In this precise, accurate, and specific liquid-chromatographic procedure for determining progesterone in serum, the serum is diluted 10-fold with water/methanol (65/35 by vol), and the progesterone is extracted from the sample by passage through a column of graphitized carbon black (Carbopack B, Supelco). After washing the column, we elute the progesterone with chloroform/methanol (90/10 by vol...

We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a...

We describe a simple method for extraction of estrogens that is efficient over the whole polarity range of estrogen metabolites in urine and plasma. A small reversed-phase C18 cartridge (Sep-Pak C18, Waters Associates, Inc.) is used for this purpose. After application of the biological fluid, followed by an aqueous wash, estrogens are eluted in 3 mL of methanol. For urine the pH should be kept ...

A simple and sensitive high performance liquid chromatographic method with electrochemical detection is described for the quantitative determination of the amoxycillin and clavulanic acid in pharmaceutical dosage forms. Separation of sample components occurs on a reversed phase C18 column with a mobile phase consisting of methanol-water-phosphate buffer. For the amperometric detection, the pote...

Sirolimus (rapamycin) has a macrolide structure and is under clinical investigation as an immunosuppressant after organ transplantation. An HPLC/mass spectrometry assay to quantify sirolimus in blood was developed. 28-O-Acetyl sirolimus was used as internal standard. Blood samples were extracted with C18 columns. The extracts were injected into an HPLC system and isocratically eluted with metha...

Poor peak shape Wrong silica type Use type B silica Blocked frit or column void Replace frit, backflush column Silanol interactions Use amine additives, change pH, use end-capped stationary phase Excessive peak width Bad column Replace column Column overload Reduce injection volume or mass High molecular weight Normal Unresolved peaks Improve separation Inadequate retention Mobile phase too str...

A selective and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS/MS) has been developed and validated for the quantification of zileuton in human plasma. Deuterated internal standard (zileuton D4) was used as the internal standard (ISTD). Zileuton was extracted by liquid-liquid extraction using methyl tert-butyl ether and separated by isocratic elution on a C18 column (10...

To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine...

Neutral glycosphingolipids were purified from human senile cataractous lenses by a combination of solvent extraction, Folch's partition, acetylation, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major glycosphingolipids (A-F) from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis, secondary ion-mass spectrometry,...