Radial Compression Separation System Research Articles

Simultaneous determination of CGP 22 848 and its major metabolite (free carboxylic acid) CGP 35 652 in plasma by HPLC

A specific method for the quantitative determination of CGP 22 848 (a basic compound) and its major metabolite CGP 35 652 (a free carboxylic acid metabolite) in plasma using CGP 17 582 as internal standard is described. Separation of components is by reversed-phase chromatography using a mobile phase consisting of pH 3 phosphate buffer-acetonitrile (76:24, v/v) at a flow-rate of 1 ml/min in conjunction with a Radialpak C18 cartridge combined with a radial compression separation system or by using a mobile phase consisting of pH 3 phosphate buffer-acetonitrile (80:20, v/v) at a flow-rate of 0.8 ml/min in conjunction with a Novapak C18 column. Rapid extraction of CGP 22 848 and CGP 35 652 from plasma is achieved using reversed-phase C18 columns (Bond-Elut or Supelco). With 250 µl of plasma, concentrations down to 0.078 µM (25 ng/ml) of CGP 22 848 B and 0.326 µM (100 ng/ml) of CGP 35 652 A can be determined. A wavelength of 221 nm was used to monitor CGP 22 848, 280 nm for CGP 35 652 and 320 nm for the internal standard.

Direct, simultaneous measurement of chloramphenicol and its monosuccinate ester in micro-samples of plasma by radial-compression liquid chromatography.

A simple method of simultaneous analysis for chloramphenicol and chloramphenicol succinate in 10-microL samples of plasma is described. We injected the plasma samples directly into a radial-compression liquid chromatograph equipped with a precolumn module and a C18 insert. A mixture of acetic acid solution (pH 3)/acetonitrile (75/25, by vol) was used as mobile phase, at a flow rate of 4 mL/min. We separated the compounds in a 10-micron (particle size) C18 cartridge with a radial compression separation system and detected them in the effluent at 280 nm. The peak height for both compounds was linearly (r greater than 0.9993) related to concentration over the range investigated, 1-50 mg/L. We also performed the analysis with use of an internal standard (methylprednisolone) and obtained equally good results (r greater than 0.9995). We observed no interference from other antibiotics or drugs in the assay, and the inter- and intra-run precision at different concentrations was good (CV, 0 to 5.6%). We analyzed microsamples of plasma from an infant treated for meningitis with chloramphenicol sodium succinate intravenously. Total analysis time for each sample was less than 8 min.

Improved assay for etoposide in plasma by radial-compression liquid chromatography with electrochemical detection.

We describe a rapid, sensitive, and expedient radial-compression liquid-chromatographic method for routine quantification of etoposide (VP-16-213) in plasma of cancer patients undergoing chemotherapy with this agent. Teniposide was used as the internal standard. The mobile phase was a mixture of 10 mmol/L phosphate solution and methanol (50.7/49.3, by vol), adjusted to pH 3 with phosphoric acid. The flow rate was 2 mL/min. We used a 5-micron particle size C18 cartridge with a radial-compression separation system. The effluent was monitored with an electrochemical detector. Plasma samples are extracted with chloroform and the chloroform is evaporated. The residue, reconstituted in mobile phase, is injected onto the cartridge. Assay results were linearly related to concentration (r greater than 0.9944) over the range examined, 0.010-28.0 mg/L. The intra-run CV was less than 4.4%, and we encountered no interference from anticancer drugs or other compounds. We present data for plasma sampled at different intervals from two cancer patients being infused with etoposide.

Analysis of n-hexane, 2-hexanone, 2,5-hexanedione, and related chemicals by capillary gas chromatography and high-performance liquid chromatography

