Nitrosamine impurities analysis solution guide
Souhrn
Význam tématu
Existence nitrosaminů v léčivých přípravcích představuje zásadní zdravotní riziko vzhledem k jejich prokázaným karcinogenním účinkům. Po odhalení vysokých hladin NDMA a dalších nitrosaminů ve valsartanu v roce 2018 se regulatorní orgány po celém světě zaměřily na stanovení maximálních přípustných mezí a na zavedení přísných testovacích postupů. Dnešní farmaceutické laboratoře potřebují robustní, citlivé a spolehlivé analytické řešení, které zajistí splnění požadavků ICH M7(R1), FDA a dalších norem bez rizika falešně pozitivních či falešně negativních výsledků.
Cíle a přehled studie / článku
Průvodce si klade za cíl představit ucelenou škálu analytických přístupů pro jednotlivé fáze analýzy nitrosaminových nečistot: od monitoringu prekurzorů během syntézy API přes rutinní screening až po potvrzovací kvantifikaci. Popisuje výhody, omezení a doporučené pracovní postupy pro LC-HRAM MS, GC-HRAM MS, LC-MS/MS, GC-MS/MS a iontově chromatografickou analýzu nitritů.
Použitá metodika a instrumentace
Pro různé aplikace a citlivostní požadavky byly navrženy následující systémy:
- LC-HRAM MS: Orbitrap Exploris 120 pro vysoké rozlišení (>45 000 FWHM) a absolutní potvrzení molární hmotnosti.
- GC-HRAM MS: Orbitrap Exploris GC pro rozlišení izobarických interferencí v plynných vzorcích.
- LC-MS/MS: TSQ Quantis/TSQ Altis pro vysoce citlivý a rychlý screening nitrosaminů v API a hotových formách.
- GC-MS/MS: TSQ 9000 pro analýzu těkavých nitrosaminů s nízkými LOQ.
- Iontová chromatografie: Dionex IonPac AS19-4 μm a ICS-6000 pro selektivní stanovení nitritů a nitridů jako předběžný screening rizika tvorby nitrosaminů.
- Chromatografická platforma a automace: Vanquish Flex/Core UHPLC, TriPlus RSH pro dávkování a Chromeleon CDS jako jednotné compliance-ready prostředí.
Hlavní výsledky a diskuse
Implementace HRAM MS potvrdila, že rozlišení ≥ 45 000 a přesnost hmotnostního měření pod 3 ppm zcela eliminuje falešné pozitivní skladby např. NDMA versus 15N izotop DMF. GC-HRAM MS dosáhlo separace 15 nitrosaminů pod 12 min při LOD < 2 ppb. Pomocí LC-MS/MS lze rutinně zpracovat stovky vzorků denně s LOQ 5 ppb (APCI) a 10 ppb (HESI). IC metoda pro nitrity dosahuje LOD ~ 0,9 ppm bez složité přípravy vzorku.
Přínosy a praktické využití metody
Tyto multidisciplinární přístupy umožňují farmaceutickým závodům:
- průběžně monitorovat rizikové prekurzory nitrosaminů během výroby,
- prokázat shodu s regulatorními limity a minimalizovat riziko stažení šarží,
- dosahovat vysoké propustnosti laboratoře s minimalizovanou pracovní zátěží personálu,
- zajistit plnou auditovatelnost a integritu dat díky jedinému softwarovému prostředí.
Budoucí trendy a možnosti využití
Vývoj se bude ubírat směrem k plné automatizaci injekčních a předúpravových kroků, integraci HRAM a MS/MS dat pro inteligentní potvrzování výsledků a nasazení strojového učení pro rychlejší vyhodnocení chromatografických a hmotnostních dat. Dále lze očekávat rozšíření spektra sledovaných genotoxických nečistot a propojení s cloudovými platformami pro globální monitoring kvality léčiv.
Závěr
Analýza nitrosaminových nečistot vyžaduje kombinaci vysokého rozlišení, citlivosti a software pro dodržení přísných regulačních požadavků. Přístup založený na HRAM potvrzení a MS/MS screeningu představuje optimální strategii pro maximální jistotu výsledků a efektivní kontrolu kvality.
