Antihistamines are widely used to alleviate the symptoms caused by allergic reactions. Most of these drugs have zwitteriónicas and/or amphoteric characteristics, which confer additional analytical challenges. This work aimed to develop a single eco-friendly and efficient chromatographic methods for analysis of seven antihistamines, namely, azelastine HCl, desloratadine, ebastine, fexofenadine HCl, ketotifen, loratadine, and olopatadine HCl. The separations were obtained using RP C-18 LUNA (150x4.6mm, 5
It is estimated that around 400 million people are affected by allergic rhinitis and 300 million by asthma in the world. In Brazil, 30 to 35% of the population presents some type of allergic reaction such as allergic rhinitis, asthma, and atopic dermatitis. Total medical expenses exceed 98 million per year with hospitalizations and are the third cause of hospitalization. These overheads outweigh tuberculosis and AIDS [
The second-generation antihistamines are considered as first-line treatment in allergic disorders and do not cause drowsiness and anticholinergic effects. The improved effectiveness, reduced adverse effects, and remarkable drug compliance of modern antihistaminic drugs may be attributed to low lipophilicity and reduced affinity for brain H1 receptors [
The second-generation antihistaminic drugs selected in this study are piperidines except for azelastine (methylazepane derivative) and olopatadine (dibenz[b,e]oxepin derivative). The chemical name, molecular structures, and dissociation constants of azelastine HCl (AZE), desloratadine (DESl), ebastine (EB), fexofenadine HCl (FEX), ketotifen (KET), loratadine (LOR), and olopatadine HCl (OLO) are described in Table
The chemical names, molecular structures, and dissociation constants of azelastine HCl, desloratadine, ebastine, fexofenadine HCl, ketotifen, loratadine, and olopatadine HCl [
Antihistamines | Chemical name | pKa | Chemical structure |
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Azelastine HCl | 4-[( | 8.88 | |
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Desloratadine | 8-Chloro-6,11-dihydro-11-(4-piperidinylidene)-5H- benzo[ | 4.33 and 9.73 | |
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Ebastine | 1-[ | 8.43 | |
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Fexofenidine HCl | 2-[ | 4.04 and 9.01 | |
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Ketotifen | 4-(1-Methyl-4-piperidinylidene)-4,9-dihydro-10H-benzo[ | 7.18 and 8.11 | |
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Loratadine | Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[ | 4.33 | |
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Olopatadine HCl | (11Z)-11-[ | 3.78 and 9.76 | |
Several HPLC methods were found in the scientific literature for quantitative determination of second-generation antihistamines, as a single drug (not cited). The British Pharmacopoeia describe monograph on AZE [
The United States Pharmacopeia describes chromatographic method for separation of azelastine and its related compounds B, D, and E [
The British Pharmacopoeia [
In the above cases, separations were obtained on a nonconventional column with nitrile groups chemically bonded to porous silica particle with mobile phase composed of buffering and ion pairing salts. Moreover, flow rate is too high, with estimated total run time over 15 minutes.
To our knowledge, no eco-friendly HPLC method was found for separation of seven second-generation antihistamines, with minor modification in mobile phase organic content.
The present study aims to develop a single chromatographic setup for analysis of seven antihistaminic drugs in pharmaceutical formulations. We intended to propose highly efficient and accurate HPLC methods that abide by the green analytical chemistry (GAC) principles. Additionally, a stability-indicating method was developed for separation of azelastine and three related impurities.
The certified pharmaceutical secondary standard (assigned purity ≥98%) of azelastine HCl, desloratadine, ebastine, fexofenadine HCl, ketotifen, loratadine, and olopatadine HCl were obtained from Sigma-Aldrich (St. Louis, MA, USA).
The azelastine impurity B (1-benzoyl-2-[(4
The HPLC grade acetonitrile and analytical grade hydrochloric acid (37%) were obtained from Merck KGaA (Darmstadt, Germany). The purified water was obtained from Millipore Milli-Q® Plus (Millipore, Bradford, USA).
The nasal solution containing 0.9 mg mL−1 of azelastine was obtained from a local pharmacy. The Rino-Lastin® (Aché Pharmaceutical Laboratories, Brazil) formulation is stated to contain following excipients: benzalkonium chloride, citric acid, sorbitol, hypromellose, purified water, disodium edetate dihydrate, and sodium phosphate dibasic dodecahydrate.
