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HPLC AND LC-MS STUDIES ON STRESS DEGRADATION BEHAVIOR OF LEVOCETIRIZINE AND DEVELOPMENT OF A VALIDATED SPECIFIC STABILITYINDICATING METHOD
Rahul P. Gunjal , G. Raju , A. Ramesh Babu , N. Mallikarjun , Nalini Shastri & R. Srinivas
a a a b a b b a

National Institute of Pharmaceutical Education and Research, IDPL Centre, Balanagar, Hyderabad, India
b

National Centre for Mass Spectrometry Division, Indian Institute of Chemical Technology, Tarnaka, Habsiguda, Hyderabad, India Available online: 15 Jun 2011

To cite this article: Rahul P. Gunjal, G. Raju, A. Ramesh Babu, N. Mallikarjun, Nalini Shastri & R. Srinivas (2011): HPLC AND LC-MS STUDIES ON STRESS DEGRADATION BEHAVIOR OF LEVOCETIRIZINE AND DEVELOPMENT OF A VALIDATED SPECIFIC STABILITY-INDICATING METHOD, Journal of Liquid Chromatography & Related Technologies, 34:12, 955-965 To link to this article: http://dx.doi.org/10.1080/10826076.2011.564704

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Journal of Liquid Chromatography & Related Technologies, 34:955965, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10826076.2011.564704

HPLC AND LC-MS STUDIES ON STRESS DEGRADATION BEHAVIOR OF LEVOCETIRIZINE AND DEVELOPMENT OF A VALIDATED SPECIFIC STABILITY-INDICATING METHOD

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Rahul P. Gunjal,1 G. Raju,2 A. Ramesh Babu,2 N. Mallikarjun,1 Nalini Shastri,1 and R. Srinivas1,2 1 National Institute of Pharmaceutical Education and Research, IDPL Centre, Balanagar, Hyderabad, India 2 National Centre for Mass Spectrometry Division, Indian Institute of Chemical Technology, Tarnaka, Habsiguda, Hyderabad, India

& The objective of the current investigation was to study the degradation behavior of levocetirizine under different ICH recommended stress conditions using HPLC and LC-MS and to establish a validated stability-indicating method. The drug was subjected to stress conditions of hydrolysis, photolysis, and thermal decomposition. Extensive degradation was found to occur in photolytic stress conditions. Mild degradation was observed in acidic, alkaline, and neutral conditions. The drug was stable to thermal stress condition. Successful separation of drugs from degradation products formed under stress conditions was achieved on a C-18 Supelco1 Column using water-acetonitrile (50:50%v=v) as the mobile phase. The flow rate was 1.0 mL=min and the detection wavelength was 230 nm. The degradant products were characterized by LC-MS. The method was validated with respect to linearity, precision, accuracy, specificity, and robustness. The developed method efficiently separated the drug and degradation product in actual samples.

INTRODUCTION The aim of the present study is to establish inherent stability of levocetirizine through stress studies under a variety of ICH recommended stress conditions.[1,2] The Levocetirizine dihydrochloride (LCT) is chemically (R)-2-(2-(4-((4-chlorophenyl) phenyl methyl) piperazin-1-yl) ethoxy) acetic acid (Figure 1) and is a selective potent H1-antihistamine compound indicated for the treatment of allergic rhinitis and chronic idiopathic urticaria. Levocetirizine dihydrochloride has anti-allergic properties when used once daily for treatment of allergic rhinitis.
Address correspondence to R. Srinivas, National Center for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500607, India. E-mail: sragampeta@yahoo.co.in; srini@iict.res.in

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FIGURE 1 Chemical structure of levocetirizine.

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Different spectrophotometric,[3,4] HPLC,[511] and LC-MS[12] methods have been reported for the determination of levocetirizine in pharmaceutical formulations and biological fluids. In the present study, attempts were made to develop a rapid, precise, and accurate method for the estimation of the ingredient in the presence of their degradants and their characterization by LC-MS study.

EXPERIMENTAL Materials Levocetirizine dihydrochloride sample was provided by Hetero Labs., Hyderabad, India and was used without purification. HPLC grade water, Millipore (Bedford, MA, USA) and Ammonium acetate and formate, Merck (Mumbai, India) were used. Acetonitrile and methanol, Merck (Mumbai, India) were used. Sodium hydroxide, hydrochloric acid, glacial acetic acid, and hydrogen peroxide were of analytical-reagent grade from S.D Fine Chemicals Ltd. (Mumbai, India).

Instrumentation Precision water baths equipped with a MV temperature controller (Julabo, Seelbach, Germany) were used for hydrolytic degradation studies. Photostability studies were done by exposing the solution to UV radiation 320400 nm in UV chamber for an appropriate period. The samples were exposed for a total period of 2 days. Thermal stability studies were performed in a dry-air oven (IB-05 G, Jeio Tech, Korea). The Schimadzu HPLC system consisted of a pressure pump LC-20AT, UV-visible dual wavelength detector (SPD-10AD), and System controller (SCL-10AV); data were acquired and processed by the use of LC-Solution software version 1.11 SP1 (Schimadzu, Kyoto, Japan). The chromatographic separations were carried out on Supelco (Bellefonte, USA) C-18 column (250 mm 4.6 mm i.d. with particle size of 0.5 mm).

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Characterization of the degradation products was carried out using Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, UK). Waters 2695 LC consisted of an auto sampler, a 2996 PDA detector, a P4000 pump, and a 5 mm Supelco-ODS column (150 4.6 mm i.d). The Quattro micro triple-quadrupole mass spectrometer was equipped with an electrospray ionization (ESI) source and the data acquisition was under the control of Masslynx v.4.1 software. The ESI mass spectra were recorded by scanning MS1 in positive mode. The capillary voltage was maintained between 3.5 and 4.0 kV, and nitrogen gas was used for both desolvation and nebulization. The source and desolvation temperatures were 110 and 300 C.
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Degradation Studies All degradation studies were done at a drug concentration of 1 mg= mL. For acid decomposition studies, drug was dissolved in 0.1 N HCl and solution was refluxed for a period of 6 hr. The study in alkaline conditions was carried out in 0.1 N NaOH and refluxed for a period of 6 hr. These were repeated at a lower temperature of 40 C, keeping all other conditions constant. For the study in neutral conditions, the drug was dissolved in water and refluxed for 10 hr. Oxidative studies were carried out at room temperature in 3% H2O2 after keeping the solution for one month in the dark at room temperature and also at 10% H2O2 for a period of 15 days in the dark at room temperature. Photo-degradation studies were performed in 0.1 N HCl, 0.1 N NaOH, and neutral conditions. The solutions were exposed to UV radiation of 320400 nm in UV chamber for 2 days. Suitable controls were kept under the darkness. Additionally, the drug powder was exposed to dry heat at 65 C for 15 days. Samples were withdrawn at appropriate times and subjected to HPLC analysis after diluting with mobile phase composition to 10ug=mL.

Separation Studies LC-MS studies were carried out on all reaction solutions individually, and then on a mixture of those solutions in which decomposition was observed. Satisfactory separations of components of the mixture were achieved by using isocratic elution mode acetonitrile-water (50:50% v=v) as a mobile phase. The mobile phase was filtered through 0.45 mm nylon membrane and degassed before use. The injection volume was 20 mL and mobile phase flow rate was kept constant at 1 mL=min.

