Nitrosamine impurities in medications: test methods

The following testing methods have been developed by Health Canada and may serve as a potential testing option for regulators and industry to detect nitrosamine impurities in certain drug substances and drug products. These methods should be validated by the user if the resulting data are used to support a required quality assessment of the API or drug product, or if the results are used in a regulatory submission. Methods are subject to change as new information becomes available.

On this page

• Test method for sartans or angiotensin II receptor blockers (ARBs)
• Test method for ranitidine products
• Test Methods for metformin products

Test method for sartans or angiotensin II receptor blockers (ARBs)

Determination of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by GC-MS/MS (direct injection) in sartan finished products and drug substances

1. Principle and Scope

The present method has been developed to detect and quantify the nitrosamine impurities N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) in valsartan, irbesartan and losartan finished products. The method is performed by gas chromatography-tandem mass spectrometry (GC-MS/MS) using direct injection.

The method can also be used to detect and quantify NDMA and NDEA in candesartan and olmesartan finished products, and in sartan drug substances (e.g., valsartan, irbesartan, losartan, candesartan and olmesartan). However, if interferences are observed, further validation may be required.

2. Reagents and Reference Standards

• Methanol, HPLC grade (CAS #: 67-56-1)
• NDMA: N-nitrosodimethylamine solution (5000 μg/mL in methanol; CAS #: 62-75-9)
• NDEA: N-nitrosodiethylamine solution, (100 μg/mL in methanol; CAS #: 55-18-5)
• NDMA-d6 solution: N-nitrosodimethylamine-d6 (1000 μg/mL in methanol; CAS #: 17829-05-9)

3. Instrument/Equipment

• Agilent GC 7890A with MS/MS 7000 with EI source or equivalent
• Agilent DB-624 25 m x 0.32 μm 1.8 μm or equivalent
• 2 mL amber screw-cap GC vials with caps (PTFE/Silicone)
• 20 x 125 mm screw cap round bottom glass tubes with caps
• Automatic pipettes, various volumes
• Top-loading balance suitable for ± 0.01 g and Analytical balance suitable for ± 0.0001 g readability
• Volumetric flasks (class A), various volumes
• Pasteur pipettes and pipette bulbs
• Spatula
• 15 mL amber glass vials with caps
• 40 mL amber glass vials with caps
• Vortex mixer, single and multi-tube
• Vial racks and storage racks
• Kimwipes
• Mortar and pestle
• Ultrasonic water bath
• Centrifuge, Beckmann-Coulter Allegra X-15R or equivalent

4. Preparation of Solutions

Standard solutions

Reference standard stock solutions (as purchased)

• NDMA standard solution in methanol (5000 ppm)
• NDEA standard solution in methanol (100 ppm)

Internal reference standard stock solution (as purchased)

• NDMA-d6 standard solution in methanol (1000 ppm)

Diluted standard solutions

NDMA standard solution (200 ppm):

Transfer 800 μL of NDMA reference solution (5000 ppm) into a 20 mL volumetric flask, dilute to volume with methanol.

Diluted standard solution (NDMA: 40 ppm, NDEA: 20 ppm):

Transfer 800 μL of NDMA standard solution (200 ppm) and 800 μL of NDEA reference solution (100 ppm) into a 4 mL volumetric flask, dilute to volume with methanol.

Internal standard solution-1 (20 ppm):

Transfer 500 μL of NDMA-d6 standard solution (1000 ppm) into a 25 mL volumetric flask, dilute to volume with methanol.

Internal standard solution-2 (0.2 ppm):

Transfer 5 mL of internal standard solution-1 (20 ppm) into a 500 mL volumetric flask, dilute to volume with methanol.

Calibration solutions

STD-12:

Transfer 500 μL of diluted standard solution (NDMA: 40 ppm, NDEA: 20 ppm) and 50 μL of internal standard solution-1 (20 ppm) into a 5 mL volumetric flask, dilute to volume with methanol. Mix well.

STD-7:

Transfer 1000 μL of STD-12 into a 20 mL volumetric flask, dilute to volume with internal standard solution-2 (0.2 ppm). Mix well. This solution will be used for the system suitability test and system drift check.

STD-11:

1:1 dilution of STD-12 with internal standard solution-2

STD-10:

1:1 dilution of STD-11 with internal standard solution-2

STD-9:

1:1 dilution of STD-10 with internal standard solution-2

STD-8:

3:2 dilution of STD-9 with internal standard solution-2

STD-6:

1:1 dilution of STD-7 with internal standard solution-2

STD-5:

1:1 dilution of STD-6 with internal standard solution-2

STD-4:

2:3 dilution of STD-5 with internal standard solution-2

STD-3:

1:1 dilution of STD-4 with internal standard solution-2

STD-2:

1:1 dilution of STD-3 with internal standard solution-2

STD-1:

2:3 dilution of STD-2 with internal standard solution-2

Concentration of calibration solutions:

Description	NDMA concentration (µg/mL)	NDEA concentration (µg/mL)	NDMA-d6 concentration (µg/mL)
STD-1	0.002	0.001	0.2
STD-2	0.005	0.0025	0.2
STD-3	0.01	0.005	0.2
STD-4	0.02	0.01	0.2
STD-5	0.05	0.025	0.2
STD-6	0.1	0.05	0.2
STD-7	0.2	0.1	0.2
STD-8	0.3	0.15	0.2
STD-9	0.5	0.25	0.2
STD-10	1	0.5	0.2
STD-11	2	1	0.2
STD-12	4	2	0.2

For NDMA, STD-1 to STD-6 are used as working range from 0.002 - 0.1 µg/mL, and STD-6 to STD-12 are used as working range from 0.1 - 4.0 µg/mL.

