Nitrosamines in Pharmaceuticals? No Problem! in Pharmaceutic… · ylamine (NDMA) impurity from...

Nitrosamines in Pharmaceuticals? No Problem!

Pharma Trends

Shimadzu commits to contribute to Pharma society through Science and Technology

Detection and control of nitrosamine impurities in drug manufacturing processes become very critical to sustain business of pharmaceutical industry. Shimadzu has standardized analytical methods capable of detecting problematic nitrosamines compounds for assisting pharma society to overcome the challenging market situation. Nitrosamine is an organic compound containing the group -NNO attached to two organic groups. Nitrosamines are found in tobacco products, tobacco smoke and many foods such as fried foods, fish, meat, beer and water. Nitrosamines are formed by reaction of secondary or tertiary amines with a nitrosating agent and some of nitrosamines are classified as probable human carcinogens. In July 2018, the U.S. Food and Drug Administration (FDA) announced that the carcinogenic impurities: N-Nitrosodimethyl-amine (NDMA) and N-Nitrosodiethylamine (NDEA) had been detected in Valsartan bulk drug substances manufactured by Chinese drug manufacturers. Valsartan is used in the treatment of high blood pressure and congestive heart failure.

Subsequently, regulatory agencies including USFDA, European Medicines Agency (EMA), Health Canada, Health Science Authority, Singapore (HSA) and Ministry of Health, Labour and Welfare, Japan (MHLW), are investigating presence of nitrosamines in medicines. As a result, many medicines, e.g. Angiotensin II Receptor Antagonists (ARBs), Ranitidine, Nizatidine and Metformin, have been recalled till now. This global trend is revealing the necessity for rugged and robust analytical methods to detect nitrosamines in APIs and medicines.Shimadzu introduces rugged and robust analytical methods for nitrosamines in Sartans and Ranitidine. The methods have been standardized on the Shimadzu GCMS-TQ8050 NX, LCMS-8045 and LCMS-9030.

[email protected]


LC-MS/MS for the Determination of NDMA in Ranitidine Drug Substance as per reference from USFDA method.

BackgroundOn September 13, 2019, the FDA announced that preliminary tests found low levels of N-Nitrosodimethylamine (NDMA) in ranitidine, a heartburn medication used by millions of people. This impurity is classified as a probable human carcinogen and is believed to have been introduced into the finished products as a result of the manufacturing process. LC-MS/MS method may be used as an alternative or confirmatory method for the liquid chromatography high resolution mass spectrometry (LC-HRMS) method detailed in FY19-177DPA-S (US FDA) can also be adopted. The TQ (Triple Quadrupole) method is more widely available than the LC-HRMS platform previously shared by the USFDA agency.

PrincipleReverse phase analysis is used for the separation of N-Nitrosodimeth-ylamine (NDMA) impurity from Ranitidine. MRM method has been developed for the NDMA wherein 75 > 43 is used as a quantifier ion

and 75 > 58 is used as a qualifier ion. Internal standard method is used for the quantification of NDMA in sample.

Conclusions

LC-MS/MS method was developed and validated following ICH Q2 (R1) for the detection and quantitation of NDMA in Ranitidine drug as per USFDA protocol. The limit of detection (LOD), limit of quantitation (LOQ) and range of the method are summarized in Table 1. NDMA

Description Unit NDMA

LODng/mL 0.300

ppm 0.010

LOQng/mL 1.000

ppm 0.033

Rangeng/mL 1.0-100

ppm 0.033- 3.33 Table 1: LOD,LOQ for NDMA

Reagents

NDMA Reference Standard • Formic acid, ULCMS make (BIOSOLVE) • Methanol, LC/MS grade (JT baker) • Water, LC/MS grade (JT Baker)

Instrument

N-Series UHPLC with LCMS-8045 system.

Highlighted Features

• Best in Class Sensitivity (UFsensitivity)

• World Fastest Scanning Speed: 30,000 u /sec (UFscanning)

• World Fastest Polarity Switching Rate: 5 msec (UFswitching)

Mobile Phase Preparation• Mobile phase A (0.1% formic

acid in water): mix formic acid and water at a volume ratio of 1:1000

HPLC Column

Shim pack GISS C18 (250 mm x 4.6 mm, 5 micron) (P/N :227-30061-07)

Column Temperature 30°C

Flow Rate 0.6mL/min

Mobile Phase A 0.1% Formic acid in Water

Mobile Phase B 0.1% Formic acid in Methanol

Gradient Time (Min) A% B%

0 95 5

5 95 5

15 10 90

18 10 90

18.1 95 5

25 95 5

Injection Volume 10uL

Autosampler Temp. 5°C

Table 2: Analytical Conditions for NDMA

Interface Parameters

Interface/Polarity : APCI/Positive

Nebulizing Gas Flow : 3.00 L

Interface Temperature : 350 ˚C

DL Temperature : 200 ˚C

Heating Block Temperature : 200 ˚C

Drying Gas Flow : 5.00 L

Target : NDMA : 75.20 > 43.00 (Quantifier); 75.20 > 58.00 (Qualifier)

ISTD : NDMA-D6 : 81.10 > 46.10

Time (Min) Divert Valve Position

0 1

10 0

MRM parameters

Divert Valve settings

Note: Position 1 – To Mass; Position 0 – To Waste.

Fig. 1: LCMS-8045 Table 3: Mass Spectrometer Conditions in LCMS-8045

Figure 1: LCMS-8045 system

Highlighted Feature

✓ Best in Class Sensitivity (UFsensitivity) ✓ World Fastest Scanning Speed: 30,000 u /sec (UFscanning) ✓ World Fastest Polarity Switching Rate: 5 msec (UFswitching)

Mobile Phase Preparation

• Mobile phase A (0.1% formic acid in water): mix formic acid and water at a volume ratio of 1:1000

• Mobile phase B (0.1% formic acid in methanol): mix formic acid and methanol at a volume ratio of 1:1000

Diluent and Blank: Water

Mixed Intermediate Stock Standard preparation (100 ng/mL)

• Prepare a 100 ng/mL intermediate stock standard solution in water using commercially available nitrosamine standards reference stock standard solution.

