP) in rat plasma using chloramphenicol as the internal standard (IS). The SSs were separated using an ACQUITY UPLC ® BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and detection of these compounds were done by using a Qtrap 5500 mass spectrometer coupled with negative electrospray ionization (ESI) source under the multiple reaction monitoring (MRM) mode. According to regulatory guidelines, the established method was fully validated and results were showed within acceptable limits. The lower limit of quantifications (LLOQs) of all analytes were 0.2 ng/mL. The validated method was successfully applied into a pharmacokinetic study of orally administered BDP in rats. [ABSTRACT FROM AUTHOR]

A sensitive, simple and rapid analytical method based on a liquid chromatography–tandem mass spetrometry (LC–MS/MS) has been established and validated for the determination of stachyose in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Separation of stachyose and nystose (internal standard, IS) was achieved using acetonitrile-water (55:45, v/v) as the mobile phase at a flow rate of 1 ml/min for 6 min on an Asahipak NH 2 P-50 4E column with an Asahipak NH2P-50G 4A guard column. Detection and quantification were conducted by LC–MS/MS method in the negative ion mode using multiple reaction monitoring (MRM) transitions at m / z [M–H] − 665.4 → 383.1 for stachyose and 665.5 → 485.0 for IS, respectively. The method was linear over the concentration ranges of 100–30000 ng/ml with a lower limit of quantification (LLOQ) of 100 ng/ml. The intra- and inter- day precision were all within 8.7% and the accuracy ranged from 97.2–108.4% and 98.3–102.4%, respectively. Stability studies indicated that stachyose was stable under short-term, long-term and three freeze-thaw storage conditions. The method was successfully applied to a pharmacokinetic study involving pulmonary administration of micronized Rehmannia glutinosa oligosaccharides (RGOS) to rats. [ABSTRACT FROM AUTHOR]

Lurbinectedin (PM01183) is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected sequences in the minor groove of DNA, inducing PM01183-DNA adducts that stall replication, DNA repair and transcription and gives rise to double-strand breaks and finally, caspase-dependent apoptotic cell death. PM01183 has demonstrated clinical antitumor activity in platinum resistant/refractory ovarian cancer patients. A rapid and sensitive liquid chromatography/tandem mass spectrometry assay was developed and validated to quantify PM01183 in plasma from nonclinical species. The bioanalysis consisted of a supported liquid extraction, followed by a gradient phase chromatography and, detection by positive ion electrospray tandem mass spectrometry. The calibration range for PM01183 was established using PM01183 standards from 0.1 to 100 ng/mL in blank plasma. The multiple reaction monitoring, based on the transition m / z 767.3 → 273.0, was specific for PM01183, and that based on the transition m / z 771.4 → 277.0 was specific for the internal standard (deuterated PM01183). No endogenous material interfered with the analysis of PM01183 and the internal standard from blank plasma. The limit of detection (LOD) of the assay was calculated as 0.025 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9937 to 0.9987. The mean inter-day accuracies for all calibration standards ranged from 92 to 108% (≤8% bias), and the mean inter-day precision for calibration standards was always less than 12%. The mean intra and inter-day assay accuracy for all quality control replicates remained between 91 and 109%. The mean intra and inter-day assay precision was less than 10% for all QC levels. The method was validated to demonstrate the specificity, recovery, limit of quantification, accuracy and precision of measurements. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies of PM01183 in nonclinical species. The main PK parameters in dogs (3 male and 3 female, respectively) were calculated as follows: maximum concentration ( C max , 12.9 ± 0.6 and 10.2 ± 3.0 ng/mL) and the area under the plasma concentration-time curve (AUC, 24.9 ± 0.7 and 22.6 ± 6.1 ng h/mL). The results showed that plasma samples could be monitored for PM01183 for long enough to accurately estimate pharmacokinetics information. [ABSTRACT FROM AUTHOR]

