Introduction
Phthalates are a class of chemicals – phthalate acid esters or PAEs – that are widely used as plasticizers in polymer industry. Plasticizers are not chemically bound to their native polymer and can therefore leach into food from packaging materials in significant amounts. The most important congener is di-(2-ethylhexyl)-phthalate (DEHP), which accounts for about 50% of the world production of phthalates.
Due to the fact that some Phthalates imitate estrogen structure they are now classified as endocrine disruptors having adverse health effects on humans, especially children. Besides, it has been recently discovered that DEHP was intentionally added as a clouding agent to sports drinks, fruit juice, other beverages and food products.
Sensitive and robust methods for the analysis of phthalates in food are needed to protect the consumers from food adulteration and phthalate migration from food packaging materials. As an example, China and Taiwan have set limits for phthalates in food products at 1 part per million (ppm), corresponding to 1000 μg/kg.
Gas Chromatography with Mass-Spectrometric detection is a method of choice for detection and quantitation of phthalates’ residues in food and beverages. The analytes of interest from a sample mix are separated by a gas chromatograph and then selectively detected as individual compounds and quantitated even at their low concentration levels in the sample.
In order to reach good separation of the phthalate peaks on chromatogram it is recommended to use a capillary column at least 30m long, otherwise structural similarity of individual phthalates may impede their mass-spectral identification as well as quantitation. In particular, high intensity ion of m/z=149 amu is present in mass-spectrum of most phthalates, thus, making it difficult for each individual compound to be reliably identified and quantitated unless completely separated chromatographically.
This goal of this work is to demonstrate capability of Q-Tek single quadrupole GC-MS to reliably detect the phthalate residues that have leached into pear juice from various types of package during the storage.
Experimental
Standards and Reagents
All reagents used in this work were HPLC grade. Phthalates standard was commercially obtained from Restek (USA) as Method 8061A Phthalate Esters Mixture at 1,000 μg/mL each in hexane : acetone (80:20) (Restek, Cat.# 33227).
The samples of bottled pear juice in various container types were obtained from a supermarket and prepared as per GB/T 21911-2008 method [4].
Table. 1. GC-MSD method parameters.
| Q-Tek GC-MS system configuration | |
| inlet | Split/Splitless (liner: Topaz 4.0 mm ID Straight Inlet Liner) |
| GC column | Rtx-5MS (30m x 0.25mm; 250um) |
| Method parameters | |
| Injected Sample volume | 1 uL |
| Inlet mode | Splitless, Purge Time = 0.7 min, Purge Flow = 50ml/min |
| Inlet temperature | 250°C |
| Carrier gas flow mode | Constant flow |
| Oven program | 60°C (1,0 min); 20°C/min up to 220°C (1,0 min);
5,0°C/min up to 280°C (4,0 min) |
| Carrier gas flow and type | (He) 1,0 mL/min |
| GC-MS transfer line temperature | 280°C |
| Detector settings | |
| Ion source | Inert, Electron impact (EI) |
| Ion source temperature | 240°C |
| Solvent delay | 250 sec |
| Data Acquisition mode | SIM (see below) created automatically by SIM Wizard® |
| Dwell Time | Automatically calculated by iDwell® Time to ensure 10 spectra/sec for all groups, except # 11 (8 spectra/sec (see fig. )) |
Q-Tek proprietary data processing algorithm SIM Wizard ® was used to automatically build SIM method based on full SCAN data from pure standard mix. The SIM-library was, then, used to facilitate choice of most informative and optimal ions to be monitored during SIM acquisition. To maximize the system sensitivity iDwell®Time algorithm was engaged. iDwell®Time algorithm automatically groups the target compounds into time segments so, that dwell time on each of the monitored ion mass is as long as needed. The both proprietary algorithms described above are nice productivity tools that finally helped to quickly optimize the data acquisition parameters and to obtain high intensity signal, ideal peak shape and reach statistics on all target masses including those for the analytes occurring at their trace concentrations in the sample.
Sample preparation
The samples of bottled pear juice from a local supermarket were prepared, as per GB/T 21911-2008 standard method [4]
The target analytes were extracted with hexane. The 5 mL volume of juice was transferred to a 10 mL glass flask then 2 mL of hexane was added. The flask was then Vortexed for 1 minute and centrifuged for 3 minutes at 2000 rpm. The mixture was hold for 10 min to settle down and separate, then 1 mL aliquote of supernatant hexane layer was sampled and transferred to an autosampler vial for the following GC-MS analysis in SIM mode.
Fig. 2 Pear juice phthalates extract prior to GC-MS analysis.
The list of ions to be monitored for each target analyte during GC-MS analysis is presented in Table 2 below.
