Method for measuring content of triclosan in absorbable suture line by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention relates to the technical field of novel detection, in particular to a method for determining the content of triclosan in an absorbable suture line by HPLC (High Performance Liquid Chromatography), which comprises the following steps: S1, preparation of a standard stock solution: adding a triclosan reference substance into a solvent to prepare a standard working solution; s2, drawing a standard curve: diluting the standard working solution into different concentrations, analyzing through HPLC (High Performance Liquid Chromatography), and drawing the standard curve by taking the concentration as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate; s3, sample pretreatment: weighing a certain amount of a sample, adding the sample into a centrifuge tube, extracting the sample with the extract, centrifuging, and taking the supernatant as a test solution; s4, measuring the content of triclosan: performing HPLC (High Performance Liquid Chromatography) detection on the test solution prepared in S3, setting detection conditions, recording a chromatogram, and detecting the content of triclosan in the sample. The method for determining the content of triclosan in the absorbable surgical suture line through high performance liquid chromatography (HPLC) is established, and the method has the advantages of being easy to operate and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for determining the content of triclosan in absorbable suture by HPLC method. Background Art

[0002] Triclosan, with the chemical name of 5-chloro-2-(2',4'-dichlorophenoxy)phenol and the chemical formula of C 12 H 7 Cl 3 O 2 , is an organic compound with broad-spectrum antibacterial efficacy. It can be absorbed into the human body through the skin, oral mucosa and gastrointestinal tract. At present, foreign studies have shown that long-term and excessive use of triclosan will cause certain harm to the human body. Literature reports indicate that triclosan can damage the integrity of the liver, and also cause infertility, obesity, slow development and cancer. It can also weaken the human immune system by changing the homeostasis of the skin, oral and intestinal flora. There is evidence that children who are exposed to products containing triclosan for a long time have a higher chance of developing allergies. The latest research finds that triclosan exposure during pregnancy will increase the risk of autism in offspring. In addition, triclosan can also cause intestinal flora imbalance and exacerbate liver injury.

[0003] In the medical field, absorbable surgical sutures, as a key material for surgical sutures, their quality and safety are directly related to the recovery and health of patients. Triclosan (TCS), as an important additive to enhance the antibacterial performance of sutures, the accurate determination of its content is crucial for ensuring the overall quality of sutures. However, the current industry's detection standards and methods for the content of triclosan in absorbable surgical sutures are not yet perfect, and there are certain gaps. Summary of the Invention

[0004] To solve the above problems, the present application provides a method for determining the content of triclosan in absorbable suture by HPLC method, which is characterized by including the following steps:

[0005] S1 Preparation of standard stock solution: Add triclosan reference substance to a solvent to prepare a standard working solution;

[0006] S2 Plotting the standard curve: Dilute the standard working solution into different concentrations, analyze it by HPLC, use the concentration as the abscissa and the corresponding chromatographic peak area as the ordinate to plot the standard curve;

[0007] S3 Sample pretreatment: Weigh a certain amount of sample and add it to a centrifuge tube, extract and centrifuge the sample with an extraction solution, and take the supernatant as the test solution;

[0008] S4 Determination of triclosan content: Perform HPLC detection on the test solution prepared in S3, set the detection conditions, record the chromatogram, and detect the triclosan content in the sample.

[0009] In one embodiment, the extraction solution is an aqueous solution of acetonitrile or an aqueous solution of methanol.

[0010] In one embodiment, the concentration of the extraction solution is 25% to 100%.

[0011] In one embodiment, the extraction time in step S3 is 15 min to 10080 min.

[0012] In one embodiment, in step S2, it is diluted step by step with acetonitrile to prepare a series of standard working solutions, and the concentrations of triclosan are 0.5, 5.0, 10.0, 20.0, 50.0, and 100.0 μg / mL.

[0013] In one embodiment, in step S3, after centrifugal vortex for 2 min and extraction, the centrifuge tube is centrifuged at 3000 rpm for 5 min, and the supernatant is taken to pass through a 0.45 μm organic filter membrane for on-machine inspection.

[0014] In one embodiment, the chromatographic detection conditions in step S4 are: ultraviolet detector; Agilent C 18 chromatographic column; the flow rate is 0.8 mL / min; the injection volume is 10 μL; the detection wavelength is 204 nm; the column temperature is 40°C; mobile phase A is acetonitrile, B is water, and gradient elution is performed.

