Isocratic RP-HPLC Method for Simultaneous Estimation of Spirinolactone and Hydrochlorthiazide in Oral Solid Dosage Form

 

ABSTRACT:

A simple, rapid, sensitive and accurate isocratic reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and subsequently validated for the simultaneous determination of Spirinolactone   and Hydrochlorthiazide in combined dosage form. The method was employed on a μ-Bondapak C18 column (150 mm × 4.6 mm, 5 μm) at ambient temperature. The mobile phase consisted of methanol: 0.02M dibasic sodium phosphate containing Trifluroacetic acid and (40:60, pH 3.2) at a flow rate of 1 ml/min. The UV detection wavelength was set at 278 nm and 20 μL of sample was injected into the HPLC system. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness and limit of quantification. The suggested method was successfully applied for the simultaneous analysis of the studied drugs in their co-formulated tablets. The results of analysis were subjected to statistical analysis.

 

 

INTRODUCTION:

Spironolactone[SPI] is a potassium-sparing diuretic (water pill) that prevents your body from absorbing too much salt and keeps your potassium levels from getting too low. It is chemically (7α, 17α)-7-(Acetylthio)-17-hydroxyl-3-oxopregn-4-ene-21-carboxylicacid-γ-lactone and used as diuretic.  Spironolactone acts from the interstitial side of the tubular cell combines with the mineralocorticoids receptor and inhibits the formation of aldosterone induced proteins (AIPs) in a competitive manner ^[1]. It is official in I.P^[2] and USP^[3].  Literature survey reveals that two quantitative spectrophotometric method ^[4],[5] and few HPLC^{[6], [7]} methods are reported for the determination of SPI.

 

Hydrochlorothiazide [HCT] is chemically 6-chloro-3,4-dihydro-2 H 1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide is  a thiazide diuretic. Thiazides affect the renal tubular mechanisms of electrolyte reabsorption, directly increasing excretion of sodium and chloride in approximately equivalent amount.it is official in I.P^[8],B.P^[9],USP^[10] . A combination of both drugs having additive effect and used to increase urinary output and lung function in infants with broncho pulmonary dysplasia^[11].

 

Literature survey shows that no isocratic RP-HPLC method has been reported so far for the simultaneous estimation of both the drugs with short analysis time. For HCT the reported methods are UV-Spectroscopic method^{[12] [13]} , RP-HPLC^{[14] [15]}  method and LC-MS^[16] method Only one RP-HPLC methods has been reported so far for simultaneous estimation of both the drugs^[17] , but the reported method is not isocratic. The objective of this work was to develop a novel, simple, precise and rapid isocratic RP-HPLC method which can be used as a assay for combination drug product of SPI and HCT.

 

MATERIAL AND METHOD:

Materials

Gift samples of spirinolactone and Hydrochlorthiazide was received from analytical research and development laboratory of International Speciality Product, Hyderabad, India. HPLC grade acetonitrile was purchased from Fischer Scientific, India. Analytical reagent grade disodium hydrogen orthophosphate was purchased from S.D. Fine Chem. Limited, India, and trifluro acetic acid   were purchased from Qualigens Fine Chemicals, India. High pure water was prepared by using Millipore Milli Q plus purification system. Aldactazide® commercial formulations were purchased from the local market.

 

Equipment

The LC system, used for method development and method validation was Waters 2695 series (manufactured by Waters Technologies, USA) LC system with a diode array detector. The output signal was monitored and processed using Empower software (designed by Waters Technologies, USA) on lenovo computer (Digital Equipment Co.).

 

Chromatographic condition

The chromatographic column used was Waters using μ-Bondapak C18 (150mm Χ 4.6mm Χ 5μ). The mobile phase consists of a mixture of methanol (solvent A) and 0.02 M disodium hydrogen ortho-phosphate, pH adjusted to 3.2 using trifluro acetic acid, (solvent B) in 40: 60 v/v ratio. The mobile phase was pumped from the solvent reservoir to the column at a flow roate of 1 ml/min for 10 min. The column temperature was maintained at 23 ± 1 °C. The eluate was monitored at 278 nm using the PDA detector. The injection volume was 20 μl. Mobile phase was used as diluent during the standard and test samples preparation.

