ES2385590B1 - METHOD OF DETERMINATION OF NUCLEOTIDES IN FOODS BY LIQUID CHROMATOGRAPHY OF IONIC COUPLE WITH PHOTODY DETECTION COUPLED TO MASS SPECTROMETRY. - Google Patents

Abstract

Method of determination of nucleotides in food by liquid chromatography of ionic pairs with detection by photodiodes coupled to mass spectrometry of flight time with dual ionization operating in negative mode (HPLC / ESI-APCI-TOF-MS). The method allows the rapid determination of the five nucleotide monophosphates authorized in baby foods (citidine 5? -Monophosphate, uridine 5? -Monophosphate, adenosine 5? -Monophosphate, inosine 5? -Monophosphate and guanosine 5? -Monophosphate), and comprises prior precipitation of food proteins and direct analysis of nucleotides in the resulting extract by HPLC of ionic pairs using isocratic elution. The recovery obtained for fortified samples was satisfactory for all analytes. The proposed procedure allows the determination of the only authorized nucleotides and has been successfully applied to the analysis of different samples of children's and functional foods, such as purees, cereals and pots.

Description

  METHOD OF DETERMINATION OF NUCLEOTICS IN FOODS THROUGH LIQUID CHROMATOGRAPHY OF IONIC PAIRS WITH DETECTION BY PHOTO DIODES COUPLED TO SPECTROMETRY OF MASSES

FIELD OF THE INVENTION The present invention relates to the determination of nucleotides in

foods, particularly in baby foods, by precipitation of the sample proteins and the determination of nucleotides in the extract

resulting by liquid chromatography (HPLC) of ionic pairs with photodiode detection (DAD) coupled to mass spectrometry (MS).

Introduction

Nucleotides are monomers consisting of a phosphate group, a sugar

of five carbon atoms (ribase or deoxyribase) and one or two rings of a nitrogen-containing base. They are an essential part of nucleic acids, perhaps the fundamental and most important constituents of cells [1].

They have been recognized as important elements in mammalian nutrition especially during periods of rapid growth or stress

physiological, and also play a crucial role in the efficient function of the system

immunological Many tissues are not able to synthesize nucleotides and

Men must incorporate them through diet [2]. In many biochemical processes, primary monophosphate nucleotides are used to produce

intermediate metabolites through a series of enzymatic reactions. The addition of primary nucleotides to the diet provides a real source in the synthesis of intermediates.

Nucleotides are abundantly found in human milk and when

Breastfeeding is not possible, an enriched formula is recommended. For healthy young people, their intake through diet is sufficient; however, in most foods, the amounts of useful nucleotides are very low compared to their needs. All ingredients of animal origin

and plant that contain cellular material are potential sources, usually in the form of nucieoproteins. The content is particularly high in

ingredients such as soluble proteins of animal origin, legumes and

yeast extracts The content, proportion and availability differs between them. Muscle is a poor source of nucieotides, as are seeds, soybeans, cereals, fruits, vegetables and processed milk products [3]. Between

marine sources, anchovies and sardines, for example, have

Very high levels of guanine. Availability is also a very important factor. Soluble proteins of fish and animal origin are highly digestive and affect total availability [4].

There is great interest about the importance of diet nucleotides in the

Child nutrition. Children's foods combine a wide variety of

matrices [5]: non-fatty foods based on fruits and vegetables (content of

fat less than 2%), fatty foods based on meat / eggs / cheese and

Cereal-based foods with different fat contents. Since foods derived from plants and milk> have low content of

Nucleotides, dietary supplementation may be of particular interest to children who not only feed on milk. The nucleotides allowed in the

Infant formulas are five compounds, cytidine 5'-monophosphate (CMP), uridine 5'-monophosphate (UMP), adenosine 5'-monophosphate (AMP), guanosine 5'monophosphate (GMP) and inosine 5'-monophosphate (IMP) [ 6]. However, it does not exist

Information on nucleotide levels in some baby foods

such as cereals and purees and there are no established levels for the supplement of infant food.

