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.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

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.     

柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛（o-phthalaldehyde，OPA）为衍生试剂，建立了柱前衍生-高效液相色谱（HPLC）同时测定血清中氨基酸类神经递质牛磺酸（Tau）、谷氨酸（Glu）、甘氨酸（Gly）、γ-氨基丁酸（γ-GABA）和单胺类神经递质多巴胺（DA）含量的分析方法。血清与乙醇以1：2的体积比混合，进行蛋白质沉淀后离心，取其上清液，氮吹至近干。前处理后的样品与OPA进行柱前衍生，衍生化产物采用Luna 5u C18色谱柱（250 mm×4.6 mm，5 μm）分离，以柠檬酸-乙酸钠缓冲溶液（pH 3.73）为流动相A、乙腈为流动相B进行梯度洗脱，流速为1.0 mL/min，柱温为30℃，检测波长为338 nm。5种神经递质在各自范围内线性关系良好（r²≥0.9866），检出限为0.10~0.40 μmol/L，不同加标水平下目标物的加标回收率为87.57%~115.31%，相对标准偏差均低于7.80%。方法操作简单，灵敏度高，精密度、线性关系和回收率等方法学指标较好，可实现血清中氨基酸类及单胺类神经递质的同时检测。     

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.     

Simultaneous determination of imidazoleacetic acid and N tau- and N pi-methylimidazoleacetic acids in human urine by high-performance liquid chromatography with fluorescence detection

A sensitive method for the simultaneous determination of urinary imidazoleacetic acid and N tau- and N pi-methylimidazoleacetic acids which employs high-performance liquid chromatography with fluorescence detection is described. The compounds were converted into the corresponding fluorescent esters by reaction with 4-bromomethyl-7-methoxycoumarin. These derivatives are separated by liquid chromatography on a Radial-Pak silica column. The detection limits for imidazoleacetic acid and N tau-and N pi-methylimidazoleacetic acids in urine are 15, 10 and 20 pmol/ml, respectively. The 24-h urinary excretion of imidazoleacetic acid and N tau-and N N pi-methylimidazoleacetic acids by healthy persons was 5.7-39.9, 4.3-24.6 and 1.5-19.3 nmol/mg of creatinine, respectively.     

Fluorometric determination of 2,3-dimercaptopropane-1-sulfonic acid and other dithiols by precolumn derivatization with bromobimane and column liquid chromatography

The increasing therapeutic use of dithiol metal binding agents, such as 2,3-dimercaptopropane-1-sulfonic acid (DMPS), has stimulated the need for a sensitive and selective method for their determination in biological fluids. A method has now been developed in which DMPS was converted to a highly fluorescent and stable derivative by reaction with bromobimane in aqueous solution at pH 8.3. The reaction was complete within 5 min. The derivative was separated by ion-pair reversed-phase column liquid chromatography. The mass spectrum of the derivative showed that two bromobimane molecules reacted with one DMPS molecule. This method is also applicable to the determination of other dithiols. The detection limit for DMPS in urine is 10 pmol per 20-microliters injection and the precision is 7.4% at the 100-pmol level. The fluorescence response was linear from 1 to 400 micron. This method was used to determine DMPS in the urine of rabbits treated with this metal binding agent. In addition, total DMPS was determined by adding sodium tetrahydridoborate to the same urine to reduce biotransformed and oxidized DMPS.     

Determination of sulfonamide residues in the tissues of food animals using automated precolumn derivatization and liquid chromatography with fluorescence detection

A liquid chromatographic method for the determination of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadoxine, sulfaethoxypyridazine, sulfamethazine, sulfaquinoxaline, and sulfathiazole residues in the muscle, liver, and kidney of food animals using sulfapyridine as internal standard is reported. Tissues are extracted using a modified version of AOAC Official Method 983.31 (Sulfonamide Residues in Animal Tissues). The sample extract is reconstituted in pH 3.0 buffer-acetonitrile (60 + 40) and filtered into an autosampler vial. Using a programmable autosampler of a liquid chromatograph, a portion of the sample is derivatized precolumn with fluorescamine. The sulfonamide derivatives are separated by liquid chromatography using a C18 column with a mobile phase of 0.02M phosphoric acid-acetonitrile (60.5 + 39.5) and detected by fluorescence (excitation, 405 nm; emission, 495 nm). The method was applied to swine and cattle muscle, liver, and kidney; sheep and horse muscle and kidney; and chicken muscle and liver. The mean values for samples fortified with sulfonamides at levels between 0.05 and 0.2 microg/g agreed within 96-99% of spiked levels, with coefficients of variation ranging from 4-10%. The limit of detection (LOD) for all sulfonamides was 0.01 microg/g, with the exception of sulfaquinoxaline, for which the LOD was 0.015 microg/g.     

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.     

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.     

Simultaneous determination of tiopronin and d-penicillamine in human urine by liquid chromatography with ultraviolet detection

d-Penicillamine and tiopronin are drugs widely used for the treatment of many diseases. Because of the relatively high frequency of side effects to these compounds, some of which are dose-related, drug monitoring in urine samples during treatment is advisable. In this paper, we describe a simple method for the determination of tiopronin and d-penicillamine in human urine. The method was based on derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet-absorbance detection. 2-S-quinolinium derivatives of thiols were detected at 355 nm. The derivatization was optimized in terms of pH and time of the reaction. Baseline separation was achieved on an analytical Zorbax SB C-18 (5 μm, 150 mm × 4.6 mm) column with a mobile phase consisting of pH 2.0 0.09 mol L⁻¹ trichloroacetic acid buffer (component A) and acetonitrile (component B) pumped at 1.0 mL min⁻¹. Gradient elution was used: 0-4 min, 12% B; 4-8 min, 12-40% B; 8-12 min, 40-12% B. The d-penicillamine and tiopronin standards added to the urine show that the response of the detector is linear within the range studied, from 1 to 200 μmol L⁻¹ urine. The imprecision ranges for tiopronin and d-penicillamine were within 1.61-8.24% and 2.92-10.60%, respectively. The analytical accuracy for determined compounds was from 97.24 to 109.39%. The lower limits of detection and quantitation were 0.5 μmol L⁻¹ and 1.0 μmol L⁻¹ urine, respectively. This method can be used for routine clinical monitoring of the title thiol-drugs. Cysteine can be measured concurrently, if needed.     

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.     

Uronic acid determination by high performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric high performance liquid chromatography (HPLC) technique for uronic acid microanalysis, a saline mobile phase and the postcolumn fluorimetric determination were combined. The detection limits of D-glucuronic, D-galacturonic and D-mannuronic acids were 7.19, 23.88 and 7.08 pmol, respectively. The proposed method was successfully applied to uronic acid microanalysis in a polysaccharide hydrolysate and a drink.     

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.     

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.