INTRODUCTION:

Cetirizine dihydrochloride (CZ) is (RS)-2-[2-[4-[(4- chlorophenyl) phenyl methyl] piperazine-1-yl] ethoxy] acetic acid dihydrochloride and is a long acting non-sedating antihistamine drug with some mast-cell stabilizing activity. CZ is therapeutically applied for the symptomatic relief of allergic conditions including rhinitis and chronic urticaria¹. Basically CZ was assayed in bulk material by acid base titration^2,3 and HPLC^4,5. In various pharmaceutical formulations^6,7–10 CZ was assayed by Various HPLC methods where C18 columns of different brands were used. The mobile phases mainly consisted acetonitrile and phosphate buffer of different pH values. The detection was carried out by UV detection at 230 nm or 254 nm. Other techniques such as ultraviolet spectrophotometry^6,11,12, spectroﬂuorimetry^13,14, calorimetry^6,14-17 and ion- selective electrodes¹⁸ were also used for CZ assay in pharmaceutical formulations . Studies also reported TLC and HPTLC methods for the determination of CZ as a bulk material and in formulations, respectively^19,20]. But there were very limited methods reported in literature for the determination of CZ related impurities^3,21. Although The European Pharmacopoeia depicted a method³ for the determination of CZ related impurities, namely: A, B, C, D, E and F. The chromatographic system used in this method was equipped with a column of 250 × 4.6mm packed with 5 µm silica gel and the mobile phase was a mixture of dilute sulfuric acid: water: acetonitrile (0.4:6.6:93, v/v/v) with the ﬂow rate set at 1ml/min and the detection made at 230 nm. A grown body of literature demonstrated several disadvantages of this particular method such as rapid column deterioration at lower ph of the mobile phase (<0.5).

Another method²¹ was also developed and validated to determine all impurities mentioned in the European pharmacopoeial method³. This particular method involved the use of LC/MS technique equipped with a cyano-column coupled to an electrospray ionization mass spectrometer and was expensive.

Thus, this present work aimed at developing a simple HPLC method for simultaneous determination of CZ and its related impurity A in the presence of formulation excipients either in solution or solid formulations based on isocratic system and a commonly used UV detector. In this study the most common CZ related impurity A was focused only.

MATERIALS AND METHODS:

Materials and equipment:

CZ, CZ related impurity A and all other formulation excipients were all provided by the Alembic Pharmaceutical Manufacturing Company. In house tablets (10mg) and solutions were used for the study. All other chemicals were of HPLC or analytical grade and purchased from E. Merck (India) Ltd. Worli, Mumbai, India. The 0.45 µm nylon filters were purchased from Advanced Micro Devices Pvt. Ltd. Chandigarh, India. Double distilled water was used throughout the experiment. The chromatographic system was consisted of chromatographic system of Waters, Alliance System, UV/VIS Detector (Waters 2487), System Software (Waters Empower). Chromatographic separation was achieved on Hypersil SS C18 (250 mm × 4.6 mm, 5µm) analytical column.

Analytical solutions:

Mobile phase:

A degassed and ﬁltered mixture of 0.4 volumes of dilute sulphuric acid, 6.6 volumes of water and 93 volumes of acetonitrile (0.4:6.6:93, v/v/v) with a pH of about 5.5 was used as the mobile phase.

Standard reference solutions:

A 0.02 % w/v solution of CZ was prepared in 100ml of the mobile phase. Further, dilutions were carried out by diluting 1 ml of this solution to 100 ml with the mobile phase. Standard solution of 0.02 % of the impurity was prepared by dissolving accurately weighed amount of the CZ impurity A in the mobile phase followed by dilution of 1 ml of this solution to 100 ml with the mobile phase.

Solutions for testing linearity and accuracy:

Standard solutions used for testing the linearity of calibration plots for CZ were prepared by separately dissolving accurately weighed quantities of CZ (10–80 mg) in 100 ml of the mobile phase. Further, dilutions with the mobile phase were carried out to prepare another set of CZ concentrations in the range of 0.5–5 µg/ml. The solutions of CZ related impurity A were prepared by dissolving accurately weighed 5 mg of the component in 200 ml mobile phase followed by further dilutions to obtain solutions having concentrations ranging from 0.5 to 5 µg/ml. Samples of CZ and its related impurity A were prepared by transferring into three 100 ml volumetric ﬂask three quantities of CZ in the range of 25–75 mg and three quantities of impurity A, respectively, in the range of 0.1–0.4 mg, followed by additions of 50 ml of the solution’s drug-matrix or 1400 mg of the tablet’s drug-matrix. Mobile phase was then added to make the volume up to the mark. The samples containing the tablet’s drug-matrix were sonicated for 15 min centrifuged at 4000 rpm for 15 min and the supernatant was used for HPLC injections. Five milliliter portions of the ﬁnal solutions were separately transferred into 25 ml volumetric ﬂask followed by additions of 5 ml of the solution’s drug-matrix into each ﬂask and adjusting the volume up to the mark by the mobile phase.

