Determination of endocrine disrupting hormones in drinking and wastewater utilizing the Atlantic® HLB SPE Disk

Scope

The purpose of this application note is to outline an automated extraction method utilizing the Atlantic® HLB SPE Disk and the Biotage automated and manual SPE solutions for the extraction of hormones in drinking water. 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® VacMaster™ Disk for this application.

Introduction

Hormones in drinking water are a concern, since the presence of these compounds can potentially be linked to serious health hazards such as human developmental and reproductive side-effects. Hormones are introduced into our environment in several ways. Natural and synthetic hormones that are given to livestock, pass through the animals, runoff into surface water, and leach into underground water supplies. Humans also produce and excrete natural hormone waste every day. An example is the natural and synthetic estrogens and androgens that are used in large quantities for medical purposes, such as oral contraceptives and hormone replacement. Human wastewater flows through to sewage treatment plants, but these plants are not designed to handle the removal of hormones, so the hormones filter though the treatment process, and can ultimately end up in drinking water.

The Biotage® Horizon 5000 extraction system provides automatic extraction of liquid samples by solid phase extraction methods, and can handle samples ranging from 20 mL to 4 L. The Biotage® Horizon 5000 software allows for the control of up to four extractors, each with three stations, allowing for up to twelve samples to be extracted simultaneously. The DryVap® Concentrator System provides automatic sample drying with patented DryDisk® Separation PTFE Membrane technology, automatically concentrating each dried extract by applying heat, vacuum, and sparge flow for up to six samples simultaneously. The Biotage® Solvent Recovery SVOC is designed to condense and collect the volume of solvent vapor generated from the DryVap® Concentrator System while in operation.

Instrumentation

Biotage instruments:

• Biotage® Horizon 5000 Automated Extraction System
• DryVap® Concentrator System
• DryDisk® Separation Membranes
• Biotage® Solvent Recovery SVOC
• Atlantic® HLB SPE Disks (47 mm)

Method summary

1. Obtain 500 mL aliquots of sample and verify they are pH 5–7. Preserve with 25 mg of ascorbic acid.
2. Agitate the bottles by hand until preservative is dissolved.
3. Spike the compound standard into any laboratory control or matrix spike samples (typically 50 ppt).
4. Place the sample bottle on the Biotage® Horizon 5000 Extraction System and place the Atlantic® HLB disk in the standard 47mm disk holder.
5. Start extraction method shown in table 1.
6. Collect the sample extract (approximately 32 mL).
7. Add the extract to the DryDisk® holder and start the concentration process on the DryVap® using the conditions in table 2. Concentrate the extract to 0.5 mL.
8. Add 2 mL of methanol into the concentrator tube and mix for solvent exchange.
9. Concentrate to 0.5 mL and transfer the extract to a GC vial for analysis via LC/MS/MS.
   
   

Table 1. Biotage® Horizon 5000 extraction method.

Table 2. Conditions used for the DryVap® concentrator system.

Acknowledgements

Our thanks go to Ali Haghani, from Montgomery Watson Labs for his time and assistance with developing this application note.

Application Modifications

Biotage® VacMaster™ Disk Method Summary

1. Repeat the following steps for each active Biotage® VacMaster™ Disk station.

2. Setup 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 center of the disk holder. Place the Atlantic® HLB 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 lure 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 3 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 indicated time (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 indicated time (in SECONDS).

V. DRAIN to the appropriate waste destination for the indicated time (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 to 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.

Table 3. Disk conditioning.

8. Elute the SPE Disk: (Please note: the elution solvent 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 40 mL 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 4 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 indicated time (in SECONDS) to “ORGANIC”.

IV. SOAK the disk for the indicated time (in SECONDS).

V. DRAIN to “ORGANIC” for the indicated time (in SECONDS). Switch to the “OFF” position.

VI. Remove the chamber lid to release the vacuum from inside the chamber.

Table 4. Disk elution.

Literature number: AN001