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.     

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE‐ immobilized microenzyme reactors (IMER) development, human flavin‐containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in‐line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE‐IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE‐IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.     

Development and validation of a capillary electrophoresis method for the characterization of protegrin IB-367.

A capillary electrophoresis (CE) method was developed to characterize protegrin IB-367, an antimicrobial peptide being developed for the treatment of oral mucositis and for other topical applications. The electrophoretic purity and levels of potential impurities/degradation products of IB-367 drug substance are determined by CE using area normalization. Electrophoresis parameters were optimized to allow optimal resolution, reproducibility and minimal analysis time. The separation and resolution between this polycationic peptide and truncated analogs determined by the CE method was much greater than those by the HPLC methods. In addition, the CE methods separates the potential impurities/degradation products from each other while the HPLC methods failed to resolve them. The CE method was validated in the aspects of accuracy, precision, linearity, range, limit of detection, limit of quantitation, specificity, system suitability and robustness. An internal standard was used for the quantitation purpose. The selection criteria of the internal standard as well as the method validation results are presented. The truncated peptide analogs were used to demonstrate the specificity of the method. These analogs were also used to evaluate the limit of quantitation of potential impurities. The relative response factors of these analogs were assessed to determine area normalization feasibility. System suitability tests were established.     

In‐capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry

Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE‐MS method for the analysis of mAbs is presented analyzing SDS‐complexed samples. To obtain narrow and intensive peaks of SDS‐treated antibodies, an in‐capillary strategy was developed based on the co‐injection of positively charged surfactants and methanol as organic solvent. For samples containing 0.2% (v/v) of SDS, recovered MS peak intensities up to 97 and 95% were achieved using cetyltrimethylammonium bromide or benzalkonium chloride, respectively. Successful removal of SDS was shown in neutral coated capillaries but also in a capillary with a positively charged coating applying reversed polarity. The usefulness of this in‐capillary strategy was demonstrated also for other proteins and for antibodies dissolved in up to 10% v/v SDS solution, and in other SDS‐containing matrices, including the sieving matrix used in a standard CE‐SDS method and gel‐buffers applied in SDS‐PAGE methods. The developed CE‐MS approaches enable fast and reproducible characterization of SDS‐complexed antibodies.     

Capillary electrophoretic determination of lignin degradation products obtained by permanganate oxidation

A new capillary electrophoretic (CE) technique was developed for the separation of lignin degradation products after permanganate oxidation, yielding information about quality and quantity of various linkages in the lignin molecule. This CE method is a promising alternative to existing gas chromatographic (GC) methods. An advantage in comparison with GC is the short separation time and the fact that the oxidation products (aromatic acids) can be analyzed without derivatization. The selectivity and sensitivity of CE combined with UV detection is adequate and makes it suited for fast routine characterization of lignins. If necessary, the CE method can be coupled with electrospray ionization mass spectrometry in order to make a clear assignment of the peaks.     

The analysis of human amniotic fluid using capillary electrophoresis

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.     

Characterization of degradation products of Ivabradine by LC‐HR‐MS/MS: a typical case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis

A validated stability‐indicating HPLC method was established, and comprehensive stress testing of ivabradine, a cardiotonic drug, was carried out as per ICH guidelines. Ivabradine was subjected to acidic, basic and neutral hydrolysis, oxidation, photolysis and thermal stress conditions, and the resulting degradation products were investigated by LC‐PDA and LC‐HR‐MS/MS. The drug was found to degrade in acid and base hydrolysis. An efficient and selective stability assay method was developed on Phenomenex Luna C18 (250 × 4.6 mm, 5.0 µm) column using ammonium formate (10 mM, pH 3.0) and acetonitrile as mobile phase at 30 °C in gradient elution mode. The flow rate was 0.7 ml/min and detection wavelength was 286 nm. A total of five degradation products (I‐1 to I‐5) were identified and characterized by LC‐HR‐MS/MS in combination with accurate mass measurements. The drug exhibited different degradation behaviour in HCl and H₂SO₄ hydrolysis conditions. It is a unique example where two of the five degradation products in HCl hydrolysis were absent in H₂SO₄ acid hydrolysis. The present study provides guidance to revise the stress test for the determination of inherent stability of drugs containing lactam moiety under hydrolytic conditions. Most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation pattern of the drug and its degradation products. In silico toxicity revealed that the degradation products ( I‐2 to I‐5 ) were found to be severe irritants in case of ocular irritancy. The analytical assay method was validated with respect to specificity, linearity, range, precision, accuracy and robustness. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a sheathless CE‐ESI‐MS interface

A sheath‐flow interface is the most common ionization technique in CE‐ESI‐MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE‐MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath‐flow CE‐MS. Because the interface is easy to construct, it is cost‐effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE‐MS‐based metabolomic analysis.     

