Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 microm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.     

pH-mediated acid stacking with reverse pressure for the analysis of cationic pharmaceuticals in capillary electrophoresis

When using capillary electrophoresis (CE) for the analysis of biological samples, it is often necessary to employ techniques to overcome peak-broadening that results from having a high-conductivity sample matrix. To improve the concentration detection limits and separation efficiency of cationic pharmaceuticals in CE, pH-mediated acid stacking was performed to electrofocus the sample, improving separation sensitivity for the analyzed cations by 60-fold. However, this method introduces a large titrated acid plug into the capillary. To overcome the limitations this low-conductivity plug poses to stacking, the plug was removed prior to the separation step by applying reverse pressure to force it out of the anode of the capillary. Employing this technique allows for roughly twice the volume of sample to be injected. A maximum sample injection time of 240 s was attainable with baseline peak resolution compared to a maximum sample injection time of 120 s without reverse pressure, leading to a twofold decrease in the limits of detection of the analytes used. Separation efficiency overall is also improved when utilizing the reverse pressure step. For example, a 60 s sample injection time results in 94,000 theoretical plates as compared to 60,500 theoretical plates without reverse pressure. This reverse-pressure method was used for detection and quantitation of several cationic pharmaceuticals that were prepared in Ringer's solution to simulate microdialysis sampling conditions.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Capillary electrophoresis of methylmercury with injection by sample stacking

A procedure for separation and quantitation of methylmercury by capillary electrophoresis using sample stacking as the injection technique is presented. The CE conditions have been optimized in order to separate the methylmercury from the excess cysteine peak and to concentrate large volumes of sample obtaining a low detection limit. Under the proposed operational conditions, the detection limit (S/N = 3) was 12 ng g⁻ and the limit of quantitation (S/N = 10) was 20 ng g⁻¹ with a linear range of 20–100 ng g⁻¹ (as methylmercury in samples). The method was tested using different reference materials with a certified methylmercury content.     

Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL⁻¹ for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10–1000 ng mL⁻¹ and 21–1000 ng mL⁻¹, respectively.     

CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Identification and quantitation of cis-ketoconazole impurity by capillary zone electrophoresis-mass spectrometry

trans-Ketoconazole was identified and quantified as impurity of cis-ketoconazole, an antifungal compound, by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS). The chirality of this impurity was demonstrated separating their enantiomers by adding heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin to the separation buffer in capillary electrophoresis (CE) with UV detection. However, MS detection was hyphenated to the CE instrument for its identification. As both compounds are diastereomers, they have the same m/z values and are needed to be separated prior to the MS identification. A 0.4M ammonium formate separation buffer at pH 3.0 enabled the separation of the impurity from cis-ketoconazole. Under these conditions, the optimization of ESI-MS parameters (composition and flow of the sheath-liquid, drying temperature, drying gas flow, and capillary potential) was carried out to obtain the best MS sensitivity. CZE-ESI-MS optimized conditions enabled the identification of trans-ketoconazole as impurity of cis-ketoconazole. In addition, the quantitation of this impurity was achieved in different samples: cis-ketoconazole standard and three different pharmaceutical formulations (two tablets and one syrup) containing this standard. In all cases, percentages higher than 2.0 were determined for the impurity. According to ICH guidelines, these values required the identification and quantitation of any impurity in drug substances and products.     

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013–2015)

This review updates and follows‐up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug‐related substances, chiral CE, and chiral CE‐MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor–ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.     

Combination of solid-phase extraction and large-volume stacking with polarity switching in micellar electrokinetic capillary chromatography for the determination of traces of nonsteroidal anti-inflammatory drugs in saliva

A reliable MEKC method for the identification and quantitation of traces of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketoprofen, fenbufen and indomethacin in saliva is proposed. Using CE to analyze biological samples often requires suppressing the interferences and peak broadening typically resulting from high-conductivity sample matrices. We addressed this problem by using Microcon, a centrifugal filter device, to reduce the viscosity of saliva and exclude most higher-molecular-mass substances. This initial pretreatment was followed by the combined used of off-line SPE to isolate and concentrate the analytes, and large-volume stacking with polarity switching (LVSS) in the capillary. These two preconcentration steps allow the determination of NSAIDs at concentrations above 0.1 microg/L; therefore, the proposed SPE/LVSS/MEKC method affords a 500-fold sensitivity enhancement relative to conventional CE injection. The LODs obtained afford the determination of NSAIDs in saliva, where analytes can be present at the microgram-per-liter level.     

