Detection method of ethylenediamine residue in deproteinized biological bone

Abstract

The invention discloses a detection method for the residual amount of ethylenediamine in a deproteinized biological bone, comprising the following steps: in a headspace bottle, first, a 3-8g / L sodium hydroxide aqueous solution is used to ultrasonically extract the deproteinized biological bone sample for 3-10min, then N, N-dimethylformamide is added thereto, the volume of N, N-dimethylformamide is greater than the volume of the sodium hydroxide aqueous solution, the bottle cap is screwed, shaken, balanced at 98-105°C for 20-40min, and then headspace gas chromatography is performed; the deproteinized biological bone is a biological bone after protein is extracted by ethylenediamine heating cycle. The method has good precision and high accuracy, and is suitable for accurate determination and quality control of the residual amount of ethylenediamine in animal deproteinized bone.

Description

Technical Field

[0001] The invention belongs to the technical field of deproteinized biological bone impurity detection, and in particular relates to a method for detecting the residual amount of ethylenediamine in deproteinized biological bone. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Bone repair materials mainly include natural bone materials and bone transplant substitute materials. Among them, deproteinized biological bone materials are prepared by defatting and deproteinizing heterogeneous natural bone materials. They have macroscopic and microscopic porous structures similar to human porous bones. Their three-dimensional porous structures are similar to human cancellous bones. They have good biocompatibility and osteoconductivity, help maintain the stability of the osteogenic environment and the vascularization of new tissues, and have a certain guiding effect on regenerated bone. They are widely used in clinical practice. In order to ensure the quality of the product, any impurities that affect the deproteinized biological bone material should be controlled. Once improperly controlled, they may be transferred to the product, thereby affecting the safety and effectiveness of the product, and even causing side effects that endanger human health.

[0004] Deproteinized bio-bone materials are mostly extracted by ethylenediamine circulation under heating conditions to remove proteins from xenogeneic natural bones. Ethylenediamine, as the main extraction reagent, may also remain as an impurity in the deproteinized bio-bone material, thereby affecting the purity of the deproteinized bio-bone material. Ethylenediamine belongs to the amine organic matter. Excessive ethylenediamine will form carcinogens in the body, so the control of ethylenediamine residue is particularly important. However, there is no relevant research on the determination of ethylenediamine residues in deproteinized bio-bone materials. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a method for detecting the residual amount of ethylenediamine in deproteinized biological bone.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A method for detecting the residual amount of ethylenediamine in deproteinized biological bone comprises the following steps:

[0008] In a headspace bottle, first, a 3-8 g / L sodium hydroxide aqueous solution is used to ultrasonically extract the deproteinized biological bone sample for 3-10 minutes, and then N,N-dimethylformamide is added thereto, the volume of N,N-dimethylformamide being greater than the volume of the sodium hydroxide aqueous solution, the bottle cap is screwed tightly, shaken well, balanced at 98-105° C. for 20-40 minutes, and then headspace gas chromatography is performed;

[0009] The deproteinized biological bone is biological bone obtained by extracting protein through heating cycle of ethylenediamine.

[0010] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0011] In the prior art, methanol and N,N-dimethylformamide are generally used as solvents for ethylenediamine detection and analysis. However, experiments have shown that when methanol, ethanol and N,N-dimethylformamide are used as solvents to extract the residual ethylenediamine in deproteinized biological bone, the target product cannot be detected. When the deproteinized biological bone sample is first ultrasonically extracted with a 3-8g / L sodium hydroxide aqueous solution and then N,N-dimethylformamide is added, free ethylenediamine can be obtained smoothly, and then the residual ethylenediamine can be detected smoothly. The reason is that the deproteinized biological bone has a certain adsorption effect, and the free calcium will also be complexed with ethylenediamine. Experiments have shown that trace amounts of residual ethylenediamine cannot be directly extracted with methanol. Adding sodium hydroxide solution provides it with a strong alkaline environment so that it takes away the H on N + , so that the organic amines are released.

