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Unlocking precision: nitrosamine and NDSRI analysis using SCIEX solutions

Parakh Sachdeva | 16 Jan 2025

Confidential – company proprietary

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Agenda

N-nitrosamines: an overview

NDSRIs: an overview

Regulatory landscapes and compliances

Analytical techniques: NDSRI detection and quantification

Case studies: NDSRI analysis in drug substances and drug products

Summary and conclusions

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Analytical insights into nitrosamine drug substance related impurities (NDSRIs):

Following the depicted approach:

Robustness and ruggedness of the analytical method

Restrict variability

Challenges associated with recovery

Stability of the analyte

Accurate estimation of impurity of interest

Instrument to instrument variability

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N-nitrosamines: an overview

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N-Nitrosamines (or simply “nitrosamines”) are a class of compounds where the amine moiety may be derived from any organic secondary amine. N-Nitrosamines are known to be carcinogenic to animals and are reasonably anticipated to be human carcinogens.

In mid-2018, N-nitroso-dimethylamine (NDMA) was abruptly detected in a valsartan active pharmaceutical ingredient (API).

This has led to significant regulatory response, including drug recalls and regulatory guidance that requires the re-evaluation of all synthetic and formulation routes for the potential presence of nitrosamine impurities.

An overview

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N-Nitrosamines are a class of compounds notorious both for the potent carcinogenicity of many of its members and for their widespread occurrence throughout the human environment, from air and water to our diets and drugs.

Although nitrosamines are typically depicted as structure 1a, their actual structures and chemical reactivity are heavily influenced by the zwitterionic resonance structure

Scheme 1 shows the mechanism of carcinogenicity for 2. Nitrosamine 2 first undergoes enzymatic α-hydroxylation with cytochrome P450 and subsequently forms the dealkylated primary nitrosamine. The unstable primary nitrosamine further decomposes to diazonium 3, a DNA alkylating agent The resulting DNA damage can lead to cancer.

Today, four years after the first detection of NDMA in valsartan, the pharmaceutical industry and regulatory authorities have not yet completed this chapter, even with the huge efforts that have been made already.

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N-Nitrosamines

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NDSRIs: an overview

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Nitrosamine drug substance related impurities

Active Pharmaceutical Ingredient (API) derived complex nitrosamines, also called nitrosamine drug substance related impurities (NDSRIs)

These are a class of nitrosamine impurities identified in many drug products and could be present in active pharmaceutical ingredients (APIs).

NDSRIs can potentially form in APIs:

Nitrosating agents are present in the API manufacturing process.

APIs undergo processing steps that can potentially induce their formation such as fluid bed drying at an elevated temperature and jet milling

The presence of high levels of NDSRIs has been associated with drug products rather than APIs because NDSRI formation usually results from a reaction between the API or API fragment and nitrosating agents in the drug formulation.

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Nitrosamine drug substance related impurities (NDSRIs)

Primary Amines

Primary amines are no nitrosamine precursors. The nitrosation of an aliphatic primary amine yields an alkyl diazonium ion and water, not a nitrosamine.

Secondary Amines

Secondary amines can be converted to nitrosamines. The reactivity depends on the basicity of the secondary amine.

Tertiary Amines

Tertiary amines can undergo nitrosation as well. This reaction is referred to as nitrosative cleavage or nitrosative dealkylation. However, the reaction necessitates the presence of at least one proton in the alpha-position to the amine nitrogen to enable the dealkylation pathway via an iminium intermediate

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Nitrosamine drug substance related impurities(NDSRIs)

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Nitrosamine drug substance related impurities(NDSRIs)

There has been a spate of withdrawals caused by nitrosamine drug substance-related impurities (NDSRIs).

Varenicline tablets
Propranolol extended-release capsules
Orphenadrine citrate extended-release Tablets
Quinapril HCl tablets
Dabigatran Etexilate capsules
Losartan Potassium tablets

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Regulatory landscapes and compliances

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USFDA and EMEAI updated guidelines

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Regulatory approach

Regulatory agencies issued guidance(s) to industry, recommending that manufacturers of APIs and drug products take steps to detect and prevent unacceptable levels of N-nitrosamine(s) impurities as well as NDSRIs in drug product(s), or to avoid their presence when feasible.

