MEDICINAL CHEMISTRY RESEARCH
Med Chem Res DOI 10.1007/s00044-009-9285-6 ORIGINAL RESEARCH
RP-HPLC method for the quantitative determination of fexofenadine hydrochloride in coated tablets and human serum M. Saeed Arayne • Najma Sultana • Hina Shehnaz Amir Haider
•
Received: 26 May 2009 / Accepted: 4 December 2009 Ó Springer Science+Business Media, LLC 2009
Abstract Fexofenadine is a non-sedative and selective peripheral H1 receptor antagonist prescribed for allergic rhinitis and chronic urticaria. This article deals with a simple, feasible, and sensitive isocratic reverse-phase high-performance liquid chromatographic method for the determination of fexofenadine hydrochloride in bulk drug, pharmaceutical dosage forms and in human serum. The chromatography was carried out at 20 ± 2°C using two different chromatographs and five different stationary phases. The isocratic mobile phase was phosphate buffer pH 7.4 and methanol (methanol–phosphate buffer, 35:65, v/v), detection was made at 218 nm and the mobile phase flowed at 1 ml min-1. Validation parameters included linearity, accuracy, precision, specificity, limit of detection (LOD), limit of quantification (LOQ), and robustness over a linearity range 5–15 lg ml-1 according to the ICH guidelines (r [ 0.9999), the inter- and intra-day precisions were relative standard deviation (RSD) \ 0.8%. The system suitability was scrutinized by capacity factor, tailing factor, and number of theoretical plates (capacity factor [ 2.0, tailing factor B 2.0, and theoretical plates [ 2000). The retention time for five different stationary phases ranged from 3.78 to 4.15 min. The LOD and LOQ for the procedure were executed on samples containing very low concentrations of analytes on two different commercial brands of detectors. Keywords Fexofenadine Urticaria High-performance liquid chromatography Isocratic Robustness Five different stationary phases
M. S. Arayne H. Shehnaz (&) A. Haider Department of Chemistry, University of Karachi, Karachi 75270, Pakistan e-mail:
[email protected] N. Sultana Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan
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Introduction Fexofenadine, a,a-dimethyl-4-[1-hydroxy-4-[4-(hydroxydiphenyl-methyl)-1-piperidinyl]butyl]-benzene acetic acid (Fig. 1), is used to relieve the allergy symptoms of seasonal allergic rhinitis (‘‘hay fever’’), including runny nose; sneezing; and red, itchy, or watery eyes; or itching of the nose, throat, or roof of the mouth in adults (Markham and Wagstaff, 1998; Simpson and Jarvis, 2000). It is carboxylic acid metabolite of terfenadine, a non-sedating selective histamine H1 receptor antagonist (Caballero et al., 1999). Unlike its precursor, fexofenadine lacks the cardiotoxic potential, effective in the management of allergic rhinitis, and chronic idiopathic urticaria for which it is a suitable option for first-line therapy (Inomata et al., 2009). Fexofenadine is a substrate of P-glycoprotein, used to explore activity in vivo because it is not metabolized in human body. Besides, no sedative or other central nervous system effects were observed and radiolabeled tissue distribution studies in rats indicated that fexofenadine does not cross the blood–brain barrier (British Pharmacoepia, 2000; Barnes et al., 1993). On pharmaceutical dosage forms, very few methods from quality control are presented to determine fexofenadine in dosage form, in urine, serum, and pharmaceutical formulations (Drescher et al., 2002; Mattila and Paakkari, 1999). Three methods were presented for determination of fexofenadine in pure form and its R(1) and S(-) enantiomers were analyzed in plasma and urine by validated performance liquid chromatographic (HPLC) methods in commercial dosage