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Sujata Rajan1 , Sashank Pillai1 , Rahul Baghla2 and Eshani Galermo 2
1SCIEX, India ; 2SCIEX, USA
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Abstract
Abstract
Key features
Key-features
Introduction
Introduction
Methods
Methods
Results and discussion
Results
Conclusion
Conclusion
References
References
Abstract

Abstract

This technical note demonstrates a sensitive method for the identification and quantitation of N-nitroso sertraline (NNSL) impurity in sertraline hydrochloride (SH) API using the QTRAP 4500 system (Figure 1). A limit of quantitation (LOQ) of 0.125 ng/mL was achieved with baseline separation of NNSL and sertraline API samples .

SH is an oral antidepressant that blocks serotonin reuptake in the central nervous system and is used to treat major depressive disorder, OCD, panic disorder, PTSD, PMDD, and social anxiety disorder .1 NNSL is a derivative of SH and a potential carcinogenic impurity . As a result, regulatory bodies have set strict limits on the daily acceptable intake (100 ng/day).2,3 This is equivalent to a maximum daily dose of SH of <200 mg/day.4 Therefore, an NNSL limit of 0.5 ng/mg is required to be quantified in the API. Sensitive assays are crucial for assessing NNSL levels in SH and ensuring drug safety and efficacy.

Key-features

Key benefits for analysis of NNSL using the QTRAP 4500 system

  • Sub- ng/mL level of quantitation: Achieve 0.125 ng/mL LOQ for the quantitation of NNSL.
  • 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 paired with library matching capability on SCIEX OS software.
  • Robust analytical performance: Achieve accurate quantitative performance with %CV <7 at all concentration levels across a linear dynamic range (LDR) of 4.2 orders of magnitude.
  • Streamlined data management: SCIEX OS software, a 21 CFR Part 11 - compliant platform, simplifies data acquisition and processing.
Figure 1. Detection and verification of NNSL using the MRM > EPI approach on the QTRAP 4500 system. Representative extracted ion chromatograms (XICs) of the API blank (left, top), and 0.125 ng/mL NNSL -spiked API sample (left, bottom) and corresponding EPI (MS/MS) spectra (right) are displayed. Verification of the impurity peak in the SH API sample was pe rformed using MRM > EPI spectra matching and the fit was 97.2 as demonstrated on SCIEX OS software.
Introduction

Introduction

Nitrosamines are highly potent carcinogens that are prone to affect various organs.3 Nitrosamines are classified into various categories (class 1 -5) using the Carcinogenic Potency Categorization Approach (CPCA). The severity is determined based on the acceptable intake and the activating or deactivating features defined in the structures. Based on the structure of NNSL, it is placed under a class 2 category following the CPCA.5

The formation of NNSL in SH is high due to the presence of the secondary amine.6-7 Since the probability of forming nitrosamine impurity is high, the EU has set a regulation limit of 100 ng/day.3 Considering the maximum daily dose of 200 mg/day and the regulation limit, NNSL should be analyzed below 0.5 ng/mg.

Methods

Methods

Standard preparation: Calibration curve dilutions of NNSL were prepared across a range of concentrations (0.125 ng/mL to 2000 ng/mL) using a 90:10 (v/v) methanol/water containing 0.1% formic acid and analyzed in triplicate.

Sample preparation: 1 mg of SH was weighed into a suitable vessel, and then 1 mL of 0.1% formic acid in 90:10 (v/v) methanol:water was added and vortexed thoroughly to obtain a 1 mg/mL concentration. The spiked API samples were prepared by spiking 1 mg/mL SH API samples with NNSL , resulting in a 0.25 ng/mg.

Chromatography: Analytical separation was performed on the ExionLC system (SCIEX) using a Phenomenex Kinetex XB C18 column (2.1 × 100 mm, 2.6 µm) at a flow rate of 0.4 mL/min. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acet onitrile. The column temperature was set to 40 °C. The gradient conditions used are summarized in Table 1. A 5 µL sample aliquot was injected for LC-MS/MS analysis. The LC flow was diverted to waste for the first 3 min to prevent SH API from entering the ma ss spectrometer.

Mass spectrometry: The optimized source and gas parameters are listed in Table 2, and the MRM parameters are included in Table 3.

Table 1. LC gradient conditions.
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Table 2. Source and gas parameters.
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Table 3. MRM parameters used for quantitation.
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Data processing: Data collection and analysis were performed using SCIEX OS software, version 4 .0. Peaks were integrated using the MQ4 algorithm and a weighting of 1/x² was used for NNSL quantitation.
Results

Results and discussion

Quantitative performance on the QTRAP 4500 system

Baseline separation with ~3.2 min difference was achieved between SH and the NNSL peaks. The NNSL eluted at 7.58 min, while the SH API eluted at 4.39 min (Figure 2) .

