Assay by reversed-phase HPLC

The degradation of plant cell walls requires fra/is-p-coumaroyl esterase, an important enzyme in the digestion of forages and dietary fiber. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reversed-phase HPLC. 

Vitamin C Vitamin C activity resides in two naturally occurring compounds ascorbic acid and its oxidation product, dehydroascorbic acid. In human tissues ascorbic acid predominates. Ascorbic acid is labile in most samples, oxidizing to dehydroascorbic acid and then degrading to 2,3-diketogluconic acid. Various reagents can be used to prevent this oxidation in plasma or whole blood samples. Extraction with 5% metaphosphoric or trichloroacetic acid is the usual initial preparation. Only ascorbic acid may be detected by UV spectrophotometry at 245-265 nm, the absorption maxima of dehydroascorbic acid being 210 nm. A similar problem exists with electrochemical detection where ascorbic acid oxidizes at +0.7V with carbon electrodes. Fluorescent derivatives may be formed with 2-4-din-itrophenylhydrazine or o-phenyldiamine. These derivatives can be assayed by reversed-phase HPLC. 

Degradation rates based on Arrhenius calculations from degradation at elevated temperatures, assayed by reverse phase HPLC. 

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. 

Normal phase silica column Chloroform-methanol-ammonia solution (86.8 12.5 0.7) 254 nm Assay of primaquine and hepatic targeting neoglycoalbumin-primaquine in whole blood and liver of mouse by reversed-phase HPLC. [105]... 

The activity of dihydropyrimidinase or /J-urcidopropionasc can only be measured in liver or kidney. The activity of dihydropyrimidinase is determined using a radiochemical assay with subsequent separation of radiolabeled dihydrouracil from radiolabeled N-carbamyl-/>-alanine with reverse-phase HPLC combined with detection of 14C02by liquid scintillation counting [11]. The activity of /1-ureidopropionase can be determined using radiolabeled N-carbamyl-/l-alanine followed by separation of radiolabeled N-carbamyl-/>-alanine from radiolabeled /1-alanine by reverse-phase HPLC [10,14]. 

A procedure to determine PIR residues in bovine milk using HPLC with the derivatization step for UV detection has also been published (211). The PIR was extracted from milk after protein precipitation and a two-step LLE procedure. The extract was evaporated to dryness, dissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (FMOC). The de-rivatized extract was analyzed by reversed-phase HPLC. Overall recovery was 89%, with 4% for coefficient of variation. A linear regression analysis of HPLC/UV results was compared with the HPLC/MS assay (209,210). The procedure takes about 2.5 hours to complete six or eight samples. Pirlimycin is stable in milk frozen to —60°C or below for at least 3 months. 

Two more recent routes to the detection and assay of ethanolamine and related compounds deserve mention here. Sundler and Akesson (1975) reported an elegant method for the analysis of ethanolamine and possible derivatives (e.g., O-phosphoethanolamine) using the dansyl(5-dimethylaminonaphtha-lene-l-sulfonyl) derivatives and their fluorescence characteristics, in the 0.05-5 xM range. McMasters and Choy (1992) outlined a procedure for the determination of ethanolamine, by reverse-phase HPLC, as the phenythio-carbamyl derivative. A quantitative evaluation could be obtained in the 0.1-10 nmol range. 

In this assay, the substrate, Dopa, and the reaction product were separated by reversed-phase HPLC and eluted isocratically with 0.1 M potassium phosphate buffer at pH 3.0. The eluent was monitored with an electrochemical detector. The separation obtained with this procedure is shown in Figure 9.4B, together with results obtained after incubation of L-Dopa with the enzyme from rat cerebral cortex for 20 minutes at 37°C (Fig. 9.44). Using a calibration curve of the type shown in Figure 9.5, it was possible to show that 1.55 nmol... 

The assay developed for this activity involves the reaction of the P5C with o-aminobenzaldehyde (OAB) to form the reaction product dihydroquinozoli-nium (DHQ). The DHQ and unreacted OAB were separated by reversed-phase HPLC (LiChrosorb C18). The column was eluted isocratically with a mobile phase of 1 part methanol to 2 parts water, and the separation shown in Figure 9.37 obtained. The reaction mixture contained L-omithine (35 mAf), a-ketoglutarate, potassium phosphate (pH 7.4), and pryidoxyl phosphate in a total volume of 2 mL. The reaction was started by the addition of the homogenate and terminated by the addition of 1 mL of 3 N HO containing the OAB. Precipitated protein was removed by centrifugation (3000 rpm), and samples of the supernatant solution (10 nL) were injected for analysis. 

In this assay the asparagine, aspartate, glutamine, and glutamate are separated by reversed-phase HPLC (Cj8) using a mobile phase of 70% sodium acetate buffer (pH 5.9), and 30% methanol as shown in Figure 9.44. 

In a study by To and Wells (1984), the glucuronic acid was transferred from UDPGA to the acceptor, a-naphthol. In this assay the substrate a-naphthol was separated from the reaction product a-naphthol glucuronide by reversed-phase HPLC (Ci8 column) with a solvent of 0.1 M acetic acid-methanol (55 45, v/v). Absorbance was monitored at 240 nm. 

The assay involves the separation of reactants by reversed-phase HPLC on a Cis (Partisil 5 ODS) column with a mobile phase of 0.02 F KH2P04 (pH... 

