Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography.

The present paper describes the development of a micellar electrokinetic chromatographic (MEKC) method for simultaneous determination of andrographolide, deoxyandrographolide and neoandrographolide in ethanol extracts of Andrographis paniculata. Separations were carried out in a fused-silica capillary tube with UV detection at 214 nm. Good separation was achieved using a 20 mM borate buffer, containing 20 mM sodium dodecyl sulphate and 10 mM sodium cholate, adjusted to pH 8.3 at an operating voltage of 25 kV, temperature of 35°C and a hydrodynamic injection of 5 s. The method was validated with good correlation coefficients obtained (0.9986–0.9989) while relative standard deviation (RSD) of migration time was between 1.14 and 2.42. It is concluded that this method could be used for speedy and accurate qualitative and quantitative analysis of bioactive diterpenoids in andrographis herb and its derived products.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

Solid-phase extraction and sample stacking-micellar electrokinetic capillary chromatography for the determination of multiresidues of herbicides and metabolites

Micellar electrokinetic capillary chromatography (MEKC) with diode array detection was used for the separation of 13 compounds (eight herbicides widely used in agriculture: metribuzin, lenacil, ethofumesate, atrazine, terbutryn, isoproturon, chlorotoluron and linuron, and five of their principal degradation products; namely, deethylatrazine, 2-hydroxyatrazine, deethyl-2-hydroxyatrazine, deisopropylatrazine and 3-chloro-4-methylphenylurea). Peak separation for the 13 analytes was not successful when a single surfactant system was employed, neither sodium dodecyl sulfate (SDS) nor dioctyl sulfosuccinate (DOSS) sodium salt. However, a mixture of these herbicides was successfully separated using a mixed micellar system involving SDS–DOSS in less than 14 min. An application study of an on-line concentration technique for MEKC was carried out to enhance sensitivity. The optimized on-line stacking procedure consisted simply of the addition of 50 mM of sodium chloride to the injection sample, the stacking effect being more intensive as analyte polarity increased. When this stacking procedure was combined with an off-line sample preconcentration step, based on solid-phase extraction, analytes could be detected in the ppb range. The whole method was applied to ultra-high-quality and natural waters. Linear relationships between the analytical signal and the initial analyte concentration were found to be independent of the type of water, except for the more polar analytes for which small differences were observed.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Determination of synthetic food dyes by capillary electrophoresis

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm × 75 μm I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of β-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.     

Micellar electrokinetic chromatography-mass spectrometry

The combination of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can be hazardous due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of off-line coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for on-line coupling MEKC with electrospray ionization (ESI)-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug and anodically migrating micelles, are reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of on-line MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactants allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z region. Alternatively, the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications.     

Capillary zone electrophoresis in the analysis of dyes and other compounds employed in the dye-manufacturing and dye-using industries

High resolution separation of several dyes and related intermediates, as well as other compounds employed in the dye-manufacturing and dye-using industries, has been achieved using capillary zone electrophoresis (CZE).

The analysis of anionic dyes and some non-coloured anionic intermediates has been achieved using 10 mM Na₂B₄O₇−40 mM sodium dodecyl sulphate (SDS) buffer; high-resolution separations of water soluble anionic, neutral and cationic intermediates were also achieved using this micellar buffer. Micellar electrokinetic capillary chromatography (MECC) has also been developed for the analysis of aqueous insoluble, electrically neutral compounds by incorporating a co-solvent, acetonitrile, into a micellar buffer. In addition, MECC has been used successfully for following all the major steps involved in the synthesis of a disperse dye.     

Simultaneous separation of the enantiomers of cizolirtine and its degradation products by capillary electrophoresis

The simultaneous enantioselective separation of (±)-cizolirtine and its impurities: (±)-N-desmethylcizolirtine, (±)-cizolirtine-N-oxide and (±)-5-(-hydroxybenzyl)-1-methylpyrazole was investigated by capillary electrophoresis. Electrokinetic chromatography with carboxymethyl-β-CD (CM-β-CD) and sulfobutyl-ether-β-CD was tried, showing good enantioseparation but poor chemical selectivity. The four racemic pairs were baseline separated, in a single run, by cyclodextrin-modified micellar electrokinetic chromatography. The migration buffer composition was: (60 mM hydroxypropyl-β-cyclodextrin–150 mM sodium dodecyl sulfate–50 mM disodium tetraborate, pH 9.2, in water)–butanol (95:5, v/v). Work was done to determine the effect of buffer components and their optimal concentration on selectivity. The method was validated with respect to enantioselectivity of cizolirtine as well as its degradation products and separation selectivity between the different components. Linearity, limit of detection, limit of quantitation and precision were also determined. This method is suitable for the enantiomeric purity determination and stability control of cizolirtine (racemic mixture or enantiomers) and its degradation products. Examples of electropherograms of (R)-cizolirtine degraded under stressed conditions are shown.     

