Microemulsion electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive microemulsion electrokinetic chromatography with laser-induced fluorescence detection method was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. By a series of optimization, a running buffer composed of 20 mM borate + microemulsion (23.3 mM Sodium dodecyl sulfate/180.85 mM 1-butanol/16.4 mM n-heptane) +8% acetonitrile was applied for the separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.058-11.58 microg.mL(-1) (correlation coefficient: 0.9993 for E, 0.9995 for PE), and the detection limits for E and PE were 5.3 and 3.9 ng.mL(-1). The method was applied to the analysis of the two alkaloids in Chinese traditional herbal preparations with recoveries in the range of 96.9-105.4%.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive micellar electrokinetic chromatography method with laser-induced fluorescence detection was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. After conducting a series of optimizations, a running buffer of 10 mM sodium borate + 16 mM SDS was used for separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.044-6.6 microg mL(-1) (correlation coefficient: 0.9943 for E, 0.9946 for PE), and the detection limits for E and PE were 0.70 and 0.30 ng mL(-1), respectively. The sensitivity of E and PE was improved by several multiples of ten over those of CZE-LIF method. The method was applied to the analysis of the two alkaloids in ephedra herbal medicine and preparations with recoveries in the range of 98.3-107.1%.     

Microemulsion electrokinetic chromatography with laser-induced fluorescence detection: as tested with amino acid derivatives

Over a decade ago, microemulsion electrokinetic chromatography was introduced as a novel mode of capillary electrophoresis. However, there has not been publication on the combination of microemulsion electrokinetic chromatography with laser-induced fluorescence detection. In this paper, a preliminary method using microemulsion eletrokinetic chromatography combined with laser-induced fluorescence detection and second derivative electrophoregram was established as a sensitive and selective assay for separation and determination of nine amino acids after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. The derivatization and separation conditions were optimized. In the investigated concentration ranges correlation coefficients were better than 0.995. The relative standard deviation (n = 5) of the migration times and peak heights were 0.56-0.76 and 2.21-7.15%, respectively. The detection limits (S/N = 3) were at a neaomolar level (0.32-2.20 nM). The method was applied for the analysis of compound amino acid injection and a Chinese traditional herbal medicine. The recoveries were 95.9-107.9%.     

Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for rapid and sensitive analysis of two new bioactive reagents using dynamic covalent coating

A simple, rapid, selective, and sensitive micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection (LIF) method was developed, using hexamethyldisilazane (HMDS) as dynamic covalent coating (DCC), for the analysis of two new bioactive agents N-n-hexyl-N'-(sodium p-aminobenzenesulfonate) thiourea (HXPT) and N-n-undecyl-N'-(sodium p-aminobenzenesulfonate) thiourea (UPT) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. MEKC methods both not using DCC and using DCC were investigated. In a series of optimization steps, DCC and a running buffer of 20 mM Na2B4O7 + 16 mM SDS + 8% acetonitrile were applied for determination of the derivatives. Linear relationships for HXPT and UPT were obtained in the range of 5 to 100 microM (correlation coefficient: 0.9986 for HXPT, 0.9978 for UPT), and the detection limits for HXPT and UPT were 16.5 and 39.0 ng mL(-1). The sensitivity was improved over that of fluorescence spectroscopy methods. The method was applied to the analysis of the two reagents in lab water waste with recoveries in the range of 95.6-107.5%.     

In-capillary derivatization and analysis of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL(-1) for E and 1.6 ng mL(-1) for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

Simple and extractionless high-performance liquid chromatographic determination of rosiglitazone in human plasma and application to pharmacokinetics in humans

A simple and extractionless HPLC method using fluorescence detection was developed for the determination of rosiglitazone in human plasma. After deproteinization using perchloric acid the plasma samples were directly injected onto the HPLC system. The mobile phase was composed of acetonitrile (52%) and 20 mm ammonium acetate (48%, pH 7.5), and analysis was run at a flow rate of 0.2 mL/min with the detector operating at 247 nm for excitation wavelength and at 367 nm for emission wavelength, respectively. The method has a mean recovery of 97%, while the intra-day and inter-day precisions were all less than 7%. This method is simple, specific, sensitive and requires only a small plasma volume with short analytical time, and is suitable for the determination of plasma rosiglitazone in routine measurements for pharmacokinetic studies.     

