Barbara Żuromska-Witek

Separation and characterization of ciprofloxacin, difloxacin, lomefloxacin, norfloxacin, and ofloxacin oxidation products under potassium permanganate treatment in acidic medium by UPLC-MS/MS.

A simple, sensitive and reproducible ultra-performance liquid chromatography method for determination of ciprofloxacin, difloxacin, lomefloxacin, norfloxacin and ofloxacin oxidation stability under permanganate treatment in acidic conditions at pH from 3.0 to 6.0, was developed. Chromatographic separations were carried out using the Acquity UPLC BEH C18 column; (2.1×100 mm, 1.7 μm particle size). The column was maintained at 40°C, and eluted under isocratic conditions using 83% of eluent A and 17% of eluent B over 6.5 min, at a flow rate of 0.3 mL min(-1). Eluent A: water/formic acid (0.1 v/v%); eluent B: acetonitrile/formic acid (0.1 v/v%). An oxidation process followed kinetic of the second order reaction and depended upon solution acidity. Oxidation of fluoroquinolones proceeded at piperazine moiety yielding respective hydroxy and oxo analogs, and remaining the quinolone fragment intact. Structures of products formed were assigned on a basis of UPLC/MS/MS fragmentation pathways.

THIN-LAYER CHROMATOGRAPHY WITH DENSITOMETRY FOR THE DETERMINATION OF DIFLOXACIN AND ITS PHOTODEGRADATION PRODUCTS. KINETIC EVALUATION OF THE DEGRADATION PROCESS AND IDENTIFICATION OF PHOTOPRODUCTS BY MASS SPECTROMETRY

TLC-densitometric method was developed for determination of difloxacin (DIF) in the presence its photodegradation products. Silica gel TLC F254 plates were used as the stationary phase and methylene chloride:methanol:2-propanol:ammonia 25% (4:4:5:2, v/v/v/v) as the mobile phase. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The photodegradation process of DIF followed kinetics of the first order reaction for the substrate. Potential photodegradation products of DIF identified by UPLC-MS/MS are: 7-(2-aminoethylamino)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 7-amino-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 6-fluoro-1-(4-fuorophenyl)-7-(3-hydroxypiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 6-fluoro-1-(4-fuorophenyl)-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid.

Determination of sparfloxacin and its photodegradation products by thin-layer chromatography with densitometry detection. Kinetic evaluation of the degradation process and identification of photoproduct by mass spectrometry

A TLC-densitometric method was developed for the determination of sparfloxacin in the presence of its photodegradation products. Silica gel TLC F254 plates were used as the stationary phase and methylene chloride : methanol : 2-propanol : ammonia 25% (4 : 4 : 5 : 2 v/v/v/v) as the mobile phase. The developed method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The linear regression analysis for the calibration curve showed a good linear correlation over the concentration range 1.00–3.00 μg per band, with the determination coefficient, R2, exceeding 0.9958. The method was shown to have good precision and intermediate precision, as reflected by the relative standard deviation values, lower than 2.72% and characterized by a recovery rate at three concentration levels from 93.60% to 103.90%. The limits of detection and quantification were respectively 0.16 and 0.48 μg per band. The photodegradation process of sparfloxacin followed the kinetics of a first order reaction for the substrate. Nine products of photodegradation were identified by UPLC-MS/MS. The structure of a product previously not identified was determined.

Lipophilicity study of fifteen fluoroquinolones by reversed-phase thin-layer chromatography

The lipophilicity of fifteen fluoroquinolones has been investigated. Reversed-phase thin layer chromatography with densitometric detection was applied to determine the RM0 factor. The RP-TLC investigations were performed in mixtures of organic modifier–water. The partition coefficients of examined fluoroquinolones were also calculated with computational programs. All the obtained data, from experimental methods and theoretical calculations, were compared and a suitable conclusion was reached. The received results demonstrate that the RP-TLC method may be the only reliable technique for fluoroquinolones in describing their lipophilic nature as well as the activity.

Determination of danofloxacin and its photodegradation products by HPLC-DAD. Kinetic evaluation of the degradation process and identification of photoproducts by mass spectrometry

A HPLC method was developed for determination of danofloxacin (DAN) in the presence of its photodegradation products. Chromatography was performed on a Gemini-NX C18 110A, 150 mm × 4.60 mm, 3 μm particle size column with 0.025 M phosphate buffer (pH = 5.00)–acetonitrile–methanol (95 : 10 : 30 v/v/v) as the mobile phase at a flow rate of 1.2 mL min−1. UV detection was performed at 280 nm. The column was thermostatted at 25 °C. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The linear regression analysis for the calibration curve showed a good linear correlation over the concentration range 0.20–0.80 mg mL−1, with determination coefficient, R2, exceeding 0.9966. The method was shown to have good and intermediate precision, as reflected by the relative standard deviation values, lower than 2.21% and characterized by a recovery rate at three concentration levels from 98.0% to 101.70%. The limits of detection and quantification were respectively 0.0055 and 0.0167 mg mL−1. The photodegradation process of DAN followed kinetics of the first order reaction for the substrate. Ten products of photodegradation were identified by UPLC/MS/MS.

