Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method
- Paloma Flórez Borges†1, 2Email author,
- Pilar Pérez Lozano†1,
- Encarna García Montoya†1,
- Montserrat Miñarro†1,
- Josep R Ticó†1,
- Enric Jo†2 and
- Josep M Suñe Negre†1
© Flórez Borges et al.; licensee BioMed Central. 2014
Received: 16 October 2014
Accepted: 2 December 2014
Published: 9 December 2014
A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride.
For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 μm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K2HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min −1. Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification.
The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products.
Regression analysis showed r2 > 0.999 for cetirizine dihydrochloride in the concentration range of 650 μg mL −1 to 350 μg mL−1 for drug substance assay and a r2 > 0.999 in the concentration range of 0.25 μg mL−1 to 5 μg mL−1 for degradation products. The method presents a limit of detection of 0.056 μg mL −1 and a limit of quantification of 0.25 μg mL−1. The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method.
The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.
This new formulation consists of an oral lyophilized dosage form, whose aim is to facilitate swallowing (in the case of patients with dysphagia, such as children and elderly, for instance), easy to administer, effective, safe and stable over time.
Several HPLC methods have been reported in literature for the determination of CTZ alone – and also determining CTZ simultaneously with other drug substances, as in multicomponents preparations –. In order to develop a new chromatographic method for the determination and quantification of CTZ and its DE generated after a forced degradation study, several chromatographic methods for CTZ were investigated in the literature. Among them, was the Ph. Eur. method for CTZ . However, the latter was discarded due to the use of a normal phase chromatographic column and mobile phase that used much organic solvent (acetonitrile, not very cost-effective). Also, the Ph. Eur. method presents a very acid mobile phase pH (pH <0.5), which is known to diminish the life span of the chromatographic column . Also, some chromatographic analytical methods , used chromatographic columns of reverse phase, usually C18 and C8. Depending on the type of separation pursued (as for instance, CTZ combined with another DS), isocratic or gradient methods were used, and also mobile phases with ionic pairing. We have developed a reverse-phase high performance liquid chromatography (RP-HPLC) method by studying the effect of the stationary phase (C18 or C8 analytical columns) on peak resolution, the influence of pH -mobile phase- when adjusting the desired retention time (t R ) for the DS. Plus, by using a reverse-phase column, we reduced the amount of organic solvent (acetonitrile) used for the identification of the DS, in comparison to the analytical method validated by Ph. Eur. , which uses a normal phase chromatographic column, requiring more organic solvent due to its characteristics –. Therefore the aim of this study is to determine all possible DE generated under stress conditions, by developing and validating a stability-indicating RP-HPLC method for cetirizine dihydrochloride in the early stage of drug development of an oral lyophilizate.
Chemicals and reagents
All chemicals were analytical grade and used as received. All solutions were prepared in Milli-Q deionized water from a Milli Q gradient A10 water purification system (Molsheim, France). CTZ bulk powder (Cetirizine dihydrochloride, Ph. Eur) was purchased from Jubilant Lifesciences Ltd (Mysore, India) and kindly provided by Reig Jofre Group (Barcelona, Spain). HPLC grade acetonitrile was obtained from Panreac (Barcelona, Spain). Ortho-phosphoric acid 85% was purchased from Panreac (Barcelona, Spain). Potassium phosphate dibasic Ph. Eur. (K2HPO4) was purchased from Fagron (Terrassa, Spain). Hydrochloric acid 37%, sodium hydroxide and hydrogen peroxide (H2O2) at 33% were purchased from Panreac (Barcelona, Spain).
Equipment and chromatographic conditions
Samples were analyzed on Dionex Ultimate 3000 HPLC Thermo Fisher Scientific (California, USA), equipped with data system Chromeleon version 6.8 SP2 Build 2284, with degasifier SR3000, LPG-3400 quaternary pump, injector WPS3000, oven 6P TCC-3100, UV–vis detector PDA-3000. For initial development studies it was used an analytical chromatographic column Kromasyl 100-5C18 150 mm × 4.6 mm, 5 μm particle size (Tecnokroma Akzonobel, Terrasa, Spain). For final development and method validation, it was used an analytical chromatographic column Eclipse XDB-C8 150 mm × 4.6 mm, 5 μm particle size (Agilent columns, Barcelona, Spain). An isocratic mobile phase consisting of acetonitrile and 0.2 M potassium phosphate dibasic Ph. Eur. buffer solution at pH 7.00 (35:65 v/v) was used, and the analysis was carried out at a flow rate of 1 mL min −1. All determinations were performed at 30°C. The injection volume was 25 μL. The detector was set at λ 230 nm. The peak homogeneity was expressed in terms of peak purity factor and was obtained directly from spectral analysis report using the above mentioned software. Other apparatus included a Crison micropH 2002 pH meter (Barcelona, Spain) and Heraeus oven T5028 for thermal degradation (dry heat at 105°C) (Hanaus, Germany).
