Vitamin C (or L-ascorbic acid) is the hexose ramification of acidity, and the lactone of saccharinic acid in enol form. VC is mainly derived from fruit and vegetables. It is also the essential nutrients for senior primates and other small numbers of creatures. There are two kinds of isomer for VC, including L-type and D-type. Only L-type has physiological function, and both reduced form and oxidized form have physiological activity.
Material and Reagents
HPLC purity of methanol; analytically grade of metaphosphoric acid, trisodium trimetaphosphate, phosphoric acid and monopotassium phosphate; Guarantee reagents of L-cysteine, and the ultrapure water.
Transfer about 0.5 g ~ 2 g (to the accuracy of 0.001 g) well mixed solid sample (containing 0.03 mg ~ 6 mg of L (+)-Ascorbic acid approximately) into a 50 mL centrifuge tube, add 20 g/L metaphosphoric acid solution and transfer the mixture into a 50 mL volumetric flask. Shake and dissolve to the constant volume, transfer all the solution into a 50 mL centrifuge tube. Ultrasonic extraction for 5 min, centrifuge at 4000 rpm for 5 min, Aspirate the supernatant and filter through 0.45 μm aqueous filter followed by HPLC analysis. (This solution can be used to test the content of L (+)-Ascorbic acid and D (+)-Ascorbic acid of the sample simultaneously).
Transfer 20 mL the above supernate into a 50 mL centrifuge tube, add 10 mL L-cysteine solution of 40 g/L, and adjust pH to 7.0 ~ 7.2 with 100 g/L trisodium phosphate anhydrous, vibrate at 200 times/min for 5 min. Adjust the pH to 2.5 ~ 2.8 with phosphoric acid, and transfer all solution into a 50 mL volumetric flask, dilute to the constant volume, mix well. Filter the solution through 0.45 μm aqueous filter and ready for HPLC testing. (This solution can be used
to measure the total amount of L (+)-Ascorbic acid including the dehydrogenated type in the sample).
HPLC column: Venusil C18 Plus; 5 μm, 120 Å, 4.6 × 250 mm;
Mobile phase: A:B = 98:2; A: dissolve 6.8 g potassium dihydrogen phosphate to certain volume of 1 L using water, adjust pH to 2.5-2.8 with phosphoric acid; B: methanol;
Detection: 245 nm;
Flow rate: 0.7 mL/min;
Column temperature: 25 °C;
Injection: 20 mL.
Results and Discussion
Table 2 Performance of Venusil C18 Plus in analysis of 20 µg/ml Ascorbic acid in spiked milk powder sample
L (+)-Ascorbic acid
D (+)-Ascorbic acid
Figure 1 Chromatogram of 50 µg/mL L (+)-Ascorbic acid and D (+)-Ascorbic acid mix standard solution
Figure 2 Chromatogram of blank milk powder sample
Figure 3 Isocratic separation of 20 mg/mL L (+)-Ascorbic acid and D (+)-Ascorbic acid in spiked milk powder sample
A robust HPLC analytical method was developed for quantifying L (+)-Ascorbic acid and D (+)-Ascorbic acid in infant formula milk powder. Exceptional separation was obtained with high resolution using Venusil C18 Plus. The method was determined to be accurate and reproducible. Typical method results were well within acceptable criteria.
Venusil C18 Plus
5 μm, 120 Å, 4.6 × 250 mm
1.5 mL vials
Screw neck vials, 12 × 32 mm
Caps and Septa
Screw neck cap, center hole; red silicone/ white PTEE septa, slitted
Syringe Filter (Nylon)
Monofilm, 13 mm, 0.22 μm
Disposable Needle-Free injection systems
2 mL, 100/pk