Brodifacoum (BDF) is a second-generation anticoagulant rodenticide structurally related to warfarin but containing two chiral centers. Highly stable, BDF can contaminate food andwater supplies causing accidental poisoning of humans and nontarget animals. To determine the distribution of BDF isomers in serumand tissues, a quantitativemethodwas developed and validated according to FDA guidelines based on high-performance liquid chromatography-tandemmass spectrometry. A single liquid-liquid extraction step provided recoveries exceeding 93%. Reversed-phase chromatographic separations required <6 min, and quantitative analysis utilized a triple-quadrupole mass spectrometer equipped with negative ion electrospray and selected reaction monitoring. The standard curve had a linear regression coefficient of 0.999 and intra- and inter-assay variations of <10%. The chromatographic method enabled the resolution and measurement of pairs of BDF diastereomers in commercial materials as well as in rat tissues. This method is suitable for measuring BDF exposure as well as basic science studies of the distribution and elimination of BDF diastereomers to various tissues. [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]

Colistin is a polypeptide antibiotic that effectively treats infections caused by multidrug-resistant Gram-negative bacteria, but its clinical use is limited due to nephrotoxicity. The purpose of the present study was to identify biomarkers of colistin-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites using untargeted metabolomic approach. Rats receiving intraperitoneal administration of colistin sodium methanesulfonate (CMS) (25 or 50 mg/kg) exhibited histopathological changes in the kidney and increased blood urea nitrogen levels. Additionally, the levels of phenylalanine, tryptophan, and tyrosine in the urine of the CMS-treated group were significantly higher than those of the control group, suggesting that colistin caused proximal tubular damage. Urinary acetylcarnitine and butyrylcarnitine levels also increased after CMS treatment, but the levels of purine metabolites and metabolites related to the tricarboxylic acid cycle were reduced. The most significant increase in the CMS-treated groups was observed in creatine levels. CMS-induced selective nephrotoxicity may be attributed to relatively high tissue concentrations of colistin in the kidney. Taken together, our results indicate that high levels of colistin in the kidney caused perturbations in the tricarboxylic acid cycle, amino acid metabolism, creatine metabolism, and purine metabolism and ultimately led to kidney injury. [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]

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [ 14 C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC–MS/MS method for the analysis of stable isotope-modified [ 13 C 12 ]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [ 13 C 6 ]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90–106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear ( r 2 ≥ 0.99) in the concentration ranges tested for the diluted blood and plasma (10–1000 ng/mL) and brain homogenate (1–200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated ( n = 5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [ 13 C 12 ]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [ 13 C 12 ]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models. [ABSTRACT FROM AUTHOR]

The objective of this research is to develop and validate a sensitive and reproducible UPLC–MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88–10000 nM, 39–5000 nM, 48.8–6250 nM and 48.8–6250 nM, respectively (R 2 > 0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25–2000 nM, 4.88–1250 nM, 9.8–1250 nM and 9.8–1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC–MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 μl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples. [ABSTRACT FROM AUTHOR]

The study was to assess the long-term toxic effects of acetochlor on rats. Two different doses (42.96 and 107.4 mg/kg body weight/day) of acetochlor were administered to Wistar rats through their food for over 24 weeks. Rat urine samples were collected at two time-points for the measurements of the metabonomics profiles with ultra-performance liquid chromatography–mass spectrometry (UPLC–MSMS). The results of clinical chemistry and histopathology suggested that long-term use of acetochlor in rats caused liver and kidney damage, and dysfunction of antioxidant system. The urinary metabonomics analysis indicated that the high and low-dose exposure of acetochlor could cause alterations of these metabonomics in urine in the rat. Significant changes of the levels of hippuric acid (0.403-fold decrease), citric acid (0.430-fold decrease), pantothenic acid (0.486-fold decrease), uracil (0.419-fold decrease), β-Alanine (0.325-fold decrease), nonanedioic acid (0.445-fold decrease), l -tyrosine (0.410-fold decrease), d -glucuronic acid (8.389-fold increase) and 2-ethyl-6-methyl-N-methyl-2-chloro-acetanilide in urine were observed. In addition, it may interfere with the fatty acid synthesis, the pyrimidine degradation and pantothenate biosynthesis. The level of 2-ethyl-6-methyl-N-methyl-2-chloro-acetanilide is detected in all treated groups which is not found in the control groups, indicating which can be used as an early, sensitive marker of acetochlor exposure in rat. This study illustrates the important utility of metabonomics approaches to understand the toxicity of long-term exposure of acetochlor. [ABSTRACT FROM AUTHOR]

