Plasma and Urine

After purification of human serum bile acids on an Amberlyst A-26 anion e.xchanger, Sjovall and co-workers (31, 120, 121) hydrolyzed the concentrated bile acids in alkali and extracted the free acids with ethyl acetate. After methylation, the methyl cholanoates were purified on an aluminum oxide column. The bile acid fraction then obtained was subjected to gas-liquid chromatography on CNSi columns after conversion to trifluoroacetate 

Nair and Garcia (122) extract bile acids from serum (adjusted to pH 11) with ethanol. The dried extract is partitioned between an alkaline aqueous phase and diethyl ether and the remaining lower phase is subjected to clostridial cholyl glycine hydrolase at pH 5.6. After further acidification free bile acids are extracted with ether. The subsequent purification procedure is similar to that above (31), and a triple component (QF-1/SE-30/NGS) column (see Table XIll) is used for quantitative analysis. With the omission of the diethyl ether extraction of the alkaline aqueous phase the same procedure was used with rat bile. 

With a purified enzyme preparation from Clostridium perfringens, Roovers et al. (36) cleave conjugated bile acids directly in plasma. Proteins are then precipitated with Ba(OH)2-saturated ethanol (123) and the supernatant is taken to near dryness. The residue is dissolved in a toluene-isopropanol-methanol-30 % aqueous NaOH (10 20 20 6, v/v) mixture, water is added, and neutral lipids removed by light petroleum extraction. Bile acids are then obtained by acidification and diethyl ether extraction. The bile acids are then methylated and analyzed as above except that the bile acid methyl esters are acetylated before chromatography on 1 % XE-60 columns at 250°C. With this column the following retention times relative to that of the diacetoxy derivative of methyl deoxycholate were found for the following acetate methyl ester derivatives lithocholic, 0.60 23-nor-deoxycholic acid (internal standard), 0.77 chenodeoxycholic acid, 1.24 and cholic acid, 1.88. The deoxycholic acid derivative was eluted after 9.0 min and methyl 5 3-cholanoate after 0.32 min. 

Heparinized whole blood could also be subjected to digestion with the bile acid conjugate hydrolase. Except for a slightly modified extraction procedure, the same method as utilized for plasma was applied to the workup of whole blood. 

---

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. 

Quantitation of the oral bronehodilator 2,5-diethyl-7-(tetrahydro-l,4-thiazin-4-yl)-l,2,4-triazolo[l,5-c]pyrimidine (R-836) (195) in plasma and urine of humans and experimental animals utilized reversed-phase HPLC and UV deteetion (88MI1). 

Figure 11.4 Chromatograms of plasma samples on a silica-chiralcel OJ coupled column system (a) plasma spiked with oxprenolol (internal standard) (b) plasma spiked with 040 p-g/ml metyrapone and 0.39 p-g/ml metyrapol (racemate) (c) plasma sample obtained after oral administration of 750 mg metaiypone. Peaks are as follows 1, metyrapone 2, metyrapol enantiomers 3, oxprenolol. Reprinted from Journal of Chromatography, 665, J. A. Chiarotto and I. W. Wainer, Determination of metyrapone and the enantiomers of its chfral metabolite metyrapol in human plasma and urine using coupled achfral-chfral liquid cltro-matography, pp. 147-154, copyright 1995, with permission from Elsevier Science.

P. O. Edlund, E. Bowers and J. Henion, Detemination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid cliromatography with ulti aviolet detection and confimation by tandem mass spectrometiy , 7. Chromatogr. 487 341-356(1989). 

K. Yamashita, M. Motohaslii and T. Yashiki, High-performance liquid cliromatograpliic determination of phenylpropanolamine in human plasma and urine, using column switching combined with ion-pair chromatography , J. Chromatogr. 527 103-114 (1990). 

J. A. Chiar otto and I. W. Wainer, Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achhal-chhal liquid cliromatogruphy , J. Chromatogr. 665 147-154 (1995). 

R. A. Coe, E. S. DeCesare and J. W. Eee, Quantitation of efletirizine in human plasma and urine using automated solid-phase exti action and column-switching high-performance liquid clrromatography , 7. Chromatogr. B 730 239-247 (1999). 

