A method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography

Abstract

The present invention relates to the technical field of drug analysis, and in particular to a method for online detection of peony lactone glycoside-6'-O-benzene sulfonate related substances by high performance liquid chromatography, the method comprising the following steps: (1) dissolving a peony lactone glycoside-6'-O-benzene sulfonate sample and set the volume to the mark, shaking well, and obtaining a test solution; (2) subjecting the peony lactone glycoside-6'-O-benzene sulfonate sample in step (1) to high performance liquid chromatography analysis, using an ultraviolet detector to determine the related substances in the peony lactone glycoside-6'-O-benzene sulfonate sample, using an area normalization method to quantify the related substances, and by comparing the content change difference of the related substances in the peony lactone glycoside-6'-O-benzene sulfonate samples at different production stages, the production process of peony lactone glycoside-6'-O-benzene sulfonate is subjected to online quality monitoring. The method is simple to operate, economical and environmentally friendly, and highly safe, and can be used as an online quality control auxiliary means for the production process of peony lactone glycoside-6'-O-benzene sulfonate products.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for online detection of substances related to paeoniflorin-6'-O-benzenesulfonate by high performance liquid phase. Background Art

[0002] Peony lactone glycoside-6'-O-benzenesulfonate, the structural formula is as follows:

[0003]

[0004] White peony root is the root of Paeonia lactiflora of the family Paeonia lactiflora, which has antipyretic and analgesic effects. Modern pharmacology shows that white peony extract also has multiple pharmacological activities such as anti-inflammatory immunomodulation, detoxification and liver protection, anti-mutagenicity and anti-tumor regulation. White peony total glycosides are also the first anti-inflammatory immunomodulator used in the clinical treatment of rheumatoid arthritis, and have the characteristics of good efficacy, few adverse reactions, and good tolerance. Albiflorin, as one of the main active ingredients of white peony, also has multiple pharmacological effects, such as anti-epileptic, analgesic, drug detoxification, anti-vertigo, etc. It can also be used to treat rheumatoid arthritis, bacterial dysentery, enteritis, viral hepatitis, senile diseases, etc.

[0005] Bifidobacterium lactone glycoside is the second most active ingredient in the traditional Chinese medicine white peony. In recent years, with the in-depth study of its pharmacodynamics, it has been found that bifidobacterium lactone glycoside has unique pharmacodynamic characteristics that are different from paeoniflorin. It has pharmacodynamic characteristics that are superior to paeoniflorin in terms of blood tonification, anti-depression, and potential treatment of diabetes, and has anticonvulsant, anti-inflammatory and liver-protecting effects. Furthermore, in the study of the pharmacokinetics of bifidobacterium lactone glycoside, it was found that after oral administration of bifidobacterium lactone glycoside to ICR mice, the content of other tissues except the stomach, intestines, and kidneys was low, and the clearance rate was fast, which may be related to the poor lipid solubility of bifidobacterium lactone glycoside. Therefore, it is necessary to derivatize bifidobacterium lactone glycoside to improve its lipid solubility, thereby improving its absorption and bioavailability, and better exerting its pharmacodynamic effects.

[0006] The peony lactone glycoside-6'-O-benzenesulfonate is a raw material rich in peony lactone glycosides. After extraction, adsorption, separation and purification, peony lactone glycosides with high purity are obtained. Organic metal catalysts, acid-binding agents and chemically active benzenesulfonyl chloride are used to react with peony lactone glycosides. The product is separated and refined by column chromatography to obtain more than 98% of peony lactone glycoside-6'-O-benzenesulfonate, which is dissolved in a mixed solvent and programmed drying is performed to finally obtain the finished product of peony lactone glycoside-6'-O-benzenesulfonate. Peony lactone glycoside-6'-O-benzenesulfonate is an innovative drug. At present, there is no report on the method of using high performance liquid phase to determine the impurities of peony lactone glycoside-6'-O-benzenesulfonate in domestic and foreign patents and literatures. 6'-O-benzenesulfonate is very easy to be degraded by alcohol. At the same time, this product is very hygroscopic and thermally unstable, so it is very easy to produce degradation products during the synthesis, purification and drying production process, resulting in a significant increase in related substances, which poses a hidden danger to the quality and safety of the raw materials. In order to monitor the related substances in the production process of 6'-O-benzenesulfonate raw materials and effectively control the related substances of the raw materials, this study aims to establish a high-performance liquid chromatography determination method that can quickly detect the related substances in the production process of 6'-O-benzenesulfonate, analyze the changes in the related substances in the manufacturing process of 6'-O-benzenesulfonate, and guide the optimization of the production process. Summary of the invention

[0007] The purpose of the present invention is to solve the problem that degradation products are easily generated in the synthesis, purification and drying production process of peony lactone glycoside-6'-O-benzenesulfonate, and the prior art lacks relevant detection means. After a large number of experiments, a method for detecting related substances in the production process of peony lactone glycoside-6'-O-benzenesulfonate raw materials is explored, and a method for online detection of peony lactone glycoside-6'-O-benzenesulfonate related substances using high performance liquid chromatography is provided. The method is simple to operate, economical and environmentally friendly, and highly safe, and can be used as an online quality control auxiliary means for the production process of peony lactone glycoside-6'-O-benzenesulfonate.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography, using acetonitrile-tetrahydrofuran mixed solvent as mobile phase A, phosphoric acid aqueous solution as mobile phase B, and a reverse phase analytical column for gradient elution to determine the related substances in a sample of paeoniflorin-6'-O-benzenesulfonate.

[0010] There are many commonly used mobile phases in liquid phase analysis, but how to mix and match mobile phases to achieve better separation effects is the focus of research in this field. Since some impurities in the objects detected by the present invention are very similar in polarity and structure, and the number of impurities is large, this causes great difficulties in the selection and optimization of the mobile phase system. After long-term research, it was unexpectedly found that using acetonitrile-tetrahydrofuran mixed solvent as mobile phase A and phosphoric acid aqueous solution as mobile phase B can achieve excellent separation effects, thereby realizing the determination of related substances in paeoniflorin-6'-O-benzenesulfonate samples.

[0011] Preferably, the gradient elution is performed with a mixed solvent of acetonitrile and tetrahydrofuran in a volume ratio of 95:5 as the mobile phase A and a 0.2% phosphoric acid aqueous solution as the mobile phase B; and the reverse phase analytical column is YMC-Triart C18.

[0012] The inventors found that the process of peony lactone glycoside-6'-O-benzenesulfonate has many impurities before purification, the separation of impurities and main components, and the separation effect between impurities with similar structures is poor, resulting in the inability to accurately detect impurities. After multiple experiments, it was confirmed that the YMC-Triart (C184.6mm×250mm, 3μm) used, the mobile phase A was acetonitrile-tetrahydrofuran (95:5) as the mobile phase A, and 0.2% phosphoric acid aqueous solution was used as the mobile phase B for gradient elution. It was unexpected that the product was developed and produced in the process. A variety of related substances and the main components achieved good separation effects, and the separation effect between the impurities was good. The chromatographic information of other mobile phase systems such as acetonitrile-phosphoric acid or methanol-phosphoric acid was worse than the detection method of this article, which was not conducive to the quality control of the product production process, resulting in the inability to provide reliable data in time to support the online real-time quality tracking of the product.

[0013] Preferably, the specific operation of the gradient elution is: at 0 to 12 minutes, the initial volume ratio of the mobile phase A to the mobile phase B is 20:80; at 12 to 15 minutes, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 20:80 to 34:66; at 15 to 40 minutes, the volume ratio of the mobile phase A to the mobile phase B is maintained at 34:66; at 40 to 50 minutes, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 34:66 to 60:40; at 50 to 60 minutes, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 34:66 to 60:40; n, the volume ratio of the mobile phase A to the mobile phase B is maintained at 60:40; from 60 to 65 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 60:40 to 80:20; from 65 to 70 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 80:20; from 70 to 75 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 80:20 to 20:80; from 75 to 85 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 20:80.

[0014] The elution procedure is as follows:

[0015]

[0016]

[0017] Preferably, the mobile phase flow rate of the gradient elution is 0.8 ml / min, the injection volume is 20 μL, the column temperature is 20-30° C., the detector is an ultraviolet absorption detector, and the detection wavelength is 220-240 nm.

[0018] Preferably, the reverse phase analytical column is YMC-Triart C18, with a specification of 4.6 mm×250 mm and a filler particle size of 3 μm; the column temperature of the gradient elution is 25° C., and the detection wavelength of the ultraviolet absorption detector is 230 nm.

[0019] Preferably, the test solution is prepared before the gradient elution, specifically: dissolving a sample of paeoniflorin-6'-O-benzenesulfonate and diluting to the mark, and shaking well to obtain the test solution.

[0020] Preferably, the paeoniflorin-6'-O-benzenesulfonate sample is dissolved in methanol; in the test solution, the solution concentration is 1 mg / ml.

[0021] An application of the above-mentioned method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography is used for online quality control of the production process of paeoniflorin-6'-O-benzenesulfonate, comprising the following steps:

[0022] A. Determine the related substances in the sample of peony lactone glycoside-6'-O-benzenesulfonate using the method described in claim 1, and record the chromatogram;

[0023] B. quantifying the related substances by area normalization method;

[0024] C. By comparing the content changes of the related substances in samples of peony lactone glycoside-6'-O-benzenesulfonate at different production stages, it is used for online quality control of the production process of peony lactone glycoside-6'-O-benzenesulfonate.

[0025] Preferably, in step B, the specific operation includes: removing the solvent peak and the main peak of paeoniflorin-6'-O-benzenesulfonate, and analyzing and calculating the sum of the impurity peak area percentage content by using the area normalization method.

[0026] Preferably, in step B, peaks less than 0.05% are ignored.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention applies conventional high performance liquid chromatography separation and detection technology to the analysis of related substances of peony lactone glycoside-6'-O-benzenesulfonate. The analytical method can better reflect the impurity change information of peony lactone glycoside-6'-O-benzenesulfonate, and can achieve better separation between each chromatographic peak. By comparing the changing trends of the content of related substances of peony lactone glycoside-6'-O-benzenesulfonate at different production stages, as an online quality control method for the production process of peony lactone glycoside-6'-O-benzenesulfonate, data support is provided for the industrialization of the product and a reliable basis is provided for the establishment of product quality standards.

[0029] 2. The method adopted by the present invention is simple to operate, economical, environmentally friendly, highly safe, has low requirements for the configuration of detection equipment, and can be promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate at the process stage of intermediate product 1 under the chromatographic conditions of the present invention in Example 1;

[0031] Figure 2 This is a liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate at the process stage of the intermediate product 1 under the same type of conventional chromatographic column as in Example 1;

[0032] Figure 3 This is a liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate in the process stage of intermediate product 1 under mobile phase system 1 (conventional mobile phase solution system) of Example 1;

[0033] Figure 4This is a liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate at the process stage of intermediate product 1 under mobile phase system 3 of Example 1;

[0034] Figure 5 This is a liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate in the process stage of the reaction solution of Example 2.

[0035] Figure 6 This is the liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate in the process stage of intermediate product 1 of Example 2.

[0036] Figure 7 This is the liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate in the process stage of intermediate product 2 of Example 2.

[0037] Figure 8 This is the liquid chromatogram of peony lactone glycoside-6'-O-benzenesulfonate in the process stage 3 of the intermediate product of Example 2.

[0038] Fig. 9 This is the liquid chromatogram of paeoniflorin-6'-O-benzenesulfonate in the finished product process stage of Example 2. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0040] Example 1

[0041] Screening of experimental parameters for HPLC detection methods

[0042] 1. Mobile phase system screening

[0043] The specific operation process is as follows:

[0044] (1) Preparation of test solution: Weigh the intermediate product 1 of peony lactone glycoside-6'-O-benzenesulfonate with relatively high impurity content, dilute it with methanol to a sample content of 1 mg / ml, mix well, and filter to prepare the test solution.

[0045] (2) Setting the HPLC detection conditions: the chromatographic column is YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), the flow rate is 0.8 ml / min for gradient elution, the detection wavelength is 230 nm, and the column temperature is 25 °C;

[0046] (3) Accurately pipette 20 μl of the test solution and inject it into a high performance liquid chromatograph. Analyze according to the gradient elution procedure in Table 1 and record the chromatogram.

[0047] (4) The area normalization method was used to analyze and calculate the content of related substances. The sum of the impurity peak area percentages was calculated, excluding the solvent peak and the main peak of paeoniflorin-6'-O-benzenesulfonate. Any peak less than 0.05% in the test solution can be ignored. The results of the mobile phase system screening are shown in Table 2.

[0048] Table 1 Gradient elution program

[0049]

[0050]

[0051] Table 2 Comparative study results of mobile phase systems

[0052] Chromatographic columns Number of impurities Minimum resolution between peaks Mobile phase system 1 19 1.57 Mobile phase system 2 25 1.41 Mobile phase system 3 19 1.11

[0053] The results show that when using mobile phase system 1, some impurities cannot achieve baseline separation. After adding an appropriate amount of tetrahydrofuran to the mobile phase system 2, the different solvent distribution coefficients and elution abilities are different, which greatly improves the separation and resolution between impurities, and also avoids the situation where some impurities with similar polarity overlap and cannot be separated. The appropriate proportion of tetrahydrofuran in mobile phase A is very important. Too much or too little tetrahydrofuran cannot effectively separate all impurities. The number of impurities detected when mobile phase A does not contain tetrahydrofuran is 19. When mobile phase A contains 10% tetrahydrofuran (the volume ratio of acetonitrile-tetrahydrofuran is 90:10), the number of impurities detected is 19. When mobile phase A contains 5% tetrahydrofuran (the volume ratio of acetonitrile-tetrahydrofuran is 95:5), the number of impurities detected is 25. Better results are achieved in the number of related substances detected and the separation between peaks. Therefore, we selected the volume ratio of acetonitrile-tetrahydrofuran in mobile phase A as 95:5. This mobile phase system has achieved significant improvement results.

[0054] 2. Chromatographic column screening

[0055] Compare the chromatographic columns under the selected mobile phase chromatography system. The specific operation process is as follows:

[0056] (1) Preparation of test solution: Weigh the intermediate product 1 of peony lactone glycoside-6'-O-benzenesulfonate with relatively high impurity content, dilute it with methanol to a sample content of 1 mg / ml, mix well, and filter to prepare the test solution.

[0057] (2) Setting HPLC detection conditions: Column 1 is DIKMA C18(2) (4.6mm×250mm, 3μm), column 2 is YMC-Triart C18 (4.6mm×250mm, 3μm), flow rate is 0.8ml / min, gradient elution is performed, detection wavelength is 230nm, column temperature is 25℃;

[0058] (3) Accurately pipette 20 μl of the test solution and inject it into a high performance liquid chromatograph. Analyze according to the gradient elution sequence in Table 3 and record the chromatogram.

[0059] (4) The area normalization method was used to analyze and calculate the content of related substances. The sum of the area percentage content of the impurity peaks was calculated, excluding the solvent peak and the main peak of paeoniflorin-6'-O-benzenesulfonate. Any peak less than 0.05% in the test solution can be ignored. The results of the chromatographic column comparison study are shown in Table 4.

[0060] Table 4 Column screening conditions

[0061]

[0062] Table 4 Column comparison study results

[0063] Chromatographic columns Number of impurities Minimum resolution between peaks DIKMA 14 1.07 YMC 25 1.41

[0064] The results showed that the number of impurities detected by column 2 (YMC-Triart C18 4.6mm×250mm, 3μm) was higher than that of column 1 (DIKMA C18 (2), 4.6mmx250mm, 3μm). The inventors have found through research that some impurities are very close in polarity to the main component. Therefore, when using the traditional C18 chromatographic column for detection, even if the mobile phase and gradient elution program are repeatedly optimized, it is difficult to effectively separate the main component and impurities, and between the impurities. Therefore, the separation between the components in the product is difficult to meet the detection requirements, and even the main peak of the product overlaps with the impurity peak, and the presence of impurities cannot be detected. When this special chromatographic column and mobile phase are used for detection, impurities with similar structures and polarities, as well as the main components of the product, can be well separated, and the impurity content can be accurately detected. Therefore, the chromatographic column finally selected is chromatographic column 2 (YMC-Triart C18 4.6mm×250mm, 3μm).

[0065] Example 2

[0066] Taking the production process of a certain product of peony lactone glycoside-6'-O-benzenesulfonate as an example, the quality control methods in the production process are explored. In this production process, there are five process stages: reaction liquid, intermediate 1, intermediate 2, intermediate 3, and finished product. The products of each process stage are taken for experiment.

[0067] (1) Preparation of test solution: Weigh appropriate amounts of the reaction solution, intermediate 1, intermediate 2, intermediate 3, and finished product from the production process of peony lactone glycoside-6'-O-benzenesulfonate, dilute with methanol to a sample content of 1 mg / ml, mix well, and filter to prepare the test solution.

[0068] (2) Setting the HPLC detection conditions: the chromatographic column is YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), the flow rate is 0.8 ml / min for gradient elution, the detection wavelength is 230 nm, and the column temperature is 25 °C;

[0069] (3) Accurately pipette 20 μl of the test solution and inject it into a high performance liquid chromatograph for analysis and record the chromatogram.

[0070] (4) The area normalization method was used to analyze and calculate the content of related substances. The sum of the area percentage content of the impurity peaks was calculated, excluding the solvent peak. Any peak less than 0.05% in the test solution can be ignored. The determination results of products of peony lactone glycoside-6'-O-benzenesulfonate at different production stages are shown in Table 5.

[0071] Table 5 Test results of products at different production stages

[0072]

[0073] As shown in Table 5, the number of chromatographic peaks of related substances in the reaction solution of peony lactone glycoside-6'-O-benzenesulfonate is 27. After the process of eliminating impurities, the number of impurities in different process stages continues to decrease, and the number of chromatographic peaks of the final product is 5. Therefore, through the above data analysis, the changing trend of the content of related substances in peony lactone glycoside-6'-O-benzenesulfonate samples at different production stages can be summarized, which can be used as an online quality control method for the production process of peony lactone glycoside-6'-O-benzenesulfonate products, providing data support for the industrialization of products and providing a basis for the establishment of product quality standards.

[0074] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography, characterized in that: The acetonitrile-tetrahydrofuran mixed solvent was used as mobile phase A, phosphoric acid aqueous solution was used as mobile phase B, and a reverse phase analytical column was used for gradient elution to determine the related substances in the sample of paeoniflorin-6'-O-benzenesulfonate. The gradient elution was performed using a mixed solvent of acetonitrile and tetrahydrofuran in a volume ratio of 95:5 as the mobile phase A and a 0.2% by volume phosphoric acid aqueous solution as the mobile phase B; the reverse phase analytical column was YMC-TriartC18; The specific operation of the gradient elution is as follows: at 0-12 min, the initial volume ratio of the mobile phase A to the mobile phase B is 20:80; at 12-15 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 20:80 to 34:66; at 15-40 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 34:66; at 40-50 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 34:66 to 60:40; at 50-60 min, The volume ratio of the mobile phase A to the mobile phase B is maintained at 60:40; from 60 to 65 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 60:40 to 80:20; from 65 to 70 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 80:20; from 70 to 75 min, the volume ratio of the mobile phase A to the mobile phase B changes linearly and uniformly from 80:20 to 20:80; from 75 to 85 min, the volume ratio of the mobile phase A to the mobile phase B is maintained at 20:

80.

2. The method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 1, characterized in that: The mobile phase flow rate of the gradient elution is 0.8 ml / min, the injection volume is 20 μL, the column temperature is 20-30° C., the detector is an ultraviolet absorption detector, and the detection wavelength is 220-240 nm.

3. The method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 2, characterized in that: The reverse phase analytical column is YMC-TriartC18, with a specification of 4.6 mm×250 mm and a filler particle size of 3 μm; the column temperature of the gradient elution is 25° C., and the detection wavelength of the ultraviolet absorption detector is 230 nm.

4. The method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 1, characterized in that: The test solution is prepared before the gradient elution, specifically, the following steps: dissolving a sample of paeoniflorin-6'-O-benzenesulfonate and diluting to the mark, and shaking well to obtain the test solution.

5. The method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 4, characterized in that: The paeoniflorin-6'-O-benzenesulfonate sample was dissolved in methanol; in the test solution, the solution concentration was 1 mg / ml.

6. An application of the method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 1, characterized in that: The method is used for online quality control of the production process of peony lactone glycoside-6'-O-benzenesulfonate, comprising the following steps: A. Determine the related substances in the sample of peony lactone glycoside-6'-O-benzenesulfonate using the method described in claim 1, and record the chromatogram; B. quantifying the related substances by area normalization method; C. By comparing the content changes of the related substances in samples of peony lactone glycoside-6'-O-benzenesulfonate at different production stages, it is used for online quality control of the production process of peony lactone glycoside-6'-O-benzenesulfonate.

7. Application of the method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 6, characterized in that: In step B, the specific operation includes: removing the solvent peak and the main peak of paeoniflorin-6'-O-benzenesulfonate, and analyzing and calculating the sum of the impurity peak area percentage content by using the area normalization method.

8. Application of the method for online detection of related substances of paeoniflorin-6'-O-benzenesulfonate by high performance liquid chromatography as claimed in claim 7, characterized in that: In step B, peaks with less than 0.05% were ignored.

Images