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A 2D HPLC-MS/MS method for several antibiotics in blood plasma, plasma water, and diverse tissue samples

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Abstract

An analytical method using 2D high-performance liquid chromatography followed by tandem mass spectrometry for the quantification of the beta-lactam antibiotics amoxicillin, flucloxacillin, piperacillin, benzylpenicillin, the beta-lactamase inhibitors clavulanic acid, and tazobactam, as well as the macrolide antibiotic clindamycin, is presented. All analytes were measured in human plasma, while amoxicillin, clavulanic acid, flucloxacillin, and clindamycin were also analyzed in human tissue samples. Because of its high-protein binding, additionally, the free fraction of flucloxacillin was measured after ultrafiltration. As internal standards, deuterated forms of the beta-lactams were used. Sample preparation for all matrices was protein precipitation followed by online extraction on a TurboFlow MAX column, while sample separation was performed on an Accucore XL C18 column. Calibration curves were linear over 0.2–25 mg/kg for the tissue samples and 0.05–20 mg/l for the free fraction of flucloxacillin. In plasma, the calibration curves for amoxicillin and piperacillin were linear over 3.125–125 mg/l, for clavulanic acid and tazobactam over 1–40 mg/l, for benzylpenicillin 0.25–40 mg/l, and for flucloxacillin and clindamycin over 1.5–60 mg/l and 0.05–8 mg/l respectively. In plasma and plasma ultrafiltrate, inaccuracy and imprecision for any analyte were always less than 15%. In tissue, the accuracy and precision varied up to 16%, respectively, 20%, when various tissues were analyzed using a calibration in water.

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References

  1. Muller AE, Huttner B, Huttner A. Therapeutic drug monitoring of beta-lactams and other antibiotics in the intensive care unit: which agents, which patients and which infections? Drugs. 2018;78(4):439–51. https://doi.org/10.1007/s40265-018-0880-z .

    Article  CAS  PubMed  Google Scholar 

  2. Huttner A, Harbarth S, Hope WW, Lipman J, Roberts JA. Therapeutic drug monitoring of the β-lactam antibiotics: what is the evidence and which patients should we be using it for? J Antimicrob Chemother. 2015;70(12):3178–83. https://doi.org/10.1093/jac/dkv201 .

    Article  CAS  PubMed  Google Scholar 

  3. Dalhoff A. Seventy-five years of research on protein binding. Antimicrob Agents Chemother. 2018;62(2):1663–17. https://doi.org/10.1128/AAC.01663-17 .

    Article  Google Scholar 

  4. Sutherland R, Croydon EA, Rolinson GN. Flucloxacillin, a new isoxazolyl penicillin, compared with oxacillin, cloxacillin, and dicloxacillin. Br Med J. 1970;4(5733):455–60. https://doi.org/10.1136/bmj.4.5733.455 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Røder BL, Frimodt-Møller N, Espersen F, Rasmussen SN. Dicloxacillin and flucloxacillin: pharmacokinetics, protein binding and serum bactericidal titers in healthy subjects after oral administration. Infection. 1995;23(2):107–12. https://doi.org/10.1007/BF01833876 .

    Article  PubMed  Google Scholar 

  6. Gordon RC, Regamey C, Kirby WM. Serum protein binding of erythromycin, lincomycin, and clindamycin. J Pharm Sci. 1973;62(7):1074–7. https://doi.org/10.1002/jps.2600620704 .

    Article  CAS  PubMed  Google Scholar 

  7. Ulldemolins M, Roberts JA, Wallis SC, Rello J, Lipman J. Flucloxacillin dosing in critically ill patients with hypoalbuminaemia: special emphasis on unbound pharmacokinetics. J Antimicrob Chemother. 2010;65(8):1771–8. https://doi.org/10.1093/jac/dkq184 .

    Article  CAS  PubMed  Google Scholar 

  8. Theuretzbacher U. Tissue penetration of antibacterial agents: how should this be incorporated into pharmacodynamic analyses? Curr Opin Pharmacol. 2007;7(5):498–504. https://doi.org/10.1016/j.coph.2007.05.003 .

    Article  CAS  PubMed  Google Scholar 

  9. Pardridge WM. Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab. 2012;32(11):1959–72. https://doi.org/10.1038/jcbfm.2012.126 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li X, Zhang Z, Chang X, Wang X, Hu J, Lin Q, et al. Disruption of blood-brain barrier by an Escherichia coli isolated from canine septicemia and meningoencephalitis. Comp Immunol Microbiol Infect Dis. 2019;63:44–50. https://doi.org/10.1016/j.cimid.2019.01.002 .

