Simple HPLC determination of colistin in medicated feeds by pre-column derivatization and fluorescence detection

Summary A simple and sensitive HPLC method was developed for the determination of colistin antibiotic in feeds employing pre-column derivatization and fluorescence detection. Extraction of colistin in feeds was by sonication and shaking with 0.1 M HCl. Pre-column derivatization was with phthaldialdehyde and 2-mercaptoethanol (ME) in borate buffer (pH 10.5) to obtain a fluorescent derivative. Elution of the derivative onto an Ultracarb 5 μm ODS column was by using acetonitrile—ultrapure water (75∶25). Detection was by spectrofluorimetry at 340 nm (excitation wavelength) and 440 nm (emission wavelength). Total elution time was <20 min. The applicability of the validated method was tested by analyzing commercial medicated feeds without any interference from the matrix.     

Dynamic ultrasound-assisted extraction of colistin from feeds with on-line pre-column derivatization and liquid chromatography-fluorimetric detection

A dynamic ultrasound-assisted extraction (UAE) method with on-line pre-column derivatization/high performance liquid chromatography (HPLC) and fluorimetric detection is proposed for the analysis of colistin in feed. A flow injection manifold is used for the development of the extraction and derivatization steps and for interfacing them with the separation/detection step, thus providing an on-line approach with the advantage of minimum sample handling. The derivatization was performed with ortho-phthaldialdehyde and 2-mercaptoethanol. The optimum conditions for colistin extraction and formation of the fluorescent derivative have been obtained by experimental design methodology. The use of a high-intensity probe sonication makes UAE an expeditious (7 min versus > 1 h) and efficient (93.1-98.2% versus 87.5-94% of recovery) alternative as compared with extraction using an ultrasonic bath. The within-laboratory reproducibility and repeatability, expressed as percentage of relative standard deviation, were 5.2 and 5.8, respectively.     

液相色谱-串联质谱法测定牛奶中5种多肤类抗生素

建立了牛奶中杆菌肽、粘杆菌素A、粘杆菌素B、维吉尼霉素和万占霉素5种多肽类抗生素的反相液相色谱-串联质谱(HPLC-MS/MS)检测方法.牛奶样品经甲醇-0.1%甲酸水提取后,用4%三氯乙酸乙睛除蛋白,液-液萃取后,采用0.1%甲酸(A)和0.1%甲酸乙腈(B)作为流动相进行梯度洗脱.质谱(ESI+)采用多离子检测模式...     

Sub-inhibitory concentration of essential oils induces antibiotic resistance in Staphylococcus aureus

Fourteen Staphylococcus aureus wild strains were stressed with sub-inhibitory concentration of five essential oils: Leptospermum scoparium (manuka), Origanum majorana (marjoram), Origanum vulgare (oregano), Satureja montana (winter savoury) and Thymus vulgaris (thyme). Antibiotics susceptibility profiles of the strains were determined by agar disk diffusion method before and after EOs treatment. The following antibiotics were employed: amoxicillin-clavulanic acid, amikacin, ampicillin, amoxicillin, aztreonam, ceftazidime, cephalothin, ciprofloxacin, colistin, cefotaxime, doxycycline, enrofloxacin, erythromycin, gentamicin, cephalexin, neomycin, piperacillin, rifampin, streptomycin, trimethoprim-sulphamethoxazole, tetracycline and tobramycin. Before EOs treatment, strains were susceptible to all antibiotics except for aztreonam and colistin. After exposure to sub-inhibitory EOs concentration of manuka, marjoram and oregano, several modifications in antibiotics susceptibility profiles were detected. Conversely, few modifications were induced by winter savoury and thyme EOs. Moreover, occurrence of resistances seems uncorrelated with drug classes as low concentration of EO could induce phenotypic changes in susceptible bacteria leading to antibiotic resistance phenomena.     

Reversed-phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre-column derivatization with phenylisocyanate

A pre-column derivatization liquid chromatographic method has been developed for the analysis of aminoglycoside antibiotics using phenylisocyanate as a derivatization reagent. Derivatives including kanamycin, neomycin and gentamicin were formed by reaction of the analytes with phenylisocyanate in the presence of triethylamine. Phenylisocyanato groups were attached to corresponding amino groups of aminoglycoside and their molecular mass was confirmed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The experimental conditions for derivatization and separation of aminoglycoside derivatives were optimized and validated. A simple liquid chromatographic method for the determination of aminoglycoside antibiotics was demonstrated.     