Validation of the method of identification and quantitative determination of bacitracin in the form of bacitracin derivative with dabsyl chloride by thin-layer chromatography and densitometry

A thin-layer chromatographic (TLC)-densitometric method has been developed for identification and quantitative determination of bacitracin derivative with dabsyl chloride. The analysis of antibiotic was achieved on the silica gel TLC plates with fluorescent indicator with n-butanol-2-butanone-25% ammonia-water (10:5:2:2, v/v) as mobile phase. The densitometric measurements were made at 460 nm. Under these conditions, good separation of chosen polypeptide antibiotic from reagent used to make a complex was obtained. The method distinguishes of high sensitivity, LOD from 8.6 ng per band and LOQ from 26.2 ng per band, wide linearity range from 0.0854 to 0.8536 µg per band for bacitracin. The precision of the method was good; relative standard deviation (RSD) varied from 1.23% to 2.47%. Satisfactory results of validation of the method were also confirmed by determination of selected antibiotic in pharmaceutical commercial preparations. The results obtained by TLC-densitometric method were compared with those obtained by spectrophotometric method.

DETERMINATION OF DANOFLOXACIN AND ITS PHOTODEGRADATION PRODUCTS BY THIN-LAYER CHROMATOGRAPHY: KINETIC EVALUATION OF DEGRADATION PROCESS

TLC-densitometric method was developed for determination of danofloxacin (DAN) in presence of its photodegradation products. Silica gel HPTLC Lichrospher 60 F254 plates were used as the stationary phase and methanol:acetone:1 M citric acid:triethylamine (2.8:2:0.2:0.5, v/v/v/v) as the mobile phase. The elaborated method meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. The linear regression analysis for the calibration curve showed a good linear correlation over the concentration range 1.25– 3.75 µg per band, with determination coefficient, exceeding 0.9806. Accuracy of the method expressed as % recovery at three concentration levels was from 95.56% to 97.30%. Good precision and intermediate precision with % RSD less than 0.92% was also observed. The limits of detection and quantification were respectively 1.10 and 3.40 ng per band. Satisfactory results of validation of the method were also confirmed by determination of DAN in veterinary commercial preparation. The photodegradation process of DAN followed kinetics of the first order reaction for the substrate.

Quantification of saponins in different plant parts of Lysimachia L. Species by validated HPTLC-densitometric method

Quantitative high-performance thin-layer chromatography (HPTLC) analysis of triterpene saponins in different parts of eight Lysimachia L. species is described. For separation, silica gel F254 plates were used with chloroform-methanol-water (8:7:1, v/v) as mobile phase 1 and with n-butanol-acetic acid-water (6:1:3, v/v) as mobile phase 2. The method was validated for selectivity, sensitivity, accuracy, precision, and indirect precision. Saponins were most abundant in the underground parts of all analyzed species, where their content ranged from 0.12% in Lysimachia clethroides to 1.18% in Lysimachia ephemerum. The saponin levels in aerial parts of the plant were generally lower, the only exception being L. ephemerum leaf (1.57%) and Lysimachia thyrsiflora stem (2.26%). The method is sensitive with good precision and accuracy and may be suitable for comparison of the differences in saponin content among samples from various related species and genera.

TLC-DENSITOMETRIC DETERMINATION OF TOLPERISONE AND ITS IMPURITIES 4-METHYLPROPIOPHENONE AND PIPERIDINE IN PHARMACEUTICAL PREPARATIONS

A chromatographic-densitometric method was developed for determination of tolperisone hydrochloride and its impurities. Silica gel TLCF 254 plates were used as the stationery phase and cyclohexane-1,4 dioxane-isopropanol–ethanol-glacial acetic acid (16:0.5:1:4:0.6 v/v/v) and n-butanol-isopropanol-water-glacial acetic acid (10:7:8:2 v/v/v/v) as the mobile phases. Densitometric measurements were done at 260 nm for tolperisone hydrochloride and 4-methylpropiophenone and at 570 nm for piperidine hydrochloride. The method is specific, sensitive, precise, and has a broad range of linearity, with potential application in pharmaceutical analysis.

Chromatographic-Densitometric Analysis of Chosen Fluoroquinolones on TLC Plates Using Mobile Phases with Different Viscosity

TLC-densitometric method was developed for the identification and quantitative determination of selected fluoroquinolones. Silica gel TLC 60 F254 plates were used as the stationary phase. For the separation of examined compounds the suitability of the following four mobile phases with different dynamic viscosity was checked: (I) methanol:ammonia:ethyl acetate:acetonitrile 1:1:2:1 v/v/v/v (1.70 mPa · s), (II) dichloromethane:methanol:isopropanol:ammonia 4:4:5:2 v/v/v/v (2.08 mPa · s), (III) dichloromethane:ethanol:toluene:ammonia:n-butanol:water 6:6:2:1.8:3:0.3 v/v/v/v/v/v (2.27 mPa · s), (IV) 1,4- dioxane:ammonia:tetrahydrofuran 6:3:2 v/v/v (3.24 mPa · s). The calculated values of retardation factors RF, capacity factors k, selectivity factors α, resolution factors Rs, and also asymmetry factors As for the evaluation of separation conditions were used. The obtained results confirmed that dynamic viscosity of the mobile phases is essential and influences the retention parameters of the studied fluoroquinolone chemotherapeutics. The validation results for the mobile phase IV indicate its suitability for the identification and also quantitative analysis of selected drugs in pharmaceutical analysis.