[0012] This method has good precision and high accuracy, and is suitable for the accurate determination and quality control of ethylenediamine residues in animal deproteinized bones. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 This is the chromatogram of ethylenediamine obtained by adding the spiked sample in Example 1;

[0015] Figure 2 This is the chromatogram of ethylenediamine obtained from the blank test of Example 1;

[0016] Figure 3 It is the calibration curve under the conditions of Example 1;

[0017] Figure 4 The chromatogram of ethylenediamine obtained in Comparative Example 1;

[0018] Figure 5 The chromatogram of ethylenediamine obtained in Comparative Example 2;

[0019] Figure 6 The chromatogram of ethylenediamine obtained in Comparative Example 3;

[0020] Figure 7 The chromatogram of ethylenediamine obtained in Comparative Example 4;

[0021] Figure 8The chromatogram of ethylenediamine obtained in Comparative Example 5;

[0022] Fig. 9 This is the chromatogram of ethylenediamine obtained in Comparative Example 6;

[0023] Fig.10 This is the chromatogram of ethylenediamine obtained in Comparative Example 7;

[0024] Fig.11 This is the chromatogram of ethylenediamine obtained in Comparative Example 8. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] A method for detecting the residual amount of ethylenediamine in deproteinized biological bone comprises the following steps:

[0027] In a headspace bottle, first, a 3-8 g / L sodium hydroxide aqueous solution is used to ultrasonically extract the deproteinized biological bone sample for 3-10 minutes, and then N,N-dimethylformamide is added thereto, the volume of N,N-dimethylformamide being greater than the volume of the sodium hydroxide aqueous solution, the bottle cap is screwed tightly, shaken well, balanced at 98-105° C. for 20-40 minutes, and then headspace gas chromatography is performed;

[0028] The deproteinized biological bone is biological bone obtained by extracting protein through heating cycle of ethylenediamine.

[0029] The ethylenediamine impurity in deproteinized bio-bone material is introduced by the deproteinized raw materials and needs to be controlled during the production process. If it is not properly controlled, it will be introduced into the final product. Therefore, in order to ensure the safety of the final product, it is necessary to use an effective detection method to study and control it and control the ethylenediamine residue within the allowable range. Therefore, based on the characteristics of the product, a headspace gas chromatography method was developed to determine the ethylenediamine residue in deproteinized bio-bone.

[0030] Selection of extraction solvent: In the prior art, methanol and N,N-dimethylformamide are mostly used as solvents for analysis. According to the chemical properties of deproteinized biological bone, the extraction rates of ethylenediamine in deproteinized biological bone materials by N,N-dimethylformamide solution containing 1g / L sodium hydroxide, N,N-dimethylformamide, and methanol were investigated respectively. The results showed that under the optimal instrument conditions, N,N-dimethylformamide and methanol were used for extraction, and no target peaks were corresponding (see Comparative Examples 6 and 8); when N,N-dimethylformamide solution containing 1g / L sodium hydroxide was used, the target peak appeared. Therefore, N,N-dimethylformamide solution containing 1g / L sodium hydroxide was finally selected as the extraction solution, which solved the problem that the general extraction method could not release ethylenediamine.

[0031] Selection of injection method: In the prior art, direct injection method is used to analyze ethylenediamine in products. The premise of this method is that the matrix of the substance to be tested can be dissolved in the extraction medium. For deproteinized biological bone, its main component is hydroxyapatite, which is difficult to dissolve in water and organic solvents, and it is difficult to use direct injection method to detect it. Therefore, this study uses headspace injection method to inject it.

[0032] In some embodiments, the volume of the added sodium hydroxide aqueous solution is 1 mL, and the volume of the N,N-dimethylformamide is 4 mL.

[0033] Preferably, the concentration of sodium hydroxide is 4-6 g / L.

[0034] In some embodiments, the ultrasonic extraction time is 3-6 min.

[0035] In some embodiments, headspace gas chromatography is performed after equilibration at 99-102° C. for 25-35 min.

[0036] In some embodiments, the conditions for headspace gas chromatography detection are: the chromatographic column is CP-Volamine, 30m×320μm×0μm; the injection port temperature is 195-205°C.

