Chromatographic Methods for Comprehensive

Nitrosamine Impurity Analysis By LC-MS/MS

By Dr. Shun-Hsin Liang, Justin Steimling, and Connor Flannery

Nitrosamines are probable human carcinogens that can form during the manufacturing of
active pharmaceutical ingredients (API) and drug products. These impurities are produced Related Products
through nitrosation reactions between amines (secondary, tertiary, or quaternary amines) • Raptor Biphenyl LC Columns
and nitrous acid. The recent discovery of nitrosamine contaminants in several classes of
drugs, such as some angiotensin II receptor blockers, metformin, and ranitidine, prompted • Reference Standards
worldwide recalls and resulted in shortages of these important medicines. In response, risk • Nitrosamine Calibration Mix
assessment strategies for the detection and prevention of nitrosamine formation in drug • N-Nitrosodiphenylamine
products have been established.
Regulatory authorities (ICH, EMA, FDA) have identified several nitrosamine impurities
that theoretically could be present in drug products [1]. The seven compounds on
the FDA’s list are N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-methyl-4-aminobutanoic acid
(NMBA), N-nitrosoisopropylethylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodibutylamine (NDBA), and
N-nitrosomethylphenylamine (NMPA). Analytical methods typically cover these compounds and a few others, but more comprehensive
methods are needed because other potential nitrosamine impurities can form from the wide range of chemicals and processes that are
used in drug production.
The goal of this study was to develop a flexible LC-MS/MS screening method that allows more comprehensive nitrosamine impurity
analysis. To that purpose, our approach was designed to accommodate different sample preparation schemes utilizing various extraction
solutions and sample diluents. In addition, we explored changes related to sample diluent and mobile phase compatibility that were
necessary to ensure good chromatographic performance.
Column Selection and Establishment of Analytical Conditions
Initial column scouting experiments should always include a diverse array of stationary phase chemistries, so we evaluated Raptor
Biphenyl, Raptor FluoroPhenyl, Raptor ARC-18, Ultra Aqueous C18, and Ultra IBD columns in the 100 x 2.1 mm format. For the mobile
phase, we used 0.1% formic acid in water and methanol because it is commonly used for nitrosamines analysis. Results showed that the
Raptor Biphenyl column had the best chromatographic performance, producing sharper peaks with higher peak intensity than the other
columns. Since effective screening methods for nitrosamines need to be sensitive down to low ppb levels, the better peak shape and
intensity seen on the Raptor Biphenyl column weighed heavily in column selection.
After the stationary and mobile phases had been determined, the method was further optimized on two column formats to provide
method flexibility based on individual labs’ sample preparation process and instrumental capability. The established MRM transitions
and optimal analytical conditions are shown in Tables I and II, respectively. Atmospheric pressure chemical ionization (APCI) was used
to improve sensitivity, and during optimization we determined that the MS source temperature was the most important parameter in
obtaining consistent MRMs and stronger signals. It also had significant impact on the peak shape of some nitrosamines. With the method
established here, all 16 analytes had suitable detection signals at 1 ng/mL or less.
Even with these optimized conditions for comprehensive nitrosamine impurity analysis, we did observe peak splitting for NDIPLA and
peak shoulders for NMBA, NMEA, and NEIPA (Figure 1). These phenomena have been reported in other nitrosamine methods and have
been studied more extensively by Chang et al. [2].

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Table I: Target Compounds and Ion Transitions for Comprehensive Nitrosamine Impurity Analysis by LC-MS/MS.

