人血浆中双氢可待因浓度HPLC-MS/MS测定方法的建立

Determination of dihydrocodeine in human plasma by HPLC-MS/MS

目的建立测定人血浆中双氢可待因浓度的高效液相色谱一串联质谱分析方法。方法血浆样品用甲醇沉淀蛋白后，选用安捷伦色谱柱XDB．C18，2．1×50mm，5μm梯度洗脱。流动相A为0．1％甲酸10mM甲酸铵水溶液，流动相B为0．1％甲酸甲醇。流动性梯度为0min（B5％）→1min（B5％）→1．3（B65％）→2．4min（B65％）→2．5min（B5％）→3．5min（B5％）。流速0．6mL／min，柱温30℃，自动进样器温度4cC，进样量10止。选用岛津LC-20A高效液相色谱系统和串联APl4000Qtrap型三重四极杆质谱仪，ESI源正离子MRM扫描，Is电压5500V，CAD气Medium，CUR气40psi，Gasl：35psi，Gas2：40psi，源温度550℃，EP电压10V，CXP电压9V。双氢可待因：m／z302．1—199．1，DP电压：105V，CE电压：47V。右美沙芬（内标）：m／z271．3→170．1，DP电压100V，CE电压54V。结果该方法测定血浆中二氢可待因的线性范围为2～1000ng／mL（r〉0．99），定量下限为2ng／mL，准确度均在85％～115％，批内和批间精密度也均〈15％。稳定性试验中，血浆样品及血浆经处理后样品中的双氢可待因在方法学要求的各种贮存条件下均比较稳定。结论该方法简单、快速、灵敏、专属性强、重现性好，适用于检测人血浆中双氢可待因浓度的研究。

Objective To establish the HPLC-MS/MS method for determination of dihydro- codeine in human plasma. Methods After protein precipitation with methanol, the analyte and inter- nal standard were separated on a agilent XDB-C18,2.1 × 50 mm, 5μm analytical column using the gradient elution method at a flow rate of 0.6 mL/min. The mobile phase A was 0.1% formic acid and 10 mM ammonium formate in water, and the mobile phase B was 0.1% formic acid in methanol. The ratio of mobile phase B was 0 min （5%）→1min（5% ）→1.3（65% ）→2.4 min （65%）→2.5 min （5 % ）→3.5 min （5 % ）. Column temperature ： 30 ~C, the autosampler temperature ： 4 %, injection volume ： 10 ~L. Detection was carried out by SHIMADZU LC-20A liquid chromatography and electros- pray positive ionization mass spectrometry in the multiple reaction monitoring （MRM） mode. IS volt- age： 5 500 V, CAD gas： Medium, CUR gas： 40 psi, Gasl ： 35 psi, Gas2：40 psi, source tempera- ture： 550 ~C, EP voltage： 10 V the CXP voltage： 9 V. Dihydrocodeine： rn/z 302.1---~199.1, DP voltage： 105 V, CE voltage： 47 V. Dextromethorphan （internal standard） ： m/z 271.3-→170.1 DP voltage： 100 V, CE voltage： 54 V. Results The calibration curve is linear over the concentration range of 2 -1000 ng/mL with the lower limit of quantitation （LLOQ） 2 ng/mL. The Accuracy is in the range of 85 -115% , inter- and intra-day precisions are both less than 15%. In the stability stud- ies, dihydrocodeine in the plasma and the prepared plasma samples was found to be stable under vari- ous storage conditions. Conclusion The established HPLC-MS/MS method is simple, rapid, sensi- tive, specific and reliable for the determination of dihydrocodeine in human plasma.