TRANSITION METAL CHELATING BEADS

Abstract

The present invention relates to a complex comprising a transition metal cation (i); a ligand (ii) comprising at least one chelating group, preferably from 1 to 4 chelating groups, more preferred 2 or 3 chelating groups, wherein the chelating group(s) is/are selected from hydroxamate group —N(O″)—C(═O)—R, catechol ate group, carboxyl ate group, partly or totally protonated forms of these chelating groups and mixtures of these chelating groups and/or their partly or totally protonated forms, wherein R is hydrogen or a C1 to C5 alkyl group; and a magnetic bead (iii); wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded. The invention also relates to the use of the complex for reduction of the content of at least one phosphor-oxy-substance, which preferably comprises a structural element —O—P(O″)(═O)—O— within its structure, in a fluid sample, as well as to a method for decreasing the content of at least one phosphor-oxy-substance, preferably a phospholipid, in a fluid sample, comprising a step of adding the complex. The invention further relates to a supernatant obtained or obtainable from this method and to the use of a supernatant obtained or obtainable from the method for qualitative and/or quantitative determination of at least one analyte in said supernatant. Furthermore, the invention relates to a method for qualitative and/or quantitative determination of at least one analyte in a fluid sample and to a method for determining the kind and/or amount of at least one phosphor-oxy-substance in a fluid sample.

Claims

1. A complex comprising i) a transition metal cation; ii) a ligand comprising at least one chelating group, wherein the chelating group(s) is/are selected from hydroxamate group N(O.sup.−)—C(═O)—R, partly or totally protonated forms of hydroxamate groups and mixtures of hydroxamate groups and their partly or totally protonated forms, wherein R is hydrogen or a C1 to C5 alkyl group; iii) a magnetic bead; wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded.

2. The complex of claim 1, wherein the ligand (ii) has the general structure (I) ##STR00011## wherein: - - - is the bond to the magnetic bead (iii); A is selected from the group consisting of a hydrogen atom, a —X—Y—(CH.sub.2).sub.m—CH.sub.2—(CHR.sup.3).sub.q—R.sup.2-group and R.sup.3; n, m are independently zero or an integer from 1 to 5; p, q are independently an integer from 1 to 10; X is —CH.sub.2— or —NH—; Y is —CH.sub.2— or —C(═O)—; R.sup.1, R.sup.2 are independently selected from the group consisting of hydroxamate group —N(O.sup.−)—C(═O)—R, wherein R is hydrogen or a C1 to C5 alkyl group, and partly or totally protonated forms of hydroxamate group; R.sup.3 is a hydrogen atom or a NHZ group, wherein Z is a —C(═O)—O—CH.sub.2—C.sub.6H.sub.5 group (benzyloxycarbonyl group, Cbz) or a tert-Butyloxycarbonyl group (Boc).

3. The complex according to claim 1, wherein the ligand (ii) has the general structure (Ia): ##STR00012## wherein - - - is the bond to the magnetic bead (iii); and A is selected from the group consisting of a hydrogen atom, a —X—Y—(CH.sub.2).sub.m—CH.sub.2—(CHR.sup.3).sub.q—R.sup.2-group and R.sup.3.

4. The complex according to claim 1, wherein the ligand (ii) has the general structure (Ia1) or (Ia2): ##STR00013## wherein - - - is the bond to the magnetic bead (iii); and Z is a —C(═O)—O—CH.sub.2—C.sub.6H.sub.5 group (benzyloycarbonyl group, Cbz) or a tert-Butyloxycarbonyl group (BOC).

5. The complex according to claim 1, wherein the transition metal cation (i) is selected from the group of platinum-, ruthenium-, iridium-, scandium-, titanium-, vanadium-, chromium-, manganese-, iron-, cobalt-, nickel-, copper- and zinc-cation.

6. The complex according to claim 1, wherein the magnetic bead (iii) comprises a polymer matrix (P), at least one magnetic particle (M) and at least one (CH.sub.2).sub.r—NH - - - group covalently bonded on the polymer matrix (P) surface (S), wherein - - - is the bond to the ligand (ii), and r is zero or an integer in the range of from 1 to 10; wherein the polymer matrix (P) comprises at least one crosslinked (co-)polymer.

