Application Note
Pharma & Biopharma

Screening of Nitrosamine Impurities
in Drug Products and Drug
Substances Using Agilent GC/MS/MS
Instrumentation

Authors

Abstract

Carole Antoine, Milan
Macé, Elodie Barrau, andEmmanuel Desmartin
Eurofins Amatsi Analytics

This application note highlights a comprehensive solution for the screening andestimation of 13 nitrosamine impurities (NDMA, NDEA, NMOR, NMEA, NPYR, NPIP,
NEIPA, NDIPA, NDPA, NDBA, NMPA, NMPEA and NDPh) in drug products and drug
substances in both organic or aqueous matrices at trace levels using an Agilent
8890 GC coupled to an Agilent 7010B triple quadrupole GC/MS/MS system. Briefly,
product is either dispersed in methylene chloride or in water following a liquid-liquid
extraction depending on its own solubility.

Laurent Pascaud
Agilent Technologies, Inc.

Calibration is made using 1 to 60 ppb calibration solutions, LOQs are obtained
between 1 and 10 ppb and satisfying recovery are obtained.
The 7010B triple quadrupole GC/MS/MS is equipped with a high-efficiency source(HES) that offers excellent sensitivity, repeatability, and precision. This method
allows for the performance of an initial screening of drug products (after risk
evaluation) to confirm the presence or absence of nitrosamines.

Introduction
For nearly four years, the concerns
regarding trace levels of N-nitrosaminesin pharmaceuticals and the associatedcancer risk have significantly expanded
and are a major issue facing the global
pharmaceutical industry. According
to FDA and EMA requirements, first, amarketing authorization holder mustcarry out a risk-based assessment ofeach product. Then, a confirmatory
testing should, therefore, be performed
using appropriately validated andsensitive methods, and Marketing
Autorisation Holders (MAHs) should
inform the competent authorities
immediately if tests confirm thepresence of one or several nitrosamine
impurities, irrespective of theamount detected.
After that, MAHs should apply fora variation in a timely manner tointroduce any required changes,
such as an amendment of the
manufacturing process or changes to
product specifications.
The screening testing can be performed
either by liquid chromatography-mass
spectrometry (LC/MS/MS) or bygas chromatography-tandem mass
spectrometry (GC/MS/MS), using
a generic sample preparation andanalysis method.
These standardized procedures can be
simplified according to the galenic form
(powder, tabs, pills, liquid, cream, and soon), drug substance (DS), or drug product(DP) properties (polarity, solubility). The
use of LC or GC technique will dependon the nitrosamines that have to bescreened, the limits to be achieved, andthe thermal stability of DP and excipient.

2

Although the screening was initially
focused on N-nitrosodimethylamine(NDMA) and N-nitrosodiethylamine(NDEA), the list used in this method
can be extended to other common or
specific nitrosamines according to therisk assessment.
Our current GC/MS/MS screening allows
to confirm 13 of them:N-nitrosodimethylamine (NDMA),
N-nitrosodiethylamine (NDEA),
N-nitrosomorpholine (NMOR),
N-nitrosomethylethylamine(NMEA), N-nitrosopyrrolidine(NPYR), N-nitrosopiperidine (NPIP),
N-nitrosoethylisopropylamine (NEIPA),
N-nitrosodiisopropylamine (NDIPA),
N-nitrosodipropylamine (NDPA),
N-nitrosodibutylamine (NDBA),
N-nitrosomethylphenylamine (NMPA),
N-nitrosomethyl-2-phenylethylamine(NMPEA), and N-nitrosodiphenylamine(NDPh).
The screening approach is to apply thisgeneric method to several batches ofthe pharmaceutical product (at least
three or four batches, representativeof the life cycle). The validity of thescreening will be assessed by checking
for each product:– The sensitivity of the method atlimit of detection (LOD) and limit ofquantification (LOQ) levels– The selectivity in the matrix– The recovery (accuracy) of thegeneric method using spiked samples
at two concentration levels
The approach for this limit assay caneither target one nitrosamine, or screen
the full list.

