Analysis of Protein Dietary Supplements

By

This project is submitted in part fulfilment of the HETAC requirements for the award of Bachelor of Science (Honours) Forensic Investigation and Analysis Degree.

MAY, 2011

Title: ANALYSIS OF PROTEIN DIETARY SUPPLEMENTS Name: ID: S00093560 Supervisor: Declaration: I hereby declare that this project is entirely my own work and that it has not been submitted for any other academic award, or part thereof, at this or any other education establishment. _________________

Abstract

Protein dietary supplements are widely used across all ages and user groups. Many analytical techniques are available to determine what is contained in protein supplements. Moisture, peptide, total protein and chiral amino acid analysis are all investigated. The moisture content of the dried products ranged from 3.14% (Sunwarrior) -7.72% (Hemp). Total protein of these dried products ranged from 37.08% (Hemp) - 83.34% (Soya 90%). All samples (Dried and hydrolysed liquid) analysed contained levels of D-amino acids with liquid Aminos having the highest level at 30.72mg/100ml D-Glutamic acid. D-amino acids cannot be assimilated in the body, and therefore no use to consumers. The D-amino acids are suspected to originate through manufacturing processes although no products states their presence on the label.

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Acknowledgements

I would like to acknowledge the ongoing support, guidance and attention of Dr. Fiona Mc Ardle who help me immensely throughout this project. I would also like to acknowledge the support, patients and understanding of my family and Carla who have supported me through this project. Sincere thanks must go to the technical staff at IT Sligo, without them this project would not have been successful.

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Table of Contents
Page Abstract Acknowledgements Contents Introduction Section 1: Literature Review 1.1 What is protein? 1.2 Amino Acids 1.2.1 Digestion of amino acids 1.3 Dietary protein requirements 1.4 Stereoisomerism of amino acids 1.4.1 Effects of production on chirality 1.4.2 Regulation on chirality 1.4.3 Forensic applications of amino acid racemisation 1.4.4 Chirality and the International Olympic Committee 1.5 Methods available for the analysis of protein 1.5.1 Total Protein 1.5.2 Peptide analysis by RP-HPLC iii 3 7 10 16 17 18 19 20 21 22 22 26 i ii iii 1

Page 1.5.3 Amino acid analysis 1.5.4 Chiral separation of amino acids by gas chromatography. Section 2: Methodology 2.1 Introduction 2.2 Experimental protocol 2.2.1 Moisture analysis 2.2.2 Biuret total protein 2.2.3 Hach Persulfate total nitrogen 2.2.4 RP-HPLC peptide analysis 2.2.5 GCFID analysis of pentafluoropropyl esters of amino acids 2.3 Results 2.4 Discussion 2.5 Conclusion 2.6 Recommendations Appendix I Appendix II Appendix III Bibliography iv 35 36 38 40 42 46 54 63 64 66 70 88 100 33 29 31

Introduction
A dietary supplement is a concentrated source of nutrients or other substances with a nutritional or physiological effect, whose purpose is to supplement the diet. The Sale of dietary supplements in Europe is worth 0.5 billion euros with dietary supplements are being used across all ages and user groups. Protein Based supplements will be investigated in this dissertation. The sale of dietary supplements as a foodstuff is controlled under the European Communities Food Supplements amendment regulations 2010 and this is enforced in Ireland by the food safety authority of Ireland. ISO 22000 is the international standard for the production of these supplements, few companies however strive to attain ISO 22000:2005 but may have other standards in place including ISO 9001 (quality system) ISO 17025 (analysis). All manufactures of Dietary supplements must adhere to food good manufacturing practices. This is primarily concerned with safety and sanitation whilst ensuring ingredients used provide a reasonable certainty of no harm. Although these regulation and guidelines are in place there are claims that there is insufficient regulation and monitoring regarding dietary supplements in Europe (Petroczi et al 2010) The EU Rapid Alert System for Food and Feed RASFF report over 10 claims per month regarding dietary supplements. These claims can be regarding contamination, higher/lower than stated levels of active ingredient and the illegal inclusion of anabolic steroids or hormones. Recent literature links the use of dietary supplement use to hepatotoxicity (Navarro 2009), renal failure (Krishann et al 2009)and toxic effects caused by an excess intake of vitamins and minerals (MacFarquhar et al 2010). If the standards regarding the manufacture and labelling of dietary supplements was that of a pharmaceutical product, these side effects could be prevented. (MacFarquhar et al 2010). Analysis of these products is required in order to ascertain whether or not these products are safe for consumption by people across all user groups.

The aim of this dissertation is 3 fold; 1. Investigate methods of analysis for protein based dietary supplements. 2. Carry out Laboratory based analysis of these products. 3. Ascertain if there are any major discrepancies between laboratory analysis and manufactures nutritional information.

What is protein?
Proteins are a diverse and plentiful class of biological molecules which make up for over half of the dry weight of the human body (Garrett 2010). Proteins are of a diverse nature and each protein needs to carry out specific biological functions. The dominant structural units of proteins are polymers (called polypeptides) with molecular weights ranging from 10,000 to several million and are described as highly complex structures. These polypeptides are formed from amino acid monomer units (Holum 1998). Polypeptides are joined up peptide molecules. Peptides are short sequences of amino acids (up to 20 amino acids). Protein structures have 4 levels of complexity: Primary structures Secondary structures Tertiary structures. Quaternary structures

Primary structure:
The primary (1o) structure is the first and most fundamental level of the protein. This entails only the sequence of amino acid residue in the proteins polypeptide(s). A peptide bond joins amino acids together to form a polypeptide. This system is considered an amide system because of the Carbonyl to Nitrogen bond. When two amino acids are joined in the body a molecule of water expels from the reaction, as shown in the figure 1 below(Holum 1998).

Fig. 1 Figure 1: Diagram of 2 amino acids joining to form dipeptide, expelling water during reaction. (www.molecularsciences.org accessed 16-03-2010) NB: Amino acids will be discussed further in this section.

Secondary structures:
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The polypeptide chain can arrange itself into conformations including pleated and helical formations (fig 2). This is achieved through non-covalent Hydrogen bonding and hydrophobic interactions between adjacent amino acids. These bonds determine how a polypeptide conforms into a specific shape. It is primarily the hydrophobic interactions that define the formation of the polypeptide shape and the hydrogen bonds stabilize the structure. Beta pleated sheets and alpha helix structures (fig2) are the common shape of polypeptides, due to the twisting and folding caused by hydrogen bonding and hydrophobic interaction. (Garrett 2010)

Fig2. Figure 2: Illustrating the hydrogen bonding in alpha helix and beta pleated sheet formations of secondary structures of a polypeptide. (www.abcte.org accessed 16-Mar-2011)

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Tertiary Structures.
The tertiary 3o structure involves the bending and folding of secondary structures so that they can assume a three-dimensional shape and become more compact as a structure. It is at this point the molecule becomes globular in shape. It is this globular shape that gives the lowest volume to surface ratio which lowers the interaction with the molecule and its environment (Garrett 2010). During the formation of tertiary structures, alpha helix and beta pleated sheets undergo extensive folding and twisting in order for the hydrophobic groups to be tucked inside the molecule and the hydrophilic groups are exposed on the outermost of the structure (Holum 1998). Ionic bonds help stabilize tertiary structures. These bonds are formed from positive or negative charges each occurring on a side chain of an amino acid. Disulphide bonds present in tertiary structures will give rise to loops in polypeptides or have the ability to join strands of polypeptide together. Van der Waals forces are also present at this point between R groups or side chains of the amino acids.

Fig. 3 Figure 3: illustration of bonds and interactions within tertiary structure of protein. (http://www-3.unipv.it accessed 18-Apr-2011)

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Quaternary structure:
Some proteins have final shapes at the tertiary level, i.e. Myoglobin because it only consists of one chain of amino acids and therefore it is a single peptide. If a protein is made up of two or more polypeptides, this results in a quaternary ( 4o) structure to the protein. Shown below (fig. 4) is an illustrated version of the 4 structures of protein complexity.

Fig 4 Figure 4: Illustration of the 4 levels of complexity in the structures of proteins.

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Amino Acids:
There are 20 -amino acids (nineteen amino acids and one imino acid) that are used in the synthesis of peptides and proteins by the human body and these are essentially the building blocks of protein. Amino acids are the primary products of protein degradation. These 20 amino acids are needed to build the various proteins that are used by the body for growth, repair and maintenance of body tissue. The syntheses of these proteins are controlled by genes (Holum 1998).

The structure of amino acids.

The term Amino Acid comes from Aminoalkanoic Acid which is an organic compound comprising of an amino group and a carboxylic acid group (Barrett 1998). The -amino acid term is used by chemists to identify the carbon atoms in sequence along a chain starting from the atom next to a major functional group, i.e. Carboxyl group (fig 5). The amino acids that occur in proteins are strictly -amino acids since their amino group is carried by the alpha carbon. At physiological pH, in addition to the positive charge on the amino group and the negative charge on the carboxyl group, five of the twenty -amino acids also carry a charge on their R-group or side chain. Other side chains are polar, but some are non-polar. See table 1 (Marks et al 1996).

Fig 5 Figure 5: Illustration of the general formula of amino acids. (www.cr4.globalspec.com accessed 20/03/2011) Nineteen of the twenty -amino acids follow this formula with proline not conforming to this formula as in the following table (table 1).

