Antimicrobial Original Research Paper
Quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma by liquid chromatography/ tandem mass spectrometry Sebastiano Barco1, Roberto Bandettini1, Angelo Maffia1, Gino Tripodi1, Elio Castagnola2, Giuliana Cangemi1 1
Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy, 2Infectious Disease Unit, Istituto Giannina Gaslini, Genoa, Italy Therapeutic drug monitoring is a cornerstone of antibacterial therapy, especially in an era of increasing antibacterial resistance in individualizing antimicrobial therapy. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was used for the simultaneous measurement of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in 50 ml plasma samples over a wide range. The overall turnaround time for the assay was 20 minutes. Intra-assay precision and accuracy for quality control samples ranged within 1.8–8.5 and 91.4–106.7%, respectively. Inter-assay precision and accuracy ranged within 1.3–14.4 and 95.8–104.6%, respectively. The lower limit of quantification was below 1.5 mg/ml for all the five antibiotics. No ion suppression due to matrix effects was found. A simple and rapid LC–MS/MS method which provides high specificity, precision and accuracy for the simultaneous quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma has been developed and validated. The present method is suitable for therapeutic drug monitoring in paediatrics.
Keywords: Piperacillin, Tazobactam, Meropenem, Ceftazidime, Linezolid, Liquid chromatography/tandem mass spectrometry
Introduction Optimization of antibiotic dosage regimens is crucial for the management of severe infectious diseases and reduction of resistant mutant selection, especially in an era of increasing antibiotic resistance. Therefore, TDM may be of fundamental importance in individualizing antimicrobial therapy, in particular in critically ill patients, in the presence of peculiar pathophysiological condition or of infections due to pathogens with reduced sensitivity to antibiotics, which may require higher doses administration to be effective.1–4 For these reasons, the availability of reliable and validated bioanalytical methods for the determination of antibiotics in human plasma is of crucial importance. In the present study, we describe a simple and fast liquid chromatography-tandem mass spectrometry (L C–MS/MS) method that can be employed for the determination of plasma levels of five different antibiotics, piperacillin, tazobactam, meropenem, ceftazidime, and linezolid, using small volume of plasma (50 ml).
Correspondence to: E. Castagnola, Infectious Diseases Unit, Istituto Giannina Gaslini, Largo G. Gaslini 3-5, 16147 Genova, Italy. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000209
Materials and Methods Chemicals and reagents Piperacillin, tazobactam, meropenem, ceftazidime, linezolid, and prasozin (as internal standard, IS) were purchased from Sigma-Aldrich (Milan, Italy). Water was purified by reverse osmosis and filtrated through a Milli-Q Purification System (Millipore, Milford, MA, USA). High-performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Sigma-Aldrich Srl (Milan, Italy) and formic acid (99.9%) from Merck (Darmstadt, Germany).
Preparation of calibrators and quality control samples Stock solutions (2 mg/ml) were prepared by dissolving each powdered antibiotic in water except linezolid that was dissolved in dimethyl sulphoxide. Calibrators and quality controls were made by spiking tazobactam, linezolid, meropenem, piperacillin, and ceftazidime from different batches of working solution into a pool of plasma. Two different six-point calibration curves were created from calibrator standards that were prepared by adding the five antibiotics to a pool of plasma to yield concentrations of 6.25–12.5–25–50–100 and 200 mg/ml
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for piperacillin and ceftazidime and 1.25–2.5–5–10–20 and 40 mg/ml for tazobactam, meropenem, and linezolid, respectively. Quality control (QC) samples were prepared by adding the five antibiotics at the following four concentration levels (QC I, II, III, and IV) to a pool of plasma: 7.5–15–30 and 60 mg/ml for piperacillin and ceftazidime and 1.5-3–6 and 12 mg/ml for tazobactam, meropenem, and linezolid.
Human samples Leftover plasma samples from routine clinical chemistry have been used for the development and validation of the present method. Plasma was obtained from peripheral blood collected in sodium heparin-containing tubes by centrifuging at 4000g for 5 minutes. Plasma samples were stored at 230uC until analysed. Blank samples were obtained from healthy adults and from paediatric patients. The model was validated (for piperacillin, tazobactam, and meropenem) in leftover plasma samples from routine clinical chemistry derived from pediatric patients, including those admitted in the neonatal intensive care unit. Ethical Committee approval was required for the use of the leftover plasma samples to validate the analytical method.