In case of a positive result for thescreening of one or more nitrosamines,
a specific quantitative method has
to be developed and validated for a
confirmatory testing.
This confirmatory method can either bean update of the generic method used
for the screening, or a method developed
specifically to the intended use (sample
preparation, analysis technique,and detection).
This study performed the screening
method using the 8890 GC coupled
to the 7010B GC/MS/MS and foundthat this system provides excellent
performances for the 13 nitrosamines
screened. The HES, with improved
ionization efficiency and 20x ion
generation characteristics, delivers
confident trace analysis. The 8890 GC
has a touch screen interface instead ofa keypad to control the GC, and offers
diagnostic tests, system monitoringalerts, and mobile access.

Experimental
Sample preparation
DS and DP have to be dispersed forproper analysis. Here, two different
matrices (water and methylene chloride)
have been tested for further drug
products and DS analysis using two
different methods of preparation.
For samples soluble in organic solvents,
500 to 1,000 mg of sample (DS or DP)
are directly dispersed into 5.0 mL of
MeCl2 and filtered through a ww-PTFE
MS 0.2 µm syringe filter.
For samples soluble in water matrices,
200 to 1,000 mg of sample are dispersed
into 8.0 mL of extraction mixture(NaOH 1 M solution in water). Then, aliquid‑liquid extraction is made using
2.0 mL of MeCl2 The organic fraction isfiltered through a ww-PTFE MS 0.2 µm
syringe filter and analyzed.

Standard preparations

Particular attention

Instrumentation

Deuterated nitrosamines are used asinternal standards: NDMA-d6, NDEA-d10,
NDIPA-d14 and NDBA-d18.

The two following technical aspects
required particular attention duringexperimental analysis:

Individual internal standard stock
solutions (IS stock) and individual
nitrosamine stock solutions (nitro stock)were prepared by weighing each pureanalytical standard in 20 mL glass tubes,
then dissolving into methanol to reach
1,000 µg/mL concentration.

1. Interferences caused by the presenceof trace amounts of nitrosamines intesting materials used (e.g. water,
airborne sources, plastic products,
rubber/elastomeric products,
smokers, etc.)

Analysis was performed using
Agilent 8890 GC equipped with an
Agilent 7693A automatic liquid sampler
coupled to an Agilent 7010B triple
quadrupole GC/MS/MS. From the inlet,
an Agilent J&W DB-1701 GC capillarycolumn (14% cyanopropylphenyl/86%
dimethylpolysiloxane) of dimensions
30 m × 0.25 mm, 1.0 µm was connectedto the MS/MS detector.

A mixed nitrosamine standard solution
and a mixed internal standard solution
at 1 µg/mL are prepared by mixing
each nitro stock (or IS stock) solution
with MeCl2.
Mixed nitrosamine standard solution isdiluted appropriately with MeCl2 to obtaincalibration solutions of the following
concentrations: 0.01, 0.05, 0.1, 0.2, 0.5,
1.0, 2.0, 3.0, 4.0 and 6.0 ng/mL (andat 3 ng/mL of each internal standard).
When samples are not soluble in organic
solvents, standard solutions undergo
the extraction procedure described
previously using 2.0 mL of MeCl2 without
filtration. Calibration solutions are thentransferred into a GC vial for analysis.

2. In situ formation of nitrosamines

Dwell was chosen to have sufficient data
points avoiding sharp peaks.
Tables 1 and 2 display the GC andMS parameters.

Table 1. GC parameters.
Parameter

Value

MMI Injection Mode

Splitless, pressure: 7.334 psi,
Total flow: 34 mL/min

Inlet Temperature

220° C

Oven Temperature Program

40 °C (0.5 min)
20 °C/min to 160 °C (0 min)
10 °C/min to 200 °C (0 min)
25 °C/min to 240 °C (2 min)
100°C/min to 280°C (10 min)Total Run Time

24.5 minMS Transfer Line Temperature

280 °CInjection Volume

1 µL

Carrier Gas

Helium, 1 mL/min

Table 2. Repeatability calculated on standards.
Repeatability
Std

DirectDispersion

Aqueous
Extraction

Compound%RSD (n = 3)

%RSD (n = 3)

NDMA

2%

1%

NMEA

1%

2%

NDEA

1%

1%

NEIPA

1%

1%

NDIPA

4%

2%

NMPA

7%

4%

NDPA

1%

1%

NMOR

1%

1%

NPYR

2%

2%

NPIP

1%

1%

NDBA

5%

3%

NMPEA

4%

2%

NDPh

5%

3%

3

Results and discussion
The nitrosamines were separated
chromatographically and detectedusing the advantage of a GC/MS/MS.
The resolution between compounds
was good enough to prevent cross‑talkissues, and the target peaks were
well resolved from solvent and matrix
compounds. Retention times of 13
compounds are presented in Figure 1.
Injection repeatability was determinedwith standard solution verifying RSD<20% at the 30 ppb level.

