Voltammetric Behaviour of Vitamins D2 and D3 at a ...

View Article Online / Journal Homepage / Table of Contents for this issue

1441

ANALYST, SEPTEMBER 1992, VOL. 117

Published on 01 January 1992. Downloaded by University of the West of England on 08/05/2015 13:31:58.

Voltammetric Behaviour of Vitamins D2 and D3 a t a Glassy Carbon Electrode and Their Determination in Pharmaceutical Products by Using Liquid Chromatography With Amperometric Detection John P. Hart and Michael D. Norman Faculty of Applied Sciences, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS 76 7QY, UK Christopher J. Lacey Liquid Inks Laboratory, Coates Lorilleux, Midsomer Norton, Bath BA3 4BQ, UK

Cyclic voltammetry was used to study the oxidation of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol)at a planar glassy carbon electrode.The electrode reaction for cholecalciferol was found to be dependent on the apparent pH between 4.95 and 6.10,and pH independent between pH 6.10 and 8.65when the solutions contained 90% methanol; this suggested a pK, value of 6.10 for vitamin D3. Similar behaviour was exhibited by ergocalciferol, and a pK, value of 6.35 was found. The peak currents for both vitamins were found to be dependent on the methanol concentration of the supporting electrolyte. The peak currents were also found to be dependent on the ionic strength of the acetate buffer (pH 6.0)over the range 0.1-1.0 rnol dm-3. Both substanceswere oxidized in one step, which was found to be an irreversible reaction; the final product for vitamin D2 can undergo absorption at the electrode surface. The parent compounds could be undergoing oxidation at the triene moieties. The optimum mobile phase for liquid chromatography with amperometric detection was found to be 95% methanol-0.05 mol dm-3 acetate buffer (pH 6.0);the detector was operated at a potential of +I .3 V (versus Ag-AgCI), and a linear response was obtained for vitamin D3 over the range from 10 to 100 ng injected; for vitamin D2the response was linear from 20 to 200 ng injected. Extracts of pharmaceutical products were separated on reversed-phase columns prior to amperometric detection of the vitamins. Cholecalciferol was successfully determined in a multivitamin tablet, and ergocalciferol in a multivitamin liquid preparation. Keywords: Vitamin D3 (cholecalciferol); vitamin D2 (ergocalciferol); cyclic chromatography; amperometric detection

Vitamin D2 [ergocalciferol; 9,10-secoergosta-5,7,10( 19), 22tetraen-3-01] and vitamin D3 [cholecalciferol; 9,lO-secocholesta-5,7,10(19)-trien-3-ol] are the main forms of vitamin D and have equal biological potency in man; they can be used in the prevention of the bone disease known as rickets.’ Pharmaceutical multivitamin preparations contain either one of these substances and, generally, the levels are much lower than those of other fat-soluble vitamins (such as A and E) and of some water-soluble vitamins (such as vitamin B1, B6 and C). Consequently, analytical methods to determine the D vitamins in these matrices need to be both selective and sensitive. Methods involving high-performance liquid chromatography (HPLC) with ultraviolet detection have been applied to analyses for vitamin D;’ however, alternative methods with even greater selectivity and sensitivity might be more advantageous. One of the most powerful techniques for trace analysis of complex matrices is liquid chromatography with electrochemical detection (LCEC).”-” It has been utilized by Hart and co-workers for the determination of two other fat-soluble vitamins, K1+8 and A1,c).10 in human blood; other workers have used LCEC to determine vitamin E in body fluids.11.1’ As vitamin D3 has been shown to undergo oxidation at a glassy carbon electrode,13 it was considered that this might also be a suitable candidate for determination by LCEC; as ergocalciferol is structurally very similar to cholecalciferol, it was considered that this might also be determined by a suitable electrochemical detection system. To the best of our knowledge, no systematic studies have been reported on the development of an LCEC assay for these two D vitamins. The effects of buffer pH and ionic strength, and also solvent, have been shown to exert a considerable influence on the electrode reactions of vitamin K,14.*Sand A1;9therefore, it is important to carry out detailed studies of these parameters in order to obtain the optimum solution conditions. The purpose of the first part of the present study was to carry out a detailed investigation on the electrochemical

voltammetry;

liquid

behaviour of vitamins D2 and D3 at a planar glassy carbon electrode by using cyclic voltammetry and a variety of solution conditions. These studies were then used in the development of LCEC assays for the vitamins in multivitamin preparations. This paper describes the results of our studies.