For NDEA, STD-1 to STD-5 are used as working range from 0.001 - 0.025 µg/mL, and STD-5 to STD-11 are used as working range from 0.025 - 1.0 µg/mL.

The working ranges of NDMA and NDEA can be adjusted as needed.

Sample Preparation

For finished product:

Weigh NLT 20 tablets and calculate average tablet weight. Carefully grind NLT 20 tablets into fine powder using a mortar and pestle.

Prepare triplicate samples for each product. Accurately weigh an amount equivalent to 250 mg of drug substance of the homogenized sample powder into a screw cap round bottom glass tube.

Using an automatic pipette, add 5 mL of NDMA-d6 internal standard solution-2 to each sample tube. Tightly cap the tubes, sonicate for 5 minutes, and then vortex the rack of tubes on the multi-tube vortex mixer at 2000 rpm for five minutes.

Centrifuge the tubes for at least 5 minutes at 1500 rpm. Carefully remove the tubes from the centrifuge. Use a Pasteur pipette to transfer an aliquot from each tube to a 2 mL GC vial and cap.

Note: Method accuracy was assessed by recovery studies. Valsartan, irbesartan and losartan finished products were spiked with reference standard solution in the following way:

For samples with NDMA above 0.3 ppm and/or NDEA above 0.08 ppm:

Spike sample solution with the reference standard solution at concentration level close to sample concentration and calculate recovery.

For samples with NDMA below 0.3 ppm and/or NDEA below 0.08 ppm:

Spike sample solution with STD-3 (1:1) to get solution containing 0.05 ppm of NDMA and 0.025 ppm of NDEA. Check S/N and calculate the LOD and LOQ.

For drug substance:

Prepare triplicate samples for each substance. Accurately weigh 250 mg of the homogenized sample powder into a screw cap round bottom glass tube.

Continue as per the instructions above for finished product. Spiking to determine method accuracy is recommended.

5. Instrument Operating Parameters:

Suggested GC parameters:

Injector Settings:

Injector mode:

Pulsed Splitless

Injector temperature:

240°C

Flow rate:

1.8 mL/min

Septum purge flow:

3 mL/min

Purge Flow:

50 mL/min after 0.75 minutes

Injection volume:

2.0 µL

Oven program:

Initial Temp: 60°C
Hold: 2 minutes

Table 1. oven program

Ramp #	Rate (°C/min)	Final Temp (°C)	Hold Time (min)
1	5	130	0
2	40	240	5
Total run time: 24 minutes

Suggested MS settings

MS transfer line (aux. temp):

250°C

Ion source:

EI

Source temperature:

250 °C

Solvent delay:

6 minutes

Stop time:

15 minutes

Quench gas:

Helium at 2.25 mL/min

Collision gas:

Nitrogen at 1.5 mL/min

Table 2. MS/MS parameters

Analyte	Retention time (min)	Segment	Retention time window (min)	Precursor ion (m/z)	Product ion (m/z)	CE (V)	Resolution	Dwell (ms)
NDMA-d6 (ISTD)	7.8	1	7.5-8.1	80	50	5	wide/wide	100
NDMA	7.8	1	7.5-8.1	74	42	15	wide/wide	100
				74	44	4		100
NDEA	12.7	2	12.4-13.0	102	44	12	wide/wide	150
				102	85	2		150

6. System Suitability

The coefficient of determination (R2) for each calibration curve is NLT 0.995.

The signal-to-noise of the STD-1 (NDMA = 0.002 µg/mL; NDEA = 0.001 µg/mL) solution should be NLT 10.

7. Calculation

Construct calibration curves for NDMA and NDEA by plotting the ratio of response factor (NDMA or NDEA peak area divided by internal standard peak area) against standard concentration (µg/mL). Using the slopes and intercepts of the calibration curves, determine the content of NDMA and NDEA in each sample using the following equations.

For finished product:

The results, in ppm relative to the declared amount of sartan drug substance in the product, are given by:

Where,

y =

ratio of peak area of NDMA or NDEA to peak area of NDMA-d6

b =

intercept of the linear curve

m =

slope of the linear curve

Wt_spl =

sample weight (g)

AVG_wt =

average tablet weight (g)

LC =

label claim of sample (g)

V =

5 mL (volume)

Text description

Parts per million equals y minus b over m, which is the difference between the ratio of the peak area of the impurity to peak area of internal standard minus the intercept of the linear curve over the slope of the linear curve. This value is multiplied by average tablet weight, then multiplied by the volume, divided by the sample weight and then divided by the label claim of the sample.

For drug substance:

The results, in ppm relative to the drug substance being tested, are given by:

Where,

y =

Ratio of Peak Area of NDMA or NDEA to Peak Area of NDMA-d6

b =

intercept of the linear curve

m =

slope of the linear curve

Wt_spl =

sample weight (g)

V =

5 mL (volume)

Text Description

Parts per million equals y minus b over m, which is the difference between the ratio of the peak area of the impurity to peak area of internal standard minus the intercept of the linear curve over the slope of the linear curve. This value is multiplied by the volume and then divided by the weight of the sample.