[0037] The selection of chromatographic columns: In the prior art, when determining ethylenediamine in piperazine products, weakly polar and medium-polar chromatographic columns such as HP-5 and DB-624 are mostly used for analysis. We investigated CP-Volamine (30m×320μm×0μm) column, HP-5 (30m×320μm×0.25μm) column, and DB-624 (30m×250μm×1.4μm) column respectively. The experimental results show that under the same analytical method and optimal equipment conditions, the response values ​​of ethylenediamine on HP-5 column and DB-624 column are low, and the tailing is serious; CP-Volamine (30m×320μm×0μm) column is more suitable for analyzing the residual amount of ethylenediamine in deproteinized biological bone by headspace gas chromatography.

[0038] Preferably, the temperature rising program is: maintain at 75-85°C for 1-5 min, increase to 120°C at 10-15°C / min, maintain for 2 min, increase to 250°C at 33°C / min, maintain for 3 min.

[0039] Preferably, the detector is a hydrogen flame ionization detector, and the detector temperature is 250°C;

[0040] Preferably, the injection volume is 0.8-1.5 mL, the column flow rate is 1.5-2.5 mL / min; the hydrogen flow rate is 25-35 mL / min; the nitrogen flow rate is 20-30 mL / min; and the air flow rate is 250-350 mL / min.

[0041] Preferably, the split ratio is 8-12:1.

[0042] Using the above gas chromatography detection conditions, the impurities and ethylenediamine in the deproteinized biological bone can be effectively separated, with a linear range of 20 μg / mL to 500 μg / mL (r=0.9990), a precision RSD of 1.20%, a repeatability RSD of 1.07%, a limit of quantification of 200 ug / g, and a spiked recovery rate of 89.6% to 110.0%, all of which meet the relevant regulatory requirements.

[0043] This method is simple to operate, highly accurate, widely applicable, precise and sensitive. It is suitable for the detection of ethylenediamine residues in deproteinized biological bone materials and can meet the detection needs of large quantities of samples.

[0044] The present invention is described in detail below with reference to specific embodiments.

[0045] Example 1

[0046] instrument

[0047] Agilent GC7890B gas chromatograph-hydrogen flame ionization detector (Agilent Technologies Co., Ltd.); Agilent 7697A headspace sampler (Agilent Technologies Co., Ltd.); SECURA224-1CN electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); KQ250DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0048] Reagents

[0049] Ethylenediamine (Tianjin Komiou Reagent Co., Ltd., AR, batch number 20200426, purity ≥ 99.0%); N,N-dimethylformamide (McLean, HPLC, batch number C11286889, purity ≥ 99.9%); sodium hydroxide (Sinopharm Reagent, AR, batch number 20180308); the water used in the experiment was first-grade water.

[0050] Material

[0051] Deproteinized biological bone: Take fresh bovine femur, remove both ends of the femur, peel off the soft tissue, saw into small pieces, soak in 30% hydrogen peroxide solution at 37°C for 24 hours, take out, rinse with running water, remove soft tissue residue, dry, and degrease with ether for 24 hours. Take out, put into a reaction container, extract with ethylenediamine for 24 hours, take out, rinse with running water, and dry. Obtain biological bone tissue.

[0052] Methods and Results

[0053] Solution preparation

[0054] Reference solution Ethylenediamine standard solution: Accurately weigh an appropriate amount of ethylenediamine and prepare a 10.0 mg / mL ethylenediamine standard stock solution with N,N-dimethylformamide. Shake well and store in a sealed container under refrigeration.

[0055] 5g / L sodium hydroxide solution: Weigh 5g of sodium hydroxide and slowly add it to 1000mL of experimental water to dissolve it.

[0056] Preparation of test sample solutions

[0057] Weigh about 0.5g of sample (accurate to 0.1mg) into a 20mL headspace bottle, accurately add 1mL of sodium hydroxide solution (5g / L), ultrasonicate for 5min, accurately add 4mL of N,N-dimethylformamide, tighten the bottle cap, shake well, and balance at 100℃ for 30min.

