Effects of exogenous zinc on the physiological characteristics and enzyme activities of Passiflora edulis Sims f. edulis seedlings.

Passionflower (Passiflora edulis Sims) is widely distributed in tropical and subtropical areas for edible, medicinal and skin care product processing, and the market demand is large. Zinc (Zn) is a necessary trace element for plant growth and development. In many countries, the content of Zn in soil is low and/or bioavailability is low. The exogenous application of Zn has become a common agronomic measure in agriculture. However, the effect of Zn on the physiological characteristics and enzyme activity of passionflower seedlings is not clear. In this study, pot experiments were conducted to analyse the effects of different concentrations of Zn (0, 200, 400, 800 mg kg-1) on the plant growth, photosynthetic pigments, osmotic regulators, membrane system and antioxidant enzyme system of purple passionflower (Passiflora edulis Sims f. edulis) seedlings, and Pearson correlation and principal component analyses were performed. The results showed that (1) the 200 mg kg-1 Zn treatment increased the contents of chlorophyll a (37.65%), chlorophyll b (41.22%), chlorophyll a+b (38.59%) and carotenoids (29.74%). The value of chlorophyll a/b changed little and had no effect on leaf growth. (2) The contents of proline (Pro) and malondialdehyde (MDA) in P. edulis Sims f. edulis seedlings treated with 400 mg kg-1 Zn increased significantly by 116.84% and 42.69%, respectively. The activities of catalase (CAT) and peroxidase (POD) increased by 16.82% and 18.70%, respectively. Superoxide dismutase (SOD), leaf area (LA), leaf perimeter (LP) and leaf width (LW) decreased significantly by 47.20%, 19.75%, 8.32% and 11.97%, respectively. (3) 800 mg kg-1 Zn significantly increased the contents of Pro (202.56%) and MDA (26.7%) and the activities of CAT (16.00%) and POD (67.00%), while the soluble sugar (SS), SOD, LA, LP and LW decreased significantly by 36.67%, 32.86%, 23.36%, 8.32% and 11.18%, respectively. (4) There was a significant positive correlation between Pro and photosynthetic pigments and between SOD and leaf growth and a significant negative correlation between POD and SS and between SOD and MDA. (5) A low concentration (200 mg kg-1) of Zn promoted the growth of P. edulis Sims f. edulis seedlings and allowed stress caused by high Zn concentrations to be tolerated. The results of this study can provide a reference for the application of Zn fertilizer to P. edulis Sims f. edulis.

Multi-Response Optimization of High-Performance Low-pH Grouting Materials by Using Taguchi-Based Grey Relational Analysis.

The most accepted approach to sealing in high-level radioactive waste repositories (HLRWs) is to develop a low-pH grouting material with a pH of the pore solution of less than 11. Currently, the most widely used binary low-pH grouting material is MCSF64, which comprises 60% microfine cement (MC) and 40% silica fume (SF). In this study, a high-performance MCSF64-based grouting material was developed by incorporating naphthalene superplasticizer (NSP), aluminum sulfate (AS), and united expansion agent (UEA) to enhance the slurry's shear strength, compressive strength, and hydration process. Orthogonal experiments were conducted to measure the flow time, yield stress, plastic viscosity, initial setting time, shear strength, and compressive strength of the MCSF64-based slurry, and the optimal mix proportion was determined using the Taguchi-Grey relational analysis method. The pH variation of the pore solution, shrinkage/expansion, and hydration products of the optimal hardened slurry were evaluated using simplified ex-situ leaching (S-ESL), a length comparometer, and scanning electron microscopy (SEM), respectively. The results demonstrate that the Bingham model effectively predicted the rheological properties of the MCSF64-based slurry. The optimum ratio for the MCSF64-based slurry was water/binder (W/B) ratio of 1.4, and the contents of NSP, AS and UEA by mass of binder were 1.9%, 3.6% and 4.8%, respectively. The optimal mix exhibited a pH value below 11 after curing for 120 days. The addition of AS and UEA facilitated hydration, shortened the initial setting time, improved early shear strength, and enhanced the expansion ability of the optimal mix under water curing conditions.

Unravel the regulatory mechanism of Yrr1p phosphorylation in response to vanillin stress in Saccharomyces cerevisiae.

Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

Capture Hi-C reveals the influence on dynamic three-dimensional chromosome organization perturbed by genetic variation or vanillin stress in Saccharomyces cerevisiae.

