Organic solvent-based assays

In situations where the analyte is poorly soluble in aqueous buffers, the use of organic solvents may even be beneficial. One such example is determination of cyclosporin, which is a potent immunosuppresive drug used in transplant surgery 

SUMMARY OF STUDIES ON COMPETITIVE LIGAND BINDING EXPERIMENTS EMPLOYING MIPS 

Analyte Incubation medium Concen- Limit of Reference 

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Andersson, L.I. (1996) Application of molecular imprinting to the development of aqueous buffer and organic solvent based radioligand binding assays for (5) propranolol. Analytical Chemistry, 68, 111 117. 

The crude ketal from the Birch reduction is dissolved in a mixture of 700 ml ethyl acetate, 1260 ml absolute ethanol and 31.5 ml water. To this solution is added 198 ml of 0.01 Mp-toluenesulfonic acid in absolute ethanol. (Methanol cannot be substituted for the ethanol nor can denatured ethanol containing methanol be used. In the presence of methanol, the diethyl ketal forms the mixed methyl ethyl ketal at C-17 and this mixed ketal hydrolyzes at a much slower rate than does the diethyl ketal.) The mixture is stirred at room temperature under nitrogen for 10 min and 56 ml of 10% potassium bicarbonate solution is added to neutralize the toluenesulfonic acid. The organic solvents are removed in a rotary vacuum evaporator and water is added as the organic solvents distill. When all of the organic solvents have been distilled, the granular precipitate of 1,4-dihydroestrone 3- methyl ether is collected on a filter and washed well with cold water. The solid is sucked dry and is dissolved in 800 ml of methyl ethyl ketone. To this solution is added 1600 ml of 1 1 methanol-water mixture and the resulting mixture is cooled in an ice bath for 1 hr. The solid is collected, rinsed with cold methanol-water (1 1), air-dried, and finally dried in a vacuum oven at 60° yield, 71.5 g (81 % based on estrone methyl ether actually carried into the Birch reduction as the ketal) mp 139-141°, reported mp 141-141.5°. The material has an enol ether assay of 99%, a residual aromatics content of 0.6% and a 19-norandrost-5(10)-ene-3,17-dione content of 0.5% (from hydrolysis of the 3-enol ether). It contains less than 0.1 % of 17-ol and only a trace of ketal formed by addition of ethanol to the 3-enol ether. 

One decade has passed since the parallel artificial membrane permeation assay (PAM PA) was first introduced in 1998 [47]. Since then, PAM PA rapidly gained wide popularity in drug discovery [3, 48-51]. Today, PAMPA is the most widely used physicochemical membrane permeation model. The term PAMPA is nowusedas the general name for a plate-based (HTS enabled), biter-supported (filter immobilized) artificial membrane. Typically, phospholipids dissolved in an organic solvent are impregnated into the filter to construct a PAMPA membrane. 

MIP assays can also be utilized in synthetic organic applications. For example, MIP-based assays have been used to measure the chiral purity of samples in organic solvents. An L-phenylalanine anilide (l-PAA) imprinted polymer was utilized as a recognition element to measure the enantiomeric excess (ee) of PAA samples (Chen and Shimizu 2002). The MIP displays greater capacity for l-PAA versus d-PAA samples of similar concentration, and this difference was used to estimate enantiomeric excess. The enantiomeric excess of an unknown solution was determined by comparing the UV absorbance of the PAA remaining in solution after equilibration against a calibration curve. This MIP assay was demonstrated to be rapid and accurate with a standard error of +5% ee. 

The assay solvent must be selected to maximize the differences between the amount of probe bound to the MIP and to the NIP (or the CP), considered to be entirely non-specific. When the assay is optimized in aqueous solutions, a small amount of nonionic surfactant such as Triton X-100 (0.5%, w/v) or miscible organic solvents, such as ethanol, may be added to increase polymer wettability. The additive may also help to reduce hydrophobic interactions, especially for MIPs based on ethylenglycoldimethacrylate (EGDMA) or divinylbenzene (DVB) [5], Competitive assays are usually carried out only with the MIP, although it is advisable to check that no competition is observed when the NIP/CP is used instead. 

Napoli KL (2006) Organic solvents compromise performance of internal standard (ascomycin) in proficiency tesing of mass spectrometry-based assay for tacrolimus. Clin Chem 52 765-766... 

Although PPE is the most efficient and inexpensive extraction technique, it is also the most nonspecific extraction procedure which is known to be susceptible to matrix effect for LC-MS/MS assay. In contrast, LLE provides a much cleaner extract. LLE, also known as solvent extraction and partitioning, separates analytes based on their relative solubility in two different immiscible solvents, usually water and an organic solvent. The most commonly used solvents for LLE are ethyl acetate (EtOAc), methyl ferf-butyl-ether (MtBE), methylene chloride (CH2C12), and hexane or the combination of the above solvents. In order to manipulate the polarity of the analytes, often a volatile acid or base such as FA or NH4OH respectively at 5-10 %... 

Incomplete solubility and associated point quenching constitute a major problem in scintillation counting. Efficient scintillation counting requires that the sample be fully soluble in the excitable organic solvents of the scintillation fluid. However, biological systems, which are usually aqueous systems or assays, frequently contain water or hydrophilic molecules that will not dissolve in standard toluene-based scintillation cocktails. 

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