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Wednesday 3 December 2008 OFFICIAL JOURNAL (Section One)

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MINISTRY OF THE ECONOMY DRAFT Official Mexican Standard PROY-NOM-173-SCFI-2008, Pre-packaged fruit juices –

Trade descriptions, physico-chemical specifications, commercial information and test methods

In the margin is a seal bearing the national coat of arms, with the words: United Mexican States – Ministry of the Economy

DRAFT OFFICIAL MEXICAN STANDARD PROY-NOM-173-SCFI-2008, PRE-PACKAGED FRUIT JUICES. TRADE DESCRIPTIONS, PHYSICO-CHEMICAL SPECIFICATIONS, COMMERCIAL INFORMATION AND TEST METHODS.

The Ministry of the Economy, through the General Directorate of Standards, on the basis of Articles 34 sections XIII and XXXI of the Organic Law of the Federal Public Administration; 39 section V, 40 sections I and XII, 47 section I of the Federal Law on Metrology and Standardisation, 33 of its Regulation and 19 sections I, XIV and XV of this Ministry’s Rules of Procedure, hereby submits for public consultation the following Draft Official Mexican Standard PROY-NOM-173-SCFI-2008 “Pre-packaged fruit juices – Trade descriptions, physico-chemical specifications, commercial information and test methods”, to enable interested parties to submit their comments within 60 calendar days to the National Advisory Committee on Standardization for User Safety, Commercial Information and Trade Practices, located at Avenida Puente de Tecamachalco number 6, colonia Lomas de Tecamachalco, Sección Fuentes, Naucalpan de Juárez, post code 53950, Mexico State, telephone 57 29 93 00, Ext. 43222, Fax 55 20 97 15 or e-mail addresses [email protected]; [email protected] and/or [email protected], to enable them to be considered in the proposing committee as required by law.

Mexico City, 14 November 2008. Director-General for Standards and Chairman of the National Advisory Committee on Standardization for User Safety, Commercial Information and Trade Practices, Francisco Ramos Gómez.- Initialled.

DRAFT OFFICIAL MEXICAN STANDARD PROY-NOM-173-SCFI-2008, PRE-PACKAGED FRUIT JUICES – TRADE DESCRIPTIONS, PHYSICO-CHEMICAL

SPECIFICATIONS, COMMERCIAL INFORMATION AND TEST METHODS

INTRODUCTION

The following firms and institutions took part in the drafting of this draft official Mexican standard:

ALMACENADORA ACCEL, S.A. DE C.V.

ASOCIACIÓN NACIONAL DE PRODUCTORES CITRICOLAS, A.C. (National Association of Citrus Growers)

ASOCIACIÓN NACIONAL DE VITIVINICULTORES, A.C. (National Association of Wine Growers)

CÁMARA NACIONAL DE LA INDUSTRIA DE CONSERVAS ALIMENTICIAS (National Chamber for the Food Preserving Industry)


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CENTRO DE CONTROL TOTAL DE CALIDADES, S.A. DE C.V. (Total Quality Control Centre)

COCA-COLA DE MEXICO, S.A. DE C.V. CONFEDERACIÓN DE CÁMARAS INDUSTRIALES DE LOS ESTADOS UNIDOS MEXICANOS (Confederation of Chambers of Industry of the United Mexican States) CONSEJO CITRICOLA MEXICANO (Mexican Citrus Growers’ Council)

CONSEJO MEXICANO DE LA INDUSTRIA DE PRODUCTOS DE CONSUMO, A. C. (Mexican Council of the Consumer Products Industry)

GERBER, S.A. DE C.V. GRUPO JUMEX

HECA, S.A. HERDEZ, S.A. DE C.V.

JUGOS DEL VALLE, S.A. DE C.V. LEFIX Y ASOCIADOS

PROCONSUMIDORES, A.C. PROCURADURIA FEDERAL DEL CONSUMIDOR (Federal Attorney for Consumer Affairs) Laboratorio Nacional de Pruebas (National Testing Laboratory)

Dirección General de Verificación y Vigilancia (Directorate-General for Testing and Monitoring)

SABRITAS, S.A. DE C.V. SECRETARIA DE SALUD (Ministry of Health)

Comisión Federal de Protección contra Riesgos Sanitarios (Federal Commission for Protection against Health Hazards)

SECRETARIA DE AGRICULTURA, GANADERIA, DESARROLLO RURAL, PESCA Y ALIMENTACION (Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food) Dirección General de Fomento a la Agricultura (Directorate-General for Agricultural Development) SECRETARIA DE HACIENDA Y CREDITO PUBLICO (Ministry of the Treasury and Public Credit) Sistema de Administración Tributaria (Tax Administration System)

SIGMA ALIMENTOS, S.A. DE C.V. SOCIEDAD COOPERATIVA TRABAJADORES PASCUAL, S.A.

SOCIEDAD MEXICANA DE NORMALIZACION Y CERTIFICACION, S. C. (Mexican Standardisation and Certification Company)

UNILEVER DE MEXICO, S.A. DE C.V. UNIVERSIDAD DE LAS AMERICAS DE PUEBLA (University of the Americas, Puebla)


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UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO (National Autonomous University of Mexico City)

Instituto de Química (Institute of Chemistry) Instituto de Geología (Institute of Geology)

VALLE REDONDO, S.A. DE C.V.

CONTENTS

Chapter

Objective and scope

References

Definitions

Classification and trade description

Product specifications

Sampling

Commercial information

Monitoring

Bibliography

Concordance with international standards

1. Objective and scope This official Mexican standard establishes the characteristics of processed and pre-packaged products and the minimum requirements that they must satisfy in order to be described as fruit juices, and also the commercial information that they are required to display.

This standard applies to products marketed as fruit juices on Mexican territory.

2. References This Mexican standard is complemented by the following current official Mexican standards and Mexican standards:

NOM-002-SCFI-1993 Pre-packaged products. Net contents, tolerance and checking methods, published in the Official Journal of the Federation on 13 October 1993.

NOM-030-SCFI-2006 Commercial information – Declaration of the quantity on the label – Specifications, published in the Official Journal of the Federation on 6 November 2006.

NOM-051-SCFI-1994 General specifications for labelling pre-packaged foodstuffs and non-alcoholic beverages, published in the Official Journal of the Federation on 24 January 1996.


