高效液相色谱-串联质谱法测定葡萄果实中的乙酰辅酶A

建立了高效液相色谱-三重串联四极杆质谱法(HPLC-MS/MS)测定葡萄果实中重要中间代谢产物乙酰辅酶A的分析方法。样品用纯水提取后离心,经C18固相萃取小柱净化,以5 mmol/L甲酸铵和乙腈（20:80,v/v）为流动相等度洗脱,经C18柱分离后,在电喷雾正离子化(ESI+)模式下,通过多反应监测(MRM)模式进行定性,以n-丙酰辅酶A为内标进行定量。该方法在1～2000 μg/L范围内线性关系良好,相关系数大于0.99,检出限(以信噪比(S/N)为3计)为0.1 μg/L,定量限(以S/N=10计)为0.5 μg/L;添加水平分别为50、500、1000 μg/L的3个样品的加标回收率为82.87%～89.67%,相对标准偏差均小于10%。该方法简便、快速、灵敏,可明显减少乙酰辅酶A在实验过程中的损失,适用于葡萄果实中乙酰辅酶A的检测分析。

Determination of acetyl coenzyme A in grape berries by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of acetyl coenzyme A in grape berries using n-propionyl coenzyme A as an internal standard (IS). The sample was extracted with water and then centrifuged for 5 min at 10000 r/min on a centrifuge at 4 ℃, and cleaned-up with a C18 solid phase extraction cartridge. The identification and quantification were carried out by using electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The total run time was 1 min and the elution of both acetyl coenzyme A and n-propionyl coenzyme A occurred at about 0.45 min. This was achieved with a mobile phase consisting of 5 mmol/L ammonium formate-acetonitrile (20:80, v/v) at a flow rate of 0.4 mL/min on a C18 column. A linear response function was established for the concentration range of 1-2000 μg/L for acetyl coenzyme A and the correlation coefficient was more than 0.99. The limit of detection of acetyl coenzyme A was 0.1 μg/L. The spiked recoveries at 50, 500, 1000 μg/L ranged from 82.87%-89.67% with the relative standard deviations less than 10%. This method is simple, rapid, sensitive and can significantly reduce the loss of acetyl coenzyme A during the experiment. It is suitable for the determination of acetyl coenzyme A in grape berries.