Measurement of Acetylcholine in Rat Brain Microdialysates by LC-Isotope Dilution Tandem MS
Measurement of Acetylcholine in Rat Brain Microdialysates by LC-Isotope Dilution Tandem MS
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DOI：
10.1365/s10337-008-0697-0
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发表时间：
2008-01-01
期刊：
影响因子：
1.7
通讯作者：
Nguyen, Vien
中科院分区：
文献类型：
--
作者：
Prokai, Laszlo;Frycak, Petr;Nguyen, Vien
开发了一种 LC-MS-MS 方法，使用反相离子对色谱法、四极离子阱仪器上的电喷雾电离和四氘代类似物 (ACh-1,l,2,2-d(4)) 作为内标来测量水介质中的乙酰胆碱 (ACh)。使用七氟丁酸 (0.1% v/v) 作为离子对剂，并需要 20 mM 甲酸铵缓冲液中的 10% v/v 乙腈，在 200 μl min(-1) 流速下进行等度洗脱，在 5 cm 长的十八烷基硅胶柱（内径 2.1 mm）上实现快速分离。使用人工脑脊液中含有已知摩尔比的 ACh 和 ACh-1,1,2,2-d(4) 的样品校准仪器的响应，得出的结论是，可以通过将测量的分析物与内标离子电流比乘以所添加的已知摩尔浓度的 ACh-1,1,2,2-d(4) 来确定分析物浓度。通过测量大鼠脑微透析液中的基础神经递质浓度来测试快速而简单的测定，在样品收集时不使用胆碱酯酶抑制剂。
An LC-MS-MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,l,2,2-d(4)) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 mu l min(-1) flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d(4) in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1, 1, 2,2-d(4) added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.