Determination of PBDEs in river water utilizing automated SPE and Atlantic® C18 SPE disks in conformance with EU standards at IRSA

Scope

This application note will outline the appropriate methods for the extraction of PBDEs as outlined by EU Standards at IRSA using Atlantic® C18 solid phase extraction disks processed using Biotage automated or manual SPE solutions. The first section will highlight the use of the Biotage® Horizon 5000 fully automated extraction system and the method used for this application. Additionally, there will be an Application Modification section that will highlight the use of the Biotage® Horizon 4790 and Biotage® VacMaster™ Disk vacuum manifold for this application.

Polybrominated diphenyl ethers (PBDEs) are a worldwide contamination problem. Structurally similar to PCBs, these compounds are long-lived in the environment and can
bio-accumulate throughout the food chain. The health hazards of these chemicals have attracted increasing scrutiny and, as such, a great deal of research and regulations have been implemented to manage and control them.
 
PBDEs have been used in a wide array of products, including building materials, electronics, furnishings, motor vehicles, airplanes, plastics, polyurethane foams, and textiles. Although many harmful PBDE congeners have been banned throughout the United States, many may still be present in equipment used today. As an international concern, the European Union has also banned multiple PBDE formulates, such as Penta- and Octa-BDE formulates due to their persistence and ability to bio-accumulate within the environment.

The importance of monitoring and controlling PBDEs became apparent as levels were seen to be rising rapidly in human tissues, as evidenced by studies of human breast milk. Data from Sweden sounded the first alarm about the potential for breast milk contamination from PBDEs. In the Swedish study, archived samples collected between 1972 and 1997 were analysed for the presence of PBDEs to ascertain an overall sum of PBDEs in milk. The data showed a drastic increase in the quantity of PBDEs detected with concentrations doubling every five years between 1972 and 1997. Although the concentrations were not considered extremely dangerous, further scientific inquiry and monitoring is necessary as PBDEs do interfere with the body’s hormone system and can pose other health risks.

The analysis of PBDEs in water has proved a challenging to both implement and to meet the data requirements of end users. Traditional methods typically involve the use of a manual separatory funnel, or continuous liquid-liquid extraction (CLLE) of the water samples with dichloromethane (DCM).

With the necessity to be able to determine trace amounts of PBDEs in water, these liquid-liquid extraction methods are labour intensive, use large amounts of solvent, and require contaminant free glassware. Furthermore, the European Union’s requirement to eliminate chlorinated solvents means that DCM cannot be used. Therefore, laboratories are limited in the number of samples they can process in a day while still obtaining reproducible results and low detection limits. Automating this process with SPE will help to reduce the difficulties associated with traditional methods while generating highly reproducible results.

Instrumentation

Biotage instruments:

» Biotage® Horizon 5000 Automated Extraction System
» Atlantic® C18 SPE Disk
» GC-MS/MS: Thermo Electron TRACE GC 2000 coupled with PTV injector and PolarisQ Ion Trap mass spectrometer.
» Restek: Rtx-5MS,60 m x 0.25 mm ID x 0.25 µm film
» GC-ECD for PBDE- 209: Thermo Electron TRACE GC Ultra equipped with a cold on-cartridge injector and ECD-40 detector
» Commercially available concentrator with external heat source and nitrogen sparge

Method Summary

HPLC Grade Water Samples

1. Approximately 1 L of HPLC water sample was used.
2. Spike the samples with 2.8 ng of native (except for 28 ng of BDE-209) and 1.1 ng of labelled PDBE compounds (Table 4 shows list of PBDE compounds).
3. Place the sample bottle on the Biotage® Horizon 5000 Extraction system and place an Atlantic®C18 disk in the disk holder.
4. Place the sample collection vial on the extractor.
5. Run the Biotage® Horizon 5000 using the method shown in table 1.
6.  After extraction is completed collect the final extract.
7. Pipette off the bottom methanol/water layer.
8. Concentrate the extract down to 1.0 mL.
9. Analyse by GC-MS/MS (GC/ECD for PBDE-209) using the conditions in table 2.
   