Table. 2. Target analytes’ Retention Times and respective mass-spectrometric detection parameters
| № | Соединение | RT, min | Quantifier Ion | Qualifier Ion 1 | Qualifier Ion 2 |
| 1 | Dimethyl phthalate (DMP) | 7.71 | 133.1 | 77.1 | 163 |
| 2 | Diethyl phthalate (DEP) | 8.57 | 150.1 | 177.1 | 149.1 |
| 3 | Di-iso-butyl phthalate (DIBP) | 10.25 | 150.1 | 149.1 | 223.2 |
| 4 | Di-butyl phthalate (DBP) | 10.97 | 150.1 | 149.1 | 223.2 |
| 5 | bis(2-methoxy ethyl) phthalate (DMEP) | 11.34 | 149.1 | 59 | 76.1 |
| 6 | bis(4-methyl-2-pentyl) phthalate (BMPP) | 12.01 | 167.1 | 149.1 | 251.2 |
| 7 | bis(2-ethoxy ethyl) phthalate (DEEP) | 12.37 | 72.1 | 149.1 | 176.1 |
| 8 | bis(n-pentyl) phthalate (DPP) | 12.72 | 149.1 | 237.1 | 150.1 |
| 9 | Dihexyl phthalate (DHXP) | 14.83 | 149.1 | 251.1 | 150.1 |
| 10 | Benzyl Butyl phthalate (BBP) | 15.015 | 149.1 | 91.1 | 206.1 |
| 11 | bis(2-n-butoxyethyl) phthalate (DBEP) | 16.4 | 101.2 | 149.1 | 176.1 |
| 12 | Dicyclohexyl phthalate (DCHP) | 17.12 | 149.1 | 167.1 | 150.1 |
| 13 | bis(2-ethylhexyl) phthalate (DEHP) | 17.27 | 279.1 | 167.1 | 149.1 |
| 14 | Diphenyl isophthalate | 17.482 | 225.1 | 77.1 | 153.1 |
| 15 | Di-n-octyl phthalate (DNP) | 19.65 | 279.1 | 150.1 | 149.1 |
Results and discussion
Linearity
Phthalates were quantitated using absolute calibration method. 7-level calibration was done for GC-MS instrument having points at 10 ppb – 20 ppb – 50 ppb – 100 ppb – 200 ppb –500 ppb – 1000 ppb for building respective calibration curves. Linear regression model was used for all the calibration curves running through zero in order to calculate concentrations if a peak area gets below first calibration point. For injection of low concentration calibration standards (10; 20; 50 ppb) 3 repeats were made, while the rest of calibration standards were injected with 2 repeats. Thus, altogether 17 calibration points were processed. All the target compound calibrations were linear having R2 ≥ 0.98 – 0.99.
The fig.4 shows calibration curves for some compounds from mix. 
Fig. 3. Extracted Ion Chromatogram of phthalate mix at 1000 ppb.
1 – DMP; 2– DEP; 3 – DIBP; 4 – DBP; 5 – DMEP; 6 – BMPP; 7 – DEEP; 8 – DPP; 9 – DHXP; 10 – BBP; 11 – DBEP; 12 – DCHP;
13 – DEHP; 14 – Diphenyl isophthalate; 15 – DNP.
Fig. 4. Linear calibration curves for some phthalates from the calibration mix.
| Dimethyl phthalate (DMP) | Diethyl phthalate (DEP) |
 |
|
| i–butyl phthalate (DBP) | bis(2-methoxy ethyl) phthalate (DMEP) |
 |
|
| bis(4-methyl-2-pentyl) phthalate (BMPP) | bis(2-ethoxy ethyl) phthalate (DEEP) |
 |
|
| Dihexyl phthalate (DHXP) | Benzyl Butyl phthalate (BBP) |
 |
Result discussion
Experimental data demonstrate that following standards sample preparation method the target phthalates are successfully extracted even at their trace concentration level in pear juice samples. The GC-MSD system is capable to reliably detect them all following the standard ssample preparation method specified in GB/T 21911-2008. Linearity of the calibrations permit to quantitate the target analytes in wide range of their concentration in real samples.
Table 3 shows phthalates concentrations found in pear juice samples bottled and stored in containers made of different types of packing material. The highest phthalates level was unexpectedly found in a pear juice stored in glass bottle. One might assume the elevated level of phthalates here can be attributed to the juice processing prior to the bottling into glass vessel. Another potential source of phthalates could presumably be the bottle cap plastics containing some phthalates that could have been leaching into the juice during its storage.
Table. 3. Concentration of phthalates extracted from pear juice samples, pg/uL (ppb).
| Compound | ||||
| glass | plastic | cartoon | foil | |
| DMP | 13.67 | 0.40 | 10.63 | 0.00 |
| DEP | 1.46 | 0.53 | 0.73 | 0.55 |
| DIBP | 1.44 | 2.92 | 0.53 | 0.56 |
| DBP | 4.26 | 1.05 | 0.63 | 0.36 |
| DMEP | 0.00 | 0.00 | 0.00 | 0.00 |
| BMPP | 0.00 | 0.00 | 0.00 | 0.00 |
| DEEP | 0.00 | 0.00 | 0.00 | 0.00 |
| DPP | 1.38 | 0.23 | 0.33 | 0.00 |
| DHXP | 0.43 | 0.00 | 0.00 | 0.00 |
| BBP | 0.00 | 0.00 | 0.00 | 0.00 |
| DBEP | 0.00 | 0.00 | 0.00 | 0.00 |
| DCHP | 0.00 | 0.00 | 0.00 | 0.00 |
| DEHP | 7.56 | 1.83 | 1.60 | 1.27 |
| Diphenyl isophthalate | 0.00 | 0.00 | 0.00 | 0.00 |
| DNP | 0.00 | 0.00 | 0.00 | 0.00 |
Conclusion
The experimental data have proven the Q-Tek single quadrupole GC-MS to be the instrument of choice for phthalates detection and quantitation in juice as per standard GB/T 21911-2008 method. The GC-MS delivers reliable results for both qualitative and quantitative analyses and features nice productivity tools to help an operator to set up the system and the acquisition method for desired results much faster. The use of GC-MS in SIM mode improves the selectivity of the analysis and greatly reduces sample preparation time required to reach expected level of system sensitivity and precision.
References
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- GB/T 21911-2008 Determination of phthalate esters in foods.