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Validation of the Method Linearity and Range A stock solution of the drug was prepared at the strength of 1 mg=mL. It was diluted to prepare solution containing 540 mg=mL of the drug. The solutions were injected in triplicate into the HPLC column, keeping the injection volume constant (20 mL). Precision Six injections, of three different concentration (15, 20, 25 mg=mL), were given on the same day and the values of relative standard deviation (R.S.D) were calculated to determine intra-day precision. These studies were also repeated on different days to determine inter-day precision. Accuracy Accuracy of the method was evaluated by spiking the drug at three concentrations in a mixture of stressed solutions and determining recovery of the added drug. Specificity and Selectivity The specificity of the method was established through the study of resolution factors of the drug peak from the nearest resolving peak, and also among all other peaks. Robustness Robustness of the proposed method was assessed with respect to small alteration in the flow rate, detection wavelength, and mobile phase composition. The degree of reproducibility obtained as a result of small deliberate variations in the method parameters like 0.1 change in flow rate, 5 nm change in detection wavelength, and 2 change in mobile phase composition has proven that the method is robust. RESULTS AND DISCUSSION Development and Optimization of the Stability-Indicating Method Initial method development was done on pure drug using water:methanol (50:50) as the mobile phase using HPLC-UV. During this trial, some of the degradant product may have interfered with the active drug, and the drug peak shows little peak tailing along. Later methanol was replaced with the acetonitrile and it was observed that satisfactory resolution was obtained

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FIGURE 2 Typical HPLC chromatogram of levocetirizine. (Color figure available online.)

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FIGURE 3 LC-ESI-MS chromatogram showing separation of degradant products from levocetirizine in mixture of stressed solution. Key: L-1, formed in all types of hydrolytic and photolytic conditions, L-2, photolytic product in acid. (Color figure available online.)

with water:acetonitrile in the ratio of 50:50 at flow rate 1.0 mL=min (Figure 2). The levocetirizine was observed to be separated from the degradent product as shown in (Figure 3).

Degradation Behavior LC-ESI-MS studies of levocetirizine (Figure 4) under different stress conditions suggested the following degradation behavior. Acidic Condition The drug gradually decreased with time after 6 hr when refluxed in 0.1 N HCl, forming degradation product (L-1) at a retention time of 2.2 min. The electrospray ionization (ESI) mass spectrum of this peak showed [MH] at m=z 189 (Figure 5). The levocetirizine eluted at a retention time of 3.9 min and was confirmed by its ESI mass spectrum, which showed [MH] at m=z 389. The rate of hydrolysis in acid

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FIGURE 4 ESI-MS spectrum of levocetirizine in untreated sample of LCT. (Color figure available online.)

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FIGURE 5 ESI-MS spectrum of LCT degradant (L-1) in acidic, alkaline, neutral, and photolytic stress condition. (Color figure available online.)

FIGURE 6 ESI-MS spectrum of LCT degradant (L-2) in photolytic acidic stress conditions. (Color figure available online.)

(6.33%) was slower compared to that of alkali degradation (15.25%). Based on the structure of the drug levocetirizine, the most probable structure of the proposed degradant is shown in Figure 9. Degradation in Alkali The drug was found more labile to alkaline condition as compared to acidic conditions. The degradation was carried out in 0.1 N NaOH when refluxed for about 6 hr. The sufficient degradation (15.25%) was seen form-

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ing similar degradation product (L-1) at a retention time of 2.7 min and its ESI spectrum showed [MH] at m=z 189 (Figure 5); the probable structure is given in Figure 9.

Neutral Degradation In neutral condition, 8.16% of degradation of the drug was observed when refluxed for 10 hr with generation of the same degradant product (L-1) which was eluted at a retention time of 2.19 min and its ESI spectrum also showed [MH] at m=z 189 (Figure 5); the probable structure is given in Figure 9.
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Oxidative Degradation As the drug did not undergo any degradation under the conditions of 3% H2O2 at room temperature and 10% H2O2 after keeping it for 15 days in the dark at room temperature, the drug seems to be stable under these conditions (Figures 7 and 8).

Photolytic Conditions The photolytic study was carried out in 0.1 N HCl, 0.1 N NaOH, and neutral conditions. In alkaline and neutral photolytic conditions the drug was found to be stable, while in 0.1 N HCl the drug underwent degradation to form two major degradation products (L-1) and (L-2) which eluted at 2.07 min and 2.7 min, respectively. The ESI mass spectra of these peaks showed [MH] at m=z 189 and m=z 345 (Figures 5 and 6), respectively. This suggests that the drug is more unstable to the photolytic acidic stress conditions. The controlled study was carried out by keeping the same solutions in dark conditions. The probable structures of the degradants are as shown in Figures 9 and 10.

