RP-UPLC METHOD DEVELOPMENT AND VALIDATION FOR QUANTITATIVE ESTIMATION OF LEVOCETIRIZINE IN LEVOCETIRIZINE DIHYDROCHLORIDE TABLETS

RP-UPLC METHOD DEVELOPMENT AND VALIDATION FOR QUANTITATIVE ESTIMATION OF LEVOCETIRIZINE IN LEVOCETIRIZINE DIHYDROCHLORIDE TABLETS

S. Shetgar *, D. Ramadevi, B. M Rao, K. Basavaiah

Department of Inorganic and Analytical Chemistry, Andhra University, Visakhapatnam, Andhra Pradesh, India.

ABSTRACT: Current analytical research and experiments provide the development of a simple, sensitive, accurate, rapid, and stability indicative RP-UPLC method for estimation of Levocetirizine in Levocetirizine dihydrochloride tablets. This development was achieved using a Waters Acquity UPLC with BEH, C18 column of dimensions of 100 mm × 2.1 mm, 1.7 µm column at 0.3 mL/min flow rate and Acquity TUV detector at 230 nm. This method was validated based on the guidelines of the International Council on Harmonization (ICH). Linearity was demonstrated at 25% to 150% levels with R² value of 0.999. Precision and accuracy were in line with the ICH guidance with a mean recovery of 99.02%. The RP-UPLC method is sensitive with levels of Limit of detection (LOD) and Limit of Quantitation (LOQ) at 0.04µg/mL and 0.12µg/mL, respectively. Degradation studies in conditions of Oxidation, Acid, Base, Temperature, Water, UV light demonstrate there is no placebo as well as degradation impurities interfering with the Levocetirizine main peak. The simple, sensitive, accurate, rapid, and stability-indicating method makes it an efficient tool in routine quality control testing of the active pharmaceutical ingredient as well as in formulations.

Keywords: Levocetirizine, RP-UPLC, Rapid, Sensitive, Stability indicating

INTRODUCTION: Levocetirizine comes under the class of antihistamine and is used to provide relief to patients who show symptoms such as sneezing, running nose, itching, and hives.

On administration, the Levocetirizine Dihydrochloride selectively blocks histamine produced by the body when an allergic reaction occurs. Levocetirizine Dihydrochloride, Fig. 1, is levorotatory enantiomer of Cetirizine with IUPAC name 2- [2-[4-[(R)-(4-chlorophenyl) - phenyl (methyl)] piperazin-1-yl] ethoxy] acetic acid; dihydrochloride. Literature review for analytical estimations reveals that different analytical methods have been used for the determination of Levocetirizine. Most of these methods are for simultaneous estimations with other drug combinations using the HPLC, HPTLC, and one RP-UPLC method ^1-8. In the literature review, came across one RP-UPLC method for simultaneous estimation of Levocetirizine and Montelukast sodium. However, it was developed for a liquid dosage form using a phenyl column with gradient elution. The objective of this study is to develop a simple, sensitive, accurate, stability-indicating and quick method for estimation of Levocetirizine in bulk as well as Levocetirizine tablets and validate it as per International council of harmonization (ICH) guidance so that it can be applied for routine analysis in the testing laboratories.

FIG. 1: STRUCTURE OF LEVOCETIRIZINE DIHYDROCHLORIDE

MATERIALS AND METHODS:

Materials: Levocetirizine dihydrochloride pure drug (API) and Levocetirizine Capsules (Xyzal) were obtained from Dr. Reddy’s Laboratories. Other reagent and solvents like acetonitrile, methanol, potassium dihydrogen ortho phosphate buffer, ortho-phosphoric acid, glacial acetic acid was procured from Rankem and are of analytical reagent grade. Water for UPLC was obtained through the Milli-Q laboratory water generation system.

Equipment Used: Waters -Acquity UPLC (Ultra performance Liquid Chromatograph system with Auto Injector and Tunable UV Detector. Software used is Empower 2, Sonicator (Ultrasonic), pH meter (Thermo scientific), Micro balance (Sartorius), Vacuum filter pump.