Protein precipitation A 50 ml aliquot of plasma was spiked with 10 ml of 220 ng/ml IS. Protein was precipitated by adding 300 ml of methanol and centrifuging for 10 minutes at 20 000g. The supernatant then was filtered through a 0.22 mm filter.
Chromatography and mass spectrometry Gradient separation chromatography was carried out using a Kinetex C18 column (10064.6 mm, i.d. 2.6 mm; Phenomenex, Bologna, Italy) with mobile phase A consisting of 0.1% formic acid in water and mobile phase B of acetonitrile. The percentage of solvent B was programmed to reach 100% in 3 minutes at a flow rate of 700 ml/min, then the column was washed with 100% B for 1 minutes and finally reconditioned at 95% A for 2 minutes for a total run time of 5.5 minutes. The column temperature was maintained at 40uC and injection volume was 10 ml. The five analytes have retention times between 2.7 and 3.42 minutes. Mass spectrometric detection was
performed using a Thermo Scientific TSQ Quantum Access MAX triple quadrupole system. Ionization was achieved using electrospray in the positive ion mode except for tazobactam which was ionized in the negative ion mode. The spray voltage was set at 3500 V in positive polarity and 3000 V in negative polarity. Nitrogen was used as the nebulizer and auxiliary gas, set at 50 and 15 arbitrary units, respectively. Vaporizer and capillary temperature settings were 350 and 275uC. For collision-induced dissociation, high purity argon was used at a pressure of 1.5 mTorr. The antibiotics were detected using multiple reaction monitoring of the specific transitions listed in Table 1.
Method validation A laboratory scheme based on the International Conference on Harmonization and Food and Drug Administration (FDA) guidelines5,6 was used for assay validation. To exclude the presence of potentially interfering substances, tests were conducted on samples obtained from consenting healthy volunteers. In addition, 30 blank samples were analysed according to the whole analytical procedure. The impact on the analyte of ion suppression and enhancement resulting from ionization of matrix components has been tested. Seven aliquots of processed blank plasma samples from different drug-free volunteers were injected into the HPLC system, while a mixture of the five analytes was continuously infused post-column and mixed with the column effluent through a ‘T’ before entering the electrospray interface.7 The concentration of each analyte was 10 mg/ml in acetonitrile. Infusion flow was 10 ml/min and HPLC flow was 690 ml/min. Linearity was evaluated on the basis of the mean of six replicate assays of each calibrator standard. The slope, intercept, and correlation coefficient for each calibration curve were estimated by plotting the peak area ratio of analyte/IS vs. the analyte concentration of each calibration standard sample using a 1/x weighting factor. Intra-assay and inter-assay imprecision and accuracy were determined at four concentration levels, using the four level QC samples with each sample being analysed five separate times. Inter-assay imprecision was estimated by repeating the test in six non-consecutive analytical sessions. Accuracy was
Table 1 LC-MS/MS parameters of the five antibiotics and IS
344
Compounds
Parent ion
Product ions
Collision energy
Tube lens
Meropenem
384.1
Positive
2.69
546.9 298.9
70 70
Positive Negative
2.7 2.72
Prasozin Linezolid Piperacillin
384.0 338.1 540
30 19 30 12 17 12 29 36 16
70
Ceftazidime Tazobactam
114.0 141.0 246.8 467.6 138.1 254.9 246.8 148.0 397.7
70 120 160
Positive Positive Positive
3.03 3.27 3.41
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Figure 1 Chromatograms of the five antibiotics and IS obtained at the LLOQ. NL, normalized level; RT, retention time; SN, signal to noise ratio.
expressed as the mean relative error (i.e. the agreement between test results and the expected value expressed as percentage). Imprecision was expressed as the coefficient of variation (CV%). The acceptance criteria for intra- and inter-assay imprecision were #15% and for accuracy was 85–115% of the nominal concentrations as specified by FDA guidelines.6 Recovery, defined as the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard, was estimated from six replicate determinations using samples prepared with QC I and QC IV samples for each analyte. In accordance with FDA guidelines, the lower limit of quantification (LLOQ) was determined as the sample concentration that provided measurements with an imprecision #20% and accuracy within 80–120% of the nominal concentration Stability was determined from three replicate assays of both the plasma samples and extracts of QCI and QC IV after maintaining plasma and extracts samples at 25uC for over 6 hours. Freeze– thaw sample stability was determined after three freeze and thaw cycles. Long-term stability was determined from a plasma sample stored at 230uC for 2 months. Stock solution (200 mg/ml) stability was evaluated after keeping it at room temperature for 6 hours.
