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ANALYST, OCTOBER 1992. VOL. 117
1635
Determination of Ascorbic Acid in Pharmaceuticalsand Urine by Reverse Flow Injection Isabel Albero, Ma.Soledad Garcia, C. Sanchez-Pedreno" and Jose Rodriguez Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Murcia, Spain
Ma.
Two reverse flow injection (FI) methods, using spectrophotometric detection, are proposed for the determination of ascorbic acid. Both methods are based on its reaction with the ethylenediaminetetraacetic acid-CoIl'complex in a medium of 5% diethylamine. In the first method, using the peak-height FI technique, ascorbic acid is determined over the range from 2 x 10-4 to 5 x 10-3 rnol dm-3 and in the second, using the peak-width FI method, the working range is extended (2 x 10-3-5 x 10-2 rnol dm-3). Both FI methods were applied to the determination of ascorbic acid in pharmaceuticals while the peak-height FI technique was also used to determine ascorbic acid in urine. Keywords: Ascorbic acid; ethylenediaminetetraacetic acid-cobalt(iir) complex; flow injection; pharmaceuticals; urine
Ascorbic acid participates in many different biological processes, and is important in human diet. There is a need for fast, selective and automated methods for its determination, particularly in routine analyses. Most of the methods proposed have been listed in reviews and monographs14 and some have been suggested as reference methods for different types of samples.5-7 Flow injection (FI) has been applied to determine ascorbic acid in a variety of samples, including foods, pharmaceuticals and biological samples. Several detection techniques have been proposed, e.g., potentiometric,g coulometric,9~~() amperometric,"11-18 spectrophotometric~9-25and spectrofluorimetric26 techniques. Few methods are found in the literature using FI gradient techniques.24.27 In this work, two simple, fast and selective methods for the determination of ascorbic acid, based on the reaction between Co"'-e t hy lenediamine tetraace tic acid (EDTA) and ascorbic acid using FI techniques are described. Peak height and peak width are used as quantitative parameters. These methods have been applied, with good results, to the routine determination of ascorbic acid in pharmaceuticals and urine.
Experimental Apparatus The FI system consisted of a Gilson HP4 peristaltic pump, an Omnifit injection valve, a Hellma 18 mm3 flow cell and a Pye Unicam spectrophotometer as detector. Connecting tubing of 0.5 mm bore, poly(tetrafluoroethy1ene) (PTFE) tubing and various end fittings and connectors (Omnifit) were used. For the peak-width measurements, a Perspex gradient tube of 2 mm inner diameter was used. Reagents
All chemicals were of analytical-reagent grade and the solutions were prepared with doubly distilled water. Cobalt(iii)-EDTA, 4 x 10-2 rnol dm-3. Exactly 50 cm3 of 0.1 rnol dm-3 cobalt(i1) nitrate (Merck) and 50 cm3 of 0.1 rnol dm-3 Na2H2EDTA(Merck) were transferred by pipette into a 250 cm3 beaker. Dipotassium peroxodisulfate (3 g, Merck) was then added and the solution adjusted to pH 6 with ammonia solution (1 + 1; d = 0.88 g cm-3) and boiled gently for about 20 min in order to decompose the excess of peroxodisulfate.28 The solution was diluted to volume with doubly distilled water in a 125 cm3 calibrated flask.
* To whom correspondcnce should bc addressed.
Ascorbic acid stock solution, 0.1 rnol dm-3. Prepared by dissolving 4.4032 mg of the acid (Sigma) in 250 cm3 of distilled water; the solution was stored at 4"C in a dark bottle. Working solutions of ascorbic acid were prepared daily by dilution of this stock solution. Diethylamine, 5% vIv. Prepared by dissolving diethylamine (Merck) in distilled water. FI Procedures The FI manifold shown in Fig. l(a) was used. An 85 3711713 aliquot of 5 x 10-3 rnol dm-3 CoI'I-EDTA solution was injected into a stream formed by the mixing of 5% diethylamine solution and the ascorbic acid sample. All streams were pumped at a rate of 0.87 cm3 min-1. The absorbance of Co'II-EDTA at 540 nm was measured, and a calibration graph was obtained by plotting the peak height versus the concentration of ascorbic acid over the range from 2 x 10-4 to 5 x 10-3 mol dm-3. For the peak-width method the manifold shown in Fig. l(b) was used. A 146 mm3 aliquot of 5 X 10-3 rnol dm-3 Co"'-EDTA solution was injected into a stream formed by the mixing of 5% diethylamine solution and the ascorbic acid sample; the absorbance was monitored at 540 nm with a plotting rate of 10 s cm-1. A calibration graph was obtained by plotting the peak width expressed in seconds, measured to a preselected absorbance level of 0.1, versus the logarithm of the ascorbic acid concentration. Determination of Ascorbic Acid in Authentic Samples
Pharmaceutical samples No sample pre-treatment was needed for these analyses. An accurately weighed or measured amount of the pharmaceutical sample was dissolved in water and the solution filtered. The C 0 2 was expelled from the samples by shaking, if necessary, and the sample diluted to a final volume of 250 cm3 with doubly distilled water. The FI procedures described above were applied to the resulting solutions. Urine samples The proposed FI method was applied to the determination of ascorbic acid in urine. The composition of the synthetic urine was: lactic acid, 5 x rnol dm-3; uric acid, 2 x 10-4 rnol dm-3; hippuric acid, 8 X 10-3 rnol dm-3; urea, 3 X 10-4 rnol dm-3; glucose, 3 X rnol dm-3; CI-, 6 mg cm-3; P043-, 0.5 mg cm-3; Na+, 4 mg cm-3; K+, 0.5 mg ~ m - and ~ ; Ca", 0.5 mg cm-3. Five aliquots of 100 cm3 each, in which different amounts of ascorbic acid had been dissolved, were
