Classifications

• • 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
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • 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
  • G01N33/02—Food

Definitions

• the present invention concerns the method and the respective analytical kit for quantitative nutritional composition analysis, and more specifically, for the simultaneous determination of the content of B-complex vitamins B l , B2, B3, B5 and B6 in food, food- and bio-supplements and feed.
• Liquid chromatography combined with mass or tandem mass spectrometry are widely used analysis techniques for the determination of water soluble vitamins.
• the vitamins are extracted from the sample along with other low molecular weight compounds and separated by LC according to their physico-chemical properties, followed by detection with MS or MS/MS using the mass to charge ratios of ionized vitamers. Each vitamer is quantified using its respective MS peak intensity.
• Leporati et al. (2005) discloses the use of LC-MS/MS to determine the simplest vitamers of six B-complex vitamins (thiamine, riboflavin, pyridoxine, nicotinamide, nicotinic acid, pantothenic acid, and folic acid) in pasta products by using various extraction methods: hydrochoric acid extraction for thiamine, riboflavin, pyridoxine, nicotinamide, nicotinic acid, and acetic acid extraction for pantothenic acid, and sodium phosphate-sodium citrate/ascorbate extraction (pH 8) for folic acid.
• SIL stable isotope labelled
• Hachey et al. (1985) and Midttun et al. (2005) disclose the quantification of vitamin B2 and B6 related vitamers (riboflavin, pyridoxal, pyridoxine, pyridoxamine, pyridoxal phosphate, pyridoxine phosphate, and pyridoxamine phosphate) by LC-MS/MS and SIL standards in liver and urine and in human blood plasma, respectively. Sample extraction was performed with trichloroacetic acid that contained a known amount of SIL internal standards of these vitamers and the concentration was calculated based on the ratio of MS peak intensities of natural and SIL forms.
• vitamin B2 and B6 related vitamers riboflavin, pyridoxal, pyridoxine, pyridoxamine, pyridoxal phosphate, pyridoxine phosphate, and pyridoxamine phosphate
• each B-complex vitamin is defined as a sum of concentration of different chemical substances (vitamers), which all have a similar biological activity of that particular vitamin.
• bioactive forms of vitamin B l and B6 in food are their phosphorylated forms, thiamine mono-, di- and triphosphates and pyridoxal, pyridoxine and pyridoxamine phosphates, respectively.
• Ndaw et al. (2000) and Jakobsen (2008) used enzymatic extraction to liberate vitamers from a food matrix and to dephosphorylate them.
• USA patent application No. US20090093009 discloses an application which limits the use of acid phosphatase to liberate different phosphorylated forms of thiamine into free (i.e. non-phosphorylated) thiamine.
• B-complex vitamins B l thiamine
• B2 riboflavin
• B3 nicotinic acid and nicotinamide
• B5 pantothenic acid
• B6 pyridoxine and pyridoxal
• GIT human gastrointestinal tract
• potential vitamin B2 sources in food are flavin coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are hydrolyzed in the small intestine by alkaline phosphatase (EC 3.1.3.1.) into riboflavin and then absorbed (Daniel et al. 1983).
• FMN flavin mononucleotide
• FAD flavin adenine dinucleotide
• potential vitamin B3 (nicotinic acid and nicotinamide) sources in food are coenzymes nicotinamide adenine dinucleotides (NAD, NADH, NADP, and NADPH), which are converted to nicotinamide mononucleotide and adenosine monophosphate by NAD pyrophosphatase (EC 3.6.1.22) found in small intestine juice secretes (and also, to a small extent, in pancreatic juice).
• NAD nicotinamide adenine dinucleotides
• NADP NADP
• NADPH NAD pyrophosphatase
• Nicotinamide mononucleotide is further hydrolyzed to nicotinamide riboside and then further to free nicotinamide by enzymes associated with the epithelial cells of the small intestine (Gross and Henderson, 1983).
• CoA Coenzyme A
• Pantetheine is hydrolyzed to free pantothenic acid by pantetheinase, an enzyme secreted by the gastrointestinal mucosa, and then absorbed in the jejunum part of the small intestine (Shibata et al. 1983).
• B-complex vitamers may be glycosylated (e.g., vitamers which possess vitamin B6 activity) or bound to proteins (e.g. vitamers which possess vitamin B2 activity) in food (Gregory 3rd, 1998; White III et al, 1986). Protein bound vitamers dissociate in the stomach due to low pH caused by hydrochloric acid.
• the resulting primary forms of these B-complex vitamins can be simultaneously quantified using LC- MS/(MS).