procedure start to the completion of the LC-MS/MS analysis. Figure 1 shows the chromatograms obtained for the five antibiotics and IS at the LLOQ. Method specificity was established by demonstrating the absence of interference at the specific ion transitions that were selected for analyte quantification by using blank samples. The ion suppression tests demonstrated that no ion suppression was found at the elution time of the analytes. The linear regression fit for the calibration curves was achieved for all the five analytes with R2.0.9954. The mean equations obtained were y52661023z5.961023x for piperacillin, y5 21.861023z4.261023x for tazobactam, y520.596 1023z6.961023x for meropenem, y5251.761023z 4.961023x for ceftazidime, and y56.861023z1146 1023x for linezolid, respectively. The LLOQ obtained for the five antibiotics were: 0.075, 0.3, 0.15, 0.015, and 1.5 mg/ml for piperacillin, tazobactam, meropenem, linezolid, and ceftazidime, respectively. Intra- and inter-assay imprecision and
Results Method development and validation
Linezolid
Piperacillin, tazobactam, meropenem, ceftazidime, and linezolid could be simultaneously quantified with an overall turnaround time of 20 minutes from the
Table 2 Recovery data Recovery Compounds Meropenem Tazobactam
Ceftazidime Piperacillin
QC concentration (mg/l)
CV (%)
RE (%)
2.4 2.1 3.5 1.5 1.2 1.1 5.6 3.2 0.6 1.4
104 100 108 101 119 104 106 95 100 111
1.5 12 1.5 12 1.5 12 7.5 60 7.5 60
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Ceftazidime Piperacillin
Meropenem Tazobactam Linezolid
Inter-assay Compounds
Ceftazidime Piperacillin
Meropenem Tazobactam Linezolid
Compounds
Intra-assay
Mean6S.D. (mg/l) 1.5 1.460.1 1.560.11 1.460.11 7.5 7.560.3 7.560.2
1.560.09 1.660.07 1.660.06 7.5 860.68 7.560.17
1.5
Mean6SD (mg/l)
3.9 2.7
6.9 7.4 7.9
Precison (%)
8.5 2.2
5.1 3.7 3.3
Precison (%)
100.5 100.6
95.8 97 96
Accuracy (%)
106.2 100.4
99.1 106.7 105.8
Accuracy (%)
14.960.85 14.960.27
360.16 360.19 2.960.2
Mean6SD (mg/l)
14.360.69 14.860.35
3.160.09 2.960.27 360.07
Mean6S.D. (mg/l)
Precison (%) 3 5.2 6.4 7 15 5.7 1.8
2.4 7.4 2 15 4.8 2.3
3
Precison (%)
99.5 99.4
101.4 100.8 97.2
Accuracy (%)
95.1 98.9
96.4 95 101.9
Accuracy (%)
Table 3 Intra-assay and inter-assay precision and accuracy obtained for the five antibiotics
27.560.51 31.361.87
6.160.37 5.960.53 6.260.26
Mean6SD (mg/l)
2860.75 28.761.19
5.660.2 5.760.61 5.660.14
Mean6SD (mg/l)
Precison (%) 6 6.1 8.9 4.1 30 1.9 6
2.9 8.4 1.9 30 2.7 4.2
6
Precison (%)
91.7 104.4
101.2 99.1 103.3
Accuracy (%)
93.3 95.6
94.1 96.6 94.1
Accuracy (%)
59.560.77 59.961.21
12.26.057 12.560.7 12.360.47
Mean6SD (mg/l)
59.762.4 57.561.2
11.160.45 10.960.98 10.760.25
Mean6SD (mg/l)
1.3 2
4.7 5.5 3.8
Precison (%)
3.2 7 1.8 60 4 2.1
12
Precison (%)
Accuracy (%) 12 101.5 104.6 102.8 60 99.1 99.8
99.5 95.8
93.8 93 91.4
Accuracy (%)
Barco et al. Antibiotics in human plasma
Barco et al.
accuracy were all within the established ranges of acceptance as summarized in Table 3. Recovery data obtained on six replicates of each QC level are shown in Table 2. The stability tests (data not shown) demonstrated that the five analytes in a stock solution and in plasma samples are stable at the pre-specified conditions. Losses were less than 6% and there was no decline in assay precision, with CV ,5.6%.