Nucleotides are unstable and relatively non-volatile compounds and the

The method generally used for food analysis has been HPLC. However, their separation is generally carried out by anion exchange or reverse phase with ion pairs, because they have a high

polarity and anionic nature. Thus, different methods have been proposed by HPLC for the separation and determination of nucleotides in

infant formulas or biological matrices [7-36] using different systems of

detection, mainly DAD and MS. However, in non-supplemented foods (such as certain infant foods), nucleotide levels are very low and, being the matrix very complex, existing methods present a problem of lack of sensitivity and safety in the

Nucleotide identification with total certainty.

Consequently, there is still a need for a method of determining the five authorized nucleotides in baby foods, to

know 5'-CMP, 5'-UMP, 5'-AMP, 5'-GMP and 5'-IMP, having a high

sensitivity for said nucleotides in addition to high safety in the

Identification of them.

This problem has been solved in the method of the invention through a

First stage of precipitation of the sample proteins followed by the analysis of the extract by means of HPLC coupling of ionic pairs with photodiode detection (DAD) coupled to time mass spectrometry

of flight (TOF-MS) with dual electrospray and chemical ionization at atmospheric pressure (HPLC / ESI-APCI-TOF-MS). In particular, the use of the volatile reagent for the formation of N-ion pairs, N-dimethylhexylamine (DMHA) has allowed to improve the HPLC compatibility of ionic pairs with ESI-APCI-TOF-MS.

Until the knowledge of the inventors, this is the first study in which

have quickly determined the five authorized nucleotides in a large

variety of children's and / or functional foods such as cereals and purees, without and with supplementation.

Consequently, a first aspect dH the invention is directed to a method of determining in a food of alnenes a nucleotide selected from the

group consisting of: citidine monophosphate 5'-monophosphate, uridine 5 '

monophosphate, adenosine 5'-monophosphate, inosine 5'-monophosphate and guanosine 5'monophosphate, alone or in any combination between them, characterized in that

It comprises the steps of: a) precipitating the food proteins with acetic acid or trichloroacetic acid and then separating the precipitate, obtaining

an extract;

b) subject the extract obtained to liquid chromatography (HPLC) of

ionic pairs with photodiode detection coupled to

dual-time ionization mass spectrometry

ESI-APCI operating in a negative mode, characterized in that the mobile phase of the HPLC system comprises N, N-dimethylhexylamine (DMHA) at a concentration between 5 and 25 mM as an ion pair phonation reagent.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Chromatograms obtained for a mixture of standard nucleotides of

1 I-Ig mL-1 using HPLC of ionic pairs. A: Chromatogram by

HPLC / DAD at 260 nm; B-F: Chromatograms of the ions extracted by HPLC / ESI-APCI-TOF-MS showing the spectra of the compounds.

Detailed description of the invention.

1. Instrumentation used The HPLC system is Agilent 1200 (Agilent, Waldbronn, Genmany) composed of a binary pump (G1312A) operating with a flow rate of 0.7

mL min1. The solvents were degassed using a membrane system

online (Agilent G1379A). The column was kept in a thermostated compartment at room temperature (Agilent G1316A). The diode detector is Agilent G1315D operating at three wavelengths: 252, 260 and 271 nm. The separation was carried out on a Gemini®-NX 5 ~ m C18 column (150 x 4.6

mm), which provided high efficiency and mechanical strength in an interval

Very wide pH of 1-12. The injection (40 ~ L) was carried out with an autosampler (Agilent G1329A). The HPLC system was coupled to a TOF / Q-TOF mass spectrometer (Agilent 6220) equipped with a

dual interface ESI-APCI. The solutions were stored in amber glass vials of 2 010 mL. An EBA 20 centrifuge (Hettich, Tuttlingen, Germany) and an ultrasonic bath (Selecta, Barcelona, Spain) were used to extract the samples.

