RESUMEN
In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core-shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box-Behnken experimental design, and the method was validated using the total error concept. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L-1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of -2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.
Asunto(s)
Colistina , Acetilcisteína , Cromatografía Líquida de Alta Presión/métodos , Colistina/orina , Humanos , o-FtalaldehídoRESUMEN
OBJECTIVES: Colistin, administered as the prodrug colistin methanesulphonate (CMS), is an antibiotic frequently administered as aerosol in cystic fibrosis (CF) patient. Our aim was to assess the plasma PK of colistin in CF patients treated with CMS administered intravenously or as aerosol and to compare these results with those previously reported in healthy volunteers. METHODS: Six CF patients were included, CMS and colistin concentrations were measured in plasma, urine and sputum. Either after single intravenous administration of 2 Million International Unit (MIU) or after repeated nebulizations of 2 MIU of CMS. PK of CMS and colistin were assessed by a mixed effect modeling approach. RESULTS: Renal clearance of CMS was lower in CF patients compared to that previously reported in healthy volunteers (64.3â¯mL/min (RSEâ¯=â¯15%) vs. 103â¯mL/min (RSEâ¯=â¯8%)). However, apparent clearance of colistin was higher in CF patients compared to healthy volunteers (124â¯mL/min (RSEâ¯=â¯13%) vs. 48.7â¯mL/min (RSEâ¯=â¯15%)), resulting in reduced systemic exposure to colistin (dose normalized AUC (2 MIU) of 7.4â¯h.mg/L/MIU vs. 11.2â¯h.mg/L/MIU). After repeated nebulizations, colistin concentrations were very low in plasma (<0.21â¯mg/L). CONCLUSIONS: Although our study suggests a lower median dose normalized colistin plasma concentrations in CF patients compared with healthy controls, this difference was not significant and a larger study is needed to substantiate this.
Asunto(s)
Administración por Inhalación , Administración Intravenosa , Colistina , Fibrosis Quística , Adulto , Antibacterianos/sangre , Antibacterianos/farmacocinética , Antibacterianos/orina , Colistina/sangre , Colistina/farmacocinética , Colistina/orina , Fibrosis Quística/sangre , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/fisiopatología , Fibrosis Quística/orina , Relación Dosis-Respuesta a Droga , Monitoreo de Drogas/métodos , Femenino , Humanos , Masculino , Mesilatos/farmacocinética , Tasa de Depuración Metabólica , Profármacos/farmacocinética , Eliminación Renal , Esputo/químicaRESUMEN
Limited information is available on the urinary excretion of colistin in infected patients. This study aimed to investigate the pharmacokinetics of colistimethate sodium (CMS) and formed colistin in urine in patients with multidrug-resistant (MDR) Gram-negative bacterial infections. A pharmacokinetic study was conducted on 12 patients diagnosed with an infection caused by an extremely drug-resistant (XDR) P. aeruginosa strain and treated with intravenous CMS. Fresh urine samples were collected at 2-h intervals, and blood samples were collected predose (Cmin ss) and at the end of the CMS infusion (Cmax ss) for measurement of concentrations of CMS and formed colistin using high-performance liquid chromatography (HPLC). CMS urinary recovery was determined as the summed amount of CMS and formed colistin recovered in urine for each 2-h interval divided by the CMS dose. There were 12 enrolled patients, 9 of whom were male (75%). Data [median (range)] were as follows: age, 65.5 (37 to 86) years; colistimethate urinary recovery 0 to 6 h, 42.6% (2.9% to 72.8%); range of concentrations of colistin in urine, <0.1 to 95.4 mg/liter; Cmin ss and Cmax ss of colistin in plasma, 0.9 (<0.2 to 1.4) and 0.9 (<0.2 to 1.4) mg/liter, respectively. In 6/12 (50%) patients, more than 40% of the CMS dose was recovered in the urine within the first 6 h after CMS administration. This study demonstrated rapid urinary excretion of CMS in patients within the first 6 h after intravenous administration. In all but one patient, the concentrations of formed colistin in urine were above the MIC for the most predominant isolate of P. aeruginosa in our hospital. Future studies are warranted for optimizing CMS dosage regimens in urinary tract infection (UTI) patients.
Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Antibacterianos/farmacocinética , Antibacterianos/orina , Colistina/análogos & derivados , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones Urinarias/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Cromatografía Líquida de Alta Presión , Colistina/farmacocinética , Colistina/uso terapéutico , Colistina/orina , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Infecciones Urinarias/microbiologíaRESUMEN
BACKGROUND: Colistin is a polypeptide antibiotic from the polymyxin E group used for the treatment of infections caused by multidrug-resistant gram-negative bacteria. The main constituents, accounting for approximately 85% of this mixture, are colistin A (polymyxin E1) and colistin B (polymyxin E2). The aim of this study was to develop and validate new and fast methods of quantification of colistin A and B and its precursors [colistin methanesulfonate sodium (CMS) A and B] by ultraperformance liquid chromatography-tandem mass spectrometry in plasma and urine with short pretreatment and run times. METHODS: Chromatography was performed on an Acquity UPLC-MS/MS system (WATERS) with a WATERS Acquity UPLC C18 column (4.6 × 150 mm, 3.5 µm particle size). The pretreatment of samples consists of precipitation and extraction into microcolumns plate and HLB 96-well plate 30 µm-30 mg (OASIS) with a Positive Pressure-96 (WATERS). RESULTS: Quantification was performed using a multiple reaction monitoring of the following transitions: m/z 390.9 â 385.1 for colistin A, m/z 386.2 â 101.0 for colistin B, and m/z 602.4 â 241.1 for polymyxin B1 sulfate. In plasma and urine, calibration curves were linear from 30 to 6000 ng/mL for colistin A and from 15 to 3000 ng/mL for colistin B. With an acceptable accuracy and precision, the lower limit of quantification were set at 24.0 ng/mL and 12.0 ng/mL for colistin A and B in plasma, and at 18.0 ng/mL and 9.0 ng/mL for colistin A and B in urine. CONCLUSIONS: These LC-MS/MS methods of quantification for colistin A and B and its precursors (CMS A and B) in plasma and urine are fast, simple, specific, sensitive, accurate, precise, and reliable. Furthermore, they are linear and repeatable. These procedures were successfully applied to a pharmacokinetic study of a critically ill patient suffering from ventilator-associated pneumonia, who was treated with nebulized CMS.
Asunto(s)
Colistina/análogos & derivados , Colistina/sangre , Colistina/orina , Antibacterianos/sangre , Antibacterianos/orina , Calibración , Cromatografía Líquida de Alta Presión/métodos , Monitoreo de Drogas/métodos , Humanos , Polimixinas/sangre , Polimixinas/orina , Espectrometría de Masas en Tándem/métodosRESUMEN
A rapid ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS and formed colistin in human plasma and urine. After extraction on a 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the eluents were mixed and injected into the UHPLC-MS/MS system directly. A Phonomenex Kinetex XB-C18 analytical column was employed with a mobile phase consisting of solution "A" (acetonitrile:methanol, 1:1, v/v) and solution "B" (0.1% formic acid in water, v/v). The flow rate was 0.4 mL/min with gradient elution over 3.5 min. Ions were detected in ESI positive ion mode and the precursor-product ion pairs were m/z 390.7/101.3 for colistin A, m/z 386.0/101.2 for colistin B, and m/z 402.3/101.2 for polymyxin B1 (IS), respectively. The lower limit of quantification (LLOQ) was 0.0130 and 0.0251 mg/L for colistin A and colistin B in both plasma and urine with accuracy (relative error, %) <± 12.6% and precision (relative standard deviation, %) <± 10.8%. Stability of CMS was demonstrated in biological samples before and during sample treatment, and in the extract. This new analytical method provides high-throughput treatment and optimized quantification of CMS and colistin, which offers a highly efficient tool for the analysis of a large number of clinical samples as well as routine therapeutic drug monitoring.
Asunto(s)
Colistina/análisis , Extracción en Fase Sólida/métodos , Resinas de Intercambio de Catión , Cromatografía Liquida , Colistina/sangre , Colistina/orina , Humanos , Límite de Detección , Espectrometría de Masas en TándemRESUMEN
Colistin is a polypeptide antibiotic that effectively treats infections caused by multidrug-resistant Gram-negative bacteria, but its clinical use is limited due to nephrotoxicity. The purpose of the present study was to identify biomarkers of colistin-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites using untargeted metabolomic approach. Rats receiving intraperitoneal administration of colistin sodium methanesulfonate (CMS) (25 or 50mg/kg) exhibited histopathological changes in the kidney and increased blood urea nitrogen levels. Additionally, the levels of phenylalanine, tryptophan, and tyrosine in the urine of the CMS-treated group were significantly higher than those of the control group, suggesting that colistin caused proximal tubular damage. Urinary acetylcarnitine and butyrylcarnitine levels also increased after CMS treatment, but the levels of purine metabolites and metabolites related to the tricarboxylic acid cycle were reduced. The most significant increase in the CMS-treated groups was observed in creatine levels. CMS-induced selective nephrotoxicity may be attributed to relatively high tissue concentrations of colistin in the kidney. Taken together, our results indicate that high levels of colistin in the kidney caused perturbations in the tricarboxylic acid cycle, amino acid metabolism, creatine metabolism, and purine metabolism and ultimately led to kidney injury.