7. The complex according to claim 6, wherein the polymer matrix (P) comprises a co-polymer obtained or obtainable by a method comprising polymerization of at least two different monomeric building blocks selected from the group consisting of styrene, functionalized styrenes, vinylbenzylchloride, divinylbenzene, vinylacetate, methylmethaacrylate and acrylic acid: ##STR00014##

8. The complex according to claim 6, wherein the (co-)polymer of the polymer matrix (P) is crosslinked, wherein the crosslinked (co-)polymer of the polymer matrix (P) is obtained or obtainable by co-polymerizing at least two different monomeric building blocks according to claim 7 in the presence of at least one monomeric building block which is a crosslinking agent, wherein the crosslinking agent is preferably selected from the group consisting of divinylbenzene, bis(vinylphenyl)ethane, bis(vinylbenzyloxy)hexane, bis(vinylbenzyloxy)dodecane and mixtures of two or more of these crosslinking agents.

9. The complex according to claim 6, wherein the at least one magnetic particle (M) comprises a compound selected from the group consisting of metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal carbide, metal chelate and a mixture of two or more thereof.

10. Use of complex according to claim 1 reduction of the content of at least one phosphor-oxy-substance in a fluid sample.

11. A method for decreasing the content of at least one phosphor-oxy-substance, in a fluid sample, comprising the steps: a) providing a fluid sample comprising at least one analyte of interest and at least one phosphor-oxy-substance; b) optionally adjusting the pH value of the fluid sample, so that the pH value of the fluid sample is in the range of from 2.5 to 12 thereby obtaining a pH adjusted fluid sample; c) adding at least one first complex, wherein the complex comprises: i) a transition metal cation; ii) a ligand comprising at least one chelating group, wherein the chelating group(s) is/are selected from hydroxamate group —N(O.sup.−)—C(═O)—R, partly or totally protonated forms of hydroxamate groups and mixtures of hydroxamate groups and their partly or totally protonated forms, wherein R is hydrogen or a C1 to C5 alkyl group; iii) a magnetic bead; wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded; thereby forming a suspension comprising a second complex, which comprises the first complex and the at least one phosphor-oxy-substance in complexed form; d) spacely separating the second complex in the suspension obtained in (c) by application of a magnetic field, thereby obtaining a supernatant substantially free of the second complex; e) removing the supernatant, thereby obtaining a separated second complex.

12. A supernatant obtained or obtainable from the method of claim 11.

13. Use of a supernatant obtained or obtainable from the method of claim 11 for qualitative and/or quantitative determination of at least one analyte in said supernatant.

14. A method for qualitative and/or quantitative determination of at least one analyte in a fluid sample, comprising the steps of the purification method according to claim 11 and further comprising a step of qualitative and/or quantitative determination of the at least one analyte in the supernatant obtained from (e) and/or (f) and/or (g).

15. A method for determining the kind and/or amount of at least one phosphor-oxy-substance in a fluid sample, comprising the steps of the purification method according to claim 11 and further comprising: h) adding an aqueous and/or organic elution solution to the (further) separated second complex obtained according to (e) and/or (f), wherein the aqueous and/or organic elution solution contains a buffer and/or a reductant, and/or wherein the adding is done in a reductive atmosphere, thereby separating the at least one phosphor-oxy-substance from the separated second complex and obtaining a solution comprising the at least one phosphor-oxy-substance; j) determining the kind and/or amount of the at least one phosphor-oxy-substance in the solution obtained according to (h).

16. The complex according to claim 1 comprising from 1 to 4 chelating groups.

17. The complex according to claim 7, wherein the at least two different monomeric building blocks are selected from the group consisting of the following monomers: ##STR00015## wherein r is zero or an integer in the range of from 1 to 10; R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5, being independently of each other selected from the group consisting of —N.sub.3, —NH.sub.2, —Br, —I, —F, —NR′R″, —NR′R″R′″, —COOH, —CN, —OH, —OR′, —COOR′, —NO.sub.2, —SH.sub.2, —SO.sub.2, —R′(OH)x, —R′(COOH)x, —R′(COOR″)x, —R′(OR″)x, —R′(NH.sub.2)x, —R′(NHR″)x, —R′(NR″R′″)x, —R′(Cl)x, —R′(I)x, —R′(Br)x, —R′(F)x, R′(CN)x, —R′(N.sub.3)x, —R′(NO.sub.2)x, —R′(SH.sub.2)x, —R′(SO.sub.2)x, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl; with R′, R″ and R′″ being, independently of each other, selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halides, hydrogen, sulfides, nitrates and amines; and wherein x is an integer in the range of from 1 to 3.

