INTRODUCTION:

Levocetirizine, the R-enantiomer of cetirizine dihydrochloride, has a negligible hepatic metabolism similar to that of cetirizine. Compared to cetirizine, Levocetirizine has double the affinity for H1 receptors. When used in patients with seasonal allergic rhinitis, it is effective and well tolerated. Levocetirizine exhibits advantageous pharmacodynamic and pharmacokinetic properties. Clinical trials have demonstrated that it is safe and effective, with few side effects, in the treatment of allergic rhinitis and chronic idiopathic urticaria in adults and children.¹

Fig.1 Structure of Levocetirizine HCl²

Third-generation, non-sedating, selective histamine H1 receptor antagonist Levocetirizine has antihistamine, anti-inflammatory, and maybe anti-angiogenic properties. When it comes to binding at peripheral H1-receptor sites on the effector cell surface, Levocetirizine and endogenous histamine compete. By doing this, the adverse effects of histamine release and an allergic reaction are avoided. Furthermore, as histamine is a key player in the angiogenesis that occurs during an allergic inflammatory response, inhibiting its activity may change the way pro-angiogenic factors are expressed, which in turn may stop angiogenesis. Levocetirizine is a third-generation histamine H1 receptor antagonist that is less likely to cause side effects than the majority of second-generation antihistamines.³ IUPAC name for Levocetirizine is 2-[2-[4-[(R)-(4-chlorophenyl)-phenylmethyl] piperazin-1-yl] ethoxy] acetic acid.³

Material identification, separation, and quantification for both natural and synthesized substances are methods used in analytical chemistry. The number of samples, the speed and expense of the analysis, the chemical properties of the analyte and its concentration sample matrix, and the kind of measurements (qualitative or quantitative) are only a few of the many variables that affect the choice of analytical methodology. A qualitative method can be used to identify the species' chemical identity in the sample. A quantitative technique provides numerical data regarding the relative concentrations of one or more analytes in the sample.⁴

Qualitative analysis is a method used in chemistry to determine the chemical composition of a sample.⁵

METHODS:

Materials and reagents: Okacet L 5mg tablets (Levocetirizine hydrochloride) provided by Cipla Ltd. as a gift sample, HPLC grade methanol, HPLC grade water.

Instrumentation and software: An Agilent 1260 Infinity II HPLC system with a quaternary pump was used. The column used as the stationary phase for the separation was Phenomenex C18 with dimensions of (250mm x 4.6mm i.d.) 5μm and an oven temperature of 40°C. The mobile phase used was Methanol: Water (80:20) with an optimized flow rate of 1.0ml/min, covering a run time of 8 minutes. The variable wavelength used for the PDA detector was set at the wavelength of 230nm.

Preparation of mobile phase: Water and methanol make up the mobile phase in 80:20 ratios. After 15 minutes of sporadic shaking, the mixture was sonicated. To facilitate additional analysis, this solution was employed in a mobile phase.

Selection of analytical wavelength

Selection of solvent: Water was selected as the solvent for dissolving Levocetirizine HCl.

Preparation of standard stock solutions: In order to prepare the stock solution, accurately weigh 10 mg of Levocetirizine HCl and transfer it into a 20-ml volumetric flask. Add 15ml of water, sonicate to dissolve the standard completely, and dilute up to the mark with water (500 PPM). Further, the solution was diluted from 1mL to 25mL with water. (20 PPM)

Selection of analytical wavelength: Water as a blank and Levocetirizine HCl Standard Solution (20 PPM) were scanned from 400nm to 200nm. Absorption maxima were determined for the drug. Levocetirizine HCl showed maximum absorbance at 230nm, as shown in the results.

Method Development by RP– HPLC:

Preparations of solutions:

Preparation of standard stock solution for Chromatographic development: Levocetirizine HCl A standard stock solution was prepared by transferring 10mg of Levocetirizine HCl into a 20-mL clean and dried volumetric flask, adding about 15mL of water to dissolve it completely, and making the volume up to the mark with water. (500 PPM). Further diluted 2mL of stock solution to 10 mL with water. (100 PPM).

Selection of analytical wavelength for HPLC method development: The analytical wavelength for the examination was selected from the wavelength of maximum absorption from the spectrophotometric analysis, which was 230nm.

Preparation of standard stock solution for system suitability test: Weighed about 10mg of Levocetirizine HCl and transferred it into a 20mL volumetric flask. Add 15mL of water, sonicate to dissolve it, and make the volume up to the mark with water. Pipette out 0.8ml of the standard stock solution and transfer it into a 20ml volumetric flask. Make the volume up to the mark with water. (20 µ/mL 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 injections of standard drug solutions, and the results were recorded.