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NOM-086-SSAI-1994 Goods and services. Foodstuffs and non-alcoholic beverages with modified composition. Nutritional specifications, published in the Official Journal of the Federation on 26 June 1996.

NOM-130-SSA1-1995 Goods and services. Foodstuffs packaged in hermetically sealed containers and subjected to heat treatment. Health provisions and specifications, published in the Official Journal of the Federation on 21 November 1997.

NMX-F-103-1982 Foodstuffs – Fruit and derivatives – determination of degrees Brix. Declaration of validity published in the Official Journal of the Federation on 14 October 1982.

NMX-F-309-NORMEX-2001 Determination of benzoates, salicylates and sorbates in foodstuffs. Declaration of validity published in the Official Journal of the Federation on 26 July 2001.

3. Definitions For the purposes of this standard, the following definitions shall apply:

3.1 Adulteration Product of a nature not matching its labelling, advertising, sale or supply or the authorised specification, even where it has been processed to conceal those circumstances or conceal defects in processing or the health quality of the raw materials used in it.

3.2 Citrus fruit Fruit of the Rutaceae family that is damaged at low temperatures, characterised by oils and pigments in the peel.

3.3 Sound fruit Fruit free of disease, cuts, rot, damage by inspects or other pests, containing no live or dead inspects or larvae thereof.

3.4 Ripe fruit Fruit that is in season or at maturity.

3.5 Degrees Brix Percentage of solids dissolved in a product derived from fruit or a liquid of sugar.

3.6 Fruit juice The unfermented but fermentable liquid product obtained by pressing fully ripened and fresh fruit in good condition, or fruit that has been kept in good condition using appropriate procedures, including surface treatments applied after harvesting, clarified or otherwise, and subject to appropriate treatment to ensure that it is preserved in the packaging. It must not contain peel or seeds or inadmissible foreign material.

Juice must be prepared using procedures that preserve the essential physical, chemical, organoleptic and nutritional properties of the original fruit. Pulp and cells obtained by means of physical processes appropriate to the type of fruit may be added. In the case of citrus fruit, the pulp and cells are the juice sacs obtained from the endocarp.


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This product may be made from frozen fruit juice and/or reconstituted concentrated fruit juice, provided that it meets the specifications laid down in this standard.

3.7 Concentrated fruit juice Fruit juice from which water has been physically removed in an amount sufficient to raise the Brix level to a value of at least 50% greater than the Brix value established for the liquid product obtained by pressing healthy and ripe fruit that has been subjected to physical treatment or stored under appropriate conditions that ensure it is preserved in the container. It must not contain peel or seeds or inadmissible foreign material.

3.8 Juice of multiple fruits (mix) A blend of two or more liquid products obtained by pressing healthy and ripe fruit of the relevant variety, clarified or not, unfermented and suitably treated to preserve it in the container. It must not contain peel or seeds or inadmissible foreign material.

This product may be produced by blending two or more frozen juices and/or purées, and/or concentrated juices and/or purées reconstituted from various kinds of fruit.

Juice must be prepared using procedures that preserve the essential physical, chemical, organoleptic and nutritional properties of the original fruit. Pulp and cells obtained by means of physical processes appropriate to the type of fruit may be added. In the case of citrus fruit, the pulp and cells are the juice sacs obtained from the endocarp.

3.9 Frozen juice Fruit juice that has undergone a thermal treatment by means of appropriate equipment until the product reaches a temperature of -15°C at the thermal centre.

3.10 Pre-packaged products Foodstuffs and non-alcoholic beverages packaged in a container of any kind in the absence of the consumer, where the quantity of product contained cannot be altered without opening or altering the container in a noticeable way.

3.11 Fruit pulp The fleshy and often juicy mass of the fruit (insoluble solids). In the case of citrus fruit, the pulp consists of a considerable number of segments full of juice.

3.12 Fruit purée The fermentable but unfermented product obtained by appropriate procedures, such as sieving, crushing or crumbling the edible part of whole or peeled fruit without removing the juice. The fruit must be in good condition, fully ripened and fresh, or preserved by means of physical procedures or treatments applied in accordance with the relevant provisions of the competent authority.

3.13 Concentrated fruit purée The product obtained by physical removal of a sufficient quantity of water from fruit purée to raise the Brix level to a value at least 50% greater than the Brix value established for juice reconstituted from the same fruit.


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3.14 Dissolved fruit solids Soluble solids from fruit, quantified in degrees Brix.

4. Symbols and abbreviations °Bx degrees Brix % per cent g gram mg milligram µg microgram kg kilogram L or l litre mL or ml millilitre µL or µl microlitre mm millimetre cm centimetre m3 cubic metre GMP good manufacturing practice pH hydrogen potential m/v solids/total volume m/m mass/mass 13C total number of atoms of carbon-13 12C total number of atoms of carbon-12 •13C delta carbon 13 VPDB Vienna Peedee belemnite ‰ parts per mille g acceleration of gravity mol mole rpm revolutions per minute h hour min minute s second °C degrees Celsius K Kelvin Pa pascal kPa kilopascal mbar millibar amu atomic mass unit σ standard deviation


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5. Classification and trade description

5.1 Juices Juices covered by this standard shall be described with the same character size in accordance with the following.

5.1.1 Fruit juices as defined in 3.6 shall be described as follows:

__________ JUICE

The name of the fruit concerned shall be written in the blank space.

5.1.2 Concentrated fruit juices as defined in 3.7 shall be described as follows:

CONCENTRATED ____________ JUICE


5.1.3 Juices of multiple fruits as defined in section 3.8 shall be described as follows:

5.1.3.1 Where juice of multiple fruit consists of a blend of two fruit juices, it shall be described as:

__________ JUICE

The name of the two fruits concerned shall be written in the blank space, beginning with the one of higher content.

5.1.3.2 Where the blend consists of juices and/or purées of three or more fruit it shall be described as:

__________ JUICE


6. Product specifications

6.1 Juices 6.1.1 Sensory characteristics

Colour: Characteristic similar to the variety used.

Smell: Characteristic of the juice concerned.

Taste: Characteristic of the juice concerned, without off-flavours.

It must not contain peel or seeds or inadmissible foreign material.

6.1.2 Physical and chemical properties

Juices covered by this standard must comply with the physical and chemical specifications set out in Table 1.


