Determination of 7 Nitrosamines in Drinking Water by GC-MS/MS Using a SUPELCOWAX Capillary GC Column Following GB 5750.10-2023

Jack Wang
R&D APAC lab, Shanghai, China

Abstract

A method for the determination of N-nitrosamines in drinking water by solid-phase extraction and subsequent gas chromatography-tandem mass spectrometry (GC-MS/MS) was established. The samples were adsorbed on a graphitized carbon solid-phase extraction (SPE) column and eluted using dichloromethane. After eluate concentration, the samples were analyzed by GC-MS/MS and quantified utilizing external calibration. The N-nitrosamines showed an excellent linear relationship in the calibration range from 25.0 to 500 µg/L (representing concentrations in the water sample of 25-500 ng/L considering the applied sample preparation) with correlation coefficients of greater than 0.9990. The recovery rates were in the range of 71.5% to 88.0%, and the relative standard deviations (RSD) were 2.29 to 3.64%. The method is simple, short, sensitive, and provides high recoveries, and it is suitable for the analysis of N-nitrosamines in drinking water according to GB 5750.10-2023.

Section Overview

• Introduction
• Experimental
• Results & Discussion
• Conclusion
• Related Products

Introduction

In recent years, chloramines have been used to disinfect drinking water instead of chlorine. However, studies have shown that chloramine disinfection produces N-nitrosamines as a by-product.^1,2 N-nitrosamines are a class of compounds with an N-N=O structure. At present, more than 80 percent of more than 130 N-nitrosamines have been found to be strong carcinogens.

N-Nitrosodimethylamine (NDMA), for example, is considered a priority pollutant, and many international, national, and local agencies have developed regulatory guidelines for limits of various nitrosamines in drinking water. In 1989, NDMA has been found in drinking water wells in northern California,³ and since then, this US state has established for NDMA a notification level of 10 ng/L (ppt).⁴ In case this response concentration is reached, it is recommended to stop the use of respective water sources. Internationally, the World Health Organization (WHO) included NDMA in its drinking water quality control guidelines in 2008.⁵ In March 2010, Health Canada proposed a maximum acceptable concentration of NDMA in drinking water of 40 ng/L.⁶

As of October 2009, the U.S. Environmental Protection Agency (EPA) has listed five nitrosamines (including NDMA) on its candidate list of drinking water contaminants to be controlled and will study whether regulatory standards are needed. If NDMA or other nitrosamines become regulated drinking water contaminants, a stable and sensitive analytical method is needed for regulatory certification monitoring. ^8-10

The new Chinese GB 5750.10-2023 method¹¹ also provides a new method for analyzing NDMAs. The scope and working principle of the method are as follows: Seven nitrosamines (NDMA, NDBA, NDPA, NMEA, NDEA, NPYR, and NPIP) in the to be tested drinking water samples are extracted by a solid-phase extraction column, eluted with dichloromethane, concentrated, and then separated on a polar capillary chromatography column. The detection is done by quadrupole mass spectrometry (MS).

At the same time, the GB national standard also makes the following provisions on the quality control data for method verification:

• Detection limits of seven N-nitrosamines: For a water sample size of 1000 mL, the minimum detection concentration (LOD, also referred to as the minimum detection mass concentration in GB 5750.10-2023) of the seven nitrosamines NDMA, NDBA, NDPA, NMEA, NDEA, NPYR, and NPIP should be ≤10 ng/L.
• Linearity of calibration: The concentration range for the external calibration is given as 25.0-500 µg/L (representing concentrations in the water sample of 25-500 ng/L considering the sample pre-treatment enrichment factor of 1:1000) and should provide linearity values R² of >0.9950.

In this study, a highly sensitive method for the analysis of seven nitrosamines in a drinking water sample according to the GB method was developed. It combines graphitized carbon solid-phase extraction with GC-MS/MS analysis and quantification with external standards. The method verification experiments were carried out in full accordance with the quality control requirements in the GB 5750.10-2023 national standard for the determination of N-nitrosamines in drinking water. The sample preparation process was carried out in accordance with the requirements of the 2024 updated GB method. A 1000 mL water sample (formerly 500 mL, changed in Aug. 2024 by the China Standardization Committee issuing a notice of corrigendum) to be tested was extracted using a Supelclean™ ENVI-Carb™, 0.5 g/6 mL (57094) SPE tube and finally concentrated to 1 mL for LC-MS/MS analysis.

Chemical structures of seven N-nitrosamines analyzed in the study

Experimental

Standard Preparation

The standards and samples were prepared following these procedures using a certified reference material (CRM) mixture.

