INTRODUCTION:

Alcaftadine, an ophthalmic medication, serves a dual purpose as both an H1-antihistamine and mast cell stabilizer. It was approved by the USFDA in July 2010 under the brand name Lastacaft for alleviating itching associated with allergic conjunctivitis. Lastacaft is a sterile solution intended for topical application in the eyes, providing targeted relief for allergic reactions affecting this area.¹

It is a broad-spectrum antihistamine known to display a high affinity for histamine H1 and H2 receptors and a lower affinity for H4 receptors’ It also demonstrates modulatory action on immune cell recruitment and mast cell stabilizing effects.

Chemistry:

The IUPAC name of alcaftadine is 6, 11-dihydro-11-(1-methyl-4-piperidinylidene)-5H-imidazo [2, 1-b]. benzazepine-3-carboxaldehyde² (figure -1)

Figure 1: Chemical Structure of Alcaftadine

The metabolism of alcaftadine is mediated by non-CYP450 cytosolic enzymes to produce the active carboxylic acid metabolite. The protein bindings of alcaftadine and the active metabolite are 39.2% and 62.7%, respectively.³

Alcaftadine, which is used to treat allergic conjunctivitis, isn't officially recognized in major pharmacopeias like the British Pharmacopoeia (BP), Indian Pharmacopoeia (IP), or United States Pharmacopeia (USP). A literature review revealed that there's no established analytical method for determining alcaftadine levels in either its raw form or in ophthalmic products. However, one clinical pharmacological review described using liquid chromatography with tandem mass spectrometry (LC/MS/MS) to measure the levels of alcaftadine and its active metabolite, R90692, in K3 EDTA-treated human plasma. This method is useful for quantifying drug concentrations in pharmacokinetic studies^.4 A study used high-performance liquid chromatography (HPLC) to figure out how 14C-Alcaftadine is broken down and transformed into Vidisha Analytical in the body. By using HPLC, researchers could separate the original drug from its byproducts (metabolites), helping them understand how the drug is metabolized and processed. This information is useful for understanding how the drug works and its safety.⁵

MATERIALS AND METHODS:

Materials:

The alcaftadine drug was received as a gift sample.

Instrumentation and software:

An Agilent 1260 Infinity II HPLC system with a DEAX02386 pump and autosampler with UV-visible detector served as the chromatographic system (DEACX16446). For data collection and processing, the chromatograms were registered using Open lab EZ Chrome Workstation on a Windows-based computer system. Alcaftadine concentrations were determined using a Kromasil C18 column (250 mm x 4.6 mm ID, 5 μm).

Methods:

Selection of an analytical wavelength

Selection of solvent: Methanol was selected as a blank, and Alcaftadine standard solution (20 PPM) was scanned from 400nm to 200nm. Absorption maxima were determined for the drug. Alcaftadine showed maximum absorbance at 282nm, as shown in the results in (figure 2).

Method Development by UHPLC:

Preparation of a standard stock solution for chromatographic development:

To prepare a stock solution, accurately weigh 20mg of alcaftadine and transfer it into a 20ml volumetric flask. Add 15ml of methanol, sonicate to dissolve the standard completely, and dilute up to the mark with methanol (1000 PPM). Further diluted 2ml of stock solution to 20 mL with mobile phase (100PPM). It was prepared in the mobile phase of each trial and injected into development trials.

Selection of analytical wavelength for UHPLC method development: The analytical wavelength for the examination was selected from the wavelength of maximum absorption from the spectrophotometric analysis, which was 282 nm. Shown in (figure 2).

Method Validation by UHPLC:

[a] Development and optimization of the HPLC method: After all experimental trials and concerning the acceptance criteria for various system suitability parameters, the conditions were optimized for the estimation of alcaftdaine bulk drug and its dosage form, and the result is shown in (Figure 3 and Table 1).

[b] Preparation of a system suitability test (Alcaftadine standard solution):

Weighed about 25mg of alcaftadine and transferred it in a 50mL volumetric flask, added 30-35mL of methanol, sonicated it to dissolve it, and made volume up to the mark with methanol. Pipette out 1 ml from the standard stock solution and transfer it into a 25ml volumetric flask, making the volume up to the mark with the mobile phase (20 PPM working concentration). Chromatograms were recorded, as shown in (Figure 3).

[A] Analysis of the marketed test sample:

Marketed test samples Having the name Alcaft 0.25% w/v Eye Drop is selected for analysis and for doing validation (Equation 1).