H. Zehender, J. Denouel, M. Roy, E. Ee Saux and P. Schaub, Simultaneous determination of terbinafine (Eamisil) and five metabolites in human plasma and urine by liigh-peifoimance liquid cliromatogi aphy using on-line solid-phase exti action , 7. Chromatogr. B 664 347-355 (1995). 

J. R. Veraait, C. Gooijer, H. Lingeman, N. H. Velthorst and U. A. Th Brinkman, Determination of phenprocoumon in plasma and urine by at-line solid-phase exti action-capillaiy electi ophoresis , 7. Pharm. Biomed. Anal. 17 1161-1166 (1998). 

Figure 32, Chromatograms of plasma and urine samples with various abnormalities, A, Phenylalaninemia B, tyrosinemia C, elevated plasma methionine seen in homocystinuria D, glycinemia E, normal urine F, argininosuccinic aciduria G, homocystinuria H, hyperglycinuria I, hyperlysinuria.

Century, B. Vorkink, W. P. and Natelson, S. Thin layer chromatographic screening of aminoacids in plasma and urine of newborns. Clin. Chem. (1974), 20,... 

RICHELLE M, PRIDMORE-MERTEN S, BODENSTAB S, ENSLEN M, OFFORD E A (2002) Hydrolysis of isoflavone glycosides to aglycones by (3-glycosidase does not alter plasma and urine isoflavone pharmacokinetics. J Nutr 132, 2587-92. 

Heath, D.D. et ah, Curcumin in plasma and urine quantitation by high-performance liquid chromatography, J. Chromatogr. BAnalyt. Technol. Biomed. Life ScL,7S3, 287, 2003. 

The positions, numbers, and types of sugars on the anthocyanin molecule influence its bioaccessibility. Indeed, a recent human study reported that the acylation of anthocyaifins resulted in a sigififlcant decrease of anthocyanin recoveries in plasma and urine. In addition, anthocyanins form linkages with aromatic acids, aliphatic acids, and methyl ester derivatives, which can also affect their passage through the intestinal barrier. 

Kurilich, A.C. et al.. Plasma and urine responses are lower for acylated versus nonacylated anthocyanins from raw and cooked purple carrots, J. Agric. Food Chem., 53, 6537, 2005. 

Domino, E.F., and Wilson, A.E. Effects of urine acidification on plasma and urine phencyclidine levels in overdosage. Clin Pharmacol Ther 22(41 421-424, 1977. 

Dear, G.J., Fraser, I.J., Patel, D.K., Long, J., and Pleasance, S., Use of liquid chromatography-tandem mass spectrometry for the quantitative and qualitative analysis of an antipsychotic agent and its metabolites in human plasma and urine, /. Chromatogr. A, 794, 27, 1998. 

Hieda et al. determined theophylline, theobromine, and caffeine in human plasma and urine by gradient capillary HPLC with frit fast atom bombardment (FAB) mass spectrometry with 7-ethyl theophylline as the internal standard.64... 

Hieda, Y., Kashimura, S., Hara, K., and Kageura, M., Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by high performance liquid chromatography frit fast atom bombardment spectrometry, J. Chromatogr., 667,241,1995... 

Roy, A., Pickar, D., Dejong, J., Karoum, F. Linnoila, M. (1998). Norepinephrine and its metabolites in cerebrospinal fluid, plasma and urine. Relationship to hypothalamic-pituitary-adrenal axis function in depression. Arch. Gen. Psychiatry, 45, 849-57. 

NOx levels are increased in plasma and urine of septic animals. Many nonse-lective NO synthase inhibitors (e.g., L-NMMA) are used in several models with experimental induced sepsis (S40). In most studies it was shown that the cardiovascular abnormalities associated with sepsis were reversed, increasing blood pressure and systemic vascular resistance (F7, K9, M26, N5), together with a improvement in renal function (B42, H24). Also, selective inhibition of iNOS prolonged survival in septic rats (A7). 

