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. 2014 Winter;13(Suppl):43-50.

Development and Validation of a Reversed-phase HPLC Method for Assay of the Decapeptide Cetrorelix Acetate in Bulk and Pharmaceutical Dosage Forms

Shirin Hooshfar  1 , Seyed Alireza Mortazavi  1 , Mohammad Piryaei  2 , Hossein Ramandi Darzi  2 , Nahid Shahsavari  3 , Farzad Kobarfard  4
Affiliations

Development and Validation of a Reversed-phase HPLC Method for Assay of the Decapeptide Cetrorelix Acetate in Bulk and Pharmaceutical Dosage Forms

Shirin Hooshfar et al. Iran J Pharm Res. 2014 Winter.

Abstract

A gradient reversed-phase high performance liquid chromatography (HPLC) method was developed for the assay of cetrorelix acetate, a synthetic decapeptide with gonadotropin-releasing hormone (GnRH) antagonistic activity used in infertility treatment. The HPLC method, which is used to determine cetrorelix in bulk and pharmaceutical dosage forms, was validated per ICH guidelines. The chromatographic separation was achieved on a C18 reversed-phase column using acetonitrile, water and trifluoroacetic acid (TFA) as mobile phase and wavelength was set at 275 nm. The calibration curve was linear (r2 = 0.999) over cetrorelix concentrations ranging from 62.50 to 12.50 μg/mL (n = 6). The limits of detection (LOD) and quantification (LOQ) were calculated from the peak-to-noise ratio as 15.6 and 62.5 μg/mL, respectively. The method had an accuracy of > 97% and intra- and inter-day RSD of < 0.3% and < 1.6%, respectively and was validated with excellent specificity, sensitivity, and stability. The validated method was successfully applied for determination of cetrorelix in bulk and pharmaceutical dosage forms.

Keywords: Assay; Cetrorelix acetate; Formulation; HPLC; UV detection.

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Figures

Figure 1
Figure 1
Chemical structure of cetrorelix
Figure 2
Figure 2
Chromatograms of: (A) blank sample (deionized water) in 230 nm (B) standard solution, 125 μg/mL cetrorelix as acetate in 230 nm (C) blank sample (deionized water) in 265 nm (D) standard solution, 125 μg/mL cetrorelix as acetate in 265 nm (E) blank sample (deionized water) in 275 nm (F) standard solution, 125 μg/mL cetrorelix as acetate in 230 nm
Figure 3
Figure 3
UV spectrum of cetrorelix acetate
Figure 4
Figure 4
Chromatograms of: (A) blank sample (deionized water) and (B) standard solution, 125 μg/mL cetrorelix as acetate (RT: 15.9 min) with gradient of 90% A for 5min, from 90% A to 70% B in 10 min, 70% B for 10 min – (C) blank sample (deionized water) and (D) standard solution, 125 μg/ mL cetrorelix as acetate (RT: 17.5 min) with gradient of 90% A for 5min, from 90% A to 70% B in 15 min, 70% B for 10 min
Figure 5
Figure 5
Chromatograms of: (A) blank sample (deionized water) (B) standard solution, 125 μg/mL cetrorelix as acetate in mobile phase with 0.05% TFA (C) standard solution, 125 μg/ mL cetrorelix as acetate in mobile phase with 0.1% TFA
Figure 6
Figure 6
Chromatograms of: (A) blank sample (deionized water) (B) blank sample (queous solution of mannitol, 54.8 mg/mL) (C) standard solution, 250 μg/mL cetrorelix as acetate (D) Assay sample solution (Cetrotide® 0.25 mg in 1 mL deionized water)
Figure 7
Figure 7
Linearity plot for cetrorelix drug substance
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