Application Note
Pharma and Biopharma

Quantification of Nitrosamine
Impurities in Metformin Using
Agilent GC/MS/MS Instrumentation

Authors
Soma Dasgupta,
Vivek Dhyani, Samir Vyas, andMelissa Churley,
Agilent Technologies, Inc.

Abstract
Since June 2018, several drugs were recalled due to the presence of nitrosamine
impurities. The impacted molecules belonged mostly to sartans, followed byranitidine and metformin. Regulatory authorities have allowed a transition
period to make these changes in manufacturing processes for manufacturers
to minimize nitrosamine impurities in finished products. During this transition
period, interim limits have been applied to products. The low levels at which
the nitrosamine impurities are expected to be analyzed creates challenges fortesting. This application note highlights a complete solution for the determinationand estimation of five nitrosamine impurities N-nitrosodimethylamine(NDMA), N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA),
N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA) in metformin
drug substances and drug products at trace levels. An Agilent 8890 GC coupled
to an Agilent 7010B triple quadrupole GC/MS system was used to perform this.
The limits of quantification (LOQs) achieved for NDMA, NDEA, NEIPA, and NDIPA
impurities were <0.001 ppm while LOQ for NDBA was <0.0025 ppm with respect todrug substance. At LOQ, recoveries ranged within 80 to 120% with <5% RSD.

Introduction
Detection of nitrosamine impurities insartans led to investigations into othermedicines for the possible presenceof nitrosamines.1,2 All products thatwere identified as having a risk ofnitrosamine formation were mandated
for further testing to confirm theabsence of nitrosamines. One such
identified drug was metformin,
available as immediate‑release (IR) andextended‑release (ER) forms.
Testing by the U.S. Food and Drug
Administration (USFDA) has indicatedthe presence of NDMA at unacceptablelevels in several lots of the ER
formulation of metformin.3 Currently,
the elevated levels of NDMA have been
detected only in some tablets of the ER
formulation but have not been detected
in drug substance.
The European Network of Official
Medicines Control Laboratories(OMCLs) has released a few methods
for the analysis of NDMA and NDEA
in metformin drug substances anddrug products.4 These methods are
based on the extraction of the drug
substance or the finished productwith dichloromethane alone or withan additional partitioning step withwater. This is followed by analysis
using a single quadrupole GC/MSor a triple quadrupole GC/MS/MS.
Interfering peaks have been observed
while analyzing drug products by single
quadrupole MS. At lower concentrationsof the impurities, this may lead tofalse positives. The relative intensityof the qualifier is much lower than thequantifier and hence it is difficult toconfirm the presence or absence of
the impurities. The higher baseline also
results in reduced sensitivity. In contrast,
GC/MS/MS methodology is better
equipped for attaining specificity andsensitivity, and as such is preferred over
GC/MS methods.

2

Although the official method fromthe OMCL network specifies only
two impurities, it is still worthwhile
to monitor other impurities such as
NEIPA, NDIPA, and NDBA, all of which
may arise unintentionally in the final
product as carryover from the use ofcontaminated solvents.
We tested the previously developed
method for nitrosamines in sartans fortesting metformin using the 8890 GC
coupled to the 7010B GC/MS/MS. The
7010B GC/MS/MS is equipped with theHigh Efficiency Source (HES) that has
an improved ionization efficiency and20x ion generation characteristics. Thisdelivered confident trace analysis andhelped to attain the very low detection
limits required for the analysis. The
8890 GC has a touch screen interface,instead of a keypad, to control the
GC and offers instrument operation
diagnostic tests, system monitoringalerts, and mobile access.

Experimental
Sample preparation
The APIs and drug products tested
for this analysis included metformin,
metformin ER (500 mg, 750 mg, and
1,000 mg). Three different methods ofsample preparation were evaluated.
Method 1: For drug substance: A portionof 500 mg of drug substance wasweighed accurately into a disposable
15 mL glass centrifuge tube, and 5 mLof internal standard solution (~50 ng/mL
NDMA:C13-d6 in dichloromethane)
was added via volumetric pipette. The
sample was vortexed for one minute,then placed in the centrifuge and spunat 4,000 rpm for five minutes. The
undissolved metformin drug substancesettled at the bottom. Using a disposable
pipette, approximately 2 mL of thedichloromethane layer was filteredthrough a 0.45 µm nylon filter andtransferred to a GC vial for analysis.

