制貂肾中黄曲霉毒素的测定方法研究及暴露风险评估

目的:本研究基于免疫亲和柱净化样品,采用高效液相色谱法光化学衍生荧光检测器测定制貂肾中黄曲霉毒素B₁、B₂、G₁、G₂的含量并对其进行暴露风险评估。方法:样品采用70%甲醇作为提取溶剂,经免疫亲和柱净化、高效液相色谱分离、光化学柱后衍生后,通过荧光检测器测定其中黄曲霉毒素的含量。采用暴露边界比对制貂肾黄曲霉毒素的有害残留物进行风险评估。结果:结果表明黄曲霉毒素B₁的线性范围为0.010 4～0.052 0 ng(r=0.999 9)、黄曲霉毒素B₂的线性范围为0.003 8～0.019 0 ng(r=0.999 8)、黄曲霉毒素G₁的线性范围为0.010 8～0.054 0 ng(r=0.999 8)、黄曲霉毒素G₂的线性范围为0.003 8～0.019 0 ng(r=0.999 8),线性关系良好,回收率在89.31%～99.54%间,RSD≤3.1%。结论:建立的制貂肾中黄曲霉毒素检测方法具有操作简单,灵敏度...     

UPLC-MS/MS法分析非酒精性脂肪肝病小鼠血清中IAA异常变化

目的：建立超高效液相色谱-串联质谱（UPLC-MS/MS）法定量检测小鼠血清中吲哚乙酸（IAA）,考察非酒精性脂肪肝病（NAFLD）小鼠血清中IAA水平。方法：采集对照组与NAFLD组小鼠的血清,UPLC-MS/MS法定量检测IAA含量。色谱条件：色谱柱为BEH C₁₈（2.1 mm×50.0 mm,1.7 μm）,流动相为0.01%甲酸乙腈-0.01%甲酸水,梯度洗脱,流速为0.4 mL·min^-1,电喷雾正离子模式,多反应监测（MRM）。结果：IAA质量浓度在5~1 000 ng·mL^-1（r²=0.996 4）范围内线性关系良好,日间和日内精密度RSD均<15%,提取回收率为76.83%~98.8%,基质效应为90.2%~103.8%,稳定性良好。对照组小鼠血清中IAA浓度为（130.65±31.23） ng·mL^-1,NAFLD组小鼠血清中IAA浓度为（28.72±4.83） ng·mL^-1。结论：本研究建立的方法简单,专属性高,稳定性好,可准确检测小鼠血清中IAA的含量。NAFLD组小鼠血清IAA表达显著低于对照组,IAA可能是NAFLD诊断的潜在标记物。     

超高效液相色谱-质谱联用法检测人全血中氯喹、羟氯喹、阿比多尔及其临床应用

目的: 建立并验证一种人全血中氯喹、羟氯喹、阿比多尔的检测方法,并将其用于临床检测。方法: 全血样本经氯化铵裂解和乙腈蛋白沉淀后,采用液相-串联质谱联用仪测定。色谱柱为BEH C₁₈,以含0.5%甲酸乙腈溶液-0.5%甲酸水溶液为流动相梯度洗脱,流速为0.4 mL·min^-1,电喷雾正离子模式,多反应监测。结果: 氯喹、羟氯喹、阿比多尔在8~1 600 ng·mL^-1线性关系良好;批内和批间精密度(RSD)均在12.71%以内,批内和批间准确度(RE)均在-7.12%~13.51%;提取回收率均在76.50%~92.49%;内标归一化的基质因子变异系数(RSD)均小于15%;在多种储存条件下,处理前后的样品稳定性良好。结论: 该方法专属性强,准确度高,重复性好,可用于3种药物治疗药物监测及中毒检测。     

柱前衍生化法UPLC-MS/MS检测肿瘤患者不同化疗周期顺铂血药浓度

目的：建立定量检测肿瘤患者血浆中顺铂浓度的柱前衍生化超高效液相色谱-串联质谱（UPLC-MS/MS）法,比较顺铂（75 mg·m^-2·d^-1） QD*1 d和（25 mg·m^-2·d^-1） QD*3 d在不同化疗周期顺铂血药浓度的变化。方法：血浆样品的顺铂与二乙基二硫代氨基甲酸钠（DDTC）络合生成Pt （DDTC）衍生化产物,经蛋白沉淀后,采用UPLC-MS/MS测定,色谱柱为BEH C₁₈（2.1 mm×50.0 mm,1.7 μm）,流动相为乙腈-0.1%甲酸水,梯度洗脱,流速为0.35 mL·min^-1,采用电喷雾正离子模式,以多反应监测（MRM）定量分析。结果：顺铂在20~3 000 ng·mL^-1（r²=0.995 4）具有良好的线性范围;批内RSD在7.21%~12.06%,批间RSD在4.64%~11.17%;提取回收率为78.48%~99.2%,基质效应在90.7%~94.8%,稳定性良好。顺铂（75 mg·m^-2·d^-1） QD*1 d在整个化疗周期中血药浓度呈上升趋势,顺铂（25 mg·m^-2·d^-1） QD*3 d血药浓度呈下降趋势。结论：本研究建立的方法专属性高、稳定性好,适用于肿瘤患者血浆中顺铂浓度的检测。     

