Determination of Pregabalin in Human Plasma by SPE-LC-MS/MS Using SOLA CX Mixed Mode Solid Phase Extraction Cartridges and an Accucore PFP HPLC Column

A simple, rapid and sensitive procedure for the determination of pregabalin in human plasma by liquid chromatography-tandem mass spectrometry was developed and evaluated. The drug was isolated from a plasma matrix using SOLA CX solid phase extraction material, and the components of the resultant extracts were separated on an Accucore PFP HPLC column under reversed-phase, gradient conditions. Detection was performed on a triple quadrupole mass spectrometer using positive polarity, heated electrospray ionisation (HESI) conditions operating in selected reaction monitoring (SRM) mode. Gabapentin was used as the internal standard. Good chromatographic peak shape and linearity over the dynamic range 1 to 250 ng/mL was achieved with excellent recovery and precision. coverage, robust phases. This coverage results in a significant reduction in secondary interactions and thus highly efficient peaks with very low tailing. The Accucore PFP phase provides extra retention for halogenated species and unique selectivity for non-halogenated compounds orthogonal selectivity to the C18 phase. Pregabalin is used to relieve neuropathic pain (pain from damaged nerves) that can occur with diabetic patients or patients who are suffering from shingles (a painful rash that occurs after infection with the herpes zoster virus). It is also used to treat fibromyalgia (a long-lasting condition that may cause pain, muscle stiffness and tenderness, tiredness, and difficulty falling asleep or staying asleep). Pregabalin is used with other medications to treat certain types of seizures in people with epilepsy. Pregabalin is in a class of medications called anticonvulsants, and works by decreasing the number of pain signals that are sent out by damaged nerves in the body1. The purpose of this particular study is to demonstrate the effectiveness of a SOLA CX solid phase extraction material and an Accucore PFP HPLC column for the determination of pregabalin in human plasma by liquid chromatography-tandem mass spectrometry using gabapentin as an internal standard. Figure 1 shows the structures of pregabalin and gabapentin. 2 Experimental Details Chemicals and Reagents Part Number Fisher Scientific Optima LC/MS grade methanol A456-1 Water, from TKA Water Purification System Formic acid Pregabalin from Ind-Swift Gabapentin from sigma Human plasma with CPD Sample Handling Equipment Part Number Thermo Scientific FinnPipette (100-1000 μL) 642090 Thermo Scientific FinnPipette (10-100 μL) 4642070 Thermo Scientific FinnPipette (1-10 μL) 4642040 Vials and Closures Part Number Thermo Scientific Micro+TM Vial 300 μL, Fused Insert 60180-507 Thermo Scientific 9mm Screw Top Cap W/ PTFE/Silicone septa 60180-516 Solid Phase Extraction Part Number Thermo Scientific SOLA CX 10 mg/1 mL cartridge 60109-002 Preparation of samples: Calibration standards: A stock solution of pregabalin was prepared in 50:50 (v/v) methanol / water at a concentration of 1 mg/mL. Secondary standards (SS1 – SS9) were prepared by subsequent serial dilution of the pregabalin stock solution in 50:50:0.1 (v/v) methanol / water / formic acid. A stock solution of the internal standard, gabapentin was prepared in 50:50 (v/v) methanol / water at a concentration of 0.1 mg/mL. Further dilutions, were prepared in 50:50:0.1 (v/v) methanol / water / formic acid. Plasma spiked calibration standards of pregabalin were prepared at nine different concentration levels (1, 2, 5, 25, 75, 125, 175, 225 and 250 ng/mL) by fortification of plasma (285 μL) with 15 μL of appropriate stock standard. The internal standard (gabapentin) was added (30 μL) at the 100 ng/mL level into each of the calibrants. Standards (S1-S9) were prepared and S1 and S9 were in duplicate. The concentration range selected was determined by targeting a lower limit of quantification as 1 ng/mL (< 1% of C max , 2.15 μg/mL). Further samples were prepared to allow for calculation of precision, recovery and matrix effect. A mid-range concentration was prepared six times and subjected to the same extraction procedure to determine Figure 1: Structures of pregabalin A. and gabapentin (IS) B. H2N O