42 Pharmaceutical ADVERTISING SUPPLEMENT THE APPLICATION NOTEBOOK – FEBRUARY 2008 Generic Procedure for the Extraction of Diuretics from Urine Using EVOLUTE ABN SPE Columns Richard Calverley, Matthew Cleeve, Helen Lodder, Scott Merriman, Joanna Smith & Lee Williams, Biotage GB Ltd, UK iuretics are prescribed to treat various conditions such as ) 100 heart, liver, kidney or lung diseases. However, their mode 90 D = 5 of action has been misused in sport and has led to the n 80 70 International Olympic Committee (IOC) and various horse rac- ry (%, 60 50 ing authorities banning their use in competition. cove 40 ™ This Application Note describes the use of EVOLUTE e Re 30 20 rag ABN 50 ␮m (new larger particle size), a water wettable resin- 10 based non-polar SPE sorbent, for the extraction of diuretics Ave 0 e de de de id ide ide ide zide ide from urine. It describes a generic approach to the extraction of ori azi azi ton c ac lam lam sem thi thi thia etan lac yni Amil tazo diuretics to which additional compounds can be included with oro hazo ume Furo ume Bum rono acr minimal method development time. The analyte suite includes Ace ochl Met ofl ofl Spi Eth Hydr Hydr Bend thiazides, carbonic anhydrase inhibitors, loop and potassium Figure 1: Recoveries for diuretics from urine using EVOLUTE ABN sparring diuretics with wide ranging pKa and logP values. tute in 50:50 (v/v) H2O/MeOH Sample Preparation Procedure (1 mL) for subsequent LC-MS/MS Analytes: analysis Amiloride, Acetazolamide, Hydrochlorothiazide, Methazolamide, HPLC Conditions: Hydroflumethiazide, Furosemide, Bendoflumethiazide, Instrument: Waters 2795 Liquid Handling Bumetanide, Spironolactone and Ethacrynic acid. System & Ultima Pt triple quadru- pole mass spectrometer (Waters EVOLUTE ABN Column Configuration: Assoc., Manchester, UK) EVOLUTE ABN 50 ␮m 100 mg/3 mL (part number 610- Column: Z orbax Eclipse XDB C18 3.5 ␮m 0010-H). analytical column (100 ϫ 2.1 mmID) (Agilent Technologies, Berkshire, UK) EVOLUTE ABN Procedure: Guard Column: C8 guard column (Agilent Column Configuration: 100 mg/10 mL Technologies, Berkshire, UK) Sample Pre-treatment: Dilute urine sample (1-2 mL, 1:1 Mobile Phase: 0.1% (v/v) aqueous formic acid and v/v) with 1% (v/v) formic acid MeCN at a flow rate of 0.25 mL/min. Column Conditioning: Methanol (3 mL) Gradient: 10 / 90 (v/v) MeCN / 0.1% (v/v) Column Equilibration: 0.1% (v/v) formic acid (3 mL) aqueous formic acid increasing to Sample Application: Apply diluted sample 90% (v/v) MeCN over 7 minutes Interference Elution: Rinse with 95:5 (v/v) water/ and held for 1 minute. methanol (3 mL) Injection Volume: 10 ␮L Analyte Elution: Methanol (3 mL) Temperature: Ambient temperature Post Extraction: Evaporate to dryness and reconsti- Conclusions Table I: Recovery and % RSD of diuretics from urine EVOLUTE ABN has been successfully used to extract a range of Analyte % Recovery % RSD diuretics from urine using a generic method (Table I). Recoveries Amiloride 100 1 for all analytes were > 80% with excellent RSD’s (n=5) as shown Acetazolamide 94 6 in Figure 1. Hydrochlorothiazide 104 1 Biotage Methazolamide 94 5 1725 Discovery Drive Hydroflumethiazide 100 2 Furosemide 102 5 Charlottesville, VA 22911 Bendoflumethiazide 94 7 Tel: (800) 446-4752 or (434) 979-2319 Bumetanide 87 7 Fax: (434) 979-4743 Spironolactone 80 10 E:mail: [email protected] Ethacrynic acid 91 8 Website: www.biotage.com.