Analysis of Four Phthalate Monoesters in Human Urine Using Liquid Chromatography–Tandem Mass Spectrometry


A method was developed using high performance liquid chromatography with tandem mass spectrometry detection for the analysis of phthalate metabolites in urine samples. The urine samples were treated using solid-phase extraction columns. Multiple reaction monitoring models were used for quantitative detection with high sensitivity and selectivity. In this work, a fast, sensitive, and accurate quantitative method is provided to detect toxic phthalate esters in urine and assess environmental toxicants.

Phthalate esters are widely used as plasticizers in manufacturing. Phthalate esters are also used as raw materials of pesticide carriers, paint, adhesives, cosmetics, lubricants, and so on (1). Because phthalate esters are noncovalently bound to the plastic and maintain relatively independent chemical properties, they can leach out of these products and regularly release into the environment as time goes on (2,3). Globally, more than six million tons of phthalates are used each year (4). Presently, phthalate esters have been detected in many different areas in the ecological environment of the world's major industrial countries; they have become one of the most popular global pollutants. This has severely threatened the environment and human health, despite the fact that phthalate esters play an important role in the production and life of mankind (5).


Figure 1: General metabolic pathways of phthalates in humans. R = H; phthalic acid = PA, C2H5; monoethyl phthalate = MEP, (CH2)3CH3; monobutyl phthalate = MBP, CH2C6H5; monobenzyl phthalate = MBzP, CH2CH (C2H5) (CH2)3CH3; mono (2-ethylhexyl) phthalate = MEHP.
Diet, skin, and inhalation are considered the major exposure routes for phthalate esters (6). When phthalate esters enter the human body they are rapidly metabolized and hydrolyzed to the corresponding phthalate monoesters before absorption, which are the biologically active molecules and recognized testicular toxicant (7,8), and then further oxidized into various metabolites (9). General metabolic pathways of phthalates in humans are shown in Figure 1 (10). Most phthalate esters and their metabolites are excreted in urine or feces. The total concentrations of phthalate metabolites in urine are normally used as biomarkers of individual intake of phthalate esters (11). The monoesters contain a free reactive carboxylic acid that can conjugate with α-D-glucuronic acid to produce more hydrophilic compounds. Phthalate esters, which mimic estrogen, have been reported to affect multiple biochemical processes as environmental hormones in humans and wildlife. It is one of the major reasons for male reproductive problems (12–14). Excessive exposure to phthalate esters can increase the risk of breast cancer for women. Many phthalate esters can damage the reproductive systems of offspring as well (15–18). For instance, studies showed that some toxicologically active metabolites, such as MEHP, MBPA, and MBzP, are capable of crossing rodent placenta, which causes adverse effects on the development of the male reproductive system and induces early embryonic death, respectively (19,20). Animal experiments have shown that the toxicities of some monoesters are greater than those of corresponding diesters (21).

Phthalate monoesters are considered to be valuable biomarkers for exposure to phthalate esters. Measuring phthalate monoesters investigates the contamination status, absorption, and metabolism of phthalate esters in the body better than phthalate diesters. Several developed methods have been used to quantify phthalate monoesters, including gas chromatography–mass spectrometry (GC–MS) (22,23). Calafat developed a sensitive method for measuring 13 phthalate metabolites in breast milk using isotope dilution high performance liquid chromatography (HPLC) with negative ion electrospray ionization tandem mass spectrometry (24). This method showed good reproducibility and accuracy. Liquid chromatography–tandem mass spectrometry (LC–MS-MS) methods with high selectivity and sensitivity show an advantage compared to GC–MS methods because of simple sample pretreatment without derivatization.

Some methods, such as on-line solid-phase extraction (SPE) coupled to HPLC–MS-MS, are considered the most advanced method to investigate phthalate esters and their metabolites, but the high cost limited their widespread application. To develop a sensitive, selective, low cost, and efficient analytical method to quantify these chemicals appears essential for understanding how human health might be affected by exposure to phthalates under general laboratory conditions. In this article, we report a method for the quantitative detection of four phthalate monoesters including MEP, MBP, MBzP, and MEHP simultaneously in human urine samples. The method involves the enzymatic dissociation of conjugated monoesters, SPE, and separation and detection using HPLC with negative ion electrospray ionization tandem mass spectrometry. The method was used to analyze human urine samples collected in the same area for evaluating potential phthalate ester exposure.