Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.     

Citrus Flavonoids in Fruit and Traditional Chinese Medicinal Food Ingredients in China

Flavonoids-enriched tissues of citrus such as peel, immature fruit and flower are consumed as culinary seasonings, tea ingredients in China for centuries. This HPLC quantitative study on the five citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin on a wide range of Chinese citrus fruits and several Traditional Chinese Medicinal food ingredients in East China, revealed a great diversity in flavonoid composition. Huyou peel (C. paradisi cv. Changshanhuyou) was found to be the best naringin (3.25%) and neohesperidin (2.76%) source; C. aurantium, a major ingredient of several citrus-related TCM, is also a suitable source of naringin and neohesperidin, and a good juice source for flavanone glycosides; the peel of Wenzhoumiju (C. unshiu) is one of the richest local species in hesperidin (up to 6.25%); Zaoju (C. subcompressa) has the highest content of nobiletin (0.59%), a polymethoxylated flavone. LC-ES-MS analysis of Zanthoxylum genus for flavonoids revealed for the first time the presence of significant amounts (0.74%) of hesperidin in the root of Liangmianzhen (Z. nitidum (Roxb.) DC), a relative of Sichuan pepper, which is a spice widely used in China.     

Clinical Pharmacokinetics and Effects of Vincristine Sulfate in Dogs with Transmissible Venereal Tumor (TVT)

This study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (TVT). Dogs with TVT were intravenously administered vincristine sulfate at a dose of 0.7 mg/m² of body surface area. Blood samples were collected starting from 5 min to 48 hr after drug administration. The plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. The volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 ± 0.210 l/kg, 21.5 ± 6.90 min, 47.6 ± 14.2 min and 0.010 ± 0.001 l/min/kg, respectively. Tumor regression was determined at weekly interval by a physical examination and histopathological analysis. In our study, three to eight administrations of vincristine at a dose of 0.7 mg/m² were able to induce a complete tumor regression without any evidence of gross lesion of disease. Therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with TVT, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug’s clinical effects. In addition, we validated the LC-MS/MS method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.     

超高效液相色谱——串联质谱内标法测定兔肉中三聚氰胺含量

本文的主要目的在于建立一种准确、可靠的方法来测定兔肉中的三聚氰胺,为此,我们在LC-MS/MS检测三聚氰胺的分析过程中引入了三聚氰胺-15N3作为内标物。它对于分析全过程中所导致的目标物损失给予了完全的校正,改善了方法的精密度。样品经乙腈水加盐酸提取目标分析物,经MCX柱净化分离,选择反应监测（SRM）正离子模式测定。该方法的检测限为0.05ug/kg,在10.0-500ng/ml时峰强度与质量浓度的线性关系良好（r为0.99995）。方法的平均回收率为80-118%。     

小麦叶片蛋白质组的2D-LC分离及Nano LC-MS/MS分析

 【目的】二维液相色谱（2D-LC）与双向电泳（2-DE）技术具有互补性,本文旨在探讨利用2D-LC和纳升级液相色谱串联质谱（Nano LC-MS/MS）技术分离鉴定小麦叶片蛋白质组的新方法。【方法】小麦叶片蛋白经提取、脱盐后,进行第一维阴离子交换分离;收集洗脱组分进行SDS-PAGE分析,并对非高丰度蛋白组分进行第二维反相液相色谱分离;随机选择含较低吸收峰的部分组分经胰蛋白酶水解后进行Nano LC-MS/MS 分析;将串联质谱数据通过MASCOT搜索NCBInr和EST数据库,并对二级质谱获得的蛋白序列进行MS BLAST分析。【结果】经第一维阴离子交换色谱分离,收集得到15个组分,其中第15组分包含小麦叶片丰度最高的蛋白——1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO）;其余14个组分经反相液相色谱分离,共收集1 551个组分,获得1 867个色谱峰;随机对其中6个含较低吸收峰的收集组分酶解和Nano LC-MS/MS 检测,9种蛋白得到鉴定。【结论】结果初步表明,利用2D-LC和Nano LC-MS/MS技术可有效地分离和鉴定小麦叶片蛋白质组,为更深入全面地研究小麦叶片蛋白质组奠定基础。     