2. Reagents Acetonltrile and methanol reagent grade from Lab-Scan (Dublin, Ireland) was used. Deionized water was obtained with a Milli-Q purification system (Millipore, Bedford, MA, USA). Commercial nucleotides 5'-monophosphate, 5'-CMP, 5'AMP, 5'-GMP, 5'-IMP and 5'-UMP, were obtained as sodium salts of Sigma (SI. Louis, MO, USA). The concentrated solutions (1000 ~ g mL "') are

prepared by dissolving commercial products, without prior purification, in water. All were kept at -20 oC in dark bottles closed with caps

PTFE / silicone. Standard working solutions were prepared

daily diluting with water. Other reagents used were formate

sodium, DMHA and trichloroacetic acid (Sigma).

3. Samples Samples of different types of baby foods and functional foods were

supplied by the company Hero España, SA in the form of purees or potitos

(from 4 months of age) containing vegetables, meat, fish and fruits,

prepared foods (from 12 months of age) of meat with vegetables, lentils, chickpea stew and spaghetti and baby cereals (multigrain

with honey and cocoa). On the other hand, a study on the

Nucleotide stability in samples with pasteurized yogurt and formulas

of follow-up over a period of 6 months in two conditions

Different experimental: at room temperature and at 30 oC. The studies of

Recovery was carried out using fortified samples with a standard mixture of nucleotides. The samples were allowed to equilibrate at room temperature for at least half an hour before performing the extraction procedure.

4. Analytical procedure

Samples were prepared by weighing 1 9 of the infant food in 1 Q-mL centrifuge tubes and diluting with 2 mL of water. Then 1 mL of 3% m / v trichloroacetic acid was added and the sample was brought to 5 mL, which meant a final concentration of 0.6% m / v, ~ I was then allowed to stand for 15

min to get precipitate proteins. It was then centrifuged at 6000 rpm for 10 min at room temperature. The supernatant was collected and was

diluted to 5 mL with water in a volumetric flask. Aliquots were filtered through

0.45 ~ m syringe filters of polyvinylidene difluoride (PVDF) with polypropylene housing and 40 ~ L were injected into the chromatograph using the vials

Amber of the autosampler. The mobile phase under isocratic conditions was a

mixture of 0.1 M formate regulatory solution (pH 5.5) at 95% (v / v) containing 0.01 M DMHA and 5% methanol (v / v). The flow rate was 0.7 mL min. "UV-vis detection was carried out in the 190--450 nm range and the diode detector was used at 252 nm wavelengths for 5'-GMP and 5'-IMP, 260 nm for 5'-AMP and 5'-UMP and 271 nm for 5'-CMP.

They performed in triplicate.

5. ES / -APC / -TOF-MS detection

The HPLC system (0.7 mL min ") was coupled to a TOF-MS device with a dual ESI-APCI interface operating in negative mode and using a capillary voltage of +1 kV. The other optimal parameters of the ESI-TOF were: temperature of

drying gas, 350 oC; drying gas flow, 11 L min-1 and gas pressure

nebulizer, 58 psi. The detection was carried out considering a mass range of 50-600 miz. The exact mass data of molecular ions

were processed using softwan ~ Mass Hunter considering the level of accuracy widely accepted for composition confirmation

elemental at 5 ppm. During the development of the HPLC method, external calibration of the instrument was performed. The calibration solution was injected at the beginning of each batch and all spectra were calibrated before the

compound identification.

EXPERIMENTAL EXAMPLES

1. HPLC separation Nucleotides have a strong acidic character as a result of the dissociation of phosphate groups in aqueous solution. Generally, they have a negative charge since their pK values are lower than 7. Ionized nucleotides have a higher polarity than neutral species and are retained to a lesser extent, which can be attributed to their weak interaction with the hydrophobic stationary phase and their higher affinity for the aqueous mobile phase. Ionized species also have ionic interactions with activated silanol groups, which impairs the shape of the beak and reproducibility. Reverse phase chromatography was first tested using columns

