Determination of organic acids by means of reversed phase chromatography/ion chromatography
This method is suitable for wine, fruit juice and other non-alcoholic beverages.
The organic acids are separated using two combined columns, reversed-phase HPLC and an ion exchange column and are then determined using a UV detector.
Determination of D-isocitric acid by enzymatic means
This analysis is suitable for non-alcoholic and beer-based beverages.
Fruit juices:
The acid spectrum typical of certain types of fruit are used, along with other criteria, as a basis for recognizing unadulterated fruit juices. Tartaric acid, citric acid and L-malic acid are recorded here, which, with a few exceptions, determine the total acidity of the fruit.
Citric acid occurs as the primary acid in citrus juices and other juices. Orange juice usually contains 3–17 g/l citric acid (AIJN).
In citrus juices, an addition of citric acid can be detected via the citric acid/D-isocitric acid ratio, as this lies within relatively narrow limits. In orange juice, values below 130 are found.
D-isocitric acid is partly present in fruit products as a lactone. The lactone must first be saponified prior to enzymatic determination in order to detect the total D-isocitric acid content.
D-Isocitrate in the sample is isolated as a barium salt through precipitation. The concentration of D-isocitrate is determined enzymatically. The enzyme isocitrate dehydrogenase (ICDH) catalyzes the oxidative decarboxylation of D-isocitrate to α-ketoglutarate with nicotinamide adenine dinucleotide phosphate (NADP):
D-Isocitrate + NADP+ \(^{\underrightarrow{ICDH}}\)α-ketoglutarate + NADPH + CO2 + H+
The amount of NADPH formed is proportional to the amount of D-isocitrate and is measured photometrically through the increase in absorbance.