Simultaneous determination of eleven quinolones in animal feed by liquid chromatography with fluorescence and ultraviolet absorbance detection

A rapid and simple multi-residue procedure is described for assaying eleven quinolones (cinoxacin, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid and sarafloxacin) in feeds at sub-additive levels (1–5 mg kg⁻¹). Five grams of sample were extracted by a metaphosphoric acid/acetonitrile mixture (70/30, v/v) and purified onto OASIS HLB cartridges. The determination was achieved by liquid chromatography (LC) using a GEMINI C18 analytical column both with fluorescence detection (FD) and photodiode-array (DAD). Limits of detection for each drug were in the range 0.04–0.8 mg kg⁻¹. Above the limit of quantification (LOQ), in poultry feed the recoveries were from 69 to 98% with relative standard deviations less than or equal 10%. Finally the measurement uncertainty was estimated using the bottom-up approach.     

Quantitative determination of cortisol in human plasma by high-pressure liquid chromatography.

A method is described for the determination of cortisol in human plasma by high-pressure liquid chromatography. The simplified extraction procedure makes the method applicable to routine clinical assays. Partition chromatography is carried out on a Zorbax-Sil column with the eluent system dichloromethane-ethanol-water. A 78% recovery was obtained for cortisol. The detection limit is 1 mug per 100 ml in 1 ml of plasma. Cortisol values were determined in samples from a random selection of patients.     

High performance liquid chromatography determination of cyanide in urine by pre-column fluorescence derivatization

A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product for reversed-phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50-1000 pmol/mL was 85-96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.     

Characterisation and quantitation of morphine in urine using high-pressure liquid chromatography with fluorescence detection.

A simple and rapid method for the identification and quantitation of morphine in urine samples is described. The procedure, which involves conversion of the drug a fluorescent product followed by liquid chromatography, is shown to be highly sensitive and specific. Levels down to 0.01 mug/ml of morphine can be quantitatively detected in urine. A large number of drugs have been tested and shown not to interfere.     

Microanalysis of methotrexate by high-performance liquid chromatography using a fluorescence detector

Methotrexate (N¹⁰-methyl-4-aminofolic acid) has been determined by HPLC after oxidizing it with permanganate to a fluorogenic derivative. The detection limit has been 0.01 g/ml. The method has been applied for the determination of methotrexate in blood serum of rats.     

Direct determination of t,t-muconic acid in human urine by two-dimensional liquid chromatography

The accurate determination of E,E-2,4-hexadiendioic acid (t,t-muconic acid), an important urinary biomarker of benzene exposure, is directly performed in human urine by a new two-dimensional liquid chromatographic method. After a first partial separation of urine components by a reversed-phase mechanism and the focalisation of the anionic analytes on a small anion-chromatographic column, a conventional ion-chromatographic analysis with UV detection is carried out. The analytical procedure is fully controlled by the HPLC instrumentation software using an eight-port switch valve. If compared with conventional one-dimensional procedures, the new method produces chromatograms containing a limited number of well resolved peaks and consequently allows better analytical performances: no interfering peaks, absence of bias, repeatability better than 5% in the concentration range 0.09-5 mg dm-3 and a detection limit of 4.0 micrograms dm-3 (alpha = beta = 0.05) for the analysis of real samples.     

Direct determination of p-hydroxymethamphetamine glucuronide in human urine by high-performance liquid chromatography

High-performance liquid chromatography (HPLC) with UV detection for the simultaneous determination of the free form of p-hydroxymethamphetamine (p-OHMA) and its metabolite, glucuronide (p-OHMAG) was accomplished for the first time. We achieved this by employing 1) an ion pair reagent for retention of sample to a solid-phase extraction (SPE) cartridge, Sep-Pak Light C18 and 2) a simple two-step stepwise elution technique for subsequent ion pair RP-HPLC. The proposed method was optimized for resolution of p-OHMAG, p-OHMA and MA. The method was successfully applied to urine samples collected from MA abusers.     

Trenbolone acetate and trenbolone: trace analysis in animal chow, wastewater and human urine by high pressure liquid chromatography and electron capture gas chromatography.

Analytical methodology is described for determining residues of the synthetic anabolic steroid trenbolone acetate (TBA) and its hydrolysis product trenbolone (TBOH) in admixture in animal chow, human urine, and wastewater. Benzene extracts of the substrates are subjected to liquid-liquid partitioning, further cleanup on a column of silica gel, and direct analysis by high pressure liquid chromotography or derivatization with pentafluoropropionic anhydride and analysis by electron capture gas chromatography. Satisfactory recoveries were obtained with both compounds from all three substrates. Residue levels of TBA and TBOH as low as 0.32 and 0.04 ppm, respectively, could be detected in chow; about 0.6 ppb of each compound could be detected in urine and wastewater. Thin layer chromatographic behavior of the two compounds in 7 solvent systems and other ancillary analytical data are also presented.     