Oishi H, Nomiyama H, Nomiyama K, et al. 1996. Fluorometric HPLC determination of delta-aminolevulinic acid (ALA) in the plasma and urine of lead workers biological indicators of lead exposure. J Anal Toxicol 20(2) 106-10. 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

Like plasma and urine, matrixes from plant or environmental sources contain a vast diversity of components. Thus, achiral-chiral LC-LC is also useful for analysis involving samples from these sources. Stalcup et al. (1991) studied the enantiomeric purity of scopolamine extracted from Datura sanguinea in both homogenized plant leaves and commercial extracts. A reverse-phase separation on a C j g column separated the scopolamine from other alkaloid and matrix components while the enantiomeric separation (also in the reverse-phase mode) was carried out on two coupled [3-cyclodextrin columns or a single acetylated (3-cyclodextrin column. The single... 

Diaz-Perez, M.J., Chen, J.C., Aubry, A.F., Wainer, I.W. (1994). The direct determination of the enantiomers of Ketorlac and parahydroxyketorlac in plasma and urine using enantiose-lective liquid chromatography on a human semm albumin-based chiral stationary phase. Chirality 6, 283-289. 

Batki S., Manfredi L., Jacob P., Jones R. Fluoxetine for cocaine dependence in methadone maintenance quantitative plasma and urine cocaine/benzoylecgonine concentrations. J. Clin. Psychopharmacol. 13 243, 1993. 

Under in vivo conditions, the free thiol has also been found in plasma and urine of patients with rheumatoid arthritis after treatment with the drug. It was also found... 

Rabenstein and Yamashita [52] determined penicillamine and its symmetrical and mixed disulfides by HPLC in biological fluids. Plasma and urine were deproteinized with trichloroacetic acid, and HPLC was performed on a column (25 cm x 4.6 mm) or Biophase ODS (5 pm) with a mobile phase comprising 0.1 M phosphate buffer (pH 3) and 0.34 mM Na octylsulfate at 1 mL/min. Detection was with a dual Hg-Au amalgam electrode versus a Ag-AgCl reference electrode. (z>)-penicillamine and homocysteine were determined at the downstream electrode at +0.15 V, and homocystine, penicillamine-homocysteine, and penicillamine disulfides were first reduced... 

Greaves et al. [74] used a selected ion-monitoring assay method for the determination of primaquine in plasma and urine using gas chromatography-mass spectrometric method and a deuterated internal standard. After freeze-drying and extraction with trichloroethylene, the sample plus internal standard was reacted with Tri Sil TBT (a 3 3 2 by volume mixture of trimethylsilylimidazole, A/O-bis-(trimethylsilylacetamide and trimethylchlorosilane) and an aliquot injected to the gas chromatograph-mass spectrometer. The gas chromatographic effluent was monitored at m/z 403, and m/z 406, the molecular ions of the bis-tetramethylsilane ethers of primaquine and 6-trideuteromethoxy primaquine. 

Parkhurst et al. [79] described a high performance liquid chromatographic method for the simultaneous determination of primaquine and its metabolites from plasma and urine samples, utilizing acetonitrile deproteinization, and direct injection onto a cyano column. Levels of 100 ng/mL per 20 pL injection could be quantitated. Preliminary pharmacokinetic analysis is reported for two human subjects after oral doses of 60 90 mg primaquine diphosphate. Two apparent plasma metabolites and two possible urinary metabolites are also reported. 

Fletcher et al. [123] used a sensitive and specific gas chromatography-mass spectrometry method for the assay of primaquine in plasma and urine for studying the plasma kinetics. Preliminary studies on the effects of single and multiple oral doses were carried out. In both cases, the drug was completely removed from plasma in 24 h. The concentration of primaquine in plasma usually reached a peak 1-2 h after oral administration. The plasma elimination half-life was about 4 h. 

Finally, hydroperoxides are reduced to trihydroxy compounds (Figure 25.6). As seen from Figure 25.6, the oxidation of AA resulted in the formation of four F2-isoprostane regio-isomers, each of which is a mixture of eight racemic diastereomers. It is important that the level of F2-isoprostanes in normal human plasma and urine are one to two orders of magnitude higher than the level of COX-derived prostaglandins. 

---