forms (Gazy et al., 2002). Another bioanalytical method was reported using solid phase extraction and liquid chromatography with electrospray (Naidong et al., 2002). Mass spectrometry (LC/MS/MS), RPLC, ionspray tandem mass spectrometry detection (Gergov et al., 2001; Fu et al., 2004), and fluorescence detection (Uno et al., 2004) are used in the pharmaceutical dosage form using ultraviolet spectrophotometry (Pratt et al., 1999; Radhakrishna and Om Reddy, 2002; Milne et al., 2000; Hamman et al., 2001; Russell et al., 1998; Pathak et al., 2008; Karakus et al., 2008). HPLC is the most widely used technique in pharmaceutical companies, clinical laboratories, and research and development laboratories. Fexofenadine is the most frequently prescribed H1 receptor antagonist manufactured by many pharmaceutical companies world wide. The methods reported in the literature for the determination of fexofenadine are time-consuming, difficult, and expensive making them unpractical for everyday clinical trials. Also, there is no specific method for the
Fig. 1 Fexofenadine
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determination of only fexofenadine in pharmaceutical dosage forms as well as in human serum. Therefore, there was a need for developing an HPLC method for the determination of fexofenadine in reference drug material, pharmaceutical formulations, and in human serum, which should have adequate sensitivity and short elution time making it suitable as a regular method for pharmaceutical and clinical labs. Various HPLC methods have been reported for the quantitation of H1-receptor antagonists like cetirizine, buclizine, and levocetirizine in dosage formulations and human serum (Sultana et al., 2009; Arayne et al., 2005; Arayne et al., 2008; Gowekar et al., 2007). The aim of this study was to present the method which should be rapid, selective, linear, precise and sensitive, and should be less timeconsuming (analysis time 3.78–4.15 min). The method presented in this article was validated according to the ICH guidelines (ICH guidelines Topic Q2 (R1) Validation of Analytical Procedures), and the low limit of quantification (LOQ) and limit of detection (LOD) values make it a better choice for the estimation of fexofenadine in human serum. This validated method was also applied on different brands of fexofenadine available in Pakistan (60 mg tablets) which supports the analysis of fexofenadine in bulk, in dosage formulations, and in human serum for therapeutic purpose using HPLC.
Experimental Reagents and chemicals Fexofenadine hydrochloride reference substance (99.6%) was obtained from Aventis Pharma (Pvt.) Limited, Pakistan. Monobasic potassium phosphate, potassium hydroxide, HPLC, and analytical grade solvent (methanol) were purchased from Merck (Germany), water for HPLC was prepared by double distillation and filtration through Millipore 0.45 lm membrane filter (Millipore, Milford, MA, USA), and degassed with Branson 3200 ultrasonic bath. Three commercial preparations, FexetÒ tablet (Getz Pharma Pakistan (Pvt.) Ltd., FexofastÒ tablet (Platinum Pharmaceuticals (Pvt.) Ltd., and TelfastÒ tablet (Sanofi Aventis Pharma (Pvt.) Ltd., Pakistan, all containing 60 mg fexofenadine/tablet were assayed. Chromatographic system and conditions Two chromatographs were used (a) Shimadzu liquid chromatograph equipped with a model LC-10AVP isocratic pump, and model SPD-10AVP UV detector. Detection was made at 218 nm. CLASS-GC chromatography software. (b) The HPLC Chromatograph Series 200 was produced by Perkin Elmer, USA, and equipped with autosampler, pump, UV/VIS detector, Totalchrom Navigator Version 6.3.1.0504 software, interface 600 series LINK were used. Five different brands of stationary phase were used: (i) 250 9 4.6 i.d. mm KROMASILÒ 100-5 C-18 column (5 lm particle size) HICHROM (UK), (ii) 250 mm 9 4.6 mm i.d. 9 5 lm particle size GEMINIÒ C18 Phenomenex (USA), (iii) 250 mm 9 4.6 mm i.d. 9 5 lm particle size NUCLEOSILÒ 100-5 C18 Macherey–Nagel (Germany),