A HPLC system consisted of two solvent delivery pumps, an autoinjector fitted with 20
Analytical conditions were optimized using a Luna-RP C18 column (250x4.6 mm, 5
All analyses were done at room temperature, and the column temperature was controlled at 25°C ± 1. The mobile phase was prepared fresh each day, vacuum-filtered through a 0.45
The stock solutions of azelastine HCl reference standard was prepared by transferring mass equivalent to 25 mg of azelastine into a 25 mL volumetric flask. The volume was completed with mobile phase to obtain 1000
The stock solutions of DESL, EB, FEX, KET, LOR, and OLO reference standards were prepared likewise, to obtain 1000
Five AZE nasal solution preparations (claimed 0.9 mg mL−1 of AZE) were pooled into a single flask. An aliquot equivalent to 10 mg of AZE was transferred to a 50 mL volumetric flask. The volume was completed with mobile phase to obtain 200.0
According to official guidelines, the system suitability tests are an integral part of method development [
To demonstrate adequateness of the proposed methods for seven antihistaminic drugs, azelastine HCl was selected as prototype drug. The proposed method was fully validated according to international guidelines on validation of a chromatographic method for pharmaceutical drug products and applied in the analysis of AZE in nasal solutions [
The selectivity of the proposed method was evaluated through the analysis of reference standards, sample, and placebo solutions. Besides, peak purity index of obtained chromatograms was accessed by UV diode array detector.
The discovery of third-generation antihistamine drugs has brought additional analytical challenges, besides the therapeutic benefits. However, there is limited information on the related impurities and their probable human cardiotoxic effects and pharmacokinetic and prolonged pharmacodynamics profiles [
The third-generation H1 antihistamines have an atypical characteristic in their molecular structures. Depending on the pH of the medium, they often are electrically neutral due to opposite charge at different nonadjacent atoms and/or amphoteric due to the presence of acid and basic groups in their molecular structures [
Besides these important aspects, it is necessary to constantly seek fast and alternative methods that increase productivity, reduced solvent, and sample consumption. Most of the solvents used in liquid chromatography are hazardous to analysts and environment due to inherent volatility, flammability, and toxicity of these solvents [
We tested several reverse-phase columns, such as conventional C18 and core-shell technology C18 stationary phase, besides, -C8, -CN, and –NH2 columns. The preliminary chromatographic parameters obtained with the RP C-18 LUNA (250x4.6mm, 5
The separations obtained with acetonitrile, as organic phase modifier, were better than those obtained with methanol. Neat improvement in peak symmetry and intensity with acetonitrile can be attributed to the solubility of antihistaminic drugs in acetonitrile [
According to the pH dependent solubility curves described in the literature [
To improve column efficiency and avoid peak tailing during chromatographic runs, in reverse phase mode, it is essential to assure a single ionic form of molecules [
Finally, most of the commercial reverse phase columns are stable at pH between 2.0 and 8.0. Thus, a mobile phase composed of acetonitrile and acidified water (pH 2.1) was selected for further method development and optimization of chromatographic conditions.
The seven antihistaminic drugs selected for study are structurally related and belong to the same therapeutic class; however chromatographic interaction with the column is dissimilar. To develop a unique eco-friendly method, factors such as mobile phase organic content, flow rate, and volume of injection were optimized through univariate experimental design approach for seven antihistaminic drugs.
The optimum conditions for AZE, DESL, EB, FEX, KET, LOR, and OLO were fixed based on method sensitivity (peak area), efficiency (theoretical plates), asymmetry factor (As), and rapid elution (retention factor). Briefly, standard reference solutions were prepared (200
Optimum chromatographic conditions for separation of seven antihistaminic drugs.
Drug | Mobile Phase | Flow variation | Injection volume variation | Detection | Retention factor | Peak Asymmetry | Theoretical plates |
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Azelastine HCl | 35:65 (v/v) | 1.0 | 20 | 287 | 1.52 | 1.34 | 14390 |
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Desloratadine | 15:85 (v/v) | 1.0 | 20 | 275 | 1.26 | 1.32 | 15052 |
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Ebastine | 45:55 (v/v) | 1.0 | 20 | 254 | 1.54 | 1.60 | 12698 |
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Fexofenidine HCl | 32:68 (v/v) | 1.0 | 20 | 217 | 1.10 | 1.04 | 14720 |
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Ketotifen | 25:75 (v/v) | 1.0 | 20 | 296 | 0.82 | 1.79 | 12512 |
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Loratadine | 35:65 (v/v) | 1.0 | 20 | 271 | 1.26 | 1.38 | 15680 |
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Olopatadine HCl | 25:75 (v/v) | 1.0 | 20 | 296 | 1.63 | 1.33 | 15412 |
Impact of mobile phase flow rate on peak area of AZE, DESL, EB, FEX, KET, LOR, and OLO.
Impact of mobile phase flow rate on peak asymmetry of AZE, DESL, EB, FEX, KET, LOR, and OLO.
Impact of sample injection volume on peak area of AZE, DESL, EB, FEX, KET, LOR, and OLO.
Impact of sample injection volume on peak asymmetry of AZE, DESL, EB, FEX, KET, LOR, and OLO.
The detection wavelength for AZE, DESL, EB, FEX, KET, LOR, and OLO was selected based on the DAD detector, and the column temperature was maintained at 25°C ± 1.