Experimental Chemicals and Reagents

All chemicals were of analytical-reagent grade unless stated otherwise; Vitamins D2 and D3 were purchased from Sigma (St. Louis, MO, USA) and were of reference grade purity. The supporting electrolytes used for the cyclic voltammetric studies were prepared by mixing stock solutions of 0.5 mol dm-3 sodium acetate and acetic acid to yield solutions of the desired pH (measured by a p H meter); the resulting acetate buffers were diluted with methanol and water to yield the desired concentrations. The methanolic acetate buffer used as the mobile phase in the liquid chromatographic studies was prepared by mixing 1 mol dm-3 sodium acetate-acetic acid (pH 6.0) and methanol to yield a final electrolyte concentration of 0.05 mol dm-3. Stock solutions of the two vitamins were prepared in ethanol and were protected from light during all investigations. Abidec drops were a gift from Parke Davis (Ann Arbor, MI, USA) and contained 400 U per 0.6 cm3 (10 pg per 0.6 cm3). Plurivite tablets are manufactured by Boots Chemist and contain 400 U per tablet (0.8 g) (10 pg per tablet). Apparatus

Cyclic voltammograms were recorded with an Eco-Chemie Autolab electrochemical analyser (Utrecht, The Netherlands) in conjunction with a Viglen SL1 PC (London, UK) and an Epson LX-400 dot matrix printer (Nagano, Japan). A

View Article Online


1442

three-electrode cell, incorporating a glassy carbon working electrode (area 0.283 cm2, unless stated otherwise), an Ag-AgC1 reference electrode and a platinum wire counter electrode, was used. Studies involving liquid chromatography with amperometric detection were carried out with use of two different systems. For vitamin D3, investigations were performed with a Spectra-Physics Isochrom LC pump (San Jose, CA, USA) and a Metrohm 641 VA-Detector (Herisau, Switzerland), together with a wall-jet cell (Metrohm 656 electrochemical detector) containing a glassy carbon working electrode, a gold counter electrode and an Ag-AgCI reference electrode. Sample injections were effected via a syringeloading Rheodyne valve (Cotati, CA, USA). Separations were carried out on a Nucleosil ODS 250 X 4.6 mm i.d. column (particle diameter 5 pm) ; chromatograms were recorded on a Servogor 120, BBC Goerz Metrawatt recorder (Austria). For vitamin D2, studies were performed with a Pye Unicam LC-XPD pump (Cambridge, UK), an Apex I1 ODS 250 x 4.6 mm i.d. column (particle diameter 5 pm) and a Gould BS-271/1 recorder (Ilford, Essex, UK), together with the detection system described above. Voltammetric Procedures Unless stated otherwise, cyclic voltammetry was performed on solutions containing 1 x 10-4 rnol dm-3 of either vitamin D2 or vitamin D3. In order to study the effect of pH, the solutions also contained 90% methanol and 0.05 rnol dm-3 pH 3.0-7.0 acetate buffers (apparent pH 4.95-8.65). The voltammetric conditions were as follows: initial potential, 0 V; scan rate, 50 mV s-1; and final potential, +1.5 V. Between successive runs the working electrode was cleaned by washing with distilled water, polishing the surface with aluminium oxide powder (0.3 pm), rinsing again with distilled water and finally drying with tissue paper. The effect of methanol was investigated by dissolving the appropriate amounts of the vitamins, in solutions containing 85-95% methanol and 0.05 rnol dm-3 acetate buffer (pH 6.0), and performing cyclic voltammetry. The effect of the ionic strength of the acetate buffer (pH 6.0) was studied in the concentration range 0.01-0.1 rnol dm-3 by using a constant concentration of 90% v/v methanol; the scan rate was 50 mV s-1. The presence of any adsorption processes was investigated by varying the scan rate over the range 20-200 mV s-1 for solutions containing the vitamins dissolved in 90% methanol-0.05 rnol dm-3 acetate (pH 6.0). Hydrodynamic voltammetry was performed by injecting 100 ng amounts of either one of the vitamins, dissolved in 95% methanol-0.05 rnol dm-3 acetate buffer (pH 6.0), and varying the potential between 0.9 and 1.5 V. For these studies the analytical column was removed from the system, and the mobile phase consisted of 95% methanol-0.05 rnol dm-3 acetate buffer, pH 6.0 (initial pH); a flow rate of 2 cm3 min-1 was used. Procedures for the Determination of D Vitamins in Pharmaceutical Products