Peak # tR (min) tR (min)

Analytes Precursor Ion Product Ion
in Figures (100 x 2.1 mm) (100 x 3.0 mm)

1 N-Nitrosodiethanolamine(NDELA) 135.1 74.0 0.90 1.15

2 N-Nitrosodimethylamine (NDMA) 75.1 58.0 1.52 1.74

3 N-Nitrosodiisopropanolamine (NDIPLA) 163.0 88.0 1.90 1.87

4 N-Nitrosomorpholine (NMOR) 117.1 45.2 2.38 2.45

5 N-Nitrosomethylethylamine (NMEA) 89.1 61.0 2.57 2.64

6 N-Nitroso-N-methyl-4-aminobutyric acid (NMBA) 147.1 117.0 2.61 2.51

7 N-Nitrosopyrrolidine (NPYR) 101.1 55.1 3.12 3.12

8 N-Nitrosodiethylamine (NDEA) 103.1 75.0 3.86 3.81

9 N-Nitrosopeperidine (NPIP) 115.1 69.0 4.66 4.60

10 N-Nitrosoethylisopropylamine (NEIPA) 117.1 75.0 4.91 4.78

11 N-Nitrosodiisopropylamine (NDIPA) 131.1 43.0 5.79 5.60

12 N-Nitrosodipropylamine (NDPA) 131.1 43.0 6.07 5.87

13 N-Nitrosomethylphenylamine (NMPA) 137.0 65.9 6.30 6.12

14 N-Nitrosodiisobutylamine (NDIBA) 159.1 57.1 7.23 6.94

15 N-Nitrosodibutylamine (NDBA) 159.1 57.1 7.46 7.16

16 N-Nitrosodiphenylamine (NDPHA) 199.1 169.0 8.17 7.88

2 www.restek.com
 Table II: Optimized Analytical Conditions for Two Column Formats.

Raptor Biphenyl 100 mm x 2.1 mm, 2.7 µm (cat.# 9309A12)

Analytical Columns
Raptor Biphenyl 100 mm x 3.0 mm, 2.7 µm (cat.# 9309A1E)

Instrument Shimadzu Nexera X2 UHPLC coupled with SCIEX Triple Quad 4500 MS

Mobile Phase A 0.1% Formic acid in water

Mobile Phase B 0.1% Formic acid in methanol

100 x 2.1 mm 100 x 3.0 mm
Time (min) %B Time (min) %B
0.00 5 0.00 15
Gradient
9.00 95 8.00 85

9.01 5 8.01 15

11.0 5 10.0 15

Flow Rate 0.4 mL/min 0.5 mL/min

Column Temp. 40 °C

Ion Mode Positive APCI

Curtain Gas 30 psi

Collision Gas 8

Nebulizer Current 1 µA

Ion Source Gas 1 50 psi

Source Temp. 220 °C

Effect of Sample Diluent, Injection Volume, and Column Format on Chromatographic Performance
Because sample preparation methods will vary based on differences in drug matrices, our goal was to produce a method that was
compatible with a wide range of sample diluents from aqueous to organic solutions. For this, we tested 100% water, 50:50 water:methanol,
and 100% methanol as diluents and evaluated their impact on chromatographic performance. Mismatch between the sample diluents and
starting mobile phase condition can greatly affect peak shape, intensity, and resolution, so we also tested different injection volumes on
different column formats to determine which combination produced the best chromatographic results.
Through these experiments, it was immediately clear that the peak shapes of early eluting compounds were greatly impacted by the
diluents used. On the smaller 100 x 2.1 mm column format, the mismatch of the starting mobile phase (5% methanol) and diluents with
higher methanol concentration (50% or 100%) required that a lower injection volume (3 µL) be used to obtain acceptable peak shapes,
especially for early eluting compounds such as NDELA and NDIPLA. When the larger column format (100 x 3.0 mm) was tested in an
attempt to reduce the mismatching effect, larger injection volumes could be used without negatively affecting the peak shapes. In addition,
with the larger column it was possible to use a higher organic starting mobile phase (15% methanol), which improved the peak shapes for
early eluting compounds when diluted in 50:50 water:methanol or 100% methanol. A comparison of the chromatographic performance of
the three diluents analyzed on 100 x 2.1 mm and 100 x 3.0 mm columns using optimized conditions for each is shown in Figures 1–3.
Use of a larger column and smaller injection volume are strategies that can reduce the impact of mismatched sample diluents and mobile
phases. However, detection sensitivity for NDMA and NMBA was slightly reduced on the larger column. All versions of the method
developed here provided good chromatographic performance for comprehensive nitrosamine impurity analysis, but which one is most
appropriate should be determined by individual labs based on the sensitivity of their MS system and list of target compounds.