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Polar properties of amino acids; Glycine, Alanine, and Proline have small, non-polar side chains and are all weakly hydrophobic. Phenylalanine, Valine, Leucine, Isoleucine, and Methionine have larger side chains and are more strongly hydrophobic. There are also eight -amino acids with polar, uncharged side chains. Serine and Threonine have hydroxyl groups. Asparagine and Glutamine have amide groups. Histidine and Tryptophan have heterocyclic aromatic amine side chains. Cysteine has a sulfhydryl group. Tyrosine has a Phenolic side chain. The sulfhydryl group of Cysteine, phenolic hydroxyl group of tyrosine, and imidazole group of Histidine all show some degree of pH-dependent ionization (Holum 1998). There are four -amino acids with charged side chains. Aspartic acid and Glutamic acid have carboxyl groups on their side chains. Each acid is fully ionized at pH 7.4. Arginine and lysine have side chains with amino groups. Their side chains are fully protonated at pH 7.4 (Helmenstine 2011).

Table 1: Diagram outlining the properties of the 20 -amino acids. (www.personal.psu.edu Accessed 20-Mar-2011)

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Although Methionine is listed in table 1 as non-polar, Marks et al 1996 states that this is somewhat polar, and also states that tryptophan is relatively polar because of its indole ring.

Isoelectric point, symbols and Hydropathy index (pI) of -amino acids

The pI of an amino acid or protein is that pH at which the average charge of the species is 0. When a protein is at its pI, it will not go into solution. When milk (casein) turns sour, a precipitate is formed because a change in pH causes the casein molecules to become Isoelectric (Holum 1998). The following shown in table 2 are the pI values for individual amino acids and their symbols One letter Name Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tryptophan Proline Serine Threonine Tyrosine Aspartic acid Glutamic acid Asparagine Glutamine Lysine Arginine Histidine Cysteine Methionine symbol G A V L I F W P S T Y D E N Q K R H C M Three letter symbol Gly Ala Val Leu Ile Phe Trp Pro Ser Thr Tyr Asp Glu Asn Gln Lys Arg His Cys Met pI 6.06 6.11 6.00 6.04 6.04 5.91 5.88 6.30 5.68 5.64 5.63 2.98 3.08 5.41 5.65 9.47 10.76 7.64 5.07 5.74 Hydropathy Index -0.4 1.8 4.2 3.8 4.5 2.8 -0.9 -1.6 -0.8 -0.7 -1.3 -3.5 -3.5 -3.5 -3.5 -3.9 -4.5 -3.2 2.5 1.9

Table 2: -amino acids and chemical properties. Essential Amino acids are marked in bold.

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At the pI value these molecules become zwitterions and will not migrate in an electric field because the amount of negative charge on the molecule is equal to that of the positive charge. The Hydropathy index is a measure of the hydrophobicity of the amino acid. The greater the value, the more hydrophobic the amino acid is. The symbols are shortened versions for writing many amino acids, in the case of a peptide being illustrated at amino acid level (Marks et al 1996 & Holum 1998).

Essential and non-essential Amino acids

There are 11 -amino acids that can be synthesised by the body by using Carbon, Ammonia, carbohydrate and/or fat as the source. These are considered non-essential amino acids, as they are not directly required from a persons diet (normal text table 1). However, the other 9 amino acids, marked in bold above, are essential amino acids and must be sought from a persons diet in order to ensure that body protein levels are not diminished.

Metabolism of amino acids in the body

When protein is ingested the digestion process starts in the stomach and travels through the intestine where the products of digestion, the amino acids and small peptides are rapidly transported across the walls of the small intestine. See fig. 6. Advances in understanding protein metabolism have been made in the last few years with the advent of dual tracer methodology for assessing differences between exogenous and endogenous amino acid contribution to the protein pool, enhancing the comprehension of amino acid absorption kinetics (Bilsborough et al 2006).

Enzymatic digestion Proenzymes- Trypsinogen, Chymotrypsinogen, Proelastase and Procarboxypeptidases (a+b) excreted from the pancreas are inactive until they reach the lumen of the digestive tract where they are cleaved to form active enzymes which digest the peptides into amino acids. No single enzyme can fully digest a protein, but by working together this is achievable (Marks et al 1996). The excretion of Hydrochloric acid in the stomach enables Pepsinogen to be cleaved to form Pepsin, which is an active protease. The acid environment also allows for the denaturation of the protein.
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Fig 6 Figure 6: Illustration of the digestion of protein in food into amino acids (Marks 1996). Amino acid catabolism Once these amino acids enter circulation, they become part of the Nitrogen pool. The nitrogen pool represents all the nitrogenous compounds throughout the body (Holum 1998). These nitrogenous compounds are used for the synthesis of proteins or they are oxidized as energy. Unlike some compounds, such as fats and carbohydrates, the body cannot store amino acids. Amino acids in the nitrogen pool, which are free (i.e.: amino acids that are not needed to make other amino acids), are soon catabolised. This is primarily carried out in the liver but, under fasting conditions, will also take place in the kidneys. The nature of amino acids and their molecular make up allow for amino acids to be used to synthesise anything the body requires. The end product of complete amino acid catabolism is Carbon Dioxide, Water and Urea. (See urea cycle fig.7)
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All the pathways for use of carbohydrate, fats and protein interact within one way or another, as visible in fig 7. The carbon skeletons of the essential amino acids outlined in table 2 cannot be synthesized by the body. It is for this reason that they are essential and required from the diet.

Figure 7: Illustration of amino acid metabolism and interaction pathways in the body. KG: ketogluarate; OAA: oxyloacetate; G-6-P: glucose 6- phosphate; G-1-P: glucose 1phosphate (Marks 1996).

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Transamination and Deamination

Amino acid metabolism involves the amino acid undergoing transamination, (fig 8) which is the transfer of an amino group from the amino acid to a keto acid (carbon skeleton). This carbon skeleton is then oxidized and most of the carbons are converted to energy in the form of pyruvate, intermediates of the tricaboxylic acid cycle or to acetyl CoA. All amino acids can undergo this type of reaction except lysine and Threonine. For the majority of these reactions -ketoglutarate and glutamate serve as an -keto/amino pair needed along with the cofactor pyridoxal phosphate. This reaction is reversible and for this reason is involved in both synthesis and degradation of amino acids (Marks et al 1996). Deamination removes amino groups from the amino acids. Three types of deamination can occur; oxidative, reductive and direct.

Fig. 8. Figure 8: Illustration of transamination. Pairs of amino acids and their corresponding keto acids are involved in this type of reaction (Marks 1996).

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Urea Cycle. The body cannot store amino acids and once the degradation/synthesis of amino acids has taken place and the excess amino acids are converted to proteins and energy, the body must remove excess ammonia groups which are the product of many reactions- See figure 9. This occurs because ammonia is toxic to the body, especially to the brain and central nervous system. There are two sources of nitrogen being converted to the urea. 1. Oxidatative deamination of glutamate which produce an ammonium ion. 2. Asparatate. The urea cycle produces urea from ammonium ion, carbon dioxide and asparatate. (Fig 9) Water soluble urea leaves the body via the kidneys in the form of urine. Amino acid catabolism must occur in a way that does not elevate ammonia levels in the blood. The rate of amino acid derived ammonia converted to urea is limited (Marks et al 1996).In addition nitrogenous compounds are also excreted in faeces and sweat but to a far lesser extent (Lemon 1991). In a specific study a maximal rate of 55mg urea N.h-1. Kg-0.75 excretion is observed when 0.53g protein N/kg-0.75 is ingested, this rate was maintained even though a further increase in protein consumption was undertaken. This rate is not specific to everyone however, because the amount of dietary protein that can be deaminated by the liver is dependent on body weight and individual variation in efficiency of this process (Bilsborough et al 2006). Problems may occur if a diet consists of protein content that is greater than the bodys ability to excrete unwanted ammonia.

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Figure 9: Illustration of the sources of ammonia (left) and the Urea cycle (right) (Marks et al 1996) Nitrogen Balance: For a person to be in nitrogen balance they will have ingested the amount of nitrogen in the form of protein which is equal to the amount of nitrogen excreted (Marks 1996). Furthermore, Stuart et al 2007 states the nitrogen balance is a likely and acceptable method for establishing nitrogen or amino acid requirements necessary to prevent deficiency. In contrast, using the nitrogen balance to determine the protein needs of the individual is flawed according to Trumbo 2002. The reason that this is flawed is twofold; 1: high nitrogen balances are observed with high protein intakes. 2: increased economy nitrogen is observed at low protein intakes. These balances are often estimated and do not take into account dermal and miscellaneous losses of nitrogen. The World Health Organisation (WHO) concurs with this theory and states that using this method results in a lower amount than is truly needed to establish daily allowances for protein consumption.

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Dietary protein requirements: Dietary protein requirements has been a long debated area of nutrition. There are many parameters that effect the dietary protein requirements (WHO 2001) of individuals. These include lifestyle, metabolic demand, growth and the efficiency of the body to use protein. The primary lifestyle influences on an individuals need for protein will depend on the level of physical activity. Whilst an increase in physical activity will increase the demand for protein the extent of this can be reduced through training, as the body will adapt to the physical activity. Physical activity increases oxidation of amino acids which leads to increased ureagenesis and the loss of nitrogen (Rennie et al 2000). The type and intensity of the physical activity can have an effect in the way that amino acids are utilized in the body. Hargreaves et al 2001 explain that during prolonged exercise, individuals show catabolism of branched chain amino acids (BCAAs). The catabolism of Isoleucine, Valine and especially Leucine, indicate that there is an increase in the uptake of BCAAs in the contracting muscle, an increase in the oxidation of BCAAs within the muscle and an increase in the production of carbon dioxide as a by-product. The World Health Organisation states that the safe protein requirement for protein is 0.83 g/kg per day, for proteins with a protein digestibility-corrected amino acid score value of 1.0. (WHO 2007)

Table 3. Table 3: The safe protein level for both men and woman (>18 yrs) according to their bodyweight. The WHO 2007 have not set an upper limit for protein consumption, but they do recommend that caution is taken if individuals are to ingest 3 or 4 times the safe limit, as these cannot be assumed to be risk free.

