Application Note
Pharma/Biopharma

Determination of Nitrosamine
Impurities Using the Agilent 6475
Triple Quadrupole LC/MS SystemAuthors
Linfeng Wu and Lee Bertram
Agilent Technologies, Inc.
Santa Clara, CA, U.S.
Shan-An Chan
Agilent Technologies, Inc.
TaiwanAbstract
Nitrosamine impurities are by-products that are produced in trace amounts during
the manufacturing process of pharmaceutical drugs. These impurities are classified
as potentially genotoxic impurities and probable carcinogens with long-term intake.
Therefore, it is important to determine their levels in final drug products with a highlevel of sensitivity and confidence. This application note evaluates the quantification
performance of eight nitrosamine impurities using the Agilent 6475 triple quadrupole
LC/MS (LC/TQ) system coupled with the Agilent 1290 Infinity II Bio LC system andatmospheric pressure chemical ionization (APCI) source.

Introduction
A recent announcement for the recall of some angiotensin II
receptor blocker (ARB) drug products made nitrosamine
impurities a focus for regulatory agencies.1 Nitrosamine
impurities are by-products in the manufacturing process ofpharmaceutical drugs. They are a member of the "cohortof concern," which classifies them as potentially genotoxic
impurities and probable carcinogens with long-term intake.
In this application note, a comprehensive analysis of eightnitrosamine compounds was carried out on the 6475 LC/TQ
coupled with the 1290 Infinity II Bio LC system and APCI
source. It is demonstrated that the following compounds canbe determined at low levels:– N-Nitrosodimethylamine (NDMA)– N-Nitrosomorpholine (NMOR)– N-Nitrosomethylethylamine (NMEA)– N-Nitrosopyrrolidine (NPYR)– N-Nitrosodiethylamine (NDEA)– N-Nitrosopiperidine (NPIP)– N-Nitrosodi-n-propylamine (NDPA)– N-Nitrosodi-n-butylamine (NDBA)

Experimental
Chemicals and standards
All reagents and solvents used for the analysis were LC/MSgrade. Ultrapure water was produced with a Milli-Q Integral
system equipped with a LC-Pak Polisher and a 0.22 µm
point‑of-use membrane filter cartridge (MilliporeSigma,Billerica, MA, U.S.). Standards containing eight nitrosamines
NDMA, NDEA, NMOR, NMEA, NPYR, NPIP, NDPA, and NDBA
were from Agilent (part number US-113N-1).

Sample preparation
Nitrosamine standards were spiked into solvent blank(10:90 methanol:water) at nine different concentrationlevels ranging from 0.0125 to 10 ng/mL. The preparation ofactive pharmaceutical ingredient (API) matrix was carriedout following these steps: 100 mg losartan potassium
drug product was dissolved in 2 mL of solvent (50:50
methanol:water), followed by sonication for 30 minutes.
Then, samples were centrifuged at 12,000 rpm for
10 minutes. Supernatants were collected and then diluted
with water at a ratio of 1:5. Nitrosamine standards werespiked into the prepared API matrix at concentrations ranging
from 0.05 to 1 ng/mL.

2

Equipment
Sample separation was performed using the Agilent 1290
Infinity II Bio LC system consisting of the following modules:– Agilent 1290 Infinity II Bio high-speed pump (G7132A)– Agilent 1290 Infinity II Bio multisampler with thermostat(G7137A)– Agilent 1290 Infinity II multicolumn thermostat (G7116B)
The LC system was coupled to the Agilent 6475 triple
quadrupole LC/MS (G6475AA) equipped with the Agilent APCI
source (G1947B). Agilent MassHunter Workstation software
12.0 was used for data acquisition.

Methods
Liquid chromatography/mass spectrometry (LC/MS)
conditions and parameters are provided in Tables 1 and 2.
The multiple reaction monitoring (MRM) settings for thecompounds are listed in Table 3. For calibration curve
analysis, linear fitting with origin ignored and 1/x weighting
was used.
Table 1. Agilent 1290 Infinity II Bio LC method.
Parameter

ValueColumn

Agilent InfinityLab Poroshell 120 EC-C18,
3.0 × 150 mm, 2.7 µm (p/n 693975-302)

Sampler Temperature

4 °CMobile Phase A

ddH2O + 0.1% formic acid

Mobile Phase B

MeOH + 0.1% formic acid

Flow Rate

0.5 mL/min

Injection Volume

20 µL

Column Temperature

40 °C

Gradient ProgramTime (min)
0.0
3.5
7.0
9.0
11.0
15.0
16.0
16.1
20.0%B
55456060
659055

Table 2. Agilent 6475 LC/TQ parameters.
Parameter

Value

Ion Source

Agilent APCI source

Polarity

Positive

Gas Temperature

300 °C

Drying Gas Flow

7 L/min

Nebulizer

25 psiAPCI Vaporizer Temperature

350 °C

Capillary Voltage

4,000 V

Corona Current

4.0 µA

Scan TypeMRM with time segments

Detector Gain Factor (+)

10LC Diverter to Waste

0 to 2 min; 12 to 13 min; 14 to 18 min(the remaining time diverter to MS)

Table 3. Detailed multiple reaction monitoring (MRM) settings and compound information on the Agilent 6475 LC/TQ.
Compound
Name
NDMA
NMOR
NMEA
NPYR
NDEA
NPIP
NDPA
NDBA

Precursor
m/z

Productm/z

Dwell(ms)Fragmentor(V)Cell Accelerator
Voltage (V)Collision Energy(V)Polarity

Measured Retention
Time (min)75435095

316+2.5175

585095

310+

2.51
3.99

117

4550

80

421+

117

87

50

80

411+

3.99

89

4350

75

312+

5.35
5.35

89

61

50

75

310+

101

4150

90

324+

5.70

101

55

50

90

319+

5.70

103

47

50

80

420+

7.48

103

75

50

80

412+

7.48

115

41

50

90

324+

7.90

115

69

50

90

312+

7.90

131

4350

80

410+

10.10

131

89

50

80

416+

10.10

159

41

50

80

320+

13.50

159

57

50

80

312+13.50

3Results and discussion

The analysis results are summarized here:– Good chromatographic separation and peak shapes for all
the analytes (Figure 1)

Calibration curve analysis
To evaluate the quantification performance of nitrosamines,
the calibration curves of the eight nitrosamine compounds
were analyzed with concentrations ranging from 0.0125 to
10 ng/mL in solvent.

– Outstanding precision and accuracy observed at all tested
levels including the lower limit of quantitation (LLOQ)
levels (Table 4)– High analytical sensitivity with an LLOQ ≤0.025 ng/mL for
all targeted analytes (Table 4)– Excellent quantitation linearity for the tested levels, with
R2 >0.99 for all eight analytes (Figure 2)

×104

1.4

NPIP
NDPA

1.2
NPYRCounts

1.00.8

NDMA
NDBA

0.6
NMEA
NDEA

0.4
NMOR

0.203456789Acquisition time (min)101112Figure 1. Total MRM chromatograms of the eight nitrosamine compounds at 1 ng/mL.

Table 4. Average response, signal-to-noise ratio (S/N) of quantifier ion, %RSD, and accuracy (%) at 0.025 and 0.05 ng/mL using theAgilent 6475 LC/TQ system (n = 3).
Concentration

0.025 ng/mL (n = 3)

0.05 ng/mL (n = 3)

Response

S/NRSD (%)

Accuracy (%)

Response

S/N

RSD (%)

Accuracy (%)

LLOQ*(ng/mL)

1,145.08

14.63

1.28

113.86

2,154.26

23.08

1.07

105.41

0.025

NMOR

328.76

52.40

15.94

104.90705.55105.41

14.94

103.87

0.025

NMEA

1,057.38

40.79

3.86

116.86

1,909.29

91.84

4.81

101.000.0125

NPYR

918.54

24.31

2.65

115.47

1,697.56

41.59

2.45

89.61

0.025

NDEA

382.15

31.11

8.14

107.67606.87

52.10

8.58

87.91

0.025

NPIP

890.01

57.47

6.46

97.45

2,106.81

111.72

5.30

98.29

0.025

NDPA

795.9395.62

12.01

106.13

1,647.60

177.47

10.77

95.97

0.025

NDBA

892.36

67.66

7.58

112.05

1,581.52

123.787.83

101.33

0.025

Name
NDMA

* LLOQ was determined as S/N >10 for quantifier, S/N >3 for qualifier, RSD <20%, and accuracy within 80 to 120%.

413

×105
1.4

NMOR

y = 41,636.234539x – 40.077594

1.2

y = 14,656.807124x –55.622367

R2 = 0.9986

1.0

R2 = 0.9980

Responses

12

3

4
5
6
7
Concentration (ng/mL)

NMEA

3.5

y = 40,029.293808x –112.126579

89R = 0.9981
21.5
1.000.50

Responses

1234567
Concentration (ng/mL)

1.2

NDEA

1.0

y = 13,191.743327x + 27.046247
R2 = 0.9968

8

90.6
0.4
01234567
Concentration (ng/mL)

