WO2013010553A1 - Method and analytical kit for simultaneous quantification of b-complex vitamin content in food - Google Patents

Abstract

The present invention relates to an analytical vitamin analysis kit, the method of its preparation, and the method of its application. The given analytical kit can be used for the simultaneous determination of the quantity of five B-complex vitamins (B l, B2, B3, B5 and B6) and individual vitamers in drugs, food, food- and bio-supplements, feed, and other biological samples using LC-MS isotope internal standard assay. The analytical kit is based on the use of sample vials containing lyophilized or dried isotope labelled internal standards of the B-complex vitamers with a mix of hydrolytic enzymes, used to convert vitamers and cofactors into the same chemical forms as the used isotope labelled internal standards, and for their following quantification by means of LC-MS or LC-MS/MS.

Description

METHOD AND ANALYTICAL KIT FOR SIMULTANEOUS QUANTIFICATION OF B-COMPLEX VITAMIN CONTENT IN FOOD

Field of the invention

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.

Background of the invention

Simultaneous determination of B-complex vitamins in food and other biological samples is challenging due to their low concentration in the sample, relatively low stability as well as diverse chemical structure and physical properties of the individual vitamers. Liquid chromatography combined with mass or tandem mass spectrometry (LC-MS or LC-MS/MS) 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.

Quantitative mass spectrometry is complicated by matrix-associated ionization effects caused by compounds that co-elute from the LC at the same retention time as the compound under study and may significantly affect the intensity of its MS peaks. To compensate the matrix- associated ionization effects, stable isotope labelled (SIL) vitamers can be used as internal standards due to their nearly identical chemical and physical properties. Thus, SIL internal standards can be added to the sample and the concentration of the vitamer under study can be calculated based on the ratio of MS peak intensities of natural and SIL forms.

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.

The activity of 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. For example, 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.

Besides the simplest chemical forms of the B-complex vitamins B l (thiamine), B2 (riboflavin), B3 (nicotinic acid and nicotinamide), B5 (pantothenic acid) and B6 (pyridoxine and pyridoxal) and their phosphorylated and glycosylated forms there are several cofactors and associated metabolites in food which also may possess the activity of the given vitamins. In the human gastrointestinal tract (GIT) these cofactors are converted into the simplest forms of vitamers and absorbed in the small intestine. For example,, potential vitamin B2 sources (riboflavin precursors) 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).

Similarly, 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). 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).

An additional source of vitamin B5 (pantothenic acid) in food is Coenzyme A (CoA) which is hydrolyzed in the small intestine to pantetheine by non-specific pyrophophatase and phosphatase reactions. 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).

Additionally, 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.

In order to express total B-complex vitamin content in food it is important to quantitatively determine all respective B-complex vitamers and their precursors which are relevant from a nutritional point of view. The individual determination of each and every compound with B- complex vitamin activity is tedious and time-consuming. To resolve this issue the relevant vitamers could be converted into the simplest chemical forms of their respective vitamins (e.g., thiamine in case of vitamin B l, riboflavin in case of vitamin B2, nicotinamide and nicotinic acid in case of vitamin B3, pantothenic acid in case of vitamin B5 and pyridoxal or pyridoxine in case of vitamin B6) using enzymes with appropriate specificity and activity. 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).

In the analysis of the nutritional value of food, the availability of user- friendly and high- throughput analytical methods is becoming increasingly important. Hence, there is an obvious need for method and supporting analytical kit allowing one to simultaneously (i.e. in the same extraction and chromatographic run) determine the content of different B-complex vitamins.

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. At the same time, 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.

Summary of the invention

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. For simultaneous quantitative determination of the given five B-complex vitamins, LC-MS (or LC-MS/MS) with stable isotope internal standard assay kit is used. In contrast to the conventional methods (microbiological or HPLC-based methods) 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.

Alternatively, the calibration mixture that contains only unlabelled B-complex vitamers can be used for calibration. In this case, 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.

After capping the vials under an inert gas atmosphere, the sample and calibration vials, described above, 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 (the method and analytical kit) 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.

The main differences between the inventions described in the "background of the invention" section are:

- the 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 β-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.

- for quantification, 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;

- 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. μηιοΐ in 1 g of sample, μg in 1 gram of sample etc).

Brief description of the drawings

The present invention is illustrated with drawings, where:

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 (^l2C and Ή) 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.

Detailed description of the invention

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:

- a fixed amount of the selected isotope labelled internal standards of B-complex vitamers with and without a mixture of hydrolytic enzymes or enzyme preparations with specific activities is dried or lyophilized into sample vials;

- in the sample vial with enzyme mixture, the vitamers and cofactors in the sample, which possess the B-complex vitamins activity, 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; - in addition to the sample vials described above, calibration vials containing the same dried or lyophilized unlabelled vitamers standard mixture at different concentrations is included in the analysis kit;

- to determine the concentration of vitamers in the sample a calibration curve is generated by using calibration vials. Alternatively, sample vials can be used, wherein only unlabelled

(natural abundance) B-complex vitamers (meaning calibration mixture) at different concentrations are added.

- the content of 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 μg of thiamine in 1 g of sample etc.);

- alternatively, 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 μιη, 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.

To generate a calibration curve, 1 ml of 0.05 M ammonium formate buffer (pH 4.5) is added into each vial in the set of 2 ml calibration vials (containing different amounts of unlabelled vitamers), the contents of the vials are mixed thoroughly, and incubated with frequent shaking at 37 °C for 18 h in the dark and analysed by LC/MS. Vitamers are determined using ACQUITY UPLC^® liquid chromatograph (Waters, Milford, MA, USA), which consists of an autosampler and gradient pumps. The liquid chromatograph instrument is coupled with an LCT Premier™ 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.

Vitamers are chromatographically separated using a reversed phase chromatography column ACQUITY UPLC HSS C- 18 1.8 μιη (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 μΐ 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 (¹³C, 2H and ¹⁵N) and unlabeled (natural abundance) of B-complex vitamers being identified.

Vitamin Rt, min m/z

Thiamine 1.57 265.1 1

Thiamine [¹³C₃] 1.61 268.12

Riboflavin 9.95 377.15

Riboflavin [¹³C₄,¹⁵N₂] 9.95 383.16

Nicotinamide 3.60 123.06

Nicotinamide [²H₄] 3.47 127.08

Nicotinic acid 3.09 124.04

Nicotinic acid [²H₄] 3.00 128.06

Pantothenic acid 7.82 220.12

Pantothenic acid [¹³C₃, ¹⁵N] 7.82 224.13 Pyridoxal 3.56 168.07

Pyridoxal [²H₃] 3.55 171.09

Pyridoxine 5.61 170.08

Pyridoxine [^l3C₄] 5.60 174.10

The concentration of each vitamin in the sample is calculated using a seven-point external calibration curve (0.05 - 3.75 μΜ in the case of thiamine, 0.03 - 2.48 μΜ in the case of riboflavin, 0.10 - 7.66 μΜ in the case of nicotinamide, 0.10 -7.63 μΜ in the case of nicotinic acid, 0.05 - 4.16 μΜ in the case of pantothenic acid, 0.07 - 5.68 μΜ in the case of pyridoxal, and 0.07 - 5.40 μΜ in the case of pyridoxine). The concentration of labelled internal standard is 1.13 μΜ in the case of thiamine, 0.53 μΜ in the case of riboflavin, 4.10 μΜ in the case of nicotinamide, 3.28 μΜ in the case of nicotinic acid, 0.91 μΜ in the case of pantothenic acid, 1.20 μΜ in the case of pyridoxal, and 1.18 μΜ 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 coefficients of linear regression are as follows: r² = 0.9999 (y = 0.7545x + 0.0105) for thiamine, r² = 0.9999 (y = 2.3417x + 0.0123) for riboflavin, r² = 0.9995 (y = 0.2068x + 0.0106) for nicotinamide, r² = 0.9998 (y = 0.3568x - 0.0082) for nicotinic acid, r² = 0.9999 (y = 1.1862x + 0.0047) for pantothenic acid, r² = 0.9993 (y = 0.9202x + 0.033) for pyridoxal, r² = 0.9999 (y = 1.0826x - 0.007) for pyridoxine.

The concentration of vitamers is calculated by the ratio of corresponding spectral lines in the sample and calibration vial using formula 1 nr — ^ std ^ ^lstd smpl

^ smpi i 2 . 13 · (1), where,

std smpl l²C_smpi - concentration of vitamer in sample vial

C_std - concentration of unlabeled vitamer standard in the calibration vial

I²istd - signal intensity of unlabelled vitamer standard in the calibration vial;

1³ id - signal intensity of isotope labelled vitamer standard in the calibration vial ^{l 2} mpi - signal intensity of vitamer in the sample vial;

¹³i_Smpi - signal intensity of isotope labelled internal standard in the extraction vial.

To calculate the content (B) of a specific vitamin, for example vitamin B l activity in the sample ^g of thiamine in 1 g of sample), the following equation is used:

msmpl where,

l²CBi_smpi is the concentration of respective vitamer possessing vitamin Bl activity in the extraction solution calculated from calibration curve, μΜ;

m_smpi 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.

