INTRODUCTION TO FOOD

ANALYSIS

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 INTRODUCTION
• All food products require analysis as part of a quality
management program.
• The nature of the sample and the specific reason for the
analysis commonly dictate the choice of analytical
methods.
• Speed, precision, accuracy, and ruggedness often are key
factors in this choice.
• Validation of the method for the specific food matrix being
analyzed is necessary.
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Purpose of Characteristics of Compound/ Characteristic
Analysis Methods of Interest

Applications:
Selecting specific method
to analyze specific
component/ characteristic
in specific food

Method selection in food analysis

3
• The success of any analytical method relies on the proper
selection and preparation of the food sample, carefully
performing the analysis, doing the appropriate
calculations and interpretation of the data.
• Official methods are critical in the analysis of foods, to
ensure that they meet the legal requirements established by
governmental agencies.

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 TYPES OF SAMPLES ANALYZED

• Chemical analysis of foods is an important part of a quality

assurance program in food processing, from ingredients and
raw materials, through processing, to the finished products.

• Chemical analysis also is important in formulating and

developing new products, evaluating new processes for
making food products, identifying the source of problems
with unacceptable products.

5
• It may be necessary to determine either just one or many
components.

• Process control samples are usually analyzed by rapid

methods.

• Nutritive value information for nutrition labelling generally

requires the use of more time-consuming methods.

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 Types of samples analyzed in a quality assurance program
for food products
Sample Type Critical Questions

Raw materials Do they meet your specifications?

Do they meet required legal specifications?
Are they safe and authentic?
Will a processing parameter have to be modified because of
any change in the composition of raw materials?
Are the quality and composition the same as for previous
deliveries?
How does the material from a potential new supplier
compare to that from the current supplier?

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Sample Type Critical Questions
Process Did a specific processing step result in a product of acceptable
control composition or characteristics?
samples Does a further processing step need to be modified to obtain a
final product of acceptable quality?
Finished Does it meet the legal requirements?
product What is the nutritive value, so that label information can be
developed? Or is the nutritive value as specified on an existing
label?
Does it meet product claim requirements (e.g., “low fat”)?
Will it be acceptable to the consumer?
Will it have the appropriate shelf life?
If unacceptable and cannot be salvaged, how do you handle it
(trash? rework? seconds?)
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 STEPS IN ANALYSIS
• Select and prepare sample:
– All results depend on obtaining a representative sample and
converting the sample to a form that can be analyzed.
– Sampling is the initial point for sample identification.
– Analytical laboratories must keep track of incoming samples and
be able to store the analytical data from the analyses.
– Analytical information often is stored on a laboratory
information management system, or LIMS, which is a
computer database program.

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CHOICE AND VALIDITY OF METHOD

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 Objective of the Assay

• Selection of a method depends largely on the objective of the

measurement.
• Methods used for rapid online processing measurements may
be less accurate than official methods used for nutritional
labelling purposes.
• Methods referred to as reference, definitive, official, or
primary are most applicable in a well-equipped and staffed
analytical lab.

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• The more rapid secondary or field methods may be more
applicable on the manufacturing floor in a food processing
facility.

• For example, refractive index may be used as a rapid,

secondary method for sugar analysis the primary method,
high-performance liquid chromatography (HPLC).

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 Consideration of Food Composition
and Characteristics
• Proximate analysis of foods refers to determining the major
components of moisture, ash, lipids, protein, and
carbohydrates.
• The performance of many analytical methods is affected by
the food matrix.
• High-fat or high-sugar foods can cause different types of
interferences than low-fat or low-sugar foods.
• Digestion procedures and extraction steps necessary for
accurate analytical results can be very dependent on the
food matrix.
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• AOAC International suggested a “triangle scheme” for
dividing foods into matrix categories.

• The apexes of the triangle contain food groups that were

either 100% fat, 100% protein, or 100% carbohydrate.

• Foods were rated as “high,” “low,” or “medium” based on

levels of fat, carbohydrate, and proteins.

• This created nine possible combinations of high, medium,

and low levels of fat, carbohydrate, and protein.

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 Schematic layout of food
matrixes based on protein, fat,
and carbohydrate content,
excluding moisture and ash.

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• General analytical methods ideally would be geared to handle
each of the nine combinations, replacing more numerous
matrix-dependent methods developed for specific foods.

• Using matrix-dependent methods, one method might be

applied to potato chips and chocolates, both of which are
low-protein, medium-fat, medium-carbohydrate foods, but
another might be required for a high-protein, low-fat,
high-carbohydrate food such as nonfat dry milk.

