IntroductionA variety of organic molecules originating from pesticides, pharmaceuticals, personal care products, and industrial chemicals used in daily life can be introduced into the environment through domestic and industrial wastewater sources. These molecules are a subclass of compounds collectively known as Trace Organic Contaminants (TOrCs), commonly found in a variety of potable and non-potable water resources. Although not acutely dangerous as highly dilute individual entities, the effects of long-term synergistic exposure due to mixtures of these compounds are still to be determined1. While characterization for long-term and chronic toxicity are yet to be established, it is important to monitor their presence in current water resources2.

Using the online SPE capabilities of the Agilent 1290 Infinity Flexible Cube with the analytical power of the Agilent Ultivo Triple Quadupole LC/MS, we have demonstrated a rapid and sensitive method for automated analysis of trace organic contaminants in drinking water without tedious offline enrichment.

AUTOMATED ONLINE SPE/TANDEM MS ANALYSIS OF TRACE ORGANIC CONTAMINANTS IN DRINKING WATER

Agilent Ultivo Triple Quadrupole LC/MS System

For more information, visit:www.agilent.com/chem/Ultivo

Figure 1B. Agilent 1290 Infinity FlexCube.

Figure 1A. Agilent Ultivo Triple Quadrupole LC/MS.

MethodologyThe Agilent 1290 Infinity Flexible Cube Online SPE dual cartridge configuration (Figure 2) allows injection volumes of up to 900 µL for sample enrichment with minimal user intervention, improving accuracy and reproducibility. The dual cartridge setup allows for enhanced sample throughput by equilibrating one cartridge as the other is cartridge is being eluted.

Figure 3. Signal response after sample enrichment of a 10 ppt spiked water sample with 750 µL injection.

Figure 2. Valve diagram for dual cartridge Online SPE.

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Analytical column

In this experiment, a selection of 31 representative TOrCs (listed in Figure 3) were analyzed in spiked water samples from 0.5–200 ng/L (ppt) in concentration. Sample volumes of 750 µL were introduced onto the SPE cartridge for enrichment, then eluted at the 4-minute mark, triggering the start of the HPLC linear gradient. Detection of analytes was carried out on the Ultivo Triple Quadrupole LC/MS using dynamic MRM for maximized sensitivity.

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Acquisition time (min)

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Compound RT (min)

Atenolol 7.92

Caffeine 8.25

Trimethoprim 8.36

Benzotriazole 8.58

Hydrochlorothiazide 8.82

Primidone 8.84

Propranolol 9.23

Meprobamate 9.30

Sulfamethoxazole 9.40

Diphenhydramine 9.51

Diltiazem 9.60

Compound RT (min)Clofibric acid 9.60

Hydrocortisone 9.97

Carbamazepine 9.98

Fluoxetine 9.98

Simazine 10.01

TCEP 10.34

BPA 10.51

Benzophenone 10.52

Naproxen 10.53

DEET 10.55

Atrazine 10.57

Compound RT (min)Propylparaben 10.57

Testosterone 10.72

Diclofenac 10.98

TCPP 11.19

Norgestrel 11.28

Ibuprofen 11.36

Gemfibrozil 11.91

Triclocarban 12.20

Triclosan 12.24

Online SPE, HPLC, and MS parameters

Table 1 lists Online SPE, HPLC, and MS instrument parameters.

Table 1. Instrument conditions.

Online SPE conditions

SPE Cartridge Agilent ZORBAX Extend C18, 4.6 × 12.5 mm, 5 µm

Agilent 1290 Infinity Flexible Cube solvents

A (Load): H2O with 0.1 % v/v formic acid B (Elute): 1:1 ACN/IPA

Sample volume 750 µL

SPE Elution gradient 0 minutes: Load - Pump for 240 seconds H2O with FA at 1.5 mL/min 4 minutes: Valve Switch (Elute) - Pump for 360 seconds 1:1 ACN/IPA at 1.0 mL/min 8 minutes: Equilibrate - Pump for 240 seconds of H2O with FA at 1.5 mL/min