C,. like Zorbax Eclipse XDB and Discovery HS. The mobile phases were mixtures of different regulatory solutions such as formate (10 mM, pH 35), phosphate (50 mM, pH 7) or Bis-Tris (10 mM, pH 7) in the absence and in the presence of methanol up to 60% v / v. Without emt, argo, due to their polar nature, the

Nucleotides were not retained in the columns in any experimental condition. Acidification of the mobile phase to neutralize silanol groups is a common practice to reduce secondary interactions and

decrease the ionization of the analytes, favoring the interaction with the phase

stationary Thus, a mobile phase of pH 2 containing acetic acid was tested

(2% v / v) and methanol (0-40% v / v), but the results were not satisfactory and

Reverse phase technique was discarded.

To increase nucleotide retention, chromatography was selected.

of ionic pairs (IPC). The ionic nature of phosphate esters facilitated the

strong interactions with cationic ion pair reagents at values of

Appropriate pH, thereby increasing retention and resolution. With the use of

HPLC / MS technique, the resolution of the column with volatile regulatory solutions is critical. A Gemini®-NX 5 ~ m C18 column has been selected,

which is able to tolerate changes in the pH of the mobile phase of 1-12,

providing high efficiency and mechanical resistance without degradation of the column. Since the nucleotides are negatively charged, the surface of the C 18 packing was coated with adsorbed molecules of a positive ion pair reagent and the charge of the matrix was linked to the stationary phase. Different reagents were tested. The critical pair of bands for this

separation was 5'-GMP and 5'-IMP. When the hydroxide reagents of

tetrapropylammonium and tetrabutylammonium hydroxide were obtained very poor resolution for said band. On the other hand, these non-volatile reagents are not compatible with MS detection. Consequently, a reagent was tested

Volatile N, N-dimethylhexylamine to cause minimal interference with MS and adequate separation. It is very common that the influence of concentration

of the ion pair forming reagent have opposite effects on HPLC and MS techniques; thus, the retention of the nucleotides was modified by varying the concentration of the reagent in the mobile phase between 5 and 25 mM. The retention reached a maximum at a concentration of 20 mM. On the other hand, the

HPLC / MS coupling can only be successful if the mobile phase allows it to be

generate enough ionized analyte in the gas phase. However, to this

such a high concentration of reagent, detection by MS is difficult since the signal

In the background it is very high and not reproducible. Therefore, a 10 mM concentration was selected that improves the balance of good separation and

MS sensitivity.

The effect of the pH of the mobile faHe in the range 2-8 on the

retention times and the shape of the peaks. At pH 2-4, nucleotides, which are predominantly as ionized species, decrease their retention. At pH 5-6 they are in neutral form and exhibit more retention, while

remain uncharged until pH 8. Therefore, a pH 5.5 value was selected

which gives the best resolution and appropriate forms of the peaks. The influence of the

concentration of the formate regulatory solution was studied between 20 and 100

mM and an increase in concentration translates into decreased retention. A 100 mM concentration was selected.

Organic modifiers such as COITIO methanol or acetonitrile added to the

regulatory can improve resolution. Changes in the percentage of methanol between O and 15% v / v were tested in order to modify the retention and

the spacing of the bands. An increase in the percentage of methanol reduces the retention of all compounds and the resolution between the two bands of

5'-GMP and 5'-IMP, as it decreases the adsorption of the peer forming reagent

of ions in the stationary phase and this causes a reduction in the retention of ionic nucleotides. The best separation was obtained with a mobile phase containing 5% v / v methanol.

2. Detection by ESI-APCI-TOF-MS

ESI is a weaker ionization method than APCI, and is normally used for different polar compounds. Therefore, the ESI-APCI dual ionization mode that is applicable to a wide variety of compounds has been selected.

