HPLC-UV method for the simultaneous determinations of ascorbic acid and dehydroascorbic acid in human plasma

Chemicals and Reagents

L-ascorbic acid was purchased from Shinyo Pure Chemical Co. (Osaka, Japan). D-isoascorbic acid, metaphosphoric acid (HPO₃), potassium dihydrogenphosphate (KH₂PO₄), hexadecyl trimethyl ammonium bromide (HTAB), and dithiothreitol reagent (DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and water were purchased at the highest grade available from Fisher Scientific (Fair Lawn, NJ, USA). All other reagents were of analytical grade and were purchased from Sigma-Aldrich.

HPLC conditions

Chromatographic analysis was performed using an HPLC system (GILSON, Wisconsin, USA) with 254 nm and 265 nm wavelengths. Chromatographic separations were performed using a C18 (symmetry® 4.6 µm × 280 mm, particle size 5 µm) (Waters, Milford, MA, USA). The mobile phase consisted of 5 mM HTAB + 50 mM KH₂PO₄. The flow rate was kept constant at 1.2 mL/min.

Preparation of standard solution and determination of AA

Standard stock solutions of AA and D-isoascorbic acid were prepared as internal standards (IS) in 50% methanol at 1 mg/mL each. The working solutions of AA were diluted with 50% methanol to prepare standards of 10, 20, 40, 100, 500, 800, and 1,000 µg/mL. Human plasma was depleted of AA by leaving it on the bench for 96 h as described in a study by Karlsen et al.[17] The plasma calibration curves were prepared at concentrations of 1, 2, 4, 10, 50, 80, and 100 µg/mL by spiking 400 µL of blank plasma (AA free status) with 50 µL of each working standard, and 50 µL of the IS (100 µg/mL) of each analyte was added at the appropriate concentration. The stability of AA in blood is a very critical issue because it rapidly degrades to DHAA, thus metaphosphoric acid was employed as a very efficient stabilizer and protein precipitator for the AA analysis.[18] Five hundred microliter of 10% metaphosphoric acid (HPO₃) was carefully added to each sample. Then, samples were vortexed for 1 min and centrifuged at 12,000 rpm for 10 min at 4℃. The supernatants were transferred to injection vials, and 10 µL were injected into an HPLC system for analysis.

Determination of total AA

The blank plasma (400 µL) was subjected to reduction with the additions of 50 µL of working standard solution (addition of 2 mM DTT at pH 7.8) and 50 µL of IS. The solution reacted for 20 min at room temperature in the dark. Five hundred microliter of 10% HPO₃ was carefully added to each sample. Then, the samples were vortexed for 1 min and centrifuged at 12,000 rpm for 10 min at 4℃. The supernatants were transferred to injection vials, and 10 µL was used for chromatographic analysis. The concentration of DHAA was determined by subtracting the concentration of AA from the total AA concentration.

Method Validation

The method for the determination of AA in human plasma was validated by using spiked solutions of AA in AA-depleted human heparin plasma. The method validation parameters studied were specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, and recovery. The specificity of the method was investigated by comparing chromatograms of extracted blank plasma obtained from six different human plasma samples spiked with AA and IS to ensure that they were free of interference around the retention time of AA. The LOD was determined by diluting solutions of known concentration until the response was three times the noise (S/N 3), and the LOQ was defined as the lowest concentration that can be calculated on the basis of minimal accepted value of S/N 10. Linearity of the method was determined with a calibration curve constructed using seven concentration points. Calibration curves were constructed by plotting the response ratios (ratios of peak areas of analytes to IS) versus concentration of each analyte using a linear least squares regression. The precision study was carried out based on injection repeatability and analysis repeatability of spiked plasma samples. Injection repeatability was determined by repeated injections of plasma samples spiked with standard mixtures equivalent to each analyte into the HPLC system. The accuracy of the method was defined by replicate analysis of samples containing known amounts of the analyte. The deviation of the mean from the true value serves as the measure of accuracy. The recovery of the method was determined as the ratio of the peak area of extracted samples after a full assay to that of a direct injection of equivalent concentration (in 50% methanol). The intra-day precision and accuracy were determined within one day by analyzing five replicate samples. The inter-day precision and accuracy were determined on five separate days at identical concentrations.

Applications in pharmacokinetic studies

The pharmacokinetic study followed the principles of the Declaration of Helsinki, and was authorized by the Institution Ethics Committee of Chonbuk National University Hospital (IRB No. 2014-09-022). Healthy male volunteers aged ≥19 and ≤55 years at screening were included in the study. Normal haematology, blood chemistry, and urinalysis results were required at screening. Subjects with evidence of drug abuse, alcoholism, or any cardiac condition or illness putting the subject at risk were not allowed to be enrolled. The bioanalytical method was applied to study the pharmacokinetics of AA and DHAA in three healthy subjects after administration of 4000 mg of vitamin C tablet. Blood samples (4 mL) were collected into heparinized tubes at pre-dose (0 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, and 8 h post-dosing. Then plasma was immediately separated by centrifuging at 3,000 rpm for 10 min. The obtained plasma was stored frozen at -80℃ until analysis.

Sample preparation

AA stability tests were conducted for the use of human plasma. Plasma samples were stored at -70℃, -4℃, 25℃, and 25℃ while masked from light masked during 60 min periods. The results showed that at -70℃, the highest relative recovery was achieved after around 10 min (Fig. 2). However, all trends indicated the instability of AA in plasma with heat and light over time; therefore, we attempted the sample preparation of AA in plasma after removing from storage at -70℃ in each case.

