AS-2300.1.8-2008.pdf

1 AS 2300.1.82008 www.standards.org.au Australian Standard ® Methods of chemical and physical testing for the dairying industry Method 1.8: General methods and principles Assessment of instrumental methodsProtein in milk and infrared spectrometric analysis of milk PREFACE This Standard was prepared by the Standards Australia Committee FT-024, Food Products and Subcommittee FT-024-05, Dairy Products, to supersede AS 2300.1.8.1, Methods of chemical and physical testing for the dairying industryMethod 1.8.1: General, methods and principlesAnalytical quality assurance of instrumental methodsProtein in milk and AS 2300.1.8.2, Methods of chemical and physical testing for the dairying industry Method 1.8.2: General methods and principlesAssessment of instrumental methods Infrared spectrometric analysis of milk. After a periodic review, the Committee recommended to amalgamate two parts into one Standard to provide procedures to assess the capabilities of instrumental methods for the determination of protein in milk. This edition confirms the method without technical changes, but updates the referenced documents and reflects the current editorial style. It includes a clause on uncertainty of measurement. AS 2300 comprises a series of methods and related Standards for chemical and physical testing of milk and dairy products, including the preparation of samples for testing. Standards in the AS 2300 series are divided into categories according to type of product to be tested, as follows: AS 2300.1 General methods and principles 2300.2 Liquid milks 2300.4 Dried milk and dried milk products 2300.5 Condensed milk 2300.6 Cheese 2300.7 Butter 2300.8 Anhydrous milk fat 2300.9 Analysis of ice-cream and frozen milk products 2300.10 Caseins, caseinates and coprecipitates 2300.11 Cultured milk products AS 2300.1.82008 Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 AS 2300.1.82008 2 © Standards Australia www.standards.org.au FOREWORD The increasing use of instrumental methods for estimation of the composition of milk has made it necessary for a standard procedure to be devised to assess the capabilities of instrumental methods compared with accepted standard chemical methods. The purpose of Section 2 of this Standard is to provide a means by which the protein content of liquid milk from an instrumental method can be related to the results obtained by a standard chemical method. When reporting results obtained by an instrumental test method, the analyst may quote that the instrument has been validated in accordance with this Standard. Section 3 of this Standard deals with the assessment of the performance of mid-infrared instruments for analyzing milk. This Standard makes no attempt to describe routine procedures for their operation; this is left to the manufacturers manual of instructions and regulatory or local requirements for calibration, with the expectation that good laboratory practice will be used. Further information on routine procedures for operation of the instruments is also given in International Dairy Federation (IDF) Provisional Standard 141 (Ref. 1). The assumption is also made that the instruments are designed to operate on sound physical and chemical principles. While instrumental methods are less susceptible to operator bias, a rigid discipline of check procedures as described in the manufacturers manual is essential to ensure that the instrument functions at all times within its design limits. METHOD S E C T I O N 1 S C O P E A N D G E N E R A L 1.1 SCOPE This Standard describes a procedure to assess the capabilities of instrumental methods with accepted standard chemical methods. 1.2 REFERENCED DOCUMENTS The following Standards are referred to in this Standard. AS 2300 Methods of chemical and physical testing for the dairying industry 2300.1.2.1 Method 1.2.1: General methods and principlesDetermination of nitrogen Reference Kjeldahl method 2300.1.2.2 Method 1.2.2: General methods and principlesDetermination of nitrogen Nitrogen fractions from milk 2300.1.3 Method 1.3: General methods and principlesDetermination of fat Gravimetric method 2300.2.6 Method 2.6: Liquid milksDetermination of lactose Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 3 AS 2300.1.82008 www.standards.org.au © Standards Australia AS/NZS 2243 Safety in laboratories 2243.2 Part 2: Chemical aspects WARNING: THE USE OF THIS STANDARD MAY INVOLVE THE USE OF HAZARDOUS MATERIALS, OPERATIONS, AND EQUIPMENT. THIS STANDARD DOES NOT PURPORT TO ADDRESS ALL THE SAFETY RISKS ASSOCIATED WITH ITS USE. IT IS THE RESPONSIBILITY OF THE USER OF THIS STANDARD TO ESTABLISH APPROPRIATE SAFETY AND HEALTHY PRACTICES AND DETERMINE THE APPLICABILITY OF LOCAL REGULATORY LIMITATIONS PRIOR TO USE. SEE AS/NZS 2243.2 FOR MORE DETAILS REGARDING LABORATORY SAFETY. 