Method:

Chromatographic Conditions: The chromatographic system used was the Waters Acquity UPLC with BEH C18 column of dimension 100 mm × 2.1 mm, 1.7 µm.

The mobile phase comprised of 0.01N Potassium dihydrogen phosphate (KH₂PO₄) buffer and Acetonitrile in the ratio of 60:40 (pH 4.80). The flow rate was set at 0.3 mL/min with 2 μL injection volume and column oven temperature of 30 °C.

The diluent used was a mixture of 0.01N KH₂PO₄ buffer and Acetonitrile in the ratio 50:50. Detection was achieved with an Acquity Tunable UV detector at 230 nm. Instrument operation, data acquiring, and processing were done using Empower 2 software.

Mobile Phase and Solution Preparation: Preparation of 0.01N KH₂PO₄ Buffer (pH-4.8): 1.36 g of potassium dihydrogen phosphate was diluted to 1000 mL with Milli-Q grade water to get 0.01N KH₂PO₄ buffer.

Preparation of Mobile Phase: Mixture of 0.01N KH₂PO₄ Buffer (pH-4.8) and acetonitrile in the ratio of 60:40

Preparation of Diluent:  Based upon the solubility of the drug, diluent was selected. 0.01N KH₂PO₄ Buffer and Acetonitrile were taken in the ratio of 60:40.

Preparation of Levocetirizine Standard stock solutions: Standard stock solution of 50 µg/mL was prepared by accurately weighing 2.5 mg of Levocetirizine Dihydrochloride in a 50 mL volumetric flask. About 30 mL of the diluent was added and sonicated for 10 min. The solution was made up to 50 mL with diluent.

Preparation of Levocetirizine Standard working solutions (100% Solution): A 5 µg/mL of Levocetirizine Dihydrochloride was prepared by diluting 1 mL of Levocetirizine Dihydrochloride stock solution to 10 mL with the diluent.

Preparation of Levocetirizine Sample Stock Solutions: 10 tablets were weighed and powdered. Then the average weight of each tablet was calculated (100.41 mg).

Weight equivalent to 1 tablet was transferred into a 100 mL volumetric flask, 50 mL of diluent was added and sonicated for 25 min, further, the volume was made up with diluent and filtered using 0.45 µ PVDF filters to get a solution of 50 µg/mL of Levocetirizine Dihydrochloride.

Preparation of Levocetirizine Sample working solutions (100% solution): 1 mL of filtered sample stock solution was transferred to 10 mL volumetric flask and made up with diluent to get a 5 µg/mL of Levocetirizine Dihydrochloride.

RESULTS AND DISCUSSION:

Optimization of Sample Preparation: The sample preparation needs to ensure complete extraction from the formulation mix. Tablet was finely powdered. Based on the solubility of the Levocetirizine, acetonitrile and potassium dihydrogen phosphate buffer combination were assessed and optimized for efficient extraction.

The filter used was the standard 0.45µ PVDF filter which didn’t demonstrate any retention of the drug on the filter.

Optimization of Chromatographic Conditions: The sensitivity of the UPLC method depends upon the selection of detection wavelength. An ideal wavelength is one that gives a good response for all impurities and analyte peaks to be detected.

The wavelength for measurement was selected as 230 nm from the absorption spectrum considering the UV maxima shown by Levocetirizine dihydrochloride in Fig. 2.

FIG. 2: UV SPECTRUM OF LEVOCETIRIZINE DIHYDROCHLORIDE

Various UPLC columns like X-Bridge, Hibar, HSS, BEH 100 mm lengths were evaluated. Mobile phase options of Water and Methanol mixture, 0.1% orthophosphoric acid and methanol mixtures, 0.1% orthophosphoric acid and acetonitrile mixtures and mobile phase combination of 0.01N potassium dihydrogen phosphate and acetonitrile mixture were evaluated.  The use of water and methanol mixture was not satisfactory due to peak split and baseline disturbance. 0.1% orthophosphoric acid and methanol also did not give appropriate peak shapes.