[0058] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0059] A capillary column CP-Volamine (30 m × 320 μm) was used; column temperature: starting temperature 80 °C, maintained for 2 min, heated to 120 °C at a rate of 11 °C / min and maintained for 2 min, heated to 250 °C at a rate of 33 °C / min and maintained for 3 min;

[0060] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0061] The detector (FID) temperature was 250°C;

[0062] The carrier gas is nitrogen;

[0063] The injection method was headspace injection, and the headspace injector conditions were as follows: the heating box temperature was 100°C for 30 min, the injection valve temperature was 110°C, and the transfer line temperature was 120°C;

[0064] The injection volume of the sample to be tested was 1 mL, and the column flow rate was 2 mL / min;

[0065] Use deproteinized biological bone as a sample, weigh 0.5g of deproteinized biological bone into a 20mL headspace bottle, add 250μL of 2000μg / mL ethylenediamine standard solution, accurately add 1mL of sodium hydroxide solution (5g / L), ultrasonicate for 5min, accurately add 4mL of N,N-dimethylformamide, press the cap, mix well, place in the headspace injector, and measure. Perform a blank test at the same time.

[0066] The chromatogram of ethylenediamine residue in the spiked sample is obtained by Figure 1 As shown, the blank test chromatogram is shown in Figure 2 shown.

[0067] Depend on Figure 1 and Figure 2 It can be seen that under the conditions of Example 1, the retention time of the ethylenediamine target peak of the deproteinized biological bone spiked sample is 5.700 min, there are no impurity peaks nearby, and the peak shape is good. This chromatographic condition can be used as the final optimization condition.

[0068] Sample determination

[0069] Deproteinized biological bone samples were taken to prepare test sample solutions, which were analyzed by gas chromatography. The results are shown in Table 1.

[0070] Table 1 Determination results of ethylenediamine residue in deproteinized biological bone samples

[0071]

[0072]

[0073] The relevant indicators of the detection method provided in Example 1 were evaluated, specifically:

[0074] (1) Limit of quantification

[0075] The ethylenediamine reference solution was diluted step by step and subjected to gas chromatography determination according to the above chromatographic analysis conditions until the signal-to-noise ratio met the quantitative limit requirement, that is, the signal-to-noise ratio S / N ≥ 10, as shown in Table 2.

[0076] Table 2

[0077] <![CDATA[LOD1]]> <![CDATA[LOD2]]> <![CDATA[LOD3]]> Signal-to-Noise Ratio 11.3 10.7 10.5

[0078] The results showed that when the concentration of ethylenediamine was diluted to 20 μg / mL, the average signal-to-noise ratio (S / N) was 11, which met the requirement of the quantification limit (S / N ≥ 10).

[0079] (2) Precision test

[0080] The precision test was carried out with spiked samples. 0.500 g (accurate to 0.1 mg) of deproteinized biological bone was accurately weighed in a headspace bottle. A total of 7 portions were weighed. 500 μg of ethylenediamine reference substance was added to each portion of the sample. 1 mL of sodium hydroxide solution (5 g / L) was accurately added. Ultrasonication was performed for 5 min. 4 mL of N, N-dimethylformamide was accurately added. The bottle cap was tightened and shaken. The temperature was 100 ° C. and the balance was 30 min. The results are shown in Table 3.

[0081] Table 3

[0082]

[0083] The results showed that after 7 repeated measurements, the peak area RSD was 1.20%, the retention time RSD was 0.01%, and the RSD values ​​were all <5%, indicating that the method had good precision under this chromatographic condition and met the requirements of system suitability.

[0084] (3) Linear relationship investigation

[0085] Accurately pipette an appropriate amount of ethylenediamine standard solution into a 20mL headspace bottle, add 1mL of sodium hydroxide solution (5g / L), add N,N-dimethylformamide to 5mL, tighten the bottle cap, and shake well. Prepare ethylenediamine standard working solutions containing 6 different concentrations (20μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, 500μg / mL) for determination. Perform linear regression analysis with the peak area A of the analyte as the ordinate and the concentration C (μg / mL) of the analyte as the abscissa to obtain the linear regression equation, as shown in Table 4, and the calibration curve is shown in Figure 3 .

