В изследователските лаборатории
CALIBRATION OF GC/MS METHOD FOR DETERMINATION OF PHTHALATES
Резюме. The calibration of method for determination of phthalates by means of GC/MS was done. The obtained working range for different phthalates is as following: DBP 58.8 – 1175,3 ppb; BBP 75,2 – 1504,6 ppb; DEHP 72,7 – 1454,2 ppb; DOP 59,1 – 1182,2 ppb; DINP 484,5 – 9690,3 ppb; DIDP 573,9 – 11477,0 ppb. Dicyclohexylphtalate (DICHP) was selected as an internal standard. Hexane is used as a solvent for preparation of standard solutions of phthalates.
Ключови думи: phthalates; GC/MS; LOD; LOQ; calibration
30 Years Chemistry Education in South-West University 19 – 21 October 2017, Blagoevgrad
30 години химическо образование в Югозападния университет 19 – 21 октомври 2017
Introduction
Phthalates are dialkyl- or diaryl- esters of 1,2-benzene dicarboxylic acids, also known in a practice as o-phthalic acid. The general method for their synthesis is reaction between phthalic anhydride and alcohol like methanol, ethanol until tridecyl alcohol. They are colorless liquids without taste and any smell. They also are water insoluble, but very well soluble in fats. The main application of phthalates is in polymers production, where they play role of plasticizers (Lao & Wong, 2000). Phthalates could be introduced in human organism by consuming different foods stored in polymeric packages as well as using of cosmetics, toys, building materials, etc (Pivnenko et al., 2016). They affect the reproductive system, so they leads to male and female sterility, polycystic ovaries, congenital defects of the fetus in pregnant women (Gao et al., 2017). Phthalates are one of the causes of asthma in children. This class of compounds affects the endocrine system and is the cause of obesity. Because of their toxicity, phthalates are the subject of demand and determination in polymeric goods, including polymer packaging for food products, which are now widely used in practice (Guo et al., 2010). Herein, we report the calibration as a part of introduction in laboratory practice of a GC/ MS standardized method for determining the content of phthalates according to the international standards and in accordance with the conditions in the selected laboratory. \({ }^{1,2)}\)
Experimental
Materials, methods and apparatus
Dibutyl phthalates (DBP), Benzyl butyl phthalates (BBP), Bis-(2-ethylhexyl) phthalate (DEHP), Di n-octyl phthalates (DOP), Di iso-nonyl phthalate (DINP), Di iso-decyl phthalate (DIDP), Dicyclohexyl phthalate (DICHP) and n-hexane with analytical grade were purchased by Sigma Aldrich, Germany.
Centrifuge tubes of 15 mL – ISO LAB, Glass tubes of 10mL – Valerus Ltd, Bulgaria, Syringe filters PTFE \(0.45 \mu \mathrm{~m}\), Vortex – Fisher Scientific, Ultrasonic bath – Apronecs Ltd, Bulgaria, Gas chromatograph Shimadzu QP 2010 Ultra.
Procedure for GS/MS determination
Before performing the calibration, the gas chromatograph shall be adjusted according to the manufacturer‘s instructions and the operating conditions specified in the measurement method.
First, a blank sample of pure solvent is injected to verify the system for the way it works and for the presence of contamination. A solution containing the individual components in the SCAN mode of the mass spectrometer is then injected and the chromatogram further used for their identification is recorded. The identification is performed by two parameters – retention time \(\mathrm{t}_{\mathrm{R}}(\mathrm{min})\) and the resulting mass spectrum, which is compared to a reference, located in a library of the apparatus.
From the obtained data, а SIM (select ion monitoring) method, which scans certain ions characteristic of the respective component is performed. This mode is used because of the higher sensitivity of the detector compared to the SCAN mode due to the scanning of a smaller number of ions, which reduces side effects and other types of interferences.
Chromatographic conditions
Column SLB-5MS \(29.5 \mathrm{~m} / 0.25 \mathrm{~mm} / 0.25 \mu \mathrm{~m}\), Carrier gas-helium, Column oven temperature \(-150.0^{\circ} \mathrm{C}\), Injection temperature \(-200.0^{\circ} \mathrm{C}\), Total flow – 40.0 \(\mathrm{ml} / \mathrm{min}\), Column flow \(-2 \mathrm{ml} / \mathrm{min}\), Injection mode – Split with split ratio \(-(-1.0)\), Ion source temperature \(-200.0^{\circ} \mathrm{C}\)
Oven program is described in Table 1. GC/MS works in SIM mode according to the regime presented in Table 2.