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Blank
(a)
g = 0.87 cm3 min-1 Colll-EDTA 5 x 10-3 mot dm-31
1
I
1 540 nm
Diethylamine 5%
D
Ascorbic acid
U
v = 86 mm3 W
Time
I (6)
-
Blank
9 = 0.5 cm3 min-1
Colll-EDTA 5 x 10-3 rnol dm-3 540 nm
Diethylamine 5% Ascorbic acid
U
v = 146 mm3
w
-
I Time
Fig. 1 FI manifolds for the determination of ascorbic acid. Recordings obtained using (a) peak-height method and (b) peak-width method
taken. The content of each aliquot was determined by following the recommended procedure. For human urine, urine samples were obtained from an individual 2 h after he had ingested a pharmaceutical compound containing 1g of vitamin C. Sampleswere collected in a polyethylene flask. Aliquots of 15 cm3 were taken, 2 cm3 of 0.1 mol dm-3 Na2H2EDTA were added and the mixture was diluted in a 25 cm3 calibrated flask with doubly distilled water. The content of each aliquot was determined by following the recommended FI procedure.
7.5 6.5 -
E
Y
2 5.5 4.5
3.5
I
1
I
Results and Discussion Ascorbic acid was found to reduce the Co"'-EDTA complex to ColI-EDTA in a water-diethylamine medium (pH 12.5) and this reaction was adapted in order to develop two FI methods for determining ascorbic acid. Detection was carried out spectrophotometrically at 540 nm, the absorption maximum of CoIII-EDTA, by reverse FI as the analyte is pumped in both methods, the peak height being measured in the first method and the peak width in the second.
Peak-height FI Method A schematic diagram of the reverse FI manifold is shown in Fig. l(a). The solution of CoIlI-EDTA was injected into a stream containing a mixture of the sample and diethylamine solution. Under these conditions the ascorbic acid reduced the Co"'-EDTA complex and the absorbance was measured spectrophotometricallyat 540 nm. In the absencc of ascorbic acid (blank), a maximum peak was obtained corresponding to the initial concentration of CoI"-EDTA. The presence of ascorbic acid caused a decrease in the analytical signal proportional to the ascorbic acid concentration [Fig. l(a)]. Flow injection and chemical variables were optimized for the proposed FI method. This study was carried out by altering each variable in turn while keeping the others constant. The optimum chemical and FI variables chosen were those that yielded maximum and constant differences between the peak height of the blank and the samples (Ah).
Fig. 2 Variation of Ah with: A, the pumping rate; B. reactor length; and C, loop size
It was found that a basic medium was necessary for the reaction between Co"'-EDTA and ascorbic acid to proceed and the best results were obtained with the use of diethylamine. The influence of the diethylamine concentration was studied in the range between 1 and 7% vlv. The concentrations of ascorbic acid and CoIIl-EDTA were 2 x 10-3 and 5 x 10-3 rnol dm-3, respectively. The best results were obtained with a concentration of 5% v/v diethylamine. Similarly, the influence of the concentration of Co"'-EDTA was studied in the range from 1 x 10-3 to 5 x 10-3 rnol dm-3. A concentration of 5 x 10-3 rnol dm-3 CoIll-EDTA was selected as this yielded suitable peak heights both for the blank and in the range of the calibration graph. Fig. 2 shows the effects of the flow rate, reactor length and loop size on the differences between the signal of the blank and the sample (Ah) for an ascorbic acid concentration of 2 x 10-3 rnol dm-3. The effect of the flow rate on Ah was studied over the range 0.5-1.2 cm3 min-1. An increase in the flow rate resulted in a decrease in Ah (Fig. 2, line A). A flow rate of 0.85 cm3 min-'
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ANALYST, OCTOBER 1992, VOL. 117
1637
was selected in order to obtain a reasonable sampling frequency. The influence of the reactor length on Ah was studied over the range 2-6 m (inner diameter, 0.5 mm). The results (Fig. 2, line B) showed that Ah is virtually constant throughout the range studied. A 4 m reactor was selected. The effect of the loop size on Ah is shown in Fig. 2, line C. An increase in loop size produced an increase in Ah, and a loop size of 85 mm3 was chosen. Determination of Ascorbic Acid
With the manifold described above and under the selected experimental conditions, viz., 5 x 10-3 rnol dm-3 Coi1'EDTA and 5% v/v diethylamine, a linear calibration graph between 2.0 x 10-4 and 5.0 x 10-3 rnol dm-3 ascorbic acid was obtained. The regression equation found was h = 11.21 X 102[ascorbic acid] + 18.4, where h is the peak height in centimetres and [ascorbic acid] is expressed in rnol dm-3, with a correlation coefficient (r) of 0.9985. The relative standard deviation (RSD) (p = 0.05) for ten determinations of 2 X 10-3 rnol dm-3 ascorbic acid was 0.94%. The detection and quantification limits were 6 x 10-5 and 1.2 x 10-4 rnol dm-3, respectively, and the sampling frequency was 60 samples h-*.
The effect of the FI experimental variables on At-At' (the difference between the peak width of the blank and the samples) was studied. The results obtained are shown in Fig. 3. The selected values for the FI variables were as follows: flow rate = 0.51 cm3 min-1, with which a suitable At without a notable decrease in the sample frequency was obtained; and injection volume = 146 mm3; this large volume of CoiliEDTA injected ensured that wide peaks were obtained. Several tubes of 2 mm inner diameter and between 1.0 and 12 cm in length were used for the study of the influence of the gradient tube volume on At. The peak width increased with the volume of the tube, and hence a tube of length 12 cm (diameter = 2 mm) was selected. The concentrations of Co'Il-EDTA and diethylamine selected were the same as in the peak-height method. The absorbance at which the peak width was measured influenced the sensitivity of the determination. It is advisable to measure the peak width at the absorbance level that yields the maximum difference between the blank and the sample. A plot of the peak width versus the logarithm of the concentration of ascorbic acid showed different linear ranges according to the value of the absorbance at which At was measured (as is shown in Table 1). An absorbance of 0.1 was selected because a wider range and a greater slope were obtained.
Peak-width FI Method
This method is based on the measurement of the peak width at a pre-determined height from the baseline. One of the problems found in routine analytical determinationsis that the analyte concentrations of the sample often do not fall within the limited range of concentration covered by the calibration graphs. As the aim of this work was the routine determination of ascorbic acid, the peak-width FI method was applied to extend the useful working range for this acid. A logarithmic relationship between the peak width and the concentration of the samples was found. The proposed FI manifold for the determination of ascorbic acid is shown in Fig. l(6).
28 1
1
20
4 a 12
I
I
0.3
0.5 0.7 0.9 Pump rate (q)/cm3 min-1
1
50
100
1.1
I
I
150
200
Table 2 Effect of various foreign species on the determination of 4 10-3 mol dm-3 ascorbic acid using the two FI methods
Limiting molar ratio [species]: [ascorbic acid] 100 50
Species assayed* N03-, C1-, CH3COO-, Po43S042-, C 0 3 2 - ,Br-, As02S032-, oxalate 40 Se032-, citrate, tartrate, Se042-, lactose, 20 starch? Ca2+, I-, F-, Mo042-, urea, thiourea, fructose, lacticacid, gelatint 10 Glucose, maltose, methionine, hippuric acid, glutamic acid, phenylalanine, NH4+, sucrose, valine, thiamine, pyridoxine, leucine 5 Nicotinamide, Zn2+$, Cu2+$, Mn2+$ 1 Uric acid§, riboflavine 0.5 NO;?-, S20.1 * Caffeine, aspartic acid and saccharin do not interfere. Mass relation. $ In the presence of H2EDTA2-. Q Maximum relation assayed by the solubility of uric acid. Table 3 Determination of ascorbic acid in pharmaceuticals Ascorbic acid content*
1
Loop size (v)lmm3
Peak-height Peak-width Reference method method method Stated lt 162.52 (0.89)$ 162.75 (0.95) 163.34(1.92) 166.67 29 50.37 (0.92) 50.46 (0.87) 50.73 (1.77) 50.0 37 200.49 (1.17) 199.46 (1.25) 200.13 (2.53) 200.0 * Values for samples 1 and 2 in mg g-I; values for sample 3 in mg cm-3. t Cebion sachets (Abbott Laboratories): vitamin C, 1g; aromatized excipient (including 8 mg of sodium saccharin) up to 6 g. $ Values in parentheses are relative standard deviations in % (n = 5) * Q Frenadol sachets (Abello Laboratories): paracetamol. 600 mg; salicylamide, 100 mg; codeine phosphate, 10 mg; caffeine citrate, 30 mg; chlorpheniramine maleate. 4 mg; ascorbic acid, 500 mg; plus excipient with attached carbohydrate groups up to 10 g. 7 Redoxon drops (Roche Laboratories): vitamin C, 200 mg; sodium saccharin, 2 mg; plus excipient up to 1 cm3. Sample
0.6
3.6
6.6 9.6 Length ( / ) l c m
12.6
Fig. 3 Variation of At' with: A, the pumping rate; B, loop size; and C, reactor length
Table 1 Calibration graphs for the determination of ascorbic acid using the peak-width method Concentration range/mol dm-3 Absorbance* 2 X 10-3-5 X 10-2 0.1 2 X 10-3-3 X 10-2 0.2 2 x 10-3-1 x 10-2 0.3 * Absorbance at which the peak width
Correlation Slope coefficient ( r ) -26.632 0.9926 -20.068 0.9888 -18.446 0.9422 (Ar) was measured.
