Title : Development and validation of a novel RP-HPLC-DAD method using a single mobile phase for the simultaneous determination of seventeen Active Pharmaceutical Ingredients (APIs) from different classes containing β-Lactam and Non-β-Lactam moieties
Abstract:
A simple, accurate, and robust reverse-phase high-performance liquid chromatography coupled with diode array detection (RP-HPLC-DAD) method was developed and validated for the simultaneous determination of seventeen active pharmaceutical ingredients (APIs) containing both β-lactam and non-β-lactam classes. The method enables the analysis of Cefotaxime Sodium, Paracetamol, Cefadroxil, Amoxicillin Trihydrate, Ibuprofen, Levofloxacin, Sulfamethoxazole, Cephradine, Cephalexin, Ampicillin Trihydrate, Cefaclor, Ceftriaxone Sodium, Cefixime, Sofosbuvir, Ciprofloxacin Hydrochloride, Cloxacillin Sodium and Flucloxacillin Sodium using a single chromatographic system.
Chromatographic separation was achieved on a Phenosphere C18 column (100 × 4.6 mm, 5 μm) using gradient elution. Mobile phases A and B consisted of tetraheptylammonium bromide (THAB) buffer and acetonitrile in different proportions at pH 6.3. The flow rate was 1.0 mL min⁻¹, column temperature 35 ± 2°C, and injection volume 20 μL. Detection was performed at 230 nm using a diode array detector.
The method was validated according to internationally recognized analytical validation guidelines. Excellent linearity was obtained over the concentration range of 50–250%. Precision studies showed relative standard deviation (RSD) values below 2.0% for all analytes. Accuracy, limits of detection (LOD), and limits of quantification (LOQ) met acceptance criteria.
All compounds were well resolved with satisfactory peak symmetry, resolution, and reproducible retention times, demonstrating the robustness of the method. The proposed method provides a cost-effective and time-efficient approach for the simultaneous analysis of chemically diverse pharmaceutical compounds. Reduced solvent consumption, simplified analytical procedures, and improved laboratory efficiency make it suitable for routine quality control applications. The method may also be applied for monitoring potential cross-contamination between β-lactam and non-β-lactam products in pharmaceutical manufacturing facilities.
