Original Article HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE SIMULTANEOUS ESTIMATION OF CEFOPERAZONE AND SULBACTAM IN RAT PLASMA AND ITS IMPORTANCE IN THERAPEUTIC DRUG MONITORING SWATI KORAKE 1# , ABHISHEK PAWAR 1# , SUSHANK SURYWANSHI 1 , C. BOTHIRAJA 1 , ATMARAM PAWAR 1* 1 Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune 411038 Email: p_atmaram@rediffmail.com Received: 05 Jun 2020, Revised and Accepted: 10 Aug 2020 ABSTRACT Objective: To study the Therapeutic drug monitoring and pharmacokinetics of marketed antibiotics formulation by developing a sensitive and specific Bioanalytical Chromatographic method. Methods: In the present study, we developed a rapid, sensitive and selective chromatographic method for simultaneous estimation of Cefoperazone (CEF) And Sulbactam (SAL) in male Wistar rat plasma. A novel liquid phase extraction method has adopted the preparation of plasma sample preparation. The CEF and SAL were eluted on a peerless Basic C18 (25 cm; 4.6 mm x 5 µm) column maintained at controlled environmental conditions. The gradient mobile phase comprised of 10 mmol ammonium acetate and acetonitrile. A UV detector was set at 250 nm and retention times for CEF and SAL were approximately 5.6 and 14.2 min, respectively. The proposed HPLC method was validated according to the US FDA guidelines with respect to the linearity, accuracy, precision, detection and quantitation limits, robustness and specificity. Results: Calibration curves of CEF and SAL were linear across the concentration range of 600-1000 and 6-10 µg/ml, with correlation coefficients (r2)>0.9977 and (r2)>0.9987, respectively. The limits of detection for CEF and SAL were 70.48 and 0.35 µg/ml, respectively. Additionally, CEF and SAL were stable in plasma for at least 24 h when stored at room temperature and 2-8 °C. Conclusion: The developed chromatographic method was effectively utilized to measure the plasma CEF and SAL concentrations in a pharmacokinetics study after intravenous injection to the healthy male Wistar rats. Keywords: Cefoperazone, Sulbactam, Pharmacokinetics study, Liquid phase extraction and HPLC © 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2020v12i10.38638. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps. INTRONDUCTION Antibiotics are chemically synthesized antimicrobial molecules regularly prescribed over the times for the prevention and treatment of various infectious diseases. Additionally, now day’s antibiotic and/or antibiotics combinations (e. g. cefoperazone/sulbactam; sulfamethoxazole/trimethoprim and amoxicillin/clavulanate) are also frequently used to treat severe infections such as abdominal, urinary tract infections and respiratory disorders [1, 2]. Appropriate dosing of the antibiotics is of utmost significance for clinical management. Besides, attaining the correct therapeutic level is of high importance for avoiding the development of antimicrobial resistance. Additionally, it also affects the drug distribution, metabolism and clearance. Therefore, guided management with therapeutic drug monitoring (TDM) helps to maximize the efficiency of the antibiotic and similarly to reduce adverse side effects, likewise enhancing the clinical outcomes. By knowing these consequences, for effective antibiotic TDM, in the present study bio-analytical method was developed and validated for commonly prescribed antibiotics combinations of cefoperazone (CEF) and sulbactam (SAL) [3-5]. Various active analysts and researchers took numerous efforts to develop the analytical method for the simultaneous estimation of the CEF/SAL in the biological matrix. A high-performance liquid chromatography (HPLC) [6-10], high-performance thin-layer chromatography (HPTLC) [11, 12] gas chromatography (GC), liquid chromatography/tandem mass spectrometry (LC-MS/MS) [13] and ultraviolet-visible spectroscopy (UV-VIS) [14, 15] spectro- photometric method have been developed and explored for simultaneous estimation of the antibiotics combinations. Among the available analytical techniques, HPLC found to be a helpful tool for quality control and assessment of antibiotics because of its simplicity, few processing requirements and low cost. Additionally, it also has been commercially used to develop a chromatographic fingerprint for various synthetic, semi-synthetic antibiotics molecules and formulations. CEF is chemically (6R,7R)-7-[[(2R)-[[(4-Ethyl-2,3-dioxo-1- piperazinyl)carbonyl]amino] (4-hydroxy phenyl) acetyl] amino]-3- [[(1-methyl-1Htetrazol-5-yl)thio]methyl]-8-oxo-5-thia-1-azabicyclo- oct-2-ene-2-carboxylic acid. CEF is a third-generation cephalosporin antibiotic routinely prescribed for Pseudomonas bacterial infections [15]. Additionally, it also used for the treatment of respiratory infection, urinary tract infection and female genital tract infection. It is a white color crystalline powder with a log P value of-0.74 and two ionizable groups (pKa = 2.55 and 9.55). Therefore, CEF exists mainly as a mono-anionic at a physiological pH. Different analytical techniques have been well documented in the scientific literature for determining the CEF in the various matrix such as reverse phase HPLC [7-10], HPTLC [11, 12] and UV spectrophotometry [14, 15]. SAL is a semi-synthetic beta-lactamase inhibitor and is chemically (2S, 5R)-3,3- dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0] heptane-2- carboxylic acid. It is administered in combination with β-lactam antibiotics (e. g. penicillin and cephalosporin) to inhibit β-lactamase [15]. It is a small molecule (233.24 g/mol) with a log P value of-0.92 and freely soluble in water (48.5 mg/ml). Several analytical techniques have been reported to determining the SAL in combination, including HPLC [7-10], HPTLC [11, 12], LC-MS/MS [13] and UV spectrophotometry [14, 15] for the quantitative determination of SAL in the pharmaceutical dosage form, bulk drug and plasma. To the finest of our information, no efforts have been made to analyze and detect the CEF and SAL combinations in plasma using a suitable chromatographic method. Therefore, in the present study, we developed and validated a bio-analytical method for the simultaneous detection of CEF and SAL in male Wistar rats. International Journal of Pharmacy and Pharmaceutical Sciences Print ISSN: 2656-0097 | Online ISSN: 0975-1491 Vol 12, Issue 10, 2020