X
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ANALYST, OCTOBER 1992. VOL. 117
Table 4 Determination of ascorbic acid in urine by the peak-height FI method
Added mg cm-3 0.296 0.501 0.672 Human urine O.OO0 0.352 0.528 0.704 * Average of five determinations. Sample Synthetic urine
Found*/ mg cm-3 0.298 0.510 0.674 0.129 0.349 0.542 0.699
Recovery (Yo)
100.8 101.9 100.3
are summarized in Table 4 and show that the components of the urine do not interfere in the determination of ascorbic acid in the samples assayed. Recovery data were obtained in a similar way to that described for synthetic urine. We gratefully acknowledge the Direccion General Cientifica y TCcnica for financial support (PB 90-0008).
99.20 101.06 99.25
Calibration Graph
The equation of the calibration graph for determining ascorbic acid is At = -26.63 x log [ascorbic acid] -9.16 ( r = 0.9926), where At is expressed in seconds and [ascorbic acid] in mol dm-3. The equation is satisfied for ascorbic acid concentrations between 2.0 x 10-3 and 5.0 x 10-2 mol dm-3. Hence, by using the peak width as a quantitative parameter, it was possible to determine greater concentrations of ascorbic acid than by using the peak height. The RSD (n = 10) for 7.5 x 10-3 mol dm-3 ascorbic acid was 2.02%. Study of Interferents in Both Methods
The effect of foreign species in both FI methods was studied. the results for the determination of 4 x 10-3 mol dm-3 ascorbic acid are listed in Table 2. The tolerance limit was taken as the concentration causing an error of not more than +3% in the determination of ascorbic acid. As can be seen, the proposed methods are sufficiently selective. Applications
The first (peak-height) method was applied to the determination of ascorbic acid in three pharmaceuticals and urine and the second (peak-width) method only to the determination of ascorbic acid in pharmaceuticals. The results obtained for pharmaceuticals are summarized in Table 3. Reproducibility was good in all instances. The results obtained with the proposed methods were compared with those given by the standard British Pharmacopoeia.5 The peak-height method was also tested for its applicability to the determination of ascorbic acid in urine. A recovery experiment was performed using a mixture to simulate human urine. The data from this study are summarized in Table 4. Recovery data were obtained by adding different amounts of standard ascorbic acid to the synthetic urine samples and subtracting the results obtained from samples prepared in a similar way but with no ascorbic acid added. The results obtained for human urine samples containing ascorbic acid, in addition to the data from the recovery study,
References 1 Looke, J. R.. and Moxon, R. E. D., in Vitamin C (Ascorbic Acid), eds. Counsell, J. M., and Horming, D. H.. Applied Science Publishers, London, 1981, p. 167. 2 Briggs, M., Vitamins in Human Biology and Medicine, CRC Press, Boca Ration, FL, 1981. 3 Zloch, Z., Chem. Listy, 1988, 82, 825. 4 Grolubkina, N. A., and Prudnik, 0.V., Zh. Anal. Khim., 1989, 44, 1349. 5 British Pharmacopoeia f98O, The Pharmaceutical Press, London, 1980, vol. 1, p. 39. 6 Officiul Methods of Analysis of the Association of Official Analytical Chemists, ed. Williams, S., Association of Official Analytical Chemists, Washington, DC, 1984, p. 844. 7 The United States Pharmacopeia X X I , The National Formulary XXVI, US Pharmacopeial Convention, Rockville, MD, 1985, p. 75. 8 Karlberg, B., and Thelander, S., Analyst, 1978, 103, 1154. 9 Strohl, A., and Curran, D., Anal. Chem., 1979,51, 1045. 10 Curran, D. J., and Tougas, T., Anal. Chem., 1984,56,672. 11 Fogg, A. G., Summan, A. M., and Ferndndez-Arciniega, M. A., Analyst, 1985, 110, 341. 12 Wang, J., and Frelha, B. A., Anal. Chem., 1983, 55, 1285. 13 Uchiyama, S., Tofuku. Y., and Suzuki, S., Anal. Chim. Acta, 1988,208, 291. 14 Bradberry, C., and Adams, R.. Anal. Chem., 1983,55,2439. 15 Ikeda, S . , Satake, H., and Yohri, Y., Chem. Lett., 1984,6,873. 16 Lunte, C., Wong, S., Redgway, T., Heineman, W., and Chan, K., Anal. Chim. Acta, 1986, 188, 263. 17 Greenway, G. M., and Ongomo, P., Analyst, 1990, 115, 1297. 