Liquid chromatographic method for extracellular Guanosine 5’-Triphosphate and Tetrahydrobiopterin pathway products analysis from cadaveric samples and human biofluids

To gain a deep insight and to obtain a superior understanding about guanosine-based pathway, this paper reports an innovative approach to study this critical subject. Firstly, after an exhaustive analysis of literature with a focus in legal medicine and extracellular vesicles, it was understood that a new method is inevitable to follow, determine, and quantify these analytes (Guanosine monophosphate - GMP, guanosine diphosphate - GDP, guanosine triphosphate - GTP, Guanosine, Neopterin and Tetrahydrobiopterin - BH4). Starting from a previously method, we implemented and validated a new HPLC-DAD method in gradient elution mode with these six target analytes fully resolved in 18 min. The HPLC-DAD method uses a stationary phase XTIMATE C18 (4.6 mm × 250 mm, 5 µm, Welch, Shanghai, China) and mobile phase’s phosphate buffer (40 mM, pH 7) (A) and Acetonitrile (B). Good correlation goes from 0.05 to 10 µg/mL with a limit of detection equal to 0.02 µg/mL and a limit of quantification equal to 0.05 µg/mL (R2 ≥ 0.9824). Method was tested on human extracellular vesicles, isolated from different human parts, like urine, saliva and muscle, giving interesting results as different quantification of analytes depending on the sample matrix used. Interesting to underline is that saliva was the poorest source of these analytes, if compared with growth medium and urine.