Presentation No. 14 - A Rapid, Easy Sample Cleanup Process utilizing Supported Liquid Extraction Plates Prior to LC-MS/MS Analysis
Lee Williams,Elena Gairloch
Biotage GB Limited, Dyffryn Business Park, Ystrad Mynach, Mid Glamorgan, CF82 7RJ, UK; Biotage, 1725 Discovery Drive, Charlottesville, VA 22911, USA
Introduction: : It is well known that traditional liquid-liquid extraction (LLE) provides very clean extracts prior to LC/MS analysis. Supported liquid extraction is analogous to traditional LLE, however, analyte partitioning takes place using an inert support material, rather than two immiscible liquids. This provides excellent extraction efficiencies while alleviating many of the tedious liquid handling issues associated with LLE.
This talk shows how more generic methods for the extraction of various acidic, basic or neutral drugs can be utilized, therefore simplifying the whole SLE+ method development process. Extraction screening protocols based on the combination of two loading pHs and four extraction solvents are compared. The loading pH is dictated by the types of analytes being tested: For acidic analytes the plasma loading pHs are approximately 3.2 and 6.1; for neutral analytes the pHs are approximately 6.1 and 8.0; and finally for basic analytes 8.0 and 10.5. Full buffer details are given in the experimental section.
Methods: Reagents: Non-steroidal anti-inflammatory drugs (NSAIDs), ?-blockers, corticosteroids and formic acid were purchased from Sigma Chemical Co. (Poole, UK). Blank human plasma was obtained through the Welsh Blood Service (Pontyclun, UK). All solvents were HPLC grade from Fisher Scientific (Loughborough, UK). Sample Preparation: Supported Liquid Extraction Procedure. Plate: ISOLUTE SLE+ Supported Liquid Extraction Plate 200 mg, part number 820-0200-P01. Sample pre-treatment: Acidic analytes (NSAIDs):- Plasma pre-treatment 1:1 v/v with either 1% formic acid or 0.1% formic acid aq. This sample dilution results in loading pH’s of approximately 3.2 and 6.1, respectively; Neutral analytes (Corticosteroids):- Plasma pre-treatment 1:1 v/v with either H2O or 0.1% formic acid aq. This sample dilution results in loading pH’s of approximately 6.1 and 8.0, respectively; Basic analytes (?-blockers):- Plasma pre-treatment 1:1 v/v with either H2O or 0.5M ammonium hydroxide. This sample dilution results in loading pH’s of approximately 8.0 and 10.5, respectively. Sample Application: The pre-treated plasma was loaded onto the plate, a pulse of vacuum applied to initiate flow and the samples left to absorb for 5 minutes. Analyte Elution: Addition of 1 mL of various water immiscible extraction solvents. The extraction solvents evaluated were DCM, 95:5 (v/v) DCM/IPA, MTBE and EtOAc. Post Extraction: The eluate was evaporated to dryness and the analytes reconstituted in 500 µL of appropriate H2O/MeOH mixtures prior to analysis. HPLC Conditons: Instrument Waters 2795 Liquid Handling System (Waters Assoc., Milford, MA, USA). Column: Zorbax Eclipse XDB C18 3.5 µm analytical column (100 x 2.1 mm id, 3.5 µm) (Agilent Technologies, Berkshire, UK). Guard Column: C8 guard column (Agilent Technologies, Berkshire, UK). Mobile Phase: 0.1% formic acid aq and MeCN (acetonitrile) at a flow rate of 0.25 mL/min using various gradients. Mass Spectrometry: Instrument: Ultima Pt triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis. Positive and negative ions were acquired in the multiple reaction monitoring mode (MRM). Desolvation Temperature: 350 °C; Ion Source Temperature: 100 °C; Collision Gas Pressure: 2.3 x 10-3 mbar.
Results: Analytes were selected to give a broad range of polarities (LogP) and pKa values within each suite. The appropriate screening procedures listed above were then carried out. Figures 2-7 show the respective recoveries obtained for the three analyte suites using various combinations of pH loading and extraction solvent conditions.
Conclusions: The acidic analytes, NSAID, recoveries for equivalent extraction solvents were generally higher for the lower pH loading conditions, while the Neutral Corticosteroid recoveries were consistently high using all extraction solvent and pH loading combinations. Triamcinolone, however, only gave 50% recoveries when using DCM as the extraction solvent. Six out of Nine ?-blockers showed consistently high recoveries for all extraction solvents at both loading pH’s. For the 3 most polar analytes (atenolol, sotalol and nadalol) better recoveries were obtained when using combinations of more polar extraction solvents at a higher loading pH. By using the suggested protocol (screening two pH loading conditions combined with four extraction solvents) good recoveries and RSD’s were obtained for the majority of analytes used in this study. For very polar analytes, assuming they have adequate solubility in the extraction solvents, more precise pH control is required (analytes must be in their unionized forms).
