EX VIVO ANALYTIC METHOD

Abstract

The present invention relates to an ex vivo analytic method for analysis of a sample (S1), comprising the steps: A) Determining Fe2.sup.+, Fe3.sup.+, Fe-Ferritin and total Fe content in sample (S1), comprising the steps: A1) Separating simultaneously Fe2.sup.+, Fe3.sup.+ and Fe-Ferritin present in a sample (S1); and A2) Quantifying simultaneously Fe2.sup.+, Fe3.sup.+, Fe-Ferritin separated in step A1) and total Fe based on sample (S1); B) Determining S and Se containing compounds content of sample (S1), comprising the steps B1) Separating simultaneously S and Se containing compounds present in sample (S1); and B2) Quantifying simultaneously S and Se containing compounds separated in step B1) of sample (S1). Further, the invention relates to a kit for performing the analytic method.

Claims

1. An ex vivo analytic method for analysis of a sample (S1), comprising the steps: A) Determining Fe.sup.2+, Fe.sup.3+, Fe-Ferritin and total Fe content in sample (S1), comprising the steps: A1) Separating simultaneously Fe.sup.2+, Fe.sup.3+ and Fe-Ferritin present in a sample (S1); A2) Quantifying simultaneously Fe.sup.2+, Fe.sup.3+, Fe-Ferritin separated in step A1) and total Fe based on sample (S1); B) Determining S and Se containing compounds content of sample (S1), comprising the steps B1) Separating simultaneously S and Se containing compounds present in sample (S1); B2) Quantifying simultaneously S and Se containing compounds separated in step B1) of sample (S1).

2. The analytic method of claim 1, wherein in step A1) and/or B1) separating is carried out with capillary electrophoresis; wherein optionally I) in step A1) the sample is injected at the inlet; II) after injection of the sample, the capillary is treated with an electrolyte (E2) in step A1), wherein the electrolyte (E2) is an aqueous HCl solution, preferably with a concentration of 0.005 to 0.2 mM, more preferably 0.01 to 0.1 mM, most preferably 0.05 mM; and/or III) wherein seperation in step A1) using capillary electrophoresis comprises a capillary preparation step before the actual separation, including purging and filling with leading electrolyte and stacking, preferably with a solution (E1) comprising HCl and MeOH; wherein optionally i) the concentration of HCl is 5 to 15 weight-%, preferably 8 to 12 weight-%, more preferably 10 weight-%; based on the overall amount of the solution and/or ii) the concentration of MeOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-% based on the overall amount of the solution and/or IV) the electrolyte (E3) in step A1) at the inlet is an aqueous HCl solution, preferably with a concentration of 20 to 80 mM, more preferably 30 to 70 mM, most preferably 50 mM; and/or V) the electrolyte (E4) in step A1) at the outlet is an aqueous HCl solution, preferably with a concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM; and/or VI) wherein separation in step B1) using capillary electrophoresis comprises a capillary preparation step before the actual separation, including purging and filling with leading electrolyte (E5) and stacking, preferably with a solution comprising a borate buffer, and CTAB Cetyltrimethylammonium bromide), wherein optionally i) the concentration of borate is 50 to 200 mM, preferably 80 to 150 mM, more preferably 90 mM; and/or ii) the concentration of CTAB is 1 to 10 mM, preferably 1 to 5 mM, more preferably 3 mM; and/or VII) the electrolyte in step B1) at the inlet is an aqueous solution (E6) comprising HCl, preferably in concentration of 5 to 100 mM, more preferably 10 to 50 mM, most preferably 20 mM; and/or VIII) in step B1) the sample is at the inlet; and/or IX) the electrolyte (E7) in step B1) at the inlet is an aqueous solution comprising TMAH (Tetramethylammoniumhydroxide), EtOH, and/or CTAB (Cetyltrimethylammonium bromide, wherein optionally i) the concentration of TMAH is 0.5 to 10 weight-%, preferably 1 to 5 weight-%, more preferably 3 weight-%; based on the overall amount of the aqueous solution and/or ii) the concentration of EtOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-%; based on the overall amount of the aqueous solution and/or iii) the concentration of CTAB is 1 to 10 mM, preferably 1 to 5 mM, more preferably 3 mM; and/or X) the electrolyte (E8) in step B2) at the outlet is an aqueous solution comprising NH.sub.4-acetate preferably in concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM.

