Pregabalin, as a new type of GABA receptor agonist, is used for not only anticonvulsant treatment and adjuvant therapy for localized epilepsy, but also the treatment of diabetic peripheral neuropathic pain as well as neuralgia caused by the herpes virus infection. It is a safe and effective antiepileptic drug with high therapeutic index, less adverse reaction and huge market potential. In this paper, the Cleanert® Protein Precipitation Plate is used to extract Pregabalin from the plasma that is a complex matrix, ensuring the high recovery rate and good experimental reproducibility of Pregabalin in analytical detection.
Figure 1. Structure of pregabalin
SPE Procedure with Positive Pressure SPE-M96
Preparation: Place the Protein Precipitation Plate wells on a collection plate.
Add Solvent: Add 500 μL methanol to all Plate wells.
Load Sample: 100 μL mixture of sample;
Mix: Mix the sample with pipette or on a vortexer;
Filter: Mixed the sample with pipette or vortexer and put the plate onto SPE-M96 positive pressure device, adjust the pressure, collect the elution into collection plate.
Reconstitute: Add 400 μL 0.1% formic acid in water to elution.
Column: Venusil® MP C18(2) (2.1 ´ 50mm, 5μm, 110 Å);
Flow rate: 200 μL/min;
Temperature: 30 °C;
Injection: 5 μL;
Mobile phase: A-0.1% formic acid in water; B-Methanol;
Mode: Sciex 4000+
Scanning mode: Positive;
Scan type: MRM;
Ion Spray Voltage: 4500V;
Curtain Gas: 30 Psi;
Ion Source Gas 1: 60 Psi; Ion Source Gas 2: 30 psi;
Temperature: 450 °C
Table 1 HPLC gradient parameters
Table 2 Quantifier and qualifier MRM transitions and retention times for Pregabalin
Results and Discussion
The chromatograms of Pregabalin with concentration of 25 ng/mL is shown in Figure 2.
Peak A: Standard Solution Peak B: Blank Plasma Peak C: Spiked Plasma
Figure 2 Chromatograms of the samples spiked with 25 ng/mL Pregabalin
Table 3 Recovery of Pregabalin in plasma
Average recovery (n=8)
Table 3 shown that the average recoveries of Pregabalin were 96.8% and 95.9%, at the concentration of 25 ng/mL and 100 ng/mL, respectively. And RSD are 3.12% and 5.05% at a concentration of 25 ng/mL and 100 ng/mL, respectively.
The result demonstrated good and recovery of Cleanert® Protein Precipitation Plate with specification of 2 mL/well. Cleanert® Protein Precipitation Plate (P/N: 96CD2025-Q) show high-performance in the sample preparation process which could be used in DMPK and clinical research.
Cleanert Protein Precipitation Plate
96 wells, 2 mL/well
96-well Collection plate
2.2 mL Square well
Cleanert® M96 Positive Pressure Device
Adapt to 96-well plate
2.1 × 50 mm, 5 μm, 110 Å