• the method described in the present invention where quantitative determination of B-complex vitamins is based on the sum of molar concentrations of their respective vitamers is unique and has not been previously disclosed in the research and patent literature.
• This method has significant advantages over conventional methods (e.g. HPLC or microbiological methods) that are currently applied for quantification of B-complex vitamins. For example, physiological differences between bacteria used in the microbiological assay and the human organism can cause over- or underestimation of some vitamin activities (Bender, 2003).
• the proposed analytical kit which supports such simultaneous quantification of B-complex vitamins contains: i) sample vials containing known quantities of lyophilized or dried isotope labelled vitamers and/or enzyme preparations, ii) calibration vials, containing, in addition to the extraction vial content, known amounts of the same unlabeled vitamers at different concentrations or alternatively iii) calibration mixture vials containing known amounts of unlabeled vitamers.
• UK patent application GB141 1381 discloses a method of making an assay kit comprising vials arranged for quantitative determination of a compound in a sample to be added to a vial wherein each vial contains a standard amount of a lyophilized labelled (radioactively or stable isotope labelled) version of the compound to be determined and a standard amount of a lyophilized specific reagent to react (for example to form a complex) with the compound to be determined.
• An assay kit may also comprise vials which are intended for use as standards, which contain, in addition to the lyophilized specific reagents and labelled standards, also a known amount of the lyophilized unlabelled version of the compound to be determined.
• the amount of a specific reagent in the vial is set to be insufficient to react with all labelled and unlabelled compound. Assuming that a specific reagent reacts with the labelled and unlabelled (natural) compound in stoichiometrically equivalent amounts, it is possible to determine the concentration of unreacted labelled compound in the sample, and, in addition, calculate the concentration of unlabeled (natural) compound in the sample.
• This invention discloses an example which is based on immunoanalysis, wherein lyophilized reagents in the vial are antibodies which form complexes with compounds of interest.
• the present invention relates to the method and analytical kit, which can be used for the simultaneous determination of B-complex vitamin (B l, B2, B3, B5 and B6) content in food, food- and bio-supplements, and feed.
• B-complex vitamin B l, B2, B3, B5 and B6
• LC-MS or LC-MS/MS
• stable isotope internal standard assay kit is used for simultaneous quantitative determination of the given five B-complex vitamins.
• the present invention considerably simplifies the determination procedure, increases the accuracy of the analysis, and allows one to reduce the analysis time and labour.
• the analytical kit described in the present invention consists of one or more sample vials and one or more calibration vials.
• the sample vials contain a known amount of appropriate lyophilized stable isotope labelled B-complex vitamers with and without lyophilized mixtures of hydrolytic enzymes or enzyme preparations with specific activities.
• the calibration vials contain a known amount of the same lyophilized stable isotope labelled B-complex vitamers with or without a mixture of lyophilized hydrolytic enzymes or enzyme preparations with specific activities and a known amount of the same unlabelled (natural abundance) B-complex vitamers.
• Sample vials which contain lyophilized stable isotope labelled internal standards of the B- complex vitamers with hydrolytic enzymes or enzyme preparations with specific hydrolytic activities, are used to convert the B-complex vitamers and their respective cofactors into the same chemical forms as the stable isotope labelled internal standards used.
• Sample vials which do not contain an enzyme mixture, are used for the quantification of vitamers, which are already in the same chemical forms as the used internal standards, for example for fortified food analysis.
• Calibration vials which contain a mixture of known amounts of isotope labelled B-complex vitamers with and without enzymes, and known amounts of unlabeled (natural abundance) B-complex vitamers at different concentrations, and/or a mixture of hydrolytic enzymes or enzyme preparations with a specific activities, are used to generate a calibration curve, when an extraction buffer of an equal volume to that of the sample extract is added to the calibration vials.
• the calibration mixture that contains only unlabelled B-complex vitamers can be used for calibration.
• their contents are dissolved in an extraction buffer of an equal volume to that of the sample extract and added to the sample vials instead of the sample extract.
• the content of B-complex vitamins (B l, B2, B3, B5 and B6) in the sample is determined as the sum of the molar concentration of the respective individual vitamers.
• Sample vials of the present analytical kit are prepared by dispensing a fixed volume of a solution of appropriate isotope labelled B-complex vitamers of a known concentration with and without a mixture of hydrolytic enzymes or enzyme preparations with specific activities under oxygen-deprived conditions (i.e., under an inert gas atmosphere) into appropriate vials and by lyophilizing the contents of the vial.
• Calibration vials of the present analytical kit are prepared by dispensing a fixed volume of a solution of the same isotope labelled B-complex vitamers and the same unlabeled B-complex vitamers at different concentrations and/or a mixture of hydrolytic enzymes or enzyme preparations with specific activities under oxygen-deprived conditions into appropriate vials and by lyophilizing the contents of the vial.
