Poster Reprint

ASMS 2020
WP 555Determination of Nitrosamine
impurities in Losartan Potassium
drug substance using Triple
Quadrupole Liquid
Chromatography Mass
Spectrometry
Chander Mani ,Saikat Banerjee and SamirVyas(Agilent Technologies, India.)Introduction

The announcement for the recall of ARB medicines
Valsartan, Losartan and Irbesartan made N-Nitroso
impurities a focus for regulatory agencies including
the FDA and the European Medicines Agency (EMA).
Nitrosamine impurities are byproducts produced intrace amounts during the manufacturing processes ofthese medicines. These impurities/compounds areclassified as probable carcinogens (i.e. potentiallygenotoxic impurities).
The liquid chromatography mass spectrometry-based
method described in this poster was carried out on
the 6470 Triple Quadrupole LC/MS (LC/TQ),
presenting a comprehensive analysis of 6 nitrosamine
impurities in Losartan Potassium drug substance atvery low detection limits. All nitrosamine impurities
are of very small molecular weight. These nitrosamine
impurities include: N-nitrosodimethylamine (NDMA),
N-nitrosodiethylamine (NDEA), N-nitroso-4-methyl-4aminobutyric acid (NMBA), Nnitrosoethylisopropylamine (NEIPA), Nnitrosodiisopropylamine (NDIPA) and Nnitrosodibutylamine (NDBA).
Instrumentation
1290 Infinity II high-speed pump (G7120A)
1290 Infinity II multisampler (G7167B)
1290 Infinity II multicolumn thermostat (G7116B)
1290 Infinity II variable wavelength detector (G7114B)
6470 triple quadrupole LC/MS (G6470A)

Table 1: Instrumentation detail

Experimental

Sample Preparation
The sample preparation procedure was optimized
using the following steps.
1.Weigh 100mg(± 2mg) Losartan Potassium drug
substance sample in a 15 mL centrifuge tube.
2. Add 5 mL sample diluent and vortex for 2minute.

3. Now put the sample in shaker at 450rpm for 40minutes.
4. Centrifuge the sample at 5000 rpm for 10 minutes.
5. Filter the supernatant using 0.2µm nylon syringe
filter into an LCMS vial.
6. Inject the sample into LC/TQ.

Needle wash
Sample diluent
Multisampler
temperature
Injection
volumeAnalyticalcolumnColumntemperature
Mobile phase A
Mobile phase B
Flow rate
Gradient

Stop time
Post time
Figure 1: 6470 triple quadrupole LC/MSLC Conditions
Methanol: Water/ 80:20
Water: Methanol 95:5
6 oC
20 µL
Zorbax Eclipse Plus Phenyl-Hexyl,
RRHD 2.1 x 100mm 1.8µm (P/N959758-912)
40 oC
0.2 % formic acid in water
Methanol
0.25 mL/min
Time (min) %B
Flow(mL/min)
0.0
50.25
5.0
25
0.25
13.0550.40
20.055
0.40
20.1
95
0.25
23.0
95
0.25
23.1
50.25
25.0
50.25
25 minutes
2 minutes
Table 2: LC conditions2

Results and Discussion

Method Optimization
The 6470 LC/TQ was used for detecting the massconditions for nitrosamine impurities in positive mode
where M+H ion were found to be predominant precursor
ions. The method was optimized using atmosphericpressure chemical ionization (APCI) source as most of
the nitrosamines give better response and low noisebackground using APCI source. MRM method wasconverted into a dynamic MRM method.

NDMA
NMBA
NDEA
NEIPA
NDIPA

MRM Transitions and Conditions
CompoundPrec.
Ion
(m/z)
103.1
103.1
75.1
75.1
147.1
147.1
117.1
117.1
131.1
131.1
159.1
159.1

NDEA
NDEA
NDMA
NDMA
NMBA
NMBA
NEIPA
NEIPA
NDIPA
NDIPA
NDBA
NDBA

NDBA

ProductIon(m/z)
75.1
47.1
58
43.1
44.2
87.2
75.1
47.1
89.1
43.1
57.2
41.1

Frag.
(V)

CE(V)

CAV ±(V)

8080
6060
60
60
7575
75759090

9171218161081861212
22333333383833++++++++++++

Table 3: MRM transitions and conditions
MS Conditions

Gas Temperature

300 0C

Gas Flow

6 L/min

Capillary Voltage

3000V

Nebulizer Pressure

55 psiAPCI Heater

350 0CAPCI Needle Positive

4 µA

Table 4: MS conditions
The chromatographic separation of Losartan Potassiumdrug substance and nitrosamine impurities was best
achieved using Zorbax Eclipse Plus Phenyl-Hexyl column
and diverter valve was programmed such that Losartan
Potassium peak was diverted to waste and monitored
using variable wavelength detector.

Losartan API in UV

Figure 2: Representative EIC of NDMA, NMBA,NDEA, NEIPA, NDIPA and NDBA at 0.1 ppm conc.
using 20mg/mL of Losartan Potassium API.

Below is presented the reproducibility data at 1ng/mL
standard concentration for 8 replicates including
bracketing standards (# 7 and 8) showing excellent peakarea RSD % of < 6 % for each 6 nitrosamine impurities.
Area % RSD at 1ng/mL# NDMA
12556
22409
32436
42442
52435
62578
72442
82434
Avg
2467
SD
63.16

NMBA NDEA NEIPA
5484 10530 36010
5609 10727 36593
4844
9962
34563
4937 10067 32146
4827 10066 32805
4996 10182 32838
4987 10145 33254
4966 10193 33108
5081 10234 33915
295.66 259.96 1629.64

RSD(%)

5.82

2.56

2.544.81

NDIPA NDBA
14023 1868613478 18853
13899 16452
13871 16342
14375 1694213822 16670
14335 16706
13868 16691
13959 17168
289.9 1005.5
092.08
5.86

Table 5: Peak area % RSD for 8 replicates at 1ng/mL

3

Results and Discussion

Method Performance Characterization
Figure 3 shows the calibration curves for the standard
calibration of all 6 nitrosamines. The relevant calibration
range for NDMA, NMBA and NDEA is from 0.05ng/mL to25ng/mL and for NEIPA, NDIPA and NDBA is from 0.1ng/mL to 25ng/mL.

Recovery Study
The recovery experiment shows excellent recovery of ±20 % of the spiked concentrations.
Nitrosamine
Impurity
NDMA
NMBA
NDEA
NEIPA
NDIPA
NDBA

Concentration(ng/mL)211112

Recovery %110113
103
100
98
91

Table 6: Recovery data in Losartan APIConclusions•••

The method provides excellent reproducibility at
USFDA defined LOQ concentrations levels as it shows
area RSDs of < 6% with bracketing standards includedin the calculations.
The method is a ready to use method for analysis ofLosartan Potassium drug substance batches as themethod shows excellent recovery.
The Losartan Potassium drug substance peak ischromatographically well separated from nitrosamine
peaks so it can easily be diverted from the MS.
Therefore, there is no contamination to the mass
spectrometer due to a high concentration of API.
References

Figure 3: Calibration curves of all 6 nitrosamines withr2 > 0.997

This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Published in USA, June 1, 2020