Analysis of naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis with sample stacking

This study systematically investigates the optimal conditions for analyzing the positional isomers of multi-charged naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis (CE). Specifically, this work employs large-volume sample injection with the electrode polarity switching technique. The most effective separation and sample stacking conditions were 15 mM borate buffer with a mixture of beta- and gamma-cyclodextrin (concentration ratio 3:7 mM) at pH 9.2, and the sample hydrodynamic injection of up to 60 s at 3 p.s.i. (around 1.8 microl, and 1 p.s.i. = 6.9 kPa). Significantly selective and sensitive improvements were observed and a more than 100-fold enrichment was achieved (based on peak area). The reproducibility of migration time and quantitative results of stacking CE can be improved by using an internal standard. The quantitation limits of these naphthalenesulfonate isomers, based on a signal-to-noise ratio above 10, can be about 4 microg/l with UV detection. This method was successfully applied to determine the trace amount of naphthalenesulfonate isomers in a spiked drinking water sample.     

Determination of copper and iron using [S,S']-ethylenediaminedisuccinic acid as a chelating agent in wood pulp by capillary electrophoresis.

A capillary electrophoresis (CE) method is described for the simultaneous determination of copper and iron after complexation with a readily biodegradable chelating agent, [S,S']-ethylenediaminedisuccinic acid (EDDS), in wood pulp. CE separation was performed in a fused-silica capillary (50 microm i.d.; total length, 65 cm) with an electrolyte containing 25 mM borate buffer and 0.5 mM CTAB at pH 7.0 and an applied voltage of -25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s, and detection of the complexes was monitored at 245 nm. The methodology performance of the methods was evaluated in terms of the linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The applicability of the method was demonstrated for the analysis of copper and iron in wood pulp.     

Application of capillary electrophoresis to the determination of various benzylpenicillin salts

The application of capillary electrophoresis for separation of benzylpenicillin, procaine, benzathine and clemizole was investigated. Phosphate-borate buffer supplemented with sodium dodecyl sulphate 14.4 g/l and electrophoresis voltage 18 kV seem to provide optimal conditions for micellar electrokinetic chromatographic assay of penicillin salts. This method is selective and precise. The results obtained from CE method recovery assay (above 98% for all but procaine-97% substances) and from determination of benzylpenicillin by CE compared with HPLC results, confirmed good accuracy.     

Field amplified sample injection-capillary electrophoresis-tandem mass spectrometry for the analysis of acrylamide in foodstuffs

This paper shows the applicability of capillary electrophoresis (CE) coupled to mass spectrometry (MS) for the analysis of acrylamide (AA) in foodstuffs. In order to obtain an ionisable compound amenable to be analysed by CE, acrylamide was derivatised with 2-mercaptobenzoic acid. Spectra in positive and negative modes were studied in order to select the best ionisation mode and multistep tandem mass spectrometry was used to obtain structural information. Maximum signal was observed when negative mode was used and MS/MS and MS3 were selected for quantitation and confirmation, respectively. For the separation, a fused-silica capillary of 80 cm and 50 microm I.D. and 35 mM ammonium formate/ammonia solution at pH 10 as running electrolyte were used. The applicability of field amplified sample injection (FASI) in reversed polarity was evaluated in order to decrease detection limits. The developed FASI-CE-MS/MS method provided a detection limit of 8 ng g(-1) and good linearity (r=0.999) and precision (day-to-day lower than 15%). The method has been applied to the analysis of different representative food products and the results were compared with those obtained by LC-MS/MS.     

Large volume sample stacking of cationic tetracycline antibiotics toward 10 ppb level analysis by capillary electrophoresis with UV detection

