Quantitation of nicotine in tobacco products by capillary electrophoresis

A simple and rapid capillary electrophoresis (CE) method was developed for the quantitation of nicotine in commercial tobacco products. The method involves a 6 min run at 30 kV, using a 50 mM phosphate buffer (pH 2.5), paraquat as internal standard, and UV detection at 260 nm. Nicotine was extracted from tobacco products in <15 min. Recoveries from spiked extracts were >95%, and the extraction efficiencies of water, 1 M HCI, 1 M acetic acid, 5 mM phosphate buffer (pH 2.5), and 1% triethanol amine were similar. Nicotine concentrations in 67 samples of cigarettes, cigars, and bidis varied between 0.37 and 2.96% (w/w). An established gas chromatography/mass spectrometry method using toluene extraction consistently yielded lower nicotine values than the CE method. Experimental evidence suggests that this is due to insufficient extraction of nicotine by toluene.     

Generic capillary electrophoresis conditions for chiral assay in early pharmaceutical development

Generic capillary electrophoresis (CE) conditions have been implemented for chiral separations in early pharmaceutical development. The chiral CE separations of several pharmaceutical samples at different stages of development, i.e., discovery, process chemistry, and investigative new drug application, have been obtained using sulfated beta-cyclodextrin (CD). Several sulfated beta-CDs have been screened to select an appropriate enantioselective agent. The use of a generic CE method allows for a convenient and rapid chiral recognition of different weak bases, with minimal or no method development. CE using sulfated beta-CD for the chiral separation of N-benzoyl methyl piperazine has been validated for linearity, precision, accuracy, limits of detection and quantitation (LOD, LOQ). Although less sensitive than a specific liquid chromatography method using a Chiralpak AD column, the overall performance of the chiral CE method was found comparable. Validation data demonstrate that a LOD of 0.1%, sufficient to fulfill regulatory requirements, is achievable by chiral CE.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

Further research on iodine speciation in seawater by capillary zone electrophoresis with isotachophoresis preconcentration

A novel, simple and highly sensitive CE method was developed to determine total iodine (TI) in seawater. The method is based on the on-capillary reduction of iodine species to iodide by a reductant, introduced into the capillary before sample injection, the preconcentration of iodide using isotachophoresis, followed by its UV detection. Under optimized conditions for reduction and CE separation, the limit of detection for TI (S/N = 3) reached 0.4 microg L(-1) (226 nm). The repeatability of migration time and peak area, expressed by relative standard deviation, was 0.46 and 1.45%, respectively (n = 19). The correlation factor was 0.9991 (n = 10) for the concentration range of 12-115 microg I L(-1). The CE results obtained for the real seawater analysis agreed with the data of ion chromatography. To determine the genuine TI by the proposed method, organic iodinated compounds in the sample were treated with H202 and irradiation with UV light before analysis.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.     

Physicochemical characterization of a PEGylated liposomal drug formulation using capillary electrophoresis

In this work, the applicability of using CE to perform a physicochemical characterization of a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was examined. Characterization of the liposomal drug formulation using CE instrumentation encompassed: determination of the electrophoretic mobilities, size determination by Taylor dispersion analysis and interaction studies. Electrophoretic mobilities determined by CE were compared with the results obtained by laser Doppler electrophoresis, which were found to be subject to larger variation. Average hydrodynamic diameters of the liposome preparations, as determined by Taylor dispersion analysis, were in the range of 61-84 nm and were compared with the results obtained by dynamic light scattering. Interactions between oxaliplatin (and paracetamol) and the PEGylated liposome were non-detectable by CE frontal analysis as well as by liposome electrokinetic chromatography. In contrast, for the more lipophilic compound propranolol, apparent liposome-aqueous phase distribution coefficients (D(lip) ) were successfully determined by both electrokinetic chromatography (log?D(lip) =2.10) and by CE frontal analysis (log?D(lip) =2.14). It is envisioned that CE and capillary-based techniques, including Taylor dispersion analysis, will be useful tools for the characterization of nanoparticulate (e.g. liposomal) drug formulations.     

Analysis of bioactive ingredients in the brown alga Fucus vesiculosus by capillary electrophoresis and neutron activation analysis

Two different types of bioactive components of the seaweed Fucus vesiculosus were analysed: (1) polyphenols (phlorotannins) by capillary electrophoresis (CE) and (2) mineral part (including bioactive microelements) by neutron activation analysis (NAA). CE experiments were carried out using a UV detector (at 210 nm) and an uncoated silica capillary. The best separation was achieved at a voltage of 20 kV using borate or acetate buffer in a methanol/acetonitrile mixture as background electrolyte. The CE analysis data were confirmed by high-performance liquid chromatography (HPLC). Determination of mineral composition of algal biomass by NAA was performed on the basis of various nuclides; the best results (from 38 elements determined) were obtained for Mn, Fe, Zn, As, Br, Sr, I, Ba, Au and Hg.     

Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary,LC-ESI-MS and MALDI-TOF-MS

The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.     

