Hydrophilic interaction chromatography (HILIC) for the determination of cetirizine dihydrochloride

1 Introduction

Cetirizine dihydrochloride (CTZ), chemically known as [2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyl] ethoxy]-acetic acid dihydrochloride, is an antagonist of a H₁ receptor. CTZ is a racemic mixture of levocetirizine, showing biological antihistamine activity, and dextrocetirizine, with no such effect (Baltes et al., 2001). The difference in the molecular structures of the enantiomers affects their pharmacological properties, including an affinity to the receptor being 30 times greater for levocetirizine (Tillement et al., 2003). CTZ is used in the treatment of hay fever, allergies, angioedema, and urticaria. It belongs to the second-generation antihistamines that are less able to cross the blood–brain barrier and thus do not cause side effects such as sedation, drowsiness or decreased cognitive processing (Berger et al., 2006; Carson et al., 2010; Garg and Thami, 2007; Golightly and Greos, 2005; Howarth et al., 1999). In previous studies, spectrophotometric, HPTLC and HPLC methods have been applied for determining cetirizine hydrochloride (Arayne et al., 2005; Haghighi et al., 2013; Kaur et al., 2014; Khan et al., 2011; Rawool et al., 2013; Souri et al., 2013). So far only one report has presented a UHPLC procedure for CTZ determination, conducted in the presence of ambroxol hydrochloride and antimicrobial preservatives in liquid pharmaceutical formulations (Trivedi et al., 2011). All of those methods utilized classical stationary phases known from reversed-phase liquid chromatography. Moreover for the separation of cetirizine enantiomers were also proposed: HPLC method developed by Kang et al. (2010) and capillary electrophoresis (CE) obtained by Eeckhaut and Michotte (2006). Recently supercritical fluid chromatography was reported as fast method for chiral separation of racemic cetirizine and levocetirizine (Eom et al., 2016). However, it is worth noting that Pharmacopeia recommended potentiometric titration as a method for cetirizine determination, while chromatographic separation was signed as method for separation of A-F impurities of cetirizine (including levocetirizine) (European Pharmacopoeia; British Pharmacopoeia).

Currently, the interest in the application of hydrophilic interaction chromatography (HILIC) in the analysis of many polar drugs, metabolites and biological important compounds was observed (Nuijs et al., 2011). According to Buszewski and Noga (2012), the application of hydrophilic interaction liquid chromatography (HILIC) is a viable alternative to high-performance liquid chromatography in the analysis of many compounds. HILIC is also applicable to charged substances (Bertuzzi et al., 2010). It is also equally important that separation by this method, while analyzing hydrophilic and ionizable drugs, can be combined with mass spectroscopy (MS) resulting in a significantly increased sensitivity of determination (Sewell et al., 2012).

Earlier studies have shown that cetirizine dihydrochloride is stable under conditions of base hydrolysis (1 M NaOH) and thermolysis (T = 43 K, RH = 76.4%), whereas strong affecting factors are required to degrade CTZ in an acidic environment (2 M HCl, T = 343 K). The greatest chemical instability of CTZ is observed on exposure to an oxidation factor (2% H₂O₂, T = 53 K), which is of particular practical importance indicating that excipients promoting an oxidative degradation path must be avoided (Khan et al., 2011).

Based on the above, the present study aimed to develop a rapid and sensitive HPLC–HILIC method for the determination of cetirizine dihydrochloride in the presence of its degradation product and excipients found in solid pharmaceutical dosage forms containing this compound. The method is useful for quality control of cetirizine dihydrochloride in biological fluids and in pharmaceutical matrix.

2 Experimental

3 Results and discussion

The HPLC procedure was developed in regard to stability-indicating assay of CTZ in bulk substance and pharmaceutical dosage forms. The chromatographic separation of CTZ and its degradation products were achieved on a Poroshell 120 Hilic column, 2.7 μm particle size (4.6 × 150 mm) at 25 °C. A mixture of acetonitrile and 0.1% formic acid (20:80 v/v) with a flow rate of 1.0 mL/min was used as a mobile phase. The wavelength of detection was set at 235 nm. The chromatograms of non-degraded and degraded samples CTZ are shown in Fig. 1. On chromatograms of degraded samples of CTZ, peaks originating from degradation product were observed at retention times from about 1.65 (Fig. 1). The degradants and excipients were not interfering with the main peaks. As a result of exploiting hydrophilic interactions in the determination of CTZ by using HPLC in this work, it was possible to obtain shorter retention times for CTZ (t_R = 1.79 min) compared to values received with HPLC (t_R = 3.8 min and t_R = 7.0 min) (Kaur et al., 2014; Souri et al., 2013). The use of a hydrophilic interactions in HILIC ensuring a high selectivity of CTZ determination in the presence of oxidation products without using phosphate buffers allowed an analysis of degradation products by means of an EIS-Q-MS mass spectrometer. Buszewski noted that the advantage of coupling HILIC with an MS detector is a tenfold increase in sensitivity as against HPLC-MS systems due to the presence of a higher organic content in the mobile phase (20% in this study) leading to rapid solvent evaporation during electrospray ionization (Buszewski and Noga, 2012).

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Figure 1

The chromatograms of non-degraded samples of CTZ in a bulk substance (A) and degraded samples of CTZ in bulk substance under oxidizing conditions (2% H₂O₂, T = 53 K, t = 40 min) (B).