Linearity standards Preparation

• Linearity standards of mixed nitrosamines were prepared from 0.25ng/mL to 100 ng/mL levels. Spiked each level with two internal standards NDMA-D6 & NDEA-D10 so that resultant concentration of the internal standards would be 10ng/mL.

Metformin drug sample preparation

• Accurately weigh 100 mg of drug substance into a 2 mL of effendroff tube. • Dissolve completely in 1mL of water. • Filter through 0.2 µm syringe filter in HPLC vial.

[email protected] 2


Figure 8: Comparison between UV Chromatogram (at 254nm) of Ranitidine and MRM of NDMA

NDMA peak is well separated from ranitidine drug. Ranitidine API peak is diverted to the waste by using divert valve to avoid the contamination of Mass spectrometer.

Figure 9A: Blank Figure 9B : NDMA (1.0 ng/mL standard)

1.36e4Q 75.20>43.00 (+)

RT (min)7.0 7.5 8.0 8.5 9.0 9.5

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.35e4 Q 75.20>43.00 (+)

R1 21.04% (NC)

RT=8.063

RT (min) 6.5 7.0 7.5 8.0 8.5 9.0 9.5 1.1e4 1.1e4 1.2e4 1.2e4 1.3e4 1.3e4 1.4e4 1.4e4 1.5e4 1.5e4 1.6e4 1.6e4 1.7e4

Figure 8: Comparison between UV Chromatogram (at 254nm) of Ranitidine and MRM of NDMA


Figure 9A: Blank Figure 9B : NDMA (1.0 ng/mL standard)

1.36e4Q 75.20>43.00 (+)

RT (min)7.0 7.5 8.0 8.5 9.0 9.5

1.2e4

1.2e4

1.3e4

1.3e4

1.4e4

1.4e4

1.5e4

1.5e4

1.6e4

1.35e4 Q 75.20>43.00 (+)

R1 21.04% (NC)

RT=8.063

RT (min) 6.5 7.0 7.5 8.0 8.5 9.0 9.5 1.1e4 1.1e4 1.2e4 1.2e4 1.3e4 1.3e4 1.4e4 1.4e4 1.5e4 1.5e4 1.6e4 1.6e4 1.7e4

Figure 9C: NDMA-D6 Figure 9D: Calibration Curve (1 – 100 ng/mL)

Blank chromatogram does not show any interference at the retention time of NDMA as shown in figure 9A and 9B. Calibration curve was performed by using internal standard method with coefficient of correlation of 0.999 over a range of 1.0 to 100.0 ng/mL (refer figure 9D). Recovery study was performed by spiking LOQ level in the ranitidine sample and it was found to be well within the criteria.

In continuation to above work, Shimadzu lab, mumbai has developed the in-house method to determine NDMA in Ranitidine by using LCMS-8045 system, wherein LOQ achieved is up to 0.005 ppm / LOD level of 0.002 ppm in ranitidine. For further information please contact us on [email protected].

LC-HRMS method for the determination of NDMA in Ranitidine drug substance as per reference from USFDA method

Background about NDMA in Ranitidine:

Ranitidine HCl is a prescription and over the counter medication used to treat acid reflux. The drug is a histamine-2 receptor antagonist (acid inhibitor or H2 blocker). Some of the common H2 receptor blockers include: Ranitidine (Zantac), Nizatidine (Acid), Famotidine (Pepcid, Pepcid AC) and Cimetidine (Tagamet, Tagamet HB). As GC based methods had been observed to elevate NDMA levels in tested materials an alternative method which prevents the degradation of ranitidine and the subsequent formation of NDMA was therefore needed. A liquid chromatography with high resolution mass spectrometer (LC-HRMS) was developed to measure the levels of NDMA in ranitidine drug substance.

Purpose

This method will be used to quantitate N-nitroso-di-methylamine (NDMA) impurity in ranitidine drug substance as per reference from USFDA protocol.

Principle

Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

5

10

15

20

25

30

35

40

45

50

55

60

65

NDMAy = 0.6699354x + 0.04647662R² = 0.9991052 R = 0.9995525

Curve Fit: Default (Linear)Weighting: 1/CZero: Default (Not Forced)

Mean RF: 6.947008e-001SD RF: 7.724404e-002%RSD: 11.119038

3.06e3 ISTD 81.10>46.10 (+)

RT=7.954

RT (min) 6.5 7.0 7.5 8.0 8.5 9.0 0.0e0 5.0e2 1.0e3 1.5e3 2.0e3 2.5e3 3.0e3 3.5e3 4.0e3 4.5e3 5.0e3 5.5e3 6.0e3

Results and Discussion

Fig. 2: Comparison between UV Chromatogram (at 254nm) of Ranitidine and MRM of NDMA


• It was observed that MRMtransition of NDMA 75


LC-MS/MS method for the determination of NDMA, NDEA, NDIPA, NMBA, NEIPA and NDBA in Valsartan Drug Substance as per reference from USFDA method

BackgroundIn July 2018, the American Food and Drug Administration (FDA) announced that the carcinogenic impurities: Nitrosamines (N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA)) had been detected in Valsartan bulk drug substances manufactured by Chinese manufacturers. Subsequently, a worldwide recall was issued of pharmaceutical products that use Valsartan bulk drug substances. Valsartan is used in the treatment of high blood pressure and congestive heart failure.

This LC-MS/MS method is used for the quantitation of six nitrosamines in Valsartan drug substance. The impurities are : N-Nitroso-dimethylamine (NDMA), N-Nitroso-dieth-ylamine (NDEA), N-Nitroso-N-methyl-4-aminobutyric acid (NMBA), N-Nitroso-ethyl isopropylamine (NEIPA), N-Nitroso-di-isopropylamine (NDIPA) and N-Nitroso-dibutylamine (NDBA)

PrincipleReverse phase analysis is used for the separation of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA impurities from valsartan. MRM method is developed for the all six nitrosamines. Two different Internal standards method is used for the quantification of NDMA in sample.

