CN109959751A - Thin-layer quantitative image recognition detection method for water-soluble natural products - Google Patents
Thin-layer quantitative image recognition detection method for water-soluble natural products Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
本发明公开了一种水溶性天然产物薄层定量图像识别检测方法,包括:(1)含有水溶性天然产物的待检测样品在薄层色谱板上进行点样;(2)利用第一展开剂和第二展开剂对点样后的薄层色谱板进行薄层色谱逐级展开;所述第一展开剂为乙酸乙酯/乙醇/水/乙酸组成的展开体系,所述第二展开剂为三氯甲烷/乙酸乙酯/甲酸组成的展开体系;(3)利用染色剂系统对展开后的薄层色谱板进行显色;(4)对显色后的薄层色谱板进行图像采集,根据图像与水溶性天然产物含量之间的关系,得出水溶性天然产物的含量。本发明的方法操作简便,不需要专用仪器。本发明的方法定量性好,准确度高。本发明的方法测试速度快,可以多个样品同时测定。
The invention discloses a thin-layer quantitative image recognition and detection method for water-soluble natural products, comprising: (1) spotting a sample to be detected containing water-soluble natural products on a thin-layer chromatographic plate; (2) using a first developing agent Carry out thin-layer chromatography step-by-step development on the thin-layer chromatography plate after spotting with the second developing agent; the first developing agent is a developing system composed of ethyl acetate/ethanol/water/acetic acid, and the second developing agent is A developing system consisting of chloroform/ethyl acetate/formic acid; (3) developing the color of the developed thin-layer chromatographic plate by using a dye system; (4) collecting the image of the developed thin-layer chromatographic plate, according to the The relationship between the image and the content of water-soluble natural products yields the content of water-soluble natural products. The method of the invention is easy to operate and does not need special instruments. The method of the invention has good quantification and high accuracy. The method of the invention has a fast test speed and can measure multiple samples simultaneously.
Description
技术领域technical field
本发明属于天然产物和食品药品领域,具体涉及一种水溶性天然产物薄层定量图像识别检测方法。The invention belongs to the field of natural products and food and medicine, and particularly relates to a thin-layer quantitative image identification and detection method for water-soluble natural products.
背景技术Background technique
水溶性天然产物包括类黄酮、黄酮苷、花色苷、皂苷是植物中重要的活性成分,具有抑菌保鲜、抗氧化、延缓衰老、抗菌、抗病毒、降血脂、抗癌等功效。类黄酮、黄酮苷、花色苷水溶性天然产物具有优良的抗氧化作用,可通过清除活性氧簇或其反应生成稳定化合物的方式有效平衡人体内自由基,并且其金属离子鳌合能力能终止自由基链式反应。大量的体内及体外实验和流行病学资料表明,皂苷水溶性天然产物具有抗炎、降脂、抗糖尿病和心血管疾病(CVD)预防等生物学和药理作用,在制药、生化、日化、食品以及精细化工等高科技领域具有广阔的应用前景。常用的水溶性天然产物检测方法有分光光度法方法、液相法,但是需要专用的仪器,操作相对麻烦。Water-soluble natural products, including flavonoids, flavonoid glycosides, anthocyanins, and saponins, are important active ingredients in plants, and have antibacterial and fresh-keeping, antioxidant, anti-aging, antibacterial, antiviral, hypolipidemic, and anticancer effects. The water-soluble natural products of flavonoids, flavonoid glycosides, and anthocyanins have excellent antioxidant effects, which can effectively balance free radicals in the human body by scavenging reactive oxygen species or their reactions to generate stable compounds, and their metal ion chelation ability can terminate free radicals. base chain reaction. A large number of in vivo and in vitro experiments and epidemiological data show that the water-soluble natural products of saponins have biological and pharmacological effects such as anti-inflammatory, lipid-lowering, anti-diabetic and cardiovascular disease (CVD) prevention. High-tech fields such as food and fine chemicals have broad application prospects. Commonly used detection methods for water-soluble natural products include spectrophotometric method and liquid phase method, but they require special instruments and are relatively troublesome to operate.
最近计算机图像处理技术迅猛发展,图像的定量分析已经可行。以ImageJ为代表的图像处理程序已广泛用于生命科学研究领域,如电泳条带定量分析、细胞计数、组织结构定量处理等。ImageJ是一个NIH开发的公共领域Java图像处理程序。它可以在任何具有Java1.4或更高版本虚拟机的计算机上作为联机applet或可下载应用程序运行。可下载的发行版适用于Windows,Mac OS,Mac OS X和Linux。它可以显示,编辑,分析,处理,保存和打印8位,16位和32位图像。它可以读取许多图像格式,包括TIFF,GIF,JPEG,BMP,DICOM,FITS和“raw”。它支持“堆栈”,一系列共享单个窗口的图像。它是多线程的,因此可以与其他操作并行执行诸如图像文件读取之类的耗时操作。它可以计算用户定义选择的面积和像素值统计。它可以测量距离和角度。它可以创建密度直方图和线轮廓图。它支持标准图像处理功能,如对比度操作,锐化,平滑,边缘检测和中值滤波。它可以进行几何变换,如缩放,旋转和翻转。所有分析和处理功能均可在任何放大系数下使用。该程序同时支持任意数量的窗口(图像),仅受可用内存的限制。空间校准可用于以毫米为单位提供真实世界的尺寸测量。还可以进行密度或灰度校准。ImageJ采用开放式架构设计,通过Java插件提供可扩展性。可以使用ImageJ的内置编辑器和Java编译器开发自定义采集,分析和处理插件。用户编写的插件可以解决几乎任何图像处理或分析问题。Recently, computer image processing technology has developed rapidly, and quantitative analysis of images has become feasible. Image processing programs represented by ImageJ have been widely used in the field of life science research, such as quantitative analysis of electrophoretic bands, cell counting, and quantitative processing of tissue structures. ImageJ is a public domain Java image processing program developed by the NIH. It can be run as an online applet or downloadable application on any computer with a Java 1.4 or higher virtual machine. Downloadable distributions are available for Windows, Mac OS, Mac OS X and Linux. It can display, edit, analyze, process, save and print 8-bit, 16-bit and 32-bit images. It can read many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and "raw". It supports "stacks", a series of images that share a single window. It is multi-threaded, so time-consuming operations such as image file reading can be performed in parallel with other operations. It can calculate area and pixel value statistics for user-defined selections. It can measure distances and angles. It can create density histograms and line contour plots. It supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering. It can perform geometric transformations such as scaling, rotation and flipping. All analysis and processing functions are available at any magnification factor. The program supports any number of windows (images) simultaneously, limited only by available memory. Spatial calibration can be used to provide real-world dimensional measurements in millimeters. Density or grayscale calibration is also possible. ImageJ is designed with an open architecture and provides extensibility through Java plugins. Custom acquisition, analysis and processing plugins can be developed using ImageJ's built-in editor and Java compiler. User-written plugins can solve almost any image processing or analysis problem.
