Metabolite sample treatment

This chapter focuses on LC—MS/MS applied to pesticide residue analysis, as this technique is the most attractive and efficient nowadays for developing MRMs [11], including both parent pesticides and metabolites. Sample treatment (mainly extraction and cleanup) are briefly commented on, with emphasis on those commonly applied in MRMs. A brief mention is made of problematic pesticides that do not fit in MRMs and consequently need to be determined with individual-specific LC—MS/MS methods. The use of HR MS in combination with LC also is briefly treated, either for the investigation of parent pesticides or for metabolite research, as this is a field of major interest at present. 

HPLC coupled to MS was used for the determination of dimethyl xanthine metabolites in plasma.82 There have also been a number of methods published on the use of HPLC with a PDA detector. In 1996, Mei published a method for the determination of adenosine, inosine, hypoxanthine, xanthine, and uric acid in microdialysis samples using microbore column HPLC with a PDA detector.63 In this method, samples were directly injected onto the HPLC without the need for any additional sample treatment. 

When male Wistar rats were exposed to -hexane at concentrations up to 3,074 ppm for 8 hours, analysis of urine showed that 2-hexanol was the major metabolite, accounting for about 60-70% of the total metabolites collected over the 48-hour collecting period (Fedtke and Bolt 1987). This is in contrast to humans, in which the major urinary metabolite is 2,5-hexanedione (Perbellini et al. 1981). The amounts of metabolites excreted were linearly dependent on the exposure concentration, up to an exposure of about 300 ppm. 2-Hexanol and 2-hexanone were detected in the first sample (obtained during the 8-hour exposure) excretion of 2,5-hexanedione was delayed and was not detected until 8-16 hours after exposure began. The amount of 2,5-hexanedione detected depended on sample treatment total excreted amounts over 48 hours were approximately 350 g/kg 2,5-hexanedione without acid treatment and 3,000 g/kg with total acid hydrolysis, indicating conversion of 4,5-dihydroxy-2-hexanone with acid treatment. 

Detectable concentrations of various antibacterials in milk attained by different microbiological tests are presented in Table 27.2. Milk constitutes a matrix that, apart from heating to destroy natural inhibitory substances, does not generally necessitate further sample treatment. Some antibiotics, however, exhibit some instability to heat treatment (54-56) and, therefore, if further confirmation is required reference frozen samples should always be available. When raw milk is directly analyzed, critical evaluation is generally required because natural inhibitors such as somatic cells, immunoglobulins, and metabolites may cause zones of inhibition (56, 57). Furthermore, several factors including marked pH-devia-tions, use of paper disks that contain inhibitory substances, and work with forceps that are too hot or have not been cleaned properly can readily lead to falsepositive readings (56, 58). 

Prior to metabolomic analysis, sample treatment is typically needed, as CSF contains approximately 0.3 mg/mL protein (114) that may hinder metabolite analysis. Consequently, CSF sample treatment is essentially directed to protein removal by means of organic solvent addition (84,88) or by ultrafiltration (85,89,90). The final metabolic extract composition will depend in a great extent on the sample treatment (115), and it will be selected mostly regarding the metabolomic approach and the analytical technique that will be afterward applied. 

Wils et al. (25,26) previously reported an entirely different approach to TDG analysis. TDG in urine was converted back to sulfur mustard by treatment with concentrated HC1. The sample treatment is less straightforward than the methods described above, but analysis as sulfur mustard is facile. Urine, plus 2H8-TDG as internal standard, was cleaned up by elution through two C18 cartridges. Concentrated HC1 was added and the sample stirred and heated at 120 °C. Nitrogen was blown over the solution and sulfur mustard isolated from the headspace by adsorption onto Tenax-TA. The method was used to detect TDG in urine from casualties of CW attacks (see below). A disadvantage of this method is that it may convert metabolites other than TDG to sulfur mustard. This is supported by the detection of relatively high levels of analytes in urine from control subjects. Vycudilik (27) used a similar procedure, but recovered the mustard by steam distillation and extraction. 

Another MEKC separation (75 mM SDS, phosphate-borate buffer, pH 9.1) was reported for die determination of ll-nor-A-9-tetrahydrocannabinol-9-carboxylic acid, the major metabolite of A-9-tetrahydrocannabinol present in urine (Wemly and Thormann, 1992a). Sample treatment included basic hydrolysis of urine (5 mL), solid phase extraction, and concentration. The resulting sensitivity was 10 to 30 ng/mL (i.e., comparable to the cutoffs of immunoassays). Again, detection was by on-line recording of peak spectra, by means erf fast-scanning UV detector. 

The gas-phase analytical techniques have been used for the analysis of urinary steroids for a long time. The determinations of urinary estrogens, progesterone metabolites, 17-ketosteroids and, to a lesser degree, corticosteroid metabolites, with packed-column GC are extensively documented in the earlier monographs on the subject [274,290]. Various sample treatments, approaches to conjugate hydrolysis, and volatile derivatives have been described. Among those steroids, aldosterone stands out as a uniquely difficult substance to derivatize and determine. 

Retrospective analysis to search for compounds not included in the initial analysis is an attractive feature of TOP MS-based methods. Without additional injection of the samples, it is feasible to investigate the presence of other contaminants or metabolites. This has allowed the detection and identification of pharmaceutical metabolites in wastewater [51]. Obviously, this possibility is also available to other compounds, pesticides included, provided that they are compatible with the sample treatment and LC—MS analysis applied. A detailed comparison of the capabilities of LC—MS using QqQ, TOP, and QTOP for quantification, confirmation, and screening in the field of PRA is given in [45]. 

No degradation of unstable metabolites due to sample treatment All metabolites are detected simultaneously in a single analysis... 

Mepanipyrim in crop samples is recovered by acetone solvent extraction. The acetone is evaporated under reduced pressure and the residual aqueous extract is hydrolyzed with enzyme (jS-glucosidases) to release hydroxylated metabolite(s). After enzyme treatment, mepanipyrim and the propanol form metabolite are extracted with dichloromethane, purified by silica gel column chromatography and quantified by gas chromatography/nitrogen-phosphoms detection (GC/NPD). 

Fungal degradation of BP1 resulted in the formation of 4HB and 4DHB. The levels of these metabolites found after 3 and 6 days of treatment were similar to those achieved in BP3 degradation experiments. Likewise in BP3 degradation tests, THB and DHMB were not detected in any of the collected samples. The levels found of these metabolites were quite low therefore, these products cannot be considered as major degradation products. 

The protocol consisted of preconditioning with methanol (1 mL) followed by water (1 mL). Urine samples (3 mL) were deconjugated by treatment with /3-glucuronidase and arylsulfatase (10 jt/L and 200 fig/fiL) in 0.1M sodium acetate (pH 5.5) and then loaded onto conditioned cartridges. After washing with water (1 mL) and methanoksodium acetate (3 mL, 4 6, pH 5.5), the PAH metabolites were eluted with dichloromethane (3 mL). The eluate was spiked with dodecane (used... 

Metaphase Analysis. Metaphase analysis can be performed in any tissue with actively dividing cells, but bone marrow is the tissue most often examined. Cells are treated with a test compound and are arrested in metaphase by the administration of colcemid or colchicine at various sampling times after treatment. Preparations are examined for structural chromosome damage. Because the bone marrow has a good blood supply, the cells should be exposed to the test compound or its metabolites in the peripheral blood supply. Additionally, these cells are sensitive to S-dependent and S-independent mutagens (Topham et al., 1983). 

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