APPLICATION NOTE

Liquid Chromatography/
Mass Spectrometry

Authors:
Dr. Narayan Kamble
Kailas Gavhane
Dr. Umesh Takelar

PerkinElmer (India) Pvt. Ltd.

Mumbai
India

Determination of
Nitrosamine Impurities in Introduction

Active Pharmaceutical Nitrosamines are a well-known

group of highly potent, mutagenic

Ingredient (API) Using impurities formed by the reaction of

secondary amines with nitrite under
QSight 220 LC-MS/MS acidic conditions1-4. In pharmaceutical
drug substances, the formation
of nitrosamines is possible in the
presence of secondary, tertiary, or quaternary amines and nitrite salts under acidic
reaction conditions. Under these conditions, nitrite salts may form nitrous acid, which
can react with an amine to form a nitrosamine. Additional sources of nitrosamines in
pharmaceutical drug substances includes the use of recovered solvents or equipment
contaminated with N-nitrosamines formed outside of the declared synthetic process2,3.
Evidence of carcinogenicity associated with nitrosamines was briefly summarized by
the National Toxicology Program in the 14th report on carcinogens1. The most probable
nitrosamine impurities are N-Nitroso-di-methyl amine (NDMA, CAS No. 62-75-9),
N-Nitroso-di-ethyl amine (NDEA, CAS No. 55-18-5), N-nitroso-N-methyl-4-aminobutyric
acid (NMBA, CAS No. 61445-55-4), N-nitroso-di-butylamine (NDBA, CAS No. 924-16-
3), N-nitroso-di-isopropyl amine (NDIPA, CAS No. 601-77-4), and N-ethyl-n-nitroso-2-
propanamine (NEIPA, CAS No. 16339-04-1).
In mid-2018, nitrosamine impurities were observed for the first time in Valsartan drugs,
which are used to treat high blood pressure and congestive heart failure2. The US Food
 and Drug Administration (US FDA) and European Medicines Agency Method Parameters
(EMA) made an announcement in July 2018 regarding the presence LC parameters, including column and mobile phase gradient
of NDMA and NDEA in generic drug substance and drug products, program, are given in Table 1. The MS/MS parameters are given in
especially in angiotensin II receptor blockers (ARBs) also known as Table 2. The MRM transition parameters for all targeted analytes
sartan family drugs2,4. The ICH guideline, ICH M7 (R1) classified are listed in Table 3.
these compounds in Class 1, which is known to be mutagenic and
carcinogenic to the human body with high risk5. Based on available Table 1. LC Parameters.
toxicity data, acceptable daily intake for these impurities, which has LC Conditions
been adopted by most of the regulatory bodies, is 96 ng/day for LC Column C18, 50 x 4.6 mm, 3 μm
NDMA and NMBA, and 26.5 ng/day for NDEA, NDBA, NDIPA Mobile Phase A 0.1% Formic acid in water
and NEIPA4. Mobile Phase B 0.1% Formic acid in Methanol
Mobile Phase Sr. No Time %A %B
For safety assessment and control of the most probable impurities Gradient 1 0.00 90 10
in pharmaceutical drug substances and products, it is critical to have
2 0.87 90 10
a specific, sensitive, and reliable method5-7. This application note 3 4.04 45 55
describes an efficient, sensitive and reproducible method developed 4 9.81 45 55
using the PerkinElmer QSight 220® LC/MS/MS for the detection and 5 9.87 10 90
quantification of NDMA, NDEA, NMBA, NDBA, NDIPA and NEIPA 6 12.12 10 90
impurities in sartan and metformin active pharmaceutical ingredients 7 12.87 90 10
(APIs). This work demonstrates that all six nitrosamine impurities can 8 14.00 90 10
be determined at the lowest concentration limits set by US and Column Oven
45 oC
European regulatory agencies. Temperature
Auto sampler
10 oC
Temperature
Experimental
Injection Volume 40 μL
Hardware/Software Flow 1 mL/ min
Chromatographic separation was carried out by a PerkinElmer LX50 Run Time 14 min
UHPLC system, with subsequent detection achieved using a PerkinElmer
QSight 220 triple quadrupole mass spectrometer equipped with an
APCI ionization source. All instrument control, data acquisition, data
Table 2. MS/MS Source Parameters
processing and result reporting were performed using Simplicity 3Q™
Parameter Setting Value
software. The QSight system with photodiode array (PDA) detector
APCI Corona Discharge Current (Positive) 3 µA
and diverter valve was used to determine the elution time of the
APIs and nitrosamine impurities during the development stage to Drying Gas 120
avoid blocking of the APCI source needle. The PDA detector may be Nebulizer Gas 350
removed once the time window is set in the MS method parameter Source Temperature 450 oC
for diverting the main API to waste through the diverter valve. This HSID Temperature 250 oC
is necessary to keep the mass spectrometer clean for extended
periods of time, thus minimizing down time.

