Determination of microcystins in water using automated SPE and concentration techniques

Application note scope

This application note will outline appropriate methods for the extraction of microcystins in water using Biotage automated or manual SPE solutions and the DryVap™* Concentrator System. 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.

*The DryVap™ system has been discontinued. We recommend using the TurboVap® evaporation systems for achieving equivalent results.

Introduction

Pollution arising from freshwater algae has become a global environmental problem. Of all the pollutants, microcystin-LR is thought to be the most toxic and the most acute hazard. There have been many serious accidents caused by pollution from freshwater algae all over the world. The microcystin-LR maximum contaminant level (MCL) in drinking water recommended by the World Health Organization (WHO) is 1.0 µg/L.

This application note combines the Biotage® Horizon 5000 Automated Extraction System and the DryVap™ Concentrator System for extraction of microcystins in water with a final analysis by HPLC. The purpose of this application note is to develop a method for automating the extraction and concentration of microcystin samples.

The Biotage® Horizon 5000 provides automated extraction of liquid samples by solid phase extraction methods. It can handle samples from 20 mL to 4 L. The operation software provides a user-friendly controller capable of interacting with up to four extractors via a standard PC. The DryVap™ Concentrator System provides automated sample drying with patented DryDisk membrane technology and automatically concentrates each dried extract by applying heat, vacuum, and sparge flow for up to six samples at one time.

Instrumentation

• Biotage Instruments and Consumables
  »  Biotage® Horizon 5000 Automated Extraction System
  » Atlantic® DVB SPE Disk
  » DryVap™ Concentrator System
  » DryDisk® Solvent Drying System
• LC600 HPLC
• Cartridge: Labtech C18 0.250 mm x 4.6 mm x 0.5 µm
• Microcystin-LR (MC-LR) standard solution
• Phosphate buffer solution
  
  

Method summary

1. Dissolve 100 µg of MC-LR standard to a constant volume of 10 mL in methanol (store solution at -20 °C) to yield a final concentration 10 µg/mL.
2. Spike 1 L of DI water with 0.1 mL of MC-LR standard solution for a final concentration of 1 µg/L.
3. Extract samples with the Biotage® Horizon 5000 extraction method shown in table 1.
4. Concentrate the extract to less than 1.0 mL using the DryVap™ settings shown in table 2 and quantitatively bring the extract volume to 1.0 mL.
5. Analyze by HPLC using the conditions in table 3.
   
   

*Preparation of phosphate buffer solution: Adjust 0.05 mol/L of potassium dihydrogen phosphate solution to pH 3.0 with 20% (volume fraction) of phosphoric acid.

Acknowledgements

Biotage would like to thank Lab Tech LTD China in Beijing, China for their assistance in developing this method.

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® 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:
   
   

• Guide for each conditioning step in table 4 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:

1. For multifunnel: quickly and efficiently angle the bottle to rest on the multifunnel upside-down.
2. For no multifunnel: pour a portion of the sample into the disk holder.
3. 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

1. Return the vacuum to -24”Hg and continue to air dry the SPE disk to “AQUEOUS” waste for an additional 120 SECONDS. Switch to the “OFF” position.
2. Remove the sample bottle from the multifunnel if it was used.
   
   

8. Elute the SPE Disk: (Please note: the elution solvent will go into the collection flask inside the chamber, not to waste containers)

1. Place a clean 125 mL 24/40 tapered Erlenmeyer flask or
   40 mL VOA vial using the VOA vial insert into the 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.
2. Guide for each elution step in table 5 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: AN057-HOR