Measuring Inhibitors Of Purines And Xanthine: Allopurinol And Oxypurinol Using HPLC

Objectives of the Study

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.

• Developing five-point marking calibration curve within distilled water meant for oxypurinol, allopurinol, xanthine, hypoxanthine, and uric acid.
• Analyzing calibration curves as well as samples provided that are unknown (each four time).
• Calculating precision of the undertaken analyses of purines as well as drugs from derived outcomes (Hoey, Butler and Halliwell, 1988).

• Effective laboratory practices as well as higher standards of work hygiene should be observed all throughout the assay.
• Every involved personnel is required to use proper protective clothing as well as safety equipment needed to perform various tasks.
• The COSHH sheets associated with all materials to be utilized throughout the assay would indicate the important precautions needed to be applied prior to the initiation of the assay (Okamoto and Nishino, 2008).

• Increasingly performing liquid chromatography (often stated as high pressure liquid chromatography), HPLC is a technique in chromatography utilized for separating a compound mixture within analytical chemistry as well as biochemistry having an aim to identify, quantify, and purify each basic component of the mixture (Kam Ming Ko and Godin, 1990). It is even considered as a technique of instrumentation of analytical chemistry rather than a gravimetric technique.

• The system of HPLC comprises of a Shimadzu LC-10AT VP pump, a Shimadzu SPD-6A variable wavelength UV detector, a Shimadzu SIL-9A sample injector and a computer with Jones JCL 6000 data intake software. The column used is a PhenomenexHypercolone 5μm, dimensions 25cm × 4.6 mm (id).The guard column is a Crawford Scientific Opti-Guard 1mm C18 column. The column is maintained at ambient temperature (Pacher, 2006)

• Flow: 1.0 ml/min.
• : UV; λ 280 nm; Absorbance 0.01; Gain (data acquisition) 12.
• Inject vol.: 50 μl.
• Temperature: ambient (22-24â°C).

• Stock standard solutions of:

Allopurinol=5 mM

Oxypurinol=10 mM

Uric acid=5 mM

Hypoxanthine =2 mM

Xanthine=2 mM

• Distilled water to construct calibration curves
• Internal Standard (IS) 5-Fluorouracil-10μm/ml distilled Hâ‚‚
• Mobile phase‒50mM Phosphate buffer, pH 6.2; no organic modifier.
• 3 “unknown” aqueous solutions for analyses four times (Springer et al., 2009).

• Preparing work standards
• Deciding upon five proper calibrations in given range for each of the compounds

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

• The amount of stock standard necessary to build 10 ml work standards within distilled water was decided by application of the given equation

• Since all the compounds are simultaneously measured, only one 10 ml volumetric flask is required to be spiked along with each stock for each standard concentration.

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

• Label 10 ml glass volumetric flask 2 – 6 and making up the work Standard concentrations that we have upon in these flasks.
• Used a clean pipette tip every time (Sun et al., 2001).
• Once added the appropriate volume of stock to volumetric flask, we used the distilled water in the wash-bottle to initially make the volume up near to the 10 ml mark in the volumetric, and then if required we use a pastille to make up the mark.

1. Label 20 HPLC vials 1 – 20:

• 1 – 6 contain standard 1 – 6
• 7 –10 contain “unknown-1”
• 11 –14 contain “unknown-2”
• 15 –18 contain “unknown-3”
• 19 – 20 contain water to prevent carryover.

1. Add 75μl from each working standard or unknown to an HPLC vial.
2. Add 25ul IS (10μg/ml 5-flourouracil/ml) to each vial.
3. Mix very briefly (3 seconds) by using vortex mixture.
4. Load vials into HPLC auto sampler and inject (Vieira, Gonçalo and Figueiredo, 2004).

All 21 chromatographs were out –

• The very first graph shows the blank sample
• The groups from S2 to S6 were presenting the standard samples.
• The groups from unknown1A to unknown1D were presenting unknown-1 sample.
• The groups from unknown2A to unknown2D were presenting unknown-2 sample.
• The groups from unknown3A to unknown3D were presenting represent unknown-3 sample.

• The five standard curves of allopurinol, oxypurinol, hypoxanthine, xanthine and uric acid would be drowning using the regression (PHR on the Y axis against Concentration on X axis).
• The peak high ratio (PHR) for the standard= height of the standard divide on the height of internal standard.

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.