Metabolite HPLC analysis

Recent studies in our laboratories have shown that newly hatched herring (Clupea harengus pal Iasi) larvae exposed to purififed 3H-naphthalene in seawater at concentrations of 10 ppb for 9 hr accumulated a variety of conjugated and non-conjugated metabolites. HPLC analysis indicates the presence of the parent compound as well as three additional compounds whose retention times are consistent with a sulfate, a dihydrodiol, and 1-naphthol (Fig. 3). 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

In maize, many phenotypic mutants have been associated with cloned genes by a combination of HPLC analysis of specific intermediate metabolite accumulation, RT-PCR and immunolocalization of candidate genes, and recombinant inbred mapping of candidate cDNAs. Psyl was cloned by transposon tagging and later shown to be functional in the color complementation system and to be the specific... 

The HPLC analysis of milkweed, the food-plant source for Monarch butterflies, demonstrates that it contains a complex mixture of carotenoids including lutein, several other xanthophylls, xanthophyll epoxides, and (3-carotene, Figure 25.3b. There is a component in the leaf extract that is observed to elute near 8min, which has a typical carotenoid spectrum but is not identical to that of the lutein metabolite observed at near the same retention time in the extracts from larval tissue. 

Fig. 3.93. The HPLC analysis on metabolites resulting from decolourization of reactive red 22 by Pseudomonas luteola (a) at the beginning of static incubation (IA = 3 639 667, /B = 130 140, Ic 116 243), (b) after static incubation for 4.7 h (/A = 2 231 542, /B = 230 559, Ic = 120 563), (c) after static incubation for 23.4 h (/A = 1 892 854, /B = 428 414, Ic = 205 169), (d) 3-amino t-methoxyphenyl /1-hydroxyl sulphone sulphonic acid ester (AMHSSAE), 90 per cent pure, 52 mg/1, and (e) products resulting from decolourization of Reactive red 22 by chemical reduction with SnCl2, (/A, /B, and 7C represent intensities of peaks A, B, and C, respectively). Reprinted with permission from J.-S. Chang et al. [154].

Fig. 3.101. HPLC analysis on metabolites resulting from decolourization of Remazol brilliant orange 3R under anaerobic-aerobic conditions (1) at the beginning of the anaerobic incubation (2) after anaerobic incubation for 24 h and (3) after aerobic incubation for 12 h. Reprinted with permission from N. Supaka et al. [158].

Queroz RH, Lanchote VL, Bonato PS, de Carvaldo D. 1995. Simultaneous HPLC analysis of tricyclic antidepressants and metabolites in plasma samples. Pharm Acta Helv 70 (2) 181-186. 

Figure 3. HPLC Analysis of Ginsenosides. Ginsenosides were isolated from spent broth in which either no organism (upper trace), Trichoderma hamatum (middle trace) or Pythium irregulare (lower trace) had been cultured for five days at 25 °C in the dark. Ginsenosides were chromatographed on a Microsorb-MV C-18 column (150 x 4.6 mm, 5 mm) using an acetonitrile H20 gradient (Nicol et al., 2002), and detected at 203 nm. The in the lower trace indicates the unknown metabolite found in the spent broth of Py. irregulare.

The separation of cimetidine and its metabolites is usually carried out by extraction of the biological medium with 1-octanol fran an aqueous alkaline pH solution followed by mixing, addition of an internal standard and centrifugation. The extraction with octanol is repeated and the combined extracts are re-extracted with dilute hydrochloric acid. The aqueous acid solution is then separated, ethanol is added and mixed. This is then followed by saturating the mixture with a large amount of potassium or sodium carbonate to "salt out" the ethanol layer which contains the cimetidine and its metabolite, the sulfoxide. Several different internal standards have been used Metiamide, 1-methyl-3-[2-[[(5-methyl-imidazole-4-yl) -methyl] thio]ethyl]-2-thiourea,19 31 39 (N-cyano-N1-methy1-N"-(3-(4-imidazolyl)-propyl)guanidine32, and 13-hydroxy-theophylline. 0 After extraction the samples are either evaporated to dryness and reconstituted with a known amount of ethanol, injected directly or dissolved in the mobile phase for the HPLC analysis. 

Relative standard deviation of repeat HPLC analysis of a drug metabolite standard was between 2 and 5%. Preliminary measurements of several serum samples via solid-phase extraction cleanup followed by HPLC analyses showed that the analyte concentration was between 5 and 15 mg/L and the standard deviation was 2.5 mg/L. The extraction step clearly increased the random error of the overall process. Calculate the number of samples required so that the sample mean would be within +1.2 mg/L of the population mean at the 95% confidence level. 

HPLC Analysis of a 9-Tetrahydrocannabinol and Metabolites in Biological Fluids... 

