Determination of Nitrosamine Impurities in Propranolol Hydrochloride Tablets by Liquid Chromatography

Keywords: Propranolol, high performance liquid chromatography, nitrosamine impurities

1. Instruments and Reagents​

1.1 Configuration List of HPLC System

1.2 Reagents and Standard Solutions

Table 2 Reagents and Standard Solutions

1.3 Experiment Materials and Auxiliary Equipment

Ultrasonic cleaner

Vortex mixer

2. Experiment Method

2.1 Solution Preparation

2.1.1 2mol/L Sodium Hydroxide Solution: Take 4g of sodium hydroxide, dissolve in 50mL of water, and mix well.

2.1.2 Acetonitrile-Water-Sulfuric Acid (55:45:0.1) Mixture: Take 1.6g of sodium dodecyl sulfate and 0.31g of tetrabutylammonium dihydrogen phosphate, add 1mL of sulfuric acid, 450mL of water, and 550mL of acetonitrile. Adjust to pH 3.3 with 2 mol/L sodium hydroxide solution, and mix well.

2.1.3 Solvent: Same as mobile phase.

2.1.4 Linear Solution: Accurately weigh an appropriate amount of N-Nitroso-propranolol reference standard, dissolve and dilute with mobile phase to obtain a solution containing approximately 200ng per 1mL as the standard stock solution. Then accurately transfer appropriate volumes of the stock solution into six 100mL volumetric flasks, dilute to the mark with mobile phase, and mix well to prepare a standard series of working curves with concentrations of 0.8ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, and 6ng/mL.

2.1.5 Reference Standard Solution: Accurately weigh an appropriate amount of N-Nitroso-propranolol reference standard, dissolve and dilute with mobile phase to prepare a solution containing approximately 2ng of N-Nitroso-propranolol per 1mL as the reference standard solution.

2.2 Sample Preparation

2.2.1 Sample Solution: accurately weigh an appropriate amount of finely powdered propranolol hydrochloride tablets, dissolve and dilute with mobile phase to prepare a solution containing approximately 0.27mg of propranolol per 1mL as the sample solution.

2.2.2 Accuracy Solution: accurately weigh approximately 297mg of finely powdered propranolol hydrochloride tablets into a 100mL volumetric flask. Add an appropriate amount of N-Nitroso-propranolol reference standard solution, dissolve and dilute to the mark with mobile phase to prepare a 100% spiked sample. Mix well to obtain the accuracy solution.

3. Experiment Result

3.1 Specificity

Fig 2 Chromatogram of Specificity

Note: The solvent does not interfere with the detection of N-Nitroso-propranolol, and the chromatographic peaks of other impurities in the test solution also do not interfere with the determination of the N-Nitroso-propranolol impurity.

3.2 Linear Test

Fig 3 Standard Curve of N-Nitroso-propranolol Impurity

Note: The experimental results show that the correlation coefficient (R) of the N-Nitroso-propranolol impurity test curve exceeds 0.999, meeting the requirements of the test.

3.3 Repeatability Test

Fig 4 Chromatograms of Six Replicate Injections for 2ng/mL Standard

Table 3 Data of Six Replicate Injections for 2ng/mL Standard

Note: As shown in the table above, the retention time repeatability of N-nitroso-propranolol is 0.043%, and the peak area repeatability is 0.464%, indicating good repeatability.

3.4 Accuracy Test

Fig 5 Overlaid Accuracy Chromatograms of N-Nitroso-propranolol in Tablets

Table 4 Accuracy of Propranolol Hydrochloride Tablets (N-Nitroso-propranolol)

Note: The recovery of N-Nitroso-propranolol in six samples ranged from 96.98% to 104.71%, with an average recovery of 101.38%. The method shows good accuracy and repeatability.

3.5 LOD Test

Fig 6 Test Chromatogram of N-Nitroso-propranolol at 2ng/mL Standard Concentration

Table 5 Reference Test Data of N-Nitroso-propranolol at 2ng/mL Standard Concentration

Note: Based on the test data for the N-nitroso-propranolol reference standard at a concentration of 2ng/mL in the table above, the theoretical detection limit for N-nitroso-propranolol is calculated as 0.151ng/mL using a 3:1 signal-to-noise ratio as the detection limit criterion.

3.6 A Brand Tablet Testing

Fig 7 Test Chromatogram of a Brand Tablets

Note: N-Nitroso-propranolol was not detected in the brand tablets.

4. Conclusion

A study on the nitrosamine impurity N-nitroso-propranolol in propranolol hydrochloride tablets was conducted in accordance with the FDA's recently published guidance document on establishing acceptable intake levels for Nitrosamine Drug Substance-Related Impurities (NDSRI). According to this document, the AI (Acceptable Intake) value is calculated as 1500ng/day. Considering the maximum daily dose of this product (200mg), the control limit in tablets shall not exceed 7.5ppm. The method validation was performed by using Wayeal’s high-performance liquid chromatography (HPLC) series equipped with a UV detector. Tablet samples were analyzed using the validated detection method, and no nitrosamine impurity N-nitroso-propranolol was detected. All the above data comply with the pharmacopoeial requirements for instrumentation.