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%.     

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%.     

Rapid and sensitive determination of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with on-line regenerating covalent coating

A rapid, sensitive and reproducible micellar electrokinetic chromatographic method using hexamethyldisilazane as on-line regenerating covalent coating was developed for the quantification of ephedrine (E) and pseudoephedrine (PE). E and PE were derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol for laser-induced fluorescence detection. The on-line regenerating covalent coating formed a combinative double coating with the subsequently produced dynamic SDS coating. The total coating can be easily removed and conveniently regenerated on-line. The simple coating procedure was described. By a series of optimization, a running buffer of 20 mm Na(2)B(4)O(7) + 16 mm SDS was applied for the separation of the derivatives. Linear relationships for E and PE were obtained in the range of 0.044-6.60 microg mL(-1) (correlation coefficients: 0.9975 for E, 0.9981 for PE), and the detection limits for E and PE were 1.71 and 0.67 ng mL(-1), respectively. The separation speed, the reproducibility and the sensitivity were much improved over those of other capillary electrophoresis methods more recently reported. The method was applied to the analysis of the two alkaloids in traditional herbal preparations with recoveries in the range 92.8-104.8%.     

Separation and determination of flavonoids using microemulsion EKC with electrochemical detection

A selective and sensitive method of microemulsion EKC (MEEKC) with electrochemical detection (ED) was developed for separation and determination of 14 flavonoids. In order to obtain the better stability for the studied flavonoids, oil (ethyl acetate) with low interfacial surface tension was employed as organic solvent. A running buffer composed of 0.9% (w/v, 30 mM) SDS, 0.9% (w/v, 21 mM) sodium cholate (SC), 0.9% (w/v, 121 mM) butan-1-ol, 0.6% (w/v, 68 mM) ethyl acetate, and 98.2% v/v 10 mM Na(2)B(4)O(7)-20 mM H(3)BO(3) buffer (pH 7.5) was applied for the separation of flavonoids. Under the optimum conditions, the relationship between peak currents and analyte concentrations was linear over about 1.3 and 1.7 orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.5 microg/mL for all analytes. This method was applied for the determination of flavonoids in real samples with simple extraction procedures, and the assay results were satisfactory.     

Analysis of plant hormones by microemulsion electrokinetic capillary chromatography coupled with on-line large volume sample stacking

A novel method of microemulsion electrokinetic capillary chromatography (MEEKC) coupled with on-line large volume sample stacking was developed for the analysis of six plant hormones including indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, abscisic acid and salicylic acid. Baseline separation of six plant hormones was achieved within 10 min by using the microemulsion background electrolyte containing a 97.2% (w/w) 10 mM borate buffer at pH 9.2, 1.0% (w/w) ethyl acetate as oil droplets, 0.6% (w/w) sodium dodecyl sulphate as surfactant and 1.2% (w/w) 1-butanol as cosurfactant. In addition, an on-line concentration method based on a large volume sample stacking technique and multiple wavelength detection was adopted for improving the detection sensitivity in order to determine trace level hormones in a real sample. The optimal method provided about 50-100 fold increase in detection sensitivity compared with a single MEEKC method, and the detection limits (S/N = 3) were between 0.005 and 0.02 μg mL(-1). The proposed method was simple, rapid and sensitive and could be applied to the determination of six plant hormones in spiked water samples, tobacco leaves and 1-naphthylacetic acid in leaf fertilizer. The recoveries ranged from 76.0% to 119.1%, and good reproducibilities were obtained with relative standard deviations (RSDs) less than 6.6%.     

Studies and application of catalytic kinetic spectrophotometric determination of trace chromium(VI) sensitized by microemulsion medium

A new catalytic kinetic spectrophotometric method for the determination of trace amount of chromium (VI) in microemulsion medium has been developed. The method is based on the catalytic effect of chromium (VI) on the oxidation of nuclear fast red in the presence of Tween-80 microemulsion and HAc-NaAc buffer solution (pH = 5.2). The linear ranges are in the range of 0 approximately 0.0480 microg mL(-1) of Cr (VI) at 528 nm and the detection limit is 1.56x10(-9)g mL(-1). The method has been applied in determination of Cr (VI) in water and food samples with satisfactory results. The relative standard deviation is less than 4.6 % and the recovery is 93.5 approximately 104 %.     