Studying the mechanisms of resistance to vanillin in microorganisms, which is derived from lignin and blocks a major pathway of DNA double-strand break repair in yeast, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. A high vanillin-tolerant Saccharomyces cerevisiae strain EMV-8 carrying site mutations compared to its parent strain NAN-27 was selected for the analyses. The dynamics of the chromatin structure of eukaryotic cells play a critical role in transcription and the regulation of gene expression and thus the phenotype. Consequently, Hi-C and transcriptome analyses were conducted in EMV-8 and NAN-27 in the log phase with or without vanillin stress to determine the effects of mutations and vanillin disturbance on the dynamics of three-dimensional chromosome organization and the influence of the organization on the transcriptome. The outcomes indicated that the chromosome interaction pattern disturbed by vanillin stress or genetic mutations in the log phase was similar to that in mouse cells. The short chromosomes contact the short chromosomes, and the long chromosomes contact the long chromosomes. In response to vanillin stress, the boundaries of the topologically associating domain (TAD) in the vanillin-tolerant strain EMV-8 were more stable than those in its parent strain NAN-27. The motifs of SFL1, STB3, and NHP6A/B were enriched at TAD boundaries in both EMV-8 and NAN-27 with or without vanillin, indicating that these four genes were probably related to TAD formation. The Indel mutation of YRR1, whose absence was confirmed to benefit vanillin tolerance in EMV-8, caused two new interaction sites that contained three genes, WTM2, PUP1, and ALE1, whose overexpression did not affect vanillin resistance in yeast. Overall, our results revealed that in the log phase, genetic mutations and vanillin disturbance have a negligible effect on three-dimensional chromosome organization, and the reformation or disappearance of TAD boundaries did not show an association with gene expression, which provides an example for studying yeast chromatin structure during stress tolerance using Hi-C technology.

Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae.

BACKGROUND: Vanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin. RESULTS: In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae. CONCLUSIONS: Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat.

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.

Identification and expression analysis of some wheat F-box subfamilies during plant development and infection by Puccinia triticina.

As one of the largest protein families in plants, F-box proteins are involved in many important cellular processes. Until now, a limited number of investigations have been conducted on wheat F-box genes due to its variable structure and large and polyploid genome. Classification, identification, structural analysis, evolutionary relationship, and chromosomal distribution of some wheat F-box genes are described in the present study. A total number of 1013 potential F-box proteins which are encoded by 409 genes was identified in wheat, and classified into 12 subfamilies based on their C-terminal domain structures. Furthermore, proteins with identical or similar C-terminal domain were clustered together. Location of 409 F-box genes was identified on all 21 wheat chromosomes but showed an uneven distribution. Segmental duplication was the main reason for the increase in the number of wheat F-box genes. Gene expression analysis based on digital PCR showed that most of the F-box genes were highly expressed in the later development stages of wheat, including the formation of spike, grain, flag leaf, and participated in drought stress (DS), heat stress (HS), and their combination (HD). Of the nine F-box genes we investigated using quantitative PCR (qPCR) following fungal pathogen infection, five were involved in wheat resistance to the infection by leaf rust pathogen and one in the susceptible response. These results provide important information on wheat F-box proteins for further functional studies, especially the proteins that played roles in response to heat and drought stresses and leaf rust pathogen infection.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

Simultaneous chemical fingerprint and quantitative analysis of Rhizoma Smilacis Glabrae by accelerated solvent extraction and high-performance liquid chromatography with tandem mass spectrometry.

Rhizoma Smilacis Glabrae (RSG) is a well-known herbal medicine with the homology of medicine and food. In this study, simultaneous chemical fingerprint and quantitative analysis of the bioactive flavonoid components of RSG were developed using accelerated solvent extraction and high-performance liquid chromatography coupled with ion trap tandem mass spectrometry. The operational parameters of accelerated solvent extraction including extraction solvent, extraction temperature, static extraction time, solid-to-liquid ratio, and extraction cycles were optimized. Hierarchical cluster analysis, similarity analysis, and principal component analysis were performed to evaluate the similarity and variation of the samples collected from several provinces in China. Subsequently, high-performance liquid chromatography fingerprints were established for the discrimination of 16 batches of RSG samples, and the major six flavonoids, namely, toxifolin, neoastilbin, astilbin, neoisoastilbin, isoastilbin, and engeletin were then quantitatively determined. The calibration curves for all the six analytes showed good linearity (r(2) > 0.999), and the limits of detection and quantification were less than 0.10 and 0.27 µg·mL(-1) , respectively. Therefore, the proposed extraction and determination methods were proved to be robust and reliable for the quality control of RSG.

Isolation and purification of diastereoisomeric flavonolignans from silymarin by binary-column recycling preparative high-performance liquid chromatography.

Silymarin extracted from Silybum marianum (L.) Gaertn consists of a large number of flavonolignans, of which diastereoisomeric flavonolignans including silybin A and silybin B, and isosilybin A and isosilybin B are the main bioactive components, whose preparation from the crude extracts is still a difficult task. In this work, binary-column recycling preparative high-performance liquid chromatography systems without sample loop trapping, where two columns were switched alternately via one or two six-port switching valves, were established and successfully applied to the isolation and purification of the four diastereoisomeric flavonolignans from silymarin. The proposed system showed significant advantages over conventional preparative high-performance liquid chromatography with a single column in increasing efficiency and reducing the cost. To obtain the same amounts of products, the proposed system spends only one tenth of the time that the conventional system spends, and needs only one eleventh of the solvent that the conventional system consumes. Using the proposed system, the four diastereoisomers were successfully isolated from silymarin with purities over 98%.