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Stereoisomerism of -amino acids

All the -amino acids, except glycine, are chiral molecules and can be present in dextrorotatory (D) or laevorotatory (L) configuration. The D- and L- enantiomers are mirror images of each other that cannot be superimposed on each other by rotating the molecule. Enantiomers are optical isomers and are known to have identical chemical and physical properties. Enantiomers cause rotation of polarized light in opposite direction. (L- rotates light to the left & D- rotates light to the right) Enantiomers react with other optical active compounds at different rates. (Zawirska-Wojtasiak 2006) Holum 1998 states that nature supplies only one of the two enantiomers. The amino acids concerned with humans and human nutrition belong to the L- optical family. All the proteins in the human body are synthesized from L-amino acids, and all these are chiral. Some Damino acids are formed in bacteria and this configuration helps the bacteria to survive in humans where the enzymes used to destroy infecting protein organisms only recognize the Lform of the organism. See figure 10 below.

Fig 10. Figure 10: Illustration of D- & L- configuration of amino acids and Glycine (Marks et al 1996). Glycine is not a chiral compound because the -Carbon is not asymmetric as it contains two groups which are identical.

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Effects of production on chirality in protein rich foodstuffs

In the production of foods, specifically protein sports supplements and infant formula the starting material is generally a protein rich food source, such as milk or whey. Production techniques may involve heat, irradiation, fermentation, addition of synthetic materials and the adjustment of pH which may induce racemisation. Racemisation is the transformation of one-half of the molecules of an optically active compound into molecules that possess exactly the opposite (mirror-image) configuration, with complete loss of rotatory power because of the statistical balance between equal numbers of dextrorotatory and levorotatory molecules (Miller-Keane 2003).

An example of racemisation is discussed by Zawirska-Wojtasiak 2006 who claims that microwave heating of reconstituted infant formula may lead to the formation of D-proline. This, however, is rebutted by Marchelli 1996 & Sibre 1996 who state that No significant racemisation occurred in either the free or protein-bound amino acids under the conditions used. The conditions used to test this theory included a domestic microwave oven, a conventional oil bath and a wave guide device. This study did not investigate other production techniques that may involve intense heat or irradiation. Marchelli 1996 elaborates, stating that there is no toxicity of D-amino acids in humans and even in infants, although their metabolic rate has not been established.

Proteins play an important role in taste perception and in all amino acids except Methionine depending on their D- of L- configuration are either neutral, bitter or sweet. (Prescott et al 1995) During production a protein based product may be chemically altered and certain amino acids may undergo racemisation to exhibit a specific taste. In the case of sports supplements, an amino acid profile may be visible on the label but, in some cases, it may not state if the amino acids are in the L-form which the body may use in the synthesis of proteins to aid the athlete.

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Regulation on chirality:

A world-wide scare occurred when Thalidomide, a sedative drug was manufactured by Chemie Grunenthal (a German chemical company). It was sold around the world from 19571961 and it caused 10,000+ birth defects (Riehl 2010).

This drug never made it into the U.S.A on a commercial basis, as the Food and Drug Administration (FDA) demanded further evidence that this was effective and safe. However, it was used on an experimental basis, causing 17 birth defects in the US.

The issue with this particular drug is that L-isomer is teratogenic and D-isomer is an active drug to treat morning sickness. A further complication with this drug is that it can racemize in vivo. Therefore, even if only one isomer is administered both will be found in the serum. Often only one isomer of a racemic chiral drug is the effective agent that is therapeutic, whilst the other isomer may display limited effects, no effects or may even be toxic like LThalidomide. 1987 saw the US Food and Drug Administration (FDA) include, for the first time, the area of stereo-chemical regulation in their drug substance guidelines. Today, this proves to be a fundamental part of drug design, as in 2006 when more than 80% of new drugs approved by the FDA were chiral compounds (Riehl 2010). However, it was not until 1992, twenty-nine years after president Kennedy signed an amendment regarding a new drug safety policy, that the FDA insisted that, in order to market a chiral drug as a racemic mixture, the company must determine the pharmacological and toxicological activities of both enantiomers in animals and in humans.

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Forensic application of Amino Acid racemisation. A valued method being used by the forensic community to estimate the age (age at death) of the remains for the past 30 years is called amino acid racemisation (AAR). This method is based on the understanding that through the racemisation process, the native protein building L- enantiomers are spontaneously converted into D- enantiomers in a time dependent manner. The most commonly used amino acid is aspartic acid (Asp). However, others have been used with success and these include glutamate, alanine and serine (Arany et al 2010). The accuracy of this technique is (+/-) 3yrs (Waite 1999, Arany et al 2010). The rate of racemisation of these amino acids is dependent on pH, temperature, protein concentration and environmental water concentration (Arany et al 2010). Dentin is used because the proteins in the dentin are metabolically isolated and are kept at a constant temperature of 37oC (+/- 1oC) during life (Waite 1999). The racemised form of the original protein can then accumulate over time and the extent of this racemisation can then be used to estimate the age at which the person died. An extensive study carried out in 1999 by Waite discovered a range of racemisation rates (dentin) published by a series of authors. These values ranged from 5.688 X 10-4 yr-1 to 8.35 X 10-4 yr-1. The reason for the extensive variation among the authors was believed to be down to the differences in the methods used to determine the rate.

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Chirality and the International Olympic Committee. In 2002 Alan Baxter an Olympic bronze medal winner was forced to forfeit his medal because he tested positive for speed after using a Vicks vapour inhaler nasal decongestant. This is because the active enantiomer in the inhaler is L-Methamphetamine. The banned substance Speed is D-Methamphetamine. IOC drug testing use standard GC and these enantiomers will have the same retention. This is why the International Olympic Committee (IOC) stripped the athlete of the medal. If a Chiral GC column was used enantiomers would have different retention times (Ahern 2004). Although it was later proven this athlete did not Speed the IOC rules required the medal to be forfeited, an in addition the IOC updated there prohibited list (class 1.Aa and 1.Ab stimulants) to include both enantiomers of prohibited drugs (Mottram 2003).

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Methods available for the analysis of protein

There are a wide range of methods available for protein analysis. Depending on the protein characteristics and what information the analyst requires will depict what method an analyst will use. For the purpose of this study, the focus will remain with total protein quantification of intact proteins, peptide identification and amino acid determination.

Total Protein quantification

Kjeldahl This method is based on the understanding that when organic nitrogen- containing compounds such as protein- is digested/heated with H2SO4 (catalyst present) the nitrogen is converted to (NH4)2SO4. The digestion process is carried out using a digestion block, as in figure 11 b, below. The digestion products are treated with excess alkali (NaOH) (Fig 11b) and ammonia, produced by distillation and collected in a flask containing boric acid. The receiving flask is then titrated to a grey colour using hydrochloric acid. Calculation: %N= ( ) Sample Weight (grams) X 10 (Royal Society of Chemistry 2005)

This has been used as a standard method for the determination of nitrogen content in protein compounds for over 100 years and is a standard method in the pharmaceutical, agricultural, food sector, biological sediments and in waste water matrices. Furthermore, Owusu-Apenten 2002 state that this method gives accurate readings, no matter what state the sample is in. This method, however, is labour intensive, time consuming and involves toxic/corrosive chemicals, although this is considered a very accurate and reliable method as it is an absolute method of analysis. Kjeldahl analysis has a reported detection limit of 0.053 mg N and a quantification limit of 0.159mg N. (www.bushi.fr Accessed 06/04/2011)

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Figure 11a: Buchi Distillation/condenser unit. (www.buchi.fr. Accessed 06/03/2011)

Figure 11b: Buchi Digestion block. (www.imlab.com Accessed 06/03/2011)

Domini et al 2009 developed a method based on the Kjeldalh method. The difference is that this method used microwave and ultrasound energy to assist the digestion. This two-step (mineralization & oxidation) digestion means that there is a significant reduction in the time taken for the analysis which is approximately 7 minutes. This closed vessel low pressure technique reduced analysis time and risk, as it resulted in less exposure time to toxic chemicals.

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Bradford assay
The Bradford assay is a dye-based assay for determining protein content in solution. The dye (Serva blue G dye) interacts with the protein and the protein dye complex is detectable using a spectrometer at 595nm. A standard curve, with samples of known concentration, must be prepared in parallel with the unknown protein solution. The concentration of the unknown protein can then be determined from the standard curve. A common standard used is Bovine Serum Albumin (BSA). This assay is fast at approximately 10 minutes (2 min development time), but does involve the use of phosphoric acid which is corrosive. Bradford assay sensitivity ranges from 25g/ml 200g/ml. If the samples are left for over 10 minutes, then they may start to precipitate due to the acidic conditions. However, this method is not widely used due to the fact that the dye interacts more or less strongly with different proteins and thus is not strictly quantitative (Bradford 1976, Bollag et al 1991). Pierce Biotechnology, a member of the Thermo scientific group has designed an assay that is quantitative, based on the Bradford method. This method utilises higher concentrations of protein samples, at 125- 1500g/ml, and uses Comaomassie G as its dye. (Fig 12) (www.qcbio.com accessed 06/04/2011)

Fig 12. Figure 12: Illustration of the expanded Bradford method, Comaomassie Plus, as sold by Pierce Biotechnology. (www.qcbio.com accessed 06/04/2011)

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Biuret assay
Compounds containing 2 or more peptide bonds react with the Biuret solution, which is dilute alkali copper sulphate solution, and gives a characteristic purple colour. The colour reaction comes from the formation of a compound, formed from the copper atom and the four nitrogen atoms, two from each of the two peptide chains (Fig 13).

