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Written by Luca Rota, Application Specialist | May 9, 2024
Innovative method of microwave assisted extraction of MOSH and MOAH in food
INTRODUCTION
In recent years, the assessment and quantification of the presence of mineral oil hydrocarbons, both aliphatic (MOSH) and aromatic (MOAH), has become a growing requirement for food quality control laboratories around the world.
While MOAH, a class of compounds similar but not identical in structure to PAHs, are established carcinogens and therefore should not be present in food, MOSH are a broad class of hydrocarbons, present in both linear and branched forms, whose toxicology is not yet fully understood and therefore no definitive regulatory limits have been established.
Since the first analytical method developed in 2009 by Biedermann and Grobb, many efforts have been made to address the occurrence of contamination in different matrices, in particular by the European Commission, and to develop analytical methods.
Following the direction given by the JRC guidance, all laboratories are expected to follow the performance criteria, but new developments are constantly being added to the method.
Particularly for samples with a high fat content, the fatty acid chain can cause some analytical problems due to rapid overloading of the LC column. To avoid this, sample preparation for this analysis requires a tedious purification step by silica or alternatively by saponification of the fatty acids.
In the present work we have developed a simple, accurate and faster method to perform the saponification in the sample preparation of contaminated food samples. The evaluation of MOSH and MOAH content was performed on different food matrices ranging from cereal products to fish and vegetable oils.
While MOAH, a class of compounds similar but not identical in structure to PAHs, are established carcinogens and therefore should not be present in food, MOSH are a broad class of hydrocarbons, present in both linear and branched forms, whose toxicology is not yet fully understood and therefore no definitive regulatory limits have been established.
Since the first analytical method developed in 2009 by Biedermann and Grobb, many efforts have been made to address the occurrence of contamination in different matrices, in particular by the European Commission, and to develop analytical methods.
Following the direction given by the JRC guidance, all laboratories are expected to follow the performance criteria, but new developments are constantly being added to the method.
Particularly for samples with a high fat content, the fatty acid chain can cause some analytical problems due to rapid overloading of the LC column. To avoid this, sample preparation for this analysis requires a tedious purification step by silica or alternatively by saponification of the fatty acids.
In the present work we have developed a simple, accurate and faster method to perform the saponification in the sample preparation of contaminated food samples. The evaluation of MOSH and MOAH content was performed on different food matrices ranging from cereal products to fish and vegetable oils.
EXPERIMENTAL
EQUIPMENT
- Milestone ETHOS X with easyTEMP
- SR-15 easyTEMP extraction rotor
STANDARD AND REAGENTS
Potassium hydroxide (KOH), Methanol and n-hexane were used for saponification. All chemicals and solvent used are analytical grade and were purchased from Sigma-Aldrich (Milan, Italy). For the test of recoveries, a printing ink solvent (containing 91% of MOSH and 9% of MOAH in the n-C14–C20 range) was provided by a producer. The working internal standard (IS) solution, was purchased from Restek (Milan, Italy) and consisted of: 5-a-cholestane (Cho) and perylene (Per) at 0.6 mg/mL, 1,3,5-tri-tert-butylbenzene (TBB), n-undecane, cyclohexyl cyclohexane (CyCy), pentyl benzene (5B), 1-methyl naphthalene (1MN), 2-methyl naphthalene (2MN) at 0.30 mg/mL, and n-C13 at 0.15mg/mL in toluene.
SAMPLES
All products were purchased in Italian local market. The bakery (cereals) products tested were dry and egg pasta, biscuits, bread and cake. Different kinds of packaging for the same product were tested with variable lifetime. Different kinds of fish with different preservation treatments (fresh, frozen, smoked) and different kinds of packaging were tested. Lastly, a group of 5 different virgin olive oils were extracted.
SAMPLE PREPARATION
Sample to be saponified was weighted (5 g) directly inside the SR-15 eT extraction vessel and 5 µL of IS was added, followed by 10 ml of saturated KOH in methanol and finally 10 mL of n-hexane. Magnetic stirring bars were added to each vessel and the SR-15 eT segment was properly closed. The following temperature method was employed, under a set constant stirring of 80 %:
At the end of the program, after cooling to room temperature, the SR-15 eT vessel was opened. The dry pasta and bread samples did not require any sample post treatment before LC–GC injection. For all the other samples a washing step was performed, adding 40 mL of deionized water inside the vessel followed by 2 mL of methanol, letting the solvent flow along the walls of the extraction cell. The vessel was let to rest for 30 minutes at –20°C. 5 mL of the organic phase of n-hexane was collected and concentrated to 1 mL for following LC-GC analyses.
ANALYTICAL CONDITIONS
On-line LC-GC-FID analysis was performed on a LC-GC. FID detector (sampling frequency of 50 Hz) and solvent vapor exit were heated at 360 and 140 °C, respectively.
The sample was eluted at 300 mL/min using a gradient starting with hexane (0.1 min hold) and reaching 30% of dichloromethane after 0.5 min. The GC run oven ramp consists of constant 20° C/min from 60 to 350 °C with hydrogen as carrier gas. The MOSH area was determined by the integration of the whole hump of largely unresolved peaks. All sharp peaks standing on the top of the MOAH “hump” were subtracted from the total area. The quantification was determined on the IS.
