1. INTRODUCTION

Validation is defined as "documented evidence which gives a high degree of confidence that a process, system, facility will consistently produce a product meeting its predetermined specifications and quality attributes.¹

Method validation:

Method validation is the process of providing an analytical method that is acceptable for its intended purpose. For pharmaceutical methods, guidelines from the International Conference on Harmonization (ICH)² and the Food and Drug Administration (FDA) provide a framework for performing such validations.

In general, methods for regulatory submission must include studies on specificity, linearity, accuracy, precision, range, detection of limit, quantification of limit, and robustness.³

1.1Accuracy:

Accuracy is the measure of exactness of an analytical method, or the closeness of an agreement between the values, which is accepted either as a conventional, true value or an accepted reference value and the value found. It is measured as the percent of analyte recovered by assay, by spiking samples in a blind study. For the assay of the drug substance, accuracy measurements are obtained by comparison of the results with the analysis of a standard reference material, or by comparison to a second, well-characterized method. For the assay of the drug product, accuracy is evaluated by analyzing synthetic mixtures spiked with known quantities of components.⁴

1.2 Precision:

Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the percent relative standard deviation for a statistically significant number of samples. According to the ICH, precision should be performed at three different levels: repeatability, intermediate precision, and reproducibility. Repeatability is the results of the method operating over a short time interval under the same conditions (inter-assay precision). It should be determined from a minimum of nine determinations covering the specified range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration. Intermediate precision is the result from within lab variations due to random events such as different days, analysts, equipment, etc.⁵

1.3 Specificity:

Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix. It is a measure of the degree of interference from such things as other active ingredients, excipients, impurities, and degradation products, ensuring that a peak response is due to a single component only i.e. that no co- elution exist. Specificity is measured and documented in a separation by the resolution, plate count (efficiency), and tailing factor. ICH also uses the term specificity, and divides it into two separate categories: identification, and assay/impurity tests.⁶

1.4 Limit of Detection:

The limit of detection (LOD) is defined, as the lowest concentration of an analyte in a sample that can be detected but not quantified. It is a limit test that specifies whether or not an analyte is above or below a certain value. It is expressed as a concentration at a specified signal-to-noise ratio, usually two-or three-to-one. LOD's may also be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve ( S) at levels approximating the LOD according to the formula.^7,8

LOD = 3. 3(SD/S)

1.5 Limit of Quantitation:

The Limit of Quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. Like LOD, LOQ is expressed as concentration, with the precision and accuracy of the measurement also reported. The calculation method is again based on the standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula:⁹

LOQ = 10(SD/S)

1.6 Linearity and Range:

Linearity is the ability of the method to elicit test results that are directly proportional to the analyte concentration within a given range. Linearity is generally reported as the variance of the slope of the regression line. Range is the interval between the upper and lower levels of analyte (inclusive) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. For assay, the minimum specified range is from 80-120% of the target concentration. For content uniformity testing, the minimum range is from 70-130% of the test or target concentration.

1.7 Ruggedness:

Ruggedness, according to the USP, is the degree of reproducibility of the results obtained under a variety of conditions, expressed as %RSD. These conditions include different laboratories, analysts, instruments, reagents, days, etc. In the guideline on definitions and terminology, the ICH did not address ruggedness specifically.¹⁰

1.8 Robustness:

Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters. The robustness of a method is evaluated by varying method parameters such as percent organic, pH, flow rate, temperature, etc., and determining the effect (if any) on the results of the method. As documented in the ICH guidelines, robustness should be considered early in the development of a method. In addition, if the results of a method or other measurements are susceptible to variations in method parameters, these parameters should be adequately controlled and a precautionary statement included in the method.¹¹

2. MATERIAL AND METHOD:

List of Instruments and chemicals:

Table 1 List of Apparatus/Instruments used:

Equipments	Company
Maxia220 electronic balance	Shinko Denshi Co Ltd, Japan
UV 150-02, Visible double beam spectrophotometer	Shimadzu corporation, Japan
Digital P^HMeter	Global, Ltd. Model No:-PGB100
Sonicator	Wenser
HPLC Binary Gradient System Model No:-3000 series HPLC pump : P-3000-M Reciprocating Column: Cosmosil C-18 (4.6 ID×250mm, Particle size 5µ) Detector- UV 3000	Analytical Technologies Ltd.
Analytical Balance Model No. PGB 100	Wenser

Table 2: List of Chemicals and Active Pharmaceutical ingredient used:

Name	Manufacturer/Supplier
Methanol	Merck
Purified Water (MilliQ)	In House Production
Irbesartan	Macleods pharmaceutical. Ltd. Sarigram Gujarat

Preparation of Mobile phase:

Prepare mixture of 80 volume of Methanol and 20 volume of Water mix well and degassed it by sonication.