18 Matuszewski, W., Torjanowicz, M., and Ilcheva, L., Electroanalysis. 1990,2, 147. 19 Karayannis, M., Talanta, 1976,23. 27. 20 Karayannis, M. I., and Farasoglou, D. I., Analyst, 1987, 112, 767. 21 Hiromi, K., Kuwamoto, C., and Onishi, M.,J. Biochem., 1980, 100,421. 22 Yamane, T.. and Ogawa, T., Bunseki Kagaku, 1987,36, 625. 23 Herndndez-Mendez, J.. Alonso, A.. Almendral, M. J., and Garcia de Maria, C., Anal. Chim. Acta, 1986. 184, 243. 24 Lazaro. F., Rios. A., Luque de Castro, M. D.. and Valcarcel, M., Analyst, 1986, 111, 163. 25 Ldzaro. F.. Luque de Castro. M. D.. and Valcarcel. M.. Analusis, 1987, 15, 183. 26 Vanderslice. J., and Higss. D., Micronutr. Anal., 1989, 6, 109. 27 Koupparis, M., and Anagnostopoulou. P.. Talanta. 1985, 32, 411. 28 Chang. F.. and Cheng. K.. Mikrochim. Acta, Part fI, 1979.219.
Paper 2/0105 7G Received February 28, 1992 Accepted May 14, 1992
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ANALYST, OCTOBER 1992. VOL. 117
1635
Determination of Ascorbic Acid in Pharmaceuticalsand Urine by Reverse Flow Injection Isabel Albero, Ma.Soledad Garcia, C. Sanchez-Pedreno" and Jose Rodriguez Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Murcia, Spain
Ma.
Two reverse flow injection (FI) methods, using spectrophotometric detection, are proposed for the determination of ascorbic acid. Both methods are based on its reaction with the ethylenediaminetetraacetic acid-CoIl'complex in a medium of 5% diethylamine. In the first method, using the peak-height FI technique, ascorbic acid is determined over the range from 2 x 10-4 to 5 x 10-3 rnol dm-3 and in the second, using the peak-width FI method, the working range is extended (2 x 10-3-5 x 10-2 rnol dm-3). Both FI methods were applied to the determination of ascorbic acid in pharmaceuticals while the peak-height FI technique was also used to determine ascorbic acid in urine. Keywords: Ascorbic acid; ethylenediaminetetraacetic acid-cobalt(iir) complex; flow injection; pharmaceuticals; urine
Ascorbic acid participates in many different biological processes, and is important in human diet. There is a need for fast, selective and automated methods for its determination, particularly in routine analyses. Most of the methods proposed have been listed in reviews and monographs14 and some have been suggested as reference methods for different types of samples.5-7 Flow injection (FI) has been applied to determine ascorbic acid in a variety of samples, including foods, pharmaceuticals and biological samples. Several detection techniques have been proposed, e.g., potentiometric,g coulometric,9~~() amperometric,"11-18 spectrophotometric~9-25and spectrofluorimetric26 techniques. Few methods are found in the literature using FI gradient techniques.24.27 In this work, two simple, fast and selective methods for the determination of ascorbic acid, based on the reaction between Co"'-e t hy lenediamine tetraace tic acid (EDTA) and ascorbic acid using FI techniques are described. Peak height and peak width are used as quantitative parameters. These methods have been applied, with good results, to the routine determination of ascorbic acid in pharmaceuticals and urine.
Experimental Apparatus The FI system consisted of a Gilson HP4 peristaltic pump, an Omnifit injection valve, a Hellma 18 mm3 flow cell and a Pye Unicam spectrophotometer as detector. Connecting tubing of 0.5 mm bore, poly(tetrafluoroethy1ene) (PTFE) tubing and various end fittings and connectors (Omnifit) were used. For the peak-width measurements, a Perspex gradient tube of 2 mm inner diameter was used. Reagents
All chemicals were of analytical-reagent grade and the solutions were prepared with doubly distilled water. Cobalt(iii)-EDTA, 4 x 10-2 rnol dm-3. Exactly 50 cm3 of 0.1 rnol dm-3 cobalt(i1) nitrate (Merck) and 50 cm3 of 0.1 rnol dm-3 Na2H2EDTA(Merck) were transferred by pipette into a 250 cm3 beaker. Dipotassium peroxodisulfate (3 g, Merck) was then added and the solution adjusted to pH 6 with ammonia solution (1 + 1; d = 0.88 g cm-3) and boiled gently for about 20 min in order to decompose the excess of peroxodisulfate.28 The solution was diluted to volume with doubly distilled water in a 125 cm3 calibrated flask.
