Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

A portable fluorescent microsphere-based lateral flow immunosensor for the simultaneous detection of colistin and bacitracin in milk.

The polypeptide antibiotics colistin (COL) and bacitracin (Baci) are extensively used as veterinary drugs and feedstock additives in the livestock industry, which inevitably causes residues in animal-origin food, which can accelerate human tolerance to antibiotics. In this study, a portable lateral flow immunoassay (LFIA) for the simultaneous determination of COL and Baci residues in milk was developed. The replacement of gold nanoparticles used in the traditional LFIA with fluorescent microspheres (FMs) to label monoclonal antibodies (mAbs) allowed qualitative and quantitative analyses within a few minutes. Based on the principle of competitive binding to FM-labelled mAbs between analytes in samples and fixed antigens on the membrane, the assay provided qualitative cut-off values of 100 and 50 ng mL-1 for Baci and COL in milk samples. Furthermore, a strip reader-based semi-quantitative detection system could detect lower limits of 7.85 and 1.89 ng mL-1 for Baci and COL, respectively. In conclusion, the proposed multiplex LFIA immunosensor provides an auxiliary analytical tool for the rapid and simultaneous screening of COL and Baci in large cohorts of samples.

A fluorescent artificial receptor with specific imprinted cavities to selectively detect colistin.

A novel and facile fluorescent artificial receptor on the basis of the molecularly imprinted polymer-coated graphene quantum dots was engineered successfully to detect colistin. The colistin imprinted graphene quantum dots (CMIP-GQDs) was synthesized by vinyl-based radical polymerization between functional monomers and crosslinker at around the template molecule on the surface of graphene quantum dots. The size of bare, CNIP-GQDs, and CMIP-GQDs was about 4.8 ± 0.6 nm, 18.4 ± 1.7 nm, and 19.7 ± 1.3 nm, respectively. The CMIP-GQDs, which showed the strong fluorescence emission at 440 nm with the excitation wavelength fixed at 380 nm, had excellent selectivity and specificity to rapidly recognize and detect colistin. The linear range of fluorescence quenching of this fluorescent artificial receptor for detection colistin was 0.016-2.0 µg mL-1 with a correlation coefficient (R2) of 0.99919, and the detection limit was 7.3 ng mL-1 in human serum samples. The designed receptor was successfully applied to detect colistin in human serum samples and it achieved excellent recoveries shifted from 93.8 to 105%. Graphical abstract.

Preliminary method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy.

Colistin use has increased in response to the advent of infections caused by multidrug-resistant organisms. It is administered parenterally as an inactive prodrug, colistin methanesulfonate (CMS). Various formulations of CMS and labeling conventions can lead to confusion about colistin dosing, and questions remain about the pharmacokinetics of CMS. Since CMS does not have strong UV absorbance, current methods employ a laborious process of chemical conversion to colistin followed by precolumn derivatization to detect formed colistin by high-performance liquid chromatography. Here, we report a method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR).

Simple in-house flow-injection capillary electrophoresis with capacitively coupled contactless conductivity method for the determination of colistin.

An in-house flow-injection capillary electrophoresis with capacitively coupled contactless conductivity detection method was developed for the direct measurement of colistin in pharmaceutical samples. The flow injection and capillary electrophoresis systems are connected by an acrylic interface. Capillary electrophoresis separation is achieved within 2 min using a background electrolyte solution of 5 mM 2-morpholinoethanesulfonic acid and 5 mM histidine (pH 6). The flow-injection section allows for convenient filling of the capillary and sample introduction without the use of a pressure/vacuum manifold. Capacitively coupled contactless conductivity detection is employed since colistin has no chromophore but is cationic at pH 6. Calibration curve is linear from 20 to 150 mg/L, with a correlation coefficient (r(2) ) of 0.997. The limit of quantitation is 20 mg/L. The developed method provides precision, simplicity, and short analysis time.

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan.

BACKGROUND: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clinical healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan. METHODS: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospitals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identified that had not been previously reported in patients from Japan. RESULTS: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As the ESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2. CONCLUSIONS: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary.

Whole-genome epidemiology and characterisation of mcr-1-encoding Escherichia coli in aquatic bird farms from the Pearl River Delta, China, 2019-2020.

Due to their unique breeding pattern, aquatic bird farms are increasingly considered as hotspots in the development and spread of antimicrobial resistance. However, comprehensive studies addressing the whole-genomic features of colistin-resistant bacteria in aquatic bird farms are scarce. Over a 2-year period, we conducted surveillance to determine the whole-genome epidemiology and characterisation of mcr-1-positive Escherichia coli in aquatic bird farms in southeastern coastal China. A total of 100 mcr-1-producing isolates among 654 E. coli strains were recovered from 781 samples collected in 11 aquatic bird farms and 1 veterinary clinic in the Pearl River Delta area. Higher resistance phenotypes to 17 antibiotics were found in mcr-1-positive isolates compared with other isolates. Subsequently, 20 mcr-1-carrying isolates were sequenced to analyse the whole-genomic features. Molecular typing as well as antimicrobial resistance gene and virulence factor profiles of the isolates showed considerable diversity. Three types of genetic backbones of mcr-1 in the isolates were assembled and were identified in diverse broad-host-range plasmids and bacterial species. Pangenome analyses revealed a large genetic pool composed of the isolates. Furthermore, phylogenetic trees both of the isolates in this study and a global data set were built, indicating the spread of the three mcr-1 backbones and the mcr-1-positive isolates among different habitats, farms and even countries. This study highlights that aquatic bird farms may act as an important reservoir for mcr-1-producing E. coli, from which colistin resistance may be spread to diverse habitats, different geographical locations and even across bacterial species.

Development of a liquid chromatography-tandem mass spectrometry method to determine colistin, bacitracin and virginiamycin M1 at cross-contamination levels in animal feed.

Cross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6-107.8% and precision ranged 12.6-21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on "real" samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin.

Evaluating the stability of colistin and colistin methanesulphonate in human plasma under different conditions of storage.

OBJECTIVES: The purpose of this study was to assess the stability of colistin and colistin methanesulphonate (CMS) in human plasma under storage conditions typically used in clinical pharmacokinetic (PK) and PK/pharmacodynamic (PD) investigations. METHODS: Human plasma (pH adjusted to 7.4) containing colistin (2 mg/L) or CMS (2 or 30 mg/L) was stored at -20, -70 or -80 degrees C for 6-12 months. At periodic intervals, the concentrations of colistin in colistin-spiked samples, and of CMS and formed colistin in CMS-spiked samples, were analysed (n = 3 replicates at each time) by HPLC. RESULTS: The time course of colistin concentrations in colistin-spiked plasma showed a substantially better stability at -80 and -70 degrees C than at -20 degrees C. With regard to CMS-spiked plasma of 2 and 30 mg/L stored at -80 and -70 degrees C, no quantifiable colistin formed over a 4 month period. However, the plasma spiked to 2 mg/L stored at -20 degrees C showed a substantial concentration of colistin ( approximately 0.4 mg/L) within 2 months. At all three storage temperatures, the stability of CMS was substantially better for the plasma spiked to contain 30 mg/L as compared with 2 mg/L. CONCLUSIONS: The results of our long-term stability study have significant implications for those involved in conducting clinical PK and PK/PD studies with CMS/colistin.

Evaluation of three ELISA kits for the screening of colistin residue in porcine and poultry muscle according to the European guideline for the validation of screening methods.

Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCß) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

Quantitative determination of colistin A/B and colistin methanesulfonate in biological samples using hydrophilic interaction chromatography tandem mass spectrometry.

Colistin (polymyxin E) is a polycation antibiotic which is increasingly used (administered as colistin methanesulfonate, CMS) as a salvage therapy in critically ill patients with multidrug resistant Gram-negative infections. Even though colistin has been used for more than 50 years, its metabolic fate is poorly understood. One of the current challenges for studying the pharmacokinetics (PK) is the precise and accurate determination of colistin in in vitro and in vivo studies. In the present study, we developed and validated a series of sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for analysing biological samples obtained from in vitro and in vivo disposition assays. After a zinc acetate-mediated precipitation, hydrophilic-lipophilic-balanced solid phase extraction (HLB-SPE) was used for the extraction of colistin. The compounds were retained on a hydrophilic interaction liquid chromatography (HILIC) column and were detected by MS/MS. CMS was quantified by determining the produced amount of colistin during acidic hydrolysis. The developed methods are sensitive with lower limits of quantification varying between 0.009 µg/mL and 0.071 µg/mL for colistin A, and 0.002 µg/mL to 0.013 µg/mL for colistin B. The intra- and inter-day precision and accuracy were within ±15%. Calibration curves of colistin were linear (0.063 µg/mL to 8.00 µg/mL) within clinically relevant concentration ranges. Zinc acetate-mediated precipitation and the use of a HILIC column were found to be essential. The developed methods are sensitive, accurate, precise, highly efficient and allow monitoring colistin and CMS in biological samples without the need for an internal standard.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

Emergence of mcr-1 conferred colistin resistance among bacterial isolates from urban sewage water in India.