Discussion Monitoring plasma concentration of antibiotic drugs and comparing the results with the MIC of microorganisms is crucial for the individualization of antimicrobial therapy.8 Several HPLC-UV or LC–MS/MS methods for the determination of antibiotics in various biological matrices can be found in the literature,9–12 but very few methods are validated for clinical use.13–15 In this study, we described a LC–MS/MS-based fast, reliable, and cost effective assay that uses a multi-parametric analytical approach for the quantification of five different antibiotics: piperacillin, tazobactam, meropenem, ceftazidime, and linezolid. The performance and suitability of the assay was evaluated by applying an extensive validation protocol based on international guidelines,5,6 and showed high degree of specificity, accuracy and reproducibility for the majority of drugs tested. Even if the method could not be very accurate for the detection of the lowest drug concentrations, especially for ceftazidime, it resulted accurate and reproducible in the concentrations around the MIC breakpoints.2,8,16,17 Furthermore, the LC–MS/MS allowed the simultaneous identification of five different drugs by using the same analytical procedure and this possibility represents an advantage for routine analysis in terms of turnaround time when different samples for different patients and drugs must be test, or in the case that some of these drugs are co-administered (e.g. meropenem and linezolid in a patient with ventilatorassociated pneumonia). Finally, and maybe of utmost important in the pediatric setting, the method allowed the use a very small amount of plasma, starting from 50 ml, which is a volume compatible with the sample limitations in pediatrics18 and could be very attractive especially in low birth weight neonates who have a small blood volume and therefore, could not be sampled frequently and/or taking large volumes for drug quantification.
Disclaimer Statements Contributors All authors have contributed to the study and approved the manuscript. Funding Istituto Giannina Gaslini and Fondazione Italiana Neuroblastoma. Conflicts of interest No conflict of interest.
Antibiotics in human plasma
Ethics approval None.
Acknowledgements SB is recipient of a Fondazione Italiana Neuroblastoma fellowship.
References 1 Adembri C, Novelli A. Pharmacokinetic and pharmacodynamic parameters of antimicrobials: potential for providing dosing regimens that are less vulnerable to resistance. Clin Pharmacokinet. 2009;48(8):517–28. 2 Petrosillo N, Giannella M, Lewis R, Viale P. Treatment of carbapenem-resistant klebsiella pneumoniae: the state of the art. Expert Rev Anti-infect Ther. 2013;11(2):159–77. 3 Roberts JA, Lipman J. Pharmacokinetic issues for antibiotics in the critically ill patient. Crit Care Med. 2009;37(3):840–51. 4 Choi G, Gomersall CD, Tian Q, Joynt GM, Freebairn R, Lipman J. Principles of antibacterial dosing in continuous renal replacement therapy. Crit Care Med. 2009;37(3):2268–82. 5 US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER). Guidance for industry: bioanalytical method validation. Silver Spring, MD: FDA; 2001. 6 International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Topic Q2 (R1): Validation of analytical procedures: text and methodology. Geneva: ICH; 2005. 7 Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003;49(7):1041–4. 8 Mouton JW, Brown DF, Apfalter P, Canto´n R, Giske CG, Ivanova M, et al. The role of pharmacokinetics/pharmacodynamics in setting clinical MIC breakpoints: the EUCAST approach. Clin Microbiol Infect. 2012;18(3):E37–45. 9 Reyns T, de Baere S, Croubels S, de Backer P. Determination of clavulanic acid in calf plasma by liquid chromatography tandem mass spectrometry. J Mass Spectrom. 2006;41(11):1414–20. 10 Reyns T, de Boever S, de Baere S, de Backer P, Croubels S. Quantitative analysis of clavulanic acid in porcine tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry. Anal Chim Acta. 2007;597(2):282–9. 11 Fagerquist CK, Lightfield AR, Lehotay SJ. Confirmatory and quantitative analysis of beta-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chem. 2005;77(5):1473–82. 12 Mastovska K, Lightfield AR. Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. J Cromatogr A. 2008;1202(2):118–23. 13 Arzuaga A, Isla A, Gascon AR, Maynar J, Martin A, Solinis MA, et al. Quantitation and stability of piperacillin and tazobactam in plasma and ultrafiltrate from patients undergoing continuous venovenous hemofiltration by HPLC. Biomed Chromatogr. 