893.0

y = 39,698.406712x –257.406634

2.5

R2 = 0.9937

Responses

Responses

×105

NDPA

2.0
1.5
1.0
0.5
0

2.0

234567
Concentration (ng/mL)

89108910

y = 48,878.903808x –492.467762
R2 = 0.9980

1234567Concentration (ng/mL)NPIP
y = 49,099.862899x –306.238333
R2 = 0.9986

010×105
4.0
3.5
3.0
2.5×105
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
01NPYR00.20×105
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
010

Responses

00.8

0.4

10

2.0×105

0.60

4.0
3.0
2.5

0.8

0.20×105

Responses

NDMA

Responses

Responses

×105
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
01234567Concentration (ng/mL)

89108910NDBA
y = 30,420.287995x + 40.207717
R2 = 0.9912

1.51.00.5001234567Concentration (ng/mL)

89100

1234567Concentration (ng/mL)

Figure 2. Calibration curves on the Agilent 6475 LC/TQ for the eight nitrosamine compounds in the concentration range of 0.0125 to 10 ng/mL.

5

Quantification in losartan potassium drug matrix
To examine the analytical performance of nitrosamine
impurities in pharmaceutical drug products, the nitrosamine
standards were spiked into the losartan potassium drug
extract at concentrations of 0.05 to 1 ng/mL. The results
show that all eight nitrosamine compounds could bequantified with high confidence at 0.05 ng/mL in drug matrix.
The analytical sensitivities for all eight analytes were well
below the acceptable nitrosamine content that is stated byregulatory agencies.2, 3

NDMA (75.0 → 58.0)

NDMA (75.0 → 43.0)

×102

API matrix(n = 2)

1.2

2.5

1.0

2.0

CountsCounts×103
1.4

NDMA is commonly a challenging compound for nitrosamine
impurity analysis. Figure 3 shows the representative
chromatograms of NDMA at matrix blank, and at 0.05
and 0.1 ng/mL in 10 mg/mL drug extract with replicates.
Excellent response, S/N, and reproducibility of NDMA at thelow concentrations in drug matrix were demonstrated on the6475 LC/TQ.

0.8

0.5

0.4
2.2

1.4

2.3

2.4

2.5
2.6
2.7
Acquisition time (min)

2.8

2.2

2.9

2.3

2.4

2.5
2.6
2.7
Acquisition time (min)

2.8

2.9

2.8

2.9

2.8

2.9

×10250 ppt(n = 3)

2.5

Area = 2,583
S/N = 35
Counts1.2Counts1.5
1.00.6×103

API matrix(n = 2)1.0
0.8

50 ppt(n = 3)2.0

Area = 350
S/N = 14

1.5
1.0

0.6

0.5

2.2×103

2.4

2.5
2.6
2.7
Acquisition time (min)

2.8

2.2

2.9

2.3

2.4

2.5
2.6
2.7
Acquisition time (min)×102

Area = 4,707
S/N = 50100 ppt(n = 3)

3.0

1.2

2.5

1.0

2.0Counts

Counts

1.4

2.3

0.8
0.6

100 ppt(n = 3)

Area = 589
S/N = 22

1.5
1.0

0.4
2.22.32.4

2.5
2.6
2.7
Acquisition time (min)

2.8

2.9

2.2

2.3

2.4
2.5
2.6
2.7
Acquisition time (min)Figure 3. Replicate chromatograms of NDMA product ions in losartan potassium drug matrix at low concentration levels.

6Conclusion

References

This application note demonstrates that the Agilent 6475
triple quadrupole LC/MS system can confidently quantify
nitrosamine impurities at the low concentration levels
specified by regulatory requirements. This method canbe used to quantify these impurities in different ARB drug
products, with some changes in chromatographic conditions
based on the elution pattern of the drug product.

1. U.S. Food and Drug Administration. FDA Updates andPress Announcements on Angiotensin II Receptor Blocker(ARB) Recalls (Valsartan, Losartan, and Irbesartan).
Last revised March 2023. https://www.fda.gov/drugs/
drug-safety-and-availability/fda-updates-and-pressannouncements-angiotensin-ii-receptor-blocker-arbrecalls-valsartan-losartan
2. Center for Drug Evaluation and Research, U.S. Food andDrug Administration, Control of Nitrosamine Impurities
in Human Drugs: Guidance for Industry. Last revised
February 2021. Control of Nitrosamine Impurities in
Human Drugs | FDA. https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/controlnitrosamine-impurities-human-drugs
3. Committee for Medicinal Products for Human Use,
European Medicines Agency. Procedure under Article
5(3) of Regulation EC (No) 726/2004. Procedure number:
EMEA/H/A-5(3)/1490. Nitrosamines Impurities in
Human Medicinal Products. Last revised June 2020.
Nitrosamines EMEA-H-A5(3)-1490 - Assessment Report(europa.eu)

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This information is subject to change without notice.
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Printed in the USA, March 21, 2023
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