• the sample and calibration vials are usable for at least one year for quantitative analysis of five B-complex vitamins (Bl, B2, B3, B5 and B6) in food, food- and biosupplements, or in feed using LC-MS (or LC -MS/MS).
• the present invention allows one to reduce the analysis time and labour used for sample preparation and analysis, allows one to conduct high throughput quantitative analysis of at least five B-complex vitamins (B l, B2, B3, B5 and B6) as well as folic acid and vitamin C in drugs, food, food- and bio-supplements, feed, and other biological samples.
• B-complex vitamins B l, B2, B3, B5 and B6
• sample and calibration vials contain solid substances with a long shelf life
• the sample vials of the present analytical kit contain isotope labelled internal standards of B- complex vitamers and/or hydrolytic enzymes or enzyme preparations, which do not chemically change themselves during the analysis process and act as catalysts (in case of enzymes) by liberating B-complex vitamers contain in the sample from phosphorylated, glycolylated and protein bound complexes and by converting them into the same chemical forms as the internal standards used.
• the mixture of enzymes or enzyme preparations possesses protease and/or a-amylase and/or acid phosphatase and/or acid pyrophosphatase and/or ⁇ -glucosidase activity and does not possess any activities, which may decrease B-complex vitamers activity and/or interfere with LC-MS (or LC-MS/MS) determination.
• a homogenised sample is added into a sample vial and for calibration the same amount of extraction buffer is added to the calibration vials, or alternatively, a calibration mixture containing unlabeled vitamer standards at different concentrations is added to another sample vial, samples and calibration solutions are incubated on a shaker, filtered, and the concentration of vitamers is determined by the LC-MS isotope internal standard method;
• B l absolute activity of five B-complex vitamins (B l, B2, B3, B5 and B6) is calculated by summarizing the molar concentrations of the respective vitamers and recalculated into respective vitamin activity units (e.g. ⁇ in 1 g of sample, ⁇ g in 1 gram of sample etc).
• Fig. 1 shows a principal scheme of the method of the present invention
• Fig. 2 - 6 show enzymatic conversion reactions of vitamers with B l, B2, B3, B5, and B6 activities;
• Fig. 7 shows an example of MS chromatograms of unlabelled ( l2 C and ⁇ ) and labelled ( 1 C or D) B-complex vitamers (left) and the respective MS spectra (right);
• Fig. 8 shows the stability of isotope labelled internal standards in the sample vials over a period of one year of storage under subdued lighting conditions at -20 °C.
• the present invention relates to the method and to an appropriate analytical kit used for the simultaneous determination of the content of five B-complex vitamins (B l, B2, B3, B5 and B6) in food, food- and bio-supplements, and feed by LC-MS (or LC-MS/MS) isotope internal standard assay.
• the method is comprised of the following steps:
• the vitamers and cofactors in the sample are converted into the same chemical forms as the respective stable isotope labelled internal standards during a sample incubation of between 2 - 24 h, preferably 18 h at a temperature of between 20 - 50 °C, preferably 37 °C, under acidic conditions where the pH is between 2.0 - 5.0, preferably 4.5, to prevent microbial growth;
• calibration vials containing the same dried or lyophilized unlabelled vitamers standard mixture at different concentrations is included in the analysis kit;
• a calibration curve is generated by using calibration vials.
• sample vials can be used, wherein only unlabelled
• B-complex vitamers meaning calibration mixture
• B-complex vitamins is calculated as the sum of the molar concentrations of the vitamers, which possess the respective vitamin activity, and is expressed in appropriate concentration units (for example ⁇ g of thiamine in 1 g of sample etc.);
• the content of one sample vial can be used for more than one sample analysis, by adding a known amount of the vial's content, which is previously dissolved with an extraction buffer, to several samples.
• the contents of calibration vials can be used in the same way.
• EXAMPLE 1 Determination of vitamins Bl , B2, B3, B5 and B6 content in inactive dried yeast.
• 0.15 g of inactive dried yeast is weighed into a 10 ml sample vial with enzymes, 0.15 g of inactive dried yeast is weighted into a 10 ml sample vial without enzymes, 10 ml of 0.05 M ammonium formate buffer (pH 4.5) is added into the vials, the contents of the vials are mixed thoroughly, and incubated with frequent shaking at 37 °C for 18 h in the dark. Following this, 2 ml from each of the incubation mixtures is centrifuged (14000 rpm/min) for 5 minutes at 10 °C and filtered (Millex-LG 13mm Philic PTFE 0.2 ⁇ , Millipore, Carrigtwohill, Ireland). 1 ml of this filtered extract is added into an empty 2 ml LC-MS vial and injected into the LC-MS system.
