Sample Glucuronidase

Several extraction techniques have also been described that use enzymatic or chemical reactions to improve extraction efficiency. A technique that has been used to increase the overall recovery of the marker residue is enzymatic hydrolysis to convert specific phase II metabolites (glucuronides or sulfates) back into the parent residue. Cooper etal used a glucuronidase to increase 10-fold the concentration of chloramphenicol residues in incurred tissue. As an example of a chemical reaction, Moghaddam et al. used Raney nickel to reduce thioether bonds between benomyl and polar cellular components, and as a result achieved a substantially improved recovery over conventional solvent extraction. In choosing to use either of these approaches, thorough characterization of the metabolism in the tissue sample must be available. 

The protocol consisted of preconditioning with methanol (1 mL) followed by water (1 mL). Urine samples (3 mL) were deconjugated by treatment with /3-glucuronidase and arylsulfatase (10 jt/L and 200 fig/fiL) in 0.1M sodium acetate (pH 5.5) and then loaded onto conditioned cartridges. After washing with water (1 mL) and methanoksodium acetate (3 mL, 4 6, pH 5.5), the PAH metabolites were eluted with dichloromethane (3 mL). The eluate was spiked with dodecane (used... 

Plasma samples were mixed (0.01 pi) with 50 pi of 2 M acetate buffer (pH = 5) containing 10 mg/ml ascorbic acid and 20 pi of 500 U a-glucuronidase. The suspension was held at 37°C for 3h then extracted with 3 X 200 pi of ethyl acetate. The combined organic extracts were evaporated to dryness and redissolved in 100 pi of methanol-water (4 6, v/v). Separation was performed in a microbore ODS column (150 X 1mm i.d. particle... 

Sample incubated with glucuronidase and sulfatase at pFl 5 and 37 C, FIjSCh added and steam distilled (total phenol) and analyzed FlPLC-electrochemical detector 2 ng/ injection 95-107% Schaltenbrand and Coburn 1985... 

Following implantation of 200 mg of radiolabeled trenbolone acetate in calves and heifers, maximum levels of residues in tissues occurred at about 30 days postimplantation (31). The highest total drug-related residues expressed as trenbolone equivalents were approximately 50 and 3 ppb in liver and muscle, respectively. Only 25% and 10% of those residues could be extracted by ether or ethyl acetate from glucuronidase-treated liver and muscle samples, respectively. Tire majority of trenbolone residues were not extractable by organic solvents, a finding suggesting that they were covalently bound to tissues (32). 

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

Following pretreatment, samples can be further treated with -glucuronidase (25-27) to achieve hydrolysis of conjugated chloramphenicol residues. It... 

Liquid samples such as urine, plasma, bile, or milk are normally incubated in the presence of -glucuronidase/sulfatase at 37 C for 2 h to deconjugate glucuronide and sulfate conjugates of the analytes (427-430). The most common preparation for this purpose is the juice of the snail Helix pomatia, which has sulfatase and glucuronidase activity. In some instances, dilution of urine with water (431), or dilution of plasma with phosphate buffer and centrifugation (432), may constitute the only pretreatment procedure applied. 

Methods for analysis of individual phthalates in saliva, blood, urine, and/or feces involve separation of metabolites by HPLC combined with GC/MS (Niino et al. 2001 Sjoberg et al. 1985c) or GC/FID (Albro et al. 19 84). Analysis for metabolites differs from analys is for DEHP mainly in sample preparation procedures (Albro et al. 1984 Sjoberg and Bondesson 1985). Metabolites from urine and/or feces are often treated with -glucuronidase to remove conjugated glucuronic acid moieties. When GC methods... 

Application of LC-MS/MS techniques to the analysis of phthalate ester metabolites in urine have also been developed. For example, Blount et al. (2000b) have developed an assay to quantify the monoester metabolites (including MEHP) of eight phthalate diesters in urine, utilizing HPLC coupled with atmospheric pressure chemical ionization and tandem mass spectrometric (APCI-MS/MS) detection techniques. Urine samples were treated with -glucuronidase to release the free phthalate monoesters followed by a two-step solid phase extraction procedure. After evaporative concentration of the eluant, the analytes in the purified samples are further separated on a phenyl reverse phase HPLC column and quantified by APCI-MS/MS, following careful optizimation of the APCI-MS/MS instrument. The limits of detection for MEHP were determined to be 1.2 ng/ml urine with recovery efficiencies of between 78 and 91%. 

Adjust the urine sample to pH 5 by the addition of dilute hydrochloric acid. For each 9 volumes of urine add 1 volume of acetate buffer pH 5) containing 5000 Fishman units/ml of mixed glucuronidase/ sulphatase (from Helixpomatia), and incubate the mixture at 31 for 24 hours. Centrifuge, pour the supernatant liquid on to a column of Amberlite XAD-2 resin, wash the column with 50 ml of water, and then elute the steroids with 100 ml of ethanol. Evaporate the ethanol using a rotary film evaporator, dissolve the residue in 0.5 ml of ethanol, and add 2 ml of cyclohexane. 

Figure 3.3 Average plasma tea catechin concentration vs. time profiles following an 800-mg dose of EGCG. A. Data obtained from plasma samples without subjecting to glucuronidase/sulfatase treatment. B. Data obtained from plasma samples treated with glucuronidase/sulfatase.

Two fairly recent papers on the metabolic face of atropine In man have been published (104,105) an excellent summary of the work was presented by Kaiser (106). The two subjects In the first of these papers excreted within 24 h 85% of an Intramuscularly Injected dose of 2 mg of alpha-[ C]atroplne. A urine saaq>le collected from one of the subjects between 1.5 and 4 h after injection contained substances that produced four small. Interconnected peaks of radioactivity on a paper chromatogram that were apparently removed by Incubation of the urine with bacterial beta-glucuronldase. Only three clear peaks of radioactivity appeared on the paper chromatograms of this urine after It had been exposed to glucuronidase. Two of these had Rfs chat agreed with those for atropine (the major peak) and for tropic acid (a small peak). The Identity of the third peak, with an Rf below that for atropine, was not determined. After alkaline hydrolysis of this sample of urine, the only radioactive substance detected In the chromatogram had an Rf similar to that of tropic acid thus, the tropic acid... 

---