Reference
1. Thermo Fisher Scientific. Nitrosamine Impurities Analysis Solution Guide. 2021.
2. International Council for Harmonisation. ICH M7(R1) Guideline on Assessment and Control of DNA Reactive (Mutagenic) Impurities. 2017.
Obsah byl automaticky vytvořen z originálního PDF dokumentu pomocí AI a může obsahovat nepřesnosti.
Nitrosamine impurities
analysis solution guide
• Find the right analytical solution for each
stage of nitrosamine impurity analysis
• Confidently detect and quantify genotoxic
impurities in active pharmaceutical ingredients
and finished drug products
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Table of contents
Monitoring genotoxic impurities in pharmaceutical products
Why is nitrosamine impurity analysis in drugs important?
Requirements for nitrosamine analysis
Enabling analytical tools for each stage of nitrosamine impurity analysis
Compliance for all analytical solutions
Absolute confidence with High Resolution Accurate Mass
Cost effective nitrosamine screening with MS/MS
Are you at a cross-roads in choosing your nitrosamine analysis strategy?
LC-HRAM MS solution for absolute mass confirmation
Learn more about our LC-HRAM MS solution—products and resources 13
GC-HRAM MS solution for nitrosamine analysis gives ultimate
Learn more about our GC-HRAM MS solution—products and resources 15
LC-MS/MS solution for your targeted monitoring needs
Learn more about our LC-MS/MS solution—products and resources
GC-MS/MS solution for volatile analysis needs with confident targeted approach 18
Learn more about our GC-MS/MS solution—products and resources
IC Solution for nitrite analysis needs with confident targeted approach
Learn more about our IC solution—products and resources
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Monitoring genotoxic impurities in pharmaceutical products
Since elevated levels of nitrosamines were detected in certain pharmaceuticals
as first reported in July 2018, finding the right solutions have become the major
focus for many pharmaceutical manufacturers. Thermo Fisher Scientific™ as a
trusted partner for pharmaceutical analysis and quality control offers several
solutions to fit your needs. We are your partner to:
• Monitoring the levels of class 1 genotoxic impurities, which are known
mutagenic carcinogens according to IARC and ICH M7(R1) regulatory
guidelines
• Avoid any risks of recalls of your drug products
• Effortlessly follow and satisfy regulatory guidelines
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Why is nitrosamine impurity analysis in drugs important?
Nitrosamines are small molecular weight chemical substances that are
probable human carcinogens, and their presence in medicines is considered
unacceptable by regulators. They are organic compounds featuring a nitroso
group and amino group. They have the generic formula R
2N-N=O where
the R groups are typically alkyl in nature, they may be formed during API
manufacturing—from starting materials, intermediates, reactants, recycling of
solvents and the presence of nitrites and secondary amines.
In 2018, nitrosamine impurities, including NDMA, were discovered in Valsartan,
an Angiotensin II receptor blocker (ARB) medication used to treat high blood
pressure and heart failure. This led to a global recall of Valsartan and several
other ARB drugs.
First mostly N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine
(NDEA), two carcinogenic impurities were the target of careful monitoring
efforts. Since then, several other N-nitrosamines have also been identified
and are being investigated by regulators: N-Nitrosodiisopropylamine (NDIPA),
N-Nitrosoethylisopropylamine (NEIPA), N-Nitrosodibutylamine (NDBA), and
N-Nitroso-N-methyl-4-aminobutyric acid (NMBA).
The outcome that all events has made clear—there’s a recognized need for
a risk assessment strategy for potential nitrosamines in any pharmaceutical
product at risk for their presence.
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Nitrosamine impurity analysis requires robust and sensitive analytical
methods to ensure confidence in the obtained results.
The wide Thermo Scientific™ portfolio is proven to be best in class for
nitrosamine analysis, ensuring your exploratory and routine methods
are performed as accurately and reliably as possible while maintaining
requirements from regulatory bodies worldwide.
