Stability Indicating RP- HPLC Method for Bumetanide in Bulk Drug and Tablet Formulation
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy,
Pune 411038, Maharashtra, India.
*Corresponding Author E-mail: janhavirao@rediffmail.com
ABSTRACT
A simple, sensitive, selective, precise and reversed-phase stability-indicating HPLC method was developed and validated for Bumetanide (BUM) in bulk drug and in formulation. The method has shown adequate separation for BUM from their associated impurities and their degradation products. Separation was achieved on a Waters Spherisorb ODS2 C18, 250 mm Ś 4.6 mm i.d., 5 ”m column using a mobile phase consisting of methanol: water (70:30, v/v) at a flow rate of 1ml/min and UV detection at 335 nm and the retention time (TR) was found to be 4.743 ± 0.05 min. The method was also applied for the determination of BUM in the presence of their degradation products formed under variety of stress conditions. Proposed method was validated for precision, accuracy, linearity range, recovery and robustness.
KEYWORDS: Reverse phase HPLC, Bumetanide, Forced degradation
INTRODUCTION:
Bumetanide (BUM) [3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid] is an important pharmaceutical compound classified as a strong diuretic agent (loop diuretic) Fig. No.1. It is indicated for the treatment of oedema associated with congestive heart failure, hepatic and renal disease, including the nephrotic syndrome 1. Few methods have been reported for the analysis of BUM in pharmaceuticals or in biological fluids. BUM has been determined using spectrophotometry 2, capillary zone electrophoresis 3, HPLC- GC/MS 4 and HPLC 5-8 in pharmaceutical preparations. As no RP-HPLC stability indicating method is reported for BUM. The aim of the present study was to develop accurate, precise and selective reverse phase HPLC assay procedure for the analysis of BUM in bulk drug and in tablet dosage form.
Bumetanide working standards was kindly supplied as a gift sample by Lupin Pharmaceuticals Ltd, Pune, India; it was certified to contain 99.85 % (w/w) on dried basis and was used without purification. All chemicals and reagent used were of HPLC grade and were purchased from Merck Chemicals, Mumbai, India. Tablet containing 1 mg of BUM was purchase from local market.
HPLC Instrumentation :
A Water HPLC system comprising of Quaternary LC- 10A VP pumps, UVVisible detector and Waters Spherisorb and Perfectsil ODS2 C18, 5 μm, 250 mm Ś 4.6 mm i.d., column was used. A Rheodyne sample injector fitted with a 20-μl loop was used for the injection of sample. The HPLC system was equipped with Jasco Borwin Software for data processing.
Chromatographic Condition:
The mobile phase containing methanol: water (70:30, v/v) was found to resolve BUM. The mobile phase was filtered through 0.45 ”m Nylon filter prior to use. The flow rate was set to1 ml/min. BUM shows good absorbance at 335 nm, which was selected as wavelength for further analysis and injection volume of 20 μl was used
Preparation of Stock and Standard solutions:
A stock solution BUM was prepared by dissolving 100 mg in 100 ml methanol (1000 mg/ml). The standard solutions were prepared by dilution of the stock solution with methanol to reach a concentration range 1-10 ”g/ml. Triplicate 20 ”l injections were made six times for each concentration and chromatographed under the conditions described above. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs
Figure 1 Structure of Bumetanide
Figure. 2 Chromatogram of standard drug (10”g/ml)