Standard Preparation

• N-Nitrosamines stock solution I (200 µg/mL): Transfer 1000 µL of a standard mixture of seven N-nitrosamines (N-nitrosodimethylamine, N-nitrosodibutylamine, N-nitrosodi-n-propylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, 1-nitrosopyrrolidine, and 1-nitrosopiperidine at 2000 µg/mL each, 40035-U) into a 10 mL amber glass volumetric flask and fill up to the mark with dichloromethane. Store the standard solution at 0 - 4 °C and protected from light.
• N-Nitrosamines stock solution II (1 µg/mL): Pipette 50 µL of N-nitrosamines stock solution I into a 10 mL amber glass volumetric flask and fill up to the mark with dichloromethane to obtain a N-nitrosamines stock solution II with an N-nitrosamines concentration of 1.00 µg/mL each. Store at 0 - 4 °C and protected from light.
• N-Nitrosamines standard solutions 1-5: Prepare a total of five standard working solutions (nos. 1-5) by pipetting 25 μL, 50 μL, 100 μL, 250 μL and 500 μL of N-nitrosamines stock solution II into five separate 1 mL vials. Fill vials up to mark with dichloromethane. The concentrations of each N-nitrosamine in the resulting solutions are 25.0, 50.0, 100, 250 and 500 μg/L, respectively.

Sample & Reagent Preparation

Transfer 1000 mL of a drinking water sample into an amber glass bottle.

Solid Phase Extraction (SPE)

1. Conditioning: A Supelclean™ ENVI-Carb™, 0.5 g/6 mL (57094) SPE tube is conditioned with 6 mL dichloromethane and 6 mL methanol, then flushed with nitrogen (application of vacuum is also possible). Subsequently, activate the column with 6 mL methanol and 9 mL water.
2. Sample loading: Pass 1000 mL of a water sample through the SPE column and keep the flow rate at 10 mL/min by properly adjusting the vacuum.
3. Drying: Remove water from the SPE tube by applying vacuum for 10 minutes.
4. Sample elution: Elute the sample with 10 mL of dichloromethane and collect the eluate in a 15 mL centrifuge tube.
5. Eluate post-treatment: Use nitrogen to concentrate the eluent to near dryness, then add 1 mL dichloromethane and transfer the solution into a sample vial for analysis.

Spiking experiments

Prepare two samples for the determination of recovery (%) and precision by mixing 1000 mL of a blank water sample with 30 μL and 100 μL of N-Nitrosamines stock solution II, respectively. The concentrations of the N-nitrosamines in the two resulting samples are 30 ng/L and 100 ng/L, respectively.

Use the sample with an N-nitrosamine concentration of 30 ng/L for the determination of analysis precision, and the sample with an N-nitrosamine concentration of 100 ng/L for the analysis of recovery rate (%).

Instruments and Equipment

• Agilent GC-MS/MS 8890GC+7000D (EI)
• ANPEL EUFO-945416 vortex mixer

GC Analysis

Standards and sample extracts were analyzed by GC-MS/MS (Tables 1 & 2).

Results & Discussion

Spiked drinking water samples were prepared and extracted by SPE as described, then analyzed by GC-MS/MS (operated in dynamic multi-reaction monitoring mode) and quantified using an external calibration.

Figures 1 and 2 show MRM chromatograms of N-nitrosamines standard solution 2 (c =50 µg/L) and of an extracted drinking water sample spiked with N-nitrosamines standard solution 3. The chromatographic data of the spiked drinking water sample and the used MS/MS transitions are displayed in Table 3.

Figure 1. GC-MS/MS chromatogram displaying the analysis of N-nitrosamines standard solution 2 (c = 50 µg/L).

Figure 2. GC-MS/MS chromatogram obtained by the analysis of drinking water sample spiked at a level of 30.0 ng/L for all N-nitrosamines.

Calibration

The results of the external calibration utilizing N-nitrosamine standard solutions 1-5 is displayed for NBMA as example in Figure 3 & Table 4, the other nitrosamines provided similar results (Table 5).

Figure 3. Calibration curve for NDMA 25-500 µg/L (injection of standard solutions).

Data Precision and Recovery (%)

For the determination of the method’s precision a sample spiked at a nitrosamine concentration of 30.0 ng/L was used (Table 6). The precisions for all nitrosamine ranged from 2.29 to 3.64 %RSD (GB criteria <15%). The determination of the recovery rates (%) was performed utilizing the sample spiked at a concentration of 100 ng/L (Table 7). The recoveries ranged from 71.5 to 88.0 % (GB criteria 60-120%). 

Sensitivity

For the sensitivity determination of the GC-MS/MS method, the baseline noise of a blank drinking water sample was employed: 3N/X was used to determine Limit of Detection (LOD), and 10N/X was used to determine Limit of Quantification (LOQ). LODs ranged from 3.05-3.27 µg/L and LOQ from 9.15-9.81 µg/L (Table 8) representing concentrations in the water samples of 3.05-3.27 ng/L and 9.15-9.81 ng/L respectively (GB criteria for LOD ≤10 ng/L).