However, 0.01N potassium hydrogen phosphate acetonitrile combination gave good peak shape as well as quick peak elution.  Different compositions of 0.01N potassium dihydrogen phosphate buffer and acetonitrile mixtures were evaluated at 50:50; 55:45; and 60:40 ratios to arrive at the optimum parameters. The final optimized condition was achieved using Waters Acquity BEH C18 column of dimensions 100 mm × 2.1 mm, 1.7 µm. with a flow rate of 0.3 mL/min with column oven temperature 30 °C and mobile phase consisting of 0.01N KH₂PO₄ buffer (pH 4.8) and Acetonitrile in the ratio of 60:40. Levocetirizine peak eluted well below 2 min, making it a quick method and hence the possibility to run multiple samples.

Method Validation: For the newly developed RP- UPLC method, validation was performed according to the ICH guideline for the analytical parameters such as system suitability test, specificity through degradation study of Levocetirizine tablets and placebo, linearity and range, precision, accuracy, the limit of detection (LOD), the limit of quantitation (LOQ) and robustness to demonstrate the adequacy of the method.

System Suitability Parameters: The system suitability parameters were determined by preparing standard solutions of Levocetirizine dihydrochloride (5 µg/mL) as six replicates and the parameters like Relative standard deviation (RSD), peak tailing and United States Pharmacopeia (USP) plate count were determined. The RSD was below

2% Retention time variation was observed in range 1.670-1.681 min with RSD of 0.2%.  Plate count was in the range 5004 to 5093 with RSD of 0.7%. The tailing factor was in the range 1.56 - 1.57, with an RSD of 0.3%. Each of these meets the USP requirements and demonstrates good chroma-tographic practice. System suitability parameters such as RSD of six replicate dilute standard injections, Number of theoretical plates, and tailing factor for Levocetirizine are tabulated under Table 1.

TABLE 1: SYSTEM SUITABILITY PARAMETERS

Specificity: Specificity for the RP-UPLC method was demonstrated by no interference from the blank, placebo and potential degradation impurities from the degradation study conducted in line with the ICH guidance. The chromatogram was reviewed for any interference from the blank, placebo, and potential degradation impurities. There were no interferences at the retention time of the levocetirizine peak. The % degradation is tabulated in Table 2.

TABLE 2: DEGRADATION DATA FOR LEVOCETIRIZINE TABLETS

Chromatograms for acid, base, oxidation, thermal, UV light, Water are shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 respectively. There were no interferences at the retention time of the Levocetirizine peak.  Chromatograms of Levocetirizine capsules placebo and Levocetirizine standard are shown in Fig. 9 and Fig. 10. respectively.

FIG. 3: ACID DEGRADATION                                                   

FIG. 4: BASE DEGRADATION

FIG. 5: OXIDATION DEGRADATION                                       

FIG. 6: THERMAL DEGRADATION

FIG. 7:  UV DEGRADATION                                                

FIG. 8:  WATER DEGRADATION

FIG. 9: FORMULATION PLACEBO                                            

FIG. 10: LEVO STANDARD

Precision: The method precision of the RP-UPLC assay method as part of repeatability was checked by injecting six replicates of Levocetirizine sample solutions of concentration 5µg/mL. The % amount of Levocetirizine dihydrochloride found was calculated, and RSD was found to be 0.6%. Tabulated details are shown in Table 3.

Six sample solutions of concentration 5 µg/mL of Levocetirizine into the chromatograph in the optimized chromatographic conditions after 24 h of preparation. The % amount of Levocetirizine found was calculated, and RSD was found to be 0.3%. Tabulated details are shown in Table 4.