Head-column field-amplified sample stacking in a capillary electrophoresis-flow injection system

A simple, effective, and continuous online concentration method for the sensitive detection of alkaloids applying CE-flow injection analysis with head-column field-amplified sample stacking was developed. A series of samples was continuously introduced into the capillary by electrokinetic means without interrupting the high voltage. A short water plug was introduced by the EOF at the capillary inlet end prior to sample introduction. Under optimum conditions, 15-fold improvement in concentration sensitivity was achieved, giving an LOD of about 0.67 and 0.73 microg/mL for ephedrine (E) and pseudoephedrine (PE), respectively. The separation could be achieved within 4 min and sample throughput rate could reach up to 7/h. The repeatability (defined as RSD) was 3.62, 1.51% with peak area evaluation and 1.30, 2.58% with peak height evaluation for E and PE, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing E and PE, and the recoveries were 92.3-102.4%.     

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%.     

Development of a NACE method for simultaneous measurement of three adenosine monophosphate isomers in biomimicking prebiotic synthesis without sample pretreatment

A practical NACE method was developed for simultaneous determination of three adenosine monophosphate (AMP) isomers. Separation of three AMP isomers was achieved using 200 mM Tris/H(3)BO(3) in acetontrile/water (2:1 v/v) at pH* 10.0 as the running buffer and +25 kV as the applied voltage over a bare fused-silica capillary of 50 microm id x 375 microm od x 54.5 cm (46 cm to the detector window). At 260 nm, the calibration curves were linear in the range of 1-100 microg/mL. The detection limits were less than 0.70 microg/mL. The recovery ranged from 94.5 to 106.4%. The intraday RSDs of the migration times were between 2.1 and 3.0%. The developed NACE method has been successfully applied for the determination of three AMP isomers in the real samples of biomimicking prebiotic synthesis reaction between N-(O,O-diisopropyl) phosphoryl amino acid and adenosine.     

Determination of the epimerization rate constant of amygdalin by microemulsion electrokinetic chromatography

A new method for separation and determination of amygdalin and its epimer (neoamygdalin) in the epimerization of amygdalin by MEEKC is proposed. For the chiral separation of amygdalin and neoamygdalin, a running buffer composed of 80 mM sodium cholate, 5.0% v/v butan‐1‐ol, 0.5% v/v heptane and 94.5% v/v 30 mM Na₂B₄O₇ buffer (pH 9.00) is proposed. Under optimum conditions, the basic separation of amygdalin and neoamygdalin can be achieved within 7 min. The calibration curve for amygdalin showed excellent linearity in the concentration range of 20–1000 μg/mL with a detection limit of 5.0 μg/mL (S/N=3). The epimerization rate constant of amygdalin in basic microemulsion was first determined by monitoring the concentration changes of amygdalin, and the epimerization rate constant of amygdalin was found to be 2×10^?3 min^?1 at 25°C under the above optimum microemulsion conditions.     

Analysis of flunarizine in the presence of some of its degradation products using micellar liquid chromatography (MLC) or microemulsion liquid chromatography (MELC)--application to dosage forms

The separation of flunarizine hydrochloride (FLZ) and five of its degradation products--1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine, 4-oxide (A), bis(4-fluorophenyl)methanone (B), bis(4-fluorophenyl)methanol (C), 1-(3-phenyl-2-propenyl)piperazine(D), and 1-[bis-4-fluorophenyl) methyl] piperazine (E)--could be accomplished by reversed phase liquid chromatography using either micellar or microemulsion mobile phases. Cyanopropyl-bonded stationary phase has been used with UV detection at 254 nm. Microemulsion mobile phase consisting of 0.15 M SDS, 10% n-propanol, 1% n-octanol, and 0.3% triethylamine in 0.02 M phosphoric acid of pH 7.0, has been used for the separation of FLZ and its degradation products (B, C, D, and E). Micellar mobile phases consisting of 0.15 M sodium dodecyl sulphate (SDS), 10% n-propanol, 0.3% triethylamine (TEA) in 0.02 M phosphoric acid of pH values either 4.0 or 6.8 have been used for the separation of FLZ from its degradation products, i.e. either from (B, C, D, and E) or from (A, B, C, and D), respectively. Micellar liquid chromatography (MLC) was applied to the determination of FLZ in pure form as well as in dosage forms; the calibration graph was linear over the concentration range of 0.15-50 microg/mL with detection limit of 0.02 microg/mL (4.19 x 10(-8)M).     