Table 3. MS parameters.
Parameter

Value

Mode

Electron ionization, 70 eV

Source Temperature

230 °C

Quadrupole Temperature

Q1 and Q2 = 150 °C

MRM Mode Conditions
MS1 Resolution

All compounds Unit

MS2 Resolution

All compounds Wide

Collision Gas Flow

Nitrogen at 1.5 mL/min,

Quenching Gas Flow

Helium at 2.25 mL/min

Detector Gain

2

The accuracy of the method wasdetermined by injecting three
preparations of sample solution spiked
at six different levels: 1, 2, 5, 10, 30, and60 ppb, verifying RSD ≤25% and recovery
within 70 and 130%.

Time segment 1
Start time: 5.00 min

NDMA
74 & 44, CE 6 V
74 & 42, CE 20 V
NDMA-d6
80 & 46, CE 25 V
NMEA
88 & 71, CE 5 V
88 & 56, CE 15 V

Time segment 2
Start time: 6.75 min

NDEA
102-> 85 CE 4V
102 -> 56 CE 18V
NDEA-d10
112 & 62, CE 15 V
NEIPA
116 & 99, CE 3 V
71 & 56, CE 7 VTime segment 3
Start time: 7.90 min

NDIPA
130 & 88, CE 3 V
130 & 42, CE 10 V
NDIPA-d14
144 & 96, CE 5 V
NMPA
107 & 106, CE 14 V
106 & 51, CE 34 V
NDPA
101 & 70, CE 3 V
130 & 43, CE 10 V

Time segment 4
Start time: 8.95 min

NMOR
116 & 86, CE 5 V
116 & 56, CE 15 V
NPYR
100 & 68, CE 10 V
100 & 41, CE 22 V
NPIP
114 & 55, CE 23 V
114 & 42, CE 30 V

Time segment 5
Start time: 9.90 min

NDBA
158 & 99, CE 7 V
84 & 56, CE 22 V
NDBA-d18
176 & 110, CE 10 V

Time segment 6
Start time: 11.50 min

NMPEA
91 & 65, CE 17 V
134 & 91, CE 14 V
NDPh
169 & 168, CE 18 V
168 & 167, CE 22 V

Quant./Qual. Transitions(FDA Method)

4

×104

13

8.5
8.0
7.5
7.0
6.5
6

6.0
5.5

Counts

5.0
4.5
4.0
3.5
3.0
2.5

1

12

4

2.0
3

1.5

5

2

1.0

8
10
11

7
9

0.5
0
6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

14.5

Acquisition time (min)Figure 1. MRM TIC chromatogram overlay of nine calibration levels for the 13 impurities in dichloromethane (Agilent 8890 GC). (1) NDMA, (2) NMEA, (3) NDEA,(4) NEIPA, (5) NDIPA, (6) NMPA, (7) NDPA, (8) NMOR, (9) NPYR, (10) NPIP, (11) NDBA, (12) NMPEA, (13) NDPh.

5

For both sample preparation methods(with water and methylene chloride),
excellent recoveries within 70 to130% were observed for each of thenitrosamines with recovery % RSD lessthan 20% at 30 ppb. The findings arereported in Tables 4 and 5.
The response linearity for nitrosamines
was evaluated over the range studied.
A mixed nitrosamine standard solution
and a mixed internal standard solution at
1 µg/mL are prepared with MeCl2.

