Urine paracetamol metabolites

Figure 13.2.6 On-flow contour plots (600 MHz) of the (a) CE-NMR, and (b) pressurized CEC-NMR separations of the paracetamol metabolites obtained from a human urine extract A, paracetamol glucoronide B, paracetamol sulfate C, hippuric acid...

Paracetamol, metabolites/urine, plasma HPLCUV Column EPS Cig (250 x 4.6 mm, 5 pm) Mobile phase 0.1 M KH2PO4 isopropanoPTHF (100 1.5 0.1 pH 3.7), adjusted with phosphoric acid (isocratic elution) Detection X = 254 nm Plasma protein precipitation (30 % perchloric acid) Urine 20 times dilution, centrifugation LOQ 0.06 pg/mL (paracetamol) 0.13 pg/mL (metabolites) [40]... 

Paracetamol, metabolites/urine UPLC MS Column ChromSpeed (50 x 4.6 mm, monolithic) Mobile phase Solvent A 0.1 % HCOOH in water Solvent B 0.1 % HCOOH in ACN (gradient elution) Detection MS, ionization ESI Five times dilution [42]... 

The further coupling of HPLC-NMR with mass spectrometry has also been used to analyse drug metabolites in urine for the human excretion of paracetamol metabolites. " This approach has also been used to identify metabolites of substituted anilines in rat urine. ... 

Ismail, S. Kokwaro, G.O. Back, D.J. Edwards, G. Effect of malaria infection on the pharmacokinetics of paracetamol in rat. Xenobiotica, 1994, 24, 527-533 [pharmacokinetics rat plasma urine extracted metabolites 3-acetamidophenol is IS]... 

Siegers, C.R Moller-Hartmann, W. Cholestyramine as an antidote against paracetamol-inducedhepato-and nephrotoxicity in the rat. Toxicol.Lett., 1989, 47, 179-184 [rat urine extracted metabolites] Lam, S. Malikin, G. An improved micro-scale protein precipitation procedure for HPLC assay of therapeutic drugs in serum. J.Liq.Chromatogr., 1989, 12, 1851-1872 [serum also amiodarone, aspirin, caffeine, chloramphenicol, flecainide, pentobarbital, procainamide, pyrimethamine, quinidine, theophylline, tocainide, trazodone fluorescence detection UV detection]... 

Spraul M, Hofmann M, Lindon JC, Farrant RD, Seddon MJ, Nicholson JK, WUson ID. Evaluation of liquid-chromatography coupled with high-field H NMR spectroscopy for drug metabolite detection and characterization the identification of paracetamol metabolites in urine and bile. NMR Biomed 1994 7 295-303. 

However, the small injection volume available to CE (a few nl) means that high sensitivity can only be achieved if concentrations of the analyte in the sample are high. Nevertheless, some results have been reported using CE-NMR and the method has been applied to the identification of paracetamol metabolites in human urine. Finally the application of capillary electrochromatography (CEC) directly coupled to NMR has been explored using the same paracetamol metabolite samples. 

The amount of futile acetylation observed for this compound in the rat, at ca. 7-10% for parent compound and metabolites, was less than that seen for phenacetin and similar to that found for paracetamol itself. The bulk of the radio-label was rapidly excreted in urine as practolol itself (albeit with 7-10% reacetylation) and the remainder as either the ring-hydroxylated metabolite or its glucuronide conjugate. As in previous examples, deuterated methanol was used in the mobile phase rather than methanol in order to be able to more easily observe the acetyl resonances of practolol and related compounds. 

Figure 4.1 Pseudo-2D plot of continuous-flow 1 H LC-NMR data obtained on human urine after dosing with paracetamol (1). The resonances from the glucuronide (2) and sulfate (3) conjugate metabolites of paracetamol and their ID slices are shown...

The same experimental setup was used in the separation of metabolites of paracetamol from a human urine extract [73] and Thomapyrin, containing acetaminophen, caffeine, and acetylsalicylic acid [74], Compared to isocratic CEC—NMR, gradient CEC—NMR offers increased sample loading capacity because of preconcentration at the front of the column and higher separation efficiency together with a reduction in analysis time [75],... 

The ability to identify metabolites and biotransformed products in biological fluids during separation is a considerable challenge. The potential application of CE-NMR and CEC-NMR for analysis of metabolites in biofluids has been demonstrated [51,52], For example, CE-NMR has successfully analyzed the major metabolites of paracetamol in human urine [51]. Two major metabolites, paracetamol glucuronide and paracetamol sulfate conjugates, as well as endogenous material (hippurate) have been characterized. Comparison of chemical shifts has confirmed the presence of these compounds. The estimated amount that can be detected in this study with a S/N of 3 is —10 ng. 

Paracetamol—Cresol-ammonia test. To 0.5 ml of urine add 0.5 ml of hydrochloric acid and heat for 10 minutes at 100° to 2 drops of the mixture add 10 ml of water, 1 ml of 1 % o-cresol in water, and 4 ml of 2M ammonium hydroxide. A blue colour appears if paracetamol is present. The test is very sensitive and can detect therapeutic concentrations. The presence of the parent drug and its conjugated metabolites can be detected for several days after overdose. If a positive result is obtained, a plasma-paracetamol determination should be carried out immediately (p. 23). 

K. Pusecker, J. Schewitz, P. Gfrorer, L.-H.Tseng, K. Albert, E. Bayer, I. D. Wilson, N. J. Bailey, G. B. Scarfe, J. K. Nicholson, and L. C. Lindon, On-flow identification of metabolites of paracetamol from hnman urine nsing directly coupled CZE-NMR and CEC-NMR spectroscopy, A aZ. Commun. 35 (1998), 213-215. 

Pusecker, K. Schewitz, J. Gfrorer, P. Tseng, L.-H. Albert, K. Bayer, E. Wilson, I.D. Bailey, N.J. Scarfe, G.B. Nicholson, J.K. Lindon, J.C. On-flow identification of metabolites of paracetamol from human urine using directly coupled CZE-NMR and CEC-NMR spectroscopy. Anal. Commun. 1998, 35, 213-215. 

Al-Obaidy, S.S. Po, A.L.W. McKiernan, P.J. Glasgow, J.F.T. Millership, J. Assay of paracetamol and its metabolites in urine, plasma and saliva of children with chronic liver disease. J.Pharm. Biomed.AnaL, 1996, 13, 1033-1039... 

Goicoechea, A.G. Lopez de Alda, M.J. Vila-Jato, J.L. A validated high-performance liquid chromatographic method for the determination of paracetamol and its mqjor metabolites in urine. J.Liq.Chromatogn, 1995,18, 3257—3268... 

Lau, G.S.N. Critchley, J.A.J.H. The estimation of paracetamol and its major metabolites in both plasma and urine by a single high-performance liquid chromatography assay. J.Pharm.Biomed.Anal., 1994, 12, 1563-1572 [extracted metabolites plasma urine blood LOD 1 ng]... 

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