Rapid High-performance Liquid Chromatography-tandem Research Articles

Comparative Study on the Pharmacokinetics of Paeoniflorin, White Peony Root Water Extract, and Taohong Siwu Decoction After Oral Administration in Rats

Taohong Siwu Decoction (TSD) is a classic traditional Chinese medicine (TCM) compound with pharmacological effects such as vasodilation and hypolipidemia. Paeoniflorin (PF) is one of the active ingredients of TSD. The aim of this study was to evaluate the pharmacokinetics of PF in herbal extracts and their purified formsin rats. A sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method for the determination of PF in rat plasma was developed. Rats were divided into three groups, and given PF solution, water extract of white peony root (WPR), or TSD by gavage. At different predetermined timepoints after gavage, blood was collected from the orbital vein. The pharmacokinetic parameters of PF in the plasma of rats in the three groups was determined. The pharmacokinetic studies showed that the time to reach maximum concentration (Tmax) of PF in the purified forms group was relatively high, while the half-lives (T½) of PF in the TSD and WPR groups were longer. Among the three groups, PF in the purified forms group had the maximum area under the concentration-time curve (AUC0-t=732.997µg/L·h) and the largest maximum concentration (Cmax=313.460µg/L), which showed a significant difference compared with the TSD group (P<0.05). Compared with the purified group, the clearance (CLz/F=86.004L/h/kg) and the apparent volume of distribution (Vz/F=254.787L/kg) of PF in the TSD group increased significantly (P<0.05). A highly specific, sensitive, and rapid HPLC-MS-MS method was developed and applied for the determination of PF in rat plasma. It was found that TSD and WPR can prolong the action time of paeoniflorin in the body.

Liquid chromatography-tandem mass spectrometry methods for quantification of roxadustat (FG-4592) in human plasma and urine and the applications in two clinical pharmacokinetic studies

Roxadustat (FG-4592) can inhibit the hypoxia-inducible factor prolyl hydroxylase (HIF-PH) enzymes that are responsible for targeting and regulating HIF for ubiquitination and proteasomal degradation. It has been approved in China as an anti-anemia drug for treating the anemia in dialysis-dependent CKD (chronic kidney disease) patients, and is also under regulatory review in Japan. Some studies are also investigating the clinical pharmacokinetics and pharmacodynamics of roxadustat in CKD patients. To support clinical investigations, a rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, with a concentration range from 1 to 5000 ng/mL, was designed for the quantification of roxadustat in human plasma and urine. Liquid-liquid extraction (LLE) was applied to sample clean-up followed by a chromatographic separation conducted on a Waters XTerra Phenyl column with isocratic elution. The mixture consisting of acetonitrile/water/formic acid (60:40:0.1[%], v/v/v) was employed as the mobile phase (flow rate: 1.0 mL/min) with 60% post-column split. The quantitation was employed in multiple reactions monitoring (MRM) mode based on positive electrospray ionization (ESI). This proposed method was fully validated and applied to the pharmacokinetic (PK) and pharmacodynamic (PD) study of roxadustat among healthy subjects in China.

Pharmacokinetic and brain distribution study of an anti-glioblastoma agent in mice by HPLC-MS/MS.

Previously compound I showed great anti-glioblastoma activity without toxicity in a mouse xenograft study. In this study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to investigate the pharmacokinetics and brain distribution of compound I in mice. The protein precipitation method was applied to extract the compound from mouse plasma and brain homogenates, and it was then separated using a Kinetex C18 column with a mobile phase consisting of acetonitrile-0.1% formic acid water (50:50, v/v). The analytes were detected with multiple reaction monitoring for the quantitative response of the compounds. The inter- and intra-day precisions were <8.29 and 3.85%, respectively, and the accuracy range was within ±7.33%. The method was successfully applied to evaluate the pharmacokinetics of compound I in mouse plasma and brain tissue. The peak concentration in plasma was achieved within 1h. The apparent elimination half-life was 4.06 h. The peak concentration of compound I in brain tissue was 0.88 μg/g. The results indicated that compound I was rapidly distributed and could cross the blood-brain barrier. The pharmacokinetic profile summarized provides valuable information for the further investigation of compound I as a potential anti-glioblastoma agent.