Separation of coumarins by micellar electrokinetic capillary chromatography

Nine coumarins were successfully separated simultaneously using micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard. A carrier composed of buffer solution (20 mM sodium dodecyl sulfate-15 mM sodium borate-15 mM sodium dihydrogenphosphate)-acetonitrile (24:1) was found to be the most suitable electrolyte for this separation. The analysis time (22 min) was shorter than that using high-performance liquid chromatography (47 min). Contents of coumarins in the crude drug of Angelicae Tuhou Radix could be easily determined by the proposed method.     

Determination of ethoxylated bisphenol A dimethacrylate monomers in dental composites by micellar electrokinetic chromatography

Separation of monomeric constituents of ethoxylated bisphenol A (BIS-EMA) with between 2 and 15 ethoxy groups per phenol in the molecule was investigated with micellar electrokinetic chromatography at different volume percentages methanol in the background electrolyte (10 mM sodium dodecylsulfate, 100 mM borate–50 mM phosphate buffer, pH 7.0). The conditions allowed the differentiation of the lower from the higher BIS-EMA homologues and of isomers, and enabled the characterisation of commercial dental composite materials. The decay curve for the light induced radical polymerisation of BIS-EMA in composite specimens was determined. The content of leachable monomers after light curing was quantified, resulting in 6% of the initial value after the recommended curing time. The method is suited to determine monomer constituents in polymerised composite material in the ppm range.     

Open tubular capillary electrochromatography of underivatized amino acids using Rh(III) tetrakis(phenoxyphenyl)porphyrinate as wall modifier

The separation of 17 “common” underivatized amino acids was attempted by open tubular capillary electrochromatography (OT-CEC) in fused-silica capillaries coated with Rh(III) tetrakis(phenoxyphenyl)porphyrinate (Rh(III)TPP(m-OPh)₄OAc) using sodium phosphate and Tris–phosphate buffers as background electrolytes (BGEs). The OT-CEC separation of amino acids was compared with that obtained by capillary zone electrophoresis in bare fused-silica capillaries using the same BGEs. The amino acids were not derivatized and the UV-absorption detection was set at 200 nm. Depending on the experimental conditions at least 15 amino acids were separated. The best separations were obtained in a Rh(III)TPP(m-OPh)₄OAc-coated capillary in 50 mM Tris–100 mM phosphate buffer at pH 2.25. Separation of the critical triplet Val–Ile–Leu was always at least indicated being better at higher BGE concentrations. Regarding the sensitivity of the method, lower concentration limits of detection (LODs) in the coated capillary were obtained for Thr, Gly, Tyr, and Val; the other amino acids exhibited lower LODs in the uncoated capillary. The separation of acidic amino acids was not achieved.     

Electrodialysis for clean-up of strongly alkaline samples in ion chromatography

An electrodialysis method is described for the off-line neutralization of strongly alkaline samples containing trace levels of common inorganic anions. This method uses an electrodialysis cell comprising three compartments separated from each other by cation-exchange membranes. These compartments comprise an anode compartment housing, a platinum wire anode and 10 ml of a suitable hydrogen ion donating medium, a sample compartment which contains 1 ml of the alkaline sample, and a cathode compartment housing a platinum wire cathode and a dilute solution of sodium hydroxide. During electrodialysis at either constant applied current or constant applied power, hydrogen ions from the anode compartment displace sodium ions from the sample, thereby effecting neutralization.