Figure 13 : Illustration of 4 Nitrogen molecules (from 2 peptides) binding to copper to form complex. Similar to the Bradford test, the Biuret test is based on the principal of spectroscopy and must also have a standard curve produced in order to quantify the protein in a sample. The intensity of the colour is proportional to the amount of protein in the sample and this is determined by measuring the absorbance at 570nm. This method is described as a quantitative estimation so, therefore, is not as reliable as an analytical scientist would require (Bollag 1991).

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Protein/Peptide analysis by liquid chromatography

Liquid chromatography is a separation technique used for the analysis of a mixture of compounds or chemicals. This technique involves 2 phases, a mobile phase which is a liquid and a stationary phase which is a solid. The sample is injected into the liquid mobile phase and this passes through a column which contains the stationary phase (fig.12). Due to chemical and/or physical interactions between the analyte, the mobile phase and the stationary phase, will determine how long the analyte is retained on the stationary phase. Compounds are separated based on retention time that is assigned to each compound as they pass through the detector at the end of the system.

Fig 12. Figure 12: Illustration of a liquid chromatographic system. (http://www.appliedporous.com accessed 11-04-2011) Several techniques have been developed to analyse proteins and peptides based on liquid chromatography. These chromatographic modes include: reverse phase high pressure (RPHPLC), hydrophobic interaction, size exclusion, affinity and isoelectric. The most widely used method is RPHPLC (Corradini 2010).

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Reverse Phase High Pressure Liquid Chromatography separation of proteins

Reverse phase means that the stationary phase is non-polar (hydrophobic) or weakly polar and the mobile phase is more polar (hydrophilic). When using RPHPLC for the analysis of proteins, the mobile phase generally consists of a mixture of water and an organic solvent. Acetonitrile is a suitable organic solvent for this purpose as it has a low ultra violet (UV) absorbance that will have minimal response with the UV detector, low viscosity and high elution strength. An organic acid is also added to the mobile phase. This acts as a counter ion of any charged amino acid which may increase peptide retention with the charged silanols of the silica support. Trifluoroacetic acid (TFA) is an organic acid that is widely used for this purpose, as well as the less popular additives such as acetic acid, formic acid, phosphoric acid and ammonium acetate. TFA is most popular due to its volatile nature, UV transparency and strong ion pairing nature (Corradini 2010). Proteins are separated using a solvent gradient system, which alters the polarity of the mobile phase over time, therefore altering the hydrophobic nature of the mobile phase. During the solvent gradient, when the amount of organic solvent reaches a precise concentration unique to each protein, the protein desorbs from the hydrophobic surface and elutes from the column, therefore being separated based on hydrophobic interaction. Where an UV detection system is unavailable, Electrospray Light Scattering Detection (ELSD) can also be used to detect proteins post separation. A refractive index detector may be used but has a detection level approximately 1000 times lower that UV (Harris 2003).

33

Other separation techniques Protein analysis can also be carried out using Capillary Electrophoresis (CE). This technique separates the proteins molecules based on charge and size. The analytes travel through a buffer solution (CE) or through a polymer immersed in a buffer (capillary gel electrophoresis). A range of slab gel electrophoresis techniques that use both denaturing and non-denaturing methods can also be used for the separation of proteins. Similar to capillary gel electrophoresis, this uses an electrical current to separate analytes based on charge and size.

34

Amino Acid analysis

Amino acid analysis can be used to quantify the protein content, determine the amino acid composition and help detect contaminants within a protein matrix. Hydrolysis: Before the amino acids in the protein can be analysed, the protein must undergo a hydrolysis step (fig.13) which breaks down the protein into individual amino acids by way of breaking the peptide link. Hydrolysis can be achieved by using acid, alkaline or enzymatic methods. The more common method is by means of acid. This is usually carried out in the presence of 6M hydrochloric acid over a 24 hour period at a constant 110oC, although microwave methods are being used as they can speed up this hydrolysis step to under 30 minutes. (www.milesonesci.com Accessed 10/05/2011) The amino acids are in the form of their positive ion state due to the hydrogen ions from Hydrochloric acid. Acid hydrolysis impedes the ability to detect tryptophan, aspartic acid and glutamine posthydrolysis as these are destroyed (www.nhis.go.jp Accessed 05/05/2011). Cysteine is partially destroyed. For this reason, acid hydrolysis limits the analyst to use 16 amino acids for quantification and identification. (www.sigmaaldrich.com Accessed 10-05-2011)

Fig. 13 Fig.13: Illustration of an Acid hydrolysis mechanism. (www.chemguide.com accessed 19Apr-2011) Once the peptide is hydrolysed into its constituent amino acids they usually need to be derivatized. The type of derivatization that is required will depend on, and be determined by, the analysis technique that is used. Generally, 2 methods are used to analyse amino acids: Liquid chromatography (HPLC) or Gas chromatography (GC)

35

HPLC detection systems that use spectrometric methods require the analyte to contain a chromophore, which once excited at one wavelength, will emit light at a different wavelength, specific to the analyte. The problem with amino acids is that they do not all have aromatic systems or a series of single/double bonds; therefore, they are not chromophores. Pre-column and post-column derivatization is available for the detection of amino acids using HPLC. Pre-column derivatives include 9-fluorenylmethyloxycarbonyl (FMOC) and 0phthalaldehyde (OPA) (Jambor et al 2009). Gas Chromatography: In gas chromatography (GC) the sample (gas or liquid) containing the analyte of interest is injected through a septum. The analyte is then vaporised in a heated chamber before it is carried onto the column by a carrier gas. The carrier gas, as the name suggests, is only used to move the analyte through the GC system (fig.14) and does not interact with the analyte, unlike liquid chromatography. The column in this case is in the form of either solid particles or a non-volatile viscous liquid on a solid support. A detector detects the analytes as they leave the column and reports this onto a computer. A range of detectors are available and cater for the varying amounts of information that is required from the analysis and the available budget. These detectors include Thermal conductivity; Flame ionization; Electron capture; Mass spectrometry (Harris 2003).

Fig14. Figure 14: Illustration of a basic Gas chromatography system.

36

Chiral separation of Amino Acids

Amino acids are not volatile; therefore the amino acid analyte must undergo derivatization. Derivatization makes the amino acid volatile so it can be used in the GC system. An example of derivatization is by creating pentafluoropropyl isopropyl esters of amino acids. This involves esterification and acetylation steps to change the native amino acids into esters of amino acids (Drozd 1981). Enantiomers of amino acids are separated on a Chiral Colum, a column modified to contain a chiral molecule that is specific to amino acids. A commonly used column is the CHIRASILVal Colum. This modified column contains L-Valine-tert.-butylamide polycyanopropylmethylsiloxanes which interact with the derivitized amino acids through hydrogen bonding between analyte and stationary phase. L- amino acids are retained slightly longer than D-amino acids when using this column due to the strategic positioning of the Lvaline-tert butylamide inside the stationary phase which allows two hydrogen bonds to form where as D-amino acids may only form one, enabling less retention on the column (Rotzsche 1991)

37

Methodology

38

Introduction
Many food supplements have been reported as potentially dangerous to consumers especially when large amounts of dietary supplements are consumed. The manufactures of these dietary supplements only need to include basic information on the label such as macromolecule composition. No consideration is given to how certain components of the dietary supplements may interact with other drugs or medication consumers have taken. The European Union Rapid Alert System for Food and Feed has called for routine thorough analysis as the number of notifications to them has increased 6 fold in last 7 years. This indicates this problem is getting worse and the need for better quality control and compliance in this sector is required to ensure consumers are not at risk. In order to find out if the supplements sold on the Irish high street are what the manufactures claim them to be, analysis of 5 products will be carried out. Of these products 4 will be highly concentrated dried protein products and one will be a liquid amino acid product. This report will look at protein dietary supplements covering 3 major areas; moisture content; total protein content and individual amino acids composition. Further to the amino acid analysis, chiral characterisation of the amino acids will also be investigated as this is an area not routinely studied at in food industry. Protein analysis and characterisation of proteins using hydrophobic interaction will also be investigated. Chiral characterisation of food stuffs and proteins in particular are important as, this analysis proves manufacturing technologies can cause racemisation of amino acids that were in their native L- form prior to manufacture of the product.

39

In this section 4 areas of analysis will be discussed. These relate to Moisture content, total protein concentration, HPLC Peptide analysis and Gas chromatography of amino acid derivatives. All protein powder subjects purchased from Holland & Barrett, Ireland except Sunwarrior which was sourced by Dr. Fiona Mc Ardle.

40

Moisture analysis Subjects: Hemp Natural Protein powder (Unflavoured) 90% Soya Protein instant powder ( Strawberry) Instant Milk & Egg protein. (Strawberry) Sunwarrior Raw Vegan protein (Chocolate)

Equipment: Binder ED11 Convection Oven (105oC) 12 * Aluminium dishes with lids. Desiccator. Oven proof gloves. Shimadzu AX 200 Analytical balance

Experimental protocol. 1. Weigh in triplicate approximately 1 gram of sample into a tared dish on an analytical balance (the dishes must have been heated for at least one hour at 100oC and subsequently, cooled and stored in a dessicator prior to use. 2. Distribute the sample over the bottom of the dishes. 3. Place the dishes in the oven ensuring they are identifiable. 4. After 2 hours remove the dishes from the oven using oven proof gloves. 5. Cool in a dessicator for 30 minutes. 6. Weigh the dishes and dried powder on an analytical balance. 7. Place back into oven for 30 minutes and repeat steps 5-7 until constant weight is achieved. Analysis. Calculations: % Moisture = Loss in weight (g)*100 / weight of sample (g) Report results on a column chart.