[0015] In one embodiment, according to the step S2, the prepared standard working solutions with different concentrations are injected into a high performance liquid chromatograph, and a standard curve is plotted with the peak area Y as the ordinate and the reference substance concentration X as the abscissa. The linear regression equation is: Y = 149051X + 27089.8, and the correlation coefficient r when fitting the regression equation is 0.9999.

[0016] In one embodiment, it further includes: using a blank sample as the sample matrix to conduct a standard addition recovery test. The addition levels of triclosan are 1.5, 5.0, and 15.0 mg / kg. The content of the sample after addition is measured and the standard addition recovery rate is calculated. Each addition concentration is determined in parallel 6 times, and the standard addition recovery rate and relative standard deviation are calculated.

[0017] In one embodiment, ultrasonic extraction is performed in step S3, and the ultrasonic extraction time is 10 min to 60 min.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention relates to a method for determining the content of triclosan in absorbable suture by HPLC. A method for determining the content of triclosan in absorbable surgical suture by high performance liquid chromatography (HPLC) is established. This method has the advantages of simple operation and high accuracy. The results of the standard addition recovery experiment show that this method has a high recovery rate, good precision, and relatively high detection sensitivity. The detection limit of this method is 0.03 μg / mL, and the technical indicators meet the relevant requirements of the analysis method, which can meet the needs of daily laboratory inspection and detection. The establishment of this method aims to fill the blank of the current industry standard and provide a scientific basis for the quality control of absorbable surgical suture, so as to better control the product quality and improve the safety and effectiveness of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the chromatogram of triclosan standard solution;

[0021] Figure 2 It is the typical chromatogram of absorbable suture sample;

[0022] Figure 3 It is the content of triclosan under different extraction times;

[0023] Figure 4 It is the chromatogram of triclosan standard solution under different mobile phases. DETAILED DESCRIPTION OF THE INVENTION

[0024] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0025] 1 Method and Results

[0026] 1.1 Instruments and Reagents

[0027] 1.1.1 Instruments

[0028] High performance liquid chromatograph (LC-20AT, Shimadzu Corporation, Japan, equipped with ultraviolet detector); high-speed centrifuge (G-16C, Sartorius Corporation); electronic analytical balance (XPR204 / A, Mettler-Toledo Corporation); numerically controlled ultrasonic cleaner (KQ5200DB, Kunshan Ultrasonic Instrument Co., Ltd.); vortex oscillator (Eppendorf MixOne, Hefei Eppendorf Scientific Instrument Co., Ltd.); pure water machine (Milli-Q IQ 7000, Millipore (China) Co., Ltd.).

[0029] 1.1.2 Reagents

[0030] Triclosan reference substance (National Institutes for Food and Drug Control, with C12 H 7 Cl 3 O 2 (calculated, content is 100.0%, CAS No. 3380 - 34 - 5); acetonitrile (chromatographic grade, Merck, Germany); the test water is secondary water meeting the national standard (GB / T 6682 - 2008).

[0031] 1.2 Preparation of standard stock solution

[0032] Accurately weigh 10 mg of triclosan reference substance (accurate to 0.1 mg) into a 10 mL volumetric flask, dissolve it with acetonitrile, and make up the volume to prepare a standard stock solution with a mass concentration of 1 mg / mL.

[0033] 1.3 Standard curve

[0034] Preparation of standard working solution: Gradually dilute with acetonitrile to prepare a series of standard working solutions with triclosan concentrations of 0.5, 5.0, 10.0, 20.0, 50.0, 100.0 μg / mL.

[0035] Drawing of standard curve: Inject the triclosan standard series, use the concentration of the triclosan standard series as the abscissa and the corresponding chromatographic peak area as the ordinate to draw the standard curve.

[0036] 1.4 Sample pretreatment

[0037] Take 2 g (at least 30 packages) of the sample and cut it into segments with a length of 5 mm. Weigh 0.5 g of the sample (accurate to 0.01 g) and add it to a centrifuge tube. Add 25 mL of 50% acetonitrile - water extraction solution (or change the volume of the extraction solution according to the ratio of 0.1 g of the sample to 5 mL of 50% acetonitrile - water). Cover the centrifuge tube, vortex for 2 min, extract at room temperature (25 °C) for 48 h, then centrifuge the centrifuge tube at 3000 rpm for 5 min, take the supernatant and filter it through a 0.45 μm organic filter membrane, and then conduct on - machine inspection.

