Method Development and validation for assessment of sulfadoxine and pyrimethamine by HPLC-UV

E. Marilly, A. Martelin, E. Seidel, D. Salmon, S. Filali, C. Merienne, C. Pivot, F. Pirot.
Service Pharmaceutique – Groupement Hospitalier Edouard Herriot – Hospices Civils de Lyon - France


Context
Our hospital pharmacy produces capsules of sulfadoxine-pyrimethamine, indicated in congenital toxoplasmosis. There are four different formulations according to weight and frequency of administration : 17,5 mg / 0,875 mg; 87,5 mg / 4,375 mg; 25 mg / 1,25 mg; 125 mg / 6,5/25 mg.

Objective
The discharge controls of those formulations include a quantitatification of drug content, for which an analytical, separative, quantitative method by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) must be used.

Material and method
The calibration range was done with 3 calibration standards : 12,5, 125 and 200 μg/mL for sulfadoxine, and 0,625, 6,25 and 10 μg/mL for pyrimethamine. The validation standards were 15, 100 and 175 μg/mL for sulfadoxine and 0,75, 5 and 8,75 μg/mL for pyrimethamine. A C18 ( 2,6 μg, 100 x 4,6 mm) column was used for separation maintained at 35°C. The mobile phase was composed of acidified water using acetic acid (pH=3,7) and acetonitrile (60 :40 v/v). The volume of injection was 10 µL and the flow rate was 1,2 mL/min, for a detection wavelengh at 220 nm. The accuracy profile, used in accordance to the recommendations of SFSTP, allowed to determine trueness, precision, linearity and limits of quantification, and to estimate inaccuracy of measures depending of the amount.

Results
The validation criteria of the method were all conform to the specifications : lack of interference from the mobile phase at retention times of sulfadoxine and pyrimethamine (chromatographic resolution > 1,5), linearity (R² > 0,99), trueness intra and inter day (CV < 5% and CV < 8% by ANOVA) and precision (CV < 10%). The tolerance interval limits were all included into the predefined (10%) tolerability interval. No matrix effect was detected, and retention times were stable for all the injections (1,2 min ± 5% for sulfadoxine and 6,2 min ± 5% for pyrimethamine).

Conclusion
The method were validated in accordance to the recommendations of ICH and SFSTP guidelines and this specific, simple and fast technic allows the achievement of the discharge quality control of the capsules.

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