INTRODUCTION:

Due to the increasing expansion of pharmaceutical businesses and medication production across the globe, there is a greater need for novel analytical techniques in the pharmaceutical sector.¹ Creating a framework for procedures that record and verify the identification, quality, and purity of drug items is the fundamental aim of validation.²

Chemical structure of Acetazolamide

Acetazolamide is carbonic anhydrase inhibitor. Acetazolamide is a drug used to treat heart failure, idiopathic intracranial hypertension, periodic paralysis, and urine alkalinization. It is also diuretic, anticonvulsants, antiglaucoma. ^{3, 4}

The major goal is to create a new method using UV and HPLC for drug Acetazolamide. This technique can also be used to analyse drugs and their dosage forms. At the formulation development stage, the new approach we will create can be employed to detect contaminants. By using the least amount of organic solvent possible, an economical strategy was developed in this study, which decreased the cost of the procedure.

EXPERIMENTAL WORK:

Method-

Standard stock solution of acetazolamide by UV-visible spectrophotometry-

10.0mg of pure acetazolamide drug sample was precisely weighed, moved to a 10ml volumetric flask, and the medication was dissolved with prepared 5:5 solution of distilled water and ethanol and sonicate for 15 min.

Selection of wavelength for analysis of acetazolamide- From the above solution 0.1ml of standard stock solution of acetazolamide was transferred into 10ml of volumetric flask and diluted to a mark with a prepared 5:5 solution of distilled water and ethanol to give a concentration of 2μg/ml, 4μg/ml, 6μg/ml, 8μg/ml, 10μg/ml. The final product was scanned between 200 and 400nm in the UV spectrum. In the spectrum acetazolamide showed an absorbance maximum at 265nm.

Preparation of stock solution of tablet formulation- Twenty tablets of Diamox containing 23.48mg of acetazolamide was weighed and finely powdered separately. Powder equivalent to 10µg/ml was weighed and transferred into 10ml of volumetric flask and diluted to a mark with prepared 5:5 solution of distilled water and ethanol.

HPLC method-

SOP for washing HPLC columns-

100ml triple distilled water for 20 minutes.

25ml ethanol and 75ml triple distilled water for 20 mins.

50ml ethanol and 50ml triple distilled water for 20 mins.

75ml ethanol and 25ml triple distilled water for 20 mins.

100ml ethanol 20 mins.

Analysis wavelength selection-

The preparation of different concentrations of acetazolamide required the proper dilution of the standard stock solution with the mobile phase. The solution was scanned between 200 and 400nm. At 265nm, the final wavelength chosen for investigation, the medication had considerable absorption. As a result, 265nm was chosen as the detection wavelength for the RP-HPLC method of drug quantification.

Mobile phase selection-

For mobile phase selection, a variety of mobile phases in a range of ratios were tested. Varied mobile phases were injected into acetazolamide at various flow rates until a strong peak without any interference peak-containing spectrum was attained. A ratio of 80:20 between acetonitrile and water produced positive results, with a retention time of 2.044.

Mobile phase preparation-

Acetonitrile (80.0ml) was combined with water (20.0ml).

The mobile phase's degassing-

The mobile phase was degassed in an ultrasonicator for 10 minutes to prevent the disturbance caused by dissolved gases.

Mobile phase filtration-

To get rid of any minuscule particles that might be in the mobile phase and lead to column obstruction, the mobile phase was degassed and then filtered using a 0.45m membrane nylon filter.

Preparation of standard stock solution-

100mg of acetazolamide was weighed and dissolved in ethanol in a 50ml volumetric flask. Pipette out 5ml of stock solution and dissolve with 100ml of distilled water.

Mobile phase loading-

Before being poured into the vial, the mobile phase underwent filtering and degassing. Every newly manufactured mobile phase had been prepped.

Stabilization of baseline-

The detector was turned on for an hour prior to the actual run in order to get consistent UV light. At the proper flow rate, the mobile phase run was started and continued until a stable baseline was established.