Weight per ml of the test sample (Alcaft 0.25% w/v Eye Drop):

Calculated weight per mL of the test sample by the following formula:

Weight per mL	=	W3 - W1	X	Density of Water at 25°C
		W2 - W1

(1)

Sample preparation for the marketed test sample:

Weighed accurately 1020mg from Alcaft eye drop 0.25 % w/v equivalent to 2.5mg of Alcaftadine and transferred to a clean and dried 25mL volumetric flask. I added 15mL of methanol and sonicated for 10 minutes with intermittent shaking. After 10 minutes, allow the solution to cool to room temperature and make the volume up to the mark with methanol. Filtered the solution through a suitable 0.45µ syringe filter, discarding 3-5mL of the initial filtrate. Use 20mcg of Alcaftadine as mobile phase to dilute 2ml of filtered stock solution to 10ml. Then, inject the resulting solution and record chromatograms. Results are shown in (Table 2) (Equation 2).

Formula for % Assay Calculation

[B] Method Validation Parameters:

FILTRATION STUDY:

Filtration studies of an analytical procedure check the interference of extraneous components from the filter, deposition on the filter bed, and compatibility of the filter with the sample. This study was conducted with an alcaftadine test sample (eye drop solution). The filtration study was carried out with an unfiltered and filtered test solution. During filtration activity, 0.45µm PVDF and 0.45µm Nylon syringe filters were used by discarding 5 mL of the aliquot sample. (Table 3).

STABILITY ANALYTICAL SOLUTION:

A stability analysis was performed on test sample and standard solution. A stability study was performed under normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed after 12 and 24hours. A standard and test solution stability study was performed by calculating the difference between the results of the test solution at each stability time point and those of the initial (Table 4).

SPECIFICITY:

Specificity is the capacity to clearly detect the analyte in the presence of potentially predicted constituents.

The following solution shall be prepared and injected to prove the specificity of the method. (Checked peak purity for standard and test sample solutions) (Table 5)

· Blank (mobile phase as a diluent)

· Placebo

· Alcaftadine Standard Solution

· Test the sample solution.

Placebo sample solution preparation:

Weighed 1017.5mg of placebo material (which is equivalent to 2.5mg of alcaftadine) and transferred it to a clean and dried 25mL volumetric flask. I added 15mL of methanol and sonicated for 10minutes with intermittent shaking. After 10minutes, allow the solution the solution to cool to room temperature and make the volume reach the mark with methanol. Filtered the solution through a suitable 0.45µ PVDF syringe filter, discarding 3-5mL of the initial filtrate. Further, 2ml of filtered stock solution was added to 10ml of mobile phase, and the resultant solution and chromatograms were recorded.

LINEARITY AND RANGE:

Linearity of the alcaftadine stock solution:

Weighed 20mg of alcaftadine and dissolved it in 20mL of methanol. Further diluted 5ml of stock solution to 50 mL with mobile phase. (100PPM) (Figure 4).

LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ):

Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantified as an exact value.

Quantitation limit:

The lowest concentration of analyte in a sample that can be quantitatively identified with appropriate precision and accuracy is known as the quantitation limit of a particular analytical process.

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

LOD = 3.3 σ/S

LOQ = 10 σ/S

Were,

σ = represents the regression line's residual standard deviation.

S = is the regression line's slope.

ACCURACY (% Recovery):

Accuracy will be conducted in the range of 50% to 15% of working concentration. The solution for 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. (Table 6)

PRECISION:

The term "precision" describes how closely two or more measurements match up. Repeatability and intermediate precision are the two categories of precision.

Repeatability: Weighed 1020mg of Alcaft 0.25% eye drop (2.5mg of Alcaftadine) and put it into a 25mL volumetric flask. I added 15mL of methanol, sonicated for 10minutes, cooled, and topped up with methanol to 25mL. Filtered with a 0.45µm PVDF syringe filter, discarding the first 3-5mL. Dilute 2mL of the filtered stock solution with the mobile phase to a final volume of 10mL (yielding 20mcg of alcaftadine). This was then injected for chromatography analysis. Six samples were prepared.

Intermediate precision: It is performed by analyzing the results the results on another day to check their reproducibility. Samples prepared in the same manner as the repeatability parameter (Table 7).

ROBUSTNESS:

An analytical procedure's robustness indicates how reliable it is and is a measure of its ability to withstand slight but intentional changes in technique parameters. during normal usage. Blank and the standard solution was injected under different chromatographic conditions. Changes in flow rate by ±10%. (± 0.1ml/min) Change in column oven temperature. (± 2ºC) Change in wavelength (± 3nm) (Table 8)

RESULT AND DISCUSSION:

Selection of analytical wavelength:

Figure 2: UV spectrum of Alcaftadine solution 20 PPM

Development and optimization of the UHPLC method.

