不同种植基地滇重楼根茎中甾体皂苷有效成分差异的研究

目的:探讨不同种植基地滇重楼根茎中甾体皂苷有效成分的差异。方法:采用超高效液相色谱法(UHPLC)测定不同种植基地滇重楼根茎中重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ及总皂苷的含量,并用液质联用(LC-MS)技术定性分析其甾体皂苷有效成分的差异。结果:不同种植基地滇重楼根茎中重楼皂苷Ⅰ及总皂苷含量差异较小,重楼皂苷Ⅱ、Ⅵ、Ⅶ含量差异较大;重楼皂苷Ⅰ、重楼皂苷Ⅲ(薯蓣皂苷)、重楼皂苷Ⅴ、重楼皂苷Ⅶ是不同种植基地滇重楼根茎中甾体皂苷的主要存在形式,不同种植基地滇重楼根茎中重楼皂苷Ⅱ、重楼皂苷H、重楼皂苷Ⅵ、纤细薯蓣皂苷差异较大;根据不同种植基地滇重楼根茎正离子模式下液质数据的主成分分析(PCA)模型可将其分为四类。结论:不同种植基地滇重楼根茎中重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ及总皂苷的含量、甾体皂苷有效成分及化学成分种类差异较大。     

A bioactivity-guided study on the anti-diarrheal activity of Polygonum chinense Linn

Ethnopharmacological relevance

Polygonum chinense Linn., a folk medicine, has long been used for the treatment of diarrhea and enteritis in southwestern China. However, the components responsible for its anti-diarrheal activity are still poorly understood.

Aim of the study

To determine anti-diarrheal activities of Polygonum chinense L. and to identify its active components through bioactivity-guided isolation technique.

Materials and methods

Animals were orally administered with the extract of Polygonum chinense L. The anti-diarrheal effects of 75% ethanol extract, four fractions with different polarities from 75% ethanol extract, different eluates collected from Diaion HP-20 macroporous resin chromatography, ellagic acid and corilagin, were examined based on mouse models of castor oil- and magnesium sulfate-induced diarrhea.

Results

The results showed that the 75% ethanol extract of Polygonum chinense L. exhibited significant anti-diarrheal activities in a dose-dependent manner in two mouse models. Through in vivo bioactivity-guided fractionation processes, n-butanol and aqueous fractions were found to exhibit prominent anti-diarrheal activities, and two major compounds, ellagic acid and corilagin, from these active fractions were found to possess anti-diarrheal effects.

Conclusion

Present study provides evidence of the utilization of Polygonum chinense L. for diarrhea, and ellagic acid and corilagin are two components contributing to the anti-diarrheal effect.     

In vitro characterization of potential CYP‐ and UGT‐derived metabolites of the psychoactive drug 25B‐NBOMe using LC‐high resolution MS

One of the main challenges posed by the emergence of new psychoactive substances is their identification in human biological samples. Trying to detect the parent drug could lead to false‐negative results when the delay between consumption and sampling has been too long. The identification of their metabolites could then improve their detection window in biological matrices. Oxidative metabolism by cytochromes P450 and glucuronidation are two major detoxification pathways in humans. In order to characterize possible CYP‐ and UGT‐dependent metabolites of the 2‐(4‐bromo‐2,5‐dimethoxy‐phenyl)‐N‐[(2‐methoxyphenyl)methyl]ethanamine (25B‐NBOMe), a synthetic psychoactive drug, analyses of human liver microsome (HLM) incubates were performed using an ultra‐high performance liquid chromatography system coupled with a quadrupole‐time of flight mass spectrometry detector (UHPLC‐Q‐TOF/MS). On‐line analyses were performed using a Waters OASIS HLB column (30 x 2.1 mm, 20 µm) for the automatic sample loading and a Waters ACQUITY HSS C18 column (150 x 2 mm, 1.8 µm) for the chromatographic separation. Twenty‐one metabolites, consisting of 12 CYP‐derived and 9 UGT‐derived metabolites, were identified. O‐Desmethyl metabolites were the most abundant compounds after the phase I process, which appears to be in accordance with data from previously published NBOMe‐intoxication case reports. Although other important metabolic transformations, such as sulfation, acetylation, methylation or glutathione conjugation, were not studied and artefactual metabolites might have been produced during the HLM incubation process, the record of all the metabolite MS spectra in our library should enable us to characterize relevant metabolites of 25B‐NBOMe and allow us to detect 25B‐MBOMe users. Copyright © 2015 John Wiley & Sons, Ltd.     