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 Validity of the method
• Must consider certain characteristics of any method, such as
specificity, precision, accuracy, and sensitivity.
• Also must consider the variability of data.
• Must consider the nature of the samples collected for the
analysis, how representative the samples were of the whole,
and the number of samples analyzed.
• Must ask whether details of the analytical procedure were
followed adequately, such that the results are accurate,
repeatable, and comparable to data collected previously.
• Equipment to conduct the analysis must be standardized and
appropriately used, the performance limitations of the
equipment must be recognized.
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 Criteria for choice of food analysis methods
Characteristic Critical Questions
Inherent properties
• Specificity/ Is the property being measured the same as that claimed to
selectivity be measured, and is it the only property being measured?
Are there interferences?
What steps are being taken to ensure a high degree of
specificity?
• Precision What is the precision of the method? Is there within-batch,
batch-to-batch, or day-to-day variation?
What step in the procedure contributes the greatest
variability?
• Accuracy How does the new method compare in accuracy to the old
or a standard method?
What is the percent recovery?
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Characteristic Critical Questions
Applicability of method to laboratory
• Sample size How much sample is needed?
Is it too large or too small to fit your needs?
Does it fit your equipment and/or glassware?
Can you obtain representative sample?
• Reagents Can you properly prepare them?
What equipment is needed? Are they stable? For how long and
under what conditions?

• Equipment Is the method very sensitive to slight or moderate changes in the

reagents?
Do you have the appropriate equipment?
Are personnel competent to operate equipment?
• Cost What is the cost in terms of equipment, reagents, and personnel?
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Characteristic Critical Questions

Usefulness

• Time How fast is it? How fast does it need to be?

required
• Reliability How reliable is it from the standpoints of precision and stability?

• Need Does it meet a need or better meet a need?

Personnel

Is any change in method worth the trouble of the change?

• Safety Are special precautions necessary?

• Procedures Who will prepare the written description of the procedures and
reagents?
Who will do any required calculations?
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• The analysis of materials used as controls, often referred to as
standard reference materials or check samples.
• Analyzing check samples concurrently with test samples is an
important part of quality control.
• AACC International has a check sample service in which a
subscribing laboratory receives specifically prepared test
samples from AACC International.
• The subscribing laboratory performs the specified analyses
on the samples and returns the results to AACC
International.
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• AACC International provides a statistical evaluation of the
analytical results, compares the subscribing laboratory’s
data with those of other laboratories to inform the
subscribing laboratory of its degree of accuracy.
• The AACC International offers check samples such as flours,
semolina, and other cereal-based samples, for analyses such
as moisture, ash, protein, vitamins, minerals, sugars,
sodium, total dietary fibre, soluble and insoluble dietary
fibre, and β-glucan.

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• The American Oil Chemists’ Society (AOCS) has a
reference sample program for oilseeds, oilseed meals,
marine oils, aflatoxin, cholesterol, trace metals, specialty
oils suitable for determination of trans fatty acids, and
formulations for nutritional labelling.
• Laboratories from many countries participate in the program
to check the accuracy of their work, their reagents, and
their laboratory apparatus against the statistical norm
derived from the group data.

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 OFFICIAL METHODS

• Official methods have been carefully developed and

standardized.

• They allow for comparability of results between different

laboratories that follow the same procedure and for
evaluating results obtained using new or more rapid
procedures.

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 AOAC International
• An organization begun in 1884 to serve the analytical
methods needs of government regulatory and research
agencies.
• The goal is to provide methods that will be fit for their
intended purpose.
• This volunteer organization functions as follows:
1. Methods of analysis from published literature are selected or
new methods are developed by AOAC International
volunteers.
2. Methods are collaboratively tested using multi-laboratory
studies in volunteers’ laboratories.
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 3. Methods are given a multilevel peer review by expert scientists,
and if found acceptable, they are adopted as official methods of
analysis.
4. Adopted methods are published in the Official Methods of
Analysis, which covers a wide variety of assays related to foods,
drugs, cosmetics, agriculture, forensic science, and products
affecting public health and welfare.
5. AOAC International publishes manuals, methods compilations in
specific areas of analysis, monographs, and the monthly
magazine Inside Laboratory Management.
6. AOAC International conducts training courses of interest to
analytical scientists and other laboratory personnel.
• Methods must be successfully validated in a formal inter-
laboratory collaborative study before being accepted as an
official first action method by AOAC.
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Table of Contents of 2007 Official Methods of Analysis of AOAC International

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 Table of Contents of 2010
Approved Methods of AACC
International

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Table of Contents of 2009 Official
Methods and Recommended
Practices of the American Oil
Chemists’ Society

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 Contents of Chemical and
Physical Methods in Standard
Methods for the Examination of
Dairy Products

• Standard Methods for the

Examination of Dairy
Products includes methods
for the chemical analysis of
milk and dairy products.

