Validation and Measurement Uncertainty of an UPLC-PDA Stability Indicating Method Applied to Degradation Kinetics of Caspofungin
Daniela Dal Molim Ghisleni, Rogerio Takao Okamoto, Felipe Rebello Lourenco and Terezinha de Jesus Andreoli Pinto
Affiliation: Departamento de Farmacia, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Av. Prof. Lineu Prestes, 580, Cidade Universitaria, 05508-000, Sao Paulo, SP, Brasil.
Keywords: Caspofungin, UPLC, uncertainty measurement, kinetics of degradation, validation.
This work aims to develop and validate a UPLC stability indicating method for quantitative analysis of Caspofungin,
grounded in statistical analysis able to estimate measurement uncertainty, and determine drug degradation kinetics.
The validation was performed as recommended in ICH guidelines. The chromatographic method was conducted applying
the following conditions: 220 nm wavelength, 10 μL injection volume, 25 °C oven temperature, 0.6 mL/minute
flow, and gradient elution involving mobile phases A and B, composed of 0.1 % solution of triethylamine pH adjusted to
2.5 with the aid of phosphoric acid and acetonitrile, respectively. The validation of the UPLC method demonstrated the
selectivity, robustness, linearity, precision, accuracy and the ability to indicate caspofungin stability. Statistical analysis
demonstrated the method is linear in the concentration range between 20 and 80 μg/mL (y= 30908912.1196x – 11246.7143;
r= 0.9997), precise (repeatability: 1.09%, intermediate precision: 1.79%) and accurate (recovery range: 95.61–101.36%). According
to these results, the uncertainties associated with accuracy and precision were the most significant, contributing to
57% overall uncertainty. On the other hand, repeatability of sample and standard peak areas were almost insignificant
(less than 5% of overall uncertainty). The analysis of measurement uncertainty proved to be useful for validating the results
and a very useful tool in routine analysis. The degradation kinetics were calculated using the UPLC method, which is
a validated and well-established technique, capable of indicating thermal stability (80 °C) of caspofungin in solution. The
drug kinetics of degradation showed a first order reaction (y = - 0.0078 x + 2.0168, r = 0.9993) and half-life (t1/2) estimated at
3.9-minute analysis under the conditions tested, indicating that caspofungin is strongly sensitive to heat stress condition.
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