INTRODUCTION Ampicillin(AMP) a derivative of 6- aminopenicillanic acid has a wide therapeutic use. But it is ineffective against organisms producing β-lactamase. Hence it needs to be administered with a β -lactamase inhibitor such as Sulbactam. Sulbactam(SB) 1 is a molecule that is given in combination with beta-lactam antibiotics to inhibit beta- lactamase, an enzyme produced by bacteria that destroys the antibiotics. Sulbactam is an irreversible inhibitor of beta-lactamase, it binds the enzyme and does not allow it to interact with the antibiotic Sulbactam is able to inhibit the most common forms of beta-lactamase but is not able to interact with the ORIENTAL JOURNAL OF CHEMISTRY www.orientjchem.org ISSN: 0970-020 X CODEN: OJCHEG 2011, Vol. 27, No. (4): Pg. 1659-1664 Est. 1984 An International Open Free Access, Peer Reviewed Research Journal Simultaneous Estimation of Ampicillin Sodium and Sulbactam Sodium in Injectable Dosage Form by High Performance Liquid Chromatography MADHUKAR A. BADGUJAR and KIRAN V. MANGAONKAR* Analytical Chemistry Research Laboratory SVKM’s Mithibai College, Vile-Parle (West), Mumbai - 400 056 (India). *Corresponding author: E-mail: bmbadgujar@yahoo.com (Received: September 10, 2011; Accepted: October 22, 2011) ABSTRACT A reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of Ampicillin sodium and Sulbactam sodium in injectable formulation. The separation was achieved by Inertsil C 18 column (250mm × 4.6mm i.d 5μm particle size) and Acetonitrile: Ammonium acetate buffer pH = 6.0 (17 : 83) as mobile phase at a flow rate of 1.0mL/min. The retention times of Ampicillin sodium and Sulbactam sodium were found to be 5.86mins & 4.33mins respectively. The proposed method was validated for linearity, accuracy and precision. The calibration curve was linear over the range of 50-150 μg/mL for Sulbactam sodium and 100 - 300 μg/mL for Ampicillin sodium. The proposed method can be applied successfully in routine analysis. Key words: Ampicillin Sodium (AMP), Sulbactam Sodium (SB), RP-HPLC, Validation. ampC cephalosporinase. Thus, it confers little protection against bacteria such as Pseudomonas aeruginosa, Citrobacter, Enterobacter, and Serratia, which often express this gene. A detail Literature survey for AMP and SB revealed that few analytical methods are reported for the determination of AMP with SB by HPLC 1-9 , but these methods are time consuming. Hence we focused on developing rapid, sensitive and cost effective method. The manuscript describes the development and subsequent validation of a screening method to simultaneously quantify AMP and SB by HPLC.