Introduction

Environmental contamination by ethylene thiourea (ETU) is

due primarily to the use of several fungicides categorized as

ethylene bisdithiocarbamates (EBDCs), which can be applied

to ornamental plants, vegetables, fruits, and field crops. ETU

is an environmental degradation product, metabolite, and syn-

thesis contaminant of EBDCs [1]. ETU is also used as an

accelerator for vulcanizing neoprene and polyacrylate rubbers

as well as in electroplating baths, synthetic resins,

pharmaceuticals, dyes, and as a scavenger in waste water

treatment.

ETU has the potential for contaminating drinking water from

both surface water and groundwater sources, due to its high

solubility in water, and its mobility in the environment.

According to US Environmental Protection Agency (EPA)

reports, ETU has been measured in one public drinking-water

well at 0.21 parts per billion (ppb), but it was not detected in

any of 84 finished drinking water sources that were sampled.

In addition, a targeted study did not find ETU in surface water

at a detection limit of 0.1 ppb [2,3,4]. Following testing of US

groundwater wells, the EPA has estimated that 0.1 % of rural

wells are contaminated with ETU.

There is adequate evidence of carcinogenicity from experi-

mental animal studies for ETU to be reasonably anticipated to

be a human carcinogen. Epidemiological study data in

humans are not adequate to evaluate the relationship

between exposure specific to ETU and human cancer [5].

However, the US EPA has established a Drinking Water

Equivalent Level of 0.7 ppb in drinking water. In addition, EPA

Method 509.1 has recently been released as a draft for the

direct detection of ETU in water using liquid chromatogra-

phy/electrospray ionization tandem mass spectrometry

(LC/ESI-MS/MS). The method allows flexibility in LC

columns, LC conditions, and MS conditions, as long as

method performance is not affected.

This application note describes the use of the Agilent 1290

Infinity Series LC and the Agilent 6460A Triple Quadrupole

LC/MS with Agilent Jet Stream technology to meet the strin-

gent quality control requirements of EPA draft Method 509.1,

using direct injection of the sample. This analysis platform

provided Lowest Concentration Minimum Reporting Levels

(LCMRLs) and Detection Limits (DLs) that exceeded EPA

requirements. Accuracy and precision were also well within

the requirements of the draft EPA method.

Table 1. HPLC and MS Conditions

HPLC

Analytical column Agilent ZORBAX SB-Aq, 3.0 × 150 mm, 3.5 µm

(p/n 863954-314)

Column temperature 40 °C

Injection volume 60 µL

Mobile phase A) 1 mM Ammonium fluoride

B) MeOH

Flow rate 0.5 mL/min

Elution 0 %B isocratic

Column flush 100 %B, for 20 minutes following each batch of

samples

Run time 4 minutes, injection to injection

Acquisition parameters ESI mode, positive ionization, MRM

Sheath gas temperature 380 °C

Sheath gas flow rate 12 L/min

Drying gas temperature 200 °C

Drying gas flow rate 4 L/min

Nebulizer pressure 40 psig

Nozzle voltage 0 V

Vcap 2,000 V positive

Experimental

Reagents and materials

Ethylene thiourea, glycine hydrochloride, cysteine

hydrochloride, and ammonium fluoride were obtained from

Sigma-Aldrich, Oakville, Ontario. ETU-d4 was obtained from

CDN Isotopes in Pointe-Claire, Quebec. Solvents were

LC grade, obtained from Caledon Laboratories, Georgetown,

Ontario.

Instruments

The draft EPA Method 509.1 was run using an Agilent 1260

Infinity High Performance Autosampler and an Agilent 1290

Infinity LC system, which was coupled to an Agilent 6460A

Triple Quadrupole LC/MS with Agilent Jet Stream technology.

The instrument conditions are shown in Table 1.