A method for determining the content of silk fibroin by PITC-HPLC pre-column derivatization

Abstract

The application discloses a method for determining the content of silk fibroin by PITC-HPLC pre-column derivatization, which comprises the following steps: first, hydrolyzing the sample to make the silk fibroin hydrolyzed into amino acids; after the treatment, performing pre-column derivatization by using phenyl isothiocyanate, and after the extraction and purification of n-hexane, taking alanine as a characteristic amino acid, and detecting by using a high performance liquid chromatograph; the method adopts binary mobile phase gradient elution, combines the alanine standard curve and the conversion coefficient to calculate the content of the silk fibroin; it is verified that the method has good specificity, excellent precision and repeatability; the application effectively avoids the interference of a complex matrix, the result is accurate and reliable, is suitable for various dosage forms such as gels, sprays and dressing patches, is simple to operate, is convenient for enterprises and inspection institutions to carry out normal quality detection, and can be popularized to the content determination of other protein medical products.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for determining silk fibroin content using PITCHPLC pre-column derivatization. Background Technology

[0002] Silk fibroin is a natural high-molecular-weight protein extracted from silkworm silk. It effectively achieves wound hemostasis, accelerates epithelial cell proliferation and tissue remodeling, and effectively alleviates local inflammatory responses, creating a suitable microenvironment for the repair and healing of damaged tissues. This makes it widely used in medical wound dressings. As a core active ingredient in medical dressings, the content of silk fibroin directly determines the clinical efficacy of the product. Currently, the detection methods for silk fibroin content in medical silk fibroin products mainly refer to the Kjeldahl method specified in YY / T 1950-2024 "Tissue Engineering Medical Devices - Silk Fibroin", or the colorimetric methods in General Chapter 0731 of the Chinese Pharmacopoeia, such as the biuret method and the Folin-Ciocalteu method, for the determination of protein content. The Kjeldahl method estimates protein content by measuring total nitrogen in a sample. The procedure is simple and suitable for determining fibroin content in products where the formulation contains no nitrogen-containing substances other than silk fibroin. However, for complex matrix preparations like medical fibroin dressings, which often contain high-molecular-weight excipients such as carboxymethyl chitosan, sodium hyaluronate, and carbomer, the results of the Kjeldahl method can be interfered with. Carboxymethyl chitosan and sodium hyaluronate contain nitrogen, which can lead to an overestimation of the fibroin content and fail to reflect the actual fibroin content in the sample. Colorimetric methods use bovine serum albumin (BSA) as a reference. However, BSA differs structurally from fibroin, causing discrepancies between the measured structure and the actual result. While using fibroin raw materials as a reference can overcome this limitation, other dressing components, such as glycerol and sugars, can also interfere with the accuracy of the colorimetric results. Therefore, a more accurate method for determining the fibroin content in medical fibroin dressings is urgently needed. Summary of the Invention

[0003] To address the shortcomings of the existing technologies, this invention aims to provide a PITCHPLC pre-column derivatization method for determining silk fibroin content. This method addresses the deficiencies of existing technologies: the Kjeldahl method and colorimetric methods are easily interfered with by nitrogen-containing excipients such as carboxymethyl chitosan and sodium hyaluronate in dressings, leading to large deviations from the true values; the colorimetric method is easily interfered with by glycerol, polyols, buffer salts, preservatives, etc., resulting in large deviations from the true values. This invention enables the detection of silk fibroin content from medical silk fibroin containing one or more of carboxymethyl chitosan, carbomer, phenoxyethanol, glycerol, and sodium hyaluronate.

[0004] To solve the above problems, the present invention adopts the following technical solution: A method for determining silk fibroin content using PITC-HPLC pre-column derivatization includes: The medical silk fibroin sample was hydrolyzed, and the medical silk fibroin sample contained one or more of carboxymethyl chitosan, carbomer, phenoxyethanol, glycerol, sodium hyaluronate, and silk fibroin. The hydrolyzed medical silk fibroin sample was subjected to PITC derivatization. The alanine content in PITC-derived medical silk fibroin samples was determined by high performance liquid chromatography, and the silk fibroin content was calculated.

[0005] As one possible implementation method, the hydrolysis of the medical silk fibroin sample includes: placing the medical silk fibroin sample in a 6 mol / L hydrochloric acid system and hydrolyzing at 110°C for 24 h; after evaporating the hydrolysate under reduced pressure, reconstitute it with 0.1 mol / L hydrochloric acid to obtain the hydrolyzed medical silk fibroin sample, wherein the concentration of silk fibroin in the hydrolyzed medical silk fibroin sample is 0.01~0.1 mg / mL.

