Extraction of Cocaine and Metabolites From Urine Using ISOLUTE SLE+ prior to LC-MS/MS Analysis

Transcription

1 Extraction of Cocaine and Metabolites From Urine Using ISOLUTE SLE+ prior to LC-MS/MS Analysis Application Note AN772 Introduction This application note describes the extraction of cocaine and a full range of cocaine metabolites from human urine prior to analysis and quantitation by LC-MS/MS This application note describes effective and efficient ISOLUTE SLE+ protocols optimized for sample (urine) volumes of either 2 µl or ml. Procedures for high throughput (96-well plate) and column format are included. The simple sample preparation procedure delivers clean extracts and analyte recoveries of 67- with RSDs of <5 for all analytes. Sub LOQs can be achieved. Figure. Structure of Benzoylecgonine ISOLUTE SLE+ Supported Liquid Extraction plates and columns offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation Analytes Cocaine, Norcocaine, Cocaethylene, Benzoylecgonine, Ecgonine Methyl Ester (EME) and Anhydroecgonine Methyl Ester (AEME). Sample Preparation Procedure Sample pre-treatment: Dilute urine ( ml) with. ammonium hydroxide (aq) ( ml). Spike ISTD (µl of BZE-d3; total concentration 5). Vortex mix thoroughly. ISOLUTE SLE+ 2 µl Supported Liquid Extraction Plate, part number 82-2-P Sample loading: Analyte extraction: Load the pre-treated urine (2 µl) onto the plate and apply a pulse of vacuum or positive pressure to initiate flow. Allow the sample to adsorb for 5 minutes. Apply DCM/IPA (95:5, v/v) ( ml) and allow to flow under gravity for 5 minutes. Apply vacuum or positive pressure to pull through any remaining extraction solvent. ISOLUTE SLE+ ml Sample Volume columns, part number 82-4-C Sample loading: Analyte extraction: Post-extraction: Load the pre-treated urine ( ml) onto the column and apply a pulse of vacuum or positive pressure to initiate flow. Allow the sample to adsorb for 5 minutes. Apply DCM/IPA (95:5, v/v) (2.5 ml) and allow to flow under gravity for 5 minutes. Apply a further aliquot of DCM/IPA (95:5, v/v) (2.5 ml) and allow to flow for another 5 minutes. Apply vacuum or positive pressure to pull through any remaining extraction solvent. Add 5mM HCl in MeOH ( µl) to stabilize AEME and EME during evaporation. Evaporate to dryness at 4 C. Reconstitution: 82-2-P: Water/Methanol (9/, v/v) (2 µl) 82-4-C: Water/Methanol (9/, v/v) (5 µl)

2 UPLC Conditions Instrument: Column: Waters Acquity UPLC fitted with 2 µl loop Waters UPLC BEH C8 (.7 μm, x 2. mm id) Sample temperature: 2 C Column temperature: 4 C Injection volume: Mobile phase: μl (partial loop with overfill) A=. ammonium hydroxide (aq) B=. ammonium hydroxide/meoh Gradient: Time (min) A B Curve Mass Spectrometry Conditions Instrument: Premier XE triple quadrupole mass spectrometer equipped with an electrospray interface for mass analysis. Desolvation temperature: 45 C Ion source temperature: 5 C Table. Positive ions acquired in the multiple reaction monitoring (MRM) mode Analyte Transition Dwell Time (s) Cone voltage (V) Collision energy (ev) EME 2 > 8.9 QUANT 2 > 82. QUAL BZE 29. > 68. QUANT 29. > 49.9 QUAL BZE-d3 ISTD 293. > AEME 82. > 22. QUANT 82. > 9.9 QUAL Norcocaine 29. > 35.9 QUANT 29. > 7.9 QUAL Cocaine 34.2 > 82. QUANT 34.2 > 49.9 QUAL Cocaethylene 38. > 96. QUANT 38. > 49.9 QUAL Biotage. 2

3 Results This ISOLUTE SLE+ protocol demonstrates analyte recoveries ranges from 67- as shown in figure 2. RSDs were below 5 for all analytes. Figure 3 shows an example calibration curve for the cocaine metabolite, benzoylecgonine. Figure 4 shows the MRM transitions, both quantification and qualification ions, for analytes extracted using the supported liquid extraction protocol Analyte recovery Figure 2. Typical analyte recoveries for a extracted Cocaine and metabolites (n=7) using the ISOLUTE SLE+ protocol Compound name: BZE Quant Correlation coefficient: r =.99974, r^2 = Calibration curve:.769 * x Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: /x, Axis trans: None Compound name: BZE Qual Correlation coefficient: r = , r^2 =.9998 Calibration curve: * x Response type: Internal Std ( Ref 5 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: /x, Axis trans: None Residual Residual 2.5. Response 5 Response Figure 3. Example calibration curve for the cocaine metabolite Benzoylecgonine Table 2. Estimated Limits of Quantitation for all analytes using the ISOLUTE SLE+ ml sample volume column Analyte EME BZE AEME Norcocaine Cocaine Cocaethylene Estimated LOQ < pg/ml < pg/ml < 2 pg/ml < 4 pg/ml, restricted by qualifier ion < 25 pg/ml < 25 pg/ml 22 Biotage. 3

4 Cocaethylene Qual > 49.9.e Cocaine Quant > 82.e Cocaine Qual > e Norcocaine Quant > e Norcocaine Qual > e : MRM of 2 Channels ES+ AEME Quant.9 82 > 22.4e : MRM of 5 Channels ES+ EME Qual.5 2 > e : MRM of 5 Channels ES+ EME Quant.5 2 > e : MRM of 5 Channels ES+ BZE ISTD > 7.3e Cocaethylene Quant AEME Qual BZE Qual BZE Quant Figure 4. MRM transitions chromatograms for Cocaine and metabolites at > e5 2: MRM of 2 Channels ES+ 82 > e5 : MRM of 5 Channels ES+ 29. > e5 : MRM of 5 Channels ES+ 29. > e4 Time 22 Biotage. 4

5 Ordering information Part number Description Quantity 82-2-P 82-4-C 82-4-CG PPM-96 PPM ISOLUTE SLE+ 2 Supported Liquid Extraction Plate ISOLUTE SLE+ ml Sample Volume columns ISOLUTE SLE+ ml Sample Volume columns (tabless for use with PPM-48) PRESSURE+96 Positive Pressure 96 Well PRESSURE+48 Positive Pressure 48 position VacMaster 96 Sample Processing VacMaster- Sample Processing VacMaster-2 Sample Processing 3 3 SD-96-DHS-UK SPE Dry V NORTH AMERICA Main Office: Toll Free: Fax: Order Tel: Order Fax: ordermailbox@biotage.com US--pointsupport@biotage.com To search and download more of Biotage s extensive database of sample preparation applications please visit or scan the QR code with your smart phone to go direct. EUROPE Main Office: Fax: Order Tel: Order Fax: order@biotage.com EU--pointsupport@biotage.com JAPAN Tel: Fax: jp_order@biotage.com JP--pointsupport@biotage.com CHINA Tel: Fax: CN_order@biotage.com CN--pointsupport@biotage.com Distributors To locate a distributor please visit our Web site at 22 Biotage. All rights reserved. All brand and product names are trademarks or registered trademarks of their respective companies. The information contained in this document is subject to change without notice. AN-772.OCT.2