18. The complex according to claim 8, wherein the crosslinking agent at least comprises divinylbenzene.

19. The complex according to claim 9, wherein the at least one magnetic particle (M) comprises an iron oxide selected from the group consisting of Fe.sub.3O.sub.4, α-Fe.sub.2O.sub.3, γ-Fe.sub.2O.sub.3, MnFe.sub.xO.sub.y, CoFe.sub.xO.sub.y, NiFe.sub.xO.sub.y, CuFe.sub.xO.sub.y, ZnFe.sub.xO.sub.yCdFe.sub.xO.sub.y, BaFe.sub.xO and SrFe.sub.xO, wherein x and y vary depending on the method of synthesis, and wherein x is preferably an integer of from 1 to 3, and wherein y is preferably 3 or 4.

20. Use of complex according to claim 1 for reduction of the content of at least one phosphor-oxy-substance comprising a structural element —O—P(O.sup.−)(═O)—O— within its structure in a fluid sample.

Description

EXAMPLES

1. Chemicals

[0131]

TABLE-US-00001 Abbreviation Chemical name magnetic bead superparamagnetic beads (Fe.sub.3O.sub.4) with crosslinked 1 polystyrene matrix having primary amino groups on the outer surface (propyl amino groups) DMF N,N-Dimethylformamide Ligand 1 -di- benzyl ((8S,11S)-4,15-diacetyl-8-(ethylcarbamoyl)- acetylated form 2,10,17-tri-oxo-3,16-dioxa-4,9,15-triazaoctadecan- 11-yl)carbamate HOBt 1-Hydroxybenzotriazole DIC Diisopropylcarbodiimide — N-methylpiperidine MeOH methanol ZrCl.sub.4 Zirconium (IV) tetrachloride DIPEA N,N-Diisopropylethylamine K.sub.3PO.sub.4/K.sub.2HPO.sub.4 Tri potassium phosphate/di potassium hydrogen phosphate HCOOH Formic acid mCPBA m-chloroperoxybenzoic acid

2. Experimental Design

[0132] Zr-complexed magnetic beads that remove residual matrix, i.e. impurities, from semi-cleaned up Human serum were synthesized and subsequently evaluated for their propensity to i) yield increase in signal to noise ratio (S/N) of analytes that are quantified using LCMS/MS methods and ii) remove lysophosphatidylcholine, an important phospholipid.

[0133] S/N increase is a direct implication of less interference from substances other than the analyte of interest when measured via LC-MS/MS. Although this technique usually removes large portions of interfering matrixes via LC and subsequently selects for defined MRM transitions, quantitation accuracy and sensitivity is in most cases still compromised. Hence, a cleaner sample allows for less ion suppression, and thereby allowing for a higher sensitivity and a higher accuracy. Indeed, the implications of a clean sample on system robustness are numerous. One direct measurable parameter that is known to compromise the longevity of an accurate and sensitive measuring LC-MS/MS system are phospholipids. To this end, one lysophosphatidylcholine (18:0) (LPC 18:0) was chosen as a representative substance that was quantified to evaluate if the new beads would be capable of removing this substance.

[0134] 2.1 Conjugation of Zirconium Chelating Ligands to Magnetic Beads and Chelation with Zr

[0135] As suitable bead, magnetic bead 1 having free amino groups were selected. To the free amine, ligand 1 in its diacetylated form, i.e. a dipeptide of O-Acetyl hydroxamate derivatized ornithine, was coupled using standard peptide chemistry. Subsequently, the hydroxy groups were deprotected and the ligands were complexed with Zr (see Scheme 2) thereby obtaining Zr-complexed magnetic beads.

##STR00009##

[0136] Conjugation of ligand 1 in its diacetylated from to the magnetic bead resulting in a hydroxamate bead, Deprotection of the hydroxamate groups of ligand 1, and complexation with Zr(IV) resulting in a Zr-complexed magnetic bead.

2.2 Evaluation of Zr-Complexed Magnetic Beads for their Capacity to Remove Phospholipids and Improve Signal to Noise Ratio (S/N) in LC-MS/MS Quantitation.

[0137] The use of the Zr-complexed magnetic beads was to remove as many substances as possible from (semi-clean) biological matrices. The goal of this experiment was to show that these beads are capable of removing important matrix components and thereby improve i) the quantitation (i.e. S/N) of clinically relevant analytes and ii) system robustness (less residual matrix obviously adds to the longevity of the LC-MS/MS system as a whole).

[0138] To this end two experiments were carried out. The first experiment was related to examining the S/N from cleaned up serum and the second was related to measuring lysophosphatidylcholine 18:0 (LPC 18:0).