The Guidance(s) introduced a three-step process that manufacturers and applicants should take to mitigate nitrosamine impurities in their drug products:

(1) conduct risk assessments for nitrosamines in their APIs and drug products;
(2) conduct confirmatory testing if risks are identified; and
(3) report changes implemented to prevent or reduce the presence of nitrosamine impurities in APIs and drug products in approved and pending NDAs and ANDAs.

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Challenges in deciding limits

Challenges in Determining AI Limits for NDSRIs

Determining an AI limit for NDSRIs is often more challenging than determining AI limits for small molecule nitrosamines.
Includes evaluation of the mutagenic and carcinogenic potential of the impurity and represents the level at or below which FDA has determined that the impurity or impurities would not pose a safety concern for patients taking the drug product.

A compound-specific AI can be calculated based on rodent carcinogenic potency data such as TD50 values
When the mutagenic potential of an NDSRI is not adequately characterized, FDA and applicants have used (quantitative) SAR methods.

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Guidelines recommended (acceptable intake) AI limits for NDSRIs

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Analytical insights into NDSRIs

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Analytical methods: status and prospects

National regulatory agencies as well as researchers have released many versions of analytical methods for nitrosamine determination:

LC coupled with spectrophotometric detection
Ultraviolet–visible (UV–Vis) spectrophotometers
Photodiode array (PDA) detectors
Spectro-fluorescence detectors
Chemiluminescence detectors
Fluorescence sensor-based methods
GC-based methods with various detectors

LC–MS based methods:

Triple quadrupole mass spectrometry (QQQ MS)
High-resolution mass spectrometry (TOF HRMS)

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Challenges in analytical methods.

Limits of NDSRIs has been convincingly achievable at levels around 50 to 100 ppb (ng/g drug product). Perhaps, in the case of NDSRIs for which control in categories 1-3 is required by the CPCA, this may not be sufficient and pose analytical challenges.

Reproducibility: Requirement to reproduce on a daily basis in a routine QC laboratory.

Chromatography: Challenge may get aggravated if the NDSRI co-elutes with the API and/or Structurally related impurities (formyl impurity) due to high physicochemical similarity.

Sensitivity:

Analytical methods are required to have an LOQ at 10 % of the content limit derived from the NDSRI’s acceptable intake and the API’s maximum daily dose.

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Estimation of NDSRIs��Challenges faced during NDSRI method development:��

Sensitivity

a) ESI (+) Polarity

b) Mobile phase pH & additives

c) Optimizing Ratio of Organic content.

Stability

a) Impurity Response was increased on storage

b) Hydrolytic and pH dependent Degradation

c) Diluent (Basified)

d) Autosampler Temperature

Recovery

a) Failed at lower level (LOQ & Specification)

b) Higher Recovery Due to Stability concern

c) Interfering peaks

Carry Over

a) Mostly associated with Column Stationary Phase

b) Worked on Gradient Profile to minimize

c) Possible to Nullify if we further work on Gradient

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Method development approach: diluent selection�

^$$ Assuming this ratio has given optimum results

Aqueous to organic ratio (80:20 v/v)

Aqueous to organic ratio (50:50 v/v)

Aqueous to organic ratio (20:80 v/v)

Step-1

Aqueous to organic ratio (50:50 v/v)

Aqueous to organic ratio (50:50 v/v)-acidified

Aqueous to organic ratio (50:50 v/v): basified

Step-2

Samples to analyze:

Impurity in Neat Diluent (specification limit)
Drug substance control
Recovery sample (specification limit)

LC-MS/MS method:

LC flow directed to MS
LC flow directed to waste

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Method development approach: LC condition set-up�

