Multi‐site binding of fenoprofen to human serum albumin studied by a combined technique of microdialysis with high performance liquid chromatographyWang, Hamlin; Zou, Hanfa; Zhang, Yukui
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<4::AID-BMC707>3.0.CO;2-Kpmid: 9470966
A simple and fast method for the determination of the multi‐site binding of fenoprofen (FP) to human serum albumin (HSA) has been developed by utilizing microdialysis sampling techniques combined with high performance liquid chromatography (HPLC). The drug and protein were mixed in different molar ratios in 0.067 Mol potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water‐bath. Then the microdialysis probe was put in the FP‐HSA solution and sampled at the perfusion rate of 1 μL/min. The concentrations of FP in microdialysates were determined by the reversed‐phase high performance liquid chromatography. Relative recovery (R) was also determined in vitro on similar condition, R is about 56.03±1.11% (n=3). Fenoprofen was found to bind to two classes of sites, the association constant (K1) and the number of the binding sites on primary binding sites of a HSA molecule (n1) for fenoprofen are 3.4×105/m and 2.5, respectively, and those for secondary binding are 1.0×104/m and 10.0, respectively. The competitive interaction of ibuprofen (IP) and palmitic acid with fenoprofen to HSA were also studied, both compounds significantly decrease the binding degree of fenoprofen to HSA. © 1998 John Wiley & Sons, Ltd.
Investigation of far red dyes for use in peroxyoxalate chemiluminescence detection and analysis of the CY5 derivative of amantadine hydrochloride in human plasmaEllingson, Andy; Thomas Karnes, H.
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<8::AID-BMC709>3.0.CO;2-2pmid: 9470967
Peroxyloxalate chemiluminescence is well established as a tool for improvement of selectivity and sensitivity for chemiluminophores and their derivations in HPLC eluates. Chemiluminescence in the far‐red spectral region was investigated in this work to further enhance the sensitivity of chemiluminescence through more efficient singlet excitation energy transfere and to enhance the selectivity of the approach through a reduction in matrix and scatter interference. A number of fluorescent compounds that can be excited in the UV, visible and far red spectral regions were investigated for chemiluminescence yield using the bis(2,4,6‐trichlorphenyl) oxylate reaction. It was found that a trend of increasing chemiluminescence with increasing excitation wavelength could be observed with CY5 providing the most efficient chemiluminescence. The succinate ester of CY5 was used to derivatize amantadine hydrochloride, an antiparkinsons drug, to form the derivative. The derivative was separated from reaction by products by C18 reversed phase HPLC and detected using a Soma S‐3400 chemiluminescence detector. The detection limit for the diluted derivative was 200 femtomoles on column and sufficient for plasma analysis. Selectivity in plasma was demonstrated through derivatization of extracts of plasma that had been spiked with amantadine hydrochloride. © 1998 John Wiley & Sons, Ltd.
Development of highly sensitive and specific HPLC assay for plasma morphine using direct injection technique and post‐column derivatizationEmara, Samy
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<15::AID-BMC713>3.0.CO;2-Rpmid: 9470969
An extremely simple, rapid and reproducible analytical method was developed for the determination of morphine in human plasma using a high performance liquid chromatography utilizing a column‐switching technique and protein‐coated precolumn. Morphine from plasma (500 μL) was preconcentrated on the protein‐coated pre‐ column without sample pre‐treatment. This column acted at the same time as a clean up device. The drug was transferred on‐line to the analytical column followed by post‐chromatographic derivatization and fluorimetric detection. Post‐column derivatization was based on the oxidative dimerization of morphine to fluorescent pseudomorphine by potassium hexacyanoferrate (III). The average morphine recoveries over a concentration range of 10 to 100 ng/mL ranged from 94.84 to 100.70%, and relative standard deviations ranged from 1.36 to 2.13%. © 1998 John Wiley & Sons, Ltd.
