Separation and determination of D-malic acid enantiomer by reversed-phase liquid chromatography after derivatization with (R)-1-(1-naphthyl) ethylamine

Abstract L-Malic acid is the Active Pharmaceutical Ingredient of the latest generation of compound electrolyte injection (STEROFUNDIN ISO, Germany) and plays a very important role in the rescue of critically ill patients. The optical purity of L-malic acid is a Critical Quality Attributes. A new reversed-phase high performance liquid chromatography (RP-HPLC) method for pre-column derivatization of D-malic acid enantiomer impurity in L-malic acid bulk drug was established. The derivatization reaction was carried out using (R)-1-(1-naphthyl)ethylamine ((R)-NEA) as a chiral derivatization reagent. The Kromasil C18 column was used with a detection wavelength of 225 nm, a flow rate of 1.0 mL·min-1, and a column temperature of 30 °C. The mobile phase was acetonitrile-0.01 mol·L-1 potassium dihydrogen phosphate solution (containing 20 mmol·L-1 sodium heptanesulfonate, adjusted to pH 2.80 with phosphoric acid) (at a ratio of 45:55) and the resolution of D-malic acid and L-malic acid derivatization products reached 1.7. The proposed method possesses the advantages of simple operation, mild conditions, stable derivatization products and low cost. Also it gave better separation and was more accurate than previous methods

RESEARCH ON BATCH QUANTITIVE DETERMINATION OF L-MALIC ACID IN FERMENTATION LIQUOR / 微生物学通报

A simple and rapid method was established for the determination of the content of L-malic acid in fermentation liquor. This procedure includes six steps: (1) Use a microi- njector to inject the fermentation liquor quantitively on the filter paper, then use the chromatography solution (n-butanol: formic acid: water=8:1.5:1) for paper chromatography. (2) Atomize the display reagent (10ml 0.02?10~(-2) bromocresol green: 0.2ml 10?10~(-2) sodium hydroxide) on the chromatography paper. (3) Scissor off L-malic acid spots from the paper and put in into 5 ml distilled water for three hours. (4) In 1ml eluent, add 6 ml 96?10~(-2) sulphuric acid and 0.1ml 1.0?10~(-2) ?-naphthol.(5) Heat the reaction system in 100℃ water for sixty minutes. (6) After cooling determine the optical density at the wavelength 476 am in the colorimeter.

The Study of Enzymatic Activity of Mutant Strain Rhizopus oryzae with L-Malic Acid Accumulation / 微生物学通报

A stable and efficient L-Malic acid accumulation mutant strain Rhizopus oryzae ME-M15 was discovered occasionally in the mutation breading for fumaric acid producers. Rhizopus oryzae ME-M15 gave a L-Malic acid output of 16.3 g/L on average after fermentation for 96 hours, more than 3 times than that of the parent strain ME-F10. In addition, other metabolites such as ethanol and fumaric acid were re-markably decreased in accordance with the depressed activity of the cytosolic isoenzyme of fumarase and alcohol dehydrogenase in strain ME-M15, while the activity of the pyruvate carboxylase had no significant difference.