Analysis after Alkaline Hydrolysis

A study of the degradation of ceftiofur at elevated temperature in kidney extract and in alkaline environment has been reported. Degradation products in kidney extract and in kidney extract after addition of ammonia were identified using a combination of triple-quadrupole MS, LC-time-of-Flight MS (TOF/MS), nuclear magnetic resonance, and microbial techniques. After addition of ammonia to kidney extract at elevated temperature, ATMA 

A clean-up procedure for the analysis of ATMA as a marker compound for ceftiofur and ceftiofur-related metabolites, based on ion exchange SPE, has been developed. Separation was carried out using reversed phase FIPLC and detection by LC-MS/MS. At the time when this chapter went to press (December 2010), this method had not yet been tested using ceftiofur incurred tissue material, and therefore this new approach has not yet been compared to current approaches. 

For ceftiofur, the EU MRL includes ceftiofur and all active ceftiofur metabolites. To be able to comply with this MRL definition, all active metabolites of ceftiofur have to be included in the analysis method. 

Chloramphenciol (CAP) is a broad-spectrum antibiotic with historical veterinary uses in all major food-producing 

Thiamphenicol and fiorfenicol, which have structures similar to that of CAP (Fig. 7.3), are permitted as substitutes within the EU. In the EU, MRLs for thiamphenicol are 50 p-g/kg for bovine and chicken tissues, and for fiorfenicol, 100 pg/kg for muscle to 3000 pg/kg for bovine liver. Within the EU fiorfenicol also has a complex MRL definition, and this will be discussed in Section 7.9.2.1. Because of the ban on CAP, methods with very low detection limits have been developed. A minimum required performance limit (MRPL) of 0.3 pg/kg was assigned by the European Commission for the analytical methods testing for CAP in products of animal origin.  

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The amidine bond is quite stable at acid pH however, it is susceptible to hydrolysis and cleavage at alkaline pH. Derivatized proteins may be assayed by amino acid analysis after acid hydrolysis without loss of imidate modifications. 

Tyrosine (9-sulfate is stable under alkaline conditions, thus allowing for its quantification by amino acid analysis upon alkaline hydrolysis [0.2 M Ba(OH)2, 110 °C, 24 h] of sulfated tyrosine peptides and proteins.[6 331 Conversely, more than 95% of the ester is hydrolyzed after five minutes in 1M hydrogen chloride at 100 °C. Despite this pronounced acid lability, sulfated tyrosine peptides are sufficiently stable to short exposures of TFA134 35 or aqueous TFA[36 as required in peptide synthesis for removal of add-labile protecting groups. 

An HPLC method using a 90-min binary gradient with (a) acidified water, pH 2.4, and (b) acetonitrile on an Adsorbosphere C]8, 3-/zl cartridge (Alltech) was also developed for pheno-lics in barley (127). Seven phenolic compounds, including vanillic acid, p-coumaric acid, ferulic acid, and their derivatives, were separated by HPLC after alkaline hydrolysis in order to evaluate the role of bound phenolic acids in their antioxidant activity in beer. In this method, cis and trans isomers of p-coumaric and ferulic acids are quantified by HPLC, although cls-p-coumaric acid was not well separated from its trans isomer in this analysis. 

For colorimetric analysis, plasma and urine samples are prepared by alkalinization and chloroform extraction. Methocarbamol couples with diazotized dinitroaniline to give an orange-red compound33. Alternatively, colorimetric determination with chromotropic acid is possible after alkaline hydrolysis (10 mins, in boiling water) of the carbamate and periodate oxidation to formaldehyde34. 

Detection After alkaline hydrolysis, enzymatically ly means of [ P]ATP and choline kinase (EC 2.7.1.32). Lit Biochem. Soc. Trans. 17, 76-79 (1989). Bergmeyer, Methods of Enzymatic Analysis (3.), vol. 8, p. 462-473, Wein-heim Verl. Chemie 1985. 

