# Phthalates Exposure and Occupational Symptoms among Slovakian Hairdressing Apprentices
## 2 Materials and Methods
### Phthalate Analyses  
Hairdressing apprentices provided first spot urine samples (2 \(\times\) 2 mL) at the beginning of work, not earlier than 6:00 am, at the end of the workweek (Friday, work duration at least 8 h per shift), which gives information about individual exposures during last 24 h. Samples were stored in a transport box at 2-6 \({}^{\circ}\)C and then in the deep freezer at -73 \({}^{\circ}\)C until analysis. We used high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) (Infinity 1260 and 6410 triplequad, Agilent, Santa Clara, CA, USA) to quantified urinary concentration of compounds: MEP, MnBP, MEHP, MEHHP, MEOHP by the method of [PERSON] et al. [42]. The method was certificated for occupational/environmental medical--toxicological analysis for the Environmental medical field by EQUAS in German External Quality Assessment Scheme.  
The analytical standards were purchased from Cambridge isotope laboratories (Tewksbury, MA, USA). Briefly, 1 ml of urine was thawed, buffered with ammonium acetate, spiked with isotope-labeled phthalate standards, \(\beta\)-glucuronidase enzyme (K12 E. [PERSON], Roche, Mannheim, Germany) and incubated during 60 min at 37 \({}^{\circ}\)C. After deconjugation, samples were diluted with phosphate buffer (NaH\({}_{2}\)PO\({}_{4}\) in H\({}_{3}\)PO\({}_{4}\)) and loaded on SPE cartridges (ABS Elut Nexus, Agilent, Santa Clara, CA, USA). Cartridges were conditioned with acetonitrile followed by phosphate buffer before extraction. To remove the hydrophilic compound, SPE cartridges were flushed by formic acid (Merck KGaA, Darmstadt, Germany) and HPLC grade water. Elution of analytes was performed by acetonitrile (Merck KGaA, Darmstadt, Germany) and ethylacetate (Sigma-Aldrich, Steinheim, Germany). Eluate was dried by nitrogen gas and reconstituted with 200\(\mu\)L of H\({}_{2}\)O and acetonitrile (1:1). For HPLC, an Agilent Infinity 1260 liquid chromatography equipped with ZORBAX Eclipse phenyl-hexyl column (2.1 \(\times\) 150 mm, 3 \(\mu\)m) was used. The separation was done using a nonlinear gradient program of mobile phases C\({}_{2}\)H\({}_{3}\)N and 0.1% acetic acid (Fisher Scientific, Loughborough, UK) in H\({}_{2}\)O. For mass specific detection of phthalate metabolites we use triplequad with electro-spray ionization (Agilent 6410), according instrumental settings as follows: Spray ion voltage (-3800 V), nitrogen nebulizer gas pressure (8 psi), and nitrogen curtain gas pressure (7 psi), capillary temperature (430 \({}^{\circ}\)C), and collision gas (nitrogen) pressure (1.5 mTor). Chromatographic and mass spectrometric parameters are described in Table 1. The sum of the di(2-ethylhexyl) phthalate (DEHP) metabolites (\(\Sigma\)MEHP) was calculated by adding the corresponding concentrations of MEHP, MEHHP, and MEOHP.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Compound Name**} & \multicolumn{2}{c}{**Precursor**} & \multicolumn{2}{c}{**Product**} & \multirow{2}{*}{**F (V)**} & \multirow{2}{*}{**CE (V)**} & **RT** & **LOD** \\  & **Ion (m/z)** & & & & **(min)** & **(ng/mL)** \\ \hline MEP & 193 & 77.1 & 60 & 15 & 6.7 & 4.41 \\ MEP—labeled & 197.1 & 79 & 60 & 15 & 6.7 & \\ MnBP & 221.1 & 76.9 & 90 & 10 & 9.8 & 1.1 \\ MBP—labeled & 225.1 & 78.8 & 90 & 10 & 9.8 & \\ MEHP & 277.1 & 133.9 & 90 & 14 & 14.7 & 0.81 \\ MEHP—labeled & 281.1 & 137.1 & 90 & 14 & 14.7 & \\ MEHHP & 291 & 121.2 & 90 & 10 & 9.7 & 0.54 \\ MEHHP—labeled & 294.9 & 124 & 90 & 10 & 9.7 & \\ MEOHP & 293.1 & 121.2 & 95 & 12 & 9.7 & 0.64 \\ MEOHP—labeled & 297 & 124.1 & 95 & 12 & 9.7 & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Phthalate monoesters: Chromatographic and mass spectrometric parameters.