FU Yan, WANG Ying-jian
(Kunming Maternal and Child Health Care Hospital, Kunming 650031, China)
Abstract: Objective To establish a bacterial endotoxin test method for Magnesium Sulfate Injection. Methods Tachypleus Amebocyte Lysate (TAL) sensitivity re-verification, sample interference tests, and sample measurements were conducted in accordance with the regulations in the Appendix of the Chinese Pharmacopoeia (2000 Edition, Volume II). Results For samples with a 25% concentration, bacterial endotoxins can be detected using TAL reagent with a labeled sensitivity of 0.25 EU⋅mL−1 after a 2-fold dilution. The experimental results were consistent with clinical applications. Conclusion The TAL method for detecting bacterial endotoxins in Magnesium Sulfate Injection is reliable, practical, and feasible. It can be used as a replacement for the rabbit pyrogen test.
Keywords: Magnesium Sulfate Injection; Tachypleus Amebocyte Lysate (TAL); Bacterial Endotoxin
Magnesium Sulfate Injection is an anticonvulsant. In addition to its clinical use in obstetrics for the prevention and treatment of eclampsia, it is also used for arrhythmia, bronchial asthma, cerebral infarction, and uterine relaxation. It is typically administered via intravenous infusion or injection. This study utilizes the TAL method to detect bacterial endotoxins to replace the rabbit pyrogen test.
1. Reagents and Equipment
- TAL Reagent: 0.1 mL /ampoule, labeled sensitivity λ=0.25 EU⋅mL−1 . Batch numbers: 020810 (Xiamen TAL Plant); 030423 (Zhanjiang Ocean Biological Products Plant); 030615 (Zhanjiang Andes Biological Co., Ltd.).
- Bacterial Endotoxin Test Water (BET Water): 2 mL/ampoule, Batch No.: 020917, Xiamen TAL Plant.
- Bacterial Endotoxin Working Standard: 50 EU/ampoule, Batch No.: 2002-9, National Institute for the Control of Pharmaceutical and Biological Products.
- Magnesium Sulfate Injection: Batch No.: 010417 (Changzhou No. 6 Pharmaceutical Plant); 020401 (Hangzhou Minsheng Pharmaceutical Group Co., Ltd.); 030927 (Tianjin Kingyork Amino Acid Co., Ltd.). (Clinical application of these three batches showed no pyrogenic reactions).
- Equipment: 100-level laminar flow clean bench (Beijing Haidong Air Purification Equipment Co., Ltd.). Glassware was baked at 250°C for 1 hour to remove pyrogens.
2. Methods and Results
2.1 Determination of Theoretical Endotoxin Limit and Maximum Valid Dilution (MVD)
The rabbit pyrogen threshold (K) for injections is 5 EU⋅kg−1. According to the quality standards for Magnesium Sulfate Injection, the rabbit dose for pyrogen testing (diluted to 0.10 g⋅mL−1) is 1 mL⋅kg−1. Therefore, for Magnesium Sulfate Injection with a concentration of 0.25 g⋅mL−1, the equivalent dose (M) is 0.4 mL⋅kg−1. The theoretical endotoxin limit is: L=K/M=5 EU⋅kg−1/0.4 mL⋅kg−1=12.5 EU⋅mL−1. The Maximum Valid Dilution is: MVD=L/λ=12.5 EU⋅mL−1/0.25 EU⋅mL−1=50 folds.
2.2 TAL Sensitivity Re-verification
The bacterial endotoxin working standard was diluted with BET water into a series of concentrations: 0.5, 0.25, 0.125, and 0.063 EU⋅mL−1. Sensitivity re-verification was performed according to the ChP 2000 Edition Volume II Appendix XI E. Results are shown in Table 1.
Tab 1: The results of sensitivity test for TAL
| TAL Batch | Labeled Sensitivity (EU⋅mL−1) | Re-verified Result ( EU⋅mL−1) | Conclusion |
| 020810 | 0.25 | 0.25 | Compliant |
| 030421 | 0.25 | 0.25 | Compliant |
| 030605 | 0.25 | 0.23 | Compliant |
2.3 Preliminary Sample Interference Test
Sample batch 030927 was diluted 2, 4, 10, 20, and 50 folds with BET water. These dilutions were then used to dilute the endotoxin standard to concentrations of 0.5, 0.25, 0.125, and 0.063 EU⋅mL−1
. Interference tests were performed using TAL from three different manufacturers. Results showed that after a 2-fold dilution, the sample did not interfere with the TAL gelation reaction. See Table 2.
Tab 2: The result of inhibition or enhancement test for magnesium sulfate injection
This is the English translation and digital recreation of the provided image (Table 2).
