Internal Validation of the Methods for Determination of Water-Soluble Vitamins from Frozen Fruits by HPLC-HRMS

The analytical methods were developed and validated for separation, detection and quantification of water-soluble vitamins (C, B2, B5, B6 and B7) from frozen fruits by high performance liquid chromatography coupled with high resolution mass spectrometry. Extraction of vitamin C was achieved in 0.1% formic acid solution at room temperature, and extraction of B-group vitamins in 0.1% formic acid solution at 70 degrees C. Extracts purification was achieved on SPE C18 cartridges, 500 mg/3 mL. Water-soluble vitamins and hippuric acid (IS) were separated on a reverse-phase C18 Hypersil GOLD aQ 150 x 2.1 mm, 3 um particle size, using mobile phases consisting of 995 mL water LC-MS, 5 mL 2M ammonium formate, 1 mL formic acid (A) and 995 mL metanol LC-MS, 5 mL 2M ammonium formate, 1 mL formic acid (B). Vitamin C was detected and quantified in the Electrospray Ionization negative ion mode (ESI-), and B-group vitamins in the Electrospray Ionization positive ion mode (ESI+). Hippuric acid (internal standard) was detected both the ESI- and ESI+ ion modes. The methods have a good sensitivity (vitamin C: LOD = 340.32 mu g/L and LOQ = 1031.27 mu g/L; vitamins B: LOD = 0.862-8.451 mu g/L and LOQ = 2.585-25.355 mu g/L). The methods showed a good precision, RSD < 10% for water-soluble vitamins analysis in frozen fruits. Methods developed and validated were applied for determination of water-soluble vitamins content of frozen fruits. Frozen Aronia melanocarpa fruits have the highest vitamin C content (39.785 mg/100g), and the frozen strawberries the highest vitamin B2 content (1.735 mg/kg), vitamin B5 content (3.703 mg/kg) and vitamin B6 content (3.553 mg/kg), respectively. The vitamin B7 content of the frozen fruits taken into study was low, ranging from 0.375 to 0.615 mg/100g.