Forced degradation studies and preparation of samples
Acid and basic hydrolysis: 5 mg of bulk powder was treated with 5 mL of 0.1 M HCl and 0.1 M NaOH. The flasks were placed in a dry air oven at 105°C. Another 5 mg of bulk powder was also treated with 5 mL of 0.1 M HCl and 0.1 M NaOH at room temperature, for 24 hours.
Oxidation with H2O2 at 33%: 5 mg of bulk powder was exposed to 5 mL of hydrogen peroxide at 33% (W/v). The vial was kept at room temperature for 24 hours.
Infrared (IR) and Ultraviolet (UV) light: 5 mg of bulk powder was exposed under an infrared lamp and another 5 mg of bulk powder was exposed under an ultraviolet lamp, for 24 hours.
Humidity HR 79%: the 5 mg bulk powder sample was placed inside a humidifier with HR 79%, for 24 hours.
Heat at 105°C: 5 mg of bulk powder sample was placed inside a 105°C dry air oven for 24 hours.
Shed sunlight for 15 days: 5 mg of bulk powder was kept in a vial for 15 days, at room temperature and exposed to direct sunlight.
Once the stress conditions were complete, 10 mL of 0.2 M phosphate buffer (pH 7.00) was added to the samples in order to achieve the standard solution concentration of 500 μm mL-1. Moreover, all the solutions and blanks were filtered with a 0.45 μm syringe filtration disk PVDF. Results were compiled in terms of relative retention times (rtR) found during the analysis.
Validation of the analytical method
In order to validate the RP-HPLC method developed, ICHQ2B guideline recommendations were followed, in terms of selectivity, linearity, range, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) . In order to fulfill ICH specifications in terms of linearity and range for the analytical method (content uniformity and assay of DS and finished product), a linear range within 70-130% was studied, by analyzing a series of three replicates, i.e., three independent sets (k = 3), each with seven different concentrations (n = 6): 350 μg ml −1 - 650 μg ml −1, considering 500 μg ml −1 as 100% (standard solution), in order to provide information on the variation in peak area values between samples of the same concentration. For evaluation of the precision estimates, repeatability and intermediate precision were performed at three concentration levels (650, 500 and 350 μg ml −1, corresponding to 130, 100 and 70%), and 10 injections of each sample (K = 10), per day. Mean average, standard deviation (SD) and relative standard deviation (RSD) of tR and the peak area achieved individually of day 1 and 2 were calculated. After the HPLC analysis, the response factor (RF) was calculated between the response (Y) and concentration achieved (X), as Y/X. Therefore, mean average, SD and RSD were calculated using the response factors obtained with an Excel 2007 spread sheet. The response factors results must comply with a RSD ± 2%. For accuracy the concentration found expressed by function of repeatability of the standard solution, relative error in percentage and the percentage of recovery, with mean average, SD and RSD deviation of each of the three concentrations studied (650, 500 and 350 μg ml −1, was considered of three replicates) were calculated. For the DS, 98-102% percentage of recovery was considered as being acceptable .
For the determination and quantification of the DE, linearity, precision, accuracy and LOD and LOQ were calculated. In order to carry out this validation, further dilutions from a stock solution of 500 μg ml −1 with the specified mobile phase were carried out in order to achieve the correspondent concentrations: 5 μg mL−1, 2.5 μg mL −1, 1.25 μg mL −1, 0.5 μg mL−1, 0.25 μg mL−1, 0.125 μg mL −1. A total of seven independent calibration curves, i.e., seven replicates ( k = 7) were prepared. The LOD and LOQ were calculated by the ratio between the standard deviation of y-intercepts of regression lines of the seven calibration curves mentioned before by averaging the slopes of calibration curve multiplied by 3.3 and 10, respectively ,. Each serial dilution (k = 7) was analyzed, with n = 6 (level of concentrations).
