Study of the matrix effects of tetrodotoxin and its content in cooked seafood by liquid chromatography with triple quadrupole mass spectrometry

Tetrodotoxin is a marine biotoxin with high acute toxicity. The levels in cooked seafood will help us to assess its intake in humans and may help assess the risk of toxicity. However, heavy matrices hinder the direct quantitation of tetrodotoxin. A quantitative method of measuring tetrodotoxin in cooked seafood using liquid chromatography with triple quadrupole mass spectrometry was established in this study. Tetrodotoxin was extracted from the sample matrix using 2% formic acid in methanol and cleaned using a cation exchange cartridge. The cleanup conditions were optimized. The matrix effects were determined using the postextraction spiking method and by comparing the slope of the linear regression equation in sample matrix to that in solvent. The limit of detection in the sample matrix was 5 μg/kg and the limit of quantification was 10 μg/kg. The mean recoveries at three spiking levels were 66.9–89.2% with relative standard deviations of 5.0–10.8% (n = 6) in five different matrices. Tetrodotoxin was found at concentrations of 26.1–2462 μg/kg in nine of 83 cooked seafoods tested in this study. Eight analogs of Tetrodotoxin were detected in the samples studied.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.     

Toward the total synthesis of tetrodotoxin: stereoselective construction of the 7-oxanorbornane intermediate

The stereoselective synthesis of 7-oxanorbornane regarding as a key synthetic intermediate of tetrodotoxin (TTX) is reported. The bicyclo ring bearing various functional groups was successfully elaborated by a furan Diels–Alder (DA) reaction. The stereoselective installation of a quaternary amino carbon center to the DA product was achieved with the Tsuji–Trost allylation. The stereochemistry was unambiguously confirmed with X-ray.     

免疫亲和柱净化-超高效亲水色谱-三重四极杆质谱联用法测定人尿液和血浆中河豚毒素

建立了测定人尿液和血浆中河豚毒素( TTX)的超高液相色谱-三重四极杆质谱联用分析方法。尿液和血浆样品经免疫亲和柱净化,以0.1%甲酸-乙腈和0.1%甲酸溶液作为流动相进行梯度洗脱,在UPLC BEH Amide柱上实现分离,正离子电喷雾多反应监测( MRM)模式检测,溶剂标准外标法定量。尿液和血浆中TTX的测量范围为0.05~400μg/L,平均加标回收率分别为92%~95%和91%~96%,相对标准偏差在3.3%~7.2%和3.9%~7.8%(n=5)之间,样品中 TTX的检出限(S/N=3)均为0.02μg/L,定量限(S/N=10)为0.05μg/L。本方法适用于尿液和血浆中TTX的中毒检测和临床监测。     

Rapid Determination of Tetrodotoxin in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive analytical method was developed to determine tetrodotoxin(TTX) in human plasma samples using protein precipitation, followed by ultra performance liquid chromatography(UPLC) analysis coupled with tandem mass spectrometry(MS/MS) using 11-deoxytetrodotoxin(11-deoxyTTX) as an internal standard. The plasma samples were prepared using protein precipitation prior to being analyzed by UPLC-MS/MS to identify TTX over a zwitterionic-hydrophilic interaction liquid chromatography column. The retention time values of TTX and 11-deoxyTTX were 4.12 and 3.67 min, respectively. TTX and 11-deoxyTTX were monitored and quantitated on the basis of their ion transitions for their respective precursor ions to their product ions(i.e., m/z 320.0→162.1 for TTX and m/z 304.0→176.0 for 11-deoxyTTX) in the multiple reaction-monitoring mode. The lower limit of quantification of this method was determined to be 0.0199 ng/mL. This method showed good linearity for plasma samples that contained TTX concentrations in the range of 0.0199-1.99 ng/mL. The specificity, precision, accuracy, matrix effect, and stability characteristics of this method were also examined. The intra-assay precision and accuracy ranged from 1.89% to 6.00% and from 92.21% to 100.00%, whereas the inter-assay precision and accuracy ranged from 0.64% to 7.75% and from 99.38% to 101.26%, respectively. This new method therefore represents a rapid, accurate, reliable, and highly sensitive method for the qualitative and quantitative analyses of a trace amount of TTX in human plasma samples.     

离子交换固相萃取高效液相色谱联用法检测河豚鱼中的河豚毒素

优化建立了采用混合模式阳离子交换固相萃取柱代替传统的C18固相萃取柱进行前处理,联用高效液相色谱检测河豚鱼中河豚毒素的方法.样品经合1％乙酸的乙腈甲醇(3:1,V:V)溶液提取,混合模式阳离子交换固相萃取柱(MCX)净化,5％氨水/甲醇溶液洗脱,采用高效液相色谱-二极管阵列检测器(HPLC-PAD)测定,外标法定量.河...     

Screening of tetrodotoxin in puffers using gas chromatography-mass spectrometry

Tetrodotoxin (TTX), a toxic compound found in some puffers can cause death to humans through consumption. We have developed a simplified method for the screening of TTX in puffers using GC-MS. A puffer tissue of 0.5g was treated with 5mL of 0.1% acetic acid, followed by alkaline hydrolysis, LLE or liquid-liquid extraction and N-methyl-N-TMS-trifluoroacetamide derivatization. The developed method used only a small sample and solvent, simplified LLE and derivatization procedures and short chromatographic analysis (8.2min). All of these contribute to cost-saving, enhanced sample throughput and high sensitivity of the screening assay. The developed method was validated and proved to be within the acceptable range.