Poster Reprint

ASMS 2020
TP 343Ultra-Fast Analysis of
Nitrosamines Using SPE-QQQ

Kevin Truempi, Kevin McCann
Agilent Technologies, Santa Clara , CA

Introduction

Experimental

Several highly sensitive quantitative methods havebeen developed for the analysis of nitrosamines usingmass spectrometry1. However, these methods rely on
chromatographic separations that take several
minutes per sample. Rapid, robust screening andquantitation of impurities is an essential analytical
tool for a wide variety of laboratories. High-throughput
environments must be able to perform these analyses
in a way that ensures productivity, minimizes costs,and eliminates backlog. The use of Solid Phase
Extraction Triple Quadrupole Mass Spectrometry(SPE-QQQ) allows for the ultra-fast analysis ofsamples without compromising analytical fidelity.
This work explores the simultaneous quantitation of apanel of nitrosamines in less than 15 seconds perinjection. An existing U.S. Food and Drug
Administration (US FDA) analytical method2 wasreproduced and then additional nitrosamines wereadded to assess feasibility and ease of expanding thepanel.

N-Nitrosoethylmethylamine (NMEA)

Instrumentation
Instrumentation for the SPE-QQQ analysis consistedof a RapidFire High-Throughput Mass Spectrometry
System coupled to an Ultivo Triple Quadrupole LC/MS.
Online solid phase extraction (SPE) was performed
using a graphitic carbon cartridge to separate target
analytes from salts and any other interferences
present in the samples.

Figure 2. Agilent RapidFire 400 High-Throughput Mass
Spectrometry System coupled to an Agilent Ultivo
triple quadrupole LC/MS
Chemicals and Reagents
Nitrosamine standards, LC/MS grade methanol, andformic acid were purchased from Sigma-Aldrich,St. Louis, MO, USA.

N-Nitrosopyrrolidine (NPyR)

MethodThe automated trap, wash, and elute cycle was
optimized to achieve an analysis time of less than 15
seconds per injection.

N-Nitrosopiperidine (NPIP)
https://www.chemspider.com/

Figure 1. Three nitrosamines studied as proof-ofconcept additions to FDA’s RapidFire method for theanalysis of nitrosamine impurities.

Figure 3. RapidFire injection cycle.

2Instrument Settings

RapidFire Conditions
Buffer A: Water + 0.1% Formic Acid
Buffer B: Methanol + 0.1% Formic Acid
SPE Cartridge: Graphitic Carbon, Type D (G9206A)
StateAspirate
Load/Wash
Elution
Re-Equilibrate

Ultivo Conditions
Parameter
Ion ModePolarity
Drying Gas Temp
Drying Gas Flow
Nebulizer
APCI Heater
APCI Needle
Capillary Voltage

Time (ms)
600
2000
7000
2000

Value
APCI
Positive
300 °C
6 L/min55 psi
350 °C
4 µA
3000 V

Results and Discussion

Ultra-Fast Data Acquisition
Injections were made at a rate of approximately 12.5
seconds per sample while data was acquired by triple
quadrupole mass spectrometry. Figure 4 shows 72
injections acquired in under 15 minutes; several blankswere run between calibration levels to assess carryover.

Reproducible and Accurate Results
Triplicate injections of each calibrator demonstrated
excellent reproducibility. Coefficients of variation range
from 4.5 to 9.0% for NPIP (Figure 4) and are
representative of all analytes. Excellent linearity is also
observed, with R2 ranging from 0.997 to 0.999 (Figure 5).
100 ng/mL

50 ng/mL

20 ng/mL

1 ng/mL

5 ng/mL

10 ng/mL

Figure 4. Triplicate injections of a 6-point calibration curve for NPIP. Six blank injections were made between eachcalibration level to evaluate carryover.

3Results and Discussion

Figure 5. Calibration from 1-100 ng/mL for NDBA and NPIP. Calibration from 5-100 ng/mL for NPyR and NMEA.
Conclusions

The US FDA’s method for rapid analysis of nitrosamine impurities has been replicated with consistent results. Further
proof-of-concept work demonstrates the simplicity of adding additional nitrosamine analytes, without any significant
method development.

References
1 FDA Updates and Press Announcements on Angiotensin II Receptor Blocker (ARB) Recalls. (2019, November 13). FDA.

Retrieved April 15, 2020. https://www.fda.gov/drugs/drug-safety-and-availability/fda-updates-and-pressannouncements-angiotensin-ii-receptor-blocker-arb-recalls-valsartan-losartan
2 Development and validation of a RapidFire-MS/MS method for screening of nitrosamine carcinogen impurities…in ARBdrugs. (2019, July 24). FDA. Retrieved April 15, 2020. https://www.fda.gov/media/125477/download
3 Information about Nitrosamine Impurities in Medications. (2020, February 3). FDA. Retrieved April 15, 2020.
https://www.fda.gov/drugs/drug-safety-and-availability/information-about-nitrosamine-impurities-medications

This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Published in USA, June 1, 2020