Ig Columns Research Articles

Chiral Hydroxy Metabolite of Mebendazole: Analytical and Semi-Preparative High-Performance Liquid Chromatography Resolution and Chiroptical Properties.

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 μm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water-acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase.

Chiral separation of terbutaline by supercritical fluid chromatography with peak purity determination by UPLC–MS and modeling for chiral recognition mechanism

AbstractBACKGROUNDTerbutaline is the drug of choice for asthma patients but it exists in racemic mixture. (R)‐(−)‐terbutaline is 200 times more active than (S)‐(+)‐terbutaline yet it is not advisable to prescribe racemic mixture due to certain adverse effects of (S)‐(+)‐terbutaline. Therefore, a fast, effective and reproducible separation method is necessary.RESULTSChiral separation was achieved on Chiralpak‐IE and Chiralpak‐IG columns (250 × 4.6 mm, 5 μm) using carbon dioxide‐methanol (CO2‐MeOH, 60:40) with a 0.2% triethylamine mobile phase. The flow was 1.0 mL min−1 with detection at 223 nm using a photodiode array (PDA) detector. The values of retention, separation and resolution factors were in the ranges 1.88–2.38, 1.14–1.26 and 0.91–1.17, respectively; with best separation with Chiralpak‐IE. The tailing factors and number of theoretical plates were in the range of 1.0–1.23 and 487–3699. The purity of the separated peaks was determined by ultra‐performance liquid chromatography–mass spectrometry (UPLC–MS); indicating 100% purity of the peaks. The chiral recognition was determined by modeling with binding affinities −5.0 and −6.0 for S‐ and R‐enantiomers, respectively; indicating S‐enantiomer elution first followed by R‐enantiomers. The major forces responsible for the chiral resolution were hydrogen bonding and π‐π interactions.CONCLUSIONOwing to the great demand for optically active pure drugs and high economic pressure on analytical techniques, the chiral separation of terbutaline was achieved via inexpensive supercritical fluid chromatography. The reported method may be used to prepare optically active pure terbutaline drugs (R‐enantiomers) at a pilot scale. © 2024 Society of Chemical Industry (SCI).

Two-Dimensional Chiral HPLC Analysis of Lactate, Hydroxybutyrates and Malate in Human Plasma

For the simultaneous determination of lactate (LA), 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB) and malate (MA) enantiomers, a new separation technique in a two-dimensional high-performance liquid chromatography (2D-HPLC) system following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been developed. The 2D-HPLC system was composed of the reversed-phase (1D) and enantioselective (2D) separations. In the first dimension, the target hydroxy acids were separated as their D plus L mixtures on a Singularity RP18 column (1.0 mm i.d. x 250 mm), and the enantiomers were separated in the second dimension using a polysaccharide-based chiral stationary phase, Chiralpak IG (2.0 mm i.d. x 250 mm). By using the Chiralpak IG column, the LA, 2HB, 3HB and MA enantiomers were separated with resolution values of 3.47, 3.63, 6.81 and 3.72, respectively. The developed 2D-HPLC system was applied to human plasma, and trace levels of D-LA and L-3HB in addition to their major antipodes were determined. The acquired %D of LA (the percentage of D-LA over total LA) and %L of 3HB (the percentage of L-3HB over total 3HB) were 1.0 and 2.7, respectively. For 2HB, only the L-form was detected, while the MA enantiomers were not found in the human plasma.

Chiral separation of β-blockers by supercritical fluid chromatography using Chiralpak-IG and Chiralpak IBN-5 columns.

Chiral separation of β-blockers is performed by utilizing the supercritical fluid chromatographic method. The chiral columns utilized were Chiralpak IG and Chiralpak IBN-5. The finest mobile phase was CO2 -0.2% TEA in methanol (60:40). The values atenolol enantiomers retention factors were 6.39 and 8.98. These values for propranolol enantiomers were 3.39 and 4.06. These values for betaxolol enantiomers were 4.08 and 4.68. The separation and resolution factor values for atenolol, propranolol, and betaxolol were 1.41 and 3.33, 1.19 and 2.23, and 1.15 and 1.87, separately and respectively. By comparison, it was observed that Chiralpak IG column is better than Chiralpak IBN-5 column. Supercritical fluid chromatography has been found as the best analytical technique due to its high speed, being eco-friendly, and being economic. The various most probable interactions responsible for the chiral resolution are hydrogen bonding, dipole-dipole interactions, steric effect, and π-π interactions. The reported methods are effective, efficient, and reproducible and may be used to separate and identify atenolol, propranolol, and betaxolol in any unknown samples.