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 SUMMARY
• Food scientists and technologists determine the chemical
composition and physical characteristics of foods routinely
as part of their quality management, product development,
or research activities.
• For example, the types of samples analyzed in a quality
management program of a food company can include raw
materials, process control samples, finished products,
competitors’ samples, and consumer complaint samples.
• Consumer, food industry, and government concern for food
quality and safety has increased the importance of analyses
that determine composition and critical product
characteristics.
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• To successfully base decisions on results of any analysis, one
must correctly conduct all three major steps in the analysis:
1. select and prepare samples,
2. perform the assay,
3. calculate and interpret the results.
• The choice of analysis method is usually based on the
objective of the analysis, characteristics of the method itself
(e.g., specificity, accuracy, precision, speed, cost of
equipment, and training of personnel), and the food matrix
involved.

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• Validation of the method is important, as is the use of
standard reference materials to ensure quality results.
• Rapid methods used for quality assessment in a production
facility may be less accurate but much faster than official
methods used for nutritional labelling.
• Methods allow for comparison of results between different
laboratories and for evaluation of new or more rapid
procedures.

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 REGULATIONS AND
INTERNATIONAL
STANDARDS RELATED TO
FOOD ANALYSIS

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 Regulations
Good Manufacturing Practice in Manufacturing, Packing, or
Holding Human Food
• The GMP regulations define requirements for acceptable
sanitary operation in food plants and include the following
relevant to food processing:
1. General provisions that define and interpret the detailed
regulations.
2. Requirements and expectations for maintaining grounds, buildings,
and facilities.
3. Requirements and expectations for design, construction, and
maintenance of equipment.
4. Requirements for production and process controls.
5. Defect action levels (DALs) for natural and unavoidable defects.
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 Hazard Analysis Critical Control Point
• HACCP is an internationally recognized systematic approach
that is used to prevent and/or control microbial, chemical,
and physical hazards within the food supply.

• The “farm to the fork” approach was originally designed to

be used by the food processing industry to produce zero
defect (no hazard) food for astronauts to consume on
space flights.

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• HACCP program approach is based on seven principles
identified below:
1. Determine potential microbial, chemical, and physical hazards in
each step of the process flow.
2. Identify critical control points in the process.
3. Establish control limits for each critical control point.
4. Establish procedures to monitor control points.
5. Establish corrective actions when limits of control point are
exceeded.
6. Establish appropriate system of record keeping.
7. Establish program to verify and validate efficacy of program.
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 Food definitions and standards
• The standards of identity are most relevant to the chemical
analysis of foods because they specifically establish which
ingredients a food must contain.
• They limit the amount of water permitted in certain products.
• The minimum levels for expensive ingredients are often set,
and maximum levels for inexpensive ingredients are
sometimes set.
• The kind and amount of certain vitamins and minerals that
must be present in foods labelled “enriched” are specified.
• The standard of identity often includes the recommended
analytical method for determining chemical composition.
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 Selected Chemical Composition
Requirements of Some Foods with
Standards of Identity

bOfficial Methods of Analysis of AOAC International.

cIU, International units.
dWithin limits of good manufacturing practice.
eIf pasteurized dairy ingredients are used.

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bOfficialMethods of Analysis of AOAC International.
fExcluding ash resulting from any added iron or salts of iron or calcium or

wheat germ.
gdwb, moisture-free or dry weight basis.

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bOfficial Methods of Analysis of AOAC International.
gdwb, moisture-free or dry weight basis.
hExclusive of added sugars, without added water. As determined by refractometer at 20 ◦C

uncorrected for acidity and read as ◦Brix on International Sucrose Scales. Exception stated for
juice from concentrate.
iDetailed titration method given in 21 CFR, 145.180 (b)(2)(ix).
jCalculated from ◦Brix and total acidity values, as described in 21 CFR 146.185 (b)(2)(ii).
kDetailed method given in 21 CFR 146.185 (b)(2)(iv).

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 REGULATION AND RECOMMENDATIONS
FOR MILK

• The standards for Grade A pasteurized milk and milk

products and bulk-shipped heat-treated milk products
under the PMO.

