Lipophilic shellfish toxins pose significant threats to the health of seafood consumers and public health. The symptoms of these kinds of toxins include severe diarrhea, abdominal cramps, nausea and gastrointestinal disorders. These symptoms could be hardly distinguished with many other symptoms of food poisoning and diseases. Therefore, a fast and accurate determination method in human biological samples is urgently needed for the accurate judgement of food poisoning incident, which is important for the investigation of public health emergencies and clinical treatment of poisoned patients. However, there were several flaws of the previous studies reported on the analysis of lipophilic shellfish toxins: (1) limited target compounds were covered; (2) the pre-treatment process was complex; (3) the sensitivity of the compound was low. In this study, a simple extraction method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 12 lipophilic shellfish toxins, including azaspir acid 1 (AZA1), azaspir acid 2 (AZA2), azaspir acid 3 (AZA3), dinophysistoxin 1 (DTX1), dinophysistoxin 2 (DTX2), gymnodimine (GYM), hyessotoxin (HYTX), okadaic acid (OA), pinnatoxin (Pntx), pectenotoxins 2 (PTX2), spirolides 1 (SPX1), yessotoxin (YTX), in plasma and urine. Firstly, the instrument conditions were optimized. Different additions in mobile phase were compared and 0.05% (v/v) ammonia solution was selected since it can improve the peak shape of YTX and HYTX, and increase the respondence by four times. Secondly, the volume of acetonitrile (0.2, 0.4, 0.6, 0.8, 1.0 mL) use for the extraction of the target compounds in plasma was optimized. Satisfactory recoveries were obtained when 0.6 mL of acetonitrile was used. At the same time, satisfactory recoveries were obtained when 0.9 mL of acetonitrile was used in urine samples. Finally, under the optimized conditions, the 12 compounds in plasma and urine samples were ultrasonically extracted with acetonitrile. Chromatographic separation was performed on a Phenomenex Kinetex C18 column (50 mm×3 mm, 2.6 μm) with 90% (v/v) acetonitrile aqueous solution and water containing 0.05% (v/v) ammonia as mobile phases. Gradient elution with a flow rate of 0.40 mL/min was employed. The 12 compounds were monitored in the multiple reactions monitoring (MRM) mode with electrospray ionization (ESI) under both positive and negative conditions. The matrix effects of the 12 compounds ranged from 0.8 to 1.1. Therefore, external standard calibration curves were used for the quantification. The 12 shellfish toxins showed good linear relationship in the range of 0.03-36.25 μg/L with the correlation coefficients greater than 0.995. The limits of detection (LODs, S/N=3) were 0.08-0.21 ng/mL for the urine samples and 0.10-0.28 μg/L for the plasma samples, respectively. The limit of quantitations (LOQs, S/N=10) were 0.23-0.63 μg/L for the urine samples and 0.31-0.84 μg/L for the plasma samples, respectively. The recoveries of the 12 compounds were in the range of 72.7%-124.1% at three spiked levels (i. e., LOQ, three times LOQ, and ten times LOQ). The intra-day and inter-day precisions were 2.1%-20.0% and 2.1%-15.3%, respectively. The method was applied in the detection of the 12 lipophilic shellfish toxins in the urine and plasma samples of healthy humans and mice previously injected with the 12 shellfish toxins intraperitoneally. None of the 12 toxins were found in the samples from healthy human, while all of the 12 lipophilic shellfish toxins were found in the urine and plasma samples collected from the poisoned mice in the range of 1.14-2.35 μg/L and 1.01-1.17 μg/L, respectively. The established method has the advantages of sensitive, quick, easy to operate, and of low sample volume. It can be used for the simultaneous determination of 12 lipophilic shellfish toxins in urine and plasma samples.
生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持。目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题。该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法。实验对提取试剂以及流动相的选择进行了优化,采用乙腈对尿液和血浆样品进行提取。采用Phenomenex Kinetex C18色谱柱(50 mm×3 mm, 2.6 μm)进行分离,以0.05%(v/v)氨水水溶液、90%(v/v)乙腈水溶液为流动相,以流速0.40 mL/min梯度洗脱时,12种目标化合物分离效果最好。串联质谱的离子源为电喷雾离子(ESI)源,采用多反应监测(MRM)模式检测。12种目标物的基质效应均在0.8~1.1之间,表明该前处理方法的基质干扰低,采用外标法可对化合物进行准确定量。12种贝类毒素的线性范围为0.03~36.25 μg/L,相关系数均大于0.995。尿液检测的方法定量限为0.23~0.63 μg/L,血浆检测的方法定量限为0.31~0.84 μg/L。3个加标水平的回收率为72.7%~124.1%,日内精密度为2.1%~20.0%,日间精密度为2.1%~15.3%。利用该方法检测健康人尿液和血浆样本,以及经腹腔注射12种贝类毒素的小鼠尿液和血液样本。20份健康人样本中未检出目标物,20份小鼠样本中12种贝类毒素均有检出。该方法操作简便,样品取样量少,方法灵敏高,适用于血浆和尿液中脂溶性贝类毒素的快速检测。
Keywords: lipophilic shellfish toxins; plasma; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); urine.