Food Analysis Manual-15-11-2014

Inspection Food And Drugs Labs Section No.
: 01
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FOOD ANALYSIS Date: 30-06-2013
Chemistry & Microbiology Page 1 of 86
FOOD ANALYSIS
IFAD Labs
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Reviewed By: Quality Manager Approved By: IFAD General Manager

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FOOD ANALYSIS
Food Composition/Nutrition Labeling
Carbohydrates (sugars, saccharides) and Dietary Fiber
Fats (fatty acids, FAMES, glycerides), Edible Oils, and Sterols
Proteins (amino acids, peptides, proteins, nitrogen)
Nutritional (other than carbohydrates, fats, proteins)
Non-Nutritional Ingredients and Additives
Water/Moisture Content
Genetically Modified Organisms (GMO)
Physical Characteristics
Food Safety
Microbiology Quality Control
Pesticide and Metabolite Residues
Veterinary Drug Residues
Toxins (other than pesticide/drug residues)
Processing/Packaging Contaminants
Adulterants
Beverages
Beverage Testing

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Food Composition/Nutrition Labeling

1. Carbohydrates (sugars, saccharides), Dietary Fiber
The determination of the carbohydrate and dietary fiber content of food and beverage
products is one measure of nutritional quality.
 Carbohydrate content is typically determined by measuring sugars and digestible
saccharides, often using HPLC
 Dietary fiber is a measure of non-digestible components, and includes non-digestible

saccharides and lignin
Sugars and Saccharides
Simple sugars using hydrophilic interaction chromatography (HILIC):

Chromatographic analysis of simple sugars can be challenging because these compounds
are highly polar, uncharged, and lack a chromophore. The preferred mode of HPLC
separation is HILIC because it provides the advantage of retaining these highly polar
compounds that are otherwise hard to separate.
 Polymeric columns bonded with aminopropyl groups offer improved stability and MS
compatibility. Sigma pHera NH2 column is based on covalently bonded polyamine to
co-polymer which offers stability from pH 2-12, mechanical and chemical strength,
and high column efficiency.
 For faster analysis on any HPLC system, turn to Ascentis Express. These columns,
based on Fused-Core technology, offer the performance of sub-2 μm particles but
exhibit the backpressure of 3 μm particles. Several phases (HILIC, OH5, and F5) are
available that are suitable for operation in the HILIC mode.
 The Ascentis Express F5 phase is also available in a more traditional 5 μm particle.

The Fused-Core technology results in increased efficiency, whereas the 5 μm
particles exhibit limited backpressure, allowing longer column lengths than that
possible with the standard 2.7 μm Ascentis Express particles.
Simple sugars using ion exclusion HPLC: Ion exclusion HPLC is another method
employed for the determination of simple sugars. SUPELCOGEL resin-based ion exchange
HPLC columns contain spherical particles of sulfonated polystyrene/divinylbenzene, each
with specific counter ions. Each counter ion (Ca, H, Pb, K, or Ag) imparts a unique selectivity
for the analysis of sugars or organic acids. When the sample matrix is a beverage, limited

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sample preparation is required. For example, a grape juice cocktail can simply be passed
through a 0.45 μm syringe filter prior to analysis.
Saccharides using ion exclusion HPLC: Because the chemical and physical properties of
various saccharides are similar, they are more difficult to analyze than many other classes of
compounds. HPLC is typically the mode of analysis, relying on differences in conformation,
configuration, and bonding mode. However, no single HPLC column or method is capable of
separating all saccharides. For this reason, SUPELCOGEL ion exclusion columns which are
specifically prepared for various saccharide analyses. One of these, SUPELCOGEL C-611,
contains two divalent cations rather than one, and provides a different selectivity compared
to the other SUPELCOGEL columns.
Enzymatic food kits: Enzymatic methods for food analysis are highly specific and offer
considerable time and cost savings over other methods, especially from the sample
preparation standpoint.

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Carbohydrate and dietary fibre levels
Qualitative and quantitative analysis in the food and beverage industry is extremely
important from quality, storage, nutrition and safety standpoints. The levels of certain
carbohydrates, like glucose, lactose, fructose, sucrose and starch, affect intolerance
conditions, diabetes and obesity. The presence of unwanted carbohydrates or their
hydrolysis products can alter the manufacturing process or reduce product shelf life. They
can also indicate microbial contamination (e.g. yeasts) or improper processing (e.g.
overheating). For fruit juice and wine, raw materials that have variable sugar content
influence the quality of the finished product and should therefore be monitored.
Determination of dietary fibre is another important food analysis. Consuming high-fibre

foods, like fruits, vegetables, nuts and grains, is recommended to treat or prevent such
maladies as constipation, haemorrhoids and diverticulitis. Water-soluble fibre also helps
decrease blood cholesterol levels. From a chemical perspective, dietary fibre is a mixture of
complex organic substances, including hydrophilic compounds, like soluble and insoluble
polysaccharides and non-digestable oligosaccharides, and a range of non-swellable,
relatively hydrophobic compounds, like cutins, suberins and lignins. Verifying a high content
of dietary fibre in food permits a higher quality grading and access to higher-end product
markets.

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2- Fats (fatty acids, FAMEs, glycerides), Edible Oils, Sterols

Fats play an important part in the food nutrition and food chemistry areas of study. The
compound classes, sample types, and analytical techniques of interest include:
 Short chain, volatile fatty acids, typically analyzed in their free acid form using GC
 Larger (C8-C24+) fatty acids (such as omega fats and trans fats), typically converted
to fatty acid methyl esters (FAMEs) prior to GC analysis
 Mono-, di-, and triglycerides, analyzed by GC or HPLC
 Edible oil characterization by GC
 Sterols by GC analysis
 IR and NMR procedures

Free Fatty Acids
Short chain, volatile fatty acids are typically analyzed in the free form using specialized
columns. This group of compounds may be referred to as free fatty acids (FFAs), volatile
fatty acids (VFA), or carboxylic acids. The analysis of fatty acids in the free form instead of
as fatty acid methyl esters results in easier and quicker sample preparation. Additionally,
artifact formation that may result from a derivatization procedure is eliminated.
For the GC analysis of free fatty acids, a specialized column that will not allow the
adsorption of active carboxyl groups is required. The Nukol, with its acidic characteristic, is
well-suited for this application, allowing chromatography with excellent peak shapes.

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Derivatization of Fatty Acids to FAMEs
GC can be used to analyze fatty acids either as free fatty acids or as fatty acid methyl
esters. The primary reasons to analyze fatty acids as fatty acid methyl esters include:
 In their free, underivatized form, fatty acids may be difficult to analyze because these
highly polar compounds tend to form hydrogen bonds, leading to adsorption issues.
Reducing their polarity may make them more amenable for analysis.
 To distinguish between the very slight differences exhibited by unsaturated fatty

acids, the polar carboxyl functional groups must first be neutralized. This then allows
column chemistry to perform separations by boiling point elution, and also by degree
of unsaturation, position of unsaturation, and even the cis vs. trans configuration of
unsaturation.
The esterification of fatty acids to fatty acid methyl esters is performed using an alkylation
derivatization reagent. Methyl esters offer excellent stability, and provide quick and
quantitative samples for GC analysis. The esterification reaction involves the condensation of
the carboxyl group of an acid and the hydroxyl group of an alcohol. Esterification is best

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done in the presence of a catalyst (such as boron trichloride). The catalyst protonates an
oxygen atom of the carboxyl group, making the acid much more reactive. An alcohol then
combines with the protonated acid to yield an ester with the loss of water. The catalyst is
removed with the water. The alcohol that is used determines the alkyl chain length of the
resulting esters (the use of methanol will result in the formation of methyl esters whereas
the use of ethanol will result in ethyl esters). The following typical esterification procedure
(using BCl3-methanol) is intended as a guideline. It may need to be altered to meet the
needs of a specific application.
 Samples can be derivatized neat or after dissolving in solvent. If appropriate, dissolve

sample in a nonpolar solvent (such as hexane, heptane, or toluene). If the sample is
in an aqueous solvent, first evaporate to dryness then use neat or dissolved in an
organic, non-polar solvent.
 Weigh 1-25 mg of sample into a 5-10 mL micro reaction vessel.
 Add 2 mL BCl3-methanol, 12% w/w. A water scavenger (such as 2,2-

dimethoxypropane) can be added at this point.
 Heat at 60 °C for 5-10 minutes. Derivatization times may vary, depending on the
specific compound(s) being derivatized.
 Cool, then add 1 mL water and 1 mL hexane.
 Shake the reaction vessel (it is critical to get the esters into the non-polar solvent).
 After allowing the layers to settle, carefully transfer the upper (organic) layer to a
clean vial. Dry the organic layer by either a) passing through a bed of anhydrous
sodium sulfate during the transfer step to the clean vial, or b) adding anhydrous
sodium sulfate to the clean vial then shaking.
 To determine the proper derivatization time, analyze aliquots of a representative

sample using different derivatization times. Plot peak area (y-axis) vs derivatization
time (x-axis). The minimum time to use is when no further increase in peak area is
observed with increasing derivatization time (where the curve becomes flat).

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 If it is suspected that complete derivatization is never achieved, use additional

reagent or re-evaluate temperature.
 It is important to prepare a reagent blank, along with the samples, to identify any
issues that may arise.
It is important to use only high quality derivatization reagents, to ensure that no artifacts
are present during analysis. Additionally, only derivatization reagents with low moisture
should be used, as the esterification reaction will be hindered by the presence of water. The
storage conditions of derivatization reagents should be strictly adhered to, as some are
susceptible to degradation during long-term storage.
FAMEs by Degree of Unsaturation
Saturated, monounsaturated, polyunsaturated, and cis/trans configuration all refer to the

structure of fatty acid moieties. Each group is believed to have the following effects on
human health:
 Saturated fatty acids (no double bonds) raise LDL cholesterol (increases risk of
cardiovascular disease).
 Mono- and poly-unsaturated cis fatty acids (≥1 cis double bond) lower LDL
cholesterol (reduces risk of cardiovascular disease).
 Mono- and poly-unsaturated trans fatty acids (≥1 trans double bond) raise LDL
cholesterol (increases risk of cardiovascular disease) and also lower HDL (increases
risk of type II diabetes).
Because of this, it is important for food manufacturers to measure and report their levels so
consumers have the chance to establish healthy dietary strategies. Nutritionally, saturated
fats are of particular concern, because an excess in the diet leads to their accumulation in
the cardiovascular system, resulting in several health-related problems. Due to this, food
manufacturers typically report the saturated fat vs. unsaturated fat content on the
nutritional panel, allowing consumers wishing to have a healthier diet to make food choices
with less saturated fat.
To confirm identification, very efficient capillary GC columns with the ability to resolve a
large number of peaks are required. SIGMA SPB-PUFA columns utilize a specially-developed

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phase for the analysis of polyunsaturated fatty acids (PUFAs) as FAMEs. It has a lower
phase polarity than commonly used ′wax′ columns, resulting in a column with a slightly
different selectivity. Another choice is SIGMA Omegawax, which provides highly
reproducible analyses, being specially tested for reproducibility of FAME equivalent chain
length (ECL) values and resolution of key components.
Omega 3 and Omega 6 FAMEs

Essential fats are nutrients that must be obtained from the diet because humans lack the
anabolic processes for their synthesis. Essential fats serve multiple purposes in the body
including:
 Production of eicosanoids, which affect inflammation and cellular function.
 Production of lipoxins and resolvins, which affect inflammation.
 Production of endogenous cannabinoids, which affect mood and behavior.
 Influencing cell signaling.
 Regulation of blood pressure, blood clotting, lipid levels, immune response, and gene
expression.
There are two closely related groups of essential fats, the omega 3 and the omega 6 fatty
acids. Both are unsaturated fatty acids, with the initial double bond located directly after the
third (omega 3) or the sixth (omega 6) carbon atom as measured from the methyl end.
Omega 3 fatty acids are found in fish oils and some nut oils. Seed oils are the primary
dietary source of omega 6 fatty acids.
Before the advent of agriculture, human diets were thought to have consisted of an equal
amount of omega 3 and omega 6 fatty acids. In contrast, the current western diet has a 1:7
ratio of omega 3 to omega 6 fatty acids. Low levels of omega 3 fatty acids, or an altered
ratio of omega 3 to omega 6 fatty acids, may play a key role in a number of human
diseases:
 Increased consumption of omega 3 fatty acids has been linked with reducing
coronary heart disease.

