基于UHPLC-MS/MS的药用植物内源性激素含量检测方法及黄花蒿不同器官激素的测定

目的 激素是调控植物活性次生代谢产物合成途径的核心调控因子，内源激素结构多样、含量极低，检测难度高，缺乏高效、准确的检测方法。本研究利用超高效液相色谱串联质谱（UHPLC-MS/MS）技术构建了对黄花蒿中茉莉酸、细胞分裂素、赤霉素、生长素、脱落酸等多类植物内源性激素高效定量检测方法。方法 使用Agilent Poroshell 120 EC-C18（2.7 μm， 3.0×150 mm）色谱柱，流动相为0.05%甲酸水-乙腈溶液，梯度洗脱，采用多反应监测模式验证方法可行性，并对黄花蒿不同器官中植物激素进行定量分析。结果 本方法中检测的16种植物激素均呈良好线性，加样回收率、精密度和稳定性均符合植物样品的测定要求。利用此方法测定青蒿不同器官的激素含量，结果表明脱落酸、茉莉酸、茉莉酸甲酯均在叶中含量较高，赤霉素和生长素在根部含量高，细胞分裂素在叶和茎中含量高。结论 青蒿不同器官的激素测定结果可进一步印证脱落酸、茉莉酸、茉莉酸甲酯在调控青蒿素合成中起着重要作用的相关报道，同时表明该方法检测性良好。本文构建的植物内源性激素的检测方法为进一步探究植物生长发育、抵御外界胁迫的相关机制及次生代谢产物的合成等方面提供了手段。     

Hepatotoxicity of cantharidin is associated with the altered bile acid metabolism

Cantharidin (CTD) is an effective antitumor agent. However, it exhibits significant hepatotoxicity, the mechanism of which remains unclear. In this study, biochemical and histopathological analyses complemented with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabolomic analysis of bile acids (BAs) were employed to investigate CTD-induced hepatotoxicity in rats. Sixteen male and female Sprague–Dawley rats were randomly divided into two groups: control and CTD (1.0 mg/kg) groups. Serum and liver samples were collected after 28 days of intervention. Biochemical, histopathological, and BA metabolomic analyses were performed for all samples. Further, the key biomarkers of CTD-induced hepatotoxicity were identified via multivariate and metabolic pathway analyses. In addition, metabolite–gene–enzyme network and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify the signaling pathways related to CTD-induced hepatotoxicity. The results revealed significantly increased levels of biochemical indices (alanine aminotransferase, aspartate aminotransferase, and total bile acid). Histopathological analysis revealed that the hepatocytes were damaged. Further, 20 endogenous BAs were quantitated via UHPLC-MS/MS, and multivariate and metabolic pathway analyses of BAs revealed that hyocholic acid, cholic acid, and chenodeoxycholic acid were the key biomarkers of CTD-induced hepatotoxicity. Meanwhile, primary and secondary BA biosynthesis and taurine and hypotaurine metabolism were found to be associated with the mechanism by which CTD induced hepatotoxicity in rats. This study provides useful insights for research on the mechanism of CTD-induced hepatotoxicity.     

Analytical method for detection and quantification of new emerging drug etaqualone in human blood and urine by gas chromatography tandem mass spectrometry

Analytical procedure for detection and quantification of etaqualone in human blood and urine using GC–MS/MS was established and applied to authentic human samples obtained from volunteers. A liquid–liquid extraction method was employed. Each 1.0 mL of blood or urine was alkalized and extracted with diethyl ether. The solvent layer was evaporated to dryness and reconstituted with methanol then analyzed by GC–MS/MS. linear relationships within the concentration range of 1–100 ng/mL were obtained in calibrators for both blood and urine, demonstrating correlation coefficients values being>0.999. For blood and urine samples, the intra-day assay precision and accuracy values are each less than 3.65%, 7.13%, and 6.02%, 9.12%; those values of the inter-day assay are each less than 1.82%, 6.74%, and 3.99%, 7.41%. The extraction recovery rates for etaqualone ranged from 98.7% to 106%. The lower limit of quantifications was 1.0 ng/mL in both blood and urine. Stabilities of etaqualone in blood and urine were satisfactory under various temperatures within 15 days. 8.51 and 2.06 ng/mL of etaqualone in blood and urine were detected at 4 h later oral ingestion; 6.91 and 3.94 ng/mL of etaqualone were also detected 30 min and 2 h later smoking from blood and urine.     

