Dl isocitric acid

All chemicals used in this study are commercially available except 2-Methylisocitrate which was synthesized in the laboratory of Dr. Sébastien Gouin, University of Nantes France. The genomic DNA of Mycobacterium tuberculosis H37Rv, Luria-Bertani (LB) medium for bacterial growth and Isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Hi-Media Laboratories, India. Primers used for gene amplification were synthesized through Eurofins Genomics India Pvt. Ltd. Restriction enzymes (NheI and HindIII), Alkaline Phosphtase and T4 DNA Ligase were procured from Fermentas, US. DL-Isocitric acid and Phenylhydrazine were obtained from Sigma (India).

Working solution (WS) for calibration curves and quality control solutions were prepared from two separate mother solutions (100 µg/ml in water) of each quantified compound: L-glutamic acid, L-aspartic acid, L-glutamine, succinic acid, alpha-ketoglutaric acid, trans-aconitic acid, L-(-)-malic acid, D,L-isocitric acid, D-glyceric acid, fumaric acid, citric acid, pyruvic acid, D-alpha-hydroxyglutaric acid disodium salt, D-(-)-lactic acid, D-(-)−3-phosphoglyceric acid, phosphoenolpyruvic acid and itaconic acid (all from Sigma). Several diluted solutions of calibration standard solutions (CSS) and quality control solutions (QCS) were prepared by successive two-fold dilutions of WS in ultrapure water. Then, a three-fold dilution in a BSA solution (7200 µg/mL), of each previous diluted solution (CSS1-8 and QCS1-3) was applied to prepare standards for the calibration curve (from 33.33 to 0.26 µg/mL), and quality control (from 53.33 to 1.51 µg/mL). A volume of 350 µL of cold methanol was added to each calibration curve and quality control solution and followed the metabolite extraction process.