INTRODUCTION:

In pharmaceutical industries, the validation of analytical method is used to demonstrate that the method is fitted for its purpose; it must follow a plan which includes scopes, Performance characteristics, and acceptance limits. Analytical methods need to be validated or revalidated prior to their introduction into routine analyses. Chromatography is an analytical techniques based on the separation of molecules due to differences in their structure and/or composition.

In general, Chromatography involves moving a sample through the system over a stationary phase. The molecules in the samples will have different affinities and interaction with the stationary support, leading to separation of Molecules. Samples components that display stronger interaction with the stationary phase will move more slowly through the column than components with weaker interaction. Different compounds can be separated from each other as they move through the column. Chromatographic separation can be carried out using a variety of stationary phases. High-Performance liquid chromatography (HPLC) is types of liquid Chromatography used to separate and quantify compounds that have been dissolved in Solution. HPLC can be used to determine the amount of a specific compound in a solution¹.

MATERIALS AND METHODS:

Material:

Alcaftadine were purchased from the Vidisha Laboratories Ltd, India. All other chemicals were of analytical grade purchased from local suppliers.

Method Development by RP – HPLC:

Preparation of standard stock solution for Chromatographic development:

In order to prepare stock solution, weighed accurately 10 mg Alcaftadine and transferred into 20ml volumetric flask, added15 ml of methanol and sonicated to dissolve the standard completely and diluted up to the mark with methanol (500 PPM).

Further diluted 2ml of stock solution to 10ml with mobile phase (100 PPM). It was prepared in mobile phase of each trial and injected in development trials^2-4.

Selection of analytical wavelength for HPLC method development: Analytical wavelength for the examination was selected from the wavelength of maximum absorption from the spectrophotometric analysis and it was 282nm.

Optimization of HPLC method:

After all experimental trials and with reference to the acceptance criteria for various system suitability parameters, the conditions were optimized for the estimation of Alcaftadine bulk drug and its dosage form. Result shown in table no 3 and figure no 2

Preparation of System suitability test (Alcaftadine standard solution):

Weighed about 10.1mg of Alcaftadine and transferred in 20 ml volumetric flask, added 15ml of methanol, sonicated to dissolve it, made volume up to the mark with methanol Pipette out 0.1ml from standard stock solution and transferred into 10ml volumetric flask and made volume up to the mark with mobile phase (5.05 µ/ml≈ 5.00 working concentration), chromatograms were recorded. System suitability is a Pharmacopoeial requirement and is used to verify, whether the chromatographic system is adequate for analysis to be done. The tests were performed by collecting data from five replicate injection of standard drug solution and the results are recorded³.

Acceptance criteria

1) RSD should not be more than 2.0 % for five replicate injections of standard.

2) USP Tailing Factor/Asymmetry Factor is not more than 2.0.

3) The column efficiency as determined for Plate Count should be more than 2000.

A) Analysis of marketed Test sample:

Marketed test sample Having Name Cafta 0.25% w/v Eye drop are selected for analysis and for doing validation.

Weight per ml of test sample (Cafta 0.25% w/v Eye drop): Calculated weight per ml of test sample by following formula:

W3- W1

Weight per ml = -------------- x Density of water at 25⁰ C

W2- W1

Sample preparation of Marketed test sample:

Weighed accurately 1010mg from Cafta eye drop 0.25% w/v equivalent to 2.5mg of Alcaftadine and transferred to clean and dried 20ml of volumetric flask. Added 15 ml of methanol, sonicated for 10minutes with intermittent shaking. After 10minute allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5ml of initial filtrate Further diluted 0.4ml of filtered stock solution to 10 ml with mobile phase. (5mcg of Alcaftadine), injected the resultant solution and chromatograms were recorded and results are recorded²¹. In Table No: 1.

Table No: 1 Sample Prepared in duplicate. Summary of sample preparation as follows:

Sample	Sample (mg)	Diluted to (ml)	Volume taken	Diluted to (ml)
Sample 1	1010.2	20	0.4	10
Sample 2	1010.1	20	0.4	10

B) VALIDATION OF RP-HPLC METHOD:

The developed method for estimation of Alcaftadine was validated as per ICH guidelines for following parameters⁹.

1) FILTRATION STUDY:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. This study was conducted with Alcaftadine Test sample (Eye Drop solution).

Filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45 µm PVDF and 0.45 µm Nylon syringe filters used by discarding 5 ml of aliquot sample^5-6.

2) STABILITY OF ANALYTICAL SOLUTION:

Stability study was conducted for standard and test sample solution. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed after 12 hours and 24 hours.

Standard and Test solution stability study was performed by calculating the difference between results of test solution at each stability time point to that of initial⁷.

3) SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Following solution shall be prepared and injected to prove the specificity nature of the method. (Checked peak purity for standard and test sample solution)

I. Blank (Mobile phase as a diluents)

II. Placebo

III. Alcaftadine Standard solution

IV. Test sample solution

Placebo Sample solution preparation:

Weighed 1007.5mg of placebo material (Which is equivalent to 2.5mg of Alcaftadine) and transferred to clean and dried 20ml of volumetric flask. Added 15ml of methanol sonicated for 10 minutes with intermittent shaking. After 10 minutes allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5ml of initial filtrate. Further dilute 0.4ml of filtered stock solution to 10ml with mobile phase, injected the resultant solution and chromatograms were recorded^11-12.

4) LINEARITY AND RANGE:

Preparation of linearity solution:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.5 levels of Linearity was performed from 10% to 150% of working concentration

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

As per ICH Q2R1^8-9 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line S = Slope of regression line

6) ACCURACY (% RECOVERY):

Accuracy will be conducted in the range from 50 % to 150 % of working concentration. Solution of each accuracy level was prepared in triplicate. Calculated% Recovery for each sample, Mean % recovery for each level and overall recovery and also calculated % RSD for each level and % RSD for overall recovery^14-15.

Procedure for preparation of Accuracy sample solution:

Take clean and dried 9 volumetric flasks of 20 ml, Weighed aprrox 1007.5 mg of placebo and transfer in each 20 ml volumetric flask. Add stock solution of Alcaftadine API as per accuracy level in same 20 ml volumetric flask. Add 10 ml of methanol sonicated it for 15 minutes with intermittent shaking after every 5 minutes. Make the volume up to the mark with methanol. Filter the solution through suitable 0.45 µ syringe filter discarding 3-5 ml of filtrate. Further dilute 0.4 ml of filtrate to 10 ml with mobile phase^16-17.

7) PRECISION:

Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous test under the prescribed conditions. Precision is of two types, Repeatability and Intermediate precision. It is performed on tablet test sample.

8) REPEATABILITY:

Preparation of sample solution (6 Samples prepared):

Weighed accurately 1010mg of sample solution from Cafta eye drop 0.25% w/v equivalent to 2.mg of Alcaftadine and transferred to clean and dried 20ml of volumetric flask. Added 15ml of methanol, sonicated for 10 minutes with intermittent shaking. After 10 minutes allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5ml of initial filtrate. Further diluted 0.4ml of filtered stock solution to 10ml with mobile phase. (5mcg of Alcaftadine), injected the resultant solution and chromatograms were recorded^17-20. Six samples prepared.

Intermediate precision:

It is performed by doing analysis on another day to check reproducibility of results. Samples prepared in same manner as that of Repeatability parameter (6 Samples prepared).

9) ROBUSTNESS 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 usage²².

Determination:

Blank and Standard solution were injected under different chromatographic conditions as shown below.

A). Changes in flow rate by ±10%. (±0.1ml/min)

B). Change in column oven temperature. (± 2ºC)

C) Change in wavelength (± 3 nm)

Sample preparation of Marketed test sample:

Weighed accurately 1010 mg from Cafta eye drop 0.25 % w/v equivalent to 2.5 mg of Alcaftadine and transferred to clean and dried 20 ml of volumetric flask. Added 15 ml of methanol, sonicated for 10 minutes with intermittent shaking. After 10 minute allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45 µ syringe filter discarding 3-5 ml of initial filtrate Further diluted 0.4 ml of filtered stock solution to 10 ml with mobile phase. (5 mcg of Alcaftadine), injected the resultant solution and chromatograms were recorded and results are recorded²¹. In Table No: 1

Table No: 1 Sample Prepared in duplicate. Summary of sample preparation as follows:

Sample	Sample (mg)	Diluted to (ml)	Volume taken	Diluted to (ml)
Sample 1	1010.2	20	0.4	10
Sample 2	1010.1	20	0.4	10

VALIDATION OF RP-HPLC METHOD:

The developed method for estimation of Alcaftadine was validated as per ICH guidelines for following parameters⁹.