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(iv) 250 mm 9 4.6 mm i.d. 9 5 lm DiscoveryÒ C18 SUPELCO (USA), (v) 250 mm 9 4.6 mm i.d. 9 5 lm HypersilTM ODS column Thermo Electron Corporation (UK). On both systems, mobile phase run isocratically, the mobile phase was prepared by mixing methanol and 6.8 g monobasic potassium phosphate in 1000-ml water and pH 7.4 (adjusted with potassium hydroxide), 35:65 (v/v), respectively. The injection volume was 20 ll and the run time was 10 min. The mobile phase was filtered using a 0.45-lm membrane filter (Millipore) and degassed with Branson 3200 ultrasonic bath. The mobile phase flow rate was 1.0 ml min-1. Injections were carried out using a 20-ll loop at room temperature (20 ± 2°C). Preparation of stock standard solution A 10 mg amount of fexofenadine reference substance was accurately weighed, dissolved in mobile phase, and diluted to volume in a 100-ml volumetric flask. Standard solution was obtained by diluting the above solution with mobile phase to a concentration of 10 lg ml-1. Preparation of sample solutions All the three commercially available brands of fexofenadine were analyzed separately by preparing a composite of 20 tablets by grinding them to a fine, uniform size powder, using mortar and pestle. After calculating the average tablet weight, amount corresponding to 10 mg of fexofenadine was accurately weighed and quantitatively transferred into a 100-ml volumetric flask. Approximately, 60 ml mobile phase was added and the solution was shaken mechanically for 15 min, then flask was made up to volume with mobile phase, and mixed. After filtration through Millipore 0.45 lm membrane, the solution was diluted with mobile phase to a concentration of 10 lg ml-1. Extraction and storage of blood samples Fresh blood samples from healthy volunteers were collected daily, centrifuged, and separated, and then 10 ml of acetonitrile was added in 1.0 ml of plasma and vortexes for 1 min, centrifuged for 10 min at 10,000 rpm. After that, supernatant was filtered by 0.45-lm pore size membrane filter. An aliquot serum sample was prepared with fexofenadine hydrochloride to achieve final concentration; the serum was kept at -20°C until analyzed. Human plasma samples were spiked with fexofenadine with mobile phase to 1–5 lg ml-1 final solution concentrations.
Result and discussion Method validation The method was validated according to the ICH guidelines (ICH guidelines Topic Q2 (R1) Validation of Analytical Procedures). The following validation
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characteristics were addressed: linearity, accuracy, precision, and specificity, LOD, LOQ, robustness, and their chromatograms are illustrated in Figs. 2, 3, 4, 5, 6, 7, 8, 9, and 10.
Fig. 2 Chromatogram of 10 lg ml-1 of fexofenadine reference standard
Fig. 3 Chromatogram of FexetÒ tablet placebo
Fig. 4 Chromatogram of FexetÒ tablet contains 10 lg ml-1 of fexofenadine
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Fig. 5 Chromatogram of FexofastÒ tablet placebo
Fig. 6 Chromatogram of FexofastÒ tablet contains 10 lg ml-1 of fexofenadine
Fig. 7 Chromatogram of TelfastÒ tablet placebo
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Fig. 8 Chromatogram of TelfastÒ tablet contains 10 lg ml-1 of fexofenadine
Fig. 9 Chromatogram of blank plasma sample from healthy volunteer
Fig. 10 Chromatogram of plasma sample spiked with 2.0 lg ml-1 of the fexofenadine
