Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry.
Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry.
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DOI：
10.1016/j.ab.2013.05.002
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发表时间：
2013-08-15
影响因子：
2.9
通讯作者：
Lynch CJ
中科院分区：
文献类型：
--
作者：
Olson KC;Chen G;Lynch CJ
支链酮酸（BCKAs）与几种退行性疾病的易感性增加有关。然而，组织中的BCKA浓度或可用组织的量通常处于标准血浆方法的检测限。为了准确、快速地测定组织中的BCKAs，我们开发了一种灵敏的超快速液相色谱-质谱（UFLC-MS）方法。对来自脱蛋白组织提取物的BCKA进行邻苯二胺（OPD）衍生化，乙酸乙酯提取，在真空离心机中冻干，并在200 mM乙酸铵中重构。将样品注射到与AB-Sciex 5600 Triple TOF质谱仪仪器偶联的Shimadzu UFLC系统上，该仪器使用多反应监测方法检测OPD BCKA产物的质量。OPD衍生的13 C标记的酮酸用作内标。显示了该方法在C57 BL/6 J（野生型）和PP 2Cm敲除小鼠组织（包括肾、脂肪组织、肝、腓肠肌和下丘脑）中的应用。通过该方法测量的最低组织浓度为20 nM，标准曲线覆盖宽范围（7.8- 32，000 nM）。该测定的液相色谱-质谱运行时间小于5分钟，有利于高通量，并且发现OPD衍生物在几天内是稳定的。
Branched-chain keto acids (BCKAs) are associated with increased susceptibility to several degenerative diseases. However, BCKA concentrations in tissues or the amounts of tissue available are frequently at the limit of detection for standard plasma methods. To accurately and quickly determine tissue BCKAs, we have developed a sensitive ultra fast liquid chromatography–mass spectrometry (UFLC–MS) method. BCKAs from deproteinized tissue extractions were o-phenylenediamine (OPD) derivatized, ethyl acetate extracted, lyophilized in a vacuum centrifuge, and reconstituted in 200 mM ammonium acetate. Samples were injected onto a Shimadzu UFLC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument that detected masses of the OPD BCKA products using a multiple reaction monitoring method. An OPD-derivatized 13C-labeled keto acid was used as an internal standard. Application of the method for C57BL/6J (wild-type) and PP2Cm knockout mouse tissues, including kidney, adipose tissue, liver, gastrocnemius, and hypothalamus, is shown. The lowest tissue concentration measured by this method was 20 nM, with the standard curve covering a wide range (7.8–32,000 nM). Liquid chromatography–mass spectrometry run times for this assay were less than 5 min, facilitating high throughput, and the OPD derivatives were found to be stable over several days.
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影响因子：
37.8
作者：
Cheng S;Rhee EP;Larson MG;Lewis GD;McCabe EL;Shen D;Palma MJ;Roberts LD;Dejam A;Souza AL;Deik AA;Magnusson M;Fox CS;O'Donnell CJ;Vasan RS;Melander O;Clish CB;Gerszten RE;Wang TJ
通讯作者：
Wang TJ
影响因子：
7.7
作者：
MACDONALD, MJ
通讯作者：
MACDONALD, MJ
影响因子：
56.9
作者：
Cota, D;Proulx, K;Seeley, RJ
通讯作者：
Seeley, RJ
DOI：
10.1016/s0378-4347(00)80946-9
发表时间：
1983-01-01
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
HAYASHI, T;TSUCHIYA, H;NARUSE, H
通讯作者：
NARUSE, H
影响因子：
7.1
作者：
HUTSON, SM;HARPER, AE
通讯作者：
HARPER, AE