天然产物的薄层分析是一种常用的高效技术,但是传统的薄层定量分析需要专用的仪器,应用不便。因此基于最新的计算机图像定量分析手段,进行类黄酮多酚的薄层定量图像识别检测能够简化操作,有力地拓宽使用范围。目前已经有一些利用图像识别进行活性成分定量检测的研究。例如申请号200510115774.X专利文献,公开了一种基于图像处理技术的薄层色谱定量分析方法,包括步骤:(1)对载有样品薄层板拍照形成数字图像;(2)对拍摄图像预处理:镜头畸变校正、滤除噪声;(3)利用点样均匀分布、纵向距离等分原则确定控制点;(4)根据控制点插值方法构造图像背景;(5)从镜头畸变校正图像去除背景,根据背景对像素点亮度归一化;(6)对薄层图像进行分割(7)对分割后的区域灰度进行积分、定量分析。但该方法只对图像分析的一般原理进行了描述,无法直接应用到具体天然产物的定量检测。申请号201510163742.0的专利文献,公开了一种巴戟天耐斯糖含量测定方法,利用梯形积分法对色谱峰进行积分,根据峰面积和样品浓度进行定量分析。但该方法仅限于一种特定糖的测定。申请号201810451846.5的专利文献,公开了一种鉴别蜂蜜真伪的方法,用ImageJ软件对蜂蜜样品中的寡糖进行分析。但该方法仅限于一类糖的分析。因此开发适用于不同类型的薄层图像识别定量分析方法,对于天然产物的快速分析鉴别具有重要的意义。Thin-layer analysis of natural products is a commonly used high-efficiency technique, but traditional thin-layer quantitative analysis requires special instruments and is inconvenient to use. Therefore, based on the latest computer image quantitative analysis methods, the thin-layer quantitative image recognition and detection of flavonoid polyphenols can simplify the operation and effectively broaden the scope of use. At present, there have been some studies using image recognition for quantitative detection of active ingredients. For example, the patent document with application number 200510115774.X discloses a thin-layer chromatography quantitative analysis method based on image processing technology, which includes the steps: (1) photographing a thin-layer plate carrying a sample to form a digital image; (2) pre-processing the photographed image Processing: lens distortion correction and noise filtering; (3) use the principle of uniform distribution of spot samples and equal vertical distance to determine control points; (4) construct the image background according to the control point interpolation method; (5) remove the background from the lens distortion corrected image , normalize the brightness of the pixel points according to the background; (6) segment the thin-layer image; (7) integrate and quantitatively analyze the gray level of the segmented area. However, this method only describes the general principles of image analysis, and cannot be directly applied to the quantitative detection of specific natural products. The patent document with the application number 201510163742.0 discloses a method for measuring the content of Morinda officinalis Nessaccharide. The trapezoidal integration method is used to integrate the chromatographic peaks, and quantitative analysis is performed according to the peak area and the sample concentration. But the method is limited to the determination of one specific sugar. The patent document of application number 201810451846.5 discloses a method for identifying the authenticity of honey, using ImageJ software to analyze oligosaccharides in honey samples. But this method is limited to the analysis of one class of sugars. Therefore, the development of quantitative analysis methods for different types of thin-layer image recognition is of great significance for the rapid analysis and identification of natural products.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种水溶性天然产物薄层定量图像识别检测方法工艺简便,不需要专用仪器,适用面广。The invention provides a thin-layer quantitative image identification and detection method for water-soluble natural products, which is simple in process, does not require special instruments, and has wide application.
一种水溶性天然产物薄层定量图像识别检测方法,包括:A water-soluble natural product thin-layer quantitative image recognition detection method, comprising:
(1)将含有水溶性天然产物的待检测样品在薄层色谱板上进行点样;(1) spotting a sample to be detected containing a water-soluble natural product on a thin-layer chromatographic plate;
(2)利用第一展开剂和第二展开剂对点样后的薄层色谱板进行薄层色谱逐级展开;所述第一展开剂为乙酸乙酯/乙醇/水/乙酸组成的展开体系,所述第二展开剂为三氯甲烷/乙酸乙酯/甲酸组成的展开体系;(2) using the first developing agent and the second developing agent to carry out thin-layer chromatography step-by-step development on the thin-layer chromatography plate after the spotting; the first developing agent is a developing system composed of ethyl acetate/ethanol/water/acetic acid , the second developing agent is the developing system that chloroform/ethyl acetate/formic acid is formed;
(3)利用染色剂系统对展开后的薄层色谱板进行显色;(3) using the dye system to develop the color of the expanded TLC plate;
(4)对显色后的薄层色谱板进行图像采集,根据图像与水溶性天然产物含量之间的关系,得出水溶性天然产物的含量。(4) Image collection of the color-developed thin-layer chromatography plate, and the content of water-soluble natural products is obtained according to the relationship between the image and the content of water-soluble natural products.
具体讲,一种水溶性天然产物薄层定量图像识别检测方法,基于计算机图像识别的对不同水溶性天然产物(类黄酮、黄酮苷、花色苷、皂苷)及其混合物进行同时定量分析,包括以下步骤:Specifically, a thin-layer quantitative image recognition and detection method for water-soluble natural products, based on computer image recognition for simultaneous quantitative analysis of different water-soluble natural products (flavonoids, flavonoids, anthocyanins, saponins) and their mixtures, including the following step:
(1)利用乙酸乙酯/乙醇/水/乙酸和三氯甲烷/乙酸乙酯/甲酸展开剂系统对不同大类天然产物进行薄层色谱逐级展开;(1) Utilize ethyl acetate/ethanol/water/acetic acid and chloroform/ethyl acetate/formic acid developing solvent system to carry out thin-layer chromatography step-by-step development of different large classes of natural products;
(2)利用统一的染色剂系统对不同水溶性天然产物进行薄层色谱显色;(2) Utilize a unified dye system to carry out TLC color development for different water-soluble natural products;
(3)对水溶性天然产物薄层色谱图像电子化及定量分析,得出最终水溶性天然产物薄层定量结果。(3) Electronization and quantitative analysis of water-soluble natural product thin-layer chromatographic images to obtain the final water-soluble natural product thin-layer quantitative result.
本发明中,水溶性天然产物在检测前,可以利用溶剂溶解,配制为水溶性天然产物样品溶液,然后再进行薄层色谱展开;所述的水溶性天然产物样品溶液浓度为0.01-10mg/mL;进一步优选为0.5-5mg/mL。采用薄层色谱时,点样量为0.1-10微升;优选为0.1~1微升。所述待检测水溶性天然产物样品可以利用多种溶剂溶解,一般采用溶解性较好,易挥发的溶剂,作为优选,所述溶剂为乙醇、二氯甲烷、丙酮、甲醇或乙酸乙酯等中的一种或多种;进一步优选为乙醇或乙酸乙酯中的一种或两种。In the present invention, before detection, the water-soluble natural product can be dissolved in a solvent, prepared into a water-soluble natural product sample solution, and then developed by thin-layer chromatography; the water-soluble natural product sample solution has a concentration of 0.01-10 mg/mL ; further preferably 0.5-5 mg/mL. When thin-layer chromatography is used, the amount of the sample to be spotted is 0.1-10 microliters; preferably, it is 0.1-1 microliter. The water-soluble natural product sample to be detected can be dissolved in a variety of solvents, generally a solvent with good solubility and volatile nature is used. Preferably, the solvent is ethanol, dichloromethane, acetone, methanol or ethyl acetate. One or more of; further preferably one or both of ethanol or ethyl acetate.
所述的水溶性天然产物样品点样至薄层色谱后在展开剂系统中逐级展开,首先用乙酸乙酯/乙醇/水/乙酸展开剂板上展开至R1的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸展开剂重新展开至R2的长度。The water-soluble natural product samples are spotted in thin-layer chromatography and then developed step by step in the developing solvent system, firstly using ethyl acetate/ethanol/water/acetic acid developing agent plate to develop to the distance of R1, then air-drying, and then Re-expand to R2 length with chloroform/ethyl acetate/formic acid developing solvent.
本发明中,所述的R1、R2均是以初始点样中心点为起始点、以展开剂展开后最前端线为终点表示的距离。作为优选,所述R1为15~25毫米;所述R2为35~45毫米。当然,对于特定的展开体系,也可以通过控制展开时间对两种展开体系的展开作用进行控制。作为优选,所述R1为20毫米;所述R2为40毫米。In the present invention, the R1 and R2 are the distances expressed by taking the initial spotting center point as the starting point and the frontmost line after the developing agent is developed as the end point. Preferably, the R1 is 15-25 mm; the R2 is 35-45 mm. Of course, for a specific deployment system, the deployment effects of the two deployment systems can also be controlled by controlling the deployment time. Preferably, the R1 is 20 mm; the R2 is 40 mm.
所述第一展开剂中各溶剂的体积比为乙酸乙酯/乙醇/水/乙酸=5:2.5~3/0.1~0.2/0.1~0.15;进一步优选的体积比为:乙酸乙酯/乙醇/水/乙酸=5/2.85/0.15/0.12;所述第二展开剂中各溶剂的体积比为三氯甲烷/乙酸乙酯/甲酸=1/0.8~1.2/0.01~0.05;进一步优选的体积比为三氯甲烷/乙酸乙酯/甲酸=1/1/0.03。The volume ratio of each solvent in the first developing agent is ethyl acetate/ethanol/water/acetic acid=5:2.5~3/0.1~0.2/0.1~0.15; further preferred volume ratio is: ethyl acetate/ethanol/ Water/acetic acid=5/2.85/0.15/0.12; the volume ratio of each solvent in the second developing agent is chloroform/ethyl acetate/formic acid=1/0.8~1.2/0.01~0.05; further preferred volume ratio It is chloroform/ethyl acetate/formic acid=1/1/0.03.
所述的水溶性天然产物样品在薄层色谱上展开后,用对甲氧基苯甲醛显色剂进行显色,显色时间0.1-10分钟,显色温度150-250℃。After the water-soluble natural product sample is developed on the thin layer chromatography, the color is developed with a p-methoxybenzaldehyde color developing agent, the color developing time is 0.1-10 minutes, and the color developing temperature is 150-250°C.