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 An excess of omega 6 fatty acids can interfere with the health benefits of omega 3
fatty acids, and has also been linked with several detrimental health conditions.
As a result of consumers′ desire to have ′healthier fat′ in the diet, the analysis of the omega
3 and omega 6 fatty acid content of food products is a very active area of research for many
food companies.
The omega 3 and omega 6 FAMEs may have very similar physical (such as boiling point)
and chemical (such as chain length) properties as other FAMEs that may be present in the
sample. Therefore, specialized GC columns with the ability to resolve these specific FAMEs
are required for proper identification. SIGMA Omegawax columns provide highly
reproducible analyses, being specially tested for reproducible resolution of key FAMEs.
Conditions: column 1: Omegawax, 30 m x 0.25 mm I.D., 0.25 µm (24136); column 2:

SLB-IL60, 30 m x 0.25 mm I.D., 0.20 µm (29505-U); oven: 170 °C, 1 °C/min to 225 °C;
inj. temp.: 250 °C; carrier gas: helium, 1.2 mL/min; det.: FID, 260 °C; injection: 1 µL, 100:1

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split; liner: 4 mm I.D., split/splitless type, single taper wool packed FocusLiner™ design;
sample: Supelco 37-Component FAME Mix (47885-U) + C22:5n3, in methylene chloride
(17269)
1. C4:0 20. C18:2n6t

2. C6:0 21. C18:3n6
3. C8:0 22. C18:3n3
4. C10:0 23. C20:0
5. C11:0 24. C20:1n9
6. C12:0 25. C20:2
7. C13:0 26. C20:3n6
8. C14:0 27. C21:0
9. C14:1 28. C20:3n3
10. C15:0 29. C20:4n6
11. C15:1 30. C20:5n3
12. C16:0 31. C22:0
13. C16:1 32. C22:1n9
14. C17:0 33. C22:2
15. C17:1 34. C23:0
16. C18:0 35. C22:5n3
17. C18:1n9c 36. C24:0
18. C18:1n9t 37. C22:6n3
19. C18:2n6c 38. C24:1n9
cis/trans FAME Isomers
Fatty acids in the cis configuration are the dominant form in nature. Correspondingly,
enzymes have evolved to efficiently digest and metabolize them with a high degree of
specificity. Conversely, trans fatty acids are relatively rare in nature. However, because they
can increase the shelf life and flavor stability of foods containing them, they have become
predominant synthetic additives to processed foods, especially fried foods and baked goods.
Unfortunately, trans fatty acids, formed by partial hydrogenation of vegetable oil, interfere
with natural metabolic process, resulting in an imbalance of the LDL:HDL ratio, and also

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increasing lipoprotein(a) levels. Studies have linked their nutritional contribution to be

similar to that of saturated fatty acids, possibly playing a role in the heightened risk of
coronary artery disease.
Because trans fatty acids have adverse health consequences and no known nutritional
benefits over other fats, consumer groups have pressured manufacturers and restaurants
for their elimination. Many regulatory agencies worldwide now require content labeling to
inform buyers of ′trans fat′ levels of foods and some dietary supplements.
The differences between cis isomer FAMEs and trans isomer FAMEs of the same carbon
length and degree of unsaturation are very small. Therefore, very efficient capillary GC
columns with highly polar phases are required.
 The highly polar SP-2560 column was specifically designed for the separation of
geometric-positional (cis/trans) isomers of FAMEs, and is extremely effective for
special FAME applications including the separation of FAMEs in hydrogenated
vegetable oil samples. This well-established column is specified in many methods.
 The extremely polar SLB-IL111 column exhibits the highest polarity of any GC
phase, providing an alternative selectivity for FAME applications typically performed
on SP-2560 and SP-2380 columns. It is able to provide resolution of some key
isomers that cannot be resolved on the SP-2560 or SP-2380 columns.
 The highly polar SP-2380 column allows the separation of geometric (cis/trans)
isomers as a group. The phase is stabilized, providing a maximum temperature
slightly higher than the popular SP-2560 column. It is available in shorter column
lengths than the SP-2560, therefore, useful for short analyses where detailed
resolution is not necessary.

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Analytical Challenge: Improved Throughput of Detailed cis/trans FAME Analyses
To increase throughput of the detailed cis/trans FAME analysis, Fast GC principles were
applied by reducing column length, column I.D., film thickness, and carrier gas viscosity.
The result is a significant reduction in analysis time compared to the 100 m column method:
30% reduction of the 37-component FAME sample (Figures 1 and 2), and nearly 50%
reduction of the detailed analysis of the C18 isomer mix (Figures 3 and 4). Note that in both
cases, peak shape and resolution does not suffer, even with the shorter analysis times.

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Figure 1. 37-Component FAME Mix on the 100 m SP-2560 Column (24056)
Figure 2. 37-Component FAME Mix on the 75 m SP-2560 Column (23348-U)

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Figure 3. Detailed Analysis of C18 FAME Isomers on the 100 m SP-2560 Column
(24056)

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Figure 4. Detailed Analysis of C18 FAME Isomers on the 75 m SP-2560 Column (23348-U)

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Mono-, Di-, and Triglycerides
Triglycerides (also called triacylglycerol, triacylglyceride, or TAG) are the main constituent of
vegetable oil and animal fat, and make up most of the fats digested by humans. They are
important in that they allow the uptake and transport of fat-soluble vitamins. Plus, they play
a role in metabolism (unused saturated or monounsaturated fatty acids are stored by the
body as triglycerides). However, triglyceride intake should be monitored because high levels

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of triglycerides have been linked to an increased risk of heart disease and stroke. Mono-, di-
and tri-glycerides are related to fatty acids:
 A monoglyceride is the condensation of one fatty acid and glycerol.
 A diglyceride is the condensation of two fatty acids and glycerol.
 A triglyceride is the condensation of three fatty acids and glycerol.

Efficient capillary GC columns with the ability to provide ample resolution are required for
proper identification. Triglycerides are large compounds requiring a relatively high final oven
temperature for elution in a reasonable time. Cool-on-column (COC) injection should be
used. Use of a heated injection port can lead to sample discrimination and is not suggested.
The syringe needle used must have a diameter small enough to fit inside the 0.53 mm I.D.
guard column. Automated injection is highly recommended for consistency.
HPLC using a C18 phase is an alternative technique for the analysis of glycerides.
 For faster analysis on any HPLC system, turn to Ascentis Express. These columns,
based on Fused-Core technology, offer the performance of sub-2 μm particles but
exhibit the backpressure of 3 μm particles.
The Ascentis Express C18 phase is also available in a more traditional 5 μm particle. The
Fused-Core technology results in increased efficiency, whereas the 5 μm particles exhibit
limited backpressure, allowing longer column lengths than that possible with the standard
2.7 μm Ascentis Express particles.
The MET-Biodiesel, in combination with cool on-column injection, fast carrier gas flow
rates, rapid oven temperature ramp rates, and high final oven temperatures, was able to
provide significant resolution for both applications with relatively short run times. As
evident, the triglyceride profile of butter is much more complex than that of lard. The profile
differences can be attributed to the fact that these products are from different sources;
butter is processed from cow milk or cream whereas lard is rendered from pig fatty tissues.

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The mono-, di-, and triglyceride composition of vegetable oils can be used as a measurement of quality and
purity. If the oil is adulterated with inferior oil, the peak patterns and/or ratios will not match up to historical
data, potentially falling outside of acceptable control limits.
Sterols:
Olive oil, eggs, margarine, soybean oil, and chocolate are a few of the foods that contain
sterols, which occur naturally in plants and animals, and are ingested as part of the diet.
These compounds perform many roles in human, such as various cellular functions and as
precursors to several hormones and vitamins. Sterols of interest include brassicasterol,
campesterol, cholestanol, cholesterol, coprostanol, desmosterol, ergosterol, lanosterol, b-
sitosterol, and stigmasterol.
Fats in a sample typically must be extracted and saponified to isolate the sterols, which will
be in the non-saponifiable fraction, along with other large molecular weight alcohols, fat-
soluble vitamins, and hydrocarbons. Vegetable oils, which are almost pure fat, do not
require extraction prior to saponification. The general saponification procedure outlined by
the AOAC Method 970.51 is useful for preparing most samples. Analysis by GC can typically
be performed without derivatization.
SAC-5 Column: Application: This column is an application specific non-polar column,

designed for reproducible analyses of plant sterols, cholesterol, and other animal sterols.
Phase: Bonded; poly(5% diphenyl/95% dimethyl siloxane)
Temp. Limits: -60 °C to 320 °C (isothermal or programmed).