* To whom correspondcnce should bc addressed.
Ascorbic acid stock solution, 0.1 rnol dm-3. Prepared by dissolving 4.4032 mg of the acid (Sigma) in 250 cm3 of distilled water; the solution was stored at 4"C in a dark bottle. Working solutions of ascorbic acid were prepared daily by dilution of this stock solution. Diethylamine, 5% vIv. Prepared by dissolving diethylamine (Merck) in distilled water. FI Procedures The FI manifold shown in Fig. l(a) was used. An 85 3711713 aliquot of 5 x 10-3 rnol dm-3 CoI'I-EDTA solution was injected into a stream formed by the mixing of 5% diethylamine solution and the ascorbic acid sample. All streams were pumped at a rate of 0.87 cm3 min-1. The absorbance of Co'II-EDTA at 540 nm was measured, and a calibration graph was obtained by plotting the peak height versus the concentration of ascorbic acid over the range from 2 x 10-4 to 5 x 10-3 mol dm-3. For the peak-width method the manifold shown in Fig. l(b) was used. A 146 mm3 aliquot of 5 X 10-3 rnol dm-3 Co"'-EDTA solution was injected into a stream formed by the mixing of 5% diethylamine solution and the ascorbic acid sample; the absorbance was monitored at 540 nm with a plotting rate of 10 s cm-1. A calibration graph was obtained by plotting the peak width expressed in seconds, measured to a preselected absorbance level of 0.1, versus the logarithm of the ascorbic acid concentration. Determination of Ascorbic Acid in Authentic Samples
Pharmaceutical samples No sample pre-treatment was needed for these analyses. An accurately weighed or measured amount of the pharmaceutical sample was dissolved in water and the solution filtered. The C 0 2 was expelled from the samples by shaking, if necessary, and the sample diluted to a final volume of 250 cm3 with doubly distilled water. The FI procedures described above were applied to the resulting solutions. Urine samples The proposed FI method was applied to the determination of ascorbic acid in urine. The composition of the synthetic urine was: lactic acid, 5 x rnol dm-3; uric acid, 2 x 10-4 rnol dm-3; hippuric acid, 8 X 10-3 rnol dm-3; urea, 3 X 10-4 rnol dm-3; glucose, 3 X rnol dm-3; CI-, 6 mg cm-3; P043-, 0.5 mg cm-3; Na+, 4 mg cm-3; K+, 0.5 mg ~ m - and ~ ; Ca", 0.5 mg cm-3. Five aliquots of 100 cm3 each, in which different amounts of ascorbic acid had been dissolved, were
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Blank
(a)
g = 0.87 cm3 min-1 Colll-EDTA 5 x 10-3 mot dm-31
1
I
1 540 nm
Diethylamine 5%
D
Ascorbic acid
U
v = 86 mm3 W
Time
I (6)
-
Blank
9 = 0.5 cm3 min-1
Colll-EDTA 5 x 10-3 rnol dm-3 540 nm
Diethylamine 5% Ascorbic acid
U
v = 146 mm3
w
-
I Time
Fig. 1 FI manifolds for the determination of ascorbic acid. Recordings obtained using (a) peak-height method and (b) peak-width method
taken. The content of each aliquot was determined by following the recommended procedure. For human urine, urine samples were obtained from an individual 2 h after he had ingested a pharmaceutical compound containing 1g of vitamin C. Sampleswere collected in a polyethylene flask. Aliquots of 15 cm3 were taken, 2 cm3 of 0.1 mol dm-3 Na2H2EDTA were added and the mixture was diluted in a 25 cm3 calibrated flask with doubly distilled water. The content of each aliquot was determined by following the recommended FI procedure.
7.5 6.5 -
E
Y
2 5.5 4.5
3.5
I
1
I
Results and Discussion Ascorbic acid was found to reduce the Co"'-EDTA complex to ColI-EDTA in a water-diethylamine medium (pH 12.5) and this reaction was adapted in order to develop two FI methods for determining ascorbic acid. Detection was carried out spectrophotometrically at 540 nm, the absorption maximum of CoIII-EDTA, by reverse FI as the analyte is pumped in both methods, the peak height being measured in the first method and the peak width in the second.
Peak-height FI Method A schematic diagram of the reverse FI manifold is shown in Fig. l(a). The solution of CoIlI-EDTA was injected into a stream containing a mixture of the sample and diethylamine solution. Under these conditions the ascorbic acid reduced the Co"'-EDTA complex and the absorbance was measured spectrophotometricallyat 540 nm. In the absencc of ascorbic acid (blank), a maximum peak was obtained corresponding to the initial concentration of CoI"-EDTA. The presence of ascorbic acid caused a decrease in the analytical signal proportional to the ascorbic acid concentration [Fig. l(a)]. Flow injection and chemical variables were optimized for the proposed FI method. This study was carried out by altering each variable in turn while keeping the others constant. The optimum chemical and FI variables chosen were those that yielded maximum and constant differences between the peak height of the blank and the samples (Ah).