 

Standard Solutions

Standard stock solutions of SPI and HCT were prepared separately by dissolving 50 mg of each drug in 50 mL volumetric flask with 10 mL of mobile phase and the solutions were sonicated for about 15 min. Then the volume was made up to the mark with mobile phase to get 1 mg/ml standard stock solution. Several aliquots of these standard stock solutions were taken in different 10 mL volumetric flask and diluted up to the mark with mobile phase such that the final linearity concentrations of SPI and HCT were 10-70 µg/ml and  respectively.

 

Assay standard solution preparation

A working standard solution containing 50 µg/ml of SPI and 50 µg/ml of HCT were prepared from the above standard stock solution.

 

Preparation of Aldactazide® tablets for assay

Twenty tablets of the formulation were weighed and the average weight of one tablet was calculated. All 20 tablets were crushed and grounded to a fine powder. Powder equivalent to 50 mg of spironolactone and 50 mg of hydrochlorthiazide (one tablet weight) was transferred in to a 50 mL of volumetric flask and diluted up to the mark with the mobile phase and mixed well, then the solution was filtered through a 0.45 μm filter to obtain a clear filtrate. This solution was suitably diluted and used for analysis. After setting the chromatographic conditions and stabilizing the instrument to obtain a steady baseline, a fixed volume of 20 μL of the sample solution was loaded by an automatic sampler. The solution was injected, and chromatograms were recorded. The injections were repeated six times, and the peak areas were recorded.

 

Method development and optimization

The primary target in developing this method is to achieve simultaneous determination of SPI and HCT in the tablet formulation under isocratic conditions that will be applicable for routine quality control of the product in laboratories because it is not possible to have gradient HPLC in small scale pharmaceutical company. To optimize the proposed RP-HPLC method, all of the experimental conditions were investigated. For the choice of the stationary phase, reversed-phase separation was preferred due to the drawbacks of the normal phase, e.g., hydration of silica with water that can cause peak tailing. To optimize the mobile phase, different systems were tried for chromatographic separation of the two components by combining homogenous design and solvent polarity optimization. Methanol was selected as organic modifier because it was economic when compare to acetonitrile. The selection of trifluroacetic acetic acid in buffer and the composition of mobile phase were based on providing good baseline, adequate separation and sharp peaks in reasonable time. The resolution was achieved using a mobile phase consist of methanol: 0.02M dibasic sodium phosphate containing Trifluroacetic acid (40:60, pH 3.2), which gave good resolution and sensitivity of both drugs. For the quantitative analytical purpose the wavelength was set at 278 nm. The typical chromatogram of sample was shown in  Figure.1.

 

 

Figure 1: Chromatogram of standard solution

 

Method validation

The developed method was validated as per ICH guidelines ^[18] in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ) and system suitability. The system suitability of the HPLC method was determined by making six replicate injections from freshly prepared standard solutions and analyzing each solute for their peak area, theoretical plates (N), resolution (R), and tailing factors (T).

 

Limit of quantification (LOQ) and limit of detection (LOD) were calculated according to the definitions of the ICH topic Q2B.   

 

Accuracy of the method was determined by recovery studies. Analytical recovery experiments were carried out by standard addition method, at 50, 100 and 150% level.

The precision of the method was investigated with respect to repeatability. To determine intermediate precision, standard solutions of the drug at the 100% concentration level were analyzed three times within the same day (intra-day variation) and on three different days (inter-day variation).

 

Robustness studies were performed on method precision by making slight variations in flow rate, amount of the mobile phase and pH changes. The ruggedness of an analytical method is the degree of reproducibility of the test results obtained by the samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, and different days.

 

RESULTS AND DISCUSSION:

System suitability

The column efficiency, resolution and peak symmetry were calculated for the standard solutions. Table.1. The RSD of system suitability factors was satisfactory that is less than 2% and resolution was satisfactory. The peaks obtained for SPI and HCT were sharp and have clear base line separation.