8. LOD and LOQ results

LOD/LOQ can be calculated using the S/N of the spiked sample solution (spiked with STD-3 at 1:1).

Theoretical LOD/LOQ:

If no spiked results are available, the theoretical LODs and LOQs can be calculated by using the S/N of STD-1 (NDMA: 0.002 µg/mL; NDEA 0.001 µg/mL).

For reference, the theoretical LOD/LOQ results at Health Canada are as follows:

Table 3. S/N of NDMA and NDEA of STD-1

Drug substance conc. µg/mL	NDMA				NDEA
	µg/mL	S/N	LOD (calc.) ppm	LOQ (calc.) ppm	µg/mL	S/N	LOD (calc.) ppm	LOQ (calc.) ppm
50	0.002	74	0.002	0.0054	0.001	55	0.002	0.0073

9. Sample Chromatograms

Text description

The figure shows chromatograms of a sample showing typical peaks for NDMA and NDEA. The chromatograms are arranged in 4 rows.

• The first row shows the Total Ion Current chromatogram from 6.0 to 15.0 minutes. There is a large peak at 8.5 minutes (labelled "matrix") and smaller peaks at 7.7 minutes (NDMA, NDMA-d6) and 12.6 minutes (NDEA).
• The second row shows a Multiple Reaction Monitoring chromatogram (m/z 80.0 → 50.0) with a single peak at 7.735 minutes for the NDMA-d6 internal standard.
• The third row shows two Multiple Reaction Monitoring chromatograms (m/z 74.0 → 44.0 and m/z 74.0 → 42.0) each with a single peak at 7.787 minutes for NDMA.
• The fourth row shows two Multiple Reaction Monitoring chromatograms (m/z 102.0 → 85.0 and m/z 102.0 → 44.0) each with a single peak at 12.561 minutes for NDEA.

Text description

The figure shows chromatograms of the STD-1 standard solution. The chromatograms are arranged in 4 rows.

• The first row shows the Total Ion Current chromatogram from 6.0 to 15.0 minutes. There is a large peak at 7.7 minutes (NDMA, NDMA-d6) and a small peak at 8.5 minutes (labelled "matrix").
• The second row shows a Multiple Reaction Monitoring chromatogram (m/z 80.0 → 50.0) with a single peak at 7.715 minutes for the NDMA-d6 internal standard.
• The third row shows two Multiple Reaction Monitoring chromatograms (m/z 74.0 → 44.0 and m/z 74.0 → 42.0) each with a single peak at 7.767 minutes for NDMA.
• The fourth row shows two Multiple Reaction Monitoring chromatograms (m/z 102.0 → 85.0 and m/z 102.0 → 44.0) each with a single peak at 12.515 minutes for NDEA.

Test method for ranitidine products

Determination of N-nitrosodimethylamine (NDMA) in ranitidine products by UPLC-MS/MS

1. Principle and Scope

The present method has been developed to detect and quantify N-nitrosodimethylamine
(NDMA) in ranitidine products by UPLC-tandem mass spectrometer (UPLC-MS/MS).

2. Reagents and Reference Standards

• NDMA, N-nitrosodimethylamine solution, 200 ppm, (CAS #62-75-9)
• Methanol, LC-MS grade (CAS #67-56-1)
• Formic acid, LC-MS grade (CAS #64-18-6)
• De-ionized water

3. Instrument/Equipment

• Waters UPLC-TQ-S micro with APCI source or equivalent
• Column: ACQUITY UPLC HSS T3 Column, 100Å, 1.8 µm, 3 mm x 150 mm or equivalent.
• 16 x 125 mm screw cap glass tubes
• 2.0 mL micro centrifuge tubes
• Eppendorf pipettes, various volumes, or equivalent
• Analytical balance
• Volumetric flasks (class A), various volumes, or equivalent
• Vortex mixer, single and multi-tube
• Mortar and pestle
• Ultrasonic water bath
• HPLC vials
• Centrifuge

4. Preparation of Solutions

Storage

Calibration and sample solutions are stored in refrigerator.

Mobile Phase Preparation

• Mobile Phase A: 0.1% formic acid in water
  • Preparation: Mix formic acid and water at a volume ratio of 1:1000.
• Mobile Phase B: 0.1% formic acid in methanol
  • Preparation: Mix formic acid and methanol at a volume ratio of 1:1000.

Diluent Preparation

• Diluent: 5% methanol in water
  • Preparation: Mix methanol and water at a volume ratio of 5:95.

Standard Solutions

Reference standard stock solutions (as purchased)

NDMA standard solution in methanol (200 ppm)

Calibration standard solutions

STD-1:

Transfer 50 µL of NDMA reference standard stock solution (200 ppm) into a 50 mL volumetric flask, dilute to volume with diluent. Mix well. This solution will be used as system suitability test and system drift check.

STD-2:

1:1 dilution of STD-1 with diluent. Mix well.

STD-3:

1:1 dilution of STD-2 with diluent. Mix well.

STD-4:

1:1 dilution of STD-3 with diluent. Mix well.

STD-5:

2:3 dilution of STD-4 with diluent. Mix well.

STD-6:

1:1 dilution of STD-5 with diluent. Mix well.

STD-7:

1:1 dilution of STD-6 with diluent. Mix well.

STD-8:

2:3 dilution of STD-7 with diluent. Mix well.

STD-9:

3:1 dilution of STD-8 with diluent. Mix well (LOD).

Table 1. Concentration of calibration solutions

Calibration Standard Solution	NDMA Concentration (ng/mL)
STD-1	200
STD-2	100
STD-3	50
STD-4	25
STD-5	10
STD-6	5
STD-7	2.5
STD-8	1
STD-9	0.75

R² for calibration curve should be NLT 0.99.

Sample Preparation

• Weigh at least 20 tablets, calculate average weight per tablet.
• Carefully grind the weighed tablets into fine powder using mortar and pestle.

Suggested sample preparation:

• Prepare in triplicate for each sample.
• Accurately weigh homogenized sample powder equivalent to about 150 mg of the API into separate glass centrifuge tubes.
• Add 5.0 mL of diluent to each sample tube. Tightly cap the tubes, sonicate for 10 minutes, and then vortex at 2500 rpm for 5 minutes.
• Transfer 2.0 mL solution to 2 mL centrifuge tube, centrifuge at 12 000 rpm for 5 minutes. Carefully remove the tubes from the centrifuge.
• Filter the supernatant using 0.2 µm PTFE syringe filter, discard the first 0.5 mL of filtrate. Collect and transfer the filtered sample solution into a 2 mL HPLC vial and cap.

The concentration of samples (weight and/or volume) may be modified as required.

Suggested Recovery Spiking:

Spike samples: Spike sample solutions with reference standard solution in triplicate and check recoveries.

5. Instrument Operating Parameters

Suggested UPLC Parameters:

Flow rate:

0.4 mL/min

Injection volume:

10.0 µL

Run time:

12.5 minutes

Table 2. Gradients

Time (min)	Mobile Phase A%	Mobile Phase B%
0	95	5
5	95	5
7	0	100
10	0	100
10.1	95	5
12.5	95	5

Suggested MS Parameters:

APCI probe temperature:

450°C

Source temperature:

150°C

Corona:

4.0 kV

Type:

MRM

Ion mode:

Positive

Span (Da):

0.3

Start time:

3.3 minutes

End time:

5.0 minutes

Run events:

Event 1:

0.00 min flow state: Waste

Event 2:

3.30 min flow state: LC

Event 3:

5.00 min flow state: Waste

Note: Flow state setting may be adjusted as necessary depending on the system to divert ranitidine to waste.

MRM Parameters:

Table 2. MRM parameters

Name	Parent Ion (m/z)	Daughter Ion (m/z	Dwell (s)	Cone (V)	Collision Cell (eV)
NDMA	75.1	42.9	0.3	22	10
NDMA	75.1	58.1	0.3	22	10

Note: Transition 75.1→58.1 is used for quantification; transition 75.1→42.9 usually has a higher background. It is possible to quantify by using another parent ion/daughter ion pair transition.

6. Calculation

The results, in ppm relative to the declared amount of drug substance in the product, are given by:

NDMA (ppm) = [(y-b)/m] x AVG_wt x V ÷ Wt_spl ÷ LC

Where,

y =

peak area of NDMA

b =

intercept of the calibration curve

m =

slope of the calibration curve

Wt_spl =

sample weight (mg)

AVG_wt =

average tablet weight (mg)

LC =

label claim of sample (mg)

V =

5 mL (volume)

Equation - Text Description

NDMA in parts per million equals y minus b over m, which is the difference between the peak area of the impurity minus the intercept of the linear curve over the slope of the linear curve. This value is multiplied by average tablet weight, then multiplied by the volume, divided by the sample weight and then divided by the label claim of the sample.

7. Range, limit of detection (LOD), and limit of quantification (LOQ)

Table 3. Range, LOD and LOQ

	NDMA (ng/mL)	NDMA (ppm)Table 3 Footnote *
LOQ	1	0.033
LOD	0.75	0.025
Range	1 – 200	0.03 – 6.7
Table 3 Footnote * ppm relative to the declared amount of drug substance in the product Table 3 Return to footnote * referrer

8. Example of Calibration Curve and Chromatograms

Text description

The figure shows a calibration curve with concentration of the NDMA standard solutions on the x axis and response (in arbitrary units) on the y axis. There is a data point for each of the nine standard solutions listed in the method, connected by a straight line. The correlation coefficient is r² = 0.999761, and the calibration curve equation is y = 257.934 x – 15.8397.

Text description

The figure includes two panels. The panel on the left shows two Multiple Reaction Monitoring chromatograms (m/z 75.1 → 58.1 and m/z 75.1 → 42.9) of the STD-1 standard solution, each with a single peak at 4.05 minutes for NDMA. The panel on the right shows the UV chromatogram of the STD-1 standard solution. The portion of the chromatogram between 3.3 and 5.0 minutes (corresponding to the period when flow is routed to the MS detector) is relatively flat, while the rest of the chromatogram contains multiple peaks and baseline shifts.

Text description

The figure includes two panels. The panel on the left shows two Multiple Reaction Monitoring chromatograms (m/z 75.1 → 58.1 and m/z 75.1 → 42.9) of a typical sample solution, each with a single peak at 4.01 minutes for NDMA. The panel on the right shows the UV chromatogram of a typical sample solution. The portion of the chromatogram between 3.3 and 5.0 minutes (corresponding to the period when flow is routed to the MS detector) is relatively flat, while the rest of the chromatogram contains multiple peaks and baseline shifts.