Ionization suppression is a complex phenomenon associated with MS detection that can be attributed to deprotonation, presence of nonvolatile components and, in complex samples, large amounts of species

competing with the available ions Thus, the suppression of ionization was evaluated by applying the two modes of operation, positive and negative. In mode

positive, the spectra produced are more complex, with great background interference from the protonated DMHA ion; while in negative mode, the

5 intensities of negative molecular ions were generally stronger and with less background signal. Therefore, the operation in

positive mode since DMHA itself produced a very strong signal that suppressed other positive ions, the ionization mode being selected

negative that gave higher sensitivity. Figure 1 shows the chromatogram

10 UV obtained at 260 nm for a mixture of the five standards of nucleotides and chromatograms of ions extracted by HPLC / ESIAPCI-TOF-MS.

Table 1 summarizes retention times, UV bands and TOF15 MS data including experimental and calculated miz values for formulas

Molecules provided, error, molecular formula and compounds identified. Tooos nucleotides were identified by interpreting the spectra

of masses obtained by ESI-APCI-TOF-MS considering the molecular formula provided and its exact mass and the absorption spectra in the UV region.

The representative TOF-MS spectra of the compounds are also shown in Fig. 1.

Table 1. TOF-MS data and UV bands for nucleotides

(mln)

Amax (nm) miz Experimental miz Calculated Error (ppm) Molecular formula Compound

one

3.75 271 322.0437 322.0446 2.56 C, H, 4N30, P 5'-CMP

2

4.77 260 323.0293 323.0: 286 -2.04 C9H13N2Ü9P 5'-UMP

3

6.81 252 362.0510 362.0! 507 -0.80 C1oH14 NsOsP 5'-GMP

4

7.04 252 347.0390 347.0: 398 2.30 C1 oH13 NsÜ7P 5'-IMP

5

14.36 260 346.0543 346.0 ~ j58 4.46 C1oH14 NsÜ7P 5'-AMP

3. Analytical data and validation 25 Linearity, detection limit, selectivity, accuracy and precision were validated

of the method. Calibration curves were obtained by linear regression analysis representing peak area versus analyte concentration using six concentration levels. Quantification was carried out by the external standard method. Validation parameters, coefficients of

Correlation and the linearity interval for nucleotides are shown in Table 2. The values of,.: 'were good (,.:'> 0.99) and excellent linearity was obtained throughout the entire studied range. Detection limits were calculated.

based on three times the standard deviation of the ordinate at the origin of the calibration graphs and the dH quantification limits using ten times the

standard deviation of the ordinate at the origin. Table 2 also shows

this values. To check the repeatability of the method, they were carried out

10 ten identical analyzes of a sample of fermented milk; the values of R.SD. they were between 2.1 and 6.8%. These values indicated that the accuracy of the method is satisfactory for control analysis.

A great selectivity of the method was achieved due to the high resolution and the 15 exact mass measurements of LCITOF ·· MS. The identification was carried out

using the widely accepted level of accuracy for confirmation of elementary composition as a tolerance of 5 ppm. No impurity peaks appeared at retention times corresponding to nucleotides.

Table 2. Analytical data for nucleotides using ion pair HPLC

Limit Limit of

Slope Coefficient Linearity

Analyte detection quantification

(mL ~ g-l) correlation (~ g mL-1)

(ng mL-1¡ (ng mL-1¡

HPLC-DAD

5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-AMP

31 30 33 29 40 0.9972 0.9964 0.9992 0.9991 0.9995

0.02-5 0.02-5 0.02-5 0.02-5 0.02-5

6.3

5.5

4.5

7.6

4.5

21 18 15 25 15

HPLC-MS

5'-CMP

14752 0.9987 0.1-5 eleven 35

5'-UMP

11019 0.9991 0.1-5 13 42

5'-GMP

3917 0.9985 0.1-5 19 62

5'-IMP

4107 0.9982 0.1-5 17 58

5'-AMP

3526 0.9992 0.1-5 16 55

Matrix effects were studied in different types of samples

analyzed to evaluate the suppression of signal produced by co-elution of

matrix compounds that may affect the ionization of the analyte. The 5 matrix effect for nucleotides in infant foods was evaluated

comparing the slopes of the calibration charts of the standards

aqueous and standard additions for the different matrices, obtained

representing concentration (at five levels) versus peak area and carrying

Outline linear regression analysis. Table 3 shows the data obtained.

