Application Note

Determination of Nitrosamine
Impurities Using the Agilent 6495D
Triple Quadrupole LC/MS SystemAuthors

Abstract

Lee Bertram and Linfeng Wu
Agilent Technologies, Inc.

Nitrosamine impurities are byproducts produced in trace amounts during themanufacture of pharmaceutical drugs. These impurities are classified as potentiallygenotoxic, and as probable carcinogens with long-term intake. Therefore, it isimportant to determine their levels in final drug products with a high level ofsensitivity and confidence.
This application note describes the evaluated quantification performance ofeight nitrosamine impurities using an Agilent 6495D triple quadrupole LC/MS(6495D LC/TQ) system coupled with an Agilent 1290 Infinity II bio LC system andAgilent APCI source.

Introduction
The recall of several pharmaceutical drugs, including
Angiotensin II receptor blocker (ARB) drug products, madenitrosamine impurities a focus for regulatory agencies.1
Nitrosamine impurities are byproducts that arise during themanufacturing of pharmaceutical drugs. They are a memberof the "cohort of concern," classifying them as potentiallygenotoxic impurities and probable carcinogens withlong-term intake.
In this application note, a comprehensive analysis of eightnitrosamine compounds was carried out on the Agilent
6495D triple quadrupole LC/MS system coupled with theAgilent 1290 Infinity II LC system and Agilent APCI source.
It demonstrated that the following compounds can be
determined at very low levels:

Sample preparation
Nitrosamine standards were spiked into solvent blank(methanol:water 10:90) at nine different concentrationlevels ranging from 0.0125 to 10 ng/mL. Preparation of theactive pharmaceutical ingredient (API) matrix was carriedout following these steps: 100 mg losartan potassium
drug product was dissolved in 2 mL of solvent (methanol:water 50:50), followed by sonication for 30 minutes. Thesamples were then centrifuged at 12,000 rpm for 10 minutes.
Supernatants were collected and then 1:5 diluted with water.
Nitrosamine standards were spiked into the prepared API
matrix at concentrations ranging from 0.005 to 20 ng/mL.

Equipment
Sample separation was performed using the 1290 Infinity II
bio LC system, consisting of the following modules:– N-Nitrosodimethylamine (NDMA)– Agilent 1290 Infinity II bio high-speed pump (G7132A)– N-Nitrosomorpholine (NMOR)– Agilent 1290 Infinity II bio multisampler withthermostat (G7137A)– N-Nitrosomethylethylamine (NMEA)– N-Nitrosopyrrolidine (NPYR)– N-Nitrosodiethylamine (NDEA)– N-Nitrosopiperidine (NPIP)– N-Nitrosodi-n-propylamine (NDPA)– N-Nitrosodi-n-butylamine (NDBA)

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

2– Agilent 1290 Infinity II multicolumn thermostat (G7116B)
The liquid chromatography system was coupled to theAgilent 6495D triple quadrupole LC/MS (G6495D) mass
spectrometer equipped with the APCI source (G1947B).
Agilent MassHunter Workstation software version 12.1 wasused for data acquisition.
The testing in this application note can be carried out using
either a 1290 Infinity II bio LC system or a 1290 Infinity IILC system.

Methods
The LC/MS conditions and parameters are provided inTables 1 and 2. The MRM settings for the compounds arelisted in Table 3. Using the MassHunter 12.1 MRM-specific
iFunnel mode feature, it was determined that the Fragile modesetting allowed for the highest abundance for all compounds
in this study. For calibration curve analysis, linear fitting withorigin ignored and 1/x weighting was used.

Liquid chromatography

Mass spectrometry

Table 1. Agilent 1290 Infinity II bio LC method.