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Figure 1: 10 ng/mL Nitrosamines Diluted in Water
(See Table I for peak identifications.)

5 µL Injection on 100 x 2.1 mm Raptor Biphenyl (Initial Mobile Phase: 5% B, 0.4 mL/min)

10

11

14

3
15
5
7 9
12
13

8
16
2
4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0542

5 µL Injection on 100 x 3.0 mm Raptor Biphenyl (Initial Mobile Phase: 15% B, 0.5 mL/min)
10

11
14

15

9
5 7 12

8
13
16
2
4
6
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0539

4 www.restek.com
Figure 2: 10 ng/mL Nitrosamines Diluted in 50:50 Methanol:Water
(See Table I for peak identifications.)

3 µL Injection on 100 x 2.1 mm Raptor Biphenyl (Initial Mobile Phase: 5% B, 0.4 mL/min)

10

11
14

15

3 5 9
1 7 12
13

8
16
2 4

6
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0537

5 µL Injection on 100 x 3.0 mm Raptor Biphenyl (Initial Mobile Phase: 15% B, 0.5 mL/min)
10

11
14

3 15
1

9 12
5 7

8 13
16
2
4
6
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0540

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Figure 3: 10 ng/mL Nitrosamines Diluted in Methanol
(See Table I for peak identifications.)

3 µL Injection on 100 x 2.1 mm Raptor Biphenyl (Initial Mobile Phase: 5% B, 0.4 mL/min)

10

11 14

15

9
12
3 5 7 13

1 8 16

2 4
6
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0538

5 µL Injection on 100 x 3.0 mm Raptor Biphenyl (Initial Mobile Phase: 15% B, 0.5 mL/min)
10

11 14

15

3
12
9

5 7
1
13
8
2 16
6
4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 50.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
LC_PH0541

6 www.restek.com
Conclusion
Due to the wide range of APIs, drug matrices, and manufacturing processes, demand is growing for more comprehensive nitrosamine
impurity analysis methods. Sample solubility, as well as compatibility between the sample diluent and starting mobile phase composition,
are critical factors in method success. For that reason, we developed an approach that can accommodate disparate sample diluents (100%
water, 50:50 water:methanol, 100% methanol) and still provide good chromatographic performance for 16 nitrosamines. The LC-MS/
MS methods described here use a Raptor Biphenyl column in two formats (100 x 2.1 mm and 100 x 3.0 mm) with injection volumes and
mobile phases that are optimized to minimize solvent mismatch for each diluent. While all versions produced good chromatographic
results, the choice of method should be determined by each lab based on their specific target nitrosamines list and the sensitivity
capabilities of their LC-MS/MS instrumentation.

References
1. U.S. Food and Drug Administration. FDA Guidance Document: Control of Nitrosamine Impurities in Human Drugs, 2020. https://www.fda.gov/regulatory-information/
search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs
2. S-H. Chang, C-C. Chang, L-J. Wang, W-C. Chen, S-Y. Fan, C-Z. Zang, Y-H. Hsu, M-C. Lin, S-H. Tseng, and D-Y. Wang, A multi-analyte LC-MS/MS method for screening and quantification of
nitrosamines in sartans, Journal of Food and Drug Analysis, 28 (2020) 292-301. https://www.jfda-online.com/cgi/viewcontent.cgi?article=1063&context=journal

Raptor Biphenyl LC Columns (USP L11)

• Ideal for bioanalytical testing applications like drug and metabolite analyses.
• Heightened selectivity and retention for compounds that are hard to resolve or elute early
on C18 and other phenyl chemistries.
• Limits ionization suppression and allows simple, MS-friendly mobile phases.
• Part of Restek’s Raptor LC column line featuring 1.8, 2.7, and 5 µm SPP core-shell silica.

