Profiling of bile acids in bovine follicular fluid by fused-core-LC–MS/MS
Profiling of bile acids in bovine follicular fluid by fused-core-LC–MS/MS
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DOI：
10.1016/j.jsbmb.2016.02.020
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发表时间：
2016-09
期刊：
影响因子：
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通讯作者：
A. Sánchez-Guijo;C. Blaschka;Michaela F. Hartmann;Christine Wrenzycki;Stefan A. Wudy
中科院分区：
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作者：
A. Sánchez-Guijo;C. Blaschka;Michaela F. Hartmann;Christine Wrenzycki;Stefan A. Wudy
胆汁酸(BAs)存在于人和牛的卵泡液中。这一事实引发了人们对bas在卵泡发生中可能扮演的角色以及它们与生育能力的可能联系的兴趣。为了更好地了解这一问题，需要新的方法来提供关于最重要的苯系物在FF中浓度的可靠信息。在此背景下，液相色谱-串联质谱仪(LC-MS/MS)以相对简单的样品处理提供了高度的专属性。我们开发并验证了一种新的方法来快速分析牛卵泡液中含量最丰富的9种BA。该方法采用FF200μL，可对胆酸、鹅去氧胆酸、脱氧胆酸及其甘氨酸、牛磺酸结合物进行定量。石胆酸(LCA)、其结合物GLCA和TLCA以及硫酸形式存在于一些样品中，但与其他BAs相比，它们的浓度较低(LCA、GLCA或TLCA的平均浓度低于60 ng/ml，相应的硫酸盐低于20 ng/ml)。方法采用三种质控方法对每种化合物的生理浓度进行考察。所有化合物在各对照水平上均具有良好的线性关系和回收率。所有化合物的日内和日间精密度(%CV)和准确度(相对误差)均低于15%。对于大多数分析物来说，基质效应可以忽略不计。经过冻融处理的样品没有显示出其BAS的降解。该方法利用熔核苯柱与三重四极串联质谱仪耦合，在5分钟内实现了色谱分离。我们量化了四种不同大小的BAs(3-5 mm，6-8 mm，9-14 mm，15 mm)，得到了所有大小的相似的相对BA分布，CA总是处于较高的浓度，范围在1,600-18000 ng/ml之间，其次是它的结合型甘胆酸，范围在800-9000 ng/ml。据我们所知，这是第一次在牛卵泡液中检测到bas亚种并进行了定量。
Bile acids (BAs) are present in follicular fluid (FF) from humans and cattle. This fact has triggered an interest on the role BAs might play in folliculogenesis and their possible association with fertility. To achieve a better understanding about this subject, new methods are needed to provide reliable information about concentrations of the most important BAs in FF. In this context, liquid chromatography-tandem mass spectrometry (LC–MS/MS) offers high specificity with a relatively simple sample workup. We developed and validated a new assay for the quick profiling of the 9 most abundant BAs in follicular fluid from cattle. The method uses 200 μl of FF and can quantify cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and their glycine (G) and taurine (T) conjugates. Lithocholic acid (LCA), its conjugates GLCA and TLCA, and sulfated forms, were present in some samples, but their concentration was low compared to other BAs (in average, below 60 ng/ml for LCA, GLCA or TLCA and below 20 ng/ml for their corresponding sulfates). Method performance was studied at three quality controls for each compound in consonance with their physiological concentration. Excellent linearity and recovery were found for all compounds at every control level. Intra-day and between-day precisions (%CV) and accuracies (relative errors) were below 15% for all the compounds. Matrix effects were negligible for most of the analytes. Samples undergoing freeze-thaw showed no degradation of their BAs. The method makes use of a fused-core phenyl column coupled to a triple quadrupole tandem mass spectrometer to achieve chromatographic separation within 5 min. We quantified BAs grouped in four different follicle sizes (3–5 mm, 6–8 mm, 9–14 mm, >15 mm), obtaining a similar relative BA profile for all the sizes, with CA always in higher concentration, ranging between 1600 and 18000 ng/ml, approximately, followed by its conjugate glycocholic acid, GCA, which ranged between 800 and 9000 ng/ml. The highest concentration in CA, DCA or CDCA was always detected in FF stemming from follicles of 6–8 mm. To our knowledge, this is the first report in which BAs subspecies have been detected and quantified in bovine follicular fluid.