3. The analytic method of claim 1 or 2, wherein in step A2) and/or B2) quantifying is carried out with mass spectroscopy, preferably with application of inductively coupled plasma mass spectroscopy (ICP), wherein optionally the inductively coupled plasma mass spectroscopy (ICP) is I) selected from the group consisting of ICP-DRC-MS, ICP-qqq-MS and ICP-sf-MS or II) ICP-DRC-MS, wherein optionally i) in step A2) the DRC gas is NH.sub.3 and/or ii) in step B2) the DRC gas is CH.sub.4.

4. The analytic method of any one of claims 1 to 3, wherein I) the one or more S containing compounds are selected from the group consisting of oxidized glutathione (GSSG), reduced glutathione (GSH), cystine and/or cysteine; and/or II) the one or more Se containing compounds are selected from the group consisting of consisting of Se.sup.4+, Se.sup.6+, Se-methionine, Se-cystine, Se-cysteine, GPX4, Selenoprotein P, and/or Seleno sugars.

5. The analytic method of any one of claims 1 to 4, wherein the sample (S1) comprises material obtained from a subject, preferably a human subject.

6. The analytic method of any one of claims 1 to 5, wherein the sample (S1) comprises a biofluid, preferably selected from the group consisting of a cell lysate, a tissue lysate, CSF, serum, and urine.

7. The analytic method of any one of claims 1 to 6, wherein I) the volume of sample (S1) is not more than 20 l, preferably not more than 15 l, more preferably not more than 10 l; and/or II) the volume used in step A1) and A2) combined is not more than 30 nL, preferably not more than 20 nL, most preferably not more than 15 nL; and/or III) the volume used in step B1) and B2) combined is not more than 30 nL, preferably not more than 20 nL, most preferably not more than 15 nL.

8. The analytic method of any one of claims 1 to 7, wherein I) step A1) and A2) and/or Il) step B1) and B2) are carried out within 1 to 15 minutes, preferably within 3 to 10 minutes, more preferably 4 to 7 minutes, most preferably within 5 minutes.

9. The analytic method of any one of claims 1 to 8, wherein step A) and B) are carried out sequentially, preferably step A) before step B).

10. The analytic method of any one of claims 1 to 9, further comprising providing information on the results of the analytic method, preferably to a subject.

11. Use of the analytic method of any one of claims 1 to 10 for an ex vivo diagnosis of oxidative stress.

12. Use of the analytic method of any one of claims 1 to 10 for an ex vivo diagnosis of ferroptosis.

13. Use of the analytic method of any one of claims 1 to 10 for an ex vivo diagnosis of ferroptosis-associated diseases, preferably hemochromatosis, or sideroblastic anemia.

14. The use of the analytic method of any one of claims 1 to 10 for an ex vivo diagnosis of prostate cancer, melanoma, multiple sclerosis, diabetes and myelodysplastic syndrom.

15. A kit for performing the analytic method of any one of claims 1 to 10, comprising i) devices for performing the analytic method; and ii) a HCl solution, preferably with a concentration of 20 to 80 mM, more preferably 30 to 70 mM, most preferably 50 mM; for performing step A1) and iii) a HCl solution, preferably with a concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM; for performing step A1) and iv) CH, gas for performing step B2).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1 Iron speciation in a cellular extract.

[0025] FIG. 2: Sulfur speciation of GSH and GSSG standard compounds.

[0026] FIG. 3: Selenium speciation of Se (IV), Se (VI), selenomethionine (SeM) and selenocystine (SeC) standard compounds.

