LU504848B1 - in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva - Google Patents
in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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Abstract
The invention relates to An in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva obtained from a subject, said method comprising -Mixing said sample with a determined amount of an acidic aqueous solution and therefore obtaining an acidified sample ; -Detecting and quantifying said micronutrient in said acidified sample by injecting said acidified sample in an active coupled plasma mass spectrometer and/or -Detecting and quantifying said vitamin in said acidified sample by agitating said acidified sample with ultrasound for 10 minutes and injecting said acidified sample in an ultra-high performance liquid chromatographer coupled with a triple quadrupole mass spectrometer.
Description
Title of the invention: in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva
[001] The present invention relates to an in vitro method for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva and the associated kit. More specifically, the method relates to the determination of the amount of trace elements and vitamins in an organism and in particular applied to a non- invasive saliva test for trace elements and vitamins. Once the concentrations are known, an algorithm adapts the quantities to be provided to the subject to avoid deficiencies but also to avoid overdoses of these trace elements and vitamins.
[002] Trace elements and vitamins remains a public health concern despite efforts to avoid deficiencies for these elements. The symptoms of trace elements and vitamin’s deficiencies are large and various: fatigue, difficulty concentrating, up to muscle and nerve damage and dysfunction of biological functions such as kidney, reproduction, liver.
[003] Upon identification of these deficiencies’ loadings within an individual, lifestyle changes and the administration of customized nutriments serve to minimize the deleterious effects. However, these treatments can only be put in place after testing of an individual for nutriments.
[004] While various public health organizations and agencies have been
Successful in performing universal and routine testing of people for some nutriments, but large numbers of individuals are never tested or tested at a frequency that is less than ideal. Compliant testing for nutriments has met with limited success owing in part to the necessity for a blood draw requiring skilled personnel, parental consent for children, blood draw trauma to the child, and sophisticated infrastructure to process samples. Saliva has previously been studied as an attractive alternative bodily fluid for the measurement of nutriments concentrations.
[005] Thus, there exists a need for a saliva-based assay for nutriments in order to increase screening, especially of athletes, pregnant women, populations with an unvaried diet. Additionally, there exists a need for a reproducible saliva sample collection methodology to facilitate correlation between clinic status (deficiency) and saliva nutriments concentrations.
[006] Current methods for detecting and quantifying micronutrients or vitamins lack stability and continuity of results, which is often caused by a wide diversity in sampling methods, sample quality and methods sensitivity and accuracy.
[007] The invention compensates for those disadvantages.
[008] The present invention relates to an in vitro method for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva obtained from a subject.
[009] According to a general definition of the invention, the in vitro method comprises: -Mixing said sample with a determined amount of an acidic aqueous solution and therefore obtaining an acidified sample ; -Detecting and quantifying said micronutrient in said acidified sample by injecting said acidified sample in an active coupled plasma mass spectrometer and/or -Detecting and quantifying said vitamin in said acidified sample by injecting said acidified sample in an ultra-high performance liquid chromatographer coupled with a triple quadrupole mass spectrometer.
[010] In one embodiment, said at least one vitamin is chosen among Vitamin
B1, Vitamin B3, Vitamin, B5, Vitamin B6, Vitamin B8, Vitamin B12, Vitamin A,
Vitamin C, Vitamin D3 and Vitamin E and said at least one micronutrient is chosen among manganese, copper, calcium, zinc, iron, iodine, chromium, selenium.
[011] In one embodiment, said sample of a determined amount of saliva is contained in a determined amount of a polymer able to absorb said determined amount of saliva.
[012] In one embodiment, the determined amount of said polymer forms a polymer ball.
[013] According to one embodiment of the invention, said determined amount of polymer containing said determined amount of saliva is contact with said acidic aqueous to extract said at least one vitamin and/or said at least one micronutrient.