Analytical methods, using capillary gas chromatography and normal-phase high-performance liquid chromatography, were developed for the analysis of the neurotoxic chemicals n-hexane, 2-hexanone, and 2,5-hexanedione and their suspected metabolites. Two gas chromatographic methods, using a 50-m glass capillary OV 101 column and cyclohexane as an internal standard, were employed. In both methods, the injector and detector temperatures were 220 and 280°C, respectively. In method I the following temperature program was used: isothermic at 50°C for 30 min, followed by a temperature increase of 10°C/min to a final temperature of 180°C, which was then maintained for 7 min. This method was used to analyze the following compounds: n-hexane, 2,5-dimethylfuran, 2-hexanone, 3-hexanone, hexanal, 1-hexanol, 2-hexanol, 3-hexanol, 5-hydroxy-2-hexanone, γ-valerolactone, 2,5-hexanedione, and 2,5-hexanediol. Method II, which was developed for n-hexane and eight of its more common metabolites, used the following temperature program: isothermic at 70°C for 15 min, followed by a temperature increase of 40°C/min to a final temperature of 220°C, which was maintained for 5 min. A linear relationship between peak area and amount injected was observed over a 100-fold range. The minimum detectable amounts ranged from 0.05 to 1 μg, depending on the compound. Normal-phase HPLC, using a 5-μm silica cartridge fitted into an RCM-100 radial-compression separation system, was utilized to analyze 2-hexanone and its metabolites 2,5-dimethylfuran, γ-valerolactone, 5-hydroxy-2-hexanone, and 2,5-hexanedione. The mobile phase was a linear gradient of 3–55% 2-propanol in n-hexane in a period of 1 min at a solvent delivery rate of 0.8 ml/min. Compounds were detected by monitoring the ultraviolet absorbance, at 254 nm, of the column eluates and quantitation was achieved by measurement of the peak heights. This method showed linearity over a 100-fold range. The minimum detectable amounts ranged from 2–10 μg depending on the compound. These methods were tested by analysis of chicken plasma to which a mixture of the chemicals was added. The recoveries ranged from 30 to 70% depending on the compound.

Rapid Analysis of Doxorubicin in Plasma by Radial Compression Liquid Chromatography

A reversed-phase liquid chromatographic method for the analysis of doxorubicin (Adriamycin) in plasma is described. The analysis was performed on a radial compression separation system with a 10-μm phenyl cartridge. The mobile phase consisted of methanol:acetonitrile:0.001M tetrabutylammonium phosphate (36.4:8.6:55) adjusted to pH 3.5 with 85% phosphoric acid. The flow rate was 2.8mL/min. Fluorometric detection at excitation and emission wavelengths of 478 and 594 nm, respectively, was used. C18 sample cleanup cartridges were utilized for extraction. A doxorubicin-related compound, 4-demethoxydaunorubicin (IMI 30), was used as an internal standard. The retention times for doxorubicin and IMI 30 were 3.34 and 7.21 min, respectively. The assay was highly linear (r>0.997) in the concentration range utilized in this study (i.e., 10–5,000ng/mL). No interferences from doxorubicin metabolites or other anticancer drugs were observed. The assay is currently used for monitoring doxorubicin levels in high-risk patients.

Determination of propranolol in plasma by radial compression liquid chromatography with fluorometric detection.

This radial compression liquid-chromatographic assay for propranolol in plasma is rapid, reproducible, and suitable for use in routine monitoring. A 10-micron particle, 8 mm X 10 cm CN cartridge is used in conjunction with a radial compression separation system. The mobile phase is monobasic sodium phosphate (pH 3) solution/methanol/acetonitrile (760/84/156 by vol), the flow rate 6 mL/min. Propranolol was detected by use of a spectrofluorometer equipped with a 20-microL flow-through cell, at excitation and emission wavelengths of 250 and 336 nm. The retention times for propranolol and metoprolol (the internal standard) are 3.13 and 1.42 min, respectively. A one-step extraction with chloroform yields "clean" chromatograms, with greater than 90% of the drug being analytically accounted for. Under these conditions, results are precise and accurate. Currently we are using this method to monitor propranolol in hypertensive neonates. Data on changes in the concentrations of propranolol in plasma with time are presented for one such patient.

A sensitive and simplified radioimmunoassay for 1,25-dihydroxyvitamin D 3

A sensitive radioimmunoassay system for 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] with an improved extraction procedure has been developed. Following one-step extraction and prepurification of 1,25(OH) 2D 3 by ‘Extrelut-1’ minicolumns final purification was achieved by high-performance liquid chromatography (HPLC) using a radial compression separation system equipped with a μPorasil cartridge. The HPLC method applied allows the purification of 4 extracts/h. Recovery of 1,25(OH) 2[ 3H]D 3 after HPLC was 77 ± 2.6% (mean ± sd, n = 51). Since the recovery of 1,25(OH) 2[ 3H]D 3 was very reproducible, addition of labelled steroid to each single serum sample for monitoring recovery was omitted. The sensitivity of the assay was 0.8 pg/tube resulting in a detection limit of 3 ng/1, when 1 ml of serum was extracted. Intra-assay and inter-assay coefficients of variation were 12% and 16.8%, respectively. Serum 1,25(OH) 2D 3 concentration in 30 normal subjects (mean age: 25 yr) was 55 ± 12 ng/1 ( mean ± SD) . In 55 elderly patients (mean age: 77 yr) the 1,25(OH) 2D 3 serum level was 32 ± 12 ng/1 ( mean ± SD) and in three patients with chronic renal failure on 1,25(OH) 2D 3 therapy 146 ± 67 ng/1 ( mean ± SD ). Patients with chronic renal failure had reduced 1,25(OH) 2D 3 serum

Liquid-chromatographic determination of indomethacin in blood from newborns with patent ductus arteriosus.