Figures
Chromatographic separation of azelastine HCl. Chromatographic conditions described in Table
Chromatographic separation of desloratadine. Chromatographic conditions described in Table
Chromatographic separation of ebastine. Chromatographic conditions described in Table
Chromatographic separation of fexofenadine HCl. Chromatographic conditions described in Table
Chromatographic separation of ketotifen. Chromatographic conditions described in Table
Chromatographic separation of loratadine. Chromatographic conditions described in Table
Chromatographic separation of olopatadine HCl. Chromatographic conditions described in Table
As stated earlier, separation of seven antihistaminic drugs was far better with acetonitrile as compared to other organic solvents tested. Thus, we estimated the greenness of the proposed methods based on the GAC principles, described in the literature [
Eco-scale scores for proposed methods for analysis of FEX, EB, LOR, DESL, AZE, KET, and OLO (adopted from [
Factor | Penalty points |
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Reagents (HCl) | 8 |
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Acetonitrile | 4 |
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Instruments (HPLC) | 1 |
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Occupational hazards | 3 |
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Waste | 8 |
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Total penalty points | 24 |
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Analytical Eco-Scale total score | 76 |
Linear regression data in the analysis of AZE.
STATISTICAL PARAMETERS | |
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Concentration range ( | 10.0 – 60.0 |
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Slope of the curve ( | 34754 |
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Intercept ( | 91113 |
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Correlation coefficient (R2) | 0.9959 |
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Standard deviation of the residuals | 1.4% |
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DL ( | 1.10 |
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QL ( | 3.32 (RSD < 2%) |
DL= detection limit; QL= quantitation limit.
The inter- and intraday precision data obtained in the analysis of AZE sample solutions (40.0
Accessed AZE ( | |
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| 38.11 ± 0.58 |
RSD (%) | 1.52 |
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Day 1 | 38.43 ± 0.54 |
RSD (%) | 1.40 |
Day 2 | 38.26 ± 0.42 |
RSD (%) | 1.10 |
Day 3 | 37.71 ± 0.54 |
RSD (%) | 1.44 |
The recovery of the proposed method was accessed at three concentration levels (low, intermediate, and high). Table
Recovery of standard AZE added to fortify sample solutions and analyzed by the proposed method.
Sample | Added concentration | Found concentration | Recovery | |
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Sample | Standard | Standard | (%) | |
AZE | 15.00 | 10.0 | 9.31 ± 0.12 | 93.10 |
15.00 | 25.0 | 24.49 ± 0.22 | 97.97 | |
15.00 | 40.0 | 39.31 ± 0.18 | 98.27 |
The robustness of proposed method for analysis of AZE was evaluated by deliberate change in mobile phase flow rate (0.9, 1.0, and 1.1 mL mL−1), injection volume (18, 20, and 22uL), and mobile phase organic content (ACN 33, 35 e 37%). There was an insignificant impact on peak retention, peak area, and peak asymmetry (Figures
The chromatographic separation of azelastine and impurities B, D, and E was obtained using the Luna C18 column (150 × 4.6mm x 5
The optimum conditions for AZE and related impurities B, D, and E were fixed based on official system suitability parameters, peak asymmetry <2.0, and peak resolution > 2.0 [
The system suitability parameters are described in Table
System suitability parameters obtained in the separation of azelastine and related impurities.
Retention factor | Peak resolution (Rs) | Peak asymmetry | |
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Impurity B | -0.35 | 1.36 | |
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Azelastine | 0.43 | 9.0 (B-Aze) | 1.36 |
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Impurity D | 3.04 | 17.14 (Aze-D) | 1.14 |
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Impurity E | 5.04 | 13.14 (D-E) | 1.05 |
k’: relative retention (retention factor).
Rs: resolution between adjacent peaks.
Chromatographic separation of azelastine HCl (3.3 min), impurity B (1.5 min), impurity D (9.3 min), and impurity E (13.9 min).
The selectivity of the method was accessed by peak purity (>0.99) and relative retention (k’) of impurity B (-0.35), AZE B (0.43), impurity D (3.04), and impurity E (5.04) standard solutions, separately.
The system suitability parameters such as peak resolutions between adjacent peaks and peak symmetry were according to the specifications for separation of azelastine and its related impurities described in official compendiums [
The proposed ecologically correct methods may contribute in high-throughput analysis of seven antihistaminic drugs, with total organic residue < 2.5 mL/analysis. The proposed method for separation of azelastine and related organic impurities B, D, and E is highly efficient and economical. The total analysis time was 15 minutes and the mobile phase is free of ion pairing agents and buffer salts. All separations were obtained on a conventional reverse phase HPLC systems and can be considered excellent in terms of greenness. The proposed methods may subsidize in the evaluation of quality, efficacy, and safety of antihistaminic drugs.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was supported by the São Paulo Research Foundation (FAPESP), Brazil, under Grant no. 2015/11210-1.