P.J.P. Cardot

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography-electrospray mass spectrometry (LC-ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C18 Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform-2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)-acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C18, 3.5 microm (150 x 1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)-acetonitrile (70:30, v/v) as mobile phase, delivered at 50 microl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r > or = 0.992) and 200 ng/ml for the active metabolites (r > or = 0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.

Chromatographic study of terpene derivatives on porous graphitic carbon stationary phase with β-cyclodextrin as mobile phase modifier

The stoichiometric coefficients and apparent formation constants (Kf) of alpha-terpineol, thymol, geraniol and linalool complexes with beta-cyclodextrin (beta-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol-water mobile phases. All the terpene derivatives under study form 1:1 guest-CD complexes. Graphs of Kf as a function of the mobile phase composition appeared different from those classically described for RP-C18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute-CD inclusion and solute-stationary phase interactions may be involved in this specific behavior.

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

SEDIMENTATION FIELD-FLOW FRACTIONATION: METHODOLOGICAL BASIS AND APPLICATIONS FOR CELL SORTING

ABSTRACT As a cell sorter, sedimentation field-flow fractionation (SdFFF) can be defined as an efficient tool for cell separation and purification which respects cell functional integrity, viability, as well as provides enhanced recovery and purified sterile fraction collection. SdFFF elution should be performed under strictly defined conditions concerning apparatus construction (channel wall materials) and set up (bio-compatible “Hyperlayer” mode) to obtain rapid cell elution, high recovery (negligible cell trapping), short- and long-term viability, and sterile conditions (cleaning and decontamination procedures). As shown recently in various reports, specific characterization of time-dependent collected cells have demonstrated the effectiveness of SdFFF to provide, in a few minutes, purified, viable, sterile cells which can be used for many investigations such as transplantation.

Age-Dependent Elution of Human Red Blood Cells in Gravitational Field-Flow Fractionation

Abstract The technique of field-flow fractionation (FFF) which combines the earth gravitational force with a carrier liquid flow in a horizontal, ribbon-like channel is well suited for the separation of micron-sized particulate species such as cells. We investigated the selective elution, in a phosphate buffer, of human red blood cells (RBC) which migrate along the FFF channel more slowly than the carrier. Fractions of the channel effluent were collected and the activities of various intracellular enzymes, which either reveal the presence of white cells or are known to be related to cell age, were evaluated for each fraction. The hemoglobin sub-fraction composition was also determined. From analysis of these biochemical determinations, nucleated cells and reticulocytes appeared to be eluted as unretained species while mature RBC form a well defined, retained peak. The steady variation of the activity of age-related enzymes within this peak demonstrates that RBC are separated according to age. These observ...

Hyphenation of sedimentation field flow fractionation with flow cytometry

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).

Analysis of relationship between cell cycle stage and apoptosis induction in K562 cells by sedimentation field-flow fractionation.

Recently, sedimentation field-flow fractionation (SdFFF) was used to study the specific kinetics of diosgenin-induced apoptosis in K562 cells. Here, we propose a new SdFFF cell separation application in the field of cancer research concerning the correlation between induction of a biological event (i.e. apoptosis) and cell status (i.e. cell cycle position). SdFFF isolated subpopulations depending on the cell cycle position allowing the study of apoptosis kinetics and extent. Results showed that cells in G0/G1 phases (F3 cells) underwent significant and earlier apoptosis than cells in the active part of the cell cycle (S/G2/M phases). Results shed light on the correlation between differences in apoptosis kinetics and cell cycle stage when exposure to the inducer began. SdFFF monitoring and size measurement also led to the description of different subpopulations demonstrating complex variations in density between fractions associated with differences in biological processes.

TiO2 colloidal suspension polydispersity analysed with sedimentation field flow fractionation and electron microscopy

Sedimentation field flow fractionation (SdFFF) operated at multi gravitational field is used to analyse a highly polydisperse TiO2 colloidal suspension. From the initial sample, time dependent eluted fractions are collected and submitted to electron microscopy (EM) shape and size analysis. To assess the accuracy of FFF in determining the average size of the different fractions, these are re-introduced into the channel by means of two different procedures, the on-channel concentration of the fractions and the direct re-injection of pre-concentrated fractions (DRI). Both methods appear accurate to determine the average size of every fraction, associated to a lower recovery in the case of DRI. The fractogram band spreading characteristics of the re-introduced fractions are correlated to the particle size distribution measured by EM. After density determination of fractionated particles, the fractogram is calibrated in terms of size and size distribution using data obtained from EM for each fraction. Quantitative analyses, based on particle counting showed high recovery (80-90%) of the eluted species. However, this loss limited the possibility to extend signal information to a quantitative one.

Sedimentation field-flow fractionation application to Toxoplasma gondii separation and purification

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii, an intracellular protozoa of micronic size range (4-10 microm). Its classical purification processes are complex and often associated with low recovery. All investigation procedures concerning this parasite require its isolation and purification from at least the mouse ascitic fluid. For this purpose, a recently developed laboratory technology was used, i.e. sedimentation field-flow fractionation. This chromatographic-like separation technology was demonstrated to be particularly selective for isolation and separation of micron-sized biological particles. Sedimentation field-flow fractionation operated on the steric-hyperlayer mode was used to isolate the parasite from the remanent ascitic contaminants of different origins and from red blood cells. With this technology, 86% recovery with 97% viability was obtained in less than 30 min.

Validation procedures of sedimentation field-flow fractionation techniques for biological applications.

Sedimentation field-flow fractionation (SdFFF) offers great potential for the separation of submicrometer and micrometer-sized species. The availability of commercial instrumentation and the versatility of this method originated its success. At this stage of development, SdFFF techniques are mature enough for use in analytical research, development and even routine work. However, prior to their use, these techniques like any other methodologies, have to be validated. As the application of SdFFF techniques to cell separation is being constantly developed, we have investigated separation performance according to validation rules classically defined for separation methods (chromatography) in the case of cellular materials.