Stationary Phase Category: Phenyl (L11)

Ligand Type: Biphenyl
Particle: 1.8 µm, 2.7 µm, or 5 µm superficially porous particle (SPP or “core-shell” particle) silica
Pore Size: 90 Å
Carbon Load: 7% (1.8 µm); 7% (2.7 μm); 5% (5 μm)
End-Cap: yes
Surface Area: 125 m2/g (1.8 µm); 130 m2/g (2.7 µm); or 100 m2/g (5 µm)
Recommended Usage:
pH Range: 2.0 to 8.0
Maximum Temperature: 80 °C
Maximum Pressure: 1034 bar/15,000 psi* (1.8 μm); 600 bar/8700 psi (2.7 μm); 400 bar/5800 psi (5 μm)
* For maximum lifetime, recommended maximum pressure for 1.8 μm particles is 830 bar/12,000 psi.
Properties:
• Increased retention for dipolar, unsaturated, or conjugated solutes.
• Enhanced selectivity when used with methanolic mobile phase.
• Ideal for increasing sensitivity and selectivity in LC-MS analyses.
Switch to a Biphenyl column when:
• Limited selectivity is observed on a C18.
• You need to increase retention of hydrophilic aromatics.

ID Length qty. cat.# ID Length qty. cat.#

similar phases
Phenomenex Kinetex Biphenyl; Supelco/Millipore
1.8 µm Particles 5 µm Particles
Sigma Ascentis Express Biphenyl (2.7 μm)
30 mm ea. 9309232 50 mm ea. 9309552
50 mm ea. 9309252 2.1 mm 100 mm ea. 9309512
2.1 mm
100 mm ea. 9309212 150 mm ea. 9309562 ordering notes
150 mm ea. 9309262 30 mm ea. 930953E Certificates of analysis for new Restek LC columns
50 mm ea. 930925E 50 mm ea. 930955E are now provided electronically. To view and
3.0 mm 3.0 mm
100 mm ea. 930921E 100 mm ea. 930951E download, visit www.restek.com/documentation
2.7 µm Particles 150 mm ea. 930956E then enter your cat.# and serial #.
30 mm ea. 9309A32 50 mm ea. 9309555
50 mm ea. 9309A52 100 mm ea. 9309515
2.1 mm 4.6 mm
100 mm ea. 9309A12 150 mm ea. 9309565
150 mm ea. 9309A62 250 mm ea. 9309575
30 mm ea. 9309A3E
50 mm ea. 9309A5E
3.0 mm
100 mm ea. 9309A1E
150 mm ea. 9309A6E
30 mm ea. 9309A35
50 mm ea. 9309A55
4.6 mm
100 mm ea. 9309A15
150 mm ea. 9309A65

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Restek Reference Standards

Nitrosamine Calibration Mix, Method 521

(7 components)
N-Nitrosodiethylamine (55-18-5) N-Nitrosomethylethylamine (10595-95-6)
N-Nitrosodimethylamine (62-75-9) N-Nitrosopiperidine (100-75-4)
N-Nitrosodi-n-butylamine (924-16-3) N-Nitrosopyrrolidine (930-55-2)
N-Nitroso-di-n-propylamine (621-64-7)
Min Shelf Life Max Shelf Life Shipping Storage
Conc. in Solvent CRM? qty. cat.#
on Ship Date on Ship Date Conditions Temp.
1000 µg/mL each in methylene 10 °C or
Yes 6 months 36 months Ambient ea. 31898
chloride, 1 mL/ampul colder

N-Nitrosodiphenylamine
N-Nitrosodiphenylamine (86-30-6)
Min Shelf Life on Max Shelf Life Shipping Storage
CAS # Conc. in Solvent CRM? qty. cat.#
Ship Date on Ship Date Conditions Temp.
1000 µg/mL in methanol, 10 °C or
86-30-6 Yes 6 months 36 months Ambient ea. 31429
1 mL/ampul colder

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