Current Analytical Chemistry - Volume 10, Issue 2, 2014

Capillary Electrophoresis (CE) has demonstrated its wide potential to solve many analytical problems. Recent developments in CE with contributions for sample preparation, improvements in detection and aspects associated with application and productivity requirements in routine laboratories are reviewed and discussed. Although these capabilities of CE, the transference of CE methods to routine analytical work is very limited. The main contributions and advantages of CE with respect other instrumental separation techniques are summarized in this article, and also the scarce use in routine laboratories is critically discussed. After this balance, the arguments to identify the ‘analytical niche’ of CE in applied analytical laboratories are reported.

An on-line preconcentration capillary electrophoresis (CE) methodology, which combines a normal stacking mode (NSM) with a dynamic pH junction technique, is introduced in this paper. A systematic study of the parameters (concentration and pH value of background electrolyte, composition of sample matrix, injection pressure and time, separation voltage and temperature) affecting on-line concentration of seven related phenolic compounds was investigated and optimized. Under the optimum focusing conditions, about 54-fold improvement in the detection sensitivity was obtained compared with usual hydrodynamic sample injection (0.5 psi, 5 s) without detriment in separation efficiency. In particular, the concentration limits of detection (LOD) (S/N = 3) for the phenolic compounds obtained after preconcentration were from 6.0 to 11.0 ng mL-1 with UV detection without any pretreatment procedure. The proposed method has been validated with RSD values between 1.13% and 1.98% for migration times and between 1.28% and 4.65% for peak areas. Developed NSM-dynamic pH junction method was applied for determination of related phenolic compounds in labeled M. officinalis medicinal herbal products commercialized in our country.

A rapid and simple analytical capillary zone electrophoresis (CZE) method has been developed and validated for quantitative determination of atypical antipsychotic drug risperidone in commercially available film-coated tablets. CZE was performed in an uncoated fused-silica capillary 40.2 cm total length x 75 μm i.d. (30 cm effective length) using a 20 mmol mL-1 sodium phosphate solution containing 0.1% triethylamine (v/v), pH 2.5 adjusted with ortho-phosphoric acid as background electrolyte. The applied voltage was +28 kV and UV detection at 236 nm, nitrazepam was used as internal standard (IS). Under these conditions, the separation of risperidone was achieved in less than 2.5 min. The method was validated and response was found to be linear with determination coefficient better than 0.999. The repeatability and intermediate precision expressed as relative standard deviation were less than 1.5 %. The accuracy, resulting from recovery experiments, was between 98.84 and 101.24 %. The active pharmaceutical ingredient was subjected to acid, alkaline and neutral hydrolysis and oxidative stress conditions. No interference from degradation products and also from tablet excipients was observed. The method was simple, precise and fast and could be successfully applied for determination of risperidone in pharmaceutical dosage forms and can be used for routine analysis in pharmaceutical industries. Furthermore, the method can be used as a stability-indicating method.

A rapid and simple microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of ethinylestradiol and levo-norgestrel in a commercial tablet formulation. The analysis was performed with an electrolyte composed of 0.5% (w/w) ethyl acetate, 1.2% (w/w) butan-1-ol, 0.6% (w/w) sodium dodecyl sulfate, 15% (v/v) ethanol and 82.7% (w/w) 12 mmol L-1 sodium tetraborate aqueous buffer at pH 9.23 and direct UV detection at 200 and 247 nm. For quantitative purposes, estrone was used as an internal standard. Retention time for levo-norgestrel and ethinylestradiol was approximately 2.1 and 2.5 min respectively. Acceptable precision (<0.90% RSD, linearity (r2 > 0.99; concentration range from 24.0 to 36.0 μg mL-1), limits of detection (LOD) and quatification (LOQ) for ethinylestradiol of 1.0 μg mL-1 and 3.2 μg mL-1, respectively, and for levo-norgestrel of 1.4 μg mL-1 and 4.1 μg mL-1, respectively, and recovery of 100 ± 2 % at three concentration levels were obtained. Based on the performance characteristics, the proposed methodology was found suitable for the determination of ethinylestradiol and levo-norgestrel in tablet formulations.

This article focuses on the main developments in element speciation analysis by capillary electrophoresis (CE) since the 1990s. A brief description of the main techniques employed in CE analysis has been reviewed with emphasis in the chemical species derived from inorganic forms and organic compounds bound to metal and non-metal elements. Analysis of the most important elemental species existing in the environment and biological fluids has been discussed employing the many different separation techniques available in CE analysis. Special attention has been paid to simple analytical methods but multiple element analysis of chemical species has also been considered. The great advances of modern CE equipments coupled to atomic absorption, inductively plasma emission and mass spectrometry as detection systems make possible to compete with HPLC for this purpose. Analysis of arsenic, chromium, iron, mercury, lead and tin species in their inorganic and organic forms has been discussed in more detail for their relevance in environmental chemistry.