Adenosine kinase catalyzes the transfer of phosphate from ATP to adenosine (Ado) to form AMP and ADP. The separation of the reactants, Ado and ATP, from the products, AMP and ADP, can be accomplished by reversed-phase HPLC (Ci8) with isocratic elution with a mobile phase of 0.1 M potassium phosphate (pH 5.5) and 10% methanol. Detection depends on the substrate. In this assay, it is useful to replace the substrate adenosine with the fluorescent analog formycin A (FoA) and to monitor the column eluent with a fluorescence detector. Thus, ATP and any of its hydrolytic products will not be detected. 

In the assay described by Rossomando et al. (1981b), cAMP is separated from the ATP substrate by reversed-phase HPLC on a C 8 (/LiBondapak) column with a mobile phase of 0.01 M potassium phosphate (pH 5.5) with 10% methanol. The column was eluted isocratically. The detectors for the eluent depended on the substrate (see below). 

In the HPLC assay of Rossomando et al. (1981a), the compounds AMP, cAMP, and adenosine are separated by reversed-phase HPLC on Clg (yuBondapak) with a mobile phase of 10 mJW KH2PO4 (pH 5.5) containing 1% methanol. The separation of cAMP and AMP obtained with a mobile phase of a phosphate buffer and 10% methanol is shown in Figure 9.107. 

The separation of the substrate and the product was accomplished by reversed-phase HPLC on an ODS column. The column was eluted isocratically with a mobile phase of methanol-water (33 67 v/v). The compounds were detected at 254 nm. The separations obtained are shown in Figure 9.121. Radiolabeled substrates were also used, and the eluent was assayed for radioactivity on fractions collected during the elution. 

High-performance liquid chromatography can be used as a separation method of volatile compounds, followed by GC or GC-MS for further separation and analysis. The most common assay is performed by reversed-phase HPLC, usually on Cig, when the separation of the compounds is according to their hydrophobicity, i.e., according to chain length and polar groups. 

DHA can be separated from AA and most ionic compounds by ion exchange columns. Such columns do not separate DHA from other AA metabolites and neutral carbohydrates. DHA can be separated from these compounds by reverse phase HPLC using water or water-acetonitrile eluants. Good separations of DHA from biological samples can be expected to be achieved by HPLC. Detection is a problem since UV absorption is inadequate. The red chromophore with amino acids is not very sensitive. Perhaps DHA could be reduced to AA after separation and detected by the strong 263-nm absorption of AA or by an electrochemical detector. DHA levels and DHA/AA ratios are probably quite important in biology and medicine, and good procedures for these assays are of considerable interest. 

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. ... 

To optimize the conditions for the maximum binding capacity assay, different concentrations of receptors (0.83-6.63 mg/mL) were incubated with a fixed amount of purified labelled molecule (up to 50 000 counts/min per tube). Also, for the 3.3 mg/mL receptor concentration, different amounts of the DOTATATE purified by reversed phase HPLC (2000-70 000 counts/min per tube) were used, and experiments for inhibition studies were initiated. 

T7 RNA polymerase incorporates biotinylated nucleotide analogues efficiently (Fenn and Herman, 1990). They noted that this enzyme did not discriminate significantly between UTP and BIO-4-UTP, but its turnover number was reduced from 130 to 28 pmol/min and the from 32 to 77 p.M for UTP and BIO-4-UTP, respectively). T3 RNA polymerase incorporates BIO-ll-UTP most efficiently (3 X SP6 and 2 X T7 RNA polymerase) (D Alessio, 1985). Fenn and Herman (1990) separated the nucleotides obtained after alkaline hydrolysis of the transcripts by reverse-phase HPLC. Gel retardation assays (Theissen et al., 1989) are simpler alternatives (change in mobility of transcripts on polyacrylamide if streptavidin is bound to biotin moieties) although RNA with secondary structures may not allow the streptavidin to bind under native conditions. 

A simple assay based on potent and specific inhibition of jack bean a-mannosidase has been devised for determining low concentrations of 162 (up to 0.5 cm ) in M anisopliae cultures (110). The new assay was used to demonstrate that the addition of L-lysine (163) to the culture medium stimulated production of the alkaloid by approximately fourfold. Other early metabolic precursors of 162 in this fungus, including a-aminoadipic acid, saccharopine (164), L-pipecolic acid (165), and L-lysine itself, were quantified by reverse-phase HPLC analysis of mycelial extracts derivatised with 9-fluorenylmethyl chloroformate (FMOC) (111). 

Chan-Palay et al. (1981) found eoexistence of GAD and motilin in 10-20% of the Purkinje cells of the rat. The presenee of motilin in Purkinje cells has, however, been disputed by Lange (1986), who was unable to demonstrate the presence of motilin using radioimmuno-assay and reversed phase HPLC in extracts of rat cerebellum. Only one of Lange s anti-motilin antibodies, all of which had been demonstrated to be effective in demonstrating motilin-like immunoreactivity in rat duodenum, was found to im-munoreaet with Purkinje cells in immunocytochemical studies with rat cerebellum. 

The experiment was conducted in a light cabinet specially constructed in-house to provide a high intensity of artificial light (2x Philips white 35 fluorescent bulbs) at a constant temperature. Constant temperature was achieved by fitting the cabinet with a powerful fan and situating the light cabinet in a constant temperature storage facility. Samples were taken over the course of the reaction and assayed for compound A content by reverse phase HPLC analysis. The reaction was allowed to proceed to a sufficient extent to allow first-order rate constants to be determined. 

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