Clinical Analysis of Vitamin D and Metabolites

Vol. 12, No. 1 - Jan - June 2009 J. Baqai Med. Univ.

* Tania Mirza, * Kiran Qadeer and * Iqbal Ahmad

ABSTRACT Vitamin D (mainly ergocalciferol and cholecalciferol) and their metabolites exist in a complex environment in biological systems. The identification and determination of the individual vitamin Ds in these systems require specific methods. The purified components of vitamin D can be determined by UV spectrometry and colorimetry. However the best method for the separation and determination of ergocalciferol and cholecalciferol and their metabolites is high-performance liquid chromatography. This technique in combination with mass spectrometry (LC-MS) can be used for the simultaneous separation and identification of vitamin D components and metabolites. The technique is also useful for the identification of vitamin D in steroidal mixture.

INTRODUCTION and soluble in benzene, chloroform, ethanol and acetone. The molecule is unstable in light and is Vitamin D is a group of fat-soluble compounds oxidized on exposure to air. containing ergocalciferol (vitamin D 2) and cholecalciferol (vitamin D 3). The D vitamins possess Cholecalciferol (C21 H44O) antirachitic activity and are involved in the maintenance It possesses four double bonds and exhibits and of calcium and phosphorous homeostasis. Calcium absorption maximum at 265 nm in ethanol or hexane 1 1 and phosphorous are required for a wide variety of with a value of molar absorptivity as 19,400 M· cm· • biological processes including muscle contraction, The solubility and stability characteristics are the same nerve pulse transmission, blood clotting, and as that of ergocalciferol 1. membrane structure. Vitamin D is also important for insulin secretion, hair growth, muscle function, METHODS 1 5 immune response and melanin synthesis · . Several methods have been developed for the analysis of vitamin D and metabolites using spectro Vitamin D is largely biologically inert and is photometric, electrochemical and chromatographic 6 10 metabolized to its dihydroxy active forms la, 25(OH)2 methods · . However, these methods lack the

D 3 and 24,25(OH)2 D 3• Both of these metabolites are sensitivity and selectivity of biological assays. The obtained in vivo by hydroxylation at carbon 25 of the important methods of the analysis of vitamin D and 1 vitamin D 3 molecule in the liver . metabolites are presented in the following sections:

CHEMICAL CHARACTERICTICSl Spectrometric Methods:

Ergocaldferol (C28 H440) a. Ultraviolet Spectrometry The molecule is unsaturated containing three double The conjugated triene system possessed by vitamin D bonds. In alcoholic and hexane solutions, it exhibits secosteroids imparts high absorption around 264 nm an absorption maximum at 264~265 nm with a molar which can be used for the determination of the vitamin. \ 1 1 absorptivity of 18,300 M· cm· • It is insoluble in water The high molar absorptivity of the vitamin D 2 and

• Institute of Pharmaceutical Sciences, Baqal Medical University, Gadap Road, Near Toll Plaza, Super Highway, Karachi. Vol. 12, No. 1 - Jan - June 2009 J. Baqai Med. Univ.

D 3 makes the method very sensitive. This technique and cholecalciferol are determined by liquid is simple and rapid, however, it require a pure sample, chromatography. free of UV-absorbing impurities, for accurate 1 determination . The method has been used for the High performance liquid chromatography has been determination of high potency vitamin D after used in combination with mass spectrometry (HPLC 11 chromatographic separation • MS) for the simultaneous separation and identification of vitamin D components and other steroidal 23 25 b. Colorimetry mixtures - • The urinary metabolites of cholecalciferol Colorimetric methods have extensively been used for in man have been identified by Higashi et al. 26 using