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Table 1 Specifications for juices and reconstituted concentrated juices

Parameter Orange Mandarine Apple Grapefruit Pineapple Grape Multiple fruits

Test methods

* Minimum dissolved solids of the fruit concerned (°Brix)

11.2 11.8 11.5 10 12.8 16 10 Ref. section 8.1 and NMX F-103-1982

Isotope ratio of carbon (13C/12C), expressed in 13CVPDB (‰).

<-24 to -28

-24 to -28 --24 to -28

--24 to -28 N.A. --24 to -28

Ref. section 8.1

* This specification shall be complied with even if the product composition is altered.

N.A.: Not applicable

It is prohibited to add both sugars and acidifying agents to the same fruit juice since it is regarded as an adulteration of the composition of the product, as established in the Codex Alimentarius.

Note: Where a juice comes from a fruit not mentioned in this table, the minimum Brix level of the fruit shall be as laid down internationally by the Codex Alimentarius.

**Additives for Table 1

Permitted additives and preservatives shall be those referred to in NOM-130-SSA1, Goods and services. Foodstuffs packaged in hermetically sealed containers and subjected to heat treatment. Health provisions and specifications.

7. Sampling For official purposes, sampling shall be subject to the applicable statutory and regulatory provisions.

8. Test method Determination of the addition of cane sugar or corn syrup using the carbon isotope ratio (13C/12C) in sugars from fruit of C3 plants using stable-isotope mass spectroscopy.

8.1 Objective and field of application This methodology aims to quantify the addition of exogenous industrial sweeteners such as those listed in CODEX Standard for Sugars CODEX STAN 212-1999 amendment 1-2) to the various classifications of industrial fruit juices from C3 plants, produced from sound and ripe fruit or prepared from fruit concentrates obtained by means of an industrial process for removing the water contained in the juices. This method is not applicable to pineapple juice.

The following exogenous sugars may be considered: cane sugar, corn syrups with a high fructose or glucose content.


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8.2 Basis The method is based on determining the carbon isotope ratios (13C/12C) using stable-isotope mass spectrometry, expressed as the quotients of the abundances of stable carbon-12 and -13 atoms as (13C/12C) using •13CVPDB units (‰), which are referred to an international standard that is physically a calcium carbonate (CaCO3) of marine origin, taken from the Peedee cretaceous formation in South Carolina, known as PDB (Peedee belemnite limestone). The reporting unit is •, expressed in parts per mille (‰), in accordance with the following equation (Craig, 1957):

∂13CVPDB =(13C /12C)Sample

(13C /12C)VPVD

−1

•103 Eq. 1

The bodies internationally responsible for establishing, maintaining and developing the metrology of stable isotopes are the International Union of Pure and Applied Chemistry (IUPAC), Commission on Isotopic Abundances and Atomic Weights (CIAAW); IUPAC has designated the Subcommittee on Isotope Abundance Measurements (SIAM) as operationally responsible for the metrology of stable isotopes, which is composed of a group of experts who publish reports and issue metrological recommendations on isotopic abundances and atomic weights. At its recent meeting in the context of the 43rd General Assembly of the IUPAC that took place in August 2005 in Beijing, China, it made the following recommendations: that delta carbon-13 values of all carbon-bearing materials be measured and expressed relative to VPDB (Vienna Peedee belemnite), b) the VPDB scale be normalised using the reference materials NBS-19 and L-SVEC by assigning the consensus values of •13CVPDB = -46.6‰ relative to L-SVEC lithium carbonate and •13CVPDB = +1.95‰ relative to NBS 19 calcium carbonate. c) authors analysing these materials should clearly state so in their reports. (Coplen, T et al 2006).

8.3 Principle of the method All organic compounds forming living beings contain the chemical element carbon. Two stable carbon atoms exist in nature that differ among themselves only in their atomic mass, all their other chemical properties being identical. These atoms are known as isotopes and are represented as carbon-13 (13C) and carbon-12 (12C).

The proportion of stable isotopes (13C/12C) contained in foodstuffs is typical and set mainly by their origin or provenance and to a much lesser extent by the industrial processes they subsequently undergo. The original isotope proportion is generally maintained, which enables us to identify their source.

The basis of the method consists of relating the quantity of stable isotopes of a given product, expressed in delta units, to the corresponding photosynthesis cycle of the plant from which the raw material originates. The reason for this is that plants have three different photosynthesis mechanisms for fixing atmospheric CO2.

The various types of origin of plants are established on the basis of photosynthesis, and are classified in three main groups depending on the photosynthesis process they use to fix atmospheric CO2, namely: type C3 plants which use the Calvin cycle, type C4 plants which follow the Hatch Slack cycle and Crasulacean acid metabolism (CAM) plants.


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In nature, the Calvin cycle is used by type C3 plants; members of this photosynthesis cycle are trees, shrubs and some fruits such as: apple, pear, peach, banana, grape, mango, guava, plum, apricot, cherry, bilberry, orange, grapefruit, mandarine, lemon, tomato, etc. The vast majority of industrially-produced juices and nectars are extracted from these fruits.

In nature, the Hatch Slack cycle is used by C4 plants, such as sugar cane and maize. Plants of this type are used to make industrial sweeteners such as cane sugar and corn syrup with a high fructose or glucose content; such sweeteners are mostly used as exogenous sugars for adding to juices and nectars.

The third group of plants is called Crasulacean Acid Metabolism (CAM). This group contains agaves, vanilla and plants such as the pineapple.

There is a considerable and measurable difference in the proportion of stable •13C isotopes (13C/12C) contained in plants of groups C3 and C4. Therefore, by measuring it in juices or concentrated juices it is possible to identify the photosynthesis process of the original plant and quantify the blend, if any.

The proportion of a blend of sugars from C3 plants with C4 plants (exogenous sugars from cane sugar or corn syrup) can be quantified using a linear proportion constructed by taking as extremes the •13C isotope composition of the exogenous sugars and the •13C isotope composition of the soluble solids contained in the fruit juice.

8.4 Description of test method The •13CVPDB is determined in a stable-isotope mass spectrometer using the CO2 obtained from the combustion of the main organic material contained in a sample of juice such as dissolved solids and pulp. The results obtained from both are reported as •13CVPDB expressed as ‰ evaluated in accordance with equation 1.

To carry out the analysis, an aliquot of juice is taken and the pulp and dissolved solids are separated out mechanically (by centrifuging); once the organic material has been separated it is oxidised by quantitative combustion to form mainly CO2 and H2O. The CO2 produced by the oxidation reaction is purified by means of a cryogenic separation process and using vacuum systems as per section 8.7.2.1.1) or a separation process using a chromatography column such as an elemental analyser for separating the N2 from the CO2.