Determination of chloramphenicol in biological matrices by solid‐phase membrane micro‐tip extraction and capillary electrophoresis

Solid‐phase membrane micro‐tip extraction (SPMMTE) and capillary electrophoresis (CE) methods were developed and validated for analysis of chloramphenicol in human plasma and urine samples. Iron composite nanoparticles were prepared using green technology. CE was carried out using a silica capillary (60 cm × 50 μm i.d.), phosphate buffer (50 mm , 8.0 pH)–acetonitrile (95:5, v/v) as the background electrolyte, 10 kV voltage, 280 nm detection, 20 s injection time and 27 ± 1°C temperature. Frusemide was used as an internal standard. The values of migration time, electrophoretic mobility, electrophoretic velocity and theoretical plates of chloramphenicol were 12.254 min, 4.44 × 10, 7.41 × 10 and 11,227. The limits of detection and quantitation of chloramphenicol were 0.1 and 1.0 μg/mL. Recovery of chloramphenicol in the standard solution was 95%. Solid‐phase membrane micro‐tip extraction and capillary electrophoresis methods may be used to analyze chloramphenicol in human plasma and urine samples of any patient.     

Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection

Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE‐C⁴D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm ) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non‐UV‐absorbing L‐valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28°C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused‐silica capillary (75 μm, i.d. × 52 cm). The CE‐C⁴D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5–250 μg/mL for the studied analytes. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2‐microglobulin as a simple tool for misfolding and stability studies

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild‐type β₂‐microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25–42°C is induced inside the capillary for the two pathogenic proteins (wtβ₂‐microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.     

Heart‐cutting 2D‐CE with on‐line preconcentration for the chiral analysis of native amino acids

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on‐line preconcentration of native amino acids in heart‐cutting 2D‐CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart‐cutting 2D‐CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid). This on‐line tMCRB preconcentration coupled with heart‐cutting 2D‐CE was applied with success to the chiral separation of D ,L ‐phenylalanine, and D ,L ‐threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 μM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 μM was determined for L ‐threonine.     

Advantages and limitations of coupling isotachophoresis and comprehensive isotachophoresis-capillary electrophoresis to time-of-flight mass spectrometry

Capillary isotachophoresis (ITP) and comprehensive isotachophoresis-capillary electrophoresis (ITP-CE) were successfully coupled to electrospray ionization (ESI) orthogonal acceleration time-of-flight mass spectrometry (TOF-MS) using angiotensin peptides as model analytes. The utility of ITP-TOF-MS and ITP-CE-TOF-MS for the analysis of samples containing analyte amounts sufficient to form flat-top ITP zones (30 microM) as well as for samples with trace analyte amounts (0.3 microM) was studied. Separations were performed in 150 microm internal diameter (I.D.) capillaries for the ITP experiments, and in 200 microm I.D. (ITP) and 50 microm I.D. (CE) capillaries for ITP-CE experiments. The fused-silica columns were coated with poly(vinyl alcohol) to suppress electroosmotic flow that can disrupt ITP zone profiles. The sample loading capacity in both ITP and comprehensive ITP-CE was greatly enhanced (up to 10 microl) compared with typical nanoliter-sized injection volumes in CE. It was concluded that ITP-TOF-MS alone was adequate for the separation and detection of high concentration samples. The outcome was different at lower analyte concentrations where mixed zones or very sharp peaks formed. With formation of mixed zones, ion suppression and discrimination could occur, complicating quantitative determination of the analytes. This problem was effectively overcome by inserting a CE capillary between the ITP and TOF-MS. In such an arrangement, samples were preconcentrated in the high load WTP capillary and then injected into a CE capillary where they were separated into non-overlapping peaks prior to their detection by TOF-MS. The advantage of this comprehensive arrangement, which we have described previously, is that there is no need to discard portions of the sample in order to avoid overloading of the CE capillary. The whole sample is analyzed by multiple injections from ITP to CE. Thus, this method can be used for the analysis of complex samples with wide ranges of component concentrations.     

An innovative impurity profiling of Avanafil using LC and LC-MS/MS with in-silico toxicity prediction