[014] According to one embodiment of the invention pertaining to vitamin detection and quantification, the acidic aqueous solution is a 1 % (v/v) formic acid aqueous solution and wherein at least one vitamin is detected and quantified.
[015] Advantageously, the acidified sample allows the permeation of the cells in the sample, allowing the extraction from any cellular structure of the targeted vitamin into the acidified sample.
[016] Furthermore, the acidified sample is filtered through a 0.2 um filter before injection into said an ultra-high performance liquid chromatographer.
[017] According to one embodiment, said ultra-high performance liquid chromatographer comprised a chromatography column packed with particles of 1.9 um diameter.
[018] In one embodiment, a binary gradient of acetonitrile and water with 5mM ammonium acetate is used for column elution.
[019] According to one embodiment of the invention pertaining to micronutrient detection and quantification, the acidic solution is a 0.2%(v/v) nitric acid solution with Triton X100. and wherein at least one micronutrient is detected and quantified.
[020] In an example of the previous embodiment, said acidic aqueous solution is prepared with one liter of Ultrapure water with a resistivity greater than 18
MQ, 1 ml of Triton X100 is added, i.e. a Triton X100 concentration of 0.1% (v/v),
and 2 ml nitric acid (HNO3) 67-69% Trace Metal™ quality, i.e. a concentration of 0.2%(v/v).
[021] Advantageously, the acidified sample allows the mineralization effect, allowing the extraction from any cellular structure of the targeted micronutrient into the acidified sample.
[022] As an example, the subject is a mammalian in particular chosen among cow, horse, sheep, dog and cat, and more particularly a human being or an avian in particular a chicken.
[023] The invention further relates to a kit for implementing the method described.
[024] According to another general definition of the invention, the kit comprises: - a pipette or a swab ; and - a 1% (v/v) aqueous formic acid solution ; and/or - a 0.2%(v/v) nitric acid solution with a surfactant able to permeate cell membrane, in particular an anionic surfactant of formula
C8H17C6H4(0C2H4)9-100H
[025] In one embodiment, said swab comprises a determined amount of polymer able to absorb the determined amount of saliva.
[026] In another embodiment, said swab is a spherical swab.
[027] The method according to the invention comprises a first step of mixing a saliva sample with a determined amount of an acidic aqueous solution and therefore obtaining an acidified sample.
[028] Regardless of the saliva collection method, the sample is placed in a package and receives a chain of custody number corresponding to the sample (QR code barcode, etc.). Preferably, the sample is collected in the form of a polymer ball calibrated to receive a precise volume of saliva, comprising a determined amount of said polymer forming said polymer ball.
[029] Said first step comprises a sub step of preparation of the sample followed by a sub step of extraction of the targeted compound.
[030] A targeted compound according to the invention is at least one vitamin chosen among Vitamin B1, Vitamin B3, Vitamin, B5, Vitamin B6, Vitamin B8,
Vitamin B12, Vitamin A, Vitamin C, Vitamin D3 and Vitamin E and/or at least one micronutrient is chosen among manganese, copper, calcium, zinc, iron, iodine, chromium, selenium.
[031] According to one embodiment, a determined amount of polymer containing said determined amount of saliva is contact with said acidic aqueous to extract said at least one vitamin and/or said at least one micronutrient.
[032] The sub-step of extraction comprises:
[033] For vitamins, the support is extracted with an acidified water solution with
LCMS quality formic acid at 1% (v/v), then agitated with ultrasound for 10 minutes. The extract is filtered on a 0.2 m membrane before injection into the
LCMS.
[034] For micronutrients, the sample support is placed in a 0.2% nitric acid solution with Triton X100 and left mineralized for a few minutes.
[035] The method according to a second step comprises a Saliva Assay analysis step, said step being configured to identify and quantify the targeted compound(s).
