Determination of 16 Free Amino Acids in Cigar Tobacco Leaves by Pre-column Derivatization-high Performance Liquid Chromatography

doi:10.11924/j.issn.1000-6850.casb2025-0752

Abstract

Abstract:

Free amino acid is an important flavor chemical component in cigar leaves, and its accurate and rapid determination is of great significance for cigar quality evaluation. A method for the simultaneous determination of 16 amino acids in cigar leaves was established based on pre-column derivatization-reversed-phase high performance liquid chromatography. The optimal detection conditions were determined by optimizing the key parameters such as column temperature, flow rate, ultrasonic extraction time and derivatization time. The methodological performance was systematically investigated and applied to the analysis of 40 cigar leaf samples from different producing areas, parts and grades. The correlation between amino acid content and sample attributes was revealed by radar map. The results showed that the contents of 16 amino acids were in the range of 2.5-400 mg/L, the linear correlation coefficient R²≥0.998, the detection limit was 0.014-0.096 mg/L, the quantitative limit was 0.047-0.320 mg/L, the recovery rate was 89.05%-107.08%, and the relative standard deviation was 1.25%-5.72%. The method was accurate and reliable. The analysis of actual samples showed that the amino acid content in cigar leaves had significant correlations with the producing area (the overall content in B area was higher), the position (upper leaf> middle leaf> lower leaf), and the grade (second grade > third grade > fourth grade). The method has the advantages of simple operation, good separation effect and high sensitivity, and can be used for the detection of amino acid content and quality grading evaluation of cigar tobacco leaves.

Key words: precolumn derivatization, high performance liquid chromatography, cigar, amino acid, extraction, assaying, quality evaluation

ZHOU Ting, CHEN Qi, ZHANG Jing, LI Ping, YUE Xiaolin, TAN Wenyuan, WANG Lan, JIN Yile, GAO Yunpeng. Determination of 16 Free Amino Acids in Cigar Tobacco Leaves by Pre-column Derivatization-high Performance Liquid Chromatography[J]. Chinese Agricultural Science Bulletin, 2025, 41(34): 129-136.

Figures/Tables 9

References 30

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时间/min	流动相A/%	流动相B/%
0.00	92.0	8.0
2.00	92.0	8.0
10.00	91.0	9.0
16.00	83.0	17.0
23.00	76.0	24.0
25.00	67.0	33.0
35.00	54.0	46.0

时间/min	流动相A/%	流动相B/%
0.00	92.0	8.0
2.00	92.0	8.0
10.00	91.0	9.0
16.00	83.0	17.0
23.00	76.0	24.0
25.00	67.0	33.0
35.00	54.0	46.0

氨基酸	保留时间/min	R²	回收率/%	RSD/%	检出限/(mg/L)	定量限/(mg/L)
天冬氨酸(Asp)	4.02	0.9992	104.36	2.12	0.046	0.153
谷氨酸(Glu)	4.27	0.9989	94.61	3.64	0.078	0.260
丝氨酸(Ser)	7.01	0.9995	96.76	4.42	0.031	0.103
甘氨酸(Gly)	7.79	0.9994	97.76	1.25	0.037	0.123
组氨酸(His)	11.58	0.9997	92.24	2.53	0.034	0.113
精氨酸(Arg)	14.05	0.9990	100.14	1.32	0.061	0.203
苏氨酸(Thr)	14.53	0.9997	95.46	2.59	0.043	0.143
丙氨酸(Ala)	15.39	0.9992	91.74	1.68	0.029	0.097
脯氨酸(Pro)	16.96	0.9987	100.15	2.01	0.023	0.077
酪氨酸(Tyr)	23.05	0.9998	97.74	5.72	0.042	0.140
缬氨酸(Val)	24.21	0.9990	101.34	4.79	0.041	0.137
蛋氨酸(Met)	25.57	0.9994	89.05	2.48	0.047	0.157
胱氨酸(Cys)	27.33	0.9991	98.90	3.10	0.096	0.320
异亮氨酸(Ile)	28.27	0.9998	107.08	3.89	0.061	0.203
亮氨酸(Leu)	28.47	0.9994	90.07	3.34	0.041	0.137
苯丙氨酸(Phe)	29.70	0.9981	105.47	3.31	0.014	0.047

氨基酸	保留时间/min	R²	回收率/%	RSD/%	检出限/(mg/L)	定量限/(mg/L)
天冬氨酸(Asp)	4.02	0.9992	104.36	2.12	0.046	0.153
谷氨酸(Glu)	4.27	0.9989	94.61	3.64	0.078	0.260
丝氨酸(Ser)	7.01	0.9995	96.76	4.42	0.031	0.103
甘氨酸(Gly)	7.79	0.9994	97.76	1.25	0.037	0.123
组氨酸(His)	11.58	0.9997	92.24	2.53	0.034	0.113
精氨酸(Arg)	14.05	0.9990	100.14	1.32	0.061	0.203
苏氨酸(Thr)	14.53	0.9997	95.46	2.59	0.043	0.143
丙氨酸(Ala)	15.39	0.9992	91.74	1.68	0.029	0.097
脯氨酸(Pro)	16.96	0.9987	100.15	2.01	0.023	0.077
酪氨酸(Tyr)	23.05	0.9998	97.74	5.72	0.042	0.140
缬氨酸(Val)	24.21	0.9990	101.34	4.79	0.041	0.137
蛋氨酸(Met)	25.57	0.9994	89.05	2.48	0.047	0.157
胱氨酸(Cys)	27.33	0.9991	98.90	3.10	0.096	0.320
异亮氨酸(Ile)	28.27	0.9998	107.08	3.89	0.061	0.203
亮氨酸(Leu)	28.47	0.9994	90.07	3.34	0.041	0.137
苯丙氨酸(Phe)	29.70	0.9981	105.47	3.31	0.014	0.047

氨基酸	范围	平均值
天冬氨酸(Asp)	0.378~6.228	2.505
谷氨酸(Glu)	0.062~1.243	0.618
丝氨酸(Ser)	0.034~3.604	0.320
甘氨酸(Gly)	0.011~2.459	0.312
组氨酸(His)	0.000~0.386	0.153
苏氨酸(Thr)	0.000~8.181	0.616
精氨酸(Arg)	0.000~13.098	1.079
丙氨酸(Ala)	0.043~15.663	9.401
脯氨酸(Pro)	0.000~3.666	0.330
酪氨酸(Tyr)	0.027~0.405	0.189
缬氨酸(Val)	0.003~0.246	0.064
蛋氨酸(Met)	0.000~0.575	0.100
胱氨酸(Cys)	0.000~0.820	0.265
异亮氨酸(Ile)	0.000~0.774	0.263
亮氨酸(Leu)	0.029~0.256	0.109
苯丙氨酸(Phe)	0.000~1.115	0.150