INTRODUCTION:

Bumetanide (BUM) is an important pharmaceutical compound classified as a strong diuretic agent (loop diuretic) of the sulfamyl category. Chemically, it is 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid. 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 far as we are aware, however, no stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of BUM in pharmaceutical dosage forms has been reported in the literature. In this work, BUM was exposed to different stress conditions and subsequent HPTLC analysis with ICH guidelines ^9-11.

EXPERIMENTAL:

Chemicals and materials:

BUM was supplied by Lupin Pharmaceuticals Ltd Pune, India, as a gift. All solvents were of analytical grade (Merck).

Tablets containing 1 mg of BUM were obtained from commercial sources within their shelf life.

HPTLC:

Chromatography was performed on 20 cm × 20 cm aluminum plates precoated with silica gel G 60F₂₅₄. TLC plates were pre-washed with methanol and activated by keeping in an oven at 110°C for about 5 min. The solutions of BUM were spotted in the form of bands of width 6 mm at 6 mm interval with Hamilton 100 μl syringe under a stream of nitrogen by means of a Camag Linomat IV. The mobile phase used was toluene: ethyl acetate: formic acid (7: 3.5: 0.5, v/v/v). Linear ascending development was performed in a twin-trough glass chamber previously saturated with mobile phase vapour for 20 min at room temperature and migration distance allowed was 80 mm. Densitometric scanning was performed using a Camag TLC scanner 3 in absorbance/reflectance mode at 335 nm operated by CATS software (V 3.15, Camag); using slit dimensions were 5 × 0.45 mm. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum in the range 190–400 nm.

Calibration:

A stock solution containing 1000 ng/μl BUM was prepared in methanol. Different volumes of this solution were applied to the plate resulting in application of 100 to 800 ng/spot to the plate. Each concentration was applied six times to the plate and the plate was developed as described above. Peak areas were plotted against corresponding concentrations to furnish the calibration plot.

Fig.1. Typical HPTLC chromatogram obtained from bumetanide (R_F0.45 ± 0.02)

Fig.2. HPTLC chromatogram obtained from acid-degraded bumetanide

METHOD VALIDATION:

Precision:

Intra-day and inter-day variation for determination of BUM was measured at three different concentrations (200, 400, and 600 ng/spot).

Robustness:

Small changes in the chromatographic conditions were introduced and the effects on the results were examined.

Limits of Detection and Quantification:

To determine the limits of detection and quantification, concentrations in the lower part of the linear range of the calibration plot were used. Stock solution of BUM (1000 μg/ml) was prepared and different volumes in the range 100 to 800 ng were applied in triplicate. Amounts of BUM per band were plotted against average response (peak area) and the regression equation was determined. The standard deviations (SD) of responses and the average standard deviations (ASD) were calculated. Detection limit was calculated as (3 × ASD)/b and quantification limit was calculated as (10 × ASD)/b, where ‘b’ denotes the slope obtained in the linearity study.

Fig.3. HPTLC chromatogram obtained from base-degraded bumetanide

Fig.4. HPTLC chromatogram obtained from hydrogen peroxide-degraded bumetanide

Specificity:

The specificity of the method was determined by analysis of drug standards and samples. The band for BUM in the sample was identified by comparing the R_F value and spectrum of the band with those of the band from a standard. The peak purity of BUM was assessed by comparing spectra acquired at three different positions on the peak, i.e. the peak start (S), peak apex (M), and peak end (E) positions of the band.

Recovery:

To check the recovery of the drug at different levels in formulations analysed samples were spiked with an extra 80, 100, and 120% of BUM standard and the mixtures were reanalysed by the proposed method. The experiment was conducted in triplicate.

Analysis of the Marketed Formulation:

To determine the BUM content of conventional tablets, twenty tablets were weighed and powdered in a glass mortar. An amount of powder equivalent to 1mg BUM was transferred to a 10 ml volumetric flask, extracted with methanol, sonicated for 30 min, and diluted to volume with same solvent. The solution was centrifuged at 3000 rpm for 5 min and the drug content of the supernatant was determined (100 μg/ml). The resulting solution was filtered through a 0.45-μm filter (Millifilter; Milford, MA, USA). The solution (5 μl, 500 ng BUM) was applied to a plate for assay of BUM and the BUM bands at R_F 0.45 were observed in the densitogram obtained from tablets. There were no interferences from the excipients commonly present in the tablets.

Table I. Linear regression data

Linear range (ng/spot)	100-800
r2	0.9996
Slope ± RSD	0.9987 ± 0.965
Intercept ± RSD	23.471 ± 1.24

Table II. Intra-day and Inter-day precision of the HPTLC method

Concentration (ng/spot)	Intra-day precision		Inter-day precision
Concentration (ng/spot)	Mean ± S.D, (ng/spot)	RSD (%)	Mean ± S.D, (ng/spot)	RSD (%)
200	196.34 ± 1.84	0.94	194.64 ± 2.04	1.04
400	395.33 ± 3.54	0.25	393.27 ± 2.64	0.67
600	594.27 ± 4.75	0.79	589.79 ± 5.64	0.94

Fig. 5. HPTLC chromatogram obtained from photo-degraded bumetanide

FORCED DEGRADATION OF BUM STANDARD:

A stock solution containing 10 mg BUM in 10 ml methanol was prepared. This solution was used for forced degradation to provide an indication of the stability-indicating property and specificity of the method. In all degradation studies the average peak area of BUM after application (100 ng/spot) of seven replicates was obtained after development and scanning of the plate as described above.

Acid and Base-Induced Degradation:

Initially 0.1N hydrochloric acid at 80°C for 8 hour was used but the degradation was not observed. Then 10-20 % degradation was observed by exposing drug solution with 1 N hydrochloric acid at 80°C for 2 h. The drug was found to undergo alkaline degradation slowly as compared with that in acid degradation. The reaction in 0.1N and 1N sodium hydroxide was used at room temp for 8 hour but since no degradation was observed, the strength of base was increased. Subsequently, studies were performed in 2 N sodium hydroxide at 80 °C for 24 h.

Hydrogen Peroxide-Induced Degradation:

The drug was found to undergo oxidative degradation slowly the drug was found to be stable in (6 %, v/v) H₂O₂ for 12 hour at room temperatures. Subsequently, studies were performed in (30 %, v/v) H₂O₂ for 48 hour at room temperature.

Photochemical 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 (5 µl) was prepared in methanol. Tablet was prepared in the same way. All samples for photostability testing were placed in a sun light for 4 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 sunlight, all samples were prepared for HPTLC analysis.

Dry Heat Degradation:

The BUM powdered drug was stored for 3 h under dry heat conditions at 50°C to see the effect of dry heat degradation. A solution of the treated powder was then prepared and applied to a plate in triplicate. The plate was then chromatographed and treated as described above.

RESULTS AND DISCUSSION:

HPTLC Method Optimization and Validation:

The TLC procedure was optimized to develop a stability-indicating method. Both pure drug and the degraded products were spotted on the plates and chromatographed with different mobile phases. Initially toluene– ethyl acetate in different ratios was tried. The mobile phase toluene: ethyl acetate: formic acid 7: 3.5: 0.5(v/v/v) enabled good resolution, and a sharp and symmetrical peak of R_F 0.45 ± 0.02 was obtained for BUM (Fig. 1) from a compact and non-diffuse band. It was observed that prewashing of TLC plates with methanol (followed by drying and activation) and pre-saturation of TLC chamber with mobile phase for 20 min (the optimum saturation time) ensured good reproducibility and peak shape of BUM.

VALIDATION:

Linearity and range:

The linear regression data for the calibration plots revealed a good linear relationship over the concentration range 100-800 ng/spot (correlation coefficient, r², 0.9996). There were no significant differences between slopes of standard curves. The results are shown in Table I.

Precision:

The precision of the method was expressed as relative standard deviation (RSD, %). The results obtained (Table II) revealed the high precision of the method.

Fig.6. HPTLC chromatogram of dry heat-degraded bumetanide

Robustness:

When the standard deviation of peak area was calculated for each change of conditions RSD was found to be less than 2%. These low RSD values (Table III) indicated the method is robust.

Recovery:

When the method was used for extraction and subsequent analysis of BUM from pharmaceutical dosage forms after spiking with80, 100, and 120% of additional drug, recovery was 98.88–99.09% (Table IV).

Limits of Detection and Quantification:

The limits of detection and quantification calculated as described above were 30 and 80 ng/spot, respectively. This indicates the sensitivity of the method is adequate.

The results obtained from validation of the method are summarized in Table V.

Assay of Marketed Formulation:

A single spot of R_F 0.45 was observed in chromatograms obtained from drug samples extracted from conventional tablets. There was no interference from the excipients commonly present in the tablets. The drug content was found to be 99.60 ± 0.654

%. It may, therefore, be inferred that degradation of BUM had not occurred in the marketed formulations analysed by this method. The low value of RSD indicates the method is suitable for routine analysis of BUM in pharmaceutical dosage forms.

Table V. Summary of validation data

Method characteristic	Value
Linear range (ng/spot)	100-800
Correlation coefficient	0.9996
Limit of detection (ng/spot)	30
Limit of quantification (ng/spot)	80
Recovery (n = 3)	98.82
Precision (%RSD)
Inter-day (n = 3)	0.88
Intra-day (n = 3)	0.66
Robustness	Robust
Specificity	Specific

ACCELERATED DEGRADATION:

Acid and Base-Induced Degradation:

The chromatograms obtained from BUM contained additional peaks at R_F 0.58 in the acid-degraded samples and at R_F 0.61 in the base-degraded samples (Fig. 2 and 3). The concentration of the drug was different from the initial concentration, indicating that BUM undergoes degradation under acidic and basic conditions.