[0027] FIG. 4: Example for the application of the inventive method in various in vitro and in vivo models: FIG. 4A shows early passages of SV40-immortalized App+/+ and App/ MEFs and E1A/Ha-RasV12-transformed murine embryonal fibroblasts (MEF) variants. Respective lysates were analyzed via CE-ICP-MS analysis presented as absolute content of Fe.sup.2+ and Fe.sup.3+ in cell lysates of conditions in (A). Data are presented as Fe.sup.2+/Fe.sup.3+ ratio (n=3, meanSD). FIG. B App/ MEF cells stably expressing Dox-induced FPN-GFP vector (App/ FPN-GFP). MEF cells were either left untreated (-Dox), incubated with either Dox (xx ng/ml) to force membrane-bound FPN1 re-expression Untreated cells served as untreated controls, while untransfected App/ and App+/+ MEF lysates served as additional controls. CE-ICP-MS analysis showing absolute content of Fe.sup.2+ and Fe.sup.3+ in cell lysates of conditions. FIG. 4C Human PrCa cell models (LNCaP, 22Rc1, DU-145, PC3). CE-ICP-MS analysis showing absolute content of Fe.sup.2+ and Fe.sup.3+ in cell lysates of conditions. Data are presented as Fe.sup.2+/Fe.sup.3+ ratio (n=3, meanSD). FIG. 4D CE-ICP-MS analysis showing absolute content of Fe.sup.2+ and Fe.sup.3+ in cell lysates of App+/+, App/ and doxycycline (DOX)-induced re-constitution human APP751-isoform in App/ (APP/resc MEF) murine embryonal fibroblasts (MEF) following 24-h treatment with 100 M ferric ammonium citrate (FAC, 200 M) or left untreated. FIG. 4E The human PrCa xenograft model was constructed by subcutaneously injecting the indicated PC3 cells in 5 mice. Twenty days after injection, mice were intraperitoneally treated with imidazole keton erastin (IKE, 50 mg/kg body weight) or vehicle (Veh) for 10 days and tumor growth was further monitored until day 40 (n=10, each group). The tumor growth was measured by electronic caliper daily, calculated using the formula volume=0.5lengthwidth2 and are plotted as meanSD. Fe.sup.2+/Fe.sup.3+ ratio from lysates of PC3 xenografts analyzed by CE-ICP-MS. FIG. 4F PMN treated with scramble siRNA (siScr) or App-siRNA (siApp) under normoxia (Nor) and OGD condition (left panel). CE-ICP-MS analysis showing absolute content of Fe.sup.2+ and Fe.sup.3+ of PMN treated with scramble siRNA (siScr) or App-siRNA (siApp) under normoxia (Nor) and OGD condition or App+/+ and App/ mice with or without MCAO surgery. FIG. 4G FAC (200 M) treatment for 5 and 10 days in PC3-scr and APPshRNA #1 and #2 cells. Fe.sup.2+/Fe.sup.3+ ratio in analyzed in cell lysates analogous to (B) analyzed by CE-ICP-MS (n=3). Values are presented as meanSD

[0028] FIG. 5: Example for the application of the inventive method on the progressive development of prostate cancer cells: FIG. 5A shows that with increasing development time: 1. The Fe.sup.2+/Fe.sup.3+ ratio increases due to increase of ROS/oxidative stress (OS). 2. The GSH/GSSG ratio decreases due to depletion of GSH as defense against oxidative stress. 3. GPX decreases due to exhaustion of GSH recovery mechanism 4. The Fe-load of ferritine is not affected. FIGS. 5B to 5E show the changes of certain markers in prostate cancer cells over 10 days in the presence of ROS promoters (#11043, #utr') or absence of ROS promoters (scr). FIG. 5B: Fe.sup.2+/Fe.sup.3+ Ratio as signature for oxidative stress (OS). FIG. 5C: GSH/GSSG ratio decreases due to depletion of GSH as defense against oxidative stress; FIG. 5D: Relative Quantification of the GPX4 share of the selenium compounds. GPX4 decreases due to exhaustion of GSH recovery mechanism. FIG. 5E: Fe-load of total Fe of ferritine. The Fe-load of ferritine is not affected.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.

Definitions

[0030] It is noted that as used herein, the singular forms a, an, and the, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a reagent includes one or more of such different reagents and reference to the method includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0031] Unless otherwise indicated, the term at least preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0032] The term and/or wherever used herein includes the meaning of and, or and all or any other combination of the elements connected by said term.

[0033] For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, f.e. more than 80% means more than or greater than the indicated number of 80%.

[0034] Throughout this specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term comprising can be substituted with the term containing or including or sometimes when used herein with the term having. When used herein consisting of excludes any element, step, or ingredient not specified.

[0035] DRC is dynamic reaction cell technology. The dynamic reaction cell or collision reaction cell, is a chamber placed before the traditional quadrupole chamber of an ICP-MS device, for eliminating isobaric interferences. See for example Yip, Y.; Sham, W (2007). Applications of collision/reaction-cell technology in isotope dilution mass spectrometry. TrAC Trends in Analytical Chemistry. 26:727. doi: 10.1016/j.trac.2007.03.007.