International literature reports three basic quantitative combustion methods. The first uses dynamic combustion. The second method is the most accurate of the three and uses high-temperature combustion in a sealed quartz tube; the combustion products are taken to a vacuum line where the CO2 is separated and purified quantitatively; the vacuum line has been purpose-designed for this method; the •13CVPDB is determined using the CO2, cryogenically purified using the spectrometer’s dual gas inlet system. The third method is a fast quantitative combustion and purification system using an elemental analyser and the continuous flow technique with helium with an interface that leads the CO2 from the sample to the stable-isotope mass spectrometer for determining the •13CVPDB.

8.5 Instrumentation The equipment needed to implement this method is a mass spectrometer to determine stable-isotope ratios with the analysis capability to determine the •13CVPDB in the range of natural abundances in CO2 with an internal accuracy of the order of 0.02‰ (defined here as the


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difference between two consecutive measurements of isotope ratios of a CO2 sample) and is the external accuracy of 0.05‰ (expressed in delta values – Eq 1). The linearity must be < 0.05‰ per nanoampere of ion current. While the spectrometer is operating, the plateaus of the peaks for universal CNOS collector must be m/∆m = 95 (10% trough).

The mass spectrometer must be capable of carrying out isotope determinations by simultaneously reading molecular masses 44, 45 and 46 amu. The spectrometer must have a dual sample inlet system for simultaneous determinations between the sample gas and the gas of a calibration standard. The dual-inlet method is intrinsically more accurate. However, specific instrumentation has been developed to connect an elemental analyser and an analysis method has been devised for it that uses a continuous flow of helium to transport the CO2 combustion product into the isotope-ratio mass spectrometer. The results of this technique are just as reliable, but it requires a set of aliquots of reference materials to be analysed in series with the samples.

Where the elemental analyser (EA) is used in line with the stable-isotope mass spectrometer, the EA must be connected directly to the spectrometer by means of an interface specially designed for this method. The EA must be capable of handling both the combustion gases from the sample or reference materials and the gas used as a calibration standard at the same time. The EA must also be capable of quantitatively converting all the carbon in the sample into carbon dioxide and of eliminating the other combustion products of the sample, principally oxides of nitrogen, oxides of sulphur and water.

8.6 Other equipment needed for the method Centrifuge for physically separating the dissolved solids of the fruit and the pulp; a centrifuge must be used with a rotor of 45o. for six centrifuge tubes with a volumetric capacity in each arm of at least 50 ml with a force of at least 1400 G.

Electric grill with magnetic stirrer: with temperature control and stirring control.

Drying oven with a temperature of 50°C to 60°C.

Hand refractometer for determining °Brix.

Sample preparation as described in section 8.7 is applicable to all types of juices, whether in fresh natural form or in commercial presentations. Concentrated juices must be diluted to 50% with distilled water. The procedure is as follows.

8.7 Procedures 8.7.1 Preparing the sample

8.7.1.1 Reagents and materials

Only the following analysis-grade reagents must be used to prepare the sample:

• Calcium hydroxide {Ca(OH)2}, analysis grade. A 25% solution is prepared with grade III water.

• Sulphuric acid (H2SO4) 95-97%, analysis grade. Concentration 1M

• Water of at least grade III as per EN ISO 3696:1995


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• pH paper

• Centrifuge tubes, 40 ml or 50 ml

• Graduated pipettes, 10 ml

• Propipette

• Precipitation beaker, 100 ml

• Precipitation beaker, 1 l

• Agate mortar and pestle

8.7.1.2 Procedure for separating and preparing soluble solids (sugars) and insoluble solids (pulp) for isotope analysis

To physically separate the constituents of a juice into dissolved solids and pulp, take 50 ml of juice of any specification and place in a centrifuge tube, centrifuging at 1400 g for 10 minutes.

a) Preparing soluble solids (sugars) for isotope analysis

• After centrifuging as above the soluble solids are contained in the supernatant, so this supernatant is decanted and 10 ml of it is placed in a 100 ml precipitation beaker.

• Add 25% calcium hydroxide solution until the pH is adjusted to between 8.5 and 9. Heat the mixture in a bain-marie at 90°C for 10 minutes while stirring.

• The organic acids, amino acids and other components are precipitated in this step; centrifuge the solution for 10 minutes at 1400 g to separate them.

• Decant the supernatant and place it in a 100 ml precipitation beaker, acidify with 1M H2SO4 until the pH is 5, namely approximately when the solution changes colour and check the pH by placing a drop of the solution onto pH paper, being careful not to bring the paper into contact with the sample.

• Refrigerate at 4°C for 12 hours and decant the supernatant liquid.

• Take an aliquot of supernatant for •13C isotope analysis. The approximate concentration in °Brix of the dissolved solids containing mainly the sugars of the juice can be measured with a hand refractometer.

b) Preparing insoluble solids (pulp)

The insoluble solids (pulp) obtained from the first centrifuging are precipitated on the bottom of the centrifuging tube, are separated and prepared as follows:

• The pulp is washed with hot distilled water at 90°C in the centrifuging tube, and is centrifuged for a further 10 minutes at 1400 G. The supernatant is decanted and broken up; this step should be repeated five times.


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• The pulp is washed a second time, with 40 ml of acetone, is centrifuged for 10 minutes at 1400 g; the supernatant is decanted and broken up (this step should be repeated at least twice). Once the pulp has been washed with water and acetone, it is freeze-dried or may be dried in an oven at 60°C–80°C for a whole night.

• The dry pulp is homogenised, pulverising it in an agate mortar.

• Weigh out an aliquot of pulp to determine the •13C.

8.7.2 Techniques for combustion of samples of dissolved solids or pulp from the juice samples for subsequent determination of •13CVPDB in the stable-isotope mass spectrometer

8.7.2.1 Purpose of combustion

The purpose of the various combustion techniques is to transform quantitatively the carbon from the organic material (dissolved solids or pulp) prepared as per 8.7.1.2 a) and 8.7.1.2 b) from the juice samples with carbon dioxide CO2. After combustion and subsequent separation of the other combustion products, the carbon dioxide CO2 is purified to measure their •13CVPDB in the stable-isotope mass spectrometer.