[036] In one embodiment for micronutrients, the sample solution is then aspirated into an Active Coupled Plasma (ICP) Mass Spectrometer (MS) (ICP
MS 7800, Agilent). ICP MS uses inductively coupled plasma for superheating elements passing through the sample analysis chamber to destroy any molecular bonds and ionize residual atoms. This liquid - the sample - is formed as an aerosol by the application of a vacuum before meeting the plasma in preference to entraining air to form an aerosol in order to improve the signal to noise ratio. lons formed by exposure to plasma travel through a modulated quadrupole mass specification to collect isotopic ions associated with the trace element of interest.
[037] According to the present invention, the trace elements are detected as low or deficient when the target value determined either by literature study or by internal data is not reached.
[038] For the dosage of vitamins, the solution extracted from acidified water is filtered through a 0.2 um filter then injected into an LCSMTQ system (UHPLC 1690 coupled to an MS TQ 6570 Agilent) on a C18 column BEH Acquity Waters 50mm x 2 .1 mm with 1.9 um particles. The gradient is a binary gradient of acetonitrile and water with 5mM ammonium acetate.
[039] According to the present invention, the vitamins are detected as low or deficient when the target value determined either by bibliographic study or by internal data, is not reached.
Metals and vitamins screening:
[040] The detectable trace elements as targeted compounds according to the present invention are appreciated for being easily identified in a precise dosage by choosing for each the most widespread natural isotope by modulating the mass spectrometer accordingly.
[041] The trace elements sought according to the present invention include, by way of illustration, are manganese, copper, calcium, zinc, iron, iodine, chromium, selenium.
[042] The vitamins sought according to the present invention include, by way of illustration, are Vitamin B1, Vitamin B3, Vitamin, B5, Vitamin B6, Vitamin BS,
Vitamin B12, Vitamin A, Vitamin C, Vitamin D3 and Vitamin E.
[043] Other metals and vitamins could be included in research depending on the legislation on the supply of trace elements as food supplements.
[044] The following values of hydrophilic vitamins, are given as an example of the saliva concentration obtained in different patients:
[045] [Table 1]
Sample NIACINE | PYRIDOXINE | ASCORBIC | PANTO- | RIBO- | CYANO-
Results | Results ACID THENOIC | FLAVIN | COBALAMIN
Results ACID Results | Results
Results 2283.7 7.6451 3757.9 1391 [06 _ |237 143.7 4973.2 11652.8 | 1169.1 3130.6 281.3 213.2
[046] The following values of lipophilic vitamins, are given as an example of the saliva concentration obtained in different patients: [Table 2]
RETINOL Results TOCOPHEROL Results
Final Concentration ng/ml Final Concentration ng/ml
[047] The following values of trace elements, are given as an example of the saliva concentration obtained in different patients:
[048] [Table 3]
Isotop | 24 Mg | 52 Cr | 55 Mn | 56 59 63 66 Zn | 78 Se | 95 Mo e ug/L ug/L ug/L F C C |ug/L | ug/L ug/L e O u ug/L | ug/L | ug/L
Sampl | 6920.20 | 250.9 |69.53 | 799.9 | 3.433 | 283.2 | 494.2 | 1.925 | 2.304 el 2 49 2 4 7 22
Sampl | 3330.04 | 267.9 |37.90 | 1663. | 2.798 | 85.76 | 538.3 | 0.551 | 3.592 e2 6 62 8 5 1 13
[049] In one embodiment according to the invention, the results are obtained by first using a standard solution for calibration wherein said solution comprises the following traces elements:
[050] [Table 2]
Analyte |ug/mL Analyte |ug/mL Analyte | ug/mL
Ba j10 [se [10 [sr [10
Eu 10 |Gd |10 [B [10
Ga [10 Ho [10 [Na wo
Po 10 Jtu [10 [Th [10
Mg [10 [Mn |10 JU = [10
Nd 10 Ni [10 [yb [10
P 10 |K J10 [cs [10
Pr 10 Rp [10
[051] This standard solution is used as a calibration method verifying the precision of the results with a standard deviation of 2.5%.