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System suitability Having optimized the efficiency of a chromatographic separation, the quality of chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor, and of theoretical plates. The system suitability method acceptance criteria set in each validation run were capacity factor [ 2.0, tailing factor B 2.0, and theoretical plates [ 2000 (CDER: Center for Drug Evaluation and Research, 1994). In all cases, the RSD for the analyte peak area for two consecutive injections was \2.0%. LOD and LOQ The LOD and LOQ studies for the developed procedure were performed on samples containing very low concentrations of analytes on two different commercial brands of detectors according to the ICH guidelines as shown in Table 1. According to the visual evaluation method, LOD was uttered by establishing the minimum level at which the analyte can be reliably detected. LOQ was considered as the lowest concentration of analytes in standards that can be reproducibly measured with acceptable accuracy and precision. Linearity Standard curves were constructed daily, for three consecutive days, using five standard concentrations in a range 5–15 lg ml-1 for fexofenadine. This concentration range corresponds to 50–150% w/w levels of the nominal analytical concentration (Table 2). The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The regression equation and correlation coefficient (r) were y = 24340x ? 120 (y peak area, x concentration) and 0.9999, respectively. Accuracy The accuracy of the method was evaluated by determination of the recovery of fexofenadine on 3 days at three levels of concentrations. Commercial preparation C TelfastÒ tablet (60 mg) was spiked with fexofenadine standard solution, corresponding to 50–150% of the nominal analytical concentration (10 lg ml-1). The results showed good recoveries ranging from 98.77 to 101.45%. The mean recovery data obtained for each level as well as for all levels combined (Table 2) were within
Table 1 LOD and LOQ on different brands of detectors Shimadzu SPD-10AVP UV detector (ng ml-1)
Perkin Elmer UV/VIS detector Series 200 (ng ml-1)
LOD
20
10
LOQ
35
20
Med Chem Res Table 2 Accuracy and linearity of method determined by recovery of fexofenadine from tablets solutions spiked with standard solution 5 lg ml-1 50%
7.5 lg ml-1 75%
10 lg ml-1 100%
12.5 lg ml-1 125%
15 lg ml-1 150%
Day 1
99.98
98.77
100.21
101.45
99.88
Day 2
99.96
98.88
100.08
100.33
100.60
Day 3
100.24
98.78
100.78
100.44
99.96
Mean (n = 3)
100.06
99.14
100.36
100.74
100.15
0.15
0.54
0.37
0.67
0.39
%RSD Total mean (n = 15)
100.022
%RSD
0.743 Ò
Telfast tablet 60 mg (Sanofi Aventis Pharma (Pvt) Ltd.) were spiked with fexofenadine standard solution, corresponding to 50–150% of the nominal analytical concentration (10 lg ml-1)
2.0% of the label claim for the active substance with an RSD \ 2.0%, which satisfied the acceptance criteria set for the study. Precision Precision of the method was determined by measuring the repeatability (intra-day precision) and intermediate precision (inter-day precision), both expressed as %RSD. The repeatability was evaluated by assay of six samples from each pharmaceutical commercial preparation (Table 3), at the same concentration (10 lg ml-1), on the same day. The intermediate precision was calculated from the results obtained on three different days. Specificity FexetÒ tablet contains 60 mg of the drug and the following excipients: pregelatinized starch, lactose, croscarmellose sodium, and microcrystalline cellulose. FexofastÒ tablet contains 60 mg of the drug and the following excipients: lactose, croscarmellose sodium, and microcrystalline cellulose. TelfastÒ tablet contains
Table 3 Intra-day (n = 6) and inter-day (n = 18) precision results from each commercial preparation tablets assay on three consecutive days Day FexetÒ tablet (60 mg) Intra-day
Inter-day
FexofastÒ tablet (60 mg)
TelfastÒ tablet (60 mg)
Intra-day
Intra-day
Inter-day
Inter-day
Mean %RSD Mean %RSD Mean %RSD Mean %RSD Mean %RSD Mean %RSD 1
99.94 0.49
2
99.97 0.44
99.96 0.45
99.91 0.65 99.93 0.56
99.71 0.66
99.89 0.52 99.82 0.55
3
99.87 0.24
99.89 0.22
99.80 0.67
99.81 0.77
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60 mg of the drug and the following excipients: lactose, croscarmellose sodium, and microcrystalline cellulose. Specificity was evaluated by preparing placebo samples of each placebo of the commercial formulation of tablets containing the same excipients and specificity was performed by preparing placebo tablets of the commercial products. The solutions were prepared using the same procedure described for the sample solutions and injected thrice (Table 3) and the characteristic chromatograms are shown in Figs. 2–8. Robustness International variations in liquid chromatographic conditions were used to evaluate the robustness of the assay method. In this study, the chromatographic parameters monitored were retention time, area, capacity factor, tailing factor, and theoretical plates. The robustness acceptance criteria set in the validation was the same established on system suitability test described earlier. Different brand of chromatographic columns Three injections of TelfastÒ tablet (Sanofi Aventis Pharma (Pvt.) Ltd., containing 60 mg fexofenadine/tablet) solutions having a concentration of 10 lg ml-1 were injected on each brand of column to evaluate robustness (Table 4). Different instruments Three injections of each commercial preparation were injected on two different brands of liquid chromatograph by two different analysts data established in Table 5. Different personnel Variation in results due to different analyst’s data is established by taking the %RSD of recovered % as shown in Table 5, which is found 0.28 and 0.45 for analysts A and B, respectively. Table 4 Chromatographic parameters of robustness evaluation Resolutiona Tailing factor
Theoretical plates
242798 3.15
3.12
1.21
2366
243375 1.68
3.48
1.08
3365
4.15
241624 2.39
3.54
1.02
3687
4.12
239963 2.17
3.02
1.34
2489
Thermo Electron Corporation 3.98 HypersilTM
241840 2.36
3.21
1.22
2562
Different columns brands (n = 3)
Retention time
Peak area
HICHROM KROMASILÒ
3.78
Phenomenex GEMINIÒ
3.80
Macherey–Nagel NUCLEOSILÒ SUPELCO DiscoveryÒ
a
In relation to the nearest peak
Capacity factor
Med Chem Res Table 5 Different instrument and different personnel variation Commercial preparation (n = 3)
Shimadzu liquid chromatograph
Perkin Elmer Series 200 liquid chromatograph
Analyst ‘‘A’’
Analyst ‘‘B’’
Found ± (mg) Ò
%RSD %Recovery Found ± (mg)
% Difference in instruments
%RSD %Recovery
Fexet
59.79 ± 1.1245 1.12
99.65
60.10 ± 1.0132 1.01
100.16
0.51
FexofastÒ
59.89 ± 1.0325 1.03
99.81
59.60 ± 1.2261 1.22
99.33
0.48
TelfastÒ
60.12 ± 1.0576 1.05
100.20
60.05 ± 1.0463 1.04
100.08
0.12
Table 6 Recovery of fexofenadine from human serum (n = 3) S. no
Concentration (lg ml-1)
Mean (lg ml-1)
1
1
0.995
0.5
0.9
2
2
2.0
100
-0.15
1.21
100.3
-0.3
3
3
3.009
4
4
3.998
5
5
5.085
Correlation coefficient (r2)
0.9997
LOD
0.03
LOQ
0.1
(%) Recovery
99.5
99.95 101.7
Relative error (%)
0.05 -1.7
CV (%)
0.8 0.68 0.16
Recovery and regression characteristics of fexofenadine, in human serum Different concentrations of human plasma samples (1–5 lg ml-1) were linear, accurate, precise, and selective by running three replicates of each concentration measured for 5 days; typical chromatogram for the concentration of 2 lg ml-1 is shown in Fig. 10. The mean recoveries, coefficient of variation, LOD, and quantification values are summarized in Table 6.
