INTRODUCTION:

Allopurinol (AL) is an uricostatic drug, employed mainly for treatment of hyperuricemia, hyperuricosuria, gout and related complications such as kidney stones etc. Although it is well known that the renal diseases get worsened by conditions like hyperuricemia and for this reason allopurinol is administered to the patients having renal diseases. Hypersensitivity to allopurinol can cause serious side effects, therefore, monitoring AL level in kidney and serum is required¹. As per literature, several HPLC methods are reported for quantification of AL in pharmaceutical dosage form, plasma and human serum but none of the HPLC method was earlier reported for quantification of AL in kidney homogenates and serum of mice. Therefore, in our present study we have utilized already developed HPLC method, for the quantitative estimation of AL in human serum, with slight modifications for quantifying AL in mice serum and kidney homogenates.

Subsequently, validation of the method for its linearity, precision, accuracy, sensitivity, specificity and robustness was carried out as per ICH guidelines².

MATERIALS AND METHODS:

Allopurinol (AL) was generously supplied as gift sample by Indoco Remedies Ltd., (India). Aciclovir (internal standard) was purchased from Sigma Aldrich (India). Acetic acid, water for HPLC and sodium acetate were purchased from Thomas Baker (India). Trichloroacetic acid was procured from SRL Pvt .Ltd., (India). All the chemicals used in this work were of HPLC grade. Animal study was performed after taking proper approval of experimental protocol from Institutional Animals Ethics Committee (Registration No: 0436) and thereafter healthy swiss albino mice, weighing 25-30 g were purchased from Disease Free Small Animal House, LUVAS, Hisar, Haryana, India.

Preparation of Standard Solutions of AL and internal standard:

Standard stock solution of AL (100 µg/ml) was prepared in HPLC grade water. Further, working solutions for calibration were prepared by adequately diluting the standard stock solution and spiking them into blank serum and kidney homogenates samples of albino mice. Aciclovir stock solution (10 µg/ml) in HPLC grade water was prepared and employed as an internal standard.

Collection and preparation of serum and kidney samples:

The mice blood samples were obtained from retro orbital sinus in tubes and allowed to clot by placing the tubes in undisturbed position at room temperature for 1 h. The clotted blood was centrifuged at 9000 rpm (4^oC, 15 min) for separation of serum³. After that animals were anaesthized and sacrificed via decapitation for removing their kidneys. The collected kidneys were washed with saline solution, weighed, homogenized in dimethylsulfoxide (5 volumes) and centrifuged at 9000 rpm (4^oC, 15 min) for obtaining clear supernatant⁴. The serum and supernatant obtained from kidney samples were preserved at -20^oC for carrying further studies. The serum and supernatant of kidney samples (100 µl each) were spiked with internal standard (aciclovir, 10 µl) and trichloroacetic acid (10%, 100 µl) as protein precipitating agent¹. The samples solution were then again centrifuged at 9000 rpm for 15 min (4^oC) and the supernatant so obtained were further filtered using 0.22 μm membrane filter and analysed in HPLC⁵.

HPLC system conditions:

HPLC chromatographic system employed for the study was Agilent Technologies 1260 Infinity consisting of 1260 QuaternaryPump VL, 1260 Diode Array Detector VL and PN 5190-1501 manual injector (all from Agilent). The chromatographic separation of samples was carried out using C18 column (ZORBAX, Agilent, SB C₁₈, 5 micron; 4.6 x 150 mm, ). The mobile phase consisting of solution of 0.02 M sodium acetate (pH 4.5; using 30% acetic acid) which was filtered, degassed and pumped at 1 ml/min. 30 µl of samples were injected into HPLC system and analysis was carried for 3 min (run time) at wavelength of 254 nm.

Validation:

The above described method for HPLC was validated for different parameters i.e. linearity, specificity, LOD, LOQ, precision, accuracy and robustness.