Use of SDS micelles for improving sensitivity, resolution, and speed in the analysis of beta-lactam antibiotics in environmental waters by SPE and CE

This study dealt with the potential of MEKC with LIF detection involving derivatization with sulfoindocyanine succinimidyl ester (Cy5) for the separation and determination of beta-lactam antibiotics (ampicillin, amoxicillin, cephradine, and cephalexin) in environmental water samples. Water samples of 50 mL were enriched by SPE by passage through a weak base-cation Amberlite(R) IRA-93 exchange column. SDS micelles play important roles in the whole analytical process by improving the yield (sensitivity) and the kinetics of the labeling reaction, the elution of the retained antibiotics from the SPE preconcentration system and the electrophoretic resolution of their Cy5-derivatives. The optimum procedure includes a derivatization step of the antibiotics at 25 degrees C for 10 min and direct injection for MEKC analysis, which is conducted within about 15 min using 15 mM SDS in the running buffer (35 mM sodium borate at pH 9.3). LODs from 30 to 45 ng/L and RSDs (within-day precision) from 3.5 to 5.9% were obtained for the antibiotics in water samples with average recoveries ranging from 96.4 to 99.4%. These results indicate that the method proposed is a straightforward and sensitive tool for the determination of these antibiotics in environmental water samples providing similar quantitative results to those using more expensive equipment like LC-electrospray MS/MS.     

Quantitative analysis of polypeptide antibiotic residues in a variety of food matrices by liquid chromatography coupled to tandem mass spectrometry

A quantitative LC–MS/MS method was developed for the determination of five polypeptide antibiotics (bacitracin, colistin A, colistin B, polymyxin B1 and polymyxin B2) in a variety of food matrices (muscle, liver, kidney, egg and milk). The described method is sufficiently sensitive, selective and provides acceptable recoveries for all compounds. The extraction is based on acidified methanolic solvent. This is followed by a reversed phase solid phase extraction step to clean-up and concentrate the extracts. The use of a modern core shell column in combination with an eluent consisting of trifluoroacetic acid, formic acid and acetonitrile provides chromatographically well resolved analyte peaks The single-step clean-up is fast and produces a sufficiently clean extract in order to control matrix-related signal suppression in the electrospray interface.     

气相色谱-质谱联用同时分析新型细菌多糖中的单糖和糖醛酸

对纯化的新型细菌多糖进行酸水解,用乙硫醇-三氟乙酸和醋酐-吡啶体系先后对酸水解物进行衍生,与之前报道不同的是糖醛酸得到有效衍生化。以木糖为内标,采用气相色谱-质谱联用(GC-MS)定量分析该多糖酸水解物中单糖和糖醛酸衍生物发现,该多糖的糖链由岩藻糖、葡萄糖、葡萄糖醛酸和半乳糖组成,其相对物质的量比为1.50:1.0:0.79:2.06;中性糖比例与糖醇乙酸酯化分析岩藻糖、葡萄糖和半乳糖的相对物质的量比(1.76:1.0:1.98)接近;糖醛酸咔唑法与该方法分析葡萄糖醛酸的含量分别为16.19%和14.85%。以上结果表明所建立的衍生化方法及GC-MS同时定量分析多糖酸水解物中单糖和糖醛酸的方法可行。此外还对葡萄糖醛酸的质谱裂解机理进行了阐述。     

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.     

Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.     

