Analytical Solutions for Accurate

Determination of Nitrosamine
Impurities
Hendy Dwi Warmiko
Sales Support Supervisor
Div. Thermo Scientific
PT GeneCraft Labs
25th March 2021

The world leader in serving science

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 Brief background
Why all the fuss?

• Nitrosamine impurities in Valsartan reported in 2018,

product recalled due to NDMA contamination.

• Nitrosamines are classified by the IARC and ICH

M7(R1) guideline as “known mutagenic carcinogens”.

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Nitrosamine – an old foe of other concerns globally
 Brief background

4
Changes in Regulatory Landscape
Timeline (Data from USP)
Bloomberg Business Week
September 2019

https://www.bloomberg.com/news/features/2019-09-12/how-carcinogen-
tainted-generic-drug-valsartan-got-past-the-fda
 Most recent guidelines from EMA, FDA, and WHO
Today
• Widening of scope for testing

• Widening of nitrosamines of interest along

with TOTAL nitrosamine content

• More universal approach to method limits

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 Changes in Regulatory Landscape
What new challenges now face?

“If nitrosamines without published AI

“If more than one of the nitrosamine impurities identified in limits are found in drug products,
Table 1 is detected and the total quantity of nitrosamine manufacturers should use the
impurities exceeds 26.5 ng/day … the manufacturer approach outlined in ICH M7(R1) to
should contact the Agency for evaluation. For drug determine the risk associated with the
products with an MDD of less than 880 mg/day, a nitrosamine and contact the Agency
recommended limit for total nitrosamines of 0.03 ppm is about the acceptability of any
considered acceptable.” proposed limit”

API manufacturers should review their API manufacturing “Manufacturers should establish methods for
processes and perform risk assessments to identify the which the LOQ and limit of detection (LOD) are
potential for nitrosamine impurities. If a risk of nitrosamine as low as reasonably practical”
impurities is identified, confirmatory testing of batches
should be conducted using sensitive and appropriately “All authorized human medicinal products
validated methods. containing chemically synthesized APIs are
to be reviewed, including generics and over-
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs the counter (OTC) products”
https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf

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 Changes in Regulatory Landscape
Timelines for risk assessments

FDA May 2021

EMA April 2021 July 2021

Step 1 : Assesment of Theoretical Risk

Step 2: Confirmation of The Risk

https://www.fda.gov/drugs/news-events-human-drugs/nitrosamines-impurities-drugs-health-risk-
assessment-and-mitigation-public-workshop-03292021

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 Review of Drug Product Recalls

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02120

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 Indonesia?

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Nitrosamine Impurities in Drugs by
GC-MS and GC-MS/MS

The world leader in serving science

12 WS73815
 U.S. FDA Testing Methods
U.S. FDA has been investigating the presence of nitrosamine impurities in drug products since 2018
Since then, US FDA published several analytical methods including both GC-MS, GC-MS/MS, LC-MS/MS and
LC/HRMS

USFDA Method Technique Compounds

Method
117843 Combined headspace method GC-MS NDMA, NDEA
117807 Combined direct injection method GC-MS/MS NDMA, NDEA
123409 Combined direct injection method GC-MS/MS NDMA, NDEA, NDIPA, NEIPA, NDBA
124025 Headspace GC-MS/MS method GC-MS/MS NDMA, NDEA, NDIPA, NEIPA
125478 LC-HRMS method LC-HRMS NDMA, NDEA, NEIPA, NDIPA, NDBA, NMBA
131868 LC-MS/MS method LC-MS/MS NDMA

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 U.S. FDA Testing Methods

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Available GC-MS Analytical Solutions
GC-MS coupled to HS GC-MS/MS coupled to direct
sampling liquid and HS sampling

• Targeted screening
• Targeted screening
• Compliance with U.S. FDA regulation
• Compliance with U.S. FDA
• Minimal sample preparation
• Dual configuration with liquid and headspace
• Low chemical background sampling for expanded sampling flexibility
• SIM acquisition for selectivity and • SRM acquisition for higher selectivity and
sensitivity lower detection limits
• AEI ion source for improved sensitivity and
Thermo Scientific Chromeleon CDS Software is
robustness

compliant with 21 Code of Federal Regulations

(CFR) part 11
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 Thermo Scientific TSQ-9000 GC-MS/MS (Sartan)
Method Performance

Data from Thermo Fisher Scientific, Hyderabad, India

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General Notes from FDA and USP

LC-HRMS (Q-Orbitrap) LC-MS/MS GC-MS/MS

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 LC-HRMS and LC-MS/MS solutions for
nitrosamine impurity analysis in drug
substances and products

The world leader in serving science

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 US FDA validated methods
FDA Method Method Technique Drug product Compounds