ConclusionsAn LC-MS/MS method was developed and validated following ICH Q2 (R1), for the detection and quantitation of six nitrosamines in Valsartan drug as USFDA protocol. The limit of detection (LOD), limit of quantitation (LOQ) and range of the method are summarized in Table 4.

ReagentsReference Standards for NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA • Formic acid, ULCMS make (BIOSOLVE) • Methanol, LC/MS grade (JT baker) • Water, LC/MS grade (JT Baker)


NDMA Intermediate Stock Standard preparation (100 ng/mL)

• Prepare a 100 ng/mL intermediate stock standard solution in water using commercially available NDMA reference stock standard solution.

Working Standard Solution Preparation (1.0 ng/mL)

• Transfer a 1.0 mL aliquot volume of the intermediate stock standard into a 100 mL volumetric flask and dilute to volume with water. Prepare fresh daily.

Ranitidine drug sample preparation

• Accurately weigh 120 mg of drug substance into a 15 mL centrifuge tube. Add 4.0 mL of water and mix the solution using a vortex mixer until dissolved.

• Filter it using a 0.22 μm PVDF syringe filter in HPLC vial.

Mass Spectrometer conditions

• Instrument

Shimadzu LCMS-8045 Triple Quad LC/MS system with APCI source (Figure 1).

Blank chromatogram does not show any interference at the retention time of NDMA as shown in Figures 3A and 3B. Calibration curve was performed by using internal standard method with coefficient of correlation of 0.999 over a range of 1.0 to 100.0 ng/mL (Figure 3D). Recovery study was performed by spiking LOQ level in the ranitidine sample and it was found to be well within the criteria.

In continuation to above work, Shimadzu Labs, Mumbai have developed the in-house method to determine NDMA in Ranitidine by using LCMS-8045 system, wherein LOQ achieved is up to 0.005 ppm / LOD level of 0.002 ppm in Ranitidine.

Description Unit NDMA NDEA NMBA NDIPA NEIPA NDBA

LODng/mL 0.250 0.100 0.250 0.100 0.050 0.250

ppm 0.0025 0.0010 0.0025 0.0010 0.0005 0.0025

LOQng/mL 0.50 0.25 0.50 0.25 0.10 0.50

ppm 0.0050 0.0025 0.0050 0.0025 0.0010 0.0050

Rangeng/mL 0.50-100 0.25-100 0.50-100 0.25-100 0.10-100 0.50-100

ppm 0.005-0.10 0.0025-0.10 0.005-0.10 0.0025-0.10 0.001-0.10 0.005-0.10

Table 4: LOD, LOQ Values for 6 Nitrosamines in Valsartan

[email protected] 4


InstrumentN-Series UHPLC with LCMS-8045 system.

Mobile Phase Preparation• Mobile phase A (0.1% formic acid in water): mix formic acid and

water at a volume ratio of 1:1000 • Mobile phase B (0.1% formic acid in methanol): mix formic acid

and methanol at a volume ratio of 1:1000

Diluent and Blank: Water: Methanol (50:50) v/vMixed Intermediate Stock Standard preparation (100 ng/mL) • Prepare a 100 ng/mL intermediate stock standard solution in water

using commercially available nitrosamine standards reference stock standard solution.

Working Standard Solution Preparation (1.0 ng/mL) • Transfer a 1.0 mL aliquot volume of the intermediate stock

standard into a 100 mL volumetric flask and dilute to volume with water. Prepare fresh daily.

Valsartan drug sample preparation • Accurately weigh 100 mg of drug substance into a

2 mL eppendorf tube.• Dissolve completely in water: Methanol mixture.• Further add 0.050 mL of mixture of internal standards of having

concentration 100 ng/mL and vortex.• Centrifuge at 10000 RPM at 150C for 10 minutes.• Filter through 0.2 µm PVDF syringe filter in HPLC vial.

HPLC Column

Shim pack Velox PFPP (100 mm x 4.6 mm, 2.7 micron) (P/N :227-32023-03)

Column Temperature 40 °CFlow Rate 0.5mL/minMobile Phase A 0.1% Formic acid in WaterMobile Phase B 0.1% Formic acid in MethanolGradient Time (Min) A% B%

0.00 60 403.00 45 556.50 45 558.20 35 65

10.00 35 6510.10 10 9013.00 10 9013.10 60 4015.00 60 40

Injection Volume 20uLAutosampler Temp. 5 °C

Table 5: Analytical Conditions (LC) for 6 Nitrosamines in Valsartan

Time (Min) Divert Valve Position

0 1

7.5 0

Table 6: Analytical Conditions (MS) for 6 Nitrosamines in Valsartan

Target : NEIPA: 117.20 > 75.10 (Quantifier); 117.20 > 27.10 (Qualifier) Target : NDBA: 159.00 > 29.20 (Quantifier); 159.00 > 103.00 (Qualifier) ISTD 1 : NDMA-D6: 81.10 > 46.10 ISTD 2 : NDEA-D10: 113.10 > 81.15

• Divert Valve settings

Time (Min) Divert Valve Position 0 1

7.5 0 Note: Position 1 – To Mass; Position 0 – To Waste.

Table 11. Analytical Conditions (MS) for 6 Nitrosamines in Valsartan


Figure 13: Comparison between UV Chromatogram (at 254nm) of Valsartan and MRM of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA

All six nitrosamine peaks are well separated from valsartan drug. Valsartan API peak is diverted to the waste by using divert valve to avoid the contamination of Mass spectrometer.