• 10 ml of 0.05 M ammonium formate buffer pH 4.5
• the liquid chromatograph instrument is coupled with an LCT PremierTM XE ESI TOF MS System (Waters, Milford, MA, USA), which consists of an electrospray ionization (ESI) source, a time-of-flight (TOF) mass analyzer, and a dual microchannel plate detector assembly detector.
• ESI electrospray ionization
• TOF time-of-flight
• Vitamers are chromatographically separated using a reversed phase chromatography column ACQUITY UPLC HSS C- 18 1.8 ⁇ (2.1 x 150 mm) (Waters, Milford, MA, USA) and two eluents (A: water + 0.1 % formic acid and B: acetonitrile + 0.1 % formic acid) applied in the gradient mode as follows: 0 - 3 min 100% A; 3 - 8.5 min 80% A and 20% B; 8.5 - 10 min: 5% A and 95% B; 10 - 15 min 100% A.
• One analysis run time is 15 minutes, with an applied flow rate of 0.25 ml/min, a sample injection of 5 ⁇ and a column temperature of 25 °C.
• Mass spectrometry is carried out in a positive ionization mode using a capillary voltage of 2000 V, a cone voltage of 30 V, a temperature source of 120 °C, and a desolvation temperature of 300°C.
• Natural vitamers and their labelled forms are identified by retention times and mass to charge ratios (m/z) (Tabel 1, Fig 7). Data are collected and processed using Mass Lynx 4.0 software (Waters, Milford, MA, USA). Table 1. Retention times (Rt) and mass to charge ratios (m/z) of isotope labelled ( 13 C, 2H and 15 N) and unlabeled (natural abundance) of B-complex vitamers being identified.
• Nicotinic acid [ 2 H 4 ] 3.00 128.06
• Pantothenic acid [ 13 C 3 , 15 N] 7.82 224.13 Pyridoxal 3.56 168.07
• the concentration of each vitamin in the sample is calculated using a seven-point external calibration curve (0.05 - 3.75 ⁇ in the case of thiamine, 0.03 - 2.48 ⁇ in the case of riboflavin, 0.10 - 7.66 ⁇ in the case of nicotinamide, 0.10 -7.63 ⁇ in the case of nicotinic acid, 0.05 - 4.16 ⁇ in the case of pantothenic acid, 0.07 - 5.68 ⁇ in the case of pyridoxal, and 0.07 - 5.40 ⁇ in the case of pyridoxine).
• the concentration of labelled internal standard is 1.13 ⁇ in the case of thiamine, 0.53 ⁇ in the case of riboflavin, 4.10 ⁇ in the case of nicotinamide, 3.28 ⁇ in the case of nicotinic acid, 0.91 ⁇ in the case of pantothenic acid, 1.20 ⁇ in the case of pyridoxal, and 1.18 ⁇ in the case of pyridoxine.
• the calibration curves are composed by relating each concentration of unlabelled external standard to its relative response factor as determined by the ratio of the peak intensity of the respective unlabelled external standard to that of the corresponding labelled internal standard.
• the concentration of vitamers is calculated by the ratio of corresponding spectral lines in the sample and calibration vial using formula 1 nr — ⁇ std ⁇ l std smpl
• I 2 istd - signal intensity of unlabelled vitamer standard in the calibration vial
• l 2 CBi_smpi is the concentration of respective vitamer possessing vitamin Bl activity in the extraction solution calculated from calibration curve, ⁇ ;
• m smp i is the sample weight in the sample vial, g;
• V is the volume of extraction solution in the sample vial, L;
• M B is the molecular weight of the vitamer used to express the results (for example in case of vitamin Bl, molecular weight of thiamine is used), Da.
• Vitamin A ⁇ g "1 B, ⁇ g '1
• Nicotinic acid 1 10.9 ⁇ 1.4 117.2 ⁇ 3.3 B3 (as nicotinic acid) , 265.7 ⁇ 14.5
• EXAMPLE 2 Determination of thiamine, riboflavin, nicotinamide, pantothenic acid, and pyridoxine content in the reference sample SRM 1849 (Infant/Adult Nutritional Formula).