Results obtained by using extraction vials with and without enzyme preparations are shown in Table 2.

Table 2. Vitamer concentrations and the respective B-complex vitamin content in inactive dried yeast (mean ± S.D., n = 3), by using extraction vials without enzyme preparation (A) and with enzyme preparation (B).

Vitamin A, μ g^"1 B, μ g^'1

Thiamine 34.2 ± 1.7 47.2 ± 1.7

B l (as thiamine) 47.2 ± 1.7

Riboflavin 12.3 ± 0.4 40.4 ± 0.5

B2 (as riboflavin) 40.4 ± 0.5

Nicotinamide 130.1 ± 2.3 149.7 ± 1 1.2

Nicotinic acid 1 10.9 ± 1.4 117.2 ± 3.3 B3 (as nicotinic acid) , 265.7 ± 14.5

Pantothenic acid 140.3 ± 4.1 130.1 ± 7.3

B5 (as pantothenic acid) 140.3 ± 4.1 130.1 ± 7.3

Pyridoxal 2.00 ± 0.26 3.07 ± 0.17

Pyridoxine 1.23 ± 0.03 1.33 ± 0.01

B6 (as pyridoxine) 4.36 ± 0.18

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 μηι, 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.

To generate a calibration curve, 1 ml of 0.05 M ammonium formate buffer (pH 4.5) is added into each vial in the set of 2 ml calibration vials (containing different concentrations of labelled and unlabelled vitamers), the contents of the vials are mixed thoroughly, and incubated with frequent shaking at 37 °C for 18 h in the dark and analysed by LC/MS. Vitamers are analysed and calculated as in Example 1.

Results are shown in Table 3.

Table 3. Vitamin concentrations in the SRM 1849.

Vitamin, mg/kg Our results³ Certified Concentration

Thiamine (expressed as

thiamine CI HC1) 15.5 ± 0.3 15.8 ±1.3

Riboflavin 17.2 ± 0.3 17.4 ± 1.0

Nicotinamide 99.7± 1.9 97.5 ± 2.3 Pantothenic acid 63.9 ± 0.9 64.8 ± 2.2

Pyridoxine (expressed as

pyridoxine HC1) 14.2 ± 0.2 14.2 ± 1.5

^a 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 expanded uncertainty is calculated as U=k*u_c, where u_c is the uncertainty of the measurement results and k is a coverage factor (k=2).

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.

As used herein, a "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.

As used herein, 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 N₂ 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. Preferably, 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. As used herein, 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 N₂ 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.

thiamine, TMP, TDP, TTP and ATDP 0.002 - 200 nmol;

riboflavin, FMN, and FAD 0.01 - 150 nmol;

· 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;

pyridoxine, pyridoxal, pyridoxamine, PNP, PLP, and PMP 0.01 - 300 nmol.

As used herein, 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 dinucleotide phosphate reduced (NADPH) and/or N-ribosyl nicotinamide and/or nicotinic acid adenine dinucleotide (NAAD) and/or nicotinic acid adenine dinucleotide phosphate (NAADP) and/or N-ribosyl nicotinic acid; B5: pantothenic acid and/or Coenzyme A (CoA) and/or 4- phosphopantetheine and/or pantetheine; B6: pyridoxine and/or pyridoxal and/or pyridoxamine and/or pyridoxine phosphate (PNP) and/or pyridoxal phosphate (PLP) and/or pyridoxamine phosphate (PMP).

As used herein, an "isotope label" refers generally to ¹³C, ¹⁵N, ²H 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:

• thiamine, TMP, TDP, TTP and ATDP 0.002 - 200 nmol;

• riboflavin, FMN, and FAD 0.01 - 150 nmol;

• 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;

• pyridoxine, pyridoxal, pyridoxatnine, PNP, PLP, and P P 0.01 - 300 nmol.

As used herein, "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 β-glucosidase (0.1 - 2 000 U/vial) activities. Selection of enzymes can depend on the sample.

As used herein, the "activity of 1 U of acid phosphatase" refers to the activity that is required to liberate 1 μηιοΐβ of phosphate from /?-nitrophenyl phosphate at pH 4.8 at 37 °C per 1 min.

As used herein, the "activity of 1 U of acid pyrophosphatase" refers to the activity that is required to liberate 1 μιηοΐε of phosphate from 7-nitrophenyl phosphate at pH 4.8 at 37 °C per 1 min.

As used herein, the "activity of 1 U of a-amylase" refers to the activity that is required to liberate 1 μιηοΐε of maltose from starch at pH 6.0 at 25 °C per 1 min.

As used herein, the "activity of 1 U of β-glucosidase" refers to the activity that is required to liberate 1 μιηοΐε of glucose from salicin at pH 4.0 at 37 °C per 1 min.

