Fluorescent Labeling Reagent Research Articles

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

9-Fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: A human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography–tandem MS/MS (LC–MS/MS) method. Following liquid–liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01μg/mL. Also the method is linear over the concentrations range of 0.01–32μg/mL of VPA in human serum using 100μL serum sample and 5μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.

Development of a fluorescence-based microplate method for the determination of volatile fatty acids in anaerobically digested and sewage sludges

This paper presents a simple, accurate and multi-sample method for the determination of volatile fatty acids (VFAs) thanks to a 96-well microplate technique. A procedure using an activating reagent of the carboxylic function (water-soluble carbodiimide EDC) and a fluorescent amino labeling reagent (N-(1-naphthyl)ethylenediamine, EDAN) allows the formation of an isoindole derivative that needs to be separated from initial fluorescent amine for efficient VFAs determination. Isolation of these fluorescent VFA-derivatives was carried out by use of the fluorescent quenching of EDAN with o-phthaldialdehyde (OPA). Quenching was most efficient at pH around 7 and by heating at 40°C within the microplate reader. This optimized procedure has been applied to various carboxylic acids and other organic compounds, demonstrating that VFA exhibit the highest fluorescence responses with homogeneous results for the main ones (acetic, propionic and butyric acid, all mass concentration expressed as acetic acid equivalents). This protocol was calibrated against acetic acid and determination of VFA was thus possible in the range 3.9–2000mgL−1 (acetic acid equivalents). Subsequent application to real samples (sewage sludges or anaerobically digested samples) and comparison to gas chromatography analyses gave accurate results, proving the great potential of our high-throughput microplate-based technique for the analysis of VFA.

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn–Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose–aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.

Highly selective and sensitive determination of free and total amino acids in Apocynum venetum L. (Luobuma tea) by a developed HPLC–FLD method coupled with pre-column fluorescent labelling

Amino acids (AA) are important chemical constituents of tea leaves remarkably influencing the quality of tea. In this study, free AA and total AA in Apocynum venetum L. (Luobuma tea) were estimated by HPLC equipped with fluorescent detector using 2-[2-(7H-dibenzo[a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEEC) as a fluorescent labelling reagent. Different parameters for derivatization and separation were optimized. AA were rapidly derivatized within 3 min at room temperature with DBCEEC. In conjunction with a gradient elution, a baseline resolution of 20 analytes was achieved on a reversed-phase Hypersil BDS C18 column. LC separation for the derivatized AA showed good reproducibility. Twenty AA were detected and showed significant linear responses with correlation coefficients (>0.9992). This developed method offered the low detection limit of 2.88–23.4 fmol.

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for HPLC determination of amino acid neurotransmitters in the cerebral cortex of mice

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L(-1) pH 7.8 boric acid buffer. The separation was performed on a C(18) column with methanol-water-buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L(-1) H(3)Cit-0.10 mol L(-1) NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L(-1). The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.

Development of a New HPLC Method with Precolumn Fluorescent Derivatization for Rapid, Selective and Sensitive Detection of Triterpenic Acids in Fruits

Triterpenic acids are widespread in plants and have multiplicity of biological properties. Unfortunately the method for accurate analysis of these compounds remains poorly investigated. This study proposed a highly sensitive and selective precolumn derivatization method for accurate determination of five triterpenic acids (betulinic acid, betulonic acid, maslinic acid, ursolic acid and oleanolic acid) in fruits using acridone-9-ethyl-p-toluenesulfonate (AETS) as fluorescent labeling reagent by HPLC with fluorescence detection (FLD). Response surface methodology was employed to optimize the derivatization reaction, ensuring the sufficient labeling of the analyzed components. The rapid separation of five triterpenic acids could be achieved in as little as 16 min. This developed method offered the exciting detection limits of 1.68-2.04 ng/mL. When applied to several popular fruits in China, it revealed satisfactory applicability and reproducibility. This developed method also exhibits powerful potential for accurate detection of triterpenic acids from other foodstuffs and nature products.

