Development and validation of an assay method for the determination of pyrimethamine in an oral suspension by HPLC-UV

S. Muhammad, Z. Ribier, G. Benoit, J. Bordenave
Hôpital Armand Trousseau, Paris, France

Introduction
Pyrimethamine (PYR), combined with sulfadiazine and folinic acid, is the first line treatment of congenital toxoplasmosis in neonates and infants. No pharmaceutical form is available to this age group in France: the development of an oral suspension of pyrimethamine is therefore essential.

Objective
Development and validation of a stability-indicating method by HPLC-UV.

Materials & Methods:
The analyses were performed by HPLC-UV with a Kinetex Coreshell C18 column, thermostated at 40°C, coupled to a diode array (λ=230 nm, 280 nm, and scan from 190 to 400 nm) with a gradient of acetonitrile/methanol/KH2PO4 buffer (pH 3). The method was performed according to the analytical validation criteria (linearity, accuracy, precision, and specificity) specified by the ICH [1].
A forced degradation (FD) study of PYR was performed to evaluate the stability indicator character. PYR was subjected to acidic (pH=2 and pH=6), alkaline (pH=12), thermal (60°C and 5+/-3°C), H2O2 oxidation (3% and 15%), and photolysis (UV and natural light) hydrolysis at Day 0, 3 and 16. Oxidation by Magnesium MonoPeroxyPhthalate (MMPP 0.01 mM) after 6 hours was also studied.

Results
The analytical method is linear for a concentration range of 40-120 mg/L, with correlation coefficients (r²) curves, with and without excipients, of 0.997 and 0.99, respectively. The method is accurate with a recovery rate between 99.41 and 100.54.
The coefficients of variation obtained in the repeatability and intermediate precision studies are less than 5% (1.77 and 2.11 respectively): the method is precise. For specificity, the excipients tested (xanthan gum, citric acid/sodium citrate, sucralose, and potassium sorbate) did not interfered with PYR, and the retention times of PYR remained stable during the study (4.98+/-0.02 min).
FD assays at Day 16 showed that PYR is sensitive to oxidation (MMPP and H2O2), poorly sensitive to light and not sensitive to hydrolysis. H202 3% stress leads to degradation products (DP) (area normalization>0.1% PYR area) at a relative retention time (RRT) of 0.57; 0.75; 0.89 and 2.0 and 11.3% decrease in PYR area. UV stress leads to DP at RRT of 0.53; 0.54; 0.71; 0.77 and 1.4% decrease in PYR area while MMPP stress leads to DP at RRT 1.25 and 1.95 but the PYR area is uninterpretable. (RRT MMPP = 1).

Conclusion
The analytical method is validated and the FD studies demonstrated the stability indicator character. This allows a stability study to be carried out on the formulation under development in order to determine the duration and conditions of storage.

[1] International Conference on Harmonisation (ICH Q2 R1).

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