Fig. 2 Variation of Ah with: A, the pumping rate; B. reactor length; and C, loop size
It was found that a basic medium was necessary for the reaction between Co"'-EDTA and ascorbic acid to proceed and the best results were obtained with the use of diethylamine. The influence of the diethylamine concentration was studied in the range between 1 and 7% vlv. The concentrations of ascorbic acid and CoIIl-EDTA were 2 x 10-3 and 5 x 10-3 rnol dm-3, respectively. The best results were obtained with a concentration of 5% v/v diethylamine. Similarly, the influence of the concentration of Co"'-EDTA was studied in the range from 1 x 10-3 to 5 x 10-3 rnol dm-3. A concentration of 5 x 10-3 rnol dm-3 CoIll-EDTA was selected as this yielded suitable peak heights both for the blank and in the range of the calibration graph. Fig. 2 shows the effects of the flow rate, reactor length and loop size on the differences between the signal of the blank and the sample (Ah) for an ascorbic acid concentration of 2 x 10-3 rnol dm-3. The effect of the flow rate on Ah was studied over the range 0.5-1.2 cm3 min-1. An increase in the flow rate resulted in a decrease in Ah (Fig. 2, line A). A flow rate of 0.85 cm3 min-'
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was selected in order to obtain a reasonable sampling frequency. The influence of the reactor length on Ah was studied over the range 2-6 m (inner diameter, 0.5 mm). The results (Fig. 2, line B) showed that Ah is virtually constant throughout the range studied. A 4 m reactor was selected. The effect of the loop size on Ah is shown in Fig. 2, line C. An increase in loop size produced an increase in Ah, and a loop size of 85 mm3 was chosen. Determination of Ascorbic Acid
With the manifold described above and under the selected experimental conditions, viz., 5 x 10-3 rnol dm-3 Coi1'EDTA and 5% v/v diethylamine, a linear calibration graph between 2.0 x 10-4 and 5.0 x 10-3 rnol dm-3 ascorbic acid was obtained. The regression equation found was h = 11.21 X 102[ascorbic acid] + 18.4, where h is the peak height in centimetres and [ascorbic acid] is expressed in rnol dm-3, with a correlation coefficient (r) of 0.9985. The relative standard deviation (RSD) (p = 0.05) for ten determinations of 2 X 10-3 rnol dm-3 ascorbic acid was 0.94%. The detection and quantification limits were 6 x 10-5 and 1.2 x 10-4 rnol dm-3, respectively, and the sampling frequency was 60 samples h-*.
The effect of the FI experimental variables on At-At' (the difference between the peak width of the blank and the samples) was studied. The results obtained are shown in Fig. 3. The selected values for the FI variables were as follows: flow rate = 0.51 cm3 min-1, with which a suitable At without a notable decrease in the sample frequency was obtained; and injection volume = 146 mm3; this large volume of CoiliEDTA injected ensured that wide peaks were obtained. Several tubes of 2 mm inner diameter and between 1.0 and 12 cm in length were used for the study of the influence of the gradient tube volume on At. The peak width increased with the volume of the tube, and hence a tube of length 12 cm (diameter = 2 mm) was selected. The concentrations of Co'Il-EDTA and diethylamine selected were the same as in the peak-height method. The absorbance at which the peak width was measured influenced the sensitivity of the determination. It is advisable to measure the peak width at the absorbance level that yields the maximum difference between the blank and the sample. A plot of the peak width versus the logarithm of the concentration of ascorbic acid showed different linear ranges according to the value of the absorbance at which At was measured (as is shown in Table 1). An absorbance of 0.1 was selected because a wider range and a greater slope were obtained.
Peak-width FI Method
This method is based on the measurement of the peak width at a pre-determined height from the baseline. One of the problems found in routine analytical determinationsis that the analyte concentrations of the sample often do not fall within the limited range of concentration covered by the calibration graphs. As the aim of this work was the routine determination of ascorbic acid, the peak-width FI method was applied to extend the useful working range for this acid. A logarithmic relationship between the peak width and the concentration of the samples was found. The proposed FI manifold for the determination of ascorbic acid is shown in Fig. l(6).
