Urine extracts, direct HPLC analysis

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. 

Preliminary extraction of 5-HIAA may be used as an initial purification step before HPLC analysis. Organic solvents, anion-exchange resins, and other solid phase extraction procedures have aU been used. For many systems, direct injection of urine onto the analytical column is a common practice,and samples are often merely diluted with a buffer to protect the HPLC system from contamination. Methods that analyze 5-HIAA without prior sample cleanup rely on the selectivity of the HPLC separation combined with fluorescence or electrochemical detection to provide the requisite specificity. 

Levels of radioactivity were highest in the liver and kidneys of male and female rats at two hours post final dose and averaged 3.A63 ug equivalents/g and 4.073 ug equivalents/g in male and female rat liver, respectively, and 2.383 and 2.132 yg equivalents/g in male and female rat kidney. Urine samples were analyzed by direct injection onto the HPLC column, whereas feces and liver samples were homogenized and extracted with methanol and subjected to solid phase extraction (if necessary) prior to HPLC analysis of the organic extract. The major radiolabeled component in male and female rat urine co-chromatographed with flunixin. Lesser amounts of the two hydroxylated metabolites, 5-hydroxyflunixin and 2 -methyIhydroxyflunixin, were also detected. Two major radiolabeled residues, identified by retention characteristics as flunixin and 5-hydroxyflunixin, were observed in rat feces. The major radiolabeled component in male and female rat liver co-chromatographed with authentic flunixin standard. The monohydroxymetabolite, 5-hydroxyflunixin was also detected in male rat liver. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

Analysis of urinary trans, trans-muconic acid (t,t-MA) seems to be a better indicator than phenol for assessing exposure to low levels of benzene (Ducos et al. 1990). However, muconic acid is a minor metabolic route and background levels of muconic acid in urine are much lower than levels of phenolic metabolites and are frequently below the limit of detection of the method used to determine them (Inoue et al. 1989). The detection of low levels of t,t-muconic acid in urine was difficult by earlier methods because of low recovery of t,t-muconic acid (37% with ether) by the commonly used solvent extraction method (Gad-El Karim et al. 1985). An improved method for the determination of urinary t,t-muconic acid utilizes solid phase extraction with SAX sorbent in combination with the HPLC/UV for quantitation. The detection limit is 0.06-0.1 mg/L, and recovery is very good (90%) (Boogaard and van Sittert 1995 Ducos et al. 1990). The relative standard deviation of the method was 5% in the concentration range 1-20 ng/L. t,t-muconic acid has been determined directly by HPLC/UV with similar sensitivity (detection limit =... 

The bioanalyst can be required to analyse most biofluids although the most common are urine and the aqueous phase of blood, i.e. plasma or serum. Other samples may be cell and tissue extracts, synovial fluid, cerebrospinal fluid (CSF) and saliva. In the case of urine and CSF with their very low protein content it might be possible to directly inject the sample into an HPLC column. With most silica-based packing materials, direct injection of blood proteins will rapidly lead to column deterioration. HPLC columns are expensive and their efficiency is easily lost so correct preparation of samples will not only improve column life but also improve the results. At its simplest it is only necessary to remove particulate matter from samples to prevent clogging of the column and frits. Modern HPLC packings are very susceptible to contamination by proteins, fats and other macromolecules from biological samples and it is necessary to remove these (except of course for protein analysis). 

In the beginning, for the most part the metabolites of drugs could only be identified in urine when a spectrum of the isolated (or independently synthesized) substance was known. For example, oxpentifylline, extensively used in the treatment of vascular diseases, and its acidic metabolite l-(4 -carboxybutyl)-3,7-dimethylxanthine can be directly identified and quantified in a freeze-dried urine sample using a 250-MHz spectrometer [6], The medication of lymphatic filariasis with diethylcarbamazepine has to be monitored for several reasons [7]. Since the drug lacks an absorbing chromophore (HPLC/UV-detection) and GC needs a special detector and troublesome extraction method, H NMR spectroscopy appears to be suitable for the urine analysis the urine samples were mixed with 10% D2O and directly measured afterwards. 

Figure 5 The 600 MHz H NMR spectra of (A) a sample of human urine collected after the oral ingestion of 200 mg flurbiprofen, (B) the 30.5 min retention time species corresponding to the /3-D-glucuronic acid conjugate of 4 -hydroxyflurbiprofen. Reprinted from (A) Spraul ef a/(1993). Coupling of HPLC with and H NMR spectroscopy to investigate the human urinary extraction of flurbiprofen metabolites. Journal of Pharmaceutical and Biomedical Analysis, 11 (10), 1009-1015 (B) Lindon et al (1996) Direct coupling of chromatographic separations to NMR spectroscopy. Progress In NMR Spectroscopy, 29, 1-49, with kind permission from Elsevier Science-NL, Sara Burgerhartstraat 25,1055 KV Amsterdam, The Netherlands.

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