Vitamin 0 3 in Plurivite tablets Tablets were weighed, then ground to a fine powder with a pestle and mortar. Portions of the powder (about 0.2 g) were extracted by using a modification of the method described previously.2 Briefly, 5.0 cm3 of ethanol and 5.0 cm3 of 0.1 rnol dm-3 orthophosphoric acid were added to the powder, and the mixture was thoroughly agitated in a vortex mixer. The resulting solution was extracted by shaking with 10 cm3 of hexane; an aliquot of the hexane phase (5.0 cm3) was evaporated to dryness at 60 "C under a stream of nitrogen. The residue was reconstituted in 0.10 cm3 of mobile phase by thorough vortex mixing, and finally 0.010 cm3 of the resulting solution were injected onto the Nucleosil ODS column.


Vitamin D2 in Abidec drops An aliquot (1.5 cm3) of Abidec drops was treated with 1.0 cm3 of 0.3 rnol dm-3 orthophosphoric acid and 5.0 cm3 of ethanol and thoroughly agitated in a vortex mixer. The resulting solution was extracted in a similar manner to that described by Stary et a1.2 This involved shaking the solution with 10 cm3 of hexane, followed by evaporation of a 5.0 cm3 aliquot of the hexane phase at 60°C under nitrogen. The residue was dissolved in 2.0 cm3 of mobile phase by thorough vortex mixing, and 0.020 cm3 of the resulting solution was injected onto the Apex I1 ODS column. Calibration Graph and Linear Range for LCEC A series of external vitamin D3 standards were prepared in the mobile phase, 95% methanol-0.05 rnol dm-3 acetate buffer (pH 6.0), and from these a calibration graph of peak current versus mass of vitamin D3 injected was established over the range 10-100 ng. In this study, the detector was set at an operating potential of +1.3 V versus Ag-AgCl; the mobile phase flow rate was 2.0 cm3 min-1. The calibration graph for vitamin D2 was prepared in a similar fashion and covered the range 20-200 ng injected.

Results and Discussion Cyclic Voltammetric Behaviour of Vitamins D2 and D3 The cyclic voltammetric behaviour of vitamin D3 was studied over the pH range 3.0-7.0 (apparent pH 4.95-8.65) in 90% methanol containing acetate buffer at a concentration of 0.05 rnol dm-3. One anodic peak was obtained over the potential range investigated (0.0 to +1.5 V). This peak was found to be pH dependent between apparent pH 4.95 and 6.10 (Fig. 1); the equation relating peak potential to pH was calculated to be

E,

=

2.257

-

0.180 pH (V)

Between pH 6.10 and 8.65 the E, was found to be independent of pH; the break in the plot shown in Fig. 1 indicates a pK, value of 6.10. Fig. 2 shows a typical cyclic voltammogram for vitamin D3, and clearly no cathodic peaks are obtained on the reverse scan. In fact, this was found to be the case under all conditions studied and, therefore, the oxidation process appears to be irreversible. The cyclic voltammetric behaviour of vitamin D2 was very similar to that observed for vitamin D3, A break in the E, versus pH plot occurred for the former compound at an apparent pH of 6.35; above this value the E, was pH independent, which suggests a pK, value of 6.35 for this vitamin. The equation below pH 6.35 was calculated to be

E,

3 .-rn

1.3

-

1.2

-

=

2.260

-

0.170 pH (V)