For finished drug product: Approximately
10 tablets were crushed andhomogenized. From the homogenizedmixture, a portion equivalent to 500 mg
of drug substance was weighed andprocessed for extraction as above.
Method 2: For drug substance: A portionof 500 mg of drug substance wasweighed accurately into a disposable
15 mL glass centrifuge tube, and 5 mLof internal standard solution (~50 ng/mL
NDMA:C13-d6 in water) was added via
volumetric pipette. The sample wasvortexed for one minute followed by anaddition of 5 mL of dichloromethane.
The tubes were again vortexed for two
minutes, then placed in the centrifuge
and spun at 4,000 rpm for five minutes.
This resulted in the formation of alighter aqueous layer and a heavierorganic layer. Using a disposable
pipette, approximately 2 mL of thedichloromethane layer at the bottom waswithdrawn and filtered through a 0.45 µm
nylon filter and transferred to a GC vial
for analysis.
For finished drug product: Approximately
10 tablets were crushed andhomogenized. From the homogenizedmixture, a portion equivalent to 500 mg
of drug substance was weighed andprocessed for extraction as above.
Method 3: For drug substance: A portionof 500 mg of drug substance wasweighed accurately into a disposable
50 mL glass centrifuge tube, and 5 mLof internal standard solution (~50 ng/mL
NDMA:C13-d6 in 1 N HCl) was added
via volumetric pipette. The sample wasvortexed for one minute followed by anaddition of 5 mL of dichloromethane.
The tubes were again vortexed for two
minutes, then placed in the centrifuge
and spun at 4,000 rpm for five minutes.
This resulted in the formation of alighter aqueous layer and a heavierorganic layer. Using a disposable
pipette, approximately 2 mL of thedichloromethane layer at the bottom waswithdrawn and filtered through a 0.45 µm
nylon filter and transferred to a GC vial
for analysis.
For finished drug product: Approximately
10 tablets were crushed andhomogenized. From the homogenizedmixture, a portion equivalent to 500 mg
of drug substance was weighed andprocessed for extraction as above.

Standard preparation
The standard stock was diluted
appropriately to obtain calibration
solutions of the following concentrations:
100, 80, 40, 20, 10, 5, and 2.5 ng/mL,
each prepared in dichloromethane
containing NDMA:C13-d6 asinternal standard.

Table 1. GC parameters.
Parameter

Value

MMI Injection Mode

Pulsed splitless: 12.285 psi until 0.5 min

Inlet Temperature

250 °C

Oven Temperature Program

40 °C (1.5 min)
20 °C/min to 200 °C (0 min)
60 °C/min to 250 °C (3 min)Total Run Time13.33 minutes

MS Transfer Line Temperature

250 °CInjection Volume

2 µL

Carrier Gas

Helium, 1 mL/min

Table 2. MS parameters.
Parameter

Value

Mode

Electron ionization, 70 eV

Source Temperature

250 °C

Quadrupole Temperature

Q1 and Q2 = 150 °C

Instrumentation

MS1 Resolution

All compounds unit

Analysis was performed using the8890 GC equipped with the 7693A
automatic liquid sampler coupled to the7010B triple quadrupole GC/MS/MS.
From the inlet, an Agilent J&W DB-WAX
GC capillary column (30 m × 0.25 mm,
0.5 µm) was connected to the MS.

MS2 Resolution

All compounds unit

Collision Gas Flow

Nitrogen at 1.5 mL/min

Quenching Gas Flow

Helium at 4 mL/min

MRM Mode Conditions

Tables 1 and 2 display the GC and MS
parameters.

MS acquisition method
Using the MRM optimizer tool, the MRMs
for all five impurities were developed andwere used for data acquisition.

Quantifier/Qualifier
Transitions

Start time: 6.5 min

NDMA
74 → 44.1, CE 6, dwell 150 ms
74 → 42.1, CE 22, dwell 50 ms
NDMA:C13-d6 82 → 48, CE 20, dwell 100 ms

Start time: 7.60 min

NDEA
102 → 85, CE 4 V, dwell 80 ms
102 → 56.1, CE 18 V, dwell 80 ms
102 → 44.1, CE 14 V, dwell 80 ms

Start time: 8.03 min

NEIPA
116 → 99.1, CE 4 V, dwell 80 ms
71 → 56.1, CE 4 V, dwell 80 ms
116 → 44.1, CE 14 V, dwell 80 ms

Start time: 8.25 min

NDIPA
130 → 88, CE 4 V, dwell 150 ms
130 → 42, CE 10 V, dwell 150 ms

Start time: 8.70 min

NDBA
158 → 99.1, CE 8 V, dwell 75 ms
84 → 56.1, CE 20 V, dwell 75 ms
84 → 42.1, CE 14 V, dwell 75 ms
158 → 141.2, CE 2 V, dwell 75 ms

3

Results and discussion

The optimized MRMs were exported
to the method and sample acquisition
was carried out. Figure 1 describes the
optimized MRMs and the chromatogram
obtained is displayed in Figures 2 and 3.

The MRMs for this method weredeveloped using the TQ optimizer.
Automated MRM development was
carried out to obtain the optimized
MRMs of all five impurities. The process
for automated MRM development is
described in another application note.5

The compounds were separated
sufficiently, and the target peaks(Figure 2) were well resolved fromsolvent and matrix species.

Calibration curves were generated using
a linear fit. Excellent linearities with
R2 >0.999 were obtained in this study for
all five impurities, as shown in Figure 4.
The repeatability of injections wastested by consecutive injection of a
40 ng/mL standard. Six consecutive
injections resulted in <2% RSDs for all
five impurities.

EϭϬϮїϰϰ͘ϭ͕ϭϰsEϭϬϮїϱϲ͘ϭ͕ϭϴsE/Wϭϭϲїϰϰ͘ϭ͕ϭϰsE/WϭϯϬїϴϴ͕ϰsE/WϭϯϬїϰϮ͕ϭϬs

NDBAϴϰїϰϮ͘ϭ͕ϭϰs

NDBAϭϱϴїϭϰϭ͘Ϯ͕Ϯs

NDMAϳϰїϰϰ͘ϭ͕ϲ

NDMAϳϰїϰϮ͘ϭ͕ϮϮEϭϬϮїϴϱ͕ϰ

E/Wϭϭϲїϵϵ͘ϭ͕ϰsE/Wϳϭїϱϲ͘ϭ͕ϰs

NDBAϭϱϴїϵϵ͘ϭ͕ϴs

NDBAϴϰїϱϲ͘ϭ͕ϮϬs

Figure 1. Optimized MRM transitions of five nitrosamine impurities using the TQ optimizer.
×106+ TIC MRM (** -> **) Std01.D
NDMA
7.276

1.6

NEIPA

1.4

NDEA
7.885

1.2
Counts

2 3 8.148 3 4

1 2

4 5

NDIPA
8.341

1.0

NDBA
9.965

0.8
0.6
0.4
0.2
0
7.0

7.2

7.4

7.6

7.8

8.0

8.2

8.4

8.6

8.8

Acquisition time (min)Figure 2. TIC chromatogram overlay in MRM mode of 100 ng/mL of five impurities in dichloromethane.