Predator-prey interactions between the ciliate Blepharisma americanum and toxic (Microcystis spp.) and non-toxic (Chlorella vulgaris, Microcystis sp.) photosynthetic microbes

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 83:211-224 (2019) - DOI: https://doi.org/10.3354/ame01913 Predator-prey interactions between the ciliate Blepharisma americanum and toxic (Microcystis spp.) and non-toxic (Chlorella vulgaris, Microcystis sp.) photosynthetic microbes Ian J. Chapman1,2, Daniel J. Franklin1, Andrew D. Turner3, Eddie J. A. McCarthy1, Genoveva F. Esteban1,* 1Bournemouth University, Department of Life and Environmental Sciences, Faculty of Science and Technology, Dorset, BH12 5BB, UK 2NSW Shellfish Program, NSW Food Authority, Taree, NSW 2430, Australia 3Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth, Dorset, DT4 8UB, UK *Corresponding author: gesteban@bournemouth.ac.uk ABSTRACT: Despite free-living protozoa being a major factor in modifying aquatic autotrophic biomass, ciliate-cyanobacteria interactions and their functional ecological roles have been poorly described, especially with toxic cyanobacteria. Trophic relationships have been neglected and grazing experiments give contradictory evidence when toxic taxa such as Microcystis are involved. Here, 2 toxic Microcystis strains (containing microcystins), 1 non-toxic Microcystis strain and a non-toxic green alga, Chlorella vulgaris, were used to investigate predator-prey interactions with a phagotrophic ciliate, Blepharisma americanum. Flow cytometric analysis for microalgal measurements and a rapid ultra high performance liquid chromatography-tandem mass spectrometry protocol to quantify microcystins showed that non-toxic photosynthetic microbes were significantly grazed by B. americanum, which sustained ciliate populations. In contrast, despite constant ingestion of toxic Microcystis, rapid egestion of cells occurred. The lack of digestion resulted in no significant control of toxic cyanobacteria densities, a complete reduction in ciliate numbers, and no observable encystment or cannibalistic behaviour (gigantism). Individual B. americanum morphological responses (biovolume and cell width) showed a significant decrease over time when sustained on non-toxic Microcystis compared to grazed C. vulgaris populations, supporting previous studies that cyanobacteria may be a relatively poor source of nutrition. Results here provide insight into the ecological interactions of ciliates and cyanobacteria, and for the first time B. americanum is shown to have the capacity to suppress potentially bloom-forming cyanobacteria. However, grazing can be significantly altered by the presence of microcystins, which could have an impact on bloom dynamics and overall community structure. KEY WORDS: Ciliate-cyanobacteria interactions · Toxic cyanobacteria · Ciliate · Grazing · Microcystis · Microcystins Full text in pdf format PreviousNextCite this article as: Chapman IJ, Franklin DJ, Turner AD, McCarthy EJA, Esteban GF (2019) Predator-prey interactions between the ciliate Blepharisma americanum and toxic (Microcystis spp.) and non-toxic (Chlorella vulgaris, Microcystis sp.) photosynthetic microbes. Aquat Microb Ecol 83:211-224. https://doi.org/10.3354/ame01913 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 83, No. 3. Online publication date: September 19, 2019 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2019 Inter-Research.

Simultaneous determination of six bioactive saponins from Rhizoma Panacis Japonici in rat plasma by UHPLC-MS/MS: Application to a pharmacokinetic study

A specific, sensitive and rapid ultra high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed and validated for simultaneous determination of six major bioactive constituents in Rhizoma Panacis Japonici (RPJ), including oleanolic acid-type chikusetsusaponin V, IV, hemsgiganoside B, damarane-type ginsenoside Rb1, Rg1 and Re in rat plasma, using estazolam as the internal standard (IS). Plasma samples were pretreated with methanol/acetonitrile (1:1, V/V) for protein precipitation. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column, using a gradient mobile phase consisting of methanol and 0.1% formic acid aqueous solution. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. For all the six analytes of interest, the calibration curves were linear in the concentration range of 2.00–500 ng/mL with r ≥ 0.9956. The intra- and inter-day precisions (in terms of relative standard deviation, RSD) were all below 10.2% and the accuracies (in terms of relative error, RE) were within −5.0% to 6.3% for all six analytes. Extraction recovery, matrix effect and stability data all met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method was applied to the pharmacokinetic study in rat. After oral administration of the total saponins from RPJ, six analytes were quickly absorbed into the blood and presented the phenomenon of double peaks. Among the six analytes, ginsenoside Rb1 showed slowest elimination from plasma with a t1/2z of 16.00 h, while that of the others were between 1.72 and 5.62 h. In conclusion, the developed method was successfully used to simultaneously analyze major oleanolic acid-type and damarane-type saponins of RPJ in rat plasma after oral administration.

