Establish a gas chromatography method for testing the residual monomer dioxanone in polydioxanone raw materials. A DB-624 capillary column (30.0 m ×535 μm ×3.00 μm) was used, and the temperature was programmed (146 ℃ for 5 min, 30 ℃/min to 200 ℃, and maintained for 2 min). The carrier gas was nitrogen, the hydrogen flame ionization detector (FID), the injection port temperature was 240 ℃, the detector temperature was 250 ℃, the split ratio was 5:1, and the injection volume was 2μL. The results showed that the monomer had a good linear relationship in the range of 0.0239 to 0.1794 mg/mL, the average recovery rate was 100.3%, the RSD was 1.39, the detection limit concentration was 0.2 μg/mL, and the quantification limit concentration was 0.5 μg/mL. The method is simple, fast, highly accurate and sensitive.

Polydioxanone (PPDO) is a linear biodegradable aliphatic polyester-ether. Its main molecular chain contains hydrolyzable ester bonds and unique ether bonds, which endow the polymer material with good biodegradability, biocompatibility and mechanical properties (i.e. excellent mechanical strength and toughness), and is an ideal biodegradable medical material. PPDO can be used for surgical sutures, orthopedic fixation materials, cell scaffolds and drug carriers, tissue repair materials, etc. As a new material for studying intravascular stents, PPDO has been certified by the FDA and approved for use in the human body. As an orthopedic fixation material, PPDO does not require the participation of special enzymes in the degradation process in the body. The metabolic products undergo the tricarboxylic acid cycle and are then excreted from the body through the respiratory tract, feces and urine.

PPDO is mostly synthesized using p-dioxanone (PDO) as raw material through ring-opening polymerization. The sample may contain unreacted PDO monomer. Although PDO monomer is not toxic, remaining in PPDO products will affect Sustained release effect and mechanical properties of PPDO. In this study, PPDO was used as the raw material and processed into products with a certain load-bearing function through injection molding. In order to ensure the mechanical properties of the products, the gas chromatography method was established to perform quality control detection of PDO monomer residues in PPDO raw materials in reference to the requirement of YY/T 0661-2008 standard that the monomer residue of polymers that need to be injection molded or load-bearing should not exceed 0.1%. At present, there is no report on the test method and residue limit of PDO monomer residue in PPDO.

I. Instruments and Reagents

Agilent 6890N gas chromatograph, flame ionization detector (FID), Agilent, USA; BS210S electronic analytical balance, d = 0.1 mg, Sartorius, Germany; Spectrafuge 6c centrifuge, Labnet, USA; KQ5200B ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; HH-2 digital constant temperature water bath, Changzhou Boyuan Experimental Analytical Instrument Factory.

PDO, purity greater than 99%; P-dioxanone (PDO), purity greater than 99%; poly-p-dioxanone (PPDO), company A; poly-p-dioxanone (PPDO), company B; dimethylformamide (DMF) ), chromatographically pure, Tianjin Comeo Chemical Reagent Co., Ltd.; methanol, chromatographically pure, Tianjin Saferui Technology Co., Ltd.; dimethyl sulfoxide (DMSO), chromatographically pure, Sinopharm Chemical Reagent Co., Ltd.

II. Methods and results

2.1. Solution preparation

2.1.1. Preparation of reference substance stock solution

Take about 10 mg of PDO, weigh it accurately, place it in a 100 mL volumetric flask, dissolve it in DMF and dilute it to volume, and use it as a reference substance stock solution.

2.1.2. Preparation of reference solution

Take 1 mL of the stock solution, place it in a 10 mL volumetric flask, dilute it with DMF and make up to volume as the reference solution.

2.1.3. Linear solution

Take 0.2, 0.5, 0.8, 1.0, 1.2, and 1.5 mL of the reference substance stock solution respectively, place them in a 10 mL volumetric flask, dilute with DMF and adjust to volume to serve as a linear solution.

2.1.4. Test solution

Take about 0.1 g of the test sample PPDO, weigh it accurately, put it in a 10 mL volumetric flask, add an appropriate amount of DMF to dissolve it, slowly add methanol dropwise, shake, and make up the volume; take an appropriate amount in a centrifuge tube, centrifuge at 3000 rpm for 30 min, and take the supernatant as the test solution.

2.2. Chromatographic conditions and system suitability test

2.2.1. Chromatographic conditions

A DB-624 capillary chromatographic column (30.0m x 535 μm x 3.00 μm) was used, with programmed temperature rise (146°C held for 5 minutes, 30°C/min heated to 200°C, held for 2 minutes), inlet temperature 240°C, detector temperature 250 ℃, split ratio 5:1, flow rate 3.0 m/min, injection volume 2 μL.

