Meat is a rich source or proteins.

Meat proteins are considered high quality proteins because of their balanced content in amino acids, especially in all essential amino acids necessary for physical and mental well-being. Free amino acids in meat also contribute to meat taste and indirectly to aroma by generation of volatile compounds through Maillard reactions and Strecker degradations. Thus, the analysis of amino acids is important for several reasons including the evaluation of nutritive value and/or sensory quality of a given meat. The strategy to follow for the analysis of amino acids depends on the final goal which include 1) the analysis of the amino acids profile: free amino acids or total (free plus hydrolyzed) amino acids profile 2) the analysis of whole amino acid content (amino nitrogen), or 3) the analysis of a single amino acid or a group of amino acids Analysis of the amino acids profile Sample preparation This depends on whether free or total amino acids have to be analysed. Sample preparation for free amino acids profile Sample preparation for free amino acids profile includes their extraction and cleanup or deproteinization of the extract. Ground meat sample is homogenised in a suitable solvent like hot water, diluted phosphate buffers or 0.01-0.1N hydrochloric acid. Sometimes concentrated strong acid solutions like 4% 5-sulfosalicylic acid, 5% trichloroacetic acid, or rich alcohol-containing solutions like > 75% ethanol or methanol are used. This has an added advantage that further cleaning is not required, since proteins are not extracted. Once extracted, the solution is centrifuged at more than 10,000 X g under refrigeration to separate the supernatant from the nonextracted pellet and filtered through glass wool to remove any fat material. Sample cleanup is done by deproteinization procedures which include both physical (cut-off membrane filters in centrifugation) and chemical (protein precipitation by concentrated acids like phosphotungstic acid, sulfosalicylic acid etc. with free amino acids remaining in solution) methods. All these procedures yield a solution rich in amino acids. Sample preparation for total or hydrolysed amino acids profile Total amino acid profile is needed to evaluate the nutritive value of meat and includes free amino acids plus those from muscle proteins. In this case, protein hydrolysis into constituent amino acids is needed. Acid digestion is the most common method for hydrolysing meat proteins in which digestion is done at 1100C for 20-96 h in 6N hydrochloric acid. To minimise degradation, nitrogen atmosphere and sealed

vials are used. Hydrolysis is achieved by liquid-phase or vapour-phase method. Liquid-phase is used to hydrolyse large amounts of sample and in this acid directly comes into contact with sample. In vapour-phase hydrolysis, only the acid vapour comes into contact with the sample, thus excluding non-volatile contaminants. In both types, hydrolysis is done in an atmosphere of nitrogen or any inert gas. Hydrolysis can be improved by optimising the temperature and time of incubation or by addition of amino acid oxidation protective compounds like 0.1% sodium sulphite or 1% phenol. This prevents the loss of most of the essential and labile amino acids like tyrosine, serine, threonine, methionine and tryptophan. Microwave hydrolysis, alkaline hydrolysis, enzymatic hydrolysis etc. have also been suggested. Derivatization Once amino acids are extracted, they are separated from each other by highperformance liquid chromatography (HPLC), capillary zone electrophoresis (CZE) or gas liquid chromatography (GLC) for individual analysis. Before or after this, amino acids may be derivatized to allow their separation or enhance their detection. Derivatization is a standard procedure in amino acid analysis. A derivatizing agent must have the following functions: It must be able to 1) react with both primary and secondary amino acids 2) give a quantitative and reproducible reaction 3) yield a single derivative of each amino acid under mild and simple reaction conditions 4) have the possibility of automation with good stability of the derivatization products and 5) no interference due to by-products or excess reagent. Two types of derivatives are obtained depending on the chosen detection/separation technique. The first type are those derivatives that enhance amino acid detection which include derivatives for spectroscopic or electrochemical detection. The choice of derivative is important because their spectral or electrochemical characteristics will affect the sensitivity and selectivity of detection. The derivatives formed will be separated by HPLC or CZE. The second type are those derivatives that allow GLC amino acid separation by increasing their volatility and temperature stability or the efficient separation of racemic mixtures of the amino acids. Derivatives for spectroscopic detection These derivatives are prepared by labelling the amino acids with reagents that enable ultraviolet or visible absorption or by adding fluorescent properties to the

