Describe about measuring inhibitors of Purines and Xanthine, Allopurinol and Oxypurinol within aqueous solution with the help of HPLC?
Allopurinol (Zuloprim as well as generics) refers to a drug that is used for treating the increased amount of uric acid in blood plasma (hyperuricemia) as well as chronic gout. This Allopurinol is said to reduce the level of uric acid within the body by simply blocking one of the many processes that make it. It is helpful in ceasing the extent of uric acid within the blood from getting higher and leading to problems such as gout and kidney stone. When allopurinol is missing, usual urinary letting outcome of oxypurines is completely in the form of uric acid. Once allopurinol is administered, it includes hypoxanthine, uric acid, and xanthine, each having variant solubility properties (Eisenberg et al., 1990). As a result, the uric acid concentration within plasma is lessened without much exposure of urinary tract towards an excess load of uric acid, thereby reducing the risk occurrence of crystalluria. By reducing the concentration of uric acid within the plasma far below its solubility limits, allopurinol enables dissolution of tophi. Despite increasing levels of hypoxanthine as well as xanthine, risk of their disposition is lesser than that of uric acid since these are more soluble and also quickly cleaned up by kidney. In order to ignore disposition of xanthine stones, it has been an advice to all individuals to carry on with increased intake of fluid and also an alkaline or neural urinary pH, particularly when concentration of initial uric acid is quite high that leads to the patient getting symptomatic. When allopurinol is metabolized by xanthine oxidoreductase to form Oxypurinol, it itself acts as a xanthine oxidoreductase inhibitor, thus decreasing formation of urate or uric acid (HAMANAKA et al., 1998). Oxypurinol as well as Allopurinol are identified to improve the function of endothelium by means of the ability to lessen the oxidative stress within the blood vessels apart from their authentic function as uric acid reducing agents. The uric acid is referred to as a chemical which is formed naturally within the body. Often, the level of this uric acid of the body may increase greatly leading to the development of gout, formation of kidney stones or other issues.
In Figure below, it reflects action mechanism for Allopurinol and Oxypurinol:
The main objective of this study is to quantitatively determine the concentration of unknown samples that bears allopurinol, uric acid, oxypurinol, xanthine and hypoxanthine within the acqueous solution by utilizing HPLC.
Allopurinol=5 mM
Oxypurinol=10 mM
Uric acid=5 mM
Hypoxanthine =2 mM
Xanthine=2 mM
Table 1: The stock standard of the compounds
Compound |
Stock Std. Conc. (mM) |
Std. Curve range (μM) |
Total volume (mls) |
Allopurinol |
5 |
0-500 |
10 |
Oxypurinol |
10 |
0-500 |
10 |
Uric acid |
5 |
0-500 |
10 |
Hypoxanthine |
2 |
0-200 |
10 |
Xanthine |
2 |
0-100 |
10 |
Table 2: The five calibration concentrations of oxypurinol, allopurinol, xanthine, hypoxanthine and uric acid
Flask No. |
Allopurinol conc. (μM) |
Oxypurinol conc. (μM) |
Uric acid conc. (μM) |
Hypoxanthine conc. (μM) |
Xanthine conc. (μM) |
2 |
100 |
100 |
100 |
25 |
20 |
3 |
200 |
200 |
200 |
50 |
40 |
4 |
300 |
300 |
300 |
75 |
60 |
5 |
400 |
400 |
400 |
100 |
80 |
6 |
500 |
500 |
500 |
125 |
100 |
Table 3: The volume of Standard stock for each compound in each flask
Flask No. |
Allopurinol vol.ml |
Oxypurinol vol.ml |
Uric acid vol.ml |
Hypoxanthine vol.ml |
Xanthine vol.ml |
water vol.ml |
2 |
0.2 |
0.1 |
0.2 |
0.125 |
0.1 |
9.275 |
3 |
0.4 |
0.2 |
0.4 |
0.25 |
0.2 |
8.55 |
4 |
0.6 |
0.3 |
0.6 |
0.375 |
0.3 |
7.825 |
5 |
0.8 |
0.4 |
0.8 |
0.5 |
0.4 |
7.1 |
6 |
1 |
0.5 |
1 |
0.625 |
0.5 |
6.375 |
All 21 chromatographs were out –
HPLC or High performance liquid chromatography is often used as a potential tool in quantitative as well as qualitative analysis that aids an enhanced understanding of the outcomes of chemical reaction, and also enabling identification of every comprising component of mixture solutions including their concentrations (Kannangara et al., 2012). Thus, it was utilized while performing the experiment for determining the actual concentrations of allopurinol, purines and oxypurinol in unknown samples. The linearity, specificity, and intra precision are significant measurements that need to be assessed during practical sessions. By using excel program it became easy to ascertain the existing linearity amidst the concentrations as well as peak height ratio for producing the calibration curve as well as regression equation. The values of R-sq for calibration curves of compounds exhibit the existence of a perfect linear relationship amidst concentration as well as peak height ratio (O’Regan, Phillis and Walter, 1989). The intra precision identified to be correct since CV values were lesser than 10%.
