First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level.

In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a ß subunit, so-called hCGα and hCGß, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms. To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100-1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000-2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.

Kinetic study of niobium and tantalum hexameric forms and their substituted ions by capillary electrophoresis in alkaline medium.

In this work a capillary electrophoretic (CE) method is used for the kinetic study of the intermetallic substitutions in hexameric ions of two strategic metals, tantalum and niobium in an alkaline medium. Recently proposed processes for the production and analytical separation of tantalum and niobium that are faster, more economical and environmental friendly are based on the use of highly alkaline media. It was previously established that in these media, tantalum and niobium exist as hexameric species, HxTa6O19X-8 (Ta6) and HxNb6019x-8 (Nb6), which can be analysed with a CE method using an alkaline electrolyte and UV detection. However, when using the above method on an industrial sample a minor species that should correspond to the substituted Ta1Nb5 form was observed. The purpose of the present study is to probe, by means of CE, the kinetic of the formation of substituted niobate-tantalate ions, Ta6-xNbx (1 ≤ x ≤ 5), starting from mixtures of pure hexaniobate and hexatantalate ions. This study required the development of a new CE method allowing the separation of all the five substituted ions and their two non-substituted hexameric parent ions in less than seven minutes. In details, a previously developed separation method was transferred to a Beckman instrument and the separation improved by adjusting the total length, the applied voltage, the injection volume, the rinsing steps and the internal standard. The kinetic study shows that samples initially containing non-substituted hexameric forms of tantalum and niobium in a 1:1M ratio naturally form the five possible substituted species Ta6-xNbx (1 ≤ x ≤ 5) after only a few hours which may represent an issue for future Nb-Ta separation processes operated in alkaline media. The developed method was also transferred to an Agilent instrument and the kinetic study repeated. Results obtained with the Agilent instrument corroborate those obtained with the Beckman instrument. The proposed electrophoretic separation method lays the ground for new analytical techniques that could help assessing the presence of substituted species that can be deleterious for Nb-Ta purification processes.

Interinstrumental transfer of a fast short-end injection capillary electrophoresis method: Application to the separation of niobium, tantalum, and their substituted ions.

The interinstrumental transfer of a short-end CE method was studied. A model separation of the hexameric forms of niobium, tantalum, and their substituted ions (Nb6-x Tax with 0 ≤ x ≤ 6) was selected as test case. The method was first optimized on a Beckman instrument and in a second step transferred to an Agilent instrument. The transfer needed updated guidelines that tackled differences in effective capillary length, 8.5 (Agilent) versus 10 cm (Beckman), because of instrumental different capillary cartridges. Differences in effective length lead to migration time and separation efficiency inequalities, illustrated by a decrease in resolution between the substituted ions. The difference in effective length was overcome by adapting the lift offset parameter of the Agilent instrument. The lift offset default setting is 4 mm and by increasing this parameter both the inlet and outlet lifts are lowered and thus the detection window can be displaced and consequently the effective length was increased. The decrease in effective length difference and the effect on the separation efficiency was investigated and led finally to a restored separation of the substituted ions. The adaptation of the lift offset parameter during short-end injection methods was added to earlier developed guidelines to facilitate interinstrumental method transfer of CE methods.

Interinstrumental method transfer of a capillary electrophoretic separation of angiotensin II and five derivatives: Evaluation and update of earlier developed guidelines.

The increased interest in the separation of peptides, proteins, immunoglobulins, and polynucleotides, led to an increased demand for appropriate analytical methodologies and instrumentation. CE, because of its unique separation mechanism and high efficiency, is frequently used in the analysis of those molecules. In this study, a CE method for the separation of six angiotensin analogues was developed in the first step. In the second step, the method was transferred to a CE instrument of another brand, taking into account guidelines defined earlier about the interinstrumental transfer of CE methods. Although previously successful, the application of these guidelines during this study was not able to maintain the baseline separation. Further research on the instrumental differences revealed that the electrical resistance on both instruments differed. At constant current, the electrical resistance, generated voltage, and separation efficiency on the Agilent instrument were lower than on the Beckman instrument. Increase of the electrical resistance, by reducing the capillary temperature, leads to an increased applied voltage and separation efficiency on the Agilent system. The guidelines developed earlier were, therefore, updated with an additional step equalizing the electrical resistances, which led to a successful interinstrumental analytical method transfer for the separation of six angiotensin derivatives.

Inter-instrumental method transfer of chiral capillary electrophoretic methods using robustness test information.

Capillary electrophoresis (CE) is an electrodriven separation technique that is often used for the separation of chiral molecules. Advantages of CE are its flexibility, low cost and efficiency. On the other hand, the precision and transfer of CE methods are well-known problems of the technique. Reasons for the more complicated method transfer are the more diverse instrumental differences, such as total capillary lengths and capillary cooling systems; and the higher response variability of CE methods compared to other techniques, such as liquid chromatography (HPLC). Therefore, a larger systematic change in peak resolutions, migration times and peak areas, with a loss of separation and efficiency may be seen when a CE method is transferred to another laboratory or another type of instrument. A swift and successful method transfer is required because development and routine use of analytical methods are usually not performed in the same laboratory and/or on the same type of equipment. The aim of our study was to develop transfer rules to facilitate CE method transfers between different laboratories and instruments. In our case study, three ß-blockers were chirally separated and inter-instrumental transfers were performed. The first step of our study was to optimise the precision of the chiral CE method. Next, a robustness test was performed to identify the instrumental and experimental parameters that were most influencing the considered responses. The precision- and the robustness study results were used to adapt instrumental and/or method settings to improve the transfer between different instruments. Finally, the comparison of adapted and non-adapted transfers allowed deriving some rules to facilitate CE method transfers.

Precision evaluation of chiral capillary electrophoretic methods in the context of inter-instrumental transfer: constant current versus constant voltage application.

Capillary electrophoresis (CE) is an electrophoretic separation technique that was rapidly increasing in popularity some years ago and that led to high expectations. Because of their different separation mechanisms, CE and HPLC are alternative and complementary separation techniques. Chiral molecules can be directly separated with CE by simply adding a chiral selector to the running buffer solution, leading to flexible and cheap methods. Major drawbacks of capillary electrophoretic separation methods are, however, the lower precision compared to HLPC methods and a more problematic analytical method transfer. Both above stated disadvantages limit the generalized use of CE methods in the pharmaceutical industry. Multiple solutions have been suggested to improve the precision of CE methods. In this work the application of a constant current during the electrophoretic separation instead of the more commonly used application of a constant voltage was studied on two CE instruments with different cooling mechanisms. This was done in the context of optimizing method transfer conditions. A constant current may reduce the generation of heat in the capillary and the consequentially radial and axial temperature fluctuations that both negatively influence the precision of the peak areas, migration times and resolutions of a CE method. The repeatability and time-different intermediate precision of both electrophoretic separation modes were compared on two different CE instruments after a successful analytical method transfer. The chiral separations of three beta-blockers, propranolol, sotalol and betaxolol, were used as test cases. A constant current led to a general improvement of the repeatability and time-different intermediate precision of the relative Area Under the Curve of all three beta-blockers, while that of the migration times remained rather constant. It also led to more similar electropherograms than the application of a constant voltage.

Development of a validated capillary electrophoresis method for enantiomeric purity control and quality control of levocetirizine in a pharmaceutical formulation.

A chiral capillary electrophoresis method has been developed for the quantification of 0.1% of the enantiomeric impurity (dextrocetirizine) in levocetirizine and determination of both in pharmaceuticals using sulfated-ß-cyclodextrins (CDs) as chiral selector. Several parameters affecting the separation were studied such as the type and concentration of chiral selectors, buffer composition and pH, organic modifier, mixtures of two CDs in a dual system, voltage, and temperature. The optimal separation conditions were obtained using a 50 mM tetraborate buffer (pH 8.2) containing 1% (w/v) sulfated-ß-CDs on a fused-silica capillary. Under these conditions, the resolution of two enantiomers was higher than 3. To validate the method, the stability of the solutions, robustness (two level half fraction factorial design for 5 factors using 19 experiments [2(n-1)+3]), precision, linearity (dextrocetirizine 0.25-2.5 µg/ml, R(2) = 0.9994, y = 0.0375x + 0.0008; levocetirizine 15-100 µg/ml, R(2) = 0.9996, y = 0.0213x + 0.0339), limit of detection (0.075 µg/ml, 0.03% m/m), limit of quantification (0.25 µg/ml, 0.1% m/m), accuracy (dextrocetirizine 84-109%, levocetirizine 97.3-103.1%), filter effect, and different CD batches were examined. The validated method was further applied to bulk drug and tablets of levocetirizine.

Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages.

The effect of ovotransferrin (ovoTF), human lactoferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellular pathogen Chlamydophila (Cp.) psittaci was evaluated using a model of Buffalo Green Monkey kidney (BGM) cells and HD11 chicken macrophages as artificial hosts. Firstly, the effect of transferrins on the infectivity of the bacteria was evaluated. Pre-incubation of Cp. psittaci with 0.5 to 5 mg/mL ovoTF prior to infecting BGM cells significantly lowered the infection rate (P < 0.05). For both lactoferrins, the infection rate could only be reduced with 5 mg/mL, albeit not significantly as compared to the infection rate created by the untreated bacteria. Secondly, transferrins were tested for their ability to influence bacterial adhesion and entry in HD11 cells. Maximal non-cytotoxic and non-bactericidal concentrations of 0.05 mg/mL ovoTF and 0.5 mg/mL hLF and bLF were used. Overall, ovoTF was more effective than human and bovine LF in inhibiting bacterial irreversible attachment and cell entry and the latter was accompanied by a dose-dependent reduction of actin recruitment at the bacterial entry site. However, once bacteria had entered HD11 cells, transferrins had apparently no effect on intracellular replication. The present findings suggest a possible role for transferrins and especially ovoTF, in preventing avian Cp. psittaci infections.