Indirect fluorescence detection of amino sugars with the use of copper complexes of tryptophan and its analogues following high-performance liquid chromatographic separation

A simple, indirect fluorescence detection method has been developed for detecting specific mono-amino sugars (D-glucosamine, D-galactosamine, D-mannosamine) following chromatographic separation. The eluting amino sugars release L-tryptophan (L-Trp) from a copper-tryptophan complex which is introduced postcolumn. Analyte detection is based on measuring the increase in L-Trp fluorescence, which is quenched when complexed with copper. Two tryptophan analogues, 5-hydroxy-L-tryptophan (5-HTP) and DL-5-methoxytryptophan (5-MTP), were also evaluated as postcolumn reagents. 5-MTP was found to be a suitable alternative to L-Trp for the detection of these mono-amino sugars. Detection limits for D-glucosamine, D-galactosamine, and D-mannosamine are in the range of 0.15-0.30 nmol injected.     

High-performance liquid chromatography with sequential injection for online precolumn derivatization of some heavy metals

HPLC was coupled with sequential injection (SI) for simultaneous analyses of some heavy metals, including Co(II), Ni(II), Cu(II), and Fe(II). 2-(5-Nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (nitro-PAPS) was employed as a derivatizing reagent for sensitive spectrophotometric detection by online precolumn derivatization. The SI system offers an automated handling of sample and reagent, online precolumn derivatization, and propulsion of derivatives to the HPLC injection loop. The metal-nitro-PAPS complexes were separated on a C(18)-muBondapak column (3.9x300 mm(2)). Using the proposed SI-HPLC system, determination of four metal ions by means of nitro-PAPS complexes was achieved within 13 min in which the parallel of derivatization and separation were processed at the same time. Linear calibration graphs were obtained in the ranges of 0.005-0.250 mg/L for Cu(II), 0.007-1.000 mg/L for Co(II), 0.005-0.075 mg/L for Ni(II), and 0.005-0.100 mg/L for Fe(II). The system provides means for automation with good precision and minimizing error in solution handling with the RSD of less than 6%. The detection limits obtained were 2 microg/L for Cu(II) and Co(II), and 1 microg/L for Ni(II) and Fe(II). The method was successfully applied for the determination of metal ions in various samples, including milk powder for infant, mineral supplements, local wines, and drinking water.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

Precolumn derivatization liquid chromatography method for analysis of dietary supplements for glucosamine: single laboratory validation study

A precolumn derivatization liquid chromatography (LC) method was developed for the analysis of various dietary supplement formulations and raw materials for glucosamine. A 1 mL sample or standard water solution (containing about 0.05 mg glucosamine) was mixed with 1 mL pH 8.3 buffer, 1 mL 5% phenylisothiocyanate methanolic solution, and derivatized at 80 degrees C in a water bath for 30 min. After derivatization, the solution was cooled in a cold water bath and centrifuged at 3000-5000 rpm. The clear upper layer was ready for injection. The LC system was equipped with a C18 reversed-phase column and an ultraviolet detector set at 240 nm. The column was developed with a linear gradient composed of 0.1% phosphoric acid in deionized water and 0.1% phosphoric acid in methanol. The method was subjected to Single Laboratory Validation. The method precision was 0.50% relative standard deviation, accuracy was less than +/-1.5%, method linearity in the range 0-2 mg glucosamine/mL was 1.00, the detection limit was 0.0705 microg/mL, and the quantitation limit was 0.235 microg/mL. Chondroitin sulfate, amino acids, and excipients did not interfere with glucosamine testing. After derivatization, both standard and sample preparations were stable for at least 48 h. Due to its high sensitivity, this method can be used to assay glucosamine in functional foods and pet foods. The validation data will be published separately.     

Determination of zinc diethyldithiocarbamate released into artificial sweat from natural rubber latex vulcanizates by HPLC

A simple high-performance liquid chromatography method is adopted in order to quantitate the amount of zinc diethyldithiocarbamate (ZDEC) released into artificial sweat from natural rubber latex vulcanizates. The artificial sweat is extracted with dichloromethane, and the residue is recovered and re-dissolved in a known quantity of dichloromethane. ZDEC is quantitated as its copper complex by reacting with copper(II) sulphate. A reversed-phase C18 column and detection wavelength of 435 nm are used to measure the copper-dithiocarbamate complex. The procedure is repeated with cobalt(II) chloride, and the amount of ZDEC obtained by both the methods is compared. It is found that the recovery of ZDEC from the artificial sweat is high when copper(II) sulphate is used, indicating that the copper(II) sulphate is a better complexing agent than cobalt(II) chloride under the conditions used in the present study. The limits of detection and the quantitation of ZDEC are found to be 0.25 and 0.86 microg/mL, respectively. The present method, based on precolumn derivatization using copper(II) sulphate, facilitates the quantitation of ZDEC in latex products.     

