The efficacy and quality of drugs are closely related to human health and safety. The substance which consisted of internal medicine and packaging material of the direct contact drug is called medicine [
When selecting and confirming the packing seals, the pharmaceutical-producing enterprises should protect the quality of drugs and achieve the purpose of drug delivery in the process of packaging, storage, and transportation. Rubber is a kind of linear flexible macromolecule polymer. Its molecular chain is flexible and can produce large deformation under the external force. It can quickly restore its original shape when external force disappears [
Antioxidant 264 (2,6-di-tert-butyl-4-methylphenol) is an excellent generic phenolic antioxidant, which is most widely used as an antioxidant in plastics. It has the advantages of good antioxidant effect, high thermal stability, good compatibility with plastics, nonpollution to plastics, and so on [
Based on the necessity of the aforementioned control of antioxidant 264, this study established a reflux extraction method of organic solvents to extract antioxidant 264 and determine it by HPLC. The method was simple and accurate for the determination of antioxidant 264 in brominated butyl rubber stoppers. Then, the developed method was successfully applied to the compatibility between brominated butyl rubber stopper and Novaferon to evaluate the safety of migration of antioxidant 264 [
Antioxidant 264 (99% purity) was obtained from Sigma Company (St. Louis, MO, USA). The batch number of the brominated butyl rubber stopper for injection was 16111006. Novaferon samples (lot numbers: 201708001, 201708002, 201708003, 201708004, 201708005, and 201708006) were provided from Jiehua Biotechnology Group Co., Ltd. (Qingdao, China).
Chromatographic-grade ethanol and dichloromethane were obtained from Dikma Technologies (Beijing, China). Acetonitrile was purchased from ANPEL Laboratory Technologies (Shanghai, China). Deionized water was purchased by China Resources C’estbon Beverage Co., Ltd. (Shenzhen, China).
Liquid chromatographic analysis was performed using the Agilent Series HPLC system (Agilent, San Jose, CA, USA) equipped with a diode array detector (DAD). Separations were carried out on an Ultimate XB-C18 (150 mm × 4.6 mm, 5
Accurately weighed amount of antioxidant 264 was taken and placed in a 100 mL volumetric flask; then, the standard stock solution of antioxidant 264 was prepared with anhydrous ethanol at the concentration of 0.4 mg/mL. An amount of the antioxidant 264 standard stork solution to a series of reference substance dilutions contained S1, S2, S3, S4, S5, S6, and S7 and then were diluted by anhydrous ethanol to the antioxiant 264 concentration of 0.004, 0.012, 0.04, 0.08, 0.12, and 0.20 mg/ml. Serial dilutions of the standard stock solution of antioxidant 264 were used for subsequent experimental.
The validation study consisted of selectivity, limit of detection (LOD), limit of quantitation (LOQ), precision, repeatability, and recovery, as well as stability.
The specificity of the method was proved by comparing typical liquid chromatograms of the blank solution and reference standard solution of antioxidant 264, as well as sample solutions of rubber stoppers and Novaferon. The results showed that there was no interference in the detection of antioxidant 264 in the blank solution, rubber stopper test solution, and Novaferon test solution, and the method presented good specificity and resolution. The chromatograms are shown in Figure
HPLC chromatogram of the blank solvent (a), reference standard solution of antioxidant 264 (b), sample solution of the rubber stopper (c), and recombinant potent antitumor and antivirus protein injection for the test (d).
The linear solution was injected into the liquid chromatograph with 20
The LOD and LOQ were determined at a signal-to-noise ratio of 3 : 1 and 10 : 1 by injecting a series of diluted standard solutions with known concentrations, respectively. When the response values were about 3 and 10-fold signal-to-noise ratio, the LOD of antioxidant 264 was 192.2 ng/mL, and the LOQ was 434.0 ng/mL.
The precision of the method was obtained by injecting the same reference solution of antioxidant 264 for six times continuously. The results showed that the RSD of the antioxidant 264 peak area was 0.07%, indicating high instrument precision.
Six test solutions of plastic stopper and Novaferon were prepared in parallel. Under the above chromatographic conditions, 20
Accurately weighed 2.5 g medicinal rubber stopper and was cut into small pieces in a 100 mL mill borosilicate glass flask, parallel operation 3 times, and then added the antioxidant 264 reference substances 0.8 mL, 1 mL, and 1.2 mL, respectively, and then added 10 mL methyl chloride-ethanol (1 : 1) of each sample. Mixture was boiled under a reflux condenser for 4 h and cooled down to room temperature, supplemented with methylene chloride-ethanol (1 : 1). Finally, the solution was filtered by the 0.45
Three parts of 2 mL Novaferon, which was filled into the rubber stopper, were added, respectively. Then, the antioxidant 264 standard stock solution (0.8 mL, 1 mL, and 1.2 mL) was extracted by the solid-phase extraction column which was activated with 4 mL methanol and 4 mL water, respectively. Then, Novaferon was eluted with anhydrous ethanol solution, and the volume was fixed to 10 mL by anhydrous ethanol solution. Finally, the solution was filtered by the 0.22
The sample-adding recoveries for antioxidant 264 in samples are shown in Tables
Recovery of antioxidant 264 from the rubber stopper.
Sample | Recovery (%) | Average recovery (%) | RSD (%) | |||
---|---|---|---|---|---|---|
Antioxidant 264 (1) | 320.24 | 354.79 | 670.27 | 98.51 | 97.82 | 1.56 |
320.40 | 366.11 | 670.27 | 94.93 | |||
320.16 | 360.66 | 670.88 | 96.89 | |||
Antioxidant 264 (2) | 400.30 | 346.54 | 744.07 | 99.31 | ||
400.50 | 355.80 | 744.07 | 96.95 | |||
400.20 | 349.91 | 739.82 | 97.43 | |||
Antioxidant 264 (3) | 480.36 | 364.79 | 838.82 | 98.68 | ||
480.60 | 367.99 | 837.31 | 97.65 | |||
480.24 | 360.01 | 840.34 | 100.02 |
Recovery of antioxidant 264 in recombinant potent antitumor and antivirus protein injection.
Sample | Recovery (%) | Average recovery (%) | RSD (%) | ||
---|---|---|---|---|---|
Antioxidant 264 (1) | 320.24 | 337.11 | 105.27 | 102.19 | 2.23 |
320.40 | 336.81 | 105.12 | |||
320.16 | 337.11 | 105.29 | |||
Antioxidant 264 (2) | 400.30 | 403.32 | 100.75 | ||
400.50 | 402.71 | 100.55 | |||
400.20 | 403.01 | 100.70 | |||
Antioxidant 264 (3) | 480.36 | 483.49 | 100.65 | ||
480.60 | 484.40 | 100.79 | |||
480.24 | 483.19 | 100.61 |
The reference solution (S4) and the sample solutions of Novaferon and the rubber stopper were used for testing the stability of this method, which were placed for 0, 10, and 22 h at room temperature, and the results showed that the RSD was 1.86% for antioxidant 264 in the rubber stopper, and it was not detected in the injection, which indicated that both the test solutions were stable in 22 h.
The method of preparation of the rubber stopper for the test solution is the same as the established detection method, and 20
Migration test was used to detect the migration of antioxidant 264 in the rubber stopper into Novaferon. In this experiment, the migration of antioxidant 264 from the rubber stopper into the product was investigated by the accelerated test (25°C ± 2°C, RH 60% ± 10%) for 0, 1, 2, and 3 months and long-term stability (2°C–8°C) for 0 and 3 months.
According to the established detection method, Novaferon was prepared at each time point, and 20
According to the accelerated test and the long-term stability test, the content of antioxidant 264 was not detected in the six-batch samples of Novaferon after storage for a period of time under certain conditions. From a safety point of view, the potential migrant in the injection could be predicted, and the amount of antioxidant 264 was 192.2 ng/mL on the basis of the limit of detection.
According to the European Chemicals Agency (ECHA) reports of the International Uniform Chemical Information Database (IUCLID), the daily oral intake of antioxidant 264 in humans was 0–0.3 mg/kg/day. The equation of permitted daily exposure (PDE) for humans is as follows [
The median lethal dose (LD50) value is 6000 mg/kg/BW (rat, oral, 24 hr) for 6000 mg/kg/BW antioxidant 264 [
The specification of Novaferon was 20
Brominated butyl rubber stopper can be widely used in primary packaging of various drugs. Due to the compatibility issues that may arose in the process of direct contact with drugs, compatibility studies are necessary to ensure the safety, stability, and efficacy of drugs. In this experiment, the content of antioxidant 264 and its migration in Novaferon were determined by HPLC. The method had high precision, good repeatability, high accuracy, and wide applicability.
This method was used to detect one batch of the rubber stopper and six batches of Novaferon. The results showed that the above used brominated butyl rubber stopper contained antioxidant 264, but its content was within the limit of the European Pharmacopoeia. Antioxidant 264 was not detected in six-batch samples, which were stored under accelerated (0, 1, 2, and 3 months) and long-term conditions (0 and 3 months); meanwhile, the daily intake was lower than the PDE value, indicating that the compatibility between Novaferon and the rubber stopper was good.
The method successfully demonstrated that rubber stoppers used as a drug-packaging material can be assessed during injectable product stability studies, thereby ensuring the safety of the drug.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.