An efficient strategy based on MAE, HPLC-DAD-ESI-MS/MS and 2D-prep-HPLC-DAD for the rapid extraction, separation, identification and purification of five active coumarin components from Radix Angelicae Dahuricae.

INTRODUCTION: Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low-level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods. OBJECTIVE: To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE. METHODOLOGY: First, active coumarins in AE were extracted with microwave-assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high-performance liquid chromatography-diode array detection-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) method was applied for the preliminary on-line identification and screening of the main coumarins in AE extract. Finally, a two-dimensional preparative high-performance liquid chromatography-diode array detection (2D-prep-HPLC-DAD) system was developed for further preparative separation of those target components. RESULTS: Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods. CONCLUSION: The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.

Isolation and purification of isoflavonoids from Rhizoma Belamcandae by two-dimensional preparative high-performance liquid chromatography with column switch technology.

A two-dimensional column-switching system without sample loop trapping, where two columns were operated via a six-port switching valve, was employed in the isolation and purification of five isoflavonoids from Rhizoma Belamcandae: tectoridin, iridin, tectorigenin, irigenin and irisflorentin. The introduction of the six-port switching value, instead of a sample loop, assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Two configurations were tested in this study. In the first mode, only one column was used in the second dimension and two 'heart-cutting' fractions were transported to the same second-dimensional column. In the second mode, two parallel columns were used in the second dimension and two fractions were transported to the two columns. Between the two configurations, the one with two second dimensional columns had double sample size, better resolution and one more purified compound. Both two-dimensional configurations consumed less solvent with even greater efficiency and shorter cycle time compared with conventional gradient methods. All of the isoflavonoids were isolated at high purities of greater than 95% with yields of above 82%.

Isolation and purification of iridoid glycosides from Gardenia jasminoides Ellis by isocratic reversed-phase two-dimensional preparative high-performance liquid chromatography with column switch technology.

A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-beta-D-gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.

Isolation and purification of lignans from Magnolia biondii Pamp by isocratic reversed-phase two-dimensional liquid chromatography following microwave-assisted extraction.

The dried flower buds of Magnolia biondii Pamp are one of the most widely used medicinal plants officially listed in the Chinese Pharmacopoeia. A 2-D column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of five lignans including pinoresinol dimethyl ether, magnolin, epi-magnolin A, fargesin, and demethoxyaschantin from M. biondii Pamp after microwave-assisted extraction. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 12 mL. In this mode of operation, the solvent consumption of the 2-D approach was less than 30% that of conventional gradient methods with even larger sample size. The simultaneous operations of the two dimensions allowed the cycle time to be less than 16 min, compared to 90 min in the gradient elution single-dimension mode of operation. All of the five lignans were isolated at high purities of over 99% with approximately 95% recoveries.

Development of a quality evaluation method for Fructus schisandrae by pressurized capillary electrochromatography.

A pressurized CEC (pCEC) method with postcolumn detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effects of different experimental conditions, such as the ACN content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (schisandrin, gomisin A, schisantherin C, deoxyschizandrin, schisandrin B) were baseline separated using a mobile phase of ACN-phosphate buffer (pH 5.4; 5 mM) (40:60 v/v) under -4 kV applied voltage. The method showed the satisfactory retention time and peak area repeatability. The calibration curves were linear in the range 50.0-1000.0 microg/mL for schisandrin, 20.0-500.0 microg/mL for gomisin A, 10.0-200.0 microg/mL for schisantherin C, 20.0-500.0 microg/mL for deoxyschizandrin, and 20.0-500.0 microg/mL for schisandrin B. The correlation coefficients were between 0.9978 and 0.9989. With this pCEC system, fingerprints of F. schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of F. schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study.

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.

Development of a liquid chromatography/tandem mass spectrometry assay for the determination of bestatin in rat plasma and its application to a pharmacokinetic study.

Bestatin is a low molecular weight aminopeptidase inhibitor originally isolated from culture filtrates of Streptomyces olivoreticuli. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of bestatin in rat plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 50 microL plasma samples by solid phase extraction (SPE). Reverse-phase HPLC separation was accomplished on a Lichrospher C18 column (4.6 mm x 50 mm, 5 microm) with a mobile phase composed of methanol-water-formic acid (70:30:0.5, v/v/v) at a flow rate of 0.8 mL/min. The method had a chromatographic total run time of 3 min. A Varian 1200L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 309.2-->120.0 (bestatin) and 313.4-->138.0 (granisetron) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 5 ng/mL, with good linearity (r2 >or= 0.999) over the linear range of 5-2000 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of bestatin in rats.

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study.

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.