Determination of Vitamin C in Small Volumes of Blood by HPLC/EC

1. Introduction

     The physiologic importance of vitamin C to human health is well known, e.g., as a potent antioxidant, ascorbic acid (AA) has the capacity to eliminate reactive oxygen species [1, 2]. The total vitamin C content in blood plasma and leucocytes is widely accepted as an indicator of tissue status of vitamin C [3]. A number of techniques have been used to quantify vitamin C in foods and biological samples including: a colorometric assay the ascorbic acid reaction with 2,4- dichlorophenol-indophenol [4], or folin-phenol reagent [5], capillary zone electrophoresis [6], several HPLC methods using UV detection [2, 7] and HPLC with electrochemical detection (EC), which is currently the method of choice for quantification of ascorbic acid in   foods, tissues and biological fluids [8-11]. The current study reports a method suf- ficiently  sensitive  to  determine  AA  plus dehydroascorbic acid (DHA) in 50 l of blood using HPLC-EC.

2. Materials and methods

2.1. Chemicals

   Ascorbic acid, D-isoascorbic acid (IAA), DL-homocysteine,  potassium  dihydrogen phosphate,  and  trichloroacetic  acid  were obtained  from  Sigma-Aldrich  Chemical Company,  Gillingham, Dorset.  Vitamin  C tablets  (500  mg,  Pharmadass)  were  purchased locally.

2.2. Finger tip blood samples

     One or two drops of blood were collected from healthy volunteers using a lancet directly into eppendorf tubes. Samples were taken before and one h, two h, and six h after the administration of a 500 mg vitamin C tablet.  Immediately  after  collecting  blood samples, 50 l blood was measured from the drop  using  a  Hamilton  syringe  and  was transferred  into  another  eppendorf  tube. Then 100 l of phosphate buffer (100 mM, pH 4.7), containing 1 mM  EDTA was added to the tubes followed by 30 l of 1% DL-homo- cysteine solution. The samples were left for 30  minutes at  the  room  temperature, and then 40 l of a 30% trichloroacetic acid solution was added to the samples. The samples were then centrifuged to remove the precipitated cells and proteins, and 200 ng of  IAA (20 µl of 10 µg/ml) was added to 100 l of the supernatant, and 50 l of the final solution was injected into the HPLC system.

2.3. Instrumentation

     A P100 Spectra-Physics isocratic HPLC system was used. The Rheodyne injection valve was fitted with a 50 l loop. Separation was  achieved by  using two  Prodigy 5  m ODS C18 reversed-phase columns (250  4.6mm i.d., Phenomenex UK, Macclesfield ) which were connected in series. Detection was carried out using a LC-4A electrochemical detector (Bioanalytical Systems). The potential of detector was set at 0.4 V versus an Ag-AgCl reference electrode . The mobile phase was composed of 100 mM phosphate buffer, pH 3, containing 1 mM EDTA, and the flow rate was 1 ml/min.

2.4. Calibration and precision

    A calibration curve was prepared by dissolving different amounts of AA + 2 µg of IAA in 1 ml of the mobile phase. The standard curve between 1-6 g/ml of AA was linear (r = 0.998). If required, samples were diluted with the mobile phase to fall within the range of the calibration curve. The precision of the method was determined by injecting 5 aliquots of the same blood sample containing 13.1 µg/ml of AA which gave precision of   1.4 % for the measurement of the sample.

3. Results

    By using two C18 reversed columns in series, baseline separation between the L- ascorbic acid present in the blood and IAA used as an internal standard was possible. The exact mechanism permitting this separation is unclear, but IAA is the ideal internal standard for this assay because of its close similarity to AA. The vitamin C level was measured in six healthy volunteers (5 male and 1 female). Typical chromatograms are shown  in Figure 1 for vitamin C in blood samples obtained from one of the volunteers. The mean  concentrations for vitamin C in the blood samples before and at 1 h, 2 h, and 6 h after the administration of a vitamin C tablet are shown in Table 1.

Table 1. Mean (SD) concentrations of vitamin C (AA+DHA) in the blood samples before and at different time points after taking a 500 mg vitamin C tablet.

       In order to determine whether any peaks corresponding to DHA appeared in the chro- matogram, a sample containing 5 g/ml DHA and  2  µg/ml  IAA  was  injected  into  the HPLC.  Consequently, only the peak corre- sponding to the IAA was observed, and the injection of DHA alone did not reveal any peak.

Figure 1. HPLC chromatogram of ascorbic acid (AA), measured in a blood drop. Samples were taken before and at 1, 2 and 6 h (left to the right, respectively ) after taking a 500 mg vitamin C tablet by volanteers. Two hundred nanograms of isoascorbic acid (IAA) was added to 100 l of each sample prior to the injection to the HPLC.

4. Discussion

      The  method  described  above  has  the potential to detect Previous  work  using  HPLC  with  UV detection determined vitamin C (AA+DHA) concentration in human plasma to be 15.3 g/ml without supplementation [3]. In anoth- er  study, it was shown that in fresh blood plasma samples, the DHA concentration was less than 5% total AA [8]. Various methods have  been  described for  the  reduction of DHA to AA e.g. incubation of DHA with dithiothreitol  [3],  2-mercaptoethanol  [12], and  DL-homocysteine  [9].  In  the  current study,  2-mercaptoethanol  in sodium phosphate buffer, pH 7.4, with 1mM thiourea and 0.1 mM EDTA [12] was tested but the efficiency of reduction was not high, and better results  were obtained with   DL-homocysteine as a reducing agent.