Experimental parameters, such as the magnitude of the applied current or power, the type of cation-exchange membrane used and the design of the cell have been studied and optimum results were obtained using a Neosepta CM-2 membrane, of area 616 mm² supported between two perspex discs, with an applied current of 150 mA or applied power of 3 W. Under these conditions, a 1 ml sample of 1 M sodium hydroxide could be neutralized in 11 min. The most effective hydrogen ion donating medium consisted of a 2:1 (w/v) slurry of BioRad AG 50W-X8 (200–400 mesh, H⁺ form) cation-exchange resin in 1 mM octanesulfonic acid. Recoveries of solute anions (3–10 μg/ml) from the dialysed solution were close to quantitative, except for fluoride and nitrite, which gave recoveries of less than 60%. It is suggested that low recoveries for these ions are due to formation of neutral, protonated species within the membrane with subsequent loss by diffusion.     

Determination of theophylline and its metabolites in biological samples by liquid chromatography-mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is a powerful tool for analysis of drugs and their metabolites. We used a column-switching system in combination with atmospheric pressure chemical ionization LC–MS (LC–APCI–MS) for the determination of theophylline and its metabolites in biological samples. The separation was carried out on a reversed-phase column using methanol–20 mM ammonium acetate as a mobile phase at a flow-rate of 1 ml/min in 30 min. In the mass spectrum, the molecular ions of these drugs and metabolites were clearly observed as base peaks. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs.     

Phase-transfer catalytic determination of phenols as methylated derivatives by gas chromatography with flame ionization and mass-selective detection

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

On-line pretreatment and determination of parabens in cosmetic products by combination of flow injection analysis, solid-phase extraction and micellar electrokinetic chromatography

A convenient and automated method for on-line pretreatment and determination of three parabens (i.e. methyl, ethyl and propyl p-hydroxybenzoate) in cosmetic products is proposed by using flow injection analysis (FIA), solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC). An improved split–flow interface is used to couple SPE on C₈-bonded silica with MEKC separation, which can avoid running buffer contamination and reduce buffer consumption, especially, containing expensive reagents. The analytes are loaded onto a C₈ column at 0.6 mL/min for 60 s and eluted with a mixed eluent of 40% (v/v) 10 mmol/L sodium tetraborate buffer (pH 9.3) and 60% (v/v) ethanol at 0.75 mL/min. The MEKC separation is accomplished with a running buffer of 20 mmol/L sodium tetraborate (pH 9.3) containing 100 mmol/L sodium dodecyl sulfate (SDS) at 15 kV. For butyl p-hydroxybenzoate did not be detected in the cosmetic products, it was used as an internal standard (IS) added into the real samples. This FIA–SPE–MEKC method using IS allows the sample separation within 12 min and the sample throughput of five samples per hour with the relative standard deviation (R.S.D.) less than 2.3% (n = 5). The limits of detection (LOD) are in the range from 0.07 to 0.1 μg/mL (S/N = 3 and n = 11). The proposed method has been used to determine three parabens in real cosmetic products satisfactorily.     

Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. Application to human hair follicles.

A cost-efficient process was specifically designed for the preparation of gram amounts of highly pure murine immunoglobulin (Ig) G₁ monoclonal antibodies (mAbs). This rapid, simple and scalable purification process employs a unique binding and elution protocol for IgG₁ mAbs on a silica-based, mixed-mode ion-exchange resin followed by conventional anion-exchange chromatography. mAbs are bound to BakerBond ABx medium at pH 5.6 directly from serum-supplemented hybridoma culture supernatants. Contamining proteins and nucleic acids are removed by an intermediate wash at pH 6.5, followed by the specific elution of IgG₁ mabs with 100 mM Tris-HCL (pH 8.5). The mAb eluate is then loaded directly on to QAE-Sepharose Fast Flow medium and eluted with 10 mM sodium phosphate buffer (pH 7.4), containing 150 mM sodium chloride. The resulting IgG₁ mAbs are greater than 98% pure, free from measurable endotoxin, formulated in a physiological buffer and suitable for in vivo applications.     

