Simultaneous Determination of Isopyrazam and Azoxystrobin in Cucumbers by Liquid Chromatography-Tandem Mass Spectrometry.

A rapid and sensitive analytical method based on high-performance liquid chromatography-tandem mass spectrometry was developed and validated for the determination of isopyrazam (IZM) and azoxystrobin (AZT) in cucumbers. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used as the pretreatment procedure. The samples were extracted with acetonitrile and cleaned up with octadecylsilyl silica (C18) and graphite carbon black. The proposed method resulted in satisfactory recovery of IZM and AZT (91.48 to 114.62%), and relative standard deviations were less than 13.1% at fortification concentrations of 1, 20, and 500 µg kg-1 (n = 3). The limits of quantification for IZM and AZT were 0.498 and 0.499 µg kg-1, respectively, which are far below the maximum residue level (0.5 mg kg-1) established for this type of sample. Matrix effects were also evaluated. This study established a sensitive and fast method for the detection of IZM and AZT in cucumber samples.

Unexpected hydrated ions in the detection of sodium adduct coumarins using electrospray tandem mass spectrometry.

A series of coumarins were analyzed using electrospray ionization quadrupole time-of-flight tandem mass spectrometry in the positive-ion mode. Unexpected hydrated ions ([M + H2O + Na]+) was observed upon collision-induced dissociation of the sodiated ions ([M + Na]+) of eight coumarins. Several factors which affected relative abundance of [M + H2O + Na]+ ions such as collision energy, concentration and solvent were investigated. None of them have effect on the relative abundance of [M + H2O + Na]+. However, the peak of hydrated ions was not detected in the further collision-induced dissociation of protonated ions of coumarins. Apigenin and Quercetin sharing similar benzopyrone structural unit with coumarins are selected for tandem mass spectrometry analysis. There were no hydrated ions in their tandem mass spectrometry spectra of the precursor [M + Na]+ ions. Thus, both coumarins and sodium were necessary for the formation of [M + H2O + Na]+. Together with the result that hydrated ions are not formed by hydrolysis reactions, a six-membered ring structure which involves with the formation of [M + H2O + Na]+ was presented. And D-labeling experiment indicates that the H2O molecule did not come from solvent.

[The Study of Methodology Validation in Multicomponent Quantitative Analysis of Raman Spectroscopy].

Raman spectroscopy with the help of sophisticated data analysis techniques based on multivariate analysis have made it possible to study the full information of Raman spectra and to draw conclusions about the chemical structure and composition of very complex systems. As Raman spectroscopy has the advantage to provide rich information of the sample, so it was first included in Chinese Pharmacopoeia (2010) as "Annex XIX L Guidelines of Raman spectroscopy". However the validation method for multicomponent quantitative analysis of Raman spectroscopy was not discussed in this pharmacopoeia. The purpose of this study was to develop innovatively the validation method for the Raman model in multicomponent quantitative analysis. Two kinds of samples with different matrix background were used to study the self-performance test of the instrument, linearity, limit of quantitation, accuracy and precision, respectively. As the sample was complex composition in our study, so there is no need to provide sample blank and rupture test for specificity study which was carried out in the traditional method of experiments. As the result showed, we propose a validation method for Raman spectroscopy in multicomponent analysis for the further study and improvement of standards of the method.

UDP-glucosyltransferase71c5, a major glucosyltransferase, mediates abscisic acid homeostasis in Arabidopsis.

Abscisic acid (ABA) plays a key role in plant growth and development. The effect of ABA in plants mainly depends on its concentration, which is determined by a balance between biosynthesis and catabolism of ABA. In this study, we characterize a unique UDP-glucosyltransferase (UGT), UGT71C5, which plays an important role in ABA homeostasis by glucosylating ABA to abscisic acid -: glucose ester (GE) in Arabidopsis (Arabidopsis thaliana). Biochemical analyses show that UGT71C5 glucosylates ABA in vitro and in vivo. Mutation of UGT71C5 and down-expression of UGT71C5 in Arabidopsis cause delay in seed germination and enhanced drought tolerance. In contrast, overexpression of UGT71C5 accelerates seed germination and reduces drought tolerance. Determination of the content of ABA and ABA-GE in Arabidopsis revealed that mutation in UGT71C5 and down-expression of UGT71C5 resulted in increased level of ABA and reduced level of ABA-GE, whereas overexpression of UGT71C5 resulted in reduced level of ABA and increased level of ABA-GE. Furthermore, altered levels of ABA in plants lead to changes in transcript abundance of ABA-responsive genes, correlating with the concentration of ABA regulated by UGT71C5 in Arabidopsis. Our work shows that UGT71C5 plays a major role in ABA glucosylation for ABA homeostasis.