Suitability Criteria Summary

In Table 9 a summarized overview on the suitability criteria of the GB method and the determined values is provided, showing that the developed method complies with the GB 5750.10-2023

Conclusion

A method for the determination of seven N-nitrosamines in drinking water by solid phase extraction (SPE) and subsequent gas chromatography-tandem mass spectrometry (GC-MS/MS) according to GB 5750.10-2023 was established. The analytes were adsorbed on a graphitized carbon solid phase extraction (SPE) column (Supelclean™ ENVI-Carb™) and eluted using dichloromethane. After eluate concentration, the samples were analyzed on a SUPELCOWAX column by GC-MS/MS operated in multi-reaction monitoring (MRM) mode and quantified utilizing external standard calibration. The investigated N-nitrosamines showed for the chromatographic method an excellent resolution and linear relationship in the calibration range from 25.0 to 500 µg/L (equivalent to 25-500 ng/L in a water sample) with correlation coefficients greater than 0.9990. The recoveries for a spiked water sample (100 ng/L) were in the range of 71.5 % to 88.0 %, and the relative standard deviations (RSD) were 2.29 to 3.64%. The limits of detection (LOD) were for all compounds under 10 ng/L. By that the method complied with the suitability criteria of the GB method. The sample preparation and analysis process were simple and provided excellent linearity, sensitivity, accuracy, and precision and has hence proved suitability for the quantitative analysis of N-nitrosamines in drinking water according to the GB 5750.10-2023 national standard.

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References

1. Li-Qun Z, Quan J, Han T, et al. 2020. Simultaneous determination of 8 N-nitrosamines in drinking water by gas chromatography-tandem mass spectrometry. [Internet]. Chinese Journal of Health Inspection. Available from: https://d.wanfangdata.com.cn/periodical/zgwsjyzz202024004

2. D. Alexandrou L, Bowen C, Jones OAH. Fast analysis of multiple haloacetic acids and nitrosamines in recycled and environmental waters using liquid chromatography-mass spectrometry with positive–negative switching and multiple reaction monitoring. Anal. Methods. 11(30):3793-3799. https://doi.org/10.1039/c9ay01276a

3. Mitch WA, Gerecke AC, Sedlak DL. 2003. A N-Nitrosodimethylamine (NDMA) precursor analysis for chlorination of water and wastewater. Water Research. 37(15):3733-3741. https://doi.org/10.1016/s0043-1354(03)00289-6

4. California State Water Resources Control Board. Nitrosamines. [Internet]. Waterboards.ca.gov.[cited 23 Jan 2025]. Available from: https://www.waterboards.ca.gov/drinking_water/certlic/drinkingwater/NDMA

5. 2008. N-Nitrosodimethylamine in Drinking-water - Background document for development of WHO Guidelines for Drinking-water Quality WHO/HSE/AMR/08.03/8. [Internet]. World Health Organization (WHO). Available from: https://cdn.who.int/media/docs/default-source/wash-documents/wash-chemicals/ndma-2add-feb2008.pdf?sfvrsn=122aa3d3_4

6. Guidelines for Canadian Drinking Water Quality Guideline Technical Document N-Nitrosodimethylamine (NDMA). [Internet]. Available from: https://publications.gc.ca/site/eng/430928/publication.html

7. Munch JW. 2005. EPA Method 521:Determination of Nitrosamines in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography with Large Volume Injection and Chemical Ionization Tandem Mass Spectrometry (MS/MS) . [Internet]. Washington, DC: U.S. Environmental Protection Agency. Available from: https://cfpub.epa.gov/si/si_public_record_report.cfm?Lab=NERL&dirEntryId=103912

8. Gao P, Fan J, Lin C. 2013. Determination of Ten N-nitrosamines in Drinking Water by Solid Phase Extraction coupled with Gas Chromatography/Electron Ionization Tandem Mass Spectrometry. [Internet]. American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics. Available from: https://asms.org/docs/default-source/Past-Annual-Conference-Programs/asms_61st_5-17_fullprogramlow.pdf

9. Wang C, Yu J, Guo Q, Zhao Y, Cao N, Yu Z, Yang M. 2019. Simultaneous quantification of fifty-one odor-causing compounds in drinking water using gas chromatography-triple quadrupole tandem mass spectrometry. Journal of Environmental Sciences. 79100-110. https://doi.org/10.1016/j.jes.2018.11.008

10. GB/T 5750.10-2023 Standard test methods for drinking water - Part 10: Indicators of disinfection by-products. [Internet]. Available from: https://std.samr.gov.cn/gb/search/gbDetailed?id=F78920660F93B223E05397BE0A0AE533