Direct injection determination of theophylline and caffeine in blood serum by high-performance liquid chromatography using an ODS column coated with a zwitterionic bile acid derivative

Abstract

An ODS column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was evaluated for direct injection determination of drugs in blood serum by HPLC. Polar functional groups such as sulfonate, ammonium and the three hydroxyl groups in CHAPS protruding towards an aqueous mobile phase formed a hydrophilic layer over the ODS reversed-phase surface, which resulted in high molecular mass compounds such as proteins being prevented from penetrating into the internal hydrophobic region. The bulk of the proteins were eluted as an unretained or nearly unretained band by using 0.2 mM sodium hydrogenphosphate solution (pH 7.4) as the mobile phase. In contrast, small molecules such as some inorganic anions and aromatic compounds were retained and thereby separated from one another. It was confirmed that the ODS column modified with CHAPS acts as a restricted access-type column with a hydrophobic interior and a hydrophilic exterior. Hence biological fluids could be directly injected into the CHAPS-coated ODS column. The present HPLC system using the CHAPS-coated ODS column was applied to the determination of theophylline and caffeine in human blood serum. The detection limits for the two drugs with UV absorption at 273 nm were 0.2 and 0.5 mg l-1 (injection volume 20 microliters) and the relative standard deviations of peak area measurements were < 1.4% and 2.2%, respectively, for 10 replicate measurements of serum spiked with 5 mg l-1 of each of the drugs.

Methylcellulose-immobilized Reversed-phase Precolumn for Direct Analysis of Drugs in Plasma by HPLC

“…As a protein-immobilized stationary phase capable of plasma direct injection, octadecylsilane (ODS) treated with human plasma was developed and plasma direct injection capability was shown by Yoshida et al 12,13 α1-Acidglycoprotein (α1-AGP)-, ovomucoid-and avidin-coated columns had been developed, [14][15][16] and are capable of plasma direct injection. [17][18][19][20] Furthermore, direct analysis of drugs in serum using zwitterionic bile acid derivative-coated ODS was reported by Umemura et al 21 Several polymer-modified reversed-phase column materials such as the shielded hydrophobic phase (SHP) and the semipermeable surface (SPS) had also been reported. 8,[22][23][24] SHP consists of a hydrophilic, polymeric, water-solvated phase, covalently bonded to a silica bed.…”

Speciation of Small Molecules and Inorganic Ions in Salmon Egg Cell Cytoplasm by Surfactant-Mediated HPLC/ICP-MS

“…7,8 The surfactant-mediated HPLC allowed rapid separation of large and small molecules based on the mixed-mode separation properties; large molecules were eluted by a size-exclusion mode, and small molecules were separated by electrostatic or hydrophobic interactions. As a result, large molecular species with molecular weights larger than 10000 Da were eluted within 2.5 min, while small molecular species with molecular weights smaller than 10000 Da were eluted after 2.5 min, but within 10 min.…”

Speciation of Trace Elements, Binding and Non-binding with Proteins in Human Blood Serum, by Surfactant-Mediated HPLC with Element-Selective Detection by ICP-MS

“…proteins, as reported in our previous paper. 4 In the present experiment, 0.2 mM Tris-HCl buffer solution (pH 7.4) was used as the mobile phase solution, instead of 0.2 mM Na2HPO3 solution (pH 7.4). 4 In addition, 0.2 mM CHAPS was added in the mobile phase to prevent the degradation of the separation column.…”

“…The CHAPS-coated ODS column was prepared in a manner similar to that used in the previous paper. 4 The mobile phase of 0.2 mM Tris-HCl buffer solution (pH 7.4) was used at a flow rate of 0.7 ml min -1 , into which 0.2 mM CHAPS was added to prevent the degradation of the separation column.For the on-line element-selective detection, an ICP-MS instrument (Model SPQ8000A, Seiko Instruments, Chiba) was used with direct introduction of the eluent from the column into the nebulizer of ICP-MS via a Teflon tubing. The ICP-MS instrument was operated using a time-sequential program, which allowed simultaneous multielement chromatogram measurements.…”

“…The CHAPS-coated ODS column was prepared in a manner similar to that used in the previous paper. 4 The mobile phase of 0.2 mM Tris-HCl buffer solution (pH 7.4) was used at a flow rate of 0.7 ml min -1 , into which 0.2 mM CHAPS was added to prevent the degradation of the separation column.…”