117843 Combined headspace method GC-MS Valsartan NDMA, NDEA

117807 Combined direct injection method GC-MS/MS Valsartan NDMA, NDEA
123409 Combined direct injection method GC-MS/MS Valsartan NDMA, NDEA, NDIPA, NEIPA, NDBA
124025 Headspace GC-MS/MS method GC-MS/MS Valsartan NDMA, NDEA, NDIPA, NEIPA
125478 LC-HRMS method LC-HRMS Losartan NDMA, NDEA, NEIPA, NDIPA, NDBA, NMBA
130801 LC-HRMS method LC-HRMS Ranitidine NDMA
131868 LC-MS/MS method LC-MS/MS Ranitidine NDMA
138617 LC-HRMS method LC-HRMS Metformin NDMA, NDEA, NEIPA, NDIPA, NDPA, NDBA,
NMBA, NMPA,
142092 LC-HRMS method LC-HRMS Rifampin & MNP, CPNP
Rifapentine

Thermo Scientific Q Exactive LC-HRMS

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 Solvent Background Peaks Around NDMA Peak

Thermo Scientific Q Exactive LC-HRMS

Data from Thermo Fisher Scientific

China-Shanghai COE Lab

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 NDMA Detection Limit and Dynamic Range

Data from Thermo Fisher Scientific

Paris COE Lab

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 Sensitivity Comparison for Scan Modes

Thermo Scientific Q Exactive

LC-HRMS

Data from Thermo Fisher Scientific

Singapore COE Lab
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 LC-HRMS Method

US-FDA LC-HRMS Method for ARB Nitrosamines Singapore HSA LC-HRMS Method for 6 Nitrosamines
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 LC-HRMS Method

US-FDA LC-HRMS Method for ARB Nitrosamines

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 LC-HRMS Method

China FDA LC-HRMS Method for NDMA

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 USP General Chapter 1469 Timeline

General Chapter <1469> Nitrosamine Impurities published in Pharmacopeial Forum

Volume 46 Issue 5, available on-line on September 1st, 2020, for public comments.

The comment period end on November 30, 2020.

The JSC is responsible for addressing public comment and revising

the standard as needed.

The Standard is balloted for approval by General Chapter Chemical

Analysis Expert Committee.

Planning to publish the chapter in Compendia-USP 2021 Issue 3,

available on-line on May 1st, 2021 with official date December 1st,
2021.

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 Nitrosamine panel lists for Orbitrap Exploris 120

1 N-Nitrosodimethylamine (NDMA) 6 N-Ethyl-N-nitroso-2-propanamine (NEIPA)

2 N-Nitrosomethylethylamine (NMEA) 7 N-Nitroso-di-isopropylamine (NDIPA)

3 N-Nitrosopyrrolidine (NPYR) 8 N-Nitroso-di-n-propylamine (NDPA)

4 N-Nitrosodiethylamine (NDEA) 9 N-Nitrosodi-n-butylamine (NDBA)

5 N-Nitrosopiperidine (NPIP)

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 Retention time and injection reproducibility

Vanquish Horizon UHPLC system and

Acclaim Polar Advantage II column, N = 50, Neat, APCI
2.1 x 100mm, 2.2µm Average RT: 0.87 min
• Excellent retention time and injection RT reproducibility: 0.69%
reproducibility Average Peak area: 11700
• Minimum carryover Peak area reproducibility: 2.1%

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 Sensitive quantitation of nitrosamine impurities
LOD/LLOQ/Linearity for both neat and excipient standards, HESI
LOD LLOQ
Matrix Linearity
ng/ml PPB ng/ml PPB
Neat 0.2 6.8 0.5 17
NDMA
Excipient 0.5 17 0.5 17
Neat 0.5 17 0.5 17
NMEA
Excipient 0.5 17 0.5 17
Neat 0.1 3.4 0.1 3.4
NPYR
Excipient 0.1 3.4 0.2 6.8
Neat 0.2 6.8 0.5 17
Orbitrap Exploris 120 MS NDEA
Excipient 0.5 17 0.5 17
• High selectivity with 120,000 mass Neat 0.2 6.8 0.2 6.8 LLOQ –
NPIP
resolution and sub ppm mass accuracy Excipient 0.2 6.8 0.2 6.8 50 ng/ml

