Development and validation of an LC tandem MS assay for the quantification of β-lactam antibiotics in the sputum of cystic fibrosis patients

Forier, Katrien; Heck, Virginie Van; Carlier, Mieke; Braeckel, Eva Van; Daele, S. Van; Baets, Frans De; Schelstraete, Petra; Haerynck, Filomeen; Stove, Veronique; Simaey, Leen Van; Vaneechoutte, Mario; Verstraete, Alain G.

doi:10.1093/jac/dkx331

Cited by 3 publications

(4 citation statements)

References 24 publications

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“…Based on the FDA recommended criteria [ 20 ] for stability as defined by compound loss not exceeding 15%, our stability study has shown acceptable values for any of the tested antibiotic combinations with fosfomycin for up to 15 h at 4 °C, apart from piperacillin (MQC-piperacillin failed FDA criteria at the 7-h time point). Our findings concurred with Forier et al, who reported limited piperacillin stability in sputum on ice [ 27 ]. Long-term storage of plasma samples was demonstrated for up to 4 weeks either at −30 °C or −80 °C; CAMHB samples can be stored at −80 °C for up to 4 weeks.…”

Section: Discussionsupporting

confidence: 93%

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

et al. 2022

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Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller–Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at −80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications.

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Section: Discussionsupporting

confidence: 93%

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

et al. 2022

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“…Run conditions, including comparison of mobile phases (see Text S1 and Table S2 in the supplemental material), were first optimized on the Waters Quattro Premier XE UPLC-MS/MS at the University of California, Irvine Mass Spectrometry Facility. A mobile phase consisting of a water-methanol gradient with 2 mM ammonium acetate and 0.1% acetic acid (57) chromatographically separated 16 of 18 antibiotics with peak areas at least 100 times higher than background (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Pharmaceutical grade or high-performance liquid chromatography (HPLC) grade antibiotics were dissolved in water, methanol, or an acetonitrile/acetic acid solution to make 1 mM or 10 mM stocks (see Table S1 in the supplemental material). Stocks of the external standards were made directly before each run due to reported low stability of some antibiotics at Ϫ20°C and Ϫ80°C (57,65). The antibiotics were then diluted with water and pooled to make a 10 M stock.…”

Section: Methodsmentioning

confidence: 99%

Liquid Chromatography Mass Spectrometry Detection of Antibiotic Agents in Sputum from Persons with Cystic Fibrosis

Gallagher

Riedel

Kapcia

et al. 2021

Antimicrob Agents Chemother

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Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at sub-nanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.

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“…20 There are several methods described to homogenize and liquefy sputum, either by chemical or mechanical methods. [21][22][23][24][25] In this study, sonication, a mechanical method, was chosen without the addition of chemicals to prevent possible interactions with the components to be measured.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Method for the Analysis of Ivacaftor, Hydroxymethyl Ivacaftor, Ivacaftor Carboxylate, Lumacaftor, and Tezacaftor in Plasma and Sputum Using Liquid Chromatography With Tandem Mass Spectrometry and Its Clinical Applicability

Vonk¹,

Meer-Vos²,

Bos

et al. 2021

Therapeutic Drug Monitoring

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Background: The novel cystic fibrosis transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, and tezacaftor, are the first drugs directly targeting the underlying pathophysiological mechanism in cystic fibrosis (CF); however, independent studies describing their pharmacokinetics are lacking. The aim of this study was to develop a quantification method for ivacaftor and its 2 main metabolites, lumacaftor and tezacaftor, in plasma and sputum using liquid chromatography with tandem mass spectrometry. Methods: The developed method used a small sample volume (20 µL) and simple pretreatment method; protein precipitation solution and internal standard were added in one step to each sample. Liquid chromatography with tandem mass spectrometry was performed for a total run time of 6 minutes. The method was validated by assessing selectivity, carryover, linearity, accuracy and precision, dilution, matrix effects, and stability. Results: The selectivity was good as no interference from matrices was observed. In the concentration range from 0.01 to 10.0 mg/L, calibration curves were linear with a correlation coefficient >0.9997 for all compounds. The within-run and between-run accuracy were between 99.7% and 116% at the lower limit of quantitation (LLOQ) and between 95.8% and 112.9% for all concentrations above LLOQ for all analytes in plasma and sputum. Within-run and between-run precisions were <12.7% for LLOQ and <6.7% for the higher limit of quantitation. Samples were stable, with no significant degradation at examined temperatures and time points. Clinical applicability was revealed by analyzing samples from 2 patients with CF. Conclusions: The presented method enables simultaneous quantification of ivacaftor, lumacaftor, and tezacaftor in plasma and sputum and is an improvement over previous methods because it uses smaller sample volumes, a simple pretreatment protocol, and includes tezacaftor. In future studies, it can be applied for examining pharmacokinetics characteristics of new CF transmembrane conductance regulator modulators.

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Development and validation of an LC tandem MS assay for the quantification of β-lactam antibiotics in the sputum of cystic fibrosis patients

Cited by 3 publications

References 24 publications

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

Liquid Chromatography Mass Spectrometry Detection of Antibiotic Agents in Sputum from Persons with Cystic Fibrosis

Quantitative Method for the Analysis of Ivacaftor, Hydroxymethyl Ivacaftor, Ivacaftor Carboxylate, Lumacaftor, and Tezacaftor in Plasma and Sputum Using Liquid Chromatography With Tandem Mass Spectrometry and Its Clinical Applicability

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