Rapid and ultrasensitive determination of ephedrine and pseudoephedrine derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper presents a micellar electrokinetic chromatography method with laser-induced fluorescence detection to analyze ephedrine (E) and pseudoephedrine (PE) after derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein. The optimum derivatization conditions were: 0.05 M Na2CO(3/NaHCO3 (pH 9.5), reaction time 30 min at 45 degrees C, molar ratio of DTAF to E and PE mixture 20:1. The baseline separation was achieved within 8 min with running buffer composed of 20 mM borate+20 mM SDS+15% acetonitrile (v/v) (adjusted pH 9.8), and applied voltage of 20 kV. Good linearity relationships (correlation coefficients: 0.9906 for E and 0.9941 for PE) between the peak heights and concentration of the analytes were obtained (2.5-50 ngmL(-1)). The detection limits for E and PE were 3.85 x 10(-4) and 1.41 x 10(-4)ngmL(-1), respectively, which indicated that the proposed method surpassed other chromatographic alternatives in terms of limit of detection by at least 10(3) folds. The method was applied to the analysis of the two alkaloids in ephedra herb plants and its preparations with recoveries in the range of 89.6-107.0%.     

Liquid chromatographic determination of nine N-methylcarbamates in drinking water

A multi-residue method for the simultaneous extraction from drinking water using solid-phase extraction on LiChrolut EN [poly(styrene-divinylbenzene), PSDVB] and determination of nine N-methylcarbamate pesticides (NMCs) (aldicarb, its metabolites i.e. aldicarb sulfone and aldicarb sulfoxide and carbaryl, carbofuran, dioxacarb, ethiofencarb, methomyl and propoxur) using reversed-phase liquid chromatography was studied. A 1000-fold pre-concentration was achieved and the method was used for determination of the nine pesticides in water, with limits of detection in the range 3-15 ng L(-1). For all compounds the recoveries determined at the 0.1 and 1 microg L(-1) level generally ranged from 85 to 104% with relative standard deviations (RSD) of 1.4-8.8%.     

Quantitative determination of DRF-1042 in human plasma by HPLC: validation and application in clinical pharmacokinetics

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.     

Simultaneous determination of catecholamines and amino acids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A selective and sensitive method was developed for separation and simultaneous determination of catecholamines and amino acids by MEKC with LIF. Interestingly enough, such work has been firstly performed on catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole and the detailed derivatization mechanism was discussed. After derivatization at 60 degrees C for 20 min, NBD-labeled catecholamines and amino acids were separated in a buffer system containing 10 mM sodium tetraborate-Na2HPO4, 20 mM SDS, and 10% v/v ACN at pH 9.75. SDS micelles were employed to improve the fluorescence intensity of catecholamine derivatives efficiently. Under optimum conditions, two catecholamines and 11 amino acids were separated in a short 13 min analysis time and the RSDs for migration time and peak area were less than 0.60 and 6.50%, respectively. The method was successfully applied for the quantification of catecholamines and amino acids in Portulaca oleracea L., human urine sample, and mixed injection sample.     

Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.     

Specific determination of cysteine in human urine by capillary micellar electrokinetic chromatography

This paper describes a method for the analysis of cysteine in human urine using capillary micellar electrokinetic chromatography and on‐column reaction with 2,2′‐dipyridyl disulfide. In this reaction cysteine is quantitatively transformed into a mixed disulfide concomitantly with formation of an equimolar amount of 2‐thiopyridone that is further separated by capillary micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of cysteine is thus estimated indirectly from the result of 2‐thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 5 mM (correlation coefficient 0.989) with a detection limit of 2.5 μM and a limit of quantitation of 8.5 μM. The inter‐day reproducibility of the peak area was 2.18% and the inter‐day reproducibility of the migration time 0.51%. The method is relatively rapid, simple, and can be easily automated. Moreover, its detection limit covers the concentration range at which cysteine is present in biological samples such as human urine.     

Subminute and sensitive determination of the neurotransmitter serotonin in urine by capillary electrophoresis with laser-induced fluorescence detection

In this work, a sub-minute and sensitive capillary electrophoresis with laser-induced fluorescence (CE-LIF) method was developed for the analysis and quantitation of the neurotransmitter 5-hydroxytryptamine (5-HT) or serotonin in urine. The method involves precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) using an excitation light from an argon ion laser of 488 nm and a 520 nm band pass emission filter. Different variables that affect derivatization (pH, FITC concentration, reaction time and temperature) and separation (buffer concentration, pH, applied voltage and injection time) were studied. The linear dynamic range obtained was between 0 and 188 nM with a detection limit of 16 nm with a RSD between 2 and 9%. The applicability of the proposed method was demonstrated by analysis of 5-HT in human urine, establishing a concentration of 57 nM in control urine. The method was validated by standard-addition methodology.     

Analysis of ecdysteroids by micellar electrokinetic chromatography with on-line preconcentration

In order to enhance the UV detection sensitivity, an application study of an on-line preconcentration technique for micellar electrokinetic chromatographic (MEKC) was carried out. The simultaneous determination of four test ecdysteroids, 20-hydroxyecdysone, ajugasterone C, polypodine B and ponasterone A has been investigated by using the normal stacking mode in MEKC with UV detection. The effects of anionic surfactant composition and concentration, the applied voltage, the pH buffer, the kind and the amount of organic solvent and the injection time on the analyte resolution were evaluated. The optimised conditions for the separation involved the use of a 50 mM borate as the running buffer containing 50 mM of a mixture of sodium dodecyl sulphate (SDS) and sodium cholate (SC) in the ratio of 1:1 together with a concentration of 10% (v/v) of 2-PrOH at pH 9.0. Hydrodynamic injection of 12 s at 50 mbar and separation voltage of 20 kV at temperature of 20 degrees C were employed. These conditions allowed a repeatability separation within 21 min. Concentration detection limit for the neutral analytes studied improve about an order of magnitude. The method was also applied to the determination of ecdysteroids in a real sample.     

Rapid determination of aniline metabolites of chlorpropham in potatoes by micellar electrokinetic chromatography using negative-charged mixed micelles and laser-induced fluorescence detection

A rapid, reliable method has been developed for the multi-residue analysis of aniline metabolites of chlorpropham in potato samples. The method involves the precolumn derivatization of aniline metabolites with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF) and their subsequent separation and determination by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). The optimum procedure includes a derivatization step of the aniline metabolites (3-chloroaniline, 3-chloro-4-hydroxyaniline and 3-chloro-4-methoxyaniline) at 40 degrees C for 40 min and a 5-fold dilution prior to MEKC analysis, which is conducted within about 7 min using negative-charged mixed micelles (SDS/Triton X-100) in the running buffer. Under these conditions, the DTAF-anilines were readily detected at 0.3-3.1 microg/L level with a precision of 4.8-6.4%. These results indicate that negative-charged mixed surfactant MEKC-LIF is useful as a selective, rapid, and sensitive tool for the determination of these anilines and surpasses other electrophoretic alternatives based on the use of fluorescein-isothiocyanate (FITC) as label reagent. Finally, the potato matrix showed no significant effects on the derivatization and determination of these analytes, since the analytical figures of merit for the real samples were similar to those obtained in aqueous solutions, and the average recovery at fortification levels of 10-250 microg/kg was over 97%.     

Determination of 3-n-butylphthalide in rabbit plasma by HPLC with fluorescence detection and its application in pharmacokinetic study

A rapid, sensitive and specific reversed-phase high-performance liquid chromatographic method was developed for the determination of 3-n-butylphthalide, a drug currently being developed for treatment of stroke, in rabbit plasma. Fluorescence detection at an excitation wavelength of 280 nm and an emission wavelength of 304 nm was used for quantification of 3-n-butylphthalide. Ibuprofen was used as internal standard. Plasma samples were extracted with diethyl ether under acidic conditions. After evaporation of the organic phase, the extract was dissolved in mobile phase and injected into the chromatograph with C(18) column and a mobile phase of 0.05 mol/L sodium acetate buffer (pH 4.5)-acetonitrile (400:600). The peak area ratio vs concentration in plasma was linear over the range of 0.0212-4.24 microg/mL (correlation coefficient r = 0.9984) and the limit of quantification was 0.0212 microg/mL. Mean recovery was determined as 101.0% by analysis of plasma standard samples containing 0.0424, 0.424, 2.12 and 4.24 microg/mL of 3-n-butylphthalide. The intra-day relative standard deviations (RSDs) ranged from 3.6 to 8.9% and inter-day RSDs were within 8.0%. Pharmacokinetics of a single intravenous dose of 3-n-butylphthalide to the rabbits was presented to illustrate the applicability of this method. 3-n-Butylphthalide exhibited linear pharmacokinetics after intravenous administration to rabbits over the dose range 1-10 mg/kg.     

Separation of doxorubicin and doxorubicinol by cyclodextrin-modified micellar electrokinetic capillary chromatography

Doxorubicinol (DOXol) is a human metabolite of the chemotherapy agent doxorubicin (DOX), and is associated with dose-dependent cardiotoxicity and decreased drug efficacy. Due to the structural similarities and equal molecular charges of DOXol and DOX, their electrophoretic separation is commonly ineffective. A method for separating and detecting DOX and DOXol, as well as two DOXol enantiomers, was established using cyclodextrin-modified micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. Differential DOXol production was detected in a DOX-sensitive and resistant pair of cell lines, with a 0.08 +/- 0.01 fmol limit of detection.