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3- Proteins (amino acids, peptides, proteins, nitrogen):
Proteins are a major source of energy in foods. Additionally, they contain essential amino
acids (such as lysine, methionine, and valine) that cannot be synthesized in the body.
Proteins are also major structural food components and determine the texture of meat and
fish. Because of this, adding isolated proteins to foods is a technique to provide desirable
appearance, texture, and stability. Areas of interest for food analysts include:
 Individual amino acid determination
 Peptide composition
 Protein identification and quantification, using HPLC or molecular spectroscopy

techniques (IR and UV)
 Nitrogen content (Kjeldahl method) as a measure of protein content.
Amino Acids
To determine the amino acid composition of a protein, the sample is first hydrolyzed to
release the constituent amino acids. Analysis is typically performed using HPLC. It is
sometimes helpful to derivatize the amino acids to aid in separation, and to permit detection
at lower concentrations. Dabsyl chloride is an effective derivatization reagent for this
application. SUPELCOSIL LC-DABS columns feature a specially treated and tested
octadecyl bonded phase, for the reversed-phase separation of pre-column derivatized dabsyl
amino acids. More than 30 amino acids and ammonia can be separated in less than one
hour.
Some amino acids exist in nature as enantiomers, and require specialized HPLC columns for
proper separation. The Astec® CHIROBIOTIC TAG, T, and T2 columns have
demonstrated excellent selectivity and effective resolution of the enantiomers of several
underivatized amino acids (such as serine and isoleucine) with simple mobile phases.
Typically, high performance liquid chromatography (HPLC) is used for the analysis of amino
acids. However, GC can also be used, and in some cases availability of instrumentation or
operation costs can make it a better choice. The polar nature of amino acids requires

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derivatization prior to GC analysis. The goal of derivatization is to make an analyte more

volatile, less reactive, and thus improve its chromatographic behavior. In the case of amino
acids, derivatization replaces active hydrogens on OH, NH2, and SH polar functional groups
with a nonpolar moiety.
Silylation is a very common derivatization technique, and is useful for a wide variety of
compounds. The main disadvantage of this method is its sensitivity to moisture. The
presence of moisture results in poor reaction yield and instability of the derivatized analytes.
For this study, we evaluated the use of the silylation reagent N-tert-butyldimethylsilyl- N-
methyltrifluoroacetamide (MTBSTFA) for the derivatization of amino acids. MTBSTFA, forms
tert-butyl dimethylsilyl (TBDMS) derivatives when reacted with polar functional groups
containing an active hydrogen:
Experimental
A 50 μL aliquot of a solution containing a mix of L-amino acids at 91 μg/mL in 0.1 N HCl was
dried, and 100 μL of neat MTBSTFA, followed by 100 μL of acetonitrile, were added. The
mixture was heated at 100 °C for 4 hours. The sample was then neutralized with sodium
bicarbonate and subjected to GC-MS analysis on a 20 m x 0.18 mm I.D. x 0.18 μm SLB™-
5ms capillary column.

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GC-MS Analysis of Amino Acid Derivatives on the SLB-5ms (28564-U)

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Mass Spectrum of TBDMS Derivative of Valine (MW of the derivative = 345)
Peptides
The tryptic digest of proteins can be used to make smaller peptides, which can be
chromatographed using reverse phase HPLC columns. This is possible because trypsin will
cleave a protein the same way each time. Peptide maps can then be used to assist with
identification of the source protein.
Peptide mapping is an established technique for assessing changes to the primary structure
of a protein. This has applications in areas of quality control or fundamental research in
which changes to the protein sequence or to the chemical modification of amino acids is to
be monitored. Essentially, the protein(s) is cleaved (digested) in a sequence-dependent
manner to generate a finite number of peptide fragments. The mixture of peptide fragments
is then resolved chromatographically. The exact pre-treatment protocol of the protein prior
to digestion varies and may depend on the sort of questions the researcher is posing.
However, a general protein pre-treatment protocol involves denaturation, reduction, and
alkylation. Alkylation is performed to control the oxidation state of free sulfhydryls that
otherwise can cause undesirable heterogeneity of the resulting chromatograms. Reduction is
performed to ensure the sulfhydryls are reduced prior to alkylation. Denaturation is
performed to ensure all sulfhydryls are accessible for chemical modification, as well as make
the polypeptide chain fully accessible for digestion.

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Peptide mapping is generally done by reversed-phase liquid chromatography. Traditionally

this was done in conjunction with low-UV detection. A good match with the low-UV
detection, is the use of perfluorinated organic acids as ion pairing reagents. TFA was ideal in
this regard. As a strong acid, at low levels, it maximizes retention of polypeptides on silica-
based columns by keeping the pH well below that of peptidyl carboxyls, and at the same
time it ion pairs with basic moieties to optimize peak shape. Thus, TFA-mobile phases
became the default method for peptide mapping with UV detection. However, the advent of
mass-spectrometry as a preferred method of detection for peptide mapping necessitated the
redevelopment of a standard mobile phase; TFA at levels typical for UV-based peptide
mapping (0.1%) cause severe reduction in sensitivity as compared to other organic acids, as
the mobile phase additive (1). This has been best described as a consequence of higher
surface tension of the charged droplet in the ESI source and strong ion pairing in the gas
phase, between TFA and peptide basic moieties (1). Acetic acid and formic acid have been
used as alternatives to TFA for LC-MS of peptides, but formic acid has become more
common. This is likely due, again, to it being a stronger acid, thus minimizing the ionization
of peptidyl carboxyls so as to enhance retention.
Sensitivity Comparison of Tryptic Digest with Different Ion Pair Reagents

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Nitrogen Determination by Kjeldahl Method
Since 1883, the Kjeldahl method has been the official worldwide standard for the
determination of nitrogen in all kinds of food and beverage samples. The Kjeldahl digestion
converts nitrogen compounds (proteins, amines, organic compounds) into ammonia
compounds. Free ammonia is released by the addition of caustics, which are then expulsed
by distillation and subsequently titrated. The Kjeldahl method is also employed in
environmental analysis and the agricultural industry for the determination of nitrates and
ammonium. The method has been approved by various scientific associations including
AOAC International, AACC International, AOCS, DIN, EPA, ISO, JAS, and USDA.

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4- Nutritional (other than carbohydrates, fats, proteins)
In addition to the major nutritional food components (carbohydrates, fats, and proteins),
several minor nutritional components are also of great interest to food analysts. Areas of
interest include:
 Nutraceuticals/antioxidants (polyphenolics, catechins, flavonoids, natural
compounds, saponins/ginsenoside, and phytoestrogens)
 Vitamins, co-factors, and enzymes
 Organic acids
 Minerals (calcium, iron, sodium, potassium, etc.)

Nutraceuticals/Antioxidants
The study of the putative health benefits of plant-derived polyphenolic compounds is an

active research area in food chemistry. HPLC and LC-MS play an important role in the
characterization of plant extracts, and both benefit from the sensitivity and resolving power
provided by highly efficient Ascentis Express HPLC columns. These columns, along with our
sample prep products and analytical standards, have been used to analyze a wide-range of
nutraceuticals, such as vanillin, catechin, resveratrol, Withania, ginsenosides, taxols,
steroildal glycosides, digoxins, silymarin, phytonutrients, spice cannabinoids, flavonoids,
catechols, resorcinals, ephedrine alkaloids, hyerpericum, and tamoxifen.
Catechins
Catechins have become a hot topic in today’s health conscious world. Current research
suggests that catechins aid health by:
 Reducing formation of athersclerotic plaques
 Suppressing the growth of tumors by inhibiting enzymes involved in the spread of

cancer cells, eradicating tumor promoting substances and blocking chemical
carcinogens
 Reducing high blood pressure
 Protecting against digestive and respiratory infections

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 Lowering cholesterol levels
 Lowering blood glucose levels
 Prevention of kidney stones
 Reducing the chance of developing rheumatoid arthritis
 Producing stronger bones
 Reducing inflammation
The catechins are a group of polyphenolic compounds exhibiting strong anti-oxidant, as well
as, remarkable antibacterial, antiviral, and anti-inflammatory properties. They are found in
high concentrations in the leaves of Camellia sinensis (tea) and in smaller amounts in
chocolate, grapes, raspberries, apples, pears, and wine. The very young leaves and buds of
Camellia sinensis used to make white tea have the highest concentrations; followed by the
slightly more mature leaves used to make green tea. Older leaves used to make Oolong and
black teas are more oxidized and contain higher concentrations of other polyphenols
including theaflavanins and thearubigins.
Catechins, like other antioxidants, help protect cells from oxidative stress. Oxygen is vital to
life; however, it is also incorporated into reactive oxygen species including hydrogen
peroxide, hypochlorous acid and free radicals such as the hydroxyl radical and the
superoxide anion. Reactive oxygen species damage cells and have been implicated in the
slow chain reaction of damage leading to heart disease, cancer, and many other ailments.
Antioxidants function by preventing the formation of reactive oxygen species, or by reacting
with them before they can damage cells.
Leaves of Camellia sinensis contain at least eight polyphenol catechins. The six predominant
catechins in tea leaves are catechin, gallocatechin gallate(GCG), epigallocatechin (EGC),
epicatechin (EC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), with EGCG
being the most abundant.

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Anthocyanins and Flavonol Glycosides
Flavonols and anthocyanins are water-soluble vacuolar flavonoid compounds that are
synthesized by organisms of the plant kingdom. Flavonoids are widely distributed in plants
fulfilling many functions including pigmentation and protection from UV light and attack by
microbes and insects.
The chemical structure of the anthocyanidins is based upon the flavonoid family of
molecules that is in turn based on the C6-C3-C6 configuration in the flavan nucleus. Figure
1 illustrates the structure of the flavylium ion which makes up the backbone of the
anthocyanidins. The chemical structure of the flavonols is also based upon the flavonoid
family of molecules where flavonols use the 3-hydroxyfl avone backbone. Anthocyanins are
anthocyanidins linked with one or more sugars that are sometimes acylated. Flavnols and
flavones are glycosylated and acylated similarly to anthocyanins.
Figure 1 Flavylium ion backbone of anthocyanidins & anthocyanins
Since 1992 more than 277 anthocyanins have been reported and more recent literature
estimates the total number of identified anthocyanins at 550. Consumers and food
manufacturers have become interested in flavonoids for their medicinal properties,
especially their potential role in the prevention of cancers and cardiovascular disease. The
beneficial effects of fruit, vegetables, and tea or even red wine have been attributed to
flavonoid compounds rather than to known nutrients and vitamins.
Both anthocyanins and flavonols are typically characterised through the utilisation of
reversed-phase HPLC with UV-VIS detection coupled to electrospray ionisation mass
spectrometry (ESI-MS). Due to the complexity of the flavonol and anthocyanin isomeric
structures that may exist in plant tissues, the separation of these molecules by RP-HPLC can
lead to co-eluting compounds. The separation of the flavonols and anthocyanins prior to
analysis by LC-ESI-MS would greatly facilitate the identification.

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The DSC-MCAX mixed-mode cation SPE cartridges were used in the separation of
anthocyadin-O-glycosides (positively charged) and fl avonol-O-glycosides (neutral) in red
tulip fl ower petals.
Extraction of Anthocyanins from Tulip
Figure 2 3-Hydroxyflavone backbone of flavonols & flavonol glycosides
Red tulip blooms (Tulipa darwin hybrid ‘Apeldoorn’) were treated with 50/50
methanol/water with 0.1 % formic acid, ground with a glass stirring rod and placed in
sonicator for several minutes to extract the anthocyanins and flavonols. The mixture was
filtered to remove large particles.
SPE Fractionation of Flavonol Glycosides and Anthocyanins
A DSC-MCAX SPE cartridge, 100 mg/3 mL (52783-U), was conditioned with 1.5 mL of
methanol and equilibrated with 1.5 mL of water containing 0.1 % formic acid. A small
aliquot of tulip extract was diluted with an equal volume of water with 0.1 % formic acid
and loaded onto the cartridge. The cartridge was rinsed with 1.5 mL water with 0.1 %
formic acid solution (0.5 mL x 3). The fl avonol glycosides were eluted with 1.5 mL
methanol (0.5 mL x 3). The anthocyanins were eluted with 1.5 mL (0.5 mL x 3) of a 50/50
solution of potassium phosphate buffer pH 6.0 and methanol.