10 As can be seen, the slopes of standard additions to the graphs

of calibration for the samples were different from those of aqueous solutions for the five nucleotides, confirming that it is necessary to carry out the calibration by the method of standard additions to compensate the

matrix effect 15 To verify the accuracy of the proposed method, a study was carried out

of recovery fortifying three samples (yogurt with milk of follow-up, infantile cereals and potito of vegetables) to two levels of concentration

corresponding to approximately two and sometimes the limits of

20 quantification. Sample nucleotide recoveries

Fortified varied from 85 to 96%, with a mean recovery ± S.D. (n; 30) of 92 ± 3.

Table 3. Calibration pending for different infant foods,

mL ~ g-l

Sample 5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-AMP

Aqueous standards 14752 11019 3917 4107 3526

Infant foods 4-6 months old Infant foods 12 months old

Lamb with vegetables

3729 3495 3345 3743 954

Turkey with vegetables

2733 48, '1 1345 2588 1368

Ham with peas

2031 4501 3563 4182 1455

Veal with vegetables

1051 36 ~ ¡4 1801 1251 757

Ham with veal

1228 4H, 8 2220 1872 1314

vegetables

Chickpea stew 1133 3302 3388 581 1011 Chicken with rice and 695 4411 2067 1077 1466

vegetables

Lentils 6638 3570 2300 782 750 Spaghetti with meat 5021 52 (; 6 2929 2706 762 Children's cereals Multigrain with honey 3654 1614 452 562 559 Cereals with cocoa 2029 1447 480 590 490

Dairy baby foods

Pasteurized yogurt and 7697 9412 2472 2267 2031 formula

tracing

4. Sample extraction

Since infant food samples are very complex biological matrices, it was necessary to carry out a method of sample preparation 5 to simplify the matrix and facilitate the interpretation with certainty of the signal. In addition, some additional precautions must be taken into account to ensure the integrity of the sample through the analytical process. This is particularly critical in the analysis of milk, since nucleotides are susceptible to enzymatic conversions by a variety of enzymes.

Endogenous as nucleotidases and phosphatases, which can rapidly degrade analytes_ Thus, the sample was treated with acids to inhibit the conversion of analytes by inactile / ation of these enzymes. To remove the proteins, the extraction was carried out with acetic acid and trichloroacetic acid (TeA) and, after centrifuging, the precipitate was washed with water

15 and the extracts were combined. The best results regarding the efficient separation of leclle proteins were achieved using TeA. The

precipitation of the proteins with acids, without neutralization, has the advantage that the preparation of the sample is simple and rapid. However, it

they can produce nucleotide losses during the storage of these with acid for a long time. Different concentrations were tested

of TeA in the range 0.1 -1% mlv and the extracts were centrifuged and filtered

for immediate chromatographic analysis. Good recovery was obtained by getting the precipitation of the proteins using 0.6% m / v TeA. Extraction was also tested with organic solvents; however, low extraction efficiencies were obtained using methanol and acetonitrile, so their use was ruled out.

Before injection into HPLC samples were filtered. Children's foods containing olive oil presented some problems for

filtration with nylon filters. However, good filtration was achieved

using polyvinylidene difluoride syringe filters (PVDF) with polypropylene housings. PVDF is hydrophilic in nature and is a membrane that can be used for proteins and peptides.

5. Analysis of infant foods

Different samples corresponding to ten infant foods were analyzed

(4-6 months old) containing vegetables, meat, fish and fruits,

baby food (12 months old) containing meat with vegetables,

chickpea stew, lentils and spaghetti and baby cereals (multigrain with honey and cocoa). All samples were analyzed in triplicate. Table 4 shows the results obtained. As you can see, the

Higher levels of nucleotides are found in fish-based foods. The lowest levels were found in infant cereal samples. Consequently, the levels found in infant food samples can be quantified properly and without

Sensitivity problems using this HPLC-MS procedure. Both

nucleotides of commercial interest such as flavor enhancers, 5'-IMP and 5'GMP, can be found in nature and have been detected in food

Children's Other nucleotides, such as 5'-AMP, are also naturally present. 5'-IMP is primarily associated with animal sources, while 5'-GMP is more prevalent in vegetable-based foods.