4-

C W c

a 0 rn

Y

2

1.1

I

I

I

4

5

6

I

7 Apparent pH

I

8

15 9

Fig. 1 Effect of pH on peak current and peak potential for vitamin D3 in 90% methanol-0.05 rnol dm-3 acetate buffers

View Article Online


1443

25

15

I l l

I I

20

1 4 t

e

15

2

4

g

2

1 9

\

13-

3

10


3

0

5

0 -5

1

I

0

0.2

I

I

I

I

I

0.6

1

0.1

0

1.6

Fig. 2 Cyclic voltammogram of 7.8 X rnol dm-3 vitamin D3 in 90% methanol-0.05 rnol dm-3 acetate buffer (pH 6.0) using a glassy carbon electrode. Initial potential. 0 V; scan rate, 50 mV s-1; electrode area, 0.071 cm2 25

I

I

I

0.025

0.05

0.075

1

I

0.8 1.0 1.2 1.4 VoltageN versus Ag-AgCI( sat)

0.4

0.1

Ionic strength/mol dm -3 Fig. 4 Variation of peak current with increasing ionic strength of acetate buffer in the supporting electrolyte for vitamin D3

I

0.2

I

I

0.3

0.4

1 .o

0.5

(Scan rateN s 'Io5 Fig. 3 Peak current versus v4 for: A, vitamin D,; and B. vitamin D?. in 90% methanol-0.05 rnol dm-, acetate buffer (pH 6.0)

Interestingly, these plots for the two vitamins were remarkably similar to that obtained by us in a previous study9 on vitamin A. The electrochemical oxidation of this fat-soluble vitamin seems to result from the presence of the conjugated double-bond system.9.16 Therefore, it seems feasible that the presence of the triene moiety at positions 5 , 7 and 10 (19) might be responsible for the electroactivity of the D vitamins. However, these vitamins also contain a hydroxy group in the 3-position, so it was considered that a compound with a similar structure might help to elucidate the position of the oxidation reaction. Cholesterol (cholest-5-en-3P-ol) is such a compound, so this was subjected to cyclic voltammetry in a supporting electrolyte consisting of 90% methanol-0.05 mol dm-3 acetate buffer, p H 6.0 (apparent p H 7.8); no anodic o r cathodic peaks were observed over the voltage range studied for the D vitamins. This behaviour strongly suggests that it is the triene moiety that undergoes oxidation in these compounds. Further evidence for the irreversible nature of the process for vitamin D3 was obtained from the variation of E, with scan rate; the peak potential shifted by 50 mV, in the anodic direction, when the scan rate was changed from 20 to 200 mV s-I. It is also known that irreversible diffusion-controlled electrochemical processes yield a linear response for a plot of i, (peak current) versus vl/z (where v is scan rate).J Fig. 3 shows that, for vitamin D2, some curvature exists in this plot, which may be as a result of some degree of product adsorption. This phenomenon might be expected to cause passivation of the electrode surfaces and could then hinder the electron-transfer process.

1.1

1.2

1.3

1.4

VoltageN versus Ag-AgClbt) Fig. 5 Hydrodynamic voltammograms for: A , vitamin D,; B, vitamin D2; and C , background current. Mobile phase, 95% methanol-0.05 rnol dm-3 acetate buffer (pH 6.0)

For vitamin D3, the plot of i, versus p H (Fig. 1) indicates that the largest currents are to be expected in methanolic acetate buffer, p H 6.0 (apparent pH of 7.80) o r greater; therefore, methanolic acetate buffer of p H 6.0 was selected for the remainder of the studies. The effect of the ionic strength of the acetate buffer o n the voltammetric peaks was investigated. As shown in Fig. 4, the peak current decreased over the range 0.01-0.1 rnol dm-3; as the gradient was lowest at 0.05 rnol dm-3 acetate, and this concentration had also proved useful in previous LCEC studies,6.7 it was used throughout the present investigation. For vitamin D2, the behaviour of peak current with respect to the solution composition was again very similar to that of D3; therefore, the same pH and ionic strength of the buffer were chosen for further studies. The effect of concentration of methanol on the peak currents of the vitamins was examined over the range 8&%% v/v; the acetate buffer composition was fixed at a p H of 6.0 and an ionic strength of 0.05 rnol dm-3. The magnitude of the currents was found to increase with the percentage of methanol; this is likely to be the result of increased solubility. From the voltammetric results discussed above, the optimum supporting electrolyte was found to consist of 95% methanol-0.05 rnol dm-3 acetate buffer (pH 6.0); therefore, this was used as the mobile phase for investigations involving LCEC.

