Extraction of Basic Drugs from Plasma Using EVOLUTE CX 96-Well Plates and Columns

This application note describes the extraction of a variety of basic drugs from plasma using EVOLUTE CX mixed-mode resin-based SPE. The analyte suite includes basic drugs with wide ranging pK_a and logP values.

The sample preparation method and suggested analytical procedures are detailed within this note. Recoveries and %RSD are shown in Figure 1.

Figure 1

Sample Preparation Procedure

Analytes

Procainamide, salbutamol, atenolol, ranitidine, naltrexone, quinidine, metoprolol, brompheniramine, mianserin, amitriptyline, and fluoxetine.

EVOLUTE CX configuration

EVOLUTE Array CX 25 mg/1 mL plate (part number 601-0025-RP).

EVOLUTE ABN procedure

Sample: Plasma spiked at 50 pg/μL concentration with the compounds listed above

Sample pretreatment: Dilute plasma sample [1:3 (v/v), 100 μL] with 0.05 M NH₄OAc, pH 6 (total analyte load 5 ng)

Column conditioning: Methanol (1 mL)

Column equilibration: 0.05 M NH₄OAc, pH 6 (1 mL)

Sample application: Apply diluted sample (400 μL)

Interference elution: Wash 1. Rinse with 0.05 M NH₄OAc, pH 6 (1 mL) Wash 2. Rinse with methanol (1 mL)

Analyte elution: Methanol/NH₄OH [95:5 (v/v), 1 mL]

Post extraction: Evaporate to dryness and reconstitute in water/methanol (80:20, 1 mL) for subsequent LC–MS–MS analysis

For general guidelines on the use of EVOLUTE CX SPE plates and columns, request chemistry data sheet TN139 EVOLUTE CX columns for solid-phase extraction in bioanalysis.

HPLC conditions

Instrument: Waters 2795 Liquid Handling system (Waters, Milford, Massachusetts)

Column: Zorbax Eclipse XDB C18 3.5 μm analytical column (100 3 2.1 mm i.d.)

Guard column: C8 guard column (both Agilent Technologies, Berkshire, United Kingdom)

Mobile phase: 0.1% formic acid aq and MeCN at a flow-rate of 0.25 mL/min

Gradient: 90:10 increasing to 20:80 (v/v) over 6.2 min. At 6.3 min initial starting conditions were applied.

Injection volume: 15 μL

Temperature: Ambient temperature

Mass spectrometry

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

Desolvation temp: 350°C

Ion source temp: 100°C

Collision gas pressure: 2.3 × 10^–3 mbar.

The base peak in each compound spectrum was attributed to the protonated molecular ions, [M+H]¹ and were subsequently used as the precursor ions in the resulting MRM transitions. Full MRM transitions and ionization conditions are shown in Table I.

Table I: Quattro Ultima Pt mass spectrometer parameters

Results

EVOLUTE is a registered trademark of Biotage AB.

Table II: Percent average recovery and RSD of basic drugs from plasma

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