[052] The calibration method uses a calibration graph obtained by preparing 3 solutions: -SF-M1 with 1 ug/L of standard solution (10 pl of standard solution with 100 mL of ultrapurewater, Triton, nitric acid at 0.2% (v/v) concentration). -SF-M2 with 50 ug/L of standard solution (500 ul of standard solution with 100 mL of ultrapurewater, Triton, nitric acid at 0.2% (v/v) concentration). -SF-M3 with 1000 ug/L of standard solution (1000 ul of standard solution with mL of ultrapurewater, Triton, nitric acid at 0.2% (v/v) concentration).
[053] The calibration solutions SF-M1, SF-M2 and SF-M3 are then used to prepare gradient solutions such as: -Level 1 Concentration o pg/L 1 ml dilution solution -Level 2 Concentration 0.001 pg/L 0.999mL dilution solution and 0.001 mL SF-
M1 -Level 3 Concentration 0.02 ug/L 0.980mL dilution solution and 0.02 mL SF-M1 -Level 4 Concentration 0.1 pg/L 0.90mL dilution solution and 0.10 mL SF-M1
-Level 5 Concentration 0.5 pg/L 0.990mL dilution solution and 0.010 mL SF-M2 -Level 6 Concentration 1 pg/L 0.980mL dilution solution and 0.020 mL SF-M2 -Level 7 Concentration 5 pg/L 0.90mL dilution solution and 0.10 mL SF-M2 -Level 8 Concentration 10 pg/L 0.990mL dilution solution and 0.010 mL SF-M3 -Level 9 Concentration 20 pg/L 0.980mL dilution solution and 0.020 mL SF-M3, and -Level 10 Concentration 100 pg/L 0.90mL dilution solution and 0.10 mL SF-M3.
[054] This solution gradient is then used to calibrate the method in order to ensure that the measurement based on a saliva sample is accurately measured.
[055] The present invention has utility as a noninvasive method of determining trace elements and vitamins levels within an individual subject. The present invention provides information about both trace elements and vitamins within an individual subject from saliva in contrast to prior art techniques that have relied on invasive sample collection techniques associated with collection of blood or tissue biopsy. The collection of saliva affords considerable advantages in requiring less skill and precaution in the collection of the sample as compared to blood or biopsy samples. The present invention in establishing a reliable correlation relation between deficiency status and saliva concentration of a trace elements and/or vitamin substance has overcome the inconsistencies associated with prior art saliva detection methodologies.
[056] As used herein a “subject' is defined to include a mammalian or avian creature specifically including a human, cow, horse, sheep, dog, cat, horse and chicken. While the collection of a saliva sample as detailed hereafter is in regard to a human Subject, it is appreciated that other saliva samples are readily obtained from nonhuman Subjects.
[057] Saliva collection is performed according to the present invention in any number of conventional procedures. Saliva is readily collected in a vial, with an absorbent swab, or pipetted from the buccal cavity onto an absorbent substrate.
The simplicity of collecting a saliva sample allows for an
[058] untrained individual to collect such a sample. Preferably, the sample is brought into contact with a preservative so as to maintain the saliva sample integrity during transport to a measurement facility.
[059] In an alternative embodiment, a kit is provided for saliva collection that includes a spherical swab which can be saturated with an exact volume of saliva. Example given are based of 0.3 milliliters of saliva onto the swab.
[060] Regardless of the method of saliva collection, the sample is placed in a bioSeal package and assigned a chain of custody number corresponding to the sample. The sample is collected in the form of an absorptive swab Strip with a predetermined quantity: as example give in tables 1, 2 and 3, the volume of saliva collected was 30 pl.