Table 4. Repeatability calculated on samples using the direct dispersion in MeCl2 procedure.
Sample Repeatability Obtained Doing a Direct Dispersion in MeCl2
Mean Recovery%RSD

Level (ppb)

1

2

5

10

30

60

30

NDMA

93

97

98

96

98

98

1.0

NMEA

101

102

103

101

102

106

2.0

NDEA

109

110

99

98

95

96

1.3

NEIPA

108

104

103

102

104

109

2.3

NDIPA

121

110

100

98

96

96

3.4

NMPA

122

108

95

85

117

NA(2)

6.5

NDPA

104

103

99

95

99

99

2.6

NMOR

116

107

97

94

97

96

2.9

NPYR

NA(1)

128

109

103

105

101

1.8

NPIP

120

117

117

104

95

NA(3)

2.5

NDBA

112

108

97

99

103

NA(2)

2.2

NMPEA

NA(1)

76

119

107

102

106

7.5

NDPh

123

108

87

80

89

87

2.5

(1) Not applicable because S/N <10 for 1 ppb.
(2) Not applicable because accuracy performed with a calibration range from 1 to 40 ppb following
out‑of‑acceptance criteria obtained for accuracy levels 1 and 2 ppb with a calibration range from 1 to 60 ppb.
(3) Not applicable because accuracy performed with a calibration range from 1 to 40 ppb following
out‑of‑acceptance criteria obtained for accuracy level 1 ppb with a calibration range from 1 to 60 ppb.

Table 5. Repeatability calculated on samples using the aqueous extraction procedure.
Sample Repeatability Obtained Doing an Aqueous Extraction
Mean Recovery
Level (ppb)

1

2

5

10

30

30

NDMA

115

99

98

95

95

2.5

NMEA

111

104

104

103

103

3.0

NDEA

95

95

97

96

98

3.3

NEIPA

107

107

102

102

104

3.9

NDIPA

126

106

102

97

98

4.5

NMPA

125

96

82

79

75

7.3

NDPA

104

108

106

103

99

3.7

NMOR

101

97

97

98

96

3.2

NPYR

NA

(1)

134

106

100

97

4.1

NPIP

107

101

105

101

98

4.0

NDBA

81

92

99

109

104

7.5

NDPh

92

91

93

92

87

5.9

(1) Not applicable because S/N <10 for this level.

6

%RSD

Linear curves were generated using
a linear fit. Correlation coefficient isequal to 1.00 for all nitrosamines on
the studied range, which complies withacceptance criteria (r ≥0.98), establishingexcellent linear response through therange (Tables 6 and 7).
To assess the sensitivity, LODs were
calculated based on the signal‑to‑noise(S/N) values for diluted standards
to determine which LODs could beachieved, enabling very trace level
detection. The LODs were in the rangeof 0.05 to 2 ppb and LOQs based on
accuracy results were in the range of
1 to 10 ppb. This result allows a goodscreening sensitivity, which should
be checked on each DS or DP matrix,
by spiking, in the run during the dayof analysis.
Calibration curves, correlation coefficient,and LOD are presented in Tables 6 and 7.

Table 6. Calibration curve obtained and validated range on the 13 nitrosamines screened with associatedLODs using the direct dispersion in MeCl2 procedure.
Compound in
MeCl2

Range Studied

NDMA

1 to 60 ppb

y = 0.073036x + 0.003881

1.00

0.1 ppb (S/N = 3)

NMEA

1 to 60 ppb

y = 0.054100x – 0.001531

1.00

0.05 ppb (S/N = 4)

NDEA

1 to 60 ppb

y = 0.065633x – 0.002706

1.00

0.05 ppb (S/N = 4)

NEIPA

1 to 60 ppb

y = 0.112296x – 0.011425

1.00

0.05 ppb (S/N = 6)

NDIPA

1 to 60 ppb

y = 0.033903x – 0.010897

1.00

0.1 ppb (S/N = 3)

NMPA

1 to 40 ppb(1)y = 0.113250x – 0.022100

1.00

0.1 ppb (S/N = 3)

NDPA

1 to 60 ppb

y = 0.014889x – 0.002761

1.00

0.05 ppb (S/N = 3)NMOR

1 to 60 ppb

y = 0.090466x – 0.020014

1.00

0.05 ppb (S/N = 6)

NPYR

(2)

2 to 60 ppb

y = 0.007464x – 0.005057

1.00

0.5 ppb (S/N = 3)NPIP

1 to 40 ppb(3)y = 0.020842x + 0.004194

1.00

0.5 ppb (S/N = 7)

NDBA

1 to 40 ppb

y = 0.061255x + 4.087211E-004

1.00

0.5 ppb (S/N = 12)

NMPEA

2 to 60 ppb(2)y = 1.188428x + 0.061221

1.00

1 ppb (S/N = 7)