molecule that improves the selectivity and sensitivity of detection method. There are two types of derivatization techniques. Post column derivatization This involves the separation of the free amino acids themselves through the liquid chromatographic column, the introduction of a suitable derivatizing reagent into the effluent system from the column, the flow of both combined liquids through a mixing manifold followed by a reaction coil, and finally the pumping of the derivatized amino acids through an online detector system. This is used in many commercial amino acid analysers like the classical Moore and Stein-type. Main drawbacks of this are that an additional pumping equipment and sometimes mixing and heating devices are needed. There may also be a peak broadening due to dead volume introduced behind the column. The reagents that have been commpnly employed in this derivatization are ninhydrin, fluorescamin and o-phthaladehyde (OPT). Ninhydrin is a reagent that reacts with all 20 amino acids at an acidic pH. Amino acids with a primary amino group give a blue reaction product with a maximum absorbance at 570 nm, while secondary amines yield a brownish reaction product that absorbs around 440 nm. The sensitivity is below 1nmol, but rarely below 100 pmol. Fluorescamine forms a fluorescent derivative with primary amino acids, but not with secondary amino acids. The fluorescence is recorded at a wavelength emission of 475 nm after excitation at 390 nm. The reaction takes place in alkaline conditions. It is used in precolumn derivatization for special applications, including the analysis of 3-methylhistidine (3-MH) as a marker content of lean meat in meat products. OPT can be detected either spectrophotometrically or with fluorescence. Pre column derivatization In this, the formed molecule improves sensitivity and selectivity at the detection, in addition, the derivatizing agent confers hydrophobicity to the amino acid molecule, making it suitable for separation by partition chromatography in a reversedphase (RP) column. Phenylisothiocyanate (PITC), butylisothiocyanate (BITC), 4dimethyl-aminoazobenzene-4-sulfonyl naphthalene-5-sulfonyl (FMOC), chloride o-phthaldialdehyde (OPA), chloride (Dabsyl-Cl), 1-dimethylaminochloroformate (Dansyl-Cl), 9-fluorenylmethyl

6-aminoquinolyl-N-hydroxysuccinimidyl

carbamate (AQC) etc are the reagents commonly used in this derivatization. PITC convertsprimary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives, which are detectable at 254 nm with detection limits around 5-50 pmol.

The PTC- amino acids are quite stable at room temperature for one day, but sample preparation is quite tedious requiring a basic medium. Currently, PITC is one of the preferred precolumn derivatizing agents for analysis of physiological amino acids from foods and feeds by HPLC, especially for the analysis of meat amino acids, due to the presence of dipeptides like carnosine, anserine and balenine, which are well separated. BITC forms butylthiocarbamyl amino acids at 400C for 30 min and the advantage of this reagent over PITC is its high volatility. Detection by dabsyl-Cl reagent is by absorption in the visible range, maximum at 448-468 nm. Derivatives are formed from both primary and secondary amino acids and are quite stable. Reaction efficiency is highly matrix dependent and variable for different amino acids. Dansyl-Cl is commonly used for N-terminus analysis of peptides and proteins. It reacts with both primary and secondary amines to give a highly fluorescent derivative, which is very stable. Eventhough sample derivatization is quite simple, reaction conditions must be carefully calibrated to optimise the product yield and to minimise secondary reactions. This is very useful for cystine and cystine-containing short chain peptides. FMOC yields stable derivatives with primary and secondary amines, which are fluorescent and can be detected at femtomole range. OPA reagent reacts with primary amino acids in the presence of a mercaptan cofactor to give highly fluorescent 1-alkylthio-2-alkyl-substituted isoindols and the fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm. The derivatization is fast, but OPA amino acids are not stable. The problem is overcome by standardizing the time between sample derivatization and column injection by automation. A disadvantage is that OPA does not react with secondary amines. This is overcome in two ways. In postcolumn derivatization, before OPA derivatization, secondary amines are converted to primary amines with hypochlorite or Cloramine T. In precolumn technique, it is normal to combine the OPA with other derivatization methods like FMOC, which should take place sequentially. AQC reacts with primary and secondary amines from amino acids, peptides and proteins, yielding very stable derivatives with fluorescent properties that are separated by reversed phase-HPLC. Sensitivity is in the femtomole range. This reaction is not affected by the presence of salt, detergents, lipids and other compounds naturally present in food.