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 Pasteurized Milk Ordinance Standards for Grade A Pasteurized Milk
and Milk Products and Bulk-Shipped Heat-Treated Milk Products

Criteria Requirement
Temperature Cooled to 7 °C (45 °F) or less and maintained thereat
Bacterial limitsa 20,000 per ml °
Coliformb Not to exceed 10 per ml. Provided, that in the case of bulk
milk transport tank shipments, shall not exceed 100 per ml.
Phosphataseb Less than 350 milliunits/L for fluid products and other milk
products by the Fluorometer or Charm ALP or equivalent.
Drugsc No positive results on drug residue detection methods.
aNot applicable to acidified or cultured products.
bNot applicable to bulk-shipped heat-treated milk products.

cReference to specific laboratory techniques.

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• Nonfat dry milk is graded based on flavour, physical
appearance, and various laboratory analyses.

US Standards for Grades of Nonfat Dry Milk (Spray Process)

US Standards: [Link]
dDocName_STELDEV3004466.
aAll values are maximum allowed.

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INTERNATIONAL STANDARDS AND POLICIES
• Codex Alimentarius:
– Codex Alimentarius is Latin for “code concerned with
nourishment”.
– The Codex Alimentarius is published in 13 volumes.
– Codex has efforts to validate and harmonize methods of food
safety analysis among countries and regions, to help maintain
the smooth flow of international commerce, and ensure
appropriate decisions on food exports and imports.

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Content of the Codex
Alimentarius

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• ISO Standards:
– The International Organization for Standardization (ISO)
has the series of standards on quality management and quality
performance.
• Other Standards:
– The European Commission sets standards for foods and food
additives for countries in the European Economic Community
(EEC).
– The chemical composition of foods is often an important factor
in determining the quality, grade, and price of a food.
– Government agencies that purchase foods for special programs
often rely on detailed specifications that include information
on food composition.
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– International organizations have developed food standards and
safety practices to protect consumers, ensure fair business
practices, and facilitate international trade.
– The Codex Alimentarius Commission is the major international
standard-setting group for food safety and quality.
– The International Organization for Standardization has a series of
standards that focus on documentation of procedures, with
some relevant to food analysis.

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 SUMMARY

• Various kinds of standards set for certain food products by

federal agencies make it possible to get essentially the
same food product whenever and wherever purchased.

• The standards of identity set define what certain food

products must consist of.

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NUTRITION LABELLING

50
 INTRODUCTION

• A major reason for analyzing the chemical components

of foods is nutrition labelling regulations.

• Nutrition label information is not only legally required in

many countries, but also is of increasing importance to
consumers as they focus more on health and wellness.

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Mandatory (Bold) and Voluntary Components for Food Label

• Nutrition panel will have the

heading “Nutrition Facts.”
• Only components listed are
allowed on the nutrition
panel, and they must be in
the order listed.
• Components are to be
expressed as amount and/or
as percent of an established
“Daily Value.”

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An example of the nutrition label

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 FOOD LABELLING REGULATIONS
Mandatory Nutrition Labelling
• Basic Format:
The standard format for nutrition information on food labels
food labels consists of the following:
– Serving size and servings per container.
– Quantitative amount per serving of each nutrient or dietary
component except vitamins and minerals.
– Amount of each nutrient, except sugars and protein, as a percent
of the Daily Value for a 2000 Calorie (Cal) diet.
– Footnote with Daily Values for selected nutrients based on
2000 Cal and 2500 Cal diets.
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• Daily Values and Serving Size:
– Daily Value (DV) is a generic term used to describe two
separate terms that are (1) Reference Daily Intake (RDI)
and (2) Daily Reference Value (DRV).
– The term RDI is used for essential vitamins and minerals.
– The term DRV is used for food components (total fat, saturated
fat, cholesterol, total carbohydrate, dietary fibre, sodium,
potassium, and protein).

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Reference Daily Intakes (RDIs) for
Vitamins and Minerals Essential in
Human Nutrition

• Values are for adults and

children 4 or more years of
age.
• RDI values have also been
established for infants,
children under 4 years of age,
and pregnant and lactating
women.

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Daily Reference Values (DRVs) of Food Components Based
on the Reference Calorie Intake of 2000 Calories

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– These data in the table are based on national food consumption
surveys as well as the serving size used in dietary guidance
recommendations, serving sizes recommended in comments,
serving size used by manufactures and grocers, or serving
sizes used by other countries.

– The labelled serving size and reference amount are important

since the use of nutrient content claims is dependent on the
serving size and the reference amount.

