Identification and sensitive quantitation of N-nitroso N-desmethyl orphenadrine impurity in orphenadrine citrate API


Lakshmanan D1, Sashank Pillai1, Rahul Baghla2 and Eshani Galermo2
1SCIEX, India and 2SCIEX, USA

Abstract


This technical note demonstrates a sensitive method for the identification (Figure 1) and quantitation of N-nitroso N-desmethyl orphenadrine (NNDO) impurity in orphenadrine citrate (OC) API using the QTRAP version of the SCIEX 7500 system. A limit of quantitation (LOQ) of 2.5 pg/mL was achieved with baseline separation of NNDO and OC (Figure 2).

OC is an anticholinergic drug of the etholamine antihistamine class, which can be chemically synthesized.1  OC has a secondary amine in the structure, which is more reactive in the formation of nitrosamines than the tertiary amines.2 The regulation has amended the maximum permissible daily intake limit for NNDO as 18 ng/day3 which applies to a maximum daily dose of 200 mg/day.4 Given that OC has a maximum daily dose of 200 mg/day, a nitrosamine limit of 90 pg/mg is required to be quantified in the API.

This technical note presents a reliable and sensitive workflow for the identification and sensitive quantitation of NNDO in OC API below the calculated specification limit using the QTRAP 7500 system.

Figure 1: Detect and verify nitrosamine impurity using the MRM > EPI approach on the QTRAP 7500 system. Representative extracted ion chromatograms (XICs) of the control sample and the spiked control sample (left) and the EPI (MS/MS) spectra are displayed (right). Here, verification of the detected impurity peak in the OC API sample was performed using EPI (MS/MS) spectra with library matching against the standard NNDO on SCIEX OS software.

Key benefits for analysis of NNDO using the QTRAP 7500 system
 

  • Low pg/mL level of quantitation: Achieve 2.5 pg/mL LOQ for the quantitation of NNDO

  • Identification of unknown impurity in API: Identify unknown impurity using reliable full scan MS/MS data acquisition with IDA driven MRM > EPI (enhanced product ion) scan

  • Baseline chromatographic separation: Achieve accurate quantitation with baseline separation of NNDO and OC API

  • Robust analytical performance: Achieve accurate quantitative performance with %CV <9 at all concentration levels across a linear dynamic range (LDR) of 3.6 orders of magnitude

  • Streamlined data management: SCIEX OS software, a 21 CFR Part 11-compliant platform, simplifies data acquisition and processing

Introduction


Nitrosamines are highly potent carcinogens classified into various drug categories (class 1-5). Categorization was performed by the Carcinogenic Potency Categorization Approach (CPCA), where the severity was determined based on the acceptable intake, activating or deactivating features defined structurally. NNDO can be formed in OC because of the presence of the secondary amine, placing it under a class 1 category as per the CPCA approach. 6

Since NDSRIs have become a more common risk in therapeutics, the EU has set a regulation limit of 18 ng/day for NNDO.3 Considering the daily dosage and the regulation limit, NNDO should be analyzed below the 90 pg/mg limit.

Methods


Standard preparation: Calibration curve dilutions of NNDO were prepared across a range of concentrations using a 50:50 (v/v) acetonitrile/water diluent (2.5, 5, 10, 50, 100, 500, 1000, 5000 and 10000 pg/mL).

Sample preparation: A 2 mg of OC API was weighed into a suitable vessel. A 20 mL aliquot of diluent (50:50 (v/v) acetonitrile/water) was added and vortexed thoroughly to yield a 0.1 mg/mL concentration. A 100 pg/mL solution of NNDO was spiked in 0.1 mg/mL of OC API solution to achieve a final concentration of 5 pg/mL.

Chromatography: Analytical separation was performed on the ExionLC system using a Phenomenex Kinetex C8 (2.1 × 100 mm, 2.6 μm) column at a flow rate of 0.5 mL/min. Mobile phase A was 5mM ammonium formate with 0.1% (v/v) formic acid in water and mobile phase B was acetonitrile. The column temperature was set to 40°C. The gradient conditions used are summarized in Table 1. A 3 μL aliquot of the sample was injected for LC-MS/MS analysis.

The LC flow was diverted to waste for the first 2 min to prevent OC API from entering the mass spectrometer and after 5 min during column wash.

Table 1: LC gradient conditions.

Mass spectrometry: The optimized source and gas parameters are listed in Table 2. MRM parameters and MRM > EPI parameters are included in Tables 3 and 4, respectively.

Table 2: Source and gas parameters.

Table 3: MRM parameters used for quantitation.

Table 4: MRM > EPI parameters used for identification.

Data processing: Data collection and analysis were performed using SCIEX OS software, version 3.3.1. Peaks were integrated using the MQ4 algorithm and a weighting of 1/x was used for NNDO quantitation.

Quantitative performance on the QTRAP 7500 system


Baseline chromatographic separation was achieved between OC and the NNDO. The NNDO retained at the column much longer with a retention time of 3.8 min, while the OC API eluted at a retention time of 1.9 min (Figure 2).

Figure 2: Good chromatographic separation was achieved between NNDO and OC API. Extracted ion chromatogram (XIC) of NNDO (top) and the UV chromatogram (235 nm) of OC (bottom) are shown.

NNDO was analyzed across the concentration range of 2.5 to 10000 pg/mL. To evaluate reproducibility, each calibration standard was analyzed in triplicate.

Linearity was achieved across concentrations ranging from 2.5 to 10000 pg/mL with a correlation of determination (r2) of >0.999 for both quantifier and qualifier ions (Figure 3). An LDR of 3.6 orders of magnitude was achieved.