Simultaneous determination of thirteen aminoalcohol-diterpenoid alkaloids in the lateral roots of Aconitum carmichaeli by solid-phase extraction-liquid chromatography-tandem mass spectrometry.

Aminoalcohol-diterpenoid alkaloids have been reported as the cardioactive components in the lateral roots of Aconitum carmichaeli (Fuzi) according to recent studies. Determination of these effective components is of great significance for quality control purposes for Fuzi. Here we report, for the first, the development and validation of a new method to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi by using a simple and accurate solid-phase extraction-liquid chromatography-tandem mass spectrometry. The chromatographic analysis was performed on an ODS column with methanol-0.1 % formic acid (80 : 20, v/v) as the mobile phase. The quantification was performed using MS/MS detection in the positive ion mode with multiple reaction monitoring. Linearity was observed within a range of concentrations of 20-2,000 ng/mL. For all the analytes, the r value was greater than 0.9990. The limit of detection and the limit of quantitation were less than 0.5 ng/mL and 2.0 ng/mL, respectively. The intraday and interday precisions were less than 5% and 10%, respectively. The accuracy was within the range of 90 to 105%. This method was successfully applied to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi from different origins and with different processing methods.

Profiling the dynamics of abscisic acid and ABA-glucose ester after using the glucosyltransferase UGT71C5 to mediate abscisic acid homeostasis in Arabidopsis thaliana by HPLC-ESI-MS/MS.

The HPLC-MS/MS method was developed to profile the dynamics of abscisic acid (ABA) and ABA-glucose ester (ABA-GE) after cloning glycosyltransferase enzyme family gene AtUGT71C5 into Arabidopsis thaliana. By constructing over-expression lines (OE) and down-expression lines (DN), we acquired mutant strains to analyze the function of AtUGT71C5. The multiple-reaction monitoring (MRM) was used for quantitative determination in negative mode. The transition was m/z 263.1→153.0 for ABA ([M-H]+), m/z 425.1→263.0 for ABA-GE ([M-H]+), and m/z 321.0→152.0 for chloramphenicol. The linear range was 0.8684-217.1 ng/mL for ABA and 0.3920-196.0 ng/mL for ABA-GE. The accuracy was 88.0-109.0% for ABA and 86.6-113.0% for ABA-GE; the inter-day and intra-day precisions were less than 5.4% for ABA and 8.9% for ABA-GE, respectively. This method is simple and sensitive enough for determination of ABA and ABA-GE in A. thaliana leaves. All the evidence confirmed the speculation that AtUGT71C5 can mediate abscisic acid homeostasis.

Release test of alliin/alliinase double-layer tablet by HPLC-Allicin determination.

A simple, precise and accurate method was developed and validated for the determination of allicin release from alliin/alliinase double-layer tablets. According to Appendix XC II of Chinese Pharmacopoeia 2010 edition Volume II, a small glass-method was adopted at the rotational speed of 100 r/min using 100 mL phosphate buffer (pH 6.8) as release medium. The release amount was determined by HPLC with a C18 column (250 mm×4.6 mm, 5 µm) using the mobile phase consisting of methanol -0.4% carboxylic acid (65:35) at a flow rate of 1 mL/min and UV detection at 242 nm. The current method demonstrates good linearity over the range 4.052-405.2 µg/mL (r2=0.9999) with an average recovery of 105.5%(RSD=1.25%). The accumulative release of alliin/alliinase double-layer tablets had good homogeneity for within- and between-batches. The method established is simple, accurate and repeatable for the determination of allicin release from alliin/alliinase double-layer tablets.

[Monitoring urine metabolic profiles of rat inflammation model with capillary electrophoresis].

