Reconstitution method, assay

ASSAY OF A MULTIENZYME COMPLEX BY THE RECONSTITUTION METHOD (Jahngen and Rossomando, 1984)... 

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

Method A. The rationale of method A is that HDL and LDL are separated by selective precipitation of LDL by dextran sulfate and Mg2+ after the reaction between LDL and reconstituted HDL containing radiolabeled CE by CETP. The method was originally described by Kato et al. [77], The assay mixtures consist of reconstituted [14C]CE-HDL as the donor for CE, LDL as the acceptor, 5,5 -dithiobis-2-nitrobenzoic acid, bovine serum albumin (BSA), partially purified CETP, and a test sample in Eppendorf tubes (1.5 ml). After a 30-min incubation at 37°C, the reaction is terminated by the addition of an LDL-precipitation solution. After standing for 20 min in an ice bath, the assay mixtures are centrifuged, and the supernatant solution containing [14C]CE-HDL is analyzed for radioactivity. Furthermore, the [14C]CE-LDL precipitate is also analyzed for radioactivity if necessary. Usually the blank and control transfer values are about 6% and 34% of initial [14C]CE-HDL added under the assay conditions, respectively. 

An isocratic HPLC method for screening plasma samples for sixteen different non-steroidal anti-inflammatory drugs (including etodolac) has been developed [29]. The extraction efficiency from plasma was 98%. Plasma samples (100-500 pL) were spiked with internal standard (benzoyl-4-phenyl)-2-butyric acid and 1 M HC1 and were extracted with diethyl ether. The organic phase was separated, evaporated, the dry residue reconstituted in mobile phase (acetonitrile-0.3% acetic acid-tetrahydrofuran, in a 36 63.1 0,9 v/v ratio), and injected on a reverse-phase ODS 300 x 3.9 mm i.d. column heated to 40°C. A flow rate of 1 mL/min was used, and UV detection at 254 nm was used for quantitation. The retention time of etodolac was 30.0 minutes. The assay was found to be linear over the range of 0.2 to 100 pg/mL, with a limit of detection of 0.1 pg/mL. The coefficients of variation for precision and reproducibility were 2.9% and 6.0%, respectively. Less than 1% variability for intra-day, and less than 5% for inter-day, in retention times was obtained. The effect of various factors, such as, different organic solvents for extraction, pH of mobile phase, proportion of acetonitrile and THF in mobile phase, column temperature, and different detection wavelengths on the extraction and separation of analytes was studied. 

The two methods published for the assay of sunitinib have used LLE of plasma with methyl tert-butyl ether solvent, evaporation and reconstitution in acetonitrile/ water/formic acid (20 80 0.1, v/v/v) [113] and acetonitrile [114],... 

Chemokines are stored as lyophilized powders. Make up 1 mg/mL stock solution in deionized water. This can be stored frozen and diluted in binding buffer. The nonradiolabeled chemokines, most of which are available from commercial suppliers such as R D Systems or PeproTech, or which can be prepared by methods described in other chapters in this volume (see Chapters 1,3-6,8) can be kept at -20°C after reconstitution at 1 mg/mL in water. Repeated freeze-thawing is not advisable, so the solutions should be aliquoted in small volumes suitable for single assays. 

The idea that the same cofactor species operated in all Mo enzymes originated from a reconstitution assay. In this assay method, the isolated Moco from one enzyme, such as XO, is inserted into a cofactor-free mutant (Nit-1) of nitrate reductase from Neuraspora crassa, where it can reactivate or reconstitute normal nitrate reductase catalytic activity. It is now recognized that the Mo at the active site has many different coordination environments, as has been illustrated for the three Mo families in Fig. 1. In this context, the mutant nitrate reductase assay experiment is interpreted as involving some reprocessing of the inserted molybdenum cofactor from foreign enzymes to obtain the correct form of the cofactor for nitrate reductase catalysis. The Moco designation, if it is to be used, must refer to the family of sites present in Moco enzymes. 