Test Methods for metformin products

Determination of Nitrosamine Impurities in metformin Products by UPLC-MS/MS

1. Principle and Scope

The present method has been developed to detect and quantify the following nitrosamine impurities in Metformin products by UPLC with tandem mass spectrometer (UPLC-MS/MS): N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), N-ethyl-N-nitroso-isopropylamine (NEIPA), N-nitroso-di-N-butylamine (NDBA) and N-Nitroso-N-methyl-4-aminobutyric acid (NMBA).

2. Reagents and Reference Standards

• NDMA, N-Nitrosodimethylamine solution (200 ppm), CAS # 62-75-9
• NDEA, N-Nitrosodiethylamine solution (100 ppm), CAS # 55-18-5
• NEIPA, N-ethyl-N-nitroso-isopropylamine solution (100 ppm), CAS # 16339-04-01
• NDIPA, N-Nitrosodiisopropylamine solution (100 ppm), CAS # 601-77-4
• NDBA, N-Nitroso-di-n-butylamine solution (100 ppm), CAS # 924-16-3
• NMBA, N-Nitroso-N-methyl-4-aminobutyric acid, CAS # 61445-55-4
• Methanol, LC-MS grade, CAS # 67-56-1
• Formic Acid, LC-MS grade, CAS # 64-18-6
• De-ionized water

3. Instrument/Equipment

• Waters UPLC-TQ-S micro with APCI source or equivalent.
• Column: ACQUITY UPLC HSS T3 Column, 100 Å, 1.8 µm, 3 mm X 150 mm or equivalent
• 16 x 125 mm screw cap glass tubes
• 2.0 mL micro centrifuge tubes
• Eppendorf pipettes, various volumes, or equivalent
• Analytical balance
• Volumetric Flasks (class A), various volumes, or equivalent
• Vortex mixer, single and multi-tube
• Mortar and pestle
• Ultrasonic Water bath
• HPLC vials
• Centrifuges

4. Preparation of Solutions

4.1 Storage

Calibration and sample solutions are stored in refrigerator.

4.2 Mobile Phase Preparation

• Mobile Phase A: 0.1% formic acid in water
  • Preparation: Mix formic acid and water at a volume ratio of 1:1000.
• Mobile Phase B: 0.1% formic acid in methanol
  • Preparation: Mix formic acid and methanol at a volume ratio of 1:1000.

4.3 Diluent Preparation

• Diluent: 5% Methanol in water
  • Preparation: Mix methanol and water at a volume ratio of 5:95.

4.4 Standard Solutions

Reference Standard Stock Solutions (as purchased)

• NDMA Standard Solution in Methanol (200 ppm)
• NDEA Standard Solution in Methanol (100 ppm)
• NEIPA Standard Solution in Methanol (100 ppm)
• NDIPA Standard Solution in Methanol (100 ppm)
• NDBA Standard Solution in Methanol (100 ppm)

Suggested NMBA Stock Standard Solution (100 ppm) preparation

Accurately weigh 2.0 mg of NMBA to 20 mL volumetric flask. Dissolve and dilute to volume with methanol.

Note: The concentration of NMBA stock standard solution can be varied. The dilution for the preparation of the Intermediate Stock Standard Solution -1 and -2 can be calculated and adjusted accordingly to make the final concentration close to 200 ppb and 5 ppb.

Intermediate Stock Standard Solution-1 (200 ppb) preparation

Transfer 100 µL of NDMA reference standard stock solution (200 ppm), 200 µL of the other Nitrosamine reference standard stock solutions (100 ppm) and 200 µL of NMBA Stock Standard Solution (100 ppm) into a 100 mL volumetric flask, dilute to volume with diluent. Mix well.

Intermediate Stock Standard Solution-2 (5 ppb) preparation (STD-4)

Transfer 500 µL of Intermediate Stock Standard Solution-1 (200 ppb) into a 20 mL volumetric flask, dilute to volume with diluent. Mix well.

Suggested Calibration Standard Solutions

STD Solution	Working solution	Working solution Conc. (ppb)	Volume of Working Solution (mL)	Total Vol. (mL)	Nitrosamine STD Conc. (ppb)
LOD	Intermediate Stock Standard Solution-2	5	0.5	5	0.5
LOQ	Intermediate Stock Standard Solution-2	5	0.75	5	0.75
STD-1	Intermediate Stock Standard Solution-2	5	1	5	1
STD-2	Intermediate Stock Standard Solution-2	5	2	5	2
STD-3	Intermediate Stock Standard Solution-2	5	3	5	3
STD-4	Intermediate Stock Standard Solution-2	5			5
STD-5	Intermediate Stock Standard Solution-1	200	0.25	5	10
STD-6	Intermediate Stock Standard Solution-1	200	0.375	5	15
STD-7	Intermediate Stock Standard Solution-1	200	0.5	5	20
STD-8	Intermediate Stock Standard Solution-1	200	0.625	5	25
STD-9	Intermediate Stock Standard Solution-1	200	1.25	5	50

R² for calibration curve should be NLT 0.995.

STD-9 will be used for system suitability and system drift.

4.5 Sample Preparation

• Weigh at least 20 tablets, calculate average weight per tablet.
• Carefully grind the weighed tablets into fine powder using mortar and pestle.