[0036] sf-MS is a sector field mass spectrometer, as known to the person skilled in the art.

[0037] Qqq-MS is a triple quadrupole mass spectrometer, as known to the person skilled in the art.

[0038] The invention is related to an ex vivo analytic method for analysis of a sample (S1).

[0039] The sample (S1) may comprise any fluid. The sample (S1) may comprise material obtained from a subject, preferably a human subject. Preferably the sample (S1) comprises a biofluid. More preferably, the biofluid is selected from the group consisting of a cell lysate, a tissue lysate, cerebrospinal fluid (CSF), serum, and urine. Preferably, samples are stored under inert gas immediately after collection or extract preparation at 0 to 100 C., more preferably at-20 to 90 C., most preferred at 75 to 80 C., until analysis. Preferably, the inert gas is selected from the group consisting of nitrogen, and argon. Preferably, serum sample collection, in particular CSF is performed according to standard guidelines, such as e.g. Stanford Lumbar Puncture Guidelines (see: https://dx.stanford.edu/procedures/Procedures_LumbarPuncture.pdf)

[0040] In one embodiment, respective cell material is lysed into a buffer with gentle agitation, preferably cooled on ice. Preferably, the buffer is lysed for 10 to 60 minutes, more preferably for 20 to 50 minutes, most preferably 30 minutes. Preferably, the cell lysates were centrifuged at 5000 to 20000 g, more preferably at 8000 to 15000 g, most preferably at 10000 g. Preferably, the buffer contains: [0041] i) PBS (phosphate buffered saline: for example comprising

TABLE-US-00001 NaCl 1.37M KCl 27 mM Na.sub.2HPO.sub.4 100 mM KH.sub.2PO.sub.4 20 mM pH 7.2-7.6);
and/or [0042] ii) sodium deoxycholate;
preferably in a concentration of 0.1 to 1 wt.-%, more preferably 0.3 to 7wt.-%, most preferably 0.5 wt.-% based on the overall mass of the sample (S1); and/or [0043] iii) NP-40 (Nonoxynol-40, CAS: 9016-45-9);
preferably in a concentration of 0.1 to 2% wt.-%, more preferably 0.2 to 1.5 wt.-%, most preferably 1% wt.-% based on the overall mass of the sample (S1).

[0044] Preferably, the protein concentration of the cell lysates is determined. Preferably, the protein concentration in the final sample (S1) is diluted, preferably with buffer as described above to a protein concentration of 0.1 to 5, more preferably to 0.5 to 3, most preferred 1 g protein/L (proteins as a result of the lysis of cells) in the sample (S1). Preferably, serum is diluted 1:10 to 1:100, more preferably 1:60 to 1:90, most preferred 1:80. Preferably, serum or urine are diluted 1:1 to 1:4, more preferably 1:1.2 to 1:3, most preferably 1:1.5.

[0045] Preferably, the volume of sample (S1) is not more than 20 l, more preferably not more than 15 l, most preferably not more than 10 l. Preferably, sample (S1) is the basis for analysis in step A) and B). Preferably, in step A) a part of sample (S1) is used and another part of sample (S1) is used in step B).

[0046] The ex vivo analytic method comprises the steps: [0047] A) Determining Fe.sup.2+, Fe.sup.3+, Fe-Ferritin and total Fe content in sample (S1) and [0048] B) Determining S and Se containing compounds content of sample (S1).

[0049] Step A) comprises the steps: [0050] A1) Separating simultaneously Fe.sup.2+, Fe.sup.3+ and Fe-Ferritin present in a sample (S1); and [0051] A2) Quantifying simultaneously Fe.sup.2+, Fe.sup.3+, Fe-Ferritin separated in step A1) and total Fe based on sample (S1).

[0052] Fe-Ferritin means in the context of the present invention, the amount of iron or iron ions bound to the iron storage protein Ferritin.

[0053] Total Fe content means the overall amount of iron present in the sample independent from its form, such as solved or bound to proteins etc.

[0054] Step A1) and/or A2) are preferably carried out within 1 to 15 minutes, more preferably within 3 to 10 minutes, most preferably 4 to 7 minutes, particular preferred within 5 minutes.

[0055] Seperating in step A1) is preferably carried out with capillary electrophoresis.

