中毒患者血液中褪黑素浓度的气相色谱-串联质谱联用检测

Detection of melatonin in poisoned patients'blood by gas chromatography coupled with tandem mass spectrometry

目的建立血液中褪黑素的气相色谱-串联质谱联用(GC-MS-MS)检测方法,为临床中毒患者的救治提供依据。方法乙酸乙酯液-液提取血液中的褪黑素,进样1μl,色谱柱:BR-5MS,载气为氦气,流速为1.0 ml/min;柱温起始温度60℃,保持2 min,以速率20℃/min升至280℃,保持10 min,EI离子源。通过Full Scan确定保留时间(RT)、多反应监测模式(MRM)采集数据,进行定性定量检测。结果褪黑素色谱峰保留时间为13.998 min,以基峰m/z 173为母离子进行MS-MS分析,最终确定子离子m/z 129.7的最佳CID为18 eV,子离子m/z 158.5的最佳CID为15 eV。配制系列褪黑素浓度(x)和峰面积(y)进行线形回归,得标准曲线y=230.44x-9535.2,r=0.9984。选择信噪比(S/N)3∶1定性、S/N 10∶1定量,得到褪黑素定性检出限为1.0 ng/ml,定量检出限为3.5 ng/ml;日内精密度RSD为2.9%,日间精密度RSD为5.3%。结论与GC-MS相比,GC-MS-MS方法选择性好、灵敏度高、排除干扰强。

Objective The gas chromatography coupled with tandem mass spectrometry(GC-MS-MS)was established to detect melatonin in human blood,so as to provide basis for the rescue of clinical poisoned patients Methods Melatonin was extracted from blood using ethyl acetate.The injection volume was 1μl,and BR-5MS capillary column was used for separation.The helium was used as carrier gas,and the flow rate was set at 1.0 ml/min in constant current mode;using EI ion source,the temperature programming of oven was employed with the initial temperature at 60℃and maintained for 2 minutes,then increased to 280℃at the speed of 20℃/min and maintained for 10 min.The retention time(RT)was determined by using full scan,and the data was collected using multiple reaction monitoring(MRM)mode and qualitative and quantitative detection were conducted using these two modes.Results The retention time of the melatonin was 13.998 min.MS/MS was analyzed by setting the base peak at m/z 173.It was found that the best CID of the daughter ion m/z 129.7 was 18 eV,and the best CID of the daughter ion m/z 158.5 was 15 eV.The standard curve(y=230.44x-9535.2,r=0.9984)was obtained by linear regression of concentration(x)and peak area(y)of the prepared series of melatonin.The signal to noise ratio(S/N)was selected to be 3∶1 qualitative and S/N 10∶1 quantitative,and the limit of qualitative detection of melatonin was 1.0 ng/ml and 3.5 ng/ml respectively.Intraday precision RSD was 2.9%and intraday precision RSD was 5.3%.Conclusion Compared with GC-MS,the GC-MS-MS method has good selectivity,high sensitivity and strong resistance to interference.