Suggested Sample Preparation:

• Prepare in triplicate for each sample.
• Accurately weigh homogenized sample powder equivalent to about 500 mg of the Metformin API into separate glass centrifuge tubes.
• Add 10.0 mL of diluent to each sample tube. Tightly cap the tubes, sonicate for 20 minutes, and then vortex at 2500 rpm for 10 minutes.
• Centrifuge sample at 2500 rpm for 10 minutes.
• Transfer 2.0 mL of the supernatant to 2 mL centrifuge tube, centrifuge at 12 000 rpm for 5 minutes. Carefully remove the tubes from the centrifuge.
• Transfer sample solution to HPLC vials for analysis.

The concentration (weight and/or volume) of samples may be modified as required.

4.6 Suggested Recovery Spiking:

• If no nitrosamines were found in the sample, prepare one spiked sample solution and check S/N. Otherwise, prepare spiking sample in triplicate.
• Accurately weigh homogenized sample powder equivalent to about 500 mg of the Metformin API into separate glass centrifuge tubes.
• Add 10.0 mL of Diluent, then 125 µL of Intermediate Stock Standard solution -1 (200 ppb), to each sample tube. Tightly cap the tubes, sonicate for 20 minutes, then vortex at 2500 rpm for 10 minutes.
• Centrifuge samples at 2500 rpm for 10 minutes.
• Transfer 2.0 mL solution to 2 mL centrifuge tube, centrifuge at 12 000 rpm for 5 minutes. Carefully remove the tubes from the centrifuge.
• Transfer sample solution to HPLC vials for analysis.

5. Instrument Operating Parameters

5.1 Suggested UPLC Parameters:

Flow Rate:

0.4 mL/min

Injection Volume:

10.0 µL

Run Time:

30 min

Gradient Table:

Time (min)	Mobile Phase A %	Mobile Phase B %
0	95	5
5	95	5
22	0	100
27	0	100
28	95	5
30	95	5

5.2 Suggested MS Parameters:

APCI probe Temp:

400°C

Source Temperature:

150°C

Corona:

4.0 kV

Type:

MRM

Ion mode:

Positive

Span (Da):

0

Start time:

3.5 min

End time:

21 min

Run Events:

Event 1: 0.00 min

Flow State: Waste

Event 2: 3.5 min

Flow State: LC

Event 3: 21 min

Flow State: Waste

Note: Flow state setting may be adjusted as necessary depending on the system to divert metformin to waste.

5.3 MRM Parameters:

Name	Time period (min)	Parent Ion (m/z)	Daughter Ion (m/z)	Dwell (s)	Cone (V)	Collision Cell (eV)
NDMA	3.5 – 6.0	75.0	43.1	0.1	30	10
		75.0	58.0Table 11 Footnote *	0.1	30	8
NMBA	6.0 – 10.0	146.9	44.0	0.1	22	12
		146.9	117.0Table 11 Footnote *	0.1	22	4
NDEA	10.0 – 13.0	103.0	47.1	0.1	30	12
		103.0	75.0Table 11 Footnote *	0.1	30	10
NEIPA	12.8 – 15.0	117.0	47.0	0.1	26	14
		117.0	75.0Table 11 Footnote *	0.1	26	10
NDIPA	14.6 – 17.0	131.0	43.1	0.1	26	10
		131.0	89.0Table 11 Footnote *	0.1	26	6
NDBA	17.0 – 21.0	159.0	57.1	0.1	32	12
		159.0	103.0Table 11 Footnote *	0.1	32	10
Table 11 Footnote * Target ions used for calculation. Table 11 Return to footnote * referrer

6. Calculation

The results, in ppm relative to the declared amount of drug substance in the product, are given by:
Nitrosamine impurity (ppm) = [(y-b)/m] × AVG_wt × V ÷ Wt_spl ÷ LC

Where: Nitrosamine impurity refers to NDMA, NDEA, NEIPA, NDIPA NDBA or NMBA

y =

Peak Area of the nitrosamine impurity

b =

Intercept of the Linear Curve

m =

Slope of the Linear Curve

Wt_spl =

Sample Weight (mg)

AVG_wt =

Average Tablet Weight (mg)

LC =

Label claim of Sample (mg)

V =

10 mL (volume)

Equation - Text Description

Nitrosamine impurity in parts per million (ppm) equals y minus b over m, which is the difference between the peak area minus the intercept of the calibration curve over the slope of the calibration curve. This value is multiplied by average tablet weight, then multiplied by the volume, divided by the sample weight and then divided by the label claim of the sample.

7. Range, limit of detection (LOD) and limit of quantification (LOQ)

Comp. Name	Conc. (ng/mL)			Conc. (ppmTable 12 Footnote *)
	LOD	LOQ	Range	LOD	LOQ	Range
NDMA	0.5	0.75	0.75 – 50	0.010	0.015	0.015 – 1.0
NDEA	0.1	0.5	0.5 – 50	0.002	0.010	0.010 – 1.0
NEIPA	0.1	0.5	0.5 – 50	0.002	0.010	0.010 – 1.0
NDIPA	0.1	0.5	0.5 – 50	0.002	0.010	0.010 – 1.0
NDBA	0.1	0.5	0.5 – 50	0.002	0.010	0.010 – 1.0
NMBA	0.25	0.5	0.5 – 50	0.005	0.010	0.010 – 1.0
Table 12 Note de bas de page * ppm (ng/g) relative to the declared amount of Metformin in the product Table 12 Retour à la référence de la note de bas de page *

8. Example Chromatograms

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 75.0 → 58.0) of the 5 ppb NDMA standard while the bottom row shows an MRM chromatogram (m/z 75.0 → 43.1) of the 5 ppb NDMA standard. Each chromatogram displays a single peak at approximately 4.03 minutes.