[0086] Table 4

[0087]

[0088] The results showed that the peak area and concentration of ethylenediamine had a good linear relationship within the range of 20 to 500 μg / mL.

[0089] (4) Accuracy test

[0090] The accuracy test is expressed by the spike recovery rate. Weigh 0.500g (accurate to 0.1mg) of deproteinized biological bone accurately into a headspace bottle, weigh 9 portions in total, and divide the 9 samples into three groups, A, B, and C. Add 1 times the quantitative limit, 2 times the quantitative limit, and 10 times the quantitative limit of ethylenediamine standard to each group of samples in turn, accurately add 1mL of sodium hydroxide solution (5g / L), ultrasonicate for 5min, accurately add 4mL of N,N-dimethylformamide, tighten the bottle cap, and shake well. Place in the headspace injector and measure. The results are shown in Table 5.

[0091] Table 5

[0092]

[0093] The results showed that the recovery of ethylenediamine was in the range of 89.56% to 110.0%, and the average recovery of 9 spiked samples was 101%. This method has good accuracy for the determination of ethylenediamine residues in deproteinized biological bone.

[0094] The gas chromatograph used was an Agilent 7890B equipped with an Agilent 7697A headspace sampler.

[0095] Comparative Example 1

[0096] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0097] A capillary column HP-5 (30m×320μm×0.25μm) was used; column temperature: starting temperature 40°C, maintained for 1 min, heated to 150°C at a rate of 10°C / min and maintained for 2 min, heated to 250°C at a rate of 33 / min and maintained for 3 min;

[0098] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0099] The detector (FID) temperature was 250°C;

[0100] The carrier gas is nitrogen;

[0101] The injection method is direct injection, the sample injection volume is 1 μL, and the column flow rate is 2 mL / min. The chromatogram of the ethylenediamine reference solution 200 μg / mL is obtained. Figure 4 shown.

[0102] Depend on Figure 4It can be seen that under the chromatographic conditions of Comparative Example 1, the retention time of ethylenediamine is 5.894 min, the response value is low, and the tailing is severe; therefore, the chromatographic conditions need to be optimized.

[0103] Comparative Example 2

[0104] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0105] A capillary column HP-5 (30m×320μm×0.25μm) was used; column temperature: starting temperature 40°C, maintained for 1 min, heated to 200°C at a rate of 20°C / min and maintained for 3 min, heated to 250°C at a rate of 33 / min and maintained for 3 min;

[0106] The injection port temperature was 240 °C, and the split ratio was 20:1;

[0107] The detector (FID) temperature was 250°C;

[0108] The carrier gas is nitrogen;

[0109] The injection method is direct injection, the injection volume of the sample to be tested is 0.5μL, and the column flow rate is 2mL / min

[0110] The chromatogram of ethylenediamine reference solution 200 μg / mL is obtained as shown in Figure 5 shown.

[0111] Depend on Figure 5 It can be seen that under the chromatographic conditions of Comparative Example 2, the retention time of ethylenediamine is 4.434 min, the peak shape is good, but the response value is low and cannot meet the expected detection limit requirements of the product; therefore, the chromatographic conditions need to be optimized.

[0112] Comparative Example 3

[0113] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0114] A capillary column DB-624 (30 m × 250 μm × 1.4 μm) was used; column temperature: starting temperature 45 °C, maintained for 5 min, heated to 250 °C at a rate of 10 °C / min and maintained for 10 min;

[0115] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0116] The detector (FID) temperature was 250°C;

[0117] The carrier gas is nitrogen;

[0118] The injection method is direct injection, the sample injection volume is 1 μL, and the column flow rate is 1 mL / min. The chromatogram of the ethylenediamine reference solution 200 μg / mL is obtained. Figure 6shown.

[0119] Depend on Figure 6 It can be seen that under the chromatographic conditions of Comparative Example 3, the retention time of ethylenediamine is 10.215 min, the peak shape is good, but the response value is low; therefore, the chromatographic conditions need to be optimized.