Table 3. MRM transition parameters for the six nitrosamine analytes.

Sr. No Name Q1 Mass Q2 Mass Entrance Voltage Collision Cell Lens 2 Voltage Collision Energy
1 NDMA-1 75.1 43.1 10 -21 -22
2 NDMA-2 75.1 58.1 18 -25 -16
3 NDEA-1 103 75.1 9 -25 -15
4 NDEA-2 103 47 18 -25 -24
5 NDBA-1 159.1 103.1 15 -37 -15
6 NDBA-2 159.1 57.2 8 -37 -19
7 NDBA-3 159.1 41.2 12 -33 -31
8 NDIPA-1 131.1 89.1 15 -29 -12
9 NDIPA-2 131.1 43.1 12 -29 -24
10 NDIPA-3 131.1 47.1 4 -21 -22
11 NEIPA-1 117.1 75.1 12 -29 -13
12 NEIPA-2 117.1 43 10 -25 -22
13 NEIPA-3 117.1 29.1 13 -29 -23
14 NMBA-1 147.1 44.1 9 -28 -11
15 NMBA-2 147.1 87.2 9 -29 -11
16 NMBA-3 147.1 117.2 8 -68 -3

2
Materials and Methods Results and Discussion
Solvents and Standards A single LC/MS/MS method was developed for six nitrosamine
All the nitrosamine impurity standards (NDMA, NDEA, NDBA, impurities. Specificity is the critical factor in this analysis, which
NDIPA, NEIPA and NMBA) used in this study were obtained from decides the ruggedness of the method developed. The specificity
Cleanchem laboratories. LC/MS grade solvents and reagents were parameter, as defined by separation between API and the closely
used in the preparation of solutions. eluting nitrosamine target analyte, was optimized using a C18
column and gradient program. The method performance was
Stock Solutions and Calibration Standards
established based on the limit of detection (LOD), limit of
The stock solutions and calibration standards were prepared
quantitation (LOQ), linearity, reproducibility and recovery. The
as follows:
developed method covers a linearity range from 0.1 ppb to 100
• Individual standard stock solution of NDMA, NDEA, NDBA, ppb (0.1 ppb, 0.3 ppb, 0.5 ppb, 1.0 ppb, 5.0 ppb, 10 ppb, 20 ppb,
NDIPA NEIPA (100 ppm) and NMBA (500 ppm): 10 mg of each 50 ppb and 100 ppb). All six nitrosamine impurities show linear
individual impurity (NDMA, NDEA, NDBA, NDIPA and NEIPA) response over the concentration range from 0.1 to 100 ppb, with
standard was weighed and transferred into a 100 mL volumetric correlation coefficient (R2) values greater than 0.99 for all analytes.
flask. The impurity standard was dissolved and diluted up to the The results of the LOD, LOQ, and linearity are summarized in Table
mark using methanol as the diluent. Similarly, the NMBA individual 4. The representative calibration curves for all six nitrosamine target
standard stock solution of 500 ppm concentration was prepared. analytes are shown in Figure 1.
• Mix standard stock solution-1 (1000 ppb): The 1.0 mL of The area response reproducibility data for all six nitrosamine
individual standard stock solution (100 ppm) was further diluted impurities are given in Table 5. The method shows excellent
to 100 mL in a volumetric flask using acidified water as the diluent. repeatability results at 1 ppb concentration level with peak area
• Standard stock solution-2 (100 ppb): The 1.0 mL of mix standard RSDs less than 10% for six injections.