Derivatives for electrochemical detection (ECD) These derivatives are molecules with electroactive functional groups. All of them also have spectroscopic properties, specifically, OPA/mercaptoethanol, or OPA/sulphite, and naphthalene-2,3-dicarboxaldehyde (NDA); fluorescent properties and electroactivity. Both reagents react with primary amines, and the sensitivity reached when connecting with capillary electrophoresis (CE) is at the femtomole level. 6-AQC and PITC have the advantage of reacting with secondary amines. Derivatives to allow separation Volatile derivatives The aim here is to obtain a volatile and thermostable molecule suitable for analysis by GLC. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. Chiral derivatives One of the techniques used to resolve racemic mixtures of D- and L-amino acids is the use of amino acid derivatives formed with fluorescent chiral reagents that permit both an effective separation of the isomers in an RP-HPLC column or in CE followed by an enhanced detection. Separation The analysis of individual amino acids needs prior separation unless a very selective method of detection is used. The separation of the individual amino acids in a mixture requires very efficient separation techniques such as chromatographic (liquid/gas) or CE methods. The choice mainly depends on the equipment available or personal preferences, because each possible methodology has advantages and disadvantages. Liquid chromatographic (LC) methods Cation exchange chromatography (CEC) This methodology is based on the amino acids charge; the underivatized amino acids are separated using sulfonated polystyrene beads as the stationary phase and aqueous sodium citrate buffers as the mobile phase. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. Under these

conditions, the more acidic amino acids elute first; those with one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram. The original method required two separate columns and needed about 4 h to achieve a complete analysis. After separation, amino acids were converted into coloured ninhydrin derivatives for spectrophotometric (colourimetric) detection. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to its smaller particle size, speed, pellicular packaging, and better detection systems. Currently, the separation times for the 20 amino acids naturally occurring in meat proteins take around 1 h and somewhat longer for physiological amino acids. There are many manufacturers that offer integrated commercial systems, including the column, buffer system, and an optimized methodology with the advantage of the ease of use and reliability. The advantage of this method is its accurate results for all known sample types (food, biological fluids, feed, plants), which makes it a reference method for amino acid analysis. Thus, each new methodology must compare its results with those obtained by CEC. The main drawbacks of this method are the high cost of the ion exchange amino acid analyzer and its maintenance, the very complex mobile phase composition, and the long time for analysis. Reversed phase high-performance liquid chromatography (RP-HPLC) This is widely used and has the advantage of requiring standard equipment that can be shared by different types of analysis. In this, prior amino acid derivatization is necessary to confer hydrophobicity to the amino acid molecule, making it suitable for partition based on chromatography. This derivatization allows the spectroscopic (ultraviolet or fluorescent) detection of amino acids. The most commonly used column packaging consists of alkyl-bonded silica particles, mainly octadecyl-silane. Selectivity varies with column packaging. The presence of residual uncapped silanol groups on the silica surface, accessible to sample molecules, can cause unwanted tailing of peaks (especially for basic amino acids). Addition of a strong cation (like triethylamine) to the mobile phase can overcome the problem. Nowadays columns are available with silanol groups blocked. Typical analytical column dimensions are 15 cm in length (for hydrolysed amino acids) or 25-30 cm (for physiological amino acids) packed with 5 m particles, or shorter columns, 10 or 15 cm long, packed with 3 m particles. Mobile phase consist in the ability to dissolve the sample while remaining transparent to the detection

system. Mobile phase composition combines an aqueous buffered phase with an organic phase consisting of acetonitrile and/or methanol and/or terrahydrofuran. The buffer may be constituted by less than 100mM concentration of acetate or phosphate. A finely adjusted binary or ternary gradient elution is often necessary when the overall amino acid profile from hydrolysed and, especially, physiological amino acids have to be analysed. Gas liquid chromatography (GLC) methods The high resolution capacity is the main advantage of gas chromatography as compared with liquid chromatographic techniques, especially since the capillary columns appeared. But this is not commonly used to determine meat amino acids. It has been used to determine nonprotein amino acids from edible seeds, nuts and beans, honey etc. In many cases, GLC has been combined with mass spectrometry for detection and identification, especially in the analysis of D-isomers, where the separation was achieved by using chiral-GC stationary phases. EZ:faast is a recently developed fast GC analysis technique capable of separating 50 compounds including amino acids, dipeptides and amines. The method yields a full amino acid profile (33 amino acids) in 15 min, including a 7 min extraction-derivatization step plus 8 min for the gas chromatographic separation. Protein removal is not required and the derivatives are stable and ready for GC/FID, GC/NPD, GC/MS and LC/MS. Described applications are available for the analysis of physiological amino acids in blood, plasma and urine matrices, but a meat application has not been described yet and the ability of this method to analyse the natural meat dipeptides carnosine, anserine and balenine is unknown. Capillary electrophoretic methods The technique of Capillary Zone Electropheresis (CZE) is extremely efficient for separation of charged solutes. The high efficiency, speed, and the low requirements in terms of sample amount make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. Difficulty of separating amino acids by this technique derives from their structure. Amino acids constitute a mix of basic, neutral, and acidic constituents, and even though a particular pH can significantly improve the resolution of one kind, it is likely to cause overlap with the others. Under the conditions of electroosmotic flow in CE, a species with

