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Determination of Pregabalin in Plasma by Cleanert Protein Precipitation Plate

Application Introduction

Pregabalin, as a new type of GABA receptor agonist, is used for not only anticonvulsant treatment and adjuvant therapy for localized epilepsy,

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Introduction

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

Experimental

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.

Instrumentation

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

Total Time(min)

Flow Rate(µL/min)

A (%)

B (%)

0

200

80

20

4

200

80

20

Table 2 Quantifier and qualifier MRM transitions and retention times for Pregabalin

Compound

Retention   time

Q1

Q3

DP

CE

Pregabalin

1.9   min

160.2

142.1

50

15

125

19

97

21

Results and Discussion

Chromatograms

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

Concentration (ng/mL)

Average recovery (n=8)

RSD (n=8)

 

LOD (ng/mL)

25

96.8%

3.12%

0.3

100

95.9%

5.05%

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.

Conclusions

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.

Order Information

Products

Specification

Cat.No

Cleanert  Protein Precipitation Plate

96 wells, 2 mL/well

96CD2025-Q

96-well Collection   plate

2.2 mL Square   well

96SP2036-2

Cleanert®   M96 Positive Pressure Device

Adapt to 96-well   plate

SPE-M96

Unisol C18(2)

2.1 × 50 mm, 5 μm,   110 Å

UO950502-2

 

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