薄层色谱扫描仪测枸杞子中甜菜碱

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枸杞中甜菜碱含量测定方法研究进展

枸杞中甜菜碱含量测定方法研究进展作者：闫丽丽来源：《农业科技与装备》2017年第09期摘要：综述枸杞中甜菜碱含量测定方法的研究进展，介绍薄层扫描法、高效液相色谱法、比色法、液相色谱-质谱联用法等分析测定条件、精密度、回收率，旨在为开展甜菜碱相关研究提供理论参考。

关键词：枸杞子；甜菜碱；含量测定；精密度；回收率中图分类号：R284.2 文献标识码：A 文章编号：1674-1161（2017）09-0069-02枸杞为茄科植物宁夏枸杞的干燥成熟果实，是传统的药食同源天然植物，具有滋补肝肾、益精明目的功效。

现代研究表明，甜菜碱是枸杞的主要生物碱之一，具有降脂、抗癌、抗氧化等作用。

甜菜碱是一种季铵型水溶性生物碱，易溶于水和甲醇，常温下极易吸湿潮解，因具有良好的生物活性及理化性质且无毒无害，因而被广泛用于化工制药、种植养殖及食品生产。

目前，甜菜碱检测方法主要有薄层扫描法、高效液相色谱法、比色法、液相色谱-质谱联用法等。

综述甜菜碱检测方法研究进展，为甜菜碱开发利用提供参考。

1 枸杞中甜菜碱含量测定方法1.1 层扫描法薄层扫描法是2015版《中国药典》（一部）中规定的枸杞子中甜菜碱含量测定方法。

具体的检测方法为：甲醇回流提取后，用活性炭滤过，加硫氰酸铬铵溶液搅拌，再用垂熔漏斗滤过制得供试品溶液；对照品加盐酸甲醇溶液（0.5→100）制成每1 mL含4 mg的溶液；精密吸取供试品溶液5 μL、对照品溶液3 μL与6 μL，分别交叉点于同一硅胶G薄层板上，以丙酮-无水乙醇-盐酸（10‥6‥1）为展开剂，预饱和30 min后展开，取出，挥干溶剂，立即喷新配制的改良碘化铋钾试液，放置1～3 h至斑点清晰，在波长515 nm，590 nm下扫描，测量供试品吸光度积分值与对照品吸光度积分值并计算。

按干燥品计算，含甜菜碱（C5H11NO2）不得少于0.30%。

双波长薄层扫描法是指采用两种不同波长的光束，先后扫描所要测定的斑点，并记录下此两波长吸光度之差。

枸杞子

四川诺迪康威光制药有限公司枸杞子检验原始记录【性状】标准规定：本品呈类纺锤形或椭圆形，长6～20mm，直径3～10mm。

表面红色或暗红色，顶端有小突起状的花柱痕，基部有白色的果梗痕。

果皮柔韧，皱缩；果肉肉质，柔润。

种子20～50粒，类肾形，扁而翘，长1.5～1.9mm，宽1～1.7mm，表面浅黄色或棕黄色。

气微，味甜。

本批性状为：符合规定（）不符合规定（）检验员：日期：年月日复核员：日期：年月日【鉴别】所用仪器：电子天平编号，显微镜编号，对照药材来源：由中国药品生物制品检定研究院1、本品粉末黄橙色或红棕色。

外果皮表皮细胞表面观呈类多角形或长多角形，垂周壁平直或细波状弯曲，外平周壁表面有平行的角质条纹；中果皮薄壁细胞呈类多角形，壁薄，胞腔内含橙红色或红棕色球形颗粒。

种皮石细胞表面观不规则多角形，壁厚，波状弯曲，层纹清晰。

符合规定（）不符合规定（）检验员：日期：年月日复核员：日期：年月日2、取本品0.5g，加水35ml，加热煮沸15分钟，放冷，滤过，滤液用乙酸乙酯15ml振摇提取，分取乙酸乙酯液，浓缩至1ml，作为供试品溶液。

另取枸杞子对照药材0.5g，同法制成对照药材溶液。

照薄层色谱法（《中国药典》2010年版一部附录VI B）试验，吸取上述两种溶液各5μl，分别点于同一硅胶G薄层板上，以乙酸乙酯-三氯甲烷-甲酸（3∶2∶1）为展开剂，展开，取出，晾干，置紫外光灯（365nm）下检视。

供试品色谱中，在与对照药材色谱相应的位置上，显相同颜色的荧光斑点。

对照药材批号：，对照药材溶液自编号：展距：检测结果：供试品色谱中，在与对照药材色谱相应的位置上，显相同（）色的荧光斑点。

符合规定（）供试品色谱中，在与对照药材色谱相应的位置上，未显相同颜色的荧光斑点。

符合规定（）符合规定（）不符合规定（）检验员：日期：年月日复核员：日期：年月日【检查】所用仪器：电子天平编号，干燥箱编号，高温炉编号。

1、水分照水分测定法（《中国药典》2010年版一部附录IX H第一法，温度为80℃）测定，不得过13.0%。

超高效合相色谱-串联质谱法快速测定枸杞中甜菜碱含量

超高效合相色谱-串联质谱法快速测定枸杞中甜菜碱含量张弦飞;杨军丽;陈娟;师彦平【摘要】枸杞作为传统的中药材,具有滋补肝肾、益精明目的功效,亦可作为功能性食品被广泛食用.基于超高效合相色谱-质谱(UPC2-MS),建立了一种快速、灵敏地定量测定枸杞中甜菜碱(指标化合物)含量的新方法.采用ACQUITY UPC2BEH 2-EP 色谱柱(150 mm×2.1 mm,1.7 μm),以0.7 mL/min超临界CO2-甲醇(80:20,v/v)等度洗脱,成功分离了枸杞中的甜菜碱.在该分离过程中,0.1%(v/v)甲酸作为改性剂;背压为1.31×107Pa;柱温为40 ℃;进样体积为1 μL;保留时间为3 min.质谱检测工作于电喷雾(ESI)正离子模式和选择离子监测(SIR)模式.在上述条件下得到线性回归方程.其相关系数为0.9992,检测范围为0.5~50.0 μg/mL,检出限为0.013 μg/mL.随后,通过对精度、重复性、稳定性和加标回收率(平均值96.3%)的分析,验证了该方法的有效性.最后,将所建立的方法应用于11批样品的分析.结果表明,该方法可较好地用于枸杞的质量控制与分析评价.%Over the past 2500 years,Lycii Fructus has been widely used as a functional food and tonic in Chi-nese herbal medicine. It can nourish the liver and kidneys,moisten the lungs,and improve eyesight. In this study,a new rapid and sensitive method has been developed for the quantitative determination of betaine (index compound)in Lycii Fructus by using ultra-performance convergence chromatography-tandem mass spectrometry(UPC2-MS). The separation of betaine was successfully achieved on an ACQUITY UPC2BEH 2-EP column(150 mm×2.1 mm,1.7 μm),with isocratic elution by a CO2-methanol(80:20,v/v)solvent at a flow rate of 0.7 mL/min. The conditions used in the separation process were as follows:modifier,0.1%(v/v)for-micacid in methanol;columntemperature,40 ℃;backpressure,1.31×107Pa;injection volume,1 μL;and retention time,3 min. The MS system was equipped with an electrospray ionization(ESI)ion source and oper-ated in the selected ionrecording(SIR)and positive ion mode. Under the abovementioned conditions,the cali-bration curve was obtained. The linear range of detection was 0.5-50.0 μg/mL,with a correlation coefficient of 0.999 2,and the limit of detection(LOD)was found to be 0.013 μg/mL. The validity of the method was tested by analyses of precision,repeatability,stability,and accuracy(average recovery:96.3%). Finally,the developed method was applied to analyze 11 batches of samples. The results indicated that this method was suitable for evaluating the quality of Lycii Fructus.【期刊名称】《色谱》【年(卷),期】2018(036)005【总页数】8页(P417-424)【关键词】超高效合相色谱-串联质谱;甜菜碱;枸杞;质量控制【作者】张弦飞;杨军丽;陈娟;师彦平【作者单位】中国科学院兰州化学物理研究所,中国科学院西北特色植物资源化学重点实验室和甘肃省天然药物重点实验室,兰州730000;中国科学院大学,北京100049;中国科学院兰州化学物理研究所,中国科学院西北特色植物资源化学重点实验室和甘肃省天然药物重点实验室,兰州730000;中国科学院兰州化学物理研究所,中国科学院西北特色植物资源化学重点实验室和甘肃省天然药物重点实验室,兰州730000;中国科学院兰州化学物理研究所,中国科学院西北特色植物资源化学重点实验室和甘肃省天然药物重点实验室,兰州730000【正文语种】中文【中图分类】O658Lycium barbarum L., which belongs to the Solanaceae family, is widely distributed in the wild in most regions of China and is mainly cultivated in Northwestern China, including Ningxia, Gansu, Qinghai, Inner Mongolia, and Xinjiang [1,2]. Lycii fructus is widely used as a functional health food [3] or as a valuable tonic in Chinese herbal medicine for nourishing the liver and kidneys, moistening the lungs, and improving eyesight [4,5]. Nowadays, Lycii fructus is marketed as a dietary supplement and functional tea in many countries in Asia, Europe, and North America [6]. Betaine is one of the major functional components in Lycii fructus and is a zwitterionic quaternary ammonium compound. It has many beneficial effects on the human body, including anti-atherosclerosis, anti-osteoporosis, anti-tumor effects; further, it lowers the blood pressure, heals peptic ulcers, and protects the liver against chemical injury [7].Both the Chinese Pharmacopoeia [8] and the Korean Pharmacopoeia [9] stipulate betaine as the index compound of Lycii Fructus. In the Chinese Pharmacopoeia, the method for determination of betaine in Lycii Fructus is stipulated as thin-layer chromatography (TLC) scanning. The precision of TLC is not sufficient, and it is typically used for qualitative analysis.Furthermore, tailing often occurs during chromatography. In recent years, some quantitative analysis methods have been developed. Shin et al. [10] determined betaine in Lycium chinense fruits by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and observed high baseline noise. Huang et al. [11] analyzed the betaine content in Lycii Fructus by high-performance liquid chromatography (HPLC) using a Luna 5u SCX 100A column and an evaporative light scattering detector (ELSD), but the target peak of betaine was not well separated from the other peaks. Zhao et al. [12] used an HILIC column and ELSD detector to analyze betaine in Lycii Fructus. The obtained peak shape of betaine was acceptable, but the retention time was slightly long, about 20 min. Besides Lycii Fructus, the determination of betaine in other matrices such as plasma [13], feed premixes [14], and human urine [15] was also reported, and good results were obtained.In 2012, Waters Company launched ultra-performance convergence chromatography (UPC2), which integrated the advantages of both supercritical fluid chromatography (SFC) and ultrahigh-performance liquid chromatography (UPLC). The mobile phase contained supercritical carbon dioxide, which is an inert fluid and a small amount of organic solvents [16]. In the supercritical state, the polarity of carbon dioxide is similar to that of hexane, so a small amount of modifier is commonly added to increase the separation efficiency [17]. UPC2 has many advantages, such as short analysis time, simple pretreatment, high sensitivity, good reproducibility, minimal consumption of organic reagents, and environmental friendliness.Nowadays, UPC2 is widely used in medicine [18-21], food [22,23], biology [24,25], chemistry, and other fields [26-28].Until now, there are only a few notable reports on the determination of betaine in Lycii Fructus, but there is no detailed research based on UPC2. The main purpose of this work was to establish a new quantitative analysis method based on UPC2-MS.1 Experimental1.1 Reagents and materialsIn 2016, 11 batches of dried fruits of L. barbarum were collected from different regions of Ningxia and Gansu provinces in China and identified by Prof. Lin Jin, Gansu University of Traditional Chinese Medicine, Gansu, China. The betaine standard (purity>98.0%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Chromatographic-grade methanol was obtained from Merck Co. Ltd. (Darmstadt, Germany), and all other chemicals of analytical grade were provided by Tianjin Chemical Reagent Co. (Tianjin, China). Water was purified using an OKP-TS purification system (Exceed-AC-16, Shanghai Laikie Instrument Co. Ltd., China).1.2 Chromatographic systemUPC2 analysis was performed on an ACQUITY UPC2 system (Waters, Milford, MA, USA), which was equipped with a binary solvent delivery pump, a fixed-loop autosampler, a column thermostat, and an automated backpressure regulator (ABPR). The separation of betaine was successfully achieved on an ACQUITY UPC2 BEH 2-EP column (150 mm×2.1 mm, 1.7μm) by isocratic elution with a CO2-methanol (80∶20, v/v) mixture at a flow rate of 0.7 mL/min. The following were the conditions for this separation process: modifier, 0.1% formic acid in methanol; column temperature, 40 ℃; injection volume, 1 μL; and ABPR, 1.31×107 Pa.The system was coupled to an MS apparatus equipped with an ESI source, and the quantification of betaine was performed by MS analysis in selected ion recording (SIR) mode and positive ion mode. The MS analysis conditions were optimized as follows: source temperature, 120 ℃; desolvation temperature, 350 ℃; capi llary voltage, 2.3 kV; cone voltage, 20 V; cone gas flow rate, 150 L/h; and desolation gas (nitrogen) flow rate, 600 L/h. Data acquisition and system control were performed using a MassLynx 4.1 workstation (Waters, USA).1.3 Preparation of standard solutions12.0 mg of betaine was accurately weighed and dissolved in methanol in a 25 mL volumetric flask to prepare the stock solution. In order to obtain the calibration curve, the stock solution was further diluted to 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μg/mL with methanol, filtered through a 0.22 μm millipore membrane, and stored at 4 ℃ before use.1.4 Preparation of sample solutions50.0 mg of the accurately weighed sample from each batch of Lycii Fructus was air-dried, crushed into powder, and extracted with 50 mL methanol in an ultrasonicator for 1 h. The extracts were separated in a centrifuge machine (TDL-5-A, Shanghai Anting Scientific Instrument Factory, China) at 4 000 r/min for 10 min. The obtained supernatants were concentrated to 5mL using a vacuum rotavapor (EYELA N-1001, EYELA Co. Ltd., China). The resulting sample solutions were filtered through a 0.22 μm millipore membrane filter and injected into the UPC2-MS system for analysis. The betaine contents were calculated according to the calibration curve, and the yields were the mass ratios of the extracted betaine to the weighed medicinal herbs.1.5 Method validationThe limit of detection (LOD) and limit of quantification (LOQ) were defined by the signal-to-noise ratios of 3 and 10, respectively. Precisions within one day (intraday) and within three consecutive days (interday) were analyzed by replicate injections of the same sample solution (n=6). Stability was tested at ～2 h intervals for three days. Repeatability was evaluated by the relative standard deviation of six different sample solutions, which were prepared in parallel with the same batch of sample S-01. Accuracy was investigated by spiking sample S-01 with three concentration levels of betaine standards, and the extraction and analysis were repeated three times for each level.2 Results and discussion2.1 Optimization of chromatographic conditionsTo obtain good peak shapes and shorten the retention time of betaine, the optimal chromatographic conditions were investigated. For UPC2, the mobile phase, column type, injection volume (0.5-3.0 μL), column temperature (35-55 ℃), flow rate (0.5-0.9 mL/min), and backpressure (1.03×107-1.52×107 Pa) were analyzed. For MS, the ionization mode,capillary voltage (2.3-3.0 kV), cone voltage (10-50 V), desolvation temperature (300-450 ℃), and desolvation gas (nitrogen) flow rate (500-1 000 L/h) were examined.Fig. 1 Effects of (a) column type, (b) injection volume,(c) cone voltage, and (d) column temperature on the peak shape of betaineA suitable chromatographic column plays an important role in the acceptable separation of components. As shown in Fig. 1a, three columns, Waters ACQUITY UPC2 BEH 2-EP (150 mm×2.1 mm, 1.7 μm), Waters ACQUITY UPC2 BEH C18 (100 mm×3.0 mm, 1.7 μm), and Waters ACQUITY UPC2 CSH Fluoro-Phenyl (150 mm×2.1 mm, 1.7 μm), were screened. The Waters ACQUITY UPC2 BEH 2-EP column is filled using hybrid silica with 2-ethylpyridine bonding. The hydrophilic interactions in the silica column are conducive for the elution of alkaloids [29]. As indicated in Fig. 1a, this column was proved to be the best since it possessed a basic character at its surface, and was chosen for subsequent analyses. Different modifiers (methanol, methanol: isopropanol, and methanol: acetonitrile) and additives (formic acid, ammonium formate, and ammonium acetate) were screened, and 0.1% (v/v) formic acid in methanol was found to be the best. Changing the injection volume or the cone voltage hardly influenced the retention time but significantly changed the peak shape. As shown in Figs. 1b and c, the peak height and peak width increased with an increase in the injection volume or cone voltage, and the peak shape worsened. Given that larger areas are required for quantitative analysis, the injection volume and cone volt age were selected as 1 μL and 20 V, respectively.Density is the main factor contributing to retention and selectivity, and it depends on three parameters: mobile phase composition, column temperature, and pressure [30,31]. Temperature is known to affect the distribution constant, and thereby, the retention and separation factors of the analytes. The density of CO2 decreases with an increase in temperature, so the elution capacity decreases and the retention time increases [32]. As presented in Fig. 1d, the retention time increased slightly when the temperature was increased to 55 ℃; further, the peak became wider as the temperature increased. This was because temperature variations would modify the chemical characteristics of the analytes and the stationary phase, leading to a change in selectivity. In this work, the column temperature was set at 40 ℃.The backpressure hardly impacted the peak shape but had a notable influence on the retention time in this work. A higher backpressure would increase the density of and solvation power of the mobile phase solvation power, thereby shortening the retention time [33]. As seen in Fig. 2a, the retention time decreased with an increase in backpressure, so the backpressure was selected as 1.31×107 Pa. Under operation a t high temperature and low backpressure, the fluid will behave like a gas, thus causing problems with repeatability and sensitivity [34]. Hence, intermediate values of temperature and backpressure may be more suitable. In Ref. [34], 40 ℃ and 1.50×107 Pa we re suggested as the temperature and backpressure, respectively, which were consistent with the results of our investigation.In this study, the flow rate, too, had no clear influence on the peak shape. However, as seen from Fig. 2b, the retention time would decrease with an increase in the flow rate. It is well known that the inlet pressure increases with increasing flow rate, and this leads to an increased density of the mobile phase in the column. Thus, the retention time decreases [33]. Given that increasing the flow rate by 0.1 mL/min would result in an increase of about 3.45×106 Pa in the system pressure and very high pressure would hinder system maintenance, the flow rate was selected as 0.7 mL/min. Other parameters such as desolvation gas flow rate and desolvation temperature were also investigated and demonstrated to have minor effects on the sensitivity.Fig. 2 Effects of (a) backpressure and (b) flow rate on the retention time of betaineFig. 3 UPC2-MS total ion chromatograms obtained in (a) positive ion mode and (b) negative ion modeThe detection of betaine was performed with the ESI source and in SIR mode; the obtained UPC2-MS total ion chromatograms are presented in Fig. 3. As shown in Fig. 3a, the retention times of the standard and the sample solutions in positive ion mode agreed well with one another, and good peak shapes were observed. These results indicated that this mode was suitable for betaine detection. As presented in Fig. 3b, UPC2-MS analysis in negative ion mode was also attempted, but this did not give acceptable results. The mechanism for the fragmentation pathways of betaine in the mass spectrum is shown in Fig. 4. In the positive ion mode,the mass-to-charge ratios (m/z) were detected based on the formation of [M+Na]+ ions.Fig. 4 Mass scan spectrum in positive ion mode and fragmentation pathways of betaine2.2 Optimization of sample preparation conditionsUsing a mono-factorial experimental design, four sample preparation conditions (solvent, extraction time, sample amount, and extraction method) were optimized. Methanol and ethanol at six volume percentages (50%, 60%, 70%, 80%, 90%, and 100%) were investigated in sample assays using an ultrasonicator, and 100% methanol was proved to be the best. Five extraction times (30, 45, 60, 75, and 90 min) were examined with 100% methanol as the solvent, and the extraction was saturated after 60 min. Samples of six different amounts (30, 40, 50, 60, 70, and 80 mg) were extracted with 50 mL methanol for 60 min, and the extraction efficiency for 50 mg was proved to be the best. Finally, two extraction methods (reflux and ultrasonication) were attempted, and the extraction efficiency with the ultrasonicator was found to be slightly better.2.3 Method validationThe calibration curve was established by linear regression analysis, and the calibration range was 0.5-50.0 μg/mL. The regression equation was expressed as y=563 544.9 957x-17 781.6 200, where y is the peak area and x is the concentration (μg/mL) of betaine. The correlation coefficient (r2) of 0.999 2 implied that the linear regression of the calibration curve was good within the calibration range; the low LOD (0.013 μg/mL) and LOQ (0.045μg/mL) meant that overload and pollution of the chromatographic columns could be avoided. In Ref. [12], betaine in Lycii Fructus was analyzed using HPLC-ELSD, and the LOD was 2.31 μg/mL. In Ref. [15], betaine was determinated using UPLC-MS/MS, and the LODs for plasma and urine were 0.03 and 3.00 μg/mL, respectively. In the present work, the LOD was 0.013 μg/m L, which was much lower than the values obtained with HPLC and UPLC.With the present method, the relative standard deviations (RSDs) of the intraday and interday (three days) precisions were found to be 0.9% and 1.1% (n=6), respectively, which indicated high precision of the instrument. The stability of the sample was reasonable over the tested period (RSD, 1.6%), and the repeatability of the method was acceptable (RSD, 1.4%). The accuracy was validated by the recovery (R), which was calculated using the following formula: R=[(found amount-original amount)/spiked amount]×100%. As shown in Table 1, the average recovery of 96.3% with RSD of 0.5% demonstrated the high accuracy of the method.Table 1 Study of recovery for validation of accuracyAnalyteSpikedleve l/μgRecovery/%Average/%RSD/%Betaine6096.7±0.5196.30.57596.3±0.749095.8±0.56Recovery: mean±standard deviation (SD); n=3.2.4 Sample analysisThe developed UPC2-MS method was applied to analyze the betaine contents in 11 batches of Lycii Fructus samples, which were collected from eleven different counties of Ningxia and Gansu provinces in China. In Table2, the obtained betaine contents are listed. It could be seen that the betaine contents in the Lycii Fructus samples from different counties were slightly different. The betaine content of sample S-01 was the best (1.39μg/mg), while that of S-09 was the worst (1.20 μg/mg). This was because the difference in the growing conditions, such as growing regions, growing years, and harvest seasons, would result in variations in quality [2].Table 2 Betaine contents of 11 batches of Lycii Fructus samplesSampleNo.CountyContent/(μg/mg)S-01Zhongning1.39±0.08S-02Guyuan1.36±0.21S-03Yinchuan1.31±0.09S-04Zhongwei1.36±0.14S-05Pingluo1.32±0.19S-06Shizuishan1.33±0.22S-07Tongxin1.35±0.31S-08Jingyuan1.24±0.17S-09Jiuquan1.20±0.21S-10Jinta1.29±0.18S-11Minqin1.31±0.26Content: mean±SD; n=3.3 ConclusionsIn the present study, a novel UPC2-MS method operating in positive ion mode was developed to analyze the betaine contents of Lycii Fructus samples. The results showed that the separation of betaine was successful on an ACQUITY UPC2 BEH 2-EP column, and the UPC2-MS total ion chromatograms showed good baseline resolution and peak shapes within a short time (3 min). The calibration curve was established, and the method validation indicated that the precision, repeatability, stability, and accuracy of this method were excellent. In particular, the LOD was much lower than that in previous reports. The developed UPC2-MS method was further applied to analyze the quality of eleven batches of Lycii Fructussamples. The betaine contents in samples from different areas were slightly different. The present method is thus expected to be very useful for evaluating the quality of Lycii Fructus.References:[1] Wu D T, Cheong K L, Deng Y, et al. Carbohyd Polym, 2015, 134: 12[2] Lu W, Jiang Q, Shi H, et al. J Agric Food Chem, 2014, 62: 9073[3] Seeram N P. J Agric Food Chem, 2008, 56: 627[4] Potterat O. Planta Med, 2010, 76: 7[5] Amagase H, Farnsworth N R. Food Res Int, 2011, 44: 1702[6] Duan H, Chen Y, Chen G. J Chromatogr A, 2010, 1217: 4511[7] Craig S A S. Am J Clin Nutr, 2004, 80: 539[8] The Pharmacopoeia Commission of the People’s Republic of China. Pharmacopoeia of People’s Republic of China, Part 1. Beijing: China Medical Science Press, 2015: 249[9] Korea Food and Drug Administration. Korean Pharmacopoeia IX, Part II. Seoul: Shinil Books, 2007: 913[10] Shin Y G, Cho K H, Kim J M, et al. J Chromatogr A, 1999, 857: 331[11] Huang H, Chen X S, Liao Q. Chinese Journal of Experimental Traditional Medical Formulae, 2010, 16: 59[12] Zhao B T, Jeong S Y, Hwangbo K, et al. Arch Pharm Res, 2013, 36: 1231[13] Holm P I, Ueland P M, Kvalheim G, et al. Clin Chem, 2003, 49: 286[14] Suo D, Li L, Zhang S, et al. Anal Methods, 2013, 5: 59[15] Ocque A J, Stubbs J R, Nolin T D. J Pharm Biomed Anal, 2015, 109: 128[16] Berger T A. J Chromatogr A, 2015, 1421: 171[17] Zhu L L, Zhao Y, Xu Y W, et al. J Pharm Biomed Anal, 2016, 120: 72[18] Breitenbach S, Rowe W F, McCord B, et al. J Chromatogr A, 2016, 1440: 201[19] Hicks M B, Regalado E L, Tan F, et al. J Pharm Biomed Anal, 2016, 117: 316[20] Ganipisetty V N R, Ravi B, Reddy C R, et al. Anal Methods, 2015, 7: 1092[21] Berger T A, Berger B K. Chromatographia, 2013, 76: 1631[22] Lin C, Xie X, Fan N, et al. Chinese Journal of Chromatography, 2015, 33: 397[23] Said A B, Guinot C, Ruiz J C, et al. J Supercrit Fluid, 2016, 110: 22[24] Perrenoud A G G, Guillarme D, Boccard J, et al. J Chromatogr A, 2016, 1450: 101[25] Hegstad S, Havnen H, Helland A, et al. J Chromatogr B, 2017,1061/1062: 103[26] Lesellier E, Mith D, Dubrulle I. J Chromatogr A, 2015, 1423: 158[27] Dai X, Wei B, Wang X, et al. Chinese Journal of Chromatography, 2015, 33: 1059[28] Li W, Li X, Li G, et al. Chinese Journal of Chromatography, 2016, 34: 795[29] West C, Lemasson E, Bertin S, et al. J Chromatogr A, 2016, 1440: 212[30] West C, Bouet A, Routier S, et al. J Chromatogr A, 2012, 1269: 325[31] Åsberg D, Enmark M, Samuelsson J, et al. J Chr omatogr A, 2014, 1374: 254[32] Li K, Fu Q, Xin H, et al. Analyst, 2014, 139: 3577[33] Jumaah F, Larsson S, Essén S, et al. J Chromatogr A, 2016, 1440: 191[34] Desfontaine V, Guillarme D, Francotte E, et al. J Pharm Biomed Anal, 2015, 113: 56。

枸杞中甜菜碱的含量测定

用高效液相色谱测定l峰面积值，计算回收萃，结果平均回收率为 97.17% ， RSD 为1.05%，且表 30
3 结论与讨论
拘祀作为常用的药食两用材料，在食晶和药晶的标准体系中均有收载。
食晶标准无需检测甜慕碱的含量，药品标准作为最严格的执行标准，对某有效成分甜菜暗有进行检测，用薄层色谱法，但是该方法温度和湿度对显色影响较大，重现性差。本文建立高效液相色谱方法测定相把子甜慕碱的方法撮作准确简单快速、重复性好，可用于拘把子药材];[.真制剂的质量控制。参考文献
含量 [l] ，该方法与中国药典标准方法薄层色谱法 (2015 年版第一部 P249 页)相比更加准确、简便可靠。 1 药舆法定测定法
《中国药典)) 2015 年版拘把子甜菜碱含量测定采用薄层色谱法，具体
操作方法为:取拘把子约泣，用剪刀
剪碎，精密称定，加 800/0 甲醇 50mL 做溶剂，加热回流，用该溶剂 30mL 分次洗涤残渣和滤器，将洗液与滤液合并后浓缩至 10mL，调节 pH 值至 1 ，加入活性炭 19 脱色，加入新配制的 2.5% 硫氨酸铭镀溶液 20mL ，搅匀，低温静置 3 h ，过滤。加丙嗣定量至 5mL ，作为供试晶溶液 [2]。另取甜
以质量浓度为横坐标，绘制标准曲线，
得回归方程为: Y=2323413X+29.21 , r=0.9999 (图 1) 。结果表明，甜菜碱在 0'{).300mg/mL 范围内线性关系良好。
2.3.2 精密度试验取浓度为 0.120mg/mL 的甜菜碱对照品溶液，于高效液相色谱连续进样 5 次，根据峰面积值测定结果(见
编号甜菜醺含量
1 0.82%
4 0.82%
5 0.84%

中药饮片——薄层扫描检验原始记录

电子天平型号：电子天平编号：
薄层色谱扫描仪型号:薄层色谱扫描仪编号:
（1）点样：精密吸取供试品溶液5µl、对照品溶液3µ1与6µl
（2）薄层板：G薄层板上
（3）展开剂：以丙酮-无水乙醇-盐酸（10：6：1)为展开剂
（4）展开：预饱和30分钟，展开，取出，挥干溶剂，立即喷以新配制的改良碘化铋钾试液，放置1~3小时至斑点清晰照
（5）测试条件：波长：；λS= 515nm,λR= 590nm,测量供试品吸光度积分值与对照品吸光度积分值,计算，即得。
计算：
结果：（本品按干燥品计算，含甜菜碱(C5H11NO2)不得少于0. 30%）
RSD=
□ 符合规定 □不符合规定 □ 仅作数据积累
XXXXXXXXX有限公司
薄层扫描原始记录
编号：
品名
规格
温度
编码（批号）
请验日期
湿度
甜菜碱取本品剪碎，取约g，精密称定，加80%甲醇50ml，加热回流1小时，放冷，滤过，用80%甲醇30ml分次洗涤残渣和滤器，合并洗液与滤液，浓缩至10ml，用盐酸调节pH值至1,加人活性炭lห้องสมุดไป่ตู้，加热煮沸，放冷，滤过，用水15ml分次洗涤，合并洗液与滤液，加人新配制的2.5%硫氰酸铬铵溶液20ml,搅匀，100C以下放置3小时。用G4垂熔漏斗滤过，沉淀用少量冰水洗涤，抽干，残渣加丙酮溶解，转移至5ml量瓶中，加丙酮至刻度，摇匀，作为供试品溶液。另取甜菜碱对照品适量，精密称定，加盐酸甲醇溶液（0. 5—100)制成每l m l含4 mg的溶液，作为对照品溶液。照薄层色谱法(通则0502)试验。

枸杞甜菜碱含量测定方法的比较研究

关键词: 枸杞; 甜菜碱; 高效液相色谱法; 分光光度法; 含量测定
中图分类号: R284. 1
文献标识码: A
文章编号: 1004 1389( 2007) 06 0292 04
Comparative Study on Methods for Determination of Betaine from Lycium barbarum L.
宁夏枸杞( L y ci um bar barum L . ) 作为重要的药用植物资源和药食同源的名贵中药材, 具有增强免疫力、降血压、降血脂、抗氧化、抗肿瘤等多方面的药理作用[ 1] 。其中甜菜碱是枸杞果、叶、柄中主要生物碱之一, 在体内起甲基供体作用, 是枸杞中对脂质代谢或抗脂肪肝作用的主要活性物质 [ 2, 3] 。因此, 枸杞甜菜碱的提取分离、定量测定已受到广泛的关注。
西北农业学报 2007, 16( 6) : 292~ 295 A cta A gr icultur ae Bor eali occidental is Sinica
枸杞甜菜碱含量测定方法的比较研究*
张自萍1 , 郭荣1 , 廖国玲1, 2, 马佳卉1
( 1. 宁夏大学西部特色生物资源保护与利用教育部重点实验室, 宁夏银川 2. 宁夏医学院药检系, 宁夏银川 750021)
搅匀, 10 以下放置 3 h。用 G4 垂熔漏斗过滤, 沉淀用少量冰水洗涤, 抽干, 残渣加 70% 丙酮溶解, 并转移至 25 m L 容量瓶中, 加 70% 丙酮至刻度, 摇匀, 即为枸杞甜菜碱待测溶液。
分光光度法甜菜碱标准曲线的制备: 精确称取甜菜碱对照品 0. 1000 g, 蒸馏水溶解并定容于 10m L 容量瓶中, 即得 10. 0 mg/ mL 甜菜碱标准液。分别精确移取甜菜碱标准液 0. 4, 0. 6, 0. 8, 1. 0, 1. 2 mL 至 10 m L 比色管中, 冷水浴放置 10 min。取出后分别加入 6 mL 新配置的2. 5% 雷氏盐溶液, 然后用蒸馏水补充至刻度。冷水浴放置 3 h, G4 垂熔漏斗过滤, 用少量冰水洗沉淀, 抽干, 70% 丙酮溶解沉淀, 并转移至 10 m L 容量瓶中, 用 70% 丙酮定容。

高效液相色谱-电喷雾检测器法测定枸杞子中甜菜碱的含量

１．２方法
１．２．１色谱条件色谱柱：安捷伦Ｐｏｒｏｓｈｅｌｌ１２０Ｈｉｌｌｉｃ（３．０ｍｍ ×１５０ｍｍ，
２７μｍ）；柱温３０℃；流速０．５ｍＬ／ｍｉｎ，流动相为乙腈－０．１％甲酸水溶液（８０∶２０）；进样量：１０μＬ；ＣｏｒｏｎａＵｌｔｒａＣＡＤ检测０℃，雾化气压２００ｋＰａ，采样频率１０Ｈｚ，过滤模式设为３，等度洗脱。１．２．２对照品溶液配制
甜菜碱为枸杞子的主要功效成分，目前枸杞子中甜菜碱含量的测定方法主要包括比色法［４］，非水滴定法［５］，薄层扫描法［１，６－７］，离子色谱法［８］，高效液相色谱法［９－１２］，高效液相色谱－串联质谱法等［１３］。比色法、非水滴定法虽然成本低，但操作费时，容易受到干扰，灵敏度低。薄层扫描法前处理复杂，且重复性较差。离子色谱法能准确测定枸杞子中甜菜碱的含量，但样品处理需要脱色以及除去蛋白质，使前处理变得复杂。高效液相色谱－串联质谱法虽然能保证灵敏度，但成本太高。高效液相色谱法是测定甜菜碱含量最常用的方法，使用紫外检测器时通常用１９２ｎｍ作为检测波长，容易受到干扰，而使用蒸发光散射检测器时重复性较差，灵敏度较低。
摘要：目的：建立枸杞子中甜菜碱的高效液相色谱－电喷雾检测器分析方法。方法：采用高效液相色谱－电喷雾检测器（ＨＰＬＣ－ＣＡＤ）法，安捷伦Ｐｏｒｏｓｈｅｌｌ１２０Ｈｉｌｌｉｃ色谱柱（３．０ｍｍ×１５０ｍｍ，２．７μｍ），乙腈－０．１％甲酸水溶液为流动相，流速０．５ｍＬ／ｍｉｎ。结果：甜菜碱标准曲线线性关系良好（ｒ＝０．９９９９），平均回收率为９１．１％～９５．９％，ＲＳＤ为１．１％～２．６％，重复性ＲＳＤ＝１．３％（ｎ＝６）。结论：该方法准确、简便，重现性好，适用于枸杞子中甜菜碱的含量测定。关键词：高效液相色谱－电喷雾检测器；枸杞子；甜菜碱中图分类号：Ｏ６５７．７＋３文献标识码：Ａ文章编号：１００８－０２１Ｘ（２０１８）１２－００６７－０３

双波长薄层扫描法测定不同来源枸杞子中甜菜碱的含量

双波长薄层扫描法测定不同来源枸杞子中甜菜碱的含量谭亮;冀恬;曹静亚;胡风祖【摘要】通过对枸杞子样品提取、脱色时间等前处理条件的优化,建立不同来源枸杞子中甜菜碱含量测定的双波长薄层扫描法(TLCS法).使用快速溶剂萃取仪(ASK 350)用80％甲醇提取出枸杞子中的甜菜碱,经活性炭脱色、雷氏盐沉淀、丙酮溶解沉淀,采用改进的薄层扫描法在检测波长为530 nm,参比波长为625 nm条件下对枸杞子中的甜菜碱进行含量测定.得到清晰的薄层色谱斑点,无干扰;甜菜碱点样量在3.84～38.40 μg范围内线性关系良好,r=0.9995;平均加样回收率为98.30％,RSD =2.55％(n=9).该方法简便、准确,重现性好,适用于测定枸杞子中甜菜碱的含量测定,可为枸杞的质量控制提供依据.【期刊名称】《天然产物研究与开发》【年(卷),期】2014(026)003【总页数】5页(P388-391,397)【关键词】双波长薄层扫描法;枸杞子;甜菜碱;不同来源;含量测定【作者】谭亮;冀恬;曹静亚;胡风祖【作者单位】中国科学院西北高原生物研究所,西宁810008;中国科学院西北高原生物研究所,西宁810008;中国科学院西北高原生物研究所,西宁810008;中国科学院西北高原生物研究所,西宁810008【正文语种】中文【中图分类】R284.2枸杞子(Fructus Lycii)为茄科植物宁夏枸杞(Lycium barbarum L．)的干燥成熟果实。

夏、秋二季果实呈红色时采收，热风烘干，除去果梗。

或晾至皮皱后，晒干，除去果梗［1］。

现代药理学研究表明，枸杞子有抗衰老、降血糖、降血脂、内分泌激素调节等多方面药理作用［2-4］。

近年来，有关枸杞子中多糖、甜菜碱、总黄酮、原花青素、β-胡萝卜素、东莨菪内酯等成分的含量测定屡有文献报道［5-8］。

其中，甜菜碱是枸杞果、叶、柄中主要生物碱之一，在体内起甲基供体作用，是枸杞中对脂质代谢或抗脂肪肝作用的主要活性物质［9］。

枸杞子中甜菜碱含量测定方法的建立和提取方法的优化

枸杞子中甜菜碱含量测定方法的建立和提取方法的优化作者：黄钰馨马玲李苗郑国保马小荣来源：《中国药房》2020年第14期中圖分类号 R927.2 文献标志码 A 文章编号 1001-0408（2020）14-1700-04DOI 10.6039/j.issn.1001-0408.2020.14.07摘要目的：建立枸杞子中甜菜碱的含量测定方法，并优化其提取方法。

方法：采用高效液相色谱法测定枸杞子中甜菜碱的含量，色谱柱为Waters Spherisorb NH2，流动相为乙腈-0.01 mol/L磷酸二氢钾水溶液（75 ∶ 25，V/V），流速为0.7 mL/min，检测波长为195 nm，柱温为30 ℃，进样量为10 μL。

以甜菜碱含量为指标，在单因素试验的基础上，采用L9（34）正交试验设计对枸杞子中甜菜碱超声提取的甲醇体积分数、提取时间、料液比进行筛选并进行验证。

测定10批枸杞子中甜菜碱的含量，并与2015年版《中国药典》收录的薄层色谱法测定结果进行比较。

结果：甜菜碱检测质量浓度的线性范围为2.035～2 035.04 μg/mL（R 2=0.999 3）;检测限、定量限分别为0.410、2.051 μg/mL，平均加样回收率为97.41%～98.86%（RSD为0.8%～1.4%，n=3），精密度、重复性、稳定性（24 h）试验的RSD均不大于1.2%。

最优提取方法为料液比1 ∶ 30（g/mL）投料、甲醇超声提取45 min。

3次验证试验所得提取液中甜菜碱的平均含量为2.30%（RSD=0.43%， n=3）。

10批枸杞子中甜菜碱的含量为1.91%～2.55%，与薄层色谱法的测定结果（1.88%～2.60%）无明显差异（相对误差为-1.92%～2.7%）。

结论：成功建立了枸杞子中甜菜碱的含量测定方法，并优化了其提取工艺。

关键词枸杞子;甜菜碱;高效液相色谱法;含量测定;提取方法Establishment of the Content Determination Method of Betaine in Lycium barbarum and Optimization of the Extraction MethodHUANG Yuxin1，MA Ling1，LI Miao2，ZHENG Guobao2，MA Xiaorong1（1. Ningxia Hui Autonomous Region Institute of Drug Control， Yinchuan 750002， China; 2. Agricultural Biotechnology Research Center of Ningxia Academy of Agricultural and Forestry Sciences，Yinchuan 750002， China）ABSTRACT OBJECTIVE： To establish the content determination method of betaine in Lycium barbarum， and to optimize the extraction method. METHODS：HPLC method was used to determine the content of betaine in L. barbarum. The determination was performed on Waters Spherisorb NH2 column with mobile phase consisted of acetonitrile-0.01 mol/L monopotassiun phosphate aqueous solution （75 ∶ 25， V/V） at the flow rate was 0.7 mL/min. The detection wavelength was 195 nm， and column temperature was 30 ℃. The sample size was 10 μL. Using the content of betaine as index， on the basis of single factor tests， L9（34） orthogonal test design were used to select the methanol volume fraction， extraction time and solid-liquid ratio of betaine in L. barbarum by ultrasonic extraction. The contents of betaine in 10 batches L. barbarum were determined， and compared with the results of TLC included in 2015 edition of Chinese Pharmacopeia. RESULTS： The linear range of betaine was 2.035-2 035.04 μg/mL（R2=0.999 3）. The limits of detection and quantification were 0.410 μg/mL and 2.051 μg/mL， respectively. The average recovery were 97.41%-98.86% （RSDs were 0.8%-1.4%， n=3）. RSDs of precision，reproducibility and stability （24 h） tests were not higher than 1.2%. The optimal extraction method included solid-liquid ration of 1 ∶ 30 （g/mL）， ultrasonic extraction with methanol for 45 min. The average content of betaine in the extract from the three validation tests was 2.30%（RSD=0.43%， n=3）. The contents of betaine in 10 batches L. barbarum were 1.91%-2.55%，which was no significantly different from the results of TLC （1.88%-2.60%）（RE were -1.92%-2.79%）. CONCLUSIONS： The content determination method of betaine in L. barbarum was established successfully， and the extraction process was optinized.KEYWORDS Lycium barbarum; Betaine; HPLC; Content determination; Extraction method枸杞子为茄科植物宁夏枸杞（Lycium barbarum L.）的干燥成熟果实[1]，是我国传统中药材，具有治疗糖尿病[2]、保护视网膜[3]、抗氧化[4]等作用。

HPLC 法测定枸杞子配方颗粒中甜菜碱的含量

HPLC 法测定枸杞子配方颗粒中甜菜碱的含量李晶;郑新元;王杰【摘要】目的：建立枸杞子配方颗粒中甜菜碱的含量测定方法。

方法：采用高效液相色谱法，乙腈-水（85：15）为流动相，流速为1.0 ml／min，柱温为30℃，检测波长为200 nm，色谱柱为 Kromasil NH2（250 mm ×4.6 mm，5μm）。

结果：6个厂家13批次的枸杞子配方颗粒中甜菜碱含量介于0.5428~2.0556 mg ／ g 之间。

结论：该方法操作简便，结果稳定可靠，可用于枸杞子配方颗粒的质量评价。

%Objective:To establish an HPLC method for determination of betaine in wolfberry fruit formula granule. Methods:HPLC was performedon a Kromasil NH2(250 mm × 4. 6 mm,5 μm)column at 30 ℃ with mobile phase consisting of acetonitrile -water(85: 15),the flow rate was 1. 0 ml/ min,the detection wavelength was 200 nm. Results:The content of betainein wolfber-ry fruit formula granule in 13 batches from six factories ranged between 0. 542 8 ~ 2. 055 6 mg/ g. Conclusion:The method is sim-ple and reliable,and can be used for the quality evaluation of betaine in wolfberry fruit formula granule.【期刊名称】《天津药学》【年(卷),期】2016(028)003【总页数】3页(P4-6)【关键词】枸杞子配方颗粒;HPLC;甜菜碱【作者】李晶;郑新元;王杰【作者单位】天津市药品检验所，天津 300070;天津市药品检验所，天津 300070;天津市药品检验所，天津 300070【正文语种】中文【中图分类】R927.2枸杞子是我国常用中药之一，为茄科植物宁夏枸杞Lycium barbarum L的干燥成熟果实，味甘，性平，具有滋补肝肾、益精明目的功效［1］，用于治疗虚劳精亏、腰膝酸痛、眩晕耳鸣、阳痿遗精、内热消渴、血虚萎黄、目昏不明。

枸杞中甜菜碱的测定(20版药典)

枸杞中甜菜碱的含量测定1、适用范围2020药典第一部枸杞子枸杞子，为茄科植物枸杞的成熟果实，具有多种保健功效，卫生部批准的药食两用食物，适量食用有益健康，配合菊杞茶有清肝明目效果。

其质量标准在历版中国药典中以测定枸杞多糖和甜菜碱成分为主。

由于甜菜碱极性大，C18色谱柱不能对较好保留，15版药典一直以薄层扫描测定为主，直到20版药典开始用氨基柱进行分析。

2、对照品溶液的制备取甜菜碱对照品适量，精密称定，加水制成每1mL含0.17mg的溶液，即得。

3、供试品溶液的制备取本品粉碎，取约1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，密塞，称定重量，加热回流1小时，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，精密量取续滤液2mL，置碱性氧化铝固相萃取柱ProElut AL-B2g/12mL(Cat#65207)上，用乙醇30mL洗脱，收集洗脱液，蒸干，残渣加水溶解，转移至2mL量瓶中，加水至刻度，摇匀，滤过，取续滤液，即得。

4、分析条件（两款色谱柱可供选择）4.1色谱柱：Platisil5μm NH2,250x4.6mm(Cat#99505)流速：1.0mL/min进样量：10uL柱温：30℃检测器：PDA195nm流动相：乙腈：水=85：154.2色谱柱：Inspire5um NH2,250x4.6mm(Cat#81706)流速：1.0mL/min进样量：10uL柱温：30℃检测器：PDA195nm流动相：乙腈：水=85：155、色谱图5.1柱1：Platisil 5μm NH2,250x 4.6mm (Cat#99505)1020Time (min)0.0000.010V o l t s 1对照品溶液（170ug/mL ）液相色谱图峰号保留时间min 峰面积μV*s 峰高μV 理论塔板数N USP 拖尾因子分离度116.85528914690296273.3271.273--1020Time (min)0.0000.010V o l t s 1供试品溶液液相色谱图峰号保留时间min 峰面积μV*s 峰高μV 理论塔板数N USP 拖尾因子分离度116.91510622734757945.9481.036--5.2柱2：Inspire 5um NH2,250x 4.6mm (Cat#81706)01020Time (min)0.000.020.040.060.080.0100.0120.0140.016V o l t s 1对照品溶液（170ug/mL ）液相色谱图峰号保留时间min 峰面积μV*s 峰高μV 理论塔板数N USP 拖尾因子分离度117.742307692114079978.0361.286--01020Time (min)0.000.020.040.060.080.0100.0120.0140.016V o l t s 1供试品溶液液相色谱图峰号保留时间min 峰面积μV*s 峰高μV 理论塔板数N USP 拖尾因子分离度117.987115496435811434.621.009--。