Direct determination of kanamycin in raw materials, veterinary formulation and culture media using a novel liquid chromatography-evaporative light scattering method

A novel method for the direct determination of kanamycin A and its minor component kanamycin B was developed and validated based on reversed phase liquid chromatography with evaporative light scattering detector (ELSD). ELSD response to kanamycins was found to be enhanced by: (a) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity or ratio of organic solvent to water), (b) use of ion-pairing acidic reagents of increased molecular mass, and (c) increase of mobile phase volatility. Utilizing an Spherisorb ODS-2 C₁₈ column, the selected optimized mobile phase was water-acetonitrile (60:40, v/v), containing 11.6 mM heptafluorobutyric acid (isocratic elution with flow rate of 1.0 ml min⁻¹). Kanamycin A was eluted at 3.9 min and kanamycin B at 5.0 min with a resolution of 2.7. Logarithmic calibration curves were obtained from 0.6 to 28 μg ml⁻¹ (r > 0.9998) for kanamycin A and 4-36 μg ml⁻¹ (r > 0.9994) for kanamycin B, with a LOD equal to 0.20 and 1.4 μg ml⁻¹, respectively.In kanamycin acid sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.1 min, LOD = 2.3 μg ml⁻¹, %R.S.D. = 1.7, r > 0.9998) and kanamycins was feasible. No significant difference (t-test) was found between the results of the developed LC-ELSD method and those of reference methods, while recovery from kanamycin B spiked samples ranged from 95 to 105%. The developed method was also applied with very good accuracy for the determination of kanamycin A in veterinary formulation (%recovery 95-103, %R.S.D. < 1.4, n = 3) and for the determination of kanamycins A and B in bacteria culture media (%recovery 102 and 99, respectively).     

Development of a validated HPLC method for the determination of four penicillin antibiotics in pharmaceuticals and human biological fluids

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.     

Development of an HPLC method for the determination of mexiletine in human plasma and urine by solid-phase extraction

A sensitive and specific high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of mexiletine (MEX) in human plasma and urine. It uses solid-phase extraction (SPE) followed by an automated reversed-phase HPLC with a pre-column derivatization with 4-chloro-7-nitrobenzofurazan (NBD-CI) and UV-vis Absorbance detection. The process was set as: the UV-vis Absorbance wavelength was set at 458 nm. Chromatographic separation was performed on a Phenomenex-C₁₈ Column (Aqua, 150 mm × 4.6 mm i.d. with 5 μm particle size) with the mobile phase consisting of acetonitrile and water (80:20, v/v), and the flow rate was set at 1.0 mL min⁻¹. Calibration of the overall analytical procedure gave a linear signal (r > 0.9998) over a MEX concentration range of 0.2-2.0 μg mL⁻¹ in human plasma and urine. The detection limit in plasma and urine was 0.1 μg mL⁻¹. Intra- and inter-day precision of the assay at three concentrations within this range were 0.31-2.50%. The high specificity and sensitivity have been achieved by this fast method (total run-time <6 min). The method has been successfully validated in human plasma and urine and it has been shown to be precise, accurate and reliable.     

Separation of fifteen quinolones by high performance liquid chromatography: application to pharmaceuticals and ofloxacin determination in urine

A simple chromatographic method is described for assaying 15 quinolones and fluoroquinolones (pipemidic acid, marbofloxacin, enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid, flumequine and piromidic acid), in urine and pharmaceutical samples. The determination was achieved by LC using an RP C18 analytical column. A mobile phase composed of mixtures of methanol-ACN-10 mM citrate buffer at pH 3.5 and 10 mM citrate buffer at pH 4.5, delivered under an optimum gradient program, at a flow rate of 1.5 mL/min, allows to accomplish the chromatographic separation in 26 min. For detection, diode-array UV-Vis at 280 nm and fluorescence detection set at excitation wavelength/emission wavelength: 280/450, 280/ 495, 280/405 and 320/360 nm were used. Detection and quantification limits were between 0.3-18 and 0.8-61 ng/mL, respectively. The method was validated in terms of interday (n = 6) and intraday (n = 6) precision and accuracy. The procedure was successfully applied to the analysis of human and veterinary pharmaceuticals. Also, ofloxacin was determined in human urine samples belonging to a patient undergoing treatment with this active principle, among others.     

