Supported Liquid Extraction is now an accepted bioanalytical sample preparation technique, supporting high throughput assays. The technique mimics the characteristics of liquid-liquid extraction (LLE), but unlike LLE lends itself to high throughput and automation. Packed with a diatomaceous earth, each well provides a support to absorb the aqueous sample, whilst leaving the analytes on the surface - there are no interactions between the analyte and the diatomaceous earth. Analytes are subsequently eluted with a water immiscible organic solvent.
The new Cleanert® SLE Plate is packed with 600mg of diatomaceous earth, so increasing the capacity and volume of plasma that can be extracted in a single extraction.
This application looks at the extraction of the angiotensin II receptor antagonist, Telmisartan.
Figure 1 Chemical Structure of Telmisartan
Human plasma samples were spiked with the analyte in the concentrations range 2 to 1000ng/mL. SLE Method using Cleanert® SLE Plate, 600mg.
Sample Application: 300µL Human plasma diluted by 300μL 2% formic acid aqueous solution.
Elute the cartridge by 6mL MTBE after 10min standing, collect the elute in a 10mL vial and concentrate to dry by nitrogen-blow concentrator at 35 °C, add 1mL acetonitrile :water (60:40, v/v), shake up.
*A short vacuum pulse is used to initiate sample loading. All other steps of the method use gravity flow.
HPLC Column: Venusil® XBP C18 (375px ´ 2.1mm, 5μm, 120Å);
Mobile Phase: Acetonitrile/Water, 65/35, v/v;
Flow rate: 0.2mL/min;
LC Instrument: Shimadzu LC-20A;
Detection: MS (ABI4000+) – see below for conditions;
Table 1 MS Conditions
Table 2 Precursor/Product Ions
Figure 1 Chromatogram of 10ng/mL Telmisartan
Figure 2 Chromatogram of spiked plasma sample with 10ng/mL Telmisartan
Figure 3 Chromatogram of blank sample
Result and Discussion
By the method of above, LOD of Telmisartan is 0.1 ng/mL.
Table 3 Responses for Telmisartan standard
Table 4 Recoveries of Telmisartan
Sample - % Recovery
Using the SLE method developed, the analyte, Telmisartan, was extracted from spiked human plasma (300µL) in the concentration range 2 to 1000ng/ml. The analyte was eluted with 6mL of elution solvent. This presents the analyte in a volatile solvent for evaporation and reconstitution.
Recoveries for the Telmisartan are 83.6 ~98.9%. The RSDs were <4.5%. Linearity checks across the full concentration range were good. Increasing the number of samples per concentration to develop and report a robust method. Blank plasma was also extracted and analysed using the same procedures to ensure it was drug free over the required concentrations.
Other commercially available SLE Plates have a maximum load of 400µL. In order to facilitate to loading of the increased volume for this 600mg plate, the top frit has been surface modified to a more hydrophilic nature. This modified surface is more "water friendly" and more readily accepts the diluted plasma, improving sample loading onto each well. Samples were consistently loaded across the entire plate.