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

Abstract

Branched-chain keto acids (BCKAs) are derivatives from the first step in the metabolism of branched-chain amino acids (BCAAs) and can provide important information on animal health and disease. Here, a simple, reliable and effective method was developed for the determination of three BCKAs (α-ketoisocaproate, α-keto-β-methylvalerate and α-ketoisovalerate) in serum and muscle samples using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS). The samples were extracted using methanol and separated on a 1.8 μm Eclipse Plus C18 column within 10 min. The mobile phase was 10 mmol L−1 ammonium acetate aqueous solution and acetonitrile. The results showed that recoveries for the three BCKAs ranged from 78.4% to 114.3% with relative standard deviation (RSD) less than 9.7%. The limit of quantitation (LOQ) were 0.06~0.23 μmol L−1 and 0.09~0.27 nmol g−1 for serum and muscle samples, respectively. The proposed method can be applied to the determination of three BCKAs in animal serum and muscle samples.

Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation

“…Analysis was performed on an AB Sciex 5500 QTRAP liquid chromatography/mass spectrometer (Applied Biosystems SCIEX) equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatted column compartment, and a triple quadrupole/iontrap mass spectrometer with electrospray ionization interface, and controlled by AB Sciex Analyst 1.6.1 Software. Phenomenex Luna C8 (150 mm × 2 mm, 5 μm) column was used for BCAA separation and Biodent C18 column was used for BCKA separation as described previously (68). Solvents for the mobile phase were 1 mmol/L aqueous ammonium acetate (A) and 5 mmol/L ammonium acetate in 90% acetonitrile (v/v) aqueous (B).…”

Effect of the BCAT2 polymorphism (rs11548193) on plasma branched-chain amino acid concentrations after dietary intervention in subjects with obesity and insulin resistance

“…The supernatant was dried in a Speedvac concentrator (Savant SPD121P) and the sample was then resuspended in 200 µL of water. Then, 1 uL of the extracted sample was injected and the branched chain ketoacids (α-ketoisocaproate, KIC; α-keto-β-methylvalerate, KMV; α -ketoisovalerate, KIV) were separated using a chromatographic method previously reported (20) , using a Zorbax Eclipse Plus C18 column, 4.…”

“…The supernatant was dried in a Speedvac concentrator (Savant SPD121P) and the sample was then resuspended in 200 μl of water. Then, 1 μl of the extracted sample was injected and the BCKA (α-ketoisocaproate; α-keto-β-methylvalerate and α-ketoisovalerate) were separated using a chromatographic method previously reported (20) , using a Zorbax Eclipse Plus C18 column, 4•6 × 50 mm, 1•8 μm (Agilent) at 30ºC and a flow rate of 0•3 ml/min. Buffer A consisted of 10 mM of ammonium acetate in water and Buffer B of acetonitrile.…”

Effects of branched-chain amino acids on glucose metabolism in obese, prediabetic men and women: a randomized, crossover study

“…Plasma was also used to determine circulating concentrations of BCAAs (leucine, isoleucine, and valine) and BCKAs, such as α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and αketomethylvalerate (KMV). Following derivatization of BCAA with a Waters AccQ-Fluor Reagent Kit, and BCKA with 4nitro-1,2-phenylenediamine, these compounds were analyzed by HPLC as described by Zhang et al (15).…”