2.2.2. Selection of different chromatographic columns

The experiments were conducted using GS-TEK GsBP-5 (30.0 m x 320 μm x 0.25 μm) capillary columns and Agilent DB-624 (30.0 m x 535 μm x 3.0 μm) capillary columns. The GS-TEK GsBP-5 capillary column had poor symmetry of the monomer peak; the Agilent DB-624 capillary column had a higher response value for the monomer and better symmetry, and the theoretical plate number and separation degree met the requirements. Therefore, Agilent DB-624 capillary chromatography column was selected for this study.

2.2.3. Selection of solvents

PPDO is soluble in dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and hexafluoroisopropanol, but the presence of fluorine in hexafluoroisopropanol may cause damage to the chromatographic column, so it is not used; the impurity peaks in DMSO interfere with the detection of monomer PDO, so it is also not used; DMF was selected as the solvent in this study, which does not interfere with the determination of the monomer.

At the same time, we studied the dosage of DMF and found that, taking the total volume of the solution as 10 mL, when 2 mL of DMF was added, PPDO could not be completely dissolved, while adding 4 mL and 6 mL of DMF could make PPDO completely dissolved. However, when the amount of DMF was 6 mL, the amount of methanol (about 4 mL) was not enough to completely precipitate PPDO. Therefore, in this experiment, the amount of DMF added was 4 mL, and the volume ratio of DMF to methanol was 2:3.

2.2.4. System suitability inspection

Inject samples of the reference solution and the test solution respectively, and record the chromatogram. The retention time of the PDO monomer is 6.53, the number of theoretical plates is 80414, and the separation of peaks from other impurities in the polymer (PDO oligomers) The degree is 2.399.

2.3. Methodological study

2.3.1. Specificity

Precisely measure 2 μL of blank solvent a (DMF), blank mixed solvent b (methanol and DMF volume ratio is 3:2) and PDO reference solution c. Inject samples respectively according to the chromatographic conditions under "2.2.1" and record the chromatogram (see Figure 1). As can be seen from Figure 1, each solvent has no interference with the determination of PDO monomer and has good specificity.

Figure 1. GC chromatograms of blank solvent DMF (a), blank mixed solvent of DMF and methanol (b), and PDO control solution (c).

1. DMF, 2. Methanol, 3. PDO

2.3.2. Linear range

Take the linear series solution under "2.1.3" and inject it according to the chromatographic conditions under "2.2.1" respectively, and record the chromatogram and peak area. Make a linear regression with the monomer concentration (X, mg/mL) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Figure 2, and the regression equation is: Y = -0.62964 + 11848X, R = 0.9996. The results show that the linear relationship of PDO monomer concentration is good in the range of 0.0239 ~ 0.1794 mg/mL.

Fig 2. The standard carve of PDO

2.3.3. Quantitative limit and detection limit

Take the lowest concentration linear solution, add DMF to dilute it step by step to a certain concentration solution, inject the samples according to the chromatographic conditions under "2.2.1", and record the chromatogram. The signal-to-noise ratio of 10:1 is taken as the quantitative limit of PDO, and the signal-to-noise ratio of 3:1 is taken as the detection limit of PDO. The test results show that the detection limit concentration of PDO is 0.2 ug/mL, and the quantitative limit concentration is 0.5 mg/mL.

2.3.4. Precision

Precisely draw 2 μL of the reference solution, inject 6 consecutive times according to the chromatographic conditions under "2.2.1", and record the chromatogram and peak area. The calculated RSD value for the PDO peak area was 1.62 %.

2.3.5. Repeatability

Take the same batch of samples and prepare 6 sample solutions in parallel. Inject the samples according to the chromatographic conditions under "2.2.1" and record the chromatogram and peak area. The average value of PDO residue was calculated to be 0.12% and the RSD value was 2.16%.

2.3.6. Solution stability investigation

Precisely draw the reference solution, and inject samples at 0, 2, 5, 8, and 12 hours according to the chromatographic conditions under "2.2.1". The RSD value of the PDO peak area is 2.33%, indicating that the reference solution remains stable for up to 12 hours.

2.3.7. Recovery rate

Prepare the reference solution according to the method under "2.1.2".

Precisely weigh about 0.1g of company A's sample PPDO, a total of 9 portions, and place them in 10 mL volumetric flasks. Precisely add 0.8, 1.0, and 1.2 mL of PDO reference solution respectively, 3 portions of each concentration, and add 4 mL of DMF to each. Dissolve it and then slowly add methanol dropwise, shake to completely precipitate PPDO, adjust to volume, take an appropriate amount in a centrifuge tube, centrifuge at 3000 rpm for 30 minutes, and take the supernatant as the test solution.

Precisely measure 2 μL of the reference solution and test solution, inject it into the gas chromatograph, record the chromatogram and peak area, and calculate the recovery rate of PDO. The results are shown in Table 1.