• 0.1 g of reference sample SRM 1849 is weighed into a 20 mi sample vial without enzymes, 20 ml of 0.05 M ammonium formate buffer (pH 4.5) is added, the content of the vial is mixed thoroughly, 2 ml of the incubation mixture is centrifuged (14000 rpm/min) for 5 minutes at 10 °C and filtered (Millex-LG 13mm Philic PTFE 0.2 ⁇ , Millipore, Carrigtwohill, Ireland). 1 ml of this filtered extract is added into an empty 2 ml LC-MS vial and injected into the LC-MS system.
• Results are expressed as a mass fraction for the material as received and as the mean of at least 30 measurements on five different days over one month.
• the uncertainties are expressed as an expanded uncertainty, U.
• the present invention relates to the method and analytical kit, which is comprised of both calibration and sample vials containing a known amount of lyophilized isotope labelled and unlabelled vitamer standards.
• sample vial refers to a vial, in a size of 0.5 - 50 ml, made from glass or plastic or any other material, impermeable to UV-light and oxygen, which is hermetically capped in an inert gas atmosphere, and which contains at least one dried or lyophilized isotope labelled vitamer with a vitamin B l, B2, B3, B5, and B6 activity, respectively, and/or a lyophilized or dried enzyme or enzyme preparation or mixture of enzymes with specific hydrolytic activities.
• the sample vials may be used as extraction vials (preferable size 20 ml) in which extraction of sample is carried out or as vials (preferably vials of an LC autosampler) into which clear pre-extracted and filtrated sample is added.
• a “calibration vial” refers to a vial, in a size of 0.5 - 50 ml, made from class or plastic, with UV- and oxygen protection, which is hermetically closed in the N 2 or some other inert gas atmosphere, and which contains the same lyophilized or dried isotope labelled vitamers as are containing in the sample vial and also lyophilized or dried unlabelled vitamers and/or a lyophilized or dried enzyme or enzyme preparation or enzyme (enzyme preparations) mixture with specific hydrolytic activities.
• the quantity and number of enzyme or enzyme preparation or enzyme (enzyme preparations) mixture and isotope labelled vitamers are identical in sample and calibration vials.
• a “calibration mixture” refers to a vial, in a size of 0.5 - 50 ml, made from class or plastic, with UV- and oxygen protection, which is hermetically closed in the N 2 or some other inert gas atmosphere, and which contains the same lyophilized or dried unlabelled vitamers with a vitamin B l, B2, B3, B5, and B6 activity as are in the sample vial.
• nicotinamide nicotinic acid, NAD, NADH, NADP, NADPH, N-ribosyl nicotinamide, NAAD, NAADP, and N-ribosyl nicotinic acid 0.04 - 400 nmol;
• pantothenic acid, CoA, 4-phosphopantetheine, and pantetheine 0.01 - 200 nmol;
• an "isotope labelled B-complex vitamer standards" refer to the following isotope labelled compounds (Fig 2 - 6): Bl : thiamine and/or thiamine monophosphate (TMP) and/or thiamine diphosphate (TDP) and/or thiamine triphosphate (TTP) and/or adenosine thiamine diphosphate (ATDP); B2: riboflavin and/or flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD); B3: nicotinamide and/or nicotinic acid and/or nicotinamide adenine dinucleotide (NAD) and/or nicotinamide adenine dinucleotide reduced (NADH) and/or nicotinamide adenine dinucleotide phosphate (NADP) and/or nicotinamide adenine din
• an “isotope label” refers generally to 13 C, 15 N, 2 H and other stable isotopes that contain at least two atomic positions in the respective isotope labelled B-complex vitamer standard.
• the amount of internal standard in the sample vial depends on the sample of interest and the size of the vial, but remains within the following range:
• enzymes with hydrolytic activities refer to enzymes or enzyme preparations that possess any combination of acid phosphatase (0.1 - 2 000 U/vial), acid pyrophosphatase (0.1 - 2000 U/vial), a-amylase (0.1- 7 500 U/vial), and ⁇ -glucosidase (0.1 - 2 000 U/vial) activities. Selection of enzymes can depend on the sample.
• the "activity of 1 U of acid phosphatase” refers to the activity that is required to liberate 1 ⁇ of phosphate from /?-nitrophenyl phosphate at pH 4.8 at 37 °C per 1 min.
• the "activity of 1 U of acid pyrophosphatase” refers to the activity that is required to liberate 1 ⁇ of phosphate from 7-nitrophenyl phosphate at pH 4.8 at 37 °C per 1 min.
• the "activity of 1 U of a-amylase” refers to the activity that is required to liberate 1 ⁇ of maltose from starch at pH 6.0 at 25 °C per 1 min.
• the "activity of 1 U of ⁇ -glucosidase” refers to the activity that is required to liberate 1 ⁇ of glucose from salicin at pH 4.0 at 37 °C per 1 min.

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