CLB-08830; No. of pages: 5; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
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Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma Gregori Casals a,⁎, Cristina Hernández b, Susana Hidalgo a, Blai Morales a, Yolanda López-Púa c, Pedro Castro d, Virginia Fortuna a, José Antonio Martínez b, Mercè Brunet a a
Pharmacology and Toxicology Laboratory, Biochemistry and Molecular Genetics, Centro de Diagnóstico Biomédico, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), IDIBAPS, Hospital Clínic de Barcelona, Barcelona University, C/Villarroel 170, 08036 Barcelona, Spain Department of Infectious Diseases, Hospital Clínic-IDIBAPS, Barcelona Centre for International Health Research (CRESIB, Universitat de Barcelona), Barcelona, Spain c Direcció de Qualitat i Seguretat Clínica, Hospital Clínic, Barcelona, Spain d Medical Intensive Care Unit, Hospital Clínic, IDIBAPS, Barcelona Centre for International Health Research (CRESIB, Universitat de Barcelona), Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 11 July 2014 Accepted 2 August 2014 Available online xxxx Keywords: Meropenem UHPLC Pharmacokinetics Therapeutic drug monitoring Clearance
a b s t r a c t Objectives: Meropenem is a β-lactam antibiotic frequently used to treat serious infections in intensive care unit patients. The main objective was to develop and validate a sensitive and specific ultra high performance liquid chromatography method with photodiode array detection for the quantitation of meropenem in human plasma. The applicability of the method for meropenem monitoring was also examined. Design and methods: The validation of the method was performed following the FDA's guidelines for bioanalytical methods. In parallel, the method was applied for monitoring meropenem in forty plasma samples from ten critically ill patients treated intravenously at a total dose of 1 g. Drug levels were measured in each patient at 0 h, 2 h, 4 h and 8 h after meropenem infusion. Results: With this method, intraday and day-to-day variation was below 10%; intraday and day-to-day accuracy was between 94% and 114%; the limit of quantification was 0.5 μg/mL and recovery was above 70%. The method was successfully applied to quantitate meropenem concentrations and the results showed significant pharmacokinetic interindividual variability. Of special interest is that 50% of treated patients had meropenem plasma levels below the minimum inhibitory concentration at 8 h after the start of infusion, which was strongly related to creatinine clearance N60 mL/min. Conclusions: The method meets the requirements to be applied for meropenem concentration measurements in pharmacokinetics studies and clinical routine. The results suggest the need for therapeutic drug monitoring of meropenem in treated critically-ill patients. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Meropenem is a β-lactam antibiotic of the carbapenem family with a broad spectrum of activity against Gram-negative and Gram-positive organisms. It is indicated for the treatment of a broad range of infections and is an important option for the empirical treatment of serious bacterial infections in hospitalized patients [1]. Multicenter studies have shown the clinical efficacy of meropenem in the treatment of complicated infections in hospitalized patients [2, 3]. Data from clinical trials in different patient groups indicate that meropenem is a well tolerated antibiotic whose adverse events profile is similar to that of other antibiotics used in the same clinical situations [1,4–6]. Because its bactericidal activity is time-dependent, several ⁎ Corresponding author at: Pharmacology and Toxicology, Hospital Clínic Universitari, Villarroel 170, Barcelona 08036, Spain. Fax: +34 93 22775697. E-mail address: [email protected] (G. Casals).
investigations in animal models of infection have suggested that meropenem concentrations should be maintained above the minimum inhibitory concentration (MIC) for a minimum of 40% of the dosing interval (f%T N MIC ≥ 40%; 40% of the time or more the free drug should remain above the MIC of the organism) [7] to achieve maximal bactericidal activity. However, there is evidence of wide interindividual variability in the elimination kinetics of the drug [7] and that standard meropenem dosing regimens may not be adequate in special patient populations such as critically-ill and hemato-oncological neutropenic septic patients due to altered meropenem pharmacokinetics [8,9] and the need for a more stringent pharmacodynamic target (f%T N MIC = 80%–100%) to achieve maximal clinical efficacy [10,11]. These patients may benefit from therapeutic monitoring of meropenem concentrations, which could serve as a guide to individualizing dose regimens by adjusting the dose to achieve and maintain target concentrations. Importantly, clinical response to carbapenems may be unnoticeable before 48 h of
http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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therapy [12]. Therapeutic drug monitoring could allow clinical failure due to underdosing to be distinguished from lack of organism susceptibility in a timely fashion. Various methods to quantify meropenem in human plasma have been developed and validated; these methods used high performance liquid chromatography with ultraviolet [13–24] or mass-spectrometry [15,25–27] detection and employed different extraction processes, including solid phase extraction [13,15,18,19,22,27], protein precipitation [16,17,21,23,24,26], ultrafiltration [14,24] and homogenization [19]. The main problems of HPLC-UV methods include the high plasma volume required and the difficulty of achieving adequate limits of detection. In contrast, mass spectrometric detection has the advantage of being faster and more specific and sensitive but is expensive and is not generally available in all laboratories. Ultra high performance chromatography (UHPLC) diode array detector is a less expensive methodology than mass spectrometry but allows faster chromatographic runs and improved resolution, throughput and sensitivity compared with conventional HPLC-UV. Our aim was to validate an accurate UHPLC-diode array detector method for the determination of total concentrations of meropenem in human plasma, which could be useful to evaluate pharmacokinetic and pharmacodynamic relationships in critically-ill patients treated with this drug.