Figure 2. Baseline chromatographic separation was achieved between NNSL and SH API. XIC of the 10 ng/mL of NNSL standard (top) and UV chromatogram of SH API at 220 nm (bottom) are displayed.
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NNSL was analyzed across the concentration range of 0.125 ng/mL to 2000 ng/mL. To evaluate reproducibility, each calibration standard was analyzed in triplicate.

Linearity was achieved across concentrations ranging from 0.125 ng/mL to 2000 ng/mL with a coefficient of determination (r2) of >0.998 and an LDR of 4.2 orders of magnitude (Figure 3).

An LOQ of 0.125 ng/mL was achieved for the quantitation of NNSL with no interference in the diluent blank (Figure 4).

Figure 3. Calibration curve for quantitation of NNSL using the quantifier ion (335.1→275.0). The calibration curve was generated using a weighing factor of 1/x².
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Figure 4. Representative XICs of NNSL in the diluent (left) and at LOQof 0.125 ng/mL (right).
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Conclusion

The specification limit (0.5 ng/mg) was calculated based on the maximum daily dose of 200 mg/day. Therefore, NNSL was analyzed at 0.25 ng/mg of API, below the calculated specification limit of 0.5 ng/mg.

The average recovery for the sample spiked with 0. 25 ng/mL NNSL was 103%. Since NNSL was detected in the SH API (Figure 1), recovery was calculated by subtracting the peak area from NNSL in the control API from the spiked sample (Table 4). %CV was below 8% for the API blank, NNSL in neat, and NNSL spiked in SH API, based on replicate analysis.

Table 4. Recovery and precision calculation.
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Figure 5. Quantitative performance for NNSL quantifier ion (335.1→275.0). 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 in SCIEX OS software.
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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 concentration should be <20% at the LOQ and <15% at all higher concentrations.

The assay accuracy was within ±4% of the actual concentration, and the %CV was less than 7%. The calculated percentage accuracy and %CV values were within the acceptance criteria at each concentration level (Figure 5).

Compliance -ready SCIEX OS software

Equivalent SCIEX OS software capabilities for regulated bioanalysis can be executed on the QTRAP 4500 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 6 illustrates the features of SCIEX OS software 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 multi -instrument laboratories, independent of compliance standards. The configuration module allows use rs to assign roles and access as the administrator, method developer, analyst, and reviewer.

Figure 6. 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 Con sole (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.
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Conclusion

  • Quantitation of NNSL

    • A LOQ of 0.125 ng/mL was achieved for the quantitation of NNSL. Good quantitative performance was demonstrated with accurate and highly reproducible (%CV <7%) results on the QTRAP 4500 system.
    • Linearity was achieved at concentrations ranging from 0.125 ng/mL to 2000 ng/mL with an r2 >0.998, resulting in an LDR of 4.2 orders of magnitude .

  • Analysis and identification of NNSL in SH API

    • NNSL impurity in SH API was identified and verified by comparing the impurity MS/MS spectra with the NNSL standard MS/MS spectra , easily performed using library matching on SCIEX OS software.
    • Accurate quantitation with good baseline separation of NNSL and SH API was achieved.
    • An average recovery of 103% was achieved with %CV <9 where NNSL was analyzed at 0.25 ng/mg of API, below the calculated specification limit of 0.5 ng/mg .

  • Software compliance

    • Retain data management and compliance -readiness (21 CFR Part 11) features using SCIEX OS software to support nitrosamine analysis on the QTRAP 4500 system .
References

References

  1. Sertraline Hydrochloride Analytical Profiles of Drug Substances and Excipients, 1996, 2008 Apr., Pages 443 - 486, vol 24
  2. Medicine for Europe, Review of NDSRI in pharmaceuticals drugs: Risk assessment, Acceptable intakes, and QSAR tools by Dr. George Johnson
  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. Website:
    https://www.ncbi.nlm.nih.gov/books/NBK547689/
  5. 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.
  6. Website:
    https://www.fda.gov/regulatory-information/search -fdaguidance - documents/cder-nitrosamine -impurityacceptable -intake-limits
  7. Website: https://www.tga.gov.au/how-we-regulate/monitoringsafety- and-shortages/industry -information- aboutspecific -safety - alerts-recalls- and-shortages/nitrosamine - impurities-medicines/established - acceptable -intake-nitrosamines-medicines