Increased use of colistin, a last resort drug due to failure of carbapenems, has possibly contributed in development and spread of resistance to colistin among Enterobacteriaceae. The colistin belongs to the family of polymyxins, cationic polypeptides, with broad-spectrum activity against Gram-negative bacteria. In this study, we obtained 253 non-duplicate bacterial isolates from sewage water in Delhi and phenotypically screened for colistin resistance. Of the 47 positive isolates, the colistin resistance gene mcr-1 was detected among 5 isolates. Based on 16S ribosomal RNA-based identification, bacterial isolates were found to be Escherichia coli, Aeromonas veronii, and Aeromonas dhakensis. Extended spectrum ß-lactamases (ESBL)-resistant determinants CTX-M and TEM were detected in all five mcr-1 positive isolates. On the basis of literature survey, this is the first report of mcr-1 gene from Aeromonas veronii and Aeromonas dhakensis worldwide. Furthermore, mcr-1 gene has not been reported earlier from sewage water in India. Antibiotic susceptibility test of all five isolates against 9 different classes of drugs revealed multidrug-resistant phenotype with high minimum inhibitory concentration values. In vitro transconjugation studies showed successful transfer of mcr-1 and other ESBL-resistant determinants. The occurrence of colistin resistance phenotype conferred by plasmid-based mcr-1 gene in the environment and an ever-increasing list of bacterial isolates is a cause of concern. A comprehensive survey of different water bodies and epidemiological studies are required to assess the risk of dissemination of resistance determinants.

Association of colistin residues and manure treatment with the abundance of mcr-1 gene in swine feedlots.

BACKGROUND: The extensive use of colistin in swine production may have contributed to the recent emergence of corresponding mobile resistance gene mcr-1. The use of colistin as a feed additive was banned in China in April 2017. OBJECTIVES: To examine the occurrence of colistin and dissemination of mcr-1 in swine feedlots before and after the colistin ban and effects of different manure treatments. METHODS: Environmental samples were collected from swine feedlots before (December 2016) and after (December 2017) the colistin ban. Colistin concentrations were determined by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The prevalence of mcr-1 were determined by quantitative PCR analysis, while bacterial community composition was investigated by 16S rRNA sequencing. RESULTS: In 2016, colistin was detected in feed and fresh manure samples at 67 mg/kg and 17 mg/kg, respectively, but was absent from all samples in 2017. In 2016, the relative abundance of mcr-1 in fresh manure was lower than that in solid samples after natural drying, while a higher relative abundance was detected in fresh manure samples compared with biogas slurry samples. A strong correlation between colistin concentration and relative abundance of mcr-1 was observed in fresh manure. The samples collected in 2017 showed a lower relative abundance of mcr-1 compared with those collected in 2016. Bacterial community analysis showed that the abundance of Enterobacteriaceae, which act as a vehicle and reservoir of mcr-1, increased with natural dying but decreased with anaerobic digestion. CONCLUSIONS: The presence of colistin exerts direct selection pressure for the accumulation of mcr-1 in manure, while the ban on colistin likely halted the dissemination of mcr-1 on pig farms. Anaerobic digestion is an effective waste treatment process for removing mcr-1, which might be mainly driven by the shift in bacterial community structure.

Stability of colistin methanesulfonate in pharmaceutical products and solutions for administration to patients.