Degradation of the drug can lead to the formation of toxic substance hence drug quality and stability is a major concern by pharma regulators. A Selected phosphodiesterase type 5 inhibitor drug Avanafil (AV) structure has amide, arylchloro and hydroxide as functional groups which can easily eliminated during hydrolysis. Henceforth, thoroughly chemical stability of AV was carried out according to ICH guideline Q1A (R2). The drug and marketed tablet formulation undergoes degradation in hydrolytic (acid, base, neutral), thermal, photolytic, oxidative conditions and forms a total new sixteen degradation products (D.P.s) which were identified by LC, characterized by LC-MS/MS and probable degradation mechanism for each stress conditions are proposed. All sixteen D.P.s were identified by optimized LC conditions; C18 column using 10 mM ammonium acetate: ACN (60:40, v/v), pH 4.5 as mobile phase at 0.9 mL min⁻¹ of flow rate, 239 nm wavelength at 20 °C column temperature and the method being LC-MS compatible characterized by LC-MS/MS confirmed by relative retention time (RRT). The structure of D.P.s was confirmed from the fragmentation pattern obtained by LC-MS/MS and further probable degradation mechanism for each stress condition is proposed. The drug and its marketed tablet formulation showed similar degradation peaks which were confirmed using RRT, photodiode array (PDA) and LC-MS. Drug degradation happens due to nucleophilic substitution reaction, amide hydrolysis, electron withdrawing properties of amide, dechlorination and bond cleavage due to energy. The amide group in AV structure can undergo hydrolysis, while due to aryl chloride and hydroxide group in structure it undergoes photodecomposition. A comprehensive stress study reveals that AV is more prone to degrade in light, temperature and moisture; hence AV requires proper storage condition temperature below 25 °C with protection to light and moisture. In silico toxicity prediction of physicochemical properties revealed that all the physicochemical parameters of impurities were within the acceptable limit which indicates that no impurity is at any risk of toxicity. In detail, the LC-MS/MS compatible AV degradation study is fully validated as per ICH Q2 (R1) guideline.     

Identification and Accurate Size Characterization of Nanoparticles in Complex Media

We have developed a new method for the identification and accurate size characterization of nanoparticles (NPs) in complex media based on capillary electrokinetic (CE) separation coupled to inductively coupled plasma mass spectrometry (ICP‐MS). Through mass scanning and Gaussian fitting of electropherogram peaks, we can obtain multidimensional information on chemical compositions, size distributions, and ionic species of multiple NPs in a single run. The results are more accurate than those obtained by using conventional methods. This method provides a powerful tool for investigating polydisperse NP systems and rapid screening of NP‐containing products.     

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser‐induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE‐LIF‐REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE‐LIF. The results demonstrate that the CE‐LIF‐REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.     

Galactomannans as a sieving matrix in capillary electrophoresis

Purification of galactomannans including guaran, tara gum, and locust bean gum is described as well as their use as a sieving matrix in DNA sequencing by capillary electrophoresis (CE). Three methods of galactomannan purification were developed and tested using guaran. The first method is based on hydrolysis of proteins using alkali treatment and precipitation of guaran with acetone. The second method uses ion-exchange resins QAE Sephadex A-25 and SP Sephadex C-25 together with acetone precipitation. The third method is similar to the second one, except that it uses ion-exchange resins based on polystyrene, Source 30Q and Source 30S. Capillary zone electrophoresis of acetonitrile extracts from guaran revealed 4-5 characteristic major peaks and several minor peaks. Guar gum from different suppliers differed in the content of proteins. In purified guaran, protein peaks were detectable only using a 300-fold concentrate of extract. The content of proteins in the guaran purified using the third method was 0.001% m/m as determined by CE. The weight average molecular mass of purified guaran can be as large as 2.2 x 10(6). The purified galactomannans were used as a sieving matrix in DNA sequencing by CE. M13 DNA was sequenced to read lengths of about 600 bases in less than 90 min. Separation efficiencies exceeded 1 million theoretical plates for DNA fragments shorter than about 600 bases.     

Capillary electrophoresis study of systemin peptides spreading in tomato plant

Systemin (Sys) is an 18‐aa plant peptide hormone involved in the regulation of plant's defensive response. Sys is considered as a fast‐spreading systemic wound signal. We developed a simple and rapid CE method to monitor the spreading of Sys peptides through tomato plant. A 1,2,3‐triazole‐linked AZT‐systemin conjugate was designed as a model to study the possibility of translocating small cargo molecules 3'‐Azido‐2',3'‐dideoxythymidine by systemin. The Sys peptides (Sys, N‐propiolyl Sys, and AZT‐systemin conjugate) were injected into the stem and leaves of mature tomato plant. Its transportation throughout the plant tissue was traced by CE. The peptides were clearly visible in the crude tomato exudates and an optimum separation was achieved in 25 mM phosphate “buffer” at pH 2.5 and a voltage of 20 kV using uncoated fused silica capillary. CE analysis showed that Sys peptides are well separated from tomato plant exudates ingredients and are stable in tomato stem and leaf exudates for up to 24 h. CE study revealed that the Sys peptides are effectively spreading throughout tomato stem and leaves and the peptides could be directly detected in the crude plant matrixes. The translocation was strongly inhibited by sodium azide. The results showed that the established CE method can be used to characterize plant peptides spreading under plant physiological conditions.     

A promising capillary electrophoresis–electrospray ionization–mass spectrometry method for carbohydrate analysis

A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI‐MS detection is presented. Reverse polarity separations were performed in bare fused‐silica capillaries with an acidic BGE. Under these conditions, negatively charged 8‐aminopyrene 1,3,6‐trisulfonate‐labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE‐MS interface must simultaneously back‐fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction‐at‐the‐tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.