41

Biuret Total Protein Subjects: Hemp Natural Protein powder (Unflavoured) 90% Soya Protein instant powder ( Strawberry) Instant Milk & Egg protein. (Strawberry) Sunwarrior Raw Vegan protein (Chocolate)

Equipment: Shimadzu 160U UV spectrometer 570nm Micropipette. P1000 Graduated pipettes (Grade A) 12 test tubes Vortex Mixer

Reagents: Standard protein solution (BSA 10mg/ml in 5% acetic acid/ ultrapure water) Biuret Reagent Samples diluted to 5mg/ml using 5% acetic acid/95% ultrapure water and filtered through Whatman (#54) filter paper to remove particulates. Experimental Protocol: 1. Label 18 tubes (6 standards and sample to be carried out in triplicate). 2. Pipette 0.0, 0.4, 0.8, 1.2, 1.6, 2.0ml of standard protein solution into a series of 6 test tubes. 3. Make these volumes up to 2ml with 5% acetic acid/95% ultrapure water. 4. Pipette 2ml diluted samples into each of 3 test tubes. 5. Add 8ml Biuret reagent to each standard and sample tubes. 6. Allow colour to develop for 30 minutes. 7. After 30 minutes read absorbance of standards and sample against reagent blank (ultrapure water) at 570nm.

42

Analysis: Using the data, draw a standard protein curve in Microsoft Excel (absorbance V weight of protein). From the standard protein curve get the trend line equation, Y= MX +C (i.e. y = 0.025 X + 0.1083) Use X= (Absorbance C)/M to determine (M) mg protein per sample. Calculate the concentration of protein in the protein sample as (%w/v, i.e. g/100ml), not forgetting the dilution factor. Compare concentration of protein in samples to expected protein concentrations and report on a column chart.

43

Hach Test n Tube Persulfate Total Nitrogen Subjects: Hemp Natural Protein powder (Unflavoured) 90% Soya Protein instant powder ( Strawberry) Instant Milk & Egg protein. (Strawberry) Sunwarrior Raw Vegan protein (Chocolate)

Equipment: Hach DRB 200 programmable reactor Digestion block.(105oC) Hach DR2800 UV portable spectrometer.

Reagents: Hach Test n Tube Kit# 2672245 consisting of: 1. LR Total Nitrogen Hydroxide digestion vials. 2. Total Nitrogen Persulphate reagent powder pillow. 3. Total Nitrogen reagent A Bisulfite powder pillow. 4. Total nitrogen reagent B- Indicator Powder. 5. Total Nitrogen Reagent C Acid solution vials 6. Reagent water 7. Plastic funnel (not included in kit) 8. Protein samples: 5g/L Experimental Protocol: As per Hach method #10071 Total Nitrogen by Persulfate digestion. 1. Using funnel, add contents of a total Nitrogen Persulfate reagent powder pillow to each of labelled total Nitrogen Hydroxide vials. 2. Prepared sample: Add 2ml of sample to sample vials. Blank: Add 2 ml of deionised water to a blank vials. 3. Cap vials and shake vigorously for at least 30 seconds to mix. 4. Insert the vials into the reactor and close the lid. Heat for exactly 30 minutes. 5. Remove the vials immediately and allow cooling to room temperature.

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6. On the DR2800, select the stored programme test 350 N Total LR TNT. 7. Remove caps from the digested vials and add the contents of one Total Nitrogen (TN) reagent A powder pillow to each vial. 8. Cap tubes and shake for 15 seconds. 9. Start a timer a 3 min reaction period will begin. 10. After the time expires, remove the caps from the vials and add one TN reagent B powder pillow to each vial. 11. Cap the tubes and shake for 15 seconds. 12. The solution will begin to turn yellow. 13. Start a timer, a 2 minute reaction period will begin. 14. After time expires remove caps from TN reagent C vials and add 2 ml of digested treated sample into one vial. For blank add 2 ml of digested treated sample to one reagent C vial. 15. Cap vials and invert ten times to mix. Use slow deliberate inversions for complete recovery. 16. Start a timer, a five minute reaction period will begin, the yellow colour will intensify. 17. Wipe reagent blank and insert into DR2800. Zero the instrument. Will display 0.0mg/L N. 18. Wipe the reagent vial and insert into DR2800. Read the results in mg/L N. Analysis: 1. Convert N content to protein by multiplying N value by 6.25. (conversion factor) 2. Compare the Mean protein value (samples) to the expected protein content and report on a column chart.

45

High Pressure Liquid Chromatography-Peptide analysis Subjects: Bovine Serum albumin. (3.53mg/ml)(67.0kDa) Myoglobin (2.80mg/ml)(18.8kDa) Cytochrome C (4.27mg/ml)(12.4kDa) (All subjects made up in mobile phase A and filtered through Whatman filter paper #54). Equipment: Dionex ultimate 3000 HPLC with discovery BIO wide pore C5 column (15cm*4.6mm,5m) Micropipette P1000.

Reagents: Temperature analysis: Mobile phase (A) 0.1% (v/v) TFA (ion paring agent) in ultrapure H2O: MeCN (75:25), (B) 0.1% (v/v) TFA in ultrapure H2O: MeCN (25:75) on gradient 0-100%B in 25

TFA Analysis: 0.005% TFA Mobile phase (A) 0.005% (v/v) TFA (ion paring agent) in ultrapure H2O: MeCN (75:25), (B) 0.005% (v/v) TFA in ultrapure H2O: MeCN (25:75) on gradient 0-100%B in 25 0.01% TFA Mobile phase (A) 0.01% (v/v) TFA (ion paring agent) in ultrapure H2O: MeCN (75:25), (B) 0.01% (v/v) TFA in ultrapure H2O: MeCN (25:75) on gradient 0100%B in 25 0.1% TFA Mobile phase (A) 0.1% (v/v) TFA (ion paring agent) in ultrapure H2O: MeCN (75:25), (B) 0.1% (v/v) TFA in ultrapure H2O: MeCN (25:75) on gradient 0100%B in 25

46

Experimental Protocol: 1. Allow column to equilibrate for at least 2 hours (flow rate 1ml/min) when adjusting mobile phase. If switching mobile phase from 0.1% TFA to no TFA allow overnight equilibration. 2. Pipette 2ml of sample into brown HPLC sample vial. 3. Place on carousel and note position. Assign name to position # on carousel via Chromeleon Software. 4. Ensure system has been purged, free of air bubbles and mobile phase is degassed. 5. Set UV detector to 220 nm, flow rate to 1ml/min, injection 10l. 6. For temperature analysis set to desired temperature per individual run, otherwise set to 30oC. 7. For TFA analysis Adjust TFA concentration and allow to equilibrate as above. 8. To run sample- Select sequence Fiona/Cormac protein mod 1, add sample to batch and name, ensure correct parameters are set. Connect pump, detector, auto sampler and oven. Click Start batch from upper tabs, ready check and then start. 9. Run should take 28 minutes and chromatograph will be stored on system. Analysis Note subjects retention time and any other obvious observations. Prepare a bar chart in Excel, one for temperature adjustment and one for TFA adjustment. Present parameter change Vs retention time and report observations.

47

Gas Chromatography (GC) of Pentafluoropropyl Isopropyl Esters of Amino Acids using Flame Ionisation Detection. 3 step technique: 1. Acid hydrolysis. 2. Derivatization of Amino acids. 3. GCFID analysis. Subjects: Hemp Natural Protein powder (Unflavoured) 90% Soya Protein instant powder ( Strawberry) Instant Milk & Egg protein. (Strawberry) Sunwarrior Raw Vegan protein (Chocolate) Aminos Drink (Hydrolysed amino acid liquid.)

Equipment: Binder ED11 Convection Oven 5 glass hydrolysis tubes. (COD vials soaked in 10% Nitric acid 0vernight) Heating block 115oC+. Nitrogen Stream apparatus. Shimadzu GC17 Gas chromatography system & CHIRASIL-Val(All Tech) Chiral Column. Reagents: Hydrolysis solution: 6M hydrochloric acid containing 0.5% Phenol. PFP-IPA Amino Acid Derivatization Kit. Cat #18093-Lennox. Standards: D/L- Valine, D/L -Glutamic Acid, D/L -Aspartic Acid, D-Leucine, D/LMethionine (Sigma 99% Puris grade) Diethylether (AnaLaR grade).

48

Experiment protocol: Acid Hydrolysis 1. Place 25mg of protein powder into hydrolysis tube. 2. Add 10ml of hydrolysis solution. 3. Hydrolyse samples at 110oC for 24 hours in an oven. NB: Aminos Amino Acid drink does not require hydrolysis. 4. Once cooled, open cap and place on hotplate at 115oC under stream of Nitrogen until hydrolysis solution has completely evaporated. 5. Allow to cool to ambient.

Derivatization of Amino Acids. 1. Add 3 ml of 0.2M Hydrochloric acid to sample tube, heat solution to approx 110oC for 5 minutes, and dry under stream of Nitrogen. 2. Slowly add 1.25ml Acetyl Chloride to 5ml Isopropanol. Add to dry Amino acid, cap tube and heat to approximately 100oC for 45 minutes in oven. Allow to cool. 3. Uncap and heat to approximately 115oC under a stream of Nitrogen to remove excess reagent, allow cooling to ambient then place tube in an ice bath. 4. Add 3ml Dichloromethane (Methylene Chloride) and one ampoule of PFPA, cap and heat to 100oC for 15 minutes. 5. After cooling to ambient, evaporate excess reagent under stream of Nitrogen and reconstitute in 500ul Diethylether.

