Marie Pospíšilová

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.

Simultaneous determination of methylparaben, propylparaben, sodium diclofenac and its degradation product in a topical emulgel by reversed-phase liquid chromatography

Abstract A novel reversed-phase liquid chromatographic method with UV spectrophotometric detection was developed and validated for the determination of compounds in topical emulgel. The method describes determination of active component sodium diclofenac, its degradation product 1-(2,6-dichlorophenyl)-indolin-2-one (occurring in formulation after long-term stability tests) and two preservatives presented in the emulgel, methylparaben and propylparaben, using flurbiprofen as an internal standard. The chromatographic separation was performed on a SUPELCO Discovery C18 column; the mobile phase for separation of all the compounds was methanol/phosphate buffer, pH 2.5 (65:35, v/v). The analysis time was

Determination of ambroxol or bromhexine in pharmaceuticals by capillary isotachophoresis

Expectorant drugs ambroxol (AX) and bromhexine (BX) were determined by capillary isotachophoresis (ITP) with conductimetric detection. The leading electrolyte (LE) was a buffer solution that contained 5 mM picolinic acid and 5 mM potassium picolinate (pH 5.2). The terminating electrolyte (TE) was 10 mM formic acid. The driving current was 80 microA (for approximately 200 s) or 50 microA (for approximately 350 s) and the detection current was 20 microA (a single analysis took about 8 min). The effective mobilities of AX and BX (evaluated with tetraethylammonium as the mobility standard) were 18.8 x 10(-9) m2 V(-1) s(-1) and 14.3 x 10(-9) m2 V(-1) s(-1) respectively. The calibration graphs relating the ITP zone length to the concentration of the analytes were rectilinear (r = 0.9993-0.9999) in the range 10 mg L(-1) (20 mg L(-1) for BX) to 200 mg l(-1) of the drug standard. The relative standard deviations (RSD) were 1.2 1.6% (n = 6) when determining 100 mg l(-1) of the analytes in pure test solutions. The method has been applied to the assay of AX or BX in seven commercial mass-produced pharmaceutical preparations. According to the validation procedure based on the standard addition technique the recoveries were 97.5-102.7% of the drug and the RSD values were 0.11-2.20% (n = 6).

Use of molybdate as novel complex-forming selector in the analysis of polyhydric phenols by capillary zone electrophoresis.

Molybdate was examined as a complex-forming additive to the CE background electrolytes (BGE) to affect the selectivity of separation of polyhydric phenols such as flavonoids (apigenin, hyperoside, luteolin, quercetin and rutin) and hydroxyphenylcarboxylic acids (ferulic, caffeic, p-coumaric and chlorogenic acid). Effects of the buffer concentrations and pH and the influence of molybdate concentration on the migration times of the analytes were investigated. In contrast to borate (which is a buffering and complex-forming agent generally used in CE at pH > or =9) molybdate forms more stable complexes with aromatic o-dihydroxy compounds and hence the complex-formation effect is observed at considerably lower pH. Model mixtures of cinnamic acid, ferulic acid, caffeic acid and 3-hydroxycinnamic acid were separated with 25 mM morpholinoethanesulfonic acid of pH 5.4 (adjusted with Tris) containing 0.15 mM sodium molybdate as the BGE (25 kV, silica capillary effective length 45 cm x 0.1mm I.D., UV-vis detection at 280 nm). With 25 mM 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulphonic acid/Tris of pH* 7.4 containing 2mM sodium molybdate in aqueous 25% (v/v) methanol as the BGE mixtures of all the above mentioned flavonoids, p-coumaric acid and chlorogenic acid could be separated (the same capillary as above, UV-vis detection at 263 nm). The calibration curves (analyte peak area versus concentration) were rectilinear (r>0.998) for approximately 8-35 microg/ml of an analyte (with 1-nitroso-2-naphthol as internal standard). The limit of quantification values ranged between 1.1 mg l(-1) for p-coumaric acid and 2.8 mg l(-1) for quercetin. The CE method was employed for the assay of flavonoids in medicinal plant extracts. The R.S.D. values ranged between 0.9 and 4.7% (n=3) when determining luteolin (0.08%) and apigenin (0.92%) in dry Matricaria recutita flowers and rutin (1.03%) and hyperoside (0.82%) in dry Hypericum perforatum haulm. The recoveries were >96%.

Determination of tramadol in various dosage forms by capillary isotachophoresis

Cationic capillary isotachophoresis (ITP) with conductometric detection has been used for separating and determining milligram amounts of tramadol [2-dimethylaminomethyl-1-(3-methoxyphenyl)-cyclohexanol hydrochloride] (I) in seven commercial mass-produced pharmaceutical preparations. The optimised ITP electrolyte system consisted of 5 mM potassium picolinate + 5 mM picolinic acid (pH 5.25) as the leading electrolyte and 10 mM formic acid as the terminating electrolyte. The driving and detection currents were 50 microA (for 320 s) and 10 microA, respectively (a single analysis took 12-15 min). Under such conditions the effective mobility of I was determined as 24.26 x 10(-9) m2 V(-1) s(-1) (with tetraethylammonium ion as standard); thermodynamic pKa value of I was 9.44 +/- 0.03 (n = 8) as determined by UV spectrophotometry at 25 degrees C and I = 0.01 (NaCl). The calibration graph relating the ITP zone length to the concentration of I was rectilinear (r = 0.99997) in the range 15-180 mg l(-1) of I. The relative standard deviation (RSD) was 0.21% (n = 6) when determining 60 mg l(-1) of I in pure test solution. Sample pre-treatment of the dosage forms involved dilution or extraction of I with water (for suppositories the extraction was carried out in an ultrasonic bath at 40 degrees C for 10 min). The method was suitable for determining 50 or 100 mg ml(-1) of I in injections and drops, 50 mg of I in capsules, and 100 mg of I in suppositories with RSD values 0.4 to 1% (n = 6). According to the validation procedure based on the standard addition technique the recoveries were 97.2-100.1% of I.

