Xianen Zhao

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C(18) column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2x10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N=3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry.

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M+H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of CO bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted derivatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono-1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC)=2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C(18) column. Detection limits were calculated from 1.0pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV<7.5. The mean interday precision for all standards was <10% of the expected concentration. Excellent linear responses were observed with coefficients of >0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples.

An amperometric sensor for the determination of benzophenone in food packaging materials based on the electropolymerized molecularly imprinted poly-o-phenylenediamine film

Benzophenone is one of the most commonly used photoinitiators of UV-cured inks on food packaging materials and can migrate into foodstuffs. In this study, an amperometric benzophenone sensor based on molecularly imprinted polymer (MIP) was successfully constructed for the first time. The sensor was prepared by electropolymerizing o-phenylenediamine (o-PD) on a glassy carbon electrode (GCE) in the presence of template benzophenone, and then removing the template by immersing the poly-o-phenylenediamine film-modified GCE in ethanol. The molecularly imprinted sensor was tested in the presence or absence of benzophenone by cyclic voltammetry and linear sweep voltammetry to verify the changes in the redox peak currents of potassium ferricyanide. The sensor responded sensitively to benzophenone over a linear range of 0.05-5 μM with a detection limit of 10 nM. The imprinted sensor showed high recognition ability for benzophenone and was successfully applied to the determination of benzophenone in food packaging material samples.

In situ derivatization-ultrasound-assisted dispersive liquid-liquid microextraction for the determination of neurotransmitters in Parkinson's rat brain microdialysates by ultra high performance liquid chromatography-tandem mass spectrometry.

Simultaneous monitoring of several neurotransmitters (NTs) linked to Parkinsons disease (PD) has important scientific significance for PD related pathology, pharmacology and drug screening. A new simple, fast and sensitive analytical method, based on in situ derivatization-ultrasound-assisted dispersive liquid-liquid microextraction (in situ DUADLLME) in a single step, has been proposed for the quantitative determination of catecholamines and their biosynthesis precursors and metabolites in rat brain microdialysates. The method involved the rapid injection of the mixture of low toxic bromobenzene (extractant) and acetonitrile (dispersant), which containing commercial Lissamine rhodamine B sulfonyl chloride (LRSC) as derivatization reagent, into the aqueous phase of sample and buffer, and the following in situ DUADLLME procedure. After centrifugation, 50μL of the sedimented phase (bromobenzene) was directly injected for ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection in multiple reaction monitoring (MRM) mode. This interesting combination brought the advantages of speediness, simpleness, low matrix effects and high sensitivity in an effective way. Parameters of in situ DUADLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ DUADLLME were found to be 30μL of microdialysates, 150μL of acetonitrile containing LRSC, 50μL of bromobenzene and 800μL of NaHCO3-Na2CO3 buffer (pH 10.5) for 3.0min at 37°C. Under the optimized conditions, good linearity was observed with LODs (S/N>3) and LOQs (S/N>10) of LRSC derivatized-NTs in the range of 0.002-0.004 and 0.007-0.015 nmol/L, respectively. It also brought good precision (3.2-12.8%, peak area CVs%), accuracy (94.2-108.6%), recovery (94.5-105.5%) and stability (3.8-8.1%, peak area CVs%) results. Moreover, LRSC derivatization significantly improved chromatographic resolution and MS detection sensitivity of NTs when compared with the reported studies through the introduction of a permanent charged moiety from LRSC into NTs. Taken together, this in situ DUADLLME method was successfully applied for the simultaneous determination of six NTs in biological samples.

Monitoring the contents of six steroidal and phenolic endocrine disrupting chemicals in chicken, fish and aquaculture pond water samples using pre-column derivatization and dispersive liquid-liquid microextraction with the aid of experimental design methodology.

This research established a sensitive and efficient pre-column derivatization HPLC method based on dispersive liquid-liquid microextraction (DLLME) for the simultaneous determination of six steroidal and phenolic endocrine disrupting chemicals (EDCs). In this study, EDCs were firstly labeled by the derivatization reagent 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) and then extracted by DLLME. The response surface methodology was employed to investigate the key parameters of pre-column derivatization and DLLME. Under the optimal conditions, a good linear relationship between the peak area and the concentration of analytes was observed with correlation coefficients of >0.9991. Limits of detection for all EDCs derivatives were achieved within the range of 0.02-0.07 μg L(-1). The proposed method has the advantages of simple operation, low consumption of organic solvent, saving time, low output limit and good selectivity. When applied to several food and water samples analysis, it demonstrated good applicability for the determination of EDCs.

