Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry.
A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.
Stephanson
Nikolai
N
Department of Medicine, Division of Clinical Pharmacology, Karolinska Institute and University Hospital, SE-171 76 Stockholm, Sweden.
Helander
Anders
A
Beck
Olof
O
D000428:Alcohol Drinking / Q000378:metabolism*; D015415:Biomarkers; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D020719:Glucuronides / Q000652:urine*; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000652:urine*; D006917:Hydroxytryptophol / Q000031:analogs & derivatives* / Q000652:urine; D007202:Indicators and Reagents; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
C500750:5-hydroxytryptophol glucuronide; D015415:Biomarkers; D020719:Glucuronides; D007202:Indicators and Reagents; D006917:Hydroxytryptophol; D006897:Hydroxyindoleacetic Acid