4

9.0

9.2

9.4

9.6

9.8

10.0

NEIPA: + MRM (116.0 -> 99.1) Std07.D
×104

1 2

7.279

2.5

2 3

3 4

4 5

8.150

9.968

Counts

2.0
1.5

8.344

7.888

1.0
0.5
7.189

0
7.0

7.2

7.4

7.6

7.8

8.0

8.2

8.4

8.6

8.8

9.0

9.2

9.4

9.6

9.8

10.0

10.2

Acquisition time (min)Figure 3. Extracted MRM chromatogram (quantifier and qualifier transition) of lowest calibration standard at 2.5 ng/mL mix of five impurities in dichloromethane.
×101 NDMA
4.5

4.5

3.5
3.0
2.5
2.0
1.5

3.5
3.0
2.5
2.0
1.5
1.0

0.5

0.5

0

0
0

×10

9
8

2

4

NEIPA

6
8 10 12 14
Relative concentration

16

18

20

0
×10
6

1

y = 4.544903x – 1.191693
R2 = 0.9997

5
Relative response

7
6
5
4
3
2

y = 2.679070x – 0.682552
R2 = 0.9998

4.0

1.0

1

Relative response

5.0

Relative response

Relative response

4.0

×101 NDEA

y = 2.454640x – 0.534529
R2 = 0.9997

2

4

NDIPA

6
8 10 12 14
Relative concentration

16

18

20

16

18

20

y = 3.018445x – 0.799795
R2 = 0.9997

4
3
2
1

1

0

0
0

2

4

6
8 10 12 14 16
Relative concentration

18

20

18

20

0

2

4

6
8 10 12 14
Relative concentration

Relative response

×101 NDBA
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

y = 2.981789x – 0.951226
R2 = 0.9996

0

2

4

6
8 10 12 14 16
Relative concentration

Figure 4. Calibration curves of NDMA, NDEA, NEIPA, NDIPA, and NDBA (2.5 to 100 ng/mL).

5

Samples of drug substances and drug
products were analyzed using all threemethods as described in previous
sections. When a drug substance
was analyzed using methods 1 and 3,
relatively higher peak areas were noted.
Method 2 resulted in comparatively lower
peak areas; however, the calculated
values were similar owing to internal
standard correction (Figure 5). Similar
observations were noted for drug
product as well (Figure 6).
Method 1 could be used efficiently for
all samples tested for drug substance.
When the same method was used for
drug products, the presence of certain
excipients resulted in the formation of aviscous solution. Some of the excipients(e.g., povidone K-30, povidone K-90)
present in the final drug product were
freely soluble in dichloromethane. As
a result, it was impossible to obtain aclear extract through centrifugationor filtration. Therefore method 1 was
unsuitable for most of the drug products.
Methods 2 and 3 were advantageous
in terms of phase separation where
the gel formation was limited to theaqueous layer and it was relatively easierto separate the clear organic layer after
centrifugation. No specific advantages
in terms of reduction in interfering peaks
or coelutions were noted in the samples
tested. Method 2 resulted in lesser peakareas, and therefore was associated withhigher LOQ. Methods 1 and 3 resulted inlower method LOQs.

6

Table 3. Peak areas and %RSD values of nitrosamine impurities at 40 ng/mL.
Standard Injections

NDMA (Area)

NDEA (Area)

NEIPA (Area)

NDIPA (Area)

NDBA (Area)

Std3_RepCheck_001.D

381642403839676849445496412430

Std3_RepCheck_002.D

379964402289669489445593413609Std3_RepCheck_003.D

383455404972684527451592403516

Std3_RepCheck_004.D

386450407865685165457072398952

Std3_RepCheck_005.D

389766409850694079460929408615

Std3_RepCheck_006.D

392361418186704485466412405082

1.25

1.41

1.80

1.86

1.37

Method 1 is simplest in terms ofsample preparation but unsuitable forformulations that can result in viscous
extracts of dichloromethane. Method 2
can be used to analyze both drug
substances and drug products. Lower
absolute areas indicate that recoveries
are reduced. However, quantification
based on internal standard correction
results in satisfactory recoveries.
Method 3 can be used to analyze both
drug substances and formulation.
Sample recoveries were calculated
by fortifying the drug substanceand a homogenized drug product at
0.005 ppm. The recoveries were foundsatisfactory within the range of 80 to120% for all three methods.

