Elsevier

Journal of Chromatography B

Volume 907, 15 October 2012, Pages 178-184
Journal of Chromatography B

Short communication
A method for determining the free (unbound) concentration of ten beta-lactam antibiotics in human plasma using high performance liquid chromatography with ultraviolet detection

https://doi.org/10.1016/j.jchromb.2012.09.016Get rights and content

Abstract

With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple high performance liquid chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200 μL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304 nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring programme.

Highlights

► Clinical imperative to foster research in the area of optimising antibiotic dosing. ► Determined the free (unbound) concentration of ten beta-lactam antibiotics. ► Validated method used in pathology laboratory for TDM in critically ill patients. ► Suggest as preferred assay due to antibiotic protein binding variability. ► Method is highly advantageous from a laboratory and clinical perspective.

Introduction

The beta-lactams constitute the most important antibiotic family, both in terms of the large number of compounds available and in terms of prescription volume [1]. These drugs all share a common structure and mechanism of action, but have evolved into various classes with differing spectrums of antibiotic activity and unique qualities [2]. From a pharmacodynamic perspective, the beta-lactam family are categorised as ‘time dependent’ antibiotics where the pharmacokinetic/pharmacodynamic index that best correlates with bacterial killing is the time that concentrations are maintained above the minimum inhibitory concentration in a dosing interval [3], [4], [5].

It is well accepted that the pharmacological activity of antibiotics depends on the free, or unbound, concentration at the site of infection [6], [7]. Given that previously published assays for beta-lactam therapeutic drug monitoring (TDM) measure total antibiotic concentrations [1], [8], accurately estimating the unbound antibiotic concentration can be difficult, particularly in critically ill patients for highly protein bound antibiotics such as ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin [9], [10]. For these drugs with protein binding  > 80%, small changes in protein binding can have a large effect on the unbound concentration [9].

Given the potential limitations of measuring total antibiotic concentrations, the aim of this paper is to describe a method to determine the free (unbound) concentration of ten beta-lactam antibiotics in human plasma using high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. We also aim to describe our observations relating to the variability of unbound concentrations for some of the antibiotics analysed with this method.

Section snippets

Material and methods

The free (unbound) beta-lactam antibiotics which are determined via this method include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). The method uses a common sample preparation and stationary phase for determination of all ten antibiotics. In order to maintain tolerable run times and peak shape, the antibiotics are grouped into four

Chromatography

Fig. 1, Fig. 2, Fig. 3, Fig. 4 depict the chromatography for each analyte under the specified mobile phase and wavelength conditions. Typical retention times are displayed in Table 1. Mobile phases 1A and 1B differ only in the amount of acetonitrile present (8% cf. 12%), as with mobile phases 2A and 2B (25% cf. 40%). The higher percentage of organic in both 1B and 2B was important to reduce run times and maintain peak shape. Buffering of mobile phases 2A and 2B at pH 3.0 was necessary for the

Discussion

This method possesses a number of advantageous features for application within the laboratory. The most notable of these is the ability to access a range of analyses with a simple and inexpensive platform. The analytical instrumentation is relatively commonplace compared to mass spectrometry detection or newer ultra high performance liquid chromatography systems. Indeed, whilst the photodiode array detector used has advantages of peak purity and spectral matching features, a single wavelength

Conclusion

To the best of our knowledge, the method presented here is the first published to determine the free (unbound) concentration of ten beta-lactam antibiotics in 10 min or under. It has the advantage of simplicity in sample preparation, chromatography and instrumentation, with the only variation in procedure being the selection of mobile phase. The performance of the method has been proven by validation and its application within a pathology laboratory. Given the variety of beta-lactams prescribed

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