Table 1 Results of recovery test of PDO

2.3.8. Sample determination

Accurately weigh the PPDO samples of Company A and Company B, prepare the reference solution according to the method under "2.1.2", prepare the test solution according to the method under "2.1.4", inject the samples according to the chromatographic conditions under "2.2.1", record the chromatogram and peak area, and calculate the PDO monomer content by the external standard method. The PDO monomer content in the PPDO samples of Company A and Company B was measured to be 0.12% and 0.05% respectively.

III. Discussion

PPDO has good biocompatibility and bioabsorbability, and contains unique ether bonds in the molecular chain, which makes it have good flexibility. It is the preferred biodegradable material for medical device product processing. The conditions for synthesizing PPDO are relatively harsh. At present, the only commercial PPDO material in China with qualified quality and stable batches is produced by "Dongguan Fortune Medical Technology Co., Ltd.". Batch production of synthetic PPDO brings great convenience to the development of medical devices. Whether it is synthesizing PPDO or using PPDO to process medical device products, it is necessary to control the raw materials, and the residual monomer content is a key indicator, which not only affects the mechanical properties of medical device products, but also affects its in vivo degradation performance.

The PDO monomers remaining in the PPDO material may be residual monomers that did not react when synthesizing the raw materials, or they may be monomer residues produced by the degradation of PPDO during processing or storage. Because the synthesis of PPDO is PDO ring-opening polymerization, and the structure of PDO is relatively stable, the synthesis reaction at high temperature is a balanced reversible reaction, so PDO monomers will inevitably remain, and post-treatment is required to reduce the monomer residual rate as much as possible; at the same time, PPDO is a degradable material, and monomer residues will inevitably remain during processing or storage. Therefore, it is necessary to detect the residual amount of PDO monomer in products whether before or after processing. There is currently no report on the detection method of PDO monomer residue. In this study, a measurement method for monomer PDO in PPDO materials was established through gas chromatography.

In the process of establishing the measurement methodology of monomer PDO in PPDO materials in this study, the chromatographic columns were first screened. PDO is a medium-polar component. According to the principle of similar compatibility, a medium-polar chromatographic column was selected. Through chromatographic scanning, it was found that the monomer peak of the non-polar chromatographic column CS-TEKGsBP-5 was not sharp and had poor symmetry, while the medium-polar chromatographic column Agilent DB-624 had a high monomer response value and good symmetry, so the medium-polar chromatographic column Agilent DB-624 was selected. The second is the choice of solvent. According to the solubility characteristics of PPDO and PDO monomers, a mixed solvent of DMF and methanol was selected to remove macromolecules in the polymer solution and reduce contamination of the liner and chromatographic column in the gas chromatograph, and the solvent had no effect on the monomer peak. The ratio of DMF to methanol is considered to be able to completely dissolve the polymer and completely precipitate the polymer macromolecules without affecting the test, so this study selected a solvent volume ratio of DMF to methanol of 2:3.The last step is the selection of chromatographic conditions. The purpose of selecting programmed temperature rise is to consider increasing the separation of solvent peaks and monomer peaks, as well as having symmetrical peak shapes and no tailing. After exploring multiple temperature programs, the chromatographic conditions for programmed temperature rise in the experiment were finally selected. The split ratio, flow rate and injection volume in the chromatographic conditions were selected through multiple explorations based on the response value and peak elution time of the monomer peak.

IV. Conclusion

In this study, the detection method of monomer residues in biodegradable medical material PPDO was studied. The research results show that using DB-624 capillary chromatography column, programmed temperature, DMF as the solvent, methanol as the precipitant, the volume ratio of solvent to precipitant is 2:3 to process the sample, and test the residual PDO monomer in the PPDO raw material. , the precision of the chromatographic system and the repeatability and specificity of the method are good, the sample recovery rate reaches 100.3%, and the RSD value reaches 1.39%. This method is simple, fast, and highly sensitive, and can be used to determine the residual PDO monomer in PPDO raw materials.

Dongguan Fortune Medical Technology Co., Ltd. (Introduction to absorbable polymer materials)

Dongguan Fortune Medical Technology Co., Ltd. is based on medical monomers and medical degradable polymer polyester materials (biomaterials), with interventional non-implantable devices (absorbable devices) as the core, providing customers with integrated, End-to-end high-end consumables R&D and production services continue to lower the R&D threshold for absorbable medical devices and help customers improve R&D efficiency. Bring more breakthrough treatment plans to patients, and the service scope covers the research and development and production of medical monomers, medical degradable polymer polyester materials (biomaterials), medical absorbable monofilaments/multifilaments, and absorbable medical devices.

In terms of polymer degradable materials, our technical core is based on years of accumulation, and we have mature and proven technical reserves. We have become a pioneer and leader in the Chinese absorbable materials market.