Table 1: Optimized Chromatographic Conditions

Parameter	Description
Mode	Isocratic
Column Name	Water cortex C18, 150mm X 4.6mm, 2.7µm
Detector	UV Detector
Injection Volume	20µl
Wavelength	282nm
Column Oven temp	35ºC
Mobile Phase	Acetonitrile: 0.05 % OPA in Water (60:40%V/V)
Flow Rate	1.0 ml/min
Run time	5 Minutes
Retention time	2.20 Minutes

Figure 3: Typical chromatogram of Optimized Trial

System Suitability Acceptance Criteria:

The relative standard deviation of the area of analyte peaks in standard chromatograms should not be more than 2.0%. In standard chromatograms, there should be a minimum of 2000 theoretical plates of analyte peak. The Tailing Factor (Asymmetry) of analyte peaks in Standard Chromatograms should be less than 2.0 shown in (Figure 3).

Analysis of Marketed Test Samples (Assay)

Alcaft 0.25% w/v Eye Drop:

Weight per mL Calculation:

Weight of empty pycnometer (W1): 27.47gm

Weight of pycnometer with water (W2): 37.62gm

Weight of pycnometer with test sample (W3): 37.85gm

Density of water at 25°C: 0.99602 shown in table 2

Weight per mL	=	W3 - W1	X	0.99602
		W2 - W1

Weight per mL	=	37.85 - 27.47	X	0.99602
		37.62 - 27.47

Weight per mL

1.020

gm / mL

Table 2: Assay results of Alcaft 0.25% w/v Eye Drop

Sample	Area	% Assay	Mean Assay
Sample 1	5136014	97.56	98.10
Sample 2	5188569	98.63	98.10

Validations of the UHPLC method^6-12

The optimized method for estimating Alcaftadine was validated for the following parameters using ICH Q2(R1) guidelines

FILTRATION STUDY:

Filtration study of an analytical procedure checks the interference of extraneous components from the filter, deposition on the filter bed and compatibility of the filter with the sample. Performed on eye drop test sample shown in (Table 3)

Table 3: Result of Filter Study

Sample description	Area	% Absolute difference
Unfiltered	5258960	NA
0.45 µ PVDF filter	5212369	0.89
0.45 µ Nylon filter	5199040	1.14

SOLUTION STABILITY:

A stability study was conducted for the Standard as well as the Test Sample. A stability study was performed under normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analysed initially, after 12 hours and 24 hours shown in (Table 4).

Table 4: Results of Solution stability

Sample solution			Standard solution
Time point	Area	% Absolute difference	Time point	Area	% Absolute difference
Initial	5232963	NA	Initial	5260317	NA
12 Hours	5186583	0.89	12 Hours	5217893	0.81
24 Hours	5168976	1.22	24 Hours	5206304	1.03

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. shown in (Table 5)

Table 5: 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 0.979
Test Solution	Peak purity was 0.975

LINEARITY AND RANGE:

The relationship between Alcaftadine concentration and corresponding UV intensity was found to be linear over the concentration range of 2.0-30.0µg/mL with a r²0.99998 shown in (Figure 4) and (Equation 3)

Equation of linearity: Y = M X + C (3)

Y = 264946.067 x + -11096.362

Fig 4: Calibration curve of Alcaftadine

LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ):

σ = 20776.6818 (Residual standard deviation of a regression line)

s = 264946.067 (Slope)

Detection limit (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 20776.6818 / 264946.067

LOD = 0.259 µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 20776.6818 / 264946.067

LOQ = 0.784 µg/mL

ACCURACY:

The accuracy of 50 %, 100% and 150 % was found to be 99.14, 99.83 and 99.90% respectively of 3 levels and % the relative standard deviation was not more than 2.0%. % Recovery is not hampered by changes in analyte concentration shown in (Table 6)

Overall Recovery: 99.62 %

% RSD for Overall Recovery: 0.880

PRECISION:

Precision was performed on the test sample. HPLC method was precise. The result is shown in (Table 7)

Table 7: Result of Intraday and Inter-Day Precision for Alcaftadine test sample assay

Sr. No	Parameters	Intraday Precision	Intraday Precision
1	Mean	98.54	98.75
2	STD	1.1210	1.333341
3	%RSD	1.138	1.350

ROBUSTNESS:

An analytical technique's robustness is a measure of how well it can withstand tiny but intentional changes in method parameters, indicating its dependability under typical operating conditions. shown in (Table 8)

Following changes made under Robustness:

· Change in Wavelength

· Change in flow rate

· Change in column oven temperature

Table 8: Result of Robustness study

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (285 NM)	2.20	4569231	1.38	5163
Wavelength by -3 NM (279 NM)	2.20	6942531	1.34	5308
Flow rate by +10% (1.1mL/min)	1.98	5362819	1.30	4942
Flow rate by -10% (0.9mL/min)	2.46	6106905	1.42	5518
Column oven temp by +2ºC (37 ºC)	2.19	5186035	1.27	5937
Column oven temp by -2ºC (33 ºC)	2.19	5224679	1.25	5882

CONCLUSION:

The objective of this project was to establish a straightforward, dependable, precise, and suitable UHPLC system. The developed analytical method was rigorously validated for linearity, accuracy, precision, and robustness, along with the determination of detection and quantification limits. The developed method offers numerous benefits, including consistent reproducibility of results, rapid data interpretation, straight forward sample preparation, and enhanced selectivity and sensitivity. Importantly, the retention time achieved in this study was shorter compared to previously reported methods. This newly established method holds promise for routine analysis in the pharmaceutical sector, facilitating the assessment of both bulk drug alcaftadine and pharmaceutical dosage forms due to its stability, reproducibility, and efficiency. Based on the experimental findings, the developed method demonstrates simplicity, precision, and accuracy, with the added advantage of shorter retention time, thereby enhancing its acceptability and cost-effectiveness. It holds potential for widespread application in routine analysis within research institutions, quality control departments in industries, and accredited testing laboratories.

REFERENCES:

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

2. Chemistry Review, http://www.accessdata.fda.gov/drugsatfda docs/nda/2010/022134s000ChemR.pdf. Accessed 16 Mar 2015)

3. https://go.drugbank.com/drugs/DB06766.

4. Clinical Pharmacology Review, http://www.fda.gov/downloads/Drugs/Developme nt Approval Process/Development Resources/UCM22 3817.pdf (last reviewed on 11th March 2015).

5. Bohets H, McGowan C, Mannens G, Schroeder N, Edwards-Swanson K, et al. Clinical pharmacology of alcaftadine, a novel antihistamine for the prevention of allergic conjunctivitis. J Ocul Pharmacol Ther. 2011; 27: 187-195.

6. Vidushi, Y., and Meenakshi, B. A review of HPLC method development and validation. Res J Life Sci, Bioinform, Pharm Chem Sci, 2017; 2(6): 178.

7. Nilesh S. Pendhbaje, Rupali V. Nirmal, Ashwini A. Jamdhade, Shain M. Pathan. Method Development and Validation by HPLC: A Brief Review. Research and Reviews: A Journal of Pharmaceutical Science. 2021; 12(1): 27–39p

8. Bose, A. HPLC calibration process parameters in terms of system suitability test. Austin Chromatography. 2014; 1(2): 14.

9. Sabir, A.M. HPLC method development and validation a review. International Research Journal of Pharmacy. 2013; 4(4): 39-46.

10. Pendbhaje N.S. Jamdhade A.A., Pathan S.M., Nirmal R.V. A Review on Quantification of Brexpiprazole in Its Bulk and Pharmaceutical Dosage Form by Various Analytical Methods. International Journal of Pharmaceutical Research and Applications. 2021; 6(1): 1118-1132.

11. Mishra, P. R., Satone, D., and Meshram, D. B. Development and validation of HPLC method for the determination of Alcaftadine in bulk drug and its ophthalmic solution. J Chromatogr Sep Tech. 2016; 7(312): 2.

12. Badr El-Din, K., Ahmed, A., Khorshed, A., Derayea, S., Oraby, M. Smart Spectrophotometric Methods Based on Feasible Mathematical Processing and Classical Chemometry for The Simultaneous Assay of Alcaftadine and Ketorolac in Their Recently Approved Pharmaceutical Formulation. Egyptian Journal of Chemistry. 2022; 65(2): 167-174. doi: 10.21608/ejchem.2021.82464.4098.

Received on 19.05.2024 Modified on 15.06.2024

Asian J. Res. Pharm. Sci. 2024; 14(3):221-226.

DOI: 10.52711/2231-5659.2024.00036

Level (%)	Area	Recovered conc (µg/mL)	Added conc (µg/mL)	% Recovery	Mean Recovery	% RSD
50	2591024	9.86	10.00	98.59	99.14	0.670
	2600792	9.90	10.00	98.96
	2624973	9.99	10.00	99.88
100	5246793	19.96	20.00	99.82	99.83	0.942
	5297046	20.16	20.00	100.78
	5198300	19.78	20.00	98.90
150	7804074	29.69	30.00	98.98	99.90	1.030
	7860907	29.91	30.00	99.70
	7964315	30.30	30.00	101.01