所述染色剂系统的组成为对甲氧基苯甲醛/乙酸/乙醇/硫酸的体积比为9.1~9.5/3.5~4/300~350/12.5。作为进一步优选,所述的对甲氧基苯甲醛显色剂组成为对甲氧基苯甲醛/乙酸/乙醇/硫酸(9.2/3.75/338/12.5,v/v/v/v)。The composition of the dye system is that the volume ratio of p-methoxybenzaldehyde/acetic acid/ethanol/sulfuric acid is 9.1-9.5/3.5-4/300-350/12.5. As a further preference, the p-methoxybenzaldehyde developer is composed of p-methoxybenzaldehyde/acetic acid/ethanol/sulfuric acid (9.2/3.75/338/12.5, v/v/v/v).
本发明中,所述水溶性天然产物包括类黄酮、黄酮苷、花色苷、皂苷中的一种或多种;作为进一步优选,所述水溶性天然产物包括儿茶素、槲皮素、茶皂苷、矢车菊素葡萄糖苷、齐墩果醇酸、氯化矢车菊素、芦丁中的一种或多种。In the present invention, the water-soluble natural product includes one or more of flavonoids, flavonoid glycosides, anthocyanins, and saponins; as a further preference, the water-soluble natural product includes catechin, quercetin, tea saponin , one or more of cyanidin glucoside, oleanolic acid, cyanidin chloride, and rutin.
所述的水溶性天然产物样品在薄层色谱上显色后,采用相机或扫描仪进行电子化图像采集。After the water-soluble natural product sample is colored on thin-layer chromatography, a camera or a scanner is used for electronic image acquisition.
如图1所示,显色完成后,可采用相机或扫描仪进行电子化图像采集,完成水溶性天然产物样品图像的电子化。所述的水溶性天然产物样品图像电子化后,选择合适的图像处理软件,如:ImageJ等,进行图像优化处理。由于定量分析基于灰度值,故先用软件将图片转为灰度图片。As shown in Figure 1, after the color development is completed, a camera or a scanner can be used for electronic image acquisition to complete the electronic image of the water-soluble natural product sample. After the image of the water-soluble natural product sample is electronically digitized, an appropriate image processing software, such as ImageJ, etc., is selected to perform image optimization processing. Since the quantitative analysis is based on grayscale values, the images are first converted into grayscale images with software.
所述的水溶性天然产物样品图像优化处理包括去噪声、平滑。对上述灰度图片进行去椒盐噪声,校正,锐化边缘,平滑等一系列操作,使图片更适合于定量分析。The image optimization processing of the water-soluble natural product sample includes denoising and smoothing. A series of operations such as removing salt and pepper noise, correcting, sharpening edges, and smoothing the above grayscale images are performed to make the images more suitable for quantitative analysis.
所述的水溶性天然产物样品图像优化处理后,进行背景去除。选择合适的算法,将待分析图像中干扰背景除去。After the image optimization processing of the water-soluble natural product sample, background removal is performed. Select an appropriate algorithm to remove the interfering background in the image to be analyzed.
所述的水溶性天然产物样品图像背景去除后,进行积分定量,得到定量结果。After the background of the water-soluble natural product sample image is removed, integration and quantification are performed to obtain a quantitative result.
以采用ImageJ软件进行图像处理为例,其过程为:Taking ImageJ software for image processing as an example, the process is as follows:
所述的水溶性天然产物样品在薄层色谱上显色后,采用HP M1005扫描仪进行电子化图像采集,各项参数设置为:百万种颜色输出,分辨率300dpi,亮度和对比度默认值,图片以TIFF格式保存。After the water-soluble natural product sample was developed on thin-layer chromatography, an HP M1005 scanner was used for electronic image acquisition, and the parameters were set as: millions of color output, resolution 300dpi, brightness and contrast default values, Pictures are saved in TIFF format.
所述的水溶性天然产物样品图像电子化后,采用ImageJ软件进行图像优化处理。After the images of the water-soluble natural product samples were electronically digitized, ImageJ software was used for image optimization.
首先,定量分析基于灰度值,故启动ImageJ软件,先选择“Image”-“Type”-“8-bit”,将图片转换成灰度图片,然后选择“Image”-“Adjust”-“Brightness/Contrast...”,在弹出的对话框中点击“Auto”,调整图像对比度和明度,使待分析区域可视化程度更高。First of all, the quantitative analysis is based on the gray value, so start the ImageJ software, first select "Image"-"Type"-"8-bit", convert the image to grayscale, and then select "Image"-"Adjust"-"Brightness" /Contrast...", click "Auto" in the pop-up dialog box to adjust the contrast and brightness of the image to make the area to be analyzed more visible.
其次,对灰度图像进行降噪和平滑等图像优化处理,先选择“Process”-“Noise”-“Despeckle”,通过一个中值滤波器将图像中的每个像素替换为其3*3邻域中像素的中值以去除图像中的椒盐噪声,再选择“Process”-“Noise”-“RemoveOutliers...”,将偏差超过设定阈值的像素点替换为周围像素的中间值以校正图像;然后再选择“Process”-“Filters”-“UnsharpMask...”,抽取模糊版本来锐化及增强图像边缘;最后选择“Process”-“Smooth”平滑整个图像以使积分基线更加平稳。Secondly, perform image optimization processing such as noise reduction and smoothing on the grayscale image, first select "Process" - "Noise" - "Despecle", and replace each pixel in the image with its 3*3 neighbors through a median filter. The median value of the pixels in the domain to remove the salt and pepper noise in the image, and then select "Process" - "Noise" - "RemoveOutliers..." to replace the pixels whose deviation exceeds the set threshold with the median value of the surrounding pixels to correct the image. ; Then choose "Process" - "Filters" - "UnsharpMask..." to extract the blurred version to sharpen and enhance the edges of the image; finally choose "Process" - "Smooth" to smooth the entire image to make the integral baseline more stable.
然后,所述的水溶性天然产物样品图像优化处理后,采用ImageJ软件进行背景去除。选择“Process”-“SubtractBackground...”,基于ImageJ中“Rollingball”算法,在弹出的对话框中给定合适的滚球半径,并勾选“Lightbackground”和“Slidingparaboloid”,将图像中的连续背景除去。Then, after image optimization of the water-soluble natural product samples, ImageJ software was used for background removal. Select "Process"-"SubtractBackground...", based on the "Rollingball" algorithm in ImageJ, give a suitable rolling ball radius in the pop-up dialog box, and check "Lightbackground" and "Slidingparaboloid", the continuous image in the image Background removal.
所述的水溶性天然产物样品图像背景去除后,采用ImageJ软件进行积分定量,得到定量结果。点击“RectangularSelectionTool”选择目标区域,点击“Ctrl+1”确定条带,点击“Ctrl+3”进行积分,用“StraightLineSelectionTool”将峰两端连接形成封闭图形后,用“wandtool”确定峰面积,完成定量分析。After the background of the water-soluble natural product sample image was removed, ImageJ software was used to perform integration and quantification to obtain quantitative results. Click "RectangularSelectionTool" to select the target area, click "Ctrl+1" to determine the band, click "Ctrl+3" to integrate, use "StraightLineSelectionTool" to connect the two ends of the peak to form a closed graph, use "wandtool" to determine the peak area, complete Quantitative analysis.
本发明可以使用各种规格的常见的薄层色谱板,作为优选,标准曲线与实际检测所用的薄层色谱板相同。The present invention can use common thin-layer chromatography plates of various specifications, and preferably, the standard curve is the same as the thin-layer chromatography plate used for actual detection.
本发明进行定量分析时,可预先构建积分值(对于ImageJ为峰面积)与样品含量之间的标准曲线,然后,在得到积分值后,可以直接得到样品的含量,即实现待检测样品的定量化。When the present invention performs quantitative analysis, a standard curve between the integral value (peak area for ImageJ) and the sample content can be pre-built, and then, after the integral value is obtained, the content of the sample can be directly obtained, that is, the quantification of the sample to be detected can be realized. change.
一种具体的制作标准曲线的方法如下:A specific method for making a standard curve is as follows:
(i)取设定浓度(比如可以为1.0,2.0,3.0mg/L,可以为平行设置多组)设定体积(可以为0.5微升)的标准样品,在薄层色谱板上进行点样;(i) Take a standard sample with a set concentration (for example, it can be 1.0, 2.0, 3.0 mg/L, and multiple groups can be set in parallel) with a set volume (can be 0.5 μl), and spot it on a thin-layer chromatographic plate. ;
(ii)利用第一展开剂和第二展开剂对点样后的薄层色谱板进行薄层色谱逐级展开;所述第一展开剂为乙酸乙酯/乙醇/水/乙酸组成的展开体系,所述第二展开剂为三氯甲烷/乙酸乙酯/甲酸组成的展开体系;(ii) use the first developing agent and the second developing agent to carry out thin-layer chromatography step-by-step development on the thin-layer chromatography plate after spotting; the first developing agent is a developing system composed of ethyl acetate/ethanol/water/acetic acid , the second developing agent is the developing system that chloroform/ethyl acetate/formic acid is formed;
(iii)利用染色剂系统对展开后的薄层色谱板进行显色;(iii) using a dye system to develop the color of the expanded TLC plate;
(iv)对显色后的薄层色谱板进行图像采集,得到积分值;(iv) image acquisition of the color-developed TLC plate to obtain an integral value;
(v)按照步骤(i)~(iv)的方法,得到多组标准样品对应的积分值,制作样品浓度与积分值之间的标准曲线。(v) According to the method of steps (i) to (iv), the integrated values corresponding to the multiple groups of standard samples are obtained, and a standard curve between the sample concentration and the integrated value is prepared.
第一展开剂和第二展开剂展开距离可以参考上述对检测方法的说明,比如首先用乙酸乙酯/乙醇/水/乙酸展开剂板上展开至R1的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸展开剂重新展开至R2的长度。所述R1为15~25毫米;所述R2为35~45毫米。当然,对于特定的展开体系,也可以通过控制展开时间对两种展开体系的展开作用进行控制。作为优选,实际检测采用的展开剂体系与制作标准曲线采用的展开体系相同。For the development distance of the first developing agent and the second developing agent, please refer to the above description of the detection method. For example, first use the ethyl acetate/ethanol/water/acetic acid developing agent plate to expand the distance to R1, then dry it, and then use trichloride. The methane/ethyl acetate/formic acid developer was re-developed to the length of R2. The R1 is 15-25 mm; the R2 is 35-45 mm. Of course, for a specific deployment system, the deployment effects of the two deployment systems can also be controlled by controlling the deployment time. Preferably, the developing agent system used in the actual detection is the same as the developing system used for making the standard curve.
作为优选,构建标准曲线时,展开距离与实际检测过程中展开距离相同,采用的点样条件,显色条件,以及图像采集和处理条件均与检测过程中一致。Preferably, when constructing the standard curve, the expansion distance is the same as the expansion distance in the actual detection process, and the sampling conditions, color development conditions, and image acquisition and processing conditions used are consistent with the detection process.
本发明即可以用于单个组分样品的检测,也可以用于混合组份样品的检测,用于混合组份样品的检测时,利用图像采集单元分别实现对各个组份样品点的图像采集,然后分别计算样品的浓度值。当然,利用本发明,我们还可以一次实现多个样品的检测,检测时,可将多个样品在同一个薄层色谱板上进行点样,然后分别进行图像采集和计算。如图1所示,针对六个浓度依次升高的样品采用本发明的方法进行检测,即将样品分别点样至同一薄层色谱板上的点样位置,相互之间距离只要能够避免干扰即可;然后展开,进行显色,然后利用计算机扫描仪进行扫描,分别读取各个样品对应的图像,进行积分,最终得到对应样品的浓度值,检测结果与分别单独检测相当,几乎没有干扰作用。The invention can be used for the detection of a single component sample, and can also be used for the detection of a mixed component sample. When used for the detection of a mixed component sample, the image acquisition unit is used to realize the image acquisition of each component sample point respectively, The concentration values of the samples are then calculated separately. Of course, by using the present invention, we can also realize the detection of multiple samples at one time. During detection, multiple samples can be spotted on the same thin-layer chromatographic plate, and then image acquisition and calculation are performed respectively. As shown in Fig. 1, the method of the present invention is adopted for the detection of six samples whose concentrations increase in sequence, namely, the samples are respectively spotted on the spotting positions on the same thin-layer chromatographic plate, and the distance between them can be as long as the interference can be avoided. ; Then unfold, carry out color development, and then scan with a computer scanner, read the corresponding images of each sample, integrate, and finally obtain the concentration value of the corresponding sample. The detection result is equivalent to the separate detection, and there is almost no interference.
本发明相对于现有的检测方法具有如下优点:Compared with the existing detection method, the present invention has the following advantages:
(1)本发明的水溶性天然产物薄层定量图像识别检测方法操作简便,不需要专用仪器。(1) The thin-layer quantitative image recognition and detection method for water-soluble natural products of the present invention is easy to operate and does not require special instruments.
(2)本发明的方法,采用同一展开体系,可以对多种水溶性天然产物进行检测,定量性好,操作简单,准确度高。(2) The method of the present invention, using the same development system, can detect a variety of water-soluble natural products, with good quantification, simple operation and high accuracy.
(3)本发明的方法测试速度快,可以多个样品同时测定;也可以对含有多个组份的样品进行检测,实用性强。(3) The method of the present invention has a fast test speed, and can measure multiple samples at the same time; it can also detect samples containing multiple components, and has strong practicability.
附图说明Description of drawings
图1为针对六个浓度依次升高的样品采用本发明的方法进行检测的过程图。Fig. 1 is a process diagram of the method of the present invention for the detection of six samples whose concentrations are successively increased.
具体实施方式Detailed ways
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.
实施例中采用的显色剂为对甲氧基苯甲醛染色剂,其组成为对甲氧基苯甲醛/乙酸/乙醇/硫酸(9.2/3.75/338/12.5,v/v/v/v)。The color developing agent adopted in the embodiment is p-methoxybenzaldehyde dyeing agent, and its composition is p-methoxybenzaldehyde/acetic acid/ethanol/sulfuric acid (9.2/3.75/338/12.5, v/v/v/v) .
首先制作标准曲线,方法如下:First, create a standard curve as follows:
(i)取设定浓度的标准样品,在薄层色谱板(采用实施例1同样的薄层色谱板)上进行点样;本实施例中,采用0.5,1.0,2.0,3.0,4.0mg/L五组标准样品(样品与每个实施例需要检测的样品相同),溶剂为乙醇,点样体积为0.5微升;(i) Take a standard sample with a set concentration and spot it on a thin-layer chromatography plate (using the same thin-layer chromatography plate in Example 1); L five groups of standard samples (the samples are the same as the samples to be detected in each example), the solvent is ethanol, and the spotting volume is 0.5 microliters;
(ii)干燥后,利用第一展开剂和第二展开剂对点样后的薄层色谱板进行薄层色谱逐级展开;(ii) after drying, use the first developing agent and the second developing agent to carry out thin-layer chromatography step-by-step development on the thin-layer chromatography plate after the spotting;
用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度;Develop a 20 mm distance on a developer plate with ethyl acetate/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v), then air dry and reapply with chloroform/ethyl acetate / Formic acid (1/1/0.03, v/v/v) spreader re-expanded to a length of 40 mm;
(iii)利用对甲氧基苯甲醛染色剂对展开后的薄层色谱板进行显色:250℃加热台加热15秒进行显色;(iii) using p-methoxybenzaldehyde dye to develop the color of the TLC plate after development: heating on a heating table at 250°C for 15 seconds to develop the color;
(iv)薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,分别得到0.5,1.0,2.0,3.0,4.0mg/L五组标准样品对应的积分值;(iv) Scanning with a computer scanner after color development of the thin layer, the resolution is set to 300 dpi. The scanned images were denoised and smoothed with ImageJ, then the background was removed, and finally quantitative integration was carried out to obtain the integration values corresponding to five groups of standard samples of 0.5, 1.0, 2.0, 3.0, and 4.0 mg/L respectively;
(v)制作样品浓度与积分值之间的标准曲线。(v) Prepare a standard curve between sample concentration and integrated value.
为了检测的精确度,针对每组标准样品,平行检测三次,取积分值的平均值为其对应的积分值。For the detection accuracy, for each group of standard samples, parallel detection was performed three times, and the average of the integrated values was taken as its corresponding integrated value.
针对不同组分的样品,均需要按照上述方法进行标准曲线的绘制。For samples of different components, the standard curve needs to be drawn according to the above method.