different charge can be simultaneously analysed, but resolution varies. CZE shows poor ability for the separation of neutral compounds, which is an important limitation to this technique. A modification of CZE called Micellar electrokinetic capillary chromatography (MECC) has been developed for separating neutral compounds. In this surfactant-formed micelles were included in the running buffer to provide a twophase chromatographic system for separating neutral compounds together with charged ones in a CE system. In this derivatization is usually used to improve separation, to enhance ultraviolet detection or to allow fluorescence detection of amino acids. The CE coupled to electrospray ionisation mass spectrometry (CE-ESI-MS) allows the direct amino acid analysis without derivatisation, using 1M formic acid as the electrolyte. Detection The choice of detection method depends on several factors, such as technique used for the separation or the requirements for sensitivity and selectivity. There are some detectors for GLC systems, like flame ionization detector (FID) or flame photometric detector (FPD), and others for liquid systems (flow injection analysis, HPLC, CZE), such as spectroscopic (colorimetric, ultraviolet, or fluorescence) or electrochemical detectors; mass spectrometry (MS) may be used with either gas or liquid systems. Detectors specific for GLC The FID is a universal detector for gas chromatography and is the most widely used; thermionic-N-P or FPD are selective for organic compounds containing phosphorus and nitrogen, being much more sensitive than FID for such compounds. Spectroscopic detectors Colorimetric detection This is achieved after derivatization with nonselective colorimetric reagents like ninhydrin (490 nm) or dabsyl chloride (448-468 nm).

Ultraviolet and fluorescence detection Amino acids, in their native form, absorb at 210 nm and thus cannot be used for spectroscopic detection, since this is a very unspecific detection wave length. Only three amino acids (phenylalanine, tyrosine and tryptophan) possess a chromophore moiety that confers a suitable maximum absorbance for more specific ultraviolet detection (280 nm for tyrosine and tryptophan and 254 nm for phenylalanine). Tryptophan also possesses native fluorescence that facilitates a more selective detection. Thus, the spectroscopic detection of amino acids requires their prior derivatization to obtain an ultraviolet-absorbing or fluorescent molecule. UV and fluorescent spectroscopic detection may be used after HPLC, CE, FIA, or thin layer chromatographic runs. Laser-induced fluorescence detection Laser-induced fluorescence (LIF) detection was introduced as an alternative fluorescence detection when looking for more selective and sensitive detectors with a wide linear dynamic range to cover new high-sensitivity applications (chiral analysis, 0-tyrosine analysis, biomedical or pharmaceutical research) and instrumentation (CE or microcolumn liquid chromatography). Amino acids are derivatized as in fluorescence detection. Mass spectrometry (MS) Mass spectrometry is based on the conversion of components of a sample into rapidly moving gaseous ions that can be resolved on the basis of their mass-charge ratios, which are characteristic of each ion, allowing its identification. Not only the 22 amino acids can be detected, this helps in the detection of D- and L-isomer mixtures, nonprotein amino acids etc. But the high cost of purchase and maintenance have limited the use of mass spectrometers in food industry. Mass spectrometers were first connected with GC equipment due to the good compatibility between the two techniques. These had been used in identification of nonprotein amino acids, chiral amino acids etc. MS has also been used as a spectroscopic detector after HPLC or CZE, offering the additional advantage of analysing the amino acids without derivatization. As a result only minor sample manipulation is needed and due to high specificity, there are reduced problems related to matrix interferences or poor resolution between peaks.

MS-HPLC combination is not very combatible. However, these difficulties have recently been overcome with the development of new interfaces. One of the main requirements for samples to be analysed by MS is that the analytes (i.e, amino acids) must be ionized. Three types of ionisation of analytes have been developed atmospheric pressure microwave induced plasma ionization (AP-MIPI), atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), the best results being obtained by AP-MIPI. MS-CE combination can also be used for detection of amino acids with separation of amino acids by CE and detection by MS. Electrochemical detection (ECD) ECD is based upon the electrical properties of a solution of analyte when it forms part of na electrochemical cell. It consists of one electrode or an array of electrodes mounted in a cell with an applied potential difference. Any electrical measure such as current, potential, conductance, or charge is related to the analyte concentration. Only amino acids with aromatic rings or sulphur-containing side chains are sufficiently electrochemically active to be detected by this method. There are also several methods to detect amino acids with nonelectrochemically active properties. They are 1) amino acids by attaching to them an the derivatization of amino acids by attaching to them an amine or carboxylic acid that is electrochemically active such as NDA, OPA/mercaptoethanol or OPA/sulphite, 6-AQC, or PITC 2) The generation of chemical reactions at the electrode surfaces to produce electrochemically active products. This method is inexpensive and has useful applications in analysing amino acids in foods or in process monitoring and control during the production of amino acids 3) the use of immobilized enzymes (amino acid oxidases with peroxidises) to react with amino acids, again yielding electrochemically active products (ammonia, hydrogen peroxide etc.) for detection. Very simple pretreatment is needed in this due to the high degree of selectivity. Analysis of the whole amino acid content The objective of this is the analysis of the whole amino acid amount without discriminating among them. This does not discriminate between free amino acids and small peptides and is based on the reaction of the -amino group with reagents such as OPA, cadmium-ninhydrin, or trinitro-benzene-sulfonic acid (TNBS), which are most frequently used. These reagents render chromophores that increase the amino

