Talanta 80 (2009) 117–126 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: Application to validation and quantification of cefepime, cefixime and cefoperazone Emirhan Nemutlu a,∗ , Sedef Kır a , Doruk Katlan b , M. Sinan Beksac ¸ b a Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Sıhhiye, Ankara, Turkey b Hacettepe University, Faculty of Medicine, Department of Obstetrics and Gynecology, Sıhhiye, Ankara, Turkey article info Article history: Received 30 March 2009 Received in revised form 8 June 2009 Accepted 12 June 2009 Available online 21 June 2009 Keywords: Cephalosporin Experimental design Solid-phase extraction Validation Maternal and fetal plasma Amniotic fluid abstract An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftiza- xime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experi- mental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parame- ters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250mm × 4.6 mm, 5 m i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min -1 flow rate and 32 ◦ C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cephalosporins are structurally and pharmacologically related to the penicillins. Like the penicillins, cephalosporins have a beta- lactam ring structure that interferes with the synthesis of the bacterial cell wall. They are used for the treatment of infections caused by Gram (+) and Gram (-) bacteria. They are among the safest and the most effective broad-spectrum bactericidal antimicrobial agents and therefore, they are the most frequently prescribed class of antibiotics [1,2]. It is well accepted that changes in body compartments and alter- ations in cardiac output and renal function occur during pregnancy along with subsequent subtherapeutic maternal serum levels for several antibiotics. For many drugs, absorption is decreased and elimination increased, thus tending to reduce plasma concentra- tions [3]. Therefore, the concentration of drugs in pregnancy must be determined in order to be monitored if therapeutic dosage is to ∗ Corresponding author. Fax: +90 312 311 47 77. E-mail address: enemutlu@hacettepe.edu.tr (E. Nemutlu). be achieved or over-dosage prevented. Moreover, the determina- tion of drugs in amniotic fluid is an important piece of information as an indicator of placental penetration [4]. The analysis of cephalosporins in biological materials from human origin [5–12] and in food-producing animals [10,13] foods [10,14], waters [11,15] and pharmaceuticals [16–18] was performed with liquid chromatographic [5–11,13–15], capillary electrophoretic [10,18], spectroscopic [10,14,16–18] and electro- chemical methods [7,10,12]. None of them, however, was analyzed the seven cephalosporins simultaneously. In addition, to the authors knowledge, no other article has previously been written regarding the analysis of CEP, CTA, CTI, COT, CIX and COP in amniotic fluid by HPLC. The method of changing one variable at a time to investigate the outcome of an experiment most likely dates back to the beginnings of systematic scientific research. The idea was fairly simple. In order to simplify control and interpretation of the results, only one of the factors was chosen for variation while the rest keep at constant values. However, the influence of several factors also needed to be investigated during optimization. Thus the conventional step-by- step optimization procedure was too tedious due to the number of 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.06.034