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Vitamins, Co-Factors, and Enzymes
HPLC is widely used to analyze the low molecular weight, thermally labile compounds. Ideal
HPLC columns for this application must possess four features: choice of bonded phase for
different selectivity, high efficiency for high s/n, compatibility with different mobile phase
systems and detection systems, and rugged and stable to accommodate complex, real
samples. Ascentis Express columns meet these requirements.
Determination of Additives in Beverages Using Ascentis Express Columns:
Beverages, such as sodas and energy drinks, can contain a number of polar ingredients,
which are easily soluble in the water matrix of the drinks. These ingredients include
sweeteners (sugars and sugar substitutes), caffeine, vitamin supplements, amino acids,
organic acids, and plant extracts. Because the analytes are already in solution, there is no
need for extensive sample preparation. Dilution followed by direct injection into an HPLC is
typically suitable.
In this article we present two beverage applications using Ascentis Express HPLC
columns. Ascentis Express columns offer faster analysis on any HPLC system. One benefit
is their ability to produce the resolution, efficiency, and speed that is associated with the
use of sub-2 micron columns on a UHPLC system, without generating high backpressure.
Column chemistries (RP-Amide and HILIC) were selected for this article based on their
enhanced performance with polar compounds in comparison to C18.

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HPLC Analysis of Artificial Sweeteners, Preservatives, and Caffeine in a Diet Soda

Conditions: column: Ascentis Express RP-Amide, 3 cm x 4.6 mm I.D., 2.7 µm particles
(53921-U); mobile phase: (A) 100 mM ammonium acetate, pH 5.6, titrated with acetic
acid, (B) water, (C) acetonitrile; gradient: 20% A constant, 5 to 60% C in 1 min, hold at
60% C for 0.1 min; flow rate: 3 mL/min; column temp.: 40 °C; detector: UV at 214 nm;
injection: 1 µL; sample: diet soda 100 - 500 µg/mL in buffer
HPLC Analysis of Sugars, Vitamins, Preservatives, and Caffeine in an Energy

Drink
Conditions: column: Ascentis Express HILIC, 10 cm x 3.0 mm I.D., 2.7 µm (53970-U);
mobile phase: (A) 100 mM ammonium acetate, pH 5.0; (B) water, (C) acetonitrile (9:1:90,
A:B:C); flow rate: 0.6 mL/min; pressure: 815 psi; column temp.: 35 °C; detector: UV, 254
nm or ELSD, 55 °C, 3.5 bar nitrogen; injection: 2 µL commercial energy drink diluted 1:9 in
acetonitrile

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Energy Drink on Ascentis Express RP-Amide
Organic Acids
Organic acids may be naturally formed, or added for desired benefits. They influence both
flavor and pH stability. HPLC, GC, and titration are all suitable techniques for the
determination of organic acids in various food products. Selection is based on analyte
volatility as well as the list of other analytes that may require analytical results for.
Ex. Wine Quality Analysis

When applied to the compositional analysis of wine, including naturally occurring
compounds, additives and contaminants, analytical chemistry plays an important role in

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ensuring both the quality of the wine and consumer safety. The notorious 1985 glycol-
doping scandal of Austrian and German wines is a prime example.
However, wine presents a complex matrix: Flavors, sugars, carboxylic acids, tannins,
phenols, amino acids, alcohols, esters and acetates are among the many typical wine
constituents, and many more are present as unwanted contaminants that must be
monitored. The composition and ratio of constituents combine to give each wine its unique
flavor and texture, a ratio that is easily disturbed by environmental conditions, shipping and
storage. Analysts involved in the quality control or regulation of wine require reliable
analytical methods and standards for its determination. To help analysts produce definitive,
quantitative results, Sigma- Aldrich offers three Fluka-brand standards for wine analysis.
Together, these standards include many of the most commonly analyzed sugars, acids and
alcohols in wine.
Ion chromatography (IC) is useful because it permits the simultaneous analysis of many
important wine components. The IC chromatograms of the Fluka standards, along with
samples of sweet and dry wines, appear in the accompanying fi gures. These samples were
provided by a Swiss vintner that performs in-house QA to show the quality of their wine as a
means to help ensure customer confidence in their product. Notable are the elevated sugars
in the sweet wine and elevated acids in the dry wine.
Columns: Sertec-Wine-ST3 300 x 7.8 mm with Sertec-Wine-ST1, 50 x 4.6 mm

precolumn Mobile phase: 2.5 mM sulfuric acid Flow rate: 0.65
mL/min. Temperature: 50 °C Detection: RI
Injection: 20 μL, wine sample diluted 1:9 with 10 mM sulfuric acid and filtered

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Separation of organic acids in fruit beverages
This application uses a SUPELCOGEL C-610H HPLC column and UV detection for the
simultaneous determination of organic acids in various fruit beverages. This column is a
cross-linked polystyrene divinylbenzene resin HPLC column prepared specifically for ion
exchange analysis of organic acids. It is also ideal for separating sugars, alcohols and other
fermentation products.
Organic Acid Standards Mix (10 μL injected)

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Minerals
Low levels of minerals are important for nutritional requirements, but some can be toxic at
high levels. For this reason the mineral content of food and beverages is measured and
controlled. Ion chromatography (IC), atomic adsorption spectroscopy (AAS), and inductively
coupled plasma (ICP) are the analytical techniques most often used to determine the
mineral content of foods. Fluka offers multiple certified reference materials (CRMs) and high
quality acids, bases and salts for the calibration and operation of IC, AAS, and ICP
instruments.

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5- Non-Nutritional Ingredients and Additives
Some materials with no nutritional value are also added to food and beverage products.
These ingredients and additives are designed to make the food smell better, taste better,
last longer, and/or look better. Areas of interest include:
 Flavors & Fragrances (raw materials, aroma, volatiles, essential oils, and
enantiomers)
 Artificial sweeteners
 Preservatives (other than antioxidants) such as sorbate, benzoate,

parabens, and nitrates
 Food Dyes
The European Food Safety Authority (EFSA) has assigned codes (E numbers) for food
additives used within the European Union (the "E" prefix stands for "Europe"). They are
commonly found throughout the European Union on food labels. A cross-reference of our
analytical standards to E numbers is included for convenince.
Flavors & Fragrances
Headspace solid phase microextraction (SPME) coupled with analysis on a capillary GC

column is an ideal approach for characterizing quality and composition of flavor & fragrance
components. SPME typically requires a short extraction time compared to other preparation
techniques, and minimizes interference by other components in the matrix. SPME can also
be used to chemically fingerprint the volatile components of natural foods. Multiple SPME
fiber chemistries are available, as well as convenient multi-fiber, multi-chemistry kits.
If the flavor & fragrance compound of interest is enantiomeric, a specialized analytical

column must be used for properly identification and quantitation. Supelco offers an
impressive list of chiral GC columns through its Astec® and DEX product lines.
Artificial Sweeteners
Synthetic compounds that duplicate the taste of sugar, but contain less energy, are often
added to diet foods and beverages. The logic is to maintain the desired taste, but reduce

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the caloric value. To achieve a targeted sweetness, artificial sweeteners are often used in
specific combinations to mimic the sweetness observed from natural sugars. Because
artificial sweeteners are considered additives, they are often regulated. Therefore, their
identifications and concentrations must be determined.
Preservatives
Compounds that have the ability to inhibit microbial growth are often added to food and
beverage products to prolong shelf life. Preservatives are considered additives, and are
typically regulated. Therefore, their identifications and concentrations must be determined.
HPLC Analysis of Artificial Sweeteners, Preservatives, and Caffeine in a Diet Soda

Conditions: column: Ascentis Express RP-Amide, 3 cm x 4.6 mm I.D., 2.7 µm particles
(53921-U); mobile phase: (A) 100 mM ammonium acetate, pH 5.6, titrated with acetic
acid, (B) water, (C) acetonitrile; gradient: 20% A constant, 5 to 60% C in 1 min, hold at
60% C for 0.1 min; flow rate: 3 mL/min; column temp.: 40 °C; detector: UV at 214 nm;
injection: 1 µL; sample: diet soda 100 - 500 µg/mL in buffer

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HPLC Analysis of Sugars, Vitamins, Preservatives, and Caffeine in an Energy

Drink
Conditions: column: Ascentis Express HILIC, 10 cm x 3.0 mm I.D., 2.7 µm (53970-U);
mobile phase: (A) 100 mM ammonium acetate, pH 5.0; (B) water, (C) acetonitrile (9:1:90,
A:B:C); flow rate: 0.6 mL/min; pressure: 815 psi; column temp.: 35 °C; detector: UV, 254
nm or ELSD, 55 °C, 3.5 bar nitrogen; injection: 2 µL commercial energy drink diluted 1:9 in
acetonitrile

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Energy Drink on Ascentis Express HILIC

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Energy Drink on Ascentis Express RP-Amide
Food Dyes
A sensitive HPLC method can be used for quality control testing of dyes and the
identification of by-products. Supelco′s Ascentis Express HPLC columns provide outstanding
sensitivity and resolution for such applications.

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Dyes surround us everywhere every day. They can be found in common places like the
printing ink in magazines or books and in plastics, textiles, and leather, but also in unusual
places like diesel fuel and tattoo color. Most of these synthetic colors are based on aromatic
ring structures containing heteroatoms and tend to have a high potential for causing cancer;
as a result, they are not intended for use in food coloring. But since 2003, there have been
several incidents of Sudan I contamination in chili powder. This situation necessitates the
analysis of spice mixtures to determine if they have been adulterated. Further, a sensitive
HPLC method is needed for quality control testing of dyes and the identifi cation of
byproducts. Supelco’s Ascentis®Express HPLC columns provide outstanding sensitivity and
resolution for such applications.
The chemical and physical properties of the dyes differ strongly, so the fi rst step in
developing a suitable HPLC method was the use of a gradient run ranging from 25%
acetonitrile to 100% acetonitrile (B) and 0.1% formic acid in water as an aqueous
counterpart (A). The UV chromatogram of the combined wavelengths 360, 550, and 620 nm
showed good chromatography of all compounds except for the poor peak shape of Sudan
410 at 17.35 minutes.

