Determination of 20 Free Amino Acids in Asparagus Tin by High-Performance Liquid Chromatographic Method after Pre-Column Derivatization

Abstract

A novel method was studied for determination of 20 free amino acids in asparagus tin by high-performance liquid chromatography with pre-column derivatization. Derivatization of the samples was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and the SPE cartridge was used for purification and enrichment of the analytes. The derivatization conditions and the influence of elution composition on the separation were investigated. The reaction of amino acids with CNBF was completed in pH 9.0 borate buffer. The separation of amino acids was achieved at room temperature within 60 min by gradient elution mode with triethylamine in mobile phase, and the flow rate is 0.32 mL min −1 constantly. The method correlation coefficient was from 0.9975 to 1.0000 in concentrations ranging from 20 to 2000 μmol L −1 , except asparagine (from 100 to 10000 μmol L −1 ). The detection limits of amino acids were from 1.2 to 6.0 μmol L −1 , with a signal-to-noise ratio of three times. The calculated recoveries of the proposed method were from 81.4% to 109.4%, and relative standard deviations were 0.48-3.94% in application to the quantitative determination of free amino acids in asparagus tin samples. The present method is reliable and sensitive that allows fast analysis of free amino acids in asparagus tin, which makes it suitable for further study of free amino acids in other asparagus foods.

Peptide and Amino Acids Separation and Identification from Natural Products

“…In 2010, Sun et al [15] have presented another method for the detection of amino acids from Stellera chamaejasme L., a widely-used plant in the Chinese traditional medicine; DBCEC (2-[2-(dibenzocarbazole)-ethoxy] ethyl chloro-formate) was used as derivatization reagent, and the modified amino acids were detected by means of liquid chromatography with fluorescence detection. Li et al [16] have proposed a new method for the detection of amino acids from the asparagus tin. After performing the derivatization of the samples with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), solid phase extractions on C18 cartridges have been performed.…”

Determination of Free Tryptophan in Beer Samples by Capillary Isotachophoretic Method

“…The gradient elution was employed: 0 min: 20% A, 80% B; 15 min: 38% A, 62% B; 35 min: 48% A, 52% B; 43 min: 75% A, 25% B; and 55-65 min: 20% A, 80% B (Li et al 2012). The flow rate was 0.32 mL min −1 , the detection wavelength was 260 nm and the temperature was set at 30°C.…”

“…Calibration curves were constructed using eight calibration solutions of the Trp in the range 1.63-81.69 mg•L −1 by appropriate dilution of the obtained solutions in phosphate buffer. Test for matrix effect was conducted by standard addition method (Li et al 2012). The obtained regression equations were as follows: y = 258,715•C − 35,989 (R 2 = 0.9999; detection limit, LOD = 0.31 mg L −1 and quantification limit, LOQ = 0.94 mg L −1 ) for standard solution of Trp and y = 258,239•C − 17,500 (R 2 = 0.9993; LOD = 4.35 mg L −1 ; LOQ = 13.18 mg L −1 ) for blank matrix with Trp.…”

“…For HPLC analysis, the solution of beverage samples (0.4 mL) was mixed with CNBF solution (0.60 mL, 0.2 mol L −1 ) in the presence of borate buffer (pH = 9) and incubated at 60°C by 30 min. The solution of HCl was added to stop the reaction and finally obtained solutions were made up to the 10 mL by phosphate buffer (pH = 7) (Li et al 2012). The derivatized samples were filtered (0.45 μm pore size filter) prior to HPLC analysis.…”

“…The proposed procedure was optimized and validated for linearity, limit of detection and quantification, selectivity and matrix effects, intra-and inter-day precision and recovery. The obtained results of Trp determination in beer samples by ITP were compared with chromatographic analyses of Trp derivative with 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (CNBF) (Guo et al 2015;Li et al 2012;Piasta et al 2014;Shi et al 2009). This reagent seems to be proper choice for amino acids determination due to its fast reaction with nucleophiles at moderate temperatures while obtained derivatives are stable and reveals high ultraviolet absorption.…”