Hydrogen Peroxide-Induced Degradation:

The chromatograms obtained from samples degraded with hydrogen peroxide (Fig. 4) contained an additional peak at R_F0.21. The spot of the degradation product was well resolved from that of the drug.

Photochemical Degradation:

The chromatograms obtained from photo-degraded samples contained an additional peak at R_F 0.32 (Fig. 5). Significant degradation was observed in standards left in daylight for 24 h.

Degradation by Dry Heat:

The chromatograms obtained from samples degraded by dry heat (Fig. 6) contained an additional peak at R_F 0.58. The spot of the degradation product was well resolved from the drug spot. These results indicate the drug is susceptible to acid/base hydrolysis, oxidation, and dry heat-induced degradation. The lower R_F values of the degradation components indicated they were less polar than BUM. The results from accelerated degradation studies are summarized in Table VI.

CONCLUSION:

The developed HPTLC technique is precise, specific, accurate and stability indicating. The developed method is able to discriminate between BUM and its possible degradation products. Statistical analysis proves that the method is suitable for the analysis of BUM as bulk drug and in pharmaceutical formulation without any interference from the excipients. The method can be used to determine the purity of drug available from various sources by detecting any related impurities. Because the method could effectively separate the drugs from their degradation products, it can be regarded as stability indicating.

ACKNOWLEDGEMENTS:

The authors are grateful to Lupin Pharmaceuticals Ltd, Pune (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.

REFERENCES:

1. Goodman LS, Gilman A, The Pharmacological Basis of Therapeutics, 9^th Edition, McGraw-Hill, New York, 1996, pp.701.

2. Florey K, Analytical Profiles of Drug Substances, Vol. 22, Academic Press, New York, 1995, pp. 109.

3. E.Gonzalez, A.Becerra, J.J.Laserna, Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection. J. Chromatogr. Biomed. Appl. 1996; 687: 145-150.

4. Gradeen CY, Billay DM, Chan SC, Analysis of bumetanide in human urine by high-performance liquid chromatography with fluorescence detection and gas chromatography/mass spectrometry. J Anal Toxicol. 1990; 14: 123-6.

5. Legorburu M.J. Alonso R.M, and Jimenez R.M, Ortiz E, Quantitative determination of loop diuretic bumetanide in urine and pharmaceuticals by high performance liquid chromatography with amperometric detection. J. Chromatogr. Sci. 2001; 39: 425-30.

6. Smith D.E, High performance liquid chromatographic assay of bumetanide in plasma and urine. J. Pharm Sci. 1982; 71: 520-3.

7. Gonzalez L, Lopez JA, Alonso RM, Jimenez RM, Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection. J Chromatogr. A 2002; 949: 49-60.

8. Zivanov-Stakic D, Solomun L.J. Zivanovic L.J. High-performance liquid chromatographic method for the determination of bumetanide in pharmaceutical preparations. J. Pharm. Biomed. Anal. 1989; 7: 1889-92.

9. ICH, Q2B Harmonized Tripartite Guideline, Text on Validation of Analytical Procedures Methodology, International Conference on Harmonization, Geneva, March 1996.

10. ICH Guidance on Analytical Method, International Convention on quality for the Pharmaceutical Industry, Toronto, Canada, September 2002.

11. Stability Testing of New Drug Substances and Products International Conference on Harmonization (ICH) Q1A (R2), IFPMA, Geneva, Switzerland 2003.

Received on 18.09.2009 Modified on 22.11.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 239-243

Label claim (mg/tablet)	Amount added (%)	Total amount (mg)	Amount recovered (mg)	Recovery (%)
1	80	3.6	1.78	98.88±0.57
	100	4.0	1.97	98.50±0.51
	120	4.4	2.18	99.09±0.98

Parameters	SD of peak area	RSD (%)
Mobile phase composition (± 0.1 ml)	7.1	1.4
Amount of mobile phase (± 0.5 %)	4.3	1.5
Time from spotting to chromatography (± 20 min)	2.2	0.62
Time from chromatography to scanning (± 20 min)	2.9	0.75

Chemicals and materials:

METHOD VALIDATION:

Acid and Base-Induced Degradation:

Precision:

Limits of Detection and Quantification:

Table V. Summary of validation data

ACCELERATED DEGRADATION:

Acid and Base-Induced Degradation:

Photochemical Degradation:

CONCLUSION:

REFERENCES:

1. Goodman LS, Gilman A, The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill, New York, 1996, pp.701.

1. Goodman LS, Gilman A, The Pharmacological Basis of Therapeutics, 9^th Edition, McGraw-Hill, New York, 1996, pp.701.