Method to analyze compounds in biological samples

A method to analyze target analyte compounds from a dried biological sample by using a microfluidic sample device comprising a hollow cartridge and an absorbent body unit, the absorbent body unit comprising a filter paper, a capillary pipette tip, or a modified polymeric sintered filter body that is configured to be attached to the hollow cartridge with a pipette-like geometry, the method comprising:

• a) reconstituting the dried biological sample using a reconstitution buffer composition, wherein:
• the absorbent body unit comprises one or more single layer structures;
• different functional groups densities of COOH, NH2, OH, TiO2, and/or ZrO2 are present in each of the one or more single layer structures as a binding tool for a pre-cleaning step;
• a first coating comprising Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), -ethoxyquin, polyacrylic acid (PAA), polyethyleneimine (PEI), sorbitan esters, polyethoxy sorbitan esters octylphenoxypolyethoxyethanol, Na2- EDTA, Na-citrate, and/or hydrogels is present on each of the one or more single layer structures as artificial antioxidants and as active hydrophilic compounds;
• the absorbent body unit is positioned at a distal end of the hollow cartridge and a proximal end of the hollow cartridge comprises a passage that is configured to be connected to a pipette head of an automated operation device;
• the hollow cartridge comprises a membrane to achieve a separation of the liquid of the absorbent body unit through the hollow cartridge passage;
• the membrane is removed by a mechanical force, a pressure, or a vacuum;
• the automated operation device is configured to change a position of the pipette head in sequential steps;
• and
• the reconstitution buffer composition is dispensed in a first vial or a first well;
• b) soaking and storing the reconstituted dried biological sample in the absorbent body unit;
• c) after the reconstituted dried biological sample is stored in the absorbent body unit, attaching the absorbent body unit to the distal end of the hollow cartridge;
• d) aspirating the reconstituted dried biological sample into the hollow cartridge through the absorbent body unit using a predefined amount of the reconstitution buffer composition;
• e) temporarily storing the reconstituted dried biological sample in the hollow cartridge for up to 600 seconds;
• f) releasing the reconstituted dried biological sample back into the first vial or the first well and then again aspirating the reconstituted dried biological sample back into the hollow cartridge at least one time in order to achieve a higher concentration of the target analyte compounds of the reconstituted dried biological sample in relation to a starting concentration of the target analyte compounds, wherein the reconstituted dried biological sample is used as a reconstitution buffer composition for at least one other reconstituted dried biological sample in order to achieve a higher target analyte compounds concentration in relation to the starting concentration of the target analyte compounds;
• g) transferring a predefined volume of the reconstituted dried biological sample to a second vial or a second well;
• h) removing abundant non-analytical compounds of the reconstituted dried biological sample by:
• adding an internal standard, wherein the internal standard comprises D6-25OH-Vitamin D3, D3-Thiamine diphosphate (TDP), D3-Pyridoxal-5′-phosphate (PLP), [13C,2H7]-apixaban, [13C6]-dabigatran, [13C6]-rivaroxaban, or [2H6]-edoxaban;
• adding a first set of coated magnetic beads, wherein the first set of coated magnetic beads comprise silica beads coated with a second coating, wherein the second coating comprises one or more functional groups selected from the group consisting of —OH, —COOH, —NH2, R—SO2—OH, —NH2;
• —RNH, —R2N, CH3, —C2H5, —C4H9, —C8H17, —C9H19, —C10H21, —C11H23, —C12H25, —C13H27, —C14H29, —C15H31, —C16H33, —C17H35, —C18H37, —C6H5, —ZrO2, TiO2, C6H9NO6, phenylhexyl, biphenyl, hydroxyapatite, boronic acid, activated carbon, fullerenes, latex, polyvinyl alcohol, melamine, and chitin;
• and
• adding a depletion buffer comprising mixtures of organic solvents and alkaline solutions, wherein the organic solvents or alkaline solutions comprise NaOH, KOH, NH4OH, (NH4)2SO4, (NH4)CH3COO, ZnSO4, MgSO4, K4[Fe(CN)6], CuSO4, AgNO3, NaCl, KCl, MgCl2, (CH3COO)2Pb, FeCl3, HNO3, HClO4, H2SO4, HCl, CF3COOH, CCl3COOH, CH3COOH, CHOOH, wherein the pH-value is in the range of 0 to 14, and wherein an ionic strength between the depletion buffer and the reconstituted dried biological sample is between 1 mM and 5000 mM;
• i) separating the abundant non-analytical compounds of the reconstituted dried biological sample by using a magnetic separator;
• j) receiving the target analyte compounds of the reconstituted dried biological sample in the supernatant;
• k) alternatively binding at least some of the received target analyte compounds from step j) to a second set of coated magnetic beads that are different than the first set of coated magnetic beads and eluting the received target analyte compounds thereafter;
• and
• l) analyzing the received target analyte compounds with one or more readout systems used in combination with one or more specific detectors, wherein the readout systems are selected from the group consisting of immunoassays, GC, HPLC, LC, and CE, and the specific detectors are selected from the group consisting of MS/MS, MS, FID, EDC, UV-VIS-spectrometer, IR- spectrometer, fluorescence, and chemiluminescence immunoassay.