Our major products for the analysis of nitrosamines include:
• Liquid, gas, and ion chromatography for robust separation
• High resolution, accurate mass (HRAM) mass spectrometry for ultimate
confidence avoiding false positive results
• Tandem mass spectrometry, the best tool in routine analysis
• Single, compliance-ready software solution for all our technology solutions
Performance
Confidence
Service
and support
Compliance
Process
Product
assessment
Results
Requirements for nitrosamine analysis
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Enabling analytical tools for each stage of
nitrosamine impurity analysis
Featured nitrosamine solutions
We’re here for you each step of the way...
GMP compliant
software for
all analysis
Routine Screening
• Sensitive, high throughput assays
• Complementary analysis
• LC and GC-MS/MS
• To enable fast and reproducible
• Less hands-on
• Efficient monitoring
Highly confident detection
• High resolution accurate mass detection
• Impurity assessment and exploratory analysis
• Confident confirmation
• Routine assay capable
High throughput
assay automation
and optimization
Ingredient and product
monitoring
• Monitor nitrite and amine
levels during manufacturing
process and storage
• Risk monitoring
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No matter which solution fit your needs, Thermo Scientific™ Chromeleon™ Chromatography
Data System (CDS) is going to be there to assure confidence in your compliance.
Powerful, compliance-ready single solution platform for:
• Instrument control
• Data analysis and reporting
• In a secure, audit-trailed space
Compliance for all analytical solutions
• LC-HRAM
• LC-MS/MS
• GC-HRAM
• GC-MS/MS
• IC
• Automation
• Fit within existing laboratory infrastructure of
chromatographic systems
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Matrix interferences and or common solvents, such as DMF, from the
manufacturing process can lead to false-positive results with unit mass
resolution mass spectrometry. This is especially true if the interfering
compounds aren’t separated chromatographically.
We recommend Thermo Scientific™ Orbitrap™ technology as the gold standard
mass spectrometry technique as a minimum of 45k resolution is critical to
eliminate false-positive results. Whether you want to perform fast and confident
nitrosamine impurity screening and or peace-of-mind confirmatory analysis in
combination with triple quadrupole MS, HRAM MS should be a corner stone in
your workflow.
Absolute confidence with High Resolution Accurate Mass
Ultimate confidence—eliminate false positive results with HRAM.
75.0864
75.0650
75.0600
75.0550
75.0492
m/z
3.00
2.00
1.00
0
5.50
5.00
4.00
DMF, 15N isotope
0.4 ppm
NDMA
0.0 ppm
DMF, 13C isotope
0.0 ppm
Mass spectrum of ranitidine drug
tablet when co-elution of NDMA and
DMF happens, with a resolution setting
of 120,000 confident and accurate
identification of isotope masses
possible and allows for accurate
quantitation.
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Triple quadrupole mass spectrometry is at the forefront of trace analysis
of nitrosamines in pharmaceuticals. We recommend using this technology
for screening and in harmony with HRAM for positive result confirmation to
eliminate false positive results. MS/MS can increase lab productivity and reduce
workload on your HRAM systems.
“ Did you know that this strategy is currently used in the same manner for dioxin analysis in
food and feed? Why? This is an established solution for maximal productivity and ultimate
confidence in results.”
Cost effective nitrosamine screening with MS/MS
Increase your productivity with the analytical workhorse MS/MS and screen hundreds of samples per day.
Chromatograph of valsartan extract with
NDMA spiked at 5 ppb (ng/mL) on column
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What are the advantages of each approach?
• Universal applicability
• Common availability
• All FDA nitrosamines are LC amenable
• Ultimate sensitivity
• Excellent peak shape
• Highest column efficiency, best separation
• Maximum selectivity
• Zero false positives
• Ultimate confidence
• Unknown screening
• High throughput targeted screening
• Support of a HRAM confirmatory
strategy
• Cost effectiveness and commonality
MS/MS
HRAM
GC
LC
Are you at a cross-roads in choosing your
nitrosamine analysis strategy?
THE GOLD STANDARD
Compliance is at the center
whichever route you choose!