    Article  PubMed  Google Scholar 

  11. Varatharaj A, Galea I. The blood-brain barrier in systemic inflammation. Brain Behav Immun. 2017;60:1–12. https://doi.org/10.1016/j.bbi.2016.03.010 .

    Article  CAS  PubMed  Google Scholar 

  12. Dando SJ, Mackay-Sim A, Norton R, Currie BJ, St John JA, Ekberg JAK, et al. Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion. Clin Microbiol Rev. 2014;27(4):691–726. https://doi.org/10.1128/CMR.00118-13 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dong X, Ding L, Cao X, Jiang L, Zhong S. A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring. Biomed Chromatogr. 2013;27(4):520–6. https://doi.org/10.1002/bmc.2824 .

    Article  CAS  PubMed  Google Scholar 

  14. Abdulla A, Bahmany S, Wijma RA, van der Nagel BCH, Koch BCP. Simultaneous determination of nine β-lactam antibiotics in human plasma by an ultrafast hydrophilic-interaction chromatography-tandem mass spectrometry. J Chromatogr B. 2017;1060:138–43. https://doi.org/10.1016/j.jchromb.2017.06.014 .

    Article  CAS  Google Scholar 

  15. Carlier M, Stove V, de Waele JJ, Verstraete AG. Ultrafast quantification of β-lactam antibiotics in human plasma using UPLC–MS/MS. J Chromatogr B. 2015;978-979:89–94. https://doi.org/10.1016/j.jchromb.2014.11.034 .

    Article  CAS  Google Scholar 

  16. Zander J, Döbbeler G, Nagel D, Maier B, Scharf C, Huseyn-Zada M, et al. Piperacillin concentration in relation to therapeutic range in critically ill patients - a prospective observational study. Crit Care. 2016;20(1):79. https://doi.org/10.1186/s13054-016-1255-z .

    Article  PubMed  PubMed Central  Google Scholar 

  17. Di Rocco M, Moloney M, O'Beirne T, Earley S, Berendsen B, Furey A, et al. Development and validation of a quantitative confirmatory method for 30 β-lactam antibiotics in bovine muscle using liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A. 2017;1500:121–35. https://doi.org/10.1016/j.chroma.2017.04.022 .

    Article  CAS  PubMed  Google Scholar 

  18. Lugoboni B, Gazzotti T, Zironi E, Barbarossa A, Pagliuca G. Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(21):1980–6. https://doi.org/10.1016/j.jchromb.2011.05.038 .

    Article  CAS  PubMed  Google Scholar 

  19. Reyns T, Cherlet M, de Baere S, de Backer P, Croubels S. Rapid method for the quantification of amoxicillin and its major metabolites in pig tissues by liquid chromatography-tandem mass spectrometry with emphasis on stability issues. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;861(1):108–16. https://doi.org/10.1016/j.jchromb.2007.11.007 .

    Article  CAS  PubMed  Google Scholar 

  20. Torkington MS, Davison MJ, Wheelwright EF, Jenkins PJ, Anthony I, Lovering AM, et al. Bone penetration of intravenous flucloxacillin and gentamicin as antibiotic prophylaxis during total hip and knee arthroplasty. Bone Joint J. 2017;99-B(3):358–64. https://doi.org/10.1302/0301-620X.99B3.BJJ-2016-0328.R1 .

    Article  CAS  PubMed  Google Scholar 

  21. Martin C, Mallet MN, Sastre B, Viviand X, Martin A, de Micco P, et al. Comparison of concentrations of two doses of clavulanic acid (200 and 400 milligrams) administered with amoxicillin (2,000 milligrams) in tissues of patients undergoing colorectal surgery. Antimicrob Agents Chemother. 1995;39(1):94–8. https://doi.org/10.1128/aac.39.1.94 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cross SE, Thompson MJ, Roberts MS. Distribution of systemically administered ampicillin, benzylpenicillin, and flucloxacillin in excisional wounds in diabetic and normal rats and effects of local topical vasodilator treatment. Antimicrob Agents Chemother. 1996;40(7):1703–10. https://doi.org/10.1128/AAC.40.7.1703 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gibson MJ, Karpinski MR, Slack RC, Cowlishaw WA, Webb JK. The penetration of antibiotics into the normal intervertebral disc. J Bone Joint Surg Br. 1987;69(5):784–6.