Most marine animals are good sources of 5'-IMP,

especially when it is a dry product [37].

Table 4. Nucleotide content 'in baby foods (~ g g ")

Sample 5 ·· CMP 5 ·· UMP 5 ·· GMP 5 ·· IMP 5 ·· AMP

Infant foods 4 · 6 months old

Lamb with

0.27 ± 0.02 2.0 ± 0.16 0.29 ± 0.02 3.1 ± 0.2 2.7 ± 0.2

vegetables

Turkey with vegetables

0.11 ± 0.01 1.1 ± 0.08 0.24 ± 0.02 0.13 ± 0.01 2.1 ± 0.1

Ham with

0.14 ± 0.01 0.91 ± O.1 0.12 ± 0.01 0.10 ± 0.01 0.56 ± 0.03

green peas

Veal with

0.53 ± 0.03 2.2 ± 0.18 0.51 ± 0.04 2.6 ± 0.1 2.9 ± 0.2

vegetables

Ham with veal

0.15 ± 0.01 0.36 ± O.03 0.07 ± 0.01 0.06 ± 0.01 1.2 ± 0.1

vegetables

Hake with rice

1.7 ± 0.1 2.2 ± 0.3 1.0 ± 0.09 2.2 ± 0.2 3.2 ± 0.2

Vegetables

2.5 ± 0.13 3.5 ± 0.3 1.8 ± 0.1 0.36 ± 0.02 4.2 ± 0.2

Peach and

1.0 ± 0.09 2.3 ± 0.2 0.59 ± 0.03 0.21 ± 0.01 1.0 ± 0.08

banana

12 month old baby food

Chickpea stew

Chicken with rice and vegetables

Spaguettis lentils with

meat

0.25 ± 0.02

1.7 ± 0.15

0.94 ± 0.1

1.8 ± 0.2

0.34 ± 0.02

1.5 ± 0.1

0.85 ± 0.06

1.5 ± 0.1

0.52 ± 0.04 0.65 ± 0.04 0.73 ± 0.05

0.07 ± 0.01

2.2 ± 0.1

2.1 ± 0.1

1.4 ± 0.06

1.7 ± 0.05

0.82 ± 0.07 0.57 ± 0.03

0.83 ± 0.05 0.27 ± 0.02

Children's cereals

Multigrain with

0.32 ± 0.02 0.12 ± O.07 0.13 ± 0.01 NO NO

honey

Cereals with cocoa

0.11 ± 0.01 0.18 ± O.1 0.27 ± 0.02 NO NO

, Mean ± standard deviation (n-3)

Chromatographic profiles demonstrate the absence of interfering peaks.

The peaks were identified by comparing the retention data obtained for the standards, the samples and the different fortified samples with the 5 standards under identical conditions. The peaks were also identified

using the DAD detector to continuously measure the spectrum while the

solute passes through the rain cell, thus confirming the identity and purity of the peaks. Excellent agreement was obtained between the UV spectra of the different peaks obtained for the standards, the samples and the

5 fortified samples. Finally, the identification was confirmed using the

widely accepted threshold for elementary composition of 5 ppm of

tolerance .

6. Stability of nucleotides € 'n infant foods acidified with

10 pasteurized yogurt and follow-up milk. In the production of fermented foods, microorganisms or enzymes are used to produce the desired biochemical changes and the modification of the flavor and nutrient content of the food. Beneficial foods

for health, that is to say the so-called "functional foods" of milk products, are based on the characteristics of lactic acid bacteria [38]. A study on the stability of the

nucleotides in infant foods I {acidified dairy. The samples were analyzed freshly prepared and after two months during the time of

Total life of six months. The expel'imentos were carried out in two

20 experimental conditions, at room temperature (TA) and after keeping the samples at 30 oC. As Table 5, 5'-GMP and 5'-UMP were not stable in the samples, while 5'-CMP and 5'-AMP were practically stable, especially in the case of samples stored at room temperature.