OBJECTIVE: To determine the comprehensive metabolic profiling of inflammation with capillary zone electrophoresis (CZE) in rat urine. METHODS: CZE-based metabolomics method was used to acquire urine metabolom data, with Computer Aided Similarity Evaluation System aligning peaks and analyzing urine profiling. Principle component analysis (PCA) was performed to compare and classify the phenotypes of the urine CZE spectra. RESULTS: The PCA revealed different phenotypes of metabolites between rats with and without inflammation, which were independent from gender, individual diversity, day-night diversity and diurnal variation. CONCLUSION: CZE-based metabolomics method could be used as a potential tool for urinary profiling for disease diagnosis and drug studies.

Effect of acupuncture on rats with acute gouty arthritis inflammation: a metabonomic method for profiling of both urine and plasma metabolic perturbation.

Acute gouty arthritis is a common inflammation model with multiple pathogenic mechanisms seen in clinical practice, for which acupuncture may potentially be an alternative therapy. To investigate the effect of acupuncture on acute gouty arthritis and search for its mechanism, a metabonomic method was developed in this investigation. Acute gouty arthritis model rats were induced by monosodium urate (MSU) crystals. The urine and plasma samples were collected at several time points and the endogenous metabolites were analyzed by an ultra-performance liquid chromatography coupled with a mass spectrometry (UPLC-MS). Data were analyzed using principal components analysis (PCA) and partial least squares (PLS) analysis to compare metabolic profiles of MSU crystal-induced acute gouty arthritis rats with MSU crystal-induced acute gouty arthritis, treated with acupuncture rats. The results showed that acupuncture could restore the metabolite network that disturbed by MSU administration. Our study indicates that UPLC-MS-based metabonomics can be used as a potential tool for the investigation of biological effect of acupuncture on acute gouty arthritis.

[Metabonomic study of the urine of rat acute gouty arthritis model].

UNLABELLED: [ OBJECTIVE: To analysis the metabonome characteristics of the urine of MSU crystal-induced acute gouty arthritis rats by the method of metabolomics. METHODS: Based on the method of metabolomics, which applies LC/MS as data acquisition platform, incorporating with the means of stoechiometry such as principal component analysis, we analyzed the metabonome difference between the urine of acute gouty arthritis rats and that of normal rats. RESULTS: Compare with control group, the metabolism status of acute gouty arthritis model group deviated. After that, with the time lapsed, it retrieved gradually to the incipient metabolism status. The variation of metabolism locus of rats measured by the methods of metabolomics properly reflects the genesis, development, and recuperation course of acute gouty arthritis. CONCLUSION: The whole metabolism status of rat model is able to be presented finely with the method of metabolomics. The metabolomics study could offer a satisfactory research approach to acute gouty arthritis.

Quantitative retention-activity relationship models of angiotensin converting enzyme inhibitors using biopartitioning micellar chromatography.

Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 under adequate experimental conditions and can simulate biopartioning process of many kinds of drugs and describe their biological behavior. The capability of BMC to describe and estimate pharmacokinetic and pharmacodynamic parameters of angiotensin-converting enzyme inhibitors (ACEIs) had been studied in this paper. The correlation between retention factors of ACEIs obtained using BMC and bioactivity parameters (half-life, volume of distribution, clearance, and IC(50)) was investigated utilizing a second-order polynomial model. The P-values obtained for half-life, volume of distribution, clearance, and IC(50) models were less than 0.05, and the r(2) of those four models were 0.89, 0.98, 0.94, and 0.97, with r(2)(adj) (adjusted for freedom degrees) being 0.85, 0.98, 0.91, and 0.95, respectively. The predictive and interpretative ability of the chromatographic models was evaluated in terms of cross-validated data [root mean squared error of calibration (RMSEC), root mean squared error of cross-validation (leave-one-out) (RMSECV), and root mean squared error of cross-validation (leave-one-out) for interpolated data (RMSECVi)]. The quantitative retention-activity relationship (QRAR) models of ACEIs developed in this paper may be a useful approach to screening new chemicals in the early stage of development.

Development of predictive quantitative retention-activity relationship models of alkaloids by mixed micellar liquid chromatography.

The mixed micellar liquid chromatography is a mode that uses mixed micellar system of Brij35/SDS (85 : 15) as a mobile phase under adequate experimental conditions, can simulate the resting membrane potential and the conformation of the long hydrophilic polyoxyethylene chains remains unchanged. In this article, the applications of biopartitioning micellar chromatography, using mixed micellar system to describe and estimate bioactivities of alkaloids, has been focused. The BMC(Brij35/SDS)-QRAR models of half-life time, volume of distribution, plasma clearance and area under concentration-time curve were obtained using Brij35-SDS retention data. The aim is to take a look at the capability of the mixed micellar liquid chromatography model to describe and/or estimate the bioactivity of alkaloids.