Colistin methanesulfonate (CMS) has the potential to hydrolyze in aqueous solution to liberate colistin, its microbiologically active and more toxic parent compound. While conversion of CMS to colistin in vivo is important for bactericidal activity, liberation of colistin during storage and/or use of pharmaceutical formulations may potentiate the toxicity of CMS. To date, there has been no information available regarding the stability of CMS in pharmaceutical preparations. Two commercial CMS formulations were investigated for stability with respect to colistin content, which was measured by a specific high-performance liquid chromatography method. Coly-Mycin M Parenteral (colistimethate lyophilized powder) was stable (<0.1% of CMS present as colistin) for at least 20 weeks at 4 degrees C and 25 degrees C at 60% relative humidity. When Coly-Mycin M was reconstituted with 2 ml of water to a CMS concentration of 200 mg/ml for injection, Coly-Mycin M was stable (<0.1% colistin formed) for at least 7 days at both 4 degrees C and 25 degrees C. When further diluted to 4 mg/ml in a glucose (5%) or saline (0.9%) infusion solution as directed, CMS hydrolyzed faster at 25 degrees C (<4% colistin formed after 48 h) than at 4 degrees C (0.3% colistin formed). The second formulation, CMS Solution for Inhalation (77.5 mg/ml), was stable at 4 degrees C and 25 degrees C for at least 12 months, as determined based on colistin content (<0.1%). This study demonstrated the concentration- and temperature-dependent hydrolysis of CMS. The information provided by this study has important implications for the formulation and clinical use of CMS products.

Determination of colistin in human plasma, urine and other biological samples using LC-MS/MS.

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.

Simultaneous determination of aminoglycosides and colistins in food.

A novel method for the simultaneous identification and quantification of twelve aminoglycosides (AGs) and two colistins in meat and bovine milk has been developed. The analysis was carried out using liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap). Among the HILIC (Hydrophilic Interaction Liquid Chromatography) stationary phases tested, the bare silica Poroshell 120 provided the best results. The samples were extracted with an aqueous solution followed by an SPE clean up based on the weak cation exchange mechanism. The validation study was performed carrying out 72 experiments per matrix at six different concentrations in a range encompassing the Maximum Residue Limits. The recoveries were from 72 to 87% in meat (except colistins) and from 82 to 96% in milk. Repeatabilities and intra-lab reproducibilities were lower than 10 and 15%, respectively. Limits of detection were lower than or equal to 33 µg kg-1. Finally, test materials containing AGs prepared for interlaboratory studies were successfully analysed.

Determination of colistin in animal tissues, egg, milk, and feed by ultra-high performance liquid chromatography-tandem mass spectrometry.

A confirmatory method for the determination of colistin in animal tissues, egg, milk, and feed was developed and validated. Colistin A and colistin B were extracted from samples with the mixture of 10% trichloroacetic acid-acetonitrile and isolated with mixed-mode weak cation exchange cartridge. Analytes were separated from matrix components using ultra-high performance liquid chromatography, and detected with electrospray ionization on a triple quadrupole mass spectrometer. Mean recoveries ranged from 78.0% to 115.6% with intra-day and inter-day relative standard deviation lower than 8.4% and 12.4%, respectively. The quantitation limits for different matrices were between 5 and 30 µg/kg, which was satisfactory for surveillance monitoring. The developed method was applied to the analysis of real samples collected from different provinces of China, and 19 out of 348 samples were found to be contaminated, with the highest concentration of approximately 12,000 µg/kg colistin A and 10,000 µg/kg colistin B in feed.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

Application of a validated method in the stability study of colistin sulfate and methylparaben in a veterinary suspension formulation by high-performance liquid chromatography with a diode array detector.

A methodology following International Cooperation on Harmonization for Veterinary Products (VICH) guidelines for the stability evaluation of colistin sulfate in a nonaqueous suspension pharmaceutical dosage form for veterinary use (via their drinking water) is described. This method monitors the percentage of colistin sulfate during the stability study of the preparation in drinking water and establishes the shelf life of the final product by a new high-performance liquid chromatography method which was developed and validated for the simultaneous determination of colistin sulfate [colistin A (Polymixin E1) and colistin B (Polymixin E2)] and methylparaben (Nipagin) using a diode array detector (DAD). The method uses a Kromasil C18 column and isocratic elution. The mobile phase consisted of an acetonitrile-sodium sulfate anhydrous solution (25 + 75) pumped at a flow rate of 1.5 mL/min. The DAD was set at 215 nm. The validation study was carried out according to the VICH guidelines in order to prove that the new analytical method meets the reliability characteristics, which include the fundamental criteria for validation: selectivity, linearity, precision, accuracy, and sensitivity. The method was applied during the quality control or stability studies of the suspension dosage form in order to quantify the drug (colistin) and preservative, and proved to be suitable for rapid and reliable quality control.