HPLC Analysis of Fexofenadine and Related Compound B on CYCLOBOND® I 2000 Phases

Analysts operating both within and outside regulatory auspices often use established methods, such as the U.S. Pharmacopeia. In many cases, however, the older methods call for or suggest materials that may not be currently available, are cost-prohibitive, or are simply poor substitutes for modern materials. As an example, the USP® monograph for fexofenadine hydrochloride, Limit of fexofenadine related compound B, dictates the use of an HPLC column that contains packing L45 [1]. According to USP Chromatographic Columns the suggested column for the monograph is a CYCLOBOND I 2000 SP phase [2]. The L45 description is “Beta cyclodextrin bonded to porous silica particles, 5 or 10 µm in diameter”. Although the SP phase was designated at the time the method was first deployed, many variations of L45 have since been commercialized.

The purpose of this study was to investigate alternative versions of L45 as they apply to the conditions set forth in the USP method for fexofenadine. Several stationary phases meeting the L45 description including CYCLOBOND I 2000, CYCLOBOND I 2000 RSP and CYCLOBOND I2000 HPRSP were compared to CYCLOBOND I 2000 SP using the conditions set forth in the USP monograph. The columns were all 25 cm x 4.6 mm packed with 5 µm particles. Fexofenadine, reagents and solvents were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Fexofenadine Related Compound B was obtained from the USP (Rockville, MD, USA).


CYCLOBOND I 2000 SP




CYCLOBOND I 2000

Figure 1. Comparison of CYCLOBOND I 2000 SP (20224AST) and CYCLOBOND I 2000 (20024AST) for Fexofenadine and Fexofenadine Related Compound B using USP Conditions.
Sample: 100 - 10 µg/mL in mobile phase, respectively; Column dimensions: 25cm x 4.6 mm I.D., 5 µm; Mobile phase: (A) ammonium acetate buffer pH 4.0; (B) acetonitrile; (80:20, A:B); flow rate: 0.5 mL/min; column temp. 25 °C; detector UV at 220 nm
injection 20 µL; Sample: 100 - 10 µg/mL in mobile phase, respectively
Peak ID: 1) Fexofenadine Related Compound B, 2) Fexofenadine

Comparison of the stationary phases using the USP conditions revealed that CYCLOBOND I 2000, an unaltered beta cyclodextrin phase, provided the most similar retention and selectivity as compared to CYCLOBOND I 2000 SP. As shown in Figure 1, the native cyclodextrin phase shows slightly greater retention and higher resolution as compared to the SP phase. A slight mobile phase adjustment from 80:20 buffer:acetonitrile to 78:22, well within the guidelines for USP methods, results in a nearly identical separation to that obtained using the SP phase (Figure 2). The adjustment using the CYCLOBOND I 2000 resulted in a relative retention time of 0.65 for the related compound and a resolution of 6.1. These results fully meet the USP requirements of relative retention of about 0.7 and a resolution of not less than 3.

Figure 2. HPLC Separation of Fexofenadine and Fexofenadine Related Compound B using CYCLOBOND I 2000 (20024AST) using USP method with Slightly Altered Mobile Phase.
Conditions: Same as Figure 1 except mobile phase ratio 78:22, A:B).
Peak ID: 1) Fexofenadine Related Compound B, 2) Fexofenadine

It was also noted in the study that both CYCLOBOND I 2000 RSP and HPRSP phases resulted in much longer retention for both the fexofenadine and the related compound. With more major adjustments to the USP conditions, a highly efficient separation was obtained using the HPRPS column (Figure 3).

Figure 3. HPLC Separation of Fexofenadine and Fexofenadine Related Compound B using CYCLOBOND I 2000 HPRSP (24024AST) using USP method with Significant Alterations.
Column dimensions: 25 cm x 4.6 mm I.D., 5 µm; Mobile phase: (A) ammonium acetate buffer pH 4.0, (B) acetonitrile, (60:40, A:B); flow rate: 1.0 mL/min; column temp. 25 °C; detector UV at 220 nm
injection 20 µL.
Peak ID: 1) Fexofenadine Related Compound B, 2) Fexofenadine


In conclusion, methods found in sources such as the USP Pharmacopeia often call for or suggest certain stationary phases. In this study, the use of alternative stationary phases to CYCLOBOND I 2000 SP, as suggested by the USP monograph for the analysis of fexofenadine related compound B, was studied. A native beta cyclodextrin phase, CYCLOBOND I 2000, was shown to be nearly equivalent to the original phase with only slight modification to the mobile phase. In addition, a more modern hydroxypropyl-modified beta cyclodextrin phase, CYCLOBOND I 2000 HPRSP was shown to provide excellent resolution and retention for the target analytes when more major alterations to the USP conditions were made. Depending on the regulatory environment an analyst is working under, each of the investigated phases and conditions may be used as a substitute for the CYCLOBOND I 2000 SP stationary phase.

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References

1. Official monograph for Fexofenadine. United States Pharmacopeia and National Formulary, USP 34-NF 29; United States Pharmacopeia Convention: Rockville, MD; 2011, vol. 2,2823-2825.
2. USP Chromatographic Columns, 2009-2010,;United States Pharmacopeia Convention: Rockville, MD; 171.

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