Electrically driven microseparation methods for pesticides and metabolites: V. Micellar electrokinetic capillary chromatography of aniline pesticidic metabolites derivatized with fluorescein isothiocyanate and their detection in real water at low levels by laser-induced fluorescence

Anilines are important pollutants occurring in the environment as industrial discharges as well as the transformation products (i.e., metabolites) of a wide variety of commonly used pesticides. In this report, we describe the precolumn derivatization of anilines with fluorescein isothiocyanate (FITC) and their subsequent separation and detection by capillary electrophoresis-laser induced fluorescence (CE-LIF) detection. The FITC-aniline derivatives were readily detected at the 10(-10) M level. This limit of detection (LOD) was achieved in the presence of glycosidic surfactants complexed with borate at alkaline pH yielding the so-called in situ charged micelles. The glycosidic surfactants evaluated were n-octyl- and n-nonylglucopyranoside. Furthermore, and under optimum conditions, the FITC precolumn derivatization of the anilines was performed in real water (e.g., tap and lake water) spiked with anilines at the LOD level. The water matrices showed marginal effects on the extent of derivatization at the LOD level, and the possible interferents in the water samples did not affect the FITC-solute signal due to the selectivity of the derivatization and detection schemes. Besides filtration from microparticles, the real water samples did not necessitate extensive sample cleanup prior to derivatization.     

Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study

A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.     

Micellar Enhanced Analytical Application of Bismuthiol-II for the Spectrophotometric Determination of Trace Copper in Nutritional Matrices

 A simple spectrophotometric method for the determination of copper is described herewith, based on the formation of colored species of Cu (II) with 5-mercapto-3-phenyl-1,3,4-thiadiazole-2-thione (Bismuthiol II) in the presence of a neutral surfactant, Triton X-114. The yellow colored complex of Cu (II)–Bismuthiol-II–Triton X-114 shows maximum absorbance at 395 nm in water. The detection limit of the method is 0.03 mg/l while the Beer’s law is obeyed up to 1.2 mg/l of the analyte in an aqueous medium. The validity of the method has been examined for the determination of copper in wines, food supplements and raisins. The results obtained were in good agreement with those obtained using flame atomic absorption spectrometry (FAAS). The method thus constitutes a handy alternative for the determination of copper (II) in nutritional samples. Received May 16, 2001; accepted December 10, 2001; published online June 24, 2002     

Single laboratory validation of a method for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by high-performance liquid chromatography with FMOC-Su derivatization

Single laboratory validation of a method for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by with high-performance liquid chromatography FMOC-Su derivatization. Tests with 2 blank matrixes containing SAMe, vitamin C, citric acid, chondroitin sulfates, methylsulfonylmethane, lemon juice concentrate, and other potential interferents showed the method to be selective and specific. Eight calibration curves prepared over 7 working days indicated excellent reproducibility with the linear range at least over 2.0-150 microg/mL, and determination coefficients >0.9999. Average spike recovery from the blank matrix (n = 8 over 2 days) was 93.5, 99.4, and 100.4% at respective spike levels of 15, 100, and 150%, and from the sample matrix containing glucosamine (n = 3) was 99.9 and 102.8% at respective levels of 10 and 40%, with relative standard deviations <0.9%. The method was also applied to 12 various glucosamine finished products and raw materials. The stability tests confirmed that glucosamine-FMOC-Su derivative once formed is stable at room temperature for at least 5 days. Limit of quantitation was 1 microg/mL and limit of detection was 0.3 microg/mL. The method is ready to proceed for the collaborative study.     