1) FILTRATION STUDY:

Filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45 µm PVDF and 0.45 µm Nylon syringe filters used by discarding 5 ml of aliquot sample^5-6.

2) STABILITY OF ANALYTICAL SOLUTION:

Standard and Test solution stability study was performed by calculating the difference between results of test solution at each stability time point to that of initial⁷.

3) SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Following solution shall be prepared and injected to prove the specificity nature of the method. (Checked peak purity for standard and test sample solution)

V. Blank (Mobile phase as a diluents)

VI. Placebo

VII. Alcaftadine Standard solution

VIII. Test sample solution

Placebo Sample solution preparation:

Weighed 1007.5 mg of placebo material (Which is equivalent to 2.5 mg of Alcaftadine) and transferred to clean and dried 20 ml of volumetric flask. Added 15 ml of methanol sonicated for 10 minutes with intermittent shaking. After 10 minutes allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45 µ syringe filter discarding 3-5 ml of initial filtrate. Further dilute 0.4 ml of filtered stock solution to 10 ml with mobile phase, injected the resultant solution and chromatograms were recorded^11-12.

4) LINEARITY AND RANGE:

Preparation of linearity solution:

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

LOD = 3.3 σ / S LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line S = Slope of regression line

6) ACCURACY (% RECOVERY):

Procedure for preparation of Accuracy sample solution:

Table No2: Refer Following table for each sample:

Level (%)	Alcaftadine stock solution (ml)	Wt of Placebo (mg)	Diluted to (ml)	Volume taken	Diluted to (ml)	Alcaftadine Added Conc. (µg/ml)
50	0.5	1007.6	20	0.4	10	2.50
	0.5	1007.5	20	0.4	10	2.50
	0.5	1007.7	20	0.4	10	2.50
100	1.0	1007.8	20	0.4	10	5.00
	1.0	1007.6	20	0.4	10	5.00
	1.0	1007.7	20	0.4	10	5.00
150	1.5	1007.4	20	0.4	10	7.50
	1.5	1007.5	20	0.4	10	7.50
	1.5	1007.6	20	0.4	10	7.50

7) PRECISION:

8) Repeatability:

Preparation of sample solution (6 Samples prepared):

Weighed accurately 1010 mg of sample solution from Cafta eye drop 0.25 % w/v equivalent to 2.5 mg of Alcaftadine and transferred to clean and dried 20 ml of volumetric flask. Added 15 ml of methanol, sonicated for 10 minutes with intermittent shaking. After 10 minutes allow cooling the solution to room temperature and made volume up to the mark with methanol. Filtered the solution through suitable 0.45 µ syringe filter discarding 3-5 ml of initial filtrate. Further diluted 0.4 ml of filtered stock solution to 10 ml with mobile phase. (5 mcg of Alcaftadine), injected the resultant solution and chromatograms were recorded^17-20. Six samples prepared.

Intermediate precision:

It is performed by doing analysis on another day to check reproducibility of results. Samples prepared in same manner as that of Repeatability parameter (6 Samples prepared).

9) ROBUSTNESS 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 usage²².

Determination: Blank and Standard solution were injected under different chromatographic conditions as shown below.

A). Changes in flow rate by ±10%. (±0.1ml/min)

B). Change in column oven temperature. (± 2ºC)

C) Change in wavelength (± 3 nm)

RESULT:

Selection of analytical wavelength

Fig. No.1 UV spectrum of Alcaftadine 20 PPM

Table No: 3 Optimized Chromatographic Conditions

Parameter	Description
Mode	Isocratic
Column Name	Kromasil C18, 250 mm X 4.6mm ID, 5 μm
Detector	UV Detector
Injection Volume	20 µl
Wavelength	282 nm
Column Oven temp	40ºC
Mobile Phase	Methanol : 0.1 % OPA (80:20%V/V)
Flow Rate	1.0 ml/min
Run time	8Minutes

Fig. No. 2 Typical chromatogram of Optimized Trial

System Suitability Acceptance Criteria

1) Relative standard deviation of the area of analyte peaks in standard chromatograms should not be more than 2.0 %.

2) Theoretical plates of analyte peak in standard chromatograms should not be less than 2000.