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 146.9 → 117.0) of the 5 ppb NMBA standard while the bottom row shows an MRM chromatogram (m/z 146.9 → 44.0) of the 5 ppb NMBA standard. Each chromatograph displays a large peak at approximately 8.5 minutes and two smaller peaks at approximately 8.6 and 8.7 minutes. The presence of these extra peaks are likely due to the asymmetric structure of NMBA.

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 103.0 → 75.0) of the 5 ppb NDEA standard while the bottom row shows an MRM chromatogram (m/z 103.0 → 47.1) of the 5 ppb NDEA standard. Each chromatogram displays a single peak at approximately 11.7 minutes.

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 117.0.0 → 75.0) of the 5 ppb NEIPA standard while the bottom row shows an MRM chromatogram (m/z 117.0 → 47.0) of the 5 ppb NEIPA standard. Each chromatograph displays a large peak at approximately 13.8 minutes and a smaller shoulder peak at approximately 14.0 minutes. The presence of the shoulder is likely due to the asymmetric structure of NEIPA.

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 131.0 → 89.0) of the 5 ppb NDIPA standard while the bottom row shows an MRM chromatogram (m/z 131.0 → 43.1) of the 5 ppb NDIPA standard. Each chromatograph displays a single peak at approximately 15.6 minutes.

Text Description

The figure includes two rows. The top row shows an MRM chromatogram (m/z 159.0 → 103.0) of the 5 ppb NDBA standard while the bottom row shows an MRM chromatogram (m/z 159.0 → 57.1) of the 5 ppb NDBA standard. Each chromatograph displays a single peak at approximately 19.2 minutes.

Determination of N-nitrosodimethylamine Impurity in Metformin Products by GC-MS/MS

1. Principle and Scope

The present method has been developed to detect and quantify N-nitrosodimethylamine (NDMA) impurity in Metformin products by GC-MS/MS.

2. Reagents and Reference Standards

• NDMA, N-Nitrosodimethylamine solution (200 ppm), CAS # 62-75-9
• NDMA-d6, N-nitrosodimethylamine-d6 solution (1000ppm), CAS # 17829-05-9
• Dichloromethane, GC grade, CAS # 75-09-2

3. Instrument/Equipment

• Agilent GC 7890A with MSMS 7000 with EI source or equivalent
• Column: Agilent DB-624 30m x 0.32 µm x 1.8 µm or equivalent
• 16 x 125 mm screw cap glass tubes
• 2.0 mL micro centrifuge tubes
• Eppendorf pipettes, various volumes, or equivalent
• Analytical balance
• Volumetric Flasks (class A), various volumes, or equivalent
• Vortex mixer, single and multi-tube
• Mortar and pestle
• Ultrasonic Water bath
• GC vials
• Centrifuges

4. Preparation of Solutions

4.1 Storage

Calibration and sample solutions are stored in freezer.

4.2 Standard Solutions

Reference Standard Solutions (as purchased)

• NDMA Standard Solution in Methanol (200 ppm)
• NDMA-d6 Standard Solution in Methanol (1000 ppm)

NDMA Stock Solution-1 (1 ppm) preparation

Transfer 125 µL of NDMA reference standard solution (200 ppm) into a 25 mL volumetric flask, dilute to volume with Dichloromethane. Mix well.

Internal Standard Solution-1 (2 ppm) preparation

Transfer 100 µL of NDMA-d6 reference standard solution (1000 ppm) into a 50 mL volumetric flask, dilute to volume with Dichloromethane. Mix well.

Internal Standard Solution-2 (10 ppb) preparation

Transfer 2.5 mL of Internal Standard Solution-1 (2 ppm) into a 500 mL volumetric flask, dilute to volume with Dichloromethane. Mix well.

Calibration Standard Solutions

STD Solution	Volume of STD Spiking Solution (uL)	Volume of ISTD-1(uL)	Diluent	Volumetric Flask (mL)	NDMA STD Conc. (ppb)	ISTD Conc. (ppb)
STD-1	500 (NDMA Stock Solution-1)	50	Dichloromethane	10	50	10
STD-2	250 (NDMA Stock Solution-1)	50	Dichloromethane	10	25	10
STD-3	100 (NDMA Stock Solution-1)	50	Dichloromethane	10	10	10
STD-4	50 (NDMA Stock Solution-1)	50	Dichloromethane	10	5	10
STD-5	25 (NDMA Stock Solution-1)	50	Dichloromethane	10	2.5	10
STD-6	50 (NDMA Stock Solution-1)	250	Dichloromethane	50	1	10
STD-7(LOQ)	5000(STD-6)	0	ISTD-2	10	0.5	10
LOD	1000(STD-6)	0	ISTD-2	10	0.1	10

• R² for calibration curve should be NLT 0.995.
• STD-1 will be used for system suitability and system drift.

4.3 Sample Preparation

• Weigh at least 20 tablets, calculate average weight per tablet.
• Carefully grind the weighed tablets into fine powder using mortar and pestle.