Ion chromatography with the indirect ultraviolet detection of alkali metal ions and ammonium using imidazolium ionic liquid as ultraviolet absorption reagent and eluent

Indirect ultraviolet detection was conducted in ultraviolet‐absorption‐agent‐added mobile phase to complete the detection of the absence of ultraviolet absorption functional group in analytes. Compared with precolumn derivatization or postcolumn derivatization, this method can be widely used, has the advantages of simple operation and good linear relationship. Chromatographic separation of Li⁺, Na⁺, K⁺, and NH₄⁺ was performed on a carboxylic acid base cation exchange column using imidazolium ionic liquid/acid/organic solvent as the mobile phase, in which imidazolium ionic liquids acted as ultraviolet absorption reagent and eluting agent. The retention behaviors of four kinds of cations are discussed, and the mechanism of separation and detection are described. The main factors influencing the separation and detection were the background ultraviolet absorption reagent and the concentration of hydrogen ion in the ion chromatography‐indirect ultraviolet detection. The successful separation and detection of Li⁺, Na⁺, K⁺, and NH₄⁺ within 13 min was achieved using the selected chromatographic conditions, and the detection limits (S/N = 3) were 0.02, 0.11, 0.30, and 0.06 mg/L, respectively. A new separation and analysis method of alkali metal ions and ammonium by ion chromatography with indirect ultraviolet detection method was developed, and the application range of ionic liquid was expanded.     

Determination of aflatoxins and ochratoxin A in ginseng and other botanical roots by immunoaffinity column cleanup and liquid chromatography with fluorescence detection

Mycotoxins are toxic secondary metabolites produced by certain molds and are common contaminants of many important food crops, such as grains, nuts, and spices. Some mycotoxins are found in fruits, vegetables, and botanical roots. These contaminants have a broad range of toxic effects, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins, such as the aflatoxins (AFL). Our research focused on method development for 2 of these toxins, AFL and ochratoxin A (OTA), in ginseng and other selected botanical roots. Methods using an immunoaffinity column (IAC) cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Two types of IAC cleanup were evaluated: IAC for AFL, and IAC for both AFL and OTA. Three derivatization techniques to enhance the fluorescence of the AFL were compared: precolumn trifluoroacetic acid, postcolumn bromination, and postcolumn ultraviolet irradiation. No derivatization was needed for OTA. Results for AFL using the single analyte IAC cleanup and the 3 derivatization techniques were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added AFL for ginseng at levels from 2 to 16 ng/g were about 80%. Using IAC cleanup for both AFL and OTA recoveries of added AFL for ginseng at 4-16 ng/g were about 70%, and for ginger, licorice, and kava-kava were about 60%. Recoveries of added OTA for ginseng, ginger, and echinacea at 4 ng/g were about 55%.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

Using HPLC for the determination of platinum drugs in biological matrixes after derivatization with diethyldithiocarbamate

Challenges and pitfalls in the application of diethyldithiocarbamate derivatization for LC analysis of cisplatin and oxaliplatin, as well as the suitability of this method for different biological matrices with implications for use in routine practice have been identified. The LC of platinum drugs presents a significant challenge. They are polar compounds with poor retention on reverse phase packings. Cisplatin also exhibits poor absorption in UV and ionization in mass spectrometry. Therefore, we developed and optimized a derivatization approach for the LC analysis of total platinum in plasma, plasma ultrafiltrate, peritoneal fluid, and urine. Derivatization in urine proved to be difficult due to the complexity of the matrix, and extended testing was required. Our results highlight the important issues affecting the efficiency, reliability, and suitability of platinum drug derivatization. Although precolumn derivatization is less selective than its postcolumn counterpart, the application of precolumn derivatization is a simple, rapid, and universal approach for the determination of platinum drugs by HPLC. One of its major advantages is that it allows a more affordable analysis using UV detection without the need for additional high-end instrumentation such as a MS detector.     

Determination of coenzyme Q10 in over-the-counter dietary supplements by high-performance liquid chromatography with coulometric detection

A rapid and sensitive method is described for the determination of coenzyme Q10 (Q10) in over-the-counter dietary supplements by automated high-performance liquid chromatography (HPLC) with coulometric detection. Sample solutions of powder-filled capsules, oil-based softgels, and tablets were prepared by serial dilution with 1-propanol. After dilution, a known volume of sample solution containing Q10 and the internal standard, coenzyme Q9 (Q9), was directly injected into the HPLC system. Most of electrochemically active compounds in the injection were oxidized at the precolumn conditioning cell and postcolumn guard cell. Q9 and Q10 were monitored at an analytical cell that contained 2 coulometric electrodes, where Q9 and Q10 were reduced to the corresponding ubiquinol-9 and -10 and then oxidized to produce currents. This method produced a linear detector response for peak height measurements over the concentration range of 0.05-8 microg/mL (r > 0.999). The lower limit of detection was 5 ng/mL (signal-to-noise ratio, > or =3). The mean recovery was 98.9 +/- 0.6%; coefficients of variation for intra- and interday precisions were 1.8-4.0%. The proposed method was successfully applied to the determination of Q10 in marketed products.     