Solutions for testing precision, stability and effects of method’s parameters:

Solutions of CZ and its related impurity A were prepared by transferring accurately weighed portions of CZ (20 mg) and related impurity (0.25mg) into 100 ml volumetric ﬂasks followed by 50 ml additions of the solution’s drug- matrix or 1400mg of the tablet’s drug-matrix. Mobile phase was then added to make the volumes up to 100ml. The above solutions were also used to study the effect of various method’s parameters and their stability was tested by storing at ambient conditions for 24 h.

Chromatographic procedure:

20 µl samples were injected into the chromatograph; the ﬂow rate was set at about 1ml/min and the HPLC chromatograms were recorded at a detector setting of 230 nm. The tailing factor for CZ peak found was not more than 2 and the relative standard deviation for replicate injections was not more than 2.0 %. The resolution between the peaks of CZ and impurity A was not less than 3.

RESULTS AND DISCUSSION:

Development of the HPLC Method:

During the development of the method, different parameters were manipulated to obtain an acceptable resolution between the analyte components with acceptable recoveries and to satisfy the HPLC system suitability. These parameters include: flow rate (0.8–1.2 ml/min), different types of C18 columns, 0.05 M phosphate buffers of pH ranging from 5.3 to 5.7, and various mobile phase ratios. The preliminary work was conducted by using a C18 column and binary mobile phases consisting phosphate buffer and acetonitrile. The UV detection at 230 nm was found to be more sensitive where it gave high absorptivities for CZ and its related impurity A. Binary mobile phase systems of different ratios were found not suitable due to a bad resolution and low recoveries observed for some CZ impurity A. No significant effect on resolution and recoveries was obtained by changing flow rate and column trade names. When a mobile phase of multicomponents was used a better resolution was demonstrated. Various mixtures of dilute sulphuric acid, acetonitrile and water of different ratios were tried. Finally A degassed and ﬁltered mixture of 0.4 volumes of dilute sulphuric acid, 6.6 volumes of water and 93 volumes of acetonitrile (0.4:6.6:93, v/v/v) with a pH of about 5.5 was used as the mobile phase. Thus, coupling of this mobile phase, a flow rate of 1ml/min and detection at 230 nm showed a significant resolution of CZ and impurity A.

Specificity:

The specificity was demonstrated by the HPLC chromatograms recorded for mixtures of CZ, CZ related impurity A dissolved in the mobile phase. Well resolved peaks for CZ and impurity A were observed (Figure 1 and 2) with relative retention times of 2.937 and 4.115, respectively.

Linearity and accuracy:

The linearity of calibration curves was tested for the determination of CZ and CZ related impurity A and were in the ranges of 0.5-5µg/ml and 0.5-5µg/ml, respectively. The linear regression parameter (correlation coefficient) were estimated and reported in Table 1. The linearity of the curves was better than 0.999. The accuracy of the method was tested at three concentration levels within each analyte target concentration and each concentration level was analyzed by three different analysts. The average percent recoveries, R.S.D. and bias were presented in the table 2 and 3.

Limit of detection (LOD) and limit of quantization (LOQ):

Calibration curve method was used for the determination of LOD and LOQ of CZ and CZ related impurity A. Solutions of both were prepared in the range of 0.5 – 5.0 µg/ml and injected in triplicate. Average peak area of three analyses was plotted against concentration. LOD and LOQ were calculated by using following equations.

Table 1. Linearity of the calibration plots for CZ and CZ related impurity A.

Compound	Calibration range (µg/ml)	r 2	LOD (µg/ml)	LOQ (µg/ml)
CZ	0.5 -5.0	> 0.999	0.01	0.03
Impurity A	0.5 -5.0	> 0.999	0.02	0.08

Table 2. Accuracy of the method for CZ.

Compound

Quantity (µg/ml)

Recovery (%)

Bias ^b (%)

Added

Found^a

(solution’s matrix)

Average ± R.S.D.

250

500

750

246.3

497.2

726.3

98.52

99.44

96.84

98.27 ± 1.32

-1.48

-0.56

-3.16

(tablet’s matrix)

Average ± R.S.D.

300

500

700

300.3

500.8

697.0

100.1

100.16

99.57

99.94 ± 0.32

0.1

0.16

-0.43

^aAverage of three individual results

^bBias = (% recovery – 100)

Table 3. Accuracy of the method for CZ related impurity A.

