Automated solid-phase extraction and liquid chromatographic method for retinoid determination in biological samples

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was < 11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.     

Solid-phase extraction of arginine vasotocin and isotocin in fish samples and subsequent gradient reversed-phase high-performance liquid chromatographic separation

Gradient high-performance liquid chromatography (HPLC) preceded by a solid-phase extraction (SPE) step is described for determining arginine vasotocin and isotocin, the neurohypophysial nonapeptides, in fish plasma samples. The combination of these two methods significantly improves the separation and increases the sensitivity of the assay. The proposed assay may be a useful alternative for analysis of similar nonapeptides in plasma without the use of radioisotopes, while taking into consideration a difference in detection sensitivity.     

Solid-phase extraction or liquid chromatography coupled on-line with gas chromatography in the analysis of biological samples

This review provides an overview of the on-line coupling of solid-phase extraction or liquid chromatography with gas chromatography for the analysis of biological samples. Principles relevant to techniques are briefly presented and selected applications are described. Benefits of the coupled systems are discussed.     

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.     

High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

We describe a rapid, reverse-phase HPLC procedure for separating and quantifying tetrapyrroles of biological interest. This procedure uses a 5-micron C18 column and the mobile phase is ammonium phosphate (pH 3.5) with a methanol gradient that is increased from 61 to 100%. Detection is by absorbance at 405 nm or by fluorescence. Porphyrins, heme, and the heme breakdown products, biliverdin and bilirubin, can be separated from a single injection in 25 min. Injections can be made every 40 min. Limits of detection are about 0.1 pmol for porphyrins, 5 pmol for heme, and 10 pmol for biliverdin and bilirubin. We present examples of the use of the system for separating tetrapyrroles formed by primary cultures of chick embryo hepatocytes and homogenates of rat liver.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

High-performance liquid chromatographic analysis of cytosine arabinoside and metabolites in biological samples

A rapid, non-radioactive method to quantitate therapeutically realistic levels of 1-β- -arabinofuranosylcytosine (Ara-C) and its metabolites would be useful both in the clinic, for monitoring drug levels, and in the laboratory for correlating drug levels with cellular and molecular perturbations. Liquid chromatographic analysis of arabinose-nucleoside analogs in biological samples is complicated by the presence of interfering nucleosides and nucleotides. We report the development of two analytic procedures to measure Ara-C and metabolite levels in biological samples. One method uses a quaternary ammonium type anion-exchange resin to achieve isocratic separation in less than one hour. The second method utilizes a boronate-derivatized polyacrylamide column which binds cis-diols to selectively retain cytosine and uridine, while arabinose compounds are eluted with recovery approaching 100%. The eluted compounds are then easily quantitated on a reversed-phase C₁₅ column. The sensitivity of both procedures was sufficient to obtain pharmacokinetic data on Ara-C and uracil-arabinose levels in serum and urine and on Ara-C triphosphate levels in tumor cells.     

Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples

A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.     

Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum

A new method is described using solid-phase extraction (SPE) for preconcentration of articaine and the metabolite articainic acid and high-performance liquid chromatography (HPLC) for the determination of both compounds in human serum. Articaine and articainic acid were extracted in one step with SDB-RPS disk cartridges after precipitation of the serum proteins by perchloric acid. The HPLC separation was then performed on a reversed-phase C₈ column using phosphate buffer–acetonitrile (88:12, v/v). UV absorption at 274 nm was used for measuring the analytes with a low limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies of low dose submucosal injections of the local anaesthetic agent articaine hydrochloride in dentistry.     

High-performance liquid chromatographic analysis of 2',3'-dideoxyinosine in biological samples

A high-performance liquid chromatographic analysis for the anti-AIDS drug 2',3'-dideoxyinosine (ddI) in rat plasma and urine, with a limit of detection of 0.2 μg/ml and requiring a sample size of 100 μl is described. Diluted plasma or urine samples were extracted using a C₁₈ solid-phase extraction column. Retention of ddI on more polar solid-phase extraction columns was insufficient for sample clean-up. This method is useful for pharmacokinetic studies of ddI in small rodents.     

High-performance liquid chromatographic determination of histamine in biological samples after derivation with o-phthalaldehyde

A high-performance liquid chromatographic (HPLC) method for the determination of histamine in tissues, based on precolumn derivation with o-phthalaldehyde, is described. Trichloroacetic acid extracts of rat brain, but not of rat stomach or of rat peritoneal mast cells, had to be cleaned-up by a chromatographic step before HPLC. The extracts were allowed to react with o-phthalaldehyde at pH 12.5 and -20 degrees C for 12 h, followed by acidification to pH 2.0. HPLC was performed on a reverse-phase column with isocratic elution using sulfuric acid in methanol as solvent system. A fluorescence detection system was used; excitation was set at 353 nm and emission was read at 451 nm. One chromatographic run was completed in 20 min. The detection limit with the conventional procedure was 1.5 ng histamine per sample, with a scaled-down procedure it was 250 pg per sample. With extracts of rat gastric mucosa the within-run variation was 2.7% and the day-to-day variation 4.5%.     

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples

The ascorbic acid (AA)—dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

Validated liquid chromatographic ultraviolet method for the quantitation of Etoricoxib in human plasma using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (284 nm) was developed and validated for quantitation of Etoricoxib in human plasma, the newest addition to the group of nonsteroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. Following a single-step liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v), the analyte and internal standard (Zaleplon) were separated using an isocratic mobile phase of water/acetonitrile (58/42, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 5 ng/mL, with a relative standard deviation of less than 20%. A linear range of 5-2500 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 4.1-5.1% and 1.1-2.4%, respectively. The between- and within-batch bias was -3.8-4.7% and -0.6-9.4%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Etoricoxib in plasma was >90%, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive and simple with between-batch precision of <6% and was used in pharmacokinetic studies.     

Rapid and sensitive pre-column extraction high-performance liquid chromatographic assay for propofol in biological fluids

A completely automated high-performance liquid chromatographic system is described for the determination of the phenolic anaesthetic propofol. The method is based on pre-column extraction in a closed system allowing direct injection of biological samples without any sample pretreatment. The assay is sensitive (limit of quantification is 5 ng/ml serum), reliable (the variability within a series is 2%) and rapid (results are available after 6 min).     

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.