Linearity:

Linearity was assessed via spiking the serum and kidney homogenates with six different concentrations of AL ranging from 0.5-5 µg/ml and 0.5-12.5 µg/ml, respectively. The samples were then processed and analyzed using method already mentioned above. Further, the calibration curves were built between the peak area of these samples against their respective concentration and the values of regression coefficient (R²), slope, intercept were determined⁶.

Precision:

a) Repeatability (Intraday precision):

For evaluating intra-day precision, the serum and kidney homogenates were spiked with 2.5 µg/ml and 5 µg/ml of AL, respectively. The samples solutions were prepared, filtered and six times in a row injected into the HPLC system on same day and relative standard deviation (% RSD) was determined for assessing the repeatability of method in the samples^6-8.

b) Intermediate Precision

(Interday precision/Ruggedness):

For evaluating inter-day precision, samples were injected six times consecutively into HPLC system for two different days by two different analyst and % RSD was computed. The results procured from the two sets of the study were compared to assess intermediate precision⁶^,^9-11.

Acceptance criteria: The % RSD values obtained on intraday and interday study should be less than 2%.

Accuracy:

Accuracy is the assessment of exactness of any method and it is evaluated by measuring the amount of analyte recovered from added known amount. Six replicates of samples were injected on intra and interday and percent accuracy [(calculated concentration/actual concentration)*100] was determined^12-14.

Specificity:

Blank serum and kidney homogenates were analysed in order to annul the probability of interference by any of the components in the proximate region of the retention time of AL and aciclovir (internal standard)⁶^,¹⁵.

Sensitivity:

The sensitivity of system for measuring AL using the abovementioned procedure was assessed in terms of Limit of detection and Limit of Quantification^16-19.

a) Limit of detection:

LOD is the smallest concentration of AL in samples which can be detected, however can’t be quantified. LOD can be computed from standard deviation of intercept (α) and slope (β) obtained from linearity graph using below equation²⁰

3.3α

= ––––––––––––––––––––

b) Limit of Quantification:

LOQ is the smallest concentration of allopurinol in the samples which can be quantified with acceptable criteria of precision and accuracy under specified operational environment of the method. LOQ was determined using following equation²⁰^,²¹

10 α

= ––––––––––––––––––––

Where, α is standard deviation of intercept (y-axis)

β is slope of linearity graph

Robustness:

Robustness was generally carried by deliberately varying the chromatographic conditions such as pH of mobile phase, detection wavelength, flow rate etc. and evaluating their effect on responses²². In our study we had varied pH of mobile phase from 4.4-4.6.

RESULTS AND DISCUSSION:

The serum and kidney samples were prepared and analyzed at wavelength of 254 nm. Chromatograms of allopurinol with aciclovir as internal standard in serum and kidney homogenate samples are shown in Figure 1. The retention times (RT) observed for AL and aciclovir were 1.6 and 2.1 min respectively, therefore the total run time was kept as 3 min.

Validation:

Linearity:

Linear relationship was observed over concentration ranges of 0.5-5 µg/ml AL in serum and 0.5-12.5 µg/ml allopurinol in kidney homogenate. A calibration curve with R² values of 0.999 was procured for both the serum as well as kidney samples, which indicates linearity of method. The regression line equations procured for AL spiked serum and kidney homogenate samples are as follows respectively:

Y= 94210x+30975

Y= 96942x +9374

Precision

The inter-day and intra-day precisions for AL in serum and kidney homogenates are shown in Table 1 and 2. The precision values were found to lie within the acceptance range of % RSD i.e. lesser than 2%.

Accuracy:

Accuracy of the inter-day and intra-day study has been depicted in Table 1 and 2. The accuracy values were found to be well within the acceptance limit as described by ICH guidelines.

Specificity:

Specificity was evaluated by running blank serum and kidney homogenate samples in HPLC system and no interference was observed with the peaks of AL and internal standard which indicates specificity of method (Fig. 1).