A United States Pharmacopeia (USP)
signal‑to‑noise ratio (S/N) of 10 wasused as a basis for LOQ determinationin the methods. In the present study,
S/N values for samples spiked atconcentrations of 0.001 ppm were
evaluated. This demonstrates thatthe instrument meets the sensitivity
requirements easily and further lower
LOQs could be achieved, enabling very
trace level detections (Figure 7).

NDMA and NDEA in drug substances

2.0
1.8
1.6

Method 1

Counts

1.4+ MRM (74.0 & 42.1)

×102
1.1

RT = 7.278 min.
Name = NDMA
Area = 30,888.87
Final conc. = 67.3583 ppb

1.0
0.9Counts

×104

1.2
1.0

0.8

+ MRM (102.0 & 85.0)
RT = 7.883 min.
Name = NDEA
Area = 135.67
Final conc. = 24.3912 ppb

0.7
0.6

0.8
0.6

0.5

0.4

0.4

0.2

0.3

0

0.2
6.9

7.0

7.1

7.2

7.3

7.4

7.7

7.5

Acquisition time (min)+ MRM (74.0 & 42.1)

1.4

RT = 7.278 min.
Name = NDMA
Area = 22,578.70
Final conc. = 68.4608 ppb

Method 2

Counts

1.2
1.0
0.8

×101

Counts×104
1.6

Decrease in peak area by 27%

0.6
0.4
0.2
0
6.9

7.0

7.1

7.2

7.3

9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5

8.0

8.1

8.0

8.1

7.8
7.9
8.0
Acquisition time (min)8.1

RT = 7.888 min.
Name = NDEA
Area = 78.44
Final conc. = 24.3554 ppb
Decrease in peak areaby 42%

7.7

7.4

7.8

7.9

Acquisition time (min)+ MRM (74.0 & 42.1)

×102
1.2

RT = 7.278 min.
Name = NDMA
Area = 34,675.17
Final conc. = 69.2073 ppb

1.1
1.0
CountsCounts

Method 3

2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

7.9

+ MRM (102.0 & 85.0)

Acquisition time (min)
×104

7.8

Acquisition time (min)0.9+ MRM (102.0 & 85.0)

RT = 7.888 min.
Name = NDEA
Area = 113.08
Final conc. = 24.3483 ppb

0.8
0.7
0.6
0.5
0.4
0.3
0.2

7.0

7.1
7.2
7.3
Acquisition time (min)

7.4

7.5

7.7

Figure 5. NDMA and NDEA in a metformin sample using three different extraction methods.

7

NDMA and NDEA in drug substances

2.2
2
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

×102

+ MRM (102.0 & 85.0)

1.0
0.9

RT = 7.278 min.
Name = NDMA
Area = 33,632.43
Final conc. = 71.1737 ppb

0.8Counts

Method 1

Counts×104+ MRM (74.0 & 42.1)

0.7

RT = 7.888 min.
Name = NDEA
Area = 119.82
Final conc. = 24.3427 ppb

0.6
0.5
0.4
0.3
0.2

7.0

7.1

7.2

7.3

7.4

7.5

7.7

Acquisition time (min)
×104

×102

+ MRM (74.0 & 42.1)

1.8

Method 2

Counts

1.4
1.2
1.0

0.9

RT = 7.278 min.
Name = NDMA
Area = 24,904.98
Final conc. = 73.4202 ppb

0.8
0.6

Decrease in peak area by 25%

0.2

0.3
7.1
7.2
7.3
Acquisition time (min)

7.4

7.8
7.9
8.0
Acquisition time (min)8.1+ MRM (102.0 & 85.0)

RT = 7.888 min.
Name = NDEA
Area = 83.15
Final conc. = 24.3647 ppb

Decrease in peak areaby 30%

7.7

7.5

×102

+ MRM (74.0 & 42.1)+ MRM (102.0 & 85.0)

1.1
RT = 7.273 min.
Name = NDMA
Area = 37,161.88
Final conc. = 73.3600 ppb

1.0
0.9
0.8

RT = 7.883 min.
Name = NDEA
Area = 123.70
Final conc. = 24.3640 ppb

0.7
0.6
0.5
0.4
0.3
0.2

6.9

7.0

7.1
7.2
7.3
Acquisition time (min)

7.4

7.5

Figure 6. NDMA and NDEA in a metformin finished product using three different extraction methods.

8

8.1

0.2
7.0

Counts

2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

8.0

0.6

0.4

6.9

Counts

0.7

0.5

0

Method 3

0.8

0.4

×104

7.9

1.0

Counts

1.6

7.8

Acquisition time (min)

7.7

7.8
7.9
8.0
Acquisition time (min)

8.1

1.0

RT = 7.278 min
Name = NDMA
S/N = 11.06
Area = 970.56

4.0
3.5

0.7

NDMA: 0.001 ppm
S/N >11

1.5

0.4

0.5
6.7

6.8

6.9 7.0 7.1 7.2 7.3
Acquisition time (min)×103+ MRM (116.0 & 99.1)

1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

RT = 8.150 min
Name = NEIPA
S/N = 47.73
Area = 1,718.64

7.4

4.0

7.8

7.9

8.0

8.1

Acquisition time (min)
×102
6.5
6.0
5.5
5.0
4.5

+ MRM (130.0 & 88.0)
RT = 8.344 min
Name = NDIPA
S/N = 15.82
Area = 569.06

NDIPA: 0.0005 ppm
S/N >15

4.0
3.5
3.0
8.10

8.15

8.20

8.25

Acquisition time (min)4.5

NDEA: 0.0005 ppm
S/N >34

7.7

7.5

NEIPA: 0.001 ppm
S/N >47

8.05

Counts

2.0

1.0

5.0

RT = 7.888 min
Name = NDEA
S/N = 34.71
Area = 517.97

2.5

0.5

×102

+ MRM (102.0 & 85.0)

3.0

0.8

0.6

Counts×102

CountsCounts0.9+ MRM (74.0 & 42.1)

Counts

×103
1.1

2.5
8.1

8.2

8.3

8.4

8.5

8.6

Acquisition time (min)+ MRM (158.0 & 141.2)
RT = 9.972 min
Name = NDBA
S/N = 13.30
Area = 427.28

3.5
3.0
2.5
2.0

NDBA: 0.0025 ppm
S/N >13

1.5
1.0
0.5
0
9.00

9.25

9.50

9.75

10.00 10.25 10.50 10.75

Acquisition time (min)Figure 7. S/N of samples spiked at trace levels for LOQ determination.

9Conclusion

References

The Agilent 8890 GC and theAgilent 7010B GC/MS/MS systems
demonstrated excellent performance forthe determination of all five nitrosamine
drug impurities in metformin. The
8890 GC offers instrument operation
diagnostic testing and systemmonitoring alerts as well as touch
screen control and mobile access. The
mobile access option enables operatorsand managers to securely monitor
instrument status and function while
away from the lab, ensuring minimum
downtime along with unassisted
troubleshooting. These features areuseful for continued operation of thesystem. The design of the 7010B triple
quadrupole GC/MS, which includes the
HES, enables lower detection limits fortrace-level impurities when combinedwith the inert sample path provided by
the 8890 GC. These features enabledreliable quantification of all five residues.

1.

https://www.ema.europa.eu/
en/human-regulatory/postauthorisation/referral-procedures/
nitrosamine-impurities

2.

https://www.ema.europa.eu/en/
news/ema-update-metformindiabetes-medicines

3.

https://www.fda.gov/drugs/drugsafety-and-availability/fda-updatesand-press-announcements-ndmametformin

4.

https://www.edqm.eu/en/ad-hocprojects-omcl-network#Methods%20
for%20determination%20of%20
nitrosamines%20in%20metformin

5.

Andrianova, A.; Quimby, B.;
Churley, M.; Automated MRM
Method Development for US EPA
Method 8270 with the Agilent
MassHunter Optimizer for GC/TQ.
Agilent Technologies applicationnote, publication number
5994‑2086EN, 2020.

www.agilent.com/chem
DE.4620486111
This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Printed in the USA, September 21, 2020
5994-2419EN