UHPLC法同时测定复方丁香油涂剂中丁香酚和樟脑的含量

目的建立UHPLC法同时测定复方丁香油涂剂中丁香酚和樟脑的含量。方法采用Agilent Zorbax SB-C18（2.1 mm×50 mm,1.8μm）色谱柱,流动相：甲醇-0.1%甲酸水溶液（58∶42）,检测波长：285 nm,流速：0.2 ml·min^-1,柱温：25℃。结果丁香酚、樟脑分别在27.48~274.80μg·ml^-1（r=0.9995）和0.38~2.28 mg·ml^-1（r=0.9998）范围内与峰面积线性关系良好;平均加样回收率分别为99.98%和100.12%,RSD分别为1.14%和0.75%（n=9）。结论本方法操作快速,结果准确,专属性强,可用于复方丁香油涂剂中丁香酚和樟脑的含量测定。     

A UHPLC/MS/MS Assay Based on an Isotope-Labeled Peptide for Sensitive miR-21 Detection in HCC Serum

Background: MicroRNAs (miRNAs) have been identified as promising novel biomarkers for cancer diagnosis and prognosis, especially for hepatocellular carcinoma (HCC). Nowadays, the expression level of miR-21 in serum samples is a diagnostic indicator for HCC diagnosis. Thus, the quantitative determination of miRNA concentration is of significance in clinical practice. It is particularly important to establish an analytical detection method for miR-21 in patient serum. Methods: The signal readout for miR-21 was based on the mass response of a reporter peptide using an isotope dilution mass spectrometry (MS) method in this work. To be more specific, miR-21 was biotinylated before being coupled with streptavidin (SA) agarose and then hybridized with a newly synthesized DNA-peptide probe. The release and purification of the sample was based on the method including trypsin digestion, solid-phase extraction, and drying, and the detection of the reporter peptide was carried out by UHPLC/MS/MS. The miR-21 in the corresponding samples was quantified by the ratio of the chromatographic peak area of the redissolved polypeptide to that of the isotope-labeled polypeptide. Additionally, within the calibration range, the performance of the method (including precision, accuracy, linearity, and recovery) was evaluated. Results: The concentration of miR-21 was determined using the ratio of relative peak area of stable isotope-labeled internal standard and reporter peptide, yielding a linear range of 0.1∼30.0 nM (y = 0.0818x + 0.7554, R² = 0.9586, P < 0.01). The limit of detection (LOD) for miR-21 was 10 pM. For y⁵ , the recoveries (n = 3) were 91.36 ± 2.19%, 93.64 ± 3.55%, and 96.04 ± 2.02% for the levels of three miR-21 samples including RL, RM, and RH, respectively. Conclusions: Overall, this research provides a novel analytical approach for quantitative detection of miRNAs in clinical serum samples.     

HPLC法分析补肺活血胶囊中12种指标成分

目的 建立超高效液相色谱法（UHPLC）同时测定补肺活血胶囊（Bufei Huoxue Capsules,BHC）中12种指标成分没食子酸、氧化芍药苷、芍药内酯苷、补骨脂苷、芍药苷、异补骨脂苷、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、补骨脂素、异补骨脂素、苯甲酰芍药苷的含量。方法 采用Acquity UPLC BEH C18色谱柱（100 mm×2.1 mm,1.7μm）;流动相为乙腈-0.1%甲酸水溶液,梯度洗脱;体积流量0.4 mL/min,柱温35℃,进样量4μL,检测波长246 nm。结果 BHC中12种指标成分在18min内色谱峰分离良好,没食子酸、氧化芍药苷、芍药内酯苷、补骨脂苷、芍药苷、异补骨脂苷、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、补骨脂素、异补骨脂素和苯甲酰芍药苷分别在0.38～195.20(r=0.999 9)、0.17～21.44(r=0.999 6)、0.85～433.80(r=0.999 9)、0.68～345.60(r=0.999 9)、1.11～566.60(r=0.999 8)、0.47～241.40(r=0.999 9)、0.08～39.36(r=0.99...     

