固相萃取联合高效液相色谱-串联质谱测定人血浆中肌苷浓度的方法学建立

doi:10.3969/j.issn.1674-8115.2021.03.021

摘要/Abstract

摘要：

目的·建立一种使用固相萃取联合高效液相色谱-串联质谱（solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry，SPE-HPLC-MS/MS）测定人血浆中肌苷浓度的方法。方法·采用电喷雾离子化正离子模式和多反应监测扫描方式对人体血浆样本中肌苷和内标物进行定量分析。肌苷及内标物的定量离子对分别为m/z 269.1/137.1和m/z 230.2/112.2。方法学验证内容包括定量限、准确度、日内和日间精密度、提取回收率测定以及不同条件下人体血浆样品的稳定性考察。结果·方法学验证结果显示，肌苷在28.5~912.0 ng/mL的范围内线性关系良好（R²>0.999），定量下限为28.5 ng/mL，准确度为96.9%~103.8%，日内和日间精密度的相对标准偏差均小于10%，提取回收率为98.9%~102.3%。人体血浆样本在室温放置4 h、反复冻融3次、冷冻放置7 d（-70 ℃）和处理后自动进样器（4 ℃）放置24 h 4种条件下，肌苷的变化率均小于15%。结论·建立的SPE-HPLC-MS/MS法迅速、准确、专属性强、重复性好，适用于人体血浆中肌苷浓度的测定。

关键词: 肌苷, 血药浓度, 高效液相色谱-串联质谱, 固相萃取

Abstract:

Objective·To establish a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for the determination of inosine in human plasma.

Methods·Inosine and internal standard in human plasma were quantitatively analyzed with electrospray ionization positive ion mode and multiple reaction monitoring scan analysis. The ion pairs used for quantitative analysis were m/z 269.1/137.1 (inosine) and m/z 230.2/112.2 (internal standard). The results of method validation included limit of quantification, accuracy, intra-day and inter-day precision, extraction recovery, and stability tests in different conditions.

Results·The results of method validation showed that the calibration curve of inosine in human plasma exhibited a good linearity (R²>0.999) over the range from 28.5 to 912.0 ng/mL, and quantitative lower limit was 28.5 ng/mL. Accuracy was 96.9%?103.8%, and the relative standard deviations of intra-day and inter-day precision were both less than 10%. The extraction recovery was between 98.9% and 102.3%. In stability tests (samples stored at room temperature for 4 h, 3 freeze-thaw cycles, -70 ℃ for 7 d and 4 ℃ in autosampler for 24 h), the rates of change of inosine in human plasma were less than 15%.

Conclusion·The established SPE-HPLC-MS/MS method is simple, rapid, sensitive and accurate, and it is suitable for the clinical monitoring of inosine concentration in human plasma.

Key words: inosine, blood drug level, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), solid-phase extraction (SPE)

中图分类号:

R917

金冠钦, 孙黎, 夏玲红, 林厚文. 固相萃取联合高效液相色谱-串联质谱测定人血浆中肌苷浓度的方法学建立[J]. 上海交通大学学报(医学版), 2021, 41(3): 400-405.

Guan-qin JIN, Li SUN, Ling-hong XIA, Hou-wen LIN. Determination of inosine in human plasma by high performance liquid chromatography-tandem mass spectrometry combined with solid-phase extraction[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 400-405.

图/表 7

参考文献 19

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Material	Linear range/(ng·mL^-1)	Linear equation	R² value	Quantitative lower limit/(ng·mL^-1)
Inosine	28.5?912.0	y =1.183×10^-2x+2.574	0.999 7	28.5
Lamivudine	12.5?100.0	y =4.800×10^-4x-5.495	0.998 4	12.5

Material	Linear range/(ng·mL^-1)	Linear equation	R² value	Quantitative lower limit/(ng·mL^-1)
Inosine	28.5?912.0	y =1.183×10^-2x+2.574	0.999 7	28.5
Lamivudine	12.5?100.0	y =4.800×10^-4x-5.495	0.998 4	12.5

Theoretical concentration/(ng·mL^-1)	Measured concentration/(ng·mL^-1)	Intra-day precision/%	Inter-day precision/%	Accuracy/%
28.5	27.6	4.8	4.6	96.9
57.0	55.6	5.6	7.0	97.5
228.0	236.7	3.1	0.2	103.8
912.0	897.6	9.7	3.3	98.4

Theoretical concentration/(ng·mL^-1)	Measured concentration/(ng·mL^-1)	Intra-day precision/%	Inter-day precision/%	Accuracy/%
28.5	27.6	4.8	4.6	96.9
57.0	55.6	5.6	7.0	97.5
228.0	236.7	3.1	0.2	103.8
912.0	897.6	9.7	3.3	98.4

Theoretical concentration/ (ng·mL^-1)	Matrix effect		Recovery test
Theoretical concentration/ (ng·mL^-1)	Mean value/%	RSD/%	Mean value/%	RSD/%
57.0	106.1	10.9	98.9	6.0
228.0	94.0	2.2	101.3	2.9
912.0	100.4	1.6	102.3	9.1