Conclusion:
HPLC or the High performance liquid chromatography system refers to an automated process which takes just few minutes for generating results. This reflects the difference over liquid chromatography that utilizes gravity rather than high speed pump for forcing compounds in the midst of dense-packed tubing. The outcomes generated are of greater resolution that is easy to be read, and also tests are conveniently reproduced through the automated process. But, HPLC hardly detects co-elution that may lead to improper compound categorization. A demand exists for a higher cost for equipment required for conducting HPLC. The operation of it may be complex and require a thoroughly trained technician for its operation. Due to the speed of the process, this equipment possesses low sensitivity towards some compounds.
References
Eisenberg, E., Conzentino, P., Liversidge, G. and Cundy, K. (1990). Simultaneous determination of allopurinol and oxypurinol by liquid chromatography using immobilized xanthine oxidase with electrochemical detection. Journal of Chromatography B: Biomedical Sciences and Applications, 530, pp.65-73.
HAMANAKA, MIZUTANI, NOUCHI, SHIMIZU, and SHIMIZU, (1998). Allopurinol hypersensitivity syndrome: hypersensitivity to oxypurinol but not allopurinol. Clinical & Experimental Dermatology, 23(1), pp.32-34.
Hoey, B., Butler, J. and Halliwell, B. (1988). On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals. Free Radical Research, 4(4), pp.259-263.
Kam Ming Ko, and Godin, D. (1990). Inhibition of transition metal ion-catalysed ascorbate oxidation and lipid peroxidation by allopurinol and oxypurinol. Biochemical Pharmacology, 40(4), pp.803-809.
Kannangara, D., Roberts, D., Furlong, T., Graham, G., Williams, K. and Day, R. (2012). Oxypurinol, allopurinol and allopurinol-1-riboside in plasma following an acute overdose of allopurinol in a patient with advanced chronic kidney disease. British Journal of Clinical Pharmacology, 73(5), pp.828-829.
Okamoto, K. and Nishino, T. (2008). Crystal Structures of Mammalian Xanthine Oxidoreductase Bound with Various Inhibitors: Allopurinol, Febuxostat, and FYX-051. Journal of Nippon Medical School, 75(1), pp.2-3.
O’Regan, M., Phillis, J. and Walter, G. (1989). The effects of the xanthine oxidase inhibitors, allopurinol and oxypurinol on the pattern of purine release from hypoxic rat cerebral cortex.Neurochemistry International, 14(1), pp.91-99.
Pacher, P. (2006). Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol. Pharmacological Reviews, 58(1), pp.87-114.
Springer, J., Hartmann, A., Palus, S., Adams, V., von Harsdorf, R., Anker, S. and Doehner, W. (2009). The Xanthine Oxidase Inhibitors Oxypurinol and Allopurinol Reduce Wasting and Improve Cardiac Function in Experimental Cancer Cachexia. Journal of Cardiac Failure, 15(6), p.S22.
Sun, X., Cao, W., Bai, X., Yang, X. and Wang, E. (2001). Determination of allopurinol and its active metabolite oxypurinol by capillary electrophoresis with end-column amperometric detection.Analytica Chimica Acta, 442(1), pp.121-128.
Vieira, R., Gonçalo, M. and Figueiredo, A. (2004). FS09.5 Patch testing with allopurinol and oxypurinol in drug eruptions. Contact Dermatitis, 50(3), pp.156-156.
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