2005;19(8):570–8. 14 Ohmori T, Suzuki A, Niwa T, Ushikoshi H, Shirai K, Yoshida S, et al. Simultaneous determination of eight beta-lactam antibiotics in human serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(15–16):1038–42 15 Phillips OA, Abdel-Hamid ME, Al-Hassawi NA. Determination of linezolid in human plasma by LC–MS–MS. Analyst. 2001;126(5):609–14. 16 European Committee on Antimicrobial Scusceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0, 2014. Available from: http://www.eucast. org/fileadmin/src/media/PDFs/ EUCAST_files/Breakpoint_tables/ Breakpoint_table_v_3.1.pdf (accessed 30 March 2014). 17 Zavaski AP, Bulitta JB and Landersdorfer CB. Combination therapy for carbapenem-resistant Gram-negative bacteria. Expert Rev Anti-infect Ther. 2013;11(12):1333–53. 18 Clinical Investigation of Medicinal Products in the Paediatric Population E11. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Step 4 version. Geneva: ICH; 2000. Available from: http://www.ich.org/fileadmin/Public_ Web_Site/ICH_Products/Guidelines/Efficacy/E11/Step4/ E11_Guide line.pdf (accessed 30 March 2014).
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Quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma by liquid chromatography/ tandem mass spectrometry Sebastiano Barco1, Roberto Bandettini1, Angelo Maffia1, Gino Tripodi1, Elio Castagnola2, Giuliana Cangemi1 1
Clinical Pathology Laboratory Unit, Istituto Giannina Gaslini, Genoa, Italy, 2Infectious Disease Unit, Istituto Giannina Gaslini, Genoa, Italy Therapeutic drug monitoring is a cornerstone of antibacterial therapy, especially in an era of increasing antibacterial resistance in individualizing antimicrobial therapy. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was used for the simultaneous measurement of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in 50 ml plasma samples over a wide range. The overall turnaround time for the assay was 20 minutes. Intra-assay precision and accuracy for quality control samples ranged within 1.8–8.5 and 91.4–106.7%, respectively. Inter-assay precision and accuracy ranged within 1.3–14.4 and 95.8–104.6%, respectively. The lower limit of quantification was below 1.5 mg/ml for all the five antibiotics. No ion suppression due to matrix effects was found. A simple and rapid LC–MS/MS method which provides high specificity, precision and accuracy for the simultaneous quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma has been developed and validated. The present method is suitable for therapeutic drug monitoring in paediatrics.
Keywords: Piperacillin, Tazobactam, Meropenem, Ceftazidime, Linezolid, Liquid chromatography/tandem mass spectrometry
Introduction Optimization of antibiotic dosage regimens is crucial for the management of severe infectious diseases and reduction of resistant mutant selection, especially in an era of increasing antibiotic resistance. Therefore, TDM may be of fundamental importance in individualizing antimicrobial therapy, in particular in critically ill patients, in the presence of peculiar pathophysiological condition or of infections due to pathogens with reduced sensitivity to antibiotics, which may require higher doses administration to be effective.1–4 For these reasons, the availability of reliable and validated bioanalytical methods for the determination of antibiotics in human plasma is of crucial importance. In the present study, we describe a simple and fast liquid chromatography-tandem mass spectrometry (L C–MS/MS) method that can be employed for the determination of plasma levels of five different antibiotics, piperacillin, tazobactam, meropenem, ceftazidime, and linezolid, using small volume of plasma (50 ml).
Correspondence to: E. Castagnola, Infectious Diseases Unit, Istituto Giannina Gaslini, Largo G. Gaslini 3-5, 16147 Genova, Italy. Email: [email protected]
ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000209
Materials and Methods Chemicals and reagents Piperacillin, tazobactam, meropenem, ceftazidime, linezolid, and prasozin (as internal standard, IS) were purchased from Sigma-Aldrich (Milan, Italy). Water was purified by reverse osmosis and filtrated through a Milli-Q Purification System (Millipore, Milford, MA, USA). High-performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Sigma-Aldrich Srl (Milan, Italy) and formic acid (99.9%) from Merck (Darmstadt, Germany).
