Study on the bioavailability of nateglinide-hydroxypropyl-beta-cyclodextrin complex capsule in rabbits by liquid chromatographic-tandem mass spectrometry

To evaluate the bioavailability of nateglinide-hydroxypropyl-beta-cyclodextrin (HPCD) complex, a rapid and specific liquid chromatographic-tandem mass spectrometric method was developed and validated to determine nateglinide in rabbit serum. The analyte was extracted from serum samples by liquid-liquid extraction, separated on a Zorbax C(18) column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Daidzein was used as the internal standard. The method has a lower limit of quantitation of 0.25 mg/L using 200 micro L serum. The intra- and inter-day relative standard deviations calculated from quality control (QC) samples were below 4%. The inter-day relative error was within 1%. Nateglinide serum concentrations in rabbits given nateglinide-hydroxypropyl-beta-cyclodextrin complex were much higher than those given the free drug. Significant difference was observed in main pharmacokinetic parameters of t(max) and C(max) but not AUC(0-t) between the complex and free drug. It was concluded that the absorption rate of nateglinide-HPCD complex was enhanced, compared with that of nateglinide free drug.     

Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.     

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.     

Separation and determination of isoflavonoids in several kudzu samples by high-performance capillary electrophoresis (HPCE)

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from root, stem, leaf, callus and cell samples, is very important for the best, safest and most efficacious use of kudzu as a medicinal plant, and for the studies on quantitative analysis in the secondary metabolism of isoflavonoids. In this paper, a simple, rapid and precise high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD) has been developed for separation and determination of isoflavonoids in several kudzu samples. The isoflavonoids could be well separated within 15 min in a 40 cm length capillary at a separation voltage of 15kV in a 30 mmol L(-1) borax buffer (pH9.29), and this proposed method demonstrated excellent reproducibility and accuracy with relative standard deviations of less than 5% for isoflavonoid content (n = 5) of different kudzu samples. The relationship between peak areas and isoflavone concentrations, in a specified working range with linear response, was determined by first-order polynomial regression over the range 0.05-0.5 mg mL(-1) for puerarin and 2.5-50 microg mL(-1) for 3'-methoxypuerarin, daidzin and daidzein, respectively, and quantitative evaluation of those four main isoflavonoid components was determined by ultraviolet absorption at lambda = 192 nm. The differences were also illustrated by comparison of the determination of isoflavonoid components from kudzu root, stem, leaf samples and plant tissue cultures in vitro.     

High-performance thin-layer chromatographic fingerprints of isoflavonoids for distinguishing between Radix Puerariae Lobate and Radix Puerariae Thomsonii

The roots of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth have been officially recorded in all editions of Chinese Pharmacopoeia under the same monograph 'Gegen' (Radix Puerariae, RP). However, in its 2005 edition, the two species were separated into both individual monographs, namely 'Gegen' (Radix Puerariae Lobatae, RPL) and 'Fenge' (Radix Puerariae Thomsonii, RPT), respectively, due to their obvious content discrepancy of puerarin, the major active constituent. In present paper, the fingerprint of high-performance thin-layer chromatography (HPTLC) combining digital scanning profiling was developed to identify and distinguish the both species in detail. The unique properties of the HPTLC fingerprints were validated by analyzing ten batches of Pueraria lobata and P. thomsonii samples, respectively. The common pattern of the HPTLC images of the roots of Pueraria spp. and the respective different ratios of the chemical distribution can directly discern the two species. The corresponding digital scanning profiles provided an easy way for quantifiable comparison among the samples. Obvious difference in ingredient content and HPTLC patterns of the two species questioned their bio-equivalence and explained that recording both species separately in the current edition of Chinese Pharmacopoeia (2005 edition) is reasonable due to not only the content of major constituent, puerarin, but also the peak-to-peak distribution in the fingerprint and integration value of the total components. Furthermore, the HPTLC fingerprint is also suitable for rapid and simple authentication and comparison of the subtle difference among samples with identical plant resource but different geographic locations.     

Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection was developed for the determination of puerarin, daidzein and rutin. Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The working electrode was a 300-microm diameter carbon disc electrode positioned opposite the outlet of capillary. The three analytes could be well separated within 12 min in a 40 cm length capillary at a separation voltage of 9 kV in a 50 mmol/l borate buffer (pH 9.0). The relationship between peak currents and analyte concentrations was linear over about three orders of magnitude with detection limits (SIN=3) ranging from 0.241 x 10(-6) to 0.511 x 10(-6) mol/l for all compounds. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied for the determination of puerarin, daidzein and rutin in Chinese traditional drugs, the vines of Pueraria lobata (Wild.) Ohwi and Puerariae Radix.     