Table 2. LC/TQ parameters.
Agilent 6495D Triple Quadrupole LC/MS System

Agilent 1290 Infinity II Bio LC System
ColumnAgilent InfinityLab Poroshell 120 column EC-C18,
3.0 × 150 mm, 2.7 µm (p/n 693975-302)

Ion Source
Polarity

Positive

Sampler Temperature

4 °C

Gas Temperature

290 °C

ddH2O + 0.1% formic acid

Drying Gas Flow

11 L/min

Nebulizer

25 psiAPCI Vaporizer Temperature

350 °C

Capillary Voltage

1,000 V

Corona Current

4.0 µAScan 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 diverted to MS)

Mobile Phase A
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
55456060659055Agilent APCI source

Compound information and MRM settings
Table 3. Detailed MRM settings and compound information for the Agilent 6495D triple
quadrupole LC/MS.
Precursor
m/z

Productm/z

Dwell(ms)iFunnelMode

CAV(V)

CE(V)

Polarity

Measured
Retention Time(min)

75

4350

Fragile316+2.51

NDMA

75

5850

Fragile310+2.51

NMOR

117

4550

Fragile

421+

3.99

NMOR

117

87

50

Fragile411+3.99

NMEA

89

4350

Fragile

312+

5.35

NMEA

896150

Fragile

310+

5.35

NPYR

101

4150

Fragile

324+

5.70

NPYR

101

55

50

Fragile319+5.70

NDEA

103

4750

Fragile420+7.48

NDEA

103

7550

Fragile412+7.48

NPIP

115

4150

Fragile324+7.90

NPIP

115

6950

Fragile

312+

7.90

NDPA

131

4350

Fragile

410+

10.10

NDPA

131

8950

Fragile

416+

10.10

NDBA

159

4150

Fragile

320+

13.50

NDBA

159

5750

Fragile

312+

13.50

CompoundName
NDMA

3

Results and discussion

Excellent chromatographic separation and peak shapes for
all analytes were achieved, as shown in Figure 1. Excellent
quantitation linearity for the tested levels with R2 >0.99 for all
eight analytes (Figure 2) was achieved. Outstanding precision
and accuracy were observed at all tested levels, including
the lower limit of quantitation (LLOQ) levels (Table 4). Highanalytical sensitivity, with an LLOQ ≤0.01 ng/mL (10 ppt) for
all the targeted analytes (Table 4) was observed.

Calibration curve analysis
To evaluate the quantification performance of nitrosamines,
the calibration curves of the eight nitrosamine compounds
were analyzed with concentrations ranging from 0.005 to
20 ng/mL in solvent. The analysis results were as follows:
×106

1.4

NPIP

1.3
1.2
1.1
1.0
Counts

0.9
0.8
0.7
0.6

NDPA

0.5
NPYR

NDMA0.40.3
0.2NMOR

0.10123NDBA

NMEA

45NDEA

678Acquisition time (min)9101112Figure 1. Total MRM chromatograms of the eight nitrosamine compounds at 0.6 ng/mL.
×106 NMOR
7 y = 376,733.744575x – 356.613775
6 R2 = 0.9997
5432
100123456

Responses

Responses

×107 NDMA
1.2 y = 1,268,145.780765x + 805.293263
1.0 R2 = 0.9995
0.8
0.6
0.4
0.207810 111416182345679108910Responses

×107 NDPA
1.0 y = 1,000,624.566298x – 610.660205
0.8 R2 = 0.9986
0.60.40.2001234567Concentration (ng/mL)

8161820891089101618

0.4
0.2
001234567×107 NPIP
2.0y = 1,928,106.117746x – 5,991.464537
1.6 R2 = 0.9979
1.20.80.4
001234567×107 NDBA
1.0 y = 597,885.377451x + 22,336.275333
0.8 R2 = 0.9997
0.60.4
0.20Responses

120Responses

Responses

×106 NDEA
2.0y = 203,010.639960x + 144.825818
1.6 R2 = 0.9988
1.20.80.40002468 10 12 14
×107
NPYR
1.0y = 964,256.304261x – 2,965.520354
0.8 R2 = 0.9992
0.6

Responses

9Responses

×107 NMEA
1.0 y = 594,652.252869x – 319.222554
0.8 R2 = 0.9994
0.6
0.4
0.20-0.22468 10 12002468
10 12 14
Concentration (ng/mL)

Figure 2. Calibration curves on the Agilent 6495D triple quadrupole LC/MS for the eight nitrosamine compounds.