Sample loading-

After being carefully prepared and filtered, acetazolamide was loaded and administered.

Cleaning the column-

Following the completion of the sample analysis, the column was cleaned by flushing with the mobile phase for 30 minutes, followed by an hour of a 1:1 combination of triple distilled water and methanol.

Procedure for tablet formulation analysis-

Twenty tablets, each containing 250mg of acetazolamide, were taken. The tablets were broken down into a fine powder, and 235mg of acetazolamide were measured out and added to a volumetric flask with a capacity of 50ml. After pouring enough ethanol to entirely vanish the contents, the container was shaken for 20 mins. Pipette out 5 ml of the stock solution, then dissolve it in 100ml of pure water that has been membrane-filtered to remove any gas. To obtain a chromatogram, the solution was put into the vial and stored ready to be injected into the apparatus.

RESULT AND DISCUSSION:

UV method-

Fig. 1- Absorption of acetazolamide at 265nm

Method validation-

Linearity

Fig. 2- Calibration of acetazolamide by UV

Table No. 1- Calibration curve of UV

Sr. No.	Conc.	Abs.
1.	2	0.101
2.	4	0.208
3.	6	0.295
4.	8	0.39
5.	10	0.501

Precision-

Intraday-

Table No. 2- Intraday variability of acetazolamide

Sr. No.	1.	2.	3.	4.
Actual conc. (ppm)	10	10	10	10
Abs.	0.507	0.502	0.509	0.499
Conc. Found (ppm)	10.23	10.12	10.27	10.06
Mean	10.17
±SD	0.093
% RSD	0.917

Interday-

Table No. 3- Interday variability of acetazolamide

Sr. No.	1.	2.	3.
Actual conc. (ppm)	10	10	10
Abs.	0.498	0.501	0.506
Conc. Found (ppm)	10.04	10.10	10.20
Mean	10.11
±SD	0.082
% RSD	0.815

Accuracy-

Table No. 4- Recovery study of acetazolamide by UV

Sr. No.	1.	2.	3.
Abs.	0.901	0.923	0.918
Conc. Found (18 ppm)	18.19	18.64	18.54
Mean	18.45
±SD	0.233
% RSD	102.55
Abs.	1.012	1.016	1.022
Conc. Found (20 ppm)	20.44	20.52	20.64
Mean	20.53
±SD	0.101
% RSD	102.68
Abs.	1.122	1.125	1.129
Conc. Found (22 ppm)	22.67	22.73	22.81
Mean	2273
±SD	0.071
% RSD	103.35

Limit of Detection-

3.3 X S.D.

LOD = ----------------------- = 0.60

Slope

Limit of Quantitation-

10 X S.D.

LOQ = -----------------------

Slope = 1.82

DISCUSSION:

On a Jasco V-730 spectrophotometer, the aforementioned method for estimating acetazolamide was used, with ethanol: distilled water (5:5) serving as the solvent. The maximum drug absorbance at 265nm was measured while scanning standard drug solutions. For acetazolamide, the recovery analysis research indicates recoveries of 102.55%, 102.68%, and 103.35% for various concentrations of the spiked medication. The findings of the determination of linearity at various concentrations showed that the linearity requirements for acetazolamide were satisfied in the ranges of 2–10µg/ml. Using the calibration curve graph as a guide, the LOD and LOQ for acetazolamide were found to be 0.60µg/ml and 1.82µg/ml, respectively.