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UV Chromatograms of Sudan III and Sudan 410. (A) Initial Conditions, (B) Addition of
Methanol to Mobile Phase, (C) Final Conditions after Adjusting Gradient
To optimize that peak shape, methanol was added to the organic mobile phase
(acetonitrile:methanol, 90:10); the gradient run was repeated, resulting in better peak
shape for Sudan 410 (Figure 1B).

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Initial and Final HPLC Method Settings for Separation of the Seven Dyes Listed , After
Optimization
E numbers (Food Additives)
E numbers are codes for food additives that have been assessed for use within the
European Union (the "E" prefix stands for "Europe"). They are commonly found on food
labels throughout the European Union. Safety assessment and approval are the
responsibility of the European Food Safety Authority (EFSA).
Karl Fischer Titration Reagents (HYDRANAL®)
HYDRANAL reagents for water determination by Karl Fischer titration fulfill all requirements
of practical laboratory analysis, and enable analysts to choose the best reagent for their
specific application and instrumentation.
The product range consists of one-component and two-component reagents for volumetric
determinations, coulometric reagents, and specially designed reagents for the determination
of water in aldehydes, ketones and other difficult-to-solubilize substances. The product

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range is completed by water standards for titer determination (water equivalent) and buffer
solutions for maintaining an ideal pH range between 5 and 7.
6- Physical Properties
The measurement of certain physical properties is often used to determine the identity or
purity of a substance.
 Physical properties, such as, melting point, density, conductivity, viscosity, turbidity,
particle size, color, thermal conductivity, mechanical, morphological, optical, and
isotope measurement
7- GMO Reference Materials, Qualitative

Some crop types have undergone genetic engineering to impart desirable traits, such as
gaining resistance to pests, herbicides, and weather, improving shelf life, and increasing
specific nutritional indicators. Because of concerns over the safety of food, forage, and
feedstuff produced from GMO plants, their use is strictly regulated in a number of countries.
Sigma-Aldrich offers a wide range of certified reference materials (CRMs) for the most
widely used GMOs. These products are manufactured by the IRMM (Institute of Reference
Materials and Measurements) which is a part of the joint research center (JRC) of the
European Commission. Available GMO CRMs include cotton, maize (corn), potato, rapeseed,
soya bean, and sugar beet. GMO CRMs are produced gravimetrically, by mixing GMO-
containing material with GMO-free material in different ratios to achieve the target level.

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Food Safety
Microbiology Quality Control

Pesticide and Metabolite Residues
Veterinary Drug Residues
Toxins (other than pesticide/drug residues)
Processing/Packaging Contaminants
Adulterants

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1- Microbiology Quality Control
Food contaminated by microorganisms (bacteria and yeasts), viruses, and protozoa can
cause severe disease in humans. There are two categories of foodborne diseases.
 First, food poisoning is caused by the presence of microbial toxins in food products,
e.g. by Staphylococcus aureus, Clostridium perfringens (both produce enterotoxins
which elicit enteric disease such as diarrhea), and Clostridium botulinum (botulism is
the most severe type of food poisoning).
 Second, the growth of microorganisms in the body after eating contaminated food,
e.g. by Salmonella spp. (salmonellosis) and Campylobacter jejuni (high fever,
abdominal cramps). Many human pathogens are transmitted by fecal contaminated
water, the most important being Salmonella typhi (typhoid fever) and Vibrio
cholerae (cholera).
 Microbiological test procedures for the examination of foods and beverages have
been standardized and regulated, but nearly every country has its own regulations.
The Campylobacter is one of the leading causes of human gastroenteritis.

Common Campylobacter species C. jejuni, C. coli, and C. lari are responsible for most cases
of campylobacteriosis. However, other species, like C. fetus, which causes spontaneous
abortions, have also been associated with human illness.
Campylobacter are Gram-negative, spiral-shaped, microaerophilic and motile bacteria with
uni- or bi-polar fl agella (see Figures 1, 2 and 3).
Figure 1.Scanning electron microscope image: shows the characteristic spiral, or corkscrew, shape of C. jejuni cells.
Photo by De Wood; digital colorization by Chris Pooley, USDA/ARS

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Figure 2. Scanning electron micrograph of the single polar flagellum and corkscrew shape
of C. jejuni. These morphologic characteristics contribute to the characteristic darting
motility of C jejuni in the viscous mucous layer of the intestinal lumen. Sean F. Altekruse
National Cancer Institute, Rockville
Figure 3. Electron microscope image of Campylobacters by Jochen Reetz, Bundesinstitut

für Risikobewertung (BfR), Germany
Enterobacter sakazakii is ubiquitous and frequently found on vegetables, meat and dairy
products, and especially in baby food. Consumption of contaminated powdered infant
formula milk (IFM) can result in life-threatening neonatal infections caused by the pathogen.
Taxonomic studies have determined that E. sakazakii comprises a high genetic
heterogeneity and should be reclassified as a novel genus, “Cronobacter”.

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Enterobacter sakazakii colonies grown on M1 agar
Clostridia Diagnostic
Clostridia are relatively large, gram-positive, rod-shaped bacteria that can undergo only
anaerobic metabolism. Most Clostridia cannot grow under aerobic conditions and even can
be killed by exposure to O2, but they form endospores that are able to survive long periods
of exposure to air and other adverse environmental conditions. The natural sources of
Clostridia are anaerobic habitats with organic nutrients, particularly soils, aquatic sediments
and the intestinal tracts of animals. Their fermentation of organic compounds, like sugars,
produces large amounts of CO2 and H2 as well as volatile organic compounds like acetic and
butyric acid, acetone and butanol. Metabolism of substrates like amino acids and fatty acids
results in foul-smelling degradation products. Clostridia also have an extended range of
extracellular enzymes that degrade large biological molecules in the environment into
fermentable compounds. Although there are non-pathogenic Clostridia, this genus produces
some of the most potent biological toxins. Three particularly bad actors in this group are C.
perfringens, which is responsible for cooked meat-associated food poisoning and wound and
surgical infections that lead to gas gangrene, and C. tetani, which is responsible for deadly
tetanus infections, and C. botulinum, which causes botulism.
Below are the most well-known pathogenic Clostridia species with their typical properties
and occurrence:
Clostridium perfringens
 produces a huge range of invasins and exotoxins

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 enzymes: hemolysins (β-hemolysis), lecithinase, extracellular proteases, lipases

(phospholipase-C), collagenase, hyaluronidase, saccharolytic enzymes and is able to
reduce sulphite to sulphide
 enterotoxins causes food poisoning
 found in improperly sterilized canned foods (germination of endospores) and water
 nonmotile
Clostridium difficile
 produces two enterotoxins toxin A and toxin B (lethal cytopathic toxin)
 enzymes: hydrolytic enzymes, p-hydroxyphenylacetate decarboxylase, ferments

mannitol
 formation of p-cresol as the main fermentation product of tyrosin
Clostridium tetani
 toxin: tetanospasmin (causative tetanus)
 obligate anaerobe (sensitive to oxygen)
 sensitive to heat
 flagella give limited motility
 terminal spore (resistant to heat and most antiseptics)
 typical gram-positive, may stain gram-negative or gram-variable, especially in older

cells
Clostridium botulinum
 seven subtypes (A-G) produces different botulinum toxin (types C and D are not
pathogenic)
 grow best in low-oxygen conditions

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 subterminal endospores (resistant to boiling without pressure)
 occurrence: soil, aquatic sediments, decaying vegetation, found in improperly

sterilized canned foods (germination of endospores)
 acidity, high concentration of sugar, very low levels of moisture or high levels of
oxygen inhibits the growth
 enzyme: lipase production on egg yolk agars
E. coli: Indicator organism for fecal contamination
Normal constituents of the intestinal flora of animals, coliforms are rod-shaped, Gram-
negative, non-spore forming facultative anaerobes. They ferment lactose with the
production of acid and gas when incubated at 35–37°C. Although commonly found in lakes,
rivers, swimming pools and soil from faecal sources, in most cases coliforms do not cause
illness. However, they are used as indicators for other pathogenic organisms of faecal origin.
The most common genera of coliforms are Citrobacter, Enterobacter, Escherichia, Klebsiella
and Serratia.
Escherichia coli is the best-known coliform and an important indicator of faecal

contamination because it is found almost exclusively in faeces. Occasional outbreaks of food
poisoning have been linked to certain gastroenteritis-causing E. coli strains, such as
serotype O157:H7. E coli are rod-shaped bacteria, distinguished from most other coliforms
by their ability to ferment lactose at 44°C, and by their growth characteristics on certain
media. Easy to culture, E. coli is often used in molecular biology.
Escherichia coli

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Different types of E. coli are known based on their pathogenic mechanisms:

 Enteropathogenic E. coli (EPEC). Virulence mechanism is unrelated to the excretion of
typical E. coli enterotoxins. Causes gastroenteritis (childhood diarrhea).
 Enteroinvasive E. coli (EIEC) or Shiga toxin-producing E. coli (STEC). Causes a

Shigella-like dysentery.
 Enterotoxigenic E. coli (ETEC). Produce heat-labile (LT) or heat-stable (ST)

enterotoxins. Cause traveller’s diarrhea.
 Enteroaggregative E. coli (EAggEC) or Enteroadherent E. coli (EAEC). Able to attach

to tissue culture cells in an aggregative manner and may produce EAST
(EnteroAggregative ST) toxin. Subgroups, like diffusely adhering E. coli (DAEC)
strains and Cytodetaching E. coli (CDEC), are differentiated. Primarily associated with
persistent diarrhea in children in developing countries and also traveller’s diarrhea
Detection, identification, differentiation and cultivation of Pseudomonas species
HiFluoro Pseudomonas Agar under UV light
Pseudomonas are motile (one or more polar flagella), rod shaped and aerobe Gram-negative
bacteria. They are found almost everywhere, in soil, water, plants and animals. In most
cases it is not pathogenic and in fact can be beneficial. For example, P. putida is used as a
bio-scrubber to aid in the biodegradation of diverse organic compounds in polluted air and
waste water. However, P. aeruginosa is an infamous opportunistic human pathogen most
commonly affecting immuno-compromised patients. Along with P. maltophilia, it accounts

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for the majority of human infections. Pathogenic Pseudomonas are found throughout the
body, most commonly in the urinary tract, respiratory tract, blood and wounds.
Staphylococcus Aureus: a Spreading Bacteria
Staphylococci may be airborne and can occur in both animals and humans, in sewage,
water, milk or food, and on environmental surfaces or food equipment. It is still one of the
five most common causes of nosocomial infections, often causing postsurgical wound
infections. Consequently, it poses a major concern in hospitals, especially in regard to
MRSA, methicillin-resistant Staphylococcus aureus.
Staphylococcus aureus is an invasive pathogen that can cause disease in almost any tissue
or organ in the human body, primarily in compromised individuals. Staphylococcal infections
were treated using penicillin, but over the years this pathogen developed resistance to
penicillin by building penicillinase. Methicillin was the next drug of choice as it is not cleaved
by the penicillinase. While methicillin is very eff ective in treating
most Staphylococcus infections, some strains have developed resistance to methicillin by
production of penicillin-binding protein, and can no longer be killed by this antibiotic. These
resistant bacteria are called Methicillin Resistant Staphylococcus aureus (MRSA) [1]. Patients
with breaks in their skin due to wounds, indwelling catheters or burns are at high risk of
developing MRSA infection [2]. Spread of MRSA infections can be controlled to a great
extent by maintaining personal hygiene after interaction with an MRSA-infected person [1].
Today there are many innovative solutions to detect MRSA. Sigma-Aldrich® strongly
supports the microbiologist with a selective chromogenic HiCrome MeReSa Agar for
detection of MRSA from clinical isolates and other samples. The proprietary chromogenic
mixture incorporated in the medium is specifi cally cleaved by S. aureus to give bluish-green
colonies on this medium and can be clearly diff erentiated from other species. The medium
is made selective for MRSA by the addition of methicillin.
Foods that are frequently associated with staphylococcal food poisoning include meat and
egg products, milk and dairy products, and various other products that may contain these
food ingredients. Processes in the food industry that are kept at slightly elevated
temperatures must guard against staphylococcal food poisoning, one of the leading causes
of gastroenteritis. The food poisoning is due to the presence of staphylococcal enterotoxins
produced by Staphylococcus aureus in the food.