[0139] To obtain sufficient sample that contains clinically relevant analytes that were cleaned up from serum using an enrichment workflow that was found to yield high recoveries of these analytes, 100 μl portions of spiked serum were worked up 5 times, yielding 300 μl of semi-clean eluate, according to the method described below (see also FIG. 1). This eluate was then diluted once more with water (1:1). These mixtures were next aliquoted in 9 vessels. To vessels 1-3 15 μl water was charged, to vessels 4-6 15 μl magnetic bead 1 were charged and to vessels 7-9 Zr-complexed magnetic beads were charged. The tubes were next shortly vortexed and left to stand for 5 min. Subsequently, the supernatant was transferred to a HPLC vial. The samples were next measured via LC-MS/MS to determine SN for relevant analytes and to compare LPC (18:0) contents from these samples.

3. Examples

Reference Example 1 Synthesis of Ligand 1 and its Diacetylated Form Respectively (a Dipeptide of O-Acetyl Hydroxamate Derivatized Ornithine)

[0140] ##STR00010##

[0141] Synthesis of ligand 1 in its diacetylated from

[0142] To a benzyloxycarbonyl (Cbz) protected dipeptide of ornithine, benzylaldehye was added to form an imine. The resulting product was next oxidized using mCPBA, followed by a hydrolyzation and a subsequent acetylation. The resulting product ligand 1 in its diacetylated form-can be used as such to be coupled to the free amine of an appropriate bead via its free carboxylic acid, using standard peptide chemistry.

Example 1—Synthesis of a Magnetic Beads, Bonded Via an Amide Bond to a Dipeptide of O-Acetyl Hydroxamate Derivatized Ornithine (Hydroxamate Beads)

[0143] To 30 mg magnetic beads 1 about 9 μmol ligand here 1 in its diacetylated form—may be coupled, assuming an molecular weight of 1 kDa. Based on this protocol, to 30 mg of magnetic beads 1, DMF (0.25 ml) was added and stirred. Hereto, ligand 1 in its diacetylated form (20.8 mg, 4 eq. 36 μmop, HOBt (5 mg, 2 eq., 18 μmop, DIC (5.6 μl, eq. 36 μmol), N-methylpiperidine (4.3 μl, 2 eq., 18 μmop in DMF (0.25 ml) was added and the flasks gently mixed by rolling for 2 h at room temperature.

[0144] Following this amide conjugation, the reaction mixture was removed from the conjugated beads (magnetic beads 1 coupled by amide bond with ligand 1) by applying a magnetic field. The conjugated beads were washed 3 times with MeOH and 3 times with water and twice more with MeOH. The solvent was then removed and the conjugated beads resuspended in 6% N-methylpiperidine in MeOH. Again, the conjugated beads were washed 3 times with MeOH and 3 times with water. Next, the solvent was removed, and the conjugated beads dried under vacuo, yielding 30 mg of conjugated beads.

[0145] The used reaction mixture (0.5 ml) that was removed from the beads after reaction was assessed for its presence of unreacted dipeptide: 6% DIPEA in MeOH (1 ml) was added and left to stand for 30 min. To this, FeCl.sub.3 was added, giving it a yellow color.

[0146] A reaction mixture that was not brought into contact with free-amine beads, i.e. magnetic beads 1, served as a negative control. This reaction mixture underwent the same treatment (i.e. 6% DIPEA in MeOH was added, mixture left to stand for 30 min. and subsequent addition of FeCl.sub.3). The color of this mixture was dark brown. This indicated that most, if not all dipeptide that was brought into reaction with the free-amine bead had reacted. Namely, any non-reacted dipeptide would complex with Fe.sup.3+ and give a brown color, as is the case for the negative control.

Example 2—Synthesis of a Zirconium Complex of the Beads from Example 1 (Zr-Complexed Magnetic Beads)

[0147] Hydroxamate beads (30 mg) were suspended in water (1 ml) and reacted with ZrOCl.sub.2 (250 μl, 1 M in water) by rolling at room temperature for 2 h. The reaction mixture was removed from the conjugated beads by applying a magnetic field. The conjugated beads were then washed 3 times with water. Beads were then resuspended in 1 ml of water, yielding 30 mg/ml Zr-complexed magnetic beads.

Example 3—Workup of Spiked Serum and Final Purification with Zr-Complexed Magnetic Beads

[0148] A standard bead-assessment workflow was as follows (see FIG. 1): In the tube, sample to which analytes of interest have been spiked (see Table 1 for details) was charged (step 1). The analytes used were aldosterone, benzoylecgonine and nortriptyline. Next a pH adjustment reagent was added that set the pH of the mixture (step 2). To this, a bead suspension of the Zr-complexed magnetic beads from Example 2 was added and the mixture was shaken and incubated for 5 min. (step 3). Subsequently, a magnetic field was applied and the magnetic beads were drawn to the side of the vessel (step 4) and the supernatant was removed (step 5). Next, a washing solution was added and the mixture was shaken (step 6), after which the beads were again separated from the supernatant which was then again removed. This procedure was repeated once more. Subsequently, an elution solution was added (step 7) and the mixture was shaken and incubated for another 5 min. Next, the beads were separated from the supernatant which was next transferred to another vial (step 8). To this, a mixture with the internal standards of the compounds that were spiked to the serum sample was added (step 9). Details are indicated below in Table 1. Thus, no enrichment or dilution of the analytes was effected using this workflow. For comparison, the same procedure was executed with free magnetic beads 1 and without bead addition (control).