Preparation of calibrators and quality control samples Stock solutions (2 mg/ml) were prepared by dissolving each powdered antibiotic in water except linezolid that was dissolved in dimethyl sulphoxide. Calibrators and quality controls were made by spiking tazobactam, linezolid, meropenem, piperacillin, and ceftazidime from different batches of working solution into a pool of plasma. Two different six-point calibration curves were created from calibrator standards that were prepared by adding the five antibiotics to a pool of plasma to yield concentrations of 6.25–12.5–25–50–100 and 200 mg/ml
Journal of Chemotherapy
2015
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NO.
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Antibiotics in human plasma
for piperacillin and ceftazidime and 1.25–2.5–5–10–20 and 40 mg/ml for tazobactam, meropenem, and linezolid, respectively. Quality control (QC) samples were prepared by adding the five antibiotics at the following four concentration levels (QC I, II, III, and IV) to a pool of plasma: 7.5–15–30 and 60 mg/ml for piperacillin and ceftazidime and 1.5-3–6 and 12 mg/ml for tazobactam, meropenem, and linezolid.
Human samples Leftover plasma samples from routine clinical chemistry have been used for the development and validation of the present method. Plasma was obtained from peripheral blood collected in sodium heparin-containing tubes by centrifuging at 4000g for 5 minutes. Plasma samples were stored at 230uC until analysed. Blank samples were obtained from healthy adults and from paediatric patients. The model was validated (for piperacillin, tazobactam, and meropenem) in leftover plasma samples from routine clinical chemistry derived from pediatric patients, including those admitted in the neonatal intensive care unit. Ethical Committee approval was required for the use of the leftover plasma samples to validate the analytical method.
Protein precipitation A 50 ml aliquot of plasma was spiked with 10 ml of 220 ng/ml IS. Protein was precipitated by adding 300 ml of methanol and centrifuging for 10 minutes at 20 000g. The supernatant then was filtered through a 0.22 mm filter.
Chromatography and mass spectrometry Gradient separation chromatography was carried out using a Kinetex C18 column (10064.6 mm, i.d. 2.6 mm; Phenomenex, Bologna, Italy) with mobile phase A consisting of 0.1% formic acid in water and mobile phase B of acetonitrile. The percentage of solvent B was programmed to reach 100% in 3 minutes at a flow rate of 700 ml/min, then the column was washed with 100% B for 1 minutes and finally reconditioned at 95% A for 2 minutes for a total run time of 5.5 minutes. The column temperature was maintained at 40uC and injection volume was 10 ml. The five analytes have retention times between 2.7 and 3.42 minutes. Mass spectrometric detection was
performed using a Thermo Scientific TSQ Quantum Access MAX triple quadrupole system. Ionization was achieved using electrospray in the positive ion mode except for tazobactam which was ionized in the negative ion mode. The spray voltage was set at 3500 V in positive polarity and 3000 V in negative polarity. Nitrogen was used as the nebulizer and auxiliary gas, set at 50 and 15 arbitrary units, respectively. Vaporizer and capillary temperature settings were 350 and 275uC. For collision-induced dissociation, high purity argon was used at a pressure of 1.5 mTorr. The antibiotics were detected using multiple reaction monitoring of the specific transitions listed in Table 1.