Determination of amino acids in rat brain microdialysate with 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate as labeling reagent by high performance liquid chromatographic fluorescence detection and mass spectrometric identification

A high performance liquid chromatography method for the determination of 20 amino acids (AAs), using 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate (DBCEOC–Cl) as a novel fluorescent labeling reagent, has been developed and applied for the analysis of AAs in rat brain microdialysate. The simultaneous separation of 20 AA derivatives was achieved on a Hypersil BDS C 18 column with gradient elution. And the identification of AA derivatives was carried out by on-line electrospray ionization mass spectrometry in positive ion mode. The AA derivatives were detected with excitation and emission at 300 nm and 395 nm, respectively. Excellent linear responses were observed with coefficients of >0.9988. The detection limits ranged from 0.217 to 4.75 nmol/L, at a signal-to-noise ratio of 3. The intra-day and inter-day precision for each AA was <3.2% and <4.3%, respectively. The mean recoveries for all AAs studied were in the range of 92.5–105.6%. Good compositional data could be obtained from as little as 15 μL of microdialysate. Facile DBCEOC–Cl derivatization coupled with high performance liquid chromatography and fluorescence detection allowed the development of a highly sensitive method for the quantitative analysis of trace levels of AAs from microdialysate.

Fluorescence determination of d- and l-tryptophan concentrations in rat plasma following administration of tryptophan enantiomers using HPLC with pre-column derivatization

Similar to l-tryptophan ( l-Trp), d-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life ( t 1/2) between Trp enantiomers was investigated by following the alterations in the plasma concentration of d- or l-Trp after intraperitoneal ( i.p.) administration of each enantiomer to male Sprague–Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-( N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole ( R(−)-DBD-PyNCS). The t 1/2 value of d-Trp was significantly smaller than that of l-Trp, suggesting that d-Trp was eliminated from the plasma more rapidly than l-Trp. In addition, a significant increase in the plasma concentration of l-Trp was observed following administration of d-Trp, whereas no d-Trp was detected after l-Trp administration. Furthermore, the increase in the plasma concentration of l-Trp was significantly suppressed by pretreatment with an inhibitor of d-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of d-Trp to l-Trp in vivo.

Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.

Enantiomeric separation of d,l‐tryptophan and d,l‐kynurenine by HPLC using pre‐column fluorescence derivatization with R(−)‐DBD‐PyNCS

The enantiomeric separation of D,L-tryptophan (Trp) and D,L-kynurenine (KYN) was investigated by high-performance liquid chromatography using pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7- (N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS-3 column (250 x 2.0 mm; i.d., 3 μm), four fluorescence peaks of D- and L-Trp as well as D- and L-KYN derivatized with R(-)-DBD-PyNCS were clearly observed, and their chemical structures were confirmed by HPLC-time-of-flight-mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O-CH₃CN (60:40), and the separation factors of D,L-Trp and D,L-KYN derivatized with R(-)-DBD-PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3-0.5 pmol (signal-to-noise ratio of 3).

Liquid Chromatographic Determination of Endogenous Phytohormones in Vegetable Samples Based on Chemical Derivatization with 6-Oxy(acetylpiperazine) Fluorescein

In phytohormone analysis, mass spectrometry (MS)-based methods are primary and powerful tools. However, complex sample preparation and high cost are problems for their application. As a complement for MS-based methods, a new fluorescent labeling reagent for carboxylic acids, 6-oxy(acetylpiperazine) fluorescein (APF), has been used for the determination of endogenous phytohormones, including indolebutyric acid, 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. The derivatization yield was maximized by optimizing derivatization conditions in detail, and the derivatives of three phytohormones could be separated completely in 15 min on a C18 column with fluorescence detection at lambda(ex)/lambda(em) = 467/512 nm. The derivatization limits could reach 0.1 microM, and the detection limits (signal-to-noise ratio = 3) were 4.43-14.2 nM. The proposed method has been applied to the determination of the exogenous phytohormones in the crude extracts of vegetable samples without extra purification and enrichment with recoveries of 94.2-102.4%.

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 15 min in borate buffer (20 mmol l(-1), pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 degrees C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l(-1), pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7-4.6% and 0.4-5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6-125 nmol (mg creatinine)(-1) (mean+/-SD: 21.6+/-26.6 nmol (mg creatinine)(-1), n=20).

1,3,5,7-Tetramethyl-8-aminozide-difluoroboradiaza-s-indacene as a new fluorescent labeling reagent for the determination of aliphatic aldehydes in serum with high performance liquid chromatography

A BODIPY-based fluorescent derivatization reagent with a hydrazine moiety, 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), has been designed for aldehyde labeling. An increased fluorescence quantum yield was observed from 0.38 to 0.94 in acetonitrile when it reacted with aldehydes. Twelve aliphatic aldehydes from formaldehyde to lauraldehyde were used to evaluate the analytical potential of this reagent by high performance liquid chromatography (HPLC) on C(18) column with fluorescence detection. The derivatization reaction of BODIPY-aminozide with aldehydes proceeded at 60 degrees C for 30min to form stable corresponding BODIPY hydrazone derivatives in the presence of phosphoric acid as a catalyst. The maximum excitation (495nm) and emission (505nm) wavelengths were almost the same for all the aldehyde derivatives. A baseline separation of all the 12 aliphatic aldehydes (except formaldehyde and acetaldehyde) is achieved in 20min with acetonitrile-tetrahydrofuran (THF)-water as mobile phase. The detection limits were obtained in the range from 0.43 to 0.69nM (signal-to-noise=3), which are better than or comparable with those obtained by the existing methods based on aldehyde labeling. This reagent has been applied to the precolumn derivatization followed with HPLC determination of trace aliphatic aldehydes in human serum samples without complex pretreatment or enrichment method.

Critical Cysteine Residues of Kelch-Like ECH-Associated Protein 1 in Arsenic Sensing and Suppression of Nuclear Factor Erythroid 2-Related Factor 2

Arsenic activates nuclear factor erythroid 2-related factor 2 (Nrf2) to induce phase II and antioxidative genes. Here we analyzed arsenic-Kelch-like ECH-associated protein 1 (Keap1) cysteine thiol interaction in Nrf2 activation. Arsenic-based Nrf2 activators, fluorescent biarsenical labeling reagent (FlAsH) and phenylarsine oxide (PAO), were used to probe binding of arsenic to Keap1. Strong fluorescence was observed on binding of FlAsH to purified Keap1. Pretreatment with arsenic, tert-butylhydroquinone (tBHQ), or 2,3-dimercaptopropanol significantly reduced the fluorescent signal. PAO affinity beads effectively pulled down Keap1 in vitro and from hepa1c1c7 cells. Arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pulldown. Furthermore, arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1. Thus, arsenic, FlAsH, and PAO, as well as tBHQ and cadmium, bind to Keap1 cysteine thiols in a similar fashion. All the domains of Keap1 bound PAO, and the linker region exhibited the highest binding activity. The function of arsenic-Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of Cys273 or Cys288 in the linker region resulted in high level basal expression of Nrf2 protein. Mutation of Cys151 abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic-induced expression of Nrf2 target genes. The study shows an important role of Cys273 and Cys288 in the suppression of Nrf2 by Keap1 and a critical function of Cys151 in arsenic responsiveness. Our findings support a model in which arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signal transduction.

Femtomolar Concentration Detection Limit and Zeptomole Mass Detection Limit for Protein Separation by Capillary Isoelectric Focusing and Laser-Induced Fluorescence Detection