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ID flow chart for Staphylococus aureus

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Differentiation and identification flow chart of Gram-positive cocci
Yersinia species
Yersinia are rod-shaped, Gram-negative facultative anaerobic bacteria. They exhibit

fermentative metabolism and are oxidasenegative, mannitol-positive. A psychrophilic
organism, Yersinia survives and proliferates at low temperatures of 0– 4°C, for example on
food products in a refrigerator. Some Yersinia species are also relatively heat resistant. Pigs,
rodents, rabbits, sheep, cattle, horses, dogs, and cats are the natural sources of Yersinia.
Currently, Y. enterocolitica is responsible for most cases of human illness caused
by Yersinia.

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Other clinical important species of this genus are Y. pseudotuberculosis and Y. pestis, the
infectious agent of bubonic plague. Most infections are acquired from contaminated food,
like raw or undercooked pork products, sea food, vegetables, unpasteurized milk or
untreated water. However, infections also occur from contact with infected animals, faeces
or transmission by fleas.
Early Detection of Fungal Hyphae on Bread by Fluorescently Labelled Lectins
Food quality assurance and control have gained importance as factors of concern to public
health. Perishable foods are of particular focus for routine food analysis. One of the most
critical issues is the contamination of breads and fruits by fungi and moulds, as these food
items are especially susceptible to mould development. Specific fungal species and the
mycotoxins produced can adversely affect human and animal health, and early identifi
cation of negative fungi can allow contaminated products to be removed before entering the
food chain. Here, we demonstrate a highly specifi c technique for early identifi cation of
fungi in bread via direct fl uorescence detection, using fl uorescently labelled lectins.
Cultures around the world rely on herbs and spices to add flavour and zest to food. Many
spices, however, contain very high numbers of bacteria, making them a potent source for
food spoilage and pathogens.
To study the microbiological status of herbs and spices, E. de Boer et al. tested 150 samples
collected from 54 different spices, spice mixtures and herbs. They reported at least 1,000
organisms per gram, withmost spices containing 105-106 cells per gram. A high number of
psychotropic bacteria, yeasts and Enterobacteriaceae was detected mainly on herbal spices
originating in moderate climate areas. The study also reported highmould counts,
identifying Aspergillus niger, A. flavus, A. tamarii, Penicillium citrinium, P. chrysogenum, and
Absidia corymbifera as the most frequent isolated species. Since A. flavus may produce
aflatoxins, one of the most potent naturally occurring toxins, its presence should be a
matter of concern and monitored closely by the spice industry. Another serious potential
public health risk may involve the presence of pathogenic bacteria ; frequently reported
species are Clostridium perfringens, Bacillus cereus and Salmonella.

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Structure of ferrioxamine E

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2- Pesticide and Metabolite Residues
Analysis of pesticide levels in food and beverage products is important not only to insure low
levels for human consumption, but also to avoid international trade problems. At the present
time, more than 1000 pesticide and metabolite residue compounds are identified as
associated with food crops, either in current use, or used in the past. Several techniques are
commonly used:
 Extraction/Cleanup using QuEChERS (pronounced ′catchers′): The Quick,
Easy, Cheap, Effective, Rugged, Safe sample preparation approach developed by
Anastassiades and Lehotay simplifies the sample preparation of pesticides from foods
of plant origin. This dispersive SPE method uses bulk materials instead of traditional
tube-based hardware. Extraction is done using a solvent (such as acetonitrile) and
pre-weighed extraction/buffer salts. After a centrifugation step, the extract is cleaned
with bulk sorbents. Several materials are available as QuEChERS cleanup sorbents,
each effective for the removal of specific interferences, and can be used individually
or in combinations best suited to the sample matrix.
 Extraction/Cleanup using Traditional SPE: Traditional SPE can also be

employed for the cleanup of samples prior to analysis. Our Supelclean offering
includes tubes with a single sorbent, and also tubes with multiple sorbents. An SPE
tube with a multi-layer design allows all cleanups to be accomplished in a single step.
 HPLC Analysis: LC-MS/MS is able to quickly achieve proper identification and low
detection limits for large analyte lists. It is critical to use high quality columns,
solvents, and mobile phase buffers to minimize artifacts which will interfere with the
sensitivity of the instrumentation.
 GC Analysis: GC-MS is also used as an analytical technique, requiring columns with

low bleed and high inertness characteristics.
In May of 2006, the Japan Ministry of Health, Labour and Welfare (MHLW) introduced
regulations for the levels of agricultural chemical residues allowed in plant-based and
animal-based foods. This list of chemicals includes pesticides, feed additives, and veterinary

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drugs. The goal of this regulation is to prevent the distribution of foodstuffs containing these
residues at levels above specific limits. Products that exceed these limits cannot be sold in
Japan. The analysis of these chemicals involves either liquid chromatography-mass
spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS). In this article,
the applicability of the SLB™-5ms capillary GC column was determined for the analysis of
the chemicals listed in the GC-MS sections of the regulations.
A set of six standards covering the range of GC-amenable MHLW-regulated chemicals was
located and obtained from a commercial source in Japan. Each mixture contained analytes
at 20 μg/mL in acetone. An SLB-5ms column was selected because of several features,
namely its low bleed characteristics and high inertness.
GC-MS run conditions were optimized to yield good chromatography for all analytes. These
conditions are summarized in Table, and were used for the analysis of each of the six
standards. Note that these conditions differ slightly from those stated in the MHLW
methodology. Specifically, a higher initial oven temperature and a slower ramp rate were
used to obtain better peak shape and spacing. Additionally, a smaller injection volume was
used to ensure that the inlet liner contained the resulting expansion volume of the acetone
solvent, and to minimize band broadening. Table, lists the MHLW methodology condition if
different from that used for this work.

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‘Positive List’ Mix 1 on the SLB-5ms

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Analysis of Organophosphorous Pesticides in Produce by Solid/Liquid

Extraction and Dual-Layer Amino-Silica/Carbon SPE Cleanup and GC-
NPD
Introduction
Organophosphorous pesticide use in agriculture is widespread due to the fact that they are
more amenable to environmental degradation in comparison to organochlorine or
organonitrogen compounds. There is a large number of pesticides in the
organophosphorous group, and because of their potential health effects, they are of
particular concern on produce imported from areas in which they are commonly used. In
this work we analyzed the representative organophosphorous pesticides from cabbage,
green onions, apples and mushrooms. The extraction was performed using a custommade
mixture of salts and liquid-liquid partitioning, with clean-up using dual-layer
Supelclean™ ENVI-Carb™-II/PSA Solid Phase Extraction (SPE) tubes.
Sample Preparation
A 10 g homogenized food sample was spiked at 10 ng/g with pesticides, and 10 mL of

acetonitrile was added. The sample was mixed with custom-made extraction salts – 4 g dry
magnesium sulfate and 1 g sodium chloride. The sample was centrifuged and the
supernatant was mixed with 1 g of dry magnesium sulfate. 5 mL of the resulting sample
was evaporated down to 1mL for SPE loading. A Supelclean ENVI-Carb-II/PSA 500 mg/500
mg 6 mL tube was pre-conditioned with 5 mL acetonitrile:toluene (65:35). The sample was
loaded and eluted with 10 mL of acetonitrile:toluene (65:35). The elution fraction was
evaporated to 0.5 mL and reconstituted to 1mL with ethyl acetate.
Figure 2 Supelclean ENVI-Carb-II/PSA SPE Tube

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Analysis of Melamine in Milk
Introduction
Recent news stories from China1 have reported incidents of milk, milk products, and infant
formula tainted with melamine, a substance used to make plastics and fertilizers. It is not
approved for use in foods, but health officials reported melamine was added to these dairy
products to inflate their apparent protein content. Several infants died and thousands of
children were hospitalized after suffering kidney failure from ingesting the melamine-
adulterated milk. Laboratories worldwide are now developing methods to test products for
melamine.
Milk is a complex sample matrix, demanding careful sample prep to ensure valid melamine-
content results. Sigma-Aldrich offers high-purity melamine and cyanuric acid chemicals,
which allow customers to prepare their own standards to meet their particular application
needs; as well as Discovery® solid phase extraction products to ensure clean samples, and
Ascentis®HILIC columns to give superior chromatographic results.
Discussion
Sample extraction
A 5 mL aliquot of milk was spiked with melamine standard (Fluka® Cat. no. 52549). Next, 5
mL of phosphate buffer (100mM, pH 2.5) and 1 mL acetonitrile were added to the sample
and set in an ultrasonic bath for 5 minutes. The sample was centrifuged at 3500 rpm for 10
minutes and the middle supernatant separated from the top and the bottom layers.
Sample SPE preparation
Supelco® Discovery DSC-SCX 500 mg/6 mL SPE tubes (Cat. no. 52688-U) were used for
further sample cleanup. The SPE tubes were conditioned with 3 mL methanol and 3 mL
100mM phosphate buffer (pH 2.5). Then 2.2 mL of the resulting supernatant was loaded
into the SPE tube and washed with 3 mL of phosphate buffer (100mM, pH 3) and 3 mL of
methanol. The melamine was eluted with 4 mL 5% ammonia in methanol. The elution
solvent was evaporated to dryness at 50 °C in a water bath under a flow of nitrogen at 5
psi. The samples were reconstituted into 1 mL of the LC mobile phase A (see conditions
below).

