Urine Biomarkers for Toluene, Xylene and Styrene Exposure

Authors: Pierre Picard, Jean Lacoursière and Serge Auger
Themes: High-Throughput, Urine, Biomarkers, Toluene, Xylene, Styrene, LDTD-MS/MS
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In various industrial production plants, workers can be exposed to organic compounds like toluene, xylene and styrene. The chronic exposure to these compounds has been linked to severe impacts on the central nervous system. The measurement of their major metabolites in urine would provide an important indication of the overall level of occupational exposure to these widely used compounds. Toluene, xylene and styrene are metabolized and excreted in the urine as hippuric acid (HA), methylhippuric acids (MHA) and mandelic acid (MA), respectively.

Our goal for this application note is to use an automated sample preparation method for the simultaneous quantification of creatinine, hippuric acid, methylhippuric acid and mandelic acid in urine using a single operation in LDTD-MS/MS for workers that may be victims of toluene, xylene and styrene exposure.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a quick and simple sample preparation. Metabolites are analyzed simultaneously with quantitative screening results obtained in less than 8 seconds per sample. Each metabolite has been screened based on the WHO (World Health Organization) detection range.


Sample Preparation Method


Sample Collection

Urine samples were collected and transferred into barcoded tubes, readable by the Azeo extraction system.


Automated Sample Extraction

Each barcoded vial was scanned by the Azeo Liquid Handler and an automatic batch file was created. The Azeo extraction system (Figure 3) is used to extract the samples using the following conditions:


Figure 3 – Automated extraction system



LDTD®-MS/MS Parameters


Model: Luxon S-960, Phytronix

Carrier gas: 6 L/min (air)

Laser pattern:


MS model: Q-Trap System® 5500, Sciex

Scan Time: 25 msec

Total run time: 8 seconds per sample

Ionization: APCI

Analysis Method: Negative MRM mode



Table 1 – MRM transitions for LDTD-MS/MS

  Transition CE
Creatinine 112 → 68 -32
Creatinine-d3  115 → 68 -32
Mandelic acid 151 → 107 -15
Hippuric acid  178 → 134 -25
Hippuric acid-d5  183 → 139 -25
Methylhippuric acid  192 → 148 -25


Results and Discussion


Screening range (µg/mL)

The screening range for each metabolite can be found in Table 2.



Table 2 – Metabolites Calibration Ranges

Analyte (µg/mL)
Creatinine 200 – 4000
Mandelic acid (MA) 100 – 2000
Hippuric acid (HA) 100 – 2000
Methylhippuric acid (MHA) 100 – 2000



Validation Test

Calibration curves were prepared in a methanol:water (3:7) solution in which the calibration curve samples were diluted. Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.



The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:


Mandelic Acid


Hippuric Acid


Methylhippuric Acid



Figure 4 – Calibration curves



Figure 4 shows the calibration curve for MA (A), HA (B), MHA (C) and creatinine (D).


Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:


For the inter-run precision and accuracy experiment, each standard was analyzed in triplicate, on three different days. Table 3 shows the inter-run precision and accuracy results for Hippuric acid. The obtained %CV was below 15% and the accuracy was within 15% of nominal value. Similar results were obtained for the other metabolites.

Table 3 – Inter-Run Precision and Accuracy of Hippuric Acid

Hippuric acid S1 S2 S3 S4 S5 S6 S7
Conc (µg/mL) 100 200 400 800 1200 1600 2000
N 9 9 9 9 9 9 9
Mean (µg/mL) 107 190 397 792 1169 1613 2033
SD 8,2 9,4 32,8 74,8 11,1 53,9 69
%CV 7,7 4,9 8,3 9,4 1,0 3,3 3,4
%Nom 106,9 95,2 99,1 99,0 97,4 100,8 101,6


Multi-Matrix Evaluation

Urine samples were collected from six volunteers. Samples were screened to verify the endogenic value of each metabolite. To study the matrix effect, urine samples were spiked with metabolites at final concentration addition of 667 µg/mL. Table 4 shows the screening results of all metabolites. An addition of 667 µg/mL was obtained for each matrix within ± 15% of nominal value.

Table 4 – Multi-Matrix Evaluation Results

Sample MA
U1 179 942 220 962 21 776 1657
U2 137 872 75 746 75 699 2129
U3 89 802 96 755 26 599 1413
U4 116 857 418 1101 22 642 772
U5 179 928 114 802 28 622 1240
U6 38 755 152 780 23 603 647



The Luxon Ion Source combined with Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) analysis of the urine metabolites of toluene, xylene and styrene. This method allows the quantification of urine diluted biomarkers and creatinine, simultaneously.