meropenem and of 20 μg/mL for ceftazidime. Seven-point calibration curves (0.5, 2, 5, 10, 25, 50, and 100 μg/mL) were prepared for meropenem calibration by diluting meropenem working solution (higher calibrator, 100 μg/mL) in MES buffer. These diluted solutions (100 μL) were mixed with 100 μL of drug-free human plasma. For quality controls, three concentrations were prepared (6 μg/mL, 30 μg/mL and 60 μg/mL) by serially diluting meropenem working solution in MES buffer. These diluted solutions (100 μL) were mixed with 100 μL of drug-free human plasma. Sample preparation For calibrators and quality controls, 100 μL of the internal standard working solution (20 μg/mL of ceftazidime) was added to the 200-μL mixture of calibrators or quality controls in MES buffer (100 μL) with 100 μL of drug-free human plasma. Similarly, for patient samples, 100 μL of the internal standard working solution (20 μg/mL) was added to a 200-μL mixture of MES buffer (100 μL) with 100 μL of the plasma sample. Then, 500 μL of acetonitrile was added and after centrifugation (13,000 g for 5 min) the supernatant was transferred to a conical glass vial and evaporated to dryness under nitrogen at 37 °C. The residue was reconstituted in 200 μL of MES buffer and centrifuged at 1000 g for 10 min. The supernatant was transferred to an autosampler glass vial and 7 μL was injected in the chromatographic system.
Material and methods Method validation Chemical reagents Meropenem trihydrate, ceftazidime, disodiumhydrogenphosphate dihydrate (Na2HPO4 · 2H2O) and 2-(N-Morpholino)ethanesulfonic acid (MES monohydrate) were obtained from Sigma (Steinheim, Germany). Acetonitrile and methanol (HPLC grade) were purchased from Panreac (Barcelona, Spain). Ortho-phosphoric acid (H3PO4, 85%, ACS grade) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Drug-free human plasma (citrate) used as a biological matrix for calibrators and quality controls was purchased from the Banc de Sang i Teixits (Barcelona, Spain).
Calibration curve The response function of the calibration curve was evaluated in runs of seven calibrators over a range between 0.5 and 100 μg/mL. Complete calibration curves were analyzed on 5 separate days and the target back-calculated concentrations of the calibration standards were calculated.
Chromatographic conditions
Accuracy and precision Back-calculated results of multiple analysis of the three quality controls, the lower limit of quantification (LLoQ), and the upper limit of quantification (ULoQ) were used to guarantee the accuracy and precision of the analysis method. The LLoQ and ULoQ were set at the lowest (0.5 μg/mL) and highest (100 μg/mL) calibration standard values, respectively. In the case of intra-day accuracy and precision, five replicates were performed for each concentration on the same day. Inter-day accuracy and precision were calculated on 5 different days. To pass the accuracy test, the mean values had to be within 100 ± 15% of the theoretical value. Accuracy was determined as the difference between calculated meropenem concentrations with theoretical concentrations expressed in percent. Precision at each concentration level was expressed as relative standard deviation (%RSD) for each quality control and could not exceed 15%. For the LLoQ, accuracy had to be within 100 ± 20% and precision below 20%.
The mobile phase for the chromatographic separation was mixed from buffer solution (87%) and methanol (13%). The buffer solution consisted of 0.1 M Na2HPO4 · 2H2O adjusted to pH = 7 using H3PO4 85%. The flow rate was constant at 0.25 mL/min and the column temperature was set to 30 °C. The column effluent was monitored by a diode array detector in the range of 270–320 nm. For quantification, peak areas of meropenem and ceftazidime at 295 nm were evaluated.
Recovery The recovery ratio of meropenem was evaluated at the concentration levels corresponding to the three quality control values. This ratio was determined by comparing the peak areas of meropenem for the three levels of quality control samples after extraction with the peak areas of meropenem in three solutions at the same concentration that were not extracted.
Preparation of stock solutions, working solutions, calibrators and quality control samples
Selectivity and specificity Selectivity was investigated by analyzing five different blank plasma samples and was indicated by the absence of any endogenous interference at retention times of peaks of meropenem and ceftazidime in the chromatograms. Specificity was evaluated with respect to drugs commonly used in patients with severe infection in intensive care units. In addition, five different lithium heparin blank plasma samples
Instrumentation The UHPLC chromatographic system consisted of a Shimadzu Nexera system (Kyoto, Japan) comprising a quaternary pump (LC-30AD), a degasser (DGU-20A), an autosampler (SIL-30 AC), a thermostated column compartment (CTO-20AC) and a photodiode array detector (SPD-M20A). The analytical column was a Shimadzu Shimpack XRODS III (2.0 mm id × 75 mm, 1.6 μm particle size). Data acquisition and data processing were achieved with LabSolutions Software (Shimadzu, Japan).