Two methods of quantitative combustion of the organic material can be used, the first method uses a sealed quartz tube, combustion of the gases produced taking place at a temperature of 950°C; the CO2 is separated and purified and the pure CO2 is passed into the dual gas inlet system of the stable-isotope mass spectrometer. The second combustion and purification method uses an elemental analyser connected in line with the mass spectrometer.

A detailed description of each method is given below.

8.7.2.1.1 Combustion in quartz tube

Reagents and materials for combustion in quartz tube

• Reference materials: see Table 2.

• Tank of CO2 with minimum purity 99.995% in a 7 l cylinder, contained in a metal tank at a maximum pressure of 5860 kPa at 294 K (21ºC) with a two-stage regulator having a delivery pressure of 5.0 ml/minute, to operate with cylinders having a maximum pressure of 100 kPa. For use as a calibration standard in the isotope-ratio mass spectrometer.

• Cupric oxide wire, analysis reagent grade. The cupric oxide is passed through a 40 gauge mesh to remove the dust. It is then burned in a muffle at 850°C for three hours to oxidise all the organic material it contains.

• Silver, 99.9994% pure. It is rolled to a thickness of 0.1 to 0.4 mm and cut into sections 1 mm wide and 7 mm long. The sections are cleaned in 1% nitric acid solution and then washed in distilled water and heated in a muffle at 400°C for one hour. The silver is used as a catalyst in the combustion tube.

• Activated metallic copper (granules) analysis reagent grade


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• A gas-oxygen blowtorch capable of reaching the melting temperature of quartz, for sealing the quartz tubes

• A tank of industrial oxygen, with regulator and valves for use with the blowtorch

• Butane gas for domestic use, with tubing for use with the blowtorch

• Dry ice

• Absolute industrial ethanol, 95% alcohol by volume

• Liquid nitrogen

• High vacuum grease, type Apiezon N or M

• Pointed-nosed tweezers and stainless-steel spatula for handling 10 mg samples

• Industrial hot-air dryer capable of reaching 500°C, with 1500 rpm squirrel-cage fan

• Laboratory screw clamps of various sizes

• Tesla generator for detecting leaks in glass vacuum systems

• Quartz tube, external diameter 9 mm, internal diameter 7 mm and 20 cm long, sealed at one end with a blowtorch. For use as a combustion tube.

• Quartz tube, external diameter 6 mm, internal diameter 4 mm and approximately 2 cm long, sealed at one end with a blowtorch. Used to keep the cupric oxide separate from the metallic copper and as a container for solid organic samples.

• Quartz tube, external diameter 4 mm, internal diameter 2 mm and approximately 2 cm long, with a point at one end for use as a capillary tube to contain samples of organic liquids.

• Quartz tube, external diameter 4 mm, internal diameter 2 mm and approximately 2 cm long; a silver filament 1 mm x 7 mm long is placed inside and both ends are sealed leaving a small hole to support the silver filament.

• All the quartz apparatus used in this method is placed in a muffle at 600°C for one hour to remove any particles of organic matter; once purified it is kept in containers and must be kept in a drying oven between 40°C and 60°C to drive out any moisture.

• Pyrex glass flasks with high-vacuum valve and Teflon or glass piston. These flasks are used to transport the CO2 from the sample preparation line to the inlet system of the isotope-ratio mass spectrometer.

• Pyrex glass thermos flasks of various sizes (1, 1/2 and 1/4 l) for liquid nitrogen.

• 50 litre stainless-steel thermos flask for holding liquid nitrogen.


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• Ultra-Torr vacuum unions size 3/8, part number SS-6-UT-6 and gas filter SS4VCR25M.

• A system is required to break the quartz tube containing the CO2 gas from the sample; it is made up of the following parts: a) an Ultra-Torr vacuum reducer size 1/2 to 1/4, part number SS-8-UT-6-4, b) flexible vacuum tube size 1/2, part number 321-8X-1, c) two stainless-steel cones 304-8-XOA, d) one Ultra-Torr vacuum reducer size 1/2 to 3/8 and one gas filter SS4VCR25M. The equipment in this paragraph is for a single breaker. (Vacuum unions: Swagelok Companies). Described in the article by DesMaris and Hayes, 1976.

• Micropipettes of various volumes: 0 to 200 µl; 0 to 50 µl and 0 to 10 µl with removable plastic tips.

Equipment needed to achieve combustion in a quartz tube

• Muffle. The combustion of the organic material in a sealed quartz tube takes place in a muffle with a maximum operating temperature of 1100.0ºC equipped with a digital temperature control with an error not exceeding ± 5ºC and a time control with a resolution of 1 minute.

• Vacuum line 1. For de-gassing the samples. Fitted with two vacuum pumps with a throughput of 4 m3/h and a diffusing pump with a liquid nitrogen trap with a throughput of 100 l/s capable of reaching a pressure of 0.1 Pa or 10-4 mbar.

• Vacuum line 2. For cryogenic purification of the samples. Fitted with two vacuum pumps with a pumping capacity of 4 m3/h and a diffusing pump with a liquid nitrogen trap with a pumping rate of 100 l/s capable of reaching a pressure of 0.1 Pa or 10-4 mbar.

• Analysis balance: capacity 200 g, precision 0.1 mg.

• Drying oven. That maintains a constant temperature. Maximum variation ± 0.1 degrees Celsius achieved by a measuring and control system based on a thermocouple over a temperature range of 50°C–60°C.

Preparation and packaging of the material and the sample for combustion in a sealed quartz tube

a) Preparing the combustion tube. The quartz tube is heated to 600°C and 3 g of cupric oxide and a small capillary with a filament of silver are placed inside. The whole assembly is heated for one hour at 600°C in a muffle to eliminate any remaining organic material. The prepared tubes are stored in a sealed glass container in an oven at 40°-60°C that maintains a constant temperature varying no more than ±1.0 degrees Celsius.

b) Preparing samples of dissolved sugars of pulp from juices, nectars and juice concentrates for isotope analysis. Proceed as per 8.7.1.2 a) and 8.7.1.2 b).

c) Take between 40 and 90 µl of dissolved solids or 8-10 mg of dry pulp and place in a small 6 mm external diameter quartz tube and place upside down in the 9 mm external diameter quartz combustion tube containing CuO, the silver filament and the sample; finally add 3 g of


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metallic copper with a funnel. The sample is combusted using the oxygen released by the cupric oxide; the silver acts as a catalyst for the reaction while the metallic copper allows the oxides of nitrogen to convert into N2 at a temperature of 650°C.

d) The combustion tubes prepared as per the previous paragraph are placed in the vacuum line using an Ultra-Torr 3/8" union. The samples are freeze-dried at a pressure of 1.33 Pa and are finally evacuated until a pressure of 0.133 Pa is reached, whereupon the quartz tube is sealed with a high-temperature blowtorch.

e) Every time a set of samples is prepared for •13C isotope analysis, at least two internationally agreed reference materials are prepared, such as: NBS22 (5 µl), IAEA-CH-7 (4 mg) or IAEA-CH6 (8 mg) for calibrating the mass spectrometer.