Time (min)	Pump-A %	Pump-B %
0.00	90	10
3.00	90	10
6.00	70	30
9.00	70	30
12.00	50	50
15.00	50	50
18.00	20	80
21.00	20	80
22.00	90	10
25.00	90	10

Time (min)	Pump-A %	Pump-B %
0.00	80	20
3.00	80	20
6.00	60	40
9.00	60	40
12.00	40	60
15.00	40	60
18.00	10	90
21.00	10	90
22.00	80	20
25.00	80	20

Time (min)	Pump-A %	Pump-B %
0.00	100	0
3.00	100	0
6.00	70	30
9.00	70	30
12.00	40	60
15.00	40	60
18.00	05	95
21.00	05	95
22.00	100	0
25.00	100	0

Flow Gradient-1	Flow Gradient-2	Flow Gradient-3

Flow rate: 0.400 to 0.700 mL/min

Column: Phenomenex Biphenyl 150 × 4.6 mm; 2.6 µ

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Method development approach: LC condition set-up�

Mass Parameters

Mobile Phase Composition with buffering agent and additives

Preliminary knowledge on Stability of API/Impurity Available

Diluent/Reconstitution Solution

Flow Gradient Conditions

Column Selection (an art of selection…)

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Case studies

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Determination of Brimonidine nitroso-drug substance related impurity (N-Nitroso Brimonidine) in Brimonidine tartrate drug substance using QTRAP 5500 LC-MS/MS system.�

Brimonidine tartrate

N-Nitroso brimonidine

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Determination of Brimonidine nitroso-drug substance related impurity (N-Nitroso Brimonidine) in Brimonidine tartrate drug substance using QTRAP 5500 LC-MS/MS system.�

Drug substance sample:

Dissolved ~5 mg of the drug substance in 5.000 mL of acetonitrile:water (50:50) and vortexed to mix thoroughly for 10 minutes. After the extraction, samples were centrifuged for 5 minutes at 4500 RPM at room temperature. Transferred the filtered supernatant (using PVDF filter) into autosampler vials for analysis.

N-Nitroso-Brimonidine impurity:

Accurately weighed and transferred N-Nitroso-brimonidine standard in prelabelled Tarson tube and dissolved in acetonitrile : water (50:50) to give a final concentration of 1000.000 µg/mL.

These stock solution was used to prepare serial dilutions in Acetonitrile : water (50:50) for Linearity; Specification limit, and LOQ.

Specification limit (0.030 ppm)
LOQ (0.003 ppm)

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Chromatographic conditions�

Chromatographic conditions

Column:

Kinetex^® Biphenyl Column 150×3.0 mm; 2.6 µ

Mobile Phase:

Pump A: 0.1% Acetic acid in water
Pump B: 0.1% Acetic acid in Acetonitrile

Flow rate: 0.400 mL/min
Injection volume: 10 µL
Needle wash:

80:20 v/v Acetonitrile: water

Time (min)	A (%)	B (%)	B curve
0.00	95	5	0
4.00	95	5	0
12.00	75	25	0
16.00	75	25	0
18.00	10	90	0
21.00	10	90	0
21.10	95	5	0
25.00	95	5	0

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Representative chromatograms

N-Nitroso brimonidine

API

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N-Nitroso-Brimonidine representative chromatogram

Absolute Samples

Blank	LOD (0.0015 ppm)	LOQ (0.0030ppm)	Spec (0.0300 ppm)

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Repeatability and recovery analysis

Sample	Actual concentration [ng/mL (PPM)]	Area observed (Mean ± SD)	% RSD (CV)	Number of observations (N)	Recovery (%)
Specification	0.0300 (0.0300)	84375± 2656	3.15	6	110.62

N-Nitroso-Brimonidine
Injection	STD-SPEC	API- control	REC-SPEC
1	26559	56018	85901
2	26693	55661	87019
3	27215	50892	86856
4	25399	51281	82692
5	25740	57590	83413
6	26081	60380	80370
Average	26281	55304	84375
STDEV	667.83	3668.22	2656.04
Precision	2.54	6.63	3.15
	API Corrected area		29071
	% Recovery		110.62

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N-Nitroso BDA-S2 and N-Nitroso Desmethyl Bedaquiline��Standards and samples used

Impurity Preparation:

Accurately weighed 1mg of N-Nitroso BDA S2 and N-Nitroso Desmethyl Bedaquiline into two, 2 mL PP tube and dissolved with 1.000 mL Methanol v/v to give final concentration of 1000 PPM. Specification, LOQ and Linearity samples were prepared in Methanol: Water 95:05 %v/v.

