Tropical Journal of Pharmaceutical Research February 2011; 10 (1): 105-111
Research ArticleRapid and Reliable HPLC Method for the Determination of Vitamin C in Pharmaceutical Samples
Snezana S Mitić1, Danijela A Kostić1*, Danijela C NaskovićĐokić2, Milan N Mitic1
1Department of Chemistry, Faculty of Natural Sciences and Mathematics,Visegradska 33, 18000 Niš, 2D.D. ''ZdravljeActavis"-Pharmaceutical and Chemical Company, 16000 Leskovac, Serbia
*Corresponding author: Email: email@example.com; Tel: +38118533014; Fax: +38118533014
Received: 2 July 2010 Revised accepted: 24 November 2010Code Number: pr11016
develop and validate an accurate, sensitive and reproducible high
performance liquid chromatographic (HPLC) method for
the quantitation of vitamin C in pharmaceutical samples.
Keywords: Ascorbic acid; Pharmaceutical preparations; HPLC methodIntroduction
Vitamin C (ascorbic acid, ascorbate, AA) is a water-soluble organic compound involved in many biological processes . Although all the functions of AA have not been fully elucidated, it is likely that it is also involved in maintaining the reduced state of metal cofactors, for example, monooxygenase (Cu+) and dioxygenase (Fe2+). In cells, the other role of AA is to reduce hydrogen peroxide (H2O2), which preserves cells against reactive oxygen species . Primates and several other mammals are not able to synthesis ascorbic acid. The only way humans can obtain ascorbic acid is via food, but the exact daily requirements of vitamin C for humans are not yet clear. Currently, the estimated average requirement and recommended dietary allowance of ascorbic acid are 100 and 120 mg per day, respectively .
Many analytical techniques including sensors and biosensors [4-6] have been suggested for the detection of ascorbic acid in various types of samples. Integrated methods, utilizing flow injection analysis, high performance liquid chromatography [7-9] or capillary electrophoresis [10-13] and a detector, are mostly employed for the determination of vitamin C. However, some of these methods are time-consuming, while others are costly, require special training for operators of the equipment, or suffer from insufficient sensitivity or selectivity.
Vitamin C has been widely employed in pharmaceutical and cosmetic preparations to protect them against oxidation and to exert physiological/biological activities. In view of the fact that pharmaceutical dosage forms usually contain a variety of excipients that may appear as interferents, as well as the likelihood of the presence of degradation products and/or stabilizing antioxidant agents for vitamin C, HPLC method possesses advantages. HPLC is considered a sensitive and selective method and therefore suitable for active substance determination; it is also suitable for the evaluation of stability in formulations in the pharmaceutical and cosmetic industries .
The purpose of this study, therefore, was to develop and validate a HPLC method for the quantitation of vitamin C (ascorbic acid) in pharmaceutical powder or tablet preparations containing various excipients, without prior sample preparation.
ExperimentalHPLC apparatus and conditions
The HPLC system used consisted of a Hewlet Packard HPLC 1100 Series isocratic LC system with diode array detector (DAD) and flame-photometric detector (FLD). Prior to performing the validation assay, chromatographic conditions for the HPLC method were studied in order to achieve appropriate system suitability. Mobile phase composition was tested with methanol /phosphate buffer + tetrabutylammonium
(30:70 and 70:30, v/v) in C8 column (λ = 254 nm); and 0.2 % metaphosphoric acid in water solution, 0.2% metaphosphoric acid/methanol
(95:5 and 90:10, v/v), 0.2% metaphosphoric acid /acetonitrile (95:5 and 90:10, v/v) and 0.2% metaphosphoric acid/methanol /acetonitrile (90:5:5, v/v/v) in C18 column (λ = 254 nm).
The column used was Superspher RP-18 (250 x 4.6mm) while the mobile phase (pH 2.6) consisted of 1.5 g dissolved in 500 ml of acetic acid (99,8%) and mixed well. Routine degassing of the mobile phase was carried out by passing it through a 0.45 µm membrane filter (Millipore, Bedford, MA, USA). The mobile phase was pumped isocratically at a flow rate of 0.7 mL min-1 at 20 0C. This low operating temperature was used because the stability of ascorbic acid decreases with increasing temperature The injection volume was 50 µL.Reagents and chemicals
Ascorbic acid (C6H8O6) with 99.0% purity was kindly provided by F. Hoffmann–La Roche Ltd. Basel, Swiss.. Doxycycline was obtained from PromoChem, Teddington, United Kingdom. All the solvents used were of HPLC grade while the other chemicals were of spectroscopic grade and obtained from Merck (Darmstadt, Germany). Pure water was produced with a Millipore Milli-Q Plus System (Molsheim, France). All the reagents were used without any further purification. Branded pharmaceutical and veterinary formulations, in powder or tablet form, were obtained from commercial sources and used as received, without any further purification. The composition of the preparations are as follows:
Vetadox powder: Supplied by “Actavis” Leskovac, Serbia, it consists of various ingredients, among them doxycycline hyclate (the only active component; content, 250 mg), vitamin C (50 mg) as anti-oxidant and glucose as sweetner.
