Amino acids internal standards

Figure 11 Determination of panthenol in multivitamin tablets. Chromatographic conditions see Table 3. Peak identification (1) 6-aminohexanoic acid (internal standard)-fiuo-rescamine derivative (2) 3-amino-1-propanol fluorescamine derivative. (From Ref. 67.)...

Aliquots of this solution were analyzed by GC-MF using a wide-bore fiised-silica column (DB-T7, 15 m X 0.53 mm) with a flow rate of helium of 15 vaL min, and the injection port, column oven, and separator temperatures of 250°C, 200°C, and 250°C, respectively. The MS detector was operating in the electron impact mode at 70 eV, the ionization current was 100 pA and the temperature of the ion source was 200°C. The stable fragment ions [M-CHs] selected for multiple ion detection were at m/z 420,434, and 448. They were produced from the trimethylsilyl derivatives of pantothenic acid, hopantenic acid, and 5-[2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)amino]pentanoic acid (internal standard), respectively (Fig. 30). 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Figure 4.4 Release of amino acids from cortical slices exposed to 50 mM K+. Measurements by HPEC and fluorescence detection after reaction of amino acids with o-phthalaldehyde 1, aspartate 2, glutamate 3, asparagine 4, serine 5, glutamine 6, histidine 7, homoserine (internal standard) 8, glycine 9, threonine 10, arginine 11, taurine 12, alanine 13, GABA 14, tyrosine. Glutamate concentration is almost 1 pmol/gl which represents a release rate of 30 pmol/min/mg tissue...

A GC analysis of amino acids requires a derivatisation step to increase the volatility of the amino acids. Generally, norleucine and/or norvaline are the internal standards added to the hydrolysate to check the derivatisation yield. According to the experimental method applied, the limits of detection (LOD) vary in the range 10 100 pg for each amino acid. Regarding the chromatographic columns, as most of the derivatives are esters barely polar compounds the most commonly used are fused-silica capillary columns with a low... 

Figure 9.1 GC MS chromatograms acquired in the SIM mode of a laboratory blank (a) and an amino acid standard solution with concentrations at the quantitation limit (b). i.s.l, Hexadecane internal standard i.s.2, norleucine internal standard...

Figure 14.8 Partial m/z 44 and m/z 45/44 traces obtained by GC C IRMS analysis of commonly occurring amino acids (as their triflouroacetyl isopropyl ester derivatives) together with a range ofco injected internal standards...

Figure 10.19 Amino acid analyser trace. Separation of a complex physiological standard mixture of amino acids in 3.5 hours using lithium citrate buffers and ninhydrin detection 10 nmol of each amino acid, including the internal standard, nor-leucine, were applied to the column (0.3 X 35 cm) in a total volume of 50 ml. 

The colour or fluorescence produced per mole of amino acid varies slightly for different amino acids and this must be determined for each one to be quantitated. This is done by loading a mixture of amino acids containing the same concentration of each amino acid including the chosen internal standard and from the areas of the peaks on the recorder trace calculating each response factor in the usual way (Figure 10.19). These values are noted and used in subsequent calculations of sample concentrations. 

An internal standard should always be used for every analysis carried out. This is an amino acid that is known to be absent from the sample under investigation. For instance in blood plasma analysis either of the non-physio-logical amino acids, nor-leucine or a-amino-/3-guanidinobutyric acid, may be used. This should be added in a known amount to the sample prior to any sample pre-treatment (for example, removal of protein). 

If the amount of internal standard which was added to the sample is known, the concentration of the unknown amino acid can be determined using peak area relationships. These calculations must take the various response factors into account. 

Calder, A.G. Garden, K.E. Anderson, S.E. Lobley, G.E. Quantitation of Blood and Plasma Amino Acids Using Isotope Dilution GC-EI-MS with U- C Amino Acids As Internal Standards. Rapid Com-mun. Mass Spectrom. 1999, 13, 2080-2083. 

Methanolysis of standard uronic acids has been studied by Inoue and Miyawaki in regard to the depolymerization of chondroitin sulfate and dermatan sulfate. These workers found the glucosiduronic linkage to ga-lactosamine to be rather resistant to methanolysis, but that it is more efficiently cleaved after deamination of the amino galactoside, with its conversion into 2,5-anhydrotalose. For iduronic, glucuronic, and man-nuronic acids released from a polymer, it was found that the peaks monitored for these acids, relative to an internal standard, increase during the first 8 h of methanolysis (M hydrogen chloride, 100°) and remain constant for up to 20 h of methanolysis. This indicated that 8 h is required for complete methanolysis, and that the monosaccharides liberated are stable to the conditions of methanolysis. 

HPLC analysis of furosine (-peak II) in hydrolyzates of non-exposed- (bottom), buffer-exposed (middle), and glucose-exposed (top) dentin samples. Dentin was not reduced prior to hydrolysis. Only the relevant parts of the chromatograms are shown. Amino acids are visualized after post-column labelling with a fluorescent dye. I lysine, II furosine. III homoarginine (internal standard). Column Merck Polyspher AA-NA 120 x 4.6 mm flow 0.2 ml/min gradient pH 5.0 -10.2 postcolumn reagent 0.2 ml/min fluorescence Xgx 330 nm, 440 nm 100-yl injections in buffer pH 2. 

Although DIOS-MS is mainly a tool for qualitative analysis, many examples have shown that quantitative analysis is possible when internal standards are used. These may either be isotope-labelled - mostly deuterated - compounds or structurally related analogues. For example, subsequent to electrospray deposition, amino acids such as phenylalanine and tyrosine have been successfully quantified by means of DIOS-MS using their deuterated analogues as internal standards. 

Quantification of the separated amino acids is usually performed by using external calibration or the internal standard method. Due to the large differences in chemical structure exhibited by the various amino acids, there is not a single ideal standard for the overall amino acid profile. Nevertheless, a suitable internal standard must be stable to hydrolysis and offer chromatographic resolution. The most popular choices comprise norleucine, norvaline, and a-amino-n-butanoic acid (AABA) [196]. 

Following extractive deproteinization of the plasma, the amino acids (and their stable-isotope-labeled internal standards) are separated by HPLC and introduced into the mass spectrometer. Electrospray ionization results in the formation of electrically charged molecules, which are separated on the basis of their mass/charge (m/z) ratio in the first quadrupole. Following fragmentation in the collision cell, the characteristic fragment for each amino acid is selected in the second quadrupole. This process is named multiple reaction monitoring. 

Table 2.1.3 Multiple reaction monitoring of amino acids for their tandem mass spectrometry quantitation. In daily practise not all mentioned amino acids are measured in one run, but a set of ten dedicated evaluation programs has been developed, covering groups of amino acids associated with groups of disorders. Amino acids presented in italics indicate stable-isotope-labeled internal standards ... 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

Fig. 2.1.2a-c A Urine amino acids in a patient with cystinuria assayed by an amino acid analyzer (AAA). The indicated peaks are 1 glycine, 2 cystine, 3 ammonia, 4 ornithine, 5 lysine, 6 arginine, i.s. internal standard (S-amino thyl-cysteine). Cystinuria treatment is best followed-up by analyzing an early morning urine specimen, which usually shows the highest amino acid concentrations. b-c see next page... 

The internal standard is carboxymethylcysteine(10 mg in 10 mlSSA5%). This should be diluted 1 20 prior to use. Calibration standards containing the usual physiological amino acids may be purchased from Sigma or Pierce. Labile amino acids such as tryptophan, glutamine, asparagine, and homocarnosine should be prepared fresh at each calibration step, but can be stored at -20°C for at least 4 weeks. 

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