Fluorescence tends to produce the lowest detection limits for most forms of capillary electrophoresis. Two issues have discouraged its use in capillary isoelectric focusing. The first issue is fluorescent labeling of proteins. Most labeling reagents react with lysine residues and convert the cationic residue to a neutral or anionic product. At best, these reagents perturb the isoelectric point of the protein. At worse, they convert each protein into hundreds of different fluorescent products that confound analysis. The second issue is the large background signal generated by impurities within commercial ampholytes. This background signal is particularly strong when excited in the blue portion of the spectrum, which is required by many common fluorescent labeling reagents. This paper addresses these issues. For labeling, we employ Chromeo P540, which is a fluorogenic reagent that converts cationic lysine residues to cationic fluorescent products. The reaction products are excited in the green, which reduces the background signal generated by impurities present within the ampholytes. To further reduce the background signal, we photobleach ampholytes with high-power photodiodes. Photobleaching reduced the noise in the ampholyte blank by an order of magnitude. Isoelectric focusing performed with photobleached pH 3-10 ampholytes produced concentration detection limits of 270 +/- 25 fM and mass detection limits of 150 +/- 15 zmol for Chromeo P540 labeled beta-lactoglobulin. Concentration detection limits were 520 +/- 40 fM and mass detection limits were 310 +/- 30 zmol with pH 4-8 ampholytes. A homogenate was prepared from a Barrett's esophagus cell line and separated by capillary isoelectric focusing, reproducibly generating dozens of peaks. The sample taken for the separation was equal to the labeled protein homogenate from three cells.

Labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

The goal of our study was to develop a method for molecular imaging of the gastrointestinal tract using an infrared fluorescence endoscope (IRFE) and antibodies labeled with an indocyanine green (ICG) derivative to detect cancerous tissue. The IRFE comprised an infrared endoscope equipped with excitation (710-790 nm) and barrier (810-920 nm) filters. We developed ICG-N-hydroxysulfosuccinimide ester (ICG-sulfo-OSu) and 3-ICG-acyl-1,3-thiazolidine-2-thione (ICG-ATT) as infrared fluorescent-labeling reagents, and anti-human carcinoembryonic antigen (CEA) antibody and MUC1 antibody were labeled with the ICG-derivatives. Freshly resected specimens of gastric cancer were observed by IRFE after reaction with ICG-derivative-labeled antibodies. Positive fluorescence was observed at the tumor location by IRFE, and the immunofluorescent images correlated well with the tumor sites. The immunofluorescence studies suggested that the intensity of the infrared fluorescence of the ICG-ATT-labeled MUC1 antibody is stronger than the ICG-sulfo-OSu-labeled MUC1 antibody. We concluded that specific antibodies for gastrointestinal cancer labeled with an ICG-derivative accompanied by a reinforcing agent and an optimal electronic device can generate a strong enough fluorescent signal to visualize cancer proteins.

Determination of N-Ethylglycine in Urine of Cancer Patients with Metastatic Bone Disease by HPLC Using 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl Chloride as a Fluorescent Labeling Reagent

A simple and highly sensitive HPLC for the determination of N-ethylglycine in urine was developed. The labeling reaction of N-ethylglycine with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride was carried out at 70 degrees C for 15 min at pH 9.0. The fluorescent derivative was separated on a reversed-phase column and detected at excitation and emission wavelengths of 320 and 400 nm, respectively. The detection limit of N-ethylglycine was 15 fmol (S/N = 3). The recovery of N-ethylglycine added to urine was 101.9%. The concentration of N-ethylglycine in urine of cancer patients with metastatic bone disease was 11.3 +/- 22.0 nmol/mg creatinine, and that of normal subject was 0.4 +/- 0.4 nmol/mg creatinine.

Simultaneous analysis of 1,2,3,4-tetrahydroisoquinolines by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatographic method for the simultaneous analysis of 1,2,3,4-tetrahydroisoquinolines (TIQs) in the rat brain was developed. 1,2,3,4-Tetrahydroisoquinoline (TIQ), 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BeTIQ) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 50 °C for 15 min at pH 8.5. The fluorescent derivatives were separated on a reversed-phase column by gradient elution using (A) water–(B) acetonitrile/methanol (55:45) at 55 °C and detected by fluorescence measurement at 318 nm (excitation) and 398 nm (emission). The detection limits (signal-to-noise ratio = 3) were 8–9 fmol per injection. The relative standard deviations ( n = 6) of TIQs were 2.6–10.5% and the recoveries were 87.6, 101.8 and 75.2%, respectively. The concentrations of TIQ, 1-MeTIQ and 1-BeTIQ in normal rat brains ( n = 6) were 0.7 ± 0.3 (0.10 ± 0.04), 3.4 ± 1.5 (0.50 ± 0.22) and 1.3 ± 1.8 pmol/g (0.30 ± 0.41 ng/g), respectively.

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.