TABLE 3: METHOD PRECISION

TABLE 4: INTERMEDIATE PRECISION

TABLE 5: LEVOCETIRIZINE LINEARITY DATA

Linearity and Range: To demonstrate the linearity and range of Levocetirizine Dihydrochloride RP-UPLC assay method, Levocetirizine standard solution of concentration 5 µg/mL was prepared. Using this stock, six standard solutions with concentrations at 25%, 50%, 75%, 100%, 125% and 150% corresponding to range 0 µg/mL 1.25 µg/mL, 2.5 µg/mL, 3.75 µg/mL, 5 µg/mL, 6.25 µg/mL and 7.5 µg/mL of Levocetirizine dihydrochloride were prepared and injected in the RP-UPLC system with the optimized chromato-graphic condition. Concentration versus peak area was plotted. The slope obtained was 57923, Y-Intercept was at 1652.2 and Correlation Co-efficient was found to be 0.9998, indicating it is linear across the range. Details of concentration and peak area are shown in Table 5. The linearity plot is shown in Fig. 11.

FIG. 11: LEVOCETIRIZINE LINEARITY PLOT

Accuracy: To demonstrate the accuracy of the Levocetirizine dihydrochloride RP-UPLC assay method Levocetirizine sample solution of concentration 5 µg/mL was prepared by taking one tablet equivalent of the powdered Levocetirizine tablet contents followed by sonication with the diluent and filtration.  Known fixed quantities of Levocetirizine Standard stock solution were spiked onto Sample solution at levels of 50%, 100%, and 150%.    Each of the sample solution spiked with the known concentration of the standard (50%, 100%, 150% of Levocetirizine dihydrochloride) was injected in triplicate in the optimized chromatographic system, and % recovery was in the range 99.46% to 101.58%, 98.13 - 99.31% and 100.20 - 101.11% at 50%, 100%, and 150% respectively. The average recovery was observed to be 99.89%. Detailed Levocetirizine Accuracy data is tabulated as Table 6.

TABLE 6: LEVOCETIRIZINE ACCURACY

Limit of Detection (LOD) and Limit of Quantitation (LOQ):  The LOD and LOQ were established by injecting dilute solutions of Levocetirizine dihydrochloride using the signal to noise ratio approach. The LOD and LOQ values were observed to be at 0.04 µg/mL and 0.12 µg/mL, respectively. Chromatograms for LOD and LOQ are shown in Fig. 12 and 13. respectively.

FIG. 12: CHROMATOGRAM FOR LIMIT OF DETECTION

FIG. 13: CHROMATOGRAM FOR LIMIT OF QUANTITATION

Robustness: Robustness of the Levocetirizine dihydrochloride RP-UPLC Assay method was demonstrated small, deliberate changes in the method were made which could potentially occur in a laboratory environment by going on either side of the chromatographic parameters like Flow (+0.3 ml/min); Mobile phase composition (+2%); Temperature (+5 °C). Under each of these conditions, sample preparations were injected in duplicate into the optimized chromatographic conditions with the above changes. The system suitability parameters and amount of the Levocetirizine dihydrochloride content were calculated and found comparable.  The RSD was in the range of 0.7% to 1.3% for content when assessed with the deliberate changes in the chromatographic parameters.  Details are tabulated in Table 7.

TABLE 7: ROBUSTNESS - DELIBERATE CHANGES

*Note: B-Buffer; A-Acetonitrile

Assay of Marketed Formulation: Assay of the Levocetirizine Dihydrochloride tablets batch was performed as per the validated   RP-UPLC assay method. Standard and sample solutions were prepared at a concentration of 50 µg/mL of Levocetirizine dihydrochloride and chromato-graphed. Six sample sets were prepared and assayed.

The results were observed to be 99.02 %, with RSD at 0.59%. The area count, content, and RSD are tabulated in Table 8.

TABLE 8: ASSAY OF THE MARKETED FORMULATION

FIG. 14: CHROMATOGRAM OF LEVOCETIRIZINE STANDARD

FIG. 15: CHROMATOGRAM OF THE LEVOCETIRIZINE SAMPLE

Chromatograms of typical standard and sample are shown in Fig. 14 and Fig. 15 respectively.