[0120] Comparative Example 4

[0121] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0122] A capillary column DB-624 (30 m × 250 μm × 1.4 μm) was used; column temperature: starting temperature 45 °C, maintained for 5 min, heated to 250 °C at a rate of 20 °C / min and maintained for 10 min;

[0123] The injection port temperature was 200 °C, and the split ratio was 20:1;

[0124] The detector (FID) temperature was 250°C;

[0125] The carrier gas is nitrogen;

[0126] The injection method is direct injection, the sample injection volume is 1 μL, and the column flow rate is 2 mL / min. The chromatogram of the ethylenediamine reference solution 200 μg / mL is obtained. Figure 7 shown.

[0127] Depend on Figure 7 It can be seen that under the chromatographic conditions of Comparative Example 4, the retention time of ethylenediamine is 7.242 min, the peak shape is good, but the response value is low; therefore, the chromatographic conditions need to be optimized.

[0128] Comparative Example 5

[0129] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0130] A capillary column CP-Volamine (30 m × 320 μm) was used; column temperature: starting temperature 60 °C, maintained for 2 min, heated to 120 °C at a rate of 20 °C / min and maintained for 2 min, heated to 250 °C at a rate of 33 °C / min and maintained for 3 min;

[0131] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0132] The detector (FID) temperature was 250°C;

[0133] The carrier gas is nitrogen;

[0134] The injection method is direct injection, the sample injection volume is 1 μL, and the column flow rate is 2 mL / min. The chromatogram of the ethylenediamine reference solution 200 μg / mL is obtained. Figure 8shown.

[0135] Depend on Figure 8 It can be seen that under the chromatographic conditions of Comparative Example 5, the retention time of ethylenediamine is 4.419 min, the peak shape is good, the response value is improved, and there is no interference from impurities in the surrounding area. The chromatographic conditions can be used as preliminary optimization conditions.

[0136] Comparative Example 6

[0137] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0138] A capillary column CP-Volamine (30 m × 320 μm) was used; column temperature: starting temperature 60 °C, maintained for 2 min, heated to 120 °C at a rate of 20 °C / min and maintained for 2 min, heated to 250 °C at a rate of 33 °C / min and maintained for 3 min;

[0139] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0140] The detector (FID) temperature was 250°C;

[0141] The carrier gas is nitrogen;

[0142] The injection method is direct injection, the sample injection volume is 1μL, and the column flow rate is 2mL / min

[0143] Deproteinized biological bone was used as a sample. 0.5 g of deproteinized biological bone was weighed into a stoppered glass bottle. 250 μL of 2000 μg / mL ethylenediamine standard solution was added to the product. 5 mL of methanol was added, and ultrasonic extraction was performed for 5 min. The mixture was mixed and the extract was filtered through a 0.22 μm organic filter head before being measured on an analyzer.

[0144] The chromatogram of ethylenediamine residue in the spiked sample is obtained by Fig. 9 shown.

[0145] Depend on Fig. 9 It can be seen that under the chromatographic conditions of Comparative Example 6, no ethylenediamine target peak appeared at the ethylenediamine retention time of 4.419 min. The chromatographic conditions need to be further optimized.

[0146] Comparative Example 7

[0147] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0148] A capillary column CP-Volamine (30 m × 320 μm) was used; column temperature: starting temperature 60 °C, maintained for 2 min, heated to 120 °C at a rate of 20 °C / min and maintained for 2 min, heated to 250 °C at a rate of 33 °C / min and maintained for 3 min;

[0149] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0150] The detector (FID) temperature was 250°C;

[0151] The carrier gas is nitrogen;

[0152] The injection method was headspace injection, and the headspace injector conditions were as follows: the heating box temperature was 100°C for 30 min, the injection valve temperature was 110°C, and the transfer line temperature was 120°C;

[0153] The injection volume of the sample to be tested was 1 mL, and the column flow rate was 2 mL / min;

[0154] Deproteinized biological bone was used as a sample. 0.5 g of deproteinized biological bone was weighed into a 20 mL headspace bottle. 250 μL of 2000 μg / mL ethylenediamine standard solution was added. 5 mL of methanol was added. The bottle was capped and ultrasonically extracted for 5 min. The sample was mixed and placed in a headspace injector for determination.

