Penicillamine is an important detoxifying agent, commonly used for the treatment of heavy metal poisoning, rheumatoid arthritis, and other diseases. However, the thiol group (–SH) in the penicillamine molecule is chemically reactive and is easily oxidised during production and storage to form penicillamine disulfide. The presence of penicillamine disulfide affects the content and efficacy of the active ingredient and is also an important indicator of drug stability. Therefore, the core requirement of the pharmacopoeial method for penicillamine testing is that it must be able to completely separate penicillamine from its disulfide and perform accurate quantification.
1. Main changes
Method upgrade: From the TLC or HPLC UV method in the 2020 edition to an HPLC ELSD method (gradient elution);
Technical advantages: Improved specificity and sensitivity, enabling effective control of penicillamine disulfide and various other impurities;
Problems solved: Overcomes the shortcomings of the old method and of foreign pharmacopoeias regarding insufficient impurity control.
2. Standard requirements
Resolution requirement: The resolution between penicillamine and penicillamine disulfide shall be not less than 3.0.
Content standard: Calculated on the dried basis, the content of C₅H₁₁NO₂S shall be not less than 95.0%.
1. EClassical 3200L HPLC system equipped with an ELSD detector
2. Chromatographic conditions (referring to the 2025 pharmacopoeia requirements)
| Parameter | Condition |
|---|---|
| Column | SinoPak BEH T-C18 (4.6×250 mm, 5 µm) |
| Mobile phase | A: trifluoroacetic acid aqueous solution (pH=1.6); B: acetonitrile |
| Gradient | 0–17 min, 96% A; 30 min, 70% A; 31–45 min, 96% A |
| Flow rate | 0.6 mL/min |
| Column temperature | 30 °C |
| Injection volume | 20 µL |
| ELSD | Evaporation temperature 75 °C, nebulisation temperature 55 °C |
3. System suitability verification results
Separation of penicillamine and penicillamine disulfide
Figure 1 shows the HPLC separation chromatogram of penicillamine and penicillamine disulfide in a penicillamine sample (test solution) under the above conditions. The resolution between penicillamine and penicillamine disulfide reached 9.1, far higher than the pharmacopoeia requirement of 3.0.
Figure 1. Chromatogram of penicillamine and penicillamine disulfide in the test solution (Peaks: 1 – penicillamine; 2 – penicillamine disulfide)
Linearity of the calibration curves for penicillamine and penicillamine disulfide
Series of standard solutions of penicillamine and penicillamine disulfide at different concentrations were prepared. The linear regression equation for the penicillamine calibration curve was log y = 2.8829 log x + 4.9461, with a correlation coefficient r = 0.996. For penicillamine disulfide, the equation was log y = 1.3030 log x + 3.5033, with r = 0.997. Both meet the pharmacopoeia requirement of a correlation coefficient not less than 0.99.
Tailing factors of the penicillamine and penicillamine disulfide peaks
The tailing factors of the penicillamine and penicillamine disulfide peaks were calculated using the chromatographic data system. The tailing factor of the penicillamine peak was 1.505, and that of the penicillamine disulfide peak was 1.597, both meeting the pharmacopoeia requirement that the tailing factor should be between 0.8 and 1.8.
4. Results of actual sample determination
The contents of penicillamine and penicillamine disulfide in tablet samples were determined by the external standard method. The results are shown below.
| Sample | Penicillamine content (mg/g) | Penicillamine disulfide content (mg/g) |
|---|---|---|
| 1# | 465 | 2.109 |
| 2# | – | 1.998 |
| 3# | – | 3.192 |
| 4# | – | 2.871 |
| 5# | – | 3.355 |
| 6# | 440 | 2.464 |
5. Conclusion
The EClassical 3200L system equipped with the D3270L evaporative light scattering detector (ELSD) provided by Elite Technology fully meets the new requirements of the 2025 edition of the Chinese Pharmacopoeia for the determination of penicillamine related substances. The method offers:
Excellent resolution: The resolution between penicillamine and penicillamine disulfide reaches 9.1, far exceeding the pharmacopoeia requirement;
Good linearity: The correlation coefficients of the calibration curves for both compounds are greater than 0.996, meeting pharmacopoeia requirements;
Accurate quantitation: Capable of reliably determining the content of penicillamine and penicillamine disulfide in pharmaceutical samples;
Powerful impurity monitoring capability: Provides a reliable technical guarantee for the quality control of penicillamine drug products.
This solution offers pharmaceutical companies complete and reliable technical support for adapting to the pharmacopoeia standard changes, ensuring the quality control and medication safety of penicillamine products.
