Determination of glyphosate residues in plants by precolumn derivatization and coupled-column liquid chromatography with fluorescence detection

A rapid method was developed for the trace-level determination of glyphosate in olives. After extraction of the glyphosate with water-dichloromethane and simultaneous removal of the olive oil, an aliquot of the aqueous extract is derivatized with 9-fluoroenylmethyl chloroformate (9-fluorenylmethoxycarbonyl chloride; FMOC-CI) to produce a highly fluorescent derivative. A 2 mL aliquot of this extract is injected directly into a coupled-column liquid chromatography system with fluorimetric detection (LC/LC-FD). The procedure was validated by recovery experiments at 3 spiking levels; recoveries ranged from 80 to 97% with relative standard deviations of 3-6%. The limits of detection and quantitation were estimated to be 0.01 and 0.05 mg/kg, respectively. The method was also applied to other plant materials, i.e., tomato plants, strawberry plants, and pear trees (branches, leaves, and fruits) suspected to be contaminated by glyphosate. In all these cases, the extraction was performed in aqueous media. The derivatization reaction was modified by increasing the FMOC-CI concentration, to ensure a quantitative reaction between analyte and reagent in the presence of high levels of coextractives, which also react with FMOC-CI. The final determination was by LC/LC-FD, yielding a rapid, selective, and sensitive method for the determination of glyphosate residues in these samples. The method was tested with real-world samples after application of glyphosate to the surrounding area of crops.     

Determination of chlortetracycline residues in tissues using high-performance liquid chromatography with fluorescence detection

A method is described for the determination of chlortetracycline residues in tissue samples. The samples were extracted into a hydrochloric acid - glycine solution and the extracts concentrated and purified on cyclohexyl-bonded reversed-phase cartridges. Any chlortetracycline present was converted to iso-chlortetracycline at pH 12, which was then separated from interfering compounds on a reversed-phase polymeric column using high-performance liquid chromatography with fluorescence detection. The detection and determination limits of the assay were 20 and 50 ng g-1, respectively, making it suitable for statutory residue testing purposes.     

Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection

A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with a thiol-selective fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure of thiol derivatives is assessed using electrospray ionization-mass spectrometry (MS). The stability of resulting adducts in acidic medium (24 h at 10 degrees C) allows the automation of the technique and a high throughput of samples (approximately 50 per day). Separation is complete within 12 min using isocratic reversed-phase mode, and detection is operated by spectrofluorimetry (lambda ex = 385 nm and lambda em = 515 nm). Quantitation is performed by an internal standardization mode using thioglycolic acid. The LC method is fully validated, and homocysteine concentrations obtained in plasma samples are compared with values measured using either fluorescence polarization immunoassay or capillary gas chromatography-MS; a good correlation is observed between LC and both methods. The method has been applied in daily use to a large-scale study in a human healthy population, and some resulting data are discussed.     

Determination of fatty acids by high-performance liquid chromatography and fluorescence detection using precolumn derivatization with 9-fluorenylmethyl chloroformate

A new high-performance liquid chromatographic method is described for the determination of fatty acids in seed oils. The method was based on precolumn derivatization with 9-fluorenylmethyl chloroformate as a labeling agent and fluorescence detection. Fatty acids were extracted from the samples and subjected to derivatization with the reagent at 60°C for 10?min. The chromatographic separation of 14 fatty acids (C10–C22) was achieved on a combined loading compression octadecyl sulfate (CLC-ODS) column with a run time of 30?min. Three-step gradient elution of a mobile phase consisted of acetonitrile and water was used, and the signal was monitored at excitation and emission wavelengths of 265 and 315?nm, respectively. The method indicated favorable sensitivity and reproducibility for fatty acids’ derivatives. The detection limits, at a signal-to-noise ratio of 3, were 0.01–0.05?µg/ml and relative standard deviations (RSDs) were less than 0.27%. Excellent linear responses were observed with coefficients of 0.9995. This method was applied to quantify fatty acids in white, brown, and black sesame seeds’ oil.     