This paper aimed to build up a sensitive CE method for the analysis of tetracyclines (TCs) antibiotics (including tetracycline, chlorotetracycline, oxytetracycline, and doxycycline) with conventional UV detection. Here, the large volume sample stacking was applied to achieve in capillary preconcentration of the targets. To achieve large volume sample stacking, the essential step was a large volume of sample (around 83.3% of total capillary length from inlet to detection window) hydrodynamically loaded. Then, the reserved voltage was added in order to push the sample matrix out of the capillary. Due to different pH between sample solution (pH 4.6) and BGE (pH 11.0), the cationic TCs would turn into negatively charged while the sample matrix was removing from the capillary. Finally, the anionic TCs were stacked at the inlet for the subsequent separation. Although the loss of sample existed during their charge transformation, the LODs could be improved around 40 times than that obtained by normal hydrodynamic injection CE method. Here, the LODs were in the range of 8.1–14.5 μg/L, around 10 ppb that close to the level by electrochemiluminescence or laser‐induced fluorescence detection of TCs by CE. The precision was characterized by RSDs of migration times and peak areas, which were in the range of 0.19–0.24% and 0.97–2.54%, respectively. The recoveries of the developed method were in the range of 95–112% by spiking TCs in the tap water. The proposed inline preconcentration CE method could be a simple, speed, and sensitive method for the quantitative analysis of TCs.     

Detection of specific antibodies using immunosubtraction and capillary electrophoresis instrumentation

Solution-phase immunoassays based on capillary electrophoresis (CE) separations have been shown to be rapid and simple to perform. The potential for sample matrix interference and incompatibility with multiplexing conditions for antibody detection when dealing with real samples, however, has prompted the development of an assay that utilizes an immunosubtraction methodology. A model assay for the detection of specific antibodies that relies on solid-phase extraction, CE and laser-induced fluorescence (LIF) detection is described. The method, called immunocapture-immunosubtraction (ICIS), incorporates an antibody capture/purification protocol using magnetic particles. The detection of specific antibodies is achieved by CE-LIF analysis of a probe solution following incubation with the captured antibodies. As an example of the ICIS assay's capabilities, the relative quantification of anti-fluorescein in serum is presented.     

Recent advances of capillary electrophoresis in pharmaceutical analysis

This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.     

Determination of phenolic acids in olive oil by capillary electrophoresis

A CZE method for the separation and quantitation of phenolic acids (cinnamic, syringic, p-coumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic, protocatechuic), extracted from extra virgin olive oil, was developed. The sample preparation involved the LLE and SPE extraction methods. CE separation was performed in a fused silica capillary of I.D.= 50microm using as a BGE 40 mM borate buffer at pH=9.2. The separation voltage was 18kV with corresponding current of 27-28 microA. Detection was accomplished with UV-detector at lambda=200nm. The proposed method was fully validated. A good repeatability of migration time (RSD% ranged from 0.81 to 1.63) and of corrected peak area (RSD% from 2.89 to 5.77) was obtained. The linearity of detector response in the range from 5 to 50 ppm was checked, obtaining the correlation coefficient R2 values in the range: 0.9919-0.9997. Some phenolic acids in real oil samples were detected and quantified with the proposed method.     

Determination of homotaurine as impurity in calcium acamprosate by capillary zone electrophoresis.

A method is reported which allows the quantification of homotaurine as an impurity in the drug. After addition of taurine as an internal standard, the sample is derivatised with fluorescamine at ambient temperature in 10 mM borate buffer, pH 9.2. The analytes are separated by capillary zone electrophoresis in a 31.2 cm (21 cm to the detector) x 100 microns I.D. fused-silica capillary at a potential of +7 kV and 25 degrees C. A 40 mM borate buffer, pH 9.2, is used as the electrolyte and detection is carried out at 205 nm. The validation tests showed that the method is reliable between 0.01% and 0.15% (m/m) of homotaurine with respect to the active drug. The limits of quantitation (0.01%, m/m) and detection (0.004%, m/m) allows to control the homotaurine content of the drug substance for which the maximum tolerated level is 0.05% (m/m). The proposed procedure (derivatisation and separation) developed in CE is rapid (20-25 min) by comparison to that currently used in HPLC (75 min). Satisfactory agreement was found between several batches of acamprosate analysed by CE and HPLC.     

Laser-induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides

In quest for high sensitivities necessary for determining the disaccharide composition of heparin/heparan sulfate present in trace amounts in biologic samples, an ultrahighly sensitive capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection was developed. Heparin/heparan sulfate-derived Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone and resolved by a reversed-polarity CE method. Estimation of the limit of detection in concentration term and limit of quantitation showed that LIF detection of AMAC-derivatives of Delta-disaccharides resulted in 27-744 times higher sensitivity as compared to those detected by UV at 255 nm. These data suggest that CE-LIF is a powerful tool to quantify minute amounts of heparin/heparan sulfate disaccharides.     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.