®

Application Note
Nitrosamines
Introduction

N-Nitrosodibutylamine

N-Nitrosomethylethylamine

1-Nitrosopyrrolidine

1-Nitrosopiperidene
Nitrosamines have come under enormous
scrutiny in the last few years since they

N-Nitrosodimethylamine

N-Nitrosodi-n-propylamine
were first found in API's and drug prod-
ucts. Widely suspected of being a human
carcinogen, Nitrosamines must now be

N-Nitrosodiethylamine
monitored for their presence and poten-
tial introduction in manufacture since being
found in Ranitidine and Metaformin, amongst
other drugs products, by the US FDA1.

Although hundreds of nitrosamines exist

they can vary widely in their chemical na-
ture being hydrophilic or hydrophobic in
nature. Therefore presenting a challenge to

“
0
Nitrosamines
Nitrosamines
5 10
–– EPA521
EPA521 15 20 25

Nitrosamines have become Nitrosamines – EPA521 Figure 1. Separation of Nitrosamines

choices, providing the ability to enhance

a hot topic due to risk of car- resolution for critical pairs of closely related
cinogenicNitrosamines
properties, and– EPA521 compounds, ideal for the Nitrosamines which
N-Nitrosodimethylamine
Nitrosamines – EPA521 can have very similar structures.
N-Nitrosodimethylamine N-Nitrosodibutylamine
N-Nitrosodibutylamine N-Nitrosodi-n-propylamine
N-Nitrosodimethylamine N-Nitrosodi-n-propylamine

potentially being so prevai-

N-Nitrosodibutylamine
N-Nitrosodi-n-propylamine

”
Nitrosamines – EPA521
lent Experimental Conditions
developing a methodN-Nitrosodibutylamine
N-Nitrosodimethylamine that can function for N-Nitrosomethylethylamine
N-Nitrosomethylethylamine
N-Nitrosodi-n-propylamine
N-Nitrosodiethylamine
N-Nitrosodiethylamine
N-Nitrosomethylethylamine Column: 3µm Evosphere® AQUA 100x2.1mm
N-Nitrosodiethylamine
N-Nitrosodimethylamine
many of these groups. Another key crite- N-Nitrosodibutylamine
N-Nitrosodi-n-propylamine
p/n EVOAQUA-020503
N-Nitrosodimethylamine
ria was to develop a simple screening HPLC N-Nitrosodibutylamine
N-Nitrosodi-n-propylamine
Mobile phase
method that could be used quickly and ef- A: 0.1% formic acid in water
ficiently even at low concentration
N-Nitrosomethylethylamine levels
N-Nitrosodiethylamine B: 0.1% formic acid in MeOH
1-Nitrosopiperidene 1-Nitrosopyrrolidine
without the requirement for LC-MS detection.
N-Nitrosomethylethylamine N-Nitrosodiethylamine
1-Nitrosopiperidene
N-Nitrosomethylethylamine
1-Nitrosopiperidene
1-Nitrosopyrrolidine
N-Nitrosodiethylamine
1-Nitrosopyrrolidine

Time %B
0.5 5
Experimental Analysis 14 95
In1-Nitrosopiperidene
this application note we1-Nitrosopyrrolidine
show the ability
15 95
of the new Evosphere AQUA column in con-
®
1-Nitrosopiperidene
1-Nitrosopiperidene1-Nitrosopyrrolidine
1-Nitrosopyrrolidine

junction with a simple mobile phase to pro- Figure 2. Nitrosamines structures

Flow Rate: 0.4ml/min
duce full resolution and offer good sensitivity.
Evosphere is built around a new Monodis- you get a much elevated backpressure and Temp: 40oC
perse Fully Porous Particle (MFPP) which the potential for blockage and robustness is- Detection: 254nm
is designed to provide more efficiency than sues, wghich can lead to a lack of confidence
traditional polydisperse particles. So in this in the method.
The 2nd part of this method development
application note a 3µm Evosphere particle is
work was to run the Nitrosamines with a
providing the efficiency and sensitivity that The MFPP will provide better packed col-
sample of Ranitidine to see if detection of
would be expected if using a sub 2µm UHPLC umns, less band broadening and 40-50%
both species could be achieved. For this the
particle. If you run with a UHPLC particle then greater efficiency than other equivalent silica
particles in HPLC, therefore giving higher gradient was changed to:
1. Control of Nitrosamine Impurities in Human resolution and sensitivity. Bonded to this
Drugs, Guidance for Industry. February 2021 Phar- MFPP is a selection of new stationary phase
maceutical CGMP, Rev1. ® ®
Fortis and Evosphere are registered trademark of Fortis Tech-
www.fortis-technologies.com Tel: +44 151 336 2266 nologies. All columns are original manufacturers own.
Column: 3µm Evosphere® AQUA 100x2.1mm

Ranitidine
p/n EVOAQUA-020503
Mobile phase
A: 0.1% formic acid in water
B: 0.1% formic acid in MeOH

Time %B
0 5
5 5
15 45 6
4
30 95 7
1 2 3 5

Flow Rate: 0.4ml/min

Temp: 40oC
Detection: 254nm 0 5 10 15 20 25 30 35
Figure 3. Separation of Nitrosamines and Ranitidine

organic proportion of the mobile phase or in-

crease the temperature to obtain a faster run
time. If other metabolites were present then
there is also plenty of scope to shallow the
gradient to gain increased resolution.
When looking for the Nitrosamines in a drug
Figure 4. Ranitidine
substance sample, in this case Ranatidine,
good loading of the parent drug and sharp
efficient peak shapes for the nitrosamines al-
low a high sensitivity, high resolution method
Conclusion to be developed.
In this application note we have shown a ro-
The use of a monodisperse particle has pro-
bust LC-UV method for separation of some of
vided a significant gain in performance in
the Nitrosamines. The analysis is completed
terms of resolution and sensitivity for these
quickly and with good overall resolution be-
compounds. With the simple mobile phase
tween analytes, however if further gains in
utilised it would also be easy to transfer this
speed were required there is scope to de- ® ®
Fortis and SpeedCore are a registered trademark of Fortis
to LC-MS if required.
crease the size of the column, or increase the Technologies. All columns are original manufacturers own.

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