Using BioSPME for Free Fraction Analysis of Antiepileptic Drugs

By: Emily Barrey, David S. Bell, Craig Aurand and Candace Price, MilliporeSigma, Bellefonte, PA

Introduction

There are many reasons for therapeutic drug monitoring of antiepileptic drugs such as, evaluating levels when medication starts, establishing toxicity, reoccurrence of seizures after dormant period, and many more.1 Traditional sample preparation techniques are designed to determine total (bound+unbound) concentrations of drugs. However, the pharmacologically active portion of the drug is found in the fraction that is not bound to proteins (free fraction). This is the viable amount of drug that can cross the blood to brain barrier making methods to detect this concentration critical.2 Current methods for free fraction determination include rapid equilibrium dialysis, ultrafiltration, and ultracentrifugation. Ultracentrifugation is commonly employed for therapeutic drug monitoring but can be labor intensive and time consuming.2 Other sample preparation methods using saliva as the matrix enable for free fraction determination of the drugs but encounter obstacles with matrix suppression and tedious SPE methods.2

In this study, Biocompatible Solid Phase Micro Extraction (BioSPME) fibers were used to determine the free concentrations for pregabalin, levetiracetam, lamotrigine, felbamate, carbamazepine, and clonazepam in human serum and saliva samples. The BioSPME extraction eliminates the many steps in typical free concentration sample preparation methods, which reduces time of preparation and solvent use. Because this method directly measures the free fraction, various protein binding factors that impact total concentration determinations are not a concern. This microextraction technique offers a fast, simple method for the quantitation of multiple compounds at relevant therapeutic levels. BioSPME preparation followed by analysis on a biphenyl analytical column proved to be an accurate and precise method for the determination for a group of various antiepileptic drugs.

Process

BioSPME is an equilibrium extraction technique in which the analyte of interest partitions between the sample matrix and the extraction coating on a BioSPME device. The extraction coating contains functionalized silica particles that are embedded within a proprietary biocompatible binder (Figure 1). The role of this binder is to reduce or eliminate the extraction of matrix interferences during immersion, without reducing analyte extraction. This allows for the isolation of target analytes that are not protein bound, while minimizing the presence of matrix, resulting in a highly sensitive microextraction technique.

A commercially available LC tip BioSPME device which consists of a coated fiber housed within a pipette tip

Figure 1. (A) A commercially available LC tip BioSPME device which consists of a coated fiber housed within a pipette tip. (B) A basic schematic of an extraction performed with a BioSPME fiber. The fiber is coated with functionalized particles that have been embedded within a proprietary binder. The binder allows the fiber to be placed directly within a biological fluid for sampling.

 

Spikes were prepared at typical therapeutic ranges for each compound

*Spikes were prepared at typical therapeutic ranges for each compound:

 

  Conc. (ng/mL)
Carbamazepine 500
Clonazepam 50
Felbamate 5000
Lamotrigine 5000
Levetiracetam 5000
Pregabalin 2500

 

Automation

Utilizing an Apricot Designs Personal Pipettor, we were able to automate the entire extraction procedure. Figures 2 & 3 demonstrate how by using a custom head, the LC SPME tips were fixed and manipulated to their appropriate extraction stages. During the process, the condition plate had to be swapped out with the extraction plate.

Automation

 

Analytical Method

column:Ascentis® Express Biphenyl, 5 cm x 2.1 mm, 2.7 μm mobile phase: (A) 2mM ammonium formate with 0.1% formic acid in 5:95 ACN: Water, (B) 2mM ammonium formate with 0.1% formic acid in 95:5

ACN: Water
Flow rate: 400 μL/min
Column temp: 40 ºC
Det.: MS/MS, ESI (+), MRM transitions (Table 1)
Injection: 1 μL
Gradient: hold 0%B for 1 min, 0% to 80%B in 2 min, hold 80%B for 1 min, to 0%B in0.1 min and hold at 0%B for 1.9 min
instrument: Sciex 3200 Qtrap with Agilent 1290 UPLC

Representative chromatogram for Anitepileptic Drugs

Figure 2. Representative chromatogram for Anitepileptic Drugs.

 

Table 1. MRM Transitions for Anitepileptic Drugs
 

Analyte Precursor Product DP (V) CE (V) RT(min)
Carbamazepine 237.1 194.3 35 29 2.7
Carbamazepine-d10 247.1 204.2 35 29 2.7
Clonazepam 316.1 270.25 51 29 2.9
Clonazepam-d4 320.1 274.1 51 29 2.9
Felbamate^ 239.1 117 26 21 2.4
Lamotrigine^ 256 21.2 51 35 2.2
Levetiracetam 171.1 126.3 21 19 1.45
Levetiracetam-d6 177.2 133.1 21 19 1.45
Pregabalin 160.15 142 16 15 1.2
Pregabalin-d6 166.15 148 16 15 1.2

^Used carbamazepine-d10 as internal standard.

 

Calibration curve for lamotrigine prepared in saliva from 0.5 – 50 μg/mL

Figure 3. Calibration curve for lamotrigine prepared in saliva from 0.5 – 50 μg/mL.

 

Calibration curve for carbamazepine prepared in serum from 0.05 – 0.5 μg/mL

Figure 4. Calibration curve for carbamazepine prepared in serum from 0.05 – 0.5 μg/mL.

 

Results

  • Recoveries from saliva ranged from 93% to 119% for the various compounds except for pregabalin. Pregabalin was not detected in the BioSPME extracted samples. Pregabalin is very polar and probably does not extract very well on a C18 phase. Alternate chemistry may improve extraction of pregabalin.
  • Precision out of saliva was demonstrated with %RSD values less than 12% for all analytes except for pregabalin which was not detected.

Saliva

  • Recoveries from serum ranged from 92% to 108% for the various compounds except for pregabalin. As was seen in saliva, pregabalin was not detected in the BioSPME extracted samples.
  • Precision out of serum was demonstrated with %RSD values less than 11% for all analytes except for pregabalin which was not detected.

Serum

 

Summary

  • A simple 3 step extraction utilizing BioSPME fiber tips was developed for fast, reproducible detection of free fraction of antiepileptic drugs out of saliva and serum.
  • Using a biphenyl analytical column, all analytes were separated in less than 3 minutes with a total run time of 6 min.
  • Combining the automated microextraction technique with the fast analytical separation, therapeutic ranges of antiepiletic drugs can be evaluated as the free fraction with confidence.
  • Alternate chemistries may improve the extraction of pregabalin.

References

  1. Monitoring Epilepsy. (accessed November 2017)
  2. Patsalos, P., Berry, D., (2013) Therapeutic Drug Monitoring of Antiepileptic Drugs by Use of Saliva, Thera Drug Monit, 35, 4-29