Removal of Endogenous Matrix Components Using EVOLUTE® CX Mixed-Mode Cation Exchange Solid Phase Extraction

Sample preparation is an essential technique to remove unwanted matrix components prior to LC–MS-MS analysis of drugs in biological fluids. Plasma matrix components whether endogenous (salts, proteins, and phospholipids) or exogenous (dosing vehicles, e.g. PEG 400), can interfere with compounds of interest leading to regions of ion suppression or enhancement. This can lead to inaccurate quantitation and have adverse effects on sensitivity. Mixed-mode SPE provides cleaner extracts as a result of rigorous interference wash steps, afforded by the dual retention mechanism of the sorbents.

This application note from Biotage compares plasma extract cleanliness using EVOLUTE® CX with two other commercially available mixed-mode resin-based cation exchange SPE sorbents using their published generic methods.

EVOLUTE CX Sample Preparation

Analytes: Procainamide, salbutamol, atenolol, ranitidine, naltrexone, quinidine, metoprolol, brompheniramine, mianserin, aamitriptyline, and fluoxetine.

EVOLUTE CX Configuration: EVOLUTE CX 25 mg fixed-well plate (part number 601-0025-P01)

Sample Pre-treatment: Plasma sample (100 mL) was diluted with 50 mM ammonium acetate buffer at pH 6 (1:3, v/v)

Column Conditioning: Methanol (1 mL)

Column Equilibration: 50 mM ammonium acetate buffer at pH 6 (1 mL)

Sample Loading: Pre-treated plasma sample (400 mL) Interference Elution 1: 50 mM ammonium acetate buffer at pH 6 (1 mL)

Interference Elution 2: Methanol (1 mL)

Analyte Elution: 5% (v/v) NH4OH in methanol (1 mL)

HPLC Conditons

Instrument: Waters 2795 Liquid Handling System (Waters Assoc., Milford, MA).

Column: Zorbax Eclipse XDB C18 3.5 μm analytical column (50 × 2.1 mm id, 3.5 μm) (Agilent Technologies, Berkshire, UK).

Guard Column: C8 guard column (Agilent Technologies, Berkshire, UK).

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

Gradient: The gradient conditions were set to 90%, 0.1% (v/v) formic acid aq and 10% MeCN increasing to 100% MeCN over 5 min. Initial starting conditions were resumed at 5.1 min.

Injection Volume: 15 μL

Temperature: Ambient

Chromatographic Ion Suppression

Blank human plasma samples (100 μL) were extracted as in the published generic methods listed and the elution fractions evaporated to dryness (n=5). The dry extracts were spiked with 5 ng/mL of the basic analytes and reconstituted in 500 μL of 80:20 (v/v) H₂O/MeOH for LC–MS-MS analysis. Contact Biotage for full details (1).

Results

Chromatographic ion suppression experiments were run to show the effect on analytes at various retention times during the chromatographic run. Analyte suppression effects were measured and reported (see Figure 1) as a matrix factor (MF):

Figure 1: Comparison of matrix factors for various analytes (spike concentration 5 ng/mL) using different commercially available sorbents.

Peak area response in presence of matrix ions (extracted samples)

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Peak area response in absence of matrix ions (standard)

Conclusions

EVOLUTE CX Mixed-mode Cation Exchange SPE provides significant advances in extract cleanliness using a single robust generic method, compared to other commercially available mixed-mode cation exchange sorbents.

EVOLUTE is a registered trademark of Biotage.

References

(1) Poster Presented at ASMS 2008: "Evaluation of Plasma Extract Cleanliness using Various Commercially Available Mixed-mode Resin-based Cation Exchange SPE Sorbents." Lee Williams, et al.

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