HPLC determination of tobramycin in a simulated body fluid

To evaluate the release kinetics, in a simulated body fluid, of tobramycin (TOB) from carbonated apatite (CHA) in the form of granulate, a sensitive and reliable liquid chromatographic method was developed. Chromatographic separation was performed on a Metrosep Carb 1 anionic column by using 0.1 M NaOH as mobile phase at a flow rate of 1.0 ml min^− 1. TOB was quantified by pulsed amperometric detection using a 3-potential waveform on Au electrode. A linear relationship for TOB was found in the range 0.17-2.50 mg l^− 1 with a LOD of 0.05 mg l^− 1. The method exhibited good retention time and peak area reproducibility.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.     

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C₈ column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C₁₈ column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients (R²) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.     

Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation

A novel simple isocratic HPLC method with UV detection for the determination of three compounds in spray solution (active component clotrimazole and two degradation products imidazole and (2-chlorophenyl)diphenylmethanol) using ibuprofen as an internal standard was developed and validated. The complications with different acido-basic properties of the analysed compounds in HPLC separation - while clotrimazole has pK_a 4.7, imidazole has pK_a 6.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol - were finally overcome using a 3.5 μm Zorbax^® SB-Phenyl column (75 mm × 4.6 mm i.d., Agilent Technologies).The optimal mobile phase for separation of clotrimazole, degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35, v/v) with pH* conditioned by phosphoric acid to 3.5. At a flow rate of 0.5 ml min⁻¹ and detection at 210 nm, the total time of analysis was less than 6 min.The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography with tandem mass spectrometry method for the quantification of vitisin B in rat plasma and urine

A new, rapid, and sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of vitisin B and validated in rat plasma and urine using carbamazepine as an internal standard. The plasma (0.05 mL) or urine (0.2 mL) samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Eclipse Plus C₁₈ column (100 × 4.6 mm, 3.5 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid water (60:40, v/v) at a flow rate of 0.7 mL/min. Detection and quantification were performed by mass spectrometry in selected reaction‐monitoring mode with positive electrospray ionization. The calibration curves were recovered over the concentration ranges of 10?5000 ng/mL (correlation coefficients, r≥0.9833) in plasma and 5?2500 ng/mL (r≥0.9977) in urine, respectively. All validation data, including the specificity, precision, accuracy, recovery, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of vitisin B following intravenous administration of 0.5 and 1 mg/kg and intraperitoneal injection of 5, 10, and 25 mg/kg to rats. This is the first report on the pharmacokinetic properties of vitisin B. The results provide a meaningful basis to evaluate preclinical or clinical applications of vitisin B.     

Chemometrically assisted optimization and validation of a new HPLC method for the determination of paliperidone in pharmaceuticals

Paliperidone is an antipsychotic drug, which is used for the acute and maintenance treatment of schizophrenia. In this study, a new method was developed for the determination of Paliperidone in its extended-release tablets. Face-centered central composite design was applied for optimization of the method. Factors were decided as acetonitrile content, pH of the mobile phase and buffer concentration through preliminary studies. Optimal flow rate (1?mL/min), column temperature (35°C) and internal standard (Bupropion) were also determined during preliminary studies. Retention factors and tailing factors of Paliperidone and Bupropion were selected as responses. Derringer’s desirability function was applied for simultaneously optimization of these four responses. Optimal conditions were predicted as phosphate buffer (pH:3, 23?mM): acetonitrile (76:24, v:v). Developed method was validated in terms of linearity, detection and quantification limits, accuracy, precision, specificity and robustness. Method was found linear in the concentration range of 0.125-100?µg/mL. Mean equation of the calibration curve was y?=?0.0807 x - 0.0102 (R²?=?0.9999). Accuracy and precision of the method was evaluated with recovery values (98-102%) and relative standard deviation values (<2%), respectively. All other parameters were found acceptable. The method was successfully applied for the determination of Paliperidone in its extended-release tablets.     

