Application of the Reversed-phase Liquid Chromatography Coupled to Ion Trap Mass Spectrometry with Principal Components Analysis for Metabonomics Studies of Scutellarin in Rat Urine

Recently, much attention has been paid to metabonomics and great growth in it as a novel systemic approach to study metabolic profile accelerate the process of drug development1-5. Traditional Chinese Medicine (TCM) is an emerging field for medical and ph…     

Synthesis and protective effect of scutellarein on focal cerebral ischemia/reperfusion in rats

Scutellarein, the main metabolite of scutellarin in vivo, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease.     

反相高效液相色谱法测定肾复康胶囊中的野黄芩苷和芦丁

建立了肾复康胶囊中野黄芩苷和芦丁的反相高效液相色谱测定方法。以甲醇-水为提取溶剂,采用超声提取法对样品进行前处理。以0.02 mol/L磷酸二氢钾缓冲溶液(含1.0％（体积分数）冰醋酸)-甲醇（体积比为63∶37）为流动相,于330 nm波长下检测,肾复康提取液中野黄芩苷和芦丁可达基线分离,分析时间在20 min内。野黄芩苷和芦丁在10 ~300 mg/L内,其峰面积与浓度之间线性关系良好,目标物的加标回收率大于98％。该方法适用于肾复康胶囊及相关药材中野黄芩苷和芦丁黄酮类化合物的测定和质量控制。     

Microwave-assisted extraction of scutellarin from Erigeron breviscapus Hand-Mazz and its determination by high-performance liquid chromatography

An efficient microwave-assisted extraction (MAE) technique has been developed to extract scutellarin from Erigeron breviscapus for rapid determination by high-performance liquid chromatography (HPLC). The maximum yield of scutellarin reached 1.02% in 40 min under the optimal MAE conditions with 80 °C of extraction temperature and 1:10 (w/v) of the solid/liquid ratio. The MAE showed obvious advantages in terms of short duration and high efficiency to extract scutellarin in comparison with heat-flux extraction. The mechanism of the enhanced extraction by microwave assistance was discussed by detecting particle size and specific surface area of plant materials and observing cell destruction of plant material by light microscopy and scanning electron microscopy. The results showed that the plant materials were significantly destroyed due to the cell rupture after MAE treatment. Afterward, the method validation for HPLC-UV analysis was developed. Calibration range was 0.1-100 μg mL⁻¹ for scutellarin, and correlation coefficient R was 0.9993. Limit of detection was less than 0.01 μg mL⁻¹. The intra- and inter-day relative standard deviation (R.S.D.) of scutellarin detection ranged from 1.58% to 2.96% and from 3.32% to 4.19%, respectively. The recovery of the method for scutellarin ranged from 96.7% to 101.9%.     

Metabolism profile of scutellarin in urine following oral administration to rats by ultra performance liquid chromatography coupled to time-of-flight mass spectrometry

Scutellarin, a flavone glucuronide of 5,6,4′-trihydroxyflavone-7-O-glucoronide, is the main active component of the traditional Chinese botanic drug Erigeron breviscapus (Vant.) Hand.-Mazz. In this study, a method based on ultra performance liquid chromatography coupled with a time-of-flight mass spectrometer (UPLC/TOF MS) was established and validated to profile the metabolites of scutellarin in Sprague-Dawley rat urine following oral administration of single dose of scutellarin at 80.8 mg/kg. The column utilized was an Acquity BEH C18 (150 mm × 2.1 mm, 1.7 μm). The mobile phase was 0.2% formic acid and acetonitrile with gradient condition. Two standard curves of scutellarin were obtained for the concentration range of 1.065-10.65 μg/mL and 10.65-63.92 μg/mL, respectively. By automating the data processing of the software Masslynx developed by Waters Ltd., 17 metabolites of scutellarin were found and determined in rat urine, with the corresponding reactions in vivo such as isomerism, reduction, methylation, glucuronide conjugation, hydroxylation, hydroxylation and methylation, etc., most of which were discovered for the first time. For most metabolites, the time (T_p) of peak excretion was 8-12 h. Calculated as scutellarin, the cumulative urine excretion rate of the metabolites was 1.93%.     

Retracted: Determination of scutellarin isomer,a predominant metabolite of scutellarin,in human plasma by HPLC/tandem mass spectrometry and its application to a pharmacokinetic study in Chinese healthy volunteers

Retraction: The following article from Biomedical Chromatography, “Determination of scutellarin isomer, a predominant metabolite of scutellarin, in human plasma by HPLC/tandem mass spectrometry and its application to the pharmacokinetic study in Chinese healthy volunteers” by Chun‐hua Xia, Yu‐qing Xiong, and Guang‐ji Wang, published online on 21 June 2007 in Wiley Interscience (www.interscience.wiley.com/journal/bmc), has been retracted at the authors' request.     

Microwave‐assisted extraction coupled with countercurrent chromatography for the rapid preparation of flavonoids from Scutellaria barbata D. Don

As a famous Chinese herb having good inhibitory effects on numerous human cancers both in vitro and in vivo, Scutellaria barbata D. Don attracts extensive attention worldwide. In this work, four flavonoids named scutellarin, baicalin, luteolin, and apigenin were simply and rapidly prepared from S. barbata by microwave‐assisted extraction coupled to countercurrent chromatography. Extraction conditions including irradiation time, extraction temperature, liquid/solid ratio, and microwave power were optimized using an orthogonal array design method. The extract of S. barbata was separated and purified with a two‐phase solvent system composed of hexane/ethyl acetate/methanol/acetic acid/water (1:5:1.5:1:4, v/v/v/v/v) and 4.5 mg of scutellarin, 4.6 mg of baicalin, 1.1 mg of luteolin, 2.1 mg of apigenin were obtained from 2.0 g original sample in a single run. The purities of scutellarin, baicalin, luteolin, and apigenin determined by HPLC were 93.6, 97.3, 97.6, and 98.4%, respectively. The targeted compounds were identified by LC with MS and ¹H NMR spectroscopy. The total time including extraction, separation, and purification was <300 min. Compared to traditional methods, microwave‐assisted extraction coupled to countercurrent chromatography method is more simple and rapid for the extraction, separation, and purification of flavonoid compounds from natural products.     

Synthesis and Characterization of PEG-scutellarin Conjugates, a Potential PEG Ester Prodrug for the Oral Delivery of Scutellarin

Scutellarin is the active ingredient extracted from Erigeron breviscapus, a traditional Chinese herbal medicine. Pharmacological study showed that scutellarin could significantly reduce the blood viscosity, improve the blood flow, decrease the vascular re…     

Synthesis and physiochemical property evaluation of carbamate derivatives of scutellarin methyl ester

A series of carbamate prodrugs of scutellarin methyl ester (4a-4e) were prepared in the presence of bis(trichloromethyl) carbonate (BTC) with scutellarin methyl esters and L-amino acid tert-butyl ester hydrochloride as starting materials. In vitro stability and aqueous solubility of target compounds were evaluated. The results indicated that compounds 4d and 4e have higher solubility and in vitro stability than scutellarin respectively.     

Binding behaviors of scutellarin with α-, β-, γ-cyclodextrins and their derivatives

A series of cyclodextrin/scutellarin inclusion complexes were prepared from α-cyclodextrin, β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin with scutellarin (SCU), and their inclusion complexation behaviors, such as stoichiometry, complex stability constants and inclusion mode, were investigated by means of UV/Vis spectroscopy, ¹H NMR and 2D NMR. The results showed that the SCU could be efficiently encapsulated in the cyclodextrin cavity in aqueous solution to produce complexes that were more soluble than free SCU. The enhanced binding ability of cyclodextrins towards SCU was discussed from the viewpoint of the size/shape-fit and multiple recognition mechanism between host and guest.     

Simultaneous determination of baicalin,baicalein, wogonoside,wogonin, scutellarin,berberine, coptisine,ginsenoside Rb1 and ginsenoside Re of Banxia xiexin decoction in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid and high‐sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous determination of nine active constituents (baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re) in rat plasma after oral administration of Banxia xiexin decoction (BXD). Biological samples were processed wtih acetone–ethyl acetate (4:1, v/v). The mobile phase consisted of methanol and water (containing 0.1% formic acid) with gradient elution at a flow rate of 0.3 mL/min. Detection was performed on a triple quadrupole mass spectrometer using positive ion and negative ESI in the multiple reaction monitoring mode. The calibration curves for all analytes had good linearity (r > 0.9933). The mean recovery of all the nine active ingredients was >75.2%, and the intra‐ and inter‐day precisions were within 12.0%; the accuracy was between 87.4 and 110.4%. This method was successfully applied to a pharmacokinetic study after administration of BXD. The results of the pharmacokinetic study might be helpful for BXD reasonable clinical application and further studies on mechanism.     

