Simultaneous determination of five bioactive substances in mulberry leaves by LC-MS/MS

doi:10.3969/j.issn.1004-1524.20241040

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (10): 2179-2189.DOI: 10.3969/j.issn.1004-1524.20241040

• Quality and Safety of Agricultural Products • Previous Articles Next Articles

Simultaneous determination of five bioactive substances in mulberry leaves by LC-MS/MS

REN Xiaorong¹^,²(), WANG Xinquan², ZHANG Shanying³, WANG Meng³, ZHU Hongming¹, ZHANG Chenghui¹^,^*(), QI Peipei²^,^*()

1. College of Food Science and Engineering, Hainan University, Haikou 570100, China
2. Institute of Agricultural Product Quality and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. College of Plant Protection, Hainan University, Haikou 570100, China

Received:2024-11-29 Online:2025-10-25 Published:2025-11-13

Abstract

Abstract:

To establish an accurate, rapid, and simultaneous analytical method for determining five bioactive substances in mulberry leaves: 1-deoxynojirimycin (DNJ), rutin (VP), isoquercitrin (IQ), phenylalanine (Phe), and γ-aminobutyric acid (GABA), this study systematically investigated the effects of extraction solvent, solid-to-liquid ratio, and ultrasonic time on the extraction efficiency of these substances. An optimal sample pretreatment method was determined and coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis. The optimized sample pretreatment conditions were as follows: 90% aqueous methanol as the extraction solvent, a solid-to-liquid ratio of 1∶12 (g·mL^-1), ultrasonic extraction for 30 min, and two successive extractions. Under these conditions, the extraction efficiency of the five bioactive substances was significantly improved. Method validation results demonstrated excellent linearity for the target analytes within the concentration range of 1-200 μg·kg^-1 (r≥0.998 6). The limits of detection (LOD) and limits of quantification (LOQ) were 0.47-0.93 μg·kg ¹ and 1.56-3.10 μg·kg^-1, respectively. The recovery rates ranged from 76.0% to 99.5%, with relative standard deviations (RSD) below 13%, indicating high sensitivity, accuracy, and precision. When applied to authentic mulberry leaf samples, the method revealed significant differences (p<0.05) in the contents of the five bioactive substances among leaf positions. DNJ, Phe, and GABA were most abundant in the bud tips and gradually decreased toward lower leaf positions, while VP and IQ exhibited the opposite trend. These findings demonstrate that the proposed method provides reliable technical support for accurately evaluating the distribution of bioactive substances in mulberry leaves and facilitating their targeted utilization.

Key words: mulberry leaf, bioactive substance, sample-preparation method, ultra-high-performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)

CLC Number:

S789.4

REN Xiaorong, WANG Xinquan, ZHANG Shanying, WANG Meng, ZHU Hongming, ZHANG Chenghui, QI Peipei. Simultaneous determination of five bioactive substances in mulberry leaves by LC-MS/MS[J]. Acta Agriculturae Zhejiangensis, 2025, 37(10): 2179-2189.

Figures/Tables 8

References 43

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生物活性物质 Bioactive substances	分子式 Molecular Formula	先驱离子 Precursor ion(m/z)	产物离子 Product ions(m/z)	锥孔电压 Conce voltage/V	碰撞能量 Collision energy/eV	保留时间 Retention time/min
DNJ	C₆H₁₃NO₄	164	69^*/80	50	23/22	0.72
VP	C₂₇H₃₀O₁₆	611	303^*/465	50	25/10	3.07
IQ	C₂₁H₂₀O₁₂	463	300^*/255	50	25/54	3.09
Phe	C₉H₁₁NO₂	166	120^*/103	50	13/29	2.76
GABA	C₄H₉NO₂	104	87^*/69	50	8/15	0.76

生物活性物质 Bioactive substances	分子式 Molecular Formula	先驱离子 Precursor ion(m/z)	产物离子 Product ions(m/z)	锥孔电压 Conce voltage/V	碰撞能量 Collision energy/eV	保留时间 Retention time/min
DNJ	C₆H₁₃NO₄	164	69^*/80	50	23/22	0.72
VP	C₂₇H₃₀O₁₆	611	303^*/465	50	25/10	3.07
IQ	C₂₁H₂₀O₁₂	463	300^*/255	50	25/54	3.09
Phe	C₉H₁₁NO₂	166	120^*/103	50	13/29	2.76
GABA	C₄H₉NO₂	104	87^*/69	50	8/15	0.76

生物活性物质 Bioactive substances	线性方程 Linear equation	线性范围 Linear range/(μg·kg^-1)	相关系数 r	检测限 LOD/(μg·kg^-1)	定量限 LOQ/(μg·kg^-1)
DNJ	Y=782X+1 408	1~200	0.999 7	0.78	2.59
VP	Y=918X+1 516	1~200	0.998 6	0.67	2.25
IQ	Y=824X+600	1~200	0.998 8	0.47	1.56
Phe	Y=11730X+91 353	1~200	0.998 9	0.67	2.24
GABA	Y=1716X+1 677	1~200	0.999 4	0.93	3.10

生物活性物质 Bioactive substances	线性方程 Linear equation	线性范围 Linear range/(μg·kg^-1)	相关系数 r	检测限 LOD/(μg·kg^-1)	定量限 LOQ/(μg·kg^-1)
DNJ	Y=782X+1 408	1~200	0.999 7	0.78	2.59
VP	Y=918X+1 516	1~200	0.998 6	0.67	2.25
IQ	Y=824X+600	1~200	0.998 8	0.47	1.56
Phe	Y=11730X+91 353	1~200	0.998 9	0.67	2.24
GABA	Y=1716X+1 677	1~200	0.999 4	0.93	3.10

生物活性物质 Bioactive substances	本底检出值 Background/(μg·g^-1)	添加值 Added/(μg·g^-1)	检测值 Found/(μg·g^-1)	平均回收率 Average recovery/%	相对标准偏差 RSD/%
DNJ	22.5±0.6	16	35.3±0.9	80.0	7.4
	22.5±0.6	20	38.6±1.2	80.9	7.3
	22.5±0.6	24	40.7±1.5	76.0	8.0
VP	19.9±0.3	32	48.6±0.5	89.9	1.8
	19.9±0.3	40	56.4±2.5	91.4	6.7
	19.9±0.3	48	65.1±1.2	94.3	2.6
IQ	23.6±0.8	32	51.6±0.8	87.5	2.7
	23.6±0.8	40	57.1±4.2	83.7	12.6
	23.6±0.8	48	61.7±1.0	79.6	2.6
Phe	20.3±0.3	16	32.8±1.5	77.9	12.1
	20.3±0.3	20	36.5±0.4	80.6	2.5
	20.3±0.3	24	40.8±0.1	85.3	0.2
GABA	547.7±9.7	560	1 105.1±4.6	99.5	0.8
	547.7±9.7	700	1 228.6±71.8	97.3	10.6
	547.7±9.7	840	1 334.1±31.9	93.6	4.1

Simultaneous determination of five bioactive substances in mulberry leaves by LC-MS/MS

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叶位 Leaf position	含量Content
叶位 Leaf position	DNJ	VP	IQ	Phe	GABA
芽尖Bud	318.4±35.7 a	7.1±0.9 c	3.7±0.5 c	62.8±4.3 a	583.8±22.2 a
上叶位Apical leaf	253.5±18.1 b	8.0±0.7 c	4.2±0.4 c	41.3±1.0 b	547.3±26.3 b
中叶位Middle leaf	194.7±2.9 c	13.9±1.5 b	7.9±0.2 b	22.3±0.9 c	546.7±15.7 b
下叶位Basal leaf	154.1±10.0 d	18.9±1.9 a	11.1±1.0 a	21.2±1.8 c	496.0±5.7 c

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