[061] The present invention is further detailed with respect to tie following nonlimiting examples. [Table 4]
[062] Example of vitamins and trace elements formula proposed for a patient with a deficiency in different elements in one tablet per day:
Vitamina |s0 lpg vitlaminD 5 lue
Vitamnk 20 lpg
Vitamin B1
Vitamin B2
Pantothenic acid
Vitamin B6
Folates 100 lpg
Vitamin B12 02 lpg
Botn 4 0 yg
Calcium |@00 Img 1800 ug QU jlodine J100 lpg
Selenium 20 |ug
Chromium 120 lpg
Molybdenum 100 mg
[063] This formula is adapted by an algorithm to allow a personalized intake of the nutriments.
Claims (14)
- [Claim 1] An in vitro method for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva obtained from a subject, said method comprising -Mixing said sample with a determined amount of an acidic aqueous solution and therefore obtaining an acidified sample ; -Detecting and quantifying said micronutrient in said acidified sample by injecting said acidified sample in an active coupled plasma mass spectrometer and/or -Detecting and quantifying said vitamin in said acidified sample and injecting said acidified sample in an ultra-high performance liquid chromatographer coupled with a triple quadrupole mass spectrometer.
- [Claim 2] The method according to claim 1 wherein said at least one vitamin is chosen among Vitamin B1, Vitamin B3, Vitamin, B5, Vitamin B6, Vitamin B8, Vitamin B12, Vitamin A, Vitamin C, Vitamin D3 and Vitamin E and said at least one micronutrient is chosen among manganese, copper, calcium, zinc, iron, iodine, chromium, selenium.
- [Claim 3] The method according to claim 1 or 2, wherein said sample of a determined amount of saliva is contained in a determined amount of a polymer able to absorb said determined amount of saliva.
- [Claim 4] The method according to claim 3, wherein said determined amount of said polymer forms a polymer ball.
- [Claim 5] The method according to any one of claims 3 to 4, wherein said determined amount of polymer containing said determined amount of saliva is contact with said acidic aqueous to extract said at least one vitamin and/or said at least one micronutrient.
- [Claim 6] The method according to anyone of claims 1 to 5, wherein said acidic aqueous solution is a 1 % (v/v) formic acid aqueous solution and wherein at least one vitamin is detected and quantified.
- [Claim 7] The method according to anyone of claims 1 to 5, wherein said acidic aqueous solution is a 0.2%(v/v) nitric acid solution with Triton X100 and wherein at least one micronutrient is detected and quantified.
- [Claim 8] The method according to any one of claims 1 to 7, wherein said acidified sample is filtered through a 0.2 um filter before injection into said an ultra-high performance liquid chromatographer.
- [Claim 9] The method according to any one of claims 1 to 8, wherein said ultra- high performance liquid chromatographer comprised a chromatography column packed with particles of 1.9 um diameter.
- [Claim 10] The method according to any one of claims 1 to 9, wherein a binary gradient of acetonitrile and water with 5MM ammonium acetate is used for column elution.
- [Claim 11] The method according to any one of claims 1 to 10, wherein said subject is a mammalian in particular chosen among cow, horse, sheep, dog and cat, and more particularly a human being or an avian in particular a chicken.
- [Claim 12] A kit for implementing the method according to any one of claims 1 to 11, comprising - a pipette or a swab ; and - a 1% (viv) aqueous formic acid solution ; and/or - a 0.2%(v/v) nitric acid solution with a surfactant able to permeate cell membrane, in particular an anionic surfactant of formula CsH17CsH4(OC2H4)9-100H.
- [Claim 13] A kit according to claim 12, wherein said swab comprises a determined amount of polymer able to absorb the determined amount of saliva.
- [Claim 14] A kit according to any one of claims 12 and 13 wherein said swab is a spherical swab.
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| LU504848A LU504848B1 (en) | 2023-08-02 | 2023-08-02 | in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva |
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| LU504848A LU504848B1 (en) | 2023-08-02 | 2023-08-02 | in vitro method and kit for detecting and quantifying at least one vitamin and/or at least one micronutrient contained in a sample of a determined amount of saliva |
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