Determination of apramycin in animal feeds by solid-phase extraction and liquid chromatography with precolumn derivatization and fluorescence detection

A high-performance liquid chromatographic method for determining apramycin in animal feeds was developed. Apramycin in feeds was extracted with 0.1 M HCl solution and cleaned up with an MCX solid-phase extraction column. The purified extract was derivatized with o-phthaldehyde, and components were separated on a C18 column and detected with a fluorescence detector. Mass spectrometric data confirmed that apramycin was derivatized at all the 4 primary amines on the apramycin molecule. Average recoveries at 8 included levels (5, 10, 20, 40, 80, 200, 400, and 2000 mg/kg) ranged from 92.2 to 100.5%, and the coefficients of variation were < 6.5%. Standard curves were linear over the range 0.05 to 10 microg/mL. The detection and quantitation limits were determined to be 0.2 and 1.0 mg/kg, respectively.     

Determination of trans-fatty acids in food samples based on the precolumn fluorescence derivatization by high performance liquid chromatography

Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this precolumn fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography with fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25°C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C₁₈ column with isocratic elution in 14 min. With fluorescence detection at λ_ex/λ_em = 490 /510 nm, the linear range of the TFAs was 1.0-200 nM with low detection limits in the range of 0.1–0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02 to 109.22% with low relative standard deviations of 1.2–4.3% (n = 6).     

Determination of the heroin metabolite 6-acetylmorphine by high-performance liquid chromatography using automated pre-column derivatization and fluorescence detection

An improved method for the determination of 6-acetylmorphine in the urine of drug addicts receiving morphine was developed. A newly introduced reversed-phase high-performance liquid chromatographic system proved to be more sensitive than a normal-phase system used previously. By replacing the earlier manual derivatization procedure with an automated on-line pre-column method, both the reproducibility and efficiency were considerably improved. Coefficients of variation for repeated analyses typically ranged from 6 to 10% in the 1-100 micrograms/l concentration range. The detection limit was 1 microgram/l and the correction for recovery by calibration with blank urine samples spiked with 6-acetylmorphine was satisfactory. The analytical improvements achieved, however, did not increase the chance of detecting heroin use by drug addicts.     

Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection

A rapid liquid-chromatography (LC) method is presented which uses fluorescence detection (FLD) for palytoxin analogues analysis in benthic dinoflagellates of the genus Ostreopsis. The amino-acidic reagent 6-aminoquinolyl-N-hydroxisuccinimidyl carbamate (AccQ) was used for fluorescence labelling followed by LC-FLD.The efficacy of the method is exemplified by comparison of the results of the quantification obtained by LC-FLD and the hemolytic assay performed for palytoxins for which a highly significant linear correlation was achieved (r² = 0.9118). The derivatized palytoxin analogues were determined in the range of 0.75-25 ng.The proposed method was successfully applied to the determination and quantification of palytoxin analogues in 14 samples from different strains of Ostreopsis from different locations (Western Mediterranean Sea, Canary Islands, Madeira Islands and Southern coasts of Brazil). To confirm the chemical structure of the toxins, samples were also analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) with a system that has a poorer sensitivity when compared with LC-FLD detection and the hemolytic assay. The successful use of this method with dinoflagellates is a good indicator of suitability for other types of marine samples.     

柱前衍生-超高效液相色谱-串联四极杆质谱法测定茶叶中乙撑硫脲的残留

建立了茶叶中乙撑硫脲残留的柱前衍生-超高效液相色谱-串联四极杆质谱检测方法。样品采用乙腈提取,提取液经QuEChERS基质分散固相萃取净化后采用9-芴基甲基氯甲酸酯（FMOC-CL）柱前衍生;衍生溶液经BEH-C18色谱柱（100 mm×2.0 mm,1.7 μm）分离后进入串联四极杆质谱仪检测,采用同位素内标法定量;流动相为0.1%（v/v）甲酸-乙腈。该方法对茶叶样品检出限为1.3 μg/kg,定量限为4.2 μg/kg;加标回收率在97.7%~107.5%之间,相对标准偏差（RSD,n=6）在2.1%~10.0%之间;在1.0~203.4 μg/L范围内线性回归系数r为0.9993。该方法灵敏度高,重现性好,定性定量准确,可有效满足对茶叶中乙撑硫脲残留检测的要求。     