Non-aqueous capillary electrophoresis for simultaneous separation and determination of three major active components in traditional medicinal preparations

A simple and sensitive non-aqueous capillary electrophoresis method has been developed for simultaneous assay of three bioactive components (puerarin, daidzein and wogonin) in three traditional medicinal preparations for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. capillary using a non-aqueous buffer system of 20% acetonitrile, 25 mm ammonium acetate and apparent pH 9.00, with applied voltage and capillary temperature of 20 kV and 16 degrees C, respectively. The relative standard deviations (RSDs) of the migration times and the peak areas of the three analytes were in the ranges 2.5--4.0% and 3.2--3.9%, respectively. Detection limits of puerarin, daidzein and wogonin were 0.090, 0.145 and 0.090 microg mL(-1), respectively. In the tested concentration range, good linear relationships (correlation coef fi cients: 0.9998 for puerarin, 0.9998 for daidzein and 0.9978 for wogonin) between peak areas and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with recoveries from 91.0 to 114.0%.     

Pharmacokinetic study of puerarin in rat serum by liquid chromatography tandem mass spectrometry

A highly sensitive and specific atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry method was developed for serum pharmacokinetic studies of puerarin in rats. Chromatography was carried out on a reversed-phase Phenomenex Synergi 4 microm Fusion-RP80 column (150 x 2.0 mm i.d.) using a mobile phase consisting of acetonitrile-water (10:90, v/v) in 10 mm NH(4)OAc with a flow rate of 0.2 mL/min. Puerarin was analyzed in the multiple reaction monitoring mode with a precursor/product ion transition of m/z 415/267. The method was demonstrated to be specific and sensitive, and a linear response was observed over a range of 2-5000 ng/mL in rat serum. The validated method was successfully applied to the characterization of the pharmacokinetics of puerarin in rat serum after oral administration to spontaneously hypertensive rats. The blood concentration-time profile of puerarin showed a rapid initial increase, reaching a maximum and then declining within 1 h. Puerarin could not be detected after 24 h. The main pharmacokinetic parameters for puerarin after oral administration were as follows: C(max) (3.54 +/- 2.03 mg/L), T(max) (0.68 +/- 0.37 h), AUC(0-t) (7.29 +/- 3.79 mg h/L), AUC(0-infinity) (9.17 +/- 4.87 mg h/L), T(1/2) (1.7 +/- 0.6 h), CL/F (7.24 +/- 4.27 L/h/kg) and V/F (17.88 +/- 13.55 L/h/kg).     

Purification of the isoflavonoid puerarin by adsorption chromatography on cross-linked 12% agarose

The isoflavonoid puerarin in extracts of the well-known traditional Chinese drug Radix puerariae (root of the plant Pueraria lobata) can be separated from other isoflavonoids by adsorption chromatography on the cross-linked 12% agarose gel Superose 12 equilibrated in distilled water. The adsorption is totally quenched by the addition of 50% acetic acid. The separation of the isoflavonoids is tentatively ascribed to interaction with the residues of the cross-linking reagents used in the manufacturing process of Superose 12. Thus, no useful separation can be achieved with non-cross-linked 12% agarose gel media. Symmetric elution profiles at high sample loadings (16 mg on a 24 ml column) suggest linear adsorption isotherms for the isoflavonoids.     

反相高效液相色谱法用于葛根黄酮提取物的分离与主要活性成分的测定

采用反相高效液相色谱法，在C18柱上以甲酵—水为流动相进行梯度洗脱，在0-10min内将葛根黄酮提取物进行分离，并在250nm检测，采用标准加入法对实际样品中的葛根素含量进行了分析。结果表明：平均回收率为94.8％，RSD为2．4％。     

HPLC determination and pharmacokinetic study of tenatoprazole in dog plasma after oral administration of enteric-coated capsule

A simple, sensitive and selective high-performance liquid chromatographic (HPLC) method with UV detection (306 nm) was developed and validated for determination of tenatoprazole, a novel proton-pump inhibitor, in dog plasma. Tenatoprazole and internal standard (pantoprazole) were extracted into diethyl ether and separated using an isocratic mobile phase of 10 mm phosphate buffer (pH4.7)-acetonitrile (70:30, v/v) on a Diamonsil C(18) column (150 x 4.6 mm, 5 microm). The retention times for tenatoprazole and internal standard were 7.1 and 12.3 min, respectively. No endogenous interferences were observed. This HPLC method was fully validated. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 20%. A linear range of 0.02-5.0 microg/mL was established. The interday and intraday precisions were within RSD 13.4-10.1 and 4.6-1.4%, respectively. This method developed can be easily applied to the pharmacokinetic study of tenatoprazole in dog plasma after oral administration of an enteric-coated capsule. The plasma concentration of tenatoprazole from six dogs showed a mean C(max) of 2.63 microg/mL at T(max) of 1.89 h. The bioavailability of tenatoprazole was improved by administration of enteric-coated capsule.     