UPLC-MS/MS法与ELISA法测定胖大海中黄曲霉毒素的比较研究

目的 采用超高效液相色谱-串联质谱法(UPLC-MS/MS)和酶联免疫吸附法(ELISA)对胖大海中的黄曲霉毒素进行检测，比较 两种方法的检测结果以及方法学差异性。方法UPLC-MS/MS法测定：样品采用70%甲醇提取经过免疫亲和柱净化，色谱柱为C₁₈(100 mm×2.1 mm,1.7μm)，柱温为40℃，流动相为水含0.1%甲酸(A)-甲醇含0.1%甲酸(B)，梯度洗脱，流速为0.3 m L·min^-1，多反应检测扫描采集模式，正负离子同时扫描进行数据采集。同时采用ELISA法测定，样品采用甲醇提取，酶联免疫试剂盒反应。结果 UPLC-MS/MS法测定：黄曲霉毒素B₁、B₂、G₁、G₂线性关系良好，检测限分别为0.013 5,0.002 9,0.008 6,0.003 6μg·kg^-1，定量限分别为0.045 0,0.009 8,0.028 6,0.011 9μg·kg^-1，平均回收率分别为98.98%,10...     

UPLC-MS/MS与G1-熵权法多指标综合评分优化净石合剂提取工艺

目的:建立超高效液相色谱串联三重四级杆质谱法(UPLC-MS/MS)同时测定净石合剂水提物中12个指标性成分含量(京尼平苷酸、绿原酸、去甲异波尔定、苦杏仁苷、王不留行黄酮苷、毛蕊花糖苷、β-蜕皮甾酮、甘草苷、槲皮素、山柰酚、甘草酸、乌药醚内酯),并优化其水提工艺。方法:采用UPLC-MS/MS法,正负离子切换多反应监测(MRM)模式进行定量分析。色谱分离采用Waters CORTECS C₁₈色谱柱(2.1mm×100mm,1.6μm),流动相为乙腈(A)-0.1%甲酸水溶液(B),梯度洗脱,流速0.25mL·min^-1,柱温40℃,以上述12个成分的含量及浸膏得率为评价指标,在单因素试验基础上,采用正交试验联用G1-熵权法,以加水量、提取时间、提取次数为考察因素进行提取工艺优化。结果:在优化的色谱与质谱条件下,净石合剂提取物中12个成分在考察的浓度范围线性关系良好(r>0.999),平均加样回收率96.89%~101.07%,RSD<4.6%,得到最佳工艺参数为药材加15倍质量的水浸泡2h,提取2次,每次2h。结论:建立的UPLC-MS/MS同时测定净石合剂中12个成分的方法快速、简便、灵敏度高,优化的提取工艺稳定可行,可为其进一步开发利用奠定基础。     

HPLC法测定150种中药材中黄曲霉毒素G2、G1、B2、B1的含量

目的:建立了HPLC法测定150种中药材中的黄曲霉毒素G₂、G₁、B₂、B₁含量。方法:样品经70%甲醇提取、免疫亲和色谱柱净化后,用HPLC-柱后衍生-荧光检测器测定结果:黄曲霉毒素G₁、B₂在0.15～6.00 ng·ml^-1范围内,黄曲霉毒素C₁、B₁在0.5～20.00 ng·ml^-1范围内线性关系良好回收率为85.6%～92.0%结论:本法操作简便,结果准确、重复性好,可用于中药材中黄曲霉毒素G₂、G₁、B₂、B₁的测定     

正交设计优选砂炒牛蒡子的炮制工艺

目的：优选砂炒牛蒡子的最佳炮制工艺。方法：采用正交试验法考察炮制温度、炒制时间、砂用量等影响因素,以砂炒牛蒡子中牛蒡苷含量、爆裂率和煎出率作为评价指标,综合评分优选其工艺条件。结果：优选出最佳炮制工艺为：砂用量为药物重量的12倍,砂炒温度200℃,炒制时间4 min。结论：该炮制工艺可靠、爆裂率高、成品酥脆、质量好,可为工业化生产提供实验数据。     

