Applied Chemical Engineering

Optimization and validation of a rapid RP-HPLC method for the simultaneous quantification of codeine and diazepam in bulk and pharmaceutical dosage forms

Rulla Sabah

Department of Chemistry, College of Science, Mustansiriyah University, Baghdad, 10011, Iraq

Nisreen Kais Abood

Department of Chemistry, College of Science, Mustansiriyah University, Baghdad, 10011, Iraq

Mustafa Hammadi

Department of Chemistry, College of Education for Pure Sciences, University of Diyala, Baqubah, 32001, Iraq

DOI: https://doi.org/10.59429/ace.v9i2.5937

Keywords: codeine; diazepam; optimization; quantification; RP-HPLC; surface morphology (RSM)

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Abstract

This study establishes a high-performance liquid chromatography (HPLC) method that is precise and expeditious for the existing separation and quantification of codeine and diazepam, while reducing analysis time and separation efficiency. An RP-Sunfire C18 column (5 µm, 4.6 × 250 mm) was used to separate the samples. The mobile phase consisted of acetonitrile and a phosphate buffer and detection were performed at 230 nm. The approach separated the two drugs in less than 7 minutes using an isocratic elution mode. Codeine and diazepam had retention times of 4.19 and 6.09 minutes, respectively, and the resolution was a good value (Rs = 4.22). The results showed that the column was highly efficient, with theoretical plate counts passing 2579 for codeine and 2373 for diazepam. The very symmetrical peaks, with symmetry factor values ranging from 1.05 to 1.08. The method showed an excellent linearity within the range of concentration (5-25 µg. mL^-1) with high Coefficient of Determination (R²≥ 0.9998) and low limits of detection, indicating its high sensitivity. High recovery rates (99.46-100.27%) further confirmed the method's accuracy and lack of pharmaceutical interference. Separation conditions were optimized using response surface morphology (RSM), with analysis of variance (ANOVA) showing high significance for the quadratic model (p = 0.0057) and no significance for the non-conformity test (p = 0.2700). Optimal conditions were determined using a mobile phase of acetonitrile and phosphate buffer in a 40:60 (v/v) ratio at pH 4.0, with a flow rate of 1.0 mL.min^-1. These conditions confirm the method's efficiency and suitability for routine pharmaceutical laboratory applications.

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