Stock solutions of meropenem and ceftazidime were prepared at a concentration of 1 g/L and stored at −70 °C. Working solutions were prepared by diluting the stock solutions in MES buffer (MES 1 M adjusted to pH = 6 using NaOH 5 M) to final concentrations of 100 μg/mL for
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
G. Casals et al. / Clinical Biochemistry xxx (2014) xxx–xxx
and five different citrate blank plasma samples were used to prepare quality controls of meropenem (QC1 = 6 μg/mL and QC3 = 60 μg/mL) and differences between meropenem and ceftazidime (internal standard) peak area ratios among the different plasma samples were analyzed. Also, peak area ratios of meropenem and ceftazidime were compared to the same peak areas in methanolic solutions. Stability The stability of plasma extracts on the autosampler was evaluated by reanalyzing the quality control samples stored inside the autosampler at 10 °C after 24 h and by comparing the peak area response ratio (peak area meropenem/peak area ceftazidime) of the reinjections of the solution overnight over the first injections. Clinical application To investigate the applicability of the method, it was applied to the analysis of clinical samples obtained after meropenem administration to analyze plasma meropenem concentrations in patients with serious infections. Meropenem was administered intravenously at a total dose of 1 g. After administration of the first meropenem dose had finished, serial blood samples were collected for AUC analysis at 0, 2, 4 and 8 h in each treated patient. After blood extraction, containers were placed on ice and centrifuged (3000 rpm, 4 °C, 5 min) within 1 h. Afterwards, plasma samples were immediately kept at −80 °C and analyzed within 15 days. These conditions avoid influence of meropenem degradation on results according to previous evaluations [13,16,17]. Overall, 40 lithium heparin plasma samples were collected from 10 critically-ill septic patients. For each day of analysis, a set of calibration standards was analyzed together with three quality controls. The study was approved by our local Ethics Review Board. Statistical calculations Statistical calculations were performed with the SPSS software package version 15.0. All data and estimated pharmacokinetic values are expressed as median (range). Results Method validation The chromatographic conditions led to the profile shown in Fig. 1. Ceftazidime (internal standard) and meropenem were eluted from the column after 2.5 and 7.5 min, respectively. All calibration samples
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showed an accuracy above 85% as specified in the current guidelines for analysis [28]. The calibrator of 0.5 μg/mL of meropenem was chosen for the LLoQ. The accuracy of the LLoQ was above 80%, whereas precision was below 10%. Values of precision and accuracy of the LLoQ, the three quality control levels and ULoQ are summarized in Table 1. The average values for recovery calculated by comparing the peak areas of non-extracted solutions of quality control samples in MES with extracted quality control samples ranged from 71% to 78% (Table 2). Table 2 also lists the results of the 24-h autosampler stability test. The accuracy of all the quality controls was above 85%. The photodiode array detector allowed the determination of wavelength spectra (Fig. 1). Elution of ceftazidime and meropenem was free from interferences by endogenous substances. Five different samples of blank ceftazidime and meropenem-free plasma were analyzed, without detection of interfering signals. Moreover, we analyzed five more plasma samples from adult critical care patients under different treatment regimens that did not include ceftazidime or meropenem. Treatments included ceftriaxone, piperacillin/tazobactam, amikacin, fluconazole, vancomycin, tacrolimus, quetiapine, citalopram, paracetamol, omeprazole, pantoprazol, amlodipine, furosemide, gemfibrozil, noradrenaline, insulin and enoxaparin. No interferences were detected between these substances and meropenem or ceftazidime under the chromatographic conditions. Finally, there were no differences in meropenem and ceftazidime peak areas among plasma samples and between citrate and lithium heparin plasma samples. The ratios of peak areas of meropenem and ceftazidime to the same peak areas in methanolic solutions presented a CV below 15%. Clinical application As a part of the validation process, the method was applied for meropenem plasma concentration monitoring in critically-ill patients. Samples from ten patients (6 men and 4 women; age: 58.5 ± 21 years) were analyzed. The mean (SD) APACHE II score on admission was 20.6 (8.4) and the mean (SD) SOFA score at the start of meropenem therapy was 8.7 (3.4). Eight patients (80%) were on mechanical ventilation and six (60%) had septic shock. Creatinine clearance was b60 mL/min in four patients and N60 mL/min in six. Overall mortality was 30% (three patients), one due to sepsis. Most infections (80%) involved the lower respiratory tract and five were microbiologically documented (Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis). Meropenem was administered as a short infusion (mean duration 42.6 min, range 23–78 min) in three patients and as an extended infusion (mean duration 165.7 min, range 125–200 min)
Fig. 1. Chromatogram of a plasma sample from a patient receiving meropenem (plasma concentration = 49.0 μg/mL). The figure also includes absorbance spectrum of the meropenem peak.
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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Table 1 Precision expressed as relative standard deviation and accuracy values of meropenem measurement in plasma. Precision (%)
Accuracy (%)
Quality control
Meropenem (μg/mL)
Intra-day (n = 5)
Inter-day (n = 5)
Intra-day (n = 5)
Inter-day (n = 5)
LLoQ QC-1 QC-2 QC-3 ULoQ
0.5 6 30 60 100
6.5 3.7 1.7 2.7 1.4
8.4 7.9 4.2 5.3 1.5
108.2 94.0 102.7 98.0 102.1
114.0 97.8 102.9 98.6 101.0
LLoQ = low limit of quantification, ULoQ = upper limit of quantification, QC = quality control.
in the remaining seven. At the end of short and extended infusions, mean (SD) peak concentrations were 52.3 (21.7) μg/mL and 17.5 (5.8) μg/mL, respectively. The mean (SD) observed volume of distribution was 42.6 (15) L and mean (SD) meropenem clearance was 240 (139.5) mL/min. As expected, meropenem clearance was significantly correlated with creatinine clearance (Spearman correlation coefficient 0.91, p b 0.001). The estimated trough meropenem level at 8 h of starting infusion was below the breakpoint for Enterobacteriaceae and P. aeruginosa (2 μg/mL) in five (50%) patients. Notably, most patients (5 out of 6) with a creatinine clearance N 60 mL/min had an estimated meropenem level at 8 h lower than 2 μg/mL regardless of its administration as a short or extended infusion. These results indicate significant pharmacokinetic interindividual variability (Table 3), which could affect clinical outcomes (pharmacodynamic variability).