[0038] 1.5 Chromatographic conditions

[0039] Ultraviolet detector; Agilent C 18 Chromatographic column (4.6×250 mm, 5.0 μm); flow rate is 0.8 mL / min; injection volume is 10 μL; detection wavelength is 204 nm; column temperature is 40 °C; mobile phase A is acetonitrile, B is water, and the gradient elution program is shown in Table 1.

[0040]

[0041] 1.6 Linear range and detection limit

[0042] Prepare the standard use solution according to Section 1.3, and perform injection determination respectively. Plot the standard curve with the peak area (Y) as the ordinate and the concentration of the reference substance (X) as the abscissa, and the linear regression equation is obtained as: Y = 149051X + 27089.8. Triclosan has a good linear relationship in the range of 0.50 μg / mL to 100.0 μg / mL, and the correlation coefficient (r) is 0.9999 when fitting the regression equation. At a wavelength of 204 nm, at R t = 7.176 min, a sharp spectral peak corresponding to triclosan appears, and the spectrogram is shown in Figure 1 . The chromatographic peak shape has good symmetry and basically no tailing phenomenon. Add an appropriate amount of standard solution to the blank sample and dilute it step by step to 3 times the baseline noise value of the detection signal (S / N = 3), and the method detection limit of triclosan is 0.03 μg / mL.

[0043] 1.7 Recovery rate and precision

[0044] Using the blank sample as the sample matrix, perform a standard addition recovery test. The addition levels of triclosan are 1.5, 5.0, and 15.0 mg / kg. Determine the content of the sample after adding the standard and calculate the standard addition recovery rate. Each addition concentration is determined in parallel 6 times, and the standard addition recovery rate and relative standard deviation are calculated. The results are shown in Table 2.

[0045]

[0046] It can be seen from Table 2 that for the standard addition recovery test of triclosan, the addition levels are 1.5, 5.0, and 15.0 mg / kg respectively, and each level is determined in parallel 6 times. The results show that the recovery rates at different addition levels are all above 95%, the method design is reasonable and effective, and the result accuracy is good; in terms of precision, the precision at different addition levels is ≤5%, indicating that the method has good stability and reproducibility and is suitable for the detection of triclosan samples in a variety of concentration ranges.

[0047] 1.8 Sample determination

[0048] Select three models of samples from the same manufacturer for sample pretreatment according to Section 1.4, and detect them according to the optimized chromatographic conditions in Section 1.5. Triclosan is detected in all three samples, and the sample contents are 4980.7, 3802.08, and 2264.1 mg / kg respectively. The chromatogram of the sample in the 204 nm channel is shown in Figure 2 .

[0049] 2. Selection of pretreatment conditions

[0050] 2.1 Selection of extraction solvent

[0051] Under the condition that other conditions remain unchanged, only the types of extraction solutions and the proportions of organic reagents are changed. A 15 mg / kg level of triclosan standard solution was added to the blank matrix, and 9 reagents, namely 25% acetonitrile, 50% acetonitrile, 75% acetonitrile, acetonitrile, 25% methanol, 50% methanol, 75% methanol, methanol, and acetonitrile + methanol (1:1), were respectively used for extraction for 15 min, 30 min, and 60 min. The extraction solutions were taken for on-machine detection, and the detection results are shown in Table 3.

[0052] Table 3 Contents of triclosan in different extraction solutions

[0053]

[0054] It can be seen from Table 3 that when 50% acetonitrile is used for extraction, the content of triclosan in the extraction solution is the highest, and the content increases continuously with the increase of the extraction time.

[0055] 2.2 Selection of extraction temperature and time

[0056] 2.2.1 Extraction temperature

[0057] The extraction temperatures selected were room temperature of 25 °C and human body temperature of 37 °C. Through experimental verification, it was found that at the two temperatures, the difference in the content of triclosan was not significant. For the convenience of energy conservation, room temperature of 25 °C was selected as the extraction temperature.

[0058] 2.2.2 Extraction time

[0059] A 1000 mg / kg level of triclosan standard solution was added to the blank matrix, and it was extracted with 50% acetonitrile solution at room temperature of 25 °C for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d respectively. The contents of triclosan are as Figure 3 shown.

[0060] From Figure 3 it can be seen that the overall trend of the triclosan content is to increase continuously with the increase of time. The growth rate slows down at 2 d, and the incremental change is no longer obvious. Considering the time cost and inspection efficiency, the extraction time was selected as 2 d.

