LC-MS/MS method for simultaneous determination of linezolid, meropenem, piperacillin and teicoplanin in human plasma samples

https://doi.org/10.1016/j.jpba.2019.02.037Get rights and content

Highlights

  • LIN, MER, PIP and TEC can be simultaneous determined by LC–MS/MS in plasma.

  • The LC–MS/MS method can determine the major TEC isoforms.

  • The method for determining simultaneously LIN, MER, PIP and TEC is particularly suited for high throughput analysis.

Abstract

Antibiotic therapy is a crucial aspect of the management of hospitalized patients, however, current standard dosing protocols have been shown to often attain inadequate plasmatic concentrations which may impair the clinical outcome and promote the selection of multidrug-resistant bacteria. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous analysis of four commonly used antibiotics (Meropenem, Piperacillin, Linezolid and Teicoplanin) in human plasma according to the European Medicines Agency (EMA) guidelines. Samples preparation was performed using a commercially available extraction kit which needs a very small amount of sample (50 μl). Antibiotics were detected, following a 7 min gradient separation, in multiple reactions monitoring (MRM) mode using a Qtrap 5500 triple quadrupole instrument equipped with an electrospray source operating in positive ion mode. The method, covering the antibiotics’ clinically relevant concentration ranges, is also able to quantify, individually, the major teicoplanin components. The high reproducibility and the need of a small amount of sample, associated with the use of a commercial kit, together with a short chromatographic time, makes the method particularly suited for high-throughput routine analysis. Monitoring of plasma antibiotic levels, as part of the clinical routine, would result in a quick therapy adjustment leading to a higher probability of eradicating the infection as well as a potential reduction of multidrug-resistance prevalence. The method was successfully applied to monitor the antibiotic concentration of 49 patients under therapy.

Introduction

Bacterial infections represent an important source of morbidity and mortality, making antibiotic therapy a crucial aspect of the management of hospitalized patients, especially in intensive care units (ICUs) [1]. However, current antibiotic therapy based on standard dosing protocols, has been shown to often attain inadequate plasmatic concentrations with the consequence of a poor outcome frequently related to a scarce antibiotic exposure which critically impairs therapy [[2], [3], [4]] and may promote the selection of multidrug-resistant bacteria [5]. This is due to a heterogeneous patients’ pharmacokinetic responsible for a substantial inter-individual variation [6] which cannot be simply overcome by a large increase in the dosage because of the risk for dangerous toxic side effects [7]. The frequently resulting inadequate dosage [8,9] is caused by the current antibiotics’ prescription guidelines which take into account only the patients’ body weight and a possible reduction in the glomerular filtration rate, while ignoring other factors such as variations in the volume of distribution, augmented renal clearance (mostly in septic patients), drug metabolism and interaction with other compounds, which are circumstances likely to contribute to the large variation in plasma levels observed for most of the commonly used antibiotics, especially in ICU [10]. Finally, the need of customizing the desired plasmatic concentrations on the basis of the pathogen susceptibility profile (as in cases of multi-drug resistant organisms) and of the pharmacokinetic/pharmacodynamics targets may require high plasma levels of antiobiotics. In this case a strict concentration monitoring, especially for drugs with a narrow therapeutic index, is crucial [11].

A personalized antibiotic therapy might greatly improve the treatment of bacterial infections by adjusting the dosage based on the patients’ pharmacokinetic. Nevertheless, monitoring of plasma antibiotic levels, as part of the clinical routine, would result in a quick therapy adjustment leading to a higher probability of eradicating the infection as well as a potential reduction of multidrug-resistance prevalence. The main limitation remains the lack of reliable, fast and cheap methods for the quantification of antibiotics in clinically relevant specimens. In recent years the use of liquid chromatography tandem mass spectrometry (LC–MS/MS) has become the preferred method because of its low cross-reactivity from structurally related compounds and its low cost per measurements (provided the instrument is available) which results in cost savings when compared with commercial immunoassays [12,13].

