A fully validated high-throughput liquid chromatography tandem mass spectrometry method for automatic extraction and quantitative determination of endogenous nutritional biomarkers in dried blood spot samples.

Dried blood spot (DBS) sampling demonstrates multiple advantages over traditional venous blood collection in terms of quantifying biomarkers for clinical applications. The process is more convenient, less invasive and requires smaller sample size. More importantly, it lowers risk of infection and allows easier sample transportation and storage. In this study, an automated high-throughput DBS-LC-MS/MS method was developed for quantifying endogenous biomarkers in DBS (or 20 µL whole blood) and later applied in riboflavin (i.e. vitamin B2) quantification. The method consists of four steps, including internal standard spraying, high pressure sample extraction, LC-MS/MS sample analysis and automatic extraction module cleaning. The last two steps overlap, thus reducing sample preparation time and shorten the sample analysis cycle to five minutes per sample. The method was validated to be selective and sensitive (LLOQ=2 ng/mL) over a range of 2-120 ng/mL. Matrix effect was compensated by the application of internal standard, while within-run precision, between-run precision, accuracy, stability and ruggedness of the developed method were all assessed to be satisfactory. Quantitative analysis of riboflavin in 133 whole blood samples using the developed method demonstrated strong correlation compared with those quantified using traditional manual sample preparation followed by LC-MS/MS analysis (R = 0.9774). In conclusion, an automated high-throughput DBS-LC-MS/MS method was developed and validated to be sensitive, accurate and robust, suggesting great potential in the quantification of endogenous biomarkers in blood or other biofluids.