[0061] 2.2.3 Selection of ultrasonic extraction

[0062] A 1000 mg / kg level of triclosan standard solution was added to the blank matrix, and it was ultrasonically extracted with 50% acetonitrile solution at room temperature of 25 °C for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min respectively. It was found that when ultrasonically extracting for 30 min, the content of triclosan was not significantly different from that of extracting for 2 d.

[0063] 2.3 Selection and optimization of the mobile phase

[0064] Acetonitrile-water and methanol-water were selected as the mobile phases for the experiment. The chromatogram of the triclosan standard solution is as Figure 4 shown. When gradient elution was carried out with an acetonitrile-aqueous solution, at a wavelength of 204 nm, t R = 7.176 min, a sharp peak corresponding to triclosan appeared, and the signal intensity was 891256 MV; when gradient elution was carried out with a methanol-aqueous solution, at a wavelength of 282 nm, t R = 5.264 min, a sharp peak corresponding to triclosan appeared, and the signal intensity was 93411 MV. From Figure 4 this, it can be seen that the response value of triclosan when using acetonitrile-water as the mobile phase is higher, about 10 times that when using methanol-water as the mobile phase.

[0065] Advantages of the present application compared with the prior art:

[0066] The present invention relates to a method for determining the content of triclosan in absorbable suture by HPLC, and a method for determining the content of triclosan in absorbable surgical suture by high performance liquid chromatography (HPLC) is established. This method has the advantages of simple operation and high accuracy. The results of the standard addition recovery experiment show that this method has a high recovery rate, good precision, and relatively high detection sensitivity. The detection limit of this method is 0.03 μg / mL, and the technical indicators meet the relevant requirements of the analysis method, and can meet the needs of daily laboratory inspection and detection. The establishment of this method aims to fill the gap in the current industry standard, provide a scientific basis for the quality control of absorbable surgical suture, so as to better control the product quality and improve the safety and effectiveness of the product.

[0067] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0068] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can also be directly connected, or indirectly connected through an intermediate medium, and can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

Claims

1. A method for determining the content of triclosan in absorbable sutures by HPLC, characterized in that: The following steps are involved: Preparation of S1 standard stock solution: Add triclosan reference substance into solvent to prepare standard working solution; S2: Draw a standard curve: dilute the standard working solution into different concentrations, analyze it by HPLC, and draw a standard curve with the concentration as the horizontal axis and the corresponding chromatographic peak area as the vertical axis; S3 Sample pretreatment: Weigh a certain amount of sample and add it into a centrifuge tube. Use the extracting solution to extract the sample, centrifuge it, and take the supernatant as the test solution; S4 Determination of triclosan content: The test solution prepared in S3 is subjected to HPLC detection, the detection conditions are set, the chromatogram is recorded, and the triclosan content in the sample is detected.

2. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: The extract is an acetonitrile aqueous solution or a methanol aqueous solution.

3. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 2, characterized in that: The concentration of the extract is 25% to 100%.

4. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: The extraction time in step S3 is 15 min to 10080 min.

5. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: In step S2, acetonitrile is used for stepwise dilution to prepare a series of standard working solutions, and the triclosan concentrations are 0.5, 5.0, 10.0, 20.0, 50.0, and 100.0 μg / mL.

6. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: In step S3, the mixture was centrifuged and vortexed for 2 minutes. After extraction, the centrifuge tube was centrifuged at 3000 rpm for 5 minutes. The supernatant was filtered through a 0.45 μm organic filter membrane and tested on a filter.

7. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: The chromatographic detection conditions in step S4 are: UV detector; Agilent C 18 Chromatographic column; flow rate is 0.8mL / min; injection volume is 10μL; detection wavelength is 204nm; column temperature is 40℃; mobile phase A is acetonitrile, B is water, gradient elution.

8. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: According to the S2 step, the prepared standard working solutions of different concentrations were injected into the high performance liquid chromatograph, and the standard curve was plotted with the peak area Y as the ordinate and the reference substance concentration X as the abscissa. The linear regression equation was: Y=149051X+27089.8, and the correlation coefficient r was 0.9999 when fitting the regression equation.

9. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: Also includes: A spike recovery test was carried out using blank samples as the sample matrix. The addition levels of triclosan were 1.5, 5.0, and 15.0 mg / kg. The sample content after spiked was determined and the spiked recovery was calculated. Each spiked concentration was measured six times in parallel, and the spiked recovery and relative standard deviation were calculated.

10. The method for determining the content of triclosan in absorbable sutures by HPLC according to claim 1, characterized in that: In the step S3, ultrasonic extraction is performed, and the ultrasonic extraction time is 10 min to 60 min.

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