Quantification of organic acids using gas chromatography-mass spectrometry (GC-MS)

Method given by Chen et al. (2001) was modified to estimate organic acids using GC-MS. Extraction of organic acids was done by adding 0.5 ml of 0.5 N HCl and 0.5 ml of methanol to 50 mg of dried seedling powder followed by shaking for 3 h and then centrifuged at 12,000 × rpm for 10 min. To the supernatant, 300 μl of methanol and 100 μl of 50% sulfuric acid (H₂SO₄) were added followed by overnight incubation in water bath at 60°C. The mixture was cooled down to 25°C and 800 μl of chloroform and 400 μl of distilled water were added to it followed by vortexing for 1 min. The lower chloroform layer was used to estimate organic acids using GC-MS.

To estimate organic acid content, 2 μl of sample (lower chloroform layer) was injected into GC-MS system. GC conditions: Carrier gas used was helium, initial column temperature was 50°C (hold for 1 min) which was increased at 25°C/min to 125°C followed by further increase at 10°C/min to 300°C and held for 15 min. Injection temperature was 250°C, mode of injection was split, flow of gas in column was 1.7 ml/min, and analytical column used was DB-5ms. MS conditions: Ion source temperature was set at 200°C and interface temperature was 280°C, solvent cut time was 3 min, detector gain mode: relative. Organic acids viz. citrate, fumarate, malate, and succinate were detected by comparing their mass spectra using National Institute of Standard and Technology (NIST08s) and Wiley 7 library. The quantification of these organic acids was done using standard curve.