Interface Parameters



Interface Temperature : 350 ˚C

DL Temperature : 200 ˚C

Heating Block Temperature : 200 ˚C


Target : NDMA: 75.20 > 43.00 (Quantifier); 75.20 > 58.00 (Qualifier)

Target : NDEA: 103.00 > 29.00 (Quantifier); 103.00 > 75.10 (Qualifier)

Target : NMBA: 146.90 > 117.15 (Quantifier); 147.00 > 44.10 (Qualifier)

Target : NDIPA: 131.20 > 89.05 (Quantifier); 131.20 > 43.10 (Qualifier)

Target : NEIPA: 117.20 > 75.10 (Quantifier); 117.20 > 27.10 (Qualifier)

Target : NDBA: 159.00 > 29.20 (Quantifier); 159.00 > 103.00 (Qualifier)

ISTD 1 : NDMA-D6: 81.10 > 46.10

ISTD 2 : NDEA-D10: 113.10 > 81.15

MRM parameters

Divert Valve settings


Fig. 4: Comparison between UV Chromatogram (at 254nm) of Valsartan and MRM of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA

[email protected]


Figure 14A: Calibration curve of NDMA Figure 14B: Calibration curve of NDEA

Figure 14C: Calibration curve of NMBA Figure 14D: Calibration curve of NDIPA

Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70 NDMAy = 0.6263549x - 0.08186809R² = 0.9941034 R = 0.9970473

Curve Fit: Default (Linear)Weighting: 1/C^2Zero: Default (Not Forced)


Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15NDEAy = 0.1424435x - 0.004227572R² = 0.9965480 R = 0.9982725



Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

5

10

15

20

25

30

35

40

45

50

55

60NMBA y = 0.5504446x - 0.003653506R² = 0.9928172 R = 0.9964021



Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

2

4

6

8

10

12

14

16

18

20

22

24

26 NDIPAy = 0.2376013x - 0.004600544R² = 0.9960155 R = 0.9980058

Curve Fit: Default (Linear)Weighting: Default (1/C^2)Zero: Default (Not Forced)


Figure 14E: Calibration curve of NEIPA Figure 14F: Calibration curve of NDBA

Calibration curves were performed by using internal standard method with coefficient of correlation of 0.999 over the linearity range (refer figure 14). Recovery study was performed by spiking 0.005ppm in the valsartan sample and it was found to be well within the criteria.

For further information please contact us on [email protected] .

References

[1] Control of nitrosamine impurities in sartans: revision of five Ph. Eur.Monographs https://www.edqm.eu/en/news/control-nitrosamine-impurities-sartans-revision-five-ph-eur monographs [2] GC/MS Headspace Method for Detection of NDMA in Valsartan Drug Substance and Drug Products https://www.fda.gov/media/115965/download [3] LC-HRMS Method for the Determination of NDMA in Ranitidine Drug Substance and Drug Product https://www.fda.gov/media/130801/download [4] Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Determination of NDMA in Ranitidine Drug Substance https://www.fda.gov/media/131868/download [4] Analysis of Nitrosamines in Sartan-Type Bulk Drug Substances https://www.shimadzu.com/an/pharmaceuticallifescience/ndma.html

Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32NIEPAy = 0.2964796x - 0.002722954R² = 0.9946540 R = 0.9973234



Conc.Ratio (ppb)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

NDBAy = 0.1266520x - 0.004559929R² = 0.9957904 R = 0.9978930



All six nitrosamine peaks are well separated from valsartan drug. Valsartan API peak is diverted to the waste by using divert valve to avoid the contamination of Mass spectrometer. Calibration curves were performed by using internal standard method with coefficient of correlation of 0.999 over the linearity range (Figure 5). Recovery study was performed by spiking 0.005 ppm in the valsartan sample and it was found to be well within the criteria.

Liquid Chromatography- Triple Quadrupole (LC-MS/MS) method for the determination of NDMA, NDEA, NDIPA, NMBA, NEIPA and NDBA in Metformin Drug SubstanceBackground Singapore’s Health Sciences Authority (HSA) on 4th December 2019, recalled three out of 46 locally marketed metformin medicines after determining they contained NDMA 'above the internationally acceptable level' and this subject came into mainstream. Subsequently, both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have released regular updated into their investigations into the causes of medicine contamination.

Metformin is the first-line drug control of high blood sugar levels in patients with type 2 diabetes, particularly in people who are overweight.

This LC-MS/MS method is used for the quantitation of six nitrosamines in Metformin drug substance.

Purpose: This method will be used to quantitate N-nitroso-di-methylamine (NDMA), N-nitroso-di-ethyl amine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitroso-ethyl iso-propylamine (NEIPA), N-nitroso-di-iso-propylamine (NDIPA), and N-nitroso-di-butylamine NDBA impurities in Metformin drug substance.

PrincipleReverse phase analysis is used for the separation of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA impurities from metformin. MRM method is developed for all six nitrosamines. Two different Internal standards were used for the quantification of these six nitrosamines in metformin.

Conclusions A LC-MS/MS method was developed for the detection and quantitation of six nitrosamines in Metformin drug. The limit of detection (LOD), limit of quantitation (LOQ) and range of the method are summarized in Table 7:

Description Unit NDMA NDEA NMBA NDIPA NEIPA NDBA

LODng/mL 0.250 0.100 0.100 0.100 0.100 0.100

ppm 0.0025 0.0010 0.0010 0.0010 0.0010 0.0010

LOQng/mL 0.50 0.25 0.25 0.25 0.25 0.25

ppm 0.005 0.0025 0.0025 0.0025 0.0025 0.0025

Rangeng/mL 0.50-100 0.25-100 0.25-100 0.25-100 0.25-100 0.25-100

ppm 0.0050-1.0 0.0025-1.0 0.0025-1.0 0.0025-1.0 0.0025-1.0 0.0025-1.0

Fig. 5A: Calibration curve of NDMA Fig. 5B: Calibration curve of NDEA

Fig. 5C: Calibration curve of NMBA Fig. 5D: Calibration curve of NDIPA

Fig. 5E: Calibration curve of NEIPA Fig. 5F: Calibration curve of NDBA

Table 7: LOD, LOQ for NDMA, NDEA, NDIPA, NMBA, NEIPA & NDBA in Metformin

[email protected] 6


Reagents Reference Standards for NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA • Formic acid, ULCMS make (BIOSOLVE) • Methanol, LC/MS grade (JT baker) • Water, LC/MS grade (JT Baker)

InstrumentN-Series UHPLC with LCMS-8045 system.

