(T1430-08-50) Analytical Case Studies for Processed-Induced and API-Related Nitrosamines in Finished Drug Products

Purpose: In 2020, both the FDA and EMA issued guidance for control of nitrosamine impurities in human drugs. The guidance offers general strategy for risk assessments and analytical testing of nitrosamines in finished drug products, APIs, and raw materials. USP 1469 was also released and offers a similar review of the information given by the FDA/EMA and provided general guidance for the analysis of nitrosamine impurities in these drugs using LC-HRMS, LC/MS-MS or GC-MS/MS. The case studies presented a comprehensive screening strategy for up to 7 nitrosamines and method validation for quantitation of these nitrosamine impurities in finished drug products using LC-MS/MS. The robustness of the presented approach is demonstrated by method feasibility results for the screening method and validation results for quantitation of API-related nitrosamines.

Methods: A. Screening of seven FDA suggested nitrosamines in Duloxetine Capsules using LC-MS/MS:
1. Mobile Phase and Standard Solutions Preparation:
Mobile Phase A:0.1% FA in H₂O
Mobile Phase B: MeOH
7 Nitrosamines Standard Mixture (1000 ng/mL)
7 Nitrosamines Isotope Internal Standard Mixture (1000 ng/mL)
2. Sample Solutions Preparation (40 mg/mL):
Transfer all the ground content of one capsule (120 mg) into a 5-mL centrifuge tube, add 0.3mL of isotope internal standard mixture, and let it settle for 5 mins. Add 2.7 mL of diluent (0.1%formic acid in water) and mix well. Add 3 mL of hexane and shake the tube at least 50 times. Centrifuge the solution at 13000 rpm for 10 mins at 5 °C. Transfer the upper layer for LCMS analysis.
3. LC/MS-MS conditions:
AB Sciex 6500 Triple Quad Mass Spectrometer with Shimadzu LC-30AD system
Analytical Column: Agilent ZORBAX Eclipse Plus C8, 100 × 4.6 mm,
Gradient method with flow rate at 0.8 mL/min
Run time: 15 minutes
Injection volume: 50 µL
Scan Mode with Ionization: MRM with APCI Positive
MRM transition (m/z):
NDMA: 75.1 / 43.1; NDMA-D₆: 81.1 / 46.110: 113.1 / 81.1
EIPNA: 117.0 / 75.0; EIPNA-D₅ :122.0 / 80.0
DIPNA: 131.0 / 89.0; DIPNA-D₁₄ :145.0 / 97.0
NDBA: 159.1 / 57.2; NDBA-D₁₈ :177.1 / 66.1
NMBA: 147.1 / 117.1; NMBA-¹³C₄ :151.1 / 121.1
NMPA: 137.0 / 66.0; > NMPA-D₅ :142.0 / 71.1
B. Quantitation of N-Nitroso-Bumetanide (NNB) in Bumetanide Injection, USP using LC-MS/MS:
1. Mobile Phase and Standard Solutions Preparation:
Mobile Phase A: 0.1% FA in H₂O
Mobile Phase B: 0.1% FA in MeOH
Working/Check Standard Solution (0.528 ng/mL of NNB, 2.64 ppm equivalence)
>2. Sample Solutions Preparation (0.2 mg Bumetanide/mL):
Pipette 1000 µL of Bumetanide Injection (0.25 mg/mL), USP into a 1.5-mL polypropylene centrifuge tube and dilute with 250 µL of diluent (1:1 Water: Methanol) and mix well.
3. LC/MS-MS conditions:
AB Sciex 6500 Triple Quad Mass Spectrometer with Shimadzu LC-30AD system
Analytical Column: Agilent ZORBAX Eclipse Plus C18, 100 × 4.6 mm, 3.5 µm
Gradient method with flow rate at 0.8mL/min
Run time: 15 minutes
Injection volume: 50 µL
Scan Mode with Ionization: MRM with Electrospray Positive
N-Nitroso-Bumetanide MRM transition (m/z): 394 to 364

Results: Duloxetine Capsules: The results of system suitability, LOQ (22 ppb, S/N >3), accuracy (%Recovery: 99% ~ 107%) and precision (%RSD: 2% ~ 7%) at the specification level demonstrated that the method is suitable for its intended purposes for the screening of all seven recommended Nitrosamines in Duloxetine Capsules (Figure 1). Bumetanide Injection, USP: This method (Figure 2) has acceptable system suitability, specificity, linearity (Correlation coefficient > 0.99, range from 0.264 ppm to 0.528 ppm), LOD (0.132 ppm, S/N >3), LOQ (0.264 ppm, S/N >10), accuracy and precision (Figure 3), intermediate precision (%RSD:1~3%), solution stability (65 hours of stability at 2 – 8 °C), robustness, and range. It is validated for the quantitation of NNB in Bumetanide Injection, USP.

Conclusion: LC-MS/MS is a powerful tool for the detection and quantitation of nitrosamine impurities in pharmaceutical products, including finished drugs, APIs, and raw materials. The technique's high selectivity and sensitivity enable the simultaneous screening of multiple nitrosamines at very low levels, making it a direct and effective method for ensuring pharmaceutical product specificity. It is capable of identifying and quantifying a wide range of potentially harmful nitrosamine impurities in pharmaceutical products with high accuracy and precision. Its ability to detect these carcinogenic compounds at such low levels allows for early identification of potential issues and enables manufacturers to take preventive action before the products reach the market.

Figure 1. Case Study - 7 Nitrosamines Rapid Screening

Figure 2. Chemical Structures and Representative Chromatograms

Figure 3. Accuracy and Precision Results