Enantioselective Detection, Bioactivity, and Degradation of the Novel Chiral Fungicide Oxathiapiprolin

Oxathiapiprolin is a novel chiral piperidine thiazole isooxazoline fungicide that contains a pair of enantiomers. An effective analytical method was established for the enantioselective detection of oxathiapiprolin in fruit, vegetable, and soil samples using ultraperformance liquid chromatography-tandem triple quadrupole mass spectrometry. The optimal enantioseparation was achieved on a Chiralpak IG column at 35 °C using acetonitrile and 0.1% formic acid aqueous solution (90:10, v/v) as the mobile phase. The absolute configuration of the oxathiapiprolin enantiomers was identified with the elution order of R-(-)-oxathiapiprolin and S-(+)-oxathiapiprolin by electron circular dichroism spectra. The bioactivity of R-(-)-oxathiapiprolin was 2.49 to 13.30-fold higher than that of S-(+)-oxathiapiprolin against six kinds of oomycetes. The molecular docking result illuminated the mechanism of enantioselectivity in bioactivity. The glide score (-8.00 kcal/mol) for the R-enantiomer was better with the binding site in Phytophthora capsici than the S-enantiomer (-7.50 kcal/mol). Enantioselective degradation in tomato and pepper under the field condition was investigated and indicated that R-(-)-oxathiapiprolin was preferentially degraded. The present study determines the enantioselectivity of oxathiapiprolin about enantioselective detection, bioactivity, and degradation for the first time. The R-enantiomer will be a better choice than racemic oxathiapiprolin to enhance the bioactivity and reduce the pesticide residues at a lower application rate.

Separation of achiral anti-diabetic drugs using sub/supercritical fluid chromatography with a polysaccharide stationary phase: Thermodynamic considerations and molecular docking study

A simple, fast and environmental friendly supercritical fluid chromatography-photodiode array (SFC-PDA) method was developed for the simultaneous determination of metformin and three sodium-glucose co-transporter 2 (SGLT-2) inhibitors (canagliflozin, dapagliflozin and empagliflozin). The impact of different stationary phases, co-solvents, column temperature and outlet pressure was extensively evaluated for selective separation and quantitation of the drugs. Amongst several achiral and chiral stationary phases tested, the best results were obtained on amylose based Chiralpak IG column using CO2 and 0.1 % diethylamine in methanol and isopropanol 50:50 (v/v) in 60:40 ratios as the mobile phase. The separation efficiency was mainly dependent on the nature of stationary phase and the mobile phase composition. The limit of detection (LOD) and quantitation (LOQ) of the method were 0.155 and 0.469, 0.062 and 0.187, 0.015 and 0.045, 0.028 and 0.084 μg/mL for metformin, canagliflozin, dapagliflozin and empagliflozin, respectively. The chromatographic resolution between metformin-canagliflozin, metformin-dapagliflozin and metformin-empagliflozin pairs was 6.87, 3.44 and 6.47, respectively. The intra-batch and inter-batch precision of the method was ≤1.64 % while the accuracy was in the range of 96.2–103.3 %. The method was used to analyze metformin and SGLT-2 inhibitors in their binary fixed-dose formulations with acceptable accuracy (recovery) and precision. To support the experimental results, molecular docking was performed on Schrodinger software to provide a deeper insight into the interactions between the analytes and the chiral stationary phase and to understand their elution orders under optimized conditions.

Simultaneous enantiomeric analysis of six chiral pesticides in functional foods using magnetic solid-phase extraction based on carbon nanospheres as adsorbent and chiral liquid chromatography coupled with tandem mass spectrometry

This work presented a novel method for the simultaneous enantioselective analysis of six chiral pesticides in seven functional foods at enantiomeric levels. Samples were extracted from matrices using a magnetic solid-phase extraction method based on Fe3O4 modified carbon nanospheres as adsorbent. Experimental variables were optimized using one-variable-at-a-time and response surface methodology. Six chiral pesticides were simultaneously enantio-separated under one chromatographic condition using Chiralpak IG column, and a reversed-phase liquid chromatography coupled with tandem mass spectrometry was used for their sensitive and selective quantification. Under the optimum conditions, good linearity was obtained for all enantiomers with correlation coefficients (R2) greater than 0.9912. The mean recoveries of the analytes were in the range of 84.0–105.2%, with intra- and inter-batch precision less than 10.9% and 11.4%, respectively. Method quantification limits varied from 1 to 4 ng g−1 in all the tested matrices. The developed method was successfully applied to real samples to monitor the occurrence and enantiomeric composition of chiral pesticides, demonstrating its applicability for the routine analysis of chiral pesticide residues in food samples.