TABLE-US-00002 TABLE 1 pH Adjust- Super- Sample ment Bead Wash Elute natant Dilute Spiked K.sub.3PO.sub.4/K.sub.2H Bead sus- water HCOOH Transfer ISTD- serum* PO.sub.4 (250 pension 150 (100 mM) to vial Mix** 100 μl mM) (aqueous) μl in MeOH 30 μl 30 μl 40 μl 40 μl (70 wt.-%) 50 μl *serum pool from different donations to which the analytes of interest were added in a ratio of 1:40 (spike mix:serum, v/v). **ISTD (internal standard) mix with isotopically labeled analogues of the analytes of interest as added. The ISTD allowed correction of matrix effects and inaccuracies of pipetting. Also, since these concentrations were known, this allowed for analyte recovery calculations.

[0149] 3.1 Bead Assessment, Reagents and Tools

[0150] For the S/N Evaluation of analytes and comparison LPC (18:0) contents, an Agilent Infinity II multisampler and HPLC system was used in combination with an Agilent Poroshell 120 SbAq (2.1×50 mm, 2.7 μm, Serial Nr. USFAH01259) or a Thermo Fisher Hypercarb (2.1×50 mm, 3 μm, Serial Nr. 10517483) Column. As for the mobile phase water with 0.1% formic acid was used as solvent A and acetonitrile was used as solvent B. As MS/MS an AB-Sciex 6500+ TripleQuad using electronspray as ion source. For integration MultiQuant software tool was used. Data were next imported to and analyzed in JMP SAS software.

[0151] 3.2 S/N Comparison for Aldosterone, Benzoylecgonine and Nortriptyline

[0152] One criterion for bead functioning is a significant improvement of S/N (signal to noise ratio) of clinically relevant analytes. To this end S/N for the different samples were compared for aldosterone. Showing that treatment with the Zr-complexed magnetic beads from Example 2 lead to a S/N increase of factor ˜2 when compared to the non-treated sample. The “free Amine bead” refers to the magnetic bead 1 having free amino groups that was used for ligand conjugation and Zr-complexation. It is observed, that this free Amine bead also yielded a sample that allows for a better S/N, however the S/N of the Zr-complexed magnetic beads from Example 2 also outcompeted this bead. The results are graphically shown in FIG. 2. Comparable results were obtained for the other analytes nortriptyline (FIG. 3) and benzoylecgonine (FIG. 4).

[0153] 3.3 LPC (18:0) Comparison

[0154] The results of the comparison of the lysophosphatidylcholine (18:0) (LPC (18:0)) contents are shown in FIG. 5. It could be seen that the sample treated with the Zr-complexed magnetic beads from Example 2 contained significantly less LPC (18:0).

[0155] It is apparent that a cleanup of sample material that removed matrix substances had a positive effect on S/N. This allows for a more sensitive and more accurate quantitation of analytes. It was shown that by treating the sample with the new bead type, i.e. the Zr-complexed magnetic beads, at least one phospholipid is removed.

SHORT DESCRIPTION OF FIGURES

[0156] FIG. 1: shows the steps of workup of spiked serum prior to final purification with Zr-complexed magnetic beads;

[0157] FIG. 2: shows the S/N ratio's for Aldosterone samples;

[0158] FIG. 3: shows the S/N ratio's for nortriptyline samples;

[0159] FIG. 4: shows the S/N ratio's for benzoylecgonine samples;

[0160] FIG. 5: shows the LPC (18:0) area for samples w/o (with or without) active LPC removal.

CITED LITERATURE

[0161] Bylda C., Thiele, R., Kobold, U., Volmer, D. A., Analyst, 2014, 139, 2265-2276. [0162] Guerard F., Lee Y. S., Tripier R., Szajek L. P., Deschamps J. R., Brechbiel M. W., Chem. Commun., 2013, 49:1002-1004. [0163] Guérard, F., Lee, Y. S., Brechbiel, M. W., Chemistry, 2014, 20(19): 5584-5591.