Med Chem Res DOI 10.

1007/s00044-009-9285-6 ORIGINAL RESEARCH

MEDICINAL CHEMISTRY 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 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 2C using two different chromatographs and ve different stationary phases. The isocratic mobile phase was phosphate buffer pH 7.4 and methanol (methanolphosphate buffer, 35:65, v/v), detection was made at 218 nm and the mobile phase owed at 1 ml min-1. Validation parameters included linearity, accuracy, precision, specicity, limit of detection (LOD), limit of quantication (LOQ), and robustness over a linearity range 515 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 ve 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: hinashehnaz@gmail.com 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 rst-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 bloodbrain 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 uorescence 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, difcult, and expensive making them unpractical for everyday clinical trials. Also, there is no specic 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 3.784.15 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 quantication (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 ltration through Millipore 0.45 lm membrane lter (Millipore, Milford, MA, USA), and degassed with Branson 3200 ultrasonic bath. Three commercial preparations, Fexet tablet (Getz Pharma Pakistan (Pvt.) Ltd., Fexofast tablet (Platinum Pharmaceuticals (Pvt.) Ltd., and Telfast tablet (Sano 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 KROMASIL 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 GEMINI C18 Phenomenex (USA), (iii) 250 mm 9 4.6 mm i.d. 9 5 lm particle size NUCLEOSIL 100-5 C18 MachereyNagel (Germany),

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(iv) 250 mm 9 4.6 mm i.d. 9 5 lm Discovery 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 ltered using a 0.45-lm membrane lter (Millipore) and degassed with Branson 3200 ultrasonic bath. The mobile phase ow rate was 1.0 ml min-1. Injections were carried out using a 20-ll loop at room temperature (20 2C). 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 ask. 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 ne, 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 ask. Approximately, 60 ml mobile phase was added and the solution was shaken mechanically for 15 min, then ask was made up to volume with mobile phase, and mixed. After ltration 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 ltered by 0.45-lm pore size membrane lter. An aliquot serum sample was prepared with fexofenadine hydrochloride to achieve nal concentration; the serum was kept at -20C until analyzed. Human plasma samples were spiked with fexofenadine with mobile phase to 15 lg ml-1 nal 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 specicity, 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 Fexet tablet placebo

Fig. 4 Chromatogram of Fexet tablet contains 10 lg ml-1 of fexofenadine

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Fig. 5 Chromatogram of Fexofast tablet placebo

Fig. 6 Chromatogram of Fexofast tablet contains 10 lg ml-1 of fexofenadine

Fig. 7 Chromatogram of Telfast tablet placebo

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Fig. 8 Chromatogram of Telfast 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 efciency 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 ve standard concentrations in a range 515 lg ml-1 for fexofenadine. This concentration range corresponds to 50150% 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 coefcient (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 Telfast tablet (60 mg) was spiked with fexofenadine standard solution, corresponding to 50150% 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) LOD LOQ 20 35 Perkin Elmer UV/VIS detector Series 200 (ng ml-1) 10 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% Day 1 Day 2 Day 3 Mean (n = 3) %RSD Total mean (n = 15) %RSD

7.5 lg ml-1 75% 98.77 98.88 98.78 99.14 0.54

10 lg ml-1 100% 100.21 100.08 100.78 100.36 0.37 100.022 0.743

12.5 lg ml-1 125% 101.45 100.33 100.44 100.74 0.67

15 lg ml-1 150% 99.88 100.60 99.96 100.15 0.39

99.98 99.96 100.24 100.06 0.15

Telfast tablet 60 mg (Sano Aventis Pharma (Pvt) Ltd.) were spiked with fexofenadine standard solution, corresponding to 50150% 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 satised 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. Specicity Fexet tablet contains 60 mg of the drug and the following excipients: pregelatinized starch, lactose, croscarmellose sodium, and microcrystalline cellulose. Fexofast tablet contains 60 mg of the drug and the following excipients: lactose, croscarmellose sodium, and microcrystalline cellulose. Telfast 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 Fexet tablet (60 mg) Intra-day Inter-day Fexofast tablet (60 mg) Intra-day Inter-day Telfast tablet (60 mg) Intra-day Inter-day

Mean %RSD Mean %RSD Mean %RSD Mean %RSD Mean %RSD Mean %RSD 1 2 3 99.94 0.49 99.97 0.44 99.87 0.24 99.96 0.45 99.91 0.65 99.93 0.56 99.89 0.22 99.71 0.66 99.89 0.52 99.82 0.55 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. Specicity was evaluated by preparing placebo samples of each placebo of the commercial formulation of tablets containing the same excipients and specicity 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. 28. 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 Telfast tablet (Sano 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 analysts 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 Different columns brands (n = 3) HICHROM KROMASIL Phenomenex GEMINI MachereyNagel NUCLEOSIL SUPELCO Discovery Retention time 3.78 3.80 4.15 4.12 Peak area Capacity factor Resolutiona Tailing factor 3.12 3.48 3.54 3.02 3.21 1.21 1.08 1.02 1.34 1.22 Theoretical plates 2366 3365 3687 2489 2562

242798 3.15 243375 1.68 241624 2.39 239963 2.17 241840 2.36

Thermo Electron Corporation 3.98 HypersilTM

a

In relation to the nearest peak

Med Chem Res Table 5 Different instrument and different personnel variation Commercial preparation (n = 3) Shimadzu liquid chromatograph Analyst A Found (mg) Fexet

Perkin Elmer Series 200 liquid chromatograph Analyst B

% Difference in instruments

%RSD %Recovery Found (mg) 99.65 99.81 100.20

%RSD %Recovery 100.16 99.33 100.08 0.51 0.48 0.12

59.79 1.1245 1.12 59.89 1.0325 1.03 60.12 1.0576 1.05

60.10 1.0132 1.01 59.60 1.2261 1.22 60.05 1.0463 1.04

Fexofast Telfast

Table 6 Recovery of fexofenadine from human serum (n = 3) S. no Concentration (lg ml-1) 1 2 3 4 5 Mean (lg ml-1) 0.995 2.0 3.009 3.998 5.085 0.9997 0.03 0.1 (%) Recovery Relative error (%) 0.5 -0.15 -0.3 0.05 -1.7 CV (%)

1 2 3 4 5 LOD LOQ

99.5 100 100.3 99.95 101.7

0.9 1.21 0.8 0.68 0.16

Correlation coefcient (r2)

Recovery and regression characteristics of fexofenadine, in human serum Different concentrations of human plasma samples (15 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, coefcient of variation, LOD, and quantication 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 coefcients 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
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