For the identification of bile acids in bile from man and different animals, Kuksis (13) and co-workers have used ion-exchange chromatography for the separation of glycine- and taurine-conjugated bile acids. After alkaline hydrolysis, extraction, and methylation, the bile acids were analyzed on SE-30 and QF-1 columns. Tentative identifications were supported by additional gas-liquid chromatographic analysis of methyl ester acetates and methyl ester trifluoroacetates. The latter derivatives were also analyzed on OV-17 columns (117). 

Naturally occurring lipid mixtures have a composition of such extreme complexity that the analysis by chemical methods of a group of these substances or the determination of a single compound in such mixtures, appears hopeless. Up to about ten years ago, lipid mixtures were characterised by totals like acid-, saponification-, iodine-, thio-cyanogen- and diene- numbers . The determination of the amount of non-saponifiable matter after alkaline hydrolysis was a standard method in the analysis of fats. Phospholipids and sulpholipids were quantitatively determined as inorganic phosphate and sulphate after combustion. These methods were supplemented by detection of subsidiary fat constituents like lipochromes, sterols and resin acids, with the help of colour reactions. 

Analysis of carnitine and acylcarnitines by MS/MS is now considered routine in those laboratories that possess the appropriate technology. The ionization techniques commonly used with MS/MS are fast atom (or fast ion) bombardment and electrospray. Both are sufficiently sensitive to detect abnormally elevated concentrations of specific metabolites in all types of specimen, although the latter is much more widespread and is the more sensitive of the two, especially for long-chain acylcarnitines. The method is quantitative or at least semi-quantitative for most analytes, and uses stable isotope-labeled forms of the analytes as internal standards. The acylcarnitines are analyzed simultaneously in the positive ion mode as their methyl or butyl esters using a precursor ion scan function [14, 23] that detects the parent (molecular) ions. Free carnitine and total carnitine are determined by assaying the same specimen before and after alkaline hydrolysis, without derivatization, using Hs-carnitine as internal standard [17]. The value for acylcarnitine is determined by difference. This value includes the contribution of short, medium and long-chain acylcarnitines. Analysis time for each method is approximately 2 min. 

The use of pre-column HPLC method for the analysis of fatty acid with 2-nitrophenylhydrazine hydrochloride was reported (Miwa Yamamoto 1990). After alkaline hydrolysis of coconut oil free fatty acids are reacted with 2-nitrophenylhydrazine hydrochloride and then derivatized to corresponding fatty acid hydrazides. Each of the derivatives was separated on reversed-phase HPLC with isocratic elution and detected at VIS 400 nm (Miwa Yamamoto 1996). In Table 2 is presented the typical fatty acid composition of coconut oil. 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

Several techniques have been described in the past for the analysis of VLCFA, pristanic acid and phytanic acid [1, 10]. In our hands gas chromatography-mass spectrometry (GC-MS) analysis after derivatisation with N-methyl-N-(tert-butyldi-methylsilyl) trifluoroacetamide (MTBSTFA), is a robust and reliable method for the quantitative analysis of VLCFA, pristanic acid and phytanic acid, especially when combined with stable isotopes for C26 0, C24 0, C22 0, phytanic acid and pristanic acid [13]. In order to allow measurement of the total pool of VLCFA, pristanic acid and phytanic acid, samples need to be subjected to both acidic and alkaline hydrolysis, followed by extraction into hexane. After the hexane phase is washed once more, the sample is dried under nitrogen followed by addition of pyridine and MTBSTFA and heating of the samples at 80°C. The sample is subsequently dried again under nitrogen and taken up in hexane, followed by GC-MS analysis. 

Quantitative Analysis after Chemical Reaction. A large number of fluorometric analyses have been performed after converting a nonfluorescent or weakly fluorescent compound into an intensely fluorescent species. One example, which involves only hydrolysis, is the measurement of acetylsalicylic acid as salicylic acid or the salicylate anion. Because the total concentration of these two acids in blood is very important in the treatment of rheumatic disease, acetylsalicylic acid is converted to salicylic acid by hydrolysis and the total measured as salicylate. First the serum protein is removed, then the acetylsalicylic acid is hydrolyzed under alkaline conditions to salicylic acid and acetic acid (in alkaline solution, an equilibrium mixture of the anions and acids is present). The alkaline solution is then excited at 310 nm and the fluorescence emission is measured at 410 nm. 

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