3) Tailing Factor (Asymmetry) of analyte peaks in Standard Chromatograms should be less than 2.0²³

Analysis of Marketed Test samples (Assay):

a) Cafta 0.25% w/v Eye Drop:

Weight per ml Calculation:

Weight of empty picnometer (W1): 27.47 gm

Weight of picnometer with water (W2): 37.58 gm

Weight of picnometer with test sample (W3): 37.71 gm

Density of water at 25°C: 0.99602

Weight /ml

W3-W1

--------------

W2-W1

0.99602

Weight /ml

37.71-27.47

37.58-27.47

0.99602

Weight /ml

1.010

gm/ml

Table No: 4 Assay results of Cafta 0.25% w/v Eye Drop:

Sample

Area

% Assay

Mean Assay

Sample 1

12336954

98.31

98.94

Sample 2

12492805

99.56

VALIDATION OF RP-HPLC METHOD:

1) FILTRATION STUDY:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on tablet test sample.

Table no 5: Results of Filter study

Sample description	Area	% Absolute difference
Unfiltered	12634861	NA
0.45 µ PVDF filter	12538910	0.76
0.45 µ Nylon filter	12563842	0.56

2) SOLUTION STABILITY:

Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours²⁴

Table No: 6 Results of Solution stability.

Sample solution			Standard solution
Time point	Area	% Absolute difference	Time point	Area	% Absolute difference
Initial	12703987	NA	Initial	12643084	NA
12 Hours	12635879	0.54	12 Hours	12580048	0.50
24 Hours	12607038	0.76	24 Hours	12549020	0.74

3) SPECIFICITY:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present. Blank, standard solution prepared and injected to check peak purity.

Table No: 7 Results of Specificity.

Description	Observation
Blank	No interference at R.T. of Alcaftadine due to blank
Placebo	No interference at R.T. of Alcaftadine due to placebo
Standard solution	Peak purity was 0.988
Test Solution	Peak purity was 0.986

4) Linearity and Range:

Alcaftadine shows linear response in the range of 0.5-7.5 μg/ml. The Regression value was found well within the limit

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ = 41608.62962(Residual standard deviation of a regression line) s = 2597148.523(Slope)

Detection limits (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 41608.62962 / 2597148.523

LOD = 0.053 µg/ml

Quantization limits (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 41608.62962 / 2597148.523

LOQ = 0.160 µg/ml

6) ACCURACY (RECOVERY):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added.

7) PRECISION:

Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Precision was performed on Test sample.

Table 9. Precision data for Alcaftadine test sample

Sr. No	Parameters	Intraday Precision	Inter-day Precision
1	Mean	98.27	98.341
2	SD	0.897430	0.80458
3	%RSD	0.913	0.818

8) ROBUSTNESS:

The robustness of an analytical method 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. Following changes made under Robustness: Change in Wavelength, Change in flow rate, Change in column oven, Temperature.

Table No: 10 Result of Robustness study:

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (285 NM)	3.81	12572544	1.28	5660
Wavelength by -3 NM (279 NM)	3.81	12657472	1.30	5643
Flow rate by +10% (1.1mL/min)	3.47	11624985	1.27	5413
Flow rate by -10% (0.9mL/min)	4.24	13561127	1.26	5733
Column oven temp by + 2ºC (42 ºC)	3.81	12306480	1.32	5492
Column oven temp by – 2ºC (38 ºC)	3.81	12106681	1.31	5509

CONCLUSION:

The present work involved the development of simple, accurate, precise and suitable RP-HPLC method. RP-HPLC technique till now no analytical technique with the aid of using RP-HPLC turned into stated for its willpower in bulk drug and in pharmaceutical dosage forms. Hence, in the present study, a new, sensitive and suitable reversed-phase high performance liquid chromatography method was developed and validated for the determination of Alcaftadine in bulk drug and pharmaceutical dosage form²¹.

REFERENCES:

1. National PBM Drug Monograph. Alcaftadine 0.25% Ophthalmic Solution (Lastacaft). VA Pharmacy Benefits Management Services. Medical Advisory Panel and VISN Pharmacist Executives. 2015

2. US Food and Drug Administration Protecting and Promoting Your Health. 2015

3. (2015) Office of Clinical Pharmacology Review.