 

Table 1: System suitability parameters for SPI and HCT

Parameter	Spirinolactone± RSD,%	Hydrochlorthiazide ± RSD,%
Retention time, min	3.1±0.21	4.2±0.74
Tailing factor	1.12±0.15	1.20±0.89
Theoretical plates	3256.13±0.94	4156.21±0.45
Capacity factor	0.25±0.32	0.96±0.12
Resolution	6.23±0.55

 

Linearity, Limit of detection, Limit of quantification

The linearity was evaluated by linear regression analysis. The calibration graph was constructed for the proposed method from the data points over the concentration range cited in Table 2 . The linearity of the calibration graph and conformity of the HPLC method proved by the high values of the correlation coefficients (r²) of the regression equation. According to ICH recommendations the approach based on the SD of the response and the slope was used for determining the detection and quantitation limits. The detection limit and quantitation limit of SPI were found to be 1.2 μg/ml and 2.0 μg/ml and HCT 1.0 μg/ml and 2.0 μg/ml respectively.

 

Precision

Precision is a measure of the ability of the method to generate reproducible results. The precision of a method is evaluated using three separate determinations for repeatability, intermediate precision, and reproducibility. The results of intra- and inter-day variations are shown in Table 2. The results obtained from intermediate precision also indicated a good method precision. All the data were within the acceptance criteria.

 

Table 2: Summary of validation parameters of the proposed method

Parameters	Spirinolactone	Hydrochlorthiazide
Linearity	10-70 μg/ml	10-70 μg/ml
Intercept	61677	58825
Slope	98339	118465
Correlation coefficient	0.9997	0.9991
LOD	1.2 μg/ml	1.0 μg/ml
LOQ	2 μg/ml	2 μg/ml
Precision(RSD)
Intraday,( n=3),%	0.1256	0.3657
Interday,( n=3),%	0.6598	0.2547
Repeatability of injection,( n=10),%	0.7854	0.5478
Ruggedness(RSD)
Analyst 1,( n=3),%	0.1254	0.4698
Analyst 2,( n=3),%	0.3698	0.3548
Instrument 1, ( n=3),%,	0.5487	0.4578
Instrument 2,( n=3),%	0.6547	0.8974

 

Accuracy

The HPLC area responses for accuracy determination are depicted in Table .3.The results show that best recoveries (99.67% and 101.01%) of the drug were obtained at each added concentration, indicating that the method was accurate.

 

Table 3: Evaluation data for accuracy study

Drug	Amount taken (µg/ml)	Amount added (µg/ml)	Total amount found (µg/ml)	Mean Recovery* (%)	RSD (%)*
SPI	50	25	75.21	100.28	0.441
	50	50	100.10	100.01	0.253
	50	75	124.59	99.67	0.168
HCT	50	25	74.81	99.74	0.387
	50	50	99.80	99.87	0.245
	50	75	125.05	100.04	0.218

 

Robustness and Ruggedness

Robustness studies were carried out after deliberate alterations of flow rate and mobile phase compositions and pH. It was observed that the small changes in these operational parameters did not lead to changes of retention time of the peak interest. The degree of reproducibility of the results has proven that the method is robust and the data are summarized in Table.4. The ruggedness of the method was determined by carrying out the experiment on different instrument like Waters HPLC and Shimadzu HPLC and by two different operators using different columns of similar type like Phenomenex C₁₈,HypersilC₁₈ and the results were shown in Table 2,the low RSD values confirms the ruggedness of the method.

 

Table 4: Robustness testing of the method

Parameter	Modification	Spirinolactone % Recovery	Hydrochlorthiazide % Recovery
pH	3.0	100.1	99.6
	3.2	100.5	99.8
	3.4	99.9	101.4
Buffer Composition (M)	38	99.8	100.1
	40	100.6	100.2
	42	100.7	99.7
Flow rate (mL/min)	0.9	101.0	99.6
	1.0	99.3	101.3
	1.1	101.3	101.5

 

Tablet studies

The proposed method was successfully applied to the analysis of marketed products (Aldactazide®) and the results obtained are given in Table 5.