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Solution summary
Nitrosamine analysis
LC-MS
GC-MS
IC
Highly confident identification, quantitation,
and monitoring
—
Robust, high throughput routine screening
—
Laboratory workflow automation
—
Single compliance-ready software
Nitrite and nitrate assessment
—
—
Click on each listing for more information
HRAM LC-MS method for the
determination of nitrosamine
impurities in drugs
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LC-HRAM MS Solution for absolute mass confirmation
• Stable chromatography over extended period and hundreds of injections
• Confidence in analytical results allowing exceptional screening or confirmatory analysis
• Ease-of-use and compliance-ready software
• Chromatographic co-elution of NDMA and DMF may
cause over-estimation (false positive) if mass resolution
is not sufficient
• The mass difference between NDMA and DMF 15N
isotope is only 21 ppm, 0.002 amu difference—requires
minimum 45K resolution and 3 ppm mass accuracy
75.0864
75.0650
75.0600
75.0550
75.0492
m/z
3.00
2.00
1.00
0
5.50
5.00
4.00
DMF, 15N isotope
0.4 ppm
NDMA
0.0 ppm
DMF, 13C isotope
0.0 ppm
The Thermo Scientific™ Orbitrap Exploris™ 120 mass spectrometer
system provides high resolution accurate mass assuring absolute
identification and quantitation of nitrosamines.
Mass spectrum of ranitidine drug tablet when co-elution of NDMA
and DMF happens, with a resolution setting of 120,000 confident
and accurate identification of isotope masses possible and allows
for accurate quantitation.
Robust separation,
high load ability
Confident mass
detection
Fit-for-purpose
software
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Learn more about our LC-HRAM MS Solution
Products and resources
Click on each listing for more information
Contact LC-HRAM specialist now.
Robust separation, high load ability
Impurity separation with (U)HPLC platforms
• Thermo Scientific™ Acclaim™ PAII Columns
• Thermo Scientific™ Vanquish™ Flex UHPLC System
• Thermo Scientific™ Vanquish™ Core HPLC System
Confident mass detection
Mass detection
• Thermo Scientific™ Orbitrap Exploris™ 120 Mass Spectrometer
• Thermo Scientific™ Q Exactive™ Plus Mass Spectrometer
Fit-for-purpose software
Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System
Literature
• Application Note: HRAM LC-MS method for the determination of
nitrosamine impurities in drugs
• Application Note: High-resolution accurate-mass liquid
• Article: How to comply with FDA imposed nitrosamine impurity testing
• Brochure: Orbitrap Exploris 120
• Brochure: Chromeleon CDS software
Web tools
• Vanquish Selection tool
• Nitrosamine impurity analysis
Webinars
• Nitrosamine analysis; a leading CDMO’s perspective
• Virtual seminar: Nitrosamine analysis
Products
Resources
Next page
A validated method for the
rapid determination of fifteen
nitrosamines in metformin drug
substance
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GC-HRAM MS Solution for nitrosamine analysis
gives ultimate sensitivity and confidence
• Ultimate sensitivity
• Eliminate false positive results
• Ease-of-use and compliance-ready software
• Rapid separation of 15 nitrosamines in <12 minutes
• 10× <FDA regulatory limits of 30 ppb (ng/g) <2 ppb
• Excellent robustness over 2 weeks continual analysis
Retention time (minutes)
T
IC
(C
o
u
n
ts
)
5.0e7
4.0e7
3.0e7
2.0e7
1.0e7
0.0e0
4.40
5.00
6.00
7.00
8.00
9.00
10.00
11.00
1. NDMA
2. NMEA
3. NDEA
4. NIPEA
5. NDIPA
6.
NMPA
7. NDPA
8. NEBA
9. NEPhA
10. NMOR
11. NDBEA
12. NPYR
13. NPIP
14. NDBA
15. NDPhA
12.00
13.00 13.50
1
2
3
4
5
6
7
9
8 10
11, 12
13
14
15
counts
XIC of nitrosamine quantifier ions in a 50 ppb spiked sample.
The Thermo Scientific Orbitrap Exploris GC-MS system
provides high resolution accurate mass confirmation
while beating LOQ requirements.