一测多评法同时测定艾纳香油中5种成分

目的建立一测多评法同时测定艾纳香Blumea balsamifera(L.)DC.油中β⁃蒎烯、β⁃石竹烯、樟脑、α⁃石竹烯和龙脑5种成分的含有量。方法艾纳香油乙酸乙酯提取物的分析采用PEG⁃20 M柱(30 m×0.32 mm,1.0μm);程序升温;载气为高纯氮气(99.999%);FID检测器温度240℃,进样口温度240℃。以龙脑为内标,计算其他4种成分的相对校正因子,再测定其含有量。结果蒎烯、β⁃石竹烯、樟脑、α⁃石竹烯和龙脑分别在1.49~59.5μg/mL(r=0.9996)、2.22~88.8μg/mL(r=0.9996)、6.48~259μg/mL(r=0.9997)、3.64~146μg/mL(r=0.9991)和16.4~656μg/mL(r=0.9998)范围内线性关系良好,平均加样回收率(RSD)分别为97.4%(0.9%)、99.0%(1.3%)、98.9%(0.9%)、97.6%(0.9%)和99.7%(1.0%)。一测多评法所得结果接近于外标法。结论该方法准确稳定,重复性好,可用于艾纳香油的质量控制。     

基于UPLC-Q-TOF-MS/MS技术快速鉴定降香中化学成分

目的:降香为我国传统名贵中药材,然目前市场上降香来源品种复杂,质量差异较大,为进一步明确其药效物质基础,采用超高效液相色谱-串联四极杆飞行时间高分辨率质谱(UPLC-Q-TOF-MS/MS)技术快速分析降香甲醇提取物中的化学成分。方法:采用UPLC RRHD SB-C18色谱柱(3.0 mm×100 mm,1.8μm),以0.1%甲酸水溶液-乙腈为流动相进行梯度洗脱,流速0.3 mL·min^-1,柱温40℃;采用电喷雾离子源,负离子模式采集数据。结果:通过一级精确荷质比和二级碎片信息数据,结合文献资料,质谱裂解规律,Mass bank质谱数据库及对照品的保留时间等,从降香的甲醇提取物中初步鉴定83个化学成分,包括18个黄酮类,31个异黄酮类,10个新黄酮类,9个异黄烷类,7个其他类型黄酮和8个其他类成分。结论:UPLCQ-TOF-MS/MS技术方法可快捷、准确、较全面地鉴定降香甲醇提取物中的化学成分,降香的主要化学成分为异黄酮、黄酮、新黄酮、异黄烷等黄酮类,为降香的药效物质基础研究奠定基础,也为降香药材的质量标准提升提供理论依据和技术支持。     

UPLC-MS/MS测定人痰液中异烟肼、吡嗪酰胺和左氧氟沙星的浓度

目的建立超高效液相色谱串联质谱法(UPLC-MS/MS)同时检测痰液中3种药物异烟肼(isoniazid,INH)、左氧氟沙星(levofloxacin,LFX)和吡嗪酰胺(pyrazinamide,PZA)浓度的方法,冒在为实现临床个体化用药提供帮助。方法痰液样品离心后经甲醇/乙腈蛋白沉淀法预处理后进样液质联用仪进行分析。实验采用的流动相A为含0.1%甲酸的水溶液,流动相B为含0.1%甲酸的乙腈;流速0.35mL·min^-1;采用ACQUITY UPLAC HSS T31.8μm column(2.1mm×100mm,Waters公司)分离;采用电喷零电离源,采用多反应监测(multiple reaction monitoring,MRM)模式对待测物进行正离子扫描检测。此外,本试验盲态收集5例患者的痰液,用该法进行定量分析。结果测得痰液中INH、PZA和LFX分别在48~6000 ng·mL^-1(r=0.9988)、480~60000 ng·mL^-1(r=0.9993)、120~15000 ng·mL^-1(r=0.9995)内表现出良好的线性关系。INH、PZA和LFX 3种药物的日内和日间精密度均低于15%,且3种药物的提取回收率均处于97.21%~107.80%之间。7例受试者体内均检测到药物INH,质量浓度分别为44.74、120.1、301.5、481、595.5、1220及1570 ng·mL^-1;PZA的质量浓度分别为104.2、6273.34和3185 ng·mL^-1;1例受试者检出LFX,质量浓度为199.86 ng·mL^-1。结论本试验建立了痰液中INH、PZA及LFX的检测方法,具有灵敏度高、测定结果准确、快速等诸多优点,可以应用于临床治疗药物的监测。     

