C. Lucarelli

Improvements in automated analysis of catecholamine and related metabolites in biological samples by column-switching high-performance liquid chromatography☆

Previously two fully automated methods based on column switching and high-performance liquid chromatography have been described, one for plasma and urinary catecholamines and the other for catecholamine urinary metabolites. Improvements in these methods, after 3 years of routine application, are now reported. The sample processing scheme was changed in order to eliminate memory effects and, in the procedure for plasma catecholamines, a pre-analytical deproteinization step was added which enhances the analytical column lifetime. The applied voltages for the electrochemical detector have been optimized, resulting in an automated method, suitable for the simultaneous determination of vanillylmandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid. The sensitivity of the methods allows the detection of 2-3 ng/l of plasma catecholamines and 0.01-0.06 mg/l of urinary metabolites. Also, it is possible to switch from one method to the other in only 30 min. The normal values obtained from 200 healthy people are reported, together with a list of 57 potential interfering substances tested.

High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive disease. Assessment of total plasma concentration of homocysteine (tHcys) requires accurate and reproducible measurements. The aim of this study was to test a rapid isocratic HPLC method for tHcys analysis with an internal standard (I.S.) of alpha-mercaptopropionylglycine (MPG), 2-mercaptoethylamine (ME), or N-acetylcysteine (NAC) or without I.S., and to verify whether the use of an I.S. improves the precision. The method without I.S. showed an excellent linearity (y = 1.59x - 0.15, r = 1), recovery (100%) and a within-assay relative standard deviation (R.S.D.) of 1.2%. Instead, in our hands, the presence of I.S.s decreased the reproducibility (within-assay R.S.D. ranged from 4.5 to 6.5%) and lengthened the chromatogram by up to four to five times. In conclusion, HPLC measurement of plasma tHcys without I.S. improves accuracy with respect to determination with I.S.; moreover, this approach allows to routinely process larger amounts of plasma samples.

Rapid and simple high-performance liquid chromatographic determination of tricyclic antidepressants for routine and emergency serum analysis

An isocratic reversed-phase high-performance liquid chromatographic procedure is presented for the simultaneous detection of desipramine, nortriptyline, imipramine, amitriptyline and clomipramine in serum. Drugs are extracted after sample alkalinization and separated from each other on an octyl reversed-phase with n-butylamine as mobile phase modifier. Detection is achieved at 254 nm. The recovery of tricyclic antidepressants (92-110%) has good precision, with a relative standard deviation of less than 5%. Being rapid and simple, the method is suitable for the emergency clinical laboratory.

Micro high-performance liquid chromatography for the determination of nicarbazin in chicken tissues, eggs, poultry feed and litter

Abstract A micro high-performance liquid chromatography (HPLC) method has been developed for the determination of the anti-coccidial drug nicarbazin in chicken tissues, eggs, poultry feeds and litter. The 4,4′-dinitrocarbanilide (DNC) component of nicarbazin was extracted from foods, feeds and litter with acetonitrile. The extracts were purified by liquid-liquid partitioning, evaporated to dryness and taken up in methanol-acetonitrile-water (50:30:20, v/v). Micro HPLC of the 4,4′-dinitrocarbanilide (DNC) portion of nicarbazin is performed isocratically using a small bore column (1 mm I.D.) packed with reversed-phase and a UV detector set at 340 nm. The average recoveries of nicarbazin added to muscle, liver and egg were 92.8, 84.3 and 85.2%, respectively, 95.9% in poultry feed and 76.8–95.9% in different litters. The limit of detection was 25 pg, based on a detector signal-to-noise ratio of 3. These results were achieved with a simplified step extraction without the solid-phase extraction used by various researchers. This method offers a sensitive, selective, rapid, and less expensive alternative to conventional HPLC for such evaluations.

HPLC method for the simultaneous analysis of valproic acid and other common anticonvulsant drugs in human plasma or serum

SummaryThe derivatizing procedure of Moody et al. [20] for valproic acid has been simplified and applied to the simultaneous HPLC determination of valproic acid (VPA), barbital (B), primidone (PRM), phenobarbital (PB) and carbamazepine (CBZ) in serum or plasma of epileptic patients. The sample is deproteinized with acetonitrile containing esterification agents and an aliquot of the supernatant is heated to 70°C for 15 min with 4-bromophenacyl bromide. The reaction mixture is analysed on a C18 column at ambient temperature, with gradient elution and with detection at 205 nm. The time required for the chromatographic analysis is 13 min; identification is based on retention time and quantification is by peak area determination with an internal standard. The calibration curves show good linearity in the range 6.25 to 100 mg/L. The detection limits at a signal: noise ratio ≥3, ranged from 1 mg/L for B and CBZ to 2–3 mg/L for PRM, PB and VPA. The method described for the simultaneous determination of the five drugs in the same plasma pool, correlated well with isocratic HPLC methods specific for each drug. The simultaneous procedure described allows a reproducible (CVs≤6.5% within run) and rapid (25 min for sample preparation: 13 min for chromatographic run) therapeutic monitoring of patients treated with VPA and two or more antiepileptic drugs.

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).

Simultaneous measurement of l-DOPA, its metabolites and carbidopa in plasma of Parkinsonian patients by improved sample pretreatment and high-performance liquid chromatographic determination☆

A procedure is described for the determination of L-3,4-dihydroxyphenylalanine (L-DOPA), its metabolites and carbidopa (CD) in plasma of Parkinsonian patients by high-performance liquid chromatography with dual working-electrode coulometric electrochemical detection. An efficient sample preparation scheme is presented for the isolation of L-DOPA, its metabolites and the catecholamines from the same plasma aliquot. After a simple deproteinization with methanol containing 2% of 0.5 M perchloric acid and evaporation of the solvent, L-DOPA, its metabolites and CD were separated with a 5-micron Nucleosil C18 column. Catecholamines were extracted from the supernatant of the deproteinized plasma by ion exchange on small columns and adsorption on alumina. Recoveries were close to 100% for L-DOPA, its metabolites and CD and 70% for catecholamines. The use of the same mobile phase for the concurrent assay of L-DOPA, its metabolites and catecholamines considerably increased the throughput of samples in the chromatographic system. The dual-electrode coulometric detector afforded peak identification by comparing current ratios. Monitoring of data from patients under L-DOPA therapy is reported.

Analytical strategy for the assessment of the protein glycation status in uremic patients by high-performance liquid chromatography

We propose a newly integrated procedure for the analysis of furosine (early glycation product) and pentosidine (glycoxidation end-product) in plasma proteins and the simultaneous assessment of advanced glycation end-product (AGE) peptides and free pentosidine in plasma. In order to determine furosine and protein-linked pentosidine, plasma proteins were hydrolyzed in 8 M HCl and each analyte was purified by solid-phase extraction. Furosine was determined by ion-pair RP-HPLC methodology with isocratic elution and spectrophotometric detection at 280 nm and pentosidine by ion-pair RP-HPLC by using gradient elution and fluorimetric detection at 335/385 nm. To assess free pentosidine concentration and simultaneously evaluate the AGE peptides, an aliquot of plasma sample was diluted and ultrafiltered by using Centricon 10 M(r) < or = 10,000) ultrafiltration membranes. Free pentosidine and AGE peptides were analysed by ion-pair RP-HPLC, by using gradient elution and fluorimetric detection at 385 nm upon excitation at 335 nm. The HPLC methodology has been successfully used for the determination of glycation and glