A quantitative detection method based on thin-layer chromatography

Abstract

This invention discloses a quantitative detection method based on thin-layer chromatography, including steps such as sample preparation, sample loading, development, color development, and quantitative detection. Based on the linear relationship between the mass of three compounds with CAS numbers 139115-90-5, 111549-97-4, and 13035-61-5 and the area of ​​their spots on a TLC plate within a certain range, quantitative detection can be performed. This quantitative detection method has good linearity, is simple and fast to operate, and is easy to popularize and promote.

Description

Technical Field

[0001] This invention relates to the technical field of compound detection, and more particularly to a quantitative detection method based on thin-layer chromatography. Background Technology

[0002] The three compounds with CAS numbers 139115-90-5, 111549-97-4, and 13035-61-5 are used in the synthesis of a Haemophilus influenzae type b (Hib) conjugate vaccine. This Hib vaccine is a novel vaccine synthesized through a chemical reaction, and these three compounds constitute the backbone of the Hib vaccine antigenic portion. In the production process, all three compounds are quantitatively weighed before being added; therefore, quantitative detection contributes to the accuracy of the addition process.

[0003] The 2020 edition of the Pharmacopoeia of the People's Republic of China (current version) stipulates that the "content (%) of active pharmaceutical ingredients and pharmaceutical excipients, unless otherwise specified, shall be calculated by weight... The symbol '%' indicates a percentage, referring to the proportion by weight." Therefore, compounds used in drug production should also be added according to their actual weight as much as possible. However, the three compounds mentioned above are all novel compounds, and standard samples are unavailable. For compounds 111549-97-4 (ribitol derivative) and 13035-61-5 (β-ribose peracetate), existing detection methods both employ high-performance liquid chromatography (HPLC), followed by calculation of content using the area normalization method. However, the obtained content is not a weight fraction. The area normalization method only represents the percentage value of the relative area of ​​the product and impurities that are measurable by the detector under the detection conditions, not the weight percentage of the content as intended. In other words, if the detector is ultraviolet and the detection condition is set to 220nm, this method can only detect the product and impurities that have ultraviolet absorption under 220nm conditions. Furthermore, the impurities cannot be quantitatively analyzed, and their ultraviolet absorption area does not correspond to their mass. In fact, what is measured is the ratio of the area under the curve of the product and the impurity under a given ultraviolet absorption condition. This leads to a situation where we are not actually aware of how much raw material has been added during the feeding process. In particular, the amount of 111549-97-4 (ribitol derivative) is assumed to be 100% pure and used to guide the weight / volume of other raw materials to be weighed for production, resulting in uncontrollable production processes.

[0004] Another compound, 139115-90-5 (5-azido-3-oxopentanol), is currently detected using an elemental analyzer to determine its nitrogen content. Since it is a small molecule, combining it with 1H nuclear magnetic resonance spectroscopy (NMR) to determine its nitrogen content is a good method if it can be confirmed that it contains no nitrogen impurities. However, this is an indirect measurement, and the content obtained is not a proportion of its weight.

[0005] Therefore, whether a quantitative detection method for the above three compounds can be developed has become an urgent problem to be solved. Summary of the Invention

[0006] In order to develop a quantitative detection method for the three compounds mentioned in the background art, the present invention provides a quantitative detection method based on thin-layer chromatography.

[0007] The present invention provides a quantitative detection method based on thin-layer chromatography, comprising the following steps:

[0008] (1) Sample preparation: Accurately weigh the sample to be tested, dissolve the sample in dichloromethane, and prepare the sample.

[0009] (2) Sample loading, development and color development: Using a microsyringe, sample loading of 2.5 μl, 5 μl, 7.5 μl, 10 μl, 12.5 μl and 15 μl was applied to the HPTLC plate in sequence. After development with developing solvent, the sample was dried, wetted with 5% sulfuric acid ethanol, and then developed by hot air blowing.

[0010] (3) Quantitative detection: Measure the area of ​​each spot and make a standard curve. Use the standard curve as a reference to calculate the content of the sample.

[0011] Preferably, the method is applicable to the three compounds with CAS numbers 139115-90-5, 111549-97-4 and 13035-61-5.

[0012] Further preferred, the sample concentration configured in step (1) is 2 mg / ml-10 mg / ml.

[0013] Further preferably, when the sample to be tested is a compound with CAS number 139115-90-5, the developing solvent is a mixed solution of ethyl acetate, methanol and water, and the volume ratio of ethyl acetate:methanol:water is 85:10:5.

[0014] Further preferably, when the sample to be tested is a compound with CAS number 111549-97-4, the developing solvent is a mixed solution of hexane and ethyl acetate, and the volume ratio of hexane to ethyl acetate is 3:1.

[0015] Further preferably, when the sample to be tested is a compound with CAS number 13035-61-5, the developing solvent is a mixed solution of hexane and ethyl acetate, and the volume ratio of hexane to ethyl acetate is 1:3.

[0016] The present invention provides a quantitative detection method based on thin-layer chromatography, which is based on the linear relationship between the mass of three compounds with CAS numbers 139115-90-5, 111549-97-4 and 13035-61-5 and the area of ​​the spots displayed on the TLC plate within a certain range, and thus can be used for quantitative detection.

[0017] The quantitative detection method based on thin-layer chromatography provided by this invention has good linearity, is simple and quick to operate, and is easy to popularize and promote.

[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the disclosure of the present invention. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a TLC plate photograph used in Example 1 to verify whether the developing solvent can develop the compound and ultimately produce color.

[0022] Figure 2 The image shows a TLC plate photograph of the linear calculation of 139115-90-5 (5-azido-3-oxopentanol) from various manufacturers in one experiment of Example 2.

[0023] Figure 3 This is the standard curve for 5-azido-3-oxopentanol from one experiment in Example 2;

[0024] Figure 4 The images are TLC plate photographs showing the linear calculations of 139115-90-5 (5-azido-3-oxopentanol) from various manufacturers in the secondary experiments of Example 2.

[0025] Figure 5 The standard curve for 5-azido-3-oxopentanol in the second experiment of Example 2;

[0026] Figure 6 The image shows a TLC plate photograph of the linear calculation of 111549-97-4 (ribitol derivative) from various manufacturers in one experiment of Example 2.

[0027] Figure 7This is the standard curve of the ribitol derivative in one experiment of Example 2;

[0028] Figure 8 The images are TLC plate photographs showing the linear calculations of 111549-97-4 (ribitol derivative) from various manufacturers in the secondary experiments of Example 2.

[0029] Figure 9 This is the standard curve of the ribitol derivative in the second experiment of Example 2;

[0030] Figure 10 These are TLC plate images showing the linear calculations for 13035-61-5 (β-ribose peracetate) from various manufacturers in one experiment of Example 2;

[0031] Figure 11 This is the standard curve for β-ribose peracetate in one experiment of Example 2;

[0032] Figure 12 These are TLC plate images showing the linear calculations for 13035-61-5 (β-ribose peracetate) from various manufacturers in the secondary experiments of Example 2;

[0033] Figure 13 This is the standard curve for β-ribose peracetate in the second experiment of Example 2;

[0034] Figure 14 The images are linear calculation TLC plate images of 139115-90-5 (5-azido-3-oxopentanol) from Aladdin and Borren Kairun in Example 3, where (a) is Aladdin and (b) is Borren Kairun.

[0035] Figure 15 The standard curves for 5-azido-3-oxopentanol from Aladdin and Borrenkairun, two manufacturers, are shown in Example 3, where (a) is Aladdin and (b) is Borrenkairun.

[0036] Figure 16 The images are linear calculation TLC plate images of 111549-97-4 (ribitol derivative) from Aladdin and Bailingwei in Example 3, where (a) is Aladdin and (b) is Bailingwei.

[0037] Figure 17 The standard curves for ribitol derivatives from Aladdin and Bailingwei, two manufacturers, are shown in Example 3, where (a) is Aladdin and (b) is Bailingwei.

[0038] Figure 18 The images are linear calculation TLC plate images of 13035-61-5 (β-ribose peracetate) from Aladdin and Bailingwei in Example 3, where (a) is Aladdin and (b) is Bailingwei.

[0039] Figure 19 The standard curves for β-ribose peracetate from Aladdin and Bailingwei manufacturers in Example 3 are shown, where (a) is Aladdin and (b) is Bailingwei. Detailed Implementation

[0040] The present invention will be further explained below with reference to specific implementation schemes, but this is not intended to limit the scope of protection of the present invention.

[0041] Example 1: Confirming whether thin-layer chromatography (TLC) can be used for detection.

[0042] (1) Sample preparation

[0043] Take samples from each manufacturer and dissolve them in dichloromethane.

[0044] (2) Sample loading, development and color development

[0045] Samples were taken using a capillary tube and spotted onto a high-performance thin-layer chromatography (HPTLC) plate.

[0046] The development process was carried out based on the development agents used in the previous production, and the feasible development agents were estimated.

[0047] Table 1-1: Developing solvents used in confirming whether TLC can be used for detection

[0048]

[0049] After unfolding and drying the TLC plate, it was soaked in 5% sulfuric acid ethanol and then developed by blowing hot air from an electric blower.

[0050] (3) Judgment criteria

[0051] If the Rf values ​​of the main spots of products from several manufacturers are consistent (since this step is an estimate, no specific Rf calculation is performed; visual inspection determines that the products from different manufacturers are on the same level, thus determining that the Rf values ​​are consistent), then it is considered that the developing solvent can develop the sample, and further exploration can be conducted to determine whether TLC can be used for quantification.

[0052] (4) Conclusion

[0053] See Figure 1 The main spot Rf values ​​of the three novel compounds, 5-azido-3-oxopentanol, ribitol derivatives, and β-ribose peracetate, were consistent across different manufacturers, and the predicted developing solvents could be used to develop the samples. Therefore, they could all be quantitatively detected by TLC. However, 3-maleimide propionic acid N-hydroxysuccinic acid imide failed to develop color and could not be quantitatively detected by TLC.

[0054] In fact, this study conducted preliminary confirmation of four novel compounds using TLC, and three of these compounds can be detected using this TLC method.

[0055] Example 2: Confirming whether TLC can be used for quantitative detection

[0056] (1) Sample preparation

[0057] Weigh 0.02g of each of the above-mentioned compounds that can be detected by TLC, add 2ml of dichloromethane using a graduated pipette, and dissolve to obtain a sample with a concentration of 10mg / ml.

[0058] (2) Sample loading, development and color development

[0059] Gradient spotting can be performed on the TLC plate using appropriate microsyringes, allowing for a slightly wider mass distribution. Specifically, spotting 5μl, 10μl, 15μl, 20μl, and 25μl samples on the TLC plate using a 25μl microsyringe, and spotting 30μl samples on the TLC plate using a 50μl microsyringe.

[0060] The development was performed using the developing solvent from Example 1, wherein β-ribose peracetate could be used for a second development.

[0061] Table 2-1: Developing solvents used in confirming whether TLC can be used for quantitative detection

[0062] compound CAS number Developing agent and ratio 5-Azide-3-oxopentanol 139115-90-5 Ethyl acetate:methanol:water = 85:10:5 Ribitol derivatives 111549-97-4 Hexane: Ethyl acetate = 3:1 β-ribose peracetate 13035-61-5 Hexane: Ethyl acetate = 3:1

[0063] After unfolding and drying the TLC plate, it was soaked in 5% sulfuric acid ethanol and then developed by blowing hot air from an electric blower.

[0064] (3) Judgment criteria

[0065] If the mass of gradient spotting (converted to spotting volume during spotting) has a linear relationship with the spot area (converted to length × width during measurement), a more precise TLC quantitative method can be further established.

[0066] Since this step is also an exploratory confirmation, R is tentatively set. 2 When the percentage is ≥90%, the linear relationship is confirmed.

[0067] (4) TLC board photos and measurement data

[0068] To ensure the accuracy of the measurement data, data were measured twice for each compound.

[0069] Figure 2 The following are TLC images showing the linearity calculations for 139115-90-5 (5-azido-3-oxopentanol) from various manufacturers in a single experiment. The corresponding linearity calculation tables are as follows:

[0070] factory CQB Borren Kairun Apollo Aladdin <![CDATA[Mass (10 -5 g)]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> 5 9.1760 8.7792 9.9328 14.2200 10 12.6984 17.8080 22.2300 18.1148 15 24.6304 25.3988 26.8784 27.6300 20 29.3376 29.8200 34.0060 32.2472 25 56.2152 36.2292 39.4320 39.6440 30 70.0340 44.9952 46.4968 44.3300 <![CDATA[R 2 ]]> 0.9290 0.9908 0.9809 0.9900

[0071] The corresponding standard curve can be found here. Figure 3 .

[0072] Figure 4 The following are TLC images showing the linearity calculations for 139115-90-5 (5-azido-3-oxopentanol) from various manufacturers in the secondary experiment. The corresponding linearity calculation tables are as follows:

[0073] factory CQB Borren Kairun Apollo Aladdin <![CDATA[Mass (10 -5 g)]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> 5 21.9180 21.2148 21.2000 25.5200 10 32.7568 33.2220 33.3944 40.8336 15 41.7104 40.7148 40.4800 55.7280 20 52.9760 46.8520 44.5416 57.8312 25 62.0816 58.3940 52.2144 63.1800 30 66.2200 62.6400 56.6400 67.3620 <![CDATA[R 2 ]]> 0.9868 0.9856 0.9703 0.8967

[0074] The corresponding standard curve can be found here. Figure 5 .

[0075] Figure 6 The following are TLC plate images showing the linearity calculations for 111549-97-4 (ribitol derivative) from various manufacturers in a single experiment. The corresponding linearity calculation tables are as follows:

[0076]

[0077]

[0078] The corresponding standard curve can be found here. Figure 7 .

[0079] Figure 8 The TLC images show the linearity calculations for 111549-97-4 (ribitol derivative) from various manufacturers in the secondary experiment. The corresponding linearity calculation tables are as follows:

[0080] factory CQB Bailingwei Aladdin <![CDATA[Mass (10 -5 g)]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> 5 24.5160 26.6976 29.1500 10 37.5552 49.2480 42.3272 15 48.1236 56.9800 49.8336 20 60.9936 67.5120 62.6340 25 66.7320 71.3400 70.3664 30 83.9800 83.2048 70.9632 <![CDATA[R 2 ]]> 0.9897 0.9770 0.9445

[0081] The corresponding standard curve can be found here. Figure 9 .

[0082] Figure 10 The following are TLC plate images showing the linearity calculations for 13035-61-5 (β-ribose peracetate) from various manufacturers in a single experiment. The corresponding linearity calculation tables are as follows:

[0083] factory CQB Bailingwei Aladdin <![CDATA[Mass (10 -5 g)]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> 5 15.1984 13.5200 11.7504 10 26.0580 26.1504 19.6560 15 34.6104 30.1440 29.4720 20 39.4892 34.6104 34.8768 25 45.0736 41.0400 45.0072 30 53.6976 48.1664 48.7528 <![CDATA[R 2 ]]> 0.9830 0.9690 0.9880

[0084] The corresponding standard curve can be found here. Figure 11 .

[0085] Figure 12 The following are TLC plate images showing the linearity calculations for 13035-61-5 (β-ribose peracetate) from various manufacturers in the second experiment. The corresponding linearity calculation tables are as follows:

[0086] factory CQB Bailingwei Aladdin <![CDATA[Mass (10 -5 g)]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> <![CDATA[Area (mm 2 )]]> 5 24.5440 22.8816 18.1460 10 38.2800 35.4960 34.1864 15 52.7904 43.0012 43.2256 20 65.2680 52.8000 55.8900 25 74.8272 60.0000 66.0000 30 81.7440 63.0180 79.4464 <![CDATA[R 2 ]]> 0.9847 0.9720 0.9955

[0087] The corresponding standard curve can be found here. Figure 13 .

[0088] (5) Summary

[0089] Table: Linear Results

[0090]

[0091] Note: *Items are rounded to one significant figure. 2 =0.9, which also meets the linearity requirement.

[0092] (6) Conclusion

[0093] All three novel compounds can be quantitatively detected using TLC.

[0094] Example 3: Preliminary methodological establishment for quantitative detection of the above three compounds by TLC (In this step, the sample preparation, sample amount and developing solvent ratio were adjusted accordingly)

[0095] (1) Sample preparation

[0096] Accurately weigh 0.02g each of the custom-defined reference material and sample using a 0.001g balance (the reference material consists of the three compounds imported from Cuba during the previous production process, i.e., the aforementioned CQB products), and dilute to 10ml volumetric flasks with dichloromethane. This prepares a sample with a concentration of 2mg / ml.

[0097] (2) Sample loading, development and color development

[0098] Using a 25 μl microsyringe, sample solutions of 2.5 μl, 5 μl, 7.5 μl, 10 μl, 12.5 μl, and 15 μl were spotted onto a TLC plate.

[0099] The following developing solvent was used for development.

[0100] Table 3-1: Developing solvents used in the preliminary methodology establishment for quantitative detection by TLC

[0101] compound CAS number Developing agent and ratio 5-Azide-3-oxopentanol 139115-90-5 Ethyl acetate:methanol:water 85:10:5 Ribitol derivatives 111549-97-4 Hexane: Ethyl acetate 3:1 β-ribose peracetate 13035-61-5 Hexane: Ethyl acetate 1:3

[0102] After unfolding and drying the TLC plate, it was soaked in 5% sulfuric acid ethanol and then developed by blowing hot air from an electric blower.

[0103] (3) Judgment criteria

[0104] For example, the mass of a gradient sampling point (converted to sampling volume during sampling) has a linear relationship with the spot area (converted to length × width during measurement), i.e., R 2If the recovery rate is ≥95%, and the sample spot area is replaced with 7.5μl, 10μl, 12.5μl, and 15μl, and the recovery rate is within the range of 98-101% according to the provisions of the General Chapter 9101 "Guiding Principles for Validation of Analytical Methods" in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part IV, it can be determined that the TLC method can be used for quantitative detection.

[0105] (4) TLC board photos and measurement data

[0106] ①139115-90-5(5-azido-3-oxopentanol)

[0107] like Figure 14 The image shown is a TLC plate photograph of the linear calculation for 139115-90-5 (5-azido-3-oxopentanol) from Aladdin and Bailingwei manufacturers; as shown. Figure 15 The figure shows the standard curves of 5-azido-3-oxopentanol from Aladdin and Bailingwei manufacturers.

[0108] Table 3-2: Linearity and back-substitution results of 139115-90-5 (5-azido-3-oxopentanol)

[0109]

[0110]

[0111] ②111549-97-4 (ribitol derivative)

[0112] like Figure 16 The image shown is a linear computational TLC plate photograph of 111549-97-4 (ribitol derivative) from Aladdin and Borren Kairun, respectively; Figure 17 The figure shows the standard curves of ribitol derivatives from Aladdin and Borrencare.

[0113] Table 3-3: Linearity and back-substitution results of 13035-61-5 (β-riboperacetate)

[0114]

[0115] ③13035-61-5 (β-riboperacetate)

[0116] like Figure 18 The image shown is a TLC plate photograph of the linear calculation for 13035-61-5 (β-ribose peracetate) from Aladdin and Bailingwei manufacturers; as shown. Figure 19 The figure shows the standard curves for β-ribose peracetate from Aladdin and Bailingwei manufacturers.

[0117] Table 3-4: Linearity and back-substitution results of 13035-61-5 (β-riboperacetate)

[0118]

[0119]

[0120] (5) Summary

[0121] Table 3-5: Preliminary Methodological Establishment for Quantitative Detection of Three Compounds by TLC

[0122]

[0123] Note: * indicates the recovery rate closest to 98-101% on the Bailingwei TLC board.

[0124] (6) Conclusion

[0125] TLC can be used for the quantitative detection of these three novel compounds, and the methodology can be validated according to General Chapter 9101 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The conditions are shown in the table below.

[0126] Table 3-6: Preliminary Methodological Establishment for Quantitative Detection of Three Compounds by TLC

[0127]

[0128] Note: *Items require further refinement.

[0129] In TLC detection, a regression curve is constructed using mass and spot area to confirm a linear relationship between the compound content and the spot area obtained on the TLC plate. Therefore, after identifying the main component spot, the ratio of the spot area of ​​a precisely weighed reference sample to that of the sample can be compared, and the sample content can be determined using a standard curve. In this method, the mass is obtained by weighing using a 0.01 g / L balance, and the spot area is measured manually using calipers to measure its length and width. The ratio of these measurements is then obtained by multiplying them (for the same compound, spots with similar shapes, assuming they are all convex, the ratio of their length-to-width ratio to their area is consistent). With a TLC scanner, the spot area can be scanned for direct comparison with the standard curve.

[0130] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims.

[0131] It should be understood that the present invention is not limited to the content already described above, and various modifications and changes can be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for quantitative detection based on thin layer chromatography, characterized in that, Comprising the following steps: (1) sample preparation: precisely take the sample to be detected, dissolve the sample with dichloromethane as the solvent, and prepare the sample; (2) sample loading, development and coloration: use a micro-sampler to load 2.5 μl, 5 μl, 7.5 μl, 10 μl, 12.5 μl and 15 μl of the sample on the HPTLC plate in sequence, and then develop the sample with a developing agent, dry the sample, immerse it in 5% sulfuric acid ethanol, and then color it by hot air blowing; When the sample to be detected is a compound with CAS No. 139115-90-5, the developing agent is a mixed solution of ethyl acetate, methanol and water, and the volume ratio of ethyl acetate:methanol:water is 85:10:5; When the sample to be detected is a compound with CAS No. 111549-97-4, the developing agent is a mixed solution of hexane and ethyl acetate, and the volume ratio of hexane:ethyl acetate is 3:1; When the sample to be detected is a compound with CAS No. 13035-61-5, the developing agent is a mixed solution of hexane and ethyl acetate, and the volume ratio of hexane:ethyl acetate is 1:3; (3) quantitative detection: measure the area of each spot, and prepare a standard curve, and calculate the content of the sample based on the standard curve.

2. The method of quantitative detection based on thin layer chromatography according to claim 1, characterized in that, The concentration of the sample prepared in step (1) is 2 mg / ml-10 mg / ml.

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