枸杞子药材中12种有机酸类成分含量测定与分析

Content Determination and Analysis of 12 Kinds of Organic Acid in Lycium barbarum

目的:建立同时测定枸杞子药材中12种有机酸类成分含量的方法,比较不同产区、品种药材样品的成分含量差异并研究其相关性。方法:采用超高效液相色谱-三重四级杆串联质谱法测定药材样品中水杨酸、香草酸、肉桂酸、咖啡酸、对羟基苯甲酸、阿魏酸、对香豆酸、富马酸、3,4-二羟基苯甲酸、酒石酸、丁香酸和原儿茶酸的含量。色谱柱为Acquity UPLC BEH C_(18),流动相为0.1%甲酸溶液(A)-乙腈(B),梯度洗脱,流速为0.5 mL/min,柱温为40℃,进样量为1.0μL;Turbo V^(TM)离子源,电离模式为电喷雾离子化(ESI-),气帘气体积流量为30 L/min,喷雾电压为-4 500 V,雾化气体积流量为50 L/min,加热辅助气体积流量为50 L/min,采用质谱多反应监测扫描模式,离子化温度为550℃。对不同产区、品种药材样品的成分含量差异进行比较,并进行成分相关性分析。结果:水杨酸、香草酸、肉桂酸、咖啡酸、对羟基苯甲酸、阿魏酸、对香豆酸、富马酸、3,4-二羟基苯甲酸、酒石酸、丁香酸和原儿茶酸检测质量浓度线性范围分别为0.01～1.00μg/mL(r=0.999 9)、0.01～1.00μg/mL(r=0.999 8)、0.01～1.00μg/mL(r=0.996 0)、0.01～1.00μg/mL(r=0.998 2)、0.08～8.00μg/m L(r=0.999 2)、0.08～8.00μg/mL(r=0.998 9)、0.20～20.00μg/mL(r=0.996 0)、0.08～8.00μg/mL(r=0.998 4)、0.008～0.80μg/mL(r=0.996 9)、0.16～16.00μg/mL(r=0.997 5)、0.001～0.10μg/mL(r=0.993 8)、0.008～0.80μg/mL(r=0.995 1);定量限分别为3.20、10.00、8.00、10.00、0.64、0.64、1.60、2.56、8.00、16.00、1.00、8.00 ng/mL,检测限分别为0.80、6.40、0.40、1.60、0.32、0.16、0.20、0.64、0.64、1.28、0.08、1.28 ng/mL;精密度、稳定性、重复性试验的RSD均小于3%;加样回收率为95.62%～104.65%(RSD为1.23%～3.43%,n=6)。15批药材样品中对香豆酸平均含量最高;新疆产药材样品中总有机酸平均含量最高;宁杞七号总有机酸类平均含量略高于宁杞五号。香草酸含量与对羟基苯甲酸、阿魏酸和丁香酸含量存在极显著正相关(P<0.01);肉桂酸含量与富马酸和酒石酸含量存在极显著正相关(P<0.01);咖啡酸含量与阿魏酸、酒石酸和丁香酸含量存在极显著正相关(P<0.01);对羟基苯甲酸含量与丁香酸含量存在极显著正相关(P<0.01);阿魏酸含量与丁香酸含量存在极显著正相关(P<0.01);3,4-二羟基苯甲酸含量与原儿茶酸含量存在极显著正相关(P<0.01)。结论:该方法操作简便、准确,精密度、稳定性、重复性好,可用于同时测定枸杞子药材中12种有机酸类成分含量。不同品种、产区药材样品中有机酸类成分含量有所不同,且某些成分间有一定的相关性。

OBJECTIVE:To establish the method for simultaneous determination of 12 kinds of organic acid in Lycium barbarum,and to compare the component contents of samples with different production and types areas,and to study its correlation.METHODS:UPLC-triple quadrupole tandem mass spectrometry was used to determine the contents of salicylic acid,vanillic acid,cinnamic acid,caffeic acid,p-hydroxybenzoic acid,ferulic acid,p-coumaric acid,fumaric acid,3,4-dihydroxybenzoic acid,tartaric acid,syringic acid and protocatechuic acid in samples.The determination was performed on Acquity UPLC BEH C18 column with mobile phase consisted of 0.1%formic acid solution(A)-0.1%formic acid acetonitrile solution(B)for gradient elution at the flow rate of 0.5 mL/min.The column temperature was 40℃,and sample size was 1.0μL.The ion source was Turbo VTM ion source,and ionization mode was electrospray ionization(ESI-).Air curtain gas volume flow rate was 30 L/min;spray voltage was-4 500 V;atomizing gas volume flow rate was 50 L/min;heating auxiliary gas volume flow rate was 50 L/min.The mass spectrometry multiple reaction monitoring scanning mode was adopted and the ionization temperature was 550℃.The differences of component content in samples with different production areas and types were compared,and component relationship analysis was performed.RESULTS:The linear ranges of salicylic acid,vanillic acid,cinnamic acid,caffeic acid,p-hydroxybenzoic acid,ferulic acid,p-coumaric acid,fumaric acid,3,4-dihydroxybenzoic acid,tartaric acid,syringic acid and protocatechuic acid were 0.01-1.00μg/mL(r=0.999 9),0.01-1.00μg/mL(r=0.999 8),0.01-1.00μg/mL(r=0.996 0),0.01-1.00μg/mL(r=0.998 2),0.08-8.00μg/mL(r=0.999 2),0.08-8.00μg/mL(r=0.998 9),0.20-20.00μg/mL(r=0.996 0),0.08-8.00μg/mL(r=0.998 4),0.008-0.80μg/mL(r=0.996 9),0.16-16.00μg/mL(r=0.997 5),0.001-0.10μg/mL(r=0.993 8),0.008-0.80μg/mL(r=0.995 1),respectively.The limits of quantitation were 3.20,10.00,8.00,10.00,0.64,0.64,1.60,2.56,8.00,16.00,1.00,8.00 ng/mL;the limits of detection were 0.80,6.40,0.40,1.60,0.32,0.16,0.20,0.64,0.64,1.28,0.08,1.28 ng/mL,respectively.RSDs of precision,stability and reproducibility tests were all lower than 3%.The recoveries were 95.62%-104.65%(RSD were 1.23%-3.43%,n=6).Average content of p-coumaric acid was the highest in 10 batches of samples;average contents of tatol organic acid were highest in samples from Xinjiang.The total organic acid content of Ningqi No.7 was slightly higher than that of Ningqi No.5.Vanillic acid content was positively correlated with p-hydroxybenzoic acid,ferulic acid and syringic acid content(P<0.01);cinnamic acid content was positively correlated with fumaric acid and tartaric acid content(P<0.01);caffeic acid content was positively correlated with ferulic acid,tartaric acid and syringic acid content(P<0.01);formic acid content was positively correlated with syringic acid content(P<0.01);ferulic acid content was positively correlated with syringic acid content(P<0.01);and 3,4-dihydroxybenzoic acid content was positively correlated with catechuic acid content(P<0.01).CONCLUSIONS:The method is simple,accurate,precise,stable and reproducible.It can be used to determine the contents of 12 kinds of organic acid in L.bararum.The contents of organic acids in samples with different production areas and types are different;there is certain relationship among some compenents.