[Determination of sinigrin in semen Thlaspi from Sichuan and Tibet using near infrared diffuse reflectance spectroscopy].

The objective of the present study was to develop a method for the determination of sinigrin in semen Thlaspi from Sichuan using near infrared diffuse reflectance spectroscopy. Near infrared spectra (NIR) in the region of 7,502.1-5,446.2 cm(-1) were recorded for the 246 semen Thlaspi samples containing sinigrin in the content of 1.962%-3.917%. Calibration models were established using the PLS (partial least squares). Different spectra pretreatment methods were compared. The study showed that spectral information can be extracted thoroughly by minimum and maximum normalization pretreatment methods. In this calibration model, the correlation coefficient (R2) was 0.9280, the SEC (standard deviation of the calibration sets) was 0.314 and the SEP (standard deviation of the prediction sets) was 0.388. Results indicated that near infrared diffuse reflectance spectroscopy method can be used to rapidly analyze the valid component in traditional Chinese medicine, and also can be used in the quality control of traditional Chinese medicine.

Intra-herb pharmacokinetics interaction between quercetin and isorhamentin.

AIM: Quercetin and isorhamnetin are common constituents of some herb extracts, such as extracts of gingko leaves and total flavones of Hippophae rhamnoides L. The intra-herb pharmacokinetics interactions between isorhamnetin and quercetin were investigated in the present study. METHODS: Human MDR1 cDNA transfected MDCKII cells were used to validate whether isorhamnein interacted with P-gp. Caco-2 transport assays and a randomized, 3-way crossover pharmacokinetics study in rats were used to investigate the pharmacokinetics interactions. HPLC was used to determine cell transport samples. The total plasma concentrations of quercetinand isorhamnetin were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) by treatment with beta-glucuronidase and sulfatase. RESULTS: The permeability ratio (absorptive permeability/secretive permeability) of isorhamnetin across human MDR1 cDNA transfected MDCKII cells, Caco-2 cells and wild-type MDCKII cells are 0.25+/-0.02, 0.74+/-0.05, and 1.41+/-0.06, respectively. This result proved the role of P-gp in the cell efflux of isorhamnetin. While co-transporting with each other across Caco-2 cells monolayer, the permeability ratio of isorhamnetin and quercetin increased by 4.3 and 2.2 times. After coadministration with each other to rats, the C(max), AUC(0-72 h), and AUC(0-infinity) of both isorhamnetin and quercetin significantly increased compared with single administration. CONCLUSION: The above results proved intra-herb pharmacokinetics interaction between quercetin and isorhamentin. P-gp might play an important role, whereas other drug efflux pumps, such as multi-drug resistance associate protein 2 and breast cancer resistance protein, might be involved. Accordingly, besides the drug-herb interactions, intra-herb interaction might be brought into view with the wide use of herbal-based remedies.

Mixed micellar liquid chromatography methods: modelling quantitative retention-activity relationships of angiotensin converting enzyme inhibitors.

The capability of biopartitioning micellar chromatography (BMC), using pure Brij35 solution and mixed micellar system of Brij35-SDS (85:15) as mobile phase, to describe and estimate bioactivities of angiotensin converting enzyme inhibitors at different pH has been studied. Quantitative retention-activity relationships (QRAR) in BMC were investigated for these compounds. The obtained BMC(Brij35-SDS)-QRAR models were compared with the traditional BMC(Brij35)-QRAR, and better statistically models were obtained using Brij35-SDS retention data. The superiority of BMC(Brij35-SDS)-QRAR is due to the fact that the mixed micellar mobile phase can simulate the resting membrane potential and the conformation of the long hydrophilic polyoxyethylene chains remains unchanged.

[Determination of isorhamnetin in Hippophae rhamnoides Linn from West Sichuan plateau using near infrared diffuse reflectance spectroscopy].

The objective of this study was to develop a method for the determination of isorhamnetin in Hippophae rhamnoides Linn from West Sichuan plateau using near infrared diffuse reflectance spectroscopy. Applying the method of mixing with SiO2, the near infrared spectra (NIS) with the range of 12 000-4 000 cm(-1) were recorded for the Hippophae rhamnoides Linn containing isorhamnetin with the content of 0.1%-0.8%. Calibration models were established using the PLS (partial least squares). Different spectra pretreatments methods were compared. The study showed that spectral information can be extracted thoroughly by constant offset elimination (COE) pretreatments method with the correlation coefficient (r2) of 0.739 8, SEC of 0.107 (standard deviation of the calibration sets) and SEP of 0.073 (standard deviation of the prediction sets). The results indicate that near infrared diffuse reflectance spectroscopy is more rapid and convenient than conventional methods.

Biopartitioning micellar chromatography separation methods: modelling quantitative retention-activity relationships of cephalosporins.

In this article, recent applications of chromatographic systems, particularly biopartitioning micellar chromatography (BMC) systems based on amphiphilic structures, have been reported. The aim is to take a look at the capability of quantitative retention-activity relationship (QRAR) models with BMC to describe and/or estimate the bioactivity of cephalosporins. Better qualification of BMC systems was obtained according to the octanol-water partition coefficient (log P); the bioactivity parameters (Lag-T, T(1/2beta), F%, T(1/a), P%, AUC and C(max)) were correlated with the retention factors of cephalosporins processed by Alltech-chromstation software, and the classical data were compared with the predictive values based on QRAR models. The results indicate that using only one descriptor (the retention factor, k) to explain the pharmacokinetic and pharmacodynamic properties of cephalosporins is adequate, and this in vitro approach is an advanced tool for pharmacodynamics research.

Quantitative retention-activity relationship models for quinolones using biopartitioning micellar chromatography.

A simple and reproducible quantitative retention-activity relationship (QRAR) model utilizing biopartitioning micellar chromatography was developed for the biological parameter estimation of drugs. The correlation between retention factors of quinolones obtained in physiological conditions (pH, ionic strength) and biological activities was investigated using different second-order polynomial models. The predictive and interpretative ability of the chromatographic models was evaluated in terms of cross-validated data (RMSEC, RMSECV and RMSECVi). The aim was to obtain adequate QRAR models of half-life, clearance, volume of distribution, plasma protein combination rate, area under concentration-time curve and toxicity (LD50) of quinolones, and to elucidate the advantages and limitations of using a single parameter as independent variable for describing and estimating the activities.

Development of predictive quantitative retention-activity relationship models of HMG-CoA reductase inhibitors by biopartitioning micellar chromatography.

Biological fluid cell membranes are barriers for the uptake of many kinds of drugs and their metabolites, along with passive transport across membranes and bioaccumulation. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to simulate the drug's passive absorption and the transport in biological systems. The use of micellar aqueous solutions of Brij35 as mobile phases in reversed-phase liquid chromatography has proven to be valid to predict the biological activities of barbiturates, benzodiazepines, catecholamines, local anesthetics, non-steriodal anti-inflammatory drugs and tricyclic antidepressants. In this study, the relationships between the capacity factor in BMC and some pharmacokinetic and pharmacodynamic parameters of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are studied. Predictive quantitative retention-activity relationship (QRAR) models describing some of the biological activities and pharmacokinetic properties of HMG-CoA reductase inhibitors are obtained. The results indicate that QRAR model may be a useful tool during the drug discovery process.

[Study on quality evaluation methods of pretreatment for polystyrene-type macroporous absorbing resins].

OBJECTIVE: To establish a GC method using wide bore open tubular columns f or controlling pretreatment for polystyrene-type macroporous absorbing resins and its eligible standard. METHOD: A model macroporous absorbing resins made in our lab was eluted by anhydrous alcohol. The residual solvents in elution (xylene, styrene, diethylbenzene, divinybenzene, decane) were assayed by GC. When the residual solvents can not be detected, the pretreatment is eligible. Compare this method with the methods referring in the literatures. RESULT: When using this method to control the pretreatment, the resins need to be elute 11 times with anhydrous alcocol. When using "adding several times water in the alcohol elution will not be turbid" to control the pretreatment, the resins need to be elute 3 times with anhydrous alcocol. When using "the elution have no UV absorption between the wavelength 200-400 nm" to control the pretreatment, the resins need to be elute over 20 times with anhydrous alcocol. CONCLUSION: The method is simple and feasible.