Discussion The results of this validation of the UHPLC diode array detector method demonstrate that it is a convenient technique that can be implemented in clinical meropenem monitoring. Overall, the accuracy and precision of the current method were demonstrated within the entire range of the calibration standards and quality controls, while reproducibility and robustness were also demonstrated. Accordingly, the method satisfactorily allowed meropenem measurement in plasma samples obtained from critically-ill patients who received the drug because of severe infections. Indeed, the results of the method validation demonstrated that the UHPLC diode array detector method may be an acceptable alternative to mass spectrometry methods, which are expensive and not generally available in all laboratories. Sample volume and LLoQ were 100 μL and 0.5 μg/L respectively. This is in between the ranges of previously evaluated HPLC-UV and the ranges of mass spectrometry methods. Thus, sample volumes and LLoQ ranged 100–500 μL and 0.05–2.5 μg/mL, respectively, for HPLCUV methods [13,16,18,19,23] and 20–50 μL and 0.05–0.5 μg/mL, respectively, for mass spectrometry methods [15,25–27]. Previous UHPLC-UV methods presented sample volumes and LLoQ ranging 200–1000 μL and 0.05–2.5 μg/mL, respectively [17,24]. Meropenem retention time was 7.5 min. This is in the low range of previously published HPLC-UV methods (5 to 22 min) [13,16,18,19, 21–23], although slightly higher than previous UHPLC-UV (3–6 min) Table 2 Recovery and autosampler stability of quality controls at 24 h (n = 5). Quality control
Meropenem (μg/mL)
Recovery (%)
Autosampler stability at 24 ha
QC-1 QC-2 QC-3
6 30 60
71.1 74.4 77.7
0.94 ± 0.04 0.90 ± 0.07 0.96 ± 0.03
QC = quality control. a Average overnight vs initial meropenem/ceftazidime peak area ratios.
Table 3 Plasma meropenem concentrations after infusion, body weight, and creatinine clearance in treated patients. Parameter
Value
Meropenem concentration 0 h (μg/mL) Meropenem concentration 2 h (μg/mL) Meropenem concentration 4 h (μg/mL) Meropenem concentration 8 h (μg/mL) Body weight (kg) Creatinine clearance (mL/min)
19.6 (11.5–69.3) 10.3 (2.6–16.4) 4.0 (0.5–10.9) 3.8 (0.5–7.9) 70 (55–85) 95.6 (19.6–192.8)
Data are expressed as median (range).
[17,24]. Mass spectrometry methods presented retention times lower than 5 min [15,25–27]. Infection and sepsis, whether community- or hospital-acquired, are important causes of morbidity and mortality in intensive care unit patients [29], in whom meropenem, because of its wide antimicrobial spectrum and low toxicity, is among the first-line therapies. Considerable evidence demonstrates that the time above MIC is the measure of drug exposure most closely linked to the ability of β-lactam antibiotics to kill the target bacteria [30,31]. However, β-lactam antibiotic halflife time above the MIC seems to be impossible to predict in intensive care unit patients, including those with normal renal function [12]. Indeed, achieving appropriate target site concentrations in critically-ill patients remains a significant challenge for critical care physicians. Our results obtained in critically-ill infected patients treated with the same meropenem dosage showed wide variability in the pharmacokinetic parameters among patients. The immediate impact was that in 5 out of 10 patients meropenem concentrations (two patients receiving short infusion and three patients receiving extended infusion) were not maintained at target therapeutic levels during the dosage interval, favoring drug inefficacy. Although further studies are required to evaluate the pharmacokinetic/pharmacodynamic relationships of meropenem in critically-ill patients, these preliminary results suggest the need to monitor drug levels in this population in order to ensure adequate meropenem exposure in a timely and accurate manner. In agreement with our results, several studies have demonstrated the advantage of therapeutic drug monitoring in β-lactam antibiotics. Depending on the clinical situation, the pharmacokinetics of β-lactams can be highly variable and, as a consequence, plasma concentrations are difficult to predict. Critically-ill patients with severe infection show uncommon half-lives of β-lactam drugs and unusual volumes of distribution, which may require dose adjustments in a large number of treated patients [32–34]. In addition, plasma concentrations are also difficult to predict in patients with renal failure and may require dose adjustments [35]. Verdier et al. [36] evaluated data on therapeutic drug monitoring of 12 β-lactams, including meropenem, in a population of 761 treated patients and found that 15–35% of patients had inadequate βlactam concentrations, irrespective of the administration schedule and despite dose adaptation according to weight and renal function. Altogether, our results and those of previous experiences with β-lactams clearly suggest the need for personalized meropenem drug adjustment based on plasma concentration and renal function. In this study, we observed an inverse correlation between meropenem predose plasma levels and creatinine clearance, which may largely explain the observed pharmacokinetic variability. This finding is in agreement with the results obtained by Pea et al. [37] who observed a relationship between drug clearance and creatinine clearance in a retrospective cohort of patients treated with continuous meropenem infusion. Similarly, other studies have observed a close relationship between meropenem clearance and creatine clearance [38–40]. Recently, however, this relationship was observed to be much poorer in a group of intensive care unit patients in comparison with a group of febrile neutropenic hematology/oncology patients [8]. Possible explanations for this finding were a large and variable distribution volume at steady state and interindividual differences in extrarenal meropenem
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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clearance. Therefore, further studies are necessary to evaluate the effect of glomerular filtration on meropenem exposure and elimination in critically-ill patients. In summary, the current study demonstrates that the UHPLC method evaluated is robust, reliable and useful to determine plasma meropenem concentrations. Critically-ill patients showed wide interindividuality in pharmacokinetic parameters, which translated into a high proportion of patients showing low meropenem levels between dosages. Thus, the availability of this simple and rapid method could be useful for physicians to personalize drug adjustment and to identify the clinical variables related to meropenem pharmacokinetic variability.
Acknowledgments This work has been supported by a grant (Expedient PI11/00479) from the Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad, Gobierno de España, and co-financed by FEDER, Unión Europea, una manera de hacer Europa. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III.
