You Jin-mao

Determination of Free Fatty Acids by High Performance Liquid Chromatography with Fluorescence Detection and Identification with Mass Spectrometry

Abstract On a reversed-phase Eclipse XDB-C8 column, 20 fatty acids were derivatized using 2-(2-(anthracen-10-yl)-1H-naphtho [2,3-d]imidazol-1-yl) ethyl-p-toluenesulfonate (ANITS) as the pre-column derivatization reagent and were separated by high performance liquid chromatography (HPLC) in conjunction with a gradient elution with fluorescence detection and mass spectrometric identification. The reagent was reacted with fatty acids in the presence of K2CO3 catalyst at 90°C min in N,N-dimethylformamide solvent. The fluorescence excitation and emission wavelength of the derivatives were λex = 250 nm and λem = 512 nm, respectively. The identification of fatty acid derivatives was performed by online MS/APCI (atmospheric pressure chemical ionization source) in positive model detection. All linear correlation coefficients for fatty acid derivatives are over 0.9999, and the detection limits, at a signal-to-noise rate of 3:1, are 24.8-98.8 fmol for labeled fatty acids. The established method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.

Mass Spectrometric Identification of Aliphatic Amines Using 2-(11H-benzo[a]carbazol-11-yl) Ethyl Chloroformate as Fluorescence Labeling Reagent

Abstract A simple and highly sensitive method based on the precolumn derivatization has been developed for the determination of aliphatic amines in food by using a nitrogen-containing 2-(11 H -benzo[a]carbazol-11-yl)ethyl chloroformate (BCEC-Cl) as a fluorescence labeling reagent followed by high-performance liquid chromatography (HPLC) with fluorescence detection, and online postcolumn MS identification. The stable derivatives can be obtained by the labeling reaction of BCEC-Cl with amines in the presence of a borate buffer (pH 9.0) within 3 min. The maximum excitation and emission wavelengths are at λ ex = 279 nm and λ em = 380 nm respectively. Separation of 12 amine-derivatives was achieved by a gradient elution on a reversed-phase Eclipse XDB C 8 (150 mm × 4.6 mm, 5 μm) column with a good baseline resolution. The identification of amine-derivatives is carried out by online postcolumn MS using atmospheric pressure chemical ionization (APCI) as an ion source in positive-ion detection mode. The established method exhibits excellent reproducibility and recovery. Excellent linear responses are observed with coefficients > 0.9996, detection limits (at signal-to-noise ratio of 3:1) are at 1.77–14.4 f mol levels.

4-(1-METHYL-1H-PHENANTHRO[9,10-d]IMIDAZOLE-2-) BENZOIC ACID (MPIBA) AND ITS APPLICATION FOR DETERMINATION OF AMINES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION AND IDENTIFICATION WITH MASS SPECTROSCOPY/ATMOSPHERIC PRESSURE CHEMICAL IONIZATION

A simple, sensitive, and mild method for the determination of amines based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC · HCl) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acid amides with labeling reagent 4-s(1-methyl-1H-phenanthro[9,10-d]imidazole-2-) benzoic acid (MPIBA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in acetonitrile to give the corresponding sensitively fluorescent derivatives with a maximum excitation at λex 260 nm and a maximum emission at λem 446 nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI–MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH]+ under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C8 column. Liquid chromatography separation for the derivatized amines showed good reproducibility with acetonitrile–water as mobile phase. Detection limits calculated from 90.00 pmol to 88 fmol injection, at a signal-to-noise ratio of 3, were 10.5–53.4 fmol. Excellent linear responses were observed with coefficients of >0.9996. The established method for the determination of aliphatic amines from real wastewater was satisfactory.

Separation of Derivatized Carbohydrates by Capillary Zone Electrophoresis

Abstract A simple and rapid method was developed for the separation of the labeled carbohydrates using 1-naphthyl-3-methyl-5- pyrazolone (NMP) as the derivatization reagent by capillary zone electrophoresis. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary is used. To optimize the conditions, the buffer solution concentration, pH, column temperature, voltage and other factors are evaluated. The results indicate that the buffer concentration and pH value exhibit a significant impact on the separation, but no obvious influences of the temperature, voltage and additives on resolution are observed. On the basis of the optimization, the optimal separation conditions are as follows: 55 mM borate buffer (pH 9.46), voltage of 22 kV, column temperature of 20 °C and DAD detection of 254 nm. The samples are introduced atmospherically with an injection time of 10 s. The results indicate that nine NMP-labeled carbohydrates can be separated with a good baseline resolution within 18 min under the conditions proposed. The Tangutoium Nitratia sample is also analyzed under the optimum conditions. The separation is efficient and rapid with satisfactory results.

A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12μM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples.