Olga I. Shimelis, PhD Senior R&D Manager, Advanced Sample Preparation MilliporeSigma A business of Merck KGaA, Darmstadt, Germany
Candace L. Price, MBA Global Product Manager, Sample Prep – SPE, SLE, and BioSPME MilliporeSigma A business of Merck KGaA, Darmstadt, Germany
Bioavailability is a key attribute of lead compounds and drug candidates. Usually only a fraction of the substance administered to a patient exerts its therapeutic effect. Much of it binds to proteins such as serum albumin and α-acid glycoprotein in blood circulation, a phenomenon known as plasma protein binding (PPB). This disables the molecule’s therapeutic activity as it is too large and cannot pass through membranes in its protein-bound state. Determining PPB levels in early drug discovery is important to understand the pharmacokinetics, pharmacodynamics and toxicity of a drug, and the results strongly influence the eventual dosage. While liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) ultimately detects and quantifies the analyte, the method used to simulate PPB levels in humans and to generate the samples is key. Several such methods exist, among which equilibrium dialysis is the most widely used. However, a new technology based on solid-phase microextraction has emerged in recent years. This article compares the two.
Equilibrium dialysis: the traditional method
In a typical equilibrium dialysis (ED) protocol analyte-spiked plasma and buffer are loaded into corresponding dialysis chambers separated by a semipermeable membrane with a molecular weight cut-off that will retain the protein but allow the small molecule drug to pass through freely. Some of the analyte drug will bind to the protein and some will remain unbound. Equilibrium is reached when the concentration of the unbound analyte is the same in both chambers. The plasma chamber, however, will contain additional analyte that is bound to proteins, depending on the affinity of the interaction. After dialysis, there are a few pipetting steps and a centrifugation step to perform before LC-MS/MS analysis can proceed.
ED, however, has several drawbacks, among them that the entire workflow usually takes six to eight hours. Even “rapid” variants of ED, which use special devices, require about four hours for equilibration alone—how long exactly is often unknown, and terminating dialysis early risks that the system will not have come to equilibrium. Although ED is generally accurate and reproducible, its protein chamber can become diluted if volumes shift, possibly resulting in protein leakage. The method is also susceptible to the Donnan effect, whereby charged particles near a semipermeable membrane can fail to distribute evenly across its two sides, and significant amounts of phospholipids with the potential for matrix effects remain after dialysis. Furthermore, some drug molecules show an affinity to the semipermeable membrane, which can impair result quality.
BioSPME: the innovative technique
Solid-phase microextraction (SPME), when applied in bio- analytics, has become known as BioSPME. It is based on the ability of a solid-phase adsorbent to reversibly bind a certain proportion of unbound small molecule analyte, but none of the much larger protein-bound analyte. In MilliporeSigma’s
SupelTM BioSPME 96-Pin devices this adsorbent is coated onto pins as a thin layer of C18- bonded silica particles in combination with a biocompatible polymeric adhesive. These pins are configured as 96-pin devices to allow usage with 96-well microplates, and as such with automation robots. The protocol is that, after a conditioning step, the coated pins are immersed in analyte-spiked plasma for 15 minutes to adsorb free (but not bound) analyte. After a short wash step to prevent co-extracting large proteins the pins are immersed in a desorption solution, where all the bound analyte is released. Analyte- spiked buffer, in which no protein binding can occur and all the analyte is considered to be free, is simultaneously processed in the same way to calculate the percentage of protein binding. The entire workflow takes only 2 hours.
BioSPME has limitations as well. Whether or not enough of an analyte adsorbs reversibly to the coating depends on its size and hydrophobicity. This is why the SupelTM BioSPME 96-Pin device technique is less suitable for analytes with a molecular weight above 2 kDa or a logP outside the range of 1 to 5, ruling out hydrophilic molecules in particular.
How do rapid ED and BioSPME compare?
Comparative studies using APIs as the analytes have shown the PPB results obtained by BioSPME to agree closely with both rapid ED results and the average values reported in the literature, so with respect to precision and accuracy almost nothing sets the methods apart. Both are also robust and deliver reproducible results. BioSPME is at a significant advantage for time-to-result and hands-on time, and even more so when processing large numbers of samples as the method can be automated. It is also preferable where ED gives rise to concerns over volume shifts, the Donnan effect, phospholipids, or drug affinity to the dialysis membrane, all of which are no issues with BioSPME. ED, on the other hand, is the better choice for hydrophilic or larger analytes sized 2 kDa or more.
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