Determination of thiamphenicol in human plasma by micellar electrokinetic capillary chromatography

A micellar electrokinetic chromatographic method is described for the determination of thiamphenicol in human plasma. The plasma sample was basified by adding K₂HPO₄ and was then extracted with ethyl acetate. After the solvent was evaporated, the residue was reconstituted in water. Approximately 40 nl of the solution were injected hydrodynamically. The running buffer was 20 mM borate (pH 9.2) containing 40 mM sodium dodecyl sulfate and 10% acetonitrile. The applied voltage was 18 kV and the detector wavelength was set at 195 nm. On-column sample stacking was achieved during the analysis to enhance the sensitivity; the limit of quantitation was 0.1 μg/ml. Linearity was over the range of 0.2 to 10 μg/ml. Recovery was 93.7±3.3%, the intra-day precision and accuracy was 99.6±2.8%; the inter-day precision and accuracy was 98.4±3.4%. The concentration of thiamphenicol in human plasma from eight volunteers was measured after administering thiamphenicol capsules orally.     

Determination of dialysate creatinine by micellar electrokinetic chromatography

Micellar electrokinetic chromatography with UV absorbance detection has been applied for fast and selective determination of creatinine in samples of postdialysate fluid. Optimization of the method was performed, with the best results being obtained using a 30 mM borate-100 mM sodium dodecyl sulphate background electrolyte, pH 9, with the detector set at 235 nm and an applied voltage of 17 kV across a fused-silica capillary of 67 cm/75 micro m I.D. The linear range of the technique was over 2 orders of magnitude (5-1000 micro M). The developed analytical procedure is useful for the monitoring of clinical hemodialysis treatment, because creatinine levels in real undiluted samples of postdialysate range from 80 to 350 micro M. The separation system allows the analysis of about six to seven samples of spent dialysate per hour in almost real time. The determinations are not influenced by other components of dialysate fluid nor by other surrogates extracted from patient blood. The results of analysis using the developed procedure and the kinetic spectrophotometric Jaffe method conventionally used in clinical settings for creatinine determination are fully comparable. Successful clinical evaluation of the analytical system was performed. The developed system is useful for bloodless estimation of bioanalytical parameters of hemodialysis sessions such as creatinine-time profiles and total creatinine removal. Both these parameters are important in clinical models of hemodialysis therapy.     

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml (S/N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.     

Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC(50) value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.     

Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography

We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.     

Determination of alginate copolymer in pharmaceutical formulations by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method was developed for the determination and quantification of sodium alginate. The alginate peak migrated in the very short time of 1.33 min and calibrated easily though the polydisperse properties of alginates. The minimum detection limit (LOD) of the method was calculated as 0.393 mg/ml. This analysis method was successfully applied to the alginate quantification in an antacid pharmaceutical formulation. Precise and reproducible analysis results were obtained, with liquid formulations injected directly without any pre-separation process.     

Determination of mitiglinide in rat plasma by high-performance liquid chromatography with UV detection

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and 0.02 mol/l KH(2)PO(4) buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1-20 microg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.     

Determination of diterpenoid triepoxides in Tripterygium wilfordii by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic (MEKC) method has been established for the identification and determination of diterpenoid triepoxides in the Chinese herb Tripterygium wilfordii Hook F. and its preparations. Studies of the influence of boric acid and borax buffer concentration and pH, and of sodium dodecylsulphate (SDS) concentration have been carried out, and the optimum separation for the triepoxides was achieved using 20 mM boric acid and 10 mM borax with 20 mM SDS as the running buffer. MEKC was found to exhibit good accuracy, precision and repeatability. The sensitivity of the assay was sufficient to monitor the three active components in T. wilfordii and its preparations.     

Determination of aucubin and catalpol in Plantago species by micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was used for the separation and determination of two iridoid glycosides, aucubin and catalpol, in several Plantago species growing in Croatia: P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. (subsp. depauperata, subsp. holosteum and subsp. scopulorum), P. lagopus L., P. lanceolata L., and P. maritima L. Hot water extraction (HWE) was applied for the isolation of iridoid substances. Significant differences appeared between the iridoid contents in the examined species. The yield of aucubin and catalpol was up to 0.27% and 1.81% of the dry mass of the leaves, respectively. Besides aucubin and catalpol, two related compounds were determined in the plant samples.     

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection

A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi Max RP analytical column (150 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1+/-5.5% (n=6) and 72.4+/-5.9% (n=7), respectively; for OZP at 200 ng the value was 68.2+/-6.8% (n=14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n=7 in all cases) and 6.6% (n=8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n=7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of +/-20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

Determination of tadalafil in small volumes of plasma by high-performance liquid chromatography with UV detection

Tadalafil is a potent reversible phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction. This study describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of tadalafil in 50 microl of rat plasma. Tadalafil and the internal standard lamotrigine were extracted with 0.5 ml of tert-butyl methyl ether, after the samples alkalinized with 20 microl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a C18 column with the mobile phase of acetonitrile-water containing 20 mM phosphate buffer (pH 7) (35/65, v/v), at a flow rate of 1 ml/min. The eluant was detected at 290 nm. The retention time was about 4.5 min for lamotrigine and 15 min for tadalafil. No endogenous substances were found to interfere. Calibration curves were linear from 10 to 2000 ng/ml. The recovery of tadalafil from plasma was greater than 77%. The limit of quantitation was 10 ng/ml. The intra- and inter-day imprecision (expressed as coefficient of variation, C.V.) did not exceed 10.7%, and the accuracy was within 5.9% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring tadalafil concentration.     

Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 microg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552+/-152 ng/ml) and acute stressed animals showed an intermediate level (375+/-105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153+/-109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113+/-55 ng/ml for males; 128+/-25 for females) and the non-stressed humans (before exercise) show a lower concentration (63+/-37 ng/ml for male; 60+/-20 for female), respectively.     

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 μg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 μL was analyzed. The obtained peak concentrations (8.2-10.1 μg/mL) and T(max) (44-65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 μg/g.     

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration–signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC–LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU.     

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.