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The method was also validated for linearity, inter- and intra-day accuracy and precision, robustness, system suitability, LOD, LOQ and recovery for the assay of CTZ in bulk substance and in the pharmaceutical preparations. The calibration plots were linear and in CTZ column load range 0.50–3.00 μg/mL. They were described by the equations y = ax. The b values, calculated from the equation y = ax + b, were insignificant because of being lower than the critical value t_b = b/S_b (Table 1). Precision of the proposed method was characterized by relative standard deviation (RSD, %) which was calculated for six consecutive measurements at three concentration levels 80%, 100% and 120% of standard solutions during the system suitability test. All values that characterize the inter-day precision of determination of cetirizine dihydrochloride were less than 2% and meet the criteria required for the precision of the method. The accuracy of the developed HPLC method was evaluated by analyzing content of cetirizine dihydrochloride in bulk substance and in pharmaceutical preparations. Although the content of organic fraction in the mobile phase was low for the HILIC application in our studies, the high sensitivity of determination analytes was achieved. The average values of the recoveries and precision are collected in Table 2.

A comparison of validation parameters obtained in the present study with those reported previously demonstrated that the newly developed method is far more sensitive, provides a wider range of linear relation between analyte concentration and a detector signal, and allows receiving lower limits of quantitation and detection. With regard to %RSD reflecting the intra-day precision of the methods compared, HPLC procedures proved superior whereas no significant differences were found in inter-day precision. The accuracy of the HPLC method involving hydrophilic interactions was slightly worse compared to a HPLC procedure with a C18 column used as a stationary phase (Kaur et al., 2014; Souri et al., 2013; Trivedi et al., 2011).

The robustness of the HPLC–HILIC procedure was evaluated after changing the mobile phase composition (acetonitrile volume, 20 ± 5 v/v), mobile phase pH (formic acid concentration, 3–0.05%), flow rate (1.0 ± 0.1 mL/min), absorption wavelength (235 ± 5 nm) and column temperature (298 ± 10 K). No statistically significant differences were established for the parameters studied in the investigation of robustness as their deviation did not exceed the permissible range of ±2%. System suitability was examined in regard to the relative standard deviation for peak areas and peak times, peak resolution, peak asymmetry and the number of theoretical plates (Table 3).

The action of an oxidation factor (2% H₂O₂) caused changes within the bond connecting the phenyl and the piperazine rings resulting in the formation of a degradation product with a partition coefficient different from that of the analyte. The mass fragmentation of the CTZ during oxidation is shown in Fig. 2. The resulting oxidation product of cetirizine has not been previously reported as its impurity (European Pharmacopoeia; British Pharmacopoeia). As demonstrated, the degradation product of CTZ oxidation meets the criteria of Lewis base being an electron donor and thus can be assumed to interact with negatively charged silanols from the stationary phase. By contrast, CTZ is a polar compound with a tendency to form hydrogen bonds and numerous physical interactions (e.g. an ion–dipole or a dipole-induced dipole) as a consequence of being an electron acceptor. According to Yoshida, the fact of obtaining similar retention times (t_R = 1.65 min vs t_R = 1.95 min) for two different compounds, CTZ and its degradation product during separation confirms the occurrence of both hydrogen bonding (Lewis acidity/basicity) and dipole–dipole interactions (dipole moments and compound polarizability) in separation based on application of HILIC column (Yoshida, 2004). The order of elution results from a shift in the partition equilibrium toward the immobilized water layer on the stationary phase for the more hydrophilic analyte; therefore, cetirizine dihydrochloride was eluted after its degradation product.

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Figure 2

Degradation pathways of cetirizine hydrochloride during oxidation.

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Based on parameters of molecular electrostatic potential (MEP), which are useful descriptors in indicating sites for electrophilic/nucleophilic reactions and hydrogen bonding, a proposition was made as to which molecular domains of cetirizine dihydrochloride and its oxidation product participated in hydrophilic interactions with the stationary phase. In order to model behavior of CTZ and its degradation product in proximity of silanol groups, theoretical calculations were performed. Both CTZ and degradation product were subjected to pKa calculations which resulted in protonated form of CTZ in pH = 6.5 (±0.74). The protonation took place on nitrogen of piperazine ring and causes increased polarity of molecule. Dipole vectors computed for optimized geometries of protonated CTZ (Fig. 3A) and degradation product (Fig. 3B) were presented. Vector values are 13.26 Debye and 5.78 Debye for CTZ and degradation product, respectively. Electrostatic potential was visualized on ESP maps (Fig. 3C and D). Blue color indicates positive charge, which is mostly susceptible on interactions with negatively charged stationary phase. High electric dipole moment of CTZ confirms higher polarity, and thus stronger retention relatively to degradation product. Simplified model of absorption of analyte on silanol groups was done in order to measure binding energy of silanol – CTZ and silanol – degradation product systems. Application of force fields method allowed modeling of aforementioned systems with preservation of dipole effects. Models of structures are presented (Fig. 4). Calculated binding energies were −946.71 kJ/mol and −773.45 kJ/mol for silanol-CTZ and silanol-degradation product systems, respectively. Binding of silanol-CTZ is 1.2 times stronger than silanol-degradation product binding energy what stays in agreement with previous investigations of dipole moment and ESP maps.

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Figure 3

Dipole moment vectors of CTZ (A) and degradation product (B). Electrostatic potential maps of CTZ (C) and its degradation product (D).

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Figure 4

Model of CTZ – silanol stationary phase system (A) and model of degradation product – silanol stationary phase system (B).

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4 Conclusion

The proposed HPLC method exploiting hydrophilic interactions can be recommended for the determination of cetirizine dihydrochloride in the presence of its oxidation product in bulk substance and in solid pharmaceutical dosage forms. Theoretical calculations confirmed differences in hydrophilic interactions of the analytes determined the various times of their elution.