Figure 3: Calibration curve for quantitation of NNDO quantifier ion (285→181.09) and qualifier ion (285→166.11). A weighing factor of 1/x was applied for both calibration curves.

No interference in the diluent blank was observed in the calibration curve samples (Figure 4).

Figure 4: Representative XICs of the diluent (left) and LOQ, 2.5 pg/mL (right).

The specification limit (90 pg/mg) was calculated based on the maximum daily dose of 200 mg/day. The NNDO was analyzed at 5 pg/0.1 mg of API, which is below the calculated specification limit of 90 pg/mg.

Recovery was calculated against the neat solution, where the peak area from NNDO in the control OC API solution was subtracted from the peak area from spiked NNDO in OC. The average recovery was 90% with a %CV of 3.8, evaluated in triplicate (Table 5).

Table 5: Recovery and precision calculation.

Analytical performance was evaluated based on the criteria that the accuracy of the calculated mean should be between 80% and 120% at the LOQ and between 85% and 115% at the higher concentrations. In addition, the %CV of the calculated mean of the concentration should be <20% at the LOQ and <15% at all higher concentrations.

The assay accuracy was within ±9% of the actual concentration and the %CV was <9. Calculated percent accuracy and %CV values were within the acceptance criteria at each concentration level (Figure 5).

Figure 5: Quantitative performance for NNDO quantifier ion (285→181.09) and qualifier ion (285→166.11). Reproducibility and accuracy results were determined from the calibration curve standards across 3 replicates at each concentration. Statistical results were summarized using the Analytics module on SCIEX OS software.

Identification of impurity in OC API


In the OC API sample, a peak was observed at the retention time of NNDO, around 3.8 min (Figure 1).

The unknown impurity was identified by comparing its MS/MS spectra with the standard NNDO standard. Identification was performed using full scan MS/MS experiments with library searching. Data acquisition was performed using the linear ion trap feature of the QTRAP 7500 system using an MRM to EPI experiment. In this case, the selected MRM transitions for NNDO were used to create an EPI survey scan in an IDA experiment setting. Here, the MS/MS spectra from the unknown impurity was compared to standard NNDO sample spectra. MS/MS spectra matching identified the impurity as NNDO (Figure 1), but it was below the calculated specification limit of 90 pg/mg.

Compliance-ready SCIEX OS software


Equivalent SCIEX OS software capabilities for regulated bioanalysis can be executed on the QTRAP 7500 system, ensuring high fidelity when performing method transfers while retaining critical compliance features. 

SCIEX OS software is a closed system and requires records and signatures to be stored electronically, meeting the regulations outlined by 21 CFR Part 11. SCIEX OS software can open raw data files from any visible storage location within a closed network by using designated processing workstations. Figure 8 illustrates the features of SCIEX OS software that are used to monitor the audit trail, acquire and process data, and configure user access. The audit trail feature enables users to audit critical user actions and locks in data integrity. The Central Administrator Console (CAC) feature allows users to centralize acquisition and processing using a single platform to maximize efficiency for multiinstrument laboratories, independent of compliance standards. The configuration module allows users to assign roles and access as the administrator, method developer, analyst, and reviewer.

Figure 8: Features of SCIEX OS software for monitoring user access and evaluating the audit trail. The audit trail view allows users to filter for high-risk events easily and enables data integrity features to meet compliance requirements. The software features a Central Administrator Console (CAC) to manage users and groups, role definitions, workstations and projects across all systems. The CAC feature supports both regulated and non-regulated compliance standards. The configuration module enables users to quickly set up roles and levels of access for the administrator, method developer, analyst and reviewer levels.

Conclusion
 

  • An LOQ of 2.5 pg/mL was achieved for NNDO in OC API 

  • Linearity was achieved at concentrations ranging from 2.5 pg/mL to 10000 pg/mL with an r2 >0.999 for both quantifier and qualifier ions covering LDR of 3.6 orders of magnitude

  • Impurity in OC API was identified as NNDO by comparing the impurity MS/MS spectra with the NNDO standard MS/MS spectra

  • Good quantitative performance was demonstrated with accurate and highly reproducible (%CV <9) results on the QTRAP 7500 system

  • The method demonstrated the quantitation of NNDO impurity below the calculated specification limit (90 pg/mg) in the OC API

  • A single platform for streamlined data acquisition, processing, and management with SCIEX OS software was presented

  • Retain data management and compliance-readiness (21 CFR Part 11) features using SCIEX OS software to support nitrosamine analysis on the QTRAP 7500 system

References
 

  1. Stability-indicating assay for orphenadrine hydrochloride by reversed-phase high-performance liquid chromatography. Journal of Chromatography, 1984, 431-444.

  2. The Nitrosamine “Saga”: Lessons Learned from Five Years of Scrutiny. Org. Process Res. Dev. 2023, 27, 10, 1719– 1735.

  3. Nitrosamine impurities in medications: Established acceptable intake limits. Appendix 1: Established acceptable intake (AI) limits for N-nitrosamine impurities (version: 2024-05-31).

  4. Chapter 67 – analgesic agents in rheumatic disease: kelley and firestein’s textbook of rheumatology (tenth edition) Elsevier, 2017, Pages 1075-1095

  5. Prevalence of nitrosamine contaminants in drug samples: Has the crisis been overcome?Arch Pharm, 2022, 1-14.

  6. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; “ICH Harmonised Guideline - Assessment And Control Of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, M7(R1)”; March 31, 2017.
Request information