Ferveks for adults: (Bristol-Myer Squibb-New York, USA in powder form and contained pheniramin (25 mg), paracetamol (500 mg), vitamin C (200 mg) and flavor (25mg)
Eferalgan (Bristol-Myer Squibb, New York, USA): an effervescent tablet, contained paracetamol (330 mg), vitamin C (200 mg), and KHCO3, NaHCO3, sorbitol, citric acid anhydride, sodium-benzoate, sodium docusate and povidone as excipients.
Budapest, Hungary), an effervescent tablet, contained Ca (300 mg, 37.5
%) in the form of calcium carbonate, vitamin C (60 mg), as well as
sorbitol, citric acid anhydride, Na-cyclamate, Na-saccharin , synthetic
beta carotene and flavor as excipients.
Approximately 50 mg of standard ascorbic acid (99 % purity) was weighed precisely and dissolved in 50 mL of water-acetic acid clear mixture (20:1, v/v) to obtain a stock concentration of 1 mgmL−1 . Standards were freshly prepared. To obtain the working solution, aliquots of standard ascorbic acid solution were diluted to a concentration of 0.2 mg ml -1 . The working standard solutions were prepared in duplicate, filtered and degassed by passing them through a 0.45 µm membrane filter (Millipore, Bedford, MA, USA). The linearity study verifies that the sample solutions are in a concentration range where analyte response is linearly proportional to the concentration. To establish linearity of the proposed methods, five separate series of ascorbic acid solutions were prepared from the stock solutions and analyzed. Least square regression analysis was done for the data obtained. The linearity was studied over a concentration range of
0.15 – 0.25
mg mL-1 .
Replicates of three injections were performed for each sample.
Linearity data were computed on a personal computer using Microsoft
Excel program (version 2003, Microsoft Co., Redmond, USA).
Accuracy/recovery and precision assay
The accuracy of the method is the closeness of the measured value to the true value for the sample. Accuracy was assessed as percent relative error and mean % recovery.
40, 50 and 60 mg of standard ascorbic acid were weighed precisely and
dissolved, separately, in 50mL of the mobile phase. To achieve
accuracy/recovery, aliquots of these samples were diluted to
appropriate final concentrations of ascorbic acid solution , i.e.,
2 ml of each solution diluted to 10 ml with mobile phase solution. The
accuracy of the method was checked by determining recovery values.
Accuracy/ recovery was calculated for six runs of each solution. The
precision was determined by measuring five sample probes under the same
experimental conditions. To calculate precision, intra-and inter-day
tests were performed and the results were expressed as relative
standard deviation (RSD, %).
Limits of detection (LOD) and quantitation (LOQ) assay
The limits of detection and quantitation were determined by serial dilutions of ascorbic acid solutions in order to obtain signal/noise ratios of ≈ 3:1 for LOD and ≈ 10:1 for LOQ.
25 mg of standard ascorbic acid was weighed precisely and dissolved in
50 mL of the mobile phase. Appropriate amounts of standard ascorbic
acid solution were diluted to the required concentrations of 0.5, 1.0,
1.5 and 2 µg mL−1 . Working standard solutions were
prepared in triplicate.
specificity of the HPLC method for ascorbic acid quantitation in
pharmaceutical/ veterinary preparations was investigated in For
specificity and selectivity of method, ascorbic acid solutions (0.2 µg
were prepared in the mobile phase along with and without common
ingredients (doxycycline hyclate, pheniramin, paracetamol, CaCO3,
sorbitol, citric acid anhydride, sodium-benzoate, sodium docusate,
povidone, Na-cyclamate, Nasaccharin, synthetic beta carotene)
separately. All the solutions were injected into the Superspher RP-18
(250 x 4.6mm) column. In this assay, it was tested by running solutions
containing placebo (using the same quantities and conditions as for the
test samples) to verify that there is no overlapping peak at the
retention times corresponding to those of the analytes. Paired t-test
at 95 % level of significance was performed to compare the area of the
Approximately 250 mg of each topical formulation was weighed precisely and dissolved separately in 50 mL of the mobile phase. The mixtures were centrifuged at 3000 rpm for 5 min at room temperature (20 0C). The supernatants were collected and aliquots of the samples were diluted to appropriate final concentration (0,2mg ml-1).Mobile phase
The mobile phase (pH 2.6) prepared by carefully adding acetic acid (500 mL) to 1.5 g of 1-hexanesulfonic acid sodium salt thorough mixing gave good response and a retention time of 4 min for ascorbic acid. The other mobile phases tested did not present adequate response for ascorbic acid quantitation since they were not able to identify the antibiotic, doxycycline, as a component of the pharmaceutical preparations, and also provided unsuitable retention time for the active substance (≈ 1 min) .