Method validation A laboratory scheme based on the International Conference on Harmonization and Food and Drug Administration (FDA) guidelines5,6 was used for assay validation. To exclude the presence of potentially interfering substances, tests were conducted on samples obtained from consenting healthy volunteers. In addition, 30 blank samples were analysed according to the whole analytical procedure. The impact on the analyte of ion suppression and enhancement resulting from ionization of matrix components has been tested. Seven aliquots of processed blank plasma samples from different drug-free volunteers were injected into the HPLC system, while a mixture of the five analytes was continuously infused post-column and mixed with the column effluent through a ‘T’ before entering the electrospray interface.7 The concentration of each analyte was 10 mg/ml in acetonitrile. Infusion flow was 10 ml/min and HPLC flow was 690 ml/min. Linearity was evaluated on the basis of the mean of six replicate assays of each calibrator standard. The slope, intercept, and correlation coefficient for each calibration curve were estimated by plotting the peak area ratio of analyte/IS vs. the analyte concentration of each calibration standard sample using a 1/x weighting factor. Intra-assay and inter-assay imprecision and accuracy were determined at four concentration levels, using the four level QC samples with each sample being analysed five separate times. Inter-assay imprecision was estimated by repeating the test in six non-consecutive analytical sessions. Accuracy was
Table 1 LC-MS/MS parameters of the five antibiotics and IS
344
Compounds
Parent ion
Product ions
Collision energy
Tube lens
Meropenem
384.1
Positive
2.69
546.9 298.9
70 70
Positive Negative
2.7 2.72
Prasozin Linezolid Piperacillin
384.0 338.1 540
30 19 30 12 17 12 29 36 16
70
Ceftazidime Tazobactam
114.0 141.0 246.8 467.6 138.1 254.9 246.8 148.0 397.7
70 120 160
Positive Positive Positive
3.03 3.27 3.41
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ESI polarity
Retention time
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Figure 1 Chromatograms of the five antibiotics and IS obtained at the LLOQ. NL, normalized level; RT, retention time; SN, signal to noise ratio.
expressed as the mean relative error (i.e. the agreement between test results and the expected value expressed as percentage). Imprecision was expressed as the coefficient of variation (CV%). The acceptance criteria for intra- and inter-assay imprecision were #15% and for accuracy was 85–115% of the nominal concentrations as specified by FDA guidelines.6 Recovery, defined as the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard, was estimated from six replicate determinations using samples prepared with QC I and QC IV samples for each analyte. In accordance with FDA guidelines, the lower limit of quantification (LLOQ) was determined as the sample concentration that provided measurements with an imprecision #20% and accuracy within 80–120% of the nominal concentration Stability was determined from three replicate assays of both the plasma samples and extracts of QCI and QC IV after maintaining plasma and extracts samples at 25uC for over 6 hours. Freeze– thaw sample stability was determined after three freeze and thaw cycles. Long-term stability was determined from a plasma sample stored at 230uC for 2 months. Stock solution (200 mg/ml) stability was evaluated after keeping it at room temperature for 6 hours.
procedure start to the completion of the LC-MS/MS analysis. Figure 1 shows the chromatograms obtained for the five antibiotics and IS at the LLOQ. Method specificity was established by demonstrating the absence of interference at the specific ion transitions that were selected for analyte quantification by using blank samples. The ion suppression tests demonstrated that no ion suppression was found at the elution time of the analytes. The linear regression fit for the calibration curves was achieved for all the five analytes with R2.0.9954. The mean equations obtained were y52661023z5.961023x for piperacillin, y5 21.861023z4.261023x for tazobactam, y520.596 1023z6.961023x for meropenem, y5251.761023z 4.961023x for ceftazidime, and y56.861023z1146 1023x for linezolid, respectively. The LLOQ obtained for the five antibiotics were: 0.075, 0.3, 0.15, 0.015, and 1.5 mg/ml for piperacillin, tazobactam, meropenem, linezolid, and ceftazidime, respectively. Intra- and inter-assay imprecision and
Results Method development and validation
Linezolid