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 μM) and non-treatment were employed on isolated rat hearts during hypoxia/ reoxygenation treatment using an ex vivo Langendorffsystem. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptorgamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries. [ABSTRACT FROM AUTHOR]

Objective: Interactions between pre-existing differences in mesolimbic function and neuroadaptations induced by consumption of fatty, sugary foods are thought to contribute to human obesity. This study examined basal and cocaine-induced changes in striatal neurotransmitter levels without diet manipulation and D2 /D3 dopamine receptor-mediated transmission prior to and after consumption of "junk-foods" in obesity-prone and obesity-resistant rats.Methods: Microdialysis and liquid chromatography-mass spectrometry were used to determine basal and cocaine-induced changes in neurotransmitter levels in real time with cocaine-induced locomotor activity. Sensitivity to the D2 /D3 dopamine receptor agonist quinpirole was examined before and after restricted junk-food exposure. Selectively bred obesity-prone and obesity-resistant rats were used.Results: Cocaine-induced locomotion was greater in obesity-prone rats versus obesity-resistant rats prior to diet manipulation. Basal and cocaine-induced increases in dopamine and serotonin levels did not differ. Obesity-prone rats were more sensitive to the D2 receptor-mediated effects of quinpirole, and junk-food produced modest alterations in quinpirole sensitivity in obesity-resistant rats.Conclusions: These data show that mesolimbic systems differ prior to diet manipulation in susceptible versus resistant rats, and that consumption of fatty, sugary foods produce different neuroadaptations in these populations. These differences may contribute to enhanced food craving and an inability to limit food intake in susceptible individuals. [ABSTRACT FROM AUTHOR]

Pinostilbene (3-methoxyresveratrol or trans -3,4′-dihydroxy-5-methoxystilbene) is a naturally occurring monomethylether analogue of resveratrol ( trans -3,5,4′-trihydroxystilbene) that exhibits various pharmacological activities. To further examine its medicinal potential, a sensitive LC–MS/MS method was developed and validated for the determination of pinostilbene in rat plasma. Heavy Isotope labelled resveratrol was used as an internal standard. The ESI was operated in its negative ion mode while pinostilbene and resveratrol were measured by multiple reaction monitoring (MRM) using precursor-to-product ion transitions of m / z 241 → 181 and m / z 233 → 191, respectively. This LC–MS/MS method had excellent selectivity, sensitivity (LLOQ = 1 ng/ml), accuracy (both intra- and interday analytical recovery within 100 ± 15%) and precision (both intra- and interday RSD < 15%). The matrix effect was insignificant. The pharmacokinetics of pinostilbene was subsequently profiled in Sprague–Dawley rats. Upon intravenous administration (5 or 10 mg/kg), pinostilbene displayed rapid clearance (Cl = 129 ± 42 or 107 ± 31 ml/min/kg) and extremely short mean transit time (MTT = 6.24 ± 0.41 or 8.52 ± 1.38 min). After oral dosing (50 mg/kg), the bioavailability of pinostilbene was limited but highly erratic ( F = 1.87 ± 2.67%). Pharmacokinetic comparison among pinostilbene, resveratrol and some resveratrol analogues suggested that stilbenes with meta-hydroxyl group(s) may be associated with metabolic instability and subsequently suffer from rapid clearance and low oral bioavailability. The information obtained from this study will facilitate further exploration on pinostilbene as well as other resveratrol analogues. [ABSTRACT FROM AUTHOR]

A simple and rapid quantitative UPLC–MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C 18 (50 mm × 2.1 mm, 1.8 μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m / z 785.4 → 639.5, m / z 815.5 → 669.5, and m / z 763.5 → 455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5–1150 ng/mL and 13.0–1300 ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats. [ABSTRACT FROM AUTHOR]