Direct HPLC analysis of urine extracts appears feasible for A -THC. 215nm is the optimum wavelength for detection of THC-class compounds. Dual wavelength at 215 and 280nm serves as a valuable check on cannabinoid retention assignment and as a screen for unknown THC or CBN-class metabolites. The latter feature was demonstrated in the observance of CBN-class peaks in both hexane and E-I extracts. This observation suggests a CBN-metabolic route of A -THC. Evidence of a CBN-metabolic route for A -THC has been reported by McCallum (8) and Green (6) for humans and by Ben Zvi et al (9) for rhesus monkeys. 

Minano FJ, Peinado JM, Myers RD (1989) Neurotensin perfused in hypothalamus of sated or fasted rat HPLC analysis of release of DA, NE and 5-HT and their metabolites. Peptides 9 1381-1387. 

Coupling of HPLC analysis to MS (either on-line or off-line) provided the means for structure elucidation and identification of taxane metabohtes. Utilizing tandem MS (MS-MS), both aspects were significantly enhanced. Hy-droxylation, epimerization, and hydrolysis are considered to be the main routes of metabolism of taxane pharmaceuticals. The principal metabolites of both paclitaxel and docetaxel result from hydroxylation. Hydroxylation of paclitaxel was initially observed in rat and mouse, but the finding was later extended to human subjects. Nine metabolites of paclitaxel (mono or dihydro derivatives) have been identified in rat by HPLC, NMR, and LC-MS. With regard to docetaxel, 18 derivatives have been identified in the rat bile. Hydrolytic derivatives result from cleavage of the side C-13 chain, and include baccatin III and 10-DAB III. Epimerization of taxanes is mainly focused on the C-7 and C-10 atoms, and occurs in solution. Epimerization has also been detected in cell culture in both the medium and inside the cell. It should be stressed that epimers are not naturally occurring taxanes thus, they are not found in plants. 

Rosing, H. Lustig, V. Koopman, F.P. ten Bokkel Huinink, W.W. Beijnen, J.H. Bio-analysis of docetaxel and hydro-xylated metabolites in human plasma by high-performance liquid chromatography and automated solid-phase extraction. J. Chromatogr., B, Biomed. Sci. Appl. 1997, 696, 89 -98. Theodoridis, G. Laskaris, G. Verpoorte, R. HPLC analysis of taxoids in plant and plant cell tissue culture. Am. Biotechnol. Lab. 1999, 17, 40-44. 

Georga, K.A. Samanidou, V.F. Papadoyannis, I.N. Improved micro-method for the HPLC analysis of caffeine and its demethylated metabolites in human biological fluids after SPE. J. Liq. Chromatogr. Relat. Technol. 2000, 23 (10), 2975-2990. 

The parent compounds and the metabolites have very low aqueous, solubility. They have proven difficult to extract from some matrices the current method for ivermectin involves some 41 concentration and clean-up steps preceding HPLC (8). A Merck method for recovering abamectin residues from strawberries and formation of fluorescent derivatives for HPLC analysis has 18 separate steps (9). A recently published two-step solid-phase recovery procedure for ivermectin from serum indicates that it is possible to combine an abbreviated concentration and cleanup method with a sensitive and specific detection system in this case, liquid chromatography (10). An immunoassay for avermectins that could be interfaced with simplified residue recovery protocols is a promising solution to the intensifying demands on regulatory agencies to monitor these compounds. 

Figure 6.6 Chromatogram relative to the HPLC analysis of captan and its metabolites THPI, THPAM, and THPA. (Reproduced from /. Agric. Food Chem., 2003, 51, 6761-6766, Angioni et al., with permission of the American Chemical Society)...

HPLC analysis of cotton seed homogenate following the extrael scheme shown in Fig, IU.I0 showed the presence of PBO (0.086 ppm) and two polar metabolites (ca, 0.04 ppm). 

Following extraction (Fig. 10.11), analysis of an initial McOH exlraet of the cotton lint composite sample by HPLC (Fig, 10.12) revealed the presence of a major polar metabolite peak, accounting for 0,191 ppm of the radioactivity in the extract, and one nonpolar peak which appeared to be PBO, aeeounting for 0.047 ppm of the radioactivity in the extract. Metabolites in other extracts were found to be too polar for extraction and HPLC analysis. 

For the current investigation, HPLC with fluorescent detection was chosen to analyse for PBO and major metabolites. The analysis of the metabolites used... 

Robert A, Ducos P, Francin JM. 1995. Determination of urinary 4,4 -methylenedianiline and its acetylated metabolites by solid-phase extraction and HPLC analysis with UV and electrochemical detection. Int Arch Occup Environ Health 68 44-51. 

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