Optimization of LCEC Conditions In order to determine the optimum applied potential for amperometric detection, following liquid chromatography,

View Article Online

1444



T

2

I

I

I

3

4

6

8

I

1

I

0

1

2

Time/min

Fig. 7 LCEC trace of the extract from Abidec drops. Column, Apex I1 ODs; detector potential, +1.30 V I

I

16

14

I 12

I 10

I

8

I 6

I 4

I

IJ

2

0

Time/mi n

Fig. 6 LCEC trace of the extract from a Plurivite tablet. Column, Nucleosil ODs; detector potential, + 1.30 V

hydrodynamic voltammograms were constructed for both of the D vitamins (Fig. 5 ) . Both compounds exhibited one wave, which was in accord with the earlier observations performed with cyclic voltammetry (Fig. 2). The maximum currents were achieved at a potential of + 1.35 V; however, the magnitude of the background current was about 300 n A at this applied voltage. It was found that reducing the potential t o +1.3 V afforded a significant reduction in background current, which resulted in better signal-to-noise ratios; therefore, an applied potential of +1.3 V was selected for the remainder of the LCEC studies.

mean value of 248 U was found, with a relative standard deviation (RSD) of 8.4% (n = 4). It is considered that, if desirable, the recovery could be improved by including a second extraction step with hexane; however, this would, of course, increase the analysis time. As the precision of the over-all method is at a reasonable level, this would indicate that the proposed LCEC method shows promise for the determination of vitamin D3 in Plurivite tablets. It should be mentioned that a large, late eluting peak appeared, which had a retention time of about 57 min; however, it was found that it was possible to inject three samples before this peak appeared. Further samples were then injected after the late eluters had emerged from the column; this took about 35 min. Therefore, over a period of a working day (8 h) it should be possible to inject and analyse about 15 samples. Determination of Vitamin D2 in Abidec Drops by LCEC

Calibration and Linear Range The calibration graph of peak current versus the mass of vitamin D3 injected was found to be linear over the range 10-100 ng injected. For vitamin D2 the calibration graph was linear over the range 20-200 ng. These calibration ranges were considered suitable for the applications being undertaken in this study. It should be mentioned that these studies were performed with a current range of 50 nA. Lower ranges were inappropriate for the samples to be studied as these contained high levels of some other electroactive species (see later and Figs. 6 and 7); this would have resulted in large chromatographic peaks, which could obscure the signals from the two vitamins of interest. Determination of Vitamin D3 in Plurivite Tablets by LCEC The LCEC traces obtained for the multivitamin tablets, following the solvent extraction procedure described earlier, showed a well-defined peak for vitamin D3 with a retention time of 8.3 min (Fig. 6). The method was evaluated by carrying out four replicate determinations on individual portions (about 0.2 g) of the powdered tablets. The manufacturer’s stated dose is 400 U per tablet (0.8 g) and in this study a

Initial studies with the Nucleosil ODS column, used for the determination of vitamin D3 in Plurivite, showed that this stationary phase could not separate the vitamin D2 from co-extracted electro-oxidizable species present in the extract from Abidec drops. However, by simply changing the column to one containing Apex I1 ODs did result in the complete separation of vitamin D2 from these potentially interfering species, and the resulting chromatogram showed a welldefined signal at 10.0 min (Fig. 7). The method was evaluated by taking five individual aliquots (1.5 cm3) of the Abidec preparation through the complete assay. The mean concentration was calculated to be 412 U per 0.6 cm3, which compared favourably with the manufacturer’s specification (400 U per 0.6 cm3). The RSD was 7.5%; therefore, the recovery and precision data indicate that our LCEC method is reliable and shows promise for the assay of vitamin D7 in Abidec drops. It should also be mentioned that, like the previously mentioned multivitamin tablets, a large, late eluting peak appeared, which had a retention time of about 60 min; however, it was found that it was possible to analyse four samples before this peak appeared. Further samples were then injected after the late eluters had emerged from the column;

View Article Online


this took about 45 min. Therefore, over a period of a working day (8 h) it should be possible to inject and analyse about 20 samples.