UPLC-MS/MS快速测定4种抗风湿类中成药中马钱子碱与士的宁的含量

目的：建立UPLC-MS/MS法快速测定风湿马钱片、风湿安泰片、风湿骨康片和舒筋丸中马钱子碱与士的宁的含量。方法：色谱柱为Agilent Zorbax Eclipse Plus C₁₈（2.1 mm×100 mm,1.8 μm）,以0.1%甲酸铵溶液-甲醇（75：25,用甲酸调节pH至3.0）为流动相,流速为0.2 mL·min^-1,柱温为35℃,进样量为1 μL;采用电喷雾离子源（ESI）在正离子模式下,利用多反应监测（MRM）模式对马钱子碱和士的宁进行定量分析。结果：马钱子碱在2.603 7~41.659 0 μg·mL^-1线性关系良好,平均回收率为99.3%（n=9,RSD=1.9%）、士的宁在5.292 3~84.677 1 μg·mL^-1范围内线性关系良好,平均回收率为99.5%（n=9,RSD=1.1%）。结论：本方法快速、灵敏度高、准确可靠,适合多种抗风湿类中成药中马钱子碱与士的宁的定量分析。     

高效液相色谱-串联质谱法测定人腹腔积液中伏立康唑浓度及临床应用

目的: 建立高效液相色谱-串联质谱(HPLC-MS/MS)法测定人腹腔积液中伏立康唑浓度,并应用于临床样本检测。方法: 腹腔积液样品经纯乙腈沉淀蛋白后,通过Hypersil GOLD C₁₈色谱柱分离;流动相为0.1%甲酸水-纯乙腈(60∶40,V/V);流速0.4 mL·min^-1;柱温30 ℃。质谱采用电喷雾电离、正离子多反应监测模式扫描定量:m/z 350.0→281.0(伏立康唑)和m/z 353.1→284.1(伏立康唑-d3,内标)。结果: 腹腔积液样品中伏立康唑浓度在0.05~10.00 μg·mL^-1范围内线性良好(R²=0.999 9)。日内和日间精密度均小于2%,提取回收率为(100.35±4.37)% ~(107.68±3.97)%,内标归一化基质效应因子为(98.36±1.69)%~(100.57±1.10)%且RSD小于2%。此外,各项稳定性考察结果均合格。利用该方法测得4例重度肝硬化患者静脉用伏立康唑首剂给药(约滴注1 h)的腹腔积液0~12 h的浓度-时间曲线下面积(AUC_0-12)和血浆AUC_0-12的比值平均为0.54(范围0.49~0.61),提示肝硬化患者中伏立康唑具有较好的腹膜腔渗透性。结论: 该方法简单、快速、灵敏、准确,适用于人腹腔积液中伏立康唑的检测和临床药动学研究。     

Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China

The multi-mycotoxin occurrence for internal and superficial fungi contamination were comprehensively assessed in medicinal seeds used as food or beverage. Based on a polyphasic approach using morphological characters, β-tubulin and ITS gene blast, a total of 27 species belonging to 12 genera were identified from surface-sterilized seeds. Chaetomium globosporum was most predominant (23%), followed by Microascus trigonosporus (12%) and Alternaria alternata (9%). With respect to superficial mycobiota, thirty-four species belonging to 17 genera were detected. Aspergillus niger and Penicillium polonicum were predominant (12% and 15%, respectively). Medicinal seed samples and potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxins (AFB1, AFB2, AFG1, AFG2) using UPLC-MS/MS. Platycladi seeds were contaminated with AFB1 (52.0 µg/kg) and tangerine seed was contaminated with OTA (92.3 µg/kg). Subsequent analysis indicated that one A. flavus strain isolated from platycladi seed was able to synthesize AFB1 (102.0 µg/kg) and AFB2 (15.3 µg/kg). Two P. polonicum strains isolated from tangerine and lychee seeds were able to synthesize OTA (4.1 µg/kg and 14.8 µg/kg, respectively). These results identify potential sources of OTA and aflatoxins in medicinal seeds and allude to the need to establish permitted limits for these mycotoxins in these seeds that are commonly consumed by humans.     

Relaxant effects of aflatoxins on isolated guinea pig trachea.