This rapid, accurate "high-performance" liquid-chromatographic procedure is intended for measuring indomethacin in serum from neonates who are receiving indomethacin for symptomatic patent ductus arteriosus. Indomethacin and an internal standard (flufenamic acid) are extracted from serum or plasma with chloroform or diethyl ether at pH 5.0. For the chromatography we used a Waters' Radial Compression Separation System (Radial-NOVA PAK C18 reversed-phase column) and a mobile phase of methanol/sodium acetate buffer (10 mmol/L, pH 3.6), 70/30 by vol. The column effluent is monitored at 254 nm. Both indomethacin and flufenamic acid are eluted within 7 min. Indomethacin can be detected in concentrations as low as 50 micrograms/L, in 100-microL samples. Response varies linearly with indomethacin concentration to at least 2 mg/L. Analytical recovery is 75%; relative recovery is 100%. Precision is excellent. Using this method, we were able to improve the success rate for pharmacological management of symptomatic patent ductus arteriosus, especially in neonates with fast clearance rates for the drug.

Liquid Chromatographic Cleanup and Determination of Low Levels of Vitamin D3 in Sheep Plasma

A liquid chromatographic (LC) method is described for the determination of vitamin D3 in sheep plasma. Samples are extracted by one of 2 different methods, depending on the concentration of vitamin D3. The samples are purified by using either a Sep-Pak silica cartridge or a small alumina column, followed by additional cleanup on a Metalsorb LC column. Final analysis was carried out on a 5 micron C18 column using a radial compression separation system with an acetonitrile-methanol solvent system. Vitamin D3 was completely resolved from any interfering compounds in the plasma; total run time was less than 15 min, using a variable wavelength detector set at 264 nm. The method was successfully applied to samples at levels of 1-10 ng added vitamin D3 mL sheep plasma, with recoveries in the range 90-97%.

High-performance liquid chromatographic analysis on radial compression column of the neurotoxic tri- o-cresyl phosphate and metabolites

A method utilizing high-performance liquid chromatography (HPLC) has been developed for the analysis of tri- o-cresyl phosphate (TOCP) and its possible metabolites, o-cresyl dihydrogen phosphate, di- o-cresyl hydrogen phosphate, o-hydroxybenzyl alcohol, o-cresol, saligenin cyclic- o-tolyl phosphate [2-( o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one], salicylic acid, salicylaldehyde, hydroxymethyl TOCP (di- o-cresyl o-hydroxymethylphenyl phosphate), and dihydroxymethyl TOCP ( o-cresyl di- o-hydroxymethylphenyl phosphate). TOCP and its possible metabolites were analyzed on a reverse-phase C 18 cartridge fitted into RCM-100 radial-compression separation system. Compounds were separated using a linear gradient of 25 to 80% acetonitrile in 2% aqueous acetic acid at a flow rate of 1.3 ml/min in a period of 22 min. Quantification was achieved by monitoring the ultraviolet absorbance of the column eluates at 254 nm and measuring peak areas. Retention times and peak areas were highly reproducible for all compounds analyzed. The relationship between peak area and amount injected was linear over a 100-fold range for all compounds analyzed. The minimum detectable level was 3 ng for salicylaldehyde, 25 ng for o-hydroxybenzyl alcohol and salicylic acid, and 50 ng for the remaining compounds. A mixture of TOCP and its possible metabolites was added to samples of cat liver, kidney, and plasma and then extracted and analyzed. High recovery and reproducibility for most compounds was observed in tissues analyzed.

Analysis of Insect Hormones by Means of a Radial Compression Separation System

Abstract The use of a radial compression separation system for the analysis of insect hormones is described. By simple isocratic elution, both steroid molting hormones and terpenoid juvenile hormones are rapidly separated. The system is used to analyze the metabolism of juvenile hormone by an established cell line of Drosophila melanogaster.

High Pressure Liquid Chromatographic Determination of Zearalenone in Chicken Tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.