Development of a simultaneous liquid-liquid extraction and chiral derivatization method for stereospecific GC-MS analysis of amphetamine-type stimulants in human urine using fractional factorial design

A stereospecific gas chromatography-mass spectrometry analysis method for amphetamine-type stimulants in human urine was recently developed. For maximum efficiency, liquid-liquid extraction and chiral derivatization of the analytes using (R)-(-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride were performed simultaneously. The effects of (1) use of saturated sodium chloride in 2.0 m sodium hydroxide, (2) extraction solvent volume, (3) percentage of triethylamine, (4) derivatization reagent volume, (5) sample mixing time, (6) incubation temperature and (7) incubation time on method sensitivity and variability were assessed using a two-level, eight-run Plackett-Burman design followed by a fold-over design. The use of saturated sodium chloride solution and the derivatization reagent volume were significant factors (ANOVA, p < 0.01). The saturated sodium chloride solution decreased sensitivity whereas an increased volume of derivatization reagent increased sensitivity. Calibration curves for all analytes were linear between 5 and 500 microg/L, with correlation coefficients of >0.99. Detection limits were 相似文献   

Simultaneous spectrophotometric determination of phosphate and silicate ions in river water by using ion-exclusion chromatographic separation and post-column derivatization

The simultaneous spectrophotometric determination of phosphate and silicate ions in river water was examined by using ion-exclusion chromatography and post-column derivatization. Phosphate and silicate ions were separated by the ion-exclusion column packed with a polymethacrylate-based weakly acidic cation-exchange resin in the H⁺-form (TSKgel Super IC-A/C) by using ultra pure water as an eluent. After the post-column derivatization with molybdate and ascorbic acid, so-called molybdenum-blue, both ions were determined simultaneously by spectrophotometry. The effects of sulfuric acid, sodium molybdate and ascorbic acid concentrations and reaction coil length, which have relation to form the reduced complexes of molybdate and ions, on the detector response for phosphate and silicate ions were investigated. Under the optimized conditions (color-forming reactant, 50 mM sulfuric acid-10 mM sodium molybdate; reducing agent, 50 mM ascorbic acid; reaction coil length, 6 m), the calibration curves of phosphate and silicate ions were linear in the range of 50-2000 μg L⁻¹ as P and 250-10,000 μg L⁻¹ as Si. This method was successfully applied to water quality monitoring of Kurose-river watershed and it suggested that the effluent from a biological sewage treatment plant was significant source of phosphate ion in Kurose-river water.     

固相萃取-色谱测定水、沉积物及土壤中氯霉素和3种四环素类抗生素

建立了固相萃取-高效液相色谱(UV)检测水、沉积物和土壤中的氯霉素、土霉素、四环素和金霉素4种抗生素的方法。水样、土壤和沉积物样品的前处理采用EDTA-McIlvaine缓冲溶液提取,用SAX-HLB串联小柱纯化和富集,用丙酮(含10%甲醇)洗脱进一步减小天然有机质的影响。采用乙腈和0.01mol/L草酸溶液作为流动相,8min内分离4种抗生素。测定了贵阳市阿哈湖、南明河和乌江渡水库的水样及沉积物,均有抗生素检出。     

Liquid chromatography on an amide stationary phase with post-column derivatization and fluorimetric detection for the determination of streptomycin and dihydrostreptomycin in foods

The separation of streptomycin and its derivative dihydrostreptomycin using ion-pair liquid chromatography is proposed. The method is based on the use of a new stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl which avoids the appearance of tailed peaks. The isocratic mobile phase consisted of a 6:94 (v/v) acetonitrile/10 mM pentanesulfonic acid (pH 3.3) mixture at a flow-rate of 1 mL min⁻¹ and fluorescence detection involved a post-column derivatization reaction using β-naphthoquinone-4-sulfonate. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of the aminoglycoside antibiotics in different types of foods, as honey, milk, egg and liver. Extraction was carried out by acidic hydrolysis to release protein-bound antibiotics. Detection limits in the food samples are 7.5 and 15 μg kg⁻¹ for streptomycin and dihydrostreptomycin, respectively.     

Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring

Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.     