On‐line HPLC‐electrospray ionization mass spectrometry: a pharmacological tool for identifying and studying the stability of Gd 3+Behra‐Miellet, Josette; Briand, Gilbert; Kouach, Mostafa; Gressier, Bernard; Cazin, Micheline; Cazin, Jean‐Claude
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<21::AID-BMC714>3.0.CO;2-Zpmid: 9470970
The identification of MRI contrast agents (CAg) as gadolinium complexes often used at very low concentrations in Pharmacology was carried out by ESI‐MS or HPLC‐ESI‐MS. Firstly, OmniscanTM, DotaremTM and MagnevistTM were tested. In these compounds, the Gd3+ ion must be solidly chelated by linear or macrocyclic ligands because of the severe toxicity of the free Gd3+. Spectra were obtained at low voltage, preserving the non‐covalent binding integrity of the complexes, and at various higher voltages showing the progressive destruction of the complexes. Secondly, a direct reaction of these drugs with the oxidative human neutrophil production, induced in vitro by Phorbol 12‐myristate 13‐acetate enhancing the respiratory burst, was investigated. This was done to mimic what happens in the case of inflammatory diseases, or infection, or when people are likely to develop anaphylactoid reactions, as the I.V. injection of CAg causes contact between the complexes and neutrophils in the blood. Analysis by HPLC‐ESI‐MS coupling did not show any direct reaction between Gd complexes and the chemical compounds in the neutrophil oxidative metabolism, even if uncertainty remains as regards meglumine salt. HPLC‐ESI‐MS is a good way of visualizing characteristic Gd isotopic distribution and of following its associations in biological samples. © 1998 John Wiley & Sons, Ltd.
Quantitative determination of salidroside and specnuezhenide in the fruits of Ligustrum lucidum Ait by high performance liquid chromatographyShi, Lifu; Ma, Yan; Cai, Zhen
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<27::AID-BMC715>3.0.CO;2-Epmid: 9470971
An accurate RP‐HPLC method for the quantitative determination of two water‐soluble biologically active compounds, salidroside (p‐hydroxyphenethyl‐β‐d‐glucoside) and specnuezhenide in the fruits of Ligustrum lucidum Ait (a crude drug in Chinese traditional medicine) was developed. The reversed‐phase column was Nova‐Pak C18, saturated with water before the injection of the samples and eluted with a mobile phase of methanol:water (4:6, v/v). The detection wavelength was 230 nm. The recoveries of the two compounds were 96.1 and 97.0%, respectively. The contents of salidroside and specnuezhenide in the fruits of Ligustrum lucidum Ait and two traditional Chinese patent medicines in which this crude drug was an important component, Erzhi Pills and Anshenbuxin Pills, were determined. © 1998 John Wiley & Sons, Ltd.
Derivatization methodology for the analysis of butorphanol by gas chromatography‐mass spectrometryAndraus, Maristela Haddad; de Siqueira, Maria Elisa Pereira Bastos
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<34::AID-BMC718>3.0.CO;2-Dpmid: 9470973
Butorphanol is an opioid used as analgesic in humans and other species. In horses, it can cause locomotor stimulation at low doses. This drug is not well chromatographed by GC and so, it is necessary to transform it into a more suitable compound, which can be done by derivatization. The derivatization of a drug is used to impart volatility, masking polar groups to improve the results in gas chromatographic analysis. We have evaluated N,O‐bis(trimethylsilyl)‐trifluoracetamide (BSTFA)+1% trimethylchlorsilane (TMCS) and N‐methyl‐N‐trimethylsilil‐trifluoroacetamide (MSTFA) as derivatizing reagents for butorphanol at 30, 60 and 80°C during 15, 30 and 60 min. The effects of dilution of these reagents with toluene and the evaporation before the derivatization were tested. Both reagents can be used for butorphanol derivatization and analysis and the dilution and evaporation steps did not alter the final results. The best derivatization conditions were 15 min at 80°C, although 60°C, although 60°C during 60 min were also suitable. © 1998 John Wiley & Sons, Ltd.
Quantitative reverse transcription‐PCR‐HPLC for nerve growth factor mRNA using a deletion RNA as an internal standardShimizu, Hiroki; Kuroki, Jun; Ogura, Hiroo; Yamanishi, Yoshiharu; Arakawa, Yoshihiro
doi: 10.1002/(SICI)1099-0801(199801/02)12:1<38::AID-BMC724>3.0.CO;2-Cpmid: 9470974
We have developed a convenient method for the routine measurement of the absolute amount of nerve growth factor (NGF) mRNA in tissue samples. The method consists of RNA extraction, amplification by reverse transcription‐PCR and detection by high‐performance liquid chromatography. The addition of a deletion mutant RNA to tissue samples as an internal standard enabled correction for RNA recovery during extraction, and the target mRNA and the internal standard were both amplified with the same PCR primers. The conditions were optimized so that the procedure was conducted in the region where the calibration curve was linear, thereby allowing high reproducibility and reliability. The method was applied to the measurement of NGF mRNA in tissues such as skin and skeletal muscle, where the levels are too low to be easily detected by Northern blotting analysis: skin, 14.1±4.6 fg/mg tissue and skeletal muscle, 11.0±2.2 fg/mg tissue (mean±SD, n=10). The coefficient of variation of this method was less than 2.8%. This approach should also be applicable to the routine assay of the absolute amount of other mRNAs present at low levels in tissues. © 1998 John Wiley & Sons, Ltd.