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[34] Belzberg H, Zhu J, Cornwell III EE, Murray JA, Sava J, Salim A, et al. Imipenem levels are not predictable in the critically-ill patient. J Trauma 2004;56:111–7. [35] Lemaire-Hurtel AS, Gras-Champel V, Hary L, Masmoudi K, Massy Z, Andréjak M. Recommended dosage adaptation based on renal function is not always sufficient to avoid beta-lactam antibiotics side effects. Nephrol Ther 2009;5:144–8. [36] Verdier MC, Tribut O, Tattevin P, Le Tulzo Y, Michelet C, Bentué-Ferrer D. Simultaneous determination of 12 beta-lactam antibiotics in human plasma by high-performance liquid chromatography with UV detection: application to therapeutic drug monitoring. Antimicrob Agents Chemother 2011;55:4873–9. [37] Pea F, Viale P, Cojutti P, Furlanut M. Dosing nomograms for attaining optimum concentrations of meropenem by continuous infusion in critically-ill patients with severe gram-negative infections: a pharmacokinetics/pharmacodynamics-based approach. Antimicrob Agents Chemother 2012;56:6343–8. 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Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma Gregori Casals a,⁎, Cristina Hernández b, Susana Hidalgo a, Blai Morales a, Yolanda López-Púa c, Pedro Castro d, Virginia Fortuna a, José Antonio Martínez b, Mercè Brunet a a
Pharmacology and Toxicology Laboratory, Biochemistry and Molecular Genetics, Centro de Diagnóstico Biomédico, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), IDIBAPS, Hospital Clínic de Barcelona, Barcelona University, C/Villarroel 170, 08036 Barcelona, Spain Department of Infectious Diseases, Hospital Clínic-IDIBAPS, Barcelona Centre for International Health Research (CRESIB, Universitat de Barcelona), Barcelona, Spain c Direcció de Qualitat i Seguretat Clínica, Hospital Clínic, Barcelona, Spain d Medical Intensive Care Unit, Hospital Clínic, IDIBAPS, Barcelona Centre for International Health Research (CRESIB, Universitat de Barcelona), Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 29 April 2014 Received in revised form 11 July 2014 Accepted 2 August 2014 Available online xxxx Keywords: Meropenem UHPLC Pharmacokinetics Therapeutic drug monitoring Clearance
a b s t r a c t Objectives: Meropenem is a β-lactam antibiotic frequently used to treat serious infections in intensive care unit patients. The main objective was to develop and validate a sensitive and specific ultra high performance liquid chromatography method with photodiode array detection for the quantitation of meropenem in human plasma. The applicability of the method for meropenem monitoring was also examined. Design and methods: The validation of the method was performed following the FDA's guidelines for bioanalytical methods. In parallel, the method was applied for monitoring meropenem in forty plasma samples from ten critically ill patients treated intravenously at a total dose of 1 g. Drug levels were measured in each patient at 0 h, 2 h, 4 h and 8 h after meropenem infusion. Results: With this method, intraday and day-to-day variation was below 10%; intraday and day-to-day accuracy was between 94% and 114%; the limit of quantification was 0.5 μg/mL and recovery was above 70%. The method was successfully applied to quantitate meropenem concentrations and the results showed significant pharmacokinetic interindividual variability. Of special interest is that 50% of treated patients had meropenem plasma levels below the minimum inhibitory concentration at 8 h after the start of infusion, which was strongly related to creatinine clearance N60 mL/min. Conclusions: The method meets the requirements to be applied for meropenem concentration measurements in pharmacokinetics studies and clinical routine. The results suggest the need for therapeutic drug monitoring of meropenem in treated critically-ill patients. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Meropenem is a β-lactam antibiotic of the carbapenem family with a broad spectrum of activity against Gram-negative and Gram-positive organisms. It is indicated for the treatment of a broad range of infections and is an important option for the empirical treatment of serious bacterial infections in hospitalized patients [1]. Multicenter studies have shown the clinical efficacy of meropenem in the treatment of complicated infections in hospitalized patients [2, 3]. Data from clinical trials in different patient groups indicate that meropenem is a well tolerated antibiotic whose adverse events profile is similar to that of other antibiotics used in the same clinical situations [1,4–6]. Because its bactericidal activity is time-dependent, several ⁎ Corresponding author at: Pharmacology and Toxicology, Hospital Clínic Universitari, Villarroel 170, Barcelona 08036, Spain. Fax: +34 93 22775697. E-mail address: [email protected] (G. Casals).
investigations in animal models of infection have suggested that meropenem concentrations should be maintained above the minimum inhibitory concentration (MIC) for a minimum of 40% of the dosing interval (f%T N MIC ≥ 40%; 40% of the time or more the free drug should remain above the MIC of the organism) [7] to achieve maximal bactericidal activity. However, there is evidence of wide interindividual variability in the elimination kinetics of the drug [7] and that standard meropenem dosing regimens may not be adequate in special patient populations such as critically-ill and hemato-oncological neutropenic septic patients due to altered meropenem pharmacokinetics [8,9] and the need for a more stringent pharmacodynamic target (f%T N MIC = 80%–100%) to achieve maximal clinical efficacy [10,11]. These patients may benefit from therapeutic monitoring of meropenem concentrations, which could serve as a guide to individualizing dose regimens by adjusting the dose to achieve and maintain target concentrations. Importantly, clinical response to carbapenems may be unnoticeable before 48 h of
http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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therapy [12]. Therapeutic drug monitoring could allow clinical failure due to underdosing to be distinguished from lack of organism susceptibility in a timely fashion. Various methods to quantify meropenem in human plasma have been developed and validated; these methods used high performance liquid chromatography with ultraviolet [13–24] or mass-spectrometry [15,25–27] detection and employed different extraction processes, including solid phase extraction [13,15,18,19,22,27], protein precipitation [16,17,21,23,24,26], ultrafiltration [14,24] and homogenization [19]. The main problems of HPLC-UV methods include the high plasma volume required and the difficulty of achieving adequate limits of detection. In contrast, mass spectrometric detection has the advantage of being faster and more specific and sensitive but is expensive and is not generally available in all laboratories. Ultra high performance chromatography (UHPLC) diode array detector is a less expensive methodology than mass spectrometry but allows faster chromatographic runs and improved resolution, throughput and sensitivity compared with conventional HPLC-UV. Our aim was to validate an accurate UHPLC-diode array detector method for the determination of total concentrations of meropenem in human plasma, which could be useful to evaluate pharmacokinetic and pharmacodynamic relationships in critically-ill patients treated with this drug.