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• Simplified Format
– A simplified format for nutrition information may be used if
seven or more of the 13 required nutrients are present in only
insignificant amounts.
– For such foods, information on five core nutrients (Calories,
total fat, total carbohydrate, protein, and sodium) must be
given.
– If other mandatory nutrients are present in more than
insignificant amounts they must be listed.
– In the cases of protein, total carbohydrate, and dietary fibre,
insignificant is the amount that allows a statement of “less
than 1 gram.”
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Rounding Rules for Declaring Nutrients on Nutrition Label

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61
aTo express to the nearest 1g increment, amounts exactly halfway between two
whole numbers or higher (e.g., 2.50–2.99 g) round up (e.g., 3 g), and amounts
less than halfway between two whole numbers (e.g., 2.01–2.49 g) round down
(e.g., 2 g).
bNotes for rounding % Daily Value (DV) for total fat, saturated fat, cholesterol,

sodium, total carbohydrate, fibre, and protein:

1. To calculate %DV, divide either the actual (unrounded) quantitative
amount or the declared (rounded) amount by the appropriate RDI or
DRV. Use whichever amount will provide the greatest consistency on
the food label and prevent unnecessary consumer confusion (21
CFR 101.9 (d)(7)(iii).
2. When %DV values fall between two whole numbers, rounding shall be
as follows:
• For values exactly halfway between two whole numbers or higher
(e.g., 2.50–2.99) the values shall round up (e.g., 3%).
• For values less than halfway between whole numbers (e.g., 2.01–2.49)
the values shall round down (e.g., 2%).
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• Protein Quality
– For infant foods, the corrected amount of protein per serving is
calculated by multiplying the actual amount of protein (g) per
serving by the relative protein quality value.
– Relative quality value is the protein efficiency ratio (PER)
value of the subject food product divided by the PER value for
casein.
– The corrected amount of protein per serving is equal to the
actual amount of protein (g) per serving multiplied by the
protein digestibility-corrected amino acid score
(PDCAAS).

63
 EVALUATION OF
ANALYTICAL DATA

64
 INTRODUCTION

• The focus of this chapter on how to evaluate replicate

analyses of the same sample for accuracy and precision.

65
X

MEASURES OF CENTRAL TENDENCY

• To increase accuracy and precision the analysis of a sample
is usually performed (repeated) several times.
• At least three assays are typically performed, we determine
the mean (or average) values obtained and report the
results of the mean.
• The mean is designated by the symbol x and calculated
according to the equation.
x = mean
x1 + x 2 + x 3 + ... + xn Σx i x1, x2, etc. = individually measured
x= = values (xi)
n n
n = number of measurements
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• We measured a sample of uncooked hamburger for percent
moisture content four times and obtained the following
results: 64.53%, 64.45%, 65.10%, and 64.78%.

64.53 + 64.45 + 65.10 + 64.78

x= = 64.72%
4

• The result would be reported as 64.72% moisture.

• When we report the mean value, we are indicating that this is
the best experimental estimate of the value.

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 RELIABILITY OF ANALYSIS
• In our previous example, we did not have any indication of
how repeatable the tests were how close our results were
to the true value.

Accuracy and precision

• The first data obtained are the individual results and a mean
value ( x ).
• “How close were our individual measurements?”
• “How close were they to the true value?”

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• Accuracy refers to how close a particular measure is to the
true or correct value.
– Most of the time we are not sure what the true value is.

• The range is simply the difference between the largest and

smallest observation.
– This measurement is not too useful and thus is seldom used in
evaluating data.

69
• Precision is a measure of how reproducible or how close
replicate measurements become.
– If repetitive testing yields similar results, then we would say
that the precision of that test was good.
– The precision often is called error, when we are actually
looking at experimental variation.
– The concepts of precision, error, and variation are closely
related.

70
 Comparison of accuracy and
precision

(a)Good accuracy and good precision

(b)Good precision and poor accuracy
(c)Good accuracy and poor precision
(d)Poor accuracy and poor precision

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• The standard deviation measures the spread of the
experimental values and gives a good indication of how
close the values are to each other.
– The standard deviation is designated by the Greek letter sigma (σ).
– It is calculated according to equation:
σ = standard deviation
Σ(x i − μ) 2
σ= xi = individual sample values
n μ = true mean
n = total population of samples

– We do not know the value for the true mean, the equation becomes
somewhat simplified so that we can use it with real data.
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 Determination of the Standard Deviation of
Percent Moisture in Uncooked Hamburger
Observed % Deviation from
Measurement the Mean (x i − x )2
Moisture (x i − x )

1 64.53 – 0.19 0.0361

2 64.45 – 0.27 0.0729
3 65.10 + 0.38 0.1444
4 64.78 + 0.06 0.0036
Σxi = 258.86 Σ(xi − x )2 = 0.257

Σxi 258.86 Σ(xi − x )2 0.257

x= = = 64.72 SD = = = 0.2927
n 4 n −1 3
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• Coefficient of variation (CV), also known as the relative
standard deviation.