Determination of disulfiram and two of its metabolites in urine by reversed-phase liquid chromatography and spectrophotometric detection after post-column complexation

A selective method was developed for the determination of disulfiram and two of its metabolites, diethyldithiocarbamate (DTC) and copper (II) diethyldithiocarbamate [Cu(DTC)2], in complex (biological) samples by reversed-phase liquid chromatography (RP-LC) with post-column derivatization. In the first step, DTC is converted into lead (II) diethyldithiocarbamate [Pb (DTC)2] by adding lead (II) acetate. Disulfiram, Pb (DTC)2 and Cu (DTC)2 can be easily pre-concentrated on C18-bonded silica. After separation by isocratic RP-LC, derivation takes place in two solid state post-column reactors packed with metallic copper and copper (II) phosphate. Disulfiram reacts with metallic copper to form Cu (DTC)2. The same product is obtained by the ligand-exchange reaction between Pb (DTC)2 and copper (II) phosphate. Cu (DTC)2 can be detected selectively at 435 nm with good sensitivity (molar absorptivity, epsilon = 13,000). The derivatization reactions proceed rapidly and quantitatively, which was confirmed by comparison of absorption spectra. The applicability of this method is demonstrated for undiluted urine samples which, apart from the addition of lead (II) acetate and pre-concentration of C18-bonded silica, require no clean-up procedure.     

Liquid chromatography and postcolumn indirect detection of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] and its metabolite aminomethylphosphonic acid (AMPA) were separated and detected by a postcolumn indirect detection strategy. Separation can be done on a cation-exchange column, where glyphosate elutes before AMPA, or on an anion-exchange column, where the elution order is reversed. Detection was achieved by using a fluorescent Al(3+)-morin postcolumn reagent. When the postcolumn reagent combines with the column effluent in a mixing tee, the fluorescence decreases in the presence of both analytes. Variables affecting the postcolumn indirect fluorescence detection were established and optimized; the major factors were postcolumn pH and volume and temperature of the postcolumn reaction coil. Detection limits, defined as three times the background noise, for glyphosate and AMPA separated on an anion-exchange column were 14 and 40 ng, respectively.     

Subminute and sensitive determination of the neurotransmitter serotonin in urine by capillary electrophoresis with laser-induced fluorescence detection

In this work, a sub-minute and sensitive capillary electrophoresis with laser-induced fluorescence (CE-LIF) method was developed for the analysis and quantitation of the neurotransmitter 5-hydroxytryptamine (5-HT) or serotonin in urine. The method involves precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) using an excitation light from an argon ion laser of 488 nm and a 520 nm band pass emission filter. Different variables that affect derivatization (pH, FITC concentration, reaction time and temperature) and separation (buffer concentration, pH, applied voltage and injection time) were studied. The linear dynamic range obtained was between 0 and 188 nM with a detection limit of 16 nm with a RSD between 2 and 9%. The applicability of the proposed method was demonstrated by analysis of 5-HT in human urine, establishing a concentration of 57 nM in control urine. The method was validated by standard-addition methodology.     

毛细管电泳发光二极管诱导荧光对免疫球蛋白G的检测

采用自行设计、组装的毛细管电泳光导纤维发光二极管诱导荧光检测装置,建立了一种直接测定免疫球蛋白G(IgG)的方法。以蓝色发光二极管(LED)为荧光检测器的激发光源,荧光素异硫氰酸酯(FITC)为柱前衍生试剂,采用毛细管区带电泳,以20 mmol/L硼砂缓冲溶液(pH9.2)为背景电解液进行分离检测。通过对衍生反应条件和电泳分离条件进行优化,确定了最佳实验条件,在该条件下,IgG的线性范围为4.5×10-8~1.2×10-6g/L,检出限为2.0×10-8g/L。该方法简单、高效、选择性好,无需前处理,可用于人血清中IgG含量的测定。     

HPLC determination of copper(II), cobalt(II) and iron(II) in pharmaceutical preparations using 2-acetylpyridine-4-phenyl-3-thiosemicarbazone derivatizing agent

Summary Cu(II) Co(II) and Fe(II) in pharmaceutical preparations were determined using precolumn derivatization and solvent extraction with 2-acetylpyridine-4-phenyl-3-thiosemicarbazone (APPT) as complexing reagent. Liquid chromatography (LC) was carried out on a reversed phase C-18, column. The complexes were eluted isocratically with acetonitrile-water containing sodium acetate and tetrabutyl ammonium bromide as ion-pairing agents. The results obtained were compared with those using atomic absorption.