Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.     

Heterogeneity of human very low density lipoproteins by gel filtration chromatography

Very low density lipoproteins were separated by gel filtration on Sepharose 4B. A decrease in mean particle diameter and flotation rate was seen with increasing elution volumes. The smaller lipoproteins had relatively more protein and phospholipid and less triglyceride than the larger ones. No differences were noted in the relative contents of the various phospholipids or partial glycerides between small and large lipoproteins. Fatty acid patterns of triglycerides and cholesteryl esters were also similar for the various lipoproteins. Relatively more lecithin containing linoleoyl acyl groups was found in smaller lipoproteins of some subjects. More of the protein of smaller lipoproteins was apo-LDL protein. Apo-HDL peptide was lost from the very low density lipoprotein as a consequence of the gel filtration.     

Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography

This report describes a rapid, simple and sensitive isocratic high-performance liquid chromatography with diode array UV detection for micro-sample analysis of paclitaxel in mouse plasma. The analysis utilized a Capcell-pak octadecyl analytical column and a mobile phase consisting of acetonitrile–0.1% phosphoric acid in deionized water (55:45, v/v). Paclitaxel and n-hexyl p-hydroxybenzoic acid (internal standard) were extracted from plasma by one-step extraction with tert.-butyl methyl ether. Peak purity was determined over a UV wavelength range of 200 to 400 nm. Paclitaxel and the internal standard were eluted at 3.4 min and 5.4 min, respectively, at a mobile phase flow-rate of 1.3 ml/min. No interfering peaks were observed and the total run time was 10 min. The standard curve was linear (r=0.9999) over the concentration range of 0.010–500 μg/ml. The extraction recovery was >90% for both paclitaxel and n-hexyl p-hydroxybenzoic acid. The intra- and inter-day assay variabilities of paclitaxel ranged from 0.4 to 2.2% and 0.6 to 7.8%, respectively. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a plasma sample volume of 100 μl. This highly sensitive and simple assay method was successfully applied to a pharmacokinetic study after i.v. administration of paclitaxel 20 mg/kg to mice.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Rapid method for the determination of piroxicam in rat plasma using high-performance liquid chromatography

A previously published method was used for the determination of piroxicam in plasma samples obtained from rat. The sample preparation involved liquid extraction, centrifugation and evaporation. Separation of piroxicam from internal standard occurred on a reversed-phase C₁₈ column with a mobile phase consisting of methanol-phosphate buffer pH 2 (45:55). The detection limit of the assay was 0.02–20 μg/ml. The assay linearity was good (typically r = 0.9992). The method was applied for determination of piroxicam in rats after administration of an oral dose of 2 mg/kg piroxicam.     

Separation and size determination of human serum lipoproteins by agarose gel filtration

A method is described for the separation of the three major classes of human serum lipoproteins by gel filtration on columns of 4 and 6% agarose gel. After calibration of the columns, the elution volumes of the lipoproteins were used to calculate the molecular sizes and molecular weights of these macromolecules. The technique was employed to demonstrate aggregation of low density lipoprotein following partial delipidation, partial proteolysis, or mild heat denaturation. Agarose gel filtration shows promise as a useful method for the isolation, purification, and characterization of lipoproteins.     

Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm x 4 mm, 5 microm particles), the mobile phase consisted of acetonitrile -15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 μg/ml of succinylcholine. In a pilot study the plasma concentration—time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Rapid analytical gel filtration chromatography. 3. Apparent molecular weight distribution of peptides produced by proteolysis

A rapid gel filtration chromatography method is described for determination of the molecular weight distribution (MWD) of peptide mixtures by using calibrated Sephadex microbore columns. The method was applied to MWD analysis of peptide mixtures resulting from trypsin and pepsin digestion of glycinin—the major soybean storage protein—under different incubation conditions of pH, temperature, and time of hydrolysis. Possible sources of errors and suggestions for improvement are discussed.     

Improved method for the determination of serotonin in plasma by high-performance liquid chromatography using on-line sample pre-treatment

An improved method for the determination of serotonin in platelet-rich plasma (PRP) and platelet-poor plasma (PPP), by reversed-phase high-performance liquid chromatograpy with electrochemical detection and direct plasma injection, is described. The chromatographic system comprises a strong cation-exchange pre-column and a C₁₈ analytical column. The method is selective, rapid, simple and sensitive, and offers good reproducibility and recovery. Reference values for serotonin concentrations in healthy adults (n = 10) are 31 nM for PPP and 6 nmol per 10⁹ platelets for PRP. The conditions used for the preparation of PRP and PPP may influence the serotonin concentration in PRP.     

Rapid determination of tetracycline and lumecycline in human plasma and urine using high-performance liquid chromatography

A rapid and accurate method for the determination of tetracycline in human plasma and urine is presented. Determination of tetracycline in plasma is based on precipitation of plasma proteins with trifluoroacetic acid, followed by injection of the centrifuged plasma sample onto a μBondapak C₁₈ column. Acetonitrile in phosphate buffer pH 2.2 is used as mobile phase. Only tetracycline, and no trace of lumecycline can be detected in plasma and urine after administration of lumecycline, indicating that lumecycline is completely degraded to tetracycline, lysine and formaldehyde in the gastrointestinal tract prior to absorption.Determination of tetracycline in urine was performed by injection of urine diluted with phosphoric acid onto a μBondapak Phenyl column. The precision of determination of tetracycline in plasma, expressed as the relative standard deviation, was < 3% at tetracycline concentrations of 0.05 and 3.7 μg/ml. Urine determinations were made with a precision of < 1.5% at tetracycline concentrations of 0.5 and 6.7 μg/ml.