Simultaneous Determination of Eight β-Lactam Antibiotics, Amoxicillin, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Cloxacillin, Oxacillin, and Piperacillin, in Human Plasma by Using Ultra-High-Performance Liquid Chromatography with Ultraviolet Detection

Abstract

A simple and rapid ultra-high-performance liquid chromatography (UHPLC) method using UV detection was developed for the simultaneous determination of eight ␤-lactam antibiotics in human plasma, including four penicillins, amoxicillin (AMX), cloxacillin (CLX), oxacillin (OXA), and piperacillin (PIP), and four cephalosporins, cefazolin (CFZ), cefepime (FEP), cefotaxime (CTX), and ceftazidime (CAZ). One hundred-microliter samples were spiked with thiopental as an internal standard, and proteins were precipitated by acetonitrile containing 0.1% formic acid. Separation was achieved on a pentafluorophenyl (PFP) column with a mobile phase composed of phosphoric acid (10 mM) and acetonitrile in gradient elution mode at a flow rate of 500 l/min. Detection was performed at 230 nm for AMX, CLX, OXA, and PIP and 260 nm for CFZ, FEP, CTX, and CAZ. The total analysis time did not exceed 13 min. The method was found to be linear at concentrations ranging from 2 to 100 mg/liter for each compound, and all validation parameters fulfilled international requirements. Between-and within-run accuracy errors ranged from ؊5.2% to 11.4%, and precision was lower than 14.2%. This simple method requires small-volume samples and can easily be implemented in most clinical laboratories to promote the therapeutic drug monitoring of ␤-lactam antibiotics. The simultaneous determination of several antibiotics considerably reduces the time to results for clinicians, which may improve treatment efficiency, especially in critically ill patients.

Investigation of early antibiotic use in pediatric patients with acute respiratory infections by high‐performance liquid chromatography

“…Noticeably, some methods reported very high LLOQs for CPL (5 μg/ml; Najib et al, ) and CFD (12 μg/ml; Eshra et al, ) in urine samples. Regarding our method, for AMO, the LLOQ was lower than that mentioned in the previous published methods (varying from 0.4 to 5 μg/ml; Carlier et al, ; Colin et al, ; Legrand et al, ; Verdier et al, ).…”

“…in urine samples. Regarding our method, for AMO, the LLOQ was lower than that mentioned in the previous published methods (varying from 0.4 to 5 μg/ml;Carlier et al, 2015;Colin et al, 2013;Legrand et al, 2016;Verdier et al, 2011).…”

“…Even though the stability test was not performed as per the guidance instruction in our current HPLC methods, the stability of these antibiotics in different matrices was well established and confirmed in previous publications (Denooz & Charlier, 2008;El-Gindy et al, 2000;Legrand et al, 2016;Nemutlu et al, 2009;Samanidou et al, 2003;Verdier et al, 2011). In addition, internal standards were not applied in this assay to correct any internal bias in the whole procedures.…”

“…In addition, Cazorla Reyes et al also developed a liquid chromatography tandem mass spectrometry method for the analysis of 21 antibiotics including cephalosporins and various other antibiotics in plasma, urine, cerebrospinal fluid and bronchial aspiration matrix (Cazorla-Reyes, Romero-González, Frenich, Rodríguez Maresca, & Martínez Vidal, 2014). Although several HPLC methods have been developed, some are limited to quantification of one or two drugs (Bhinge, Malipatil, & Sonawane, 2014;Danafar, 2015;El-Gindy et al, 2000;Khan et al, 2011), while others have been developed for the detection of 5-12 cephalosporins (Denooz & Charlier, 2008;Legrand, Vodovar, Tournier, Khoudour, & Hulin, 2016;Verdier et al, 2011;Wolff et al, 2013). In addition, many methods using liquid chromatography coupled to tandem mass spectrometric detection (LC/MS-MS) were set up to measure simultaneously from five to 11 cephalosporins (Carlier, Stove, De Waele, & Verstraete, 2015;Colin, DE Bock, T'jollyn, Boussery, & Van Bocxlaer, 2013).…”

“…In addition, many methods using liquid chromatography coupled to tandem mass spectrometric detection (LC/MS-MS) were set up to measure simultaneously from five to 11 cephalosporins (Carlier, Stove, De Waele, & Verstraete, 2015;Colin, DE Bock, T'jollyn, Boussery, & Van Bocxlaer, 2013). Of note, several sample preparation procedures were applied to treat samples before injection into the HPLC systems: protein precipitation (Carlier et al, 2015;Khan et al, 2011;Legrand et al, 2016;McAteer et al, 1987;Samanidou et al, 2003;Verdier et al, 2011;Wolff et al, 2013), liquid-liquid extraction (Bhinge et al, 2014) and solid-phase extraction (SPE) (Colin et al, 2013;Nemutlu, Kir, Katlan, & Beksac, 2009). A pre-dilution step is commonly applied to treat urine samples because the high ionic strength of this matrix interferes with the analyte detection methods (Eshra, Hassan, & El-Walily, 1993;Kovach, Lantz, & Brier, 1991;Najib, Suleiman, El-Sayed, & Abdulhameed, 1987;Samanidou et al, 2003;Samanidou et al, 2004).…”