|
No.
|
Reagents and Standards
|
Purity
|
|
1
|
Acetonitrile
|
HPLC Grade
|
|
2
|
Trifluoroacetic acid
|
AR
|
|
3
|
Sodium dodecyl sulfate
|
AR
|
|
4
|
N-Nitroso-Desloratadine
|
98.9%
|
1.3 Experiment Materials and Auxiliary Equipments
Ultrasonic cleaner
Vortex mixer
2. Experiment Method
2.1 Solution Preparation
2.1.1 Sodium Dodecyl Sulfate (SDS) Aqueous Solution: Weigh 0.865g of sodium dodecyl sulfate and add 0.5mL of trifluoroacetic acid. Dilute to 1000mL with water and mix thoroughly until complete dissolution.
2.1.2 Solvent: Same as mobile phase (Precisely measure 570mL of sodium dodecyl sulfate aqueous solution and 430mL of acetonitrile, mix well, and ready for use).
2.1.3 Linear Solution: Accurately weigh an appropriate amount of N-Nitroso-Desloratadine reference standard, and dissolve and dilute with mobile phase to obtain a solution containing approximately 0.8μg/mL as the standard stock solution. Then, precisely pipette 1.0mL, 1.5mL, 2.0mL, 4.0mL, and 10.0mL of the standard stock solution into five separate 100mL volumetric flasks. Dilute to volume with mobile phase and mix well to prepare standard series working curves with concentrations of 8ng/mL, 12ng/mL, 16ng/mL, 32ng/mL, and 80ng/mL.
2.1.4 Reference Standard Solution: Accurately weigh an appropriate amount of N-Nitroso-Desloratadine reference standard, dissolve and dilute with mobile phase to prepare a solution containing approximately 16ng/mL of N-Nitroso-Desloratadine, as the reference standard solution.
2.2 Sample Preparation
2.2.1 Sample Solution
Precisely pipette an appropriate amount of desloratadine API, dissolve and dilute with mobile phase to prepare a solution containing approximately 0.2mg/mL of desloratadine, as the sample solution.
2.2.2 Accuracy Solution
Weigh approximately 20mg of desloratadine API into a 100mL volumetric flask. Add an appropriate volume of N-Nitroso-Desloratadine reference standard solution, dissolve and dilute to volume with mobile phase to prepare a 100% spiked sample. Mix well to obtain the accuracy solution.
3. Experiment Result
3.1 Specificity
Fig 2 Specificity Test Chromatogram
Note: The solvent does not interfere with the detection of N-Nitroso-Desloratadine, and other impurity peaks in the test solution also do not interfere with the determination of the N-Nitroso-Desloratadine impurity.
3.2 Linear Test
Fig 3 Overlap Chromatogram for N-Nitroso-Desloratadine Impurity Test
Fig 4 Standard Curve for N-Nitroso-Desloratadine Impurities
Note: The experimental results demonstrate that the linear correlation coefficient (R) for the N-Nitroso-Desloratadine impurity test exceeds 0.999, meeting the test requirements.
3.3 Repeatability Test
Fig 5 Repeatability Chromatograms of 16 ng/mL Standard (6 Injections)
Table 3 Repeatability Test Data for 16 ng/mL Standard (6 Injections)
|
Compound
|
Retention Time (min)
|
|
N-Nitroso-Desloratadine
|
15.426
|
|
15.432
|
|
15.414
|
|
15.416
|
|
15.432
|
|
15.417
|
|
Average
|
15.421
|
|
RSD (%)
|
0.045
|
Note: Based on the data from the table above, the retention time repeatability for N-Nitroso-Desloratadine is 0.045%, and the peak area repeatability is 2.043%, indicating good repeatability.
3.4 Accuracy Test
Fig 6 Accuracy Overlap Chromatogram for Desloratadine API
Table 4 Accuracy of Desloratadine API (N-Nitroso-Desloratadine)
|
Level
|
Known value (ng)
|
Spiked (ng)
|
Measured value (ng)
|
Recovery (%)
|
Average (%)
|
|
100%
|
0.00
|
1611.52
|
1656.365
|
99.690
|
100.11
|
|
100%
|
0.00
|
1610.546
|
96.932
|
|
100%
|
0.00
|
1686.721
|
101.517
|
|
100%
|
0.00
|
1704.476
|
102.585
|
|
100%
|
0.00
|
1615.701
|
97.242
|
|
100%
|
0.00
|
1706.194
|
102.689
|
Note: The recovery rate of N-nitroso-desloratadine in six samples ranged from 97.24% to 102.69%, with an RSD of 2.58%. These results demonstrate that the method achieves excellent accuracy and repeatability for the detection of this impurity.
3.5 Limit of Detection (LOD) Test
Fig 7 Chromatogram of N-Nitroso-Desloratadine Reference Standard at 8ng/mL
Table 5 Test Data for N-Nitroso-Desloratadine Reference Standard at 8ng/mL
|
Compound
|
Retention Time (min)
|
Peak Area (mAU*s)
|
SNR
|
|
N-Nitroso-Desloratadine
|
15.434
|
1.213
|
30.042
|
Note: Based on the test data for the N-Nitroso-Desloratadine reference standard at 8ng/mL in the table above, the theoretical limit of detection (LOD) calculated using the 3-fold signal-to-noise ratio is 0.7989ng/mL.
3.6 Raw Material Testing for a Specific Manufacturer
Fig 8 Chromatogram of API Testing for a Specific Manufacturer
Note: No N-Nitroso-Desloratadine Detected in API.
4. Conclusion
A study on the nitrosamine impurity N-Nitroso-Desloratadine in Desloratadine Active Pharmaceutical Ingredient (API) was conducted based on the recently issued FDA guidance document regarding the establishment of acceptable intake limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs). According to the document, the Acceptable Intake (AI) value is set at 400ng/day. Combined with the maximum daily dose of the product (5mg), the control limit in the API must not exceed 80ppm. The methodological validation was conducted using Wayeal's High Performance Liquid Chromatography (HPLC) system LC3400 series, equipped with an ultraviolet (UV) detector. The validated analytical method was applied to test the active pharmaceutical ingredient samples, and no nitrosamine impurity (N-nitroso-desloratadine) was detected. All of the above data meet the requirements of the pharmacopoeia method for instruments.
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