[0056] Any known capillary electrophoresis device may be used. A capillary electrophoresis device comprises at least a capillary and a vial with a buffer solution at the inlet of the capillary and a vial with a buffer solution at the outlet of the capillary. If the term inlet is used, that means at the inlet of the capillary and the term outlet, at the outlet of the capillary.

[0057] Seperation in step A1) using capillary electrophoresis may comprise a capillary preparation step before the actual separation. Capillary preparation includes purging and fill with leading electrolyte and stacking, preferably with a solution (E1) comprising HCl and MeOH; wherein optionally [0058] i) the concentration of HCl is 5 to 15 weight-%, preferably 8 to 12 weight-%, more preferably 10 weight-%; based on the overall amount of the solution and/or [0059] ii) the concentration of MeOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-% based on the overall amount of the solution.

[0060] Preferably, purging is carried out at a pressure of 2 to 8 bar, more preferably 3 to 7 bar, most preferably 4 bar and/or for 0.5 to 5 min, more preferably 0.6 to 3 min, most preferably 1 min.

[0061] Preferably, the sample (S1) is injected into the capillary in step A1) at the inlet, more preferably after the capillary preparation step. Preferably, the injected volume of sample (S1) is not more than 5 to 30 nL, more preferably not more than 10 to 20 nL, most preferred not more than 15 nL. Preferably, the sample (S1) is injected wth a pressure of 100 to 600 mbar, more preferably 200 to 500 mbar, most preferably 300 mbar.

[0062] After injection of the sample, the capillary may be treated with an aqueous HCl solution (E2), preferably with a concentration of 0.005 to 0.2 mM, more preferably 0.01 to 0.1 mM, most preferably 0.05 mM and/or at a pressure of 100 to 600 mbar, more preferably 200 to 500 mbar, most preferably 300 mbar.

[0063] The electrolyte in step A1) at the inlet may be an aqueous HCl solution (E3), preferably with a concentration of 20 to 80 mM, more preferably 30 to 70 mM, most preferably 50 mM.

[0064] Preferably, the electrolyte at the inlet has a pressure of 200 to 800 mbar, more preferably 300 to 700 mbar, most preferably 600 mbar. Preferably at at voltage of 10 to 60 kV, more preferably 20 to 50 kV, most preferably 30 kV.

[0065] The electrolyte in step A1) at the outlet (E4) may be an aqueous HCl solution, preferably with a concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM.

[0066] The quantifying in step A2) may be carried out with mass spectroscopy. The mass spectroscopy may comprise inductively coupled plasma mass spectroscopy (ICP-MS), preferably selected from the group consisting of ICP-DRC-MS, ICP-qqq-MS and ICP-sf-MS, more preferably ICP-DRC-MS; wherein optionally the DRC gas is NH.sub.3. Preferably, argon is used as plasma gas in the mass spectroscopy and/or as nebulizer gas. Preferably, the measured isotope is .sup.56Fe.

[0067] The volume of sample (S1) used in step A1) and A2) combined may be not more than 30 nL, preferably not more than 20 nL, most preferably not more than 15 nL.

[0068] Step B) comprises the steps: [0069] B1) Separating simultaneously S and Se containing compounds present in sample (S1); and [0070] B2) Quantifying simultaneously S and Se containing compounds separated in step B1) of sample (S1).

[0071] Step B1) and/or B2) are preferably carried out within 1 to 15 minutes, more preferably within 3 to 10 minutes, most preferably 4 to 7 minutes, particular preferred within 5 minutes.

[0072] The one or more S (sulfur) containing compounds may be selected from the group consisting of oxidized glutathione (GSSG), reduced glutathione (GSH), cystine, and/or cysteine, preferably oxidized glutathione (GSSG), reduced glutathione (GSH), and/or cysteine

[0073] The one or more Se (selenium) containing compounds may be selected from the group consisting of Se.sup.4+, Se.sup.6+, Se-methionine, Se-cystine, Se-cysteine, GPX4,Selenoprotein P, and/or Seleno sugars, preferably Se.sup.4+, Se.sup.6+, Se-methionine, Se-cysteine, Se-cystine and/or GPX4, more preferably Se.sup.4+, Se.sup.6+, Se-methionine, Se-cysteine and/or GPX4.

[0074] In the context of this invention, the term Se-methionine means selenomethionine, for example CAS number: 3211-76-5. In the context of this invention, the term Se-cysteine means selenocysteine, for example CAS number: 10236-58-5. GPX4 is Glutathione peroxidase 4, a selenium containing enzyme. Se-cystine means selenocystine, for example CAS number: 29621-88-3.