Dyspnea is one of the symptoms of acute aflatoxicosis. Contrary to expectations, we observed that naturally occurring aflatoxins (AF) AFB(1), AFB(2), AFG(1), and AFG(2) and their major metabolites AFM(1), AFM(2), AFP(1), AFQ(1), and AFG(2a) relaxed carbachol (C) precontracted guinea pig trachea to different degrees. The efficacies but not the potencies of AFB(1), AFB(2), AFG(1), and AFG(2) were similar to that of the beta-agonist, isoprenaline, whose activity was potentiated by the AF. Their mechanism of action is not clearly understood but several mechanistic indications were obtained with AFB(1): 1) its effect was not influenced by the beta-blocker, timolol, indicating that a direct interaction with beta(2)-adrenergic receptors was not involved. 2) AFB(1) potentiated PGE(1) and PGE(2), two relaxant prostaglandins, and its activity was reduced by indomethacin. 3) The cAMP level in the guinea pig trachea relaxed by AFB(1) increased, possibly due to inhibition of phosphodiesterase; direct interaction with PG receptors; and/or interaction with A(2) adenosinic receptors, suggested by the inhibitory activity of XAC, a specific antagonist. 4) Finally, since tetrodotoxin reduced the relaxant activity of AFB(1), it is speculated that this mycotoxin could stimulate inhibitory nonadrenergic, noncholinergic nerves (i-NANC). In conclusion, the symptoms of acute aflatoxicosis do not seem to be due to a direct activity on the tracheal muscle, but rather, to the well-known pro-inflammatory activity of the aflatoxins, which are capable of releasing arachidonic acid from cell membranes.     

Investigation of aflatoxins contamination in herbal materia medica in a Taiwan pharmaceutical factory

During the years 2005–2016, a total of 1067 samples for 24 types of herbal materia medica were investigated for the presence of aflatoxins (AFs) using immunoaffinity column cleanup and HPLC-coupled to a fluorescence detector after post-column derivatization. AFs were detected in 373 (35%) out of the total samples. Among them, Platycladi Semen (65% for total AFs and 79% for AFB1), Corydalis Rhizoma (53% for total AFs and 32% for AFB1), Corni Fructus (3% for total AFs), Coicis Semen (3% for total AFs and AFB1), Nelumbinis Semen (6% for total AFs and 9% for AFB1), Arecae Semen (18% for AFB1), Polygalae Radix (5% for total AFs and AFB1), and Cassiae Semen (25% for total AFs and 38% for AFB1) exceeded the official limits of 5 and 10 μg/kg, for AFB1 and total AFs (the sum of AFB1, AFB2, AFG1, and AFG2), respectively, set by the Taiwan government. We concluded that Platycladi Semen, Corydalis Rhizoma, and Cassiae Semen are the most commonly contaminated by AFs.     

Acute effects of aflatoxins on guinea pig isolated ileum.

Previous studies on the aflatoxins have focused mainly on their chronic toxic effects. In this study we investigated the acute gastrointestinal effects of four common aflatoxins on isolated guinea pig ileum. AFB(1) (EC(50) 4.6+/-0.4 microM) and AFB(2) (EC(50)17+/-4.4 microM) contracted isolated guinea pig ileum in a dose-dependent manner, whereas AFG(1) and AFG(2) evoked no contractions. Atropine (5.9 nM 11.8 and 23.6 nM) antagonized AFB(1)-induced contractions in a dose-dependent manner. Pretreatment with the nicotinic ganglionic blocker, hexamethonium (up to 55 microM), left AFB(1)-induced contractions unchanged. In contrast, tetrodotoxin (0.3 microM), blocked AFB(1) contractile activity. The two inhibitors of ACh release, morphine (0.3 microM) and clonidine (0.4 microM), antagonized EC(50) AFB(1)-induced contractions, and apamin, a drug that increases neuronal excitability, facilitated the EC(50) AFB(1)-induced contractile effect. The choline uptake blocker, hemicholinium (17.4 microM) markedly reduced AFB(1)-induced contractions. These results suggest that aflatoxins induce their contractile effect indirectly through the cholinergic system by stimulating acetylcholine release from the postganglionic parasympathetic nerve endings. The acute actions of aflatoxins on isolated guinea pig ileum could explain their acute gastrointestinal effects in humans and animals.     

Fumonisin contamination of a corn sample associated with the induction of hepatocarcinogenesis in rats-role of dietary deficiencies.

