Department of Pharmacy
Library of the School of Science

2021-04-23

2021

Dagla Ioanna Valentini

Eυάγγελος Γκίκας, Καθηγητής, Τμήμα Χημείας, ΕΚΠΑ
Αντώνιος Τσαρμπόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Εμμανουήλ Μικρός, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Νικόλας Θωμαΐδης, Καθηγητής, Τμήμα Χημείας, ΕΚΠΑ
Γεώργιος Θεοδωρίδης, Καθηγητής, Τμήμα Χημείας, ΑΠΘ
Ειρήνη Παντερή, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Ιωάννης Ντότσικας, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ

Evaluation of novel methodologies for the analysis of colistimethate sodium against Gram-negative bacteria

English

Evaluation of novel methodologies for the analysis of colistimethate sodium against Gram-negative bacteria

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. colistinus. Colistin was first isolated in 1949, but its use was restricted during 1980s due to neurotoxicity and nephrotoxicity. However, colistin has been re-emerged in clinical practice as a last resort antibiotic to treat life-threatening infections caused by multidrug resistant gram-negative bacteria. Colistin is administrated parenterally as colistimethate sodium (CMS) which is the inactive and less toxic prodrug of colistin that hydrolyzes in vivo in the active drug. CMS is produced by sulfomethylation of the free amine groups of colistin, but the actual structure of CMS still remains unclear, as there is conflict about the degree of sulfomethylation. However, it is stated that CMS is a very complex mixture consisting of partially sulfomethylated components. Colistin, having been discovered several years ago, has not been subjected to the drug development and regulatory approval processes that are applied today. Until now, the quality control procedure of the CMS commercial products is based on microbiological assays.
The important point here is that although the above-mentioned products are standardized by microbiological assays in vitro, the exposure to active colistin is different in vivo, emphasizing the need for the development of other methods in order to characterize and consequently standardize the content of CMS prodrugs which will eventually lead to precise control of the drug’s bioavailability.
The aim of the study is the development of an analytical methodology based on ultra-high performance liquid chromatography (UPLC) combined with mass spectrometry (MS) on a hybrid quadrupole time-of-flight (QqTOF) instrument that could be used for the quality control of CMS products. The careful optimization of the UPLC and QqTOF parameters was deemed crucial, as CMS is known to be a very complex mixture. Thus, a two stage Design of Experiments (DoE) pipeline was followed, aiming towards the separation of the mixture components. According to the DoE results, a baseline-resolved chromatogram revealing more than 20 compounds was achieved. The separation was performed using a Waters Acquity BEH C8 column employing gradient elution. The mobile phase consisted of aq. ammonium formate 0.005 M (pH 6) (solvent A) and methanol/acetonitrile 79/21 (v/v) (solvent B). A second optimization experiment for the MS signal was employed in order to achieve maximum sensitivity. The singly charged signals were monitored for the validation in the positive ion mode. The calibration curve range was 50 – 110 μg mL-1, corresponding to the 80-120% of the nominal CMS amount in the commercial products. Due to the complexity of the CMS chromatograms and the corresponding spectrum of each chromatographic peak, untargeted and targeted approaches were performed employing the MZmine software. Furthermore, apart from the classical univariate statistical analysis, a partial least squares regression (PLSR) model was also employed, as the variables were more than the observations. The developed methodology has been employed to analyze several batches and inconsistencies have been discovered.
Furthermore, a UPLC coupled to ultraviolet (UV) detector methodology was developed for the quantitation of CMS in injectable formulations. Care was taken in order to employ freely-available software in order to be adopted from pharmaceutical companies and even the research community as an easy-to-implement quality control procedure. Design of experiments was performed for the optimization of the chromatographic parameters. The chromatographic separation was achieved using a Waters Acquity BEH C8 column employing gradient elution with a mobile phase consisting of 0.001 M aq. ammonium formate (solvent A) and methanol/acetonitrile 79/21 (v/v) (solvent B). CMS compounds were detected at 214 nm. Twenty-three univariate linear regression models were constructed for the measurement of CMS compounds separately and one PLSR model for the assessment of the total CMS amount in formulations. The method was validated over the range 100 – 220 μg mL-1. The developed methodology was employed to analyze several batches of CMS injectable formulations that were also compared against a reference batch employing principal component analysis (PCA), similarity and distance measures, heatmaps and the structural similarity index.
The measurement of CMS levels in patients’ biological fluids is of great importance in order to find the optimal dose regimen reducing the drug toxicity. Until now, CMS assay methods are based on the indirect determination after its hydrolysis to colistin. Herein, the aim is to find the optimal conditions for the complete hydrolysis of CMS to colistin. The reaction was studied at accelerated conditions: 40 °C, 50 °C and 60 °C and the results were evaluated by assessing the Arrhenius equation and computation employing the Tenua software. A validated analytical methodology based on UPLC coupled to a QqTOF instrument was developed for the simultaneous measurement of CMS and colistin. The current methodology resulted in complete hydrolysis in contrast with the previous reported one.
Although the use of CMS is increasing rapidly worldwide, the structure of CMS has not been fully determined. Herein, efforts were made in order to shed light to the structure of CMS. Experiments were performed employing mass spectrometry-based approaches. Four types of mass analyzers were used: a) QqTOF, b) LTQ Orbitrap Discovery XL, c) Q-Exactive Orbitrap and d) Οmnitrap. Infusions of CMS were performed under different experimental conditions testing the dilution solvent, the pH and the desolvation temperature. The Kendrick mass effect was also tested. MS/MS experiments were also performed in QqTOF and Omnitrap. Cyclobrach tool was used for the interpretation of MS/MS spectra and the structures of three CMS components were presented.

Science

analysis, colistimethate sodium, mass spectrometry, quality control, Arrhenius, colistin, PLSR

Yes

10

Yes

89

257

File access is restricted until 2025-03-03.

https://pergamos.lib.uoa.gr/uoa/dl/object/2943754