Preparation of sample stock solution for marketed test sample: Weighed 20 tablets were transferred to a mortar and pestle and crushed to a fine powder. Mixed the contents with butter paper uniformly. Weighed the powder material equivalent to 20mg of Levocetirizine HCl and transferred it to a clean and dried 50 mL volumetric flask. I added 35 mL of water 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 water. Filtered the solution through a suitable 0.45µ syringe filter, discarding 3-5mL of the initial filtrate. Further diluted 1ml of filtered stock solution to 20ml with water. 20mcg of Levocetirizine HCl), injected the resultant solution, and chromatograms were recorded, and results are recorded.

Chromatographic Conditions:

Detector: U.V. Detector

Column: Phenomenex C18,

Column Dimension: (250mm X 4.6mm i.d.) 5μm

Column Oven temperature: 40°C

Injection Volume: 20μl

Wavelength: 230nm

Water: Methanol: Water (80:20)

Flow Rate: 1.0ml/min

Validation of RP-HPLC Method:

According to ICH criteria for the parameters listed below, the developed method for Levocetirizine HCl estimation was verified.

1. Filtration Study: In order to confirm that a pharmaceutical sterile filtering procedure truly removes all germs under actual processing conditions, it is necessary to evaluate the effect of the liquid on the filter.⁶

Analysis procedures that involve filtration studies verify that the filter is compatible with the sample, that it does not accumulate on the filter bed, and that it interferes with extraneous components from the filter. The test material for this investigation was a tablet solution containing Levocetirizine hydrochloride. Test solutions, both filtered and unfiltered, were used in the filtration experiments. 5 milliliters of the aliquot sample were discarded in order to use 0.45µm PVDF and 0.45µm Nylon syringe filters for the filtration activity.

2. Stability of Analytical Solution: Stable sample solutions can be injected directly from samples or matrix, requiring proper storage at room temperature and chilling according to the prescribed methodology.⁷

For both the standard and test sample solutions, a stability analysis was done. Under standard laboratory settings, a stability investigation was carried out. After 12 and 24 hours, the solution was examined under standard laboratory lighting conditions. The difference between the test solution's results at each stability time point and the starting results was calculated to conduct a standard and test solution stability study.

3. Specificity: Specificity refers to the ability to accurately assess an analyte in the presence of potentially predicted components such as matrix, degradants, and contaminants.⁸

Accurately obtaining the analyte in the presence of potentially anticipated components is known as specificity. To demonstrate the specificity of the technique, the following solution needs to be made and injected: (Test sample and standard solution peak purity were checked.)

· Blank (Water as Blank)

· Placebo

· Levocetirizine HCl Standard solution

· Tablet test sample solution

4. Placebo solution preparation: A 50-mL volumetric flask that had been cleaned and dried was filled with 477.20 mg of placebo material, which is equal to 20 mg of Levocetirizine HCl. I sonicated for 10 minutes while shaking occasionally after adding 35 mL of water. Ten minutes later, let the solution cool to room temperature and add water to get the volume up to the appropriate level. Using an appropriate 0.45 µ PVDF syringe filter, filter the solution; discard 3–5 mL of the filtrate initially obtained. Chromatograms were recorded after injecting the resulting solution after further diluting 1 ml of the filtered stock solution with 20 ml of water.

5. Linearity and Range: Linearity: An analytical method's linearity refers to its ability to produce test results that are directly or proportionate to the analyte concentration within a specified range.⁹

6. Range: The analytical method's range refers to the concentration range in the sample where the procedure's linearity, precision, and accuracy are suitable, often determined by linearity studies.¹⁶

7. Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The limit of detection (LOD) is the lowest concentration at which a method can reliably identify an analyte within a matrix, also known as the separation from background noise.¹⁰

The Limit of Quantitation (LOQ) is the minimum concentration at which bias and Imprecision can be controlled while maintaining reliable analyte detection.¹¹

Detection limit: The lowest concentration of analyte in a sample that can be identified but may not be precisely measured is known as the detection limit of a particular analytical technique.

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.

Accuracy (% Recovery): The accuracy of an analytical method is assessed by comparing test results to their true value, with percent recovery indicating the accuracy of the analytical process.¹²

When a value is determined and recognized as either a conventional true value or an acceptable reference value, the degree of agreement between the two values is expressed as the accuracy of the analytical technique. Between 50% and 15% of the working concentration will be used to test accuracy. We prepared the solution in triplicate for every accuracy level computed mean percent recovery for every level, overall recovery, and percentage recovery for every sample. Additionally, percentage RSD for every level and percentage RSD for the total recovery were computed.