Robust separation,
high load ability
Confident mass
detection
Fit-for-purpose
software
15
Learn more about our GC-HRAM MS Solution
Products and resources
Click on each listing for more information
Robust separation, high load ability
Impurity separation with GC platforms
• Thermo Scientific™ TraceGOLD TG-1701 MS Column
Confident mass detection
Mass detection
• Thermo Scientific™ Orbitrap Exploris™ GC Mass Spectrometer
Fit-for-purpose software
Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System
Literature
• Applicaton Note: GC Exploris HRAM validation of 15 nitrosamines
• Article: How to comply with FDA imposed nitrosamine impurity testing
• Case Study: GC solutions for nitrosamines
• Brochure: Orbitrap Exploris GC
• Brochure: Chromeleon CDS software
Web tools
• Orbitrap GC-MS
Webinars
• Nitrosamine analysis; a leading CDMO’s perspective
• Virtual seminar: Nitrosamine analysis
Products
Resources
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LC-MS/MS Solution for your targeted monitoring needs
• Stable chromatography over extended period and hundreds of injections
• Great for screening or confirmatory analysis
• Ease-of-use and compliance-ready software
• Vanquish Horizon UHPLC, Hypersil GOLD phenyl column
coupled to a TSQ Quantis Chromeleon CDS
• LOQ 5 ppb using APCI and 10 ppb using HESI
• Excellent reproducibility for over 1,000 sample injections
NDMA
NMEA
NPYR
NMBA
NDEA
NEIPA
NMPA
NDBA
NDIPA
NDPA
b) 20 ppb spiked sample
The Thermo Scientific Triple Quadrupole systems assure absolute
confidence in results for any targeted quantitation needs.
XIC of nitrosamine quantifier ions in a 20 ppb spiked sample. APCI
data are shown.
Robust separation,
high load ability
Robust and sensitive
mass detection
Fit-for-purpose
software
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Learn more about our LC-MS/MS Solution
Products and resources
Click on each listing for more information
Robust separation, high load ability
Impurity separation with (U)HPLC platforms
• Thermo Scientific™ Acclaim™ PAII Columns
• Thermo Scientific™ Vanquish™ Flex UHPLC System
• Thermo Scientific™ Vanquish™ Core HPLC System
Robust and sensitive mass detection
Mass detection
• Thermo Scientific™ TSQ Altis Triple Quadrupole
• Thermo Scientific™ TSQ Quantis Triple Quadrupole
• Thermo Scientific™ TSQ Fortis Triple Quadrupole
Fit-for-purpose software
Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System
Literature
• Application Note: Highly sensitive and robust LC-MS/MS solution for
quantitation of nitrosamine impurities in metformin drug products
• Application Note: TSQ Quantis application note
• Article: How to comply with FDA imposed nitrosamine impurity testing
• Article: Nitrosamine, a CDMO perspective
• Brochure: Chromeleon CDS software
Web tools
• Nitrosamine impurity analysis
Webinars
• Nitrosamine analysis; a leading CDMO’s perspective
• Virtual seminar: Nitrosamine analysis
Products
Resources
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GC-MS/MS Solution for volatile analysis needs with
confident targeted approach
• Faster data evaluation and increased confidence with exceptional levels of selectivity,
sensitivity, and linear dynamic range
• Versatility of full-scan, high-resolution accurate-mass data to screening and quantitation
• Intuitive instrument control and method templates
• Results that are compliant with the CFDA and U.S.
FDA standard methods for nitrosamines detection and
quantitation in Valsartan
• Three recommended instrumental approaches
(GC-MS, HS-GC-MS, GC-MS/MS)
• The static
headspace
injection technique
offers a simplified
workflow for
sample handling
Chromatograph of
valsartan extract with
NDMA spiked at
5 ppb (ng/mL) on column
For high-throughput analytical laboratories, the
Thermo Scientific TSQ 9000 GC-MS/MS system provides sensitive,
specific quantitation of target compounds.
