Karin Kipper (Presenter)
St. George
Bio: Karin Kipper obtained her Ph.D. from University of Tartu (UT) in 2012. Since 2004 she has been involved in the bioanalytical method development and validation for HPLC-UV/vis and LC–MS analysis, working at UT Institute of Pharmacology and Institute of Chemistry. Starting from 2012 Karin Kipper works as a research fellow at UT Institute of Chemistry and from 2014 a post-doctoral fellow at St. George's, University of London, UK. Her main research fields are pharmaceutical bioanalysis (pharmacokinetic/pharmacodynamic studies), pharmaceuticals’ pathways in environment and development of novel eluent additives for LC–MS in order to improve separation and peak shape of basic compounds.
Authorship: Karin Kipper(1,2), Charlotte Barker(1), Dagan Lonsdale(1), Mike Sharland(1), Atholl Johnston(3)
(1) Institute for Infection and Immunity, St. George
| Short Abstract Bioanalytical UPLC-MS/MS method was developed and validated for the most commonly used antimicrobials in England’s intensive care units. The list of the antimicrobials consisted: co-amoxiclav (amoxicillin with clavulanic acid), piperacillin with tazobactam, flucloxacillin, meropenem, ertapenem, ceftriaxone, cefotaxime, benzylpenicillin, clarithromycin and fluconazole. The separation between the compounds was obtained with reversed phase chromatography using the weak ion-pairing agent hexafluoroisopropanol and basic pH (9.5). The method was fully validated - stability, matrix effects, accuracy, precision, linearity, limits of quantification and method’s uncertainty estimation was evaluated. |
Long Abstract
Introduction
Sepsis continues to cause significant morbidity in intensive care: up to 37% of adult and 30% of paediatric intensive care admissions are sepsis-related [1, 2]. Despite technical advances in critical care, the associated mortality also remains high, 29% in one recent meta-analysis of trends of mortality in adults [3]. Provision of prompt, targeted antimicrobial therapy has long been recognised as an essential component of the treatment of sepsis [4].
However, current antimicrobial dosing strategies in intensive care units (ICU) do not typically account for the physiological effects of critical illness on drug handling. Recent research has demonstrated that ICU antimicrobial dosing regimens may not provide optimal antimicrobial concentration profiles to maximise bacterial killing and, importantly, that suboptimal plasma antimicrobial concentrations may be associated with worse clinical outcomes [5]. This supports a growing body of evidence that the optimization of antibiotic dosing is a key factor in improving overall outcomes in this setting [6-10].
On average, each child in Europe receives one course of antibiotics per year, but there is considerable variation in the rate of antimicrobial prescribing in different countries and also the doses used. Even for very common antibiotics, there remains a marked lack of information about the optimal dosing in the context of critical illness.
Beta-lactam antimicrobials are the important group of antimicrobials widely used in children and adults for over 50 years. β-lactams are especially intolerant to the stress conditions and the degradation occur in different ways in different conditions.
Methods
Current clinical study in St George’s Hospital ICUs aims to do this across all age ranges (neonates (0-27 days), children (27 days to 16 years) and adults (over 16 years)). The broad range of antimicrobials is selected to study. All the antimicrobials are representative of the most commonly prescribed antimicrobial classes in hospitals in England [11]: co-amoxiclav (amoxicillin with clavulanic acid), piperacillin with tazobactam, flucloxacillin, meropenem, ertapenem, ceftriaxone, cefotaxime, benzylpenicillin, clarithromycin and fluconazole.
The UPLC-MS/MS method (using Waters Acquity UPLC coupled with TQ detector) was using protein precipitation with acetonitrile (Penicillin G-D7 as internal standard (IS)) for sample preparation. For the separation of compounds, 5 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in water with pH 9.5 (using NH4OH) and methanol with gradient elution was used in reversed phase (analytical column: 50mm x 2.1mm; 1.7 µm Acquity UPLC BEH C18). Multiple reaction monitoring (MRM) mode was used for detection of antimicrobials. With 3Q detector transitions m/z 335 [M+1] -> m/z 160; 176 (for penicillin G); m/z 366 [M+1] -> m/z 114; 208 (for amoxicillin); m/z 518 [M+1] -> m/z 143; 160 (for piperacillin); m/z 454 [M+1] -> m/z 160; 295 (for flucloxacillin), m/z 300 [M+1] -> m/z 42; 99 (for tazobactam), m/z 306 [M+1] -> m/z 220; 238 (for fluconazole), m/z 555 [M+1] -> m/z 125; 396 (for ceftriaxome), m/z 384 [M+1] -> m/z 114; 254 (for meropenem); m/z 456 [M+1] -> m/z 167; 324 (for cefotaxime); m/z 476 [M+1] -> m/z 233; 346 (for ertapenem); m/z 748 [M+1] -> m/z 158; 590 (for clarithromycin); m/z 199 [M+1] -> m/z 125; 172 (for clavulanic acid) and m/z 342 [M+1] -> m/z 160 (for penicillin G-D7, IS) were used for quantification and qualification.