[0006] As one possible implementation, the PITC derivatization of the hydrolyzed medical silk fibroin sample includes: Take the hydrolyzed medical silk fibroin sample, add triethylamine acetonitrile solution and phenyl isothiocyanate acetonitrile solution, mix well, and derivatize at room temperature; add n-hexane for extraction and separation, and take the lower layer solution as the PITC-derived medical silk fibroin sample.

[0007] In one possible implementation, the concentration of the triethylamine acetonitrile solution is 1 mol / L, the concentration of the phenyl isothiocyanate acetonitrile solution is 0.1 mol / L, and the volume ratio of the hydrolyzed medical silk fibroin sample to the triethylamine acetonitrile solution and the phenyl isothiocyanate acetonitrile solution is 2:1:1; the amount of n-hexane extract added is 4 times that of the hydrolyzed medical silk fibroin sample.

[0008] As one possible implementation method, the chromatographic column of the high-performance liquid chromatography is a Waters XBridge Shield RP18 with dimensions of 5 μm, 4.6 mm × 250 mm; mobile phase A is acetonitrile-0.1 mol / L sodium acetate solution with a volume ratio of 7:93 and a pH of 6.5; mobile phase B is acetonitrile-water with a volume ratio of 80:20; the flow rate is 1.0 mL / min, the column temperature is 40 ℃, and the injection volume is 2 μL.

[0009] As one possible implementation, the gradient elution program of the high-performance liquid chromatography is as follows: 0–14 min, mobile phase A is 100% to 85%; 14–29 min, mobile phase A is 85% to 66%; 29–30 min, mobile phase A is 66% to 0%; 30–37 min, mobile phase A remains at 0%; 37.1 min, switch to mobile phase A at 100%; 37.1–45 min, mobile phase A remains at 100%.

[0010] As one possible implementation, the medical silk fibroin sample is at least one of silk fibroin gel, silk fibroin spray, and silk fibroin dressing.

[0011] As one possible implementation method, the detection wavelength of the high performance liquid chromatography is 254 nm, the peak area of ​​alanine is detected, a standard curve is established with alanine as the characteristic amino acid, and the alanine-silk fibroin conversion factor determined by the silk fibroin raw material is used to calculate the silk fibroin content in the sample.

[0012] As one possible implementation, the detection limit of the method is 0.4 μg / g, and the quantitation limit is 1 μg / g.

[0013] The beneficial effects of this invention are as follows: This method selects alanine, a characteristic amino acid of silk fibroin, as a quantitative indicator. Targeted detection is achieved through acid hydrolysis, pre-column derivatization with PITC, and high-performance liquid chromatography (HPLC). This effectively avoids interference from nitrogen-containing excipients such as carboxymethyl chitosan, sodium hyaluronate, glycerol, and sugars, as well as other matrix components. In principle, it solves the shortcomings of traditional methods, which suffer from high detection results and large deviations due to nitrogen-containing excipients and matrix cross-reactions. The determination results accurately reflect the actual content of silk fibroin in medical dressings. This method has a detection limit of 0.4 μg / g and a quantitation limit of 1 μg / g, demonstrating excellent detection capability. This detection method is not limited by product dosage form and can be applied to the content detection of different types of medical silk fibroin samples, such as silk fibroin gels, sprays, and dressings. Furthermore, this detection approach can be extended to the determination of protein content in other protein-containing medical products and dressings, offering significant potential for expansion. Attached Figure Description

[0014] Figure 1 The chromatogram is of reference standard S4 in Example 2.

[0015] Figure 2 The chromatogram is of the blank solution (carboxymethyl chitosan) in Example 2.

[0016] Figure 3 The image shows the chromatogram of the silk fibroin raw material in Example 2.

[0017] Figure 4 The chromatogram of the nasal irrigator sample in Example 2 is shown.

[0018] Figure 5 The chromatogram is of the blank solution (phenoxyethanol) in Example 3.

[0019] Figure 6 This is the chromatogram of the blank solution (glycerol) in Example 3.

[0020] Figure 7 The chromatogram is of the blank solution (carbomer) in Example 3.

[0021] Figure 8 This is a chromatogram of the silk fibroin gynecological gel sample from Example 3.

[0022] Figure 9 This is the chromatogram of the blank solution (hyaluronic acid) in Example 4.

[0023] Figure 10 The chromatogram is shown for the silk fibroin dressing sample in Example 4. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0026] Example 1 Overcoming the limitations of traditional protein content determination methods (such as the Kjeldahl method and colorimetric methods) which are susceptible to matrix interference leading to inaccurate results, this invention provides a PITC-HPLC pre-column derivatization method for determining silk fibroin content, comprising: The medical silk fibroin sample was hydrolyzed, and the medical silk fibroin sample contained one or more of carboxymethyl chitosan, carbomer, phenoxyethanol, glycerol, sodium hyaluronate, and silk fibroin. The hydrolyzed medical silk fibroin sample was subjected to PITC derivatization. The alanine content in PITC-derived medical silk fibroin samples was determined by high performance liquid chromatography, and the silk fibroin content was calculated.