Figure 1: LCMS-8045 system

Highlighted Feature

✓ Best in Class Sensitivity (UFsensitivity) ✓ World Fastest Scanning Speed: 30,000 u /sec (UFscanning) ✓ World Fastest Polarity Switching Rate: 5 msec (UFswitching)

Mobile Phase Preparation

• Mobile phase A (0.1% formic acid in water): mix formic acid and water at a volume ratio of 1:1000





Linearity standards Preparation

• Linearity standards of mixed nitrosamines were prepared from 0.25ng/mL to 100 ng/mL levels. Spiked each level with two internal standards NDMA-D6 & NDEA-D10 so that resultant concentration of the internal standards would be 10ng/mL.

Metformin drug sample preparation

• Accurately weigh 100 mg of drug substance into a 2 mL of effendroff tube. • Dissolve completely in 1mL of water. • Filter through 0.2 µm syringe filter in HPLC vial.

Fig. 6: LCMS-8045 system

Highlighted Feature• Best in Class Sensitivity (UFSensitivity)• World Fastest Scanning Speed: 30,000 u/sec

(UFScanning)• World Fastest Polarity Switching Rate: 5 msec

(UFSwitching)

Mobile Phase Preparation• Mobile phase A (0.1% formic acid in water):

mix formic acid and water at a volume ratio of 1:1000





Linearity standards Preparation • Linearity standards of mixed nitrosamines were

prepared from 0.25 ng/mL to 100 ng/mL levels. Spiked each level with two internal standards NDMA-D6 & NDEA-D10 so that resultant concentration of the internal standards would be 10 ng/mL.

Metformin drug sample preparation • Accurately weigh 100 mg of drug

substance into a 2 mL of effendroff tube.• Dissolve completely in 1mL of water.• Filter through 0.2 µm syringe filter in

HPLC vial.Analytical Conditions

HPLC ColumnShim-pack GIST (100 mm x 4.6 mm, 2.7 micron) (P/N :227-30017-07)

Column Temperature 30°C

Flow Rate 0.7mL/min

Mobile Phase A 0.1% Formic acid in Water

Mobile Phase B 0.1% Formic acid in Methanol

Gradient Time (Min) A% B%

2.00 95 05

6.00 90 10

12.00 10 90

15.00 10 90

15.01 95 05

18.00 95 05

Injection Volume 30uL

Autosampler Temp. 5°C

Mass Spectrometer conditions•Instrument

Shimadzu LCMS-8045 Triple Quad LC/MS system with APCI source (refer Figure 6)

• Ion Source SettingsInterface Parameters



Interface Temperature : 350˚C

DL Temperature : 200˚C

Heating Block Temperature : 200˚C


•MRM parametersTarget : NDMA: 75.20 > 43.10 (Quantifier); 75.20 > 58.10 (Qualifier)

Target : NDEA: 103.00 > 29.20 (Quantifier); 103.20 > 75.10 (Qualifier)

Target : NMBA: 146.80 > 117.25 (Quantifier); 147.00 > 44.00 (Qualifier)

Target : NDIPA: 131.10 > 89.05 (Quantifier); 131.10 > 43.20 (Qualifier)

Target : NEIPA: 117.00 > 75.10 (Quantifier); 117.20 > 27.20 (Qualifier)

Target : NDBA: 159.10 > 29.10 (Quantifier); 159.00 > 103.10 (Qualifier)

ISTD 1 : NDMA-D6: 81.10 > 46.10

ISTD 2 : NDEA-D10: 113.10 > 81.15

•Divert Valve settingsTime (Min) Divert Valve Position

0.0 0

5.0 1


Table 9: MS Parameters

Table 8: Analytical Conditions

[email protected]



All six nitrosamine peaks are well separated from metformin drug (refer Figure 7). Metformin API peak is diverted to the waste by using divert valve to avoid the contamination of Mass spectrometer.

• Divert Valve settings

Time (Min) Divert Valve Position 0.0 0 5.0 1



All six nitrosamine peaks are well separated from metformin drug (refer figure 2). Metformin API peak is diverted to the waste by using divert valve to avoid the contamination of Mass spectrometer.

Figure 2: Comparison between UV Chromatogram (at 254nm) of Metformin and MRM of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA

Fig. 7: Comparison between UV Chromatogram (at 254nm) of Metformin and MRM of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA

Calibration curves were performed by using internal standard method with regression coefficient of > 0.99 over the linearity range (refer Figure 8). Recovery study was performed by spiking 0.005 ppm in the metformin sample and it was found to be well within the criteria.




Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211

Curve Fit: Default (Linear)Weighting: Default (1/C)Zero: Default (Not Forced)


Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930

Curve Fit: Default (Linear)Weighting: 1/AZero: Default (Not Forced)


Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648

Curve Fit: Default (Linear)Weighting: 1/A^2Zero: Default (Not Forced)


Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727






Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727






Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727



Fig. 8A: Calibration curve of NDMA

Fig. 8B: Calibration curve of NDEA




Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727






Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727



Fig. 8C: Calibration curve of NMBA

Fig. 8E: Calibration curve of NEIPA

Fig. 8F: Calibration curve of NDBA

Fig. 8D: Calibration curve of NDIPA




Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NDMAy = 0.06648504x - 0.01570569R² = 0.9992424 R = 0.9996211



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0 NDEAy = 0.02903289x - 0.0007750937R² = 0.9996266 R = 0.9998133



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18 NMBA y = 0.1800639x + 0.01312147R² = 0.9961896 R = 0.9980930



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0 NDIPAy = 0.06269403x - 0.0004824934R² = 0.9975311 R = 0.9987648



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0NIEPAy = 0.06671144x - 0.00007999886R² = 0.9988484 R = 0.9994240



Conc.Ratio (ng/mL)0 10 20 30 40 50 60 70 80 90 100

Area Ratio

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0 NDBAy = 0.03943897x + 0.0009477200R² = 0.9985460 R = 0.9992727



[email protected] 8


Calibration curves were performed by using internal standard method with regression coefficient of >0.99 over the linearity range (refer figure 3). Recovery study was performed by spiking 0.005ppm in the metformin sample and it was found to be well within the criteria.