Conclusions The LOD and LOQ were carried out on two different commercial brands of detectors according to the ICH guidelines. The intra- and inter-day precision studies showed good reproducibility with coefficients of variation \0.77%. The ruggedness of the method was determined by carrying out the experiment on different instruments like Shimadzu HPLC (LC-10), Perkin Elmer Series 200 HPLC by different operators using different columns. Robustness of the method was determined by making slight changes in the chromatographic conditions. No marked changes in the chromatograms were observed. Thus, the proposed RP-HPLC method for the estimation of fexofenadine in dosage forms is accurate, precise, linear, rugged, robust, simple, and rapid. Hence,
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the RP-HPLC method is suitable for the quality control and routine testing of the raw materials, different dosage forms, different formulations, and in human serum. References Arayne MS, Sultana N, Siddiqui FA (2005) Determination and quantification of cetirizine HCl in dosage formulations by RP-HPLC. Pak J Pharm Sci 18:7–11 Arayne MS, Sultana N, Nawaz M (2008) Simultaneous quantification of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations and human plasma by RP-HPLC. J Anal Chem 63:881–887 Barnes CL, McKenzie CA, Webster KD, Poinsett-Holmes K (1993) Cetirizine: a new, non-sedating antihistamine. Ann Pharmacother 27:464–470 British Pharmacoepia (2000) Her Majesty Stationary Office, pp 342–343 Caballero E, Ocan˜a I, Azanza JR, Sa´daba B (1999) Fexofenadine: a antihistaminic review of its practical characteristics. Rev Med Univ Navarra 43:93–97 CDER: Center for Drug Evaluation and Research (1994) Reviewer guidance for validation of chromatographic methods. Disponı´velem: http://www.fda.gov/cder/guidance/cmc3.pdf Drescher S, Schaeffeler E, Hitzl M, Hofmann U, Schwab M, Brinkmann U, Eichelbaum M, Fromm MF (2002) MDR1 gene polymorphisms and disposition of the P-glycoprotein substrate fexofenadine. Br J Clin Pharmacol 53:526–534 Fu I, Woolf EJ, Matuszewski BK (2004) Determination of fexofenadine in human plasma using 96-well solid phase extraction and HPLC with tandem mass spectrometric detection. J Pharm Biomed Anal 35:837–846 Gazy AA, Mahgoub H, El-Yazbi FA, El-Sayed MA, Youssef RM (2002) Determination of some histamine H1 receptor antagonists in dosage forms. J Pharm Biomed Anal 30:859–867 Gergov M, Robson JN, Ojanpera¨ I, Heinonen OP, Vuori E (2001) Simultaneous screening and quantification of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry. Forensic Sci Int 121:108–115 Gowekar NM, Pande VV, Kasture AV, Tekade AR, Chandorkar JG (2007) Spectrophotometric estimation of ambroxol and cetirizine hydrochloride from tablet dosage form. Pak J Pharm Sci 20:250–251 Hamman MA, Bruce MA, Haehner-Daniels BD, Hall SD (2001) The effect of rifampin administration on the disposition of fexofenadine. Clin Pharmacol Ther 69:114–121 ICH guidelines Topic Q2 (R1) Validation of analytical procedures: methodology. www.emea.europa.eu/ htms/human/ich/ichquality.htm. Inomata N, Tatewaki S, Ikezawa Z (2009) Multiple H1-antihistamine-induced urticaria. J Dermatol 36:224–227 Karakus¸ S, Ku¨c¸u¨kgu¨zel I, Ku¨c¸u¨kgu¨zel SG (2008) Development and validation of a rapid RP-HPLC method for the determination of cetirizine or fexofenadine with pseudoephedrine in binary pharmaceutical dosage forms. J Pharm Biomed Anal 46:295–302 [Epub 2007 October 22] Markham A, Wagstaff AJ (1998) Fexofenadine. Drugs 55:269–274 discussion 275–276 Mattila MJ, Paakkari I (1999) Variations among non-sedating antihistamines: are there real differences? Eur J Clin Pharmacol 55:85–93 ˜ rgensen KLJ, Bastlund J, Stretch GR, Evans AM (2000) Hepatic disposition of Milne RW, Larsen LA, A fexofenadine: influence of the transport inhibitors erythromycin and dibromosulphothalein. Pharm Res 17:1511–1515 Naidong W, Shou WZ, Addison T, Maleki S, Jiang X (2002) Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases—a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE. Rapid Commun Mass Spectrom 16:1965–1975 Pathak SM, Kumar AR, Musmade P, Udupa N (2008) A simple and rapid high performance liquid chromatographic method with fluorescence detection for the estimation of fexofenadine in rat plasma—application to preclinical pharmacokinetics. Talanta 76:338–346 [Epub 2008 March 13] Pratt C, Brown AM, Rampe D, Mason J, Russell T, Reynolds R, Ahlbrandt R (1999) Cardiovascular safety of fexofenadine HCl. Clin Exp Allergy 29:212–216
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