High efficiency
separation
Robust and sensitive
mass detection
Fit-for-purpose
software
Determination of genotoxic
nitrosaminesin Valsartan with
gas chromatography and
mass spectrometry
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Learn more about our GC-MS/MS Solution
Products and resources
Click on each listing for more information
High efficiency separation
Impurity separation with (U)HPLC platforms
• Thermo Scientific™ TraceGOLD TG-1701 MS Column
Robust and sensitive mass detection
Mass detection
• Thermo Scientific™ TSQ 9000 GC-MS/MS System
Fit-for-purpose software
Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System
Literature
• Application Note: Determination of genotoxic nitrosamines in
Valsartan with gas chromatography and mass spectrometry
• GC Exploris HRAM validation of 15 nitrosamines in metformin drug
• Overcoming the challenges of nitrosamine impurities in drugs
• Brochure: Thermo Scientific TSQ 9000 Triple Quadrupole
• Brochure: Chromeleon CDS software
Web tools
• Nitrosamine impurity analysis
Webinars
• Nitrosamine analysis; a leading CDMO’s perspective
• Virtual Seminar: Nitrosamine analysis
Products
Resources
Nitrite in ranitidine drug product (S7) using a Dionex IonPac
AS19-4μm column
0
5
10
15
20
25
30
Minutes
-2
0
7
1
Peak
Analyte
Conc (µg/L)
1
Nitrite
95.5
mAU
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IC Solution for nitrite analysis needs with confident
targeted approach
• Little or no sample preparation is required
• Reagent-Free™ (RFIC™) ion chromatography system with electrolytically generated KOH eluent
• Multi drug product workflow
• The LOD of nitrite in a pharmaceutical sample is
0.918 ppm (µg/g API)
• The method is accurate and precise
• Applied to seven pharmaceutical samples, including
metformin, losartan, ranitidine, and diphenhydramine
The UV absorbance detection allows the sensitive and selective
detection of nitrite without interference from high amounts of chloride.
High capacity
columns
Selective
separations
Fit-for-purpose
software
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Learn more about our IC Solution
Products and resources
Click on each listing for more information
High capacity columns
Impurity separation with (U)HPLC platforms
• Thermo Scientific™ Dionex IonPac ™ AS19-4µm Column
Selective separation
Ion chromatography
• Thermo Scientific™ Dionex™ ICS-6000 Capillary HPIC™ System
Fit-for-purpose software
Software
• Thermo Scientific™ Chromeleon™ Chromatography Data System
Literature
• Application Note: Determination of nitrite in pharmaceuticals
• Brochure: Thermo Scientific Dionex ICS 6000 HPIC System
• Brochure: Chromeleon CDS software
Web tools
• IC home page
• Nitrosamine impurity analysis
Webinars
• Nitrosamine analysis; a leading CDMO’s perspective
• Virtual Seminar: Nitrosamine analysis
Products
Resources
For Research Use Only. Not for use in diagnostic procedures. © 2021 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. XX74154-EN 1221M
Find out more at thermofisher.com/nitrosamine
Need more information? Contact a specialist
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Fit-for-purpose,
reliable
quantitative
screening
Highly sensitive and robust
LC-MS/MS solution for quantitation
of nitrosamine impurities in
metformin drug products
Metformin
LC
Triple quad MS
Determination of genotoxic
nitrosamines in Valsartan
Valsartan
GC
Triple quad MS
Future proof your
lab with absolute
confidence and
quantitation
A validated method for the rapid
determination of fifteen nitrosamines
in metformin drug substance
Metformin
GC
High resolution
MS
HRAM LC-MS method for the
determination of nitrosamine
impurities in drugs
Ranitidine
LC
High resolution
MS
HRAM LC-MS methodology for
the determination and quantitation
of nitrosamine impurities in drug
products
Ranitidine
Valsartan
LC
High resolution
MS
Overcoming the challenges of
nitrosamine impurities in drugs:
What Pharmaceutical QA/QC
laboratories need to know
Metformin
Valsartan
GC
High resolution
MS Triple
quad MS
Ingredient
and product
monitoring
Determination of dimethylamine
and nitrite in pharmaceuticals by
ion chromatography to assess the
likelihood of nitrosamine formation
Multiple
IC
UV / CD
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