[0075] Seperating in step B1) is preferably carried out with capillary electrophoresis.

[0076] Seperation in step B1) using capillary electrophoresis may comprise a capillary preparation step before the actual separation. Capillary preparation includes purging and filling with leading electrolyte (E5) and stacking, preferably with a solution comprising a borate buffer, and CTAB Cetyltrimethylammonium bromide), wherein optionally [0077] i) the concentration of borate is 50 to 200 mM, preferably 80 to 150 mM, more preferably 90 mM; and/or [0078] ii) the borate is sodium borate or potassium borate, preferably sodium borate; [0079] iii) the concentration of CTAB is 1 to 10 mM, preferably 1 to 5 mM, more preferably 3 mM.

[0080] Preferably, purging and filling with leading electrolyte and stacking in step B1) is carried out at 2 to 8 bar, more preferably 3 to 7 bar, most preferably 4 bar and/or for 0.5 to 5 min, more preferably 0.6 to 3 min, most preferably 1 min.

[0081] Preferably, the sample (S1) is injected into the capillary in step B1), more preferably after the capillary preparation step. Preferably, the injected volume of sample (S1) in step B1) is not more than 5 to 30 nL, more preferably not more than 10 to 20 nL, most preferred not more than 15 nL. Preferably, the sample (S1) is injected wth a pressure of 100 to 600 mbar, more preferably 200 to 500 mbar, most preferably 300 mbar.

[0082] The electrolyte (E6) in step B1) at the inlet may be an aqueous solution comprising HCl, preferably in concentration of 5 to 100 mM, more preferably 10 to 50 mM, most preferably 20 mM.

[0083] The electrolyte (E7) in step B1) at the inlet may be an aqueous solution comprising TMAH (Tetramethylammoniumhydroxide), EtOH, and/or CTAB (Cetyltrimethylammonium bromide), wherein optionally [0084] i) the concentration of TMAH is 0.5 to 10 weight-%, preferably 1 to 5 weight-%, more preferably 3 weight-%; based on the overall amount of the aqueous solution and/or [0085] ii) the concentration of EtOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-%; based on the overall amount of the aqueous solution and/or [0086] iii) the concentration of CTAB is 1 to 10 mM, preferably 1 to 5 mM, more preferably 3 mM.

[0087] Preferably, the electrolyte at the inlet has a pressure of 200 to 800 mbar, more preferably 300 to 700 mbar, most preferably 500 mbar. Preferably at voltage of 10 to 60 kV, more preferably 20 to 50 kV, most preferably 30 kV.

[0088] The electrolyte in step B1) at the outlet may be an aqueous solution (E8) comprising NH.sub.4-acetate preferably in concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM.

[0089] Preferably, in step B1), the sample is at the inlet.

[0090] The quantifying in step B2) may be carried out with mass spectroscopy. The mass spectroscopy may comprise inductively coupled plasma (ICP) mass spectroscopy, preferably selected from the group consisting of ICP-DRC-MS, ICP-qqq-MS and ICP-sf-MS, more preferably ICP-DRC-MS; wherein optionally the DRC gas is CH.sub.4. Preferably, the use of CH.sub.4 results in a mass shift of the sulfur species from m/z=32 to m/z=48. Preferably, sulfur compounds are detected as m/z=48 peak. Preferably, argon is used as plasma gas in the mass spectroscopy and/or as nebulizer gas. Preferably, the measured isotopes are .sup.48[S-CH.sub.4], .sup.78Se, and/or .sup.80Se.

[0091] The volume of sample (S1) used in step B1) and B2) combined is not more than 30 nL, preferably not more than 20 nL, most preferably not more than 15 nL.