[0027] (1) Acid hydrolysis of the sample: The sample was hydrolyzed with hydrochloric acid at 110℃ for 24 h to completely hydrolyze the silk fibroin in the medical silk fibroin sample into amino acids. The final concentration of hydrochloric acid was 6 mol / L. The solution after acid hydrolysis was taken, evaporated to dryness under reduced pressure, and then transferred to a final volume with 0.1 mol / L hydrochloric acid to make the concentration of silk fibroin in the solution 0.01~0.1 mg / mL.

[0028] (2) PITC derivatization: Accurately measure 200 μL of the test solution and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and vortex to mix. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and vortex to mix. Seal and place at room temperature for 1 h. Add 0.8 mL of n-hexane, shake thoroughly for 20–30 s, allow to stand for layering, and accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph.

[0029] (3) HPLC detection: Detection conditions: Column: Waters XBridge Shield RP18 (5 μm, 4.6 mm × 250 mm); Mobile phase: Phase A was acetonitrile-0.1 mol / L sodium acetate solution (pH 6.5) (7:93), and Phase B was acetonitrile-water (80:20); the elution gradient of the mobile phase is shown in Table 1; flow rate: 1.0 mL·min⁻¹; detection wavelength: 254 nm; column temperature: 40 ℃; injection volume: 2 μL.

[0030] Table 1 Gradient elution program

[0031] Take alanine reference standard with a concentration of 0.989–39.560 μg / mL, derivatize it according to (2), and inject it under the above detection conditions to establish a calibration curve. Take a medical silk fibroin sample, derivatize it according to (2), and inject it under the above detection conditions. The measured alanine peak area is used to calculate the alanine content. Then, by adjusting the volume, dilution factor, and sample weight, the silk fibroin content in the sample is obtained.

[0032] (4) Determination of the conversion factor from alanine to silk fibroin The conversion factor from alanine to silk fibroin was determined by measuring the alanine content in the silk fibroin raw material.

[0033] Example 2 Determination of silk fibroin content in nasal irrigator 2.1 Information on test instruments, reagents and samples 2.1.1 Test Instruments Table 2 Instrument Information

[0034] 2.1.2 Test Reagents Table 3 Reagent Information

[0035] 2.1.3 Sample Information Table 4 Sample Information

[0036] 2.2 Solution Preparation 2.2.1 Reagent Preparation Acetonitrile-0.1 mol / L sodium acetate solution (7:93): Accurately weigh 13.6 g of sodium acetate, add 900 mL of water, adjust the pH to 6.5 with acetic acid, and then add water to 1000 mL to obtain a 0.1 mol / L sodium acetate solution; measure 70 mL of acetonitrile and 930 mL of the 0.1 mol / L sodium acetate solution, mix well, and filter using a 0.22 μm microporous membrane to obtain an acetonitrile-0.1 mol / L sodium acetate solution (7:93).

[0037] 1 mol / L triethylamine acetonitrile solution: Accurately weigh 10.12 g of triethylamine, place it in a 100 ml volumetric flask, add acetonitrile to make up to the mark, shake well, and you will get a 1 mol / L triethylamine acetonitrile solution.

[0038] 0.1 mol / L phenyl isothiocyanate acetonitrile solution: Accurately weigh 1.35 g of phenyl isothiocyanate, place it in a 100 ml volumetric flask, add acetonitrile to make up to the mark, shake well, and you will get a 0.1 mol / L phenyl isothiocyanate acetonitrile solution.