Case Study of Metformin (False Positive):

One of the sample showed positive to the NDMA, however it was supposed to be negative. This peak showed response for both the transitions of the NDMA in the sample with the similar ion ratio (Refer figures 4A &4B)

Figure 4A: NDMA peak (Reference ion ratio-25) Figure 4B: Interference peak in sample (Reference ion ratio-23)

The gradient method was developed to separate this interference peak from the NDMA peak (refer figure 5).

Figure 5: Comparision of NDMA mass chromatograms at different gradient programs

For the further confirmation, NDMA standard and suspected metformin sample was analysed on LCMS-9030 quadrupole time-of-flight (Q-TOF) mass spectrometer system, Shimadzu. The LCMS-9030 enhances the most important features of Q-TOF instrumentation - mass accuracy, sensitivity, and speed - to address qualitative and quantitative challenges with genuine confidence and ease.

Figure 6A: Mass chromatogram of NDMA Figure 6B: High resolution mass spectrum of NDMA peak

1.09e4Q 75.20>43.10 (+)

R1 22.92% (NC)

RT=6.169

RT (min)5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

(x10,000)1:75.0545-75.0561(+)

4244

56

70.0 72.5 75.0 77.5 80.0 m/z0

10000

20000

30000

40000

50000Inten.

75.055338

NDMA

Interference

Initial gradient with poor separation

Developed gradient with separation










1.09e4Q 75.20>43.10 (+)

R1 22.92% (NC)

RT=6.169

RT (min)5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

(x10,000)1:75.0545-75.0561(+)

424456

70.0 72.5 75.0 77.5 80.0 m/z0

10000

20000

30000

40000

50000Inten.

75.055338

NDMA

Interference



Fig. 10: Comparision of NDMA mass chromatograms at different gradient programs

For the further confirmation, NDMA standard and suspected metformin sample was analysed on LCMS-9030 quadrupole-time of flight (Q-TOF) mass spectrometer system, Shimadzu. The LCMS-9030 enhances the most important features of Q-TOF instrumentation - mass accuracy, sensitivity, and speed - to address qualitative and quantitative challenges with genuine confidence and ease.









1.09e4Q 75.20>43.10 (+)

R1 22.92% (NC)

RT=6.169

RT (min)5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

(x10,000)1:75.0545-75.0561(+)

4244

56

70.0 72.5 75.0 77.5 80.0 m/z0

10000

20000

30000

40000

50000Inten.

75.055338

NDMA

Interference



Case Study of Metformin (False Positive):One of the sample showed positive to the NDMA, however it was supposed to be negative. This peak showed response for both the transitions of the NDMA in the sample with the similar ion ratio (Refer Figures 9A & 9B)

Fig. 9A: NDMA peak (Reference ion ratio-25)

Fig. 9B: Interference peak in sample (Reference ion ratio-23)









1.09e4Q 75.20>43.10 (+)

R1 22.92% (NC)

RT=6.169

RT (min)5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

(x10,000)1:75.0545-75.0561(+)

4244

56

70.0 72.5 75.0 77.5 80.0 m/z0

10000

20000

30000

40000

50000Inten.

75.055338

NDMA

Interference



Fig. 11A: Mass chromatogram of NDMA

Fig. 11B: High resolution mass spectrum of NDMA peak









1.09e4Q 75.20>43.10 (+)

R1 22.92% (NC)

RT=6.169

RT (min)5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

0.0e0

1.0e3

2.0e3

3.0e3

4.0e3

5.0e3

6.0e3

7.0e3

8.0e3

9.0e3

1.0e4

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

(x10,000)1:75.0545-75.0561(+)

4244

56

70.0 72.5 75.0 77.5 80.0 m/z0

10000

20000

30000

40000

50000Inten.

75.055338

NDMA

Interference



NDMA has a monoisotopic mass of 74.048012. Therefore, total mass of protonated NDMA is [M+H]+ is 74.048012+1.007276=75.055288. The observed mass of NDMA on LCMS-9030 system is 75.055338 (refer Figure 11B) with the mass accuracy of 0.6 ppm.

In this, metformin sample the mass of interference peak is 75.063462 (refer Figure 12B) with the mass accuracy of 109 ppm. Secondly, this interference peak is showing no response at the mass of NDMA, thus, it confirmed that this sample is negative to the NDMA.

This case study helps us to understand the scenario wherein false positive results on the triple quadrupole system can be further investigated and confirmed on the high-resolution mass spectrometer such as Q-TOF system. Therefore, high resolution mass spectrometer become inevitable tool to identify the false positive/ suspected samples.

NDMA has a monoisotopic mass of 74.048012. Therefore, total mass of protonated NDMA is [M+H]+ is 74.048012+1.007276= 75.055288. The observed mass of NDMA on LCMS-9030 system is 75.055338 (refer figure 6B) with the mass accuracy of 0.6ppm.

Figure 7A: Overlay Extracted mass spectra of interference peak & NDMA Figure 7B: Mass spectrum of interference peak

In this, metformin sample the mass of interference peak is 75.063462 (refer figure 7B) with the mass accuracy of 109ppm.Secondaly, this interference peak is showing no response at the mass of NDMA, thus, it confirmed that this sample is negative to the NDMA.

This case study helps us to understand the scenario wherein false positive results on the triple quadrupole system can be further investigated and confirmed on the high-resolution mass spectrometer such as Q-ToF system. Therefore, high resolution mass spectrometer become inevitable tool to identify the false positive/ suspected samples.


4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0(x100,000)

1:75.0626-75.0642(+)1:75.0545-75.0561(+)

74.50 74.75 75.00 75.25 75.50 75.75 76.00 m/z0

25000

50000

75000

100000

125000Inten.

75.063462

NDMA has a monoisotopic mass of 74.048012. Therefore, total mass of protonated NDMA is [M+H]+ is 74.048012+1.007276= 75.055288. The observed mass of NDMA on LCMS-9030 system is 75.055338 (refer figure 6B) with the mass accuracy of 0.6ppm.