A corn sample associated with a field outbreak of equine leukoencephalomalacia in Pennsylvania, USA, during 1983/1984 and induced hepatotoxic and hepatocarcinogenic effects when fed to male Fischer rats was analyzed mycologically and chemically for the presence of fumonisins (FB), hydrolysed FB derivatives and aflatoxins (AFB). Fusarium verticillioides was found to be the predominant fungal contaminant in the corn sample but Aspergillus flavus was also present. Trace amounts (0.1 microg/kg) of AFB(1) and AFB(2) and a total FB level of 33.5 mg/kg (FB(1):FB(2):FB(3) ratio of 9:2.3:1) were found. No hydrolysed FB derivatives or AFG(1) and AFG(2) were detected. Based on the chemical stability of the fumonisins in different corn cultures of F. verticillioides kept at 4 degrees C over a period of 13-20 years, a level of approximately 55 mg/kg of total FB is estimated in the original corn sample. A possible role of certain dietary constituents such as the high protein content and deficiencies in certain micronutrients is evaluated to address differences in the organ-specific toxicity of FB(1) in rats using commercial, semi-purified, purified and corn-only diets.     

Inhibition of aflatoxins on UDP-glucuronosyltransferases (UGTs)

Aflatoxins have been recognized as the most harmful mycotoxins leading to various toxic effects. The present study aims to determine the inhibition behavior of aflatoxins on the activity of the important phase II metabolizing enzymes, UDP-glucuronosyltransferases (UGTs), based on in vitro incubation system of recombinant human UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU). 100 μM AFB1 and AFG1 exhibited extensive inhibition towards UGT isoforms especially UGT1A7 and UGT1A8, with the inhibition ratios to be 71.38%, 72.95% and 72.79% for AFB1 to UGT1A7, AFB1 to UGT1A8 and AFG1 to UGT1A8, respectively. Molecular docking results showed that hydrogen bonds and hydrophobic contacts of the particular structure consisting of double furan ring with double bond contributed to the interaction of aflatoxins and UGTs. Kinetics analysis, including inhibition types and kinetics parameters (K_i), and in vitro-in vivo extrapolation (IVIVE) indicated that there might be a medium possibility of inhibition on UGTs by aflatoxins in vivo. In conclusion, the present study indicated that aflatoxins could possibly disturb endogenous metabolism by inhibiting the activity of UGTs so as to exhibit toxic effects.     

基于超高效液相色谱-串联质谱法评价苯二氮胶体金试剂盒的检测性能

目的：采用UPLC-MS/MS定性检测人尿液中常见苯二氮类药物,用其评价苯二氮胶体金试剂盒的检测性能。方法：尿液样品液液萃取后,采用液相-串联质谱联用仪测定,色谱柱为BEH C₁₈,以含0.01%甲酸乙腈溶液-含0.01%甲酸水溶液为流动相梯度洗脱,流速为0.4 mL·min^-1,电喷雾正离子模式,多反应监测。以该UPLC-MS/MS法为对照,参考美国临床实验室标准化委员会发布的EP12-A2文件,分别对苯二氮胶体金试剂盒的灵敏度、准确度、精密度及一致性进行评价。结果：UPLC-MS/MS法检测限在0.41~1.22 ng·mL^-1之间,日内精密度RSD小于<10.81%,日间精密度RSD小于<19.22%,提取回收率均在53.90~69.32%之间。胶体金法阿普唑仑和地西泮阈值浓度为300 ng·mL^-1,艾司唑仑、劳拉西泮、氯硝西泮、三唑仑、咪达唑仑阈值浓度大于360 ng·mL^-1,硝西泮阈值浓度在240~300 ng·mL^-1之间。68例临床样本,采用两种方法检测后,Kappa值为0.12。劳拉西泮、氯硝西泮、阿普唑仑、艾司唑仑、地西泮的阳性预测值在85.7%~100%之间,劳拉西泮、阿普唑仑、艾司唑仑的阴性预测值分别为28.5%、33.3%、25%。结论：两种检测方法的一致性强度较弱。UPLC-MS/MS法具有较高灵敏度和准确性,但样品前处理复杂。苯二氮胶体金试剂盒对于不同的药物具有差异化的检测效能,阳性预测结果的准确率高,而阴性预测结果的准确率较低。     

Measurement of aflatoxin and aflatoxin metabolites in urine by liquid chromatography-tandem mass spectrometry

Automated immunoaffinity solid-phase extraction followed by liquid chromatography-tandem mass spectrometry and chemical analogue internal standardization is employed to detect and quantify the aflatoxins AFB(1), AFB(2), AFG(1), AFG(2), and the metabolites AFM(1) and AFP(1) in urine. The dynamic range of the method is nearly three orders of magnitude with limits of detection in the low femtogram on column range. The method was validated over a 12-day period by eight analysts. This method is suitable for agricultural, forensic, and public health laboratories during an accidental outbreak or a chemical terrorism event where a rapid and accurate diagnosis of aflatoxicosis is needed.