Tab 2: The result of inhibition or enhancement test for magnesium sulfate injection
| TAL Batch | Dilution Factor | Bacterial Endotoxin Concentration (EU·mL⁻¹) | Negative Control | Sample Negative Control | EsE_sEs | EtE_tEt |
| 0.5 | 0.25 | 0.125 | 0.063 | |||
| 020810 | BET Water | + + + + | + + + + | – – – – | – – – – | – – – – |
| 2 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 4 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 10 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 20 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 50 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 030421 | BET Water | + + + + | + + + + | – – – – | – – – – | – – – – |
| 2 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 4 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 10 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 20 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 50 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 030605 | BET Water | + + + + | + + + + | – – – – | – – – – | – – – – |
| 2 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 4 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 10 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 20 | + + + + | + + + + | – – – – | – – – – | – – – – | |
| 50 | + + + + | + + + + | – – – – | – – – – | – – – – |
Terminology Key:
- BET Water: Bacterial Endotoxin Test Water (used as a control).
- +: Indicates a positive gelation reaction (gel formed).
- -: Indicates a negative reaction (no gel formed).
- Replicates: The four symbols (e.g., + + + +) represent the results of the four parallel test tubes for each concentration.
-
EsE_sEs: Labeled sensitivity of the TAL reagent in BET water. -
EtE_tEt: Measured sensitivity of the TAL reagent in the presence of the test sample (Magnesium Sulfate).
2.4 Main Sample Interference Test
Three batches of samples were diluted 2-fold with BET water. The endotoxin standard was diluted with BET water and these sample dilutions to concentrations of 0.5, 0.25, 0.125, and 0.063 EU⋅mL−1. See Table 3.
Tab 3: The result of inhibition or enhancement test for magnesium sulfate injection
Tab 3: The result of inhibition or enhancement test for magnesium sulfate injection
| TAL Batch | Sample Batch | Dilution | 0.5 (EU/mL) | 0.25 (EU/mL) | 0.125 (EU/mL) | 0.063 (EU/mL) | Negative Control | Sample Negative Control | Es | Et |
| 020810 | — | BET Water | ++++ | ++++ | —- | —- | — | — | 0.25 | |
| 010417 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 020401 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 030927 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 030421 | — | BET Water | ++++ | ++++ | —- | —- | — | — | 0.25 | |
| 010417 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 020401 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 030927 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 030605 | — | BET Water | ++++ | ++++ | —- | —- | — | — | 0.25 | |
| 010417 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 020401 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 | ||
| 030927 | 2 | ++++ | ++++ | —- | —- | — | — | 0.25 |
Key:
- BET Water: Bacterial Endotoxin Test Water.
- + / -: “+” indicates positive gelation; “-” indicates negative reaction. Four symbols represent results of four parallel tubes.
- Es: Measured sensitivity of TAL reagent in water.
- Et: Measured sensitivity of TAL reagent in the sample solution.
2.5 Sample Bacterial Endotoxin Measurement
Three batches of samples were diluted 2-fold with BET water and measured according to regulations. Results are in Table 4.
Tab 4: The result of bacterial endotoxin test for magnesium sulfate injection
| Sample Batch | TAL Batch | Sample Tube | Sample Positive Control | Negative Control | Positive Control |
| 010417 | 020810 | – | + | – | + |
| 030421 | – | + | – | + | |
| 030605 | – | + | – | + | |
| 020401 | 020810 | – | + | – | + |
| 030421 | – | + | – | + | |
| 030605 | – | + | – | + | |
| 030927 | 020810 | – | + | – | + |
| 030421 | – | + | – | + | |
| 030605 | – | + | – | + |
Key:
- – : Negative reaction (no gelation).
- + : Positive reaction (gelation).
- Sample Tube: The test solution (2-fold dilution of the sample).
- Sample Positive Control: Test solution with added standard endotoxin (2λ).
- Negative Control: BET Water.
- Positive Control: BET Water with added standard endotoxin (2λ).
3. Discussion
3.1 In this experiment, the samples were subjected to serial dilutions and tested with TAL reagents from three different manufacturers to find the non-interfering concentration for each batch, making the experimental results more reliable and accurate.
3.2 As seen in Table 2, Magnesium Sulfate Injection, when diluted 2-fold and tested with TAL reagent of sensitivity 0.25 EU⋅mL−1, shows no interference. The detection results are consistent with clinical use. This method is simple, fast, accurate, practical, and feasible.
References
- XIA Zhen-min. Limit for bacterial endotoxin test of drugs [J]. Chinese Journal of Pharmaceutical Analysis, 1995, 15(3): 54.
- Chinese Pharmacopoeia 2000 Edition, Vol II [S]. 2000: 901; Appendix XIX F; Appendix XI E.
- ZHENG Yong-chu, BAI Wen-ting. Magnesium sulfate in the treatment of bronchial asthma [J]. Journal of Practical Internal Medicine, 1983, 3(2): 72.
- MA Li-guo. Application of magnesium sulfate as a uterine relaxant [J]. Foreign Medical Sciences, Section of Obstetrics and Gynecology, 1991, 18(3): 135.