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Biotage Application Note
This application note describes an optimized and effective supported liquid extraction sample preparation protocol for the extraction of a range of opiates from human urine using ISOLUTE SLE+. Supported liquid extraction is an efficient and cost effective alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced sample preparation time.
This application note outlines the procedure using the ISOLUTE SLE+ 1 mL sample volume column (part number 820-0140-C) optimized for a 1 mL pretreated urine sample volume (other formats are available). Method parameters and dilution factors have been optimized to maximize recoveries and minimize ion suppression.
Sample pre-treatment: To 1 mL of urine add 100 mM ammonium acetate pH 5 (950 µL) and β-glucuronidase (50 µL) enzyme (5000 µ/mL). Hydrolyse with heat in a water bath at 60 °C for 2 hours. Cool and add 25% aqueous ammonium hydroxide (from a 28–30% concentrated solution) (10 µL) and mix.
Sample load: Load pre-treated sample (1 mL) to plate followed by a pulse of vacuum to initiate flow and leave for five minutes.
Analyte elution: Elute with dichloromethane:isopropanol (95:5, v/v) (2.5 mL). Leave to flow under gravity for 5 minutes, then follow with a further aliquot of dichloromethane:isopropanol (95:5, v/v) (2.5 mL), apply a short pulse of vacuum.
Post extraction: Evaporate to dryness at room temperature (80 L/min) and reconstitute in 0.1% formic acid (aq) (500 µL).
Instrument: Waters Acquity UPLC interfaced to a Quattro Premier XE triple quadrupole MS using electrospray ionization
Column: Acquity BEH C18 100 × 2.1 mm × 1.7 µ
Mobile phase: A: 0.1% formic acid (aq)
B: 0.1% formic acid in methanol
For gradient see Table 1
Flow-rate: 0.40 mL/min
Injection: 15 µL (partial loop, needle overfill)
Sample temp: 20 °C
Column temp: 40 °C
Source temp: 150 °C
Desolvation temp: 450 °C
Sample recoveries of over 90% for all analytes except 6-monoacetylmorphine (>70%) with RSDs below 10% were achieved at 2 ng/mL LOQ. Figure 1 shows the mass chromatogram for morphine.
Table 1: UPLC Gradient.
This method demonstrates a robust and effective extraction of a full range of opiates from the challenging biological matrix of human urine with hydrolysis. A significantly lower LOQ than 2 ng/mL could be achieved with more sensitive mass spectrometry and/or lower reconstitution volumes.
Figure 1: Example mass chromatogram for morphine extracted at 2 ng/mL.
1. R. Jones, L. Williams et al., Application Note AN741 (available from www.biotage.com/applications).
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