HPLC method

Method validation-

Linearity-

Fig. 3- Calibration of acetazolamide by RP-HPLC

Sr. No.	Conc.	Response (Area)
1.	20	704.47
2.	40	1376.66
3.	60	2243.21
4.	80	2911.49
5.	100	3491.18

Precision-

Table No. 5- Precision values

Sr. No.	Area	Mean	±SD	% RSD
1.	3456.82	3445	41.1	1.2
2.	3496.25
3.	3467.36
4.	3398.56
5.	3408.36

Accuracy-

Table No. 6- Recovery study of acetazolamide by RP-HPLC

Sr. No.	80%	Mean	±SD	%RSD
1.	2934.23	2933.68	20.85	0.710
2.	2954.26
3.	2912.56

Sr. No.	100%	Mean	±SD	%RSD
1.	3452.65	3430.60	20.30	0.591
2.	3412.69
3.	3426.47

Sr. No.	120%	Mean	±SD	%RSD
1.	3860.27	3852.051	13.45	0.349
2.	3836.52
3.	3859.35

Fig. 4- Chromatogram of working standard containing acetazolamide

Fig. 5- Chromatogram of the marketed formulation containing acetazolamide

Table No. 7- Analysis of acetazolamide

Sr. No.	Average weight (mg)	Sample weight (mg)	Label claim (mg)	Sample area	Assay (% w/w)
1.	588.30	235.46	250.00	3392.48	98.6
2.	588.30	236.42	250.00	3402.35	98.5
3.	588.30	234.96	250.00	3396.75	98.9

Table No. 8- Details of working standard containing acetazolamide

Drug

(mins)

Area

(µV.sec)

Height

Acetazolamide

2.044

2378.68

348.32

Limit of Detection-

3.3 X S.D.

LOD = ------------------------ = 123.64

Slope

Limit of Quantitation-

10 X S.D.

LOQ = ------------------------- = 374.66

Slope

DISCUSSION:

For quantifying acetazolamide in bulk and tablet dosage form, an RP-HPLC method was created and validated with mobile phase acetonitrile: water (80:20). For acetazolamide, the assay (% w/w) was found 98.6 %, 98.5 %, 98.9 %. The flow rate was 1ml/min. The sample volume was 20.00µl. Run time 5min and retention time was 2.044. The calibration curve graph was used as a guide, the LOD and LOQ for acetazolamide were found to be 123.64µg/ml and 374.66µg/ml, respectively.

SUMMARY AND CONCLUSION:

UV method selection and optimization-

Table No. 9- Parameters of UV spectrophotometer

Validation parameters	Acetazolamide
Solvent	Ethanol and distilled water
Detection wavelength	265nm
R²	0.999
LOD	0.60µg/ml
LOQ	1.82µg/ml
Precision	% RSD<2
Recovery	102-104

HPLC method selection and optimization-

Table No. 10- Parameters of HPLC method

Parameters	Chromatographic
HPLC system	Agilent
Pump	Reciprocating quaternary pump
Detector	UV
Column	C18, waters (250X4.6mm) 5µ
Column temperature	30°c
Mobile phase	Acetonitrile: Water
Concentration of standard solution	100µg/lml
Detection of wavelength	265nm
Flow rate	1ml/min
Sample volume	20.00µl
Run time	5min
Retention time	2.044

REFERENCES:

1. Ravisankar P, Gowthami S, Rao GD. A review on analytical method development. Indian Journal of Research in Pharmacy and Biotechnology. 2014 May 1; 2(3): 1183.

2. Reddy PS, Dayakar M, Swathi P, Srinivasulu D, Reddy NV, Akhila G et al. Method Development and Validation of Acetazolamide by Using UV Spectroscopy. International Journal of Engineering Science and Computing. 2020; 10(6): 26146.

3. Indian Pharmacopoeia, Volume-II 2014, Pg No: 985

4. https://en.wikipedia.org/wiki/Acetazolamide

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Received on 09.04.2024 Modified on 25.04.2024

Asian J. Pharm. Ana. 2024; 14(2):60-64.

DOI: 10.52711/2231-5675.2024.00011

Linearity

Sr. No.

Average weight (mg)

Sample weight (mg)

Label claim (mg)

Sample area

Assay

(% w/w)

1.

588.30

235.46

250.00

3392.48

98.6

2.

588.30

236.42

250.00

3402.35

98.5

3.

588.30

234.96

250.00

3396.75

98.9