The aim of development and validation the bioanalytical method for those antimicrobials in blood plasma was the later use of it for the measurement of ICU patients’ plasma samples, in order to use the data for the population PK modelling and dose optimization of the abovementioned drugs.
In the method validation step the stability of the analyte must be carefully examined. Through the validation of the bioanalytical method according EMA guideline [12].
Results
The separation between the analytes was obtained using reversed phase chromatography with weak ion-pairing agent HFIP in basic conditions (pH 9.5). The retention times ranged from 1.8 – 5.7 minutes for all the compounds. The method’s runtime was 8.5 minutes including post-time. The calibration graphs for all of the analytes with peak area versus concentration in the concentration range 0.05-200 μg/mL were linear with r2>0.9996. Matrix effects were evaluated for all the compounds and varied from 90-123%. Beta-lactams are infamous for their stability and rapid degradation. Degradation of beta-lactams occurred in the cooled (+10 ˚C) autosampler over the 24 h time-period, maintaining approximately 85-99% of their original content, depending on the compound. Stability of the clarithromycin and fluconazole in autosampler was greater ranging 97-99%. The freeze-thaw stability of beta-lactams indicated degradation in plasma samples, ranging 92-99% of the original content after 3 freeze-thaw cycles. Bench-top stability of the beta-lactams at the room temperature (23 ± 2˚C) for 24 h indicated the degradation for all of beta-lactams ranging 40-89% of the initial drug concentration.
The method was fully validated with accuracy, precision and method’s uncertainty estimation.
Conclusion
Method was applied for the clinical study samples to characterise the pharmacokinetic profiles of these compounds in intensive care patients and link it with clinical outcomes, in order to further understanding of the extent of interindividual pharmacokinetic variability within a critically ill patient population.
References & Acknowledgements:
References:
1. Vincent J-L, Sakr Y, Sprung CL, Ranieri VM, Reinhart K, Gerlach H, et al. Critical Care Medicine. 2006;34(2):344-53 10.1097/01.CCM.0000194725.48928.3A.
2. Pérez DV, Jordan I, Esteban E, García-Soler P, Murga V, Bonil V, et al. The Pediatric Infectious Disease Journal. 2014;33(2):152-7 10.1097/01.inf.0000435502.36996.72.
3. Stevenson EK, Rubenstein AR, Radin GT, Wiener RS, Walkey AJ. Critical Care Medicine. 2014;42(3):625-31 10.1097/CCM.0000000000000026.
4. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al. Intensive care medicine. 2013;39(2):165-228.
5. Roberts JA, Paul SK, Akova M, Bassetti M, De Waele JJ, Dimopoulos G, et al. Clinical Infectious Diseases. 2014:ciu027.
6. Roberts JA, Norris R, Paterson DL, Martin JH. Br J Clin Pharmacol. 2012;73(1):27-36.
7. van Lent-Evers NA, Mathot RA, Geus WP, van Hout BA, Vinks AA. Ther Drug Monit. 1999;21(1):63-73.
8. Moise-Broder PA, Forrest A, Birmingham MC, Schentag JJ. Clin Pharmacokinet. 2004;43(13):925-42.
9. Sime FB, Roberts MS, Peake SL, Lipman J, Roberts JA. Ann Intensive Care. 2012;2(1):35.
10. Goncalves-Pereira J, Paiva JA. J Crit Care. 2013;28(4):341-6.
11. Ashiru-Oredope D, Sharland M, Charani E, McNulty C, Cooke J. J Antimicrob Chemother. 2012;67 Suppl 1:i51-63.
12. European Medicines Agency. 2011. Guideline on bioanalytical method validation.
Acknowledgments
The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n° 608765. This work was supported by PUTJD 22 from Estonian Research Council and by Analytical Services International Ltd.
| Description | Y/N | Source |
| Grants | yes | EU's 7FP under REA grant agreement n° 608765 and PUTJD 22 grant from Estonian Research Council |
| Salary | yes | EU's 7FP under REA grant agreement n° 608765 and PUTJD 22 grant from Estonian Research Counci |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |