A reversed-phase ion-pairing liquid chromatographic (RP-IP-HPLC) method for simultaneous assay of theophylline (TH), phenobarbital (PB), codeine (CD), and ephedrine (EP) in an extemporaneous (magistral) suppository was developed and validated and used to investigate a reported serious adverse event. Samples were dissolved in dichloromethane and extracted by two-step liquid-liquid extraction with acetate buffer (pH 5.0) and, subsequently, 0.1 m NaOH solution. Separation was performed on an end-capped C18 silica column with stepwise gradient elution. Sample preparation and chromatographic conditions were optimized on the basis of the pKa values of the analytes. Freedom from interference from the excipients, linearity (r2 > 0.999 for all the active ingredients), range (0.01–0.08 mg mL−1 for TH, CD, and EP; 0.0025–0.02 mg mL−1 for PB), intra-day and inter-day precision, and accuracy (recovery >95% for TH, CD, and EP and >90% for PB) of the method were demonstrated. Non-compliance of the examined product was confirmed.
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