[0039] 2.2.2 Preparation of reference solution Accurately weigh 9.90 mg of alanine and place it in a 25 mL volumetric flask. Add 0.1 mol / L hydrochloric acid and dilute to the mark to obtain alanine standard stock solution S. 贮 The concentration was 395.60 μg / mL; Accurately measure the stock solution S of alanine reference standard. 贮 1.00 mL was placed in a 10 mL volumetric flask, and 0.1 mol / L hydrochloric acid was added to dilute to the mark to obtain alanine reference solution S6 with a concentration of 39.560 μg / mL. Accurately measure the stock solution S of alanine reference standard. 贮 0.50 mL was placed in a 10 mL volumetric flask, and 0.1 mol / L hydrochloric acid was added to dilute to the mark to obtain alanine reference solution S5 with a concentration of 19.780 μg / mL; Accurately measure the stock solution S of alanine reference standard. 贮 0.25 mL was placed in a 10 mL volumetric flask, and 0.1 mol / L hydrochloric acid was added to dilute to the mark to obtain alanine reference solution S4 with a concentration of 9.890 μg / mL. Accurately measure 2.50 mL of alanine reference standard stock solution S5, place it in a 10 mL volumetric flask, and add 0.1 mol / L hydrochloric acid to dilute to the mark to obtain alanine reference standard solution S3 with a concentration of 4.945 μg / mL. Accurately measure 1.00 mL of alanine reference solution S5 and place it in a 10 mL volumetric flask. Add 0.1 mol / L hydrochloric acid to the mark to obtain alanine reference solution S2 with a concentration of 1.978 μg / mL. Accurately measure 1.00 mL of alanine reference solution S4 and place it in a 10 mL volumetric flask. Add 0.1 mol / L hydrochloric acid to the mark to obtain alanine reference solution S1 with a concentration of 0.989 μg / mL. Accurately measure 200 μL each of reference solutions S1 to S6 and place them in centrifuge tubes. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0040] 2.2.3 Preparation of test solution for nasal irrigator Accurately weigh 2g of the nasal irrigator sample, place it in a hydrolysis tube, add 1mL of water and 3mL of concentrated hydrochloric acid, hydrolyze at 110℃ for 24h, remove, distill under reduced pressure to dryness, transfer with 0.1mol / L hydrochloric acid and dilute to a 50mL volumetric flask. This yields the nasal irrigator test solution.

[0041] Accurately measure 200 μL of the nasal irrigator test solution and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0042] 2.2.4 Preparation of the test solution for silk fibroin raw material Accurately weigh 10 mg of dried silk fibroin raw material to constant weight, place it in a hydrolysis tube, add 3 mL of water and 3 mL of concentrated hydrochloric acid, and hydrolyze at 110 °C for 24 h. Remove the hydrolysate, distill under reduced pressure to dryness, transfer to a 50 mL volumetric flask with 0.1 mol / L hydrochloric acid, and dilute to the mark with 0.1 mol / L hydrochloric acid. This yields the silk fibroin raw material test solution.

[0043] Accurately measure 200 μL of the silk fibroin raw material test solution and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0044] 2.2.5 Preparation of blank solution Accurately weigh 10 mg of carboxymethyl chitosan raw material and place it in a hydrolysis tube. Add 3 mL of water and 3 mL of concentrated hydrochloric acid, and hydrolyze at 110 °C for 24 h. Remove the solution, distill under reduced pressure to dryness, transfer to a 50 mL volumetric flask with 0.1 mol / L hydrochloric acid, and dilute to the mark. Take 2 mL of each of the above hydrolysates and place them in a 10 mL volumetric flask. Dilute to the mark with 0.1 mol / L hydrochloric acid. This is the blank solution of carboxymethyl chitosan.

[0045] Accurately measure 200 μL of the blank solution of carboxymethyl chitosan and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0046] 2.2.6 Preparation of the recovered test solution Preparation of silk fibroin solution: Accurately weigh 258.00 mg of dried silk fibroin raw material to constant weight, place it in a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid to dissolve and dilute to the mark to obtain silk fibroin solution with a concentration of 10.32 mg / mL.

[0047] Preparation of the recovered test solution: Take 9 hydrolysis tubes, add 2g of sample to each, and divide them into three groups. Add 0.16mL silk fibroin solution, 0.84mL water, and 3mL hydrochloric acid to the first group (low recovery test solution). Add 0.20mL silk fibroin solution, 0.80mL water, and 3.0mL hydrochloric acid to the second group (medium recovery test solution). Add 0.24mL silk fibroin solution, 0.76mL water, and 3.0mL hydrochloric acid to the third group (high recovery test solution). Hydrolyze at 110℃ for 24h, remove, distill under reduced pressure to dryness, transfer with 0.1mol / L hydrochloric acid, and dilute to a 50mL volumetric flask to obtain the recovered test solution.

[0048] Accurately measure 200 μL of each recovered test solution and place them in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 mL of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0049] 2.3 Methodology for testing silk fibroin content in nasal irrigators 2.3.1 Specificity Take the reference solution prepared in "2.3.2", the nasal irrigator test solution prepared in "2.3.3", the silk fibroin raw material test solution prepared in "2.3.4", and the blank solution prepared in "2.3.5", and perform the determination according to the test conditions of Example 1. Record the chromatograms. See [reference section]. Figure 1-4 .

[0050] The results showed that the blank solution did not elute at the alanine chromatographic peak position, while the silk fibroin raw material and nasal irrigator sample eluted at the alanine chromatographic peak position. This indicates that the method has good specificity.

[0051] 2.3.2 Linear Take the reference solution prepared in “2.2.2” and measure it under the experimental conditions in Example 1. With the concentration of alanine (μg / mL) as the x-axis and the corresponding peak area as the y-axis, perform linear regression, calculate the regression equation and correlation coefficient R, as shown in Table 5.