[0092] In one embodiment, ex vivo analytic method for analysis of a sample (S1), comprising the steps: [0093] A) Determining Fe.sup.2+, Fe.sup.3+, Fe-Ferritin and total Fe content in sample (S1), comprising the steps: [0094] A1) Separating simultaneously Fe.sup.2+, Fe.sup.3+ and Fe-Ferritin present in a sample (S1) with capillary electrophoresis; wherein the electrolyte (E3) at the inlet is an aqueous solution comprising HCl, wherein [0095] the HCl solution (E3), has a concentration of 20 to 80 mM, preferably 30 to 70 mM, more preferably 50 mM. [0096] A2) Quantifying simultaneously Fe.sup.2+, Fe.sup.3+, Fe-Ferritin separated in step A1) and total Fe based on sample (S1) with ICP-DRC-MS; [0097] B) Determining S and Se containing compounds content of sample (S1), comprising the steps: [0098] B1) Separating simultaneously S and Se containing compounds present in sample (S1) with capillary electrophoresis; [0099] B2) Quantifying simultaneously S and Se containing compounds separated in step B1) of sample (S1) with ICP-DRC-MS, wherein the DRC gas is CH.sub.4.

[0100] In a further embodiment, ex vivo analytic method for analysis of a sample (S1), comprising the steps: [0101] A) Determining Fe.sup.2+, Fe.sup.3+, Fe-Ferritin and total Fe content in sample (S1), comprising the steps: [0102] A1) I) Capillary preparation, including purging and filling with leading electrolyte (E1) and stacking, with a solution comprising HCl and MeOH; wherein [0103] i) the concentration of HCl is 5 to 15 weight-%, preferably 8 to 12 weight-%, more preferably 10 weight-%; based on the overall amount of the solution and [0104] ii) the concentration of MeOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-% based on the overall amount of the solution; [0105] purging is carried out at a pressure of 2 to 8 bar, more preferably 3 to 7 bar, most preferably 4 bar; [0106] II) Separating simultaneously Fe.sup.2+, Fe.sup.3+ and Fe-Ferritin present in a sample (S1) with capillary electrophoresis; wherein separating comprises [0107] i) injection of a part of sample (S1); [0108] ii) wherein the electrolyte at the outlet (E4) is an aqueous HCl solution, with a concentration of 1 to 15 mM, preferably 3 to 8 mM, more preferably 5 mM;and [0109] iii) wherein the electrolyte (E3) at the inlet is an aqueous solution comprising HCl, wherein the concentration of HCl is 20 to 100 mM preferably 30 to 80 mM, more preferably 50 mM, based on the overall amount of the aqueous solution; [0110] A2) Quantifying simultaneously Fe.sup.2+, Fe.sup.3+, Fe-Ferritin separated in step A1) and total Fe based on sample (S1) with ICP-DRC-MS; [0111] B) Determining S and Se containing compounds content of sample (S1), comprising the steps: [0112] B1) I) Capillary preparation including purging and filling with leading electrolyte (E5) and stacking, with an aqueous solution (E5) comprising a borate buffer and CTAB, wherein the concentration of borate is 50 to 200 mM, preferably 80 to 150 mM, more preferably 100 mM. [0113] II) Separating simultaneously S and Se containing compounds present in sample (S1) with capillary electrophoresis; [0114] i) injection of a part of sample (S1); [0115] ii) the electrolyte (E7) at the inlet is an aqueous solution comprising TMAH, EtOH and CTAB (Cetyltrimethylammonium bromide), wherein the concentration of TMAH is 1 to 10 mM,, preferably 2 to 5 mM, more preferably 3 mM,; the concentration of TMAH is 0.5 to 10 weight-%, preferably 1 to 5 weight-%, more preferably 3 and weight-%; and/or the concentration of EtOH is 5 to 20 weight-%, preferably 7 to 15 weight-%, more preferably 10 weight-% based on the overall amount of the aqueous solution. [0116] B2) Quantifying simultaneously S and Se containing compounds separated in step B1) of sample (S1) with ICP-DRC-MS, wherein the DRC gas is CH.sub.4.

[0117] The analytic method may further comprise providing information on the results of the analytic method, preferably to a subject.

[0118] The invention further comprises the use of the analytic method for an ex vivo diagnosis of oxidative stress, ferroptosis, and ferroptosis-associated diseases. Preferably, the ferroptosis-associated disease is hemochromatosis, or sideroblastic anemia.

[0119] The invention further comprises the use of the analytic method for an ex vivo diagnosis of prostate cancer, melanoma, diabetes multiple sclerosis, and myelodysplastic syndrome, preferably prostate cancer.

[0120] The invention further comprises a kit for performing the analytic method, as described above, comprising [0121] i) devices for performing the analytic method; and [0122] ii) a HCl solution, preferably with a concentration of 20 to 80 mM, more preferably 30 to 70 mM, most preferably 50 mM; for performing step A1) and [0123] iii) a HCl solution, preferably with a concentration of 1 to 15 mM, more preferably 3 to 8 mM, most preferably 5 mM; for performing step A1) and [0124] iv) CH.sub.4 gas for performing step B2).

