The document discusses the challenges with targeted nitrosamine analysis in pharmaceutical products and introduces Ellutia's 800 Series TEA Detector for total nitrosamine analysis as a screening tool. It provides examples of total nitrosamine analysis of samples like shampoo and tablets. The conclusion is that total nitrosamine screening allows more efficient use of resources to identify positives for further targeted analysis compared to targeted analysis of all samples.

2. The need for non-targeted analysis of
Nitrosamines within the Pharmaceutical
Industry

3. Agenda
History of nitrosamines in pharmaceutical
products
Challenges with a targeted approach
Ellutia’s 800 Series TEA Detector
Total Nitrosamine Analysis
Examples
Conclusion

4. History of Nitrosamines in
Pharmaceutical products

5. History

6. History

7. History
Name CAS Number ng/day
N-Nitrosodimethylamine, NDMA 62-75-9 96
N-Nitrosodiethylamine, NDEA 55-18-5 26.5
N-Nitrosoethylisopropylamine, EIPNA 16339-04-1 26.5
N-Nitrosodiisopropylamine, DIPNA 601-77-4 26.5
N-Nitroso-N-methyl-4-aminobutyric acid, NMBA 61445-55-4 96
1-Methyl-4-nitrosopiperazine, MeNP 16339-07-4 26.5
N-Nitroso-di-n-butylamine, NDBA 924-16-3 26.5
N-Nitroso-N-methylaniline, NMPA 614-00-6 34.3
N-Nitrosomorpholine, NMOR 59-89-2 127
N-Nitrosovarenicline, NNV 37
N-Nitrosodipropylamine, NDPA 621-64-7 26.5
N-Nitrosomethylphenidate, NMPH 55557-03-4 1300
N-Nitrosopiperidine 100-75-4 1300
N-Nitrosorasagiline 2470278-90-9 18
7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3- a]pyrazine 37
N-Nitroso-1,2,3,6-tetrahydropyridine, NTHP 55556-92-8 37
N-Nitrosonortriptyline 55855-42-0 8
N-Methyl-N-nitrosophenethylamine, NMPEA 13256-11-6 8
N-Nitrosodabigatran 18
4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone, NNK 64091-91-4 100
N-nitrosoduloxetine 2680527-91-5 100
N-nitroso-fluoxetine 150494-06-7 100
N-nitrosoparoxetine 2361294-43-9 1300
N-nitroso-diphenylamine, NDPhA 86-30-6 78000
N-nitroso-mefenamic acid 2114-63-8 78000
N-nitroso-pyrrolidine, NPYR 930-55-2 1700
N-nitroso-diethanolamine, NDELA 1116-54-7 1900
Regulation (EC) No 726/2004

8. Challenges with a Targeted
Approach to Nitrosamine
Analysis

9. Targeted Testing -
Challenges
• Methods have been developed to meet requirements of
sensitivity and other typical validation criteria
• These are for specific known nitrosamines
• Sample prep guidelines are generally for specific matrixes

10. Targeted Testing -
Challenges
• Creating a target list is critical to the risk assessment process
• This assumes a that you know what you are looking for
• This may be limited by current understanding
• Potential to exclude “unexpected nitrosamines”
• Standards are available

11. Targeted Testing -
Challenges
• Analytical Challenges
- Detecting any nitrosamine present, not just those that you have standards for.
- Concentrating the sample sufficiently to meet the required LOD.
• Resources – Speed vs Cost
- Where analyte resolution is required, this takes time.
- If the throughput is low, are only finished products tested?
- Sample numbers can be increased by buying more analysis systems (GC/LC-MS-MS)
- Expensive equipment, specialist gases, convoluted extraction procedures.

12. Ellutia’s 800 Series TEA
Detector

13. 800 Series TEA
Detector
• Nitroso, nitro and
nitrogen
chemiluminescence
detector.
• Used for the detection of
nitrosamines in many
industries.
 Rubber products.
 Smoked foods and
meats.
 Brewing and
malting.
 Agrochemicals.
 Cosmetics.
 Pharmaceutical Pyrolyser

14. 800 Series TEA
Detector
• One result for apparent total nitrosamine content (ATNC).
 Both volatile and non-volatile nitrosamines detected in a single injection.
• No separation of compounds – quantitative, not qualitative analysis.
• Positive samples can be swiftly identified for further speciated analysis.
• Ideal for screening and quality checking.
• Result usually given as N-N=O concentration.
• Independent of ‘R’ groups, therefore not critical which nitrosamine is used for calibration purposes.
 1.0mg/L NDMA = 0.60mg/L N-N=O
 1.0mg/L NDPA = 0.34mg/L N-N=O
 1.0mg/L NDELA = 0.33mg/L N-N=O

15. 800 Series TEA
Detector
The chemical strip reaction.
The 800 TEA reaction.
The 800 TEA detection.

16. Total Nitrosamine Analysis

17. Total Nitrosamine
Analysis
1. Immersion Chiller
2. Lab Jack
3. Heater/Magnetic Stirrer
4. Reaction Vessel
5. Double Surface Condenser
6. Peristaltic Pump
7. Lab Jack
8. Coolant Reservoir
9. Cold Trap
10. 800 Series TEA
Vacuum pump – not shown
Ellutia has been selling a manual/glassware chemical stripping
system globally for decades.

18. Automated Total
Nitrosamine Analysis as a
Screening Tool
• Much larger sample volume
• Sharper peaks
• Better signal to noise ratio
• As low as .5 ppb

19. Total Nitrosamine
Analysis
Calibration Standards
Chromatograms of the standards for 0 to 250ng/mL of NDMA in methanol (0 to 297ng of the N-N=O molecule; 2mL sample volume).
[min]
Time
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
[mV]
Voltage
0
100
200
300
400

20. Total Nitrosamine
Analysis
Calibration Standards
[min]
Time
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
[mV]
Voltage
-5
0
5
10
15
20
25
Chromatograms of the standards for 0 to 10ng/mL of NDMA in methanol (0 to 11.9ng of the N-N=O molecule; 2mL sample volume). Highlighting the sensitivity at
the lower limits of detection in this system.

21. Total Nitrosamine
Analysis
Calibration Curve

22. Total Nitrosamine
Analysis
Reproducibility

23. Total Nitrosamine
Analysis
Reproducibility
Conc N-N=O (ng) Area
1 1.18918919 108.462 MEAN 107.4363
1 1.18918919 104.735 SD 2.36
1 1.18918919 109.112 %RSD 2.20
Conc N-N=O (ng) Area
5 5.94594595 219.417 MEAN 220.0883
5 5.94594595 222.351 SD 2.01
5 5.94594595 218.497 %RSD 0.91
Conc N-N=O (ng) Area
50 59.4594595 1059.494 MEAN 1067.97
50 59.4594595 1114.368 SD 42.79
50 59.4594595 1030.047 %RSD 4.01
As this is an external standard headspace method, these %RSD values are excellent

24. Examples

25. Examples
Shampoo
Sample
Sample Vol
(mL)
Extraction Volume
(mL)
Inj Vol
(µL)
Area
ng N-N=O
injected
ng N-N=O in
sample
ng/g or mL N-N=O
in sample
Example Shampoo 2 2 2000 343.411 31.2 31.2 15.6
Example Shampoo 2 2 2000 339.180 30.8 30.8 15.4
Example Shampoo 2 2 2000 346.413 31.5 31.5 15.7
Sample Preparation – Add 2mL shampoo to a 10mL headspace vial, add Reaction Mixture, cap

26. Examples
Tablets
Sample
Sample Mass
(g)
Extraction Volume
(mL)
Inj Vol
(µL)
Area
ng N-N=O
injected
ng N-N=O in
sample
ng/g or mL N-N=O
in sample
Example Tablets 20.558 20 2000 903.762 85.1 850.6 41.4
Example Tablets 20.558 20 2000 949.476 89.5 894.5 43.5
Sample Preparation – Weigh ground tablets into an extraction vessel. Add extraction solvent (MeOH). Vortex and place on a roller to
extract. Centrifuge and then filter the supernatant. Add 2mL supernatant to a 10mL headspace vial, add Reaction Mixture, cap

27. Examples
Removing Nitrites/Nitrates - Experimental Plan
An SPE Cartridge has been developed at Ellutia with the intention to remove nitrites/nitrates from a
simulated extraction of a pharmaceutical product containing a mixture of nitrites and nitrosamines.
10ng/ml standards of NDMA and NaNO₂; and a mixture of NDMA and NaNO₂, both at a
concentration of 10ng/ml in methanol were prepared and analysed with no further sample
preparation.
Three aliquots of 5ml of methanol was passed through three cartridges.
Three aliquots of 5ml of 10ng/ml NDMA in methanol was passed through three cartridges.
Three aliquots of 5ml of 10ng/ml NaNO₂ in methanol was passed through three cartridges.

28. Examples
Chromatogram of Blank Standard
An example chromatogram showing the blank standard with %RSD of less than 10%. It is expected
to see a rise in baseline from the injection pulse
[min]
Time
0.0 0.5 1.0 1.5 2.0 2.5
[mV]
Voltage
-8
-6
-4
-2
0
1

29. Examples
Chromatogram of NDMA
Two example NDMA Chromatograms displayed against the blank standard. The NDMA standard and
NDMA standard that was passed through the cartridge are seen to have similar area, indicating that
the cartridge allows nitrosamines to pass through.
[min]
Time
0.5 1.0 1.5 2.0 2.5
[mV]
Voltage
-10
0
10
20
30
1

30. Examples
Chromatogram of NaNO₂
[min]
Time
0.5 1.0 1.5 2.0 2.5
[mV]
Voltage
-10
-5
0
5
10
15
20
25
Two example NaNO₂ Chromatograms displayed against the blank standard. Passing the NaNO₂
standard through the cartridge results in an area similar to that of the blank standard. This indicates
that the cartridge prevents nitrites from passing through.

31. Examples
Chromatogram of NDMA with NaNO₂
[min]
Time
0.5 1.0 1.5 2.0 2.5
[mV]
Voltage
-10
0
10
20
30
40
50
60
The NaNO₂ with NDMA standard shows the largest area, as expected. Passing the NaNO₂ with
NDMA standard through the cartridge results in a peak area similar to the NDMA standard. This
indicates that the cartridge prevents nitrites from passing through, yet still has no effect on the

32. Conclusion

33. Conclusion
A rapid, simple, cheap, reliable screening tool for total nitrosamines,
allowing the more expensive extraction and identification methods to be
used solely on positive samples, is an efficient means to screen large
numbers of samples that are expected to be negative for nitrosamines.
It has been demonstrated that the prototype the Ellutia SPE Cartridge is
capable of removing nitrites from a simulated methanolic extraction of a
pharmaceutical product without removing an NDMA standard of
10ng/ml.

34. Summary
History of nitrosamines in pharmaceutical
products
Challenges with a targeted approach
Ellutia’s 800 Series TEA Detector
Total Nitrosamine Analysis
Examples
Conclusion

35. Any Questions?

36. Email:
Andrew.James@Ellutia.com
Phone: +44(0)1353 669916
Download a copy of this
presentation
Andrew James
Managing Director