Preparation of sample solution:

10mg Irbesartan tablet sample was accurately weighed and transferred it into a 100ml volumetric flask, diluents was added and sonicated for 5to10min to dissolve. The concentration become 100μg/ml. of Irbesartan. From that 6 and 8ml taken and diluted to 10ml the drug completely and finally the volume was made with diluents and mixed. The conc of Irbesartan was 60 and 80μg/ml.

Preparation of Standard stock solution:

10mg working standard Drug was accurately weighed and transferred it into a 100ml volumetric flask, diluents was added and sonicated for 5to10 min to dissolve. The concentration becomes 100μg/ml of Irbesartan. From that 6 and 8ml taken and diluted to 10ml the drug completely and finally the volume was made with diluents and mixed. The conc. of Irbesartan was 60 and 80μg/ml.

Loading of Mobile phase:

Filtered and degassed mobile phase was loaded in the reservoir. Priming was done for each freshly prepared mobile phase.

Baseline stabilization:

The detector was turned on for an hour before the actual run in order to obtain the stable UV light. The mobile phase run was started at desired flow rate and the run was continued until the stable baseline was obtained.

Loading of Samples:

Well prepared and filtered samples of Irbesartan were loaded into the Rheodyne injector port using a syringe and the sample was then injected.

Washing the column:

Once the analysis of samples was finished, the column was first washed by flushing with the mobile phase for half an hour.

3) RESULTS AND DISCUSSION:

VALIDATION OF THE DEVELOPED RP-HPLC METHOD:

3.1) Specificity:

Acceptance criteria:

It should pass the peak purity criteria.

Results:

No interference from any of the excipients was found at retention times of the examined drugs. These results demonstrate the absence of interference from other materials in the pharmaceutical formulations and therefore confirm the specificity of the proposed Method

Table No.3: Specificity data for proposed HPLC method.

Drug	Area	Amount added (mg)	Amount recovered (mg)	Percent recovery (%)	SD	RSD
Standard Drug of Irbesartan	2867513	10	9.95	99.5	1.19	1.20
Standard Drug of Irbesartan	2322410	10	9.94	99.4	1.19	1.20
Tablet Sample of Irbesartan	2109074	10	9.92	99.2	1.70	1.72
Tablet Sample of Irbesartan	2000558	10	9.99	99.9	1.70	1.72

3.2) Precision:

Acceptance criteria:

The Relative Standard Deviation should not be more than 2% for test result.

Results and Discussions:

The RSDs for intra-day and inter-day precision were not more than 2.0% for Standard Drug of Irbesartan and Tablet Sample of Irbesartan. The low RSD values indicate the repeatability and reproducibility of the method. Therefore, as per the ICH guidelines, this HPLC method for the determination of Standard Drug of Irbesartan and Tablet Sample of Irbesartan was precise.

3.3) Linearity:

Acceptance Criteria:

· The plot should be linear passing through the origin.

· Correlation Coefficient should not be more than 0.999.

Results and Discussions:

The calibration curves exhibited linear relationship of peak area to concentration in the range 60-100µg/mL for Standard Drug of Irbesartanand 60-100µg/mL for Tablet Sample ofIrbesartan. The regression coefficients (r²) for Standard Drug of Irbesartanand Tablet Sample of Irbesartan were 0.997 and 0.990, respectively, maintaining good correlation close to unity. The graph of concentration Vs Average area was plotted which is showing straight line passing through all points. So as per ICH guidelines, the proposed HPLC method for the determination of Standard Drugof Irbesartan and Tablet Sample of Irbesartanwas found to be linear.

Table No.5: Data for Standard Drug of Irbesartan.

Sr. No.	Conc. ug/ml	Area
Sr. No.	Standard Drug	Standard Drug	Tablet Sample
1.	60	1874532	1612912
2.	70	2159314	2048572
3.	80	2535040	2438745
4.	90	2910603	2735040
5.	100	3210538	3015112

Fig. 1: Linearity Graph for Standard Drug of Irbesartan

3.4) Accuracy:

Acceptance Criteria:

The Relative Standard Deviation should not be more than 2.0%.

Results and discussions:

Accuracy was checked with standard drugs by placebo spiking method at three different concentration levels (multi-level recovery). Recovery of standard drugs added was found to be 99.49-100.10% for Standard Drug of Irbesartan and 97.20-99.45% for Tablet Sample of Irbesartan with the value of RSD less than 2% indicating that the proposed method is accurate for the estimation ofStandard Drug of Irbesartan and Tablet Sample of Irbesartanfrom their combination drug products in presence of their degradation products and excipients.

