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HPLC-MS ANALYSIS OF 15 NITROSAMINES

1.1 Introduction

Docuchem has developed different chromatographic methods using columns with different stationary phases in order to have different possibilities to analyze a broad spectrum of Nitrosamines. In addition, the LOD of 5 Nitrosamines has been tested using a triple quadrupole Shimadzu 8050 MS detector.

1.2 Scope

The 15 analyzed Nitrosamines are detailed hereunder:

  • N-Nitroso-diisobutylamine (NDIBA)
  • N-Nitroso-dibenzylamine (NDBzA)
  • N-Nitroso-methylisopropylamine
  • N-Nitroso-mehtyl aniline (NMA)
  • N-Nitrosomethylethylamine
  • N-Nirosodi-n-propylamine (NDPA)
  • N-Nitrosodi-n-butylamine (NDBA)
  • N-Nitrosopyrrolidine
  • N-Nitrosomorpholine
  • N-Nitroso-N-methyl-4-aminobutyric acid
  • N-Nitrosoethylisopropylamine (NEIPA)
  • N-Nitrodiisopropylamine
  • N-Nitrosodimethylamine
  • N-Nitrosodiethylamine
  • N-Nitrosopiperidine (NPIP) 

1.3 HPLC conditions

1.3.1  Chromatographic conditions with XBridge Phenyl 150×4.6mm 3.5 µm

Column: XBridge Phenyl 150×4.6mm 3.5 µm
Detector: PDA at 254 nm
Flow rate: 0.6 mL/min
Inj. Volume: 5 µL
Column oven 40ºC
Run time: 30 min
Recording time: 30 min
Mobile phase A: H2O 0.1% HCOOH
Mobile phase B Methanol 0.1% HCOOH

Gradient:

Time (minutes) Mobile phase A% Mobile phase B%
0.00 90 10
1.5 90 10
7.0 45 55
17.0 45 55
17.1 10 90
21.0 10 90
21.1 90 10
25.0 90 10
30.0 90 10

1.3.2  Chromatographic conditions with Infinitylab Poroshell 120 PFP column

Column: Poroshell 120 PFP 100×4.6mm 2.7 µm
Detector: PDA at 254 nm
Flow rate: 0.6 mL/min
Inj. Volume: 5 µL
Column oven 40ºC
Run time: 30 min
Recording time: 30 min
Mobile phase A: H2O 0.1% HCOOH
Mobile phase B Methanol 0.1% HCOOH
Gradient:
Time minutes Mobile phase A% Mobile phase B%
0.00 90 10
1.5 90 10
7.0 45 55
17.0 45 55
17.1 10 90
21.0 10 90
21.1 90 10
25.0 90 10
30.0 90 10

1.3.4  Chromatographic conditions with XSelect CSH Phenyl Hexyl 250X4.6 mm 5 µm

 

Column: XSelect CSH Phenyl Hexyl 250×4.6mm 5 µm
Detector: PDA at 254 nm
Flow rate: 1.0 mL/min
Inj. Volume: 5 µL
Column oven 40ºC
Run time: 32 min
Recording time: 32 min
Mobile phase A: H2O 0.1% HCOOH
Mobile phase B Methanol 0.1% HCOOH
Gradient:  
Time minutes Mobile phase A% Mobile phase B%
0.0 70 30
10.0 50 50
14.0 50 50
21.0 10 90
25.0 10 90
25.1 70 30
32.0 70 30

1.4 Limit of Detection of Nitrosamines

The LOD of 5 different Nitrosamines: (N-Nitroso-Dimethylamine (NDMA), N-Nitroso-Piperidine (NPID), N-Nitroso-Diethylamine (NDEA), N-Nitroso-Ethylisopropylamine (NEIPA) and N-Nitroso-Diisopropylamine (NDIPA) has been evaluated with a triple quadrupole MS detector 8050 from Shimadzu using the HPLC method conditions with the XBridge C18 column.

Combining the HPLC methods developed by Docuchem and optimizing the MS/MS conditions, LOD of 2.5 ppb can be achieved for the 5 tested Nitrosamines. For NPID, NEIPA and NDIPA, the LOD could be lowered even more, reaching levels below 1 ppb.

Further investigations were performed obtaining a LOD of 0.1 ppb for NDMA using an UPLC method.

1.5 Conclusions

The different HPLC methods tested provides flexibility in the HPLC separation. Depending on the Nitrosamines that the costumer needs to analyze and the other substances contained in the sample, the previously listed HPLC methods can be used to achieve suitable resolutions between the analytes. For NDMA, a LOD of 0.1ppb was achieved after an optimization of the MS parameters. Therefore, LOD of all nitrosamines could be improved obtaining LOD results about 0.5ppb or even lower.