Sensitive High-performance Liquid Chromatographic Assay Research Articles

Analysis of 3′-azido-3′-deoxythymidine levels in tissues and milk by isocratic high-performance liquid chromatography

A sensitive high-performance liquid chromatographic (HPLC) assay was established to analyze levels of the antiretroviral agent 3′-azido-3′-deoxythymidine (AZT, zidovudine) in serum, milk and tissue extracts. After methanol precipitation, serum samples could be injected directly into the HPLC apparatus, whereas tissue extracts required further clarification. Recovery of AZT was virtually complete. Isocratic elution with a mobile phase consisting of 6% acetonitrile and 0.1 M ammonium acetate, pH adjusted to 4.5 with glacial acetic acid, resulted in good resolution of AZT and its metabolites; retention times for AZT and the internal standard, p-nitrophenol, were 20 and 37 min, respectively. Using this method, we have demonstrated that AZT crosses both the blood-brain and placental barriers and is excreted into milk at high levels.

Sensitive high-performance liquid chromatographic assay using 9-fluorenylmethylchloroformate for monitoring controlled-release lidocaine in plasma.

A sensitive high-performance liquid chromatographic (HPLC) assay using fluorescence detection for quantifying lidocaine levels in plasma (in the ng/ml range) was developed. This novel HPLC assay has made possible the simultaneous monitoring of lidocaine levels in coronary and peripheral plasma obtained after myocardial controlled-release matrix administration (0.92 mg/kg during 4 h) in the arrhythmic dog. The method employed extracts the drug from plasma using 1-chlorobutane and a subsequent derivatization with 9-fluorenylmethylchloroformate in acetonitrile at 110 degrees C. The derivative was chromatographed on a C18 reversed-phase column and measured with fluorescence detection (excitation 254 nm, emission 313 nm). N-Methylephedrine was found to be suitable as an internal standard, post-derivatization. The derivatization product of lidocaine was identified and characterized by mass spectral analysis. It was found to have a unique and reproducible dicarbamate structure, which was stable for at least three days at room temperature. The method was tested with human plasma as well as on dog plasma. Analytical recoveries were 88.6 +/- 3.6 and 77.4 +/- 3.0% (mean +/- S.E.), respectively, at levels ranging from 25 to 200 ng/ml. The lower detection limit was 1 ng/ml lidocaine. In conclusion, this rapid and convenient analysis was found to be suitable for the bioavailability pharmacokinetic assessment of lidocaine following low-dose regional drug administration.

High-performance liquid chromatographic assay for thymidylate synthase from the human malaria parasite, plasmodium falciparum

A rapid and highly sensitive high-performance liquid chromatographic assay for thymidylate synthase activity is described. The assay is based on the separation of the substrate, deoxyuridylate (dUMP), and its product, deoxythymidylate (dTMP), on a LiChrosorb RP-8 reversed-phase column with 44 m M triethylammonium phosphate (pH 7.0) as mobile phase and a flow-rate of 1.0 ml/min. In addition, using a μBondapak C 18 reversed-phase column with 10 m M potassium phosphate (pH 4.0) and a gradient of 0–28% methanol, dUMP, dTMP and deoxythymidine (dTdR) are well separated within 30 min. The latter system is also applied to assay thymidine kinase activity with dTdR and dTMP as substrate and product, respectively. This method is sensitive enough to measure dTMP at concentrations as low as 25 pmol, and it was used to show that crude extracts of the human malaria parasite Plasmodium falciparum contain thymidylate synthase but not thymidine kinase activity.

Sensitive high-performance liquid chromatographic assay for albendazole and its main metabolite albendazole sulphoxide in plasma and cerebrospinal fluid

Sensitive high-performance liquid chromatographic assay for albendazole and its main metabolite albendazole sulphoxide in plasma and cerebrospinal fluid

Mitomycin c determination using loop-column extraction: a rapid and sensitive high-performance liquid chromatographic assay for pharmacokinetic studies with spherex starch particles

Mitomycin c determination using loop-column extraction: a rapid and sensitive high-performance liquid chromatographic assay for pharmacokinetic studies with spherex starch particles

Sensitive high-performance liquid chromatographic assay for determination of amiloride in biologic fluids using an ion-pair extraction method.

The paucity of data on disposition of the potassium-retaining diuretic amiloride is mainly owing to the lack of sensitive and specific analytic methods for measuring the drug in biologic fluids. Poor lipophilicity and extremely low plasma concentrations impose severe constraints. A method is described for analysing plasma and urine concentrations of amiloride using an ion-pair extraction method and high-performance liquid chromatography (HPLC) with fluorescence detection. Amiloride and internal standard triamterene form an ion-pair with bromothymol blue at pH 7.6. The ion-pairs are extracted into diethyl ether: dichloromethane (2:1), and back-extraction into 0.1% tetrabutylammonium hydroxide liberates the drugs from their ion pairs. Analyses are performed using HPLC with a reverse-phase C18 column and a mobile phase of 11% acetonitrile and 0.5% triethylamine adjusted to pH 3. Amiloride is quantified using fluorescence detection (366-nm excitation, 418-nm emission cutoff). Retention times are 2.2 and 6.0 min for amiloride and triamterene, respectively. The sensitivity limit is 0.2 ng/ml, and recovery is 82% for amiloride and 71% for triamterene. Calibration curves are linear (range 0.25-25 ng/ml for plasma and 0.05-2.0 micrograms/ml for urine). Inter- and intraday assay variability is less than 8% and precision is within 5%. The assay is of sufficient sensitivity and specificity for the study of the pharmacokinetics of amiloride in humans.

