Hwang-Shang Kou

Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis.

A simple and selective capillary electrophoretic method was established for the simultaneous determination of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and polyglutamate derivatives [MTX-(Glu)n, n=2-7] in whole blood. After extraction, those analytes were separated by fused-silica capillary and a running buffer containing glycine (1.2 M, pH 9.3). The quantitative ranges were 10-50 microM for each analyte. The intra- and inter-day RSD and RE values were all less than 6 and 11%, respectively. The limits of detection (S/N= 3, injection 5 s) were found to be 1 microM for MTX, 7-OHMTX, MTX-(Glu)2, MTX-(Glu)3, and MTX-(Glu)4; 3 microM for MTX-(Glu)5 and MTX-(Glu)6; 5 microM for MTX-(Glu)7, and 8 microM for DAMPA. All recoveries were greater than 94%. This method was applied to blood MTX monitoring in a patient with acute lymphoblastic leukemia.

A fluorimetric liquid chromatography for highly sensitive analysis of very long chain fatty acids as naphthoxyethyl derivatives.

SummaryA simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with 2-(2-naphtoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were analyzed by HPLC with fluorimetric detection (λex=235 nm; λem=366 nm). The linear range for the determination of docosanoic, tetracosanoic and hexacosanoic acids was 0.028–1.4 μM with a detection limit of about 5.6 nM (S/N=3) (56 fmol per 10 μL injection). Application of the method to the analysis the non-esterified (free) very long chain fatty acids spiked in plasma proved feasible.

High-performance liquid chromatographic determination of betamethasone and dexamethasone

Abstract A simple method was developed for the determination of glucocorticoid epimers of betamethasone (BTM) and dexamethasone (DXM) by high-performance liquid chromatography with UV detection, using silica as the stationary phase and dichloromethane-ethanol (34:1, v/v) as the mobile phase. The linear range of the method for the determination of BTM and DXM in 1.0 ml of sample solution was over 5–50 nmol. The detection limits (signal-to-noise ratio = 5) of BTM and DXM with an injection volume of 25 μl were 80 and 60 pmol, respectively. The method was satisfactorily applied to the individual determination of BTM and DXM in commercial tablets.

(2-Naphthoxy)acetyl chloride, a simple fluorescent reagent.

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.

A simple and sensitive liquid chromatographic method for the analysis of free docosanoic, tetracosanoic and hexacosanoic acids in human plasma as fluorescent derivatives.

Docosanoic (C22), tetracosanoic (C24) and hexacosanoic (C26) acids are saturated very-long-chain fatty acids (VLCFA) present at trace levels in biosamples. VLCFA can be used as potential biomarkers for the diagnosis of hereditary diseases such as X-linked adrenoleukodystrophy. Because the analytes to be detected are at trace levels, a sensitive fluorimetric liquid chromatographic method was developed to analyze VLCFA in plasma. The method is simple based on extracting VLCFA from plasma with toluene, and the obtained toluene extract was subject to the derivatization of VLCFA with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate (NOEPES) without solvent evaporation/replacement. The resulting fluorescent derivatives were monitored by fluorimetric detection (excitation at 225nm and emission at 360nm), giving a high sensitivity with the limit of detection about 5.0nM (S/N=3, 10microL injected) of the analytes. Application of the method to the analysis of VLCFA in the plasma of patients with adrenoleukodystrophy proved practical and effective.

Chemically removable derivatization reagent for liquid chromatography : 2-(2-naphthoxy)ethyl 2-[1-(4-benzyl)piperazyl]ethanesulfonate

Abstract A new sulfonate reagent, 2-(2-naphthoxy)ethyl 2-[1-(4-benzyl)-piperazyl]ethanesulfonate, was synthesized for analytical derivatization in liquid chromatography. The reagent consists of two main moieties, i.e., one with a fluorophore (naphthoxy) for sensitive detection after being tagged to an analyte; and the other with a tertiary amino function (a substituted piperazine) that can be removed after derivatization by acid treatment. The reagent was applied to the derivatization of caproic acid as a model analyte. The resulting derivative was analyzed by high-performance liquid chromatography with fluorometric detection. The linear range of the method for the determination of caproic acid was over 0.01–0.5 nmol, with a detection limit (signal-to-noise ratio=3) of about 0.1 pmol. The excess reagent was readily removed from organic solvent after treatment with an aqueous acid solution.