HPLC Conditions

Column Agilent ZORBAX RRHD Eclipse Plus C18, 3.0 × 50 mm, 1.8 µm

Mobile phase A) H2O with 0.2 mM NH4F B) ACN with 0.2 mM NH4F

Flow rate 0.350 mL/min

HPLC Elution gradient Time (min) Mobile phase 0 5 %B 4 5 %B 11 100 %B 12.5 5 %B

Run time 4 minutes (sample enrichment) + 8.5 minutes (HPLC gradient) + 2.5 minutes (post run) = 16 minutes total

MS Conditions

Gas settingsDrying gas 11 L/min at 250 °C

Sheath gas 12 L/min at 375 °C

Nebulizer 45 psi

Source voltageCapillary voltage 4,000 V(+), 3,500 V(–)

Nozzle voltage 500 V(+), 500 V(–)

Quantitative linearity over 0.5–200 ng/L concentration range

Figure 4 illustrates highly linear calibration curves (R2 >0.99) for a quantitation range of 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 ng/L after Online SPE sample enrichment.

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Concentration (ng/L)

GemfibrozilR2 = 0.9987

10 ppt

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Concentration (ng/L)

MeprobamateR2 = 0.9974

10 ppt

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Concentration (ng/L)

PropylparabenR2 = 0.9973

10 ppt

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Concentration (ng/L)

AtrazineR2 = 0.9983

10 ppt

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Concentration (ng/L)

HydrocortisoneR2 = 0.9981

10 ppt

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Concentration (ng/L)

SimazineR2 = 0.9989

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Concentration (ng/L)

TestosteroneR2 = 0.9991

10 ppt

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Concentration (ng/L)

NorgestrelR2 = 0.9983

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Figure 4. Selected calibration curves demonstrating a high degree of sensitivity and linearity for quantitative analysis.

Chromatographic peak reproducibility over various concentration levels

Chromatographic peak overlays in Figure 5 demonstrate the robust retention time stability and peak shape of the dual cartridge Online SPE setup over a large concentration range (0.5–200 ng/L) for a total of 54 injections.

Figure 5. Chromatographic peak overlays and retention times of selected TOrCs.

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HydrocortisoneRT = 9.635 ±0.008 minutes

Acquisition time (min)

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PropylparabenRT = 10.554 ±0.007 minutes

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TestosteroneRT = 10.717 ±0.006 minutes

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NorgestrelRT = 11.272 ±0.006 minutes

Acquisition time (min)

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DEETRT = 10.558 ±0.006 minutes

Acquisition time (min)

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AtrazineRT = 10.56 ±0.01 minutes

Acquisition time (min)

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BenzophenoneRT = 11.516 ±0.007 minutes

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GemfibrozilRT = 11.916 ±0.007 minutes

Acquisition time (min)

Limits of detection

Figure 6 illustrates that, of the 31 TOrCs analyzed, 29 compounds have limits of detection that easily fall into the range of 5 ppt or less with only 750 μL of sample.

0.5–5 ng/L, 10 TOrCs

>5 ng/L, 2 TOrCs

<0.5 ng/L, 19 TOrCs

Figure 6. Distribution of the limits of detection of 31 common TOrCs found in water with 750 µL injection.

Conclusions• The Agilent 1290 Infinity Flexible Cube enables automated Online SPE sample

enrichment for trace organic contaminants.

• Enhanced throughput is achieved using a dual-cartridge setup for rapid sample turnaround.

• The Agilent Ultivo Triple Quadrupole LC/MS enables enhanced trace quantitative analysis with a minimized footprint.

• Technical innovations contained in Ultivo provide optimal sensitivity and robustness, without sacrificing the performance of larger instruments.

References1. C. G. Daughton, T. A. Terhes. Pharmaceuticals and personal care products in the

environment: agents of subtle change. Environ. Health Perspect. 107, 907-938 (1999).

2. T. Anumol, S. Snyder. Rapid analysis of trace organic compounds in water by automated online solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. Talanta 132, 77-86 (2015).

www.agilent.comInformation, descriptions and specifications in this

publication are subject to change without notice.

© Agilent Technologies, Inc. 2017 Published in USA, June 22, 2017

5991-8155ENRev. 1.0