Purity control of oxytetracycline by capillary electrophoresis

The applicability of capillary electrophoresis for the purity control of oxytetracycline (OTC) was investigated. OTC is a broad-spectrum antibiotic belonging to the group of the tetracyclines. Several related substances can be present due to fermentation or degradation, such as 4-epioxytetracycline, -apooxytetracycline, β-apooxytetracycline, anhydrooxytet racycline, 2-acetyl-2-decarboxamidooxytetracycline, tetracycline and 4-epitetracycline. Using fused-silica capillaries, the influence of buffer type, buffer pH and buffer concentration were investigated. In all cases 1 mM EDTA was added to prevent metal-ion complexation. The influence of the buffer counter-ion type was examined. Consequently, some instrumental parameters were changed such as capillary length and diameter as well as capillary temperature and applied voltage. The following method is finally proposed: fused-silica capillary, l (effective length) = 38 cm, L (total length) = 44 cm, 50 μm I.D.; buffer, sodium carbonate 20 mM-EDTA 1 mM, pH 11.25; voltage, 10 kV; temperature, 10°C. Linearity, limit of detection and limit of quantitation were determined as well as the relative standard deviations for all the analytes involved. This method is less selective then existing liquid chromatographic methods but it may be used as a complementary tool in purity control and stability studies.     

Quantitation of nifedipine in human plasma by on-line solid-phase extraction and high-performance liquid chromatography

An analytical methodology for nifedipine quantitation in plasma by on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contain C₂ and the analytes nifedipine and nitrendipine (internal standard) are separated on a C₁₈ column with a mobile phase consisting of acetonitrile–13 mM phosphate buffer pH 7 (65:35, v/v) followed by UV detection at 338 nm. Validation of the method demonstrated good recoveries (>90%), sensitivity (limit of quantification, 2 ng/ml), based on a 500 μl sample volume, accuracy and precision (<5.5% in concentrations greater than the limit of quantitation). This methodology has been used for bioequivalence studies.     

Qualitative determination of urinary morphine by capillary zone electrophoresis and ion trap mass spectrometry

A rapid, sensitive method for the determination of morphine and amphetamine was developed using capillary zone electrophoresis coupled with electrospray interface (ESI), ion-trap tandem mass spectrometry (CE-ESI-MS2). Morphine and amphetamine were separated in 20 mM ammonium acetate buffer (pH 6.6) and detected by ion-trap mass detector in different analytical time segments (0-6.25 min for amphetamine and 6.25-12.0 min for morphine) in which the tune file for each compound was used separately. Molecular ions of morphine (m/z 286) and amphetamine (m/z 136) were detected at 5.77 and 6.83 min, respectively, while product ions of MS2 for each compound (m/z 229, 201 for morphine and m/z 119 for amphetamine) were detected almost exactly at the same time with their parent compounds. The limits of detection (LOD) for MS2 determination were 30 and 50 ng/mL for amphetamine and morphine, respectively, with an S/N ratio of 3. For more sensitive detection of morphine, the sample was injected for a longer time (i.e., 80 s) and hydrodynamically transported into a CE capillary for MS detection. Morphine and its product ion appear at 0.36 and 0.39 min on the ion chromatogram, respectively, with a five-fold increase of detection sensitivity (LOD, 10 ng/mL). The CE-MS system thus established was further applied for forensic urine samples screened as morphine-positive by fluorescence polarization immunoassay (FPIA). These results indicated the feasibility of CE-ESI-MS2 for confirmative testing of morphine in urine sample.     

Separation of charged and neutral isotopic molecules by micellar electrokinetic chromatography in coated capillaries

Some unique separations are reported of pairs of deuterated and non-deuterated compounds by capillary zone electrophoresis (CZE) in coated capillaries in the absence and presence of surfactant micelles. Pyridine (pyridine-h₅) and [² H₅]pyridine (pyridine-d₅) could be separated in plain buffer (R = 1.1) and in 2% Nonidet P-40 (R = 1.5). Owing to the good separation obtained, it was possible to assess the degree of cross-contamination when “pure” isotopes were analysed. A 1:1 mixture of benzoic-h₅ and -d₅ acid was poorly separated in the absence of detergent (R = 0.39) but well separated in 50 mM sodium dodecyl sulphate (SDS) (R = 1.14). Benzyl-h₅ and -d₅ alcohol were reasonably well separated in a micellar system containing 50 mM SDS (R = 1.02) (a separation previously unreported in the literature. Benzene-h₆ and -d₆ were well separated again in presence of 50 mM SDS (R = 1.59). It appears that CZE offers a unique environment for the separation of positively and negatively charged and neutral isotope mixtures, previously reported only using RP-HPLC and GC.