Analytical biochemistry
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Analytical biochemistry · Jan 2000
The use of fluorogenic substrates to monitor thrombin generation for the analysis of plasma and whole blood coagulation.
Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. ⋯ Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood.
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Analytical biochemistry · Aug 1998
Linearized colorimetric assay for cremophor EL: application to pharmacokinetics after 1-hour paclitaxel infusions.
Cremophor EL (CrEL) is a polyoxyethylated castor oil surfactant used in the intravenous formulation of the anticancer drug paclitaxel (Taxol). Quantitative determination of CrEL in patient samples can be achieved by complexation of the compound with the Coomassie brilliant blue G-250 dye in protein-free extracts [Sparreboom, A., Loos, W. J., Verweij, J., De Vos, A. ⋯ By measurement of the ratio of absorbances at the maxima of the red (450 nm) and blue charge forms (595 nm) of Coomassie brilliant blue G-250, a full-scale linear relationship can be obtained over the entire range studied (0.500 to 10.0 microliter/mL). Validation data revealed that transformation of the detection procedure exhibits significantly improved specificity, accuracy(
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Analytical biochemistry · Jan 1998
Quantitation of Cremophor EL in human plasma samples using a colorimetric dye-binding microassay.
This paper describes an analytical procedure for the quantitative determination of the pharmaceutical vehicle Cremophor EL in human plasma samples. The procedure is based on rapid binding of Coomassie brilliant blue G-250 to Cremophor EL following plasma protein precipitation with acetonitrile and analyte extraction with n-butylchloride. ⋯ The assay permits estimation of Cremophor EL concentrations in the range 0.05-1.00% (v/v) in 50 microL of human plasma, with percentage deviation and precision of < or = 12 and < or = 15%, respectively. The assay was subsequently used to measure Cremophor EL concentrations in plasma samples in support of a project to develop a pharmacokinetic model for this compound in patients receiving paclitaxel.
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Analytical biochemistry · Aug 1993
Quantification of free carnitine, individual short- and medium-chain acylcarnitines, and total carnitine in plasma by high-performance liquid chromatography.
This paper describes a method for the quantitative determination of free carnitine, acetylcarnitine, propionylcarnitine, hexanoylcarnitine, octanoylcarnitine, and total carnitine in plasma. Carnitine and acylcarnitines were extracted from 100 microliters of plasma with acetonitrile/methanol and isolated using 0.5-ml columns of silica gel. Samples were then derivatized with 4'-bromophenacyl trifluoromethanesulfonate and quantified by high-performance liquid chromatography with detection at 260 nm. Carnitine and acylcarnitines were quantified in normal human plasma and the plasma of patients diagnosed with methylmalonic aciduria, propionic acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.
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Analytical biochemistry · Apr 1991
Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.
An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. ⋯ DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.