 

Table 5: Analysis of formulation

Drug	Labeled amount (mg)	Amount of mg/tab found*	% Label claim	% RSD
Spirinolactone	50	49.8	99.63	0.414
Hydrochlorthiazide	50	50.46	100.92	0.151

^*Average of six determinations

 

CONCLUSION:

The results showed that the method provided adequate accuracy, precision, sensitivity, reproducibility with better resolution for the analysis of SPI and  HCT in formulations either simultaneously or individually. The advantages of a proposed method are its short analysis time and a simple procedure for sample preparation. Thus it can be concluded that the proposed method can be used for the routine analysis of these two drugs in bulk as well as preparations without any interferences.

 

REFERENCES:

1.       K.D.Tripathi, Essential Medical Pharmacology, 5^th edition, Jaypee Brothers, New Delhi, 2004,525-537.

2.       Indian Pharmacopoeia. 4^th ed, New Delhi India, the controller of publications: 1996: Vol-II: 709-710.

3.       United states pharmacopoeia.28^th ed, Washington, Published by the board of trustees, 2005:Vol-II:1953.

4.       Dinc Erdal, Ustunda Ozgur. Spectrophotometric quantitative resolution of hydrochlorothiazide and spironolactone in tablets by chemometric analysis method. II Farmaco 2003;58:1151-61.

5.       Martin E, Jimenez AI, Hernandez O, Jimenez F,Arias JJ. Simultaneous kinetic spectrometric determination of spironolactone and canrenone in urine using partial least-square regression.Talanta.1999;49:143-54.

6.       De Croo F, Bossche W. vanden, Moerloose P. Simultaneous determination of spironolactone and althiazide in tablets by high-performance liquid chromatography. Fresenius’ Journal of Analyticalchemistry, 2004;323:91-93.

7.       Kenneth S. Alexander, Shyam Sunder K.S. Vangala, David Dollimore. Improved High-performance liquid Chromatography Assay for spironolactone analysis. Drug development and industrial pharmacy 1998; 24:101-7.

8.       Indian Pharmacopoeia.(2007) Vol. II  Published by the Government of India, Ministry of Health and Family Welfare, the Indian Pharmacopoeia Commission, New Delhi:  1194-1196.

9.       British Pharmacopoeia, (1993)Vol. II, Department of health and social services for Northen Ireland, London: stationary publication: 330.

10.     United States Pharmacopoeia, (2009) National Formulary USP23, Asian Edition, Rockville, MD: United States Pharmacopoeial Convention:2566-2568

11.     B. Engelhardt, W.A. Blalock, M. Rush, and T.A. Hazinski. Effect of  Spirinolactone-hydrochlorthiazide  on lung function in infants with chronic broncho pulmonary dysplasia. The Journal of Pediatrics. 1989 :27.234-238.

12.     Dinc E, Balenu D. Continuous wavelet transform and chemometric methods for quantitative resolution of a binary mixture of quinapril and hydrochlorothiazide in tablets.     Jou.braz.chem.soc2007;18:962-8.

13.     Kowalczuk D, Hopkala H. Application of derivative spectrophotometry for simultaneous determination of quinapril and hydrochlorothiazide in the combination tablets. Journal of  AOAC Int. 2004;87:847–51.

14.     Valizadeh H, Zakeri-Milani P, Islambulchilar Z, Tajerzadeh H. A simple and rapid high-performance liquid chromatography method for determining furosemide, hydrochlorothizde and phenol Red: Applicability to intestinal permeability studies. Journal of AOAC Int 2006;89:88. 

15.     Huang T, He Z, Yang B, Shao L, Zheng X, Duan, Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution. J Pharm Biomed Anal 2006;41:48-55

16.     Vonaparti A, Kazanis M, Panderi I. Development and validation of liquid chromatographic electro spray ionization mass spectrometric method for the determination of benazepril, benzeprilat and hydrochlorothiazide in human plasma. Journal of Mass Spectrum 2006;41:593-605

17.     Maha A. Hegazy, Fadia H. Metwaly, M. Abdelkawy, and Nada S. Validated chromatographic methods for determination of Hydrochlorthiazide and spironolactone in pharmaceutical formulation in presence of impurities  and degradants. Journal of Chromatographic Science,49;2011;129-135.

18.     Validation of Analytical Procedures: Methodology, ICH Harmonized tripartite Guideline, 1996; 1-8.

 

 

Received on 02.07.2012       Modified on 13.07.2012

Accepted on 29.07.2012      © RJPT All right reserved