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine tedizolid and linezolid in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a XEVO TQD triple quadruple mass spectrometer coupled with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 371.4 → 343.2 for tedizolid, and m/z 338.3 → 56.1 for linezolid. This assay method has been fully validated in terms of selectivity, linearity, recovery and matrix effect, accuracy, precision and stability. The linearity of this method was found to be within the concentration range of 5–5000 ng/mL for tedizolid, and 10–10,000 ng/mL for linezolid in rat plasma, respectively. Only 3.0 min was needed for an analytical run. This assay was used to support a preclinical study where multiple oral doses were administered to rats to investigate the pharmacokinetics of tedizolid and linezolid. [ABSTRACT FROM AUTHOR]

Psoralea corylifolia L. has long been used in traditional Chinese medicine for treating and preventing many diseases. A group of flavonoid components are regarded as the active principals within the seeds. In this research, a rapid, accurate and sensitive ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method has been established for simultaneous quantification of its 5 main components, namely, neobavaisoflavone, bavachin, isobavachalcone, bavachinin and corylifol A in rats’ plasma after the rats were orally administrated with Buguzhi extract. Negative ion electrospray mode was applied in the detection process. Multiple reactions monitoring (MRM) mode was utilized for simultaneous quantitative analyzing of neobavaisoflavone ( m / z 321.1 → m / z 265.1), bavachin ( m / z 323.1 → m / z 119.0), isobavachalcone ( m / z 323.2 → m / z 119.0), bavachinin ( m / z 337.2 → m / z 119.0), corylifol A ( m / z 389.2 → m / z 277.0) and liquiritigenin (Internal Standard, m / z 255.1 → m / z 119.0). Chromatographic separation of the above mentioned components was conducted on a Waters BEH-C18 column (100 mm × 2.1 mm, 1.7 μm) with gradient elution system at flow rate of 0.3 mL/min. The mobile phase was composed of acetonitrile and 0.1% formic acid solution. The lower limit of quantification (LLOQ) for each of the above analytes was 0.5 ng/mL. Each of the analytes exhibited good linearity within the concentration range of 0.5–100 ng/mL. The method was fully validated for its selectivity, accuracy, precision, stability, matrix effect and extraction recovery. The validated method has been successfully applied for simultaneous determination of the 5 flavonoids in rat plasma for the first time. [ABSTRACT FROM AUTHOR]

A chiral UFLC-MS/MS method was established and validated for quantifying d -threo-methylphenidate ( d -threo-MPH), l -threo-methylphenidate ( l -threo-MPH), d -threo-ethylphenidate ( d -threo-EPH), l -threo-ethylphenidate ( l -threo-EPH) and d , l -threo-ritalinic acid ( d , l -threo-RA) in rat plasma over the linearity range of 1–500 ng/mL. Chiral separation was performed on an Astec Chirobiotic V2 column (5 μm, 250 × 2.1 mm) with isocratic elution using methanol containing 0.003% ammonium acetate (w/v) and 0.003% trifluoroacetic acid (v/v) at a flow of 0.3 mL/min. All analytes and IS were extracted from rat plasma by a one-step liquid–liquid extraction (LLE) method. The intra- and inter-run accuracies were within 85–115%, and the intra- and inter-run precision were <10% for all analytes. Extraction recoveries were 55–62% for d -threo-MPH, 54–60% for l -threo-MPH, 55–60% for d -threo-EPH, 53–57% for l -threo-EPH and 25–30% for d , l -threo-RA. The validated UFLC-MS/MS method successfully applied to the pharmacokinetic interaction study of oral d -threo-MPH and l -threo-MPH (alone or in combination) in female Sprague Dawley rats. The EPH was not detected in rat plasma following oral administrated MPH without EtOH. As far as it is known to the authors, this study is the first one step liquid–liquid extraction method to extract and UFLC-MS/MS method to quantify d -threo-MPH, l -threo-MPH, d -threo-EPH, l -threo-EPH and d , l -threo-RA simultaneously. [ABSTRACT FROM AUTHOR]

