固相萃取-高效液相色谱串联质谱测定人体尿液中芳香化合物代谢物的方法研究

任建伟; 罗新月; 赵璇; 王璇; 朱婧; 别明江; 刘峻杉; 丛雪; 邹晓莉

doi:10.12182/20200960205

固相萃取-高效液相色谱串联质谱测定人体尿液中芳香化合物代谢物的方法研究

Determination of Aromatic Compounds Metabolites in Human Urine by Solid Phase Extraction-liquid Chromatography-Tandem Mass Spectrometry

摘要

摘要:
目的建立固相萃取-高效液相色谱串联质谱（high performance liquid chromatographic tandem mass spectrometric, HPLC-MS/MS）同时测定尿液中8种芳香化合物代谢物（苯巯基尿酸、甲基马尿酸、N-乙酰基-S-苯基-L-半胱氨酸、苯乙醛酸、苯乙醇酸、对氨基酚、对硝基酚和1-羟基芘）的方法。
方法取1 mL尿液，加入20 μL β-葡萄糖醛酸酶和1 mL乙酸铵缓冲溶液，在37 ℃孵箱中酶解20 h。酶解后混匀，取适量酶解液注入PrimeHLB 固相萃取柱净化，4 mL乙腈洗脱，洗脱液氮吹至干，用0.20 mL甲醇复溶后进样HPLC-MS/MS系统分析。高效液相色谱分离采用反相C₁₈色谱柱（2.1 mm×150 mm，3.5 μm），流动相A为含体积分数0.1%甲酸的水，流动相B为甲醇，采用梯度洗脱，流速为0.2 mL/min。质谱采用正、负离子交替扫描方式，多反应监测（multiple reaction monitoring, MRM）模式检测，内标标准曲线法定量。
结果 8种代谢物在 1~100 ng/mL范围内线性良好，相关系数均大于0.995，测定的精密度（relative standard deviation, RSD）在0.05%～9.95%之间，8种代谢物的检出限在0.041~0.12 ng/mL范围内。将所建立的方法用于尿样分析，加标回收率为80.1%～114.0%。
结论所建立的方法灵敏、快速、准确，适用于普通人群和职业人群的芳香化合物生物监测评估。

Abstract:
Objective To establish the method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with solid phase extraction (SPE) for simultaneous determination of the biological metabolites of aromatic compounds, including N-acetyl-S-phenyl-L-cysteine (SPMA), N-acetyl-S-benzyl-cysteine (SBMA), p-nitrophenol (PNP), methylhippuric acids (MHA), p-Aminophenol (PAP), mandelic acid (MA), phenylglyoxylic acid (PGA) and 1-hydroxypyrene (1-OHP) in urine.
Methods After adding 20 μL of β-glucuronidase and 1 mL ammonium acetate buffer solution in 1 mL of urine, the sample was digested in a 37 ℃ incubator for 20 h. After digestion, the enzymatic hydrolysate was purified by PRIME HLB solid phase extraction column. The target compounds were eluted with 4 mL of acetonitrile and blown to dryness with nitrogen, reconstituted with 0.20 mL of methanol. Injected the sample solution into LC-MS/MS system for analysis after filtering with 0.22 μm filter membrane. LC separation was carried out on a reversed-phase C₁₈ column (2.1 mm×150 mm, 3.5 μm); gradient eluting was performed at a flow rate of 0.2 mL/min. The water containing 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The mass spectrometry was performed with multiple reaction monitoring (MRM) mode, using alternating positive and negative ions, and internal standard curves were used for quantification.
Results The eight metabolites showed good linearity within the range of 1-100 ng/mL, with a correlation coefficients greater than 0.995, and the relative precision deviation (RSDs) was 0.050%-9.95%. The method detection limits (MDLs) of the eight target metabolites were 0.041-0.12 ng/mL. The proposed method was used for urine sample analysis and the spiked recoveries were 80.1%-114.0%.
Conclusion The established method is quick, sensitive and accurate; it meets the requirementof the biological monitoring of aromatic compounds for the general population and occupational population.

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