利胆软坚方降脂减肥作用及其对胆汁酸代谢轮廓谱的影响

目的:基于肥胖“从胆论治”的学术观点,研究利胆软坚方对高脂饮食诱导肥胖大鼠的降脂减肥作用及其对血清胆汁酸轮廓谱的影响,探讨其作用机制。方法:42只大鼠高脂饲料喂养9周建立肥胖大鼠模型,取24只造模成功大鼠随机分为模型组、利胆软坚方高、低剂量组(30,15 g·kg^-1),每组8只,另取8只正常大鼠作为正常组,模型组和正常组给予生理盐水,给药组给予相应剂量药物,灌胃4周。测定大鼠体质量、肝脏质量、脂肪质量等肥胖指标;胆管插管术引流监测2 h内胆汁流量;全自动生化仪测定血清总胆固醇(TC),甘油三酯(TG),高密度脂蛋白胆固醇(HDL-C),低密度脂蛋白胆固醇(LDL-C)水平;超高效液相色谱-质谱联用(UPLC-MS/MS)法测定大鼠血清胆汁酸代谢轮廓谱中各胆汁酸的含量。结果:与正常组比较,模型组体质量、肝脏质量、脂肪质量均显著升高(P<0.01),血清TC,TG,LDL-C水平明显上升(P<0.05,P<0.01),2 h内胆汁总分泌量及各测试点胆汁流量均降低,初级胆汁酸占比明显下降(P<0.05),血清总胆汁酸含量显著降低(P<0.01),血清胆汁酸轮廓谱中胆酸(CA),脱氧胆酸(DCA),鹅去氧胆酸(CDCA),猪去氧胆酸(HDCA),牛磺胆酸(TCA),牛磺去氧胆酸(TDCA),牛磺鹅去氧胆酸(TCDCA),牛磺猪去氧胆酸(THDCA),甘氨去氧胆酸(GDCA)含量明显降低(P<0.05,P<0.01);与模型组比较,利胆软坚方高、低剂量组体质量、肝脏质量明显降低(P<0.05,P<0.01),利胆软坚方高剂量组血清TC,TG,LDL-C水平明显下降(P<0.05,P<0.01),低剂量组TG水平明显下降(P<0.05),高剂量组在给药后1~1.5 h胆汁流量明显增加(P<0.05),高、低剂量组初级胆汁酸占比明显升高(P<0.05),利胆软坚方高剂量组TCA,DCA,甘氨胆酸(GCA),GDCA水平明显升高(P<0.05,P<0.01),低剂量组仅TCA,TCDCA水平明显升高(P<0.05)。结论:利胆软坚方具有降脂减肥作用,其机制可能与其增加胆汁分泌量,增加初级胆汁酸合成及调节胆汁酸轮廓谱有关。     

