INTRODUCTION:

Bumetanide¹ is chemically 3-(Butylamino)-4-phenoxy-5-sulfamoyl benzoic acid. The molecular weight of Bumetanide (C₁₇H₂₀N₂O₅S) is found to be 364.417 grams/mole. Bumetanide is a loop diuretic¹ of the sulfamyl category to treat heart failure and also in children for the treatment of core symptoms of autism spectrum disorder^2-3. It is indicated for the treatment of edema associated with congestive heart failure, hepatic and renal disease, including the nephrotic syndrome. Bumetanide interferes with renal cAMP and/or inhibits the sodium-potassium ATPase pump. Bumetanide appears to block the active reabsorption of chloride and possibly sodium in the ascending loop of Henle, altering electrolyte transfer in the proximal tubule. It is orally, or by whose therapeutic action generally begin within an hour and lasts for about six hours.

Figure 1: Chemical structure of Bumetanide

Bumetanide was earlier studied by different analytical techniques such as LC-MS/MS^4-5, HPTLC⁶, flow injection with fluorometric detection⁷, RP-UPLC⁸, RP-HPLC with fluorometric detection^9-10, RP-HPLC with PDA detection^11-12, RP-HPLC with amperometric detection¹³, RP-UFLC¹⁴ and spectrophotometry^15-16 in biological fluids as well as formulations.

In the present study a new stability indicating HPLC and LC-APCI-MS methods have been proposed for the quantification of Bumetanide and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Instrumentation and chromatographic conditions

HPLC conditions

TSQ scientific Quantis LCMS with Thermo Vanquish model HPLC with PDA detector and Simpack C18 (250 mm x 4.6 mm x 5µm) column was employed for the present study. The injection volume was 10 µL and the total run time was 25 mins (Detection wavelength 220 nm). 0.1% Formic acid: Acetonitrile was used as mobile phase on gradient mode with flow rate was 1 ml/min.

Mass Spectrophotometer conditions

Ion Source type : APCI

Spray Voltage : Static

Positive Ion discharge current (V) : 4

Negative Ion discharge current (V) : 10

Sheath Gas (Arb) : 45

Aux Gas (Arb) : 10

Sweep Gas (Arb) : 2

Ion transfer tube temperature : 275

Vaporizer temperature : 400

Scan mode : Full scan Q1

Scan Range : 50-2000 m/z (Positive mode)

: 50-2000 m/z (Negative mode)

Preparation of stock solution

50 mg of Bumetanide API was accurately weighed and carefully transferred into a 25 ml volumetric flask and was dissolved in HPLC grade Acetonitrile (1000 µg/ml) and the resulting solution was sonicated for 30 mins. Acetonitrile is used as diluent.

Method validation¹⁷

5.0-200 µg/ml Bumetanide solutions were prepared from the stock solution (1000 µg/ml) on dilution with the mobile phase consisting of a mixture of 0.1% Formic acid and Acetonitrile and each solution was injected (n=3) into the LC system and the average peak area from the respective chromatograms was calculated. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area of the chromatograms on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the inter-day precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) and the % RSD was calculated. Accuracy studies were performed by spiking the formulation solution with 50, 100 and 150% API solution and thereby the percentage recovery was calculated with the help of regression equation. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of Bumetanide

Bumetanide is available as tablets with brand names Bumex (Label claim: 0.5 mg; Genentech, Inc.) (Label claim: 1.0 mg; Validus pharmaceuticals LLC) Burinex (Label claim: 2.0 mg; Leo pharma), Edemex (Label claim: 1.0 mg) in India. Two different brands of Bumetanide were collected and extracted with acetonitrile and after sonication diluted with the diluent as per the requirement. The resulting solution was filtered through 0.24 μm membrane filter and 10 μL of these formulation solutions were injected in to the HPLC system. The peak area of the chromatogram (n =3) was noted and the percentage purity was determined.