[0155] The chromatogram of ethylenediamine residue in the spiked sample is obtained by Fig.10 shown.

[0156] Depend on Fig.10 It can be seen that under the chromatographic conditions of Comparative Example 7, no ethylenediamine target peak appeared at the ethylenediamine retention time of 4.419 min. Under this chromatographic condition, the sample pretreatment method needs to be further optimized.

[0157] Comparative Example 8

[0158] The gas chromatograph was used for determination, and the chromatographic conditions were as follows:

[0159] A capillary column CP-Volamine (30 m × 320 μm) was used; column temperature: starting temperature 60 °C, maintained for 2 min, heated to 120 °C at a rate of 20 °C / min and maintained for 2 min, heated to 250 °C at a rate of 33 °C / min and maintained for 3 min;

[0160] The injection port temperature was 200 °C, and the split ratio was 10:1;

[0161] The detector (FID) temperature was 250°C;

[0162] The carrier gas is nitrogen;

[0163] The injection method was headspace injection, and the headspace injector conditions were as follows: the heating box temperature was 100°C for 30 min, the injection valve temperature was 110°C, and the transfer line temperature was 120°C;

[0164] The injection volume of the sample to be tested was 1 mL, and the column flow rate was 2 mL / min;

[0165] Use deproteinized biological bone as a sample, weigh 0.5g of deproteinized biological bone into a 20mL headspace bottle, add 250μL of 2000μg / mL ethylenediamine standard solution, add 5mL N,N-dimethylformamide, press the cap, ultrasonically extract for 5min, mix well, place in the headspace injector, and measure. The chromatogram of ethylenediamine residue in the spiked sample is obtained. Fig.11 shown.

[0166] Depend on Fig.11 It can be seen that under the chromatographic conditions of Comparative Example 8, at the ethylenediamine retention time of 4.419 min, no ethylenediamine target peak appeared and there were impurity peaks near the retention time of the target peak. Therefore, the chromatographic conditions and sample pretreatment conditions need to be further optimized.

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the residual amount of ethylenediamine in deproteinized biological bone, characterized in that: The steps include: In a headspace bottle, first, a 3-8 g / L sodium hydroxide aqueous solution is used to ultrasonically extract the deproteinized biological bone sample for 3-10 minutes, and then N,N-dimethylformamide is added thereto, the volume of N,N-dimethylformamide being greater than the volume of the sodium hydroxide aqueous solution, the bottle cap is screwed tightly, shaken well, balanced at 98-105° C. for 20-40 minutes, and then headspace gas chromatography is performed; The deproteinized biological bone is biological bone obtained by heating and circulating ethylenediamine to extract protein; The conditions for headspace gas chromatography were as follows: chromatographic column: CP-Volamine, 30 m × 320 μm; The heating program was as follows: 75-85°C for 1-5 min, then increased to 120°C at 10-15°C / min, then maintained for 2 min, then increased to 250°C at 33°C / min, then maintained for 3 min; The detector is a hydrogen flame ionization detector.

2. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The volume of the added sodium hydroxide aqueous solution was 1 mL, and the volume of N,N-dimethylformamide was 4 mL.

3. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 2, characterized in that: The concentration of sodium hydroxide is 4-6g / L.

4. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The ultrasonic extraction time is 3-6 minutes.

5. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: After equilibration at 99-102°C for 25-35 min, headspace gas chromatography was performed.

6. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The conditions for headspace gas chromatography detection are: injection port temperature: 195-205°C.

7. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The detector temperature was 250°C.

8. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The injection volume is 0.8-1.5mL, the column flow rate is 1.5-2.5mL / min; the hydrogen flow rate is 25-35mL / min; the nitrogen flow rate is 20-30mL / min; and the air flow rate is 250-350mL / min.

9. The method for detecting the residual amount of ethylenediamine in deproteinized biological bone according to claim 1, characterized in that: The split ratio is 8-12:1.

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