Simultaneous Determination of Ritonavir and Lopinavir in Human Plasma after Protein Precipitation and LC-MS-MS Rajasekhar Damaramadugu 1,2 , Jaswanthkumar Inamadugu 1,2 , Ravi Kanneti 3 , Srinivasulu Polagani 2 , Venkateswarlu Ponneri 1,& 1 Analytical and Environmental Chemistry Division, Department of Chemistry, Sri Venkateswara University, Tirupati 517502, India; E-Mail: ponnerivenkat@rediffmail.com 2 Bioanalytical Division, Wellquest Clinical Research Laboratory, Hyderabad 500013, India 3 Department of Pharmacology, LM College of Pharmacy, Gujarat University, Ahmedabad 380015, India Received: 27 August 2009 / Revised: 21 February 2010 / Accepted: 23 February 2010 Online publication: 11 April 2010 Abstract A simple, rapid, specific and sensitive method has been developed and validated for simultaneous determination of lopinavir and ritonavir in human plasma. The analytical procedure involves a protein precipitation method using fluoxetine as internal standard Separation was carried out on an Inertsil ODS column using a mobile phase consisting of acetonitrile and 5 mM ammonium acetate buffer. The total run time of analysis was 2.0 min. A linear response function was established for the range of concentrations 50.67– 10,008.82 ng mL -1 for lopinavir and 5.066–1,000.693 ng mL -1 for ritonavir. The method was successfully applied to an oral bioequivalence study in humans. Keywords Column liquid chromatography ESI-MS-MS Protein precipitation extraction Lopinavir and ritonavir in human plasma Bioequivalence study Introduction The structures of lopinavir (LPV; CAS no: 192725-17-0), ritonavir (RTV; CAS no: 155213-67-5) and the internal stan- dard, fluoxetine, (±)-N-methyl-c-[4-(tri- fluoromethyl)phenoxy]benzenepropan- amine are shown in Fig. 1. LPV and RTV have both been proved to be human immunodeficiency virus (HIV) protease inhibitors and have sub- stantially reduced the morbidity and mor- tality associated with HIV-1 infection. Kaletra is a co-formulation of LPV and RTV and it is an important antiretroviral drug, which has been developed by Abbott Laboratories (Chicago, IL, USA). As co-formulated in Kaletra, RTV inhibits the CYP3A mediated metabolism of LPV, thereby providing increased plasma levels of LPV [1]. The therapeutic class of HIV protease inhibitors (PI) represents an important component of current highly active anti- retroviral therapy (HAART) regimes [2]. LPV co-administered with low-dose RTV significantly improves the pharmacokinetic (PK) properties and hence the activity of LPV against HIV-1 protease [3]. Advances in combination therapy for the treatment of human immunodeficiency virus type 1(HIV-1) have led to considerable improve- ments in the potent and durable suppres- sion of HIV replication [4, 5]. For the purpose of routine thera- peutic drug monitoring in clinical labo- ratories a simple and reliable analytical method that can simultaneously deter- mine plasma concentrations of PIs is highly desirable [6]. In recent years; several methods have been developed to quantify LPV and RTV with other PIs by LC [7–9] and LC-MS-MS 2010, 71, 815–824 DOI: 10.1365/s10337-010-1550-9 0009-5893/10/05 Ó 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH Original Chromatographia 2010, 71, May (No. 9/10) 815