Asunto(s)
Antibacterianos/orina , Colistina/análogos & derivados , Enfermedades Renales/orina , Metabolómica/métodos , Aminoácidos/orina , Animales , Antibacterianos/sangre , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Colistina/sangre , Colistina/orina , Creatinina/orina , Enfermedades Renales/patología , Masculino , Metabolómica/instrumentación , Análisis Multivariante , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Distribución TisularRESUMEN
A rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the routine quantification of colistins A and B and their prodrugs, colistin methanesulfonate (CMS) A and CMS B, respectively, in human plasma and urine by using polymyxin B1 as the internal standard (IS). CMS concentrations were determined indirectly by subtracting the colistin concentrations determined in biological samples from the whole colistin concentrations determined after sample treatment with sulfuric acid in order to hydrolyze CMS into colistin. After extraction on a solid-phase extraction column, the colistins were separated on an XBrigde C(18) column with isocratic elution (run time, 3.8 min). The mobile phase was 0.1% (vol/vol) formic acid in acetonitrile-0.1% (vol/vol) formic acid in water (20:80, vol/vol), run at a 0.2-ml/min flow rate. Ions were detected in the turbo-ion-spray-positive and multiple-reaction-monitoring modes. The ions monitored (precursor [M + 2H](2+) to product ions) were m/z 585.5/101.2 for colistin A, m/z 578.5/101.2 for colistin B, and m/z 602.5/241.2 for IS. Prevalidation studies demonstrated the stability of CMS in biological samples and extracts, a key point for the reliable quantification of colistin and CMS. The assay was accurate and reproducible for the quantification of colistins A and B and CMSs A and B in plasma samples over concentration ranges appropriate for pharmacokinetic studies: 0.024 to 6.144, 0.015 to 3.856, 0.029 to 7.492, and 0.010 to 2.508 microg/ml, respectively. In urine samples, the assay was validated over the same concentration ranges for colistins and over concentration ranges of 0.058 to 7.492 microg/ml and 0.020 to 2.508 microg/ml for CMSs A and B, respectively.
Asunto(s)
Antibacterianos/sangre , Antibacterianos/orina , Cromatografía Liquida/métodos , Colistina/sangre , Colistina/orina , Espectrometría de Masas en Tándem/métodos , Antibacterianos/farmacocinética , Calibración , Cromatografía Liquida/normas , Colistina/farmacocinética , Monitoreo de Drogas/instrumentación , Monitoreo de Drogas/métodos , Monitoreo de Drogas/normas , Humanos , Valores de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/normasRESUMEN
A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to quantify colistin in human plasma and urine, and perfusate and urine from the isolated perfused rat kidney (IPK). Solid phase extraction (SPE) preceded chromatography on a Synergi Fusion-RP column with a mobile phase of acetonitrile, water and acetic acid (80/19/1) at 0.2mL/min. Ions were generated using electrospray ionization and detected in the positive-ion mode. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 0.028microg/mL (human plasma, IPK perfusate and urine)/0.056microg/mL (human urine) to 1.78microg/mL (all four media) for colistin A sulfate; corresponding values for colistin B sulfate were 0.016/0.032 to 1.01microg/mL. Accuracy and precision were within 10%. The LLOQ for colistin A sulfate was 0.028microg/mL in human plasma, IPK perfusate and urine and 0.056microg/mL in human urine; corresponding values for colistin B sulfate were 0.016 and 0.032microg/mL. The low sample volume, short analysis time and low LLOQ are ideal for pre-clinical and human pharmacokinetic studies of colistin.
Asunto(s)
Colistina/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Colistina/sangre , Colistina/orina , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.
Asunto(s)
Bioensayo/métodos , Colistina/sangre , Colistina/orina , Medios de Cultivo , Fibrosis Quística , Estabilidad de Medicamentos , Escherichia coli/efectos de los fármacos , Humanos , Sensibilidad y Especificidad , TemperaturaRESUMEN
A simple and sensitive high-performance liquid chromatographic method is described for the determination of colistimethate sodium in plasma and urine. The accuracy and reproducibility was within 10.1 and 11.2% with rat plasma and urine, respectively. Several commonly coadministered antibacterial agents do not interfere with the assay.
Asunto(s)
Colistina/análogos & derivados , Colistina/sangre , Colistina/orina , Animales , Cromatografía Líquida de Alta Presión/métodos , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Inhalation therapy of colistin is widespread in patients with cystic fibrosis. To date, no pharmacokinetic data of colistin after inhalation are available. To optimize the inhalation therapy, pharmacokinetic data of colistin are necessary. In this study, the authors describe a chromatographic analysis for measurement of colistin concentrations in serum. After protein precipitation, the colistin sample is treated with orthophthalaldehyde for derivatization. The sum of the peak areas of the two main components of colistin (polymyxin E1 and E2) were used for quantitation. The performance of the analytical method was assessed by determining the lower limit of quantitation, the selectivity of the method, the intra-assay variation, the reproducibility, the interassay variation, and the accuracy. The lower limit of quantitation was 28 microg/L. Ceftazidime, aztreonam, piperacilline, or tobramycin showed no interference with the colistin assay. In a pilot study, the authors found a trough value of approximately 10 microg/L and peak values of approximately 100 microg/L after inhalation of 160 mg colistin in serum samples of a representative patient. These values show that the method can be used to design further experiments. The applicability of the method was also tested on urine and sputum samples. Colistin was detectable but further validation experiments are required to confirm the usefulness of the method in these biologic matrices. To the authors' knowledge this is the first study in which serum concentrations are described after inhalation of colistin in patients with cystic fibrosis.