Determination of aromatic amines in environmental water sample by hollow fiber-liquid phase microextraction and microemulsion electrokinetic chromatography

A novel method of microemulsion electrokinetic chromatography (MEEKC) coupled with hollow fiber-liquid phase microextraction (HF-LPME) was developed for determination of six aromatic amines including 4-methylaniline, 3-nitroaniline, 2,4-dimethylaniline, 4-chloroaniline, 3,4-dichloraniline and 4-aminobiphenyl. Baseline separation of six aromatic amines was achieved within 8 min by using the microemulsion buffer containing a 10 mM borate buffer at pH 9.0, 0.8% (v/v) ethyl acetate as oil droplets, 60 mM sodium cholate as surfactant, 5.0% (v/v) 1-butanol as co-surfactant. The influence factors relevant to the HF-LPME process were systemically investigated. The obtained enrichment factors were ranged between 70 and 157 in a 30 min extraction time, and the limits of detection ranged between 0.0021 and 0.0048 μg/mL. This purposed method was successfully applied for the analysis of aromatic amines in water sample and the recoveries were ranged from 87.2% to 99.8%.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Quantitative determination of the loop diuretic bumetanide in urine and pharmaceuticals by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of the diuretic bumetanide using a microBondapak C18 column. The mobile phase consists of a 50:50 acetonitrile-water mixture containing 5mM KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1350 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is done prior to chromatographic analysis in order to avoid the interferences found in the urine matrix. The percentages of recovery obtained are 71%+/-1% for liquid-liquid extraction and 84.2%+/-0.7% for solid-liquid extraction. The method developed has a linear concentration range from 50 to 499 ng/mL with a reproducibility in terms of relative standard deviation of 1.73% and 3.85% for a concentration level of 70 ng/mL and 237 ng/mL, respectively, and a detection limit of 0.25 ng/mL (3:1 signal-to-noise ratio). The method is applied to the determination of bumetanide in pharmaceutical formulations and urine obtained from hypertensive patients and healthy volunteers after the ingestion of a therapeutic dose of Fordiuran (1 mg bumetanide).     

Chiral separation of benzoporphyrin derivative mono- and diacids by laser induced fluorescence-capillary electrophoresis

A method for the separation of benzoporphyrin derivative mono- and diacid (BPDMA, BPDDA) enantiomers by laser induced fluorescence-capillary electrophoresis (LIF-CE) has been developed. By using 300 mM borate buffer, pH 9.2, 25 mM sodium cholate and 10% acetronitrile as electrolyte, +10 kV electrokinetic sampling injection of 2 s and an applied +20 kV voltage across the ends of a 37 cm capillary (30 cm to the detector, 50 microm ID), all six BPD stereoisomers were baseline-separated within 20 min. Formation constants, free electrophoretic and complexation mobilities with borate and cholate were determined based on dynamic complexation capillary electrophoresis theory. The BPD enantiomers can be quantitatively determined in the range of 10(-2)-10(-5) mg mL(-1). The correlation coefficients (r2) of the least-squares linear regression analysis of the BPD enantiomers are in the range of 0.9914-0.9997. Their limits of detection are 2.18-3.5 x 10(-3) mg mL(-1). The relative standard deviations for the separation were 2.90-4.64% (n = 10). In comparison with high-performance liquid chromatography (HPLC), CE has better resolution and efficiency. This separation method was successfully applied to the BPD enantiomers obtained from a matrix of bovine serum and from liposomally formulated material as well as from studies with rat, dog and human microsomes.     

