Quantitation of branched-chain α-keto acids as theirN-methylquinoxalone derivatives: Comparison ofO- andN-alkylation versus -silylation

Abstract

Quantitative estimation of isotopic enrichment and concentrations of keto analogs of branched-chain amino acids in biological fluid has been used for the study of protein metabolism in animal and human studies. At present, O-trimethylsilyl-quinoxalinol derivative is used widely in the quantification of branched-chain alpha-keto acids. In the present study, N-methyl-quinoxalone derivative was developed and its use in quantification in human studies verified. O-phenylenediamine and alpha-keto acid react in acidic media to yield phenolic and amide tautomers. O-trimethylsilyl-quinoxalinol derivative of the phenolic tautomer is used at present for quantification by chemical ionization/selected ion monitoring. We have prepared N-methyl-quinoxalone derivative using N,N-dimethyl formamide dimethyl acetal. This derivative is characterized by a major amide form and a minor phenolic form. The mass spectrum has a characteristic fragment, which facilitates easy identification and quantitation by electron impact/selected ion monitoring. Because m/z 174 was observed as the base peak for alpha-ketoisocaproate, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, "single ion monitoring' could be performed for the quantification of isotopic enrichment as well as plasma concentration of these three branched-chain alpha-keto acids. Isotopic enrichment from 0.25 to 7.5 at% excess could be measured easily, with an average coefficient of variation of less than 8%. Plasma concentrations as low as 10 microM l-1 in a 200-microliters aliquot could be measured. Methyl migration was an interesting feature of the mass spectrometric fragmentation pattern of the alpha-keto acids. The mechanism of methyl migration is proposed and discussed. This paper also describes some of the studies involved in the formation of isomeric O- and N-alkyl, -quinoxaline and -quinoxalone using a number of N,N-dimethyl formamide dialkyl acetals.

Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography–mass spectrometry

“…Previous methods have been developed to measure BCKAs in biological fluids, including reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection [27-33], gas chromatography–mass spectrometry [34], and gas–liquid chromatography [35,36]. These methods either are time-consuming or lack sensitivity for tissue as opposed to plasma BCKA concentrations.…”

Enzymatic-Chemical Preparation of Quinoxaline Derivatives from L-Amino Acids for Gas Chromatographic-Mass Spectrometric Analyses

“…Using the branchedchain quinoxaline derivatives, we performed some additional GC-MS studies with underivatized quinox- alines and with A/,N-dimefchylformamide dimethyl acetal as a derivatization reagent according to Fernandes et aL (33). When checked on the OV-1 column at our disposal, separation was not as excellent as with O-TMS derivatives.…”

Determination of Branched-Chain Keto Acids in Serum and Muscles Using High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry

“…Several analytical methods have been applied to measure BCKAs in biological fluids such as plasma and urine. These include gas-liquid chromatography (GC) [ 20 ], gas chromatography-mass spectrometry (GC-MS) [ 21 ] and high performance liquid chromatography (HPLC) with fluoresce detection [ 22 , 23 , 24 ]. However, these techniques are either time consuming or lack sensitivity such that certain BCKAs are not detectable in tissue.…”