Metabolite-residue analysis

Metabolite/residue analysis. Milk, urine and plasma samples were first analyzed by a microbiological cylinder/plate procedure against M.luteus which has a limit of detection of 0.02 ppm. A sub-sample of the milk was prepared for this assay by a centrifugation step followed by a pH adjustment to 8.5. In addition, an HPLC/RAM analysis was conducted after treating another sub-sample with FTSH (10% formic acid, 30% trifluoroacetic acid, 2% sodium chloride, 2N hydrochloric acid) followed by centrifugation to precipitate the proteins. The supernatant was basified and concentrated by C-18 solid phase extraction (SPE) techniques. The HPLC conditions were Column - 20 cm x 4.8 mm C-8 Mobile-phase -linear gradient at 5%/minute from 90 10 O.IM pH 7 phosphate buffer methanol to 20 80 Detectors - UV operated at 214 nm and a radioactivity flow detector operated in the DPM mode. 

To select and define the target analytes for the residue analysis of crops in a field trial, applicants should consider metabolites/degradation products of the test materials by conducting plant and animal metabolism studies and by assessing toxicity of the metabolites/degradation products. 

Almost 10% of the TRR level of the metabolites/degradation products will be selected and defined as the analytes for residue analysis. 

Several methods can be used for the residue analysis of anilides, especially gas chro-matography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). GC/ECD or GC/NPD for the determination of anilides has generally been used except for the unstable metabolites of naproanilide and clomeprop, which are determined by HPLC/UV, HPLC/FL or GC/ECD after derivatization. 

Y. Nakahira, O. Kimura, H. Aoshima, M. Ikeda, Y. Asano, and Y. Yusa, Analysis of the residue of a new fungicide, mepanipyrim, and its metabolites in crops, in Abstracts of the 18th Annual Meeting of the Pesticide Residue Analysis Society of Japan, pp. 1-10 (1994) (in Japanese). 

Tolerances for these compounds are generally O-.Ol ppm except for penicillin G in cattle (.05 ppm) and cephapirin in edible tissue (0.1 ppm) and milk (.02 ppm) (70). Many chromatographic methods have been described for determination of these compounds in clinical applications, but these methods are not sufficiently sensitive for residue analysis. The summary of methods in Table II includes one GLC (71), five TLC (72-76), and five HPLC methods (77-81). Four of the TLC methods use detection by bioautography. Three HPLC methods have been described for milk (77-79) and two for tissue (80,81). The HPLC methods described by Moats C7 ,80) and by Munns et al (77) are satisfactory for any penicillin with a neutral side-chain and this may be true with the procedure of Terada, et al. (81). The procedure of Terada and Sakabe (79) is also satisfactory for the aminopenicillin, amplclllin. The method of Munns et al (77) can also be used to detect the corresponding penicilloic acid metabolites. 

Regulatory requirements in the field of drug residues analysis are limited, in most cases, to the identification of only the major metabolites. Quantitative selection of major over minor metabolites is certainly devoid of rational biological ground, since several studies have shown that the toxic metabolites are usually transitory and often present in small quantities. However, isolation and identification of all these metabolites are difficult and proper assessment of the toxicity of drug residues is still a real challenge. 

Despite the resolving power of TLC-MS-MS, few applications in drug residue analysis have been reported. One application concerns the HPTLC-MS-MS analysis of a number of nonsteroidal anti-inflammatory drugs, including salicylic acid and its glycine conjugate salicylhippuric acid, diclofenac, indomethacin, naproxen, phenacetin, and ibuprofen (67). Another application describes the detection and identification of some of these compounds or their metabolites in urine by TLC-MS-MS (67). 

Pharmacokinetic and residue analysis of ENRO and its metabolite CIPRO in chicken samples were performed using HPLC after extraction with dichloromethane and sodium phosphate buffer (pH 7.4) (202). After shaking and centrifuging, the organic phases were dried and the residues dissolved in the mobile phase. Extraction recovery was 87% for ENRO and its metabolite. 

Y Ishi. Residue analysis of thiophanate methyl and its degradation products and metabolites by high performance liquid chromatography. J Pestic Sci 15 212-216, 1990. 

The insecticide acephate, applied as a water spray has a very short life in terns of biological activity and for this, as well as economic reasons, it is not used on large scale operations in Canada (12). Residue analysis of acephate foliar deposits (13) showed that more than half of the insecticide was lost within one day of spraying, and that by 32 days post spray, the amount of insecticide had decreased to less than 0.01 ppm (the detection limit for 20 gm of substrate). The metabolite of acephate known as Ortho 9006 (0,S-dimethyl phosphoramidothioate) was also assessed in this study but was found to be present only in very small amounts. At 2 hours post spray the average acephate concentration on spruce foliage was 55.15 ppm, and the average concentration of the Ortho 9006 was 0.12 ppm. There was no increase with time in the amount of the metabolite. The rapid... 