• High sensitivity with LLOQ ≤ 0.017 ppm Neat 1 34 2 68

NEIPA
Excipient 2 68 2 68
in HESI mode for both neat and
Neat 0.2 6.8 0.5 17
excipient standards NDIPA
Excipient 0.2 6.8 0.5 17
In HESI, Exploris 120 MS LOD/LLOQ Neat 0.2 6.8 0.2 6.8
NDPA
is comparable with FDA published Excipient 0.2 6.8 0.2 6.8
LOD/LLOQ using Q Exactive Neat 0.1 3.4 0.5 17
NDBA
Excipient 0.1 3.4 0.5 17
29 * PPB is calculated based on 30mg/ml of drug substance and product extract
 Sensitive quantitation of nitrosamine impurities
LOD/LLOQ/Linearity for both neat and excipient standards, APCI
LOD LLOQ
Matrix Linearity
ng/ml PPB ng/ml PPB
Neat 0.2 6.8 0.2 6.8
NDMA
Excipient 0.2 6.8 0.2 6.8
Neat 0.2 6.8 0.2 6.8
NMEA
Excipient 0.2 6.8 0.2 6.8
Neat 0.1 3.4 0.2 3.4
NPYR
Excipient 0.2 6.8 0.2 6.8
Neat 0.1 3.4 0.1 3.4
Orbitrap Exploris 120 MS NDEA
Excipient 0.1 3.4 0.1 3.4
• High selectivity with 120,000 mass Neat 0.1 3.4 0.1 3.4 LLOQ –
NPIP
resolution and sub ppm mass accuracy Excipient 0.2 6.8 0.2 6.8 50 ng/ml

• High sensitivity with LLOQ ≤ 0.017 ppm Neat 0.5 17 0.5 17

NEIPA
Excipient 0.5 17 0.5 17
in APCI mode for both neat and
Neat 0.1 3.4 0.1 3.4
excipient standards NDIPA
Excipient 0.1 3.4 0.1 3.4

APCI provides up to 5x sensitivity Neat 0.1 3.4 0.1 3.4

NDPA
Excipient 0.1 3.4 0.1 3.4
boost as compared to HESI
Neat 0.1 3.4 0.5 17
NDBA
Excipient 0.1 3.4 0.5 17
30 * PPB is calculated based on 30mg/ml of drug substance and product extract
 Nitrosamine impurity levels in ranitidine

* PPB is calculated based on 30mg/ml of drug substance and product extract

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 Presence of DMF in ranitidine drug products
DMF co-eluting with NDMA

A minimum resolution of 45,000 and 15 ppm mass tolerance setting are required to
prevent overestimation of NDMA when quantifying NDMA using the monoisotopic ion
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 Presence of DMF in metformin drug products

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 US FDA validated methods
FDA Method Method Technique Drug product Compounds

117843 Combined headspace method GC-MS Valsartan NDMA, NDEA

117807 Combined direct injection method GC-MS/MS Valsartan NDMA, NDEA
123409 Combined direct injection method GC-MS/MS Valsartan NDMA, NDEA, NDIPA, NEIPA, NDBA
124025 Headspace GC-MS/MS method GC-MS/MS Valsartan NDMA, NDEA, NDIPA, NEIPA
125478 LC-HRMS method LC-HRMS Losartan NDMA, NDEA, NEIPA, NDIPA, NDBA, NMBA
130801 LC-HRMS method LC-HRMS Ranitidine NDMA
131868 LC-MS/MS method LC-MS/MS Ranitidine NDMA
138617 LC-HRMS method LC-HRMS Metformin NDMA, NDEA, NEIPA, NDIPA, NDPA, NDBA,
NMBA, NMPA,
142092 LC-HRMS method LC-HRMS Rifampin & MNP, CPNP
Rifapentine

Triple Quad LC/MS system

with APCI source, 10 µL

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USFDA and Singapore HSA LC-MS/MS for NDMA
LC-MS/MS Solutions
Using Thermo Scientific TSQ Fortis/Quantis/Altis LC-MS/MS
LC-MS/MS Solutions
20 ppb NDMA spiked in 100 mg/ml metformin, APCI data
 Performance on TSQ Fortis MS
1 ng/ml neat standard, 10 µL injection, APCI-SRM

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Performance on TSQ Fortis MS
 Reproducibility of LC-MS/MS Method
Retention time and injection reproducibility for 1000 sample injections

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 MNP in Rifampin and CPNP in Rifapentine

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 Summary

A rapid, highly selective and sensitive LC-HRMS method for

1
quantitation of 9 nitrosamines in drug product

A fit for purpose LC-MS/MS method for quantitation of

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nitrosamines in drug product

Reliable determination of nitrosamine impurities in drug

3
products that meet new US FDA regulatory requirements

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 Acknowledgment

1 All Webinar Committee from PT GeneCraft Labs, Thermo Scientific

2 Badan Pengawas Obat dan Makanan RI

3 Thermo Fisher Scientific Singapore Centre of

Excellence Team

Orbitrap Q Exactive & Exploris Support Team

4
(Bremen, Dreiech, Paris, Shanghai for the Data)

5 TSQ LC-MS/MS Support Team (San Jose) and

GC-MS/MS Team (Hyderabad)

6 All Participants

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 Resources
Application note highlights

LC-HRMS (QE+ and OE120) LC-MS/MS (Quantis) GC-MS/MS

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 Resources
Analyte Guru highlights

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 Resources
Web page

Laurus labs, Hyderabad, India

Singapore Health Sciences Authority
Quinta-Analytica, Prague, Czech Republic
US Food and Drug Administration
Chromeleon eCDS software
LC-MS/MS
LC-HRAM
GC-MS

www.thermofisher.com/nitrosamine

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