Stress degradation studies¹⁸

During the acidic degradation study Bumetanide solution was treated with 0.1N HCl and immediately neutralized with 1ml 0.1N NaOH solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded. During the thermal degradation study Bumetanide solution was heated at 60ºC and the contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded. During the basic degradation study Bumetanide solution was treated with 0.1N NaOH for about 30 mins and then neutralized with 1ml 0.1N HCl solution. The contents were diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded. During the oxidative degradation study Bumetanide solution was treated with hydrogen peroxide for about 30 mins and then diluted with mobile phase and the resultant solution was injected into HPLC and LC-MS system and the peak area as well as the mass spectrum was recorded.

RESULTS AND DISCUSSION:

A new stability-indicating RP-HPLC and LC-APCI-MS methods have been developed for the quantification of Bumetanide. The earlier reported methods were discussed with the present proposed method and the details were given in Table 1.

Table 1: Table 1: Literature survey

Method	Mobile phase (v/v)	λ (nm)	Linearity (μg/ml)	Comment	Reference
LC–ESI-MS/MS (Human plasma)	Methanol : 5mM aq. ammonium trifluoroacetate	-		Tamsulosin as internal standard	4
LC–ESI-MS/MS (Serum and brain tissue)	-	-	0.001-1.25	-	5
HPTLC	Toluene: Ethyl acetate: Formic acid (7 : 3.5 : 0.5)	335	100-800 ng/spot		6
Flow injection		Exicitation (314) Emission (370)	0.05-10	Fluorimetric detection	7
RP-UPLC	Water: Acetonitrile (30: 70)	254	12.5-75	PDA	8
RP-HPLC	Methanol :water: glacial acetic acid (66:34:1)	Exicitation (228) Emission (418)	0.005-2.0	Fluorimetric detection (In plasma and urine)	9
RP-HPLC (Plasma and Urine)		Exicitation (338) Emission (433) 254		Fluorimetric detection and UV detection (Acetophenone as Internal standard)	10
RP-HPLC	Methanol: Water (70: 30)	335	1.0-10	Low linearity	11
RP-HPLC	Phosphate buffer (pH=7.8): Acetonitrile (70:30)	216	0.6-1.6	Low linearity	12
RP-HPLC (Urine)	Acetonitrile: KH₂PO₄ buffer (pH 4.0) (50:50)	0.050-0.499	-	Amperometric detection	13
RP-UFLC	Acetonitrile: Water: Glacial acetic acid (80: 20: 0.1)	220	0.1-100	High linearity	14
RP-HPLC and LC-APCI-MS	0.1% Formic acid: Acetonitrile	220	5.0-200	High linearity	Present method

Linearity, Precision, accuracy and robustness

Bumetanide obeys Beer-Lambert’s law over the concentration range 5.0-200 µg/ml (Table 3) and the linear regression equation was found to be y = 279.3x + 36.286 (R² = 0.9998) Figure 3). The LOD and LOQ values were found to be 1.5719 µg/ml and 4.7451 µg/ml respectively.

The % RSD in intraday precision (0.633), interday precision (0.0157-0.3517) (Table 4) was found to be less than 2.0% stating that the method is precise. In the accuracy study the % RSD was found to be 0.31-0.81 (<2) (Table 5) with a recovery of 98.93-99.86 indicating that the method is accurate.

Table 3: Linearity

Conc. (µg/ml)	*Mean peak area
0	0
5	1412.012
10	2823.102
25	7027.157
50	14059.613
75	21122.689
100	28121.339
150	41315.347
200	56211.891

*Mean of three replicates

Figure 3: Calibration curve

Table 4: Precision study

Intraday precision study
Conc. (µg/ml)	Mean peak area	*Mean peak area ± SD (% RSD)
10	2823.102	2840.657 ± 17.975 (0.633)
10	2847.215
10	2851.257
10	2871.854
10	2821.548
10	2828.967

Table 5: Accuracy study

Spiked conc. (µg/ml)	Formulation (µg/ml)	% Recovery	% RSD
25 (50 %)	50	99.81	0.31
50 (100 %)	50	99.74	0.54
125 (150 %)	50	99.73	0.81

*Mean of three replicates

Assay of Bumetanide

The assay of Bumetanide intravenous injection was performed using the proposed liquid chromatographic method with the optimized chromatographic conditions. The percentage of purity of Bumetanide was found to be 98.93-99.86 (Table 6).