A capillary zone electrophoretic (CZE) method has been developed for the determination of zoledronate and its related impurities (phosphyte and phosphate). Successful separation of the drug from the impurities was achieved using 7.5 mM of phthalic acid adjusted to pH 3.50 with TRIS, as background electrolyte with an indirect detection at 205 nm. The optimized method was validated for specificity, precision, linearity and accuracy. The limit of detection was 1.8 μg ml-1 and the limit of quantification was 5.9 μg ml-1 for phosphyte. The limit of detection was 2.8 μg ml-1 and the limit of quantification was 9.3 μg ml-1 for phosphate. The developed CZE method used to determine zoledronate, phosphyte and phosphate as bisphosphonates impurities can be used to evaluate the quality of regular production samples of zoledronate.

An estrogen selected molecular imprinted polymer (MIP) has been developed to be used as an alternative sorbent in solid phase extraction of serum samples before capillary electrophoresis analysis. Following a heat polymerization, MIP has been synthesized using methacrylic acid (MAA) as functional monomer, ethyleneglycol dimethacrylate (EGDMA) as crosslinker, benzoyl peroxide as radical initiator and 17β- estradiol as template, and toluene as porogen. The optimized molecular imprinted solid phase extraction (MISPE) was compared to traditional C18 solid phase extraction to purify estrogens from complex matrices. It was concluded that the MISPE system developed is useful for the selective extraction of 17β- estradiol and its major natural related estrogens, estriol and estrone, from serum samples with minimal interferences and high recoveries.

Optimization and validation of a capillary zone electrophoresis (CZE) method for the determination of inner and external inorganic sulfate from doxorubicin-loaded liposomes as a measurement of encapsulation efficiency are presented. Sulfate was determined using a fused silica capillary (50 cm x 75 μm ID) and background electrolyte (BGE) composed of 5 mM chromate with 1 mM of TTAB at pH 10.5 with indirect UV detection at 254 nm. The separations were achieved at temperature of 25 ºC, 5 kV of reverse applied voltage using a pressure of 0.5 psi for 5s of sample introduction. Sulfate quantitation from the outside of liposomes was obtained by simple dilution of the liposome sample but an extraction with a non-ionic surfactant and a mixture of methanol-chloroform-water were necessary to breakdown the liposomes for the quantitation of the total sulfate (inside and outside) with acceptable recovery percentages (96.3% to 102.6%). Parameters of validation were evaluated according to ICH guidelines such as specificity, linearity, LOD, LOQ, accuracy, precision and robustness. The proposed CE method resulted to be suitable for the inner and external sulfate analysis of doxorubicin-loaded liposomes for routine quality and stability control studies.

Capillary electrophoresis (CE) with organic solvents has gained special interest in pharmaceutical analysis as it offers an alternative to overcome problems with the solubility especially of lipophilic analytes in aqueous solutions. However, beside the solubility of the compounds (typical pharmaceuticals are weak acids and bases, that can be present in ionic and molecular form as well) other properties and variables relevant for capillary electrophoretic separations are influenced by the solvent. In the present paper the role of the solvent on these quantities is discussed from a more general point of view. This discussion includes the influence on the ionic mobility and its different concentration dependence for solvents like methanol, acetonitrile, propylene carbonate, formamide, N,N-dimethylformamide, N,N-dimethylacetamide and nitromethane in comparison with water. It also includes the explanation of the partially extreme change in the pK values of the weak acids and bases when water is replaced by the organic solvent, by taking into account the stabilization of the individual charged and uncharged particles involved in the protolysis equilibria. For a better comprehension of the role of the solvent on the resolution of the analytes, also its influence on the separation efficiency has to be elucidated. For this purpose, the function of the solvent concerning the inevitable peak broadening due to diffusion and the limitations of the achievable plate number is explicated based on a sound background. As the understanding of the effect of the solvent on the relevant properties of the analytes and the constituents of the background electrolyte, and on the properties of the separation system is a prerequisite for a systematic planning of the nonaqueous CE experiment and the critical evaluation of the result obtained, the present paper tries to contribute to this goal.

Extremely good enantioseparation of a kind of commonly used β-blockers, (±)-propranolol, has been achieved on silica gel layers which were impregnated with β-cyclodextrin and oxidized multi-walled carbon nanotubes. Solvent system tert-butenol-acetonitrile (1:1, v/v) successfully resolved the enantiomers of (±)-propranolol. The spots were detected at 254 nm with a UV lamp. The effect of concentration of the mobile phase variation was also studied.