stock solution-1 (1000 ppb) was diluted to 10 mL in a volumetric The spike recovery study was performed at six different concentrations:
flask using acidified water as the diluent. level-1 (0.5 ppb), level-2 (1.0 ppb), level-3 (2.0 ppb), level-4 (6.0
• Working Level standard solution (10 ppb): The 1.0 mL of ppb), level-5 (10 ppb), and level-6 (15 ppb). The spike recovery was
standard stock solution-2 (100 ppb) was diluted to 10 mL in a tested with six replicates for all of 6 nitrosamines. The average
volumetric flask using acidified water as the diluent. recovery results for analytes were in the range of 70-120%, with
peak area RSDs less than 15%, as shown in Tables 6 and 7 for
• Calibration standard solutions: The stock solution was serially
Irbesartan and Metformin API, respectively.
diluted with LC/MS grade water to prepare calibration standards
ranging from 0.1 ppb to 100.0 ppb for all five nitrosamine target The API sample concentration of 30,000 ppm of Irbesartan and
analytes, except for NMBA. The calibration standard Metformin was used to comply, as per set regulatory limits.
concentrations for NMBA were five times higher in comparison PerkinElmer QSight 220 LC/MS/MS is capable of low limits of detection,
to the other five nitrosamine target analytes. with respect to sample concentration based on the daily dose. Low
limits of detection can be achieved by increasing the sample size,
Sample Preparation
concentration and injection volume, provided specificity parameters
The study was conducted using Irbesartan and Metformin APIs.
could be passed.
The API sample of 30,000 ppm concentration was prepared in
water diluent. The sample solution was filtered through a 0.2 μm
nylon filter, and transferred to an amber colored LC vial for
LC/MS/MS analysis. Recovery for all target analytes at different
concentration levels was measured using an API solution at
30,000 ppm concentration.

Table 4. Result summary for LOD, LOQ, coefficient of regression, and calibration curve range.
Compound Neat Standard Concentration With respect to sample Concentration Correlation
LOD (ppb) LOQ (ppb) Linearity (ppb) LOD (ppb) LOQ (ppb) Linearity (ppb) Coeff. (R2)
NDMA 0.1 0.2 0.1-100 4.0 8.0 4.0-3000 0.998
NDEA 0.1 0.3 0.1-100 3.0 12.0 3.0-3000 0.999
NDBA 0.1 0.3 0.1-100 4.0 10.0 4.0-3000 0.997
NDIPA 0.1 0.3 0.1-100 4.0 9.0 4.0-3000 0.999
NEIPA 0.1 0.3 0.1-100 3.0 10.0 3.0-3000 0.997
NMBA 0.3 0.5 0.3-500 9.0 18.0 9.0-16000 0.992

3
 Figure 1. Calibration curves for all 6 Nitrosamine target analytes.

Table 5. Area reproducibility of all nitrosamine impurities was tested at 1 ppb.

Compound Name NDMA NDEA NDBA NDIPA NEIPA NMBA
Injection 1 1859 2311 4126 2822 6032 3072
Injection 2 2074 2346 3698 2379 4814 3143
Injection 3 1850 2311 3309 2766 4942 2963
Injection 4 2153 2609 3284 2332 4984 2562
Injection 5 2145 2663 3512 2802 5313 2614
Injection 6 2160 2570 4020 2477 5876 2750
Average 2040.17 2468.33 3658.17 2596.33 5326.83 2850.67
Std DEV 147.11 162.78 356.28 225.10 515.30 243.53
% RSD 7.21 6.59 9.74 8.67 9.67 8.54

