液相色谱-串联质谱法快速检测干血点样品中尿酸

建立了一种高通量液相色谱-串联质谱（LC-MS/MS）检测干血点(DBS)样品中尿酸(UA)的方法。采用自动液体操作平台对样品进行高通量自动化前处理，首先用含有UA-1,3-¹⁵N₂稳定同位素内标的Tris水溶液进行萃取，然后用含有0.1%甲酸、0.05%三氟乙酸的乙腈溶液沉淀蛋白质。处理后的样品经CN色谱柱分离，多反应监测(MRM)模式进行LC-MS/MS分析。结果表明，在DBS样品中，UA在7.8~1 000 μmol/L浓度范围内的线性关系良好(R²＝0.999)；检出限为3.1 μmol/L (S/N=3)；定量限为12.5 μmol/L(S/N=10)；平均回收率为95%~101%；日内相对标准偏差(RSD)为4.2%~12%；日间RSD为5.3%~14%。以样品中UA检测结果的总体RSD不超过15%来考察样品稳定性，分别将样品在-20 ℃保持30天、在37 ℃保持7天、反复冻融5次，样品中UA检测结果总体RSD小于10%，表明样品稳定性良好。将该方法与传统生化分析方法相比较，并分析了204份血样，相关性较好(R²＝0.946)。此方法可为有限采血条件下UA的检测及UA相关疾病的大规模筛查提供新途径。

Determination of Uric Acid in Dried Blood Spot Using Liquid Chromatography-Tandem Mass Spectrometry

Uric acid (UA) is a metabolite of purine compounds and an essential component of urine. The UA level of blood is an important clinical indicator to assist with the diagnosis of gout, kidney stones, kidney failure and other diseases. The methods of determining the UA level in serum samples include conventional enzymatic assays and recently reported methods, such as high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC/MS), isotope dilution mass spectrometry (IDMS) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Among these methods, HPLC-MS/MS stands out for clinical diagnostic applications due to its advantages of broad sample compatibility, excellent specificity and sensitivity, rapidity of analysis. The broad sample compatibility allows HPLC-MS/MS to work with a variety of sample forms including normal whole blood, serum samples and dried blood spot (DBS). DBS is a form of sampling where blood samples are blotted and dried on filter paper. It can be easily prepared and shipped to a remote clinical laboratory for analyzing. The easy-to-use feature facilitates field applications of DBS sampling which can significantly extend the scope of clinical diagnosis, such as chronic disease monitoring and health checkup. Thus, it is meaningful to combine HPLC-MS/MS with DBS in determination of important clinical diagnostic markers such as UA. In this study, a high-throughput method to measure UA in DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and validated. The DBS sample processing includes adding 0.2 mol/L tris(hydroxymethyl)aminomethane (Tris) solution as extraction solvent which contains internal standard UA-1,3-¹⁵N₂, followed by adding acetonitrile with 0.1% formic acid (FA) and 0.05% trifluoroacetic acid (TFA) as protein precipitant. This procedure was carried out in a 96-well plate on an automated liquid handling platform to facilitate high-throughput analysis. The processed sample was separated on a CN column with 95% water with 0.1% formic acid and 5% acetonitrile with 0.1% formic acid (A∶B, V/V). Quantitation was implemented using multiple reaction monitoring (MRM) mode. The results show that the linear range of UA in DBS samples are 7.8-1000 μmol/L (R²=0.999), limit of detection is 3.1 μmol/L (S/N=3), the limit of quantitation is 12.5 μmol/L (S/N=10), average recovery is 95%-101%, the intra day relative standard deviation (RSD) is 4.2%-12%, the inter-day RSD is 5.3%-14%. DBS sample stability was confirmed by the RSD of results in different cases for sample. The RSD is less than 15% within 30 days at -20 ℃, 7 days at room temperature and 37 ℃. The DBS samples are stable after 5 freeze-thaw cycles with the RSD less than 10%. The analytical method was compared with a conventional biochemistry assay using blood samples from 204 individuals, each blood sample has two forms: anti-coagulated whole blood for making DBS samples for LC-MS/MS analysis and serum for biochemistry assay. An excellent correlation (R²=0.946) of the two methods is observed.