Compound	Matrix for solutions				Matrix for tablets
	Quantity (µg/ml)		Recovery (%)	Bias (%)	Quantity (µg/ml)		Recovery (%)	Bias (%)
	Added	Found			Added	Found
Impurity A Average ± R.S.D.	1.20 2.50 3.60	1.19 2.50 3.53	99.17 100.00 98.06 99.08 ± 0.97	-0.83 0.00 -1.94	1.50 2.50 3.40	1.49 2.51 3.29	99.33 100.4 96.76 98.83 ± 1.87	-0.67 0.4 -3.24

Table 4. Precision results for CZ and CZ related impurity A.

	Amount taken (µg/ml)	Matrix for solution			Matrix for tablets
		Analyst I	Analyst II	Overall % R.S.D.	Analyst I	Analyst II	Overall % R.S.D.
		Amount found ^a (µg/ml)	Amount found ^a (µg/ml)	Overall % R.S.D.	Amount found ^a (µg/ml)	Amount found^a (µg/ml)	Overall % R.S.D.
CZ	500	498.7 (0.7)	494.3 (0.8)	0.94	497.7 (1.1)	499.2 (0.8)	0.98
Impurity A	2.50	2.43 (3.60)	2.45 (1.90)	3.10	2.44 (2.1)	2.51 (0.8)	2.40

Table 5. Stability of solutions of CZ and CZ related impurity A.

Compound	Quantity added (µg/ml)	Standard preparations			Matrix for solution			Matrix for tablets
		Quantity found ^a (µg/ml)		% D	Quantity found ^a (µg/ml)		% D	Quantity found ^a (µg/ml)		% D
		Fresh solution	Stored solution			Stored solution		Fresh solution	Stored solution
CZ	500	496.3	498.8	- 0.5	494.6	498.7	- 0.8	498.8	496.0	0.56
Impurity A	2.50	2.50	2.38	4.8	2.50	2.32	7.2	2.50	2.47	1.2

Table 6. Robustness results for CZ and CZ related impurity A.

Parameter	Condition	CZ	Impurity A
Flow rate	0.8 ml/min	98.8	89.8
	1.0 ml/min	98.9	99.0
	1.2 ml/min	98.4	88.8
Mobile phase ratio	85: 6: 0.2	95.8	98.7
	93:6.6:0.4	98.9	96.7
	100:7:0.6	96.6	95.9
pH	5.3	95.4	97.6
	5.5	99.8	96.4
	5.7	97.6	101.5

Column type	New	99.2	99.2
Column type	Old	99.5	93.5

Wavelength (nm)	225	97.6	100.6
	230	99.4	99.5
	235	98.9	98.6

Filtration system	Centrifuge	100.2	101.3
Filtration system	Nylon filter	100.5	100.2

Sonication time (min)	10	100.4	100.7
	15	100.2	101.8
	20	100.8	99.8

Stability of solutions and robustness:

The stability of the solutions of CZ and CZ related impurity A dissolved in the mobile phase and in the absence (standard preparation) or the presence of the drug matrices (matrices for solutions and for tablets formulations) were tested over a period of 24 h. The freshly prepared and stored samples were analyzed and the results are reported in Table 5. The percent differences in concentrations observed for CZ and CZ related impurity A were in the ranges of - 0.8 to 0.56 and 1.2 to 7.2, respectively. This indicates the possibility of using all analyte solutions in either standard or synthetic drug-matrices over a period of 24 h without degradation. The optimum HPLC parameters set for this method were slightly changed for samples of CZ (500 µg/ml) and CZ related impurity A (2.5 µg/ml) prepared in the presence of the drug-matrix for solution’s formulations. The parameters include: flow rate, mobile phase ratio, pH, column age (old or new), wavelength of detection, filtration system and sonication time. Percent recoveries of CZ and CZ related impurity A obtained (Table 6) under the various conditions were within 95.4–100.8 % and 88.8–101.8 %, respectively. These results indicate the ability of the method to remain unaffected by small changes in the method’s parameters, thus the method is considered robust.

CONCLUSION:

A new HPLC method is proposed for simultaneous determination of CZ and the synthetic impurity A in solution and solid dosage forms. This method can be an alternative to the European pharmacopoeial method where the mobile phase of pH <0.5 would cause column deterioration after few runs leading to inappropriate results. The method was found to be robust and showed good selectivity and thus it could be used as a stability indicating for the assay of CZ and related impurity A. All statistical values (percentage recoveries, R.S.D., percentage difference, confidence limits of the slope and intercept, LOD and LOQ) were within the acceptable limits²². Due to the presence of different interferents in solution formulations such as colors, flavors, sweetening agents (e.g. saccharin) and preservatives, the proposed method should be re-evaluated prior usage for commercial solution products containing excipients which are different than those used in this present work.

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Received on 11.11.2011 Modified on 06.12.2011

Asian J. Research Chem. 5(1): January 2012; Page 87-92