Metabolite extraction procedure

Owing to its relevancy in metabolite profiling analysis, several groups have investigated the suitability of different metabolite extraction procedures for GMO analysis. Selective extraction techniques, such as supercritical fluids... 

Homogenization of Plant Material Plant material should be finely dispersed before the metabolite extraction procedures. The homogenization step should be performed in liquid nitrogen using mortar and pestle or, preferably, with a precooled ball mill (e.g., Retsch MM series). The homogenization step should be performed as fast as possible without thawing of the tissue since endogenous hydrolytic enzymes may be activated in disrupted tissues even at low temperatures. The powdered plant material may also be stored at -SO C for several months. 

Validation of true extraction efficiency normally requires the identification and quantitation of field-applied radiolabeled analyte(s), including resulting metabolites and all other degradation products. The manufacturer of a new pesticide has to perform such experiments and is able to determine the extraction efficiency of aged residues. Without any identification of residue components the calculation of the ratio between extracted radioactivity and total radioactivity inside the sample before extraction gives a first impression of the extraction efficiency of solvents. At best, this ratio is nearly 1 (i.e., a traceability of about 100%) and no further information is required. Such an efficient extraction solvent may serve as a reference solvent for any comparison with other extraction procedures. 

The recovery is low on extracting n from soil by refluxing with water containing methanol. Metabolite n in soil can be extracted by sonication with alkaline methanol. Using this alkaline extraction procedure, another metabolite, 2-chlorobenzylsulfonic acid, is also extracted simultaneously. ... 

Bound residnes are those chemicals retained in the subsurface matrix in the form of the parent organic contaminant or its metabolites these residnes remain after subsequent extractions, dnring which the nature of the compound or of the matrix is not altered by the extraction procedure. An example of sequential extraction of... 

Situations can be imagined, in which more than one solute has to be extracted from a sample. Such situations are, for instance, the extraction of an analyte simultaneously with an internal standard or a drug simultaneously with one or more major metabolites or co-drugs. Under these conditions, the aim of an extraction procedure is to extract all substances as quantitatively as possible. However, for each solute to be extracted the optimum composition may be located in another region of the factor space there may be no such combination of mixture variables, that guarantees optimum extraction for all substances. 

Despite the fact that the preparation of chloramphenicol-specific antibodies was reported as early as in 1966 (36), it was 1984 before the first immunoassay was published for the determination of chloramphenicol residues in swine muscle, eggs, and milk (37). This first-published method was a radioimmunoassay that required an extraction procedure and special laboratory facilities to attain a quantification limit of 1 ppb. Employed polyclonal antibodies showed insignificant crossreactivity with structurally related compounds, except that thiamphenicol that did not interfere with the analysis. However, cross-reactivity was significant for metabolites deviating from the parent compound in the acyl side chain. 

Ultraviolet-VIS is still the most common detector employed for OPP- and OCP-residue determination in clean matrices such as water. The methods utilizing straightforward extraction procedures and the improved optics of diode array detection demonstrate the applicability of HPLC for analysis of OPPs and their metabolites in food. 

Motevalian et al. [62] developed a rapid, simple, and sensitive HPLC assay method for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 /d) was injected on a /rBondapak Ci8 reversed-phase column (4.6 mm x 250 mm, 10 /un). The mobile phase consisted of 0.05 M phosphate buffer (pH 7.5) and acetonitrile (75 25) at a flow-rate of 0.8 ml/min. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50-2000 ng/ml. The minimum detection limits were 10, 10, and 15 ng/ml for omeprazole, omeprazole sulfone, and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg of omeprazole. 

In order to achieve highest sensitivity of VitD metabolites detection, the efforts for method development were focused on three components of the analytical flow path (1) the selective SPE extraction procedure, (2) the ionization and SRM method for MS/MS, and (3) the pLC loading and separation method. 

Bouzas et al. [95] developed a quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to LC-MS/MS. The online extraction procedure described consists of an extraction column and an analytical column, which were coupled online. A PPT procedure was performed with zinc sulfate an aliquot of 0.1 M zinc sulfate in methanol was added to the serum sample in a proportion of 2 1 (v/v) to serum. Analytes were extracted by a short pentafluorophenyl silica column and separated on a longer analytical column with the same stationary phase. Recoveries of all analytes were above 80 %. The proposed procedure by Matuszewski et al. [64] was used for the evaluation of matrix effect ME(%)=B/Ax 100, where ME is the matrix effect (suppression or enhancement) and B corresponds to peak areas for standards spiked after extraction into sample extracts and A to peak areas obtained in neat solution standards. Authors compared this method to offline SPE coupled with GC-MS and results showed that LC-MS/... 

Jenkins KM, Young MS, Mallet CR, Elian AA (2004) Mixed-mode solid-phase extraction procedures for the determination of MDMA and metabolites in urine using LC-MS, LC-UV, or GC-NPD. J Anal Toxicol 28(l) 50-58... 

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%. 

Bakhtiar R, Khemani L, Hayes M et al. (2002) Quantification of the anti-leukemia drug ST1571 (Gleevec) and ist metabolite (CGP 74588) in monkey plasma using a semi-automated solid phase extraction procedure and liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 28(6) 1183-1194... 

With HPLC, serum concentrations of this important bronchodilator can be determined in about 10 min on approximately 5 p,L of serum, allowing quick dosage adjustment to ensure therapeutic levels while avoiding toxicity. In addition, LC eliminates the need for extraction procedures and prevents interference from metabolites, other medications, and dietary xanthines such as caffeine. Using an internal standard, the technician can obtain... 

Due to the low amount of specimen used for hair analysis and the low concentration of drugs and their metabolites in hair, extraction procedures are mostly necessary to concentrate the analytes in the tested solution. Various methods have been used for this step. The variety of described procedures show that there is no universal solution. The choice of an extraction procedure may be influenced by the kind of drug and drug metabolite(s) to be analyzed and the method used for the subsequent analysis. Preceding the real extraction, there are four treatments with more or less intermethodical differences to prepare the hair matrix for the extraction ... 

THC and its metabolites can be detected in plasma or urine samples only within a few hours to days after cannabis intake, which was documented by numerous extraction procedures. - At the opposite, hair appears to be an interesting substrate for the investigation of chronic exposure. ... 

For the accurate interpretation of a positive hair analysis result, it is necessary to subject hair to specially developed wash and extraction procedures. During the development of these procedures, we discovered several important properties of hair, properties which were essential for defining three unique wash kinetic criteria and their cutoff levels which, in conjunction with washing, enhance the acciuacy of the interpretation of a positive hair analysis result. Additional certainty can be achieved by the measurement of metabolites or metabolite/drug ratios, by the development of ultrasensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) or ion... 

Disposition in the Body. Rapidly but incompletely absorbed after oral administration bioavailability about 65%. Up to 90% of an intravenous dose is excreted in the urine, mainly as unchanged drug with up to 14% of the dose as a glucuronide conjugate. 2-Amino-4-chloro-5-sulphamoylanthranilicacid has been reported as a metabolite in several studies, but in other cases it has not been detected and it has been suggested that it is an analytical artefact produced during acid extraction procedures. In normal subjects, about 6 to 18% of a dose is eliminated in the faeces after intravenous administration this may be increased to about 60% in renal failure. 

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