- Milestone ETHOS X with easyTEMP
- SR-15 easyTEMP extraction rotor
STANDARD AND REAGENTS
Potassium hydroxide (KOH), Methanol and n-hexane were used for saponification. All chemicals and solvent used are analytical grade and were purchased from Sigma-Aldrich (Milan, Italy). For the test of recoveries, a printing ink solvent (containing 91% of MOSH and 9% of MOAH in the n-C14–C20 range) was provided by a producer. The working internal standard (IS) solution, was purchased from Restek (Milan, Italy) and consisted of: 5-a-cholestane (Cho) and perylene (Per) at 0.6 mg/mL, 1,3,5-tri-tert-butylbenzene (TBB), n-undecane, cyclohexyl cyclohexane (CyCy), pentyl benzene (5B), 1-methyl naphthalene (1MN), 2-methyl naphthalene (2MN) at 0.30 mg/mL, and n-C13 at 0.15mg/mL in toluene.
SAMPLES
All products were purchased in Italian local market. The bakery (cereals) products tested were dry and egg pasta, biscuits, bread and cake. Different kinds of packaging for the same product were tested with variable lifetime. Different kinds of fish with different preservation treatments (fresh, frozen, smoked) and different kinds of packaging were tested. Lastly, a group of 5 different virgin olive oils were extracted.
SAMPLE PREPARATION
Sample to be saponified was weighted (5 g) directly inside the SR-15 eT extraction vessel and 5 µL of IS was added, followed by 10 ml of saturated KOH in methanol and finally 10 mL of n-hexane. Magnetic stirring bars were added to each vessel and the SR-15 eT segment was properly closed. The following temperature method was employed, under a set constant stirring of 80 %:
| Step | Time (min) | Power (W) | Temperature C° |
|---|---|---|---|
| 1 | 5 | Up to 1600 | 120 |
| 2 | 15 | Up to 1600 | 120 |
At the end of the program, after cooling to room temperature, the SR-15 eT vessel was opened. The dry pasta and bread samples did not require any sample post treatment before LC–GC injection. For all the other samples a washing step was performed, adding 40 mL of deionized water inside the vessel followed by 2 mL of methanol, letting the solvent flow along the walls of the extraction cell. The vessel was let to rest for 30 minutes at –20°C. 5 mL of the organic phase of n-hexane was collected and concentrated to 1 mL for following LC-GC analyses.
ANALYTICAL CONDITIONS
On-line LC-GC-FID analysis was performed on a LC-GC. FID detector (sampling frequency of 50 Hz) and solvent vapor exit were heated at 360 and 140 °C, respectively.
The sample was eluted at 300 mL/min using a gradient starting with hexane (0.1 min hold) and reaching 30% of dichloromethane after 0.5 min. The GC run oven ramp consists of constant 20° C/min from 60 to 350 °C with hydrogen as carrier gas. The MOSH area was determined by the integration of the whole hump of largely unresolved peaks. All sharp peaks standing on the top of the MOAH “hump” were subtracted from the total area. The quantification was determined on the IS.
RESULTS AND DISCUSSION
The method accuracy was assessed with recovery tests performed on 4 different bakery foodstuffs plus one fish sample spiked with known amount of printing ink solvent. Blank repetitions were performed to quantify the MOSH and MOAH contamination already present. For pasta and cake two levels of concentration were tested. The results are reported in the following table:
After the recoveries studies the tests of contamination evaluation were performed. MOSH and MOAH are divided in groups according to the length of chain groups classification (C10-C16; C16-C25; C25-35; C>35) The results are divided for bakery, fish products and extra-virgin olive oils:
The results obtained permit to make the following observations:
After the recoveries studies the tests of contamination evaluation were performed. MOSH and MOAH are divided in groups according to the length of chain groups classification (C10-C16; C16-C25; C25-35; C>35) The results are divided for bakery, fish products and extra-virgin olive oils:
The results obtained permit to make the following observations:
- The recoveries study show that the method is efficient in extracting all the contamination present in the sample.
- Results on bakery products demonstrate the general hypothesis that food with higher fat content register higher MOSH and MOAH migration from the package and the material of the package determines the contamination level. As expected, recycled paperboard is generally the worst packaging material due to residual mineral oil from ink and processing oils.
- MOAH presence is registered only in the bakery products packaged in recycled paperboard containers.
Milestone’s ETHOS X with SR-15 eT rotor
Sample to be saponified was weighted (5 g) directly inside the SR-15 eT extraction vessel and 5 µL of IS was added, followed by 10 ml of saturated KOH in methanol and finally 10 mL of n-hexane. Magnetic stirring bars were added to each vessel and the SR-15 eT segment was properly closed.
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Microwave-assisted solvent extraction has demonstrated to be a well-established sample preparation technique that offers a reliable and efficient approach to their extraction.
Milestone™ Srl
Via Fatebenefratelli 1/5 - 24010 Sorisole (BG) - Italy
Phone: +39 035.573857 | Fax: +39 035.575498
Via Fatebenefratelli 1/5 - 24010 Sorisole (BG) - Italy
Phone: +39 035.573857 | Fax: +39 035.575498
Milestone™ Srl
Via Fatebenefratelli 1/5 - 24010 Sorisole (BG) - Italy
Phone: +39 035.573857 | Fax: +39 035.575498
Via Fatebenefratelli 1/5 - 24010 Sorisole (BG) - Italy
Phone: +39 035.573857 | Fax: +39 035.575498