We aimed at developing a quick and robust LC–MS/MS method for the simultaneous determination of four frequently used antibiotics in hospitalized patients. Piperacillin (PIP), a β-lactam penicillin, whose levels have been shown to differ extensively not only between patients but also within the same individual, with Cmin varying between 4.9 and 98 mg/L according to disease severity [14]. Meropenem (MER), a β-lactam carbapenem, whose levels have been shown to be inadequate in a large proportion of patients, especially in the ICU [15] and whose dosage might need to be increased when dealing with multi-drug resistant organisms [7]. Linezolid (LIN), an oxazolidinone, for which sub-therapeutic dose have been observed in critically ill patients with augmented renal clearance, whereas in other patients toxic levels of antibiotics were reached given its narrow therapeutic index [10,16]. Teicoplanin (TEC), a glycopeptide, which has been increasingly used, thanks to its effectiveness against Staphylococcal infections, including methicillin-resistant Staphylococcus aureus (MRSA) and its advantages compared to other molecules (e.g. vancomycin): once-daily bolus administration, intramuscular use, decreased occurrence of Red man syndrome and significantly lower adverse event rates of nephrotoxicity [[17], [18], [19]]. Teicoplanin analysis is usually performed by fluorescence polarization immunoassay (FPIA) which is simple and rapid but expensive, with a low sensitivity and prone to cross-reaction [20]. There seem to be a lack of a methodology for teicoplanin determination by LC–MS/MS, which has been applied only in few cases [[21], [22], [23], [24]], possibly due to the size of the molecule and the fact that this antibiotic is composed by a mixture of different glycopeptides with slightly different molecular weights. Our LC–MS/MS method is able to quantify the single TEC main components therefore avoiding the cross-reactivities of the antibodies possibly occurring in FPIA [24].

At the moment few methods have been developed for the simultaneous determination of different antibiotics [12,25,26], however, to the best of our knowledge, no one includes the four chosen for this study including the single TEC main components. LIN, MER, PIP and TEC are frequently used in a combined therapy (LIN/MER; LIN/PIP; PIP/TEC; MER/TEC). In contrast to immunoassay, our method can determine the concentration of the combined antibiotics in a single run.

Section snippets

Chemical and reagents

Meropenem (MER), Piperacillin (PIP) sodium salt and Linezolid (LIN) as well as their isotopically labeled counterparts 2H6-Meropenem (MER_IS), 2H5-Piperacillin (PIP_IS) and

2H8-Linezolid (LIN_IS) were from Alsachim (Illkirch, France). Teicoplanin (TEC) was from Sanofi-Aventis (France) whereas its IS daptomycin (DAP) was from Merck Sharp & Dohme (Germany).

Methanol (HPLC grade) and formic acid (HPLC grade) were from VWR International PBI Srl (Milan, Italy), dimethyl sulfoxide (DMSO) was from Sigma

Method development

TEC is actually a mixture of several compounds, the five major (named teicoplanin A2-1 through A2-5, Fig. 1), sharing similar activity, are found in the plasma of TEC treated patients together with the hydrolysis product A3-1, which is less active [30]. Although clinicians are usually interested in the total level of TEC, our LC–MS/MS method is able to determine the concentrations of the individual isoforms giving a more accurate clinical information. Moreover, modest accumulation of the A2-4

Discussion

In this study we present a validated HPLC–MS/MS multi-analyte method for the simultaneous quantification of LIN, MER, PIP and TEC. The latter one was further characterized by quantifying its major forms individually. The method exploits the corresponding isotope-labelled molecules as internal standards with the exception of TEC for which DAP was used. Human plasma from healthy volunteers was used as matrix for the preparation of calibrators and QC samples in order to guarantee the absence of

Conclusion

In conclusion, we report a robust and fast method for the simultaneous quantification of LIN, MER, PIP and TEC in human plasma that covers the clinical useful concentration ranges. The major advantages of the method are the need of a small amount of sample, ease of sample preparation, short chromatographic time and high reproducibility. Thanks to the above mentioned characteristics the described method can be applied to high-throughput routine use.

Conflicts of interest

Upon manuscript submission, all authors declare no conflict of interest.

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