Rapid and sensitive determination of selective progesterone modulator ulipristal acetate in human plasma

Ulipristal acetate is a new synthetic selective progesterone receptor modulator developed mainly as emergency contraceptive (EC) and also used for the treatment of uterine fibroids. A cost effective, sensitive, simple and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of ulipristal acetate in human plasma. Following liquid-liquid extraction, the analyte (Ulipristal acetate) and internal standard (Levonorgestrel) were chromatographed using mobile phase in an isocratic elution mode on a reverse phase C18 column. The LC-MS/MS operated in multiple reaction monitoring mode for respective [M+H] + ions, m/z 476.2/134.1 for analyte and 313.3/245.1 for internal standard. The assay exhibited linear dynamic range of 1-300 ng/mL. The lower limit of quantification was 1 ng/mL with relative standard deviation of 7.0%. The intra-batch and inter-batch results were precise with coefficient variation of 2.7 to 7.0 (%) and accuracy of 94.2-99.8 (%). The validated method was simple, fast and repeatable for bioequivalence, pharmacokinetic and therapeutic monitoring studies.

Simultaneous determination of three di-caffeoylquinic acids by UHPLC-MS/MS in rat plasma and its application to a comparative pharmacokinetic study in normal and acute lung injury rat.

Acute lung injury (ALI) is a severe inflammatory disease with high mortality rates. Di-caffeoylquinic acids (DCQAs), the bioactive components of reduning injection (RDN), may play important roles in the protective effect on acute lung injury (ALI). A selective and rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for the simultaneous determination of 3,4-, 3,5- and 4,5-DCQA in rat plasma. The DCQAs were extracted by liquid-liquid extraction with ethyl acetate-isopropyl alcohol (7:3, v/v). Chromatographic separation was accomplished on a C18 column using gradient elution. Detection was performed in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were all 2.0ng/mL for the three analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -13.8% to 10.0%, and the mean extraction recoveries of analytes from rat plasma were all more than 72.9%. Meanwhile, this method had been successfully applied to compare the pharmacokinetics of the three DCQAs in normal and ALI model rat after RDN was given intravenously administration. The pharmacokinetic parameters of the 3,4-, 3,5- and 4,5- DCQA were remarkably different from those in normal rats. It might result from the effects of the pathological status of ALI. This study presented a meaningful basis for the clinical applications of RDN when used in the treatment of ALI.

Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry.

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150mm×4.6mm, 5μm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3-0.6ng/mL), the limits of quantitation (LOQs, 1.0-2.0ng/mL) and recoveries (89.1%-108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.

Mouldy feed: A possible explanation for the excretion of anabolic‐androgenic steroids in horses

To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horse's hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of prasugrel active metabolite R-138727 in human plasma: a sensitive, highly selective and fast LC-MS/MS method.

A cost effective, sensitive, simple, and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the prasugrel metabolite in human plasma. Following solid phase extraction, the analyte (prasugrel active metabolite; R-138727) and internal standard (emtricitabine) were separated using a mobile phase in an isocratic elution mode on a reverse phase C18 column and were analyzed by an LC-MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 498.3-206.0 for R-138727 and m/z 248.2-130.1 for the internal standard. The assay exhibited a linear dynamic range of 0.2-120 ng/mL. The lower limit of quantification was 0.2 ng/mL with a relative standard deviation of 5.0%. This LC-MS/MS method was validated with intra-batch and inter-batch precision and accuracy. Results for precision and accuracy are in range of 3.9-9.6% and 95.2-102.2% respectively. This validated method is simple and repeatable to use in bioequivalence/pharmacokinetic studies.

Determination of Tetrodotoxin in Carcinoscorpius rotundicauda (Horseshoe Crab) by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

ABSTRACTIn recent years, food poisoning due to the consumption of horseshoe crabs has occurred sporadically in China, and Carcinoscorpius rotundicauda was responsible for most of these events. A poisoning incident due to the ingestion of C. rotundicauda occurred in Fuzhou, China, in August 2014. The remains of the C. rotundicauda were collected from the home of the victims, and six specimens of C. rotundicauda were obtained from the same market from where the victims’ samples were sold. In this study, a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry method was developed to determine tetrodotoxin in C. rotundicauda. The calibration curve of tetrodotoxin was linear from 5.0 to 1000 ng/mL. The limits of detection and quantification were 2.1 and 5.0 ng/mL, respectively. The average recoveries, measured at three concentrations, were from 74.8 to 83.6%, with the corresponding relative standard deviation below 3.8%. The C. rotundicauda samples responsible for poisoning and those purchased from the same market were shown to contain tetrodotoxin.

Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC-MS/MS and its application to a pharmacokinetics study.

Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5-100ng/mL for M608236 and 1-200ng/mL for other analytes with the correlation coefficients (r(2))≥0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86-105% and no significant matrix effect was observed. This simple and reproducible high-throughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse.