Figure 3. Chromatogram showing degradation in 1 N HCl
Figure 4.Chromatogram showing degradation in 2 N NaOH
Table no.1: Result of BUM in marketed formulation (n = 9)
Actual conc. (”g/ml) |
For HPLC |
||
Calculated conc. ± SD |
% Amount found |
% RSD |
|
4 6 8 |
3.93 ± 0.030 5.92 ± 0.051 7.95 ± 0.070 |
98.25 98.66 99.37 |
0.775 0.861 0.880 |
Table no. 2: System Suitability Parameters
Parameter |
BUM |
Linearity Range |
110 μg/ml |
Correlation Coefficient * |
0.9991 |
Slope* |
157348 |
Limit of detection (”g/ml) |
0.02 |
Limit of Quantitation (”g/ml) |
0.06 |
Intra Day Precision % RSD |
0.7186 |
Inter Day Precision % RSD |
0.7406 |
Accuracy (Recovery Studies)* |
98.68 ± 1.22 |
Retention time* |
4.62 ± 0.226 |
Tailing factor * |
1.13 ± 0.050 |
*Average of three readings ± Standard Deviation
Table no. 3: Precision of developed method (n = 3)
Drug |
Concentration(”g/ml) |
Intra-day Precision % RSD |
Inter-day Precision % RSD |
Bumetanide |
2 4 8 |
1.502 0.386 0.268 |
1.201 0.580 0.441 |
Table no. 4: Recovery of bumetanide (n = 9)
Actual Concentration (”g/ml) |
Label claim (mg /tablet) |
Amount added (mg) |
(%) Amount Recovered ± SD |
4 4 4 |
1 1 1 |
1.8 (80%) 2.0 (100%) 2.2 (120%) |
98.16 ± 1.37 99.49 ± 1.15 98.40 ± 1.15 |
Sample Preparation:
Twenty tablets were weighed and triturate to form fine powder. A portion of the powder equivalent to the weight of one tablet (containing 1 mg of BUM) was accurately weighed and transferred to 10 ml volumetric flask, dissolved and made upto volume with HPLC-grade methanol. The volumetric flasks were sonicated for 120 min to effect complete dissolution of the BUM and the solutions were filtered through a 0.45”m Nylon filter. Suitable aliquot of the filtered solution was added to a volumetric flask and made upto volume with mobile phase to get concentration of 4, 6 and 8 mg/ml, respectively. The diluted solution was analyzed under optimized chromatographic conditions and chromatogram is depicted in Fig. No.2.
Table no. 5. Robustness evaluation$ of the HPLC method (n = 3)
Factor |
Level |
Chromatographic Changes |
|
*TR |
#Tf |
||
A: Flow rate (mL/min) |
|||
0.9 1 1.1 |
-1 0 1 |
4.81 4.73 4.64 |
1.17 1.11 1.04 |
Mean ± SD (n=3) |
4.59 ± 0.3028 |
1.10 ± 0.0655 |
|
B: % of methanol in the mobile phase (v/v) |
|||
68 70 72 |
-2 0 2 |
4.92 4.72 4.65 |
1.23 1.10 1.15 |
Mean ± SD (n=3) |
4.59 ± 0.3995 |
1.16 ± 0.0654 |
|
C: Columns from different manufacturers |
|||
Water Spherisorb Thermo |
4.73 4.55 |
1.10 1.16 |
|
Mean ± SD (n=3) |
4.64 ± 0.1272 |
1.13 ± 0.0424 |
|
D: Solvents of different lots |
|||
First lot Second lot |
4.73 4.62 |
1.10 1.14 |
|
Mean ± SD (n=3) |
4.67 ± 0.0777 |
1.12 ± 0.0282 |
$Average of three concentrations 2, 4, 8 ”g/ml. *Retention time. # Tailing factor
VALIDATION OF THE METHOD:
The developed method was validated in terms of linearity, accuracy, specificity, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement.
Degradation Studies:
Stress testing of the drug substance can help identify the likely degradation products, the stability and specificity of the analytical procedure, BUM was stressed under various conditions to conduct forced degradation studies.
Acidic degradation studies
A 1 ml of 1 N hydrochloric acid was added to 9 ml of drug solution to get final concentration of 10 mg/ml of each drug. This solution was allowed to reflux at 80șC for 2 hrs.
Alkali degradation studies
A 1 ml of 2 N sodium hydroxide was added to 9 ml of drug solution to get final concentration of 10 mg/ml of each drug. This solution was allowed to reflux at 70șC for 24 hrs.