Comparison of HPLC and CE methods for the determination of cetirizine dihydrochloride in human plasma samples

Two methods, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC), for analysis of cetirizine dihydrochloride in small sample volumes of human plasma were compared. The CE and HPLC assays were developed and validated by analyzing a series of plasma samples containing cetirizine dihydrochloride in different concentrations using these two methods. The extraction procedure is simple and no complicated purification steps or derivatization are required. The analysis time in the HPLC method was shorter than that in the CE method, but solvent consumption was considerably lower in the CE method. The calibration curve was linear to at least 10-1000 ng/mL both for CE and HPLC with r(2) = 0.9993 and r(2) = 0.9994, respectively. The detection limits for cetirizine dihydrochloride were 3 and 5 ng/mL with CE and HPLC (a UV detector was applied in the both cases), respectively. Both methods were selective, robust and specific, allowing reliable quantification of cetirizine dihydrochloride, and could be useful for clinical and biomedical investigations.     

Analytical determination of nicotine in tobacco leaves by gas chromatography–mass spectrometry

A preliminary investigation using gas chromatography–mass spectrometry (GC–MS) to analyze the nicotine contained in tobacco leaves was carried out. Nicotine is an alkaloid and tobacco leaves was extracted with methanol and determined by GC–MS. The detection limit for nicotine was at the ppm level for non selective monitoring and the nanogram level for selective detection. This is a simple chromatography–mass spectrometry method for the analysis of nicotine in tobacco leave. Compared to other currently utilized methods for the detection of nicotine in tobacco leaves, the GC–MS provided advantages of high sensitivity, nicotine specific detection and lower instrumentation cost.     

Determination of ciprofloxacin and its impurities by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been elaborated for separation, identification and determination of ciprofloxacin and its impurities. The separation, phosphate buffer pH 6.0 was supplemented with 0.075 M pentane-1-sulfonic acid sodium salt. The elaborated method was validated. The selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision, and accuracy of capillary zone electrophoresis were evaluated. The results obtained by CZE were also compared with those obtained by liquid chromatography. Regarding the validation results the CE method fulfils the current European Pharmacopoeia (Eur. Ph.) requirements. The evaluated CE method could be applicable to the analysis of different medicinal products containing ciprofloxacin.     

A Validated Quantification of Benzocaine in Lozenges Using TLC and a Flatbed Scanner

We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We present an RP-18 phase separation on a cyanopropyl plate (Merck, Darmstadt, Germany) using water, CH₃CN, dioxane, ethanol, and NH₃ (25 %) (8 + 2 + 1 + 1 + 0.05, v/v) as the mobile phase. We also separated benzocaine in a normal phase system on silica gel 60 LiChrospere^® plates (Merck, Darmstadt, Germany) with the mobile phase MTBE/cyclohexane (1 + 1, v/v). The calibration functions for benzocaine in both separations were linear in the range from 1 to 1,000 ng per spot. The range of linearity covers two magnitudes of power because the Kubelka–Munk expression was used for data transformation. In the cyanopropyl-system, the benzocaine amount was quantified as 242.5 ± 18.2 ng in a spot or 6.86 ± 0.52 mg in a single lozenge. The amount of 7.0 mg benzocaine per lozenge was labelled. The combined uncertainty of sample and calibration measurements was statistically calculated using a significance level of α = 0.05 to a total relative uncertainty of 7.49 %. The separation method is inexpensive, fast and reliable.     

Use of dynamically coated capillaries with added cyclodextrins for the analysis of opium using capillary electrophoresis

A rapid, precise, accurate, and robust method using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of the major opium alkaloids in opium is presented. Dynamic coating of the capillary surface is accomplished using a commercially available reagent kit (polycation coating followed by polyanion coating). The addition of dual cyclodextrins (hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin) to the run buffer imparts excellent selectivity for the opium alkaloids. For the determination of morphine, papaverine, codeine, noscapine and thebaine in opium gum and opium latex samples (using tetracaine as an internal standard) good agreement with values obtained by gradient high-performance liquid chromatography is obtained. Compared to the latter technique, CE affords better resolution with significantly faster analysis time (12 min versus 29 min). Dynamically coated capillaries, which give rise to a relatively high and robust electroosmotic flow (EOF) at the background electrolyte pH of 2.5, allow for rapid analysis and excellent migration time and peak area precision (RSDs < or = 0.12% and < or = 1.2%, respectively). Reproducible separations (relative migration times) for over 500 samples have been obtained on a single capillary. The nature of the injection solvent, the injection time and the contents of the waste vials have a profound effect on the pressure injection precision of the relatively hydrophobic solutes. The CE conditions reported in this study are also applicable to the analysis of lysergic acid diethylamide (LSD) exhibits.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.     