Separation and determination of pharmaceutically important polyols in dosage forms by capillary isotachophoresis

Capillary isotachophoresis (ITP) with conductometric detection has been used for separating polyols such as mannitol, sorbitol, dulcitol and xylitol. Since the polyols behave as extremely weak acids in aqueous medium, the complex-formation equilibria between boric acid and the analytes was employed to convert the neutral analytes into ionic species. The characteristic feature of this approach is the use borate, which functioned both as the complexing agent and the terminating ion. The ITP electrolyte system, optimised with respect to the sensitivity of the ITP determination and the quality of separation, consisted of 10 mM HCl+20 mM imidazole (pH 7.1) containing 0.05% poly(vinylalcohol) as the leading electrolyte and 20 mM boric acid (pH 8.1) as the terminating electrolyte. The driving and detection currents were 50 μA (for 630 s) and 20 μA, respectively. The effective mobilities of the borate–polyol complexes were determined; efficient separation of mannitol–sorbitol, sorbitol–xylitol and sorbitol–dulcitol mixtures was achieved. The calibration graphs were rectilinear (r=0.9995–0.9999) in the ranges 10–200 mg l−1 of sorbitol or mannitol. The relative standard deviations were 1.4 to 1.8% (n=6) when determining 100 or 200 g l−1 of mannitol and/or 50 to 200 g l−1 of sorbitol in mass-produced infusion solutions. A single analysis took about 20 min. The results obtained by the ITP method were in good agreement with those of the standard pharmacopoeial iodimetric method.

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K+ in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25°C), GlAm (migrating as glucosaminium cation) was well separated from K+ that could occur in the dosage forms as excipient. The CE analysis was performed in fused‐silica capillaries (50 µm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100–300 µg/mL; r2=0.997) and K+ (15–75 µg/mL; r2=0.997) when using ethanolamine (100 µg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 µg/mL for GlAm and 2.9 µg/mL for K+. The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6–103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.

Short communicationSeparation and determination of ketoprofen, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography

Simple micellar electrokinetic chromatographic (MEKC) method was developed for the determination of ketoprofen as the active substance and methylparaben and propylparaben as preservatives in a semisolid pharmaceutical preparation. Separation was carried out with a fused silica capillary and UV detection at 200nm. Optimized background electrolyte was 50mM tricine buffer containing 30mM sodium dodecyl sulfate as surfactant and 15% (v/v) of methanol. Single separation took about 13min. No statistically significant differences were found when comparing the results with those of RP-HPLC method reported in literature.

Determination of methylparaben, propylparaben, clotrimazole and its degradation products in topical cream by RP-HPLC

SummaryReversed-phase, high-performance liquid chromatographic (RP-HPLC) methods with UV detection were developed and validated for determination of compounds in a topical cream. The first method describes determination of the active component clotrimazole and two preservatives present in the cream; methylparaben and propylparaben. The second method describes determination of two degradation products of clotrimazole, imidazole and (2-chlorophenyl) diphenylmethanol, in a topical cream after long-term stability tests.Chromatographic separation was on a Purospher RP-18e column; the mobile phase in Method1 for separation of clotrimazole, methylparaben and propylparaben comprises acetonitrile and water (70:30 v/v). For determination of degradations products-imidazole and (2-chlorophenyl) diphenylmethanol—the optimum composition of mobile phase in Method2 was acetonitrile and water (75:25 v/v) apparent pH* 2.7. Analysis time was <10 min for both methods. The methods were found to be applicable for routine analysis of the active compound clotrimazole, preservatives and degradation products in the pharmaceutical product: topical cream 1% Clotrimazol Cream.

Separation and determination of sorbitol and xylitol in multi-component pharmaceutical formulations by capillary isotachophoresis

Pharmaceutically important polyhydric alcohols sorbitol (SO) and xylitol (XY) are efficiently separated and determined by analytical capillary isotachophoresis (ITP) with conductometric detection. The on-column complex-formation equilibria between the polyols and boric acid are utilized--the terminating borate ion acts as the complexing agent. The ITP operational system used consists of 10 mM HCl + 20 mM imidazole (LE, pH 7.0) and 20 mM boric acid (TE, pH 8.0). The effective mobilities of the borated SO and XY are 8.3 x 10(-9) m2 V-1 s-1 and 7.4 x 10(-9) m2 V-1 s-1, respectively. The ITP analysis is performed with the driving and detection currents of 50 microA (for 700 s) and 20 microA, respectively. The calibration graphs are rectilinear in the range 25-250 mg l-1 of SO and 50 to 500 mg l-1 of XY. The method is applied to the simultaneous assay of SO and XY in three mass-produced multi-component infusion solutions. Favourable values of the method validation parameters obtained confirm the suitability of the proposed ITP method for the quality control of pharmaceuticals.