Quantitative determination of pimozide in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A simple, sensitive and specific LC–ESI/MS method was developed for the determination of pimozide in human plasma. Pimozide and cinnarizine (internal standard) were isolated from plasma samples by liquid–liquid extraction. The chromatographic separation was accomplished on a Thermo Hypersil-HyPURITY C18 reversed-phase column (150 mm × 2.1 mm, i.d., 5 μm) with the mobile phase consisting of 5 mM ammonium acetate (pH 3.5, adjusted with acetic acid)–methanol–acetonitrile (39:5:56, v/v/v). The lower limit of quantification was 0.02 ng/mL, and the assay exhibited a linear range of 0.025–12.800 ng/mL. The established method has been successfully applied to a bioequivalence study of 2 pimozide formulations in 32 healthy male Chinese volunteers.     

Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells

Cancer preventive reagent trans-resveratrol is intracellularly biotransformed to different metabolites. However, it is still unclear whether trans-resveratrol exerts its biological effects directly or through its metabolite(s). This issue was addressed here by identifying the metabolic pattern and the bioactive form of resveratrol in a resveratrol-sensitive human medulloblastoma cell line, UW228-3. The cell lysates and condition media of UW228-3 cells with or without 100 μM resveratrol treatment were analyzed by HPLC and LC/MS which revealed (1) that resveratrol was chemically unstable and the spontaneous generation of cis-resveratrol reduced resveratrol's anti-medulloblastoma efficacy and (2) that resveratrol monosulfate was the major metabolite of the cells. To identify the bioactive form of resveratrol, a mixture-containing approximately half fraction of resveratrol monosulfate was prepared by incubating trans-resveratrol with freshly prepared rat brain lysates. Medulloblastoma cells treated by 100 μM of this mixture showed attenuated cell crisis. The overall levels of the three brain-associated sulfotransferases (SULT1A1, 1C2 and 4A1) were low in medulloblastoma cells in vivo and in vitro in comparison with that in human noncancerous and rat normal cerebella; resveratrol could more or less up-regulate the production of these enzymes in UW228-3 cells but their overall level was still lower than that in normal cerebellum tissue. Our study thus demonstrated for the first time that trans-resveratrol is the bioactive form in medulloblastoma cells in which the expression of brain-associated SULTs was down-regulated, resulting in the increased intracellular bioavailability and anti-medulloblastoma efficacy of trans-resveratrol.     

Immunomodulatory effects of crude phenylethanoid glycosides from Ligustrum purpurascens

Ethnopharmacological relevance

Ligustrum purpurascens, named as “Ku ding cha”, has been used as a kind of functional tea in southern China for about two thousand years, which has the effects on diuresis, anti-hypertension, weight-loss and anti-inflammation.

The aim of the study

This study was aimed to investigate the immune enhancement effects of the crude phenylethanoid glycosides (CPGs) from Ligustrum. Purpurascens on mice and analyze the chemical profiles of phenylethanoid glycosides in the CPGs.

Materials and methods

The immune functions enhancing potential of CPGs was determined using serum hemolysin antibody, phagocytosis, splenocyte antibody production, and NK cells activity assays. The contents of five major constituents in the crude glycosides of Ligustrum purpurascens were determined by using liquid chromatography, other five glycosides were deduced according to their UV and MS spectra compared with the literature as well.

Results

In the immunizing experiment, mice treated with different doses of CPGs showed an increase (p<0.01) in the haemagglutination titre compared with the control group. The increases (p<0.05) were found to be significant at doses of 440 mg/kg and 1.32 g/kg in the experiments of antibody production of spleen cells, MΦ phagocytosis of chicken RBCs and NK cell activity. Further chemical characterization yielded 10 constituents from CPGs, five glycosides were quantified by HPLC and the structures of other five compounds were speculated according to their UV and MS spectra.

Conclusion

The results suggested that phenylethanoid glycosides from Ligustrum purpurascens have immunomodulatory effects on mice.     