Combustion

The samples in their respective sealed quartz tubes are heated up to 900ºC and maintained at temperature for two hours in a muffle, after which the temperature is lowered to 650ºC and maintained at that level for one hour. The tubes are then allowed to cool slowly until they reach ambient temperature.

Cryogenic purification of CO2

The CO2 combustion product of the sample is separated and purified cryogenically, beginning by separating the water by means of a cooling mixture (a mixture of dry ice and 80% alcohol or acetone). For purification another trap is used cooled with liquid nitrogen (-190ºC) that traps the CO2 while the non-condensable gases are removed with a vacuum line.

The flasks of purified CO2 are placed in the automatic dual gas inlet system of the stable-isotope mass spectroscope to determine the •13C of the samples.

8.7.2.1.2 Method for combusting samples of dissolved solids or pulp using an elemental analyser coupled to a stable-isotope mass spectrometer.

Reagents and materials

• Tin capsules, 5 mm x 9 mm

• Microspatula

• Tweezers

• Micropipette with volume variable from 0 to 10 µl with removable plastic tips

• Reference materials

• The materials used for correct operation of the elemental analyser are described in the manuals of the respective manufacturers of the equipment.

• Helium, 99.999% pure or helium, 99.995% pure with purification traps for water and hydrocarbons.

• Oxygen, 99.996% pure


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• CO2, 99.995% pure

• N2, 99.999% pure

Equipment

• Microbalance with a capacity of 5 g, sensitivity 1.0 µg

• Elemental analyser and interface: see section 8.5 “Instrumentation”

• Stable-isotope mass spectrometer: see section 8.5 “Instrumentation”

8.7.3 Obtaining •13CVPDB results for CO2 produced by any of the combustion methods.

The CO2 resulting from combustion of the samples of dissolved solids and/or pulp described in sub-sections 8.7.1.2 a) and b) is used to determine the 13C/12C isotope ratios using a mass spectrometer with the characteristics described in section 8.5.

The isotope ratios are determined with the isotope species 13C16O16O/12C16O16O from the corresponding intensities of molecular ion clusters of mass 44 and 45 amu corrected for 17O content as per Santrock et al 1985.

8.7.4 Obtaining •15NAIR results for N2 from organic material produced by any of the combustion methods and reduction with metallic copper.

Using the elemental analyser, the oxides of nitrogen obtained from combustion of the pulp are converted to N2 by reduction with metallic copper. The spectrometer parameters must be programmed to obtain the isotope analysis of N2 produced by the pulp used to determine the isotope ratios of 14N/15N by means of a mass spectrometer with the characteristics set out in section 8.5.

The isotope ratios are determined using isotope species 14N14N/14N15N from the corresponding intensities of the molecular ion clusters of mass 28 and 29 uma.

8.7.5 Calculations

The recording unit • is the system of units most commonly used to indicate the isotope content. The • values are used to indicate variations in isotope abundance.

The •13C of a compound is expressed by means of the isotope ratios of the sample with the isotope ratio of the standard in accordance with equation 1 in parts per mille (‰).

∂13CVPDB =(13C /12C)Sample

(13C /12C)VPDB

−1

•103 (Eq. 1)

Where •13C is expressed by the quotients of the stable isotopes (13C/12C) of the sample in relation to the international standard VPDB which is a carbonate of marine origin from the Peedee cretaceous formation in South Carolina (Craig, 1957). Its absolute isotope ratio is (13C/12C)PDB = 0.0112372. This value is the reference point on the PDB scale of • calculated using equation 1 (Eq. 1).


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The stable-isotope mass spectrometer gives the result of each analysis, directly in •13C units including various corrections in the result, including corrections due to pressure, parasite masses, memory effect, residual gas, mixture of gases owing to valve changes and isotope abundance (Mook, W.G. and Grootes P.M. 1973).

As mentioned previously the result given by the spectrometer will be on the VPDB scale if, and only if, the CO2 used as an internal standard is previously calibrated in relation to VPDB. However, to comply with the IUPAC recommendation of 2005, the result of the •13C analysis is subsequently normalised using the L-SVEC and NBS 19 scale, and the normalisation is verified using at least three of the reference materials in table 2, with the sole requirement that they cover the measurement scale of the samples concerned.

In the case of fruit juices, the reference materials may be IAEA-CH7 (polyethylene), NBS-22 (oil) and IAEA-CH6 (sucrose). Both normalisation and verification are carried out using a least-squares regression in which the experimental results obtained from the isotope-ratio mass spectrometer are plotted on the “x” axis and on the “y” axis the values of •13CVPDV assigned to each of the reference materials shown in table 2. The linear correlation coefficient R2 may not be less than 0.9999. For example, a calibration equation resulting from this normalisation might be: •13CVPDB = 1.004*•13CVPDB(experimental) + 0.06. Based on this verified calibration the final result of the value of •13CVPDB normalised in accordance with the IUPAC recommendation of 2005.

In the same way as for carbon, IUPAC recommends that the isotope ratio of any material containing nitrogen should be measured and expressed in d15NAIR which is calculated by the quotients of the stable isotopes (15N/14N) of the sample relative to the international standard, namely air, in accordance with equation 2, the reporting units being parts per mille (‰). In the same way as for carbon, normalisation is carried out using USGS 24; USGS 25 is used to verify the scale of d15NAIR as in the previous example based on the reference materials in table 2.

∂15NAIR =(15N /14N )Sample

(15N /14N )AIR

−1

•103 (Eq. 2)

8.7.6 Reference materials

The reference materials in Table 2 have been approved by the IUPAC Subcommittee on Isotope Abundance Measurements (SIAM) and are acquired from the International Atomic Energy Agency (IAEA) or the National Institute of Standards and Technology (NIST).