Oxidative studies
A 1 ml of 50 % hydrogen peroxide was added to 9 ml of drug solution to get final concentration of 10 mg/ml of each drug. This solution was allowed to stand for 24 hrs.
Temperature stress studies
Dry heat condition: BUM powder was exposed to dry heat of 50șC in an oven for 8 h to see the effect of dry heat degradation.
Wet heat condition: BUM solution was kept in humidity-controlled oven (R.H- 90 % ± 5%) at 70 șC for 36 days to see the effect of wet heat degradation.
BUM tablet, powder and solution were prepared and exposed to sun light to determine the effects of sun light irradiation on the stability of the BUM in solution and in the solid state. Approximately 25 mg of BUM powder was spread on a glass dish in a layer that was less than 2 mm thick. A solution of BUM (100 ”g/ml) was prepared in methanol. Tablet was prepared in the same way. All samples for photostability testing were placed in a sun light for 3 h. Control samples which were protected with aluminum foil were also placed in the UV and sun light and exposed concurrently. Following removal from the sun light, all samples were prepared for analysis as previously described. Fig. Nos. 3 to 7 shows the degradation behavior of the drug.
To develop a precise, accurate and suitable stability indicating HPLC method for the BUM, different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination. The results obtained by the assay of marketed formulation are summarized in Table 1. System suitability tests were carried out as per USP XXIV and parameters are summarized in Table 2.
Degradation Behavior:
The results of the stress studies indicated the specificity of the method that has been developed. BUM was degraded in all conditions. The degraded products appeared at retention time (TR) 8.059 in 1 N HCl, 6.415 in 2 N NaOH, 3.151 in 50 % H2O2, 6.352 in temperature degradation studies and 3.497 in photostability studies.
Method Validation:9-10
The proposed HPLC method was validated as per ICH guidelines.
Specificity
The peak purity of BUM was assessed by comparing the retention time (TR) of standard BUM. Good correlation was obtained between the retention time of standard and sample of BUM.
Linearity
Linearity was studied by preparing standard solutions at different concentration levels. The linearity range for BUM was found to be 110 μg/ml. The regression equation for BUM was found to be y = 157348x + 14222 with coefficient of correlation, (r) 0.9991.
LOQ and LOD
The LOQ and LOD were determined based on a signal-to-noise ratios and were based on analytical responses of 10 and 3 times the background noise, respectively. The LOQ was found to be 0.06 ”g/ml and LOD was found to be 0.02 ”g/ml.
Precision
Precision of the method was determined with BUM. System repeatability was determined by three replicate applications and three times measurement of a sample solution at three different concentration levels 2, 4, 8 ”g/ml Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. Results are shown in Table 3.
Figure 6. Chromatogram showing thermal degradation
Accuracy (Recovery studies)
To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard BUM was added to pre-analyzed samples and was subjected to the proposed RP-HPLC method. Results of recovery studies are shown in Table 4.
Robustness of method
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. This study was carried out on 2, 4, 8 ”g/ml of solution. Here, three factors were slightly changed as listed in Table No. 5.
CONCLUSION:
Hence, the proposed method is simple, accurate, precise, specific, and has the ability to separate the drug from its degradation products and excipients found in the tablet dosage forms. The method is suitable for use in the routine analysis of BUM in either bulk powder or in tablet dosage form. The HPLC procedure can be applied to the analysis of samples obtained during accelerated stability experiments to predict expiry dates of pharmaceuticals.
ACKNOWLEDGEMENTS:
The authors are grateful to Lupin laboratory Ltd. (Maharashtra, India), providing samples of bumetanide as gift. The authors are also grateful to Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, India, for providing excellent facilities for carrying out this research work.
Figure 7. Chromatogram showing photo degradation
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Received on 21.04.2009 Modified on 12.06.2009
Accepted on 16.07.2009 © AJRC All right reserved
Asian J. Research Chem. 2(3): July-Sept. 2009 page 266-269