Separation and Concentration of the Trimethylselenonium Ion for Its Determination in Urine by Graphite Furnace Atomic Absorption Spectrometry

The separation and concentration methods for the trimethylselenonium ion (TMSe) in urine samples were developed utilizing the formation of the ion pair of TMSe with tetrakis(4-fluorophenyl)borate. The ion pair could be quantitatively collected by a solvent extraction technique as well as a cellulose nitrate (CN) filter collection technique. The collected TMSe was determined by graphite furnace atomic absorption spectrometry with the addition of palladium as a matrix modifier. In the case of urine analysis for TMSe, the CN filter technique gave better reproducibility than the solvent extraction technique. The detection limit of this method for TMSe was 1 ngSe/ml for 20 ml of urine sample.     

The luminescence system of yttrium(III)-BPMPHD-CTMAB and the determination of yttrium(III)

The study indicated that yttrium(III) could form an ion association compound with a new synthetic reagent, 1,6-bi(1′-phenyl-3′-methyl-5′-pyrazolone-4′-)hexanedione (BPMPHD) and cetyl trimethyl ammonium bromide (CTMAB). The compound could enhance the natural fluorescence of BPMPHD by about 260 times, upon which a new fluorescence method was developed for determining yttrium in rare earth (RE) samples. The determination range was 9–900 ng/ml. The detection limit was 1.8 ng/ml. The composition of the ion association was [Y(BPMHD)₂]⁻CTMAB⁺.     

Simultaneous determination of prostaglandin E1, prostaglandin E0 and 15-keto-prostaglandin E0 in human plasma by gas chromatography/negative-ion chemical-ionization tandem mass spectrometry.

A sensitive and selective routine method for the simultaneous determination of prostaglandin E1 (PGE1), prostaglandin E0 (PGE0) and 15-keto-prostaglandin E0 (15-keto-PGE0) in human plasma is described using deuterated internal standards. The analytes were isolated from acidified human plasma by solid-phase extraction by means of Bond Elut C18 cartridges and derivatized to the pentafluorobenzyl (PFB) ester methoxime. The analytes were purified on Bond Elut Si cartridges and converted to the trimethylsilyl (TMS) ether. Quantitation was achieved by gas chromatography-negative-ion chemical-ionization tandem mass spectrometry. The precursor ion [M-PFB]- = [P]- carried more than 80% of the total ion current. Collision activated decomposition (CAD) of [P]- resulted in characteristic product ions of which the [P-2(CH3)3SiOH]- ion (PGE1) and the [P-(CH3)3SiOH]- ion (PGE0 and 15-keto-PGE0) were used for quantitation. The lower limit of quantitation (LLQ) was 2 pg/ml (PGE1 and PGE0) and 10 pg/ml (15-keto-PGE0) extracted from 2 ml of human plasma. Linear calibration curves were obtained over the concentration range 2-100 pg/ml (PGE1 and PGE0) and 10-500 pg/ml (15-keto-PGE0). In all cases, the precision and accuracy were < 17%. The present method has been applied successfully to pharmacokinetic and clinical studies in humans.     

Measurement of bisphenol A in human serum by gas chromatography/mass spectrometry

The purpose of this study is to establish an easy and accurate method for the determination of bisphenol A (BPA) in the human serum. The samples were applied to the C₁₈ solid phase extraction (SPE) column for clean up of samples. The BPA is conjugated with tetrabutylammonium hydrogen sulfate as the counter ion in alkali solution. The ion paired BPA is moves from the aqueous phase to the organic phase as an ion paired extraction. BPA extracted from human serum were derivatized with pentafluorobenzyl bromide (PFBBr). The derivative was analyzed by gas chromatography (GC)/mass spectrometry (MS) using negative chemical ionization (NCI). The instrumental detection limit of BPA was 5 pg/ml (10 fg). The instrumental response between 0.01 and 100 pg/ml of BPA standards was linear (r²=0.998). The recovery of BPA spiked into human serum was 101.0±4.63 (1 pg/ml) and 100.9±3.75 (10 pg/ml), respectively. The concentration of BPA in the human serum from 20 individuals was 0.54 pg/ml.     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.     