Table 5. Evolution of the nucleotide content in acidified dairy foods, with pasteurized yogurt and follow-up formula (mg / 1 00 g)

Nucleolide

Declared Freshly prepared After 2 months (TA) After 2 months I (30 'C) After 4 months (TA) After 4 months I (30 'c) After 6 months (TA) After 6 months I (30 'C)

5'-CMP

0.8 0.79 0.83 0.85 0.89 0.78 0. 79 0. 87

5'-UMP

1.4 1.2 0.94 0.90 0.92 0.90 0. 84 0. 94

5'-GMP

1.0 0.35 0.31 0..20 0.25 0.17 0.12 0.09

5'-IMP

NO NO NO ND NO NO NO NO

5'-AMP

0.3 0.37 0.34 0.30 0.35 0.16 0. 29 0.11

CONCLUSION In accordance with the present invention, an analytical method based on

in HPLC of ionic pairs with DAD detection coupled to ESI-APCI-TOFMS for the rapid determination of five monophosphate nucleotides: 5'-CMP, 5'UMP, 5'-AMP, 5'-GMP and 5'-IMP in food Children's The procedure

proposed allowed to determine these five authorized nucleotides and was successfully applied to a wide variety of baby foods and functional and cereals

children, including a study of the stability of nucleotides in

acidified dairy foods. The sensitivity and exact mass of ESI-APCJTOF-MS allowed the correct identification of the compounds.

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Claims (1)

1) Method of determination in a food of at least one nucleotide selected from the group consisting of: citidine 5'monophosphate monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, nasine 5'-monophosphate and guanosine 5'-monophosphate, alone or in any combination between them, characterized in that it comprises stages of: a) precipitate the food proteins with acetic acid or trichloroacetic acid and then separate the precipitate, obtaining an extract; b) subject the extract obtained to liquid chromatography (HPLC) of Ionic pairs with fatigue detection coupled to dual-ionization mass time-of-flight spectrometry ESI-APCI-TOF-MS operating in negative mode, characterized in that the mobile phase of the HPLC system comprises N, N dimethylhexylamine (DMHA) at a concentration between 5 and 25 mM as  ion pair forming reagent. 2) A method according to claim 1, wherein the mobile phase of the HPLC system comprises N, N-dimethylhexylamine (DMHA) at a concentration of approximately 20 mM. 3) Method according to any one of the preceding claims 1-2, in which the mobile phase of the HPLC system also comprises 5% (v / v) methanol and 95% (v / v) 0.1 M forrniato regulatory solution at pH  between 5 and 6, and the elution is isocratic. 4) Method according to claim 3, wherein the pH is  approximately 5.5. 5) Method according to any one of the preceding claims 1-4, wherein, in step a), the proteins of the food are precipitated with trichloroacetic acid at a concentration between 0.1 and 1% m / v. 6) Method according to claim 5, wherein, in step a), the proteins of the food are precipitated with trichloroacetic acid at a concentration of about 0.6% m / v. 5 7) Method according to any one of! the preceding claims in the that, prior to stage b), ,, 1 extract obtained from stage a) is filtered. 8) Method according to claim 7, wherein the filtrate of the extract is 10 performed by polyvinylidene difluoride filter (PVDF). 9) Method according to any one of the preceding claims, wherein the mobile phase is eluted at a flow rate of 0.7 ml min-1. 15 10) Method according to any one of dE! the preceding claims, in the that the HPLC stage is brought to c, abo with a capillary voltage of +1 kV. 11) Method according to any one of the preceding claims, wherein the food is a baby food or a functional food. 12) Method according to claim 11, wherein the food is a potito or baby puree