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LC-MS-MS method
Melamine (Figure 1) is a polar molecule with a pKa of 5.6 and a Log P value of -
1.37,2 making it a good candidate for aqueous normal phase (ANP) chromatography. In
ANP, a polar hydrophilic analyte partitions between a relatively polar stationary phase and a
relatively non-polar mobile phase. ANP is commonly referred to as HILIC, but the term
HILIC (Hydrophilic Interaction Chromatography) implies a mechanism that is one of several
that may be operative under ANP conditions. Determining melamine content using the
HILIC mode, run on Supelco’s Ascentis Express HILIC column (Cat. no. 53939-U), gives
better retention of the polar molecule than conventional reversed-phase chromatography.
Figure 1. Structure of Melamine
This HILIC mechanism describes the process of preferential solvation of the polar stationary
phase with the aqueous component of the mobile phase, and subsequent depletion of the
mobile phase of water. This sets up a biphasic system where there is a semi-immobilized
layer of water near the surface and an organic-rich mobile phase layer. A polar compound
may then partition from the moving organic-rich mobile phase into the stagnant aqueous
solvent near the surface. Water then becomes the “strong” solvent and analytes generally
elute (assuming HILIC is the dominant mechanism) in order of “decreasing” hydrophobicity
(a lower log P, indicating a more polar molecule, and consequently more retention).
Detection for melamine was accomplished using an ABI/MDS SCIEX® 3200 QTRAP® MS/MS
with the parameters below. Two multiple reaction monitoring (MRM) transitions were
monitored; 127/85 MRM was used for quantitation.
Other instrument parameters were as follows:

Curtain Gas: 20

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Ion Spray Voltage: 5000

Gas 1: 20
Temperature: 350 °C
Gas 2: 40
Quantitation
An external calibration curve was used for melamine. External calibration standards were
prepared in the mobile phase. No matrix-matched standards were used.
LC-MS-MS Analysis of Melamine Extracted from Milk Spiked at 100 ng/mL
Melamine Analysis via GC-MS

Introduction
Melamine contamination in food became an issue in recent years after the discovery of it,
and related compounds, in pet food and baby formula. It was discovered that melamine was
intentionally added to inflate nitrogen content, often the sole measure of the amount of
protein in these products. The tainted food led to numerous illnesses, several fatalities, and

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massive product recalls. Currently, imported raw materials, namely wheat gluten and rice
protein used to make these foods, as well as the actual consumer-ready foods, may
undergo testing to ensure the absence of these compounds. We have detailed preparation
and analytical procedures for these adulterants, using HPLC-MSMS instrumentation in
previous publications (1,2). In this article, we focus on the analysis of melamine and related
compounds with the use of more economical gas chromatography-mass spectrometry (GC-
MS) instrumentation.
GC-MS Method
The United States Food and Drug Administration (US FDA) adopted a screening method in
October 2008 for the GC-MS analysis of melamine and related compounds in a variety of
matrices (3). Per the method, 0.5 g of the sample is mixed thoroughly with 20 mL of an
extraction solvent mixture (10:40:50 diethylamine:water:acetonitrile). Following sonication
(30 minutes) and centrifugation (10 minutes), an aliquot is filtered and evaporated to
dryness. Sylon™ BFT and pyridine are then added along with an internal standard. The
extract is then incubated (70 °C for 45 minutes) so that trimethylsilyl (TMS) derivatives of
each analyte are formed. The resulting derivatized extract is then analyzed by GC-MS. The
method allows the operation of the MS in the scan mode (m/z from 50-450 amu) or the
selected ion monitoring (SIM) mode. Table 1 shows the structures of the four analytes
(melamine, ammeline, ammelide, and cyanuric acid) plus 2,6-diamino-4-chloropyrimidine,
the internal standard (I.S.) specified by the method.

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Melamine and Related Compounds
Experimental
For this work, we chose a common dry dog food obtained from a local grocery store. The
following samples were prepared:
1. Three calibration standards, each containing all four analytes, were made at levels of
10 ng/mL, 50 ng/mL, and 100 ng/mL (I.S. added at 1000 ng/mL in each),
derivatized, and then used to perform a three-point calibration of the instrument.
2. A laboratory blank was extracted (I.S. added at 1000 ng/mL), derivatized, and then
analyzed to show cleanliness.
3. A 0.5 g dog food sample was extracted (I.S. added at 1000 ng/ mL), derivatized, and
then analyzed to determine analyte levels.
4. A second 0.5 g dog food sample (spiked with each analyte at 10 μg/g) was extracted
(I.S. added at 1000 ng/mL), derivatized, and then analyzed to determine method
sensitivity and accuracy.
All standards and extracts were analyzed with the MS operating in the scan mode, and again
later with the MS operating in the SIM mode.

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Results
The following results were observed:

 MS Mode: Sensitivity was noticeably better when the MS was operated in the SIM
mode. All chromatograms shown are from SIM mode analyses.
 Instrument Calibration: The 100 ng/mL standard is shown in Figure 1. Note the
symmetrical peak shape for each analyte and the I.S., achieved because the activity
of amide functional groups was minimized when TMS derivatives were formed, and
also due to the inert nature of the capillary GC column.
 Laboratory Blank (Figure 2): Trace levels of each of the four target analytes were
detected in the laboratory blank only when the MS was operated in the SIM mode.
 Dog Food Sample (Figure 3): The detection of analytes was at a level consistent
with that observed in the laboratory blank. We concluded that this dog food was not
contaminated with any of the target analytes.
 Spiked Dog Food Sample (Figure 4): The percent recoveries of each the four
target analytes are summarized in Table 2. Good recovery was obtained for each
analyte.

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100 ng/mL Calibration Standard (SIM Mode) (referenced above is 28471-U)
Laboratory Blank (SIM Mode)

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Dog Food Sample (SIM Mode)
Spiked Dog Food Sample (SIM Mode)

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Recovery from Dog Food Spiked at 10 μg/g
Conclusions
Our observation is that the method is very easy to perform and provides good sensitivity. In
particular, the use of the specified extraction solvent mixture was found to be very effective
in solubilizing and extracting all target analytes. Additionally, the formation of TMS
derivatives allows these analytes to be analyzed by GC, with symmetrical peak shapes, high
signal-to-noise ratios, and low detection levels.
Improved Recovery of Fungicide Carbendazim from Orange Juice

By: Olga Shimelis and Emily Barrey, Reporter US Volume 30.2
A QuEChERS sample preparation method followed by LC-MS/MS
Introduction
The fungicide carbendazim can sometimes be used to combat mold on citrus fruits including
oranges. Recently, low residue levels of the fungicide were reported to be present in bulk
orange juice import shipments to the USA. Because this compound can persist through
processing steps and may be found in the final consumer product, many government
agencies have established maximum residue limits (MRLs) to ensure the safety of their
population. For example, the carbendazim MRL in orange juice is 10 ppb in the USA, and
200 ppb in the European Union (1-2).
Final consumer products include juice that is pulp-free, juice with pulp, or frozen
concentrate. Each of these matrices contains a different amount of solids, which may
interfere with sample preparation and/or analysis. For this work, we evaluated several
sample preparation methods to determine if a single method could 1) achieve acceptable
recoveries, and 2) reduce interference, for each matrix. These goals are desirable as they
would allow food analysts to process various matrices in a single workflow.

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Experimental
Pulp-free orange juice, orange juice with pulp, and frozen concentrate samples were
purchased from a local grocery store. Each matrix was subjected to three sample
preparation methods prior to LC-MS/MS analysis. A comparison of the three methods is
presented in Table 1. Method 1 is simple physical manipulation (centrifuge and filter), and
is not suitable for the frozen concentrate sample. Method 2 is a solvent extraction that uses
salts to drive analytes into the organic layer. Method 3 incorporates a QuEChERS cleanup
following solvent extraction.
Table 1. Comparison of Methods
Method 1 (Filter and Shoot)
1. Add 1 mL of orange juice to a 2 mL centrifuge tube
2. Centrifuge at 10,000 rpm for 5 min
3. Filter supernatant through a 0.45 µm PVDF filter
4. Analyze via LC-MS/MS
Method 2 (Acetonitrile Extraction)
1. Add 10 mL of orange juice, or 10 g of orange concentrate, to a 50 mL empty

extraction tube (55248-U)
2. Add 10 mL acetonitrile, and mix well for 1 min
3. Add contents of citrate extraction tube (55227-U), shake well, and mix for 1 min
4. Centrifuge at 3,200 rpm for 5 min
5. Dilute the acetonitrile layer with water 1:1
Method 3 (Acetonitrile Extraction + QuEChERS Cleanup)

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1. Follow Method 2 through step 4
2. Mix 0.7 mL of the acetonitrile layer with contents of the PSA/C18 cleanup tube
(55288-U)
3. Recover 0.25 mL of the supernatant and mix with 0.25 mL water
The Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) cleanup uses bulk materials

instead of traditional tube-based hardware, which simplifies the procedure (3). The main
sorbent used is a primary-secondary amine (PSA) which provides high capacity for the
removal of sugars, organic and fatty acids, and polar pigments. Depending on the sample,
C18 silica sorbent can be added for the cleanup of fat-containing samples, and/or carbon
sorbent can be added for the cleanup of highly pigmented samples. Based on the
interferences expected in orange juice samples, PSA/C18 sorbent was selected.
Unspiked and replicate spiked samples were prepared for each matrix using each method.
Following sample preparation, all calibration standards and extracts were analyzed using LC-
MS/MS on a short, narrow-bore, Ascentis® Express C18 column. Two mass transitions were
monitored to identify carbendazim (m/z 192/160 and 192/132). The abundant response of
carbendazim allowed an instrumental limit of detection of 0.1 ppb to be achieved.
Results & Discussion
Figure 1 shows chromatograms of an unspiked and a spiked replicate of the juice with pulp
sample following sample preparation using Method 3. The choice of an Ascentis Express
column allowed efficient chromatography in a short time. In fact, a benefit of this column
line is that great efficiency can be obtained on any system, regardless if used with HPLC or
UHPLC equipment.