The potential utility of mass spectrometry in evaluating the metabolic pathways of pesticidal chemicals by providing the structural identity of metabolites was stated as early as 1962 in a review by Gunther (3). More recently, several reviews 4,5,6) have considered the role of mass spectrometry in chemical structure evaluations with special reference to pesticide residue analysis. 

Pleasance et al. [23] described residue analysis of erythromycin A and its metabolites in salmon tissue using LC-ESl-MS. Detection limits of erythromycin A in salmon tissue were below 10 pg/kg in SIM and below 50 pg/kg in SRM, while confirmatory full-scan LC-MS or LC-MS-MS was achieved at the 0.5- and 1-mg/kg level, respectively. Next to erythromycin A, a variety of metabolites were detected, e.g., anhydroerythromycin and N-desmethylerythromycin. 

The first section of this book describes the application of LC/MS to the analysis of agricultural chemicals and their metabolites. Using LC/MS for residue analysis in agricultural chemistry has become routine in many laboratories. Many pesticides, such as the chlorophenoxy acid and sulfonyl urea herbicides or organophosphorus and methyl carbamate insecticides, are too polar or thermally labile for analysis via GC. The use of LC/MS for the identification of polar pesticide metabolites and conjugates, an area traditionally dominated by radiolabeled compounds, stands out as a particularly dramatic demonstration of the power of this technique. 

Thermospray LC/MS has been extensively used for the study of sulfonylurea herbicides (1-2). These compounds are thermally labile and can not be successfully analyzed by conventional GC/MS. Early applications of thermospray LC/MS included metabolite identification and product chemistry studies. We have recently evaluated the use of thermospray LC/MS for multi-sulfonylurea residue analysis in crops and have found the technique to meet the criteria for multiresidue methods. LC/MS offers both chromatographic separation and universal mass selectivity. Our study included optimization of the thermospray ionization and LC conditions to eliminate interferences and maximize sensitivity for trace level analysis. The target detection levels were SO ppb in crops. Selectivity of the LC/MS technique simplified sample extraction and minimized sample clean up, which saved time and optimized recovery. Average recovery for these compounds in crop was above 85%. 

In addition to MRM, the other scan modes available on a QqQ have occasionally been used for residue analysis as well. A precursor ion scan can be used to identify precursor ions from a product ion, and therefore to identify analytes and metabolites or impurities, which generate the same product ion, in complex matrices. For example, erythromycin B was identified in yogurt using this function. In this application, Q3 was held constant to measure a fragment ion at m/z 158, which is a typical product ion of compounds or impurities related to erythromycin A with a desosamine residue. Q1 was then scanned over an appropriate range, from which a precursor ion at m/z 718 was detected. The latter was identified as erythromycin B, which was an impurity in the erythromycin fermentation product. Constant neutral loss scan, which has rare applications for antibiotic analysis, records spectra that show all the precursor ions that have fragmented by the loss of a specific neutral mass. In this instance, both Q1 and Q3 scan together with a constant mass offset between the two quadrupoles. Both precursor ion and constant neutral loss scans can be performed only with ion beam tandem in-space mass spectrometers. 

Ceftiofur is known to rapidly metabolize after intramuscular administration, resulting in metabolite residues found in milk and tissue. Reported metabolites include desfuroylceftlofur (DEC), desfuroylceftlofur cysteine disulfide (DCCD), protein-bound DEC, and ceftiofur thiolactone. " Because these metabolites are all micro-biologically active, the EU MRL was defined as the sum of all residues retaining the f)-lactam structure, expressed as DEC, " whereas the Codex Alimentarius Commission (CAC) defined DEC as the only marker residue, simplifying the analysis. This approach is also used in the United States of America. 

The macrolide antibiotic tulathromycin also has residue limits defined in terms of a marker residue obtained by the hydrolysis of a range of metabolites. Again, analysis of incurred tissues indicates that the marker residue is not a major component of the residues present. ... 

Hutchinson MJ, Young PY, Hewitt SA, et al., Confirmation of the carbadox metabolite, quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis. Analyst 2002 127 342-346. 

Sczesny S, Nau H, Hamscher G, Residue analysis of tetracycbnes and their metabolites in eggs and in the environment by HPLC coupled with microbiological assay and tandem mass spectrometry, J. Agric. Food Chem. 2003 51 697-703. 

Development of immunoassays for residue analysis of small molecules has been well documented in the literature (8-9. 141 and by the articles in this volume. Recently, Dreher and Podratzki (101 reported the development of an immunoassay for endosulfan and its metabolites using a rabbit polyclonal antiserum. This assay however, did not readily detect other related cyclodiene insecticides. We report here the development of a monoclonal antibody that detectes all nine of the cyclodiene insecticides tested plus toxaphene, and the incorporation of this antibody to an immunoassay for detecting these compounds in meat, fish, and dairy products at, or below, the tolerance levels. 

M. Molina, D. Perez-Bendito and M. SUva, Multi-residue analysis of A-methylcarbamate pesticides and their hydrolytic metabolites in enviroimiental waters by use of solid-phase extraction and micellar electrokinetic chromatography, Electrophoresis, 20, 3439-3449, 1999. 

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