Development and validation of a simple and efficient HPLC method for the determination of zonisamide in pharmaceuticals and human plasma

A simple and efficient liquid chromatographic method has been developed and validated for the determination of zonisamide in pharmaceuticals and human plasma. Plasma samples are analyzed after one step protein precipitation with methanol, and chromatographic separation of zonisamide and chloramphenicol (internal standard) is carried out using a C₁₈ column and the optimum mobile phase of acetonitrile/methanol/distilled water (20: 10: 70, v/v/v). The method is validated in both mobile phase and human plasma, and the obtained limits of quantification values are 0.099 and 0.12 μg/mL in mobile phase and human plasma, respectively. Fully validated method is reproducible and selective for the determination of zonisamide in pharmaceuticals and human plasma.     

Development of a validated HPLC method for the determination of four 1,4-benzodiazepines in human biological fluids

A simple and sensitive HPLC method was developed and validated for the determination of four frequently prescribed 1,4-benzodiazepines: alprazolam (ALP), bromazepam (BRZ), diazepam (DZP), and flunitrazepam (FNZ). Separation was achieved on an Inertsil C8 analytical (250 mm x 4 mm, 5 microm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH3COONH4, 0.05 M CH3OH, and CH3CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/microL) as the internal standard. DSC-18 Supelco cartridges provided high absolute recoveries (81-115%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <12%. Recoveries from biological samples ranged from 81.2 to 115%. The detection limit of the method was calculated as 3.3-10.2 ng in blood plasma and 2.6-12.6 ng in urine for 20 microL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.     

Development and optimization of the determination of pharmaceuticals in water samples by SPE and HPLC with diode‐array detection

This paper describes the development, optimization, and validation of a method for the determination of five pharmaceuticals from different therapeutic classes (antibiotics, anthelmintics, glucocorticoides) in water samples. Water samples were prepared using SPE and extracts were analyzed by HPLC with diode‐array detection. The efficiency of 11 different SPE cartridges to extract the investigated compounds from water was tested in preliminary experiments. Then, the pH of the water sample, elution solvent, and sorbent mass were optimized. Except for optimization of the SPE procedure, selection of the optimal HPLC column with different stationary phases from different manufacturers has been performed. The developed method was validated using spring water samples spiked with appropriate concentrations of pharmaceuticals. Good linearity was obtained in the range of 2.4–200 μg/L, depending on the pharmaceutical with the correlation coefficients >0.9930 in all cases, except for ciprofloxacin (0.9866). Also, the method has revealed that low LODs (0.7–3.9 μg/L), good precision (intra‐ and interday) with RSD below 17% and recoveries above 98% for all pharmaceuticals. The method has been successfully applied to the analysis of production wastewater samples from the pharmaceutical industry.     

Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma

Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C₈-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.     

HPLC determination of ciprofloxacin, cloxacillin, and ibuprofen drugs in human urine samples

This paper reports, for the first time, a liquid chromatographic method for the simultaneous determination of three frequently co-administered active principles, two antibiotics, ciprofloxacin (CIPRO) and cloxacillin (CLOXA) belonging to the fluoroquinolones and beta-lactam families, respectively, and ibuprofen (IBU), a non-steroidal anti-inflammatory drug. The chromatographic separation was performed on a C-18 analytical column, using isocratic elution with methanol-acetonitrile-pH 3 formate buffer (CT = 0.1 M) (15:12:73, v/v/v) for 3 min and, after that, a linear gradient with methanol-acetonitrile (88:12, v/v) for 8 min. Several absorption spectra were obtained for each peak using a DAD detector. Chromatograms at the maximum absorption wavelength for each analyte, 220 nm for both IBU and CLOXA, and 280 nm for CIPRO were selected as the most suitable. The proposed chromatographic method requires about 15 min per sample. The presence of a urine background was tested and no interference was found. The method was satisfactorily applied to the determination of CIPRO, CLOXA, and IBU, in fortified urine, and in urine samples from a patient undergoing treatment with these three active principles, among others. Limits of quantification in urine were 1.00, 1.70, and 2.87 microg/mL for CIPRO, CLOXA, and IBU, respectively.     