[0052] Table 5 Linear Results

[0053] The results showed that the correlation coefficient (R) was 0.9998 in the concentration range of 0.989–39.560 μg / mL, indicating that the test method had good linearity.

[0054] 2.3.3 Limit of Detection and Limit of Quantification Take the reference solution, dilute it sequentially, and measure it according to the test conditions of Example 1. Calculate its signal-to-noise ratio (SNR). When the SNR is S / N = 3, the corresponding concentration of the reference solution is the limit of detection; when the SNR is S / N = 10, the corresponding concentration of the reference solution is the limit of quantitation. The results are shown in Table 6.

[0055] Table 6 Results of Limit of Detection and Limit of Quantification

[0056] Calculation formula: Method detection limit (μg / g) = ×3.184 Method limit of quantitation (μg / g) = ×3.184 In the formula: 3.184 is the conversion factor from alanine to silk fibroin.

[0057] 2.3.4 Precision Take the reference solutions S4, S3, and S2 and perform the determination according to Example 1. Inject each solution three times consecutively, record the chromatograms, and calculate the average value and RSD% of alanine concentration. The results are shown in Table 7.

[0058] Table 7 Precision Results

[0059] The results show that the test method has good precision.

[0060] 2.3.5 Stability Take the reference solution S3 and perform the test according to Example 1. The results of the injection at 0 h, 25 h, 49 h and 60 h are shown in Table 8.

[0061] Table 8 Stability Results

[0062] The results show that the test method has good stability within 60 hours.

[0063] 2.3.6 Determination of Alanine Content in Silk Fibroin Raw Materials Six samples of silk fibroin raw material test solution were prepared in parallel according to the same method used for preparing the test solution. The alanine content in the raw material was calculated, and the conversion factor from alanine to silk fibroin was obtained. The test structure is shown in Table 9.

[0064] Table 9 Results of Silk Fiber Protein Raw Materials

[0065] Calculation formula: Alanine content (%) = ×10-3×100% The results showed that the alanine content in the silk fibroin raw material was 31.4057%. That is, the conversion factor of alanine to silk fibroin is: 1 / 0.314057 = 3.184.

[0066] 2.3.7 Repeatability of Nasal Irrigator Sample Determination Take sample number 206241203, and prepare 6 test solutions in parallel according to the test preparation method in 2.2.3. The test solutions were determined according to Example 1. The results are shown in Table 10.

[0067] Table 10 Repeatability Results

[0068] Calculation formula: Silk fibroin content (mg / g) = ×3.184×10 -3 The results showed that the average silk fibroin content in the nasal irrigator samples was 0.81 mg / g, with an RSD of 1.03%, indicating good reproducibility of the method.

[0069] 2.3.8 Accuracy (Recovery Test) Take sample number 206241203, prepare the recovered test solution according to 2.2.6, and perform the determination according to Example 1. The results are shown in Table 11.

[0070] Table 11 Results of the recovery test

[0071] Calculation formula: 1. Silk fibroin assay (mg) = 3.184 10-3 2. Silk fibroin background amount (mg) = sample weight 0.81 (mg / g) 3. The results showed that the spiked recovery rate was between 92.37% and 96.42%, and the method had good accuracy.

[0072] 2.4 Sample Measurement Results Samples with batch numbers 206241201 and 206241202 were taken and prepared into test solutions according to the test preparation method in 2.2.3. The test solutions were then determined according to Example 1. The results are shown in Table 12.

[0073] Table 12 Sample Measurement Results

[0074] Calculation formula: Silk fibroin content (mg / g) = ×3.184×10 -3 Example 3 Determination of silk fibroin content in gynecological gel 3.1 Information on test instruments, reagents and samples 3.1.1 Same as 2.1.1.

[0075] 3.1.2 Same as 2.1.2.

[0076] 3.1.3 Sample Information Table 13 Sample Information

[0077] 3.2 Solution Preparation 3.2.1-3.2.2 are the same as 2.2.1-2.2.2.

[0078] 3.2.3 Preparation of Silk Fibroin Gynecological Gel Test Solution Accurately weigh 1 g of silk fibroin gynecological gel sample and place it in a hydrolysis tube. Add 2 mL of water and 3 mL of concentrated hydrochloric acid. Hydrolyze at 110℃ for 24 h. Remove the sample, distill under reduced pressure to dryness, transfer to a 50 mL volumetric flask with 0.1 mol / L hydrochloric acid, and dilute to the mark. Take 2 mL of the above hydrolysate and place it in a 10 mL volumetric flask. Dilute to the mark with 0.1 mol / L hydrochloric acid. This yields the silk fibroin gynecological gel test solution.

