Extraction of diuretics from urine using EVOLUTE® EXPRESS ABN cartridges

Introduction

This procedure is recommended for the extraction of a variety of diuretics from urine using non-polar polymeric SPE. The analyte suite includes thiazides, carbonic anhydrase inhibitors, loop and potassium sparring diuretics, with wide ranging pKa and logP values.

Analytes

Amiloride, Acetazolamide, Hydrochlorothiazide, Methazolamide, Hydroflumethiazide, Furosemide, Bendoflumethiazide, Bumetanide, Spironolactone, Ethacrynic acid.

Sample preparation procedure

Format

EVOLUTE® EXPRESS ABN 100 mg/3 mL (Tables), part number 610-0010-BXG.

Sample

Spike urine sample with at 50 ng/L concentration of the compounds listed above.

Sample pre-treatment

Dilute urine sample (1–2 mL) with 1% formic acid (1:1, v/v).

Cartridge conditioning

Methanol (3 mL).

Cartridge equilibration

0.1% (v/v) formic acid (3 mL).

Sample application

Apply diluted sample.

Interference elution

Rinse with water/methanol (95:5, v/v, 3 mL).

Analyte elution

Methanol (3 mL).

Post-extraction

Evaporate to dryness and reconstitute in water/methanol (50:50, v/v, 1 mL) for subsequent LC-MS/MS analysis.

HPLC conditions

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) (Agilent Technologies, Berkshire, UK).

Guard column

C8 guard column (Agilent Technologies, Berkshire, UK).

Mobile phase

0.1% aqueous formic acid and MeCN at a flow rate of 0.25 mL/min.

Gradient 90%

0.1% aqueous formic acid and 10% (v/v) MeCN at a flow rate of 0.25 mL/min increasing to 90% (v/v) MeCN over 7 minutes. The high concentration organic mobile phase was held for 1 minute then returned to the initial starting conditions.

Injection volume

10 μL.

Temperature

 Ambient temperature 

Mass spectrometry

Instrument

Ultima Pt triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis. Positive ions were acquired in the multiple reaction monitoring mode (MRM).

Desolvation temperature

350 °C

Ion source temperature:

100 °C

Collision gas pressure

2.9 x 10-3mbar

The base peak in each compound spectrum was attributed to the protonated, [M+H]+ or deprotonated molecular ions [M-H]-and were subsequently used as the precursor ions in the resulting MRM transitions. Positive/negative ion switching was utilized to analyse all the diuretics in this suite. Full MRM transitions and ionization conditions are shown in Table 1.

Figure 1: Recoveries (>80%, <10% RSD, n=5) for diuretics from urine. 

Note: This work was originally performed using EVOLUTE®   ABN 100 mg/10 mL cartridges.

Results

Ordering information