Table 1. Oven program
Table 2. Working mode of GC/MS
Initial and working standards
The initial DICHP concentration of 10.9 mg to 20 mL in n-hexane was prepared and further it was diluted 100-fold.
The standards used are in ppb concentrations presented in Table 3.
Table 3. Concentration in ppb of used standard solutions of determined phthalates
1 mL of all prepared standards are mixed with \(50 \mu \mathrm{~L}\) internal standard of DICHP and they are further injected to the GS/MS apparatus.
Results and discussion
The characteristic ions for monitored phthalates were determined at the beginning of analysis using \(\mathrm{t}_{\mathrm{R}}\) of chromatogram as well as their characteristic ions (Fig. 1). They all are presented in Table 4.
Figure 1. Chromatogram of target phthalates. Compounds 6 and 7 are mixture of isomers. They are manually integrated and the zone of their presents is determined by means of characteristic peaks of all isomers
Table 4. tR and m/z ration of characteristic target ions for monitored phthalates
Each of the prepared calibration solutions is injected 10 times in the SIM mode of the mass spectrometer. A blank sample of pure solvent is injected between different concentration levels in order to confirm the absence of system contamination. Each of the components is identified by both parameters retention times \(\mathrm{t}_{\mathrm{R}}\) and \(\mathrm{m} / \mathrm{z} /\) of characteristic ions (Table 2). The obtained data are used for standard curve drawing. It shows the relations of the average of the ratio between areas of each phthalate and the internal standard as a function of the respective concentrations of phtalates. As a results of obtained data, standard curves, presented on Figs. 2 – 7, are drawn.
Figure 2. Standard curve for dibutyl phthalates
Figure 3. Standard curve for benzyl butyl phthalates
Figure 4. Standard curve for bis-(2-ethylhexyl) phthalate
All obtained data show liner response of detector for the chosen concentration interval of phthalates. A measure of linearity is the correlation coefficient “R”. As its square is closer to one, the higher the degree of linearity. The correlation coefficients for aim phthalates are summarized in Table 5.
Table 5. Correlation coefficients R2 of target phthalates
Figure 5. Standard curve for di n-octyl phthalates
Figure 6. Standard curve for di iso-nonyl phthalate
Limit of quantification (LOQ)
By definition the limit of quantification (LOQ) is the smallest amount of the determinable component, which can be calculated with a certain probability [7]. If a technical instrument with a defined calibration band is used, the LOQ coincides with the first point in that range. For the specific case the values are presented in Table 3.
Figure 7. Standard curve for di iso-decyl phthalate
Moreover, LOQ can be calculated by means of Eq. (1):
(1) \[ \mathrm{LOQ}=10 \mathrm{~S}_{0} \]
where, \(\mathrm{S}_{0}\) is a standard deviation SD obtained after a test blank sample with a dimension equal to that of the measured quantity. By means of mathematical equation presented above the \(\mathrm{S}_{0}\) values can be easily calculated and for the target compounds they are summarized in Table 3.
Limit of detection (LOD)
The detection limit (LOD) represents the smallest amount of the detectable component that can be detected with a certain probability. \({ }^{3)}\) It can be calculated by means of Eq. (2).
(2) \[ \mathrm{LOD}=3 \mathrm{~S}_{0} \]
The calculated values for LOD of target phthalates is presented in Table 3.
Conclusion
The calibration of GC/MS system Shimadzu QP 2010 Ultra was successfully done for five phthalates - Dibutyl phthalates (DBP), Benzyl butyl phthalates (BBP), Bis-(2-ethylhexyl) phthalate (DEHP), Di n-octyl phthalates (DOP), Di iso-nonyl phthalate (DINP), Di iso-decyl phthalate (DIDP) using Dicyclohexyl phthalate (DICHP) as an internal standard. The linear range for further work, LOQ and LOD values were also determined succesfully.
Acknowledgments. This work is realized by the financial support of project 11688 with Science investigation center of UCTM. Authors would like also to thank to TC Globaltest for the presented opportunity to use GC/MS system.
NOTES
1. http://www.oiv.int/public/medias/2693/oiv-ma-bs-33-en.pdf
2. http://www.oiv.int/public/medias/2589/oiv-ma-as323-10.pdf
3. http://www.bds-bg.org/bg/bg/standard/?natstandard_document_id=46634
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