Simultaneous enantioselective determination of 22 chiral pesticides in fruits and vegetables using chiral liquid chromatography coupled with tandem mass spectrometry.

This paper presents a novel multi-residue method for the enantioselective analysis of 22 chiral pesticides in cucumber, tomato, cabbage, grape, mulberry, apple and pear at enantiomeric levels. Pesticides were efficiently extracted by magnetic solid-phase extraction based on graphene as sorbent. Response surface methodology was used to assist the multivariable optimization. The perfect chiral separation of the enantiomers was achieved on a Chiralpak IG column within 47 min by reversed-phase liquid chromatography coupled with tandem mass spectrometry, which is a significant improvement to resolve a large group of chiral compounds under one set of conditions. The method was validated in terms of selectivity, linearity, sensitivity, trueness and precision, which all satisfy the requirement for pesticides residue analysis. The proposed method was successfully applied for monitoring the occurrence and enantiomeric composition of pesticides in different fruits and vegetables, demonstrating its applicability for the routine analysis of chiral pesticide residues in food samples.

Multi-residue enantiomeric analysis of 18 chiral pesticides in water, soil and river sediment using magnetic solid-phase extraction based on amino modified multiwalled carbon nanotubes and chiral liquid chromatography coupled with tandem mass spectrometry

This manuscript describes, for the first time, the multi-residue analysis of 18 chiral pesticides at enantiomeric levels in both environmental liquid (river water, influent and effluent wastewater) and solid matrices (agricultural soil, forestal soil and river sediment) based on magnetic solid-phase extraction (MSPE) and chiral liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Magnetic amino modified multiwalled carbon nanotubes (m-MWCNTs-NH2) were prepared and firstly applied to adsorb pesticides from complex matrices. Response surface methodology (RSM) was applied to assist the multivariable optimization. The simultaneous enantioseparation of the chiral pesticides was performed on a Chiralpak IG column. Under the optimum conditions, the mean recoveries for pesticides enantiomers from the water matrices ranged from 81.1 to 106.3% with intra-day RSD of 2.1–11.9% and inter-day RSD of 2.6–12.7%; the mean recoveries for all enantiomers from the solid matrices ranged from 80.3 to 105.9% with intra-day RSD of 2.3–10.9% and inter-day RSD of 4.0–13.4%. Good linearity was achieved for all enantiomers with determination coefficients (r2) greater than 0.9912. Method quantification limits were below 2.04ng L−1 in liquid matrices and below 0.50ng g-1 in solid matrices. The developed method offered some advantages, such as simple operation, rapidity and high concentration factor. Therefore, it is suitable for monitoring the enantiomeric compositions of chiral pesticides in different environmental matrices.

Removal Of Baboon And Human Antiporcine Igg And Igm Natural Antibodies By Immunoadsorption

The safe and effective removal of xenoreactive antibodies in the peritransplant period is likely to be critical for the clinical application of xenotransplantation involving disparate donor species, such as the pig. In an effort to develop an improved method for antibody removal in xenotransplantation, we have studied reusable antihuman antibody (Ig) columns in vitro and in vivo. Two types of columns were tested: (1) an antihuman Ig column containing polyclonal sheep antihuman IgG (heavy- and light-chain-specific) conjugated to sepharose CL-4B (Ig-Therasorb), and (2) an antihuman Ig column using polyclonal antihuman IgM (mu-chain-specific) conjugated to sepharose. Passage of human or baboon plasma through the Ig-Therasorb column resulted in 97.5% and 78.4% mean reductions in total IgG and IgM, respectively. Reductions in total IgG and IgM correlated with lowering of antipig IgG (54-486 fold) and IgM (9-54 fold) antibody titers as assessed by pig endothelial cell ELISA. The ability of the Ig-Therasorb to significantly reduce IgM may be attributed to the light chain specificity of this column. With the anti-IgM column, marked reductions in total (82.6-83.9%) and antipig (27-54 fold) IgM in human and baboon plasma occurred, while levels of total and xenoreactive IgG were slightly affected. Other than a dilutional effect, neither column resulted in significant reduction in albumin, fibrinogen, factor 5, and factor 8. Repeated in vivo use of either column in baboons achieved reductions in IgG and IgM that closely followed the results of our in vitro studies. No subject morbidity or mortality occurred. Use of the Ig-Therasorb column with immunosuppression in two baboons receiving pig renal xenografts achieved sustained reductions in antipig antibodies and prevented hyperacute rejection. Subjects were sacrificed at 11 and 13 days posttransplant with functioning xenografts and were found to have no evidence of vascular xenograft rejection. We conclude that anti-Ig columns represent a safe and effective method for antibody removal, without several of the limitations of other antibody removal techniques. Also, columns appear to be safe for repeated antibody removal in the posttransplant period.