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants.

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak‐area ratios of the drugs to an internal standard (7‐hydroxy‐4‐methyl‐coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20–150 µM, carbocysteine over 100–1500 µM, guaiacol and guaifenesin each over 10–150 µM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0–5.0 µM (except that of carbocysteine at 30 µM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra‐day (n=3) and inter‐day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.

Trace Analysis of Very Long Chain Free Fatty Acids in Plasma by Fluorogenic Derivatization and Liquid Chromatography

Abstract A simple and sensitive liquid chromatographic method is described for the simultaneous determination of docosanoic, tetracosanoic and hexacosanoic acids as fluorogenic derivatives. These acids, spiked in plasma, were extracted with n-heptane and the resulting extract was derivatized with 4-bromomethyl-7-methoxycoumarin (BrMmC) in dichloromethane using potassium carbonate and 18-crown-6 as catalysts. The derivatives obtained were chromatographed on a reversed-phase C8 column with acetonitrile: water (91: 9, v/v) as a mobile phase and 7-methoxy-4-(tricosanoyloxymethyl)-coumarin as an internal standard. The linear ranges for the determination of docosanoic, tetracosanoic, and hexacosanoic acids were over 31-500, 36-580 and 36-580 pmol, respectively. The limit of detection for each acid was about 0.3 pmol per 10 μL injection (S/N=3). This work was presented in part at the annual symposium of the Pharmaceutical Society of the R.O.C., Taichung, Dec. 22th, 1996.

Trace analysis of ethosuximide in human plasma with a chemically removable derivatizing reagent and high-performance liquid chromatography.

A simple and sensitive liquid chromatographic method is described for the determination of ethosuximide in human plasma, as a highly sensitive derivative. Ethosuximide spiked in plasma was extracted with toluene and derivatized with a chemically removable derivatizing reagent, 2-(2-naphthoxy)ethyl 2-[1-(4-benzyl)piperazyl]ethanesulfonate, in a homogeneous system, using magnesium oxide as base catalyst. The resulting derivative was separated on a LiChrospher diol column with 1.2% isopropanol in n-hexane as the mobile phase and using coumarin as the internal standard. Several parameters affecting the extraction/derivatization of ethosuximide from spiked plasma were investigated. The linear range for the determination of ethosuximide in spiked plasma was over 30-700 nmol/ml. For ethosuximide in plasma, the detection limit (signal-to-noise ratio=3; sample size, 10 microl) was about 9 pmol; the relative standard deviation was 6.4% for intra-day assay (n=6) and 9.2% for inter-day assay (n=6) and the relative recovery was found greater than 94%.

Determination of Ethosuximide in Plasma By Derivatization and High Performance Liquid Chromatography

Abstract A simple and sensitive liquid chromatographic method is described for the determination of ethosuximide in plasma as a highly sensitive derivative. Ethosuximide in plasma, after separation with isopropanol extraction, was derivatized with strong chromophore reagent, 4-bromomethyl-7-methoxy-coumarin. The resulting derivatives were separated on a Nova-Pak C18 column with water-acetonitrile-methanol(60:20:20, v/v) as the mobile phase. The linear range for the determination of ethosuximide in spiked plasma was over 2-40 nmol. The limit of detection for ethosuximide was about 7.0 ± 1.2 pmol per 20 μL injection (S/N= 5). The intraday relative standard deviation (n = 6) and the interday relative standard deviation (n = 12) were all less than 2.5% for ethosuximide. The recovery for ethosuximide in plasma was greater than 96%.