The reference materials for calibrating CO2 to be used as a calibration standard and to determine •13CVPDB and •18OVPDB are NBS-19, L-SVEC and NBS-18 which are prepared using the technique by J. M. McCrea (1950) and normalised as per Tyler Coplen, 1988 and 2006.

A tank of calibrated CO2 or N2 may be used, such as those made by Oztech Trading Corporation.

Table 2

Reference materials •13CVPDB. (‰) ± σ •15NAIR (‰) ± σ

NBS-19 calcium carbonate +1.95


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L-SVEC lithium carbonate -46.6

NBS-18 calcium carbonate -5.01 ± 0.06

IAEA-CO-1 calcium carbonate +2.49 ± 0.06

IAEA-CO-8 calcium carbonate -5.76 ± 0.06

IAEA-CO-9 barium carbonate -47.32 ± 0.06

NBS-22 oil -30.03 ± 0.09

IAEA CH7 polyurethane -32.15 ± 0.1

USGS24 graphite -16.05 ± 0.07

IAEA CH6 sucrose -10.45 ± 0.09

USGS40 L-glutamic acid -26.39 ± 0.08 -4.52 ± 0.12

USGS41 L-glutamic acid +37.63 ± 0.1 +47.57 ± 0.22

IAEA-CH3 cellulose -24.72 ± 0.08

IAEA-600 caffeine -27.77 ± 0.09 +1 ± 0.2

IAEA-601 benzoic acid -28.81 ± 0.09

IAEA-602 benzoic acid -28.85 ± 0.09

IAEA-N1 (NH4)2SO4 +0.43 ± 0.07

IAEA-N2 (NH4)2SO4 +20.32 ± 0.09

IAEA-NO-3 KNO3 +4.69 ± 0.09

USGS32 KNO3 +180

USGS34 KNO3 -1.8 ± 0.2

USGS35 KNO3 +2.7 ± 0.2

USGS 25 (NH4)2SO4 -30.25 ± 0.38

USGS 26 (NH4)2SO4 +53.62 ± 0.25

Accuracy

The accuracy of the method for a particular juice can be derived from the results obtained via a validations study of the method carried out between several laboratories. The results of the validation can be found in European standards ENV 12140 and ENV 13070.

Repeatability (of measurement results)

This is defined as the closeness of agreement between the results of successive measurements of the same parameter, carried out applying all the same conditions, i.e. in two analyses for the same sample, the same measuring instrument used under the same conditions, in the same place and repeated with a short period of time.

The isotope analysis of •13CVPDB in organic material has a repeatability as follows:

For dissolved solids the repeatability limit is 0.21‰.

For pulp the repeatability limit is 0.38‰.


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This means that if a sample were sent to the laboratory again for a •13CVPDB analysis, there is a 95% probability that the new result for the sample submitted would be within a range of ±0.21‰ for dissolved solids and ±0.38‰ for pulp.

Reproducibility (of the measurement results)

This is defined as the closeness of agreement between the results of measurements of the same parameter, carried out under varying measurement conditions by different laboratories.

For the isotope analysis of •13CVPDB on organic material reproducibility should be less than the following values:

For dissolved solids the reproducibility limit is ±0.25‰.

For pulp the reproducibility limit is ±0.68‰.

Reporting of results

The results shall be reported using the format suggested in NMX-EC-17025-IMNC-2006 “General requirements for laboratory competence of assays and calibration”, section 8.16.5.10.

Expression of results

C3 plants (orange, apple, grapefruit, grape, mandarine, mango, pear, plum, peach, apricot, guava, papaya, soursop, strawberry, tamarind) have a range of dissolved solids of -24‰ to -28‰, in pulp of -24‰ to -28‰ and in ethanol derived from the fermentation of the corresponding dissolved solids of -25.5‰ to -28‰. These ranges of •13CVPDB do not apply to the pineapple as it is a CAM plant and must therefore be treated separately.

To estimate more accurately the percentage of fruit a juice contains, remember that this requires the •13CVPDB in dissolved solids and the •13CVPDB in pulp to be determined on the same sample, this last parameter being included for use as an internal standard.

This is designed to correct the results of •13CVPDB in dissolved solids due to various factors such as change in the type of fruit or change in the various environmental conditions in which the fruit was grown. This is based on the fact that in fruit both the soluble carbohydrates contained in the juice and other more complex organic molecules that form the pulp are generated simultaneously during the growing process of a given type of fruit and therefore have an almost identical value of •13CVPDB, which can be expressed in the following equation.

%Fruit =∂13CDissolvedSOlid sin Juice − ∂13CAverage(CaneSugar / Maize )

∂13CPulp + 0.24 − ∂13CAverage(CaneSugar / Maize )

∗100 Eq. 3

In a large number of analyses reported in the international literature and from samples analysed in the laboratory it has been found that the value of –11.45‰ is a good estimate for the average •13Caverage(CaneSugar/Maize) to be used in equations 3 and 4. These represent the historical average of various types of carbohydrates from C4 plants (cane sugar and/or sweeteners derived from maize) used as exogenous sweeteners commonly added to juices.


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The •13CVPDB for dissolved solids of fruit is denoted as •13CDissolvedSolidsinJuice. Finally the •13CVPDB for pulp is represented as •13CPulpJuice. Lastly, the values of +0.24 in the denominator of equations 3 and 4 is a constant that was obtained experimentally from the results of analyses carried out on various types of fruit; it is related to the average value of •13CVPDB for dissolved solids of the fruit and the •13CVPDB for pulp which has a correlation close to 1. This constant is used in equation 3 as follows:

%Fruit =∂13CDissolvedSolid sin Juice − (−11.45)

(∂13CPulp + 0.24) − (−11.45)∗100 Eq. 4

Applying equation 4 using the methodology described, validation studies have shown that there is a probable maximum error up to 5% in the determination of % Fruit.

For the special case of clarified juices and which are processed to contain no pulp, such as apple and grape juices, the percentage of dissolved solids in fruit can be calculated using equation 4:

%Fruit =∂13CDissolvedSolid sin Juice − (−11.45)

−11.45∗100 Eq. 5

Applying equation 5 using the methodology described, validation studies have shown that there is a probable maximum error up to 10% in the determination of % dissolved solids in fruit.