Microemulsion electrokinetic chromatography for the analysis of acrylamide in food

The influence of microemulsion electrokinetic chromatography (MEEKC) operating conditions, such as the type of water-immiscible alcohol, aqueous phosphate buffer concentration, pH, as well as the addition of methanol and 2-propanol, on acrylamide migration has been studied. These parameters have been optimized taking into account the presence of matrix signals, in order to avoid the interference of these peaks in acrylamide determination. The best separations were achieved using a microemulsion consisting of 0.8% m/v n-amyl alcohol, 3.3% m/v sodium dodecyl sulfate (SDS), 6.6% m/v 1-butanol, and 89.3% m/v 40 mM phosphate buffer at pH 6.5 working at 15 kV in uncoated silica capillaries. Linear calibration curves over the range studied (1.25-125 microg x mL(-1)), the detection limit (0.70 microg x mL(-1)), and both run-to-run (up to 3.4% for concentration and 1.6% for time values) and day-to-day precision (lower than 11.6% for concentration) have been established. Finally, the applicability of the MEEKC method developed has been demonstrated by analyzing levels of acrylamide present in samples of home-made French fries.     

Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detection

Therapeutic drug monitoring of sirolimus (rapamycin) is important for immunosuppressive therapy in solid organ transplantation. We have developed a simple and reliable method for determining blood concentrations of sirolimus using reversed-phase HPLC with electrochemical detection (ECD). The E(2) potential was set at +900 mV. The potential of guard cell was set at +950 mV and that of the E(1) cell at +400 mV. The method was linear for a concentration range of 1-50 ng/mL when 0.5 mL blood was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 0.5 ng/mL. The inter-assay precision ranged from 3.22 to 7.48%, and the coefficient of variation (CV) for a quality control sample at 10 ng/mL was 7.48% with a bias of 8.4% from the target value. The intra-assay precision ranged from 0.72 to 3.71%, and the CV for a quality control sample at 10 ng/mL was 0.72% with a bias of 6.8% from the target value. In a solid organ transplant recipient, trough concentrations of sirolimus were well within the analytic range of the HPLC/ECD procedure. The method described here is suitable for management of sirolimus therapy in solid organ transplantation.     

Determination of folic acid in tablets by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of folic acid, a water-soluble vitamin, in a commercial tablet formulation. The analysis was performed using a microemulsion containing 0.5% (w/w) ethyl acetate, 1.2% (w/w) butan-1-ol, 0.6% (w/w) sodium dodecyl sulfate, 15% (v/v) 2-propanol and 82.7% (w/w) 10 mmol L(-1) sodium tetraborate aqueous buffer at pH 9.2. Direct UV detection at 214nm led to an adequate sensitivity without interference from sample excipients. For quantitative purposes, niacin was used as internal standard. Acceptable precision (<1.2% relative standard deviation (R.S.D.)), linearity (r = 0.9992; range from 160.0 to 240.0 microg/mL), sensitivity (limit of detection (LOD) = 2.98 microg/mL; limit of quantification (LOQ) = 9.05 microg/mL) and recovery (99.8 +/- 1.8% at three concentration levels) were obtained. Based on the performance characteristics, the proposed methodology was found suitable for the determination of folic acid in tablet formulations.     

A porous graphitized carbon column HPLC method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation

A simple, rapid, and stability-indicating HPLC method has been developed, fully validated, and applied to the quantification of paracetamol, pseudoephedrine hydrochloride, and chlorpheniramine maleate in a pharmaceutical formulation, using hydrochlorothiazide as an internal standard. Chromatographic separation was achieved isocratically on an RP porous graphitized carbon analytical column (125 x 2.1 mm id, particle size 5 microm) using 5.0 mM ammonium acetate-acetonitrile (35 + 65, v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220 nm was used. The method had linear calibration curves over the range of 30-70 microg/mL for paracetamol, 1.8-4.2 microg/mL for pseudoephedrine hydrochloride, and 120-280 ng/mL for chlorpheniramine maleate. The intraday and interday RSD values were less than 3.2% for all compounds, while the relative error was less than 2.9%. Accelerated stability studies performed under various stress conditions proved the selectivity of the method. The developed method was applied successfully to QC and content uniformity tests of commercial tablets.     

Development and validation of a reversed-phase column liquid chromatographic method for the determination of five cephalosporins in pharmaceutical preparations

A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters microBondapak C18 column (250 x 4.6 mm id, 10 pm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)-acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30-300, 3-30, and 15-120 microg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.