Sujata Rajan1, Sashank Pillai1, Rahul Baghla2 and Eshani Galermo2
1SCIEX, India and 2SCIEX, USA
Sensitive quantitation of N-nitroso Pyribenzamine isomeric impurities in Tripelennamine hydrochloride API
Abstract
This technical note demonstrates a sensitive method for the quantitation of N-nitroso Pyribenzamine (NNPBA) isomeric impurities (NNPBA 1 and NNPBA 2) in Tripelennamine hydrochloride (TH) API using the QTRAP version of the SCIEX 7500+ system. A limit of quantitation (LOQ) of 0.005 ng/mL was achieved with no interference in the diluent blank and unspiked API samples for NNPBA isomers in a single method (Figure 1).
Tripelennamine hydrochloride (TH) is an ethylenediamine derivative. It is a sedating histamine H1 receptor antagonist with antimuscarinic properties. TH is used to alleviate symptoms of asthma, hay fever and both perennial and seasonal allergic rhinitis, as well as to manage other allergic conditions, such as urticaria.1 TH salt is formulated as tablets and called Pyribenzamine.2,3 It works by blocking the histamine Type 1 (H1) receptor, thereby reducing inflammatory reactions triggered by histamines.4 Regulatory bodies have amended the maximum permissible daily intake limit for NNPBA to 18 ng/day.5 This is equivalent to a maximum daily dose of less than 600 mg/day.1 Given that TH has a maximum daily dose of 600 mg/day, a nitrosamine limit of 0.03 ng/mg is required to be quantified in the API. This technical note presents a reliable and sensitive workflow to support the quantitative analysis of NNPBA in TH API below the regulation limit using the QTRAP 7500+ system.
Figure 1: Representative extracted ion chromatograms (XICs) of the quantifier ion (271.1→183.0) in the diluent, and LOQ at 0.005 ng/mL of NNPBA 1 and NNPBA 2 isomers (in sequence from left to right). An LOQ of 0.005 ng/mL was reached for the quantitation of NNPBA 1 and NNPBA 2. NNPBA 1 and NNPBA 2 were not detected in the diluent.
Methods
Standard preparation: Calibration curve dilutions of NNPBA were prepared across a range of concentrations in 0.1% (v/v) formic acid in water (0.005, 0.01, 0.025, 0.05, 0.1, 0.5, 1, 5, and 10 ng/mL) and analyzed in triplicate.
Sample preparation: 1 mg of TH was weighed into a suitable vessel. 1 mL of 0.1% (v/v) formic acid in water was added and vortexed thoroughly to obtain a 1 mg/mL concentration. Quality control samples were prepared by spiking samples of NNPBA in TH API at 3 concentration levels: 0.01 ng/mg, 0.02 ng/mg and 0.03 ng/mg. Six replicates of each concentration were analyzed.
Chromatography: Analytical separation of the isomers, and between the compound and the API, was performed on the ExionLC system using a Phenomenex Kinetex Biphenyl (3.0 × 100 mm, 2.6 μm) column at a flow rate of 0.4 mL/min. Mobile phase A was 0.1% (v/v) acetic acid in water and mobile phase B was 0.1% (v/v) acetic acid in methanol. The column temperature was set to 30°C. The gradient conditions used are summarized in Table 1. An 8 μL sample aliquot was injected for LC-MS/MS analysis. The LC flow was diverted to waste for the first 8 min to prevent TH API from entering the mass spectrometer.
Table 1: LC gradient conditions for NNPBA.
Mass spectrometry: The optimized source and gas parameters are listed in Table 2 and the MRM parameters are included in Table 3.
Table 2: Source and gas parameters.
Table 3: MRM parameters used for quantitation.
Data processing: Data collection and analysis were performed using SCIEX OS software, version 3.4. Peaks were integrated using the MQ4 algorithm and a weighting of 1/x was used for quantitation of both NNPBA isomers.
Quantitative performance on the QTRAP 7500+ system
The baseline separation of approximately 3.9 minutes and 4.3 minutes was achieved between the TH and NNPBA 1 peaks, and TH and NPBA 2 peaks, respectively. Based on the sequence of elution, they are addressed as NNPBA 1 and NNPBA 2. The TH API eluted at 5.65 min, while the isomer NNPBA 1 and NNPBA 2 was eluted at 9.62 and 9.96 min, respectively (Figure 2).
Figure 2: Baseline chromatographic separation was achieved between NNPBA 1, NNPBA 2 and TH API. XIC of the 0.01 ng/mL of NNPBA 1 and NNPBA 2 spiked in the TH API (top) and UV chromatogram of API at 250 nm (bottom) are displayed.
Linearity was achieved across concentrations ranging from 0.005 to 10 ng/mL with a correlation of determination (r2) of >0.999 for both quantifier and qualifier ions (Figure 3) of NNPBA isomers. An LDR of 3.3 orders of magnitude was achieved.
Figure 3: Calibration curve for quantitation of NNPBA 1 and NNPBA 2 using the quantifier ion (271.1→183) in blue and qualifier ion (271.1→198.1) in pink. Both calibration curves were generated using a weighing factor of 1/x.
The specification limit (0.03 ng/mg) was calculated based on the maximum daily dose of 600 mg/day. In this assay, NNPBA was analyzed at 0.01 ng/mg of API, below the calculated specification limit of 0.03 ng/mg (Figure 4). NNPBA 1 and NNPBA 2 were not detected in the TH API (Figure 1). Therefore, the recovery of NNPBA 1 and NNPBA 2 was calculated against the respective peak area of the NNPBA 1 and NNPBA 2 neat standard.
Figure 4: Representative extracted ion chromatograms (XICs) of the quantifier ion (271.1→183.0) in the API Blank, and 0.01 ng/mL NNPBA spiked in TH API solution of NNPBA 1 and NNPBA 2 isomers (in sequence from left to right). A 0.01 ng/mL spiked API sample below the specified limit was quantified for both NNPBA 1 and NNPBA 2. NNPBA 1 and NNPBA 2 were not detected in the API blank.
The average recovery of NNPBA 1 and NNPBA 2 was 102.2% and 113.4%, respectively. The %CV of NNPBA 1 and NNPBA 2 was <5% for spiked samples, evaluated in triplicate for neat standard and 6 replicates for the spiked sample (Table 4).
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 for both the isomers was within ±18% at LOQ and ±10% at higher levels of the actual concentration and the %CV was <5 throughout the batch. Calculated percent accuracy and %CV values were within the acceptance criteria at each concentration level for both NPBA 1 and NPBA 2 (Figure 5).
In the spiked quality control samples, all the parameters met the established acceptance criteria, reinforcing the method's reproducibility (Figure 6).
Table 4: Recovery and precision calculation of NNPBA 1 and NNPBA 2.
Figure 5: Quantitative performance for NNPBA 1 and NNPBA 2 quantifier ion (271.1→183.0) (top) and qualifier ion (271.1→198.1) (bottom). 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.
Figure 6 : Quantitative performance for NNPBA 1 and NNPBA 2 quantifier ion (271.1→183.0) (top) and qualifier ion (271.1→198.1) (bottom) using quality control samples Reproducibility and accuracy results were determined from the API spiked quality control samples across 6 replicates at each concentration. Statistical results were summarized using the Analytics module in SCIEX OS software.
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 7 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 users to assign roles and access as the administrator, method developer, analyst, and reviewer.
Figure 7: 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.