基于质量源于设计理念的制川乌UHPLC分析方法研究

目的 基于质量源于设计理念(quality by design, QbD)建立制川乌的超高效液相色谱( ultra high performance liquid chromatography,UHPLC)分析方法。方法 以制川乌中6个主要成分的定量分析为目标,选取柱温、醋酸铵溶液浓度、流速、pH值、初始乙腈浓度为考察因素进行Plackett-Burman试验设计,根据试验结果确定关键方法参数进行Box-Behnken实验,建立设计空间并选择置信度为95%的置信区间估计法考察设计空间的可靠性,最后选择准确性轮廓方法(accuracy profile,AP)进行方法学验证。以分离度为目标所建最佳模型为二次项式模型,以分析时间为目标所建最佳模型为线性模型。基于以上两个模型建立分析方法的设计空间,并采用置信区间估计对方法的可靠性进行评价。结果 结果显示,从设计空间内选择的30 mmol·L^-1的醋酸铵浓度(pH=10)和体积分数28%的初始乙腈比例,在10 min内可完成制川乌中6生物碱的完全分离,该分析方法的准确性、中间精密度、重复性、真实性都在可接受的范围内。结论 优化后的方法可以用于制川乌的UHPLC分析,QbD理念为开发并优化制川乌中6个生物碱的UHPLC分析方法提供了新的思路。     

基于UHPLC 技术的补骨脂指纹图谱研究

目的：建立补骨脂药材的超高效液相色谱（UHPLC）指纹图谱。方法：以Shim-pack XR-ODSⅢ（50 mmx2.0 mm,1.6 μm）为色谱柱,乙腈-0.2%冰醋酸溶液为流动相,梯度洗脱,流速为0.1 mL·min-1,检测波长为246 nm,柱温45℃。结果：建立了补骨脂药材的UHPLC 指纹图谱,标定10 个共有色谱峰,并通过对照品比对指认了其中6 个色谱峰。结论：该方法快速、可靠、重复性好,可为补骨脂药材的物质基础和质量控制方法的研究提供参考。     

Detection and characterization of the metabolites of rutaecarpine in rats based on ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer

Context: Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese), which possesses a variety of effects. However, its metabolism has not been investigated thoroughly yet.

Objective: This study develops a highly sensitive and effective method for detection and characterization of the metabolites of rutaecarpine in Sprague–Dawley (SD) rats.

Materials and methods: In this study, an efficient method was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UHPLC–LTQ-Orbitrap MS) to detect the metabolism profile of rutaecarpine in rat plasma. First, a blood sample (1?mL) was withdrawn 2?h after oral administration of rutaecarpine in SD rats (50?mg/kg). Second, the blood was centrifuged at 4000?rpm for 10?min and pretreated by solid-phase extraction method. Third, 2?μL of the plasma was injected into UHPLC–LTQ-Orbitrap MS for analysis. Finally, the metabolites of rutaecarpine were tentatively identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times.

Results: A total of 16 metabolites (four new metabolites, viz., dihydroxylation and sulphate conjugation products of rutaecarpine (M8–M11)) as well as parent drug itself, including three phase I and 12 phase II metabolites were detected and identified in rat plasma. Hydroxylation, sulphate conjugation and glucuronidation were confirmed as the primary metabolic pathways for rutaecarpine in rat plasma.

Discussion and conclusion: These results provide an insight into the metabolism of rutaecarpine and also can give strong indications on the effective forms of rutaecarpine in vivo.     

UHPLC法同时测定铁苋菜中4种成分含量

目的 采用UHPLC法构建铁苋菜中没食子酸,原儿茶酸,没食子酸甲酯,芦丁等4种成分的含量测定方法。方法 固定相为Poroshell 120 SB-C₁₈ (3.0 mm×150 mm,2.7 μm)色谱柱;流动相为A相0.1%甲酸-水,B相0.1%甲酸-乙腈,梯度洗脱（0~3 min,5%B→10%B;3~5 min,10%B→25%B;5~10 min,25%B→30%B）柱温40 ℃,流速0.6 mL·min^-1,检测波长265 nm。结果 4种成分的线性关系良好,平均加样回收率为100.5%~100.9%,RSD 均小于3.0%;10批样品中4种成分的含量分别为0.27~0.71,0.57~1.64,2.15~4.36,0.53~1.65 mg·g^-1。结论 该方法简便高效,稳定准确,有助于铁苋菜药材的全面质量控制。