1.3 ACCURACY UNCERTAINTY OF MEASUREMENT Estimate the uncertainty of test measurements based on the performance characteristics of the test method (trueness and precision), verification data, quality control data, previous experience or other relevant information. The appropriate degree of rigour applied to estimation of uncertainty depends on the purpose of the test and customer (end-user) requirements. NOTES: 1An estimate of uncertainty is required when it is relevant to the validity or application of the test result or when requested by a customer. 2Unless otherwise required, the measurement value should be stated together with the expanded uncertainty, calculated using a coverage factor of 2, to give a level of confidence of approximately 95%. 3The measurement value and its uncertainty should both be reported in the same units, the measurement value rounded to be consistent with its uncertainty. 4The form of reporting the result must not give a wrong impression of the uncertainty. Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 AS 2300.1.82008 4 © Standards Australia www.standards.org.au S E C T I O N 2 P R O T E I N I N M I L K 2.1 SCOPE This Section describes a procedure for evaluating the suitability of an individual instrument for measuring protein in liquid milk. NOTE: This is intended for instruments with multi-component capability such as those that can also determine fat and lactose. However, the Section does not validate the instruments capabilities for measuring analytes other than protein. 2.2 APPLICATION The procedure is applicable to all instruments capable of analysing for both crude and true protein (see Clause 2.3) in liquid milk. It is intended to verify that an instrument is capable of performing a single analysis on a sample of milk to produce a result of comparable precision and accuracy to that obtained when the protein content of the milk is estimated from the nitrogen content, determined by the reference Kjeldahl method set out in AS 2300.1.2.1. 2.3 DEFINITIONS For the purpose of this Standard, the following definitions apply. 2.3.1 Crude protein The total nitrogen content of the sample multiplied by the factor 6.38. 2.3.2 True protein The total nitrogen content of the sample minus the non-protein nitrogen content and multiplied by the factor 6.38. NOTE: The total nitrogen and non-protein nitrogen contents are determined by the methods set out in AS 2300.1.2.2. 2.4 PRINCIPLE After the instrumental performance checks, adjustments and calibrations have been carried out according to the manufacturers instructions, suitable samples of milk of the type to be analysed are measured for protein content by both the instrument and the reference Kjeldahl procedure. The results of both methods are compared by a defined statistical procedure. The conformance of the instrument is judged by defined criteria. 2.5 TEST SAMPLES Samples used in the comparison should be of the type to be measured. They should be prepared as described by the manufacturer for the particular instrument, and as appropriate for Kjeldahl analysis. 2.6 COMPARISON OF INSTRUMENT WITH REFERENCE METHOD 2.6.1 General It is essential that all steps in the procedure are carried out on the same day. It must be known that the instrument is performing correctly before its accuracy is assessed. Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 5 AS 2300.1.82008 www.standards.org.au © Standards Australia 2.6.2 Instrument performance checks Before calibrating the instrument, checks of zero settings, linearity, purging efficiency and repeatability are performed. Make any adjustments necessary for correct performance of the instrument according to the manufacturers instructions. NOTE: Appendix A gives recommended methods for checking repeatability and purging efficiency. 2.6.3 Instrument calibration Calibrate the instrument according to the manufacturers instructions using milk samples of the type to be measured. 2.6.4 Comparative analyses The procedure shall be as follows: (a)Analyse in duplicate a set of at least eight prepared samples covering the range of protein content normally measured, using the reference Kjeldahl method set out in AS 2300.1.2.1. Use the same procedures as those used to obtain the reference values with which the instrument is calibrated. (b)Analyse the samples by the instrumental method, in ascending order of protein content. Repeat the instrumental analysis in descending order of protein content. NOTES: 1The first test (possibly more) made with the instrument after a rest period may give an incorrect result. 2The first test (possibly more) made with the instrument after it has been used for a sample with a widely different protein content, may give a result that is biased towards that of the previous sample. 3More accurate results can be obtained with the instrument by repeating the tests on a sample until there is no drift in the readings, and by then taking the average of a number of subsequent readings. 2.7 CALCULATIONS 2.7.1 Mean difference Calculate the mean difference (MD) between reference and instrument results as follows: n D MD = . . . 