Figure 2
Recovered ascorbic acid contents in standard solutions stored at -70℃ (-▪-), -4℃ (··▴··), 25℃ (··♦··) and light shielded at 25℃ (-•-)

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To prevent oxidation of AA in plasma samples, the addition of a stabilizer like HPO₃ is crucial, and it should be added immediately after blood sampling (especially for clinical studies).

Several concentrations of DTT (reduction agent) have been employed: 2 mM, 4 mM, and 8 mM. Since similar total AA concentrations were determined for each DTT concentration, 2 mM of DTT was used for all analyses. The amount of DHAA was calculated from the following difference: Total AA minus reduced AA.

Chromatographic separation

A representative HPLC chromatogram obtained from a human plasma sample is shown in Figure 3. The identification of the corresponding peaks was performed by comparing the retention times in the samples with those of the separately run pure standards under the same analytical conditions.

Figure 3
Chromatogram of ascorbic acid and internal standards in human plasma. HPLC conditions: mobile phase, 5 mM HTAB + 50 mM KH₂PO₄; column, C18 column (symmetry® 4.6 µm × 280 mm, 5 µm particle size, Waters, USA); flow rate, 1.2 mL/min; UV detection wavelength, 254 and 265 nm.

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System suitability

The lowest limit of quantification for each validated batch (1 µg/mL, LLOQ) of the samples was measured at least three times, and the system suitability was established as being less than 20% for the measured concentration deviation to average concentration ratio (coefficient variance, CV).

Linearity

The calibration curve of the blank, zero-blank (internal standard added), and seven standards ranging with 1-100 µg/mL of concentration had a correlation coefficient (r²) of 0.999, which indicated excellent linearity (Fig. 4).

Figure 4
Standard calibration curve of ascorbic acid in human plasma

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Specificity

The retention times (RT) of AA and IS were about 9 min and 12 min, respectively. Blank plasma samples of different origins (six different blank samples, one zero-blank sample, and a LLOQ sample) were analyzed, and no interference was introduced by human plasma around the retention time of AA and IS.

Carryover

We investigated the possibility of carryover (order effects) between evaluations of the LLOQ, blank, and upper limit of quantification (ULOQ = 100 µg/mL) samples. No carryover effects were observed.

Accuracy, precision, and limit of quantification for concentration

Accuracy and precision were determined using four concentrations (1, 2, 50, 80 µg/mL) of the quality control (QC) samples for the intra-day and inter-day; measurements were performed five times for each. The accuracy (average % and its CV) was defined as the average and CV of the ratios of measured concentrations (calculated using the calibration curve) divided by the nominal concentrations. Precisions within ±15% and accuracies within 85–115% were established as qualifying conditions.[19] At the limit of quantification concentration (1 µg/mL), a separate set of qualifying conditions were established: a precision within ±20% and accuracy within 80–120%. The accuracy and precision of intra-day measurements of the analytical method were 86.43–103.52% and 1.33–15.47%, respectively. The accuracy and precision of inter-day measurements were 85.86–101.65% and 0.65–12.89%, respectively (Table 1).

Table 1
Intra- and inter-day precision and accuracy (n=5) of ascorbic acid QC samples in human plasma

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Recovery

AA recovery tests were performed for three QC concentrations, and the absolute recovery of IS (absolute recovery) was also carried out. The recovery of AA and internal standard were 98.98-118.41% and 99.99%, respectively (Table 2).

Table 2
Intra- and inter-day recovery of ascorbic acid in human blank plasma QC samples (n=5)

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DHAA concentration

The concentration of DHAA was calculated by subtracting the initial AA concentration from the total AA concentration after reductive treatment of 50 µg/mL of AA (Table 3). These results demonstrate that it is possible to utilize the described HPLC-UV method to accurately determine the amount of AA and DHAA in plasma samples, which contradicts assertions made by Li and Franke that HPLC-UV is not suitable for AA determination in plasma.[10] Since the total time required for the analysis of one sample (including the sample preparation) was less than 15 minutes, further improvement in the analytical reliability is expected in an AA assay.

Table 3
Dehydroascorbic acid concentrations

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Pharmacokinetic study

The validated HPLC method has been successfully applied to a pharmacokinetic study of AA and DHAA after oral administration of 4,000 mg AA tablet to three healthy Korean volunteers with age of (27.67±8.96) years, weight of (61.17±16.86) kg, height of (164.00±12.88) cm, and BMI of (22.37±2.45) kg/m². The mean plasma concentration-time profiles of AA and DHAA are shown in Figure 5 and the related pharmacokinetic parameters calculated using Phoenix WinNonlin 6.3 software (Pharsight Corporation, Mountain View, CA, USA) are listed in Table 4. As a results, T_max (2.67±0.58 h) of the AA was consistent with those reported before (T_max: 3 h).[20] Unlike previous reports on the assays of DHAA using HPLC-UV or HPLC-ECD,[13, 21, 22] the HPLC-UV method in this report was applied for the human pharmacokinetic study of AA and DHAA.

Figure 5
Mean plasma concentration-time profiles of ascorbic acid (A) and dehydroascorbic acid (B)

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Table 4
Pharmacokinetic parameters for ascorbic acid and dehydroascorbic acid in human plasma (mean±SD) obtained after administration of vitamin C 4,000 mg

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