    Article  CAS  Google Scholar 

  24. Frank U, Schmidt-Eisenlohr E, Schlosser V, Spillner G, Schindler M, Daschner FD. Concentrations of flucloxacillin in heart valves and subcutaneous and muscle tissues of patients undergoing open-heart surgery. Antimicrob Agents Chemother. 1988;32(6):930–1. https://doi.org/10.1128/aac.32.6.930 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Averono G, Vidali M, Olina M, Basile M, Bagnati M, Bellomo G, et al. Evaluation of amoxicillin plasma and tissue levels in pediatric patients undergoing tonsillectomy. Int J Pediatr Otorhinolaryngol. 2010;74(9):995–8. https://doi.org/10.1016/j.ijporl.2010.05.023 .

    Article  PubMed  Google Scholar 

  26. Cazorla-Reyes R, Romero-González R, Frenich AG, Rodríguez Maresca MA, Martínez Vidal JL. Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2014;89:203–12. https://doi.org/10.1016/j.jpba.2013.11.004 .

    Article  CAS  PubMed  Google Scholar 

  27. Barré J, Chamouard JM, Houin G, Tillement JP. Equilibrium dialysis, ultrafiltration, and ultracentrifugation compared for determining the plasma-protein-binding characteristics of valproic acid. Clin Chem. 1985;31(1):60–4.

    Article  Google Scholar 

  28. Wong G, Brinkman A, Benefield RJ, Carlier M, de Waele JJ, El Helali N, et al. An international, multicentre survey of β-lactam antibiotic therapeutic drug monitoring practice in intensive care units. J Antimicrob Chemother. 2014;69(5):1416–23. https://doi.org/10.1093/jac/dkt523 .

    Article  CAS  PubMed  Google Scholar 

  29. Kratzer A, Liebchen U, Schleibinger M, Kees MG, Kees F. Determination of free vancomycin, ceftriaxone, cefazolin and ertapenem in plasma by ultrafiltration: impact of experimental conditions. J Chromatogr B. 2014;961:97–102. https://doi.org/10.1016/j.jchromb.2014.05.021 .

    Article  CAS  Google Scholar 

  30. Wong G, Briscoe S, McWhinney B, Ally M, Ungerer J, Lipman J, et al. Therapeutic drug monitoring of β-lactam antibiotics in the critically ill: direct measurement of unbound drug concentrations to achieve appropriate drug exposures. J Antimicrob Chemother. 2018;73(11):3087–94. https://doi.org/10.1093/jac/dky314 .

    Article  CAS  PubMed  Google Scholar 

  31. Zeitlinger MA, Derendorf H, Mouton JW, Cars O, Craig WA, Andes D, et al. Protein binding: do we ever learn? Antimicrob Agents Chemother. 2011;55(7):3067–74. https://doi.org/10.1128/AAC.01433-10 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kawakami A, Kubota K, Yamada N, Tagami U, Takehana K, Sonaka I, et al. Identification and characterization of oxidized human serum albumin. A slight structural change impairs its ligand-binding and antioxidant functions. FEBS J. 2006;273(14):3346–57. https://doi.org/10.1111/j.1742-4658.2006.05341.x .

    Article  CAS  PubMed  Google Scholar 

  33. Fournier T, Medjoubi-N N, Porquet D. Alpha-1-acid glycoprotein. Biochim Biophys Acta. 2000;1482(1–2):157–71. https://doi.org/10.1016/s0167-4838(00)00153-9 .

    Article  CAS  PubMed  Google Scholar 

  34. Liebchen U, Kratzer A, Wicha SG, Kees F, Kloft C, Kees MG. Unbound fraction of ertapenem in intensive care unit patients. J Antimicrob Chemother. 2014;69(11):3108–11. https://doi.org/10.1093/jac/dku226 .

    Article  CAS  PubMed  Google Scholar 

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  1. Laboratory Medicine, University Hospital Basel, University of Basel, Petersgraben 4, 4031, Basel, Switzerland

    Sophia Rehm & Katharina M. Rentsch

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  1. Sophia Rehm
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Correspondence to Katharina M. Rentsch.

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Rehm, S., Rentsch, K.M. A 2D HPLC-MS/MS method for several antibiotics in blood plasma, plasma water, and diverse tissue samples. Anal Bioanal Chem 412, 715–725 (2020). https://doi.org/10.1007/s00216-019-02285-0

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