meropenem and of 20 μg/mL for ceftazidime. Seven-point calibration curves (0.5, 2, 5, 10, 25, 50, and 100 μg/mL) were prepared for meropenem calibration by diluting meropenem working solution (higher calibrator, 100 μg/mL) in MES buffer. These diluted solutions (100 μL) were mixed with 100 μL of drug-free human plasma. For quality controls, three concentrations were prepared (6 μg/mL, 30 μg/mL and 60 μg/mL) by serially diluting meropenem working solution in MES buffer. These diluted solutions (100 μL) were mixed with 100 μL of drug-free human plasma. Sample preparation For calibrators and quality controls, 100 μL of the internal standard working solution (20 μg/mL of ceftazidime) was added to the 200-μL mixture of calibrators or quality controls in MES buffer (100 μL) with 100 μL of drug-free human plasma. Similarly, for patient samples, 100 μL of the internal standard working solution (20 μg/mL) was added to a 200-μL mixture of MES buffer (100 μL) with 100 μL of the plasma sample. Then, 500 μL of acetonitrile was added and after centrifugation (13,000 g for 5 min) the supernatant was transferred to a conical glass vial and evaporated to dryness under nitrogen at 37 °C. The residue was reconstituted in 200 μL of MES buffer and centrifuged at 1000 g for 10 min. The supernatant was transferred to an autosampler glass vial and 7 μL was injected in the chromatographic system.
Material and methods Method validation Chemical reagents Meropenem trihydrate, ceftazidime, disodiumhydrogenphosphate dihydrate (Na2HPO4 · 2H2O) and 2-(N-Morpholino)ethanesulfonic acid (MES monohydrate) were obtained from Sigma (Steinheim, Germany). Acetonitrile and methanol (HPLC grade) were purchased from Panreac (Barcelona, Spain). Ortho-phosphoric acid (H3PO4, 85%, ACS grade) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Drug-free human plasma (citrate) used as a biological matrix for calibrators and quality controls was purchased from the Banc de Sang i Teixits (Barcelona, Spain).
Calibration curve The response function of the calibration curve was evaluated in runs of seven calibrators over a range between 0.5 and 100 μg/mL. Complete calibration curves were analyzed on 5 separate days and the target back-calculated concentrations of the calibration standards were calculated.
Chromatographic conditions
Accuracy and precision Back-calculated results of multiple analysis of the three quality controls, the lower limit of quantification (LLoQ), and the upper limit of quantification (ULoQ) were used to guarantee the accuracy and precision of the analysis method. The LLoQ and ULoQ were set at the lowest (0.5 μg/mL) and highest (100 μg/mL) calibration standard values, respectively. In the case of intra-day accuracy and precision, five replicates were performed for each concentration on the same day. Inter-day accuracy and precision were calculated on 5 different days. To pass the accuracy test, the mean values had to be within 100 ± 15% of the theoretical value. Accuracy was determined as the difference between calculated meropenem concentrations with theoretical concentrations expressed in percent. Precision at each concentration level was expressed as relative standard deviation (%RSD) for each quality control and could not exceed 15%. For the LLoQ, accuracy had to be within 100 ± 20% and precision below 20%.
The mobile phase for the chromatographic separation was mixed from buffer solution (87%) and methanol (13%). The buffer solution consisted of 0.1 M Na2HPO4 · 2H2O adjusted to pH = 7 using H3PO4 85%. The flow rate was constant at 0.25 mL/min and the column temperature was set to 30 °C. The column effluent was monitored by a diode array detector in the range of 270–320 nm. For quantification, peak areas of meropenem and ceftazidime at 295 nm were evaluated.
Recovery The recovery ratio of meropenem was evaluated at the concentration levels corresponding to the three quality control values. This ratio was determined by comparing the peak areas of meropenem for the three levels of quality control samples after extraction with the peak areas of meropenem in three solutions at the same concentration that were not extracted.
Preparation of stock solutions, working solutions, calibrators and quality control samples
Selectivity and specificity Selectivity was investigated by analyzing five different blank plasma samples and was indicated by the absence of any endogenous interference at retention times of peaks of meropenem and ceftazidime in the chromatograms. Specificity was evaluated with respect to drugs commonly used in patients with severe infection in intensive care units. In addition, five different lithium heparin blank plasma samples
Instrumentation The UHPLC chromatographic system consisted of a Shimadzu Nexera system (Kyoto, Japan) comprising a quaternary pump (LC-30AD), a degasser (DGU-20A), an autosampler (SIL-30 AC), a thermostated column compartment (CTO-20AC) and a photodiode array detector (SPD-M20A). The analytical column was a Shimadzu Shimpack XRODS III (2.0 mm id × 75 mm, 1.6 μm particle size). Data acquisition and data processing were achieved with LabSolutions Software (Shimadzu, Japan).
