Draisci R, Lucentini L, Giannetti L, Boria P, James K J, Furey A, Gillman M, Kelly S S
Istituto Superiore di Sanità, Laboratorio Medicina Veterinaria, Rome, Italy.
J AOAC Int. 1998 Mar-Apr;81(2):441-7.
A fast, sensitive, and specific procedure for determining toxins that cause diarrheic shellfish poisoning (DSP) using microliquid chromatography coupled with tandem mass spectrometry (micro-LC-MS-MS) is reported. The lipophylic polyether acidic toxins okadaic acid (OA), its isomer dinophysistoxin-2 (DTX-2), the 35-methylokadaic acid dinophysistoxin-1 (DTX-1), and the novel toxin dinophysistoxin-1B (DTX-2B; recently isolated from Irish mussels) were extracted from shellfish tissues with acetone and chromatographed by isocratic elution at 10 microL/min with CH3 CN-H2O, 80 + 20 (v/v), containing 0.1% trifluoroacetic acid, through a C18 reversed-phase column (1.0 mm id). The chromatograph is coupled via an ion spray interface to an atmospheric pressure ionization source. Collision-induced-dissociation (CID) ion mass spectra of the protonated molecule, [M + H]+, at m/z 805 for OA, DTX-2, and DTX-2B and at m/z 819 for DTX-1, were obtained in MS-MS experiments to identify 2 diagnostic fragment ions for each analyte that could be used for selected-reaction-monitoring (SRM) micro-LC-MS-MS analysis. The CID spectrum of DTX-2B confirmed it to be a new OA isomer, like DTX-2. Standard curves obtained by SRM micro-LC-MS-MS were linear (r2 > or = 0.9992) over the range 0.05-1.00 micrograms/mL (i.e., 0.10-2.00 micrograms toxin/g hepatopancreas), and a detection limit of 15 pg/injection was obtained for each DSP toxin. Average recoveries ranged from 95 to 101%, and coefficients of variation ranged from 1.8 to 3.4%. This novel SRM micro-LC-MS-MS method was used to confirm acidic DSP toxins in Irish and Italian toxic mussels. It offers a high degree of specificity because analyte confirmation is based on retention time, molecular weight, structural information obtained from the presence of 2 diagnostic fragments for each analyte, and ion ratios. OA was found in both Irish (< or = 0.7 micrograms/g hepatopancreas) and Italian (< or = 1.5 micrograms/g hepatopancreas) mussels. DTX-1 was found only in Italian mussels (< or = 0.3 micrograms/g hepatopancreas). DTX-2 (< or = 6.1 micrograms/g hepatopancreas) and DTX-2B (< or = 0.08 micrograms/hepatopancreas) were unique to Irish shellfish.
报道了一种使用微液相色谱-串联质谱法（micro-LC-MS-MS）测定引起腹泻性贝类中毒（DSP）毒素的快速、灵敏且特异的方法。用丙酮从贝类组织中提取亲脂性聚醚酸性毒素冈田酸（OA）、其异构体鳍藻毒素-2（DTX-2）、35-甲基冈田酸鳍藻毒素-1（DTX-1）以及新型毒素鳍藻毒素-1B（DTX-2B；最近从爱尔兰贻贝中分离得到），通过内径为1.0 mm的C18反相柱，以10 μL/min的流速用含0.1%三氟乙酸的CH3 CN-H2O（80 + 20，v/v）进行等度洗脱色谱分离。色谱仪通过离子喷雾接口与大气压电离源相连。在MS-MS实验中获得了质子化分子[M + H]+在m/z 805处的OA、DTX-2和DTX-2B以及在m/z 819处的DTX-1的碰撞诱导解离（CID）离子质谱，以鉴定每种分析物的2个诊断性碎片离子，可用于选择反应监测（SRM）micro-LC-MS-MS分析。DTX-2B的CID谱证实它是一种新的OA异构体，与DTX-2一样。通过SRM micro-LC-MS-MS获得的标准曲线在0.05 - 1.00 μg/mL（即0.10 - 2.00 μg毒素/g肝胰腺）范围内呈线性（r2≥0.9992），每种DSP毒素的检测限为15 pg/进样。平均回收率在95%至101%之间，变异系数在1.8%至3.4%之间。这种新型的SRM micro-LC-MS-MS方法用于确认爱尔兰和意大利有毒贻贝中的酸性DSP毒素。它具有高度的特异性，因为分析物的确认基于保留时间、分子量、从每种分析物存在2个诊断性碎片获得的结构信息以及离子比率。在爱尔兰贻贝（≤0.7 μg/g肝胰腺）和意大利贻贝（≤1.5 μg/g肝胰腺）中均发现了OA。仅在意大利贻贝（≤0.3 μg/g肝胰腺）中发现了DTX-1。DTX-2（≤6.1 μg/g肝胰腺）和DTX-2B（≤0.08 μg/肝胰腺）是爱尔兰贝类特有的。