Fig. 2: Linearity Graph for Tablet Sample of Irbesartan

3.5) Ruggedness:

Acceptance criteria:

· % RSD between the test result obtained should not be more than 2% for assay method.

Results and Discussions:

To evaluate the ruggedness of the proposed RP-HPLC method, the analysis was performed by different analysts and employing different brands of chemicals and solvents. Overall RSD for results obtained from different analysts are within limits. Therefore, the HPLC method for the determination of Standard Drug of Irbesartan and Tablet Sample of Irbesartan was found to be Rugged.

3.6) Robustness:

Acceptance criteria:

· % RSD should be NMT 2.0%.

Results and Discussions:

Results of the robustness study showed that the elution order and resolution for both components were not significantly affected. RSD of both components were examined and found to be well within the limit of 2.0%. The plate count and asymmetry factor was well within the acceptable USP limits, ensuring that the proposed method was robust and was capable of providing data of acceptable quality.

Table No.6: Accuracy Data of Standard Drug of Irbesartan and Irbesartan Tablet.

Drug	Conc. ug/ml	Area	Amount added (mg)	Amount recovered (mg)	Percent recovery (%)	SD	% RSD
Std. Drug of Irbesartan	0 60	2639654	10	9.95	99.5	0.91	0.92
	0 80	2867513	10	9.85	98.5
	100	3497122	10	10.12	100.1
Tablet Sample of Irbesartan	60	2113076	10	9.88	98.8	1.12	1.14
	80	2009078	10	9.77	97.7
	100	1822153	10	9.96	99.6

Table No. 7: Ruggedness Data of Standard Drug of Irbesartan and Tablet Sample of Irbesartan.

Drug	Area	Amount added (mg)	Amount recovered (mg)	Percent recovery (%)	SD	% RSD
Std Drug of Irbesartan.	2327816	10	9.93	99.94	0.0424	0.0425
Std Drug of Irbesartan.	2329450	10	10	100	0.0424	0.0425
Tablet Sample of Irbesartan.	2195713	10	9.85	99.85	0.0141	0.0142
Tablet Sample of Irbesartan.	1996358	10	9.90	99.87	0.0141	0.0142

3.7) Limit of Detection and Quantitation:

The limits of detection (LOD) and quantification (LOQ) were determined separately, on the basis of the standard deviation of the y intercept and slope of the calibration plots. The LOD were 0.074ug/ml for Standard Drug of Irbesartan. The LOQ were found to be 0.24ug/ml of Standard Drug of Irbesartan respectively. At these levels, RSD values were less than 2%, in accordance with ICH guidelines.

4. CONCLUSION:

Method Development and validation of Irbesartan by RP-HPLC is accurate, precise, robust, and specific. The method has been found to be better than previously reported method, because of its less retention time, use of an economical and readily available mobile phase, UV detection and better resolution of peaks. The run time is relatively short, which will enable rapid quantification of many samples in routine and quality-control analysis of various formulations containing Irbesartan. All these factors make this method suitable for quantification of Irbesartan in bulk drugs and in pharmaceutical dosage forms without any interference. The Results undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective and specific.

5. REFERENCES:

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Received on 26.11.2022 Modified on 18.09.2023

Asian J. Pharm. Ana. 2024; 14(1):6-10.

DOI: 10.52711/2231-5675.2024.00002

Drug	Time		Conc.	Area		Resolution	T.Plate		Asymmetry
	Intra-day	Inter-day	Intraday and Interday	Intra-day	Inter-day	Intraday and Interday	Intra-day	Inter-day	Intra-day	Inter-day
Std. Drug	3.287	3.278	60ug/ml	2329410	2335654	00	9245	9220	1.26	1.25
Tablet Sample	3.279	3.280	80ug/ml	2327221	2113070	00	8220	8220	1.24	1.23

Drug	Robustness Test ug/ml	Area	Amount added (mg)	Amount recovered (mg)	Percent recovery (%)	SD	% RSD
Standard Drug of Irbesartan	60	2327476	10	9.93	99.93	0.34	0.36
	70	2328997	10	10	100
	80	2330594	10	10	100
	90	2340641	10	10.3	100
Tablet sample of Irbesartan	60	2106135	10	10.2	100.2	1.98	1.87
	70	1900202	10	10	100
	80	2070569	10	9.96	99.6
	90	2191859	10	9.83	98.3