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Analysis of Phenolic Estrogen Mimics in River Water by Negative Ion Electrospray LC/MS/MS

Z. Yang, Varian, Inc.


Bisphenol A (BPA) is a primary raw material used for the production of polyacrylates, epoxy resin, phenolic resin, and polycarbonate plastic. 4-Nonylphenol (4-NP) is a degradation product of alkylphenol ethoxylate detergents used in household and industrial cleaning products. Alkylphenol ethoxylate is also widely used as an additive in plastics that are used for food packaging and used as carriers in the application of pesticides to increase the absorption of these substances in the target organism.

BPA and 4-NP can leach from food containers and plastic tableware and can be ingested by humans through chronic exposure during eating and drinking. The chemicals have been found in drinking water supplies and wastewater.

Both BPA and 4-NP mimic the effect of natural estrogen hormones. This phenomenon raises the level of concern about the adverse health effects of these compounds on both humans and wildlife.


Varian ProStar 430 AutoSampler

Varian ProStar 210 Isocratic Solvent Delivery Modules

Varian ProStar 510 Column Oven

Varian 1200L LC/MS equipped with ESI source

Materials and Reagents

Bisphenol A (Part No. 442840) and 4-Nonylphenol (Part No. 442873) supplied by Sigma-Aldrich Corp. (St. Louis, Missouri, U.S.A.).

All other chemicals are reagent grade or HPLC grade.

Sample Preparation

Samples were provided by a water company in Japan. The concentration of 4-NP and BPA were approximately 10 to 100 ng/mL and 1 to 10 ng/mL, respectively.

The samples were extracted with dichloromethane. The extracts were dried by a N2 stream and restored in the same volume of methanol/water (1:1, v/v) prior to analyses.

Results and Discussion

The LC method used a 12 minute cycle time. The data was acquired in SRM mode of two time segments (Figure 1).

Each standard calibration curve was generated using 6 points corresponding to a concentration range of 0.5 pg/μL* to 100 pg/μL (Figure 2). The linearity of the detector response was excellent with R2 0.999 for both BPA and 4-NP.

The method can quantitatively analyze both BPA and 4-NP at a level of 500 fg/μL in solution (Figure 1, bottom chromatogram). The limit of detection (LOD) is estimated to be about < 200 fg/μL in solution. The typical SRM chromatogram of water extract is given in Figure 1 (top chromatogram).


Estrogen mimics are included in a long list of chemicals targeted by various governmental and environmental groups. Complex matrices require instrumentation with good sensitivity and selectivity. In this evaluation, the use of a Varian 1200L LC/MS/MS system demonstrates how sensitive and selective tandem mass spectrometry is in the analyses of phenolic estrogen mimics. The 1200L can quantitatively analyze BPA and 4-NP at 500 fg/μL.



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