A sensitive high-performance liquid chromatographic assay for melphalan and its hydrolysis products in blood and plasma.

A sensitive high-performance liquid chromatographic assay has been developed for the measurement of the alkylating cytostatic drug melphalan (4-[bis(2-chloroethyl)amino]-L-phenyl-alanine, or L-phenylalanine-mustard, L-PAM) and its two hydrolysis products, monohydroxy melphalan (MOH) and dihydroxy melphalan (DOH). A reversed-phase phenyl column and a mobile phase consisting of acetonitrile/citrate buffer made possible an isocratic separation and quantification. N,N-[bis(2-hydroxy-ethyl)]toluidine has been synthesized as an internal standard structurally related to DOH. A new, accurate "kinetic" calibration procedure enabled us to determine even the concentration of the unstable MOH. The lower limit of quantification was 30 ng/ml for L-PAM and 20 ng/ml for both DOH and MOH with fluorescence detection. The use of this method is illustrated by some pharmacokinetic data in systemic and locoregional melphalan therapy.

Rapid and sensitive high-performance liquid chromatographic assay of tiaprofenic acid enantiomers in human plasma and urine

A sensitive stereospecific high-performance liquid chromatographic assay of tiaprofenic acid (TA) enantiomers in human plasma and urine was developed. The biological specimens are acidified, and the drug and internal standard, (±)-2-(4-benzoylphenyl) butyric acid, extracted with an isooctane—isopropanol (95:5) mixture (plasma) or chloroform (urine), followed by sequential reaction of the enantiomers with trichloroethyl chloroformate and l-leucinamide. The reactions were complete at ambient temperature in less than 3 min. The diastereoisomers of TA and internal standard were then extracted into chloroform. The organic layer was evaporated, and the reconstituted residue chromatographed at ambient temperature on a C 18 reversed-phase column with a mobile phase consisting of 0.06 M monopotassium phosphate—acetonitrile—triethylamine (65:35:0.02) at a flow-rate of 1 ml/min. The TA diastereoisomers were detected at 310 nm, free of interfering peaks, with a resolution factor of 2.1. Within the examined plasma and urine enantiomeric concentration ranges of 0.2–20 and 10–100 mg/l, respectively, an excellent linear relationship was obtained between the peak-area ratios and the corresponding concentrations. The assay was reproducible and sufficiently accurate to be applied to the stereoselective pharmacokinetic analysis of TA enantiomers in plasma and urine following administration of therapeutic doses of the drug.

A sensitive high-performance liquid chromatographic assay for trypsin-like enzymes

A sensitive high-performance liquid chromatographic assay for trypsin-like enzymes

Rapid and sensitive high-performance liquid chromatographic assay for midazolam and 1-hydroxymidazolam, the major metabolite, in human serum

Rapid and sensitive high-performance liquid chromatographic assay for midazolam and 1-hydroxymidazolam, the major metabolite, in human serum

Determination of clindamycin in plasma or serum by high-performance liquid chromatography with ultraviolet detection

A simple, sensitive high-performance liquid chromatographic assay for the determination of clindamycin in human plasma or serum has long been hampered by inability to separate it from endogenous compounds. We describe here such an assay. Proteins from a 200-μl sample were precipitated by addition of acetonitrile containing the internal standard, triazolam. The sample was then vortex-mixed and centrifuged at approximately 3000 g for 10 min. The supernatant was evaporated to about 250 μl under nitrogen, and 10–30 μl were analyzed using an autoinjector. An octadecylsilane column with acetonitrile-water-phosphoric acid-tetramethylammonium chloride as mobile phase and ultraviolet detection at 198 nm provided a reproducibly quantifiable peak corresponding to 0.17 μg/ml. Retention times for clindamycin and triazolam averaged 8 and 11.8 min, respectively. Replicate standard curves run over a 36-h period showed no loss of integrity; r 2 values generally exceeded 0.999. The method has successfully been applied to the analysis of samples taken up to 12 h after administration of intravenous infusions of 600–1200 mg in healthy volunteers.