2.7(1) where D= the difference between the average reference result and each single instrument result for each of the samples tested n= the number of instrument tests 2.7.2 Standard deviation Calculate the standard deviation of difference between reference and instrument results SD(diff) from the following equation: SD(diff) = () × 1 2 n MDDD . . . 2.7(2) where D, n and MD are shown in Clause 2.7.1. Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 AS 2300.1.82008 6 © Standards Australia www.standards.org.au 2.8 CRITERIA FOR ACCEPTABILITY OF AN INSTRUMENT An instrument is considered to be acceptable, i.e. provides results which are comparable with the reference method AS 2300.1.2.1, if it meets the following criteria resulting from the tests carried out in accordance with this Standard: (a)Mean difference between instrument results and reference results is not more than 0.05 percent protein. (b)Standard deviation of difference between instrument results and reference results is not more than 0.06 percent protein. 2.9 REPORT When reporting the use of this Standard for assessing the compliance of an instrument, include the following details: (a)The brand and model of instrument used. (b)The nature of the samples used in the tests. (c)Reference to this Australian Standard, i.e. AS 2300.1.8. (d)The reference method used to determine protein values, i.e. AS 2300.1.2.1. (e)The results determined as true or crude protein (see Clause 2.3). (f)Where applicable, a statement concerning the estimated uncertainty of measurement. (g)Date of the tests performed. (h)Any circumstance or unusual observations made during the course of the test which may have had an effect on the result. 2.10 BIBLIOGRAPHY Sections 972.16A to 972.16G, Official Methods of the Association of Official Analytical Chemists, 18th ed., 2005, AOAC Inc. Arlington, Virginia, USA. Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 7 AS 2300.1.82008 www.standards.org.au © Standards Australia S E C T I O N 3 I N F R A R E D S P E C T R O M E T R I C A N A L Y S I S O F M I L K 3.1 SCOPE This Section sets out a procedure for evaluating the suitability of a mid-infrared spectrometric instrument for quantitatively determining fat, protein and lactose in liquid milks. NOTES: 1The procedure may be used to assess a new instrument or an instrument after major repair, or for comparing instruments. 2The evaluation is based on a group of performance tests similar to those published in the Association of Official Analytical Chemists (AOAC) Official Methods of Analysis (Ref. 2). 3Mid-infrared instruments are usually suitable for testing whole milk, skimmed and partially skimmed milk, homogenized milk and modified milk. Some instruments are also suitable for testing cream directly. 4The instrument may also provide an estimated solids-not-fat (SNF) content by assigning a value for the minerals content, obtained by experiment, and adding the sum of the measured protein and lactose contents. 3.2 PRINCIPLE Suitable samples of milk, of the type used for calibration (but not the same samples), are analysed for fat, protein and lactose content by appropriate reference chemical methods. The same samples are analysed by the instrument, which measures the quantity of radiation absorbed by the following: (a)For fat determinationthe carbonyl bonds of the glyceryl esters at approximately 5.73 m (A Filter) or the C-H bonds at approximately 3.48 m (B Filter). (b)For protein determinationthe secondary amide groups of the peptide bonds at approximately 6.46 m. (c)For lactose determinationthe hydroxyl groups of lactose at approximately 9.61 m. The results of both analyses are compared by a defined statistical procedure. The conformance of the instrument is judged against defined criteria. 3.3 FACTORS AFFECTING THE ACCURACY OF ANALYSES 3.3.1 Instrumental factors 3.3.1.1 Linearity The linearity of the instruments responses to a range of analyte concentrations should be checked according to the manufacturers instructions before calibration checks and adjustments are made. The following procedures are recommended: (a)For fatuse eight dilutions of homogenized low-fat cream or whole milk concentrate, which have been accurately made on a mass/mass basis using skim milk as the diluent. (b)For proteinuse eight dilutions of skim-milk concentrate. (c)For lactoseuse a solution of lactose monohydrate of a concentration corresponding to the desired upper limit of instrument reading (usually 50 g/L is suitable), diluted to provide eight solutions. Accessed by UNIVERSITY OF SOUTH AUSTRALIA on 03 Apr 2009 AS 2300.1.82008 8 © Standards Australia www.standards.org.au Concentrations of the dilutions used should be evenly distributed from zero to the desired upper limits of instrument readings. If the response is not linear, the instrument should be adjusted in accordance with the manufacturers instructions. 3.3.1.2 Purging