Diffuse axonal injury (DAI) is fairly common during a traumatic brain injury (TBI) and is associated with high mortality. Making an early diagnosis, appropriate therapeutic decisions, and an accurate prognostic evaluation of patients with DAI still pose difficulties for clinicians. The detailed mechanisms of axonal injury after head trauma have yet to be clearly defined and no reliable biomarkers are available for early DAI diagnosis. Therefore, this study employed an established DAI animal model in conjunction with an isobaric tag for relative and absolute quantification (iTRAQ)-based protein identification/quantification approach. Alterations in rat cerebral protein expression were quantified using iTRAQ coupled LC–MS/MS, with differentially expressed proteins between the control groups, sham and sham-injured, and the injury groups, animals that died immediately post-injury and those sacrificed at 1 h, 6 h, 1 d, 3 d and 7 d post-injury, identified. A total of 1858 proteins were identified and quantified and comparative analysis identified ten candidate proteins that warranted further examination. Of the ten candidate DAI biomarkers, four proteins, citrate synthase (CS), synaptosomal-associated protein 25 (Snap25), microtubule-associated protein 1B (MAP1B) and Rho-associated protein kinase 2 (Rock2), were validated by subsequent Western blot and immunohistochemistry analyses. Our studies not only identified several novel biomarkers that may provide insight into the pathophysiological mechanisms of DAI, but also demonstrated the feasibility of iTRAQ-based quantitative proteomic analysis in cerebral tissue research. [ABSTRACT FROM AUTHOR]

To identify the sheep red blood cell (SRBC) surface immune-responsive peptides, immuno- reactive fraction of SRBC was detected by SDS-PAGE and western blot analysis with antisera from SRBC-immunized rats. Then the most intense immuno-reactive band on SDS-PAGE was subjected to nanoLC-ESI-MS/MS analysis, and 17 proteins were identified including membrane proteins of erythrocytes such as band 3 anion transport protein isoform 1 (Anion exchange protein 1; AE-1, CD233), Ammonium transporter Rh type A (Rh type A glycoprotein, CD241) and Ankyrin-1 (ANK-1), Spectrin beta chain. Among them, plasma protein AE-1 (CD233) and Rh type A glycoprotein (CD241) have transmembrane domain and correspond to extracellular region in their sequences. These extracellular regions of the plasma membrane proteins are supposed to be major immune-responsive peptides of SRBC in rats. These peptides are promising for the construction of an ELISA system which does not require the processing of SRBC membrane ghosts. [ABSTRACT FROM AUTHOR]

HsTX1[R14A] is a potent and selective Kv1.3 channel blocker peptide with the potential to treat autoimmune diseases. Given the typically poor oral bioavailability of peptides, we evaluated pulmonary administration of HsTX1[R14A] in rats as an alternative route for systemic delivery. Plasma concentrations of HsTX1[R14A] were measured by liquid chromatography coupled with tandem mass spectrometry in rats receiving intratracheal administration of HsTX1[R14A] in solution (1-4 mg/kg) or a mannitol-based powder (1 mg/kg) and compared with plasma concentrations after intravenous administration (2 mg/kg). HsTX1[R14A] stability in rat plasma and lung tissue was also determined. HsTX1[R14A] was more stable in plasma than in lung homogenate, with more than 90% of the HsTX1[R14A] remaining intact after 5 h, compared with 40.5% remaining in lung homogenate. The terminal elimination half-life, total clearance, and volume of distribution of HsTX1[R14A] after intravenous administration were 79.6 ? 6.5 min, 8.3 ? 0.6 mL/min/kg, and 949.8 ? 71.0 mL/kg, respectively (mean ? SD). After intratracheal administration, HsTX1[R14A] in solution and dry powder was absorbed to a similar degree, with absolute bioavailability values of 39.2 ? 5.2% and 44.5 ? 12.5%, respectively. This study demonstrated that pulmonary administration is a promising alternative for systemically delivering HsTX1[R14A] for treating autoimmune diseases. [ABSTRACT FROM AUTHOR]