Salicylic acid (SA) plays a key role against pathogens of plants and also induces resistance, hence it is an important signaling compound in plants. SA derivative, such as acetylsalicylic acid (Aspirin®), is widely used and marketed today as a non steroidal anti-inflammatory drug (NSAIDs) which is one of the first “wonder drugs” of the 20th century. Here we report a method for quantification of SA and its derivatives i.e. 3,4-dihyroxybenzoic acid (3,4-DHBA), gallic acid (GA) and gallic acid methyl ester (GAME) obtained from Primulae radix. First, the total polyphenol content (TPC) of Primulae radix extracts was determined by Folin-Ciocalteu assay. In that context reflux and microwave assisted extracts (MAE) in different solvents, i.e. water, 100% ethanol, 100% methanol, 50% ethanol and 50% methanol were evaluated. 50% ethanol MAE was taken for the quantitative analysis of SA and its derivatives as the highest amounts of total polyphenol content were yielded. This extract was pre-concentrated by solid phase extraction (SPE) using Strata-X (surface modified copolymer of styrene-divinylbenzene) cartridges. Eluted sample was analyzed by a reversed phase liquid chromatography-electrospray ionization mass spectrometry (RPLC-ESI-MS) system in selected ion monitoring (SIM) mode. Lower limits of detection (LOD) of 0.74 ng/ml for SA, 1.39 ng/ml for 3,4-DHBA, 3.00 ng/ml for GA and 0.37 ng/ml for GAME and limits of quantification (LOQ) of 2.45 ng/ml for SA, 4.62 ng/ml for 3,4-DHBA, 10.00 ng/ml for GA and 1.23 ng/ml for GAME were determined. Correlation coefficient values > 0.99 for all standards were obtained. The determined mean concentrations were 28.80 and 29.34 μg/g for SA, 9.22 and 8.61 μg/g for 3,4-DHBA, 6.17 and 7.12 μg/g for GA and 0.14 and 0.03 μg/g for GAME using intra and inter-day analyses respectively. Finally, presented data prove that introduced method is highly sensitive and reproducible for the simultaneous quantification of SA, 3,4-DHBA, GA and GAME in Primulae radix extract.

Dispersive liquid–liquid microextraction (DLLME) coupled with high-performance liquid chromatography (HPLC) equipped with UV detector was used for the extraction and determination of efavirenz in plasma sample. Acetonitrile and chloroform were used as disperser and extraction solvents, respectively. Several parameters, including extraction solvent, the type of dispersion solvent, volume of extraction and dispersion solvents, pH, ionic strength of the media, as well as centrifuging time were optimized. Samples were injected on a reversed-phase C18 analytical column. The mobile phase consisted of 20 mM phosphate buffer (pH=3.0) and acetonitrile (40:60, v/v). The calibration curve of the proposed method was linear in the range of 0.1–100 μg/L. The relative standard deviations (RSD, %) were 3.4–7.5% (n=6) and the limit of detection (LOD) was 0.01 μg/L. The proposed method was applied to the analysis of plasma sample and spiked recoveries in the range of 98.5–102.5% were obtained. The obtained results show that DLLME is a very simple, rapid, sensitive, and efficient analytical method for the determination of efavirenz in plasma.

Silica gel was modified via silanization with 3-chloropropyltrimethoxysilane and subsequently Cu(II)-5- dodecylsalicylaldoxime was grafted. Obtained sorbent was applied to on-line copper(II) preconcentration and its FAAS determination. Effective copper(II) elution was observed for 2% v/v nitric acid. The new sorbent exhibits optimal sorption efficiency within pH 5.6 and 7.5. A portion of the sorbent may be used repeatedly for not less than 100 sorptiondesorption cycles. The sorbent indicates high sorption and elution effectiveness of ca. 96 and 99%, respectively. Introduction of a preconcentration step before flame atomic absorption spectrometry (FAAS) copper determination with 60 s loading time resulted in 12-fold increase in the measured absorbance. This could be further raised by extending the loading time. Metals, like Zn(II), Mn(II) and Pb(II) caused negative interference effect while presence of Ca(II) and Mg(II) caused increase in the obtained signals. Copper(II) in samples such as: well water, mineral water, river and tap water was determined via the standard addition method. Detection limit for the proposed procedure was estimated at 1.5 μg L-1 (3σ). Accuracy of the method was assessed via comparison of the results obtained for copper determination in ground water and waste water certificate reference materials with those obtained with the use of a reference method (ICP MS).