Release Assay

Hunt et al.150 proposed the incorporation of LC-MS techniques to eliminate other identity assays required in the QC lab. A LCQ ion trap mass spectrometer was added downstream of the UV spectrometer. In addition to a UV trace, a total ion current chromatogram was generated. Comparison to a reference chromatogram was performed to define identity without extensive interpretation of the mass spectral data. This LC-MS method eliminated the need for other identity assays such as N-terminal sequencing and was validated as a release assay for three proteins.150... 

Hunt J., Meng H., Welk P., Austin F., and Buckel S. (2001), A Simple Mass Spectrometry Method for a Lot Release Assay, Poster communication. Well Characterized Biologicals Conference, 2001. 

Hence, a generic or universal HPLC method interfaced with a UV detector for BHT can be used on any drug as long as the acceptance criteria on accuracy, precision, robustness, and other necessary requirements have been met. Similar to appearance, drug release, assay, and impurities, preservative testing is also required if a certain degree of preservative has to be included in the drug product to ensure an adequate shelf life. 

These are the assays most frequently used to detect 5-HT3 receptor activity. However, there are other in vitro assays which could be used. Amongst these are neurotransmitter release assays using brain tissue for example, 5-... 

The platelet hist UIline release assay demonstrated that cotton mill dust extract, cotton bract extract, cotton leaf extract, dialyzed CMD extract, polyphenols, compound 48/80, rutin, trimethylamine HCl, quercetin, catechin, tannic acid, ellagic acid and sodium metasilicate all release histamine directly (48). Thus not only do tannin compounds induce histamine release, but they may also form higher molecular weight polymers and contain components that survive acid hydrolytic conditions (48). Tannins are widely distributed in the plant kingdom. 

Table 4.3 Comparison of MurF ASMS screening-based binding constants and MurF activities from the radiolabeled phosphate release assay. Included from [10] with permission from SAGE Publications.

All assays performed on digester samples were conducted in 100 mM Tris buffer pH 7.5 with substrate incubations at 37 C. Glucose-releasing assays used the same Tris buffer with 0.5% sodium azide added. Colorimetric products from enzyme assays were detected and recorded using a Milton-Roy model 601 spectrophotometer equipped with sipper and data printer. 

Bhunia, A. K., and Westbrook, D. G. (1998). Alkaline phosphatase release assay to determine cytotoxicity for Listeria species. Lett. Appl. Microbiol. 26,305-310. 

Fig. 1.1 Interaction between allergen and adenosine receptor agonists with respect to histamine release from guinea-pig chopped lung (left) or bronchoconstriction in the guinea pig in vivo (right). Tissues and animals were passively sensitized to ovalbumin. In the histamine release assay, a threshold response to allergen is augmented concentration-dependently by NECA. In the whole animal, a single intravenous injection of APNEA markedly enhances the bronchoconstrictor response to allergen (J.R. Fozard and H.J. Pfannkuche, unpublished observations 1994)...

By phase II, stability assays should be validated and a good faith effort made to validate all in-process tests. Release assays should also be validated. Removal of product and process-related impurities should be demonstrated. Stability of cells during growth should be validated. 

The data in Figure 10.1 show results from histamine, LTC4, and PGD2 release assays for selected botanicals. Results shown are means of two to eight replicate experiments with standard errors of the means and represent the effect of botanicals... 

Fig. 4 Superfusion neurotransmitter release assay in synaptosomes. (a) Schematic drawing of a superfusion setup. Synaptosomes are preloaded with radioactive neurotransmitter and captured on fiberglass filters in superfusion chambers under continuous superfusion with gassed physiological salt solution (e.g., Krebs bicarbonate buffer) using a peristaltic pump. After a 10- to 15-minute wash, neurotransmitter release is triggered by rapid switching of superfusion lines to a stimulating buffer (e.g., high-potassium solution). Superfusate is collected on a fraction collector, and radioactivity is measured by liquid scintillation, (b) Typical trace recording of tritium-labeled norepinephrine fractional release in rat cortical synaptosomes stimulated by high potassium and a-latrotoxin in the presence or absence of external calcium.

Double Blind Placebo Controlled Food Challenge (DBPCFC) Other immunological and nonimmunological tests Basophil histamine release assay Intestinal mast cell histamine release Intragastric provocation under endoscopy Intestinal biopsy after allergen elimination and feeding Others (see text below)... 

Ballmer-Weber, B. K., J. M. Weber, S. Vieths, and B. Wuthrich. 2008. Predictive value of the sulfidoleukotriene release assay in oral allergy syndrome to celery, hazelnut, and carrot. J Investig Allergol Clin Immunol 18 (2) 93-99. 

Kaul, S., D. Luttkopf, B. Kastner, L. Vogel, G. Holtz, S. Vieths, and A. Hoffmann. 2007. Mediator release assays based on human or murine immunoglobulin E in allergen standardization. Clin Exp Allergy 37 (1) 141—150. 

Colsky AS, Peacock JS. 1990. Sodium pyruvate inhibits the spontaneous release of 51Cr from RBC in chromium release assays. J Immunol Methods 129 139-141. 

Nouri AME, Mansouri M, Hussain RF, et al. 1995. Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity. J Immunol Methods 180 63-68. 

Of their many preformed intracellular mediators, histamine, not only plays an important physiological role, but can be used as a marker for these cells. In the human peripheral blood, the basophil is the only leukocyte that contains histamine. As a result, histamine release from human blood leukocyte preparations can be used to monitor basophil degranulation. In this chapter we will describe the histamine release assay, alternative techniques to measure the concentration of histamine and, finally, a method to purify human basophils to confirm that the chemokines are acting directly on the basophil. 

Korzeniewski, C. and Callewaert, D.M. 1983. An enzyme-release assay for natural cytotoxicity. J. Immunol. Meth. 64, 313-320. 

Nevertheless, in a previous study dealing with inert matrices of naltrexone-HCl [74], two different excipient percolation thresholds pc2 were found for the matrixforming excipient Eudragit RS-PM the site percolation threshold related to a change in the release kinetics and the site-bond percolation threshold derived from the mechanical properties of the tablet, where the excipient failed to maintain tablet integrity after the release assay. 

An evident change in the release kinetics between tablets containing 80 and 90% w/w of drug can be observed in Table 23 (from k 0.57 to k 0.7 in the Peppas equation). Therefore, the site percolation threshold of the excipient can be estimated between the matrices containing 80 and 90% w/w of dextromethorphan hydrobromide (10-20% v/v of excipient). Above this threshold, a percolating cluster of excipient particles exists. These particles are able to control the drug release kinetics, but their cohesion forces can be insufficient to maintain tablet integrity after the release assay. 

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