Coefficient of variation (CV) = SD × 100%

x
CV = 0.293 × 100% = 0.453%
64.72

– The CV tells us that our standard deviation is only 0.453% as

large as the mean.

– As a rule, a CV below 5% is considered acceptable, although it

depends on the type of analysis.

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• In a population with a normal distribution, 68% of those
values would be within ±1 standard deviation from the
mean, 95% would be within ±2 standard deviations, and
99.7% would be within ±3 standard deviations.
• For large numbers of samples, we can determine the
confidence limit or interval around the mean using the
statistical parameter called the Z value.
• The degrees of freedom is the sample size minus one (n − 1).
• The expression SD/ n is often reported as the standard
error of the mean.

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• The relative deviation from the mean is calculated
according to equation:
xi − x
Relative deviation from the mean = × 100
x
xi: individual sample value
x : mean

• The absolute error is simply the difference between the

experimental value and the true value.

Absolute error = Eabs = x − T

– x: experimentally determined value
– T: true value

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 Sources of Errors
• Several sources of error are systematic error (determinate),
random error (indeterminate), and gross error or blunders.

• Systematic or determinate error produces results that

consistently deviate from the expected value in one
direction or the other.
– Identifying the source of this serious type of error can be difficult
and time consuming, because it often involves inaccurate
instruments or measuring devices.
– We can overcome systematic errors by proper calibration of
instruments.
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• Random or indeterminate errors is due to our natural
limitations in measuring a particular system.
– These errors fluctuate in a random fashion.
– Background instrument noise is a factor in random error.
– Both positive and negative errors are equally possible.

• Blunders are easy to eliminate, since they are so obvious.

– Error is a result of using the wrong reagent or instrument or of
sloppy technique.

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 Specificity
• Specificity of a particular analytical method means that it
detects only the component of interest.
• The determination of food lipid (fat) is actually the crude
analysis of any compound that is soluble in an organic
solvent.
• We are not concerned about each individual compound.
• Determining the lactose content of ice cream would require a
specific method.
• Ice cream contains other types of simple sugars, without a
specific method we would overestimate the amount of lactose
present.
79
 Sensitivity and limit of detection
• Sensitivity relates to the magnitude of change of a measuring
device (instrument) with changes in compound concentration.
– It is an indicator of how little change can be made in the
unknown material before we notice a difference on a needle
gauge or a digital readout.
– We can adjust the sensitivity of an assay to fit our needs,
whether we desire more or less sensitivity.
– We even may desire a lower sensitivity so that samples with
widely varying concentration can be analyzed at the same
time.
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• Limit of detection (LOD), is the lowest possible increment
that we can detect with some degree of confidence.
– There is a lower limit at which point we are not sure if
something is present or not.
– The LOD often is reached when the signal to noise ratio is 3 or
greater.
– When the sample gives a value that is three times the magnitude
of the noise detection.
– Noise is the random signal fluctuation that occurs with any
instrument.

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 – A mathematical definition of LOD:
XLD = XBlk + 3 × SDBlk
XLD: minimum detectable consentration
XBlk: signal of blank
SDBlk: standard deviation of the blank readings

• Method detection limit (MDL):

– The minimum concentration of a substance that can be measured
and reported with 99% confidence that the analyte
concentration is greater than zero and is determined from
analysis of a sample in a given matrix containing the analyte.

82
 CURVE FITTING: REGRESSION ANALYSIS

• Curve fitting is a generic term used to describe the

relationship and evaluation between two variables.
• In analytical determinations we use only a small segment of
curvilinear analysis, the standard curve or regression line.
• A standard curve or calibration curve is used to determine
unknown concentrations based on a method that gives some
type of measurable response that is proportional to a known
amount of standard.

83
• We know how the observed values change with
concentration, it is easy to estimate the concentration of an
unknown by interpolation from the standard curve.

• Not all correlations of observed values to standard

concentrations are linear, there are examples of nonlinear
curves, antibody binding, toxicity evaluations, and
exponential growth and decay.

84
 Linear regression

• Traditionally, the concentration of the standards is represented

on the x-axis and the observed readings are on the y-axis.
– The x-axis data are called the independent variable (are
essentially free of error).

– The y-axis data (the dependent variable) may have error.