Sensitive high-performance liquid chromatographic assay of pelanserin, a novel antihypertensive agent, in plasma samples

Sensitive high-performance liquid chromatographic assay of pelanserin, a novel antihypertensive agent, in plasma samples

A high-performance liquid chromatography assay for measuring integrated biphenyl metabolism by intact cells: Its use with rat liver and human liver and kidney

A rapid, sensitive high-performance liquid chromatography assay with fluorescence detection for measuring biphenyl metabolism by intact cells has been developed. The assay does not require organic solvent extraction or enzymatic digestion for the measurement of hydroxybiphenyl conjugates. The lower limit of detectability for 4-hydroxybiphenyl is 5 pmol injected. Rat hepatocytes incubated with biphenyl form predominantly 4-hydroxybiphenyl sulfate with lesser amounts of 4-hydroxybiphenyl glucuronide and free hydroxybiphenyls, and small amounts of 3-hydroxybiphenyl sulfate and 3-hydroxybiphenyl glucuronide. Slices of fresh human liver incubated with biphenyl form predominantly 4-hydroxybiphenyl glucuronide with some free hydroxybiphenyl and small amounts of 4-hydroxybiphenyl sulfate. 4-Hydroxybiphenyl glucuronide formation by human liver shows a lag time that is not abolished by preincubating the liver without substrate. Human kidney slices incubated with biphenyl form 4-hydroxybiphenyl glucuronide and 4-hydroxybiphenyl sulfate at rates less than one-tenth those seen with human liver. Human kidney slices do not form detectable free hydroxybiphenyl. There is wide intersubject variability in the rates of hydroxybiphenyl metabolite formation by human liver and kidney.

Simple and sensitive high-performance liquid chromatographic assay for 5-aminosalicylic acid and acetyl-5-aminosalicylic acid in serum

Simple and sensitive high-performance liquid chromatographic assay for 5-aminosalicylic acid and acetyl-5-aminosalicylic acid in serum

Rapid, sensitive high-performance liquid chromatographic assay for isoflavonoids from cowpea ( Vigna unguiculata)

Rapid, sensitive high-performance liquid chromatographic assay for isoflavonoids from cowpea ( Vigna unguiculata)

Sensitive high-performance liquid chromatographic analysis of plasma vitamin E and vitamin A using amperometric and ultraviolet detection

A highly sensitive high-performance liquid chromatographic assay using amperometric detection has been developed for measuring α-tocopherol in plasma. Other minor components of vitamin E (β-, γ- and δ-tocopherols) can also be detected by this method. All- trans-retinol is simultaneously monitored by UV detection at 313 nm. This assay requires only 50 μl of plasma for quantification of both vitamin E and A. The minimum detectable quantities for both α-tocopherol and retinol are 1.0 ng with corresponding signal-to-noise ratios of 4.0 and 3.5. The total chromatographic analysis time for each sample is less than 15 min. This assay has been used to determine the concentrations of vitamin A and E in plasma obtained from patients with liver disease, liver transplant patients and normal healthy volunteers.

Rapid and sensitive high-performance liquid chromatographic assay for metformin in plasma and urine using ion-pair extraction techniques

Rapid and sensitive high-performance liquid chromatographic assay for metformin in plasma and urine using ion-pair extraction techniques

A sensitive high-performance liquid chromatography assay for γ-glutamyl hydrolase

A sensitive high-performance liquid chromatography assay for γ-glutamyl hydrolase

Simultaneous Quantitation of Lidocaine and Its Four Metabolites by High-performance Liquid Chromatography: Application to studies on In Vitro and In Vivo Metabolism of Lidocaine in Rats

A convenient and sensitive high-performance liquid chromatographic assay for the simultaneous quantitation of lidocaine and its four metabolites has been developed. The samples containing lidocaine and its metabolites were eluted from a microparticulate octadecylsilane column using a mobile phase of 0.1M phosphate buffer (pH 3.0) containing 10% acetonitrile. This method was applied to studies on in vitro metabolism and in vivo pharmacokinetics of lidocaine in rats. Kinetic studies of in vitro microsomal metabolism of lidocaine indicated that the apparent Km and Vmax for aromatic hydroxylation were smaller than those for N-deethylation. Lineweaver—Burk plots of the N-deethylation of lidocaine and those of its two primary metabolites indicated that at least two isozymes are taking part in these reactions. In in vivo lidocaine pharmacokinetics, the area under the blood concentration—time curve for the monodeethylated product, ω-ethylamino-2,6-dimethylacetanilide (1), varied considerably depending on the route of administration.

A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells

A highly sensitive and rapid assay for the detection of 6-mercaptopurine metabolites in the red blood cells of leukemic patients receiving the drug has been developed. The method employs a batch-chromatographic procedure using a mercurial cellulose resin to selectively absorb thiol compounds combined with separation by high-performance liquid chromatography using a Partisil-SAX column and uv detection. This method permits detection of 6-thioinosine monophosphate, 6-thiouric acid, and 6-thioguanosine mono-, di-, and triphosphates in patient samples with a sensitivity of 5–10 pmol. No 6-thioinosine di- or triphosphates were detected in patient samples. The results of our study indicate that 6-thioguanosine triphosphate is the major metabolite of 6-mercaptopurine retained by red blood cells after oral or iv administration of the drug.