NDPh

1 to 60 ppb

y = 2.326033x – 0.108300

1.00

0.05 ppb (S/N = 6)(1)

Equationr

Working LOD and AssociatedConcentration

With y = peak area ratio and x = concentration (ppb)(1) Range studied from 1 to 40 ppb due to the out-of-acceptance criteria obtained for accuracy levels 1 and 2 ppb
with a range from 1 to 60 ppb.
(2) Range studied from 2 to 60 ppb due to the S/N <10 obtained for standard solution at 1 ppb.
(3) Range studied from 1 to 40 ppb due to the out-of-acceptance criteria obtained for accuracy level 1 ppb with arange from 1 to 60 ppb.

Table 7. Calibration curve obtained and validated range on the 13 nitrosamines screened with associatedLODs using the aqueous extraction procedure.
Compound in
Water

Range Studied

NDMA

1 to 30 ppb

y = 0.089432x + 0.011075

1.00

0.05 (S/N = 4)

NMEA

1 to 30 ppb

y = 0.079262x + 0.015512

1.00

0.05 (S/N = 6)

NDEA

1 to 30 ppb

y = 0.067200x + 0.009409

1.00

0.1 (S/N = 3)

NEIPA

1 to 30 ppb

y = 0.118563x + 0.021747

1.00

0.05 (S/N = 3)

NDIPA

1 to 30 ppb

y = 0.035273x – 0.005707

1.00

0.05 (S/N = 5)

NMPA

1 to 30 ppb

y = 0.178605x – 0.055821

1.00

0.5 (S/N = 5)

NDPA

1 to 30 ppb

y = 0.016135x – 9.566144E-004

1.00

0.05 (S/N = 6)

NMOR

1 to 30 ppb

y = 0.078884x + 0.001414

1.00

0.1 (S/N = 3)

NPYR

2 to 30 ppb

y = 0.007386x – 0.004633

1.00

0.05 (S/N = 4)NPIP

1 to 30 ppb

y = 0.024298x + 0.006008

1.00

0.1 (S/N = 3)

NDBA

1 to 30 ppb

y = 0.059460x + 0.008600

1.00

0.1 (S/N = 2)

NMPEA

10 to 30 ppb(2)y = 1.082170x – 1.109961

1.00

2 (S/N = 4)NDPh

1 to 30 ppb

y = 2.278087x + 0.482510

1.00

0.05 (S/N = 3)

(1)

Equationr

Working LOD and AssociatedConcentration (ppb)With y = peak area ratio and x = concentration (ppb)(1) Range studied from 2 to 30 ppb because LOQ reached at 2 ppb for NPYR (S/N <10 obtained for standard
solution at 1 ppb).
(2) Range studied from 10 to 30 ppb because LOQ reached at 10 ppb for NMPEA (S/N <10 obtained for standard
solutions at 1, 2, and 5 ppb).

7

Conclusion

References

The results of theseexperiments demonstrate:

1. EMA/369136/2020/EMEA/
H/A-5(3)/1490: https://www.ema.
europa.eu/en/documents/referral/
nitrosamines-emea-h-a53-1490assessment-report_en.pdf– A good resolution between the
13 compounds– Accurate and precise recovery withspiking concentrations within 70 to130% recovery and % RSD <20% at
30 ppb level– Excellent linearity was observed in
the range– Low detection limits were achieved
These features enabled reliable
screening of all 13 residues. Each day ofanalysis, the reliability of the method isverified by spiking the analyzed matrices
with each required nitrosamine. This
allows estimation of the % recovery of
spiked samples.
This method highlights the capabilitiesof the 8890-7010B GC/MS/MS system
for the detection and screening of
13 nitrosamine drug impurities, attrace level, in drug substances anddrug products.

www.agilent.com
DE.6920601852
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023
Printed in the USA, February 24, 2023
5994-5795EN

2. EMA/409815/2020: https://www.
ema.europa.eu/en/documents/
referral/nitrosamines-emea-ha53-1490-questions-answersmarketing-authorisation-holders/applicants-chmp-opinion-article-53regulation-ec-no-726/2004-referralnitrosamine-impurities-humanmedicinal-products_en.pdf
3. Control of Nitrosamine Impurities in
Human Drugs. https://www.fda.gov/
media/141720/download