[0079] Accurately measure 200 μL of the silk fibroin gynecological gel test solution and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0080] 3.2.4 Same as 2.2.4.

[0081] 3.2.5 Preparation of blank solution Accurately weigh 10 mg each of carboxymethyl chitosan, glycerol, phenoxyethanol, and carbomer raw materials, and place them separately in hydrolysis tubes. Add 3 mL of water and 3 mL of concentrated hydrochloric acid, and hydrolyze at 110 °C for 24 h. Remove the solutions, distill under reduced pressure to dryness, transfer to a 50 mL volumetric flask with 0.1 mol / L hydrochloric acid, and dilute to the mark. Take 2 mL of each hydrolysate and place it in a 10 mL volumetric flask, then dilute to the mark with 0.1 mol / L hydrochloric acid. This is the blank solution of carboxymethyl chitosan, glycerol, phenoxyethanol, and carbomer.

[0082] Accurately measure 200 μL each of blank solutions of carboxymethyl chitosan, glycerol, phenoxyethanol, and carbomer, place them in centrifuge tubes, accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution, mix well, accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution, mix well, seal, and let stand for 1 hour. Add 0.8 ml of n-hexane, shake thoroughly for 20-30 seconds, allow to stand for layering, accurately measure 2 μL of the lower layer solution, inject it into the liquid chromatograph, and record the chromatogram.

[0083] 3.2.6 Preparation of the recovered test solution Preparation of silk fibroin solution: Accurately weigh 258.00 mg of dried silk fibroin raw material to constant weight, place it in a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid to dissolve and dilute to the mark to obtain silk fibroin solution with a concentration of 10.32 mg / mL.

[0084] Preparation of the recovered test solution: Take 9 hydrolysis tubes, add 1g of silk fibroin gynecological gel sample to each, and divide them into three groups. Add 0.8mL of silk fibroin solution, 1.2mL of water, and 3mL of hydrochloric acid to the first group (low recovery test solution). Add 1.0mL of silk fibroin solution, 1.0mL of water, and 3.0mL of hydrochloric acid to the second group (medium recovery test solution). Add 1.2mL of silk fibroin solution, 0.8mL of water, and 3.0mL of hydrochloric acid to the third group (high recovery test solution). Hydrolyze at 110℃ for 24h, remove, distill under reduced pressure to dryness, transfer with 0.1mol / L hydrochloric acid, and dilute to the mark in a 50mL volumetric flask. Take 2mL of each hydrolysate and place them in a 10mL volumetric flask, then dilute to the mark with 0.1mol / L hydrochloric acid. The recovered test solution is obtained.

[0085] Accurately measure 200 μL of each recovered test solution and place them in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 mL of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0086] 3.3 Methodology for testing silk fibroin content in gynecological fibroin gels 3.3.1 Specificity Take the silk fibroin gynecological gel test solution prepared in "3.2.3" and the blank solution prepared in "3.2.5", and determine them according to the test conditions of Example 1. Record the chromatograms. See [link to chromatogram]. Figure 5-8 .

[0087] The results showed that the blank solutions (carboxymethyl chitosan, carbomer, phenoxyethanol, and glycerol) did not elute at the alanine chromatographic peak position, while the silk fibroin raw material and the silk fibroin gynecological gel samples did elute at the alanine chromatographic peak position. This indicates that other components in the product do not affect the test, and the method has good specificity.

[0088] 3.3.2 Linear (same as 2.3.2).

[0089] 3.3.3 Limit of detection and limit of quantitation (same as 2.3.3).

[0090] 3.3.4 Precision (same as 2.3.4).

[0091] 3.3.5 Stability (same as 2.3.3).

[0092] 3.3.6 Determination of alanine content in silk fibroin raw materials (same as 2.3.6).

[0093] 3.3.7 Repeatability of Silk Fibroin Determination in Gynecological Gel Samples Take sample number 203240603, prepare 6 test solutions in parallel according to the preparation method of the test sample, and determine them according to the test in Example 1. The results are shown in Table 14.

[0094] Table 14 Repeatability Results

[0095] 3.3.8 Accuracy (Recovery Test) Take sample number 203240603, prepare the recovered test solution, and perform the determination according to Example 1. The results are shown in Table 15.

[0096] Table 15 Results of the recovery test

[0097] The results showed that the spiked recovery rate was between 94.10% and 104.70%, and the method had good accuracy.

[0098] 3.4 Sample Measurement Results Samples with batch numbers 203240601 and 203240602 were taken, and test solutions were prepared according to the test sample preparation method. The results are shown in Table 16.

[0099] Table 16 Sample Measurement Results

[0100] Example 4 Determination of silk fibroin content in silk fibroin dressings 4.1 Information on test instruments, reagents and samples 4.1.1 Same as 2.1.1.