Fig. 1: Typical chromatogram of (a) blank serum containing aciclovir (internal standard), (b) blank kidney homogenate containing aciclovir (internal standard), (c) serum containing AL and aciclovir and (d) kidney homogenate containing AL and acyclovir

Table 1: Precision and accuracy for serum Samples

Method

Actual concentration spiked (µg/ml)

Calculated average concentration (µg/ml)

% Accuracy

% RSD

Intraday precision

2.5

2.39

95.6%

1.19

Interday precision

2.5

Day1, Analyst1- 2.39

Day2, Analyst2- 2.41

Day1, Analyst1- 95.6%

Day2, Analyst2- 96.4%

0.84

Table 2: Precision and accuracy for kidney Homogenate Samples

Method

Actual concentration spiked (µg/ml)

Calculated average concentration (µg/ml)

% Accuracy

% RSD

Intraday precision

4.79

95.8%

0.65

Interday precision

Day1, Analyst1-4.79

Day2, Analyst2-4.86

Day1, Analyst1- 95.8%

Day2, Analyst2- 97.2%

1.08

Table 3: Robustness of the Method

Operational parameter

Actual drug concentration (µg/ml)

Calculated average concentration (µg/ml)

% RSD

pH of mobile phase-4.4

pH of mobile phase-4.6

9.7

9.8

0.50

0.28

Sensitivity:

LOD and LOQ were assessed from calibration curve using above mentioned equations. For serum samples, LOD and LOQ were calculated as 63 ng/ml and 192 ng/ml, respectively and for kidney homogenates, LOD and LOQ were calculated as 241 ng/ml and 730 ng/ml, respectively.

Robustness:

Robustness was ascertained via making deliberate alterations in pH of mobile phase. The results have been shown in Table 3, which represents that the small modifications in the operational conditions don’t lead to any significant changes in the performance of HPLC system.

CONCLUSION:

In the present study, HPLC method was successfully validated for parameters such as linearity, specificity, LOD and LOQ, accuracy, precision and robustness. This method can be further used in bioavailability studies and assessing the kidney targeting potential of allopurinol.

ACKNOWLEDGEMENTS:

Authors express their special thanks to Late Dr. Shailendra Kumar Singh, Professor, Department of Pharmaceutical Sciences (GJU S&T, Hisar) for sharing his worthy knowledge throughout the study. Authors thanks University Grants Commission (UGC, New Delhi) for giving Rajiv Gandhi National Fellowship and also thankful to DST FIST (Coordinator), Department of Pharmaceutical Sciences, (GJU S&T), Hisar for providing facilities for HPLC analysis

CONFLICT OF INTEREST:

None to be declared.

REFERENCES:

1. Tada H, Fujisaki A, Itoh K, Suzuki T. Facile and rapid high‐performance liquid chromatography method for simultaneous determination of allopurinol and oxypurinol in human serum. J. Clin. Pharm. Ther. 2003; 28:229-34.

2. Reinders MK et al., A simple method for quantification of allopurinol and oxipurinol in human serum by high-performance liquid chromatography with UV-detection. J. Pharm. Biomed. Anal. 2007; 45:312-7.

3. Hou PY et al. Pallidifloside D from Smilax riparia enhanced allopurinol effects in hyperuricemia mice. Fitoterapia. 2015; 105:43-8.

4. Degroodt JM, Bukanski BW, Srebrnik S. Multiresidue analysis of tetracyclines in kidney by HPLC and photodiode array detection. J. Liq. Chromatogr. Relat. Technol. 1993; 16:3515-29.

5. Nagpal K, Singh SK, Mishra DN. Formulation, optimization, in vivo pharmacokinetic, behavioral and biochemical estimations of minocycline loaded chitosan nanoparticles for enhanced brain uptake. Chem. Pharm. Bull. 2013; 61:258-72.

6. Hansraj GP, Singh SK, Kumar P. Sumatriptan succinate loaded chitosan solid lipid nanoparticles for enhanced anti-migraine potential. Int. J. Biol. Macromol. 2015; 81:467-76.