In terms of relative error and percentage of recovery three concentrations (5, 1.25 and 0.25 μg mL−1) from the range of DE were evaluated. All the solutions prepared were filtered with a 0.45 μm syringe filtration disk PVDF to the vials for injection in the HPLC system.
Results and discussion
HPLC method development
As an early stage study of drug development, our goal was to acknowledge all possible DE generated under stress conditions for CTZ. The information acquired in the early stage of the study will lead us to a better understanding of the DS itself and also the possible DE that we may find during the next step of the oral lyophilized development study. Therefore it was not our objective the development of a fast analytical method for the DS per se, but actually the development of an analytical method that could detect a complete profile of DE for this DS, leaving for the following studies of drug development the aim of reducing run time, for instance.
Results of forced degradation study
Summary of product degradation peaks in relative retention time ( rt R )
Acid hydrolysis *RT
Acid hydrolysis at 105°C
Ultraviolet light (UV)
Infrared light (IR)
Basic hydrolysis *RT
Basic hydrolysis at 105°C
Dry heat at 105°C
Shed sunlight 15 days
H2O2 at 33%
Peak purity determination by diode-array UV–vis spectra of CTZ and stress studies results
Forced degradation conditions
aPeak purity index match
Extent of decomposition
Acid hydrolysis *RT
Acid hydrolysis at 105°C
Ultraviolet light (UV)
Infrared light (IR)
Basic hydrolysis *RT
Basic hydrolysis at 105°C
Dry heat at 105°C
Shed sunlight 15 days
Oxidative medium *RT
CTZ (phosphate buffer solution)
Assay for drug substance method
Linearity for CTZ assay was verified by triplicate analysis of seven different concentrations, i.e., three sets of 130-70% range of CTZ. As a result, the linear regression equation was found to be Y = 769.56 X + 14.573 (r2 = 0.9994, k = 3 (number of replicates), n = 7 (level of concentrations), 650 μg mL −1 to 350 μg mL −1) for CTZ. In which, Y was the dependent variable, X was independent variable, 769.56 was the slope and which showed change in dependent (Y) variable per unit change in independent (X) variable; 14.573 was the Y-intercept i.e., the value of Y variable when X = 0.
Repeatability and intermediate precision according to retention time (t R ) and peak area for CTZ assay
Mean average ± aSD (min)bK = 10
Mean average ± aSD (mAU.min)bK = 10
5.79 ± 0.0274
512.54 ± 1.7865
5.79 ± 0.0327
399.69 ± 1.9090
5.80 ± 0.0228
281.21 ± 1.7867
5.77 ± 0.0195
511.75 ± 2.2059
5.78 ± 0.0149
399.54 ± 1.5972
5.80 ± 0.0248
281.63 ± 1.0483
Concentrations found, relative error in percentage, percentage of recovery and estimates for CTZ assay
aTheoretical concentration (μg mL−1)
Concentration found (μg mL−1)
cSD of recovery
Mean average, standard deviation (SD) and relative standard deviation (RSD%) of peak area mAU (5 – 0.125 μ mL −1 )
Theoretical concentration (μg mL−1)
Mean concentration average ± aSD (μg mL−1) (bk = 7)
4.0495 ± 0.1040
2.0121 ± 0.0251
1.0012 ± 0.0326
0.4021 ± 0.0327
0.1818 ± 0.0111
Relative error (%) and percentage of recovery
Concentration (μg mL−1)
Mean concentration found (μg mL−1) (ak = 7)
bRelative error% (mean)
dSD of recovery%
A new and simple RP-HPLC method was developed for the determination of CTZ and its DE during the early stage of drug development of an oral lyophilized dosage form. The proposed method was demonstrated to be linear, precise, accurate and specific, based on method validation. Satisfactory results were obtained in separating the peak of CTZ from the DE produced by forced degradation. Plus, it is a cost-effective method that requires a simple mobile phase (phosphate buffer solution and acetonitrile, 65:35 v/v) and also does not require the use of ion pairing, which can result in difficulty in recovering initial column properties. It was also able to separate with good specificity the DS peak from the entire DE generated during the stress condition study, which help us in the next step of the drug development of the oral lyophilizate, by adapting the validated method considering further aspects, such as the interactions between CTZ and the excipients chosen for the final medicinal product. The proposed analytical method proved to be stability-indicating and therefore useful in the following stages of drug development.
Reverse phase high performance liquid chromatography
- t R :
- rt R :
Relative retention time
Limit of detection
Limit of quantification
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