FIGURE 7 LC-ESI-MS chromatogram of levocetirizine in 10% H2O2 condition. (Color figure available online.)

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FIGURE 8 ESI-MS spectrum of LCT in 10% H2O2 condition. (Color figure available online.)

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FIGURE 9 Proposed structure of degradant L-1.

FIGURE 10 Proposed structure of degradant L-2.

Solid-State Study The solid-state studies showed that levocetirizine was stable to the effect of temperature. When the drug was exposed to dry heat at 65 C for 15 days, no decomposition of drug was seen.

Validation of Developed Stability-Indicating Method Using HPLC-UV Linearity Linearity was established by least squares linear regression analysis of the calibration curve. The constructed calibration curve is linear over the concentration range of 540 ug=mL for levocetirizine. Peak areas were plotted versus their respective concentrations and linear regression analysis performed on the resultant curves. The correlation coefficient was found to be 0.9996 the mean (RSD) values of slope and intercept were 45517 (0.508) and 75175.33 (3.49), respectively.

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Limit of Detection (LOD) and Limit of Quantification (LOQ) The LOD was determined based on signal-to-noise ratios and was determined using an analytical response of three times the background noise. The LOD was found to be 0.75 ug=mL. The LOQ was determined as the lowest amount of analyte that was reproducibly quantified above the baseline noise following triplicate injections. The LOQ that produced the requisite precision and accuracy was found to be 5 ug=mL. Precision Data obtained from precision experiments are given in Tables 1 and 2 for intra-day and inter-day, respectively. The RSD values for the intra- and inter-day precision study are <1% and <2.0%, which confirmed that the method is sufficiently precise. Accuracy As shown from the data in Table 3, good recoveries of the drug in the range from 98.00 to 102.00% were made at various added concentrations, despite the fact that the drug was fortified to a mixture that contained drug as well as degradation products, formed under various reaction conditions. Specificity Specificity was established by the determination of purity of the drug peak using a PDA detector. The degradant products were found to be separated from the active drug, which indicated the specificity of the method.
TABLE 1 Precision Data Evaluated through Intra-Day Study Intra-day measured concentration (mg=mL) S.D.; RSD (%), (n 3) 14.812 0.012; 0.079 19.629 0.078; 0.400 24.903 0.102; 0.412

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Actual concentration (mg=mL) 15 20 25

TABLE 2

Precision Data Evaluated through Inter-Day Study Inter-Day Measured Concentration (mg=mL) S.D.; RSD (%), (n 3)

Actual concentration (mg=mL) 15 20 25

Day 1 15.031 0.0566; 0.3765 19.941 0.093; 0.4701 25.002 0.111; 0.4456

Day 2 14.897 0.087; 0.5851 19.946 0.097; 0.4889 25.0220 0.131; 0.5265

Day 3 14.890 0.123; 0.8287 19.606 0.148; 0.7570 24.966 0.109; 0.4381

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Recovery Studies of Levocetirizine from Mixture of Stressed Samples (n 3) Calculated Concentration S.D. (ug=mL), RSD (%) 14.99 0.16; 0.61 20.31 0.14; 0.65 24.92 0.05; 0.13

Actual Concentration (mg=mL) 15 20 25

Recovery (%) 99.93 101.76 99.73

CONCLUSIONS In this study, levocetirizine was subjected to stress studies under various ICH recommended conditions. The drug underwent degradation in acidic, alkaline, and neutral stress conditions into the same degradant product. It was found to be stable in oxidative stress conditions, but extremely labile to photolytic stress conditions forming two degradation products. The drug showed stable behavior towards the thermal stress conditions. The developed stability-indicating method separates all degradants from the active drug, which indicates the specificity of the method. The developed method was proven to be simple, accurate, precise and specific, and selective. Hence, it is recommended as a suitable method for industrial application for stability samples.

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ACKNOWLEDGEMENTS The authors thank Dr. J. S. Yadav, Director, IICT, Hyderabad, for use of the facilities. G. R. and A.R. thank UGC, New Delhi for the award of the Senior Research Fellowships.

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