Extraction of acidic, basic and neutral drugs from plasma using ISOLUTE® PLD+

Introduction

This application note describes the use of the ISOLUTE® PLD+ Protein and Phospholipid Removal Plate for clean-up of a range of acidic, basic and neutral drugs from plasma.

Figure 1. Structures of acetaminophen (neutral), ketoprofen (acidic) and amitryptiline (basic): examples of the broad range of analytes extracted in this application
 
ISOLUTE® PLD+ Protein and Phospholipid Removal Plates provide very effective but extremely simple sample clean- up for LC-MS/MS analysis. Requiring next to no method development, ISOLUTE PLD+ can be integrated quickly and easily into routine workflow, increasing productivity and reducing instrument downtime. ISOLUTE PLD+ plates remove >99% of plasma proteins and phospholipids, the main causes of ion suppression, leading to cleaner extracts and increased sensitivity (signal to noise) for a broad range of analytes.

Analytes

Acetaminophen, amitriptyline, atenolol, bretylium tosylate, brompheniramine, fluoxetine, metoprolol, mianserin, naltrexone, procainamide, quinidine, ranitidine, salbutamol, sulindac, p-toluamide and ketoprofen.

Sample preparation procedure

Format:

ISOLUTE® PLD+ Protein and Phospholipid Removal Plate, 50 mg, part number 918-0050-P01 Sample Pre-treatment: If required, spike plasma samples with appropriate internal standards (typically 10 µL volume) and mix thoroughly. Note: no internal standards were used in this study.

Sample clean up:

Ensure collection plate is in position before processing

Step 1:

Dispense acetonitrile (400 µL) into each well

Step 2:

Dispense plasma (100 µL) into each well. Mix thoroughly using vortex mixing for 30 s or repeat aspirate/dispense steps

Step 3:

Apply vacuum (–0.2 bar) or positive pressure (2–3 psi) until sample is fully eluted (5 min). For extremely viscous samples e.g. dog plasma, the vacuum/positive pressure required for adequate flows may be higher.

Post extraction:

Evaporate to dryness (SPE Dry, 40oC, 40 mins)

Reconstitution:

Reconsititute in 0.1% Formic acid aq/MeOH (80/20, v/v, 200 µL) prior to analysis
 

HPLC conditions

Instrument:

Waters 2795 Liquid Handling Unit

Cartridge:

Phenomenex Kinetex XB-C18 (50 x 2.1mm, 2.6 µ)

Mobile phase:

A: 0.1% Formic acid aq (v/v) B: MeCN

Flow rate:

0.3 mL/min

Injection volume:

10 µL

Sample temperature:

20 °C

Cartridge temperature:

Ambient

Mass spectrometry conditions

Instrument:

Waters Ultima Pt Triple Quadrupole Mass Spectrometer

Desolvation Temperature:

350 °C

Ion source temperature:

100 °C

Collision cell pressure:

2.7 e-3 mbar


Positive ions were acquired in the multiple reaction monitoring (MRM) mode.

Results

Good analyte recovery, reproducibility and extract cleanliness were achieved for a broad range of analytes using ISOLUTE® PLD+ Protein and Phospholipid Removal Plates, allowing quantitation of analytes at low levels. Table 3 shows recovery and reproducibility (RSD generally <10%) for the range of analytes. Figure 2 illustrates the MRM chromatogram for amitryptilline at a concentration of 20 pg/mL in plasma (s/n 57:1).

Analyte recovery

*pK and logP values were obtained from the literature, or values were calculated if not available

Figure 2. Offset MRM chromatogram of acidic, basic and neutral drugs spiked at 20 ng/mL into plasma and extracted using the protocol described on page 1. Analyte elution order and analytical conditions as described in Table 2.

Phospholipid removal

The effective protein and phospholipid removal obtained using ISOLUTE PLD+ Protein and Phospholipid Removal Plates provided clean extracts with very low matrix effects. Residual phospholipids were investigated to provide an indication of extract cleanliness. We investigated the most abundant phospholipids (selected from full scan, SIR and precursor ion scanning experiments) using MRM transitions monitoring the common 184 product ion. Figure 3 demonstrates phospholipid content comparing protein precipitated plasma, solvent blank and the final extraction protocol.
Figure 3. MRM TICs showing phospholipid content of plasma prepared by a) protein precipitation at a 1:4 ratio of plasma : acetonitrile and b) ISOLUTE® PLD+ under the same conditions. >99% of plasma phospholipids are removed, allowing low level quantitation of analytes.

Conclusion

ISOLUTE® PLD+ Protein and Phospholipid Removal Plates are suitable for clean-up of a range of analytes with widely differing functionality and polarity characteristics from plasma, giving high recoveries, good reproducibility and excellent extract cleanliness.

Ordering information

Literature number: AN830