Urinary metabolites analytical methods

Determination in Biological Fluids and Tissues All the advances in pharmacokinetics and drug metabolism described in Sections 7 and 8 would not have been possible without the availability of the proper analytical methods. The following is a tabulation of publications in this field, most of which have already been discussed in Section 5. It should be mentioned that a few publications talk about aspirin blood levels, but really mean salicylate levels. The following tabulation covers only those papers where aspirin was differentiated from other salicylates by chromatography or other means. It seems that the "workhorse" for serum salicylate levels is still the colorimetric (ferric-nitrate) method of Brodie, Udenfriend and Coburn153 published in 1944, or modifications thereof. Simplified versions (cf. 206) may lead to erroneous results under certain conditions.207 The method is also applicable for urinary metabolites after proper hydrolysis (cf. 208). For other methods restricted to salicylic acid, see Section 5.61. 

Analytical methods have been reported for unchanged agent and six of the urinary excretion products described above. These are TDG, TDGO, the bis A-acetylcysteine conjugate (1), two 3-lyase metabolites (2) and (3), and the guanine adduct (6). These methods have been applied to animal and/or human exposures to sulfur mustard. 

Due to the discovery of two further urinary metabolites of ramipril (the respective diketopiperazine derivatives of ramipril and ramiprilat), the above-described method was slightly modified (Schmidt et al. 1985). Instead of the rather time consuming second extraction step by means of a disposable Si column, the sample is cleaned by a liquid/liquid extraction step. After methylation of the compounds with diazomethane, the eluate is evaporated to dryness at 40 °C under N2 gas. Subsequently the residue is dissolved in n-pentane/diethyl ether (3 2. v/v) and washed with 5% hydrogen carbonate solution. After separation of the upper organic layer, this is evaporated to dryness at 40 °C under N2 gas and then treated with 1 mL n-hexane/TFAA (5%) as described before (Hajdu et al. 1984). This method allows the selective determination of ramipril and its three metabolites in human urine the limit of quantification amounted to 0.020 xg/mL for each of all four analytes. Using this assay, thousands of urine samples originating from phase I—III clinical studies were analysed. 

The improvement in analytical methods has shown the importance of quantitative determination of more than one urinary metabolite which, alone, does not always reveal a metabolic abnormality. 

HPLC/ICPMS is a powerful analytical technique and is now the most commonly used method for determining selenium urinary metabolites [215]. Over the last 10 years, most of the reports of selenium species in urine have used HPLC/ICPMS, sometimes together with molecular MS techniques. In this period, a total of 16 selenium species (see below) have been reported in urine, most of them novel human metabolites and some of them completely new compounds. 

Wong, M.C. et al., An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 871(2), 341, 2008. 

B. A. G., Development, validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4-and 2,6-toluene diisocyanate, 1, 5-naphthalene diisocyanate and 4,4 -methylenediphenyl diisocyanate. Biomarkers, 8, 204-217, 2003. 

Styrene may be analyzed by GC, nsing a flame ionization detector. Air analysis may be performed by charcoal adsorption, followed by desorption of the analyte with carbon disnl-fide and injection of the elnant into GC-FID (NIOSH Method 1501 see Section 26.2). Styrene intake in the body may be estimated by analyzing mandelic acid in the nrine by liquid chromatography, polarography, or GC. However, the presence of other aromatics may interfere, as these componnds also generate the same urinary metabolite. Styrene in exhaled air may be analyzed by absorption over ethanol or charcoal followed by GC, UV, or IR analysis. 

Phase III is done to characterize the urinary metabolites in order to devise a urine monitoring procedure. If an oral Dietabolism study has been done, the main sietabolites are usually characterized and possibly identified. If the urinary metabolite patterns are similar in the oral and dermal study, a urinary method of metabolite analysis can be arrived at readily, if not already defined. Otherwise, converting the often observed multiple metabolites to one or two common entities is frequently desirable. The analytical siethod is of potential use in urine monitoring of workers. 

In an attempt to circumvent the avaUabihty of analytical standards, several CYP450 studies were carried out using the substrate depletion method. This approach does not provide information on the products formed downstream, and may be of limited use in developing human enviromnental exposure PBPK/PD models that require extensive urinary metabolite data. Hydrolytic standards (i.e., alcohols and acids) were available to investigators who studied the carboxylesterase-catalyzed hydrolysis of several pyrethroid insecticides. The data generated in these studies are suitable for use in developing human exposure PBPK/PD models. 

Schneider H, Ma L, Glatt H. 2003. Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 789 227. 

Dietary constituents or drugs can either cause direct analytical interference in assays or influence the physiological processes that determine plasma and urinary levels of catecholamines and catecholamine metabolites. In the former circumstances, the interference can be highly variable depending on the particular measurement method. In the latter circumstances, interference is usually of a more general nature and independent of the measurement method (Table 29-6). 

The value of electrochemical detection following high performance liquid chromatography is compared with that of the more widely used optical methods of detection. Its advantages are illustrated by its application to three areas of clinical chemistry that had previously posed analytical problems. Its sensitivity allowed its use for the measurement of plasma catecholamines. Its selectivity was employed to produce rapid methods for the determination of urinary levels of catecholamine and tryptophan metabolites. Finally, its value for the estimation of urinary oxalic acid is shown. Future developments such as increasing the range of detectable compounds by derivatization are briefly discussed. 

---