Enantioselective analysis of pheniramine in urine by charged CD‐mediated CZE provided with a fiber‐based DAD and an on‐line sample pretreatment by capillary ITP

Abstract

Application potentialities of CZE on-line coupled with capillary ITP and DAD to the identification and determination of trace concentration levels (microg/L) of pheniramine (PHM) enantiomers and their metabolites present in complex ionic matrices of biological origin (urine) are shown. An enhanced (enantio)selectivity of the CZE separation system obtained by the addition of carboxyethyl-beta-CD (CE-beta-CD) to the carrier electrolyte provided CZE conditions for a reliable identification of similar/identical DAD spectra of structurally related compounds (PHM enantiomers and their metabolites) in clinical urine samples differing in qualitative and quantitative composition of sample matrix constituents. A high sample loadability (a 30 microL sample injection volume), partial sample clean-up (removing macroconstituents from the sample), and preconcentration of the analytes in ITP stage resulted in the decrease of concentration LOD for PHM enantiomers in urine to 5.2 and 6.8 microg/L (2.2 x 10(-8) and 2.8 x 10(-8) mol/L), without using any sample pretreatment technique. The background correction and smoothing procedure applied to the raw DAD spectra provided analytically relevant DAD spectra of PHM enantiomers and their metabolites also when they were present in urine sample (30 microL injection volumes of ten-times diluted urine sample) at a 9 x 10(-) (8) mol/L concentration. DAD spectra of PHM enantiomers present in urine samples matched their reference spectra with reasonable certainties. DAD spectra of PHM metabolites were compared with the reference spectra of PHM enantiomers and a good match was found which indicates the similarities in the structures of enantiomers and their metabolites detected in the urine samples. This fact allows performing the quantitative analyses of PHM metabolites in the urine samples by applying the calibration parameters of PHM enantiomers also for PHM metabolites and the results show the possibilities of using the ITP-CZE-DAD combination for the direct analysis of PHM enantiomers and/or their metabolites in urine without any sample pretreatment. ITP-CZE-DAD method with oppositely charged selector is suggested to use in clinical research as it provides favorable performance parameters including sensitivity, linearity, precision, recovery, and robustness with minimal demands on sample preparation.

Column Coupling Electrophoresis in Biomedical Analysis

“…It is because such method provides probably one of the most acceptable ratio simplicitycost: universality-concentration LOD in comparison to other column coupling methods and detection systems. This suggestion is supported by many advanced applications of the ITP-CZE-UV method in the pharmaceutical and biomedical field (Marák et al, 2007;Mikuš et al, 2008aMikuš et al, , 2008bMikuš et al, , 2008cMikuš et al, , 2009. Jumps in voltage (conductivity) between neighboring zones result in permanently sharp boundaries between zones (Fig.…”

“…This analytical approach enabled us to obtain pure zones of the drugs enantiomers (without the need of the sample pretreatment). DAD spectra of PHM metabolites were compared with the reference spectra of PHM enantiomers (Marák et al, 2007;Mikuš et al, 2008c) and a very good match was found which indicated the similarities in the structures of enantiomers and their metabolites detected in the urine samples. This fact was utilized for the quantitative analyses of PHM metabolites in the urine samples by applying the calibration parameters of PHM enantiomers also for PHM metabolites.…”

“…Hence, the ITP-CZE method can be easily modified with a great variety of selectors implemented with the highest advantage into the CZE stage enabling to separate also the most problematic analytes (structural analogs, isomers, enantiomers). The ITP-CZE methods with chiral as well as achiral CZE mode have been successfully applied in various real situations (Mikuš et al, , 2008a(Mikuš et al, , 2008cDanková et al, 2001;Marák et al, 2007;Kvasnička et al, 2001). The most frequently used ITP-CZE system works in the hydrodynamically closed separation mode that is advantageous for the real analyses of multicomponent ionic mixtures because of the best premises for enhancing sample load capacity (enables using capillaries with very large I.D.).…”

“…Capillary (CE) and microchip (MCE) formats were taken into consideration. CE and MCE determinations of various biologically active compounds in model (spiked samples) as well as real biological matrices, employing an on-line coupling of the electrophoretic methods with other electrophoretic or non electrophoretic methods, are listed in (Mikuš et al, , 2008a(Mikuš et al, , 2008b(Mikuš et al, , 2008c(Mikuš et al, , 2009Marák et al, 2007) we illustrated possibilities of ITP-EKC method combined with diode array detection (DAD) for the direct achiral (celiprolol, CEL, amlodipine, AML) as well as chiral (amlodipine, AML, pheniramine, PHM, dimethinden, DIM and dioxoprometazine, DIO) quantitative determination of trace drugs in clinical human urine samples, see an example in Fig.15. ITP, on-line coupled with EKC, served in these cases as an ideal injection technique (high sample load capacity, preseparation and preconcentration) producing analyte zone suitable for its direct detection and quantitation in EKC stage.…”

“…Spectral DAD, used in our works, in comparison with single wavelength ultraviolet detection enhanced value of analytical information (i) verifying purity (i.e., spectral homogeneity) of drug zone (according to differences in spectrum profiles when compared tested and reference drug spectra) and (ii) indicating zones/peaks with spectra similar to the drug spectrum (potential structurally related metabolites). Very good selectivity was achieved by using a negatively charged carboxyethyl--cyclodextrin (CE--CD) as a chiral selector for enantioseparation and determination of trace (ng/mL) antihistaminic drugs (PHM, DIM, DIO) present in urine (Mikuš et al, , 2008cMarák et al, 2007). Charged chiral selector provided significantly different affinity towards the analytes on one hand and sample matrix constituents on the other hand; enabling the analytes can be transferred into the analytical stage without any spacers and multiple column-switching even if accompanied by a part of sample matrix constituents detectable in analytical stage.…”