液质联用分析葛根提取物及中药片剂中异黄酮类化合物

采用反相C18毛细管液相色谱柱,以乙腈(含0.1%(体积分数,下同)三氟乙酸)和水(含0.1%三氟乙酸)为流动相梯度洗脱,在26 min内分离了葛根异黄酮提取物以及愈风宁心片中的主要成分。采用毛细管液相色谱/四极杆飞行时间串联质谱仪对葛根提取物以及片剂中的几种主要异黄酮类化合物做了结构分析,发现葛根素是主成分(提取物中其平均质量分数是13.32%;片剂中每片含量19.28~24.34 mg)。对微量未知化合物,用它们的子离子谱图与已知化合物的谱图比较,推测其成分为3′-甲氧基葛根素和3′-甲氧基大豆苷。     

Development of bioluminescent enzyme immunoassay for s-equol using firefly luciferase and its application to the assessment of equol-producer status

In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (r=0.992) was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its diastereoisomer is <5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the results for log(10)-transformed urinary S-equol to daidzein ratio threshold of -1.75, namely, 34% of men and 19% of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of isoflavone, the maximum concentration (C(max)) of urinary equol appeared after 9.6 h of isoflavone consumption; this C(max) was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.     

RP-HPLC method with electrochemical detection for the determination of metoclopramide in serum and its use in pharmacokinetic studies.

A rapid and sensitive reversed-phase high performance liquid chromatographic method has been developed for the determination of metoclopramide in serum. The assay was performed after single extraction with ethyl ether using methyl parahydroxybenzoate as internal standard. Chromatographic separations were performed on C(18) stationary phase with a mobile phase composed of methanol-phosphate buffer pH 3 (30:70 v/v). Analytes were detected electrochemically. The quantification limit for metoclopramide in serum was 2 ng mL(-1). Linearity of the method was confirmed in the range of 5-120 ng mL(-1) (correlation coefficient 0.9998). Within-day relative standard deviations (RSDs) ranged from 0.3 to 5.5% and between-day RSDs from 0.8 to 6.0%. The analytical method was successfully applied for the determination of pharmacokinetic parameters after ingestion of 10 mg dose of metoclopramide. Studies were performed on 18 healthy volunteers of both sexes.     

Separation and purification of puerarin using beta-cyclodextrin-coupled agarose gel media

The isoflavonoid puerarin, a well-known traditional Chinese drug, has been purified in one step from an extract of Radix puerariae (root of the plant Pueralria lobata) by adsorption chromatography on an epichlorohydrin polymerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP. Acetic acid (10%) was used as the mobile phase and the optimum loading capacity was around 1.2 mg crude extract/ml packed gel. The purity of the collected puerarin was about 98% with a recovery of about 62%.     

电感耦合等离子体原子发射光谱(ICP-AES)法测定硒片中的硒、铁、钙和锌

用HNO_3-HClO_4(4∶1)溶解硒片,电感耦合等离子体原子发射光谱(ICP-AES)法测定其中的硒、铁、钙和锌,实验表明硒片中硒、铁、钙和锌的含量较为丰富,与外包装承诺的含量一致。方法灵敏可靠,测量相对标准偏差3%,加标回收率在96.5%~101%,实验结果为探讨硒片对人体的保健作用提供了参考数据。     

Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers

A simple, sensitive and selective LC-MS-MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C(18) column with a mobile phase consisting of 0.2% formic acid-methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508-5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC(0-24), AUC(0-infinity), C(max), T(max) and t(1/2) of huperzine A were 16.35 +/- 3.42 vs 16.38 +/- 3.61 ng h/mL, 17.53 +/- 3.80 vs 17.70 +/- 3.97 ng h/mL, 2.47 +/- 0.49 vs 2.51 +/- 0.51 ng/mL, 1.3 +/- 0.4 vs 1.2 +/- 0.3 h and 5.92 +/- 0.75 vs 6.18 +/- 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent.     

A New Pueroside from Pueraria lobata

Chemistry of Natural Compounds - A new constituent, named (4R)-6′′′′ -O-sinapoylpueroside B (1), was isolated from the dried roots of Pueraria lobata (Willd.) Ohwi. The...     

Effect of mesopores on solidification of sirolimus self-microemulsifying drug delivery system

The mesoporous silica materials had a high loading efficiency of sirolimus-SMEDDS. The length of the mesopores played a more important role than the pore diameter in drug dissolution and in vivo absorption.     

A Novel Medium Poly(vinyl acetate-triallyl isocyanurate-divinylbenzene) Coupled with Oligo-β-Cyclodextrin for the Isolation of Puerarin from Pueraria Flavones

Poly(vinyl acetate-triallyl isocyanurate-divinylbenzene) matrix coupled with oligo-β-cyclodextrin was synthesized to separate puerarin. The separation of three isoflavone homologues, puerarin, daidzin and daidzein, was achieved by increasing the proportion of methanol from 40 to 85%. The interaction mechanism between the crosslinkers and puerarin was investigated through molecular dynamics simulation and the mixed-mode character was demonstrated by computational and conformational methods. The research confirms that in order to enhance the recognition, the addition of triallyl isocyanurate is feasible. Meanwhile, an alternative matrix for the separation of puerarin was provided, with the advantages of moderate mechanical rigidity, ease of modification and low cost.