41314Table 4. Average response, signal-to-noise ratio (S/N) of the quantifier ion, %RSD, and percent accuracy at 0.01 and0.04 ng/mL using the Agilent 6495D triple quadrupole LC/MS (n = 3).
Concentration
0.01 ng/mL (n = 3)

0.04 ng/mL (n = 3)

Name

Response

S/N

RSD (%)

Accuracy (%)

Response

S/N

RSD (%)

Accuracy (%)

LLOQ (ng/mL)

NDMA

13,035

48.8

1.30

103.0

46,851

130.7

0.001

92.6

0.01

NMOR

3,286

102.1

0.01

89.4

12,779

357.7

0.009

86.90.01

NMEA

5,121

136.7

0.02

93.8

20,411

436.3

0.019

89.2

0.01

NPYR

6,792

66.9

0.01

103.6

31,451

317.3

0.030

91.4

0.01

NDEA

1,752

149.51

0.05

81.3

6,921

374.2

0.012

85.5

0.01

NPIP

14,630

162.5

0.02

109.5

64,325

858.2

0.008

93.4

0.01

NDPA

8,745

305.6

0.02

95.8

34,844

1,316.8

0.002

90.7

0.01

NDBA

28,666

586.8

0.02

108.4

46,273

769.8

0.005

102.5

0.01

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

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 ranging from 0.005 to 20 ng/mL.
The results showed that all eight nitrosamine compounds
could be quantified with high confidence at 0.03 ng/mL in
drug matrix. The analytical sensitivities for all eight analytes
were well below the accepted nitrosamine content required byregulatory agencies.2,3NDMA is often a challenging compound for nitrosamine
impurity analysis. Figure 3 shows the representative
chromatograms of NDMA at matrix blank, 0.03 ng/mL, and0.12 ng/mL in 10 mg/mL drug extract with replicates. The
system demonstrated excellent response, signal‑to‑noise ratio(S/N), and reproducibility of NDMA at low concentrations indrug matrix on the 6495D LC/TQ.

5×1041.00

×103
4.8Blank

4.4

0.90

4.0

Relative abundance (%)

0.95

Counts

0.85
0.80
0.75
0.70
0.65

3.6
3.2
2.82.40.60

2.0

0.55

1.6
2.35

2.40

2.45

2.50

2.55

2.60

2.65

2.70

2.75

Blank

2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80

2.80

Acquisition time (min)Acquisition time (min)×104
1.4×102
2.4

0.03 ng/mL
S/N = 22
Area = 17,234

2.0

Relative abundance (%)

Counts

1.3
1.2
1.1
1.0

0.03 ng/mL
S/N = 9Area = 1,569

1.6
1.2
0.8
0.4
0-0.4

0.9

-0.8
2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80

2.30

2.352.40

Acquisition time (min)2.0×1020.12 ng/mL
S/N = 50
Area = 57,609

1.2
1.0Counts

1.8
1.6
1.4
1.2

2.55

2.65

2.70

2.75

0.12 ng/mL
S/N = 24
Area = 7,186

0.8
0.6
0.4
0.2

-0.2
2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80

2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80

Acquisition time (min)Acquisition time (min)Figure 3. Representative chromatograms of NDMA product ions in losartan potassium drug matrix at low concentration levels.

62.60

01.00.8

2.50

Acquisition time (min)Relative abundance (%)×104
2.22.45

Conclusion

References

This application note demonstrates that the Agilent 6495D
triple quadrupole LC/MS system can quantify nitrosamine
impurities at levels as low as 0.01 ng/mL or 10 ppt, which
is below the concentration levels required by regulatory
requirements, with high confidence. This method can be used
to quantify these impurities in different ARB drug productswith some alterations in chromatographic conditions, basedon the elution pattern of the intended drug substance or drug
product of interest.

1. https://www.fda.gov/drugs/drug-safety-and-availability/
fda-updates-and-press-announcements-angiotensin-iireceptor-blocker-arb-recalls-valsartan-losartan

www.agilent.com
DE86697304
This information is subject to change without notice.
© Agilent Technologies, Inc. 2023, 2024
Printed in the USA, August 6, 2024
5994-6834EN

2. Control of Nitrosamine Impurities in Human Drugs
Guidance for Industry. U.S. Food & Drug Administration(updated 24 February 2021).
3. Assessment Report: Nitrosamines Impurities in Human
Medicinal Products. European Medicines Agency.
Procedure number: EMEA/H/A-5(3)/1490 (25 June 2020).