A rapid high-performance liquid chromatography-tandem mass spectrometry assay for unambiguous detection of different milk species employed in cheese manufacturing

The aim of the study was to investigate the possibility to differentiate the 4 most important species in Italian dairy industry (cow, buffalo, sheep, and goat), applying a bottom-up proteomic approach to assess the milk species involved in cheese production. Selective peptides were detected in milk to use as markers in cheese products. Trypsin-digested milk samples of cow, sheep, goat, and buffalo, analyzed by HPLC-tandem mass spectrometry provided species-specific peptides, some of them recognized by Mascot software (Matrix Science Ltd., Boston, MA) as derived from well-known species specific proteins. A multianalyte multiple reaction monitoring method, built with these specific peptides, was successfully applied to cheeses with different composition, showing high specificity in detection of species involved. Neither aging nor production method seemed to affect the response, demonstrating that chosen peptides well act as species markers for dairy products.

Validation of an HPLC-MS-MS assay for determination of morellic acid in rat plasma: application to pharmacokinetic studies.

A selective and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) was developed for the quantification of morellic acid in rat plasma. HPLC was performed using a Capcell MG C18 (50 × 4.6 mm, i.d., 5 µm) column, and isocratic elution with water-acetonitrile (20:80, v/v) at a flow rate of 0.5 mL/min. Sample preparation of analyte and internal standard (gambogic acid) involved liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) from 50 µL plasma. The precursor → production transitions for analyte and IS were m/z 559.4 → 471.3, and m/z 627.3 → 583.3, respectively, and were monitored on a triple-quadrupole mass spectrometer, operating in negative ion scan mode. The method was validated across the dynamic concentration range of 20-7,500 ng/mL for morellic acid, with a fast run time of 6.0 min. The analytical method measured concentrations of morellic acid with accuracy (% bias) of ≤6.4% and precision (% RSD) of ≤14.0%. Morellic acid was stable during the battery of stability studies. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic studies in Sprague-Dawley rats. This method will therefore be useful for further preclinical and clinical pharmacokinetic studies of morellic acid.

Determination and pharmacokinetic studies of artesunate and its metabolite in sheep plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneous quantify artesunate and its metabolite in sheep plasma. The plasma samples were prepared by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column (250×4.6mm, 5μm) using methanol: water (60:40, v/v) (the water included 1mM ammonium acetate, 0.1% formic acid, and 0.02% acetic acid) as the mobile phase. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear from 1ng/mL to 400ng/mL (r(2)=0.9992 for artesunate, r(2)=0.9993 for its metabolite). The intra- and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations for both artesunate and its metabolite. The recoveries ranged from 92% to 98% at the three concentrations for both. In summary, the LC-MS/MS metho described herein was fully successfully applied to pharmacokinetic studies of artesunate nanoemulsion after intramuscular delivery to sheep.

Determination of ambroxol hydrochloride in human plasma by ultra high performance liquid chromatography-tandem mass spectrometry and bioequivalence evaluation of its preparation

A rapid, simple and sensitive ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the determination of ambroxol hydrochloride in human plasma, and bioequivalence of its preparation was evaluated. The 50 μL-plasma sample was treated with methanol for protein precipitation, while ambroxol-d5 was used as an internal standard (IS). The separation was carried out on a Waters XBridge BEH C18 column (50 mm×2.1 mm, 2.5 μm) by gradient elution at a flow rate of 0.4 mL/min, with 0.1% (v/v) formic acid aqueous solution and methanol containing 0.1% (v/v) formic acid as the mobile phases. The analyte was detected using an electrospray ionization source in positive ion multiple reaction monitoring (MRM) mode. The calibration curves were linear in the range of 2-400 ng/mL (r=0.998). The intra- and inter-run accuracies were 97.1%-108.7%, the intra- and inter-run precisions were 1.0%-5.6%. The method was applied to the determination of the plasma concentration of the six healthy subjects after the oral administration of 30 mg of test and reference preparations. The bioavailability was (102.3±14.8)%. The 90% confidence intervals of the test preparation's pharmacokinetic parameters were 80.0%-125.0% of the reference preparation's corresponding parameters. Thus, it is proved that the test preparation and reference preparation are bioequivalent.

Determination of chrysotoxine in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study

Chrysotoxine (CTX), a naturally occurring bibenzyl compound isolated from Dendrobium species, has been reported to have neuroprotective effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method has been developed and validated for the quantification of CTX in rat plasma. Samples were pretreated using a simple liquid-liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column with acetonitrile-water (90:10, v/v) as the mobile phase. CTX and the internal standard (wogonin) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 0.5-1000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that CTX spiked into rat plasma was stable for 8 h at room temperature, for up to two weeks at -20 °C, and during three freeze-thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler. The method was successfully used to investigate the pharmacokinetic profile of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX showed rapid excretion and low bioavailability in rats.