   

River water samples

1. 2 L of Lambro river water was collected and mixed well before transferring to 2 x 1 L sample bottles (samples 1 and 2).
2. Spike the samples with 1.1 ng labelled PBDE compounds.
3. Place the sample bottle on the Biotage® Horizon 5000 Extraction System and place the Atlantic® C18 disk, pre-filters and coarse (or fine mesh) screen in the disk holder.
   a. Two pre-filters and a screen were used to extract Sample 1.
   b. No pre-filters or screens were used when extracting Sample 2.
4. Place the sample collection vial on the extractor.
5. Run the Biotage® Horizon 5000 using the method shown in table 1.
6. After the extraction is complete collect the final extract.
7. Pipette off the bottom methanol/water layer.
8. Clean the sample using silica gel.
9. Concentrate the sample down to 1.0 mL using a concentrator containing an external heat source.
10.  Analyse by GC/MS (GC/ECD for PBDE 209) using the conditions in table 2.
    
    

Acknowledgements

Biotage would like to thank Licia Guzzella and Claudio Roscioli of the IRSA in Milan, Italy for their help in developing this method.

Application modifications

Biotage® Horizon 4790 method summary

HPLC grade water samples

1. Approximately 1 L of HPLC water sample was used.
2. Spike samples with 2.8 ng of native (except for 28 ng of BDE-209) and 1.1 ng of labelled PDBE compounds (full compound list available in Table 4).
3. Place the sample bottle on the Biotage® Horizon 4790 Extraction System and place an Atlantic® C18 disk in the disk holder.
4. Place the sample collection vial on the extractor.
5. Run the Biotage® Horizon 4790 using the method shown in table 3 and collect the final extract.
6. Pipette off the bottom methanol/water layer.
7. Concentrate the extract down to 1.0 mL.
8. Analyse by GC/MS (GC/ECD for PBDE 209) using the conditions in table 2 in the previous section.
   
   

River water samples

1. 2 L of Lambro river water was collected and mixed well before transferring to 2 x 1 L sample bottles (samples 1 and 2).
2. Spike the samples with 1.1 ng labelled PBDE compounds.
3. Place the sample bottle on the Biotage® Horizon 4790 Extraction System and place the Atlantic® C18 disk, pre-filters and coarse (or fine mesh) screen in the disk holder.
   a. Two pre-filters and a screen were used to extract Sample 1
   b. No pre-filters or screens were used when extracting Sample 2.
4. Place the sample collection vial on the extractor.
5. Run the Biotage® Horizon 4790 using the method shown in table 3 and collect the final extract.
6. After the extraction is complete collect the final elution.
7. Pipette off the bottom methanol/water layer.
8. Clean the extract using silica gel
9. Concentrate the sample down to 1.0 mL using a concentrator containing an external heat source.
10. Analyse by GC/MS (GC/ECD for PBDE 209) using the conditions in table 2 above.

Biotage® Horizon 4790 results and conclusions

HPLC grade water samples:

To demonstrate consistency, a total of four samples were spiked with 1.1 ng of labelled standard and extracted on each of the two Biotage® Horizon 4790 units. The final extracts were dried, concentrated and analyzed. The concentrations of each of the compounds were compared to a “standard” hexane sample spiked with the same ng amount of the PBDEs that were spiked into each water sample. Once this hexane sample was concentrated it was analysed on GC/MS and acted as the standard.

As shown in Table 4, all four samples showed high average recoveries, 76.5% to 113.3% for the different PBDE compounds. The consistency and reproducibility of the samples was excellent. Furthermore, PBDE 209 has always been a difficult compound to measure but, in Table 3, the average recovery is 76.5% and the relative standard deviation of 3.6%.

Lambro river water samples:

Following the HPLC grade water sample runs, real particulate- laden water samples from the nearby Lambro River were extracted. The two 1-liter samples, which were taken from the same well-mixed sample bottle, were prepared with a 1.1 ng spike of labelled PBDE compounds. To show that pre-filters can effectively decrease the amount of time for sample processing, without affecting the chemistry, sample 1 was run with two pre-filters and a coarse (or fine mesh) screen, while sample 2 was run with an Atlantic® C18 disk only.
 
Figure 2: Chromatogram of HPLC Grade Water Sample 1, where
Peak 1 = BDE-28               Peak 4 = BDE-99
Peak 2 = BDE-47               Peak 5 = BDE-154
Peak 3 = BDE-100             Peak 6 = BDE-153


Table 5 shows the results for samples 1 and 2. Sample 1, with the pre-filters and screen, took 50% less time to complete (43:21 min) in comparison to sample 2 (95 min), which had only the C18 disk. This time difference shows that, although the results do not change, it is important to use the appropriate pre-filters to ensure optimal flow rates on the Biotage® Horizon 4790 Automated Extraction System.

