血清中氧化三甲胺的高效液相色谱串联质谱法测定研究

Measurement of trimethylamine-N-oxide in serum by liquid chromatography tandem mass spectrometry

目的 建立人血清中胆碱依赖肠道菌群代谢物——氧化三甲胺（TMAO）的高效液相色谱串联质谱法（HPLC-MS/MS）的测定方法,并研究其精密度和稳定性。方法 以d9-TMAO为内标,血清经乙腈除蛋白后,采用Si O2柱（2.1 mm×100 mm,5μm）、乙腈-甲酸铵（10 mmol/L,p H 3.0）等度洗脱,选择多反应监测模式测定,测定方法的精密度和准确度及不同保存条件下的稳定性。结果 本研究方法的检测限和定量限分别为0.003和0.063μmol/L,线性范围为0.16-20.00μmol/L（r2=0.999 9）,不同浓度点的日内变异系数为2.03%-2.64%,日间变异系数为6.00%-9.94%,回收率为89.5%-103.0%;血清在4℃保存时,24 h内TMAO浓度变化在10%以内,72 h后TMAO浓度升高明显,浓度变化为15.85%;血清保存于-80℃时,72 h后TMAO浓度变化范围均在10%以内;反复冻融4个冻融循环内浓度变化仍在15%的可接受范围内。结论 本研究建立的高效液相色谱串联质谱法检测TMAO的方法具有快速、准确且灵敏的特点,可以满足大样本人群血样检测要求。血液样本保存时应注意保存温度、时间对TMAO浓度的影响。

Objective To develop a method for measurement of trimethylamine-N-oxide （TMAO）-microbiota-generated metabolite of choline in serum by stable isotope dilntion high-performance liquid chromatography tandem mass spectrometry （HPLC-MS/MS） and to study its accuracy and stability. Methods Proteins in samples were precipitated with three volumes of 10 μmol/L internal standard d9- TMAO in acetonitrile and supernatants were analyzed after injection onto a SiO2 column （2. 1 mm×100 mm, 5μm. ） and equilibrated with 30% solution A （10 mmoL/L ammonium formate, pH = 3.0） and 70% solution B （aeetonitrile）. Results Lower limit of detection was 0.003 μmol/L and the lower limit of quantitation was 0.063 μmol/L at linear range of 0.16 - 20.00 mol/L （ r2 = 0. 999 9）. The intra- batch and inter-batch coefficients of variance were 2.03% - 2.64-% and 6.00% - 9.94% , and the re- covery rates were 89.5% - 103.0%, respectively. Stability study revealed that the storage temperature and time could affect the concentration of TMAO. TMAO in serum was stable both during storage at - 80 ℃ and throughout multiple freeze thaw cycles. Conclusion HPLC-MS/MS assay has characteristics of rapidness, accuracy, precision, and high-throughpnt nature and can meet the detection requirements for large quantity of blood samples.