GCFID Analysis. 1. Switch on GCFID instrument, Nitrogen carrier Gas, Hydrogen and Air. 2. Start GC17 software and select GC instrument. 3. Open method: class-vp\martin\cychex.met. 4. Ensure parameters are: Oven Temperature 90oC(4minute hold) to 190oC at 4oC/minute; Injection temperature 250oC; Detector temperature 275oC; Split ratio 56:1; Carrier gas Nitrogen; Flow rate 1.07ml/minute; Linear velocity 22.2cm/sec; Pressure 6.4psi; Sample size 1l. 5. Prior to injection, select single run, assign # to sample and press ok. 6. Wait until dialog box appears prompting injection, then inject and press data acquisition start immediately.
49

7. Manually assign peaks on samples by comparing retention times of standards to retention times of samples. 8. Report amino acid concentration in a bar chart and compare to information supplied with each product. NB: When available an internal standard, which should be an unnaturally occurring primary amino acid such as Nitrotyrosine should be used in order to monitor chemical and physical losses as well as variations during amino acid analysis.

50

Results

51

Oven Drying Moisture analysis:

Moisture analysis of Protein Products

10 9 8 7 6 5 4 3 2 1 0 7.72 Moisture content 4.32 3.14 3.65

% Moisture

Sun warrior

Hemp

Milk and Egg Soya 90%

Product

Fig A Figure A: Column chart illustrating the moisture content of 4 protein products following an oven drying method at 105oC.(Appendix II table 3)

52


100 90 80 % Protein Content 70 60 50 40 30 20 10 0

Biuret Total Protein & Hach total Nitrogen: Fig B 1

Buiret and Hach Analysis of Protein

79.69 67.61 73.00 52.94 45.63 37.08 47.00 83.34 86.00 81.38

71.00 49.48

Reported Buiret % On label % Reported Hach %

Sunwarrior

Hemp

Milk and Egg Protein Product

Soya

Fig B 1: Column chart illustrating the Total Protein content of 4 protein products following Biuret Total protein analysis and Hach Total Nitrogen (Nitrogen converted to protein by conversion factor of 6.38. (Appendix II table 4-5. Appendix II fig 13)

53

Hach Total Nitrogen: Fig B 2 Hach Persulfate analysis of Protein Supplements

100 90 80 70 60 50 40 30 20 10 0 79.69 71.00 52.94 45.74 45.63 47.00 39.13 Reported using food group conversion factors Sunwarrior Hemp Milk and Egg Soya 90% 86.00 Reported Hach (*6.25%) 65.62 On label %

% Protein Content

75.70 73.00

81.38

Protein Product

Figure B 2: Hach Persulfate results as in B1 except, the conversion factor for specific food types has been applied, these are; Sunwarrior *5.40; Hemp *5.36; Milk & Egg *5.94; Soya 90% *5.40. (Salovaananen et al 1996) (Appendix II table 4-5.Appendix II fig 13).

54

Reverse Phase High Pressure liquid Chromatography parameter evaluation using protein / peptide standards.
Effect of temperature on peptide separation: Fig C1

Efect of Temperature on Peptide Retention by RPHPLC

13 Retention Time (Minutes) 12 11 10 9 8 7 6 5 14 18 22 26 30 (OC) 34 38 42 Temperature Myoglobin (2.80mg/ml) Cytochrome C (4.27mg/ml) BSA (3.53mg/ml)

Figure C1: The result of adjusting the temperature from 14oC- 42oC in 4oC increments, and its effect on retention time of three peptides using RPHPLC. (Appendix II fig 4-12, Appendix II Table 2) Effect of Trifluoroaceticacid (TFA) on peptide separation: Fig C2

Effect of TFA concentration on Peptide Retention by RPHPLC.

12 11 10 9 8 7 6 5 4 3 0.005 0.010 % Trifluoroaceticacid (TFA) 0.100 Retention Time (minutes)

Cytochrome C (4.27mg/ml) BSA (3.53mg/ml) Myoglobin (2.80mg/ml)

Figure C2: The Result of the concentration adjustment of TFA, an ion pair reagent on retention of three Peptides using RPHPLC.(Appendix II fig 1-3, Appendix II table 1)

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Gas Chromatography Flame Ionisation Detection of Pentafluoropropyl Isopropyl Esters of Amino Acids following Hydrolysis and PFPA Derivatization of Protein Products.
SUNWARRIOR Amino Acid Analysis: Fig D 1

Sunwarrior Amino Acid Analysis

L- Aspartic acid D-Aspartic Acid

6.19 7.56 12.34 14.26

38.45

Amino Acid

L-Glutamic Acid D-Glutamic Acid L-Methionine D-Methionine L-Valine D-Valine

Sunwarrior mg/ml On Label* 4.29 5.22 Sunwarrior mg/ml Reported

10

20

30

40

50

mg Amino Acid/100mg product

Figure D 1: Partial GC Amino Acid profile of SUNWARRIOR protein following hydrolysis and PFPA Derivatization. (Appendix III fig 7. Appendix III table 1-2)

56

HEMP Amino acid analysis: Fig D2

Hemp Amino Acid Analysis

L- Aspartic acid D-Aspartic Acid

33.48 17.55 24.96 On Label** Reported 9.30

Amino Acid

L-Glutamic Acid D-Glutamic Acid L-Valine D-Valine

10

20

30

40

mg Amino Acid/100mg Product

Figure D 2: Partial GC Amino Acid profile of HEMP protein following hydrolysis and PFPA derivatization. . (Appendix III fig 4. Appendix III table 1-2) SOYA 90% Amino acid analysis: Fig D3

Soya 90% Amino Acid Analysis

L- Aspartic acid D-Aspartic Acid

7.41 3.15

10.00

Amino Acid

L-Glutamic Acid D-Glutamic Acid L-Methionine D-Methionine L-Valine D-Valine

16.40 7.34 Reported On label* 2.32 4.40

10

15

20

mg Amino Acid/100mg Product

Figure D 3: Partial GC Amino Acid profile of SOYA 90% protein following hydrolysis and PFPA derivatization. . (Appendix III fig 6. Appendix III table 1-2)

57

Milk & Egg Amino acid analysis: Fig D4

Milk & Egg Amino Acid Analysis

L- Aspartic acid D-Aspartic Acid

5.10 5.05

5.98

Amino Acid

L-Glutamic Acid D-Glutamic Acid L-Methionine D-Methionine L-Valine D-Valine

Reported On Label* 4.00 4.10

mg Amino Acid/100mg Product

Figure D4: Partial GC Amino Acid profile of Milk & Egg protein following hydrolysis and PFPA derivatization. . (Appendix III fig 5. Appendix III table 1-2) Aminos Drink Amino acid analysis: Fig D5

Aminos Drink Amino Acid Analysis

L- Aspartic acid D-Aspartic Acid

Amino Acid

L-Glutamic Acid D-Glutamic Acid L-Methionine D-Methionine L-Valine D-Valine

48.53 30.72 6.43 4.37 1.52 6.74 10.67 Reported On label***

20

40

60

mg Amino Acid/mg Product

Figure D5: Partial GC Amino Acid profile of Milk & Egg protein following hydrolysis and PFPA derivatization. . (Appendix III fig 3. Appendix III table 1-2)
58

Discussion

59

Moisture analysis (Fig A): Milk & Egg had 4.32% moisture. This value is within the Codex Alimentarius guidelines for moisture content of dried dairy based products. Milk & Egg contains Calcium Caseinate and Whey protein isolate as well as egg white solids. As prescribed by Codex and the FDA, Dried Caseinate has an upper limit of 8% and Whey protein isolate has an upper limit of 6 % moisture content. Milk and Egg however is within the regulation values for moisture content. Sun warrior had the lowest moisture content at 3.14%, unlike the highly regulated dairy industry which produces dried dairy products, other manufactures that product dietary supplements are not held to the same strict guidelines. Therefore comparison of Sunwarrior to regulation standards for moisture is not possible. The FDA state generally, manufacturers do not need to register their products with FDA nor get FDA approval before producing or selling dietary supplements. The FDA however have implemented a Good manufacturer practices (GMP) policy to ensure dietary supplements are made in a quality manner. This suggests that this area of manufactured is somewhat under regulated, and a lot of responsibility lies with the manufacturer to ensure these products are safe for human consumption, unlike the pharmaceutical industry. Soya has a moisture content of 3.65%, well below the recommended 6% which is usually expected in dried soya products. (Shurtleff et al 1990) This low value may be to increase over all protein value of the product per weight. Hemp has the highest moisture content at 7.72%, over twice the moisture content as compared to Sunwarrior and Soya 90%. This product claims to be made from 100% hemp, and undergoes limited processing unlike the other products. Hemp protein is a plant based product, and over drying may affect its ability to mix in a customers drink.