Use of electron spectroscopic imaging to determine element composition of the melanin granules in the stria vascularis of the guinea pig

Electron spectroscopic imaging (ESI) was used to analyze the element content of melanin granules in the stria vascularis seen in ultrathin sections of Spurr-embedded cochleae of the guinea pig. To determine element composition, ESI images were taken at different ionization edges, and non-specific background signals were subtracted digitally by an image processing system. The presence of calcium and nitrogen in the melanin granules could be demonstrated clearly. The calcium identified in the melanin granules was then compared with the spatial distributions of calcium binding sites after the application of an antimonate precipitation method, which was used to localize loosely bound calcium. Despite a high calcium concentration within the granules, only very small single scattered calcium precipitates could be detected between these structures as compared with the amount of calcium precipitates attached to the plasma membrane or located within the cell nuclei. The nearly complete absence of precipitates within the melanin granules after the application of antimonate suggests differences in calcium binding and mobility involved in various physiological processes of ion balance regulation within the stria vascularis. Received: 14 October 1997 / Accepted: 11 February 1998     

大豆皂甙A的分离与ESI/MS分析

大豆胚芽中皂甙的含量是子叶的6～14倍,soyasaponin A仅存在于胚芽中.以硅胶柱色谱和制备高效液相色谱法从大豆胚芽的乙醇提取物中分离出二种A组皂甙,进行定性反应.UV和ESI/MS分析确证其结构为soyasaponin A     

A rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of finasteride in human plasma and its application to pharmacokinetic study

A fast, accurate, sensitive, selective and reliable method using reversed-phase high-performance liquid chromatography-mass spectrometry coupling with an electrospray ionization interface was developed and validated for the determination of finasteride in human plasma. After deprotienation with acetonitrile, centrifugation, evaporation to dryness and dissolving in mobile phase, satisfactory separation was achieved on a Hypersil-Keystone C(18) reversed-phase column using a mobile phase consisting of acetonitrile-water (46:54, v/v), 0.1% acetic acid and 0.1% trifluoracetic acid. Carbamazepine (IS) was used as internal standard. This method involved the use of the [M+H](+) ions of finasteride and IS at m/z 373 and 237 with the selective ion monitoring (SIM) mode. The calibration curve was linear in the range of 0.2-120 ng ml(-1). The limit of quantification for finasteride in plasma was 0.2 ng ml(-1) with good accuracy and precision. The intra-assay precision and accuracy were in the range of 2.1-11.2% and -1.3% to 8.5%, respectively. The inter-assay precision and accuracy were in the order of 3.4-12.1% and -1.5% to 11.5%, respectively. The mean sample extract recoveries of the method were higher than 85% and 74% for finasteride and internal standard (IS), respectively. The assay has been successfully used to estimate the pharmacokinetics of finasteride after oral administration of a 5mg tablet of finasteride to 24 healthy volunteers.     

Rapid identification of emerging pathogens: coronavirus

We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.     

Identification of the degradation product of ezlopitant,a non-peptidic substance P antagonist receptor,by hydrogen deuterium exchange,electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete ¹H and ¹³C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

HPLC-MS／ESI同时测定人血浆中文拉法新及其3种代谢产物

目的:建立一种快速灵敏的反相高效液相色谱-质谱联用法同时测定人血浆中文拉法新(VEN)和其3种代谢产物:氧云甲基文拉法新(ODV)、氮去甲基文托法新(NDV)和氮、氰双去甲基文拉法新(DDV)的浓度。方法:以艾司唑仑为内标,样本碱化后用乙醚捉取2次,合并有机相,40℃氮气吹干,100μL流动相定容;用 BDS HYPERSIL C_(18)反相色谱柱(250mm×4.6mm,5μm)进行分离,以乙腈-缓冲溶液(30 mmol·L~(-1)醋酸铵 2.6mmol·L~(-1)甲酸 0.13 mmol·L~(-1)三氟乙酸)(40:60,v/v)为流动相,采用梯度洗脱程序,柱温为50℃,流速为1.0 mL·min~(-1)。采用质谱电喷雾电离源(ESI)将样品离子化,选择性离子监测(SIM)准分子离子和/或主要碎片离子峰。结果:VEN、ODV、NDV、DDV 以及内标艾司唑仑在6min 内完全分离;各物质在2～900 ng·mL~(-1)时线性关系良好,相关系数均大于0.9991;萃取回收率均大于77%;方法回收率均大于91%;最低检测浓度:VEN 0.4 ng·mL~(-1)、ODV0.2 ng·mL~(-1)、NDV 0.3 ng·mL~(-1)、DDV 0.2 ng·mL~(-1);日内日间RSD 均小于11%。结论:本方法简单快速,灵敏准确,可用于药物动力学、药物代谢机制以及血药浓度的临床监护、中毒分析的研究。