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• A typical standard curve used in the determination of caffeine
in various foods.
– When an unknown sample (e.g., coffee) is run on the HPLC, a
peak area is obtained that can be related back to the sample
using the standard curve.

– All the data points and a straight line that appears to pass
through most of the points.

– The line almost passes through the origin.

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A typical standard curve plot showing the data points and the generated best fit line.
The data used to plot the curve are presented on the graph.
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• We can mathematically determine the best fit of the line by
using linear regression.
– The equation for a straight line is y = ax + b, where a is the
slope and b is the y-intercept.

– There are often background interferences so that even at zero

concentration, a weak signal may be observed.

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 Correction coefficient

• How to draw the line through the data points and how well
the data fit to the straight line.

• We can pick out regions on the line where a linear

relationship does not exist.

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• Examples of standard curves showing the relationship between
the x and y variables.
(a)a high amount of correlation
(b)a lower amount of correlation
• Both lines have the same equation.
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Examples of standard curves showing the relationship between the
x and y variables.
(a) a high amount of negative correlation
(b) no correlation between x and y values.
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• The correlation coefficient (r) defines how well the data fit to a
straight line.
– The ideal situation would be that all data points lie perfectly on a
straight line.
– This is never the case, because errors are introduced in making
standards and measuring the physical values (observations).
– For standard curves, we want the value of r as close to +1.0000 or
−1.000 as possible, because this value is a perfect correlation.
– In analytical work, the r should be 0.9970 or better.

• The coefficient of determination (r2) gives a better perception

of the straight line it does not indicate the direction of the
correlation.
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Sampling and Sample
Preparation

93
 INTRODUCTION
• In most cases quality attributes are measured on small
portions of material that are taken periodically from
continuous processes or on a certain number of small
portions taken from a lot.
• The small portions taken for analysis are referred to as
samples, and the entire lot is called a population.
• The process of taking samples from a population is called
sampling.
• If the procedure is done correctly, the measurable
characteristics obtained for the samples become a very
accurate estimation of the population.
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 SELECTION OF SAMPLING PROCEDURES

General information
• The first step is to clearly define the population that is going
to be sampled.

Sampling plan
• Most sampling is done for a specific purpose, the purpose
may dictate the nature of the sampling approach.

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Sampling purposes:
1. Nutritional labelling
2. Detection of contaminants and foreign matter
3. Statistical process control (Quality Assurance)
4. Acceptance of raw materials, ingredients, or products
(Acceptance Sampling)
5. Release of lots of finished product
6. Detection of adulterations
7. Microbiological safety
8. Authenticity of food ingredients.
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• Sampling plan: a predetermined procedure for the selection,
withdrawal, preservation, transportation, and preparation of
the portions to be removed from a lot as samples”.
• A sampling plan should be a well-organized document that
establishes the goals of the sampling plan, the factors to be
measured, sampling point, sampling procedure, frequency,
size, personnel, preservation of the samples.
• A sampling plan should be selected on the basis of the
sampling objective.
• Samples are taken at different points of the food production
system.
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 Factors that Affect the
Choice of Sampling Plans

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99
 SAMPLING PROCEDURES
Introduction and examples
• The reliability of analytical data is compromised if sampling
is not done properly.
• The data to be obtained will determine the sampling
procedure.
• The method for sampling flour from sacks.
• The number of sacks to be sampled is determined by the
square root of the number of sacks in the lot.

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• Sampling is done by drawing a core from a corner at the top
of the sack diagonally to the centre.
• The sampling instrument is a cylindrical, polished trier with a
pointed end.
• A second sample is taken from the opposite corner in a
similar manner.
• The cores are stored for analysis in a clean, dry, airtight
container that has been opened near the lot to be sampled.
• The container should be sealed immediately after the sample
is added.
• A separate container is used for each sack.
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 Homogeneous vs. heterogeneous populations
• The ideal population would be uniform throughout and
identical at all locations.

• Such a population would be homogeneous.

• Sampling from such a population is simple, as a sample can

be taken from any location and the analytical data obtained
will be representative of the whole.

• Most populations that are sampled are heterogeneous.

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 Manual vs. continuous
sampling
• To obtain a manual sample the person taking the sample
must attempt to take a “random sample” to avoid human
bias in the sampling method.
– The sample must be taken from a number of locations within the
population to ensure that it is representative of the whole
population.
– Liquids may be sampled by pipetting, pumping, or dipping.
– When sampling grain from a rail car, mixing is impossible and
samples are obtained by probing from several points at random
within the rail car.
• Continuous sampling is performed mechanically.
– Continuous sampling should be less prone to human bias than
manual sampling.
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• An automatic liquid sampling
device that uses air under
high pressure to collect
multiple 1.5 ml samples.
• The control box (left) regulates
the sampling frequency.