Criteria for interpreting the results of •13CVPDB in fruit

The criteria for interpreting the results of applying equations 3 and 4 for the % of dissolved solids in fruit are as follows:

The application of equation 4 for juices containing pulp requires the pulp to be described as an internal standard for which the following conditions apply:

• The •13CVPDB in pulp must be in the range for C3 plants (-28.5‰ a –24‰)

• The analysis of d15NAIR in pulp must be detectable and have a value greater than zero.

• Once pulp is accepted as an internal standard, the following considerations must be taken into account:

• If the numerical difference (•13CVPDB in pulp - •13CVPDB in dissolved solids) is less than 1, and the values of •13CVPDB in dissolved solids and pulp are in the ranges for C3 plants the product can be considered as one in which 100% of the dissolved solids come from fruit and it can be regarded as authentic. In this case, applying equation gives a value of 98% or even slightly higher than 100%.

• If the numerical difference of •13CVPDB in pulp - •13CVPDB in dissolved solids is greater than 1, equation 4 is applied to determine the % of dissolved solids of the fruit which may have an error of 5%.

• If the •13CVPDB in pulp is not in the interval for C3 plants and/or there is no value d15NAIR for the presence of nitrogen then the pulp may not be used as an internal


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standard and equation 3 may not be used. However, equation 5 may be used to determine the % of dissolved solids of fruit, which may have an error of 10%. This implies that thickeners or cellulose have been added, which is prohibited under section 6.1.2 “Stabilisers and thickeners” of this standard.

To calculate the °Brix of dissolved solids of fruit, equation 6 may be applied:

°BrixFruit = °BrixPr oduct ∗%Fruit

100 Eq. 6

°BrixProduct values are determined by direct measurement of °Brix levels of the product being analysed.

9. Commercial information Labelling of products covered by this standard must comply with the provisions of NOM-051-SCFI-1994, identified in the references section.

The trade description must be printed in the same font and type size.

10. Monitoring This official Mexican standard is not certifiable, and compliance will be monitored by the Ministry of the Economy and the Federal Attorney for Consumer Affairs, in line with their respective powers.

11. Bibliography 11.1 Cienfuegos Edith, Casar Isabel and Morales Pedro, (1998). “Carbon isotopic composition of Mexican honey”. Journal of Apicultural Research 36(3/4): 169 – 179.

11.2 Craig Harmon. (1953). The geochemistry of stable carbon isotopes. Geochimica et Cosmochimica Acta, 3: 53 – 92.

11.3 Craig Harmon (1957). Isotopic standards for carbon and oxygen and correction factors for mass–spectrometric analysis of carbon dioxide. Geochimica et Cosmochimica Acta, 12:133 – 149.

11.4 DesMaris, D.J. and Hayes J.M. (1976). Tube cracker for opening glass Sealed ampoules under vacuum. Analytical Chem. 48: 1651 – 1652.

11.5 Mook, W.G. and Grootes P.M. (1973). The measuring procedure and corrections for the high precision mass–spectrometric analysis of isotopic abundance ratios, especially referring to carbon, oxygen and nitrogen. International Journal of Mass Spectrometry and Ion Physics. Vol 12, 273 – 298.

11.6 A.O.A.C. Official Method 981.09 Corn Syrup in Apple Juice Carbon Ratio Mass Spectrometric Method. Chapter 37, P. 19, Fruit and Fruit Products. A.O.A.C. 16th Edition, Vol. II. 1995.

11.7 A.O.A.C. Official Method 982.21 Corn Syrup in Orange Juice Carbon Ratio Mass Spectrometric Method. Chapter 37, P. 20, Fruit and Fruit Products. A.O.A.C. 16th Edition, Vol. II. 1995.


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11.8 J. Bricout & J. Koziet (1987). Control of the Authenticity of Orange Juices by Isotopic Analysis. J. Agric. Food Chem. 35, 758-780.

11.9 Doner Landis & Bills Donald. (1982). Mass Spectrometric 13C/12C Determinations to detect High Fructose Corn Syrup in Orange Juice: Collaborative Study”. J. Assoc. OFF. Anal. Chem 65 (3): 608 – 610.

11.10 J. Koziet, A. Rossmann, G.J. Martin & P.R. Ashurst (1993). Determination of carbon-13 content of sugar of fruit and vegetable juices. Analytica Chimica Acta, 271, 31-38

11.11 G.G. Martin, V. Hanote, M. Lees, Y.L. Martin (1996) Interpretation of combined 2H SNIF/NMR and 13C SIRA/MS analyses of fruit juices to detect added sugar, Journal of AOAC International, Vol. 79, No. 1 62-72.

11.12 M. Gensler & H. L. Schmidt. (1994). Isolation of the main organic acid from fruit juices and nectars for carbon isotope ratio measurements. Analytica Chimica Acta, 299, 231-237.

11.13 E. Jamin, J. González, G. Remaud, N. Naulet & G. Martin. (1997). Detection of Exogenous Sugar or Organic Acid Addition in Pineapple Juices and concentrates by 13C IRMS Analysis. J. Agric. Food Chem. 45, 3961-3967.

11.14 J. Koziet, A. Rossmann, G.J. Martin, P. Johnson. (1995). Determination of the oxygen-18 and deuterium content of fruit and vegetable juice water an European inter-laboratory comparison study, Analytica Chimica Acta 302 (1) pp. 29-37. Anal. Chim. Acta. (1995), 302, 29-37.

11.15 UNE-EN-12140:1997 Zumos de frutas y hortalizas. Determinación de la relación de los isótopos estables del carbono (13C/12C) en los azúcares de los zumos de fruta. Método por Espectrometría de Masas de Relaciones Isotópicas.

11.16 PNE-ENV-13070 Zumos de fruta y hortalizas. Determinación de la relación de los isótopos estables de carbono (13C/12C) en la pulpa de los zumos de fruta. Método por espectrometría de masas de relaciones isotópicas.

11.17 Code of Practice published by the AINJ – Association of the Juice and Nectar producing Industry.

11.18 Handbook of indices of Food Quality and Authenticity. Woodhead Publishing, 1997.

11.19 Reglamento de Control Sanitario de Productos y Servicios VII.1

12. Concordance with international standards This official Mexican standard concords partially with international standard CODEX STAN 247-2005, Codex general standard for fruit juices and nectars.

Mexico City, 14 November 2008.- Director-General for Standards and Chairman of the National Advisory Committee on Standardization for User Safety, Commercial Information and Trade Practices, Francisco Ramos Gómez.- Initialled.

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