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Total protein. (Fig B) The standard pharmacopeia method for total protein analysis is Kejdalh Total Nitrogen. This method was investigated, but the only available literature similar to high content dried protein products were methods relating to Infant formula. Several attempts were made, adjusting various parameters but the digestion solution kept frothing up and sample was lost. Due to the Hazardous nature of the chemicals used in Kejdalh, two safer methods were adopted namely Hach Persulfate and Biuret. It is understood now that too much protein sample was being used, as some of the protein samples contain over fifty times more protein than the infant formula. A dilution of at least one in fifty would be recommended if one is to follow the Kejdalh infant formula procedure and apply it to these types of high protein products. The Biuret method (fig B) for analysis of these dried protein products was adapted from a method used to estimate total protein in blood serum. This method isnt very sensitive, therefore samples was carried through at 10mg/ml. Samples were dissolved in ultra pure water/ 5% acetic acid to decrease the pH from around the Isoelectric point, so as they would go into solution. Prior to UV detection, samples were filtered to remove particulates which were especially evident in Hemp due to its plant material. The Biuret method found Soya 90% and Milk & Egg to both contain slightly less protein as stated on the label, as with Hemp which had almost 10% less. Sunwarrior had over 20% less protein which is a considerable decrease when this product is being sold as a high protein food supplement. There are limited interferences with this method, but the presence of high concentration of carbohydrate can lead to opalescence, which can cause the scattering of light which in turn may increase absorbance and therefore concentration. (Nielsen 2010) Hemp had the highest concentration (15%) of carbohydrates and Soya (1%) the lowest. This factor does not appear to have affected the results. The Hach Persulfate method uses a kit supplied by Hach to measure the total Nitrogen content of samples, commonly used for water analysis. This step wise digestion converts all form of Nitrogen to nitrate which then reacts with chromotropic acid to form a yellow complex. This method claims to have a minimum of 95% recovery and sensitivity of 0.5mg/L and is a standard test in waste water analytical laboratories. The recommended conversion factor of Nitrogen to protein is *6.25, based on the observation that animal proteins contain approximately 16% Nitrogen. This gives the crude
61

protein value and is widely used when converting Nitrogen to Protein. The true protein content in food is determined by the amount the twenty L-amino acids bound in the proteins. The food group specific conversion factors relate to the total nitrogen content from the individual amino acids present in the food, and are therefore more accurate. (Salovaananen 1996) When the food specific conversion factor is applied to the Nitrogen content, all protein products in this study fall below the standard *6.25 conversion values. Salovaananen 1996 suggests that to truly quantify the Total protein, one must quantify individual amino acid concentrations. (Values taken from Fig B 2 Food group conversion factor) Similar to the Biuret method, Milk & Egg and Hemp are within 10% of their labelled protein value although Milk & Egg has a higher than reported on the label. Again Sunwarrior has an over 20% difference in the reported protein content as compared to labelled value. Soya 90% had a difference of over 20% also which was not consistent with the Biuret method or the labelled value. Soya 90% also contains egg white solids as a source of protein that is not taken into account by this conversion factor, therefore contributing to the lower value. Milk & Egg and Soya contain mixtures of protein sources; therefore assigning one conversion factor for all components will lead to result discrepancies. These products do not state how much of each type of protein is present, just the total protein content.

62

Reverse Phase High Pressure liquid Chromatography. In Reverse Phase HPLC the stationary phase particle surface is very hydrophobic due to the chemical attachment of hydrocarbon groups to its surface. The retention of the peptide on the stationary phase is determined by how hydrophobic the peptide is. The discovery BIO wide pore C5 column which was being used to separate the peptides contained in the protein products produced results that indicted the column was somewhat damaged. This was discovered early in the analysis whilst running standards. As part of a familiarisation process with HPLC, analysis continued investigating the effect parameter changes had on the retention of peptide standards. Three standards were used; 1. Bovine serum Albumin(BSA), a transport protein that has 11 hydrophobic domains for binding hydrophobic compounds such as hormones and fatty acids, consisting of 585 Amino Acids. (Appendix I fig 3) 2. Cytochrome C, (Equine) a compound in the electron transport chain consisting of 104 amino acids and hydrophobic core. Cytochrome C enables carrying of electrons from Cytochrome bc1 complex to Cytochrome oxidase complex. The heme group in Cytochrome C enables the electron transport. (Appendix I fig 1) 3. Myoglobin, (Equine) oxygen binding protein consisting of 153 Amino acids, also contains a heme group and hydrophobic core.(Hunter 2003) (Appendix I fig 2) Cytochrome C was retained for the least time, because this, the smallest of the three compounds has the least amount of hydrophobic amino acids in its peptide (appendix I fig 1). BSA the largest of the three peptides was second to elute, although this has many hydrophobic regions, many of these are on the innermost side of the peptide, limiting maximum interaction with stationary phase and hydrophobic regions. BSA is 140 X 40 X 40 . (www.rcsb.org accessed 26-Apr-2011) This is relatively large in comparison to the other peptides, but will still fit in the pore size of 300 of the stationary phase. In relation to the size of BSA, its hydrophobic footprint was relatively small. The most retained peptide was Myoglobin, small enough at 25 X 35 X 45 (Meisenberg 2006) to enter the wide pores in the column, and with many hydrophobic regions as visible in appendix I fig 2, meant this compound had the greatest hydrophobic footprint, even though

63

this was not the largest compound. Due to the addition of TFA, the pH was below 2 for both mobile phases, well below the isoelectric point for all standards. Effect of temperature on peptide retention: The standard peptide samples were subject to a series of runs, where the temperature was adjusted in 4oC increments from 14-42oC. BSA and Myoglobin were retained longer on the column as the temperature increased initially, but at 26oC, this effect levels out. This is in contrast to literature (Chen et al 2003), which suggests that retention of peptides decreases as temperature increases, due to the lowering of mobile phase viscosity. Cytochrome C showed a distinct increase in retention at 18oC and then appeared to alternate slightly at each temperature increment. This may be due to the size of small molecule and the limited hydrophobic interaction with the stationary phase as compared to the other larger more hydrophobic peptides. As the temperature was raised past 18oC, an increase in resolution was observed, with all three peptides being fully resolved from each other. NB: Double peaks represent one compound; this is due to a damaged column. Effect of TFA on peptide separation: TFA is an additive used to control the pH and acts as an ion pair agent. When the ion pairs form, a reduction of interaction between peptide and stationary phase is observed, to allow separation based on hydrophobic interaction. TFA also acts to solubilise the protein, by ensuring the pH (<2) is below the isoelectric point of the peptides. At low pH, carboxylic acid groups- the carboxy terminal group and the side chains of aspartic acid and Glutamic acid become protonated and slightly polar. When adjusting the TFA from 0.1% to 0%, The HPLC system had to be equilibrated over a 12 hour period, in order to ensure all TFA residue has come off the stationary phase. When 0% TFA was added to the mobile phase, the peptides were not resolved and no identifiable peaks were observed. At 0.005% TFA, Cytochrome C was fully resolved, but BSA and Myoglobin Co-eluted. At 0.1% TFA the same was observed although it was apparent that BSA and Myoglobin had begun to separate, although not fully resolved from each other. At 0.1% TFA, the recommended concentration, all three peptides were separated. The TFA additive also had an effect on the retention time of all three peptides. Cytochrome Cs retention time increased as the TFA concentration increased. BSA and Myoglobin eluted from the column faster when 0.01% TFA was added as compared to 0.005% TFA. This
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decrease was minimal however in comparison to the increase observed by all peptides when TFA concentration was changed from 0.01% to 0.1%. All peptides were retained longer on the column and an increase in resolution of all peptides was observed as the concentration of TFA increased to 0.1%

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Gas Chromatography (GC) Flame Ionisation Detection (FID) of Pentafluoropropyl Isopropyl Esters of Amino Acids following Hydrolysis and PFPA Derivatization of Protein Products. In order to analyse the amino acid composition of proteins the proteins must be hydrolysed so the amide links between amino acids are broken, and individual amino acids are created from the peptide. Analytes must be somewhat volatile to be analysed using GC. Amino acids in their native state are not volatile and need to undergo a derivatization procedure. Therefore the analysis is actually of Pentafluoropropyl Isopropyl Esters of the amino acids. Initially the digestion step was difficult to complete because proper digestion vessels were not available and test tubes with rubber stoppers could not withstand the pressure generated during the reaction which involved 24 hours at 110OC. Digestion of standards and samples was then carried out using closed vessel microwave digestion. This was considered a clean, rapid and safe method for carrying out the hydrolysis. All samples and standards were carried through the derivatization step and analysed using GCFID. These gave results that could only be attributed to background noise. T o investigate whether the derivatization step had worked, a hydrolysed amino acid product namely Liquid Aminos was purchased from the same company as the samples. This product was not subject to hydrolysis and only subject to the derivatization step. On analysis of the hydrolysed product, distinct peaks were observed alike those expected from amino acids. This indicated that the derivatization step has been successful and that the problem lay with the Hydrolysis. On further investigation, it became apparent the temperature probe on the microwave digestion unit was broken, and although the microwave cycle would continue, no heat or pressure was being generated to aid in the hydrolysis of the protein samples. The hydrolysis reverted back to the conventional 24 hour method. Hach vials were steeped in nitric acid overnight, rinsed, dried and used as vessels as these were know to withstand the pressure of hydrolysis. The standards and samples were hydrolysed, derivitized and ran on the GCFID successfully using the conventional method. Two sets of standards were used (appendix III fig 1-2) one for the Liquid Aminos were, and another set for the rest of this samples. This was because the pressure and temperature programme had to be optimised to reduce retention time.

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Sunwarrior: Sunwarrior protein contained over 6 times the amount of L-Aspartic acid as compared to that reported on its label, with over 7mg/100mg D-Aspartic Acid. L- Glutamic acid and L-Valine were similar to expected. Hemp: Hemp protein contained all the amino acids tested for but also contained D-Aspartic acid. There was no amino acid profile with this product for comparison, but hemp does however contain a full amino acid profile which supports these results. Soya 90%: Soya 90% contains L-Aspartic Acid and L-Valine in lesser concentration than expected . The D-aspartic acid concentration indicates racemisation of L-Aspartic acid. The latter is also apparent in the case L-Glutamic Acid. Milk & Egg: Milk & Egg contain levels of L-Valine and L-Aspartic acid in quantities expected. There is over 5mg/100mg of D-Aspartic acid also present in this sample. LGlutamic acid however which counts for over 14% of this total sample is not present. Liquid Aminos: Liquid Aminos contained no Aspartic acid, although small amounts were present in the sample. The Glutamic acid in the Aminos drink was in the D form. Essential amino acid Valine was present, although in lesser amounts to what was stated on the label. The opposite was true for Methionine however which contained slightly more than stated on labelling.