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Figure 1. LC-MS/MS Analysis of Unspiked and Spiked Orange Juice with Pulp
HPLC Conditions: sample/matrix: add 10 mL of orange juice with pulp to a 50 mL empty

extraction tube (55248-U); add carbendazim at 1 ppb to ‘spiked’ sample; add 10 mL
acetonitrile; mix well for 1 min; add contents of citrate extraction tube (55227-U); shake
well; mix for 1 min; centrifuge at 3,200 rpm for 5 min; mix 0.7 mL of the acetonitrile layer
with the contents of the PSA/C18 cleanup tube (55288-U); recover 0.25 mL of the
supernatant; mix with 0.25 mL water
column: Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 µm particles (53822-U) mobile
phase: (A) 10 mM ammonium acetate in water; (B) 10 mM ammonium acetate in methanol
gradient: 0-1 min: 30% B; 1.5-3.5 min: 100% B; 3.5-7 min: 30% B flow rate: 0-1 min: 0.3
mL/min; 1.5-7 min: 0.5 mL/min temp.: 30 °C detector: MS, ESI(+), MRM, m/z 192/160,
192/132 . Injection: 5 µL
Very low levels of carbendazim were detected in two of the three unspiked samples. Note
that the levels observed are below the MRLs for both the USA and European Union. Average
recovery and %RSD values were calculated and are summarized in Table 2, by matrix and
method. As stated previously, Method 1 is unsuitable for a solid matrix, such as the frozen
concentrate. Based on the data presented, Method 3 achieves the first goal of this work–
to identify a single method which can achieve acceptable recoveries for each matrix.
Average Recovery and %RSD (in parentheses) from 1 ppb Spiked Samples
(n=3)
Matrix Method 1 Method 2 Method 3

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Juice, Pulp-Free 25% (79) 49% (7) 73% (5)
Juice, With Pulp 45% (18) 64% (14) 73% (15)
Concentrate, Frozen n/a 36% (13) 72% (20)

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3- Veterinary Drug Residues
The use of veterinary drugs may be integral to the process of raising food producing
animals and fish. These compound types include:
 Antibiotics (aminoglycosides, chloramphenicol, fluoroquinolones, malachite green
dye, etc.) used to inhibit microbial growth
 Feed additives (hormones and steroids) used to increase yield

It is possible that residues may remain in the final food or beverage product, even after
processing steps. Because elevated amounts of some of these drugs may cause detrimental
health effects to humans (including being carcinogens and allergy-triggers), it is important
to monitor their levels. Several techniques are commonly used:
 Extraction/Cleanup using QuEChERS (pronounced ′catchers′): The Quick,
Easy, Cheap, Effective, Rugged, Safe sample preparation approach developed by
Anastassiades and Lehotay simplifies the sample preparation of pesticides from foods
of plant origin. This dispersive SPE method uses bulk materials instead of traditional
tube-based hardware. Extraction is done using a solvent (such as acetonitrile) and
pre-weighed extraction/buffer salts. After a centrifugation step, the extract is cleaned
with bulk sorbents. Several materials are available as QuEChERS cleanup sorbents,
each effective for the removal of specific interferences, and can be used individually
or in combinations best suited to the sample matrix.
 Extraction/Cleanup using Mixed Mode SPE: One difficulty in the analysis of

drugs in complex matrices is that the sample preparation process tends to capture
many non-target compounds along with the target analytes. The presence of these
interferences may lead to ion-suppression effects when LC-MS is used as the
analytical technique. It is necessary to select cleanup steps that remove the
interferences from extracts, without also removing target analytes. One approach is
to use a mixed mode SPE material, such as Discovery DSC-MCAX. The two ligands
blended in this mixed mode hydrophobic-cation exchange material are C8 (provides
reversed-phase interaction) and benzene sulfonic acid (provides strong cation

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exchange interaction). This unique combination of ligands allows aggressive washing

steps to remove interferences without loss of target analytes.
 Extraction/Cleanup using Traditional SPE: Traditional SPE can also be

employed for the cleanup of samples prior to analysis. Our Supelclean offering
includes tubes with a single sorbent, and also tubes with multiple sorbents. An SPE
tube with a multi-layer design allows all cleanups to be accomplished in a single step.
 Extraction/Cleanup using Molecular Imprinted Polymers: Another approach

for the removal of interferences without the loss of target analytes is through the use
of molecular imprinted polymer (MIP) materials. MIPs are highly cross-linked
polymer-based molecular recognition elements. Their primary feature is that they can
be engineered to be highly selective to compounds that are structurally similar. The
benefit is increased affinity for the specific compound structure they are engineered
for, and decreased affinity for other structures.
 HPLC Analysis: LC-MS/MS is able to quickly achieve proper identification and low
detection limits for large analyte lists. It is critical to use high quality columns,
solvents, and mobile phase buffers to minimize artifacts which will interfere with the
sensitivity of the instrumentation.
 GC Analysis: GC-MS is also used as an analytical technique, requiring columns with

low bleed and high inertness characteristics.

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4- Toxins (other than pesticide/drug residues)
Many government agencies require food and beverage products to be monitored for the
presence of toxins that have detrimental effects on consumer health. Toxins can migrate
into crops if they are grown in contaminated air or water. Another source is naturally
occurring organisms (certain fungi, bacterial, or algal growths emit toxins) that contaminate
crops they co-exist with. Toxins may also be found in meat/dairy/poultry products if the
animals were exposed to contaminated feed. Many of these toxins remain potent even after
food processing/packaging steps, and remain in the final products. Areas of interest include:
 Mycotoxins
 Allergens
 Dioxins/Furans/PCBs
 Perchlorate
 Phycotoxins
 Phytochemicals
 Heavy Metals
Mycotoxins
Mycotoxins are a diverse group comprised of hundreds of secondary metabolic products of

various fungal species. Several show marked toxicity in humans. Contamination of the food
supply with mycotoxins is increasingly prevalent, and can occur during growth, harvest,
transportation, processing, or storage. Foods which are susceptible to mycotoxin
contamination are bread, cheese, cereal grains, oil seeds, and tree nuts. Techniques to
reduce mycotoxin concentration after contamination are expensive, unreliable, and
sometime reversible. Therefore, the removal of contaminated products from the food chain

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is a primary means of eliminating human exposure. The sensitive and accurate detection of
very low levels of these compounds is critical to governmental efforts to identify
contaminated product.
Allergens
There are two types of allergic reactions associated with food and beverage products.
 A number of people have developed sensitivity to additives (preservatives, dyes,
fragrances, etc.) and/or packaging contaminants (phthalate esters, bisphenol A, etc.).
HPLC or GC techniques are suitable for determining the presence of these
compounds.
A number of people are allergic to specific proteins found in dairy, egg, peanut, tree nuts,
fish, shellfish, soy, and wheat. Detection of specific proteins (immunoassay technique) or
DNA (PCR-ELISA technique) can be employed.
Dioxins/Furans/PCBs
These compounds may be found on crops near industrial facilities where both chlorinated
organic compounds and elevated temperatures exist. Elevated levels can also be found in
meat, dairy, and poultry products through contaminated feed. These compounds are
persistent in the environment, and several congeners are toxic. Specifically, those with
chlorine substitution at all four corners resulting in a co-planar shaped molecule are
identified as toxic to humans. A total of 17 of the 210 dioxin/furan congeners and 12 of the
207 PCB congeners are co-planar. Following extraction and cleanup, GC coupled with high
resolution mass spectrometry (HRMS) is necessary to properly measure these toxic co-
planar congeners.
Heavy Metals
Industrial effluent streams can introduce heavy metal contamination into aqueous systems.
Heavy metals (such as mercury, lead, cadmium, arsenic, copper, and nickel) can then
migrate to any plant or animal life which uses this water. Fish and other aquatic life are
obviously the most affected. Because heavy metals do not break down, they will bio-
accumulate up the food chain. Atomic adsorption spectroscopy (AAS) and inductively
coupled plasma (ICP) are the analytical techniques most often used to determine the

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presence and levels of heavy metals in foods, with hyphenated methods such as LC-ICP-MS
being often used for speciation analysis. Fluka offers multiple certified reference materials
(CRMs) and high quality acids, bases and salts for the calibration and operation of AAS and
ICP instruments.
Residual Solvents from Food Packaging
Establishing, regulating and monitoring food safety are among the most important
applications of analytical chemistry and chromatography. Food analysis covers myriad
subjects, including compositional analysis for nutritional labeling and monitoring
adulteration, contamination and compliance with regulations, among others. Foodstuffs can
become contaminated from a variety of sources from the raw materials and at any point
along the production process, including from the packaging materials.
Almost all foodstuffs consumed today are supplied encased in some form of packaging. By
forming a barrier between the food and the environment, packaging materials maintain
product quality and safety during shipping and storage. Packaging also protects the food
from other packaging materials that may have desirable physical properties but are
notorious sources of contamination, like recycled paperboard [1]. The types of packaging
range from a simple, thin coating of foodgrade wax to complex multi-layered systems that
combine paper, plastic and metallic components with adhesives.
However important the packaging is to maintaining the food quality and shelf-life, packaging
materials are also a well-known source of contamination when they leach undesirable
compounds into the food they are intended to protect. These compounds can be toxins and
pose significant health hazards. They can also be sources of off-flavors or off-odors,
diminishing the sensory quality of the product, which is a very important commercial aspect
of food science [2, 3].
The etiology of the contamination reveals that the manufacturing process used to make the
package materials, adhesives that hold multi-layer packaging together, and the varnishes,
inks and dyes applied to the finished package can all be sources of leachables or
extractables that end up in the food. Solvents derived from these sources are a major
contamination class and are of primary concern to food analysts.

Sign: Sign:
Issue No.: 01
Revision No.: 00
GC Analysis of Residual Solvent Mixes for Packaging Standards
GC Conditions: column: VOCOL, 60 m x 0.25 mm I.D., 1.5 μm df (24154); oven: 35 °C (4

min.), 4 °C/min. to 200 °C (0 min.); inj.: 200 °C; det.: MSD, 280 °C; carrier gas: helium, 30
cm/sec.; vent flow: 140 mL/min.; injection: 0.5 μL

Sign: Sign:

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Inspection Food And Drugs Labs Section No.

Chemistry & Microbiology Page 1 of 86

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Food Composition/Nutrition Labeling

 Dietary fiber is a measure of non-digestible components, and includes non-digestible

Simple sugars using hydrophilic interaction chromatography (HILIC):

 The Ascentis Express F5 phase is also available in a more traditional 5 μm particle.

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Carbohydrate and dietary fibre levels

Determination of dietary fibre is another important food analysis. Consuming high-fibre

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2- Fats (fatty acids, FAMEs, glycerides), Edible Oils, Sterols

 Mono-, di-, and triglycerides, analyzed by GC or HPLC

 Edible oil characterization by GC

 IR and NMR procedures

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Derivatization of Fatty Acids to FAMEs

 To distinguish between the very slight differences exhibited by unsaturated fatty

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 Samples can be derivatized neat or after dissolving in solvent. If appropriate, dissolve

 Weigh 1-25 mg of sample into a 5-10 mL micro reaction vessel.

 Add 2 mL BCl3-methanol, 12% w/w. A water scavenger (such as 2,2-

 Cool, then add 1 mL water and 1 mL hexane.

 To determine the proper derivatization time, analyze aliquots of a representative

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 If it is suspected that complete derivatization is never achieved, use additional

FAMEs by Degree of Unsaturation

Saturated, monounsaturated, polyunsaturated, and cis/trans configuration all refer to the

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Omega 3 and Omega 6 FAMEs

 Production of lipoxins and resolvins, which affect inflammation.

 Production of endogenous cannabinoids, which affect mood and behavior.

 Influencing cell signaling.

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Conditions: column 1: Omegawax, 30 m x 0.25 mm I.D., 0.25 µm (24136); column 2:

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1. C4:0 20. C18:2n6t

cis/trans FAME Isomers

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increasing lipoprotein(a) levels. Studies have linked their nutritional contribution to be

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