Figure 7A: Overlay Extracted mass spectra of interference peak & NDMA Figure 7B: Mass spectrum of interference peak

In this, metformin sample the mass of interference peak is 75.063462 (refer figure 7B) with the mass accuracy of 109ppm.Secondaly, this interference peak is showing no response at the mass of NDMA, thus, it confirmed that this sample is negative to the NDMA.

This case study helps us to understand the scenario wherein false positive results on the triple quadrupole system can be further investigated and confirmed on the high-resolution mass spectrometer such as Q-ToF system. Therefore, high resolution mass spectrometer become inevitable tool to identify the false positive/ suspected samples.


4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0(x100,000)

1:75.0626-75.0642(+)1:75.0545-75.0561(+)

74.50 74.75 75.00 75.25 75.50 75.75 76.00 m/z0

25000

50000

75000

100000

125000Inten.

75.063462

Fig. 12A: Overlay Extracted mass spectra of interference peak & NDMA

Fig. 12B: Mass spectrum of interference peak

[email protected]


Model N-Series UHPLC System

HPLC columnShim pack GISS C18 (250 mm x 4.6 mm, 5 u) (P/N :227-30061-07)

Column temp. 40 °CFlow rate 0.7 mL/minMobile phase A 0.1 % formic acid in waterMobile phase B 0.1 % formic acid in methanolGradient program Time (min) A % B %

0.0 95 55.0 95 515.0 10 9018.0 10 9018.1 95 525.0 95 5

Autosampler temp. 5 °C

Model LCMS-9030 Q-TOF Mass SpectrometerInterface/polarity APCI/PositiveNebulizing gas flow 3.00 L/minInterface temp. 350 ˚CDL temp. 200 ˚CHeating block temp. 200 ˚CDrying gas flow 5.00 L/minMS mode ScanScan range 70-90 m/z

Data processingNDMA: 75.0553

NDMA D6: 81.0927

Divert valve settingsTime (min)

Divert valve position

0 Towards MS

10 Towards waste

Table 10: Analytical Conditions for LC-HRMS

LC-HRMS method for the determination of NDMA in Ranitidine drug substance as per reference from USFDA method

Background

Ranitidine HCl is a prescription and over the counter medication used to treat acid reflux. The drug is a histamine-2 receptor antagonist (acid inhibitor or H2 blocker). Some of the common H2 receptor blockers include: Ranitidine (Zantac), Nizatidine (Acid), Famotidine (Pepcid, Pepcid AC) and Cimetidine (Tagamet, Tagamet HB). As GC based methods had been observed to elevate NDMA levels in tested materials, an alternative method which prevents the degradation of ranitidine and the subsequent formation of NDMA was therefore needed. A liquid chromatography with high resolution mass spectrometer (LC-HRMS) was developed to measure the levels of NDMA in ranitidine drug substance.

PrincipleReverse phase analysis is used for the separation of N-Nitroso- dimethylamine (NDMA) impurity from Ranitidine. HRMS method developed for the quantitation of NDMA using internal standard calibration method.

ConclusionHighly sensitive LC-HRMS method was developed for analysis of NDMA in Ranitidine drug substance. High mass accuracy of LCMS-9030 enables reliable quantitation of NDMA from Ranitidine drug substance.

InstrumentN-Series UHPLC with LCMS-9030

Highlighted features

• ReproducibleResultsThe LCMS-9030 Q-TOF delivers

outstanding mass accuracy over a wide mass range, enabling users to consistently produce accurate identification results. In addition, the LCMS-9030 does not require frequent calibration, making it easy to achieve high mass accuracy over long periods.

• HighSensitivityDetectionHigh efficiency ion guides, quadrupole and collision cell enable high sensitivity for the detection of trace level compounds. And, due to its robust platform, the LCMS-9030 achieves reproducible quantitative analysis over longer periods of time.

• UltrafastPerformanceThe LCMS-9030 incorporates the patented ultrafast technologies used in Shimadzu LC-MS/MS platforms to deliver outstanding speed. Innovative TOF architecture enables precise, ultra-fast pulsing of ions into the flight tube and ideal reflection back to the detector. The result is high-speed data acquisition compatible with the high-throughput laboratory.

Materials and methodMobile phase preparation• Mobile phase A (0.1% formic acid

in water): mix formic acid and water at a volume ratio of 1:1000

LCMS-9030

[email protected] 10


Figure 11: Comparison between UV Chromatogram (at 254nm) of Ranitidine and extracted ion chromatogram (EIC) in HRMS of NDMA

NDMA peak is well separated from ranitidine drug. Ranitidine API peak is diverted to the waste by using divert valve to avoid the contamination of mass spectrometer.

Figure 12A: Blank Figure 12B: NDMA (1.0 ng/mL standard)

Datafile Name:SAMPLE_01_010.lcdSample Name:SAMPLE_01Sample ID:1301_SARAKA

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

mAU (x1,000)Ch1-254nm,4nm (2.00)

12.0

430.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0

2.5

(x10,000)1:75.0541-75.0565(+)

8.95

0

1.89e2Q 75.0553+/-15.0ppm (+)

RT (min)6.5 7.0 7.5 8.0 8.5 9.0 9.5

0.0e0

5.0e1

1.0e2

1.5e2

2.0e2

2.5e2

3.0e2

3.5e2

4.0e2

4.5e2

3.58e2 Q 75.0553+/-15.0ppm (+)

RT=8.968

RT (min) 7.0 7.5 8.0 8.5 9.0 9.5 0.0e0

1.0e2

2.0e2

3.0e2

4.0e2

5.0e2

6.0e2

7.0e2


Figure 11: Comparison between UV Chromatogram (at 254nm) of Ranitidine and extracted ion chromatogram (EIC) in HRMS of NDMA


Figure 12A: Blank Figure 12B: NDMA (1.0 ng/mL standard)

Datafile Name:SAMPLE_01_010.lcdSample Name:SAMPLE_01Sample ID:1301_SARAKA

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

mAU (x1,000)Ch1-254nm,4nm (2.00)

12.0

43

0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

0.5

1.0

1.5

2.0

2.5

(x10,000)1:75.0541-75.0565(+)

8.95

0

1.89e2Q 75.0553+/-15.0ppm (+)

