Determination of plasma tocopherols by high-performance liquid chromatography with coulometric detection

An HPLC procedure has been developed for tocopherol determination with coulometric detection in human serum samples. Eluent optimization and foreign peak identification (bilirubin) by mass spectrometry are described. An extraction procedure gave yields around 98% with 1.3% coefficient of variation, and the calibration ranged from 0.1 to 200 mg/l with a correlation coefficient of 0.999. The detection limit achieved for vitamin E was 60 pg (3 ng/ml).     

Quantitation of zimelidine and norzimelidine in plasma using high-performance liquid chromatography

A method is described for the quantitation of a new non-tricyclic antidepressant, zimelidine, and its pharmacologically active, N-demethylated metabolite, norzimelidine, in plasma. The method involves a single extraction of basified plasma with diethyl ether, concentration of the ethereal extract, chromatography on a high-performance liquid chromatograph and quantitation using a variable-wavelength UV detector.The respective geometric isomers of zimelidine and norzimelidine are used as internal standards for quantitation. Resolution is affected using 5-μm silica gel column with an aqueous methanolic solution of ammonium nitrate as the mobile phase. The minimum quantitated amount was 25 ng and the coefficient of variation for the method did not exceed 7% in the range 25 to 1000 ng/ml for both compounds. The method has been applied in monitoring the plasma concentration of zimelidine and norzimelidine in plasma from depressed patients and an example of this application is presented.     

Quantitation of individual molecular species of phosphatidylcholines by reversed-phase high-performance liquid chromatography with fluorometric detection

The multiplicity of phosphatidylcholines is caused by the presence of different pairs of fatty acids in their individual molecular species and at least 27 miscellaneous fatty acids were identified in phosphatidylcholines in the serum of healthy individuals by combined gas–liquid chromatography and mass spectrometry in our present experiments. A method is described for the separation and quantitation of molecular species of phosphatidylcholine in human serum. Total phosphatidylcholine is isolated from lipids extracted from the serum with chloroform–methanol (2:1) by reversed-phase liquid–liquid extraction and subjected to reversed-phase high-performance liquid chromatography with a discontinuous descending gradient of water. Separation is monitored by fluorometry (340/460 nm) and absorption at 205 nm, if required. Up to 25 different molecular species of phosphatidylcholine may be quantified with a satisfactory reproducibility (±5–8%). Data on the distribution of individual molecular species in phosphatidylcholine of 53 normal serums are presented. The method may be used for quantitation of these phospholipids also in other biological materials (cell lines, leukemic cells from patients), and on a micropreparative scale to isolate individual compounds. The speed of separation as well as a satisfactory reproducibility are its principal advantages.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Quantitation of harringtonine and homoharringtonine in serum by high-performance liquid chromatography

Harringtonine and homoharringtonine are naturally occurring alkaloids with demonstrated antineoplastic activity against certain types of leukemias in cell cultures, experimental animals, and initial clinical trials. Sample preparation consists of addition of the internal standard (one compound used as the internal standard for the other), solvent extraction with methylene chloride, washing with ammonium formate, and evaporation to dryness. The residue is dissolved in the mobile phase (40% methanol—60% 0.1M ammonium formate) and an aliquot is chromatographed on μC₁₈ reversed-phase column (flow-rate 1.5 ml/min). Peaks are detected with a spectrophotofluorimeter by monitoring the emission at 320 nm with excitation wavelength of 280 nm. Limit of detection is 10 ng/ml (20 nM) for both compounds; reproducible quantitation can be made to 30 ng/ml (60 nM).     

Quantitation of melphalan in plasma of patients by reversed-phase high-performance liquid chromatography with electrochemical detection

An analytical procedure for the separation and determination of melphalan in human plasma was carried out. A simple high-performance liquid chromatographic method with electrochemical detection was developed taking advantage of the high sensitivity of the electrode redox reaction. The sample pretreatment consisted of a direct extraction of the interferents rather than of melphalan, owing to the difficulty of extraction of the drug, and was very simple, rapid and reproducible.     

Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.     

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.     

Rapid Quantitation of desmosine content in tissue hydrolysates by high-performance liquid chromatography

The lysine-derived crosslinks in elastin, desmosine, and isodesmosine, are quantitated in tissue hydrolysates by monitoring high-performance liquid chromatography eluents at 275 nm. The results from this method compare favorably with results from the amino acid analyzer. However, this more sensitive method (1) eliminates ninhydrin-positive artifacts which elute with the desmosines from some tissue hydrolysates on the amino acid analyzer, and (2) makes possible elastin quantitation in a tissue with the minimum amount of manipulations.     

Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography

This work describes the application of the fully automated high-performance liquid chromatographic system to the analysis of PCR-amplified products. Efficient separations of both DNA restriction fragments and PCR products were performed using an anion-exchange DEAE-NPR column, packed with 2.5-μm nonporous particles. The automated HPLC method was employed for the separation, quantitation, and purification of PCR products in less than 10 min in a single step.     

Quantitation of dimethyl sulfoxide in solutions and tissues by high-performance liquid chromatography.

We have developed a rapid and simple method to determine the level of dimethyl sulfoxide (Me2SO) in both solutions and tissue samples. For analysis of Me2SO in a cryopreservation medium, the solution is simply diluted in 10% (vol/vol) methanol and centrifuged. Then an aliquot of the supernatant is assayed by high-performance liquid chromatography. For tissue samples, the wet weight is measured and the intact sample is extracted with 10% (vol/vol) methanol (e.g., 10 ml/g wet wt) in a sealed vial. The extract is then diluted and centrifuged, and an aliquot of the supernatant is assayed. The dry weight of the tissue is measured after the methanol-extracted sample is placed into either for 2 h and air-dried overnight. The water content of the tissue is calculated as the difference between the wet and the dry weights. The concentration of Me2SO in the aqueous compartment of the tissue can then be calculated by taking into account the concentration of Me2SO in the extract and the dilution factor, based on the tissue water volume and the volume of methanol used to extract the Me2SO. The calculated values for porcine myocardium samples correlated 1:1 with the actual Me2SO concentrations in the solutions in which the tissue samples were equilibrated. Finally, we present results documenting the usefulness of this assay by following the time course of Me2SO penetration into core versus peripheral regions of 1-cm3 samples of porcine myocardium.     

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with μBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.     

Determination of medrogestone in plasma by high-performance liquid chromatography

Interference with the UV absorbance of medrogestone by endogenous steroids in plasma was prevented by reacting plasma with oxalyl chloride. The reduction of interference was effective when oxalyl chloride was in the range 10–50 μl/ml plasma. Reaction of oxalyl chloride with plasma for 10 min could reduce interference approximately 5.5-fold, and there was no significant reduction after 30 min. The limit of quantitative concentration for medrogestone in HPLC was 1 ng/ml. The standard curves were linear with the correlation coefficient greater than 0.999 in the range of 1–30 ng/ml. The coefficients of variation of both intra- and inter-day mean values were <12% and <10% of the actual values, respectively. The developed method for plasma sample preparation and the evaluated HPLC condition were further applied to an in vivo pharmacokinetic study.