制作标准曲线过程中,以及实施例中的样品显色后,后续图像处理过程为:In the process of making the standard curve, and after the color of the sample in the embodiment, the subsequent image processing process is as follows:
采用HP M1005扫描仪对显色后的薄层色谱板进行电子化图像采集,各项参数设置为:百万种颜色输出,分辨率300dpi,亮度和对比度默认值,图片以TIFF格式保存。An HP M1005 scanner was used to collect electronic images of the color-developed TLC plates. The parameters were set as: millions of colors output, resolution 300dpi, default values of brightness and contrast, and pictures were saved in TIFF format.
采用ImageJ软件进行图像优化处理。首先,定量分析基于灰度值,故启动ImageJ软件,先选择“Image”-“Type”-“8-bit”,将图片转换成灰度图片,然后选择“Image”-“Adjust”-“Brightness/Contrast...”,在弹出的对话框中点击“Auto”,调整图像对比度和明度,使待分析区域可视化程度更高。Image optimization was performed using ImageJ software. First of all, the quantitative analysis is based on the gray value, so start the ImageJ software, first select "Image"-"Type"-"8-bit", convert the image to grayscale, and then select "Image"-"Adjust"-"Brightness" /Contrast...", click "Auto" in the pop-up dialog box to adjust the contrast and brightness of the image to make the area to be analyzed more visible.
其次,对灰度图像进行降噪和平滑等处理,先选择“Process”-“Noise”-“Despeckle”,通过一个中值滤波器将图像中的每个像素替换为其3*3邻域中像素的中值以去除图像中的椒盐噪声,再选择“Process”-“Noise”-“RemoveOutliers...”,将偏差超过设定阈值的像素点替换为周围像素的中间值以校正图像;然后再选择“Process”-“Filters”-“Unsharp Mask...”,抽取模糊版本来锐化及增强图像边缘;最后选择“Process”-“Smooth”平滑整个图像以使积分基线更加平稳。Secondly, perform noise reduction and smoothing on the grayscale image, first select "Process" - "Noise" - "Despecle", and replace each pixel in the image with its 3*3 neighborhood through a median filter. The median value of the pixels to remove the salt and pepper noise in the image, and then select "Process" - "Noise" - "RemoveOutliers..." to replace the pixels whose deviation exceeds the set threshold with the median value of the surrounding pixels to correct the image; then Then select "Process" - "Filters" - "Unsharp Mask..." to extract the blurred version to sharpen and enhance the edges of the image; finally select "Process" - "Smooth" to smooth the entire image to make the integral baseline more stable.
然后,采用ImageJ软件进行背景去除。选择“Process”-“SubtractBackground...”,基于ImageJ中“Rollingball”算法,在弹出的对话框中给定合适的滚球半径,并勾选“Lightbackground”和“Slidingparaboloid”,将图像中的连续背景除去。Then, ImageJ software was used for background removal. Select "Process"-"SubtractBackground...", based on the "Rollingball" algorithm in ImageJ, give a suitable rolling ball radius in the pop-up dialog box, and check "Lightbackground" and "Slidingparaboloid", the continuous image in the image Background removal.
图像背景去除后,采用ImageJ软件进行积分定量,得到定量结果。点击“RectangularSelectionTool”选择目标区域,点击“Ctrl+1”确定条带,点击“Ctrl+3”进行积分,用“StraightLine SelectionTool”将峰两端连接形成封闭图形后,用“wandtool”确定峰面积,得到对应的积分值。After the image background was removed, ImageJ software was used for integration and quantification, and the quantitative results were obtained. Click "RectangularSelectionTool" to select the target area, click "Ctrl+1" to determine the band, click "Ctrl+3" to integrate, use "StraightLine SelectionTool" to connect the two ends of the peak to form a closed graph, and use "wandtool" to determine the peak area, Get the corresponding integral value.
实施例1Example 1
将(+)-儿茶素乙醇溶液,用毛细管点样0.5微升至薄层色谱待溶剂挥干后,用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度。然后取出待溶剂挥干后用对甲氧基苯甲醛染色剂染色,放置到250℃加热台加热15秒进行显色。薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,积分值通过与标准值比较得到测量结果。实际值为1.20mg/mL的(+)-儿茶素溶液,测量值为1.22mg/mL,误差为2%。The (+)-catechin ethanol solution was spotted with a capillary for 0.5 microliters to thin layer chromatography. /v/v) The developer plate was developed to a distance of 20 mm, then air-dried and re-developed to 40 mm with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) developer length. Then take out the solvent and use p-methoxybenzaldehyde dye to dye it after evaporation of the solvent, and place it on a heating table at 250°C for 15 seconds for color development. After the thin layer was developed, it was scanned with a computer scanner, and the resolution was set to 300 dpi. The scanned images were denoised and smoothed with ImageJ, and then the background was removed. Finally, quantitative integration was performed, and the integrated value was compared with the standard value to obtain the measurement result. The actual value was 1.20 mg/mL of a (+)-catechin solution, the measured value was 1.22 mg/mL, with a 2% error.
采用的薄层色谱板从市场购置,规格(硅胶材料,涂层厚度:0.2-0.25毫米,硅胶粉粒度:10-40微米)。The thin-layer chromatography plate used was purchased from the market, with specifications (silica gel material, coating thickness: 0.2-0.25 mm, silica gel powder particle size: 10-40 microns).
实施例2Example 2
将槲皮素乙醇溶液,用毛细管点样0.5微升至薄层色谱待溶剂挥干后,用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度。然后取出待溶剂挥干后用对甲氧基苯甲醛染色剂染色,放置到250℃加热台加热15秒进行显色。薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,积分值通过与标准值比较得到测量结果。实际值为1.51mg/mL的将槲皮素乙醇溶液,测量值为1.53mg/mL,误差为1.5%。The quercetin ethanol solution was spotted with a capillary at 0.5 microliters to thin layer chromatography. ) developer plate was developed to a distance of 20 mm, then air-dried and re-developed to a length of 40 mm with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) developer. Then take out the solvent and use p-methoxybenzaldehyde dye to dye it after evaporation of the solvent, and place it on a heating table at 250°C for 15 seconds for color development. After the thin layer was developed, it was scanned with a computer scanner, and the resolution was set to 300 dpi. The scanned images were denoised and smoothed with ImageJ, and then the background was removed. Finally, quantitative integration was performed, and the integrated value was compared with the standard value to obtain the measurement result. The actual value of quercetin in ethanol solution was 1.51 mg/mL, the measured value was 1.53 mg/mL, and the error was 1.5%.
