Endocrine disruptor engaged: Bisphenol A in saliva and urine by two LC/MS methods

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  • Published: Jun 20, 2011
  • Author: Steve Down
  • Channels: HPLC
thumbnail image: Endocrine disruptor engaged: Bisphenol A in saliva and urine by two LC/MS methods


Ubiquitous bisphenol A from plastic products

Bisphenol A (BPA) is an industrial chemical that has attracted the attention of environmentalists and health professionals due to its ubiquitous presence in the environment and its detection in human biological fluids. The problem with BPA is its weak estrogenic activity which makes it act as an endocrine disruptor.

This chemical attacks the endogenous hormone system within the human body but the specific effects are still widely debated. BPA has displayed adverse effects in animal testing but there are those who argue that its effects on humans are negligible due to the low concentrations at which it is encountered. On the other hand, the long-term effects might be more serious.

The doubt over BPA safety has led the Endocrine Society to declare that "exposure to BPA, particularly in the development range, increases the risk of mammary cancer, obesity, diabetes and reproductive and neuroendocrine disorders."

BPA is used to produce polycarbonates which are used in plastic bottles, food packaging, water pipes, eyeglass lenses, and toys. It is also a plasticizer in other polymers. The compound is known to leach from the plastic into the contents, raising the risk of oral exposure.

Although there are a number of published methods for measuring BPA in human biological fluids, two research teams have each devised novel procedures, one for saliva and the other for urine.


Salivary bisphenol A

Saliva is a useful matrix because it can be sampled in a non-invasive fashion and it should provide a good indication of oral exposure. This matrix was chosen by Kevin Schug and colleagues from The University of Texas at Arlington and Benjamin Figard from Shimadzu North America, Houston, Texas, in their paper published in Environmental Toxicology and Chemistry.

Saliva was collected in glass vials and contact with plastic apparatus was avoided to prevent contamination from leached BPA. Traditionally, saliva analysis is difficult due to the complex nature of the fluid, and sample preparation can become long-winded.

The Schug solution was to incorporate bulk derivatisation of the saliva with dansyl chloride under carefully optimised conditions which minimised formation of the mono-dansyl derivative and maximised the yield of the bi-dansyl derivative. This ensured that the subsequent signal intensity of derivatised BPA was concentrated in one peak.

The derivative was extracted for analysis by LC/MS with electrospray ionisation in positive-ion mode. It was separated from the reaction mixture on a C18 column using an increasing gradient of isopropanol in acidified aqueous acetonitrile. The BPA retention time was around 23.5 minutes.

BPA was measured from the extracted ion chromatograms to give a method detection limit of 3.25 ng/mL, equivalent to 16 pg on-column.

Upon application to real-world samples, the BPA signals were below the detection limit, so the team had to resort to methods to enhance the sensitivity. Three methods were compared and the best was a combination of multiple injection using a trap column as preconcentration device, combined with the addition of deuterated BPA as an internal standard.

The increased signal response due to multiple injections had to be balanced against the subsequent band broadening but the system delivered an improved detection limit of 49.0 pg/mL, or 2.9 pg on-column.

The method was tested on saliva following the ingestion of several canned food products in a proof-of-concept study. In one case, a can of Taiwanese hot and sour soup produced high levels of BPA contamination at 2.5 ng/mL.


Urinary bisphenol A

The method for urinary BPA was developed by Haleem Issaq, Stephen Fox and Timothy Veenstra from SAIC-Frederick, Inc., and Roni Falk from the NCI, Frederick, MD and published in the Journal of Separation Science.

This procedure allowed for the determination of free BPA, bound BPA and total BPA, by incorporating an enzymatic hydrolysis step. Urine was spiked with deuterated BPA then processed with and without reaction with beta-glucuronidase to measure total and free BPA, respectively.

The key step was extraction of endogenous BPA with toluene which, along with the use of precleaned glassware, minimised the contribution of exogenous BPA. The extracted analyte was once again converted to the bis-dansyl derivative for analysis by HPLC-tandem MS.

Separation was carried out on a C8 column with an increasing gradient of acetonitrile in aqueous formic acid, to give a total run time of 10 minutes. A triple quadrupole mass spectrometer in positive-ion electrospray ionisation mode was used for BPA quantitation, following the transition from the singly protonated molecule to a product ion at m/z 170.

This led to quantification and detection limits in urine of 50 and 5 pg/mL, respectively.

In studies on the urine from 14 individuals, BPA was found over a 100-fold range, from 0.05-5 ng/mL, all of it present as the glucuronide conjugate. This was expected because BPA is known to metabolise quickly.

Both of these LC/MS methods show improvements on existing methodology, with detection limits sufficiently low as to be applicable to real-world samples of saliva and urine. They will be suitable for use in health screening programs.



The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 

 The endocrine disruptor bisphenol A has been determined in human saliva and urine by two contrasting methods, both employing liquid chromatography-mass spectrometry following derivatisation with dansyl chloride. 

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