Determination of mercury in industrial workplaces by collection on activated carbon,solvent extraction and cold-vapour atomic-absorption spectrometry

A method for the determination of mercury in workplace environments in a chlor-alkali plant was optimized. Mercury was collected on activated carbon with a personal sampling pump at a flow-rate of 0.5 l/min, then the carbon was mineralized by heating with potassium permanganate and sulphuric acid. The mercuric ion was next chelated with ammonium pyrrolidinedithiocarbamate and extracted with methyl isobutyl ketone at acidic pH (1.6–5). The chelate in the extract was decomposed with sulphuric acid and the mercuric ion reduced to the metal with stannous chloride. The mercury was determined by coldvapour atomic-absorption spectrometry (CVAAS). The calibration graph was linear up to 5 ng/ml Hg in the initial solution, the relative standard deviation was 4.5% (for 2 ng/ml) and the detection limit was 0.14 ng/ml. All the figures of merit are referred to the initial sample. The proposed method gave good accordance with CVAAS without extraction.     

Negative ion chemical ionization for the determination of methylphenidate in human plasma by stable isotope dilution gas chromatography/mass spectrometry

A sensitive and specific method for the determination of methylphenidate in human plasma is presented. Methylphenidate was extracted from plasma by solvent extraction with hexane at pH 9.3 and derivatized to its heptafluorobutyrate derivative. The derivative was measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostically useful fragment ion at m/z 369 was obtained at high relative abundance. (18)O(2)-labelled methylphenidate was used as an internal standard and its rapid and facile preparation from the unlabeled compound is described. Calibration graphs were linear within the range 0.14-18.25 ng ml(-1). The inter-assay precision was 8.7% (0.14 ng ml(-1)) and 3.1% (4.56 ng ml(-1)) and the intra-assay variability was 1.3% (0.14 ng ml(-1)) and 0.4% (4.56 ng ml(-1)). Accuracy determinations showed deviations of +0.7% (0.14 ng ml(-1)) and -2.5% (4.56 ng ml(-1)). The method is rugged, rapid and robust and has been applied to the batch analysis of methylphenidate during pharmacokinetic profiling of the drug.     

Enhanced mass resolution tandem mass spectrometry method for 2,3,7,8-tetrachlorodibenzo-p-dioxin detection with ion trap mass spectrometry using high damping gas pressure

Damping gas flow was optimized for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) determination using ion trap mass spectrometer. A tandem mass spectrometry (MS-MS) method with better than unit-mass resolution (mass width, 0.3 u) was developed at a damping gas flow of 1.5 ml/min and a collision-induced dissociation (CID) voltage of 3.30 V. The relative standard deviation (R.S.D.) at the enhanced resolution was 2.9% in 24 h of consecutive injections. The detection limit was significantly improved because the efficiency of both precursor ion trapping and fragmentation increased with the damping gas flow. Product ion yield was 4.5 times higher and limit of detection was 3.2 times lower than at the default flow (0.3 ml/min and 1.65 V).     

Comparison of gas chromatographic and gas chromatographic/mass spectrometric techniques for the analysis of TNT and related nitroaromatic compounds

The use of capillary column gas chromatography and gas chromatography/mass spectrometry for the analysis of a series of standard solutions (0.1 to 10 μg/ml) of 2,4,6-trinitrotoluene (TNT) and eight other nitroaromatic components was evaluated. The techniques included gas chromatography with electron capture detection (GC/ECD), full scan and selected ion monitoring gas chromatography/mass spectrometry with electron impact ionization (EI/FS and EI/SIM), full scan and selected ion monitoring gas chromatography/mass spectrometry with positive ion chemical ionization using methane reagent gas (PICI/FS and PICI/SIM), and full scan and selected ion monitoring gas chromatography/mass spectrometry with negative ion chemical ionization using methane reagent gas (NICI/FS and NICI/SIM). The performance of the techniques was comapared by determining the linear response range, precision, and detection limits of the analyses.