Stock solutions of meropenem and ceftazidime were prepared at a concentration of 1 g/L and stored at −70 °C. Working solutions were prepared by diluting the stock solutions in MES buffer (MES 1 M adjusted to pH = 6 using NaOH 5 M) to final concentrations of 100 μg/mL for
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
G. Casals et al. / Clinical Biochemistry xxx (2014) xxx–xxx
and five different citrate blank plasma samples were used to prepare quality controls of meropenem (QC1 = 6 μg/mL and QC3 = 60 μg/mL) and differences between meropenem and ceftazidime (internal standard) peak area ratios among the different plasma samples were analyzed. Also, peak area ratios of meropenem and ceftazidime were compared to the same peak areas in methanolic solutions. Stability The stability of plasma extracts on the autosampler was evaluated by reanalyzing the quality control samples stored inside the autosampler at 10 °C after 24 h and by comparing the peak area response ratio (peak area meropenem/peak area ceftazidime) of the reinjections of the solution overnight over the first injections. Clinical application To investigate the applicability of the method, it was applied to the analysis of clinical samples obtained after meropenem administration to analyze plasma meropenem concentrations in patients with serious infections. Meropenem was administered intravenously at a total dose of 1 g. After administration of the first meropenem dose had finished, serial blood samples were collected for AUC analysis at 0, 2, 4 and 8 h in each treated patient. After blood extraction, containers were placed on ice and centrifuged (3000 rpm, 4 °C, 5 min) within 1 h. Afterwards, plasma samples were immediately kept at −80 °C and analyzed within 15 days. These conditions avoid influence of meropenem degradation on results according to previous evaluations [13,16,17]. Overall, 40 lithium heparin plasma samples were collected from 10 critically-ill septic patients. For each day of analysis, a set of calibration standards was analyzed together with three quality controls. The study was approved by our local Ethics Review Board. Statistical calculations Statistical calculations were performed with the SPSS software package version 15.0. All data and estimated pharmacokinetic values are expressed as median (range). Results Method validation The chromatographic conditions led to the profile shown in Fig. 1. Ceftazidime (internal standard) and meropenem were eluted from the column after 2.5 and 7.5 min, respectively. All calibration samples
3
showed an accuracy above 85% as specified in the current guidelines for analysis [28]. The calibrator of 0.5 μg/mL of meropenem was chosen for the LLoQ. The accuracy of the LLoQ was above 80%, whereas precision was below 10%. Values of precision and accuracy of the LLoQ, the three quality control levels and ULoQ are summarized in Table 1. The average values for recovery calculated by comparing the peak areas of non-extracted solutions of quality control samples in MES with extracted quality control samples ranged from 71% to 78% (Table 2). Table 2 also lists the results of the 24-h autosampler stability test. The accuracy of all the quality controls was above 85%. The photodiode array detector allowed the determination of wavelength spectra (Fig. 1). Elution of ceftazidime and meropenem was free from interferences by endogenous substances. Five different samples of blank ceftazidime and meropenem-free plasma were analyzed, without detection of interfering signals. Moreover, we analyzed five more plasma samples from adult critical care patients under different treatment regimens that did not include ceftazidime or meropenem. Treatments included ceftriaxone, piperacillin/tazobactam, amikacin, fluconazole, vancomycin, tacrolimus, quetiapine, citalopram, paracetamol, omeprazole, pantoprazol, amlodipine, furosemide, gemfibrozil, noradrenaline, insulin and enoxaparin. No interferences were detected between these substances and meropenem or ceftazidime under the chromatographic conditions. Finally, there were no differences in meropenem and ceftazidime peak areas among plasma samples and between citrate and lithium heparin plasma samples. The ratios of peak areas of meropenem and ceftazidime to the same peak areas in methanolic solutions presented a CV below 15%. Clinical application As a part of the validation process, the method was applied for meropenem plasma concentration monitoring in critically-ill patients. Samples from ten patients (6 men and 4 women; age: 58.5 ± 21 years) were analyzed. The mean (SD) APACHE II score on admission was 20.6 (8.4) and the mean (SD) SOFA score at the start of meropenem therapy was 8.7 (3.4). Eight patients (80%) were on mechanical ventilation and six (60%) had septic shock. Creatinine clearance was b60 mL/min in four patients and N60 mL/min in six. Overall mortality was 30% (three patients), one due to sepsis. Most infections (80%) involved the lower respiratory tract and five were microbiologically documented (Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis). Meropenem was administered as a short infusion (mean duration 42.6 min, range 23–78 min) in three patients and as an extended infusion (mean duration 165.7 min, range 125–200 min)
Fig. 1. Chromatogram of a plasma sample from a patient receiving meropenem (plasma concentration = 49.0 μg/mL). The figure also includes absorbance spectrum of the meropenem peak.
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
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Table 1 Precision expressed as relative standard deviation and accuracy values of meropenem measurement in plasma. Precision (%)
Accuracy (%)
Quality control
Meropenem (μg/mL)
Intra-day (n = 5)
Inter-day (n = 5)
Intra-day (n = 5)
Inter-day (n = 5)
LLoQ QC-1 QC-2 QC-3 ULoQ
0.5 6 30 60 100
6.5 3.7 1.7 2.7 1.4
8.4 7.9 4.2 5.3 1.5
108.2 94.0 102.7 98.0 102.1
114.0 97.8 102.9 98.6 101.0
LLoQ = low limit of quantification, ULoQ = upper limit of quantification, QC = quality control.
in the remaining seven. At the end of short and extended infusions, mean (SD) peak concentrations were 52.3 (21.7) μg/mL and 17.5 (5.8) μg/mL, respectively. The mean (SD) observed volume of distribution was 42.6 (15) L and mean (SD) meropenem clearance was 240 (139.5) mL/min. As expected, meropenem clearance was significantly correlated with creatinine clearance (Spearman correlation coefficient 0.91, p b 0.001). The estimated trough meropenem level at 8 h of starting infusion was below the breakpoint for Enterobacteriaceae and P. aeruginosa (2 μg/mL) in five (50%) patients. Notably, most patients (5 out of 6) with a creatinine clearance N 60 mL/min had an estimated meropenem level at 8 h lower than 2 μg/mL regardless of its administration as a short or extended infusion. These results indicate significant pharmacokinetic interindividual variability (Table 3), which could affect clinical outcomes (pharmacodynamic variability).