CONCLUSION: In conclusion, a simple, sensitive, accurate, and quick method was developed for the estimation of Levocetirizine Dihydrochloride in Levocetirizine Dihydrochloride Tablets by RP-UPLC technique. System suitability parameters were studied by injecting the standard six times, and results were well under the acceptance criteria. Linearity study was carried out between 25% to 150% levels, R² value was found to be > 0.999. Precision was found to be at an RSD of 0.6% for repeatability and 0.3% for intermediate precision. LOD and LOQ were established at 0.04 µg/mL and 0.12 µg/mL, respectively. Recovery of Levocetirizine was found to be 99.89%. By using the validated RP-UPLC Assay method, the marketed formulation was tested, and the content of Levocetirizine dihydrochloride was observed to be in the range of 98.60 to 99.76%.  Degradation studies of Levocetirizine conducted on lines on ICH demonstrated that the Levocetirizine peak purity thresholds were more than the purity angle. While the method is suitable for the estimation of Levocetirizine Dihydrochloride for routine and stability testing, it can also be used for estimation of Levocetirizine residues after cleaning of equipment and can be extended for impurity estimation in quality controls laboratory.

ACKNOWLEDGEMENT: The author thanks Dr. Reddy’s Laboratories for providing gift samples of Levocetirizine dihydrochloride and Levocetirizine dihydrochloride tablets.

CONFLICTS OF INTEREST: The authors have no conflict of interest to declare.

REFERENCES:

1. J. Bharati, B. Chinnappa Naidu, M. Sumanth and Nagaraju Rajana: A rapid, RP-UPLC assay method for simultaneous determination of montelukast sodium and levocetirizine dihydrochloride in pharmaceutical dosage forms. World J Pharm Pharm Sci 2015; 4(11): 1409-21
2. Devangi K. Patel, Amit J. Vyas, MN. Noolvi, Ashok B. Patel and Nilesh K. Patel: Estimation of four drugs: Ambroxol hydrochloride, Levocetirizine hydrochloride, Phenylephrine hydrochloride and Paracetamol by RP-HPLC in tablet dosage form. IJPCA 2018; 5(1): 31-38
3. Jitendra K Sonawane, Dilip A Patil, Balasaheb S Jadhav, Satish L Jadhav and Prashant B Patil: Stability Indicating RP-HPLC method development and validation for simultaneous quantification of antihistaminic & anti-asthmatic drug in bulk and tablet dosage form. J Pharm 2020; 8(1): 12-22.
4. Anna Pratima G, N Ramesh Gadikar and Shankar Turwale: A simple stability indicating HPLC method for simultaneous determination of levocetirizine dihydrochloride, phenylephrine hydrochloride and paracetamol in pharma.   IJPSR 2019; 10(5): 2497-03
5. Venkatakrishna Akula, Barij Nayan Sinha and Han Jeong Seok: Stability indicating hplc method using core shell stationary phase for the determination of related substances in levocetirizine dihydrochloride oral solution. IJPER 2018; 52(4): 684-90.
6. N Jayasimha, V Krishna Reddy and E Sasi Kiran Goud: Development and validation of RP-HPLC method for simultaneous determination of montelukast sodium and levocetirizine dihydrochloride tablet. Der Pharmacia Sinica 2015; 6(9): 8-14.
7. P Srividya, M Tejaswini, D Shanvanti and Bachu N Nalluri: Simultaneous analysis of levocetirizine dihydrochloride, ambroxol hydrochloride, and montelukast sodium by RP-HPLC - PDA method. J Liq Chromatogr Relat Technol 2013; 36: 2871-81.
8. Sushma Somkuwar and A. Pathak: Simultaneous estimation of levocetirizine dihydrochloride and montelukast sodium by. RP-HPLC Method Pharmacia 2012; 1(3): 90-94.
   

   ---

   How to cite this article:

   Shetgar SS, Ramadevi D, Rao BM, Basavaiah K: RP-UPLC method development and validation for quantitative estimation of levocetirizine in levocetirizine dihydrochloride tablets. Int J Pharm Sci & Res 2021; 13(2): 987-95. doi: 10.13040/IJPSR.0975-8232.13(2).987-95.

   ---

   All © 2022 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.