泥附子商品等级划分合理性分析

目的分析泥附子商品等级划分的合理性。方法采用HPLC-MS/MS法同时测定不同等级附子中乌头碱、新乌头碱、印乌头碱、次乌头碱、苯甲酰乌头原碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱、乌头原碱、尼奥林、附子灵、宋果灵、塔拉乌头胺、去甲乌药碱、去甲猪毛菜碱14种生物碱的含有量,并采用聚类分析初步验证附子分级的科学性;采用冰醋酸扭体法,以小鼠扭体次数和扭体潜伏期为考察指标,研究不同等级附子水提液的镇痛效果;采用大鼠尾静脉推注测定最小致死量,研究不同等级附子70%醇提液的毒性差异。结果 14种生物碱在不同等级附子中含有量存在显著差异。随着附子质量的增大,双酯型生物碱的含有量呈降低趋势,强心成分呈升高趋势,单酯类生物碱变化不明显;各等级镇痛效果无显著性差异,但小鼠扭体潜伏期存在缩短趋势;质量大于40 g的附子毒性明显低于小于40 g附子。结论附子分级在毒性评价上具有显著意义,质量大的附子具有毒性小的特点,而在镇痛疗效上并无明显差异。大个头附子强心成分具有升高趋势,但体内活性还需进一步验证。     

Comparative single dose pharmacokinetics and metabolism of racemic primaquine and its enantiomers in human volunteers

Primaquine (PQ) is a racemic drug used in treatment of malaria for six decades. Recent studies suggest that the two enantiomers of PQ are differentially metabolized in animals, and this results in different pharmacological and toxicological profiles. The current study characterizes the pharmacokinetic (PK) properties, metabolism and tolerability of the individual enantiomers of PQ in healthy human volunteers with normal glucose-6-phosphate dehydrogenase (G6PD) activity. Two cohorts (at two dose levels), each with 18 subjects, participated in three study arms in a crossover fashion: a single dose of the (−)-R enantiomer (RPQ), a single dose of the (+)-S enantiomer (SPQ), and a single dose of racemic PQ (RSPQ). PQ and its key metabolites carboxyprimaquine (cPQ) and PQ-N-carbamoyl glucuronide (PQ-N-CG) were analyzed. Clear differences were observed in PK and metabolism of the two enantiomers. Relative PQ exposure was higher with SPQ as compared to RPQ. PQ maximum plasma concentration (C_max) and area under the plasma concentration-time curve were higher for SPQ, while the apparent volume of distribution and total body clearance were higher for RPQ. Metabolism of the two enantiomers showed dramatic differences: plasma PQ-N-CG was derived solely from SPQ, while RPQ was much more efficiently converted to cPQ than was SPQ. C_max of cPQ and PQ-N-CG were 10 and 2 times higher, respectively, than the parent drugs. The study demonstrates that the PK properties of PQ enantiomers show clear differences, and metabolism is highly enantioselective. Such differences in metabolism suggest potentially distinct toxicity profiles in multi-dose regimens, especially in G6PD-deficient subjects.     

A fatal poisoning case of acetone cyanohydrin and citalopram

Acetone cyanohydrin (ACH) is a readily available source of cyanide and is widely used in basic and applied sciences. In toxicology, ACH is classified as extremely hazardous as it readily decomposes on contact with water, with the potential rapid release of highly toxic hydrogen cyanide (HCN). We report the case of a young woman found dead from the intentional ingestion of ACH and citalopram, an antidepressant of the selective serotonin reuptake inhibitor class. The autopsy findings included bright reddish-purple hypostasis and mild pulmonary edema. As ACH can decompose to acetone and HCN, we quantified the concentration of each compound and thiocyanate separately in various body fluids and organs and determined their whole-body distributions by using gas chromatography–mass spectrometry (GC–MS). We observed high concentrations of both acetone and cyanide in the blood (0.63 mg/mL and 17.99 mM, respectively) and gastric contents (9.76 mg/mL and 472.44 mM). The whole-body distributions of acetone and cyanide were similar (i.e., the concentration of each compound was the highest in the lung, followed by the heart, and then the liver). Our results suggest that not only the route of administration but also the dose taken could greatly affect the body distributions of cyanide in humans. In addition, as toxicological screening detected citalopram, which was not prescribed to the deceased, we performed a chiral analysis by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We determined that only (S)-citalopram was ingested antemortem; its concentration was 0.36 μg/mL, which is in the toxic range.