Discussion The results of this validation of the UHPLC diode array detector method demonstrate that it is a convenient technique that can be implemented in clinical meropenem monitoring. Overall, the accuracy and precision of the current method were demonstrated within the entire range of the calibration standards and quality controls, while reproducibility and robustness were also demonstrated. Accordingly, the method satisfactorily allowed meropenem measurement in plasma samples obtained from critically-ill patients who received the drug because of severe infections. Indeed, the results of the method validation demonstrated that the UHPLC diode array detector method may be an acceptable alternative to mass spectrometry methods, which are expensive and not generally available in all laboratories. Sample volume and LLoQ were 100 μL and 0.5 μg/L respectively. This is in between the ranges of previously evaluated HPLC-UV and the ranges of mass spectrometry methods. Thus, sample volumes and LLoQ ranged 100–500 μL and 0.05–2.5 μg/mL, respectively, for HPLCUV methods [13,16,18,19,23] and 20–50 μL and 0.05–0.5 μg/mL, respectively, for mass spectrometry methods [15,25–27]. Previous UHPLC-UV methods presented sample volumes and LLoQ ranging 200–1000 μL and 0.05–2.5 μg/mL, respectively [17,24]. Meropenem retention time was 7.5 min. This is in the low range of previously published HPLC-UV methods (5 to 22 min) [13,16,18,19, 21–23], although slightly higher than previous UHPLC-UV (3–6 min) Table 2 Recovery and autosampler stability of quality controls at 24 h (n = 5). Quality control
Meropenem (μg/mL)
Recovery (%)
Autosampler stability at 24 ha
QC-1 QC-2 QC-3
6 30 60
71.1 74.4 77.7
0.94 ± 0.04 0.90 ± 0.07 0.96 ± 0.03
QC = quality control. a Average overnight vs initial meropenem/ceftazidime peak area ratios.
Table 3 Plasma meropenem concentrations after infusion, body weight, and creatinine clearance in treated patients. Parameter
Value
Meropenem concentration 0 h (μg/mL) Meropenem concentration 2 h (μg/mL) Meropenem concentration 4 h (μg/mL) Meropenem concentration 8 h (μg/mL) Body weight (kg) Creatinine clearance (mL/min)
19.6 (11.5–69.3) 10.3 (2.6–16.4) 4.0 (0.5–10.9) 3.8 (0.5–7.9) 70 (55–85) 95.6 (19.6–192.8)
Data are expressed as median (range).
[17,24]. Mass spectrometry methods presented retention times lower than 5 min [15,25–27]. Infection and sepsis, whether community- or hospital-acquired, are important causes of morbidity and mortality in intensive care unit patients [29], in whom meropenem, because of its wide antimicrobial spectrum and low toxicity, is among the first-line therapies. Considerable evidence demonstrates that the time above MIC is the measure of drug exposure most closely linked to the ability of β-lactam antibiotics to kill the target bacteria [30,31]. However, β-lactam antibiotic halflife time above the MIC seems to be impossible to predict in intensive care unit patients, including those with normal renal function [12]. Indeed, achieving appropriate target site concentrations in critically-ill patients remains a significant challenge for critical care physicians. Our results obtained in critically-ill infected patients treated with the same meropenem dosage showed wide variability in the pharmacokinetic parameters among patients. The immediate impact was that in 5 out of 10 patients meropenem concentrations (two patients receiving short infusion and three patients receiving extended infusion) were not maintained at target therapeutic levels during the dosage interval, favoring drug inefficacy. Although further studies are required to evaluate the pharmacokinetic/pharmacodynamic relationships of meropenem in critically-ill patients, these preliminary results suggest the need to monitor drug levels in this population in order to ensure adequate meropenem exposure in a timely and accurate manner. In agreement with our results, several studies have demonstrated the advantage of therapeutic drug monitoring in β-lactam antibiotics. Depending on the clinical situation, the pharmacokinetics of β-lactams can be highly variable and, as a consequence, plasma concentrations are difficult to predict. Critically-ill patients with severe infection show uncommon half-lives of β-lactam drugs and unusual volumes of distribution, which may require dose adjustments in a large number of treated patients [32–34]. In addition, plasma concentrations are also difficult to predict in patients with renal failure and may require dose adjustments [35]. Verdier et al. [36] evaluated data on therapeutic drug monitoring of 12 β-lactams, including meropenem, in a population of 761 treated patients and found that 15–35% of patients had inadequate βlactam concentrations, irrespective of the administration schedule and despite dose adaptation according to weight and renal function. Altogether, our results and those of previous experiences with β-lactams clearly suggest the need for personalized meropenem drug adjustment based on plasma concentration and renal function. In this study, we observed an inverse correlation between meropenem predose plasma levels and creatinine clearance, which may largely explain the observed pharmacokinetic variability. This finding is in agreement with the results obtained by Pea et al. [37] who observed a relationship between drug clearance and creatinine clearance in a retrospective cohort of patients treated with continuous meropenem infusion. Similarly, other studies have observed a close relationship between meropenem clearance and creatine clearance [38–40]. Recently, however, this relationship was observed to be much poorer in a group of intensive care unit patients in comparison with a group of febrile neutropenic hematology/oncology patients [8]. Possible explanations for this finding were a large and variable distribution volume at steady state and interindividual differences in extrarenal meropenem
Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
G. Casals et al. / Clinical Biochemistry xxx (2014) xxx–xxx
clearance. Therefore, further studies are necessary to evaluate the effect of glomerular filtration on meropenem exposure and elimination in critically-ill patients. In summary, the current study demonstrates that the UHPLC method evaluated is robust, reliable and useful to determine plasma meropenem concentrations. Critically-ill patients showed wide interindividuality in pharmacokinetic parameters, which translated into a high proportion of patients showing low meropenem levels between dosages. Thus, the availability of this simple and rapid method could be useful for physicians to personalize drug adjustment and to identify the clinical variables related to meropenem pharmacokinetic variability.
Acknowledgments This work has been supported by a grant (Expedient PI11/00479) from the Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad, Gobierno de España, and co-financed by FEDER, Unión Europea, una manera de hacer Europa. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III.
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Please cite this article as: Casals G, et al, Development and validation of a UHPLC diode array detector method for meropenem quantification in human plasma, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.08.002