the determination of vitamin D. One of the sensitive HPLC-MS: A clinical assay of 25(OH)2D 3 metabolite methods involves isomerisation of vitamin D to in biological fluids have been developed using HPLC 27 isotachysterol and development of color with antimony MS technique • trichloride. The detection limit of vitamin D with the 12 method is 1-1000 mcg • The various colorimetric methods for the determination ergocalciferol and DISCUSSION 13 14 cholecalciferol have been reviewed • • These methods This review presents a literature survey on the are based on the development of color with sulphuric physicochemical characteristics and the methods of acid, antimony trichloride trifloroacetic acid stannous analysis of vitamin D components, ergocalciferol and chloride, iodine-ethylene-dichloride and other reagents. cholecalciferol and their various metabolites. These compounds exist as a complex mixture and the c. High Performance Liquid Chromatography determination of a particular component can not be (HPLC) carried out by the UV spectrophotometric method as HPLC is nowadays a most widely used technique for a result of interference from other components. In the determination of vitamin D, its metabolites and some cases colorimetric methods are suitable for related compounds. This technique has the advantage selective determination of individual vitamin D of separation of vitamin D components and their components. The HPLC method could be considered metabolites followed by determination. The sensitivity to be the best method for separation, identification 1 of detection of vitamin D is ~ 5 ng • HPLC methods and determination of various vitamin D components, have been developed for the determination of the their metabolites and related compounds using UV hydroxyl metabolites of ergocalciferol and detector. The application of HPLC method has 15 18 cholecalciferol and biological fluids - . Ergocalciferol, facilitated accurate determination of ng quantities of and cholecalciferol in their 25-hydroxy metabolites vitamin D in biological fluids. A combination of liquid have been determined by UV detection in plasma with chromatography-mass spectrometry (LC-MS) has the 19 a detection limit of 500 ng/L . HPLC has been advantage of separation as well as identification of considered as the best method of separation of vitamin individual vitamin D components in a mixture. The 20 D components and their metabolites . According to technique may be useful in the detection of vitamin 21 the British Pharmacopeia method , ergocalciferol D metabolites in blood circulation.

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REFERENCES 11. Mader, W.J. Vitamins. In: Higuchi, T. and Brochmann-Hanssen, E. (Eds.), Pharmaceutical 1. Norman; A.W. and Henry, H.L. Vitamin D. In Analysis, Intersciences, New York, Chap. I 1. Zempleni, J., Ruker, R.B. McCormick, D.B. and Suttie, J.W. (Eds.), Handbook of Vitamins, 4th 12. Official Methods of Analysis of the AOAC ed., CRC Press, Boca Raton, FL, 2007, Chap.2. . International, Deutsch,.J. (Eds.), AOAC International, Arlingten, VA, 1995; p.19. 2. Norman, A.W and Henry, H.L. Vitamin D, 2nd ed., Elsevier Academic Press, San Diego, CA, 13. Hashmi, M.H. Assay of Vitamins in 2005. Pharmaceutical Preparation, John Wiley, New York, 1973, Chap.14. 3. Norman, A.W. Vitamin D. The Calcium Homeostatic Steroid Hormone, Academic Press, 14. Singh, L. Vitamin D In: Augustin, J., Kleni, B.P., New York,1979. Becker, D., Venngopal, P.B. (Eds.). Methods of Vitamin Assay, 4th ed., John Wiley, New York, 4. Heaney, R.P., The vitamin D requirement in health 1985; Chap.9. and disease. J. Steroid Biochem. Mol.Biol., 2005; 97 (1-2): 13-19. 15. Glendenning, P. and Fraser, W.D. 25-OH-Vitamin D assays. J. Clin. Endocrinol. Metab., 2005; 5. Prie, D., Beck, L., Urena, P. and Friedlander, G. 90:3129-3135. Recent finding in phosphate Homeostatsis. Curr. Opin. Nephrol. Hypertens, 2005; 14:318-324. 16. Shimada, K., Mitamura, K., Kitama, N.and Kawasaki, M. Determination of 25-hydroxy

