New techniques for high efficiency on BioTrap 500

The ionpair technique

The displacement technique

The ionpair technique

A technique has been developed to obtain high separation efficiency for basic analytes and to increase recovery for more hydrophilic basic analytes, when using the BioTrap 500 column. These effects are created by the addition of an ionpair agent, for example the sodium salt of an alkylsulfonic acid, in the extraction mobile phase. Very low concentrations of the ionpairing agent is used, i.e. 1-10 mM.

In the figure, atenolol is extracted and chromatographed using the ionpair technique with BioTrap 500 C18. 200 µl of the serum is injected. If atenolol is extracted on the BioTrap 500 C18 column without the addition of the ionpair agent, the recovery is around 50%. When 5 mM sodium octanesulfonic acid is added to the extraction mobile phase, the recovery is increased to ~100%.

Injection vol.: 200 µl serum Extraction column: BioTrap 500 C18, 20 x 4.0 mm Mobile phase (extraction): 2% 2-propanol in 30 mM sodium phosphate buffer, pH 7.0 + 5mM sodium octanesulfonic acid Flow: 0.8ml/min Analytical column: Zorbax SB-CN, 150 x 4.6 mm, 5 µm + guard Analytical mobile phase: 25% acetonitrile in 30 mM sodium phosphate buffer, pH 3.0 + 2mM sodium octanesulfonic acid Flow: 1.0 ml/min Detection: Ex=230 nm, Em=300 nm

Analysis Program

Next figure shows a chromatogram obtained after online-extraction of 500 µl of serum, containing 12 ng/ml of propanolol. In this method 5 mM octanesulfonic acid sodium salt is added to the extraction mobile phase and 2 mM of the same agent added to the analytical mobile phase. The result is an increase in separation efficiency, giving a more sensitive method. It has also been shown that the addition of an ionpairing agent will have a positive effect on the lifetime of the extraction column.

Injection vol.: 500 µl serum Extraction column: BioTrap 500 C18, 20 x 4.0 mm Mobile phase (extraction): 4% 2-propanol in 20 mM sodium phosphate buffer, pH 7.0 + 5mM sodium octanesulfonic acid Flow: 0.8ml/min Analytical column: Hypersil Elite C18, 150 x 4.6 mm, 5 µm+ guard 10x4.0 mm Analytical mobile phase: 33% acetonitrile in 116 mM sodium phosphate buffer, pH 2.8 + 2mM sodium octanesulfonic acid Flow: 1.0 ml/min Detection: Ex=220 nm, Em=340 nm

Analysis Program

If you have any questions regarding the above, don´t hesitate to contact Chrom Tech!

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The displacement technique

An acid can be extracted in two different ways, as the protonated acid and as an ion-pair. Normally a very good recovery can be obtained by extraction of the protonized acid at low pH. Another way to increase the recovery is to use neutral pH and extract the acid as an ion-pair with positively charged buffer ions like sodium, with a hydrofobic acid ( for example octanoic acid) present in the extraction mobile phase.

The hydrophobic acid will compete with the analyte for the protein binding and displace the analyte. This can also have a very positive effect on the efficiency. Furthermore, in this way the peaks can be compressed and the recovery can increase. This is very favourable in low concentration analysis.

Such results are shown in figure for ibuprofen, where 10µl of serum is injected. The concentration is 20 µg/ml, however, as can be seen from the chromatogram, much lower concentrations can be detected due to the very good chromatographic performance, which means that the method can easily be used for determination of the concentration after single doses of ibuprofen.

Injection vol.: 10 µl serum Extraction column: BioTrap 500 C18, 20 x 4.0 mm Mobile phase (extraction): 2% 2-propanol in 30 mM sodium phosphate buffer, pH 7.0 + 10mM octanoic acid Flow: 0.8ml/min Analytical column: CT-sil C18, 100 x 4.6 mm, 5 µm + guard Analytical mobile phase: 35% acetonitrile in 30 mM sodium phosphate buffer, pH 7.0 Flow: 1.0 ml/min Detection: Ex=225 nm, Em=535 nm

Analysis Program

Switching valve position	Analysis step	Time
1. Extraction position	sample extraction	5 min
2. Elution position	transfer of analyte	4 min
3. Extraction position	separation and reequilibration	4 min

If you have any questions regarding the above, don´t hesitate to contact Chrom Tech!

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