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Sensitive determination of plasma protein binding of cationic drugs using mixed-mode solid-phase microextraction.

Journal of pharmaceutical and biomedical analysis (2015-08-28)
Hester Peltenburg, Ingrid J Bosman, Joop L M Hermens
ABSTRACT

Freely dissolved concentrations are considered to be the most relevant concentration in pharmacology and toxicology, as they represent the active concentration available for interaction with its surroundings. Here, a solid-phase microextraction (SPME) coating that combines octadecyl and propylsulfonic acid groups as strong cation exchange sites, known as C18/SCX or "mixed-mode" SPME, is used to measure freely dissolved concentrations of amitriptyline, amphetamine, diazepam and tramadol to different binding matrices, including bovine serum albumin (BSA), human serum albumin (HSA), human plasma and human whole blood. A potential confounding factor in binding studies is that proteins may sorb to the fiber coating leading to incorrect measurement of protein sorption or changes in uptake kinetics to the fiber coating. Sorption of bovine serum albumin (BSA) was observed and quantified using a Lowry assay. BSA binds to the C18/SCX fiber in small amounts, but large changes in uptake kinetics were not observed. All experiments were performed at equilibrium. In addition, however, the effect of depletion and non-equilibrium extraction on the estimation of protein binding affinities was also studied. Binding affinities to BSA and human serum albumin (HSA) were calculated as log KBSA or log KHSA. These values were very similar to reported literature values. Sampling at either equilibrium or non-equilibrium resulted in similar binding affinities. Furthermore, SPME fibers were used to measure freely dissolved concentrations in undiluted human plasma and whole blood. Analysis of SPME extracts could be performed using HPLC-UV or HPLC with fluorescence detection without prior clean-up of the samples. Measured bound fractions in plasma using this SPME approach were comparable to literature reference values. Bound fractions in whole blood were always higher than in plasma, due to red blood cell partitioning. This work shows the potential of SPME as sampling tool for freely dissolved concentrations, especially for highly protein-bound compounds. Conventional SPME coatings such as polyacrylate (PA) or polydimethylsiloxane (PDMS) might be lacking sensitivity when sampling the small neutral fraction of highly protein-bound positively charged compounds, but the C18/SCX fiber is able to sorb the charged species of organic cations, thereby improving sensitivity for these types of compounds.

MATERIALS
Product Number
Brand
Product Description

Supelco
SPME LC Tips, PDMS/DVB, pkg of 96 ea
Supelco
SPME-OC Fiber Assembly, Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), df 75 μm (Coating thickness includes 65 μm coating + 10 μm OC (overcoating)), needle size 23 ga, pkg of 3 ea, fiber L 1 cm
Supelco
IonSense® C18 SPE-it Tips, pkg of 96 ea
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), metal alloy fiber, df 100 μm (PDMS), needle size 23 ga, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 23 ga, for use with autosampler
Supelco
SPME fiber assembly polyacrylate (PA), df 85 μm (PA), for use with autosampler, needle size 23 ga
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 30 μm (PDMS), needle size 24 ga, for use with autosampler
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), for use with manual holder, needle size 23 ga
Supelco
SPME fiber assembly polyacrylate (PA), df 85 μm (PA), for use with manual holder, needle size 24 ga
Supelco
SPME fiber assembly polyacrylate (PA), df 85 μm (PA), for use with autosampler, needle size 24 ga
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 30 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 24 ga, for use with autosampler
Supelco
SPME Portable Field Sampler, coating PDMS
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 7 μm (PDMS), needle size 24 ga, for use with manual holder
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), for use with autosampler, needle size 24 ga
Supelco
SPME Portable Field Sampler, coating CAR/PDMS
Supelco
SPME Sampling Stand, for use with 4 mL vials
Supelco
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (PDMS), for use with autosampler, needle size 23 ga
Supelco
SPME Portable Field Sampler, coating PDMS/DVB
Supelco
SPME Sampling Stand, for use with 15 mL vials
Supelco
SPME fiber assortment kit 5, needle size 23 ga, for use with autosampler
Supelco
SPME fiber assembly, Carbowax-Polyethylene Glycol (PEG) Coating, needle size 23 ga, df 60 μm (PEG), for use with autosampler
Supelco
SPME fiber assembly, Carbowax-Polyethylene Glycol (PEG) Coating, needle size 23 ga, df 60 μm (PEG), metal alloy fiber, for use with manual holder
Supelco
SPME StableFlex fiber assortment kit, for use with autosampler, needle size 24 ga
Supelco
SPME fiber assortment kit 2, for use with manual holder, needle size 24 ga
Supelco
SPME fiber assortment kit 4, for use with autosampler, needle size 23 ga
Supelco
SPME fiber assortment kit 2, for use with autosampler, needle size 23 ga
Supelco
SPME Fiber Assembly Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), df 65 μm (PDMS/DVB), needle size 23 ga, PDMS/DVB StableFlex, for use with autosampler
Supelco
SPME StableFlex fiber assortment kit, needle size 23 ga, for use with autosampler