[0101] 4.1.2 Same as 2.1.2.

[0102] 4.1.3 Sample Information Table 17 Sample Information

[0103] 4.2 Solution Preparation 4.2.1-4.2.2 are the same as 2.2.1-2.2.2.

[0104] 4.2.3 Preparation of the test solution for silk fibroin dressing Accurately weigh 2g of silk fibroin dressing sample, place it in a hydrolysis tube, add 1mL of water and 3mL of concentrated hydrochloric acid, hydrolyze at 110℃ for 24h, remove, distill under reduced pressure to dryness, transfer with 0.1mol / L hydrochloric acid and dilute to a 50mL volumetric flask. This yields the silk fibroin dressing test solution.

[0105] Accurately measure 200 μL of the silk fibroin dressing test solution and place it in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal and let stand for 1 hour. Add 0.8 mL of n-hexane, shake thoroughly for 20-30 seconds, and allow to stand for phase separation. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0106] 4.2.4 Same as 2.2.4.

[0107] 4.2.5 Preparation of blank solution Accurately weigh 10 mg each of carboxymethyl chitosan, glycerol, sodium hyaluronate, and carbomer raw materials, place them in a hydrolysis tube, add 3 mL of water and 3 mL of concentrated hydrochloric acid, and hydrolyze at 110 °C for 24 h. Remove the solution, distill under reduced pressure to dryness, transfer to a 50 mL volumetric flask with 0.1 mol / L hydrochloric acid, and dilute to the mark. Take 2 mL of each of the above hydrolysates, place them in a 10 mL volumetric flask, and dilute to the mark with 0.1 mol / L hydrochloric acid. This is the blank solution of carboxymethyl chitosan, glycerol, sodium hyaluronate, and carbomer.

[0108] Accurately measure 200 μL each of blank solutions of carboxymethyl chitosan, glycerol, sodium hyaluronate, and carbomer, place them in centrifuge tubes, accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution, mix well, accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution, mix well, seal, and let stand for 1 hour. Add 0.8 mL of n-hexane, shake thoroughly for 20-30 seconds, allow to stand for layering, accurately measure 2 μL of the lower layer solution, inject it into the liquid chromatograph, and record the chromatogram.

[0109] 4.2.6 Preparation of the recovered test solution Preparation of silk fibroin solution: Accurately weigh 54.95 mg of dried silk fibroin raw material to constant weight, place it in a 25 mL volumetric flask, add 0.1 mol / L hydrochloric acid to dissolve and dilute to the mark to obtain silk fibroin solution with a concentration of 2.198 mg / mL.

[0110] Preparation of the recovered test solution: Take 9 hydrolysis tubes, add 2g of silk fibroin dressing sample to each, and divide them into three groups. Add 0.4mL silk fibroin solution, 0.6mL water, and 3mL hydrochloric acid to the first group (low recovery test solution). Add 0.5mL silk fibroin solution, 0.5mL water, and 3.0mL hydrochloric acid to the second group (medium recovery test solution). Add 0.6mL silk fibroin solution, 0.4mL water, and 3.0mL hydrochloric acid to the third group (high recovery test solution). Hydrolyze at 110℃ for 24h, remove, distill under reduced pressure to dryness, transfer with 0.1mol / L hydrochloric acid, and dilute to a 50mL volumetric flask. The recovered test solution is obtained.

[0111] Accurately measure 200 μL of each recovered test solution and place them in a centrifuge tube. Accurately add 100 μL of 1 mol / L triethylamine acetonitrile solution and mix well. Accurately add 100 μL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and mix well. Seal the tube and let it stand for 1 hour. Add 0.8 mL of n-hexane and shake thoroughly for 20-30 seconds. Allow the layers to separate. Accurately measure 2 μL of the lower layer solution and inject it into the liquid chromatograph. Record the chromatogram.

[0112] 4.3 Methodology for testing silk fibroin content in silk protein dressings 4.3.1 Specificity Take the protein dressing test solution prepared in "4.2.3" and the blank solution prepared in "4.2.5", and perform the determination according to the test conditions of Example 1. Record the chromatogram. See [link to chromatogram]. Figure 9-10 .

[0113] The results showed that the blank solutions (carboxymethyl chitosan, carbomer, hyaluronic acid, and glycerin) did not elute at the alanine chromatographic peak position, while the silk fibroin raw material and silk fibroin dressing samples did elute at the alanine chromatographic peak position. This indicates that other components in the product do not affect the test, and the method has good specificity.

[0114] 4.3.2 Linear (same as 2.3.2).

[0115] 4.3.3 Limit of detection and limit of quantitation (same as 2.3.3).

