INTRODUCTION:

The principles and uses of chromatography techniques such as gas chromatography, paper chromatography, TLC, HPLC, HPTLC, RP-HPLC, Stability inicating and others for a range of substances. Various physical and chemical characteristics of a broad variety of test samples to be separated using the chosen chromatographic techniques, which list the separations, identifications, and analyses with the best results for specific samples. The examination of both traditional and sophisticated methods for specific samples is the focus of this paper.

The study shows how chromatography is used at different phases of drug development and discovery.¹The measurement and analysis of electromagnetic radiation that is absorbed, scattered, or released by atoms, molecules, or other chemical species is known as spectroscopy. Since each species has distinct energy states, spectroscopy can be used to detect the interacting species. This absorption or emission is linked to changes in the energy states of the interacting chemical species.²

Analytical Method Development:

Analytic method development is the process of developing an accurate assay approach to determine the composition of a formulation. The development of an analytical technique is the process of demonstrating that an analytical method is suitable for use in a laboratory. Analytical procedures must be utilized within GMP and GLP settings and created utilizing the protocols and acceptance criteria in the ICH recommendations Q2 (R1).

Validation:

Validation is a term that developed in the United States in 1978. The idea of validation has evolved over time to encompass a wide range of activities, including analytical methods for controlling the quality of therapeutic ingredients and drug products, as well as computerized systems for clinical trials, process control, and labelling. Validation is best seen as an essential and critical component of cGMP. The term validation refers to the examination of validity or the act of demonstrating efficacy. Validation is a collaborative effort that involves individuals from several plant branches. Method validation is a "process of establishing documented evidence" that gives a high level of certainty that the product (equipment) will fulfil the requirements of the planned analytical applications.

Importance of validation:

· Quality of Assurance.

· Minimizes batch failures.

· Reduces Rejections.

· Improves productivity and effectiveness.

· Increases production.

· It also reduces testing in process and final items.

Parameters of Method Validation:

1) Accuracy

2) Precision

3) Linearity

4) Limit of detection

5) Limit of quantitation

6) Specificity

7) Range

8) Robustness

1) Accuracy: Accuracy is defined as the closeness of the test results to the true value.

2) Precision: Precision is defined as the measurement of closeness of agreement for multiple measurements on the same sample. The precision is expressed as the relative standard deviation. % RSD = Standard deviation/Mean ×100

3) Linearity: Linearity is the ability of analytical procedure to obtain a response that is directly proportional to concentration (amount) of analyte in the sample. Linearity is expressed as the confidence limit around the slope of the regression line.

4) Limit of Detection (LOD): LOD is defined as lowest amount (concentration) of analyte in a sample that can be detected or identified, not quantified. LOD is expressed as a concentration at a specified signal: noise ratio, usually 3:1. LOD = 3.3 × S/ SD

5) Limit of Quantitation (LOQ): LOQ is defined as lowest amount (concentration) of analyte is a sample that can be quantified. For LOQ, ICH has recommended a signal: noise ratio 10:1. LOQ = 10 × S/SD

6) Specificity: Specificity is defined as the ability of an analytical method to measure the analyte clearly in the presence of other components. This definition has following implications:

a. Identification

b. Purity tests

c. Assay.

7) Range: The range of the method is the interval between upper level and lower level of analyte that have been determined with acceptable accuracy, precision and linearity. It is determined on either a linear or nonlinear response curve and expressed in the same unit as the test results are expressed

8) Robustness: Robustness is defined as the measurement of capacity of analytical procedure to remain unaffected by small variations in method parameters³

METHODS AVAILABLE ON ALCAFTADINE:

1) UV Spectrophotometric method:

a) Priyanka R. Mishra and her co-workers published the paper in the year 2015. The development of easy, specific, precise, and accurate UV-Visible Spectroscopic techniques for estimating Alcaftadine in bulk and in its ophthalmic dose form. Two approaches for analytically estimating Alcaftadine have been developed. Methanol was utilized as a solvent. The absorption maxima approach (method A) yielded a value of 282nm as the maximum wavelength. Method B uses first derivative spectroscopy to measure the response (dA/dλ) of a reference solution at 267nm. The new technique was tested for linearity, accuracy, precision, specificity, LOQ, and LOD. The approaches were linear over Beer's range of 1-16μg/ml. The correlation coefficients for methods A and B were 0.999 and 0.997, respectively. The percent assay for Alcaftadine in ophthalmic formulation was determined to be 101.01% and 112.5%, respectively.⁴

b) Priyanka R. Mishra and her co-worker also published the paper in the year (2015) The current study wants to develop and validate two simple UV spectrophotometric approaches, the area under curve method of zero order and the first-order derivative, for estimating Alcaftadine in bulk medication and its application to commercial formulations. The solvent employed in this investigation was methanol. Two wavelengths, 266nm and 296nm, and 257nm and 277nm, were used for the estimation of integrated areas in the AUC of the zero-order derivative approach (approach A) and the first order derivative method (METHOD B). Linearity was seen between 1-16 μg/ml for both procedures, with correlation coefficients of 0.999 and 0.998 for methods A and B, respectively.⁴

2) High Performance Liquid Chromatography:

Mishra PR. And her co- worker published the paper in the year (2016) For the purpose of estimating alcaftadine in bulk medication and its ophthalmic dosage form, a straightforward, accurate, and precise stability indicating RP-HPLC method was created. The target analyte and degradation products were separated chromatographically using an Enable HPLC ODS C18 G (250 × 4.6mm, 5μm) column and a mobile phase consisting of 50:50 v/v methanol: water at a flow rate of 1.2ml/min with UV detection at 282nm. It was discovered that in the concentration range of 1–16 μg/ml, the correlation coefficient (r2) was 0.999. 3.15 minutes was determined to be the retention time. It was discovered that the limits of quantitation and detection were, respectively, 0.75 and 0.25μg/ml. Stress conditions such as acid and base were used to carry out the stress degradation of alcaftadine In Table No.1.⁵

Optimized Chromatographic Conditions for RP-HPLC.⁵

Table No. 1: Optimized Chromatographic Conditions for RP-HPLC.

Chromatographic Parameters	Optimized Condition
Stationary Phase	Enable HPLC C18 G 120A Column 250 × 4.6 mm, 5 μ
Mobile Phase	Methanol: water, 50:50 (%v/v)
Flow Rate	1.2 ml/min
Injection Volume	20 μl
Detection Wavelength	282 nm
Run Time	10 minutes

3) Reversed Phase Hight Performance Liquid Chromatography:

(RP-HPLC)

Kunde Monali Pundlik, and her co- worker published the paper in the year (2022) This study developed and validated a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for determining alcaftadine in bulk medication and ophthalmic formulation that is robust, economical, sensitive, appropriate, and accurate. Utilizing an Open lab EZchrome workstation programme and a UV detector, the separation was carried out using an HPLC technique with a Kromasil C18, 250mm X 4.6mm ID, 5μm Acetonitrile: 0.1percent OPA (90:10 percent V/V) at a flow rate of 1.0mL/min and detected at 282nm. After trial and error, the developed RP-HPLC method produced a satisfactory retention duration of 3.05 min for alcaftadine. Over the concentration range of 0.57.5μg/mL, the correlation coefficient (r2) of 0.9999 demonstrated the linearity of the determined technique. The precision of the method's percentage RSD was discovered in Table No.2.⁶

Optimized Chromatographic Conditions of HPLC (RP-HPLC)

Table No.2: Optimized Chromatographic Conditions of HPLC (RP-HPLC)

Parameter	Description
Mode	Isocratic
Column Name	Kromasil C15,250mmX 4.6mm ID,5μm
Detector	UV Detector
Injection Volume	20 μl
Wavelength	282 nm
Column Oven Temp	40ºC
Mobile Phase	Acetonitrile: 0.1 % OPA (90:10%V/V)
Flow Rate	1.0 ml/min
Run Time	6 Minutes
Retention Time	3.05 Minutes

4) High-Performance Thin-Layer Chromatographic (HPTLC)

Bhushan J. Mali, and her co- worker published the paper in the year (2021) for the determination of alcaftadine in bulk and eye drops, a quick, high-performance thin-layer chromatographic approach that is straightforward, exact, and accurate has been devised and validated. Aluminium plates that had been previously coated with silica gel 60 F254 as a stationary phase were used for the chromatographic development. As the mobile phase, a blend of methanol, triethylamine, and chloroform (4:1:0.5 v/v/v) provided a thick and compact Alcaftadine spot. Densitometric detection was used at a wavelength of 282nm. The drug's Rf value was determined to be 0.45±0.02. It was discovered that the calibration curve was linear between 250 and 1500ng/μl in Table No.3⁷

Optimization of mobile phase of HPTLC

Table No.3 Optimization of mobile phase of HPTLC

Sr. No	Solvents	Proportional	Rf of ALC
1	Methanol	5.0	Tailing
2	n- propanol	5.0	0.85
3	Toluene	5.0	0.20
4	Toluene: n-propanol	3.0: 2.0	0.75
5	Toluene: Methanol	3.5: 1.5	0.80
6	Chloroform: methanol	4.0: 1.0	0.43 with tailing
7	Chloroform: Methanol: Triethylamine	4: 1: 0.5	0.45

5) Stability-indicating UPLC, TLC-densitometric and UV-spectrophotometric:

Sawsan A Abdel Razeq, and her co- worker published the paper in the year (2021) Three sensitive and precise stability-indicating methods have been designed to identify alcaftadine in the presence of degradation products. The UPLC-UV-MS method was employed for efficient separation, with gradient elution using 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B), with concentrations ranging from 0.10-1.00 μg mL 1. The detection accuracy for UV and MS was 100.89%±0.74 and 99.73%±0.78, respectively. The developing system included methanol, chloroform, and glacial acetic acid (5:4:0.1, v/v/v), with a detection wavelength of 282nm. Alcaftadine's acidic and oxidative degradants have Rf values of 0.35, 0.65, and 0.88, respectively. The technique had a linearity range of 2.00-27.00μg/band and a mean accuracy of 100.58%±0.86. The mean recovery was 100.15%±0.70 in Table No.4.

Chromatographic conditions.⁸

Table No.4: Chromatographic Condition of Stability Indicating UPLC

COLUMN	ACQUITY BEH C18 column (50 mm × 2.1 mm × 1.7 μm) (Waters, Ireland
Time intervals	Mobile phase composition
0 min	95% (0.1% formic acid in H2O) A + 5% (0.1% formic acid in acetonitrile) B
0.5 min	95% A + 5% B
4.5 min	0% A + 100% B
6 min	0% A + 100% B
7 min	95% A + 5% B
7.5 min	95% A + 5% B
Mobile Phase	Phase Filtered and degassed mixture of water containing 0.1% formic acid (TFA) (A) and 0.1% formic acid (TFA) in acetonitrile (B) with different ratios at each time interval
Flow rate	0.4 mL /min
Injection volume	0.4 μL
Temp	Ambient
Detector	MS-SIM (single ion monitoring) and UV 282 nm detector.

6) Stability Indicating Reverse Phase High Performance Liquid Chromatography:

a) Ishani Patel, and her co- worker published the paper in the year (2021) A reverse phase high performance liquid chromatography technique for simultaneous quantification of alcaftadine and ketorolac tromethamine in pharmaceutical dose form has been developed. The separation was accomplished using a Hyperchrom ODS C18 Column (250*4.6mm) with Buffer 0.05M Potassium dihydrogen orthophosphate Buffer (pH-4.0): Methanol (60:40) as a mobile phase, with a flow rate of 1ml/min. The detection wavelength was 220nm. The retention times for Alcaftadine and Ketorolac Tromethamine were 3.187 and 5.497, respectively. Linearity was reported for Alcaftadine and Ketorolac Tromethamine (12.5-37.5μg/ml and 20-60μg/ml, respectively).

Optimize Chromatographic condition⁹

Table No. 5: Optimize Chromatographic condition Stability Indicating RP-HPLC

Parameters	Description
Column	Hyperchrom ODS C18 column (250*4.6mm)
Flow Rate	1.0ml/min
Injection Volume	20μL
Mobile Phase	0.05M Potassium dihydrogen ortho phosphate buffer (pH-4.0): Methanol (60:40)
Column Temp	40°C
Run Time	8 min

7) LC–QTOF‑MS/MS:

Balasaheb B. Chavan, and his co- worker published the paper in the year (2018) To selectively separate alcaftadine degradation products, a quick, precise, and accurate gradient UPLC technique was devised. Forced degradation tests of the medicine were conducted by investigating parameters recommended by ICH recommendations. The breakdown products generated under acidic, alkaline, and peroxide environments, while the medication remained stable in neutral, thermal, and photolytic degradation settings. The degradation products and drug were separated using an ACQUITY CSH C18 (100 × 2.1mm, 1.7μm) column, with a mobile phase of ammonium acetate buffer (10mM, pH 5.0) and methanol. The UPLC method was expanded to include UPLC-quadrupole time-off light tandem mass spectrometry for the characterization of degradation products. PDA detected at 284nm.¹⁰

CONCLUSION:

In summary, this review underscores the crucial role of chromatography techniques in pharmaceutical analysis, highlighting their versatility and importance in ensuring reliable analytical procedures. The focused analysis of Alcaftadine methods exemplifies the evolving landscape of pharmaceutical research. The constant emphasis on validation and the exploration of analytical methods underscore the adaptability and significance of chromatography in advancing drug development. This review provides valuable insights for researchers and practitioners, encapsulating the essence of analytical techniques in pharmaceutical research. On the basis of these review data we have found that UHPLC method is not available till now. So thats why we select UHPLC for method development.

REFERENCES:

1. Lodha, L.; Sharma, Neha; Viswas, Amit; Khinchi, M. P. A Review on Chromatography Techniques. AJPRD. 2017; 5(2): 1–8.

2. Mishra, P. R.; Inamdar, P.; Jamdar, P.; Patel, N.; Rohit, M.; Satone, D.; Meshram, D. B. Development and Validation of Uv-Spectroscopic Methods For The Estimation Of Alcaftadine In Bulk And Its Ophthalmic Dosage Form. 2015; 4 (1).

3. Rina, R.; Baile, M.; Jain, A. A Review: Analytical Method Development and Validation. Systematic Review Pharmacy. 2021; 12 (8): 450–454.

4. Mishra, P. R.; Inamdar, P.; Jamdar, P.; Patel, N.; Rohit, M.; Satone, D.; Meshram, D. B. Development and Validation of UV- Spectrophotometry and The First Order Derivative Using The Area Under Curve Method For The Estimation Of Alcaftadine In Bulk And Its Ophthalmic Dosage Form. 2015; 4 (1).

5. Pr, M.; D, S. Development and Validation of HPLC Method for the Determination of Alcaftadine in Bulk Drug and Its Ophthalmic Solution. J Chromatogr Sep Tech. 2016; 7(1). https://doi.org/10.4172/2157-7064.1000312.

6. Pundlik, K. M.; V. Shelke, Prof. A.; Jadhav, A. G. Development and Validation of RP-HPLC Method for Estimation of Alcaftadine in Bulk Drug and Dosage Form. IJPSRR. 2022: 134–139. https://doi.org/10.47583/ijpsrr.2022.v76i01.024.

7. Mali, B. J.; Wagh, R. D. Development and Validation of Hptlc Method for Estimation of Alcaftadine in Bulk and Its Formulation. Indo American Journal of Pharmaceutical Sciences. 2021; 8(1): 2267–2273.

8. Abdel Razeq, S. A.; Abdel Aziz, S. E.; Ahmed, N. S. Stability-Indicating UPLC, TLC-Densitometric and UV-Spectrophotometric Methods for Alcaftadine Determination. Journal of Chromatography B. 2021; 1177: 122804. https://doi.org/10.1016/j.jchromb.2021.122804.

9. Patel, I.; Mehta, D. N.; Dalwadi, D. P.; Dharu, N. Stability Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Alcaftadine and Ketorolac Tromethamine in Pharmaceutical Dosage Form. International Journal of All Research Education and Scientific Methods. 2021; 9 (5).

10. Chavan, B. B.; Vijaya Jyothi, P.; Kalariya, P. D.; Srinivas, R.; Talluri, M. V. N. K. Alcaftadine: Selective Separation and Characterization of Degradation Products by LC–QTOF-MS/MS. Chromatographia. 2018; 81(4): 631–638. https://doi.org/10.1007/s10337-018-3489-1.

Received on 10.02.2024 Modified on 12.03.2024

Asian J. Pharm. Ana. 2024; 14(2):104-108.

DOI: 10.52711/2231-5675.2024.00018