Table 6: Assay of Bumetanide

S. No.	Brand name	Label claim (mg/ml)	Observed amount (mg/ml)*	% Recovery*
1	Brand I	1	0.9912	99.12
2	Brand II	1	0.9893	98.93
3	Brand III	2	1.9972	99.86

*Mean of three replicates

Stress degradation studies

Bumetanide (100 µg/ml) was exposed to different stress conditions under the optimized chromatographic conditions and then injected in to the system. During the acidic degradation, Bumetanide was eluted at Rt 13.967 min and about 0.20 % has undergone decomposition. The mass spectra at Rt 14.09 min and 14.12 min were shown in Figure 4.

Figure 4: Representative chromatograms and mass spectra of Bumetanide during acidic degradation

During the thermal degradation, Bumetanide was eluted at Rt 13.967 min and about 0.33 % has undergone decomposition. The mass spectra at Rt 14.12 min and 14.16 min were shown in Figure 5.

Figure 5: Representative chromatograms and mass spectra of Bumetanide during thermal degradation

During the basic degradation, Bumetanide was eluted at Rt 13.967 min and about 62.28 % has undergone decomposition. Bumetanide is chemically 3-(Butyl amino)-4-phenoxy-5-sulfamoyl benzoic acid and therefore the benzoic acid moiety may be responsible for the major degradation of Bumetanide. The mass spectra at Rt 14.06 min and 14.09 min were shown in Figure 6.

Figure 6: Representative chromatograms and mass spectra of Bumetanide during basic degradation

During the oxidative degradation, Bumetanide was eluted at Rt 13.967 min with a degradant peak at 13.267 min and about 10.11 % has undergone decomposition. The mass spectra at Rt 14.12 min and 14.16 min were shown in Figure 7.

Figure 7: Representative chromatograms and mass spectra of Bumetanide during oxidative degradation

The details of the stress degradation studies of Bumetanide were shown in Table 7. It is observed that Bumetanide is highly sensitive towards alkaline conditions (62.28% degradation) and less than 15% degradation was observed in other stress conditions such as acidic degradation (0.20%), thermal degradation (0.33%) and oxidative degradation (10.11%).

Table 7: Stress degradation studies

Condition	R_t (min)	Mean peak area*	% Recovery*	% Drug degradation
Standard drug	13.967	28121.339	100	-
Acidic hydrolysis	13.967	28064.880	99.80	0.20
Thermal degradation	13.967	28029.091	99.67	0.33
Alkaline hydrolysis	13.967	10606.123	37.72	62.28
Oxidative degradation	13.267	25279.485	89.89	10.11

*Mean of three replicates

CONCLUSION:

The authors have established a new stability indicating RP-HPLC as well as LC-MS method coupled with APCI and triple quadrupole analyser for the estimation of Bumetanide. The method is simple, precise and accurate and used for the routine analysis of Bumetanide in pharmaceutical formulations and no interference of excipients was observed during the assay.

ACKNOWLEDGEMENT:

The authors are grateful to Mylon Laboratories (India) for providing the gift samples of Bumetanide and the authors declare no conflict of interest.

REFERENCES:

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Received on 22.06.2023 Modified on 17.07.2023

Research J. Pharm. and Tech 2023; 16(8):3809-3817.

DOI: 10.52711/0974-360X.2023.00629

Interday precision study
Conc. (µg/ml)	Day 1	Day 2	Day 3	Mean peak area ± SD (% RSD)*
10	2823.102	2847.325	2832.547	2834.325 ± 9.969 (0.3517)
50	14059.613	14087.269	14101.584	14082.822 ± 17.421 (0.1237)
100	28121.339	28131.547	28129.457	28127.448 ± 4.403 (0.0157)

Time (minutes)	Mobile phase A%	Mobile phase B%
0.0	70	30
5.0	70	30
15.0	5	95
20.0	5	95
20.1	70	30
25.0	70	90