高效液相色谱-串联质谱法检测糖尿病患者血浆甲基乙二醛和乙二醛浓度
网络出版日期: 2013-09-16
基金资助
国家自然科学基金(30872727,81000332)
Determination of plasma methylglyoxal and glyoxal concentrations in patients with diabetes mellitus by high-performance liquid chromatographic/tandem mass spectrometry
Online published: 2013-09-16
Supported by
National Natural Science Foundation of China, 30872727,81000332
目的 建立一种测定糖尿病患者血浆甲基乙二醛和乙二醛浓度的高效液相色谱-串联质谱法(HPLC-MS/MS)。方法 采用HPLC-MS/MS技术测定1型(n=15)和2型糖尿病(n=17)患者以及正常受试者(n=10)血浆甲基乙二醛和乙二醛的浓度。待测血浆加入2,3-二氨基萘(2,3-DAN)和内标乙二酮,充分反应及用乙腈沉淀蛋白后,以乙腈-0.1%甲酸水溶液(57∶43,v/v)为流动相,通过Hypersil GOLD色谱柱(50 mm×2.1 mm,5 μm)分离;采用电喷雾电离源,选择正离子检测,甲基乙二醛-DAN结合物、乙二醛-DAN结合物和乙二酮-DAN结合物用于定量分析的选择性离子分别为m/z 195.18→m/z 126.25、m/z 181.14→m/z 126.10和m/z 237.34→m/z 209.34。结果 甲基乙二醛-DAN结合物和乙二醛-DAN结合物线性范围分别为2~1 000 ng/mL和50~1 000 ng/mL,平均回收率均>70%。1型和2型糖尿病患者血浆甲基乙二醛和乙二醛浓度显著高于正常受试者。结论 成功建立测定糖尿病患者血浆中甲基乙二醛和乙二醛浓度的HPLC-MS/MS方法,灵敏度、准确度和回收率均较高,专属性强,适用于大批临床样本的分析。
关键词: 甲基乙二醛; 乙二醛; 高效液相色谱-串联质谱法; 糖尿病
孔 祥 , 黄 凯 , 张洪梅 , 等 . 高效液相色谱-串联质谱法检测糖尿病患者血浆甲基乙二醛和乙二醛浓度[J]. 上海交通大学学报(医学版), 2013 , 33(8) : 1182 . DOI: 10.3969/j.issn.1674-8115.2013.08.030
Objective To develop a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for the determination of plasma methylglyoxal and glyoxal concentrations in patients with diabetes mellitus. Methods HPLC-MS/MS method was employed to measure the plasma methylglyoxal and glyoxal concentrations in patients with type 1 diabetes mellitus (n=15) and type 2 diabetes mellitus (n=17) and normal subjects (n=10). 2,3-diaminonaphthalene (2,3-DAN) and hexanedione (using as internal standard) were added to plasma samples. After sufficient reaction and precipitation with acetonitrile from the plasma, methylglyoxal-DAN, glyoxal-DAN and hexanedione-DAN were isolated chromatographically on a Hypersil GOLD column (50 mm×2.1 mm, 5 μm). The mobile phase consisted of acetonitrile-0.1% formic acid (57∶43, v/v). Electrospray ionization source was applied and operated in the positive ion mode. The transitions of m/z 195.18→m/z 126.25, m/z 181.14→m/z 126.10 and m/z 237.34→m/z 209.34 were used to quantify methylglyoxal-DAN, glyoxal-DAN and hexanedione-DAN respectively. Results The linear calibration curves were obtained in the concentration range of 2-1 000 ng/mL and 50-1 000 ng/mL for methylglyoxal-DAN and glyoxal-DAN respectively. The average recovery rate was more than 70%. The plasma methylglyoxal and glyoxal concentrations in patients with type 1 and type 2 diabetes mellitus were significantly higher than those in normal subjects. Conclusion The established HPLC-MS/MS method is proved to be suitable for determination of plasma methylglyoxal and glyoxal concentrations in patients with diabetes mellitus, which offers advantages of high sensitivity and selectivity.
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