Assay time, reversed-phase HPLC

With this change in chemical composition, a series of statistically designed experiments was performed. Variables of interest were identified as reaction time and the concentrations of rSLPI, cystine and BME. Using the Box-Behnken statistical design, a series of 27 experiments was done. Activity assays and reversed phase HPLC analyses generated data on yield of active rSLPI, relative purity, and relative levels of specific contaminants of interest. The results were modeled with quadratic equations by the X-Stat program and projections of maximum yield and purity, and minimum production of the contaminants, were obtain. ... 

Release studies. Drug-loaded beads were placed in 10 ml glycine/HCl buffer, pH 2.0,0.15 M NaCl at 37 C for two hours and then in 10 ml isotonic PBS, pH 7.4 at 37 so as to mimic in vivo conditions in the GI tract. At different time points, one ml of the release medium was collected and replaced by the same volume of buffer. Drug released was assayed by reversed phase HPLC as described above. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Rezk et al. [74] developed and validated a reversed-phase HPLC assay method for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma. The method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17-min run time. The simple extraction method results in a clean baseline and relatively high extraction efficiency. The method was validated over the range of 2-2000 ng/ml. The resolution and analysis for the four analytes omeprazole, hydroxyome-prazole, omeprazole sulfone, and omeprazole sulfide and the internal standard utilized a Zorbax C18 (15 cm x 3 mm, 5 /im) with a Zorbax C18 (12.5 cm x 4.6 mm) guard column. The mobile phase consisted of two components. Mobile phase A was 22 mM phosphate monobasic, adjusted to a pH of 6 with diluted sodium hydroxide. This solution was filtered through a 0.45-/im membrane filter, then mixed as 900 ml buffer to 100 ml methanol. Mobile phase B was composed of 100 ml of the phosphate buffer as mobile phase A, mixed with 800 ml of acetonitrile, 100 ml of methanol, and 100 /A of trifluoroacetic acid with an initial flow-rate of 0.55 ml/min and detection at 302 nm. 

Zhao and Lou [164] studied the metabolism of omeprazole to its two major metabolites, hydroxyomeprazole and omeprazole sulfone, in rat liver microsomes by a reversed-phase HPLC assay. The formation of metabolites of omeprazole depended on incubation time, substrate concentration, microsomal protein concentration, and was found to be optimal at pH 7.4. The Pmax and Km of omeprazole hydroxylation in the rat liver microsomal preparation were 2033 nmol /(min mg protein), and 46.8 ymol/l, respectively. The effects of seven drugs on omeprazole metabolism were tested. Mephenytoin, five benzodiazepines and pava-verine caused inhibition of omeprazole metabolism. 

Initial work on the assay for this metabolite using normal phase HPLC as was developed for I was impractical due to the polar nature of VI. Using such systems resulted in long analysis time, broad eluting bands, as well as poor sensitivity for the polar metabolite. These findings led then to an investigation of reverse-phase HPLC for analysis of this metabolite. 

An attractive alternative to the above procedure would have been one extraction to remove both I and VI followed by HPLC analysis. As a prelude to this study it was found that the reverse phase HPLC conditions with slight modifications in the gradient program was capable of assay for both VI and I which had retention times of... 

The hypothesis that allatostatins In the brain are transported to the corpora allata (CA) by axons was substantiated by the binding of allatostatln antibodies to corpora cardlaca-CA complexes and by the demonstration of allatostatlc activity In fractions from extract of 6000 pairs of CA that cochromatograph with synthetic allatostatins 1-4 on reverse phase HPLC. These fractions Inhibited Juvenile hormone (JH) synthesis In test CA In an in vitro assay. Two truncated peptides containing only the 6 and 5 C-terminal amino acids of allatostatln 4 (8 amino acids) were 10 and 100 times, respectively, less Inhibitory than the complete peptide. 