[0116] 4.3.4 Precision (same as 2.3.4).

[0117] 43.3.5 Stability (same as 2.3.3).

[0118] 4.3.6 Determination of alanine content in silk fibroin raw materials (same as 2.3.6).

[0119] 4.3.7 Repeatability of Silk Fibroin Dressing Sample Determination Take sample number 202240603, prepare 6 parallel test solutions according to the test sample preparation method, and perform the determination. The results are shown in Table 18.

[0120] Table 18 Repeatability Results

[0121] The results showed that the average silk fibroin content in the six silk fibroin dressing samples was 0.51 mg / g, with an RSD of 1.90%, indicating good reproducibility of the method.

[0122] 4.3.8 Accuracy (Recovery Test) Take sample number 202240603, prepare the recovered test solution, and perform the determination. The results are shown in Table 19.

[0123] Table 19 Results of the recovery test

[0124] The results showed that the spiked recovery rate was between 96.21% and 106.59%, and the method had good accuracy.

[0125] 4.4 Sample Measurement Results Samples with batch numbers 202240601 and 202240602 were taken, and test solutions were prepared according to the test sample preparation method. The results are shown in Table 20.

[0126] Table 20 Sample Measurement Results

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for determining silk fibroin content using PITC-HPLC pre-column derivatization, characterized in that, include: The medical silk fibroin sample was hydrolyzed, and the medical silk fibroin sample contained one or more of carboxymethyl chitosan, carbomer, phenoxyethanol, glycerol, sodium hyaluronate, and silk fibroin. The hydrolyzed medical silk fibroin sample was subjected to PITC derivatization. The alanine content in PITC-derived medical silk fibroin samples was determined by high performance liquid chromatography, and the silk fibroin content was calculated.

2. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 1, characterized in that, The process of hydrolyzing the medical silk fibroin sample includes: placing the medical silk fibroin sample in a 6 mol / L hydrochloric acid system and hydrolyzing it at 110°C for 24 h; after evaporating the hydrolysate under reduced pressure, reconstituteing it with 0.1 mol / L hydrochloric acid to obtain the hydrolyzed medical silk fibroin sample, wherein the concentration of silk fibroin in the hydrolyzed medical silk fibroin sample is 0.01~0.1 mg / mL.

3. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 2, characterized in that, The step of PITC derivatization of the hydrolyzed medical silk fibroin sample includes: Take the hydrolyzed medical silk fibroin sample, add triethylamine acetonitrile solution and phenyl isothiocyanate acetonitrile solution, mix well, and derivatize at room temperature; add n-hexane for extraction and separation, and take the lower layer solution as the PITC-derived medical silk fibroin sample.

4. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 3, characterized in that, The concentration of the triethylamine acetonitrile solution was 1 mol / L, the concentration of the phenyl isothiocyanate acetonitrile solution was 0.1 mol / L, and the volume ratio of the hydrolyzed medical silk fibroin sample to the triethylamine acetonitrile solution and the phenyl isothiocyanate acetonitrile solution was 2:1:1; the amount of n-hexane extract added was 4 times that of the hydrolyzed medical silk fibroin sample.

5. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 4, characterized in that, The high-performance liquid chromatography (HPLC) used a Waters XBridge Shield RP18 column with dimensions of 5 μm, 4.6 mm × 250 mm. Mobile phase A consisted of acetonitrile-0.1 mol / L sodium acetate solution at a volume ratio of 7:93 and a pH of 6.

5. Mobile phase B consisted of acetonitrile-water at a volume ratio of 80:

20. The flow rate was 1.0 mL / min, the column temperature was 40 ℃, and the injection volume was 2 μL.

6. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 5, characterized in that, The gradient elution program for the high-performance liquid chromatography is as follows: 0–14 min, mobile phase A is 100% to 85%; 14–29 min, mobile phase A is 85% to 66%; 29–30 min, mobile phase A is 66% to 0%; 30–37 min, mobile phase A is maintained at 0%; 37.1 min, switch to mobile phase A at 100%; 37.1–45 min, mobile phase A is maintained at 100%.

7. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 6, characterized in that, The medical silk fibroin sample is at least one of silk fibroin gel, silk fibroin spray, and silk fibroin dressing.

8. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 7, characterized in that, The high-performance liquid chromatography method uses a detection wavelength of 254 nm to detect the peak area of ​​alanine. A standard curve is established using alanine as the characteristic amino acid. The alanine-silk fibroin conversion factor determined by the silk fibroin raw material is used to calculate the silk fibroin content in the sample.

9. The method for determining silk fibroin content using PITC-HPLC pre-column derivatization according to claim 8, characterized in that, The detection limit of the method is 0.4 μg / g, and the quantitation limit is 1 μg / g.

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