4
Table 6. Spike recovery results of the six impurity’s neat standards spiked in an Irbesartan API at 30,000 ppm concentration.
Name Level-1 (0.5 ppb) Level-2 (1 ppb) Level-3 (2 ppb) Level-4 (6 ppb) Level-5 (10 ppb) Level-6 (15 ppb)
NDMA 101.83 95.13 84.67 83.22 72.14 74.47
NDEA 96.07 88.77 88.23 77.46 70.47 71.57
NDBA 103.43 95.88 77.94 78.24 71.62 73.80
NDIPA 94.20 91.08 91.17 77.04 72.13 70.98
NEIPA 97.97 77.77 83.78 74.26 70.58 71.24
NMBA 91.17 81.53 75.35 78.52 74.37 74.27

Table 7. Spike recovery results of six impurity’s neat standards spiked in a Metformin API at 30,000 ppm concentration.
Name Level-1 (0.5 ppb) Level-2 (1 ppb) Level-3 (2 ppb) Level-4 (6 ppb) Level-5 (10 ppb) Level-6 (15 ppb)
NDMA 88.50 78.90 80.63 120.23 100.44 86.25
NDEA 90.40 95.57 102.13 116.65 92.51 91.11
NDBA 88.50 86.40 88.90 102.67 95.35 93.61
NDIPA 87.80 95.33 96.33 110.74 99.89 90.79
NEIPA 116.70 111.57 95.45 108.27 95.38 89.86
NMBA 104.10 119.33 107.04 107.21 91.96 87.40

Figure 2 (A and B) represents the total ion chromatogram (TIC) for Irbesartan and Metformin. Figure 3 shows MRM chromatograms for all
six analytes in Irbesartan, using a QSight 220 LC/MS/MS. All six impurities spiked into the Irbesartan API at a concentration of 6 ppb were
baseline separated from each other and Irbesartan in 9 the min runtime.

A B

Figure 2. Total ion chromatograms (TICs) of (A) Irbesartan sample, API shown eluting at 8.20 min and (B) Metformin sample, API shown eluting at 0.66 min.

Figure 3. MRM chromatogram of 6 nitrosamine impurities well separated in less than 9 minutes.

5
Conclusion References
The PerkinElmer QSight 220 LC/MS/MS can be used to determine 1. National Toxicology Program. 14th Report on carcinogens (RoC). 14th
nitrosamine impurities at the lowest levels set by regulatory Edition. 2015. https://ntp.niehs.nih.gov/annualreport/2015/glance/roc/
agencies. This application note describes a fast, selective, sensitive index.html.
and robust method for the detection and quantification of 2. European Medicines Agency, Assessment report ,14 February 2019,
nitrosamine impurities in Metformin and Irbesartan. This method EMA/217823/2019.
can be extended to the analysis of these impurities in other drug
3. European Medicines Agency, Questions and answers on
products with some modifications to the chromatography to
“Information on nitrosamines for marketing authorization holders”
achieve separation of the API from these impurities.
EMA/CHMP/428592/2019 Rev. 3, 27 March 2020.
4. ICH M7(R1) Assessment and Control of DNA Reactive (Mutagenic)
Impurities in Pharmaceuticals To Limit Potential Carcinogenic Risk,
March 2018, https://www.ich.org.
5. US FDA, Liquid Chromatography-High Resolution Mass Spectrometry
(LC-HRMS) Method for the Determination of Six Nitrosamine
Impurities in ARB Drugs, 21 May 2019.
6. Application Note, Estimation of N-nitroso dimethyl amine (NDMA) in
ranitidine drug substance by QSight™ UHPLC-MS/MS. IN_EH_
APP_17, 2019, PerkinElmer India Pvt. Ltd, Mumbai.
7. Experiment Note, Quick Determination of NDMA in metformin by
QSight™ UHPLC-MS/MS, EN_LM_20_02_1, PerkinElmer India Pvt.
Ltd, Mumbai.

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