Conclusions In this investigation, it has been shown that vitamins D2 and D3 undergo electrochemical oxidation, at a glassy carbon electrode, under a variety of solution conditions; it is likely that the conjugated double-bond system in these compounds is responsible for their electroactivity. It was possible to exploit these oxidation processes for the electrochemical detection of both vitamins, following separation on a reversed-phase HPLC column. The studies described here indicate that LCEC shows promise for the assay of vitamin D3 in Plurivite, a multivitamin tablet, and of vitamin D2 in Abidec drops, a multivitamin liquid preparation. Simple solvent extraction was the only separation step required to isolate the fat-soluble vitamins from the samples prior to LCEC; this was carried out in the isocratic mode. It should be feasible to determine these two vitamins in other multivitamin preparations as they are likely to contain similar mixtures of vitamins. In addition, it is possible that the methods could be adapted to the analysis of other matrices, including foods.

References 1 Martin, D. W . . Jr., in Harpers Review of Biochemistry, eds. Martin, D. W . , Jr., Mayes, P. A., and Rodwell. V. W . , Lange, Los Altos, CA, 19th edn., 1983, p. 114. 2 Stary. E.. Cruz, A. M. C., Donomai, C. A . . Monfardini, J . L., and Vargas, J . T. F . , J. High Resolut. Chromatogr. Chromatogr. Commun.. 1989. 12. 421.

1445 3 Stulik, K., and Pacakova, V., Electroanalytical Measurements in Flowing Liquids, Ellis Horwood, Chichester. 1987. 4 Kissinger, P. T., in Laboratory Techniques in Electroanulytical Chemistry, eds. Kissinger, P. T., and Heineman, W. R., Marcel Dekker. New York. 1984, pp. 611-634. Hart, J. P., Electroanalysis of Biologically Important Compounds, Ellis Horwood, Chichester, 1990. Hart, J. P., Shearer, M. J . , McCarthy, P. T . , and Rahim, S . , Analyst, 1984, 109, 477. Hart, J . P.. Shearer, M. J., and McCarthy, P. T., Analysf, 1985. 110, 1181. Hart, J . P., Shearer, M. J., Klenerman, L., Catterall, A . , Reeve, J., Sambrook, P. N., Dodds, R. A.. Bitensky, L., and Chayen, J . , J . Endocrinol. Metab., 1985, 60, 1268. 9 Wring. S. A., Hart, J . P., and Knight, D. W . , Analyst, 1988, 113, 1785. 10 Wring, S. A . , and Hart. J . P . , J. Med. Lab. Sci., 1989,46,367. 11 Castle, M. C., and Cooke, J. W . , Ther. Drug Monit., 1985.7, 364. 12 Chou. P. P., Jaynes, P. K . , and Bailey, J . L., Clin. Chem. (Winston-Salem, N . C . ) , 1985, 31, 880. 13 Hernandez Mendez, J., Sanchez Perez, A., Delgado Zamarreno, M., and Hernandez Garcia, M. L., J. Pharm. Biomed. Anal., 1988, 6, 737. 14 Hart, J . P.. and Catterall, A., in Electroanalysis in Hygiene, Environmental, Clinical and Phurmaceutical Chemistry, ed. Smyth, W. F., Elsevier, Amsterdam, 1980, pp. 145-153. 15 Hart, J . P., and Catterall, A., Anal. Chim. Acta, 1981,128,245. 16 MacCrehan, W. A., and Schonberger, E . , J. Chromatogr., 1987, 417, 6.5.

Paper 2/01406H Received March 17, I992 Accepted April 28, 1992