Ezchrome elite

L-Lysine hydrochloride (Merck, Germany), L-carnitine-L-tartrate (KHF Chemicals, China), potassium dihydrogen phosphate (RDH, Germany), triethylamine (Merck, Germany), acetonitrile (Sigma Aldrich, USA), methanol (Sigma Aldrich, USA) and HPLC grade water was prepared in house using Milli-Q system (Millipore, MA, USA). To develop this technique chromatographic system used is Hitachi Chromaster comprising of autosampler (5210), pump (5110), column oven (5310) and diode array detector (5430) set at 214 nm using EZChrome Elite for data acquisition.
The chromatography performed for the separation of L-lysine and LCLT was performed using Purospher star C₁₈ (250 mm×4.6 mm), 5 μm reverse phase column. The mobile phase comprising of 10 mM of potassium dihydrogen phosphate and pH was adjusted with triethylamine at 7.50. The detection was carried at 214 nm by pumping the mobile phase at flow rate of 0.5 ml/min while the temperature of column maintained at 25°. The injection volume was 20 μl and each test required just 6 min. and before injection all the standards, sample solutions as well mobile phase were filtered through membrane filter of 0.45 μm then sonicated.

The analysis was carried out using a Hitachi HPLC–DAD system equipped with a solvent delivery unit, autosampler, column oven, and diode-array detector. The acquired data were processed using EZChrome Elite for Hitachi. Separation was performed on a Gemini C₁₈ column (4.6 mm × 250 mm, 5 μm; Phenomenex, Torrance, CA, USA) at 35°C. The mobile phase, consisting of solvent A (1% aqueous acetic acid, v/v) and solvent B (acetonitrile with 1% acetic acid, v/v), was eluted using the gradient procedure, which was as follows: 5-40% (B) over 0-30 min, 40-100% (B) over 30-40 min, held for 5 min, and then re-equilibrated to 5% for 15 min. The flow rate was 1.0 mL/min and the injection volume was set to 10 μL. The optimized detection wavelengths for standard compounds were set at 230, 272, and 280 nm.