RT (min)6.5 7.0 7.5 8.0 8.5 9.0 9.5

0.0e0

5.0e1

1.0e2

1.5e2

2.0e2

2.5e2

3.0e2

3.5e2

4.0e2

4.5e2

3.58e2 Q 75.0553+/-15.0ppm (+)

RT=8.968

RT (min) 7.0 7.5 8.0 8.5 9.0 9.5 0.0e0

1.0e2

2.0e2

3.0e2

4.0e2

5.0e2

6.0e2

7.0e2


Figure 12C: NDMA-D6 Figure 12D: Calibration curve (1 – 100 ng/mL)

Blank chromatogram does not show any interference at the retention time of NDMA as shown in figure 12A and 12B. Calibration curve was performed by using internal standard method with coefficient of correlation of 0.999 over a range of 1.0 to 100.0 ng/mL (refer figure 12D). Recovery study was performed by spiking LOQ level in the ranitidine sample and it was found to be well within the criteria.

In continuation to above work, Shimadzu lab, mumbai has developed the in-house method to determine NDMA in Ranitidine by using LCMS-8045 system, wherein LOQ achieve is up to 0.005ppm/LOD level of 0.002ppm in ranitidine.

LC-MS/MS method for the determination of NDMA, NDEA, NDIPA, NMBA, NEIPA and NDBA in Valsartan Drug Substance as per reference from USFDA method

Background about nitrosamines in Valsartan:

In July 2018, the American Food and Drug Administration (FDA) announced that the carcinogenic impurities: Nitrosamines (N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA)) had been detected in Valsartan bulk drug substances manufactured by Chinese manufacturers. Subsequently, a worldwide recall was issued of pharmaceutical products that use Valsartan bulk drug substances. Valsartan is used in the treatment of high blood pressure and congestive heart failure.

This LC-MS/MS method is used for the quantitation of six nitrosamines in Valsartan drug substance. The impurities are : N-nitroso-di-methylamine (NDMA), N-nitroso-di-ethyl amine (NDEA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), N-nitroso-ethyl iso-propylamine (NEIPA), N-nitroso-di-iso-propylamine (NDIPA), and N-nitroso-di-butylamine (NDBA

Principle

Reverse phase analysis is used for the separation of NDMA, NDEA, NMBA, NDIPA, NEIPA and NDBA impurities from valsartan. MRM method is developed for the all six nitrosamines. Two different Internal standards method is used for the quantification of NDMA in sample.

Conclusions

1.88e3ISTD 81.0927+/-15.0ppm (+)RT=8.773

RT (min)7.0 7.5 8.0 8.5 9.0 9.5

0.0e0

2.0e2

4.0e2

6.0e2

8.0e2

1.0e3

1.2e3

1.4e3

1.6e3

1.8e3

Conc.Ratio (ppb)0 20 40 60 80 100

Area Ratio

0

2

4

6

8

10

12

14

NDMAy = 0.1464784x + 0.01384017R² = 0.9994579 R = 0.9997289

Curve Fit: Default (Linear)Weighting: 1/CZero: Default (Not Forced)


Results and Discussion• Mobile phase B (0.1% formic acid in

methanol): mix formic acid and methanol at a volume ratio of 1:1000

Diluent and blank: Water NDMA intermediate stock standard preparation (100 ng/mL) • Prepare a 100 ng/mL intermediate

stock standard solution in water using commercially available NDMA reference stock standard solution.

Working standard solution preparation (1.0 ng/mL) • Transfer a 1.0 mL aliquot volume of the

intermediate stock standard into a 100 mL volumetric flask and dilute to volume with water. Prepare fresh daily.

Ranitidine drug sample preparation • Accurately weigh 120 mg of drug substance

into a 15 mL glass centrifuge tube. Add 4.0 mL of water and mix the solution using a vortex mixer until dissolved.

• Filter it using a 0.22 μm PVDF syringe filter in HPLC vial.

Blank chromatogram does not show any interference at the retention time of NDMA as shown in Figures 14A and 14B. Calibration curve was performed by using internal standard method with coefficient of correlation of 0.999 over a range of 1.0 to 100.0 ng/mL (Figure 14D). Recovery study was performed by spiking LOQ level in the Ranitidine sample and it was found to be well within the criteria.

Outstanding mass accuracy over a wide mass range, high efficiency ion guides, quadrupole, and collision cell of LCMS-9030 Q-TOF enabled high sensitivity quantitation with good reproducibility over longer periods of time without need of frequent calibration.

In continuation to above work, Shimadzu Labs, Mumbai has developed the in-house method to determine NDMA in Ranitidine by using LCMS-8045 system, wherein LOQ achieve is up to 0.005 ppm/LOD level of 0.002 ppm in ranitidine.

Fig. 13: Comparison between UV Chromatogram (at 254nm) of Ranitidine and extracted ion chromatogram (EIC) in HRMS of NDMA

Fig. 14A: Blank

Fig. 14C: NDMA-D6 Fig. 14D: Calibration curve (1-100 ng/mL)

Fig. 14B: NDMA (1.0 ng/mL standard)


[email protected]

The LCMS-8045 accomplishes the highest sensitivity in its class, thanks to the superior design with a heated ESI probe. With the world's fastest scan speed (30,000 u/sec) and polarity switching time (5 ms), high speed data acquisition is a reality now for increased throughput and shortened method development time.

LCMS-8045Liquid Chromatograph Mass Spectrometer

Best in Class Sensitivity

[email protected] 1 09 281-20

40Yearsof Service to Customers

and continuing…

Accomplishes both high sensitivity and ultra-high-speed detection

Superior robustness maintaining sensitivity over the long term

High Performance for a variety of analyses - veterinary drugs,

residual pesticides, water quality analysis, bioanalysis etc.

Upgradable to LCMS-8060, the world's highest sensitivity triple quad MS.

Date post:	20-Feb-2021
Category:	Documents
Upload:	others
View:	0 times
Download:	0 times

Nitrosamines in Pharmaceuticals? No Problem! in Pharmaceutic… · ylamine (NDMA) impurity from...

Documents