acids ultraviolet response at a higher wavelength or confer visible or fluorescent characteristics on them. This method can be used in meat and meat products and involves steps like precipitation of proteins, reagent addition, and colorimetric, UVabsorption, or fluorescent determination of the amine nitrogen in the supernatant. Special applications in meat and meat products This involves the analysis of one amino acid or a group of amino acids in meat for various purposes like index of adulteration or fraud, detection of contamination, processing control, detection of bad processing practices etc. Some are as follows. 1) Determination of 3-Methylhistidine (3-MH) There is widespread use of nonmeat proteins in meat products and this has necessitated the development of methods for quantification of meat proteins. Proteinbound 3-MH has been detected only in the contractile meat proteins myosin and actin, and is absent in collagen and all nonmeat proteins, and therefore it has been used as an index of meat content in meat products. Any of the above methods can be used for the analysis of this amino acid, but the RP-HPLC separation of the fluorescamine precolumn derivatives and fluorescence detection has been the most frequently used. Due to the cationic nature of the amino acid, cation exchange HPLC is also used, where good separation of underivatized basic amino acids and naturally occurring dipeptides present in meat is achieved with postcolumn OPA detection. A GC method for detection of 3-MH has also been reported. Careful removal of the sarcoplasmic proteins is necessary before hydrolysis of myofibrillar proteins to avoid the interference of 3-MH resulting from the hydrolysis of fraction of some animal skeletal muscles, including pork. Determination of OH-proline and OH-lysine Collage is a low quality protein, since it is poor in essential amino acids and hence has less nutritional significance. 4-hydroxyproline and 5-hydroxylysine are specific amino acids located in the primary structure of collagen and have been used as an index of the collagen content in meat and meat products. The analysis of these amino acids can be done by any of the above methods, RP-HPLC being the most common. 4-hydroxyprolineis a secondary amino acid that the naturally occurring dipeptide balenine ( alanine-3-methylhistidine), which is present in the sarcoplasmic

does not react with some derivative agents like OPA. Specific colorimetric methods and GC-MS of the N(O)-trifluoroacetyl n-propyl ester derivatives are also employed for detection of these amino acids. Determination of o-Tyrosine o-Tyrosine is an indicator of food irradiation. When phenylalanine is irradiated with gamma rays, it is oxidised, yielding o- and m-tyrosine isomers. The conversion yield is proportional to the absorbed dose and temperature during irradiation. As many foods contain constant levels of phenylalanine in proteins, the level of o-tyrosine may be a good indicator of food irradiation. But, only low levels of naturally occurring o-tyrosine are present in some foods. The application is especially common in pork, chicken, fish and shrimp. Highly sensitive and selective methods like GC-MS, HPLC with fluorescence detector, HPLC-LIF and HPLC-ED are used. Determination of sulphur containing amino acids The importance of sulphur containing amino acids is due to their high reactivity, reducing power and their influence in flavour. Furthermore, methionine is an essential amino acid and cyst(e)ine is essential in premature infants. Special sample preparations and care are needed for their determination. Determination of essential amino acids and taurine CHECK IN CHAPTER FOR ESSENTIAL AA Thus, to obtain the total amino acid profile of a given meat, important factors to be taken into account are high resolution power and selectivity. High resolution is obtained by gas chromatography with capillary column techniques, but tedious and time consuming sample derivatization is required. Cation-exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are preferred methods. Very careful control of the derivatization reactions and chromatographic conditions is necessary for a consistent and reproducible analysis. Many peaks corresponding to protein and nonprotein amino acids, nucleosides, small peptides etc. may appear in the chromatogram and a complete resolution of these peaks is very difficult.