The adrenal gland is the most common toxicological target in the endocrine system, and inhibition of adrenal steroidogenesis by drugs can be fatal in humans. However, methods to evaluate the drug effect are limited. Recently, simultaneous measurement of multiple steroids, including precursors, has become possible. Here, we evaluated the usefulness of this simultaneous measurement for the evaluation of drug effects on adrenal steroidogenesis in vivo. For this purpose, we measured plasma concentrations of adrenal steroids in rats dosed with ketoconazole, a known inhibitor of adrenal steroidogenesis, and examined its relationship with the changes in histopathology and mRNA expression of steroidogenic enzymes in the adrenal gland. Ketoconazole (150 mg/kg/day) was orally administered to male rats for 7 days. The adrenal weight was high, and the zona fasciculata/reticularis were hypertrophic with an accumulation of lipid droplets. mRNA expression of CYP11A1, a rate-limiting enzyme in adrenal steroidogenesis, was slightly high in the adrenal gland. Plasma concentration of deoxycorticosterone was markedly high, while there were no significant changes in that of corticosterone, progesterone, or pregnenolone. The changes in the adrenal gland and plasma concentration of steroids were thought to reflect inhibited metabolism of deoxycorticosterone to corticosterone through inhibition of CYP11B1, and compensatory reaction for the inhibition. The compensatory reaction was thought to have masked decrease of corticosterone. These results suggest that simultaneous measurement of multiple steroids can enable sensitive evaluation of drug effects on adrenal steroidogenesis in vivo, while providing insight into the underlying mechanism of the effect. [ABSTRACT FROM AUTHOR]

TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug–protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we elucidated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 °C. After liquid–liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLC ® C 18 (2.1 mm×50 mm, 1.7 µm), and acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The assay was linear over the concentration rang of 5–2000 ng/mL. The intra- and inter-day precisions were 0.1%–14.8%, and the accuracy was from −6.4% to 7.0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4%±6.5% (rat), 87.9%±3.6% (human) and 79.4%±4.0% (beagle dog). The results revealed that there was an insignificant difference among the three species. [ABSTRACT FROM AUTHOR]

A liquid chromatography–high resolution mass spectrometry (LC–HRMS) method was developed and validated for the determination of piperine (PPR) on dried blood spots (DBS). DBS samples were prepared by spiking the whole blood with analyte to produce 30 µL of blood spots on specimen collection cards. Chromatographic separation was achieved on an Atlantis dC 18 column using acetonitrile and water (0.1% formic acid) (85:15, v/v) as mobile phase in an isocratic mode of elution at a flow rate of 0.75 mL/min. MS detection was carried out in electrospray positive ion mode for the target ions and monitored at m/z 286.1465 for PPR and 272.1303 for the internal standard (IS). The developed method exhibited a linear dynamic range over 0.01–2000 ng/mL for PPR on DBS. The overall extraction recovery of PPR from DBS was 92.5%. Influence of hematocrit and spot volume on DBS was also evaluated and found to be well within the acceptable limits. The method was successfully applied to pharmacokinetic studies of PPR in rats. [ABSTRACT FROM AUTHOR]

3- O -Acetyloleanolic acid (OAA) is a triterpenoid compound, and exerts an apoptosis in cancer cell lines, an inhibition of both atopic and allergic contact dermatitis in murine model, and a suppression of inflammatory bone loss in mice. OAA can be converted into oleanolic acid (OA) by hydrolysis in vivo , and OA exhibits several pharmacological effects as well. A liquid chromatographic method using tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of OAA and OA in rat plasma. After liquid–liquid extraction with ethylacetate, both substances were chromatographed on a reversed phase column with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile (1:9, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of both substances over time following an intravenous administration of OAA in rats. [ABSTRACT FROM AUTHOR]