Determination of amoxicillin residues in animal tissues by solid-phase extraction and liquid chromatography with fluorescence detection

Trace levels of amoxicillin residues were determined in animal tissues by liquid chromatography (LC) with fluorescence detection. An improved solid-phase extraction (SPE) procedure requiring less flammable solvent (diethyl ether) was developed for sample preparation. Muscle samples of beef, pork, chicken, and tilapia were extracted with a phosphate buffer followed by the modified SPE procedure for cleanup and concentration prior to the LC-fluorescence analysis. Average recoveries of fortified amoxicillin at 5, 10, and 20 micrograms/kg ranged from 83.9 to 85.8% in beef, 86.1 to 88.1% in pork, 81.7 to 82.9% in chicken, and 92.5 to 95.4% in tilapia. Relative standard deviations were < 4%.     

Determination of streptomycin residues in food by solid-phase extraction and liquid chromatography with post-column derivatization and fluorometric detection

A reliable and sensitive procedure is presented for the analysis of streptomycin (STP) in food of animal origin, like meat, milk and honey. The method is based on a separation by ion-pair liquid chromatography with β-naphthoquinone-4-sulfonate (NQS) postderivatization and fluorescence detection. The clean-up of the extract is done by solid-phase extraction, firstly with a cation-exchange cartridge and secondly with an octadecyl cartridge. The selectivity is very good and not many interfering peaks are observed for various food matrices. The streptomycin recovery of the total procedure is superior to 80%. The procedure is quantitatively characterized and repeatability, linearity, detection and quantification limits are very satisfactory. A special focus is given to STP residues in honeys and a survey on 64 commercial honeys is presented. For honey analysis, the HPLC method is compared with an immunoassay test (ELISA), and the possibility of using this test for screening with and without solid-phase extraction clean-up is also discussed.     

柱后衍生高效液相色谱法测定虾中14种磺胺类药物残留量

建立了虾中14种磺胺类药物残留量的柱后衍生高效液相色谱检测方法。样品在加入内标物磺胺吡啶后用乙酸乙酯提取,提取液浓缩后用4 mL乙酸乙酯溶解残余物,用盐酸溶液反萃取,正己烷去脂,盐酸溶液经滤膜过滤后,加入乙腈、甲醇和3.5 mol/L乙酸钠溶液（体积比为5：5：20）的混合溶液混匀后,经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。采用基质标样添加法绘制标准曲线,内标法定量。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50℃。14种磺胺类药物在5~200 μg/L范围内具有良好的线性。磺胺类药物的定量限（LOQ,S/N=10）为1.0~5.0 μg/kg。在1.0~100.0 μg/kg添加水平内,磺胺类药物的平均回收率为77.8%~103.6%,相对标准偏差（RSD）为2.9%~9.1%（n=6）。实验结果表明该方法灵敏、准确,重复性好,适用于虾中磺胺类药物的残留检测。     

Determination of sulfonamide residues in cultured sea cucumber by pre-column derivatization capillary electrophoresis with fluorescence detection

A simple and mild method for the separation of sulfonamide residues based on a condensation reaction with O-phthalaldehyde solution (OPA) as labeling reagent with capillary electrophoresis has been developed. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary was used. To optimize the separation conditions, the background electrolyte concentration, pH, column temperature, voltage and other factors were evaluated. The optimal separation conditions were as follows: 20 mmol L^?1 borate buffer; pH 9.1; column temperature 20 °C; separation voltage 18 kV, pressure 50 mbar and injection time 8 s. Under the optimal conditions, 10 kinds of sulfonamide derivatives could be well-separated within 8 min, and the linear ranges were 0.35–100 μg kg^?1. The detection limit (at a signal-to-noise ratio of 3) was in the range of 0.12–0.25 μg kg^?1, and the quantification limit (at a signal-to-noise ratio of 10) was in the range of 0.35–0.70 μg kg^?1. The sulfonamide residues from cultured sea cucumber samples were determined under the optimal conditions with satisfactory results.     

Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid-ethanol fluorescence derivatization and column switching.

An assay method for serum cortisol, using precolumn sulphuric acid-ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence intensity increased. Among the sulphuric acid-ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 micrograms/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.     

Stereoselective determination of demethyl- and didemethyl-citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.     

柱前衍生高效液相色谱-荧光检测法测定血浆中同型半胱氨酸

建立了一种柱前衍生高效液相色谱-荧光检测法用于测定血浆中同型半胱氨酸(Hcy)。使用三(2-羧乙基)膦盐酸盐(TCEP)为还原剂,N-(1-芘)马来酰亚胺(NPM)为衍生剂进行样品预处理,Agilent Hypersil C-18柱(250 mm×4.0 mm, 5 μm)进行分离,流动相为15 mmol/L醋酸钠-乙腈-混合酸(300 mL水中含1 mL醋酸和1 mL磷酸)混合溶液,采用梯度洗脱,荧光检测激发波长为330 nm,发射波长为380 nm。Hcy的回收率为(102.08±4.94)%。线性范围为0.500～100 μmol/L,检出限（以信噪比为3计）为0.016 μmol/L。日内与日间相对标准偏差均小于5%。利用该方法对7例高血压患者和7例健康志愿者的血浆进行了测定,结果表明两组间的Hcy含量存在显著的差异(p＜0.05)。本方法简单、快速、灵敏、特异,适用于血浆Hcy的临床定量测定。     

Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection.

An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)-N-acetyl-D-penicillamine (NAP) or cyanide (CN-) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA-CN- derivative of aldendronate has been demonstrated, and a HPLC-ED assay in human urine has been validated in the concentration range 2.5-50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA-NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA-NAP derivatization and HPLC-FD has been developed and fully validated in the concentration range 1-25 ng/ml. Based on the same NDA-NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC-FD assays were utilized to support various human pharmacokinetic studies with alendronate.     

Determination of tetracycline antibiotics in salmon muscle by liquid chromatography using post-column derivatization with fluorescence detection

A simple and accurate cleanup procedure using polymeric sorbent was developed for the determination of oxytetracycline (OTC) and tetracycline (TC) residues in salmon muscle. It was applied to the analysis of 20 salmon samples during a month period. The OTC and TC residues were extracted with ethylenediaminetetracetic acid (EDTA)-McIlvaine buffer acidified at pH 4.0 and cleaned up by solid-phase extraction with a polymeric sorbent. The advantages of the polymeric sorbent over the silica-based sorbent in the cleanup of salmon muscle samples are described. A liquid chromatographic method with post-column derivatization and fluorescence detection is proposed because of its sensitivity and specificity. The average recoveries of OTC and TC from muscle salmon tissue fortified at 50, 100, and 200 microg/kg levels, ranged from 83.9 to 93.4% with a coefficient of variation between 4.09 and 5.80%. The limit of quantitation for OTC and TC in salmon muscle was 50 microg/kg.     

Determination of sugars by liquid chromatography with post-column catalytic derivatization and fluorescence detection

Summary A method for the determination of reducing sugars such as fructose and glucose and nonreducing sugar such as sucrose by high performance liquid chromatography followed by an acidic hydrolysis and a derivatization with benzamidine has been developed. After separation of sugars on a gel column packed with a polymer-based cation exchange material (Sugar-Pak I, Waters-Millipore), the sucrose is first hydrolysed in a solid phase reactor to convert it into reducing subunits. A post-column fluorigenic reaction with benzamidine under alkaline condition allows the selective determination of both natural and converted reducing carbohydrates.This procedure has proven to be selective (fluorigenic detection) and highly sensitive (allowing detection as little as picomoles amounts), reproducible and linear over a broad range of concentrations: 5×10^–4 to 1.0×10^–2 M.The applicability of this method to natural matrices such as plant extracts and beverages is also described. The sugar content of a barley extract has been determined and compared with a specific enzymatic test. The determined sugar content of natural and commercial lemon juices as well as of Cola beverages has been compared with those found by the conventional LC refractive index analytical procedure. In all cases, the results were comparable and were within the experimental errors of the methods.