Simultaneous detection of bacitracin and polymyxin B in livestock products using liquid chromatography with tandem mass spectrometry

With the overarching aim to develop a simple and reliable method for the quantitative analysis of polypeptide antibiotics in various livestock products, the content of bacitracin, and polymyxin B in pork, beef, chicken, milk, and eggs was analyzed using colistin sulfate as an internal standard. The extracted samples were eluted via solid‐phase extraction using 2% formic acid in acetonitrile/methanol (1:1, v/v). The two polypeptides were identified and quantified based on the intensities of mass fragments from the respective triply charged precursor ions (bacitracin: 474.97 amu and polymyxin B: 402 amu) at the defined retention time windows using liquid chromatography with electrospray ionization tandem mass spectrometry in time‐scheduled multiple reaction monitoring mode. The calibration curves showed good linearity over the concentration range 50–2500 ng/mL with determination coefficients ≥ 0.991. The mean recoveries were in the range 80.3–88.8% with relative standard deviations <13% for all samples. The limits of quantitation ranged from 30–250 ng/g. The developed method was applied to market samples, but the target analytes were not detected in any of the samples. The developed method is reliable for the simultaneous detection of bacitracin and polymyxin B in pork, beef, chicken, milk, and eggs.     

Determination of superabsorbent polyacrylate dust in workplace atmospheres after derivatization with ethanol and using HPLC with pulsed electrochemical detection

Superabsorbent polyacrylates (SAPs) have been used in the hygiene industry for many years. A derivatization and analytical method was developed for routine analysis of trace levels of SAP dust in workplace atmospheres. In comparison with existing methods, which are based on the sodium content or the ion exchange properties of the polymer, this method is more specific. It has the advantage of not being influenced by any sodium containing contaminants. Air samples are collected on Teflon filters using air monitoring sampling cassettes. The filters are subsequently placed in quartz vials and a reaction mixture containing hydrochloric acid in ethanol is added. The hydrochloric acid-ethanol solution, when heated, converts the carboxylic acid groups on the backbone of the insoluble polyacrylate into ethyl esters. After reaction, the excess of ethanol and hydrochloric acid is completely removed under vacuum. The sample is then treated with aqueous sodium hydroxide at 80 degrees C to release the bound ethanol. The solution is analyzed by HPLC on an anion exclusion stationary phase using dilute perchloric acid as mobile phase. Ethanol is identified and quantified with a pulsed electrochemical detector. Several environmental samples in addition to laboratory spiked samples were successfully analyzed with this technique. Recoveries averaged > 85% for spiked blank filters at levels from 5 to 50 micrograms per filter with relative standard deviations up to 7%. The instrument's limit of detection (LOD) for ethanol was 0.1 mg l-1. The LOD for derivatization and analysis corresponds to 3 micrograms of SAP per filter (assuming an esterification factor of 0.30 microgram of ethanol per microgram of SAP).     

快速柱前衍生HPLC法检测曲格列汀中(R)-3-氨基哌啶

以邻苯二甲醛(OPA)和3-巯基丙酸为衍生试剂,建立了柱前衍生高效液相色谱(HPLC)测定曲格列汀中(R)-3-氨基哌啶含量的分析方法。(R)-3-氨基哌啶与衍生剂在碱性(p H 10.5)条件下于室温反应30s,进行柱前衍生,并利用高效液相色谱-质谱对衍生产物进行定性分析。采用YMC-Triart C18色谱柱(150 mm×4.6 mm,5μm)分离,流动相采用0.05 mol/L乙酸钠(p H 6.0)-甲醇溶液,梯度洗脱,流速1.0 mL/min,检测波长331 nm,柱温30℃。(R)-3-氨基哌啶质量浓度在0.08~2.0μg/mL范围内线性关系良好(r=0.9993),定量限为1.6 ng,加标回收率在98.0%~10⁶.9%之间,相对标准偏差(RSD)为2.8%。该方法可用于曲格列汀中(R)-3-氨基哌啶含量测定。     

Potassium permanganate-glyoxal chemiluminescence system for flow injection analysis of cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium in pharmaceutical preparations

A sensitive flow injection chemiluminescence (FL-CL) method for the determination of cephalosporin antibiotics, was developed. The method was based on that cephalosporin antibiotics could enhance the CL reaction of glyoxal and KMnO₄ in sulfuric acid. Method development included the optimization of reagent concentrations and flow-rate. Under the optimized conditions, three cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium, were determined. The detection limits of the method are 10 ng ml⁻¹ cefalexin, 2 ng ml⁻¹ cefadroxil, and 2 ng ml⁻¹ cefazolin sodium. The method was successfully applied to the determination of three cephalosporin antibiotics in pharmaceutical preparations.