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Abstract
Simple and rapid reverse-phase ultra-performance liquid chromatography with UV-detection (RP-UPLC-UV) was developed and validated for the simultaneous analysis of 2-isopropylmalate (2-IPM), 3-isopropylmalate (3-IPM), ketoisovalerate (KIV), ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV) in the culture fluid (CF) of branched-chain amino acid (BCAA) Escherichia coli strain producers. The RP-UPLC-UV method was developed using a Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of phosphate monobasic salt adjusted to a pH of 3.5 and methanol solution. The flow rate of the mobile phase was 0.5 mL min−1 with gradient elution. The RP-UPLC-UV method was compared with reverse-phase high-performance liquid chromatography with UV-detection (RP-HPLC-UV), ion-exchange high-performance liquid chromatography with UV-detection (IE-HPLC-UV), and ion-exchange high-performance liquid chromatography with electrochemical detection (IE-HPLC-ECD) and a postcolumn buffer for organic acid analysis. The proposed RP-UPLC-UV method was highly sensitive (LOD for KIV was 2.5 × 10−3 mg mL−1, for 2-IPM – 4.5 × 10−3 mg mL−1, for 3-IPM – 4.4 × 10−3 mg mL−1, for KIC – 3.4 × 10−3 mg mL−1, and for KMV – 2.8 × 10−3 mg mL−1) and successfully applied for the analysis of organic acids in the CF of E. coli strain producers.
Graphical abstract
Disclosure statement
No potential conflict of interest was reported by the authors.