The mobile phase (pH 2.6) prepared by carefully
adding acetic acid (500 mL) to 1.5 g of 1-hexanesulfonic acid sodium
salt thorough mixing gave good response and a retention time of 4 min
for ascorbic acid. The other mobile phases tested did not present
adequate response for ascorbic acid quantitation since they were not
able to identify the antibiotic, doxycycline, as a component of the
pharmaceutical preparations, and also provided unsuitable retention
time for the active substance (≈ 1 min) .
The linearity was checked on samples of
standard ascorbic acid at five different concentrations (0.25 – 1.5 mg
mL−1). The regression
equation derived was: y = 10245x - 89.95 with
a correlation coefficient (R2) of 0.9998, where x
represents concentration in µg
ml−1, and y represents
the HPLC peak area, which was automatically measured by an integrator
of the HPLC instrument. Linearity data were computed on a personal
computer using Microsoft Excel program (version 2003, Microsoft Corp.,
Accuracy/recovery and precision
was calculated for three runs of each solution. The results of
accuracy/recovery and precision experiments are recorded in Table 1.
The data indicate an adequate percentage of accuracy/recovery for the
HPLC method for the quantitation of ascorbic acid in the pharmaceutical
Limits of detection (LOD) and quantitation (LOQ)
which is defined as the lowest active substance concentration that can
be determined by a method, usually cannot be calculated precisely and
accurately. On the other hand, LOQ is the concentration of the sample
used in analysis that can be obtained with adequate precision and
accuracy. An estimation of the limits, which was achieved by the
determination of the signal/noise ratios of 3:1 (LOD) and 10:1 (LOQ),
were ≈ 1.95 and ≈ 6.5 µgmL−1 , for LOD and LOQ,
specificity of the HPLC method for vitamin C quantitation in the
pharmaceutical formulations is an indication of possible interference
from excipients in the preparations. The presence of other ingredients,
including the antibiotics, in the formulations did not cause any
interference with the ascorbic acid peak. Under the test conditions,
ascorbic acid was observed to be well resolved from the other
components of the formulations and potential degradation products of
vitamin C. Thus the method is specific for vitamin C.
Application of the developed method to vitamin C formulations
The outcome of the application of the developed method to the determination of vitamin C in some pharmaceutical formulations are shown in Figures 1 and 2. The representative chromatograms of the standard sample of vitamin C and the test preparations show identical retention times.
Assay results for the determination of ascorbic acid in commercial pharmaceutical are shown in Table 3. RSD (%) indicates the accuracy of determination of ascorbic acid in the investigated pharmaceutical preparations Table 3: Assay results for the determination of ascorbic acid in some commercial pharmaceutical preparations.
The mobile phase used increased retention time to ≈3.5 min, providing a satisfactory resolution of ascorbic acid from doxycycline and the excipients present in the formulations, with sharp and symmetrical peaks, and also minimal interferences from equipment noise. In addition, the mobile phase maintained long column life.
The results demonstrated satisfactory and consistent performance of the HPLC method. Least-squares regression analysis used to evaluate the concentration range data indicate excellent linearity over the interval studied (0.25 -1.5 mg mL-1), The R2 obtained for this ascorbic acid concentration range was ≥ 0.99.
The low values of RSD (0.38 and 1.22 %, respectively) are evidence of the good precision of the developed HPLC method and also indicates non-variability of the data. LOD and LOQ values of ≈1.95 and ≈6.5 µg mL−1 , respectively, indicate that the method is sensitive.
The presence of the other ingredients in the formulations, including an antibiotic (doxycycline) did not cause any interference with ascorbic acid peak. Ascorbic acid was well resolved from the other components of the formulations and the potential degradation product of vitamin C. Thus, the method is selective for vitamin C.
The relative standard deviation (RSD) of 1.87 % was low, being less than < RSDmax. Thus the method is precise. Overall, the results show that the proposed method can be successfully applied for the determination of vitamin C in pharmaceutical preparations.
ConclusionThe developed HPLC method for the determination of ascorbic acid in pharmaceutical preparations containing various other ingredients including excipients (doxycycline hydrochloride, pheniramin, paracetamol, etc) has been validated for linearity, sufficient accuracy/recovery and precision, as well as low values of limits of detection and quantitation. The method provides a rapid, sensitive, accurate, and reproducible means of determining vitamin C in pharmaceutical and veterinary formulations without prior sample preparation.
This research was supported partly by the European Union Project (FP7–REGPOT2007–3-01, KBBE: Food, Agriculture, and Biotechnology, “Chromolab-Antioxidant”, 204756) and partly by the Ministry of Science and Technological Development of the Republic of Serbia (Project ON 142015).
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
The following images related to this document are available:
Photo images[pr11016f1.jpg] [pr11016t3.jpg] [pr11016f2.jpg] [pr11016t1.jpg]