Capillary liquid chromatography-mass spectrometry and micellar electrokinetic chromatography as complementary techniques in environmental analysis

Applications of capillary electrophoresis (CE) and capillary liquid chromatography (LC) to environmental analysis have been limited. In this work we present applications of micellar electrokinetic chromatography (MEKC) to the analysis of environmental matrices for synthetic dyes. Separations obtained by capillary LC are compared with those obtained under MEKC for seven selected dyes. Both techniques are capable of resolving the subject compounds at high efficiency. Recovery data for spiked water and soil matrices were obtained for four dyes using solid-phase extraction cartridges and disks with determination by MEKC-UV detection. Both pH adjustment via acid and ion-pairing via a cationic surfactant were investigated for isolating dyes. Capillary LC detection was by continuous-flow liquid secondary ion mass spectrometry (CF-LSI-MS) whereas MEKC used UV detection (214 nm). Application of peak-profiling at high mass resolution is illustrated with the capillary LC-MS technique. Interfacing capillary LC under CF-LSI-MS using the coaxial arrangement is easier than interfacing CE with this arrangement. MEKC provides a powerful screening and determinative technique, while capillary LC-MS provides a confirmatory tool.     

Drug-plasma protein binding assay by electrokinetic chromatography-frontal analysis

We developed a rapid, microscale and reliable analytical method for binding of drugs to plasma proteins using capillary electrophoresis (CE) with ionic cyclodextrins (CD) combined with frontal analysis. These CDs were used as pseudostationary phases of electrokinetic chromatography (EKC). The CD-modified EKC (CDEKC) approach allowed us to separate anionic drugs from plasma proteins, whereas CZE could not separate these drugs from plasma proteins because they had a similar mobility like plasma proteins. CDs uniquely interact with these drugs but not with plasma proteins. Therefore, CDEKC could be coupled with frontal analysis to measure the binding of anionic drugs to plasma proteins. The binding values obtained by CDEKC were highly consistent with those determined by the ultrafiltration method. Our CDEKC approach should expand the applicability of CE to protein binding analysis.     

Evaluation of capillary electrophoresis for the analysis of nicotine and selected minor alkaloids from tobacco

Summary Difficulties encountered in the gas or liquid chromatographic analysis of nicotine and other alkaloids in tobacco are largely due to the ionic character of these compounds. The potential of using capillary electrophoresis (CE) as an alternative analytical tool to eliminate these problems was evaluated. Parameters including electroosmotic flow, ionic forms of the analytes, buffer composition and applied voltage were studied using nicotine as a model compound. Ionic forms and electrophoretic mobility, as well as UV absorbance, of nicotine were controlled by varying the pH of an aqueous buffer solution. Thus the separation was optimized based on the characters of alkaloids and the nature of capillary electrophoresis. For tobacco samples in which nicotine accounts for more than 98% of the total alkaloid content, a quick method for the determination of nicotine in an aqueous tobacco extract within 100 seconds can be achieved.     

Urtica dioica agglutinin: separation, identification, and quantitation of individual isolectins by capillary electrophoresis and capillary electrophoresis-mass spectrometry

With benign prostatic hyperplasia (BPH) being a major health problem in ageing men, alternative therapeutic approaches (e.g., with phytopharmaceuticals) are of great interest. Based on pharmacological evidences, one of the most promising options in that respect are the lectins found in Urtica dioica (stinging nettle) roots. In this study the qualitative and quantitative analysis of individual isolectins in U. dioica extracts is described, which is the first report on using capillary electrophoresis (CE) for the analysis of lectins in plant material at all. By utilizing a 200 mM sodium acetate buffer (pH 3.75) a baseline separation and determination of four closely related isolectins was feasible within 20 min in the aqueous plant extracts. The individual compounds were identified based on reference compounds as well as data obtained from CE-mass spectrometry (MS) experiments. After modifying the optimized CE conditions to 100 mM ammonium formate buffer with pH 3.75 and a voltage of 15 kV, the isolectins were clearly assignable in positive electrospray ionization (ESI) mode. The quantitative results obtained by CE (the total lectin content varied from 0 to 0.42% in the samples) were accurate (recovery rates of spiked samples between 92.5 and 96.2%), precise (relative standard deviation < 5%) and in good agreement to those obtained by High-performance liquid chromatography (HPLC). As for peak resolution, assignable compounds and required separation time the newly developed CE method was clearly advantageous over the determination achieved by LC.     

Trace determination of short-chain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

In this study, a new capillary electrophoresis (CE) method is described originally for the sensitive and selective determination of short-chain aliphatic amines in biological samples. These amines were converted into their N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) derivatives and measured by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. The derivatization conditions and separation parameters for the aliphatic amines were optimized in detail. The SIFA-labeled amines were fully separated within 8.5 min using 25 mM pH 9.6 boric acid electrolyte containing 60 mM sodium dodecyl sulfate (SDS). The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.02 to 0.1 nM, which was the lowest value reported by CE methods. The developed method was successfully employed to monitor aliphatic amines in serum and cells samples. After comparison of other CE methods using different fluorescent probes, the present method represents a powerful tool for the trace determination of aliphatic amines in complex biological samples.     

不同缓冲体系对毛细管电泳法分离15种核苷类化合物效果的比较

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(BGE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(DEA)为背景电解质,对毛细管区带电泳(CZE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOF/MS)以及胶束电动毛细管电泳(MEKC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CZE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 mmol/L DEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEKC模式下,以25 mmol/L磷酸氢二钠(添加70 mmol/L十二烷基磺酸钠(SDS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOF/MS分析中,以20 mmol/L乙酸铵(pH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用DEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。