Determination of aflatoxin B1, B2, G1, G2 in Armeniacae Semen Amarum by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in Armeniacae Semen Amarum and the application was performance in 11 samples collected from different markets, medical stores and hospitals. The sample was extracted with 84% acetonitrile/water and 250 microL extraction was directly injected into a LC-MS/MS system without further purification procedure after being redissolved with methanol. The LC separation was performed on a C18 column with a linear gradient elution program of 4 mmol x L(-1) NH4 Ac-0.1% formic acid solution and menthol as the mobile phase. Selected reaction monitoring (SRM) was used for selective determination of the four aflatoxins on a triple quadruple mass spectrometer, which was operated in positive ionization modes. All the four aflatoxins showed a good linear relationship with r > 0.999 0, the average recoveries were between 87.88% and 102.9% and the matrix effect was ranged from 90.71% to 99.30% in low, intermediate and high levels. Furthermore, the higher recovery was obtained by the method reported in this study, comparing to the cleanup procedure with the Mycosep 226 purification column. Eleven samples collected were detected and the contamination levels of the AFB1 were between 1.590-2.340 microg x kg(-1) and the AF (B1 + B2 + G1 + G2) was ranged from 2.340 to 3.340 microg x kg(-1). In summary, the developed method was suitable to detect and screen AFB1, AFB2, AFG1, AFG2 in Armeniacae Semen Amarum.

Determination of withaferin A and withanolide A in mice plasma using high-performance liquid chromatography-tandem mass spectrometry: Application to pharmacokinetics after oral administration of Withania somnifera aqueous extract

Withania somnifera (WS) is one of the popular botanical medicines widely used in Ayurveda. Withanolides such as withaferin A (WA) and withanolide A (WLD) are its bioactive constituents reported as promising drug candidates in cancer and neurological disorders respectively. A new, selective and rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for simultaneous determination of WA and WLD in mice plasma. Simple liquid–liquid extraction procedure was followed using ter-butyl methyl ether (TBME) for plasma sample pretreatment. Analytes were separated on Hypurity C18 column using methanol and ammonium acetate (95:5, v/v) as a mobile phase and detected by electrospray ionization in the multiple reaction monitoring (MRM) mode. The mass transition ion-pair was followed as m/z 471.3→281.2 for WA; m/z 437.2→292.2 for tianeptine (IS) and m/z 488.3→263.1 for WLD; m/z 315.9→270 for clonazepam (IS). The method showed excellent linearity (r2>0.997) over the concentration range of 0.484–117.880ng/ml for WA and from 0.476–116.050ng/ml for WLD. The lower limits of quantification (LLOQs) were found to be 0.484ng/ml and 0.476ng/ml for WA and WLD respectively. Precision (% CV) and accuracy (% bias) were found in the range of 3.7–14.3% and −14.4–4.0%, respectively. The validated method was successfully applied to a pharmacokinetic (PK) study for estimation of WA and WLD in mice plasma following oral administration of W. somnifera root aqueous extract (WSE). The PK study suggested rapid oral absorption of these withanolides. The PK study revealed that withaferin A has one and half times more relative bioavailability as compared to withanolide A.

Simultaneous determination of limonin, dictamnine, obacunone and fraxinellone in rat plasma by a validated UHPLC–MS/MS and its application to a pharmacokinetic study after oral administration of Cortex Dictamni extract

A rapid and selective ultra high performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of four major ingredients in Cortex Dictamni extract, including limonin, dictamnine, obacunone and fraxinellone in rats plasma. Nimodipine was used as the internal standard. Following extraction by methyl tert-butyl ether, the analytes were separated on a Thermo Syncronis C18 column by a gradient elution within a runtime of 9min. The mobile phase consisted of A (methanol) and B (2mmol/L ammonium acetate in water). The detection was accomplished by using positive ion electrospray ionization in multiple reaction monitoring mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.998. The lower limits of quantification were 9.18ng/mL for limonin, 12.0ng/mL for dictamnine, 16.05ng/mL for obacunone and 4.59ng/mL for fraxinellone. The intra- and inter-day precision (RSD%) was within 10% and the accuracy (RE%) ranged from -12.9% to 9.7%. This rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of limonin, dictamnine, obacunone and fraxinellone in the rat plasma after oral administration of Cortex Dictamni extract.