Drug Substance Sample:

Dissolved ~10 mg of the drug substance in 1.000 mL of Methanol: Water 95:05 %v/v and vortexed to mix thoroughly for 10 minutes. After the extraction, samples were centrifuged for 5 minutes at 4500 RPM at room temperature. Transferred the filtered supernatant (using PVDF filter) into auto sampler vials for analysis.

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LC Conditions

Time (min)	A %	B %	B Curve
0.00	90	10	0
3.00	90	10	0
6.00	70	30	0
9.00	70	30	0
12.00	50	50	0
15.00	50	50	0
19.00	30	70	0
31.00	30	70	0
31.10	5	95	0
34.00	5	95	0
34.10	90	10	0
37.00	90	10	0

Chromatographic conditions

Column:

Kinetex Biphenyl 150 X 3.0 mm; 2.6 um

Mobile phase:

Pump A: 2mM ammonium formate + 0.1% formic acid in water
Pump B: Acetonitrile:Methanol (50:50 % v/v)

Flow rate: 0.600 mL/min
Injection volume: 10 µL
Column oven temperature: 40°C
Autosampler temperature: 10°C

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Representative chromatograms

API Sample

BDA S2

N Nitroso Desmethyl Bedaquiline

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N-Nitroso BDA S2

Blank

Specification limit

LOQ

API

Placebo

Formulation

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N Nitroso Desmethyl Bedaquiline

Blank

Specification limit

LOQ

API

Placebo

Formulation

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Repeatability and recovery analysis

Sample	Actual concentration [ng/mL (PPM)]	Area observed (Mean ± SD)	% RSD (CV)	Number of observations (N)	Recovery (%)
Specification	0.4507 (0.0450)	153172± 4316	2.82	6	99.80

N-Nitroso-BDA S2
Injection	STD-SPEC	API- control	REC-SPEC
1	158242	917	156774
2	155317	479	157950
3	152491	815	152532
4	156150	736	151785
5	146384		156013
6	150450		146535
Average	153172	737	153598
STDEV	4316	187	4223
Precision	2.82	25.40	2.75
	API corrected area		737
	% Recovery		99.80

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Repeatability and recovery analysis

Sample	Actual concentration [ng/mL (PPM)]	Area observed (Mean ± SD)	% RSD (CV)	Number of observations (N)	Recovery (%)
Specification	0.4507 (0.0450)	345990± 5170	1.49	6	109.71

N-Nitroso-Desmethyl Bedaquiline
Injection	STD-SPEC	API- control	REC-SPEC
1	342183	29806	402355
2	343721	29148	406890
3	341401	29705	410152
4	351675	30070	411253
5	343567		410968
6	353391		414022
Average	345990	29682	409273
STDEV	5170	388	4090
Precision	1.49	1.31	1.00
	API corrected area		29682
	% Recovery		109.71

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Conclusion

SCIEX technologies for Triple Quad can successfully be implemented in quantitative analysis of cohorts of concern under discussion.

Regulatory limits can easily be achieved using SCIEX instruments.

The software used for Acquisition, processing and reporting the analytical data is SCIEX OS which is 21 CFR part 11 compliant.

SCIEX OS provides a platform which is not only user friendly for users but also for Quality control professional by means of event logs and audit trail tracking.

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Trademarks/ Licensing

The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales representative or refer to www.sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures.

Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries (see www.sciex.com/trademarks).

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The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales representative or refer to www.sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures.

Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries �(see www.sciex.com/trademarks).

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