Isocorynoxeine (ICOR) is one of the bioactive oxindole alkaloids inUncariaspecies. This study presents the pharmacokinetics and mass balance of ICOR in rats after oral dose of 40.0 mg/kg and intravenous dose of 4.0 mg/kg through detection of ICOR and its in vivo metabolites, using an optimized LC-MS with recoveries ranged from 94 to 104%, accuracy varied from 96 to 103%, and relative standard deviation for assay being less than 5%. ICOR reached its Cmaxof 336.7 ng/mL in plasma 3 hr after oral dose. The phase I metabolites of ICOR, 11-hydroxyisocorynoxeine (M1), and 10-hydroxyisocorynoxeine (M2) were detected in rat heart, kidneys, urine, and feces; whereas, in rat liver and bile, M1, M2, along with phase II metabolites, 11-hydroxyisocorynoxeine 11-O-β-D-glucuronide, and 10-hydroxyisocorynoxeine 10-O-β-D-glucuronide were identified. ICOR (77.0%) was excreted into feces and urine after oral administration within 72 hr, 0.93% drained into bile in 8 hr, 17.9% biotransformed into M1 and M2 at a ratio ofca. 1:1, and 0.028% detected in main organs attmax, in which brain uptake index is 3.2 ng/g. This work affords a developed and validated LC-MS for simultaneous determination of ICOR and itsin vivometabolites with improved precision and accuracy. [ABSTRACT FROM PUBLISHER]

Microbiota-host co-metabolites are well-known to play important physiological roles, and their dysregulation has been found to be closely related to various diseases, including but not limited to inflammatory disorders. We developed herein an original and feasible method using ultra performance liquid chromatography—tandem mass spectrometry (UPLC–MS/MS). The method developed enables rapid quantification of 11 key gut microbiota-host co-metabolites spanning the succinate, phenylacetylglutamine, hippurate and trimethylamine metabolic pathways within 10 min. With this method, we were able to simultaneously monitor inflammation-induced alterations of these metabolites in rat serum, urine and feces matrices. The measured levels for this panel of endogenous metabolites ranged from 0.001 to 172.8 μg mL −1 . The intra- and inter-day precision of three analytes was less than 13.1% and the accuracy was between −13.0 to 11.2% for all QC levels. The extraction recoveries in serum ranged from 85.4 to 103.2%, while the RSD was 9.0% or less for all recoveries. In addition, extraction recoveries of 11 analytes in urine and feces samples were between 85.7% and 102.0% and RSD was less than 9.5%. The method developed here has been successfully applied to the analysis of real samples from 2,4,6-trinitrobenzenesulfonic acid-induced Crohn’s disease in rats. All of these results suggest that the presently developed method is sufficiently sensitive and robust to simultaneously monitor co-metabolites with diverse properties and a range of different concentrations. Therefore, this method will be expected to be useful for comprehensive studies of the pathophysiological roles and mechanisms of these key microbiota-host co-metabolites, which reflect the function of the intestine, consequently offering novel opportunities for evaluating the occurrence, development and therapeutic effects of diseases related to microbiota disturbances. [ABSTRACT FROM AUTHOR]

To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the determination of concentration of nintedanib and its metabolite BIBF 1202 in rat plasma. The nintedanib and its metabolite and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.30 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m / z 540.3 → 113.1 for nintedanib, m / z 526.3 → 113.0 for BIBF 1202 and m / z 285.3 → 193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated for 1.0–200 ng/mL for nintedanib and 0.5–100 ng/mL for BIBF 1202 using 100 μL of plasma sample. Total time for each chromatograph was 3.0 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) <10.8% and the accuracy values ranged from −11.9% to 10.4%. The method was successfully applied to a pharmacokinetic study of nintedanib and BIBF 1202 in rats after oral administration of nintedanib. [ABSTRACT FROM AUTHOR]

