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.