Unusual Derivatization of Methylmalonic Acid with Pentafluorobenzyl Bromide to a Tripentafluorobenzyl Derivative and Its Stable-Isotope Dilution GC-MS Measurement in Human Urine

Abstract

Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CH₃CH(COOH)₂; pK_a1, 3.07; pK_a2, 5.76) associated with vitamin B₁₂ deficiency and many other patho-physiological conditions. In this work, we investigated several carboxylic groups-specific derivatization reactions and tested their utility for the quantitative analysis of MMA in human urine and plasma by gas chromatography-mass spectrometry (GC-MS). The most useful derivatization procedure was the reaction of unlabeled MMA (d₀-MMA) and trideutero-methyl malonic acid (d₃-MMA) with 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br) in acetone. By heating at 80 °C for 60 min, we observed the formation of the dipentafluorobenzyl (PFB) ester of MMA (CH₃CH(COOPFB)₂). In the presence of N,N-diisopropylamine, heating at 80 °C for 60 min resulted in the formation of a tripentafluorobenzyl derivative of MMA, i.e., CH₃CPFB(COOPFB)₂). The retention time was 5.6 min for CH₃CH(COOPFB)₂ and 7.3 min for CH₃CPFB(COOPFB)₂). The most intense ions in the negative-ion chemical ionization (NICI) GC-MS spectra of CH₃CH(COOPFB)₂ were mass-to-charge (m/z) 233 for d₀-MMA and m/z 236 for d₃-MMA. The most intense ions in the NICI GC-MS spectra of CH₃CPFB(COOPFB)₂ were mass-to-charge (m/z) 349 for d₀-MMA and m/z 352 for d₃-MMA. These results indicate that the H at C atom at position 2 is C-H acidic and is alkylated by PFB-Br only in the presence of the base N,N-diisopropylamine. Method validation and quantitative analyses in human urine and plasma were performed by selected ion monitoring (SIM) of m/z 349 for d₀-MMA and m/z 352 for the internal standard d₃-MMA in the NICI mode. We used the method to measure the urinary excretion rates of MMA in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). The creatinine-corrected excretion rates of MMA were 1.50 [0.85-2.52] µmol/mmol in the black boys and 1.34 [1.02-2.18] µmol/mmol in the white boys (P = 0.85; Mann-Whitney). The derivatization procedure is highly specific and sensitive for MMA and allows its accurate and precise measurement in 10-µl of human urine by GC-MS.

Keywords: C-H acidity; GC-MS; alkylation; base; dicarboxylic acids; esterification; ethnicity; pentafluorobenzyl bromide; stability; toluene; urine.