Mounting evidence suggests that pulmonary exposure to nanoparticles (NPs) has a toxic effect on biological systems. A number of studies have shown that exposure to NPs result in systemic inflammatory response, oxidative stress, and leukocyte adhesion. However, significant knowledge gaps exist for understanding the key molecular mechanisms responsible for altered microvasculature function. Utilizing comprehensive LC-MS/MS and comparative proteomic analysis strategies, important proteins related to TiO 2 NP exposure in rat plasma have been identified. Molecular pathway analysis of these proteins revealed 13 canonical pathways as being significant (p ≤ 0.05), but none were found to be significantly up or down-regulated (z > |2|). This work lays the foundation for future research that will monitor relative changes in protein abundance in plasma and tissue as a function of post-exposure time and TiO 2 NP dosage to further elucidate mechanisms of pathway activation as well as to decipher other affected pathways. [ABSTRACT FROM AUTHOR]

3,4-Divanillyltetrahydrofuran is the main active ingredient of nettle root which can increase steroid hormones in the bloodstream for many of bodybuilders. To better understand its pharmacological activities, we need to determine its pharmacokinetic profiles. In this study, a rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–MS/MS) method has been developed for the determination of 3,4-divanillyltetrahydrofuran in the plasma of rats. Chromatographic separation was performed on a C 18 column at 40 °C, with a gradient elution consisting of methanol and water containing 0.3% (v/v) formic acid at a flow rate of 0.8 mL/min. The detection was performed using an electrospray triple-quadrupole MS/MS via positive ion multiple reaction monitoring mode. The lower limits-of-quantification determined were 0.5 ng/mL. The intra- and inter-day precision (RSD%) was found to be within 15% and the accuracy (RE%) ranged from −4.0% to 7.0%. This simple yet sensitive method was fully validated and could be successfully applied to the study on pharmacokinetics of 3, 4-divanillyltetrahydrofuran. [ABSTRACT FROM AUTHOR]

A sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of tulobuterol in rat plasma for the first time. Plasma samples were extracted by liquid–liquid extraction method with methyl tert -butyl ether and the analyte and clenbuterol (IS) were separated on a Venusil MP C 18 column (100 mm × 2.1 mm, 3 μm) using 0.1% formic acid–water–methanol as mobile phase, with a runtime of 5 min. The analyte was detected in multiple reaction monitoring (MRM) mode with positive electrospray ionization. Transitions of m / z 228.2 → 154.0 for tulobuterol and m / z 277.1 → 203.0 for the clenbuterol were monitored. The linear range was 0.5–100 ng/ml ( r = 0.9967) for tulobuterol with the lower limit of quantitation of 0.5 ng/ml. The intra-day and inter-day precisions were less than 10.3% for the analyte and the accuracy was less than −8.6%. The RSD of matrix effect and recovery yield were within ±15% of nominal concentrations and tulobuterol was stable during stability studies. The validated method has been successfully applied to a pharmacokinetic study of three doses of tulobuterol patch in rats for the first time. [ABSTRACT FROM AUTHOR]

We have developed a sensitive and specific LC–MS/MS method for the simultaneous determination of alisol A ( A ), alisol A 23-acetate ( A23 ) and alisol A 24-acetate ( A24 ), the major active components in Rhizoma Alismatis extract ( RAE ), in rat plasma. In brief, plasma samples were extracted by methyl tert -butyl ether and chromatographically separated by using a C 18 column. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated for specificity, linearity, accuracy (within ±15.4%), intra- and inter-day precision (CV < 11.4%) over the concentration range of 25–5000 ng/mL for A, and 5–1000 ng/mL for both A23 and A24 . The significantly lower detection limit was determined as 25 ng/mL for A , 5 ng/mL for A23 and A24 . This validated method of ours was then used to study the pharmacokinetics of RAE in rat. The elimination half-lives ( t 1/2 ) of A , A23 and A24 was determined as 0.75, 0.83 and 0.82 h respectively after intravenous injection, and the oral absolute bioavailability of A , A23 and A24 was 43.1 ± 18.1%, 6.3 ± 1.5% and 7.9 ± 1.2%. This new determination method of us for alisols is proven to very useful to study the pharmacological activities of RAE in future. [ABSTRACT FROM AUTHOR]