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The presence of D- amino acids were observed in all samples. This presence of these nonnutritional amino acids may be attributed to manufacturing or technological processes which involve the use of heat, irradiation, homogenisation, heat sterilisation and powder drying. (Csapo et al 2009) This can subject these protein powders to extreme heat (over 250oC) which can cause the racemisation of the amino acids. Hemp which is subject to the least technological process and still contained D-Aspartic acid. A study carried out analysing a ranges of food stuffs subject to technological processes such as heat and alkali environments, found the highest degree of racemisation occurred in aspartic acid (Masters & Freeman 1979). This suggests Aspartic acid has a tendency to racemize easily. This supports the evidence that shows 4 protein samples containing D-Aspartic acid. Hemp is derived from plant matter therefore the presence of bacteria cannot be ruled out, and because Hemp has a high percentage of moisture bacteria could also survive there. Bacteria have high levels of D-Aspartic acid in their cell wall peptidoglycans which may be attributed to the presence of D-Aspartic acid in this product. Further microbiological tests would be required to confirm this. The presence of the D-amino acids in these samples shows that processes such as heat and alkali environments can cause amino acids to racemize. The presence of D-Methionine and DGlutamic Acid in the Liquid Aminos supports the findings that D- amino acids are also present in hydrolysed products, and may also be a direct result of the production methods. In some cases D-amino acids have also been known to act as growth inhibitors (Csapo et al 2009) to mammals, the opposite effects to what people supplementing their diet with protein are expecting. At the very least D-amino acids will be excreted un-metabolised. The presence of D-amino acids in protein can leads to a decrease in digestibility and availability of other amino acids present. (Csapo et al 2009) When the manufactures analyse these products for total protein, the D-amino acids, will contribute to the overall protein content, even though they are not assimilated in the body, therefore total protein should be carried out by L-amino acid analysis to determine true Total protein present.

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Conclusion All dairy based samples were within the range for acceptable moisture with Hemp, a dried plant derived product having the highest moisture content. Sunwarrior protein had the least amount of protein as compared to its label with a difference of approximately 20%. All other dried products were within 10% of their labelled protein concentrations. As you raise the temperature in RP-HPLC an increase in resolution is observed. As the TFA concentration of mobile phase is raised from 0.001% to 0.1% an increase in retention time and resolution of proteins from each other is observed. All the samples analysed contained racemic mixtures of amino acids with the highest concentration with liquid Aminos having the highest level at 30.72mg/100ml D-Glutamic acid. This is a hydrolysed product that has to go through harsh treatments which may indicate why Liquid Aminos has the highest racemisation of all the products.

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Recommendations
When carrying out all analysis again include BSA through all experiments to ensure accurate and reliable results.

Ensure the use of an internal standard when using GC to analysing stereoisomerism of amino acids to ensure any losses through preparation and derivatization can be corrected

When using the Kejdalh method, use *100 less sample than stated in the baby formula protocol for Kejdalh total protein.

Carry out amino acid analysis using a complete set of 20 amino acids. From this analysis total protein can also be determined.

Carry out all analysis in triplicate to ensure accurate Results. Ensure HPLC system / column is serviceable. Use proper digestion vials with Teflon caps for digestion and derivatization of samples to ensure appropriate pressure and heat can build up without the loss of sample.

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Appendices

71

Appendix I

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To supplement HPLC separation of peptides. These illustrations help explain how hydrophobic interaction can be used to separate these proteins using RP-HPLC. The highly hydrophobic areas are deep red, these areas will have the highest affinity for the stationary phase and will determine the size of the hydrophobic footprint. Due to the nature and properties of these proteins, some have mainly hydrophobic centres and less hydrophobic regions facing out which will reduce hydrophobic interaction with stationary phase of RPHPLC meaning they will be retained less on the system.

Fig 1: Cytochrome C (Equine), colour coded as per hydrophobicity. 104 amino acid residue starting at Glycine and ending with Glutamic acid. A single Heme group is visible centrally. (Produced using PDB protein workshop 3.9) Molecular weight: 12kDa. Isoelectric point 10.5 (Equine). Cytochrome C is primarily known as an electron carrying mitochondrial protein. (Sigma Aldrich 2011)

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Figure 2: Myoglobin (Equine Heart) colour coded as per hydrophobicity. 153 amino acid residue starting and ending with Glycine. The heme group is also visible centrally encased in a hydrophobic sphere of amino acids. (Produced using PDB protein workshop 3.9) Isoelectric point 7.2. Molecular weigh:17kDa. Myoglobin is an iron and oxygen binding protein. (Sigma Aldrich 2011)

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Figure 3: Bovine Serum Albumin colour coded as per hydrophobicity. 583 Amino acid residue. (Produced using PDB protein workshop 3.9) Isoelectric point: 4.7 (25oC) mol wt. 66 kDa. BSA is water soluble Protein whos primary function is to regulate osmotic pressure. (Sigma Aldrich 2011)

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Appendix II Raw Data from High Pressure Liquid Chromatography separation of proteins and the effect altering Trifluoroaceticacid concentration has on retention time. Fig 1:

Figure 1: Chromatograph of 0.005% TFA separation of BSA, Cytochrome C and Myoglobin using a HPLC-UV system on a gradient profile.
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Fig 2:

Figure 2: Chromatograph of 0.01% TFA separation of BSA, Cytochrome C and Myoglobin using a HPLC-UV system on a gradient profile.

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Fig 3:

Figure 3: Chromatograph of 0.1% TFA separation of BSA, Cytochrome C and Myoglobin using a HPLC-UV system on a gradient profile.

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Retention time (Min) TFA Concentration (%) 0.005 0.010 0.100 Cytochrome C (4.27mg/ml) 3.750 4.167 6.417 BSA (3.53mg/ml) 8.750 8.167 9.167 Myoglobin (2.80mg/ml) 9.667 9.417 11.330

Table 1: Raw data used to generate line graph (Fig C2 experimental report) displaying the effect TFA has on retention time.

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Raw Data from High Pressure Liquid Chromatography separation of proteins and the effect altering temperature has on retention time. Fig 4:

Figure 4: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 14oC using a HPLC-UV on a gradient profile.
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Fig 5:

Figure 5: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 18oC using a HPLC-UV on a gradient profile.

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Fig 6:

Figure 6: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 22oC using a HPLC-UV on a gradient profile.

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Fig 7:

Figure 7: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 26oC using a HPLC-UV on a gradient profile.

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Fig 8:

Figure 8: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 30oC using a HPLC-UV on a gradient profile.

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Fig 9:

Figure 9: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 34oC using a HPLC-UV on a gradient profile.

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Fig 10:

Figure 10: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 38oC using a HPLC-UV on a gradient profile.

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Fig 11:

Figure 11: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 42oC using a HPLC-UV on a gradient profile.

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Fig 12

Figure 12: Chromatograph of the separation of BSA, Cytochrome C and Myoglobin at 46oC using a HPLC-UV on a gradient profile.

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Table 2: Retention time (Min) Temperature oC Cytochrome C (4.27mg/ml) 14 18 22 26 30 34 38 42 6.667 7.000 6.667 6.583 6.667 6.583 6.583 6.667 BSA (3.53mg/ml) 9.250 9.250 9.417 9.500 9.583 9.667 9.667 9.667 Myoglobin (2.80mg/ml) 11.583 11.667 11.833 11.917 12.000 12.000 12.000 12.000

Table 2: Raw data used to generate line graph (Fig C1 2.3 experimental report) displaying the effect Temperature has on retention time.

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90

91

92

93

Appendix III

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Raw data from Gas Chromatography Flame Ionisation Detection of Pentafluoropropyl Isopropyl Esters of Amino Acids following Hydrolysis and PFPA Derivatization of Protein Products. Fig 1: Standard set 1

Figure 1: GC chromatograph and report for Standard set 1. (D/L Valine, D/L Methionine and D/L Glutamic acid).
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Fig 2: Standard set 2.

Figure 2: GC chromatograph and report for Standard set 2. (D/L Valine, D/L Aspartic acid and D/L Glutamic acid).

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Fig 3: Liquid Aminos

Figure 3: GC chromatograph and report for Liquid Aminos amino acid drink.

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Fig 4: Hemp.

Figure 4: GC chromatograph and report for Hemp protein product.

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Figure 5: Milk & Egg.

Figure 5: GC chromatograph and report for Milk & Egg protein product.

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Fig 6: Soya 90%

Figure 6: GC chromatograph and report for Soya 90% protein product.

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Fig 7: Sunwarrior.

Figure 7: GC chromatograph and report for Sunwarrior protein product.

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PRODUCT/STD RETENTION TIME STD SET Amino Acid D-Valine L-Valine D-Methionine L-Methionine D-Glutamic acid L-Glutamic acid D-Aspartic acid L-Aspartic Acid u/k 6.97 7.03 7.08 6.95 7.02 u/k 6.50 6.57 6.60 6.48 6.55 23.98 18.87 18.85 18.82 23.98 23.78 18.67 18.12 18.12 1 14.83 15.02 18.87 19.02 STD SET 2 4.15 4.32 u/k u/k 4.35 4.42 4.25 4.33 15.02 18.88 19.22 Sunwarrior Hemp Milk & Egg Soya 90% Amino Acid Drink

Table 1: Raw data used to determine the origin of peaks shown in the chromatograms. Amino acid drink (liquid Aminos) was compared against standard set 1 shown in red. The other 4 samples were compared against standard set 2.

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103

Table 2: (Page 97) Raw data used to generate Bar charts displayed in experimental section Fig D1-D5. *NB: On label figures are derived from amino acid content/100mg (product) as this was the weight of product used for digestion and later suspended in 1ml Diethylether. **Hemp On label figures are not available from the distributer. 300mg hemp powder used due to low protein content therefore reported concentration /3.

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Table 3: Raw data used to quantify amino acid concentrations in samples from the standards analysed.

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