Liquid–liquid microextraction leads to GC satisfaction

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  • Published: Jan 16, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Liquid–liquid microextraction leads to GC satisfaction

Improved methods needed for plasma phthalate assays

Phthalate esters are used as plasticisers in a wide range of products, such as plastic items, cosmetics and paints. These compounds are believed to be endocrine disruptors, affecting the hormone system and possibly giving rise to harmful effects on reproduction and on the development of babies and children; however, the exact effects of phthalates remain a subject of controversy. The EU has introduced restrictions on the use of phthalates in childcare items and toys, while further general restrictions are also being considered.

Accurate methods are needed to measure the low levels of phthalates typically present in human plasma. Traditional liquid–liquid extraction methods and HPLC lead to the use of high volumes of solvents when large numbers of samples are examined, so methods that minimise solvent consumption are needed.

The Isfahan researchers used dispersive liquid–liquid microextraction (DLLME) to extract four phthalates, diethyl phthalate, di-n-butyl phthalate, benzyl butyl phthalate and di-2-ethylhexyl phthalate, from human plasma. This relatively new extraction method involves adding two solvents to an aqueous solution: a dense extraction solvent, which is usually a chlorinated solvent or a dense ionic liquid, and a water-miscible disperser solvent, such as acetonitrile. The resulting cloudy mixture is then centrifuged to give a lower layer containing the analytes.

DLLME and GC-MS used to measure phthalates in plasma

Phthalate-free plasma was spiked with phthalates for calibration purposes. Protein was then precipitated by vortexing with trichloroacetic acid for 30 seconds and standing for 30 minutes at ca. 4 °C. Trichloroacetic acid was shown to be superior to methanol, ethanol and acetonitrile for this step. The precipitated protein was then removed by centrifugation.

DLLME was carried out on 5-mL portions of the treated plasma by adding 10 μL of chlorobenzene (extraction solvent) and 750 μL of acetonitrile (disperser solvent) and then centrifuging for 5 minutes at 4500 rpm. Chlorobenzene was shown to be superior to other chlorinated solvents, while acetonitrile performed better than methanol or acetone. The volumes, temperature and centrifugation time were also carefully optimised. It was found there was no significant difference between centrifugation at 10, 20 or 30 °C. Times longer than 5 minutes did not improve recovery and there was no beneficial effect from adding salt.

GC employed an Agilent 7890A instrument fitted with a DB-5MS column. The oven temperature was held at 100 °C for 5 min, ramped at 20 °C/min to 300 °C and kept at the latter temperature for 5 min. Helium was used as the carrier gas with a flow rate of 1 ml/min. An Agilent 5975C inert mass selective detector (MSD) was used with electron ionisation (EI) and the identities of the peaks were confirmed by comparison of their mass spectra with those from spectral libraries. For the quantitative analysis, in order to increase sensitivity, selected ion monitoring (SIM) was used, looking at the typical m/z 149 phthalate peak for all four analytes.

The method gave a wide linear range of 50–1000 ng/mL for all four phthalates and recoveries ranging from 91 to 97%. The limits of detection (LODs) ranged from 1.06 to 1.60 ng/mL, while the limits of quantification (LOQ) were 3.5 to 5.3 ng/mL. Replicates gave reasonable repeatability, with a ‘between day’ relative standard deviation (RSD) ranging from 4.7 to 6.1%, depending on the phthalate.

New microextraction and GC-MS method gives fast phthalate analysis

The combination of DLLME and GC-MS allows for fast analysis of plasma phthalates, using minimal amounts of organic solvent, thus making the technique suitable for monitoring multiple samples. The authors carefully optimised the extraction conditions, giving a quick method that can detect low levels of phthalates. It is also possible that the method could be adapted to other trace pollutants.

Related Links

Journal of Separation Science, 2017, 40, 4403-4410. Ebrahim et al. Development of a simple and valid method for the trace determination of phthalate esters in human plasma using dispersive liquid–liquid microextraction coupled with gas chromatography–mass spectrometry.

Applied Spectroscopy Reviews, 2017, 52, 267-415. Campillo et al. Ten years of dispersive liquid–liquid microextraction and derived techniques.

Wikipedia, Selected Ion Monitoring

Article by Ryan De Vooght-Johnson

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

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