Precision: The degree of agreement between a set of measurements taken during successive samplings of the same homogenous test conducted under the specified conditions is expressed as the precision of an analytical method. Repeatability and intermediate precision are the two types of precision. The test sample is a pill.

Repeatability: Repeatability in analytical processes is determined by covering a defined range of nine determinations, including three concentrations and three replicates, at 100% of the test concentration.¹³

Intermediate precision: Intermediate precision, also known as within-laboratory or within-device, is a measure of precision measured under specific conditions like the same procedure, system, location, and repeated measurements.¹⁴

Robustness: An analytical procedure's dependability under typical operating conditions and its ability to remain unaffected by slight modifications in method parameters is a measure of its reliability.¹⁵An analytical procedure's robustness indicates how reliable it is under typical operating conditions and can withstand intentional, modest changes in method parameters.

RESULTS:

UV spectrum of Levocetirizine HCl Method Development by RP – HPLC

Fig.2 UV spectrum of Levocetirizine HCl

Fig.3 Optimized Chromatograph

Results for System Suitability Test of Levocetirizine HCl:

The plain evaluation of five standard solutions showed that the average figures are the same, with a mean area under the peaks of 14, 553, 320 and an asymmetry of 1.36. The theoretical plates were 7965, thereby suggesting that the system provides excellent separation, and the chromatography efficiency is also considerable. This research has fully established the dependability and perfection of chromatographic methods.

VALIDATION OF RP-HPLC METHOD:

1. Filtration Study: The tablet sample test showed varying impacts on chromatographic analysis based on filter types. A 0.45µ PVDF filter cut the area to 14, 366, 925 units. A 0.45µ Nylon filter shrank it further to 14, 313, 625 units. This means a drop of 0.82 percent and 1.18 percent, respectively.

2. Solution Stability: The sample solution's concentration fell from 14, 532, 036 to 14, 395, 603 units in area over 24+ hours. The standard solution dipped from 14, 596, 810 to 14, 475, 823 units. This equals a decrease of 0.94% and 0.83%, respectively.

3. Specificity: Blank and placebo didn't mess with the retention time. The standard solution peak purity hit 0.997. The test solution lagged a bit at 0.994.

4. Linearity and Range: Scientists pitted concentration levels (10%, 50%, 100%, 125%, and 150% µg/mL) against their peak areas in the chromatogram to check linearity.

Summary HPLC linearity of Levocetirizine HCl: The beer's linearity range for this parameter is 2.0–30.0 µg/mL, with the corresponding correlation coefficient being 0.99997; this value is beyond the set criterion of 0.98. Uncombined intercept and slope values share the CV limitation, and they are only thrown out when CV > 1%. Chromatographic analysis is helpful.

Fig.4 Calibration curve of Levocetirizine HCl

1. Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ =66600.36324 (Residual standard deviation of a regression line)

s = 713548.823 (Slope)

Detection limit (LOD): LOD = 0.308 µg/mL

Quantitation limit (LOQ): LOQ = 0.933 µg/mL

2. Accuracy (Recovery): Results were 10.18-10.21 with 10.20-10.10 added. At 100% 19.66-20.14 with 98.74% recovery and 0.4616% RSD.

3. Precision: The summary analytical data is presented for intra-day precision with an average assay percentage of 98.19%, intermediate precision of 1.239%, and inter-day precision of 98.76%. All tests combined to give an assay percentage of 98.478% with a combined standard deviation of 1.23962% and a relative standard deviation of 1.259%.

4. Robustness: Experimental changes included wavelength variation, adjustment of flow rate, and column oven temperature change. The following results for pregnancy are presented: The wavelength variation increased by 2.3 nm, the flow rate was adjusted by 1.1 mL/min, the column oven temperature changed by -2 °C, and the retention time decreased by 0.3 ms. The experimental results deviated a lot from the performance of the system.

CONCLUSION:

In the field of pharmacological analysis, RP-HPLC was developed for estimating levocetirizine HCl. The method was developed to be very robust, accurate, and specific; therefore, it includes pain-taking procedures for material identification, separation, and quantification in compliance with the prescribed programs of ICH-compliant validation. All technique-specificity, peak purity results, low quantitation and detection limits, and high sensitivity have been assessed properly. The method's intermediate precision and its repeatability were also checked. The reliability against changes in chromatographic factors makes routine analysis beneficial. RP-HPLC methods are reliable for pharmaceutical research and quality control in different experimental settings.

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16. Validation of Analytical Methods Document ID IPC/GD/04 Version 1.0 Issue Date 16 th September 2021 https://ipc.gov.in/images/GD-04-Method_Validation.pdf

Received on 05.07.2024 Revised on 09.09.2024

Accepted on 17.10.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):256-260.

DOI: 10.52711/2231-5675.2024.00046