This chapter describes the use of the HPLC method to assay the activity of several enzymes simultaneously. The examples include several different enzymes that can use the same substrate and form the same product, a single enzyme that can use different substrates to form different products and two different activities using the same substrate to form different products. In another example the use of the HPLC method to study metabolic pathways is described through a series of reconstitution studies, and finally the HPLC method is applied to the anabolism of adenosine. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

Providing the product is reconstituted in a small volume of diluent, the resulting solution is more concentrated than the formulated solution before freeze-drying. It is therefore easier to assay minute amounts of drug or impurities and the analytical methods used for QC release can be implemented. 

Transhydrogenase was also purified somewhat later and independently by a more complicated procedure which, however, also produced the 115000 molecular weight polypeptide [102]. Subsequently, additional improved methods have been published, which involve the use of immobilized antibodies [103] and affinity chromatography on immobilized NAD" [104] or NADP" [105]. With all preparations reconstituted transhydrogenase was shown to be a proton pump by both an indirect assay using 9-aminoacridine as pH probe for the interior space of the vesicles [106], and a direct assay using a pH electrode [107]. 

Hoja et al. [74] reported a method for LSD and A-desmethyl-LSD in urine. The analytes were extracted from urine by means of an Extrelut-3 SPE cartridge. The eluate of the cartridge was evaporated to dryness and reconstituted in mobile phase prior to LC-ESl-MS analysis. LOQ were 0.05 and 0.10 ng/ml, with linearity up to 20 ng/ml. Intra-assay and inter-assay precision at 0.1 ng/ml were better than 9 % and 14%, respectively... 

Deflazacourt is an inactive prodrag which after oral administration is rapidly converted to the active 21-hydroxydeflazacort. It has anti-inflammatoiy activity, and is used in the treatment of rheumatoid arthritis. Ifa et al. [87] developed a method for the determination of 21-hydroxydeflazacort in human plasma. The analyte and dexamethasone-21-acetate as the IS are extracted from plasma using a diethyl ether LLE. After evaporation to dryness and reconstitution in mobile phase, the compoimds are analysed using LC-MS with positive-ion APCl in SRM mode, monitoring the transitions m/z 400 124 for the analyte and m/z 435 397 for the IS. The linearity was tested in the range of 1 00 ng/ml. The intra-assay precision and accuracy were better than 5.5% and 7.1%, respectively. 

Valentine et al. applied, in principle, the method of Midha and Charette for a quantitative assay of quinidines (quinidine and hydroquinidine) in plasma, using methylene chloride instead of benzene for the extraction of a small volume of plasma after basification. To the extract was added the internal standard, cinchonine evaporation to dryness and reconstituting in a methanolic solution of trimethylanilinium hydroxide followed. An aliquot of this solution was analyzed by GLC via on-column methylation reaction. Evaluation of the method over the range 0.5 - 10 ug/ml in human plasma gave a precision and accuracy overall of 4.5 %... 

In the following examples, HP7680 SFE instruments (Hewlett-Packard) were used. With these instruments, samples are input to the instrument via containers referred to as extraction thimbles. Extracted components are collected and concentrated on soUd traps. The temperature of the solid trap can be independently set for extraction and reconstitution steps in a method. The chemical functionahty (none to quite polar) of the trap is selected by the choice of the packing material. A reconstitution solvent is used to move extracted components from the solid trap to automatic liquid sampler vials (or to waste). Depending upon the appUcation, the vials are presented to a subsequent analytical instrument or possibly manipulated for a gravimetric assay. With this instrument implementation, samples are input and fractions of extracted components in hquid solvents in 2-mL vials are output with no manual intervention between input and output steps. 

For every sample clean-up method, it is important to investigate the recovery of the parent drug as well as the potentially interfering compounds. Any organic solvent must be evaporated completely and the residue reconstituted in the assay buffer to avoid altering the antibody-binding activity. 

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