4. Church MK, McGill JI. Human ocular mast cells. Curr Opin Allergy Clin Immunol. 2002; 2(5): 419–422.

5. Abelson MB, Udell IJ. H2 -receptors in the human ocular surface. Arch Ophthalmol. 1981; 99(2): 302–304.

6. Chigbu DI. The pathophysiology of ocular allergy: a review. Cont Lens Anterior Eye. 2009; 32(1): 3–15. [quiz 43–4].

7. Wong AH, Barg SS, Leung AK. Seasonal and perennial allergic conjunctivitis. Recent Pat Inﬂamm Allergy Drug Discov. 2014; 8(2): 139–153.

8. Beckett, A. H., Stenlake, J. B. Practical Pharmaceutical Chemistry, 4th ed.; CBS Publishers and Distributors, India, 2007. [2] Alcaftadine 0.25% ophthalmic solution (Lastacaft) National PBM Drug Monograph http://www.pbm.va.gov/PBM/clinicalguidance/drugmonographs/Alcaftadine_Drug_Monograph. docx

9. ICH, Q2A, Hamonised Tripartite Guideline, Test on Validation of Analytical Procedures, IFPMA. In: Proceedings of the International Conference on Harmonization, Geneva. March, 1994.

10. ICH, Q2B, Hamonised Tripartite Guideline, Validation of Analytical Procedure: Methodology. IFPMA, In: Proceedings of the International Conference on Harmonization, Geneva. March, 1996.

11. Christian. C. Analytical Chemistry, 6th Edn, 2008; 822-828

12. Ahmad M.Z., Mohammad Ali, Textbook of Pharmaceutical Drug Analysis, 1st Edn. 2009. 2287

13. Parimoo P., Pharmaceutical Analysis. 2005:365-370

14. Connors. K.A. A textbook of Pharmaceutical Analysis, John Wiley and sons, 3rd ed. 1999: 277-288

15. Kasture A.V., Wadodkar S.G., Mahadik K.R., More H.N., Pharmaceutical Analysis: Instrumental methods II, 11th Edn, 2004

16. Gurdeep R, Chatwal S, Anand K. Instrumental methods of chemical analysis. Himalaya Publishing House. 2016

17. Willard HH, Merritt Jr LL, Dean JA, Settle Jr FA. Instrumental methods of analysis 7th Edn. 1995: 1-3

18. Mendham J, Denney R.C., Barnes J.D., M. Thomas, Vogel’s, Textbook of Quantitative Analysis. Pearson Education, Singapore. 2008: 387

19. Jack V Greiner 1, Kimberly Edwards-Swanson, Avner Ingerman - Evaluation of alcaftadine 0.25% ophthalmic solution in acute allergic conjunctivitis at 15 minutes and 16 hours after instillation versus placebo and olopatadine 0.1%.

20. Mishra PR, Satone D2 and Meshram DB. Development and Validation of HPLC Method for the Determination of Alcaftadine in Bulk Drug and its Ophthalmic Solution. 2016.

21. DeGaulle I Chigbu and Alissa M Coyne- Update and clinical utility of Alcaftadine ophthalmic solution 0.25% in the treatment of allergic conjunctivitis.

22. Jaime Hornecker et al., to evaluate the safety and efficacy of Alcaftadine for the prevention of itching associated with allergic conjunctivitis. One trial compared Alcaftadine to placebo, and another trial compared Alcaftadine to placebo and olopatadine HCl to placebo. 2019.

23. Arya T. Soudi et,al., Stability indicating methods are essential to determine the stability of newly developed dosage forms. 2020.

24. M S Smitha Gowda1, Kiran Kumar L et, al., Comparison of efficacy of topical Alcaftadine (0.25%) versus olopatadine (0.1%) in allergic conjunctivitis. 2021.

Received on 10.01.2024 Modified on 08.02.2024

Asian J. Pharm. Ana. 2024; 14(1):26-32.

DOI: 10.52711/2231-5675.2024.00006

Level (%)	Area	Recovered conc. (µg/ml)	Added conc. (µg/ml)	% Recovery	Mean Recovery	% RSD
50	6209071	2.47	2.50	98.98	98.88	0.090
	6201358	2.47	2.50	98.85
	6198254	2.47	2.50	98.81
100	12500441	4.98	5.00	99.63	99.18	0.745
	12336482	4.92	5.00	98.33
	12494854	4.98	5.00	99.59
150	18641803	7.43	7.50	99.06	99.13	0.373
	18730694	7.46	7.50	99.53
	18594309	7.41	7.50	98.80