基于LC-MS技术的代谢组学方法研究根肿菌对拟南芥代谢的影响

为探寻根肿菌侵染拟南芥后期寄主代谢的生物标志物,将罹患根肿病的油菜根切块进行组织培养,获得较为纯化的、单一的根肿菌接种体,与拟南芥种子在1/2MS培养基上共培养,利用液相色谱-质谱联用(LCMS)技术的非靶向代谢组学方法分析根肿菌侵染后期拟南芥根部代谢物差异。组织培养结果表明,根肿菌可以在愈伤组织中生长和繁殖;共培养结果显示45d后拟南芥侧根尖端肿大,显微观察显示根肿菌能完成侵染循环;代谢组数据PCA和PLS-DA分析结果显示试验组(接种根肿菌)和对照组(未接种根肿菌)明显分离;OPLSDA分析和t检验结果表明重要的差异代谢物包括油菜素内酯、类黄酮、木质素、萜类、甾体皂苷和磷脂等物质。提示这些物质可能在根肿菌侵染后期时拟南芥与根肿菌的相互作用中起重要作用。     

QuEChERS-高效液相色谱-串联质谱法分析甘蓝和土壤中的鱼藤酮

采用QuEChERS-液相色谱-串联质谱法(QuEChERS and LC-MS/MS)建立了鱼藤酮在甘蓝及土壤中的残留检测方法。样品前处理采用以乙腈作提取剂、PSA(primary sec-ondary amine,N-丙基乙二胺)为分散净化剂的QuEChERS方法,利用高效液相色谱-串联质谱(HPLC-MS/MS)在多反应离子检测模式下进行检测。在甘蓝和土壤中的添加水平为0.002-0.1mg/kg条件下,鱼藤酮在甘蓝中的添加回收率为78.36%～88.16%(相对标准偏差为6.05%～13.38%),在土壤中的添加回收率为77.94%～89.50%(相对标准偏差为4.70%～12.35%)。鱼藤酮在甘蓝和土壤中的最低检测浓度为0.002mg/kg,最小检出量为1.0×10-11g。该方法样品前处理简单、快速,准确度、精密度和灵敏度均符合农药残留检测要求,适用于甘蓝和土壤中鱼藤酮残留的检测。     

质谱法在食品样本农药残留分析中的应用进展

质谱法(Mass spectrometry,MS)是当前农药残留分析中的主导检测技术。对气相色谱-质谱法(GC-MS)、气相色谱-串联质谱法(GC-MS/MS)、液相色谱-串联质谱法(LC-MS/MS)和飞行时间质谱法(time-of-flight mass spectrometry,TOF-MS)在食品样本农药残留分析中的应用进行了综述,重点对质谱技术在国内外多残留分析方法研究中的应用进展进行了总结和归纳,简要比较了质谱分析时常用的几种样品前处理技术的优缺点及其适用性,展望了质谱法在农药残留分析研究领域的发展前景。     

LC-MS/MS法研究甲磺隆和氯磺隆在不同条件土壤中的残留行为

在建立液相色谱三重串联四极杆质谱（LC-MS/MS）检测技术的基础上,采用PVC材料的圆柱形盆钵栽培方法,研究了不同土壤环境条件下甲磺隆和氯磺隆的残留特性。结果表明,甲磺隆和氯磺隆在水稻根际和非根际土壤中的残留量均呈不断下降趋势。处理后15d,根际和非根际土壤中两种农药残留量分别下降了44.7%和41.5%（甲磺隆）及38.7%和40.1%（氯磺隆）,根际和非根际的残留差异不显著（P〉0.05）;处理后31d,残留量分别下降了77.7%和64.8%（甲磺隆）及62.7%和50.1%（氯磺隆）,差异达极显著水平（P〈0.01）;处理后63d,残留量分别下降了96.4%和85.1%（甲磺隆）及90.0%和79.4%（氯磺隆）,残留差异达极显著水平（P〈0.01）。甲磺隆的降解趋势和氯磺隆基本一致,但下降幅度比氯磺隆大。二者在水稻根际和非根际土壤中的残留量均符合一级动力学方程式C=C0e-λ（tC代表浓度;C0代表初始浓度;t时间）,决定系数范围在0.9342～0.9957之间。在种植水稻的条件下,下层土的农药残留量呈先上升后下降的趋势,处理后数日内达最高点,之后不断下降,122d后低于检测限。水稻下层土残留的从无到有说明农药在土壤中的淋溶可能是水田环境农药残留降解的原因之一。干旱土壤条件下,土壤的淋溶作用不明显。说明水旱轮作有利于农药残留在土壤环境中的降解。     

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为探明烯酰吗啉在不同土壤中的降解状况,采用LC-MS/MS检测方法,对烯酰吗啉在不同土壤中的降解进行测定.结果表明:烯酰吗啉在土壤中的添加回收率为85.55％～99.52％,相对标准偏差(RSD)为0.58％～1.75％,土壤中含水率越多,微生物越多,有机质越多,烯酰吗啉在土壤中的降解越快.烯酰吗啉在土壤中的降解主要受微生物和有机质含量影响.