Sample Preparation:

• Prepare in triplicate for each sample.
• Accurately weigh homogenized sample powder equivalent to about 500 mg of the Metformin API into separate glass centrifuge tubes.
• Add 10.0 mL of Internal Standard Solution-2 to each sample tube. Tightly cap the tubes, sonicate for 20 minutes, and then vortex at 2500 rpm for 10 minutes.
• Centrifuge sample at 2500 rpm for 15 minutes.
• Transfer 2.0 mL of the supernatant to 2 mL centrifuge tube, centrifuge at 12 000 rpm for 5 minutes. Carefully remove the tubes from the centrifuge.
• Transfer sample solution to GC vials for analysis.

4.4 Recovery Spiking:

• If no nitrosamine impurity is found in the sample, prepare one spiked sample solution and check S/N. Otherwise, prepare spiking sample in triplicate.
• Accurately weigh homogenized sample powder equivalent to about 500 mg of the Metformin API into separate glass centrifuge tubes.
• Add 10.0 mL of Internal Standard Solution-2. Add 25 µL of NDMA Standard Solution-1 (1 ppm) to each sample tube. Tightly cap the tubes, sonicate for 20 minutes, then vortex at 2500 rpm for 10 minutes.
• Centrifuge samples at 2500 rpm for 15 minutes.
• Transfer 2.0 mL solution to 2 mL centrifuge tube, centrifuge at 12 000 rpm for 5 minutes. Carefully remove the tubes from the centrifuge.
• Transfer sample solution to GC vials for analysis.

5. Instrument Operating Parameters

5.1 GC Parameters:

Injector Settings:

Injector Mode:

Pulsed Splitless

Injector temperature:

240°C

Flow Rate:

1.3 mL/min

Septum Purge Flow:

10 mL/min

Purge Flow:

50 mL/min after 1 minute

Injection Volume:

2.0 µL

Oven Program:
Initial temperature:

60°C; Hold: 2 min

Ramp#	Rate (°C /min)	Final Temp (ºC)	Hold Time (min)
1	20	220	2
2	60	240	3

Total Run Time:

15.33 min

5.2 MS Parameters:

MS transfer line (Auxiliary temperature):

240°C

Ion Source:

EI

Source Temperature:

250°C

Solvent delay:

4 min

Stop time:

7 min

Quench gas:

Helium at 2.5 mL/min

Collision gas:

Nitrogen at 1.5 mL/min

5.3 MRM Parameters:

Name	Retention Time (min)	Precursor Ion (m/z)	Product Ion (m/z)	Left delta RT	Right delta RT	Resolution (MS1 and MS2)	CE	Dwell (ms)
NDMA	5.8	74	42	1.0	1.2	Unit/wide	7	165.8
		74	44Footnote *	1.0	1.2	Unit/wide	4	165.8
NDMA-d6	5.8	80.1	50.1	1.0	1.2	Unit/wide	5	165.8
Table notes Table Note * Target ions used for calculation. Return to tablenote 1 referrer

6. Calculation

The results, in ppm relative to the declared amount of drug substance in the product, are given by:

Where:
Nitrosamine impurity, NDMA, (ppm) = [(y-b)/m]× 〖AVG〗_wt × V ÷〖Wt〗_spl ÷LC

y =

ratio of peak area of NDMA to peak area of NDMA-d6

b =

Intercept of the Linear Curve

m =

Slope of the Linear Curve

Wt_spl =

Sample Weight (mg)

AVG_wt =

Average Tablet Weight (mg)

LC =

Label claim of Sample (mg)

V =

10 mL (volume)

Equation - Text Description

Nitrosamine impurity in parts per million (ppm) equals y minus b over m, which is the difference between the peak area minus the intercept of the calibration curve over the slope of the calibration curve. This value is multiplied by average tablet weight, then multiplied by the volume, divided by the sample weight and then divided by the label claim of the sample.

7. Range, limit of detection (LOD) and limit of quantification (LOQ)

Comp. Name	Conc. (ng/mL)			Conc. (ppmTable x Footnote *)
	LOD	LOQ	Range	LOD	LOQ	Range
NDMA	0.1	0.5	0.5-50	0.002	0.01	0.01-1.00
Table Note ppm (ug/g) relative to the declared amount of Metformin in the product Return to footnote * referrer

8. Example Chromatograms

Text Equivalent

The figure shows chromatograms of a sample showing typical peaks for NDMA and NDMA-d6. The chromatograms are arranged in three rows.

• The first row shows the Total Ion Chromatogram from 5.6 to 6.25 minutes. There is a large peak at 5.853 minutes (NDMA-d6) and another large peak at 5.872 minutes (NDMA).
• The second row shows a Multiple Reaction Monitoring chromatogram (m/z 74.0 → 44.0) with a peak at 5.872 minutes for NDMA.
• The third row shows a Multiple Reaction Monitoring chromatogram (m/z 80.1 → 50.1) with a single peak at 5.853 minutes for the NDMA-d6 internal standard.

Text Equivalent

The figure shows chromatograms of a sample showing typical peaks for NDMA and NDMA-d6. The chromatograms are arranged in three rows.

• The first row shows the Total Ion Chromatogram from 5.7 to 6.4 minutes. There is a large peak at 5.861 minutes (NDMA-d6), a small peak at 5.889 minutes (NDMA), and a matrix related peak at 6.32 minutes.
• The second row shows a Multiple Reaction Monitoring chromatograms (m/z 74.0 → 44.0) with a peak at 5.889 minutes for NDMA and a large matrix related peak at 6.3 minutes.
• The third row shows a Multiple Reaction Monitoring chromatogram (m/z 80.1 → 50.1) with a single peak at 5.861 minutes for the NDMA-d6 internal standard.