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 PREPARATION OF
Stream of material SAMPLES

Rotating A rotating tube divider for

tube reducing a large sample
(ca. 880 kg) of dry, free-
flowing material to a
laboratory size sample
(ca. 0.2 kg).

Laboratory Remainder of
sample material
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 Grinding
• Grinding is important both for sample preparation prior to
analysis and for food ingredient processing.
• Various mills are available for reducing particle size to
achieve sample homogenization.
• To homogenize moist samples, bowl cutters, meat mincers,
tissue grinders, mortars and pestles, or blenders are used,
while mortars and pestles and mills are best for dry
samples.
• Some foods are more easily ground after drying in a
desiccator or vacuum oven.
• Grinding wet samples may cause significant losses of
moisture and chemical changes.
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• Grinding frozen samples reduces undesirable changes.
• The grinding process should not heat the sample.
• The grinder should not be overloaded because heat will be
produced through friction.
• Contact of food with bare metal surfaces should be avoided if
trace metal analysis is to be performed
• A number of slicing devices are available: bowl cutters can
be used for fleshy tubers and leafy vegetables, meat
mincers may be better suited for fruit, root, and meat.

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• Applications for Grinding Equipment
– Mills differ according to their mode of action, being classified as
a burr, hammer, impeller, cyclone, impact, centrifugal, or
roller mill.
– Hammer mills wear well, and reliably and effectively grind
cereals and dry foods, while small samples can be finely
ground by ball mills.
– A ball mill grinds by rotating the sample in a container that is
half filled with ceramic balls.
– A chilled ball mill can be used to grind frozen foods without
predrying and also reduces the likelihood of undesirable heat-
initiated chemical reactions occurring during milling.
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• Determination of Particle Size
– The mesh number is the number of square screen openings per
linear inch of mesh.

– The final particles of dried foods should be 20 mesh for

moisture, total protein, or mineral determinations.

– Particles of 40-mesh size are used for extraction assays such as

lipid and carbohydrate estimation.

109
– A Rototap is an instrument that can achieve separation by size
of small particles, to then determine the size distribution of
the sample.
– This is a horizontal rotating hollow cylinder into which materials
of varying particle sizes are introduced.
– The internal surface of the cylinder has many indentations of a
specific size that hold particles of a specific size or smaller.
– In this way the rotating cylinder carries smaller particles away
from the mass of material that remains at the bottom of the
cylinder.

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 US Standard Mesh with
Equivalents in Inches and
Millimeters

111
 Enzymatic inactivation
• Food materials often contain enzymes that may degrade the
food components being analyzed.
• Enzyme activity therefore must be eliminated or controlled
using methods that depend on the nature of the food.
• Heat denaturation to inactivate enzymes and freezer storage
(−20 °C to −30 °C) for limiting enzyme activity are
common methods.
• Some enzymes are more effectively controlled by changing
the pH, or by salting out.
• Oxidative enzymes may be controlled by adding reducing
agents.
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 Lipid oxidation protection
• High-fat foods are difficult to grind and may need to be
ground while frozen.
• Unsaturated lipids are sensitive to oxidative degradation and
should be protected by storing under nitrogen or vacuum.
• Antioxidants may stabilize lipids and may be used if they do
not interfere with the analysis.
• Light-initiated photooxidation of unsaturated lipids can be
avoided by controlling storage conditions.

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 Microbial growth and contamination

• Microorganisms are present in almost all foods and can alter

the sample composition.

• Microorganisms are present on all but sterilized surfaces, so

sample cross-contamination can occur.

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 SUMMARY
• Food quality is monitored at various processing stages but
100% inspection is rarely possible, or even desirable.
• To ensure that a representative sample of the population is
obtained for analysis, sampling and sample reduction
methods must be developed and implemented.
• Increasing the sample size will generally increase the
reliability of the analytical results.
• Sampling is a vital process, as it is often the most variable
step in the entire analytical procedure.

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• Sampling plans are determined by whether the population is
homogeneous or heterogeneous.
• Sampling from a homogeneous population is simple, it rarely is
found in practical industrial situations.
• Sampling from heterogeneous populations is most common, and
suitable sampling plans must be used to obtain a representative
sample.
• Sampling methods may be manual or continuous.
• Each sample must be clearly marked for identification and
preserved during storage until completion of the analysis.
• Samples can be preserved by limiting enzyme activity, preventing
lipid oxidation, and inhibiting microbial growth/contamination.
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