28 1
1
20
4 a 12
I
I
0.3
0.5 0.7 0.9 Pump rate (q)/cm3 min-1
1
50
100
1.1
I
I
150
200
Table 2 Effect of various foreign species on the determination of 4 10-3 mol dm-3 ascorbic acid using the two FI methods
Limiting molar ratio [species]: [ascorbic acid] 100 50
Species assayed* N03-, C1-, CH3COO-, Po43S042-, C 0 3 2 - ,Br-, As02S032-, oxalate 40 Se032-, citrate, tartrate, Se042-, lactose, 20 starch? Ca2+, I-, F-, Mo042-, urea, thiourea, fructose, lacticacid, gelatint 10 Glucose, maltose, methionine, hippuric acid, glutamic acid, phenylalanine, NH4+, sucrose, valine, thiamine, pyridoxine, leucine 5 Nicotinamide, Zn2+$, Cu2+$, Mn2+$ 1 Uric acid§, riboflavine 0.5 NO;?-, S20.1 * Caffeine, aspartic acid and saccharin do not interfere. Mass relation. $ In the presence of H2EDTA2-. Q Maximum relation assayed by the solubility of uric acid. Table 3 Determination of ascorbic acid in pharmaceuticals Ascorbic acid content*
1
Loop size (v)lmm3
Peak-height Peak-width Reference method method method Stated lt 162.52 (0.89)$ 162.75 (0.95) 163.34(1.92) 166.67 29 50.37 (0.92) 50.46 (0.87) 50.73 (1.77) 50.0 37 200.49 (1.17) 199.46 (1.25) 200.13 (2.53) 200.0 * Values for samples 1 and 2 in mg g-I; values for sample 3 in mg cm-3. t Cebion sachets (Abbott Laboratories): vitamin C, 1g; aromatized excipient (including 8 mg of sodium saccharin) up to 6 g. $ Values in parentheses are relative standard deviations in % (n = 5) * Q Frenadol sachets (Abello Laboratories): paracetamol. 600 mg; salicylamide, 100 mg; codeine phosphate, 10 mg; caffeine citrate, 30 mg; chlorpheniramine maleate. 4 mg; ascorbic acid, 500 mg; plus excipient with attached carbohydrate groups up to 10 g. 7 Redoxon drops (Roche Laboratories): vitamin C, 200 mg; sodium saccharin, 2 mg; plus excipient up to 1 cm3. Sample
0.6
3.6
6.6 9.6 Length ( / ) l c m
12.6
Fig. 3 Variation of At' with: A, the pumping rate; B, loop size; and C, reactor length
Table 1 Calibration graphs for the determination of ascorbic acid using the peak-width method Concentration range/mol dm-3 Absorbance* 2 X 10-3-5 X 10-2 0.1 2 X 10-3-3 X 10-2 0.2 2 x 10-3-1 x 10-2 0.3 * Absorbance at which the peak width
Correlation Slope coefficient ( r ) -26.632 0.9926 -20.068 0.9888 -18.446 0.9422 (Ar) was measured.
X
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Table 4 Determination of ascorbic acid in urine by the peak-height FI method
Added mg cm-3 0.296 0.501 0.672 Human urine O.OO0 0.352 0.528 0.704 * Average of five determinations. Sample Synthetic urine
Found*/ mg cm-3 0.298 0.510 0.674 0.129 0.349 0.542 0.699
Recovery (Yo)
100.8 101.9 100.3
are summarized in Table 4 and show that the components of the urine do not interfere in the determination of ascorbic acid in the samples assayed. Recovery data were obtained in a similar way to that described for synthetic urine. We gratefully acknowledge the Direccion General Cientifica y TCcnica for financial support (PB 90-0008).
99.20 101.06 99.25
Calibration Graph
The equation of the calibration graph for determining ascorbic acid is At = -26.63 x log [ascorbic acid] -9.16 ( r = 0.9926), where At is expressed in seconds and [ascorbic acid] in mol dm-3. The equation is satisfied for ascorbic acid concentrations between 2.0 x 10-3 and 5.0 x 10-2 mol dm-3. Hence, by using the peak width as a quantitative parameter, it was possible to determine greater concentrations of ascorbic acid than by using the peak height. The RSD (n = 10) for 7.5 x 10-3 mol dm-3 ascorbic acid was 2.02%. Study of Interferents in Both Methods
The effect of foreign species in both FI methods was studied. the results for the determination of 4 x 10-3 mol dm-3 ascorbic acid are listed in Table 2. The tolerance limit was taken as the concentration causing an error of not more than +3% in the determination of ascorbic acid. As can be seen, the proposed methods are sufficiently selective. Applications
The first (peak-height) method was applied to the determination of ascorbic acid in three pharmaceuticals and urine and the second (peak-width) method only to the determination of ascorbic acid in pharmaceuticals. The results obtained for pharmaceuticals are summarized in Table 3. Reproducibility was good in all instances. The results obtained with the proposed methods were compared with those given by the standard British Pharmacopoeia.5 The peak-height method was also tested for its applicability to the determination of ascorbic acid in urine. A recovery experiment was performed using a mixture to simulate human urine. The data from this study are summarized in Table 4. Recovery data were obtained by adding different amounts of standard ascorbic acid to the synthetic urine samples and subtracting the results obtained from samples prepared in a similar way but with no ascorbic acid added. The results obtained for human urine samples containing ascorbic acid, in addition to the data from the recovery study,
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Paper 2/0105 7G Received February 28, 1992 Accepted May 14, 1992
