Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet, vertebrate blood. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, indicating that levels of B vitamins vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood and hemolymph can provide valuable informative background and understanding of general principles of insect symbiosis. In this work chromatographic separation of a mixture of eight B vitamins (B1 thiamine, B2 riboflavin, B3 niacin, B5 pantothenic acid, B6 pyridoxine, B7 biotin, B9 folic acid and B12 cyanocobalamine), four B vitamin derivatives (B3 niacinamide, B6 pyridoxal-5-phosphate, B6 4-pyridoxic acid and B9 tetrahydrofolic acid) and 3 stable isotope labelled internal standards (B3 niacin-13C6, B5 pantothenic acid (di-beta-alanine-13C6,15N2) and B7 biotin-(ring-6,6-d2)) on C30 column was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Matching internal standards with analytes was done according to the results of linearity, accuracy and precision. Except for vitamin B9 (tetrahydrofolic acid) instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 ug/L, correlation coefficients from 0.9997 to 1.0000 and QC coefficients from 0.53 to 3.2 %. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. Samples of whole blood prepared in six independent replicates were spiked at 10 ug/L and 100 ug/L level. The best results were obtained for zinc sulphate in methanol, but only nine analytes (B1 thiamine, B2 riboflavin, B3 niacin, B3 niacinamide, B5 pantothenic acid, B6 pyridoxine, B6 4-pyridoxic acid, B7 biotin and B12 cyanocobalamine) were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120 %, precision from 0.5 to 13 % and process efficiency from 65 to 108 %.

中文翻译：

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用于全血中B维生素及其某些衍生物测定的LC-MS / MS方法的开发和验证

据推测，与仅以脊椎动物血液为食的昆虫相关的专性共生细菌可补充饮食中可能缺乏的维生素B，即脊椎动物血液。最近的基因组分析表明，不同共生体在生物合成能力方面存在相当大的差异，这表明不同脊椎动物宿主中维生素B的含量也有所不同。但是，尚未严格测定各种脊椎动物血液中B族维生素的含量。一种针对血液和淋巴中B维生素复合物的可靠分析方法可以提供有价值的信息背景和对昆虫共生一般原理的理解。在这项工作中，对八种B族维生素（B1硫胺素，B2核黄素，B3烟酸，B5泛酸，B6吡ido醇，B7生物素，B9叶酸和B12氰钴胺），四种B维生素衍生物（B3烟酰胺，B6吡ido醛-5-磷酸，B6 4-吡啶氧酸和B9四氢叶酸）和3种稳定的同位素标记的内标（B3烟酸-13C6，B5泛酸（开发了C30色谱柱上的di-beta-丙氨酸13C6,15N2）和B7生物素-（ring-6,6-d2））。使用双压力线性离子阱质谱仪以FullScan MS / MS和SIM模式进行检测。根据线性，准确性和精密度的结果，将内标物与分析物进行匹配。除维生素B9（四氢叶酸）仪器定量限外，所有分析物的定量限为0.42至5.0 ug / L，相关系数为0.9997至1.0000，QC系数为0.53至3.2％。全血样品制备步骤的优化尤其着重于评估两种类型的蛋白质沉淀剂：三氯乙酸和甲醇中的硫酸锌。将六次独立重复制备的全血样品加标到10 ug / L和100 ug / L的水平。甲醇中的硫酸锌获得了最佳结果，但仅成功验证了九种分析物（B1硫胺素，B2核黄素，B3烟酸，B3烟酰胺，B5泛酸，B6吡ido醇，B6 4-吡啶氧酸，B7生物素和B12氰钴胺）。 。使用这种蛋白质沉淀剂的过程的准确度为89％至120％，精度为0.5％至13％，过程效率为65％至108％。将六次独立重复制备的全血样品加标到10 ug / L和100 ug / L的水平。甲醇中的硫酸锌获得了最佳结果，但仅成功验证了九种分析物（B1硫胺素，B2核黄素，B3烟酸，B3烟酰胺，B5泛酸，B6吡ido醇，B6 4-吡啶氧酸，B7生物素和B12氰钴胺）。 。使用这种蛋白质沉淀剂的过程的准确度为89％至120％，精度为0.5％至13％，过程效率为65％至108％。将六次独立重复制备的全血样品加标到10 ug / L和100 ug / L的水平。甲醇中的硫酸锌获得了最佳结果，但仅成功验证了九种分析物（B1硫胺素，B2核黄素，B3烟酸，B3烟酰胺，B5泛酸，B6吡ido醇，B6 4-吡啶氧酸，B7生物素和B12氰钴胺）。 。使用这种蛋白质沉淀剂的过程的准确度为89％至120％，精度为0.5％至13％，过程效率为65％至108％。