There are several caveats associated with this assay that may affect accuracy and precision. (+)-Catechin is the natural form in proanthocyanidins. Part of (+)-catechin epimerizes at the C2 position to form (+)-epicatechin during depolymerization. Similarly, part of (—)-epicatechin epimerizes to form (—)-catechin as an artifact. (+)-Catechin and (+)-epicatechin are an epimer pair in solution (similar to a- and 3-glucose in solution), i.e. they are chiral isomers that cannot be separated on a common reversed-phase HPLC column. The degree of epimerization increases with reaction temperature and time. Depolymerization at room temperature for 10 h caused less than 10% of flavan-3-ols to undergo epimerization. Toluene-a-thiol also causes the heterocyclic ring fission of flavan-3-ols to form adducts that... 

Based on the concept that the retention time of a polypeptide chain in reverse-phase HPLC increases with the degree of unfolding of the chain 2,4,18,19) we infered that HPLC peaks 1, 2, 3, and 4 corresponded to the isoforms so numbered, respectively, in Fig. 3. In order to determine which of the isoforms is biologically active, the eluted peaks from HPLC were assayed on C6 glioma cells for suppression of proliferation. Table II shows that only... 

H]W1N55,212-2. A solution of 25 mg (0.044 mmol) of precursor 56 in 2 mL of ethanol with 25 mg of 10% Pd/C and 0.025 mL of triethylamine was vigorously stirred with 80 Ci of tritium gas at ambient temperature and atmospheric pressure for 4 h. After this time the catalyst was filtered, labile tritium was ronoved by several evaporations of methanol, and the crude product (1960 mCi) was dissolved in 20 mL of ethanol. Purification was accomplished on two 500-mm silica gel plates eluted with hexaneiethyl acetate 50 50 to afford 988 mCi (a 37% radiochemical yield based on precursor 56) of product that was found to be >98% radiochemically pure and to coelute with authentic WIN55,212-2 (49) both on silica gel TLC (hexane ethyl acetate 50 50) and reverse-phase HPLC (water acetonitrile 35 65). The specific activity of product [ H]WIN55,212-2 was measured to be 60 Ci/mmol by UV assay (where E246 = 20,958) and the UV of product was superimposable on that of unlabeled compound. A proton-decoupled tritium NMR (CDCI3) of product 49 showed two peaks at 7.49 and 7.98 ppm. 

A reverse phase HPLC thermospray MS method for detection of impurities in AZT found some of the 5 -trityl derivative and 3 -chloro-3 -deoxythymidine in tiny amounts these are process impurities. The AZT metabolite 3 -amino-3 deoxythymidine can be assayed in plasma samples following derivatization with 9-fluorenylmethylchloroformate followed by fluorimetric detection. This metabolite is five times more toxic to bone marrow cells than is AZT. 1-P-d-AtabinofuranosyI- -5-(2-bromovinyI)uracil has been determined in urine using reverse phase columns and both manual and automated methods. ... 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

HPLC separations were carried out with a reverse-phase column (pBondapak C-18) using methanol-water (45 55) at 1.8 mL/min for 13.5 min followed by methanol-water (60 40) for 20 min. A Waters 6000A pump, Model 440 UV absorption detector and a fraction collector (LKB Multirac) were used. Fractions eluting from the column were collected and assayed by LSC. Retention times of fenitrothion, AF, and MNP under these conditions were 27.0, 11.0, and 7.5 minutes, 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. 

Figure 4.5 HPLC analysis of enzymatic assay with ATP in free and metal-bound forms. Separations were carried out on a reversed-phase C 8 column with a potassium phosphate mobile phase containing 10% methanol. The flow rate was 2 mL/min. Assay mixture of 100 /xL contained 8 mAf Tris-HCl (pH 7.5), 2 mM ATP, and 2 mM MgCl2 and enzyme preparation containing ATP pyrophosphohydrolase (10 /xg of protein). Chromatograms of 20 /xL samples illustrate incubation of (A) zero time and (B) 2 hours. (From Jahngen and Rossomando, 1983).

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