Determination of urinary mevalonic acid using isotope dilution technique

A simplified and highly accurate, stable isotope dilution method using gas chromatograph/mass spectrometry (GC/MS) with deuterated (2H7) or (2H7) or (2H3)mevalonic acid (MVA) as internal standard was developed for the measurement of MVA in urine samples. MVA was converted to its lactone (MVL) and transferred with the total water of the sample (100 microliters) to a mixture of acetone/methyl t-butyl ether (MTBE). This solution was concentrated and dried by azeotropic removal of MTBE/water in special vessels (used for volume reduction under partial reflux for solutions containing volatile compounds). After producing MVA by adding NaOH followed by azeotropic drying, MVA was converted to its tris-t-butyldimethylsilyl (tri-TBDMS) derivative. GC/MS selected ion monitoring measurements at m/z 317, m/z 320 and m/z 324 were performed in the electron impact ionization (EI) mode. Overall recoveries of about 70% were obtained as shown by following the procedure with (14C)MVL, (2H7)- and (2H3)MVL as external and/or internal standards. Six replicate analyses of one urine sample revealed a coefficient of variation of 3.3%. Based on this experience we developed two other methods. The second method was based on a fluid/fluid extraction and the third one on an extraction with total water transfer. Both methods are combined with azeotropic drying and showed improved precision, handling and speed.