EXAMPLES OF THE INVENTION

Capillary Zone Electrophoresis (CZE)

[0125] A PrinCe 706 CE system from PrinCe Technologies B.V. (Emmen, The Netherlands) is employed. Temperature settings for sample/buffer tray and capillary are set at 20 oC by air cooling, each. An uncoated capillary with dimension 100 cm50 m ID (CS-Chromatographie Service GmbH, Langerwehe, Germany) was used for separation and hyphenation to the ICP-MS. A CE-ICP-MS interface was installed which provided the electrical connection between CE capillary end and outlet electrode. The self-aspiration mode allowed for best flow rate adjustment and reduced (avoided) suction flow.

[0126] Table 1 and 2 show the consecutive steps of the analysis: [0127] First shot for speciation of Fe-ferritin, Fe.sup.2+ and Fe.sup.3+. [0128] Section A: capillary preparation steps before measurement; [0129] Section B: electrolytes and conditions during analysis. [0130] Section C: Detection: ICP-DRC-MS conditions during analysis

TABLE-US-00002 TABLE 1 First shot A Capillary preparation Purge and fill with leading 10% HCl/10% 4 bar, 1 min electrolyte and stacking MeOH Injection 300 mbar, 6 s Stacking with terminating 0.0 5mM HCl 300 mbar, 6 s electrolyte B Analysis by CZE separation Inlet electrolyte 50 mM HCl 600 mbar, 5 min, + 30 kV Outlet/sheath electrolyte at 5 mM HCl Interface working ICP-MS interface with self-aspiration ca. 100 L/min C ICP-DRC-MS detection Conditions RF power 1,250 W Plasma gas 16 L Ar/min Nebulizer gas 0.94 L Ar/min. Dwell time 50 ms DRC gas NH.sub.3 DRC gas flow 0.58 mL/min DRC rejection 0.58 parameter .sup.56Fe Measured isotope [0131] Second shot for speciation of GSH, GSSG, cysteine, and GPX4 and other seleno species [0132] Section A: capillary preparation steps before measurement; [0133] Section B: electrolytes and conditions during analysis. [0134] Section C: Detection: ICP-DRC-MS conditions during analysis

TABLE-US-00003 TABLE 2 Second Shot A Capillary preparation Purge and fill with 100 mM borate buffer, 4 bar, 1 min background electrolyte 3 mM CTAB, pH 7.8 Injection 300 mbar, 10 s B Analysis Inlet electrolyte 3% TMAH, 10% 500 mbar, 5 EtOH, 3 mM CTAB min, + 30 kV Outlet/sheath 5 mM NH.sub.4-acetate Interface working electrolyte at ICP-MS with self-aspiration interface ca. 100 L/min ICP-DRC-MS detection Conditions RF power 1,250 W Plasma gas 16 L Ar/min Nebulizer gas 0.94 L Ar/min. Dwell time 50 ms DRC gas CH.sub.4 DRC gas flow 0.6 mL/min DRC rejection 0.60 parameter .sup.48[SCH.sub.4], Measured isotopes .sup.78Se, .sup.80Se

[0135] Inductively coupled plasma mass spectrometry (ICP-MS) as CE detector

[0136] A Nexlon 300 D (Perkin Elmer, Sciex, Toronto, Canada) was operated as ICP-MS system for the on-line detection of CE-efflux. The RF power was set to 1250 W, the plasma gas was 16 L Ar/min. The nebulizer gas was optimized and finally set to 0.98 L Ar/min.

[0137] During the first analysis shot (iron speciation) the isotope .sup.56Fe was measured in dynamic reaction cell (DRC) mode. The dwell time was 50 ms. Ammonia was used as DRC gas (0.58 ml NH3/min) and DRC rejection parameter was set to 0.58.

[0138] During the second analysis shot (sulfur and selenium speciation) the isotopes .sup.48[S-CH.sub.4], .sup.78Se, .sup.80Se were measured in dynamic reaction cell (DRC) mode. The dwell time was 50 ms. Methane was used as DRC gas (0.60 ml CH.sub.4/min) and DRC rejection parameter was set to 0.60.

REFERENCES

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