References:

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Hachey, D. L., Coburn, S. P., Brown, L. T., Erbelding, W. F., DeMark, B., & Klein, P. D. (1985). Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry. Analytical Biochemistry, 151, 1, 159-168.

Midttun, 0., Hustad, S., Solheim, E., Schneede, J., & Ueland, P. M. (2005). Multianalyte Quantification of Vitamin B6 and B2 Species in the Nanomolar Range in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry. Clinical Chemistry, 51 , 7, 1206-1216.

Ndaw, S., Bergaentzle, M, Aoude- Werner, D., & Hasselmann, C. (2000). Extraction procedures for the liquid chromatographic determination of thiamin, riboflavin and vitamin B6 in foodstuffs. Food Chemistry, 71, 129-138. Jakobsen, J. (2008). Optimisation of the determination of thiamin, 2-(l-hydroxyethyl)thiamin, and riboflavin in food samples by use of HPLC. Food Chemistry, 106, 1209-1217.

Daniel, H., Binninger, E. & Rehner, G. (1983). Hydrolysis of FMN and FAD by alkaline phosphatase of the intestinal brush-border membrane. International Journal of Vitamin and Nutrition Research, 53, 109-1 14.

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Claims

1. A method for simultaneous quantification of B-complex vitamins in drugs, food, food- and bio-supplements, feed, and other biological samples using LC-MS or LC -MS/MS and isotope labelled internal standard method, comprising:

- simultaneous bottling of a known amount of isotope labelled B-complex vitamers possessing vitamin B l, B2, B3, B5 and B6 activity into sample and calibration vials;

- bottling of a known concentration of the same unlabelled vitamers into the calibration vials;

- simultaneous bottling of a mixture of enzyme preparation, which may possess either acid phosphatase, pyrophosphatase, a-amylase, and β-glucosidase activity or all these activities into the sample and calibration vials, lyophilisation and hermetic enclosure of sample vials and calibration vials under oxygen deprived conditions;

- incubation of acidic extract of a sample under study in a sample vial to convert all the containing vitamers into the same forms as the isotope labelled internal standards used;

- calibrating the LC-MS system with calibration vials or a calibration mixture, which are incubated under the same conditions as the sample extract;

- determination of the content of B-complex vitamins as the sum of the molar concentrations of their respective vitamers.

2. The method of claim 1, wherein said the content of at least five B-complex vitamins is determined simultaneously.

3. The method of claim 2, wherein said the content of vitamins B l, B2, B3 B5, B6 is determined.

4. The method of claim 1, wherein said the mixture of isotope labelled vitamers, internal standards, and/or a mixture of enzymes or enzyme preparations with specific hydrolytic activities, are lyophilized or dried simultaneously into the sample vials.

5. The method of claim 1 , wherein said the enzyme preparations with hydrolytic activities is lyophilized or dried into the sample vials.

6. The method of claim 1, wherein said the pH of the said sample is in the range of 2.0 - 5.0.

7. The method of claim 6, wherein said the pH of the said sample is preferably pH 4.5.

8. The method of claim 1, wherein said the sample extract is incubated for 2 - 24 h within the temperature range of 20 - 50 °C.

9. The method of claim 8, wherein said the sample extract is incubated, preferably for 18 h at 37 °C.

10. The method of claim 1, wherein said the concentration of vitamers in the said sample is determined by LC-MS using a calibration mixture, which is added to the sample vials instead of a sample with an equal volume of the sample extract.

11. The method of claim 1, wherein said the mixture of isotope labelled vitamers as internal standards, and unlabelled vitamers, and a mixture of enzymes or enzyme preparations with specific hydrolytic activities, are lyophilized or dried simultaneously into the calibration vials.

12. The method of claim 1, wherein said a mixture of enzymes or enzyme preparations with specific hydrolytic activities in the calibration vials are lyophilized or dried simultaneously.

13. The method of claim 1, wherein said the concentration of vitamers in the internal standard is brought to the same value in the sample and in the calibration vials.

14. The method of claim 1, wherein said a known amounts of isotope labelled vitamers is bottled into sample and calibration vials of the analysis kit and lyophilized or dried.

15. The method of claim 1 , wherein, instead of vitamers, said a known amounts of isotope labelled biomass or its extract can be lyophilized or dried into the sample and calibration vials.

16. An analytical kit for simultaneous quantification of the content of five B-complex vitamins (Bi, B₂, B₃, B₅ and B₆) in vitamin containing drugs, food, food- and bio-supplements, feed, and other biological samples comprising of a pair of calibration and sample vials, which contain an equal amount of isotope labelled vitamers.