6. Moffort, A.C., Osselton, M.D. and Widdop, B. vitamin D 3 in human plasma by reversed-phase Clarke's Analysis of drugs and Poison, 2nd ed., high performance liquid chromatography with Vol.2, Pharmaceutical Press, London, pp.849,981. ultraviolet detection. J. Chromatogr. B. Biomed. Sci. Appl., 1997; 689(2): 409-414. 7. Ahmad, I. Usman Ghani, K. Analysis of Medicinal Compounds and Plant Drugs, Research 17. Meta-Cranados, J.M., Lukue, D.C. and Quesada, Institute of Indusyunic Medicine, Karachi; J.M. Fully automated method for the

pp.109-157. determination of 24, 25(OH)2 and (OH)2D 3 hydroxyvitamins, and vitamin A and E in human ( 8. Modern analytical methodologies in fat- and water- plasma by HPLC. J. Phann. Biomed. Anal, 2004; soluble vitamins/ edited by Won 0. Song, Gary 35(3):575-582. R. Beecher, Ronald R. Eitenmiller. John Wiley, New York, 2000; Chap.2. 18. Frolich, A., Storm, T., and Thode, J. Does the plasma concentration of 25-hydroxyvitamin D 9. Ball, G.F.M. Vitamins in Food Analysis, determine the level of 1, 25-dihydroxyvitamin D Bioavailability and Stability. CRC Press, Boca in primary hyperparathyroidism? Miner. Raton, FL, 2000; Chap.4. Electrolyte Metab., 1996; 22:203-206.

10. Eitenmiller, R.R., Lin, Ye and Landen, W.O. Jr. 19. Turpeinen, U., Hohenthal, U. and Stenman, U. Vitamin Analysis for the Health and Food Sciences, H. Determination of 25-hydroxyvitamin D in 2nd ed., CRC Press, Boca Raton, FL, 2008; serum by HPLC and immunoassay. Clin. Chem., Chap.2. 2003; 49:1521-1524. Vol. 12, No. 1 - Jan - June 2009 J. Baqai Med. Univ.

20. Jones, G., Assay of Vitamins D 2 and D 3 and 25- tandem mass spectrometry. Biochem. hydroxyvitamins D2 and D3 in human plasma by· Pharmachol., 1995; 49:1099-1110. HPLC. Clin. Chem., 1978;24; 287-298. 25. lshigai M., Asoh Y., Kumaki K., Determination 21. Halloran B.P., Bikle D.D., J.O., Separation of of 22-oxacalcitriol, a new analogue of la, 25-

isotopically labeled vitamin D metabolites by high dihydroxyvitamin D 3, in human serum by liquid performance liquid-chromatography. J. chromatography mass spectrometry. J. Chromatogr., 1984; 303: 229-233. Chromatogr. B. Biomed. Sci. Appl., 1998; 706:261- 267. 22. British Pharmacopoeia, Her Majesty's Stationary Office, London, Electronic Version. 26. Higashi T., Hoinma S., Iwata H., and Shimada ~-, Characterization of urinary metabolites of

23. Wilson S.R., Tulchinsky, M.L. , and Wu Y., vitamin D 3 in man under physiological conditions Electroscopy ionization mass spectrometry of using liquid chromatography- tandem mass vitamin D derivatives. Bioorg. Med. Chem. spectrometry. J. Pharm. Biomed. Anal., 2002; Lett.,1993;3: 1805-1808. 29:947-955.

24. Yeung B., Vouros P., Siu-Caldera M.L ., and 27. Vogeser M., Kyriatsoulis A., Huber E., Kobold Satyanarayana Reddy G., Characterization of the U., Candidiate reference method for quantificastion

metabolic pathway of 1,25-dihydroxy-16-ene of circulating 25-hydroxyvitamin D 3 by liquid

vitamin D 3 in rat kidney by one-line high chromatography-tandem mass spectrometry. Clin. performance liquid chromatography-electrospray Chem., 2004; 50:1415-1417.