实施例3Example 3
将茶皂苷乙醇溶液,用毛细管点样0.5微升至薄层色谱待溶剂挥干后,用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度。然后取出待溶剂挥干后用对甲氧基苯甲醛染色剂染色,放置到250℃加热台加热15秒进行显色。薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,积分值通过与标准值比较得到测量结果。实际值为2.5mg/mL的将茶皂苷乙醇溶液,测量值为2.62mg/mL,误差为5%。The tea saponin ethanol solution was spotted with a capillary at 0.5 microliters to thin layer chromatography. After the solvent was evaporated, ethyl acetate/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) The developer plate was developed a distance of 20 mm, then air-dried and re-developed to a length of 40 mm with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) developer. Then take out the solvent and use p-methoxybenzaldehyde dye to dye it after evaporation of the solvent, and place it on a heating table at 250°C for 15 seconds for color development. After the thin layer was developed, it was scanned with a computer scanner, and the resolution was set to 300 dpi. The scanned images were denoised and smoothed with ImageJ, and then the background was removed. Finally, quantitative integration was performed, and the integrated value was compared with the standard value to obtain the measurement result. The actual value of tea saponin ethanol solution of 2.5mg/mL, the measured value of 2.62mg/mL, the error is 5%.
实施例4Example 4
将矢车菊素葡萄糖苷乙醇溶液,用毛细管点样0.5微升至薄层色谱待溶剂挥干后,用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度。然后取出待溶剂挥干后用对甲氧基苯甲醛染色剂染色,放置到250℃加热台加热15秒进行显色。薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,积分值通过与标准值比较得到测量结果。实际值为1.50mg/mL的矢车菊素葡萄糖苷溶液,测量值为1.53mg/mL,误差为2%。The cyanidin glucoside ethanol solution was spotted with a capillary for 0.5 microliters to thin layer chromatography. v/v) The developer plate was developed to a distance of 20 mm, then air-dried and re-developed to 40 mm with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) developer length. Then take out the solvent and use p-methoxybenzaldehyde dye to dye it after evaporation of the solvent, and place it on a heating table at 250°C for 15 seconds for color development. After the thin layer was developed, it was scanned with a computer scanner, and the resolution was set to 300 dpi. The scanned images were denoised and smoothed with ImageJ, and then the background was removed. Finally, quantitative integration was performed, and the integrated value was compared with the standard value to obtain the measurement result. The actual value of cyanidin glucoside solution was 1.50 mg/mL, and the measured value was 1.53 mg/mL, with an error of 2%.
实施例5Example 5
齐墩果醇酸、槲皮素、(+)-儿茶素、氯化矢车菊素、芦丁乙醇溶液(实际含量分别为:齐墩果醇酸:1.21mg/mL;槲皮素:1.31mg/mL;(+)-儿茶素:1.17mg/mL;氯化矢车菊素:2.54mg/mL;芦丁:1.12mg/mL),用毛细管点样0.5微升至薄层色谱待溶剂挥干后,用乙酸乙酯/乙醇/水/乙酸(5/2.85/0.15/0.12,v/v/v/v)展开剂板上展开20毫米的距离,然后晾干,再用三氯甲烷/乙酸乙酯/甲酸(1/1/0.03,v/v/v)展开剂重新展开至40毫米的长度。然后取出待溶剂挥干后用对甲氧基苯甲醛染色剂染色,放置到250℃加热台加热15秒进行显色。薄层显色后用计算机扫描仪进行扫描,分辨率设置为300dpi。扫描得到的图像用ImageJ进行去噪声、平滑处理,然后去除背景,最后进行定量积分,积分值通过与标准值比较得到测量结果,得到含量值为齐墩果醇酸:1.17mg/mL;槲皮素:1.27mg/mL;(+)-儿茶素:1.15mg/mL;氯化矢车菊素:2.46mg/mL;芦丁:1.09mg/mL。误差3%以下。Oleanolic acid, quercetin, (+)-catechin, cyanidin chloride, rutin ethanol solution (the actual contents are: oleanolic acid: 1.21 mg/mL; quercetin: 1.31 mg/mL; (+)-catechin: 1.17 mg/mL; cyanidin chloride: 2.54 mg/mL; rutin: 1.12 mg/mL), spotted by capillary 0.5 μL to TLC After the solvent is evaporated, use ethyl acetate/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) to spread out a distance of 20 mm on the developer plate, then air dry, and then use three The methyl chloride/ethyl acetate/formic acid (1/1/0.03, v/v/v) developer was re-developed to a length of 40 mm. Then take out the solvent and use p-methoxybenzaldehyde dye to dye it after evaporation of the solvent, and place it on a heating table at 250°C for 15 seconds for color development. After the thin layer was developed, it was scanned with a computer scanner, and the resolution was set to 300 dpi. The scanned image was denoised and smoothed with ImageJ, then the background was removed, and finally quantitative integration was performed. The integrated value was compared with the standard value to obtain the measurement result, and the content value was oleanolic acid: 1.17 mg/mL; quercetin cyanidin: 1.27 mg/mL; (+)-catechin: 1.15 mg/mL; cyanidin chloride: 2.46 mg/mL; rutin: 1.09 mg/mL. The error is less than 3%.
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