Kinetics of Class II MHC expression on cytotoxic T cells generated by skin allograft

In the present work the kinetics of class II MHC expression on OX8+ lymphocytes generated by skin allograft and its relationship to the lytic activity were studied. Mononuclear cells from the spleen of LEW (RT1(1) rats bearing BN (RT1n) skin graft for 3, 5 or 7 days were sorted out by sequential immune affinity using columns of Degalan-V26 beads treated with anti-rat or anti-mouse Ig. After depletion of B cells, T cells were precoated with W3/25 MoAb (anti-CD4 equivalent) and sorted out using an anti-mouse Ig column. The W3/25-/OX8+ cells (CD8 equivalent) were then coated with OX4 MoAb (anti-RT1.B) or murine A.TH anti-A.TL alloantiserum (anti I-E, cross-reacts with RT1.D) and were passed through a new anti-mouse Ig column in order to obtain the four subpopulations, RT1.B+, .B-, .D+ and .D-. Their specific lytic activity against BN Con A-stimulated cells increased from the 3rd to the 7th d after the skin graft. The lytic activity observed on the 3rd and 5th d was associated with all four subpopulations analyzed. In contrast, on the 7th d, the lytic activity was concentrated in the RT1.B+ subpopulation. These results, associated with the increase in the number of OX8+/RT1.B+ cells along with days after graft, suggest that RT1.B expression is not essential but is associated with the effectiveness of the cytotoxic activity. It is also possible that RT1.B expression is a marker of cytotoxic T-cell differentiation.

Xenoserum-Induced Cytolytic «T» Cells: Polyclonal Specificity with an Apparent «Anti-Self» Component, and Cooperative Induction

Mice were primed in vivo by injection of fetal calf serum (FCS) and their spleen cells were incubated in vitro for 5 days in medium containing 10% FCS. This resulted in the development of cytolytic activity, which was most probably due to «T» cells, since effector cells 1) were sensitive to anti-Thy 1 antiserum or monoclonal antibodies in the presence of complement, 2) were not retained on Ig-anti Ig columns, 3) did not develop from «nude» spleen cells. Further arguments for the T cell nature of these effector cells came from their specificity. Blocking experiments using unlabeled competitor cells demonstrated that FCS-induced cytolysis was polyclonal, with clones recognizing allogeneic or syngeneic determinants possibly related to allo or self H-2. In keeping with polyclonality, cytolysis tested on any given target cell was greatly increased by adding Concanavalin A during the cytolysis test. Experiments were made to investigate whether in particular the anti-self cytolytic activity was directed against FCS determinants. We feel that this possibility, although not formally excluded, was made unlikely. The polyclonal specificity at the effector stage stood in sharp contrast to the serum specificity at the induction stage (reported elsewhere). We demonstrated that these two sets of specificities corresponded to two sets of specific cells. A first population of FCS-primed cells had promoter .. activity, in the sense that it could trigger a second population of «precursor» cells to differentiate into polyclonally cytolytic T cells.

Sera from lipopolysaccharide (LPS)-injected mice exhibit complement-dependent cytotoxicity against syngeneic and autologous spleen cells

To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearance of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.

Optimal conditions for the separation of rat T lymphocytes on anti-immunoglobulin-immunoglobulin affinity columns

The separation of rat T lymphocytes was investigated on anti-IgIg columns. A simple and efficient method for the purification of rat Ig by precipitation of rat serum with sodium sulfate is presented. Protein binding characteristics of glass and plastic beads, as solid support of affinity columns, are described, as well as optimal parameters for coating beads with rat Ig (with BSA, ribonuclease and lysozyme, as comparison). Binding of Ig was primarily dependent on the concentration of the Ig solution. Maximal strong binding of Ig (6.2 × 10 3 molecules per μ 2 of bead surface) was reached at 400 μg per ml concentration of purified Ig solution during 20 min of incubation. Higher concentrations increased only the amount of loosely bound Ig on the surface of beads whereas the amount of firmly bound Ig remained unchanged. Fractionation of lymphoid cell suspensions on anti-IgIg affinity columns prepared at optimal conditions resulted in highly purified T-cell suspensions containing less than 1% of lymphocytes bearing surface Ig receptors.

Separation of cells with different histocompatibility (H‐2) antigens by passage through cellular immunoadsorbent columns

The anti-Ig column technique of Wigzell et al. (Scand. J. Immunol. 1972. 1:75) was adapted to the separation of mouse cells with different H-2 antigens. Fractionation was achieved by a two-step procedure: first the cells were treated with anti-H-2 sera, and thereafter passed over anti-mouse Ig columns. Antibody-coated cells were selectively retained in the anti-Ig column. The method could be applied to the separation of normal mouse lymphocytes or lymphoma cells with different H-2 antigen complexes and for the selective isolation of H-2 loss variants from a mouse carcinoma/fibroblast hybrid.

Lymphoid cell fractionation by aggregated immunoglobulin-agarose columns

Fractionation by columns of aggregated rat immunoglobulin (Agg Ig)-agarose was investigated as a method of separating different populations of lymphoid cells. With rat spleen cells, Agg Ig columns retained phagocytes, IgM- and IgG-antibody-forming-cells, cells mediating antibody- or PHA-induced lysis of chicken erythrocytes, and specifically immune splenocytes lytic to chicken erythrocytes without exogenous antibody. Agg Ig columns did not selectively remove ‘B lymphocytes’ (surface-Ig-bearing lymphocytes with or without EAC′ receptors), or T lymphocytes capable of PHA-induced proliferation or graft-versus-host reactivity. With mouse spleen cells, Agg Ig columns retained alloimmune cytotoxic T cells.

Antibody-dependent cell-mediated cytotoxicity in the mouse. I. Surface characteristics of an effecter cell.

The surface characteristics of the mouse spleen cells mediating antibody‐dependent cytotoxicity (ADCC) against antigen‐coated chicken erythrocytes have been studied by several different column fractionation methods The major effector cells in this system were shown to be surface‐adherent and could be depleted from spleen cells by passage through glass‐bead ovalbumin/anti‐OA immune complex columns (Fc receptor‐binding), glass‐bead immunoglobulin/anti‐mouse Ig columns (Fc receptor and surface immunoglobulin‐binding), and glass‐bead mouse Ig/rabbit (Fab')2‐anti‐mouse Ig or Sephadex G‐200/rabbit anti‐mouse Ig columns (surface immunoglobulin binding) The concentration of EDTA in the medium used to fractionate the cells played a significant role in determining whether surface immunoglobulin could be demonstrated on the ADCC effector cells. From these results, the conclusion was drawn that ADCC on the part of mouse spleen cells could be mediated by surface‐adherent, Fc receptor‐positive cells bearing surface immunoglobulin of unknown origin. The possibility that ADCC can be mediated by a heterogenous population of cells in the mouse spleen is discussed.

Cytotoxic immune cells with specificity for defined soluble antigens. IV. Antibody as mediator of specific cytotoxicity.

Spleen cells from mice immunized against ovalbumin (OA) or dinitrophenylated mouse serum albumin (DM) were found to be specifically cytotoxic in vitro towards target cells (chicken red blood cells) coated with these antigens. Inhibition of specific cytotoxicity was observed when free soluble antigen was added to the incubation mixtures. DM-immune cell cytotoxicity could be specifically and completely inhibited by DNP-lysine and was thus shown to be hapten specific. Complete and specific inhibition was also observed for OA-immune cell cytotoxicity using OA as inhibitor, but compared with the inhibition curves obtained with DNP-lysine, the OA cytotoxicity inhibition curves were shifted by a factor of about one hundred towards lower molar inhibitor concentrations. Very similar results were observed when the serum antibodies of DM- and OA-immune animals were analyzed by passive hemagglutination inhibition. With increasing time after immunization, both cytotoxicity inhibition curves and agglutination inhibition curves, shifted to lower antigen or hapten concentrations. Specific cytotoxicity against antigen-coated target cells was induced in nonimmune spleen cells (a) by serum from immune animals, and (b) by supernatants from in vitro immune cell cultures. In both instances, the factor which induced antigen-specific cytotoxic activity could be absorbed on anti-mouse Ig columns, thus demonstrating its immunoglobulin nature. The ability of target cell bound antibodies to induce cytotoxicity in nonimmune spleen cells was restricted to the 7S antibody class.