Determination of bisphenol A, 4‐octylphenol, and 4‐nonylphenol in soft drinks and dairy products by ultrasound‐assisted dispersive liquid–liquid microextraction combined with derivatization and high‐performance liquid chromatography with fluorescence detection

A novel hyphenated method based on ultrasound-assisted dispersive liquid-liquid microextraction coupled to precolumn derivatization has been established for the simultaneous determination of bisphenol A, 4-octylphenol, and 4-nonylphenol by high-performance liquid chromatography with fluorescence detection. Different parameters that influence microextraction and derivatization have been optimized. The quantitative linear range of analytes is 5.0-400.0 ng/L, and the correlation coefficients are more than 0.9998. Limits of detection for soft drinks and dairy products have been obtained in the range of 0.5-1.2 ng/kg and 0.01-0.04 μg/kg, respectively. Relative standard deviations of intra- and inter-day precision for retention time and peak area are in the range of 0.47-2.31 and 2.76-8.79%, respectively. Accuracy is satisfactory in the range of 81.5-118.7%. Relative standard deviations of repeatability are in the range of 0.35-1.43 and 2.36-4.75% for retention time and peak area, respectively. Enrichment factors for bisphenol A, 4-octylphenol, and 4-nonylphenol are 170.5, 240.3, and 283.2, respectively. The results of recovery and matrix effect are in the range of 82.7-114.9 and 92.0-109.0%, respectively. The proposed method has been applied to the determination of bisphenol A, 4-octylphenol, and 4-nonylphenol in soft drinks and dairy products with much higher sensitivity than many other methods.

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.

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH(2)O-CO and CH(2)-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate (FMOC-Cl) as labeling reagents. The ratios I(BCEC)/I(BCEOC)=1.94-2.17 and I(BCEC)/I(FMOC)=1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C(8) column. Detection limits calculated from 0.50pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4fmol. The relative standard deviations for within-day determination (n=11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were <3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of >0.9996.

Determination of dopamine, serotonin, biosynthesis precursors and metabolites in rat brain microdialysates by ultrasonic-assisted in situ derivatization–dispersive liquid–liquid microextraction coupled with UHPLC-MS/MS

This paper, for the first time, reported a simple, rapid, sensitive and environmental friendly ultrasonic-assisted in situ derivatization-dispersive liquid-liquid microextraction (in situ UAD-DLLME) method followed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of dopamine (DA), serotonin (5-HT) and their biosynthesis precursors and metabolites in rat brain microdialysates. In this work, a commercial reagent, Lissamine rhodamine B sulfonylchloride (LRSC), was proposed as a derivatization reagent. The ionization efficiency of neurotransmitters was greatly enhanced through the introduction of a permanent charged moiety of LRSC into their derivatives during electrospray ionization MS (ESI-MS) analysis. Parameters of in situ UAD-DLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ UAD-DLLME were found to be as follows: a mixture of 150μL of acetonitrile (dispersant) containing LRSC (derivatization reagents) and 50μL of low toxic bromobenzene (extractant) was rapidly injected into an aqueous sample containing 30μL of microdialysate and 800μL of NaHCO3-Na2CO3 buffer solution (pH 10.5) at 37°C. After ultrasonication for 3min and centrifuging for 2min, the sedimented phase was conveniently injected for UHPLC-MS/MS analysis. Under the optimized conditions, good linearity was observed with the limits of detection (LODs, S/N>3) and limits of quantification (LOQs, S/N>10) in the range of 0.002-0.008 and 0.015-0.040nmol/L, respectively. Meanwhile, it also brought good results of precision (3.2-13.0%, peak area RSDs %), accuracy (86.4-112%), recovery (73.9-105%), matrix effect (86.2-105%), and stability (3.1-8.8%, peak area RSDs %). The developed method was successfully applied for the simultaneous determination of multiple neurotransmitters, their precursors and metabolites in brain microdialysates of normal and L-DOPA induced dyskinesias (LID) rats.

Determination of Free Fatty Acids from Soil and Bryophyte by HPLC with Fluorescence Detection and Identification with Mass Spectrometry

Abstract A simple and sensitive method for the determination of free fatty acids using acridone-9-ethyl- p -toluenesulfonate (AETS) as tagging reagent by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed. Studies on derivatization conditions indicate that free fatty acids react rapidly and smoothly with AETS in the presence of K 2 CO 3 catalyst in N , N -dimethylformamide (DMF) solvent at 85°C to give the corresponding sensitive fluorescence derivatives. The maximal derivatization yields are close to 100% with a six fold molar reagent excess. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Separation of the derivatized fatty acids exhibits a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C 8 column. The identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode. The contents of 19 free fatty acids in soil and three kinds of bryophytes ( Tree Moss, Entodon macropodus, Dicranum caesium ) were determined. The results indicate that the bryophyte plants enrich an abundance of free fatty acids from soil. The fluorescence excitation and emission wavelengths of derivatives were 404nm of λ ex and 440 nm of λ em , respectively. Excellent linear responses were observed with the linear correlation coefficients over 0.9996, and detection limits (at signal-to-noise of 3:1) are 12.3–43.7 fmol. The proposed method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.