High Pressure Liquid Chromatographic Determination of Aflatoxins by Using Radial Compression Separation

A rapid method for the determination of aflatoxins was developed using high pressure liquid chromatography and a radial compression separation system. A standard solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed at flow rates of 2.0 and 6.0 mL/min. Retention times, peak heights, and peak areas were reproducible over a 3-day period. Coefficients of variation for aflatoxin B1 at 2.0 and 6.0 mL/min were, respectively, 1.04 and 0.87% (retention time); 2.9 and 4.7% (peak height); and 8.2 and 4.7% (peak area). At 6.0 mL/min there was an approximate 25% loss in sensitivity but a greater than 50% reduction in retention time. Separation of all the aflatoxins was excellent using a dual flow rate of 2.0 mL/min with a change to 8.0 mL/min at 15 min post-injection. The applicability of the radial compression separation system for the rapid determination of aflatoxins in human tissues was also tested. Spiked samples of liver, serum, and urine-showed good resolution of all aflatoxin peaks at the higher flow rates.

A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system

A new HPLC method for analyzing conjugated bile acids in bile and especially in serum is presented. For separation a novel radial compression system with a 10-cm flexible-walled reverse phase column (RCM-100 Module, Waters Ass.) packed with μ-Bondapak C 18 was used. Without any laborious hydrolysis and derivatization steps, 10 conjugated bile acids are separated easily with a mobile phase of methanol/0.01 mol/l KH 2PO 4 (150:50, v/v; pH 6.0) in about 30 min, determined with a variable UV detector at 200 nm and a flow rate of only 0.5 ml/min. Dexamethasone was used as internal standard. Conjugated bile acids are extracted from serum with Sep-pak C 18 cartridges after a 1:8 dilution with a mixture of the mobile phase and 0.2 mol/l NaOH (6:8, v/v). Extraction from bile was also performed using Sep-pak cartridges after a 1:20 dilution with 0.5 mol/l phosphate buffer (pH 7.0). The complete analysis time of the method, with Sep-pak extraction, is less than one hour. Recoveries for the different conjugated bile acids varied from 89.4% to 96.7% in serum. The HPLC method for serum and bile was validated for the primary bile acids cholate and chenodeoxycholate against the previously described GLC method. Agreement between the two methods was excellent. Duplicate analyses in serum and bile samples also showed that the reproducibility was good.

A high pressure liquid chromatographic method for the analysis of zearalenone in chicken blood.

A high pressure liquid chromatographic (HPLC) method is described to determine zearalenone in chicken blood. Samples are extracted with acetonitrile, followed by a hexane cleanup procedure and extracted further with ethyl acetate. The analysis of zearalenone is by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector and a mobile phase of acetonitrile-water 60:40 (v/v). Recoveries of zearalenone in blood at levels of 50-200 ng/ml are in the range of 66.8-72.6%.

High Pressure Liquid Chromatographic Determination and Fluorescence Detection of Aflatoxins in Corn and Dairy Feeds

A high pressure liquid chromatographic (HPLC) method is described for the determination of aflatoxins B1, B2, G1, and G2 in animal feeds at levels as low as 2.5 ppb. Samples are extracted with acetonitrile-water and initially purified by using a Sep-Pak silica cartridge. The aflatoxins are then reacted with trifluoracetic acid and acetonitrile-water (1 + 1). After filtration, the aflatoxins are completely resolved on a 10 micrometer C18 column using a radial compression separation system with an acetonitrile-water solvent system. Aflatoxins G2a, B2a, G2, and B2 were reported in less than 30 min, using fluorescence detection. The method was successfully applied to samples at levels of 20-2.5 ppb added aflatoxins with recoveries in the range of 82-99%.

Hypnotic-Sedative Screen by High Performance Liquid Chromatography

Abstract In this procedure some of the most commonly abused sedatives and hypnotics can be identified and quantitated using 100 ul of serum. Ethosuximide, primidone, methyprylon, phenobarbital, butabarbital, butalbital, glutethimide, mephobarbital, phenytoin, carbamazepine, secobarbital, phensuximide, pentobarbital, amobarbital, and methaqualone are extracted into a chloroform-ethanol solvent containing either 5-(p-methylphenyl)-5-phenyl-hydantoin or hexobarbital as an internal standard, evaporated to dryness, reconstituted with mobile phase and injected into a high performance liquid chromatograph in conjunction with a radial compression separation system. Peak heights are measured at 195 nm and 254 nm and sensitivity for all drugs is one μg/ml. Day to day precision obtained CV's ranging from 4.5 to 10.4%.