L-column3 Application Data Sheet for LC
Application No. L3050

Nitrosamines in Pharmaceuticals
Nitrosamines are known to be carcinogenic. In 2018, N-Nitrosodimethylamine (NDMA) was found in preparations of valsartan, asartan drug. As a result, voluntary inspections were conducted. Due to factors such as the synthesis process and storage
conditions, there is a limit for NDMA and other nitrosamines in the drug substance. LC-MS/MS analysis by ESI and APCI was used
to detect 7 nitrosamines.

Key wordsColumn: L-column3 Ammonia eluent LC-MS/MS Nitrosamines NDMA: USP category: L1

It has been reported in two studies that ranitidine can be transformed into NDMA by a slight increase in temperature. In
one study, ranitidine was heated at 130°C for 1 hour, and the formation of NDMA was confirmed through testing.

10000

Sample 1Ranitidine 50 mg

Ranitidine
5000 mg/L

Add water; 5 mL
Fixed volume
By acetonitrile; 10 mL

5000

Sample 2Ranitidine 50 mg

010000
10000

0123456

Heated Ranitidine
5000 mg/L

8000

Heated at 130℃; 1 hour
Add water; 5 mL
Fixed volumeby acetonitrile; 10 mL

6000
500040002000000123456012345610000

HeatNDMA2 mg/L
5000

Fig.2 Formation process of NDMA. ※200123Time (min)Fig.1 Chromatogram of ranitidine samples.

456L-column3 Application Data Sheet for LC

Application No. L3050[ Analytical conditions ]Column: L-column3 C18 (2 μm, 12 nm); 3.0 mm I.D. × 30 mm L.; Cat. No. 823650
Eluent: A: 0.1% HCOOH in H2O ; B:0.1% HCOOH in CH3OHA/B, 90/10(0-0.5 min)-5/95(5-6 min)Flow rate: 0.5 mL/minTemperature: 40℃
Detection: APCI-MS/MS(+), ESI-MS/MS(+)
Injection volume : 5 μL
System: LC: Ultimate 3000 Bio RS (Thermo Fisher Scientific K.K.); MS/MS: 3200 QTRAP (SCIEX)
Sample: Nitrosamines 2 mg/L in H2O/CH3OH (9/1 v/v) each
1. N-Nitrosodimethylamine(NDMA)
N-ニトロソジメチルアミン
m/z 75.0 → 58.2

2. N-Nitrosodiethylamine(NDEA)
N-ニトロソジエチルアミン
m/z 103.0 → 75.1

5. N-Nitrosomethylethylamine(NMEA)
N-ニトロソメチルエチルアミン
m/z 89.1 → 61.0

3. N-Nitrosodipropylamine(NDIPA)
N-ニトロソジイソプロピルアミン
m/z 131.0 → 89.0

6. N-Nitrosopyrrolidine(NPYR)
N-ニトロソピロリジン
m/z 101.0 → 55.0

4.N-Nitrosodi-n-butylamine(NDBA)
N-ニトロソジ-n-ブチルアミン
m/z 159.2 → 103.2

7. N-Nitrosopiperidine(NPIP)
N-ニトロソピペリジン
m/z 116.0 → 70.3

2.9e5
6

Intensity/cps

234

1.5e5
5

No.

Component
Name

1.
2.
3.

NDMA
NDEA
NDIPA

1333
8524
14274

1867
644

4.
5.
6.

NDBA
NMEA
NPYR

18602
2790
5133

7321
511133

7.

NPIP

1300

164

Signal / Noise
APCI
ESI

1703Time (min)6Fig. 3 Chromatogram of nitrosamines standard solution (APCI)LC-MS/MS analysis by ESI and APCI was performed using seven nitrosamines.Here, high-speed analysis
was performed using a 30 mm long column. While ESI has low sensitivity for low-molecular-weightcomponents, APCI was able to detect even low-molecular-weight components with high sensitivity.

L-column3 Application Data Sheet for LC

Application No. L3050

In September 2020, the FDA issued guidelines for nitrosamines that stipulate the use of a highly sensitive analysis method with alower limit of quantification of 0.03 ppm or less to meet regulatory requirements. To comply with these guidelines, ionization by
APCI was performed in this study, resulting in good linearity at concentrations of 20 to 200 ng/mL for all seven components. By
using a SIM, the S/N ratio can be improved by 2 to 5 times. However, quantifying NDMA, which has a small molecular weight, canbe challenging due to its susceptibility to the baseline and matrix, resulting in poor quantification.

NDEA

NDMA

0100

y = 336.94x
R² = 0.9998

Peak area

Peak areay = 53.733x
R² = 0.9981200 (ng/L)

0100

NDIPA

NDBA
y = 535.96x
R² = 0.9996

Peak area

Peak areay = 494.19x
R² = 0.9993

0100

0200 (ng/L)

100

y = 437.27x
R² = 0.9992

100200 (ng/L)

NPYR
y = 501.4x
R² = 0.9991

Peak area

Peak area

NMEA

0200 (ng/L)

200 (ng/L)

0100200 (ng/L)

Peak area

NPIP

0y = 43.322x
R² = 0.9987

100*1 Ministry of Health, Labor and Welfare. (2021). Director of Pharmaceutical Evaluation Division,
Pharmaceutical Safety and Environmental Health Bureau, Ministry of Health, Labor and Welfare,
dated October 8, 2021.
・Pharmaceutical Safety Division Director, Supervisory Guidance,
Narcotics Countermeasures Division Director・Pharmaceutical Safety Notification
No. 1008 No. 1 ・Pharmaceutical Narcotics Notification
No. 1008 No. 1"Voluntary inspection on the risk of contamination
by nitrosamines in pharmaceuticals.
*2 Valisure Citizen Petition on Ranitidin, Varisure,200 (ng/L)