高效液相色谱法分离/蒸发光散射和紫外检测法测定天麻中天麻甙含量

天麻（Gastrodia elata Blume）系兰科多年生寄生植物,用于治疗头错、眩晕、肢体麻木等症,冯孝章和周俊等分离并鉴定出天麻的活性成分有天麻甙（对羟甲基苯β－D－吡喃葡萄糖式,亦称天麻素）、天麻甙元（对羟基苯甲醇）等,其中天麻甙为主要成分,一些药理实验也证实了这一点,在测定天麻甙含量的方法中,采用高效液相色谱法9HPLC）最多,正相PHLC和反相HPLC都可用于天麻及其占天麻甙的分离,-通常采用紫外检测法,检测波长在220nm或270nm处。     

Optimization and validation of a method for the direct determination of catechin and epicatechin in red wines by HPLC/fluorescence

The present paper describes a direct procedure for the determination of catechin and epicatechin concentrations in red wines employing reverse-phase high performance liquid chromatography (RP HPLC) and detection by fluorescence. The method was performed using a sample volume of 10 µL without dilution. The separation process employed a Chromolith performance RP-18e (100 mm × 4.6 mm) column, and the mobile phase was composed of solvent A: methanol-acetic acid-water (90:8:2) and solvent B: water-acetic acid-methanol (10:2:88) at a flow rate of 1.0 mL min^− 1. Linearity was observed in the range of 1 to 30 mg L^− 1, with limits of detection and quantification of 0.27 and 0.89 mg L^− 1, respectively, for catechin and 0.33 and 1.01 mg L^− 1, respectively, for epicatechin. The precisions estimated by the relative standard deviation were 3.34 and 1.09% for catechin concentrations of 0.5 and 20 mg L^− 1 respectively and 2.82 and 0.49% for epicatechin concentrations of 0.5 and 20 mg L^− 1, respectively. The evaluation of the accuracy was done using an addition/recovery assay. Four wine samples were used, and the recoveries varied from 105 to 108% for catechin and from 97 to 119% for epicatechin. The method was applied to the analysis of red wine samples collected from the São Francisco region, Bahia State, Brazil. Nine samples were analyzed, and the catechin and epicatechin concentrations varied from 7.51 to 73.20 and from 5.08 to 43.32 mg L^− 1, respectively. The concentrations found agree with data reported in the literature.     

Flow-through optosensor combined with photochemically induced fluorescence for simultaneous determination of binary mixtures of sulfonamides in pharmaceuticals, milk and urine

A sensitive and selective flow-through optosensor implemented with photochemically induced fluorescence (PIF) is proposed for the simultaneous determination of mixtures sulfamethoxazole/sulfanilamide and sulfathiazole/sulfanilamide. The resolution was accomplished by placing in the flow system a minicolumn filled with an appropriate solid support. Whereas one of the sulfonamides is not retained in the minicolumn and is determined by measuring its native fluorescence on the solid surface of the sensing microbeads in the detection area, the other one is retained and, after its elution, it is photochemically converted into a strongly fluorescent photoproduct which is transitorily retained on the sensing support in the flow cell and monitored. Linear calibration graphs were obtained over a concentration range of 2–3 orders of magnitude. The detection limits for the determination of sulfamethoxazole, sulfanilamide and sulfathiazole are 8.1, 2.9 and 5.7 ng mL⁻¹, respectively. The method was applied to pharmaceuticals, milk and human urine. The recovery of sulfamethoxazole from pharmaceuticals was 102.5% indicating no interference from trimethoprim which is not photochemically active. The recoveries for urine and milk samples fortified with sulfonamides at levels between 0.1 and 0.7 μg mL⁻¹ agreed within 95.0–107.5% of spiked levels.     

Development and validation of a capillary zone electrophoresis method for the determination of propranolol and N-desisopropylpropranolol in human urine

A simple, rapid and sensitive procedure using solid phase extraction and capillary zone electrophoresis for the determination of propranolol (a beta-blocker) and one of its metabolites, N-desisopropylpropranolol, has been developed and validated. The optimum separation of both analytes was obtained in a 37 cm × 75 μm fused silica capillary using 20 mmol/L phosphate buffer (pH 2.2) as electrolyte, at 25 kV and 30 °C, and hydrodynamic injection for 5 s. Prior to the electrophoretic separation, the samples were cleaned up and concentrated using a C₁₈ cartridge and then, eluted with methanol, allowing a concentration factor of 30.Good results were obtained in terms of precision, accuracy and linearity. The limits of detection were 28 and 30 μg/L for N-desisopropylpropranolol and propranolol, respectively. Additionally, a robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments.The presented method has been applied to the determination of both compounds in human urine.