Piperacillin, tazobactam, meropenem, ceftazidime, and linezolid could be simultaneously quantified with an overall turnaround time of 20 minutes from the
Table 2 Recovery data Recovery Compounds Meropenem Tazobactam
Ceftazidime Piperacillin
QC concentration (mg/l)
CV (%)
RE (%)
2.4 2.1 3.5 1.5 1.2 1.1 5.6 3.2 0.6 1.4
104 100 108 101 119 104 106 95 100 111
1.5 12 1.5 12 1.5 12 7.5 60 7.5 60
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Ceftazidime Piperacillin
Meropenem Tazobactam Linezolid
Inter-assay Compounds
Ceftazidime Piperacillin
Meropenem Tazobactam Linezolid
Compounds
Intra-assay
Mean6S.D. (mg/l) 1.5 1.460.1 1.560.11 1.460.11 7.5 7.560.3 7.560.2
1.560.09 1.660.07 1.660.06 7.5 860.68 7.560.17
1.5
Mean6SD (mg/l)
3.9 2.7
6.9 7.4 7.9
Precison (%)
8.5 2.2
5.1 3.7 3.3
Precison (%)
100.5 100.6
95.8 97 96
Accuracy (%)
106.2 100.4
99.1 106.7 105.8
Accuracy (%)
14.960.85 14.960.27
360.16 360.19 2.960.2
Mean6SD (mg/l)
14.360.69 14.860.35
3.160.09 2.960.27 360.07
Mean6S.D. (mg/l)
Precison (%) 3 5.2 6.4 7 15 5.7 1.8
2.4 7.4 2 15 4.8 2.3
3
Precison (%)
99.5 99.4
101.4 100.8 97.2
Accuracy (%)
95.1 98.9
96.4 95 101.9
Accuracy (%)
Table 3 Intra-assay and inter-assay precision and accuracy obtained for the five antibiotics
27.560.51 31.361.87
6.160.37 5.960.53 6.260.26
Mean6SD (mg/l)
2860.75 28.761.19
5.660.2 5.760.61 5.660.14
Mean6SD (mg/l)
Precison (%) 6 6.1 8.9 4.1 30 1.9 6
2.9 8.4 1.9 30 2.7 4.2
6
Precison (%)
91.7 104.4
101.2 99.1 103.3
Accuracy (%)
93.3 95.6
94.1 96.6 94.1
Accuracy (%)
59.560.77 59.961.21
12.26.057 12.560.7 12.360.47
Mean6SD (mg/l)
59.762.4 57.561.2
11.160.45 10.960.98 10.760.25
Mean6SD (mg/l)
1.3 2
4.7 5.5 3.8
Precison (%)
3.2 7 1.8 60 4 2.1
12
Precison (%)
Accuracy (%) 12 101.5 104.6 102.8 60 99.1 99.8
99.5 95.8
93.8 93 91.4
Accuracy (%)
Barco et al. Antibiotics in human plasma
Barco et al.
accuracy were all within the established ranges of acceptance as summarized in Table 3. Recovery data obtained on six replicates of each QC level are shown in Table 2. The stability tests (data not shown) demonstrated that the five analytes in a stock solution and in plasma samples are stable at the pre-specified conditions. Losses were less than 6% and there was no decline in assay precision, with CV ,5.6%.
Discussion Monitoring plasma concentration of antibiotic drugs and comparing the results with the MIC of microorganisms is crucial for the individualization of antimicrobial therapy.8 Several HPLC-UV or LC–MS/MS methods for the determination of antibiotics in various biological matrices can be found in the literature,9–12 but very few methods are validated for clinical use.13–15 In this study, we described a LC–MS/MS-based fast, reliable, and cost effective assay that uses a multi-parametric analytical approach for the quantification of five different antibiotics: piperacillin, tazobactam, meropenem, ceftazidime, and linezolid. The performance and suitability of the assay was evaluated by applying an extensive validation protocol based on international guidelines,5,6 and showed high degree of specificity, accuracy and reproducibility for the majority of drugs tested. Even if the method could not be very accurate for the detection of the lowest drug concentrations, especially for ceftazidime, it resulted accurate and reproducible in the concentrations around the MIC breakpoints.2,8,16,17 Furthermore, the LC–MS/MS allowed the simultaneous identification of five different drugs by using the same analytical procedure and this possibility represents an advantage for routine analysis in terms of turnaround time when different samples for different patients and drugs must be test, or in the case that some of these drugs are co-administered (e.g. meropenem and linezolid in a patient with ventilatorassociated pneumonia). Finally, and maybe of utmost important in the pediatric setting, the method allowed the use a very small amount of plasma, starting from 50 ml, which is a volume compatible with the sample limitations in pediatrics18 and could be very attractive especially in low birth weight neonates who have a small blood volume and therefore, could not be sampled frequently and/or taking large volumes for drug quantification.
Disclaimer Statements Contributors All authors have contributed to the study and approved the manuscript. Funding Istituto Giannina Gaslini and Fondazione Italiana Neuroblastoma. Conflicts of interest No conflict of interest.
Antibiotics in human plasma
Ethics approval None.
Acknowledgements SB is recipient of a Fondazione Italiana Neuroblastoma fellowship.
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