The data shows that, even though PBDE 28 and 47 showed a decrease in recoveries, all labelled PBDE compounds displayed a consistency in their results indicating that they were not affected by the particulate matter in the real samples. Since both PBDE 28 and 47 are lower molecular weight, it is likely that they formed a strong bond with the particulate matter in the water sample and were unable to be recovered fully.

Table 6 shows the results of a PBDE background study done on the Lambro River and indicates that the small differences that occurred between the spiked runs can be attributed to the sample not being completely homogeneous.

Biotage® VacMaster™ Disk method summary

1. Repeat the following steps for each active Biotage® VacMaster™ Disk station.
2. Set up the VacMaster Disk manifolds ensuring all waste lines and vacuum lines are attached. Set the vacuum pump to -24”Hg.
3. Prepare the disk holder assembly (47 mm): ensure the support screen is flat in the centre of the disk holder. Place the Atlantic® C18 Disk on top of the support screen with the ripples of the disk on top and add any prefilters on top of the disk. Place the disk holder assembly on the VacMaster Disk manifold ensuring there is a tight seal with the luer fitting.
4. If using the multifunnel, place onto the disk holder assembly. If not using the multifunnel, omit those directions throughout the method.
5. Condition the SPE Disk:
a. Guide for each conditioning step in table 7 below:
i. Measure the appropriate VOLUME of SOLVENT into a graduated cylinder and pour into the disk holder assembly.
ii. Using a Nalgene Wash Bottle (phthalate free), rinse the multifunnel and disk holder in a circle for about 3 seconds using the same SOLVENT (approximately 5 additional mL).
iii. SATURATE the disk for the time
indicated (in SECONDS). (Saturate means: quickly turn the knob to the appropriate waste destination and back to the “OFF” position. This brings the solvent into the disk media bed).
iv. SOAK the disk for the time indicated (in SECONDS).
v. DRAIN to the appropriate waste destination for the time indicated (in SECONDS). Switch to the “OFF” position.

6. Load the Sample:
a. For multifunnel: quickly and efficiently angle the bottle to rest on the multifunnel upside-down.
b. For no multifunnel: pour a portion of the sample into the disk holder.
c. Adjust the vacuum between -10”Hg and -15”Hg for sample load (please note, if the sample is flowing too slowly,
the vacuum can be increased). Drain the sample to “AQUEOUS” waste. Continue to pour the sample into the disk holder ensuring the disk does not go dry or overflow for the duration of sample load.

7. Air Dry the SPE Disk:
a. Return the vacuum to -24”Hg and continue to air dry the SPE disk to “AQUEOUS” waste for an additional 600 SECONDS. Switch to the “OFF” position.
b. Remove the sample bottle from the multifunnel if it was used.
8. Elute the SPE Disk: (Please note: the elution solvents will go into the collection flask inside the chamber, not to waste containers).
a. Place a clean 125 mL 24/40 tapered Erlenmeyer flask or 40mL VOA vial using the VOA vial insert into the Biotage® VacMaster™ Disk collection chamber. Place the cover on the chamber. Remove the disk holder assembly and place the disk holder assembly into the luer fitting on top of the collection chamber. Attach the luer fitting of the collection chamber assembly onto the manifold.
b. Guide for each elution step in table 8 below:
i. Measure the appropriate VOLUME of SOLVENT into a graduated cylinder, pour into the sample bottle, and swirl around. Pour the solvent in the sample bottle into the disk holder assembly.
ii. Using a Nalgene Wash Bottle (phthalate free), rinse the multifunnel and disk holder in a circle for about 3 seconds using the same SOLVENT (approximately 5 additional mL).
iii. SATURATE the disk for the time indicated (in SECONDS) to “ORGANIC”.
iv. SOAK the disk for the time indicated (in SECONDS).
v. DRAIN to “ORGANIC” for the time indicated (in SECONDS). Switch to the “OFF” position.
vi. Remove the chamber lid to release the vacuum from inside the chamber.
 

Literature number: AN055-HOR