7. Rajavel R, Ganesh M, Jagadeeswaran M, Srinivasan K, Valarmathi J, Sivakumar T. RP-HPLC method for the simultaneous determination of aspirin, atorvastatin and pioglitazone in capsule dosage form. Asian J. Research Chem. 2008; 1(1):40-2.

8. Jain N, Jain R, Swami H, Jain DK. RP-HPLC method for simultaneous estimation of simvastatin and ezetimibe in bulk drug and its combined dosage form. Asian J. Research Chem. 2008; 1:29-31.

9. Rezazadeh M et al. A rapid and sensitive HPLC method for quantitation of paclitaxel in biological samples using liquid-liquid extraction and UV detection: application to pharmacokinetics and tissues distribution study of paclitaxel loaded targeted polymeric micelles in tumor bearing mice. J. Pharm. Pharm. Sci. 2015; 18:647-60.

10. Rajkumar B, Bhavya T, Kumar AA. Reverse phase HPLC method development and validation for the simultaneous quantitative estimation of alpha lipoic acid and allopurinol in tablets. Int. J. Pharm. Pharm. Sci. 2014; 6:307-12.

11. Bagyalakshmi J, Vijayaraj S, Ravi TK. Method Development and Validation of Erythrosine (E127) Using RP-HPLC Coupled With PDA Detector. Asian J. Research Chem. 2008; 1(2):95-00.

12. Li H et al. Development and validation of an HPLC method for quantitation of BA‐TPQ, a novel iminoquinone anticancer agent, and an initial pharmacokinetic study in mice. Biomed. Chromatogr. 2011; 25:628-34.

13. Wei Y, Bachu RD, Sari Y, Boddu SH. Development and Validation of a HPLC Method for MS-153 Quantification: Assessment of its Stability in Rat Plasma and Brain Homogenate. Pharm. Anal. Acta. 2018; 9:2.

14. Battu PR, Reddy MS. Development and validation of RP-HPLC for the rabeprazole sodium in pharmaceutical formulations and human plasma. Asian J. Research Chem. 2009 Jan; 2(1):49-51.

15. Phale MD, Hamrapurkar PD. A validated and simplified RP-HPLC of metoprolol succinate from bulk drugs. Asian J. Research Chem. 2009; 2(2):119-22.

16. Vani R, Kumar BV, Mohan GK. Analytical Method Development and Validation for the Determination of Allopurinol and Alphalipoic Acid Using Reverse Phase HPLC Method in Bulk and Tablet Dosage Form. Res. J. Pharm. and Technol. 2015; 8:207.

17. Battu PR, Reddy MS. RP-HPLC method for simultaneous estimation of paracetamol and ibuprofen in tablets. Asian J. Research Chem. 2009; 2(1):70-2.

18. Godse VP, Deodhar MN, Bhosale AV, Sonawane RA, Sakpal PS, Borkar DD, Bafana YS. Reverse phase HPLC method for determination of aceclofenac and paracetamol in tablet dosage form. Asian J. Research Chem. 2009; 2(1):37-40.

19. Ashwini SJ et al. A Validated Chiral HPLC Method for the Enantiomeric separation of Mefloquine. Res. J. Pharm. and Technol. 2019; 12(5):2304-2308.

20. Godge RK, Damle MC, Pattan SR, Kendre PN, Lateef SN, Burange PJ. RP-HPLC Method for Simultaneous Estimation of Pseudoephidrine Sulphate and Desloratidine from Bulk and Tablets. Asian J. Research Chem. 2009; 2(2):139-42.

21. Raju GV, Ganapathy S, Sankar DG, Naidu PY. Development and Validation of an HPLC Method for Analysis of Levetiracetam in Human Plasma. Asian J. Research Chem. 2010 Sep 28; 3(3):776-80.

22. Chitlange SS, Bagri K, Sakarkar DM. Stability Indicating RP-HPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation. Asian J. Research Chem. 2008; 1(1):15-8.

Received on 25.06.2019 Modified on 30.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 373-376

DOI: 10.5958/0974-360X.2020.00074.8