Determination of diclazuril in animal feed by liquid chromatography.

A method is described for the determination of diclazuril (Janssen Research Compound R64433; trademark Clinacox) in chicken feed at the mg kg(-1) level. Compound R062646, a structure analogous to diclazuril, was used as the interna standard. The drug was extracted from food with acidified methanol. Diclazuril was then isolated by means of solid-phase extraction with a cartridge containing a C18 phase. The eluate was evaporated and the residue redissolved in dimethylformamide. An aliquot was injected onto a reversed-phase high-performance liquid chromatographic column and the drug substance quantified at 280 nm by an ultraviolet detector. Extraction (absolute) recoveries of 85% for both internal standard and diclazuril were obtained. The method is suitable for diclazuril concentrations ranging from 0.1 to 1.5 mg kg(-1). Method validation data are presented.     

Determination of sodium phenobarbital in animal chow by high-pressure liquid chromatography

An analytical procedure is described for determining residues of sodium phenobarbital in animal chow at levels as low as 0.14 ppm. The methanol extract is subjected to a liquid-liquid cleanup at pH 13 and 1, further cleaned up on a silica gel column and assayed by high-pressure liquid chromatography by using an ultraviolet absorption detector at 210 nm. Data concerning extraction efficiency, partition values and stability of the chemical in animal chow are also presented.     

A liquid chromatography method using a monolithic column for the determination of corticoids in animal feed and animal feeding water

An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 °C, with acetonitrile:H₂O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.     

固相萃取-液相色谱荧光测定动物组织中盐酸莱克多巴胺

采用固相萃取(SPE)前处理净化技术,高效液相色谱(HPLC)测定动物组织中盐酸莱克多巴胺残留量,通过对样品中待测组分的提取、净化,建立了反相高效液相色谱(RP-HPLC)测定动物组织中盐酸莱克多巴胺残留量。线性范围为1.0~100μg/L;相关系数大于0.9998;检出限0.3μg/L;3个不同水平标准添加回收率(n=7)为89.3%~100.5%;相对标准偏差(RSD)为3.9%~5.0%。     

Determination of N1-methylnicotinamide in urine by high-pressure liquid chromatography.

This paper reports a precise method that is shorter than previously reported methods for the quantitative determination of N1-methylnicotinamide (MNA) in urine. The method employs a single column chromatographic isolation step, followed by high-pressure liquid chromatographic (HPLC) analysis. Potential interfering substances present in urine are removed during the column chromatography step. The combined MNA fractions eluted from this column were collected and concentrated for quantitative assay of MNA by HPLC. HPLC analysis was effected in less than 15 min using a strong cation- exchange column eluted with 0.25 M ammonium dihydrogen phosphate (pH 4.3). Linearity of MNA detection by HPLC at 254 nm extended below 20 ng, with an average recovery of 101% for 150, 250 and 500 microgram MNA added to 5 ml or urine.     

Determination of 21-hydroxycorticosteroids in human urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method with fluorescence detection for the determination of nineteen 21-hydroxycorticosteroids is described. The corticosteroids are oxidized by cupric acetate to form the corresponding glyoxal derivatives. The derivatives are converted into fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-dicarbonyl compounds. The quinoxalines are separated within 70 min on a reversed-phase column (TSK gel ODS-120T) by stepwise elution with mixtures of methanol, acetonitrile, and 1.0 M ammonium acetate. The detection limits are 0.14-29.4 pmol at a signal-to-noise ratio of 3 in a 50-microliter injection volume. This sensitivity permits precise determination of hydrocortisone, cortisone, corticosterone, and their tetrahydro derivatives in 500 microliters of normal human urine.     

Trace determination and characterization of some proteins in blood serum by high-performance liquid chromatography with a time-resolved laser fluorimetric detector

Protein in human serum is measured by high-performance liquid chromatography with a time- resolved laser fluorimetric detector. The detection limit is 2.3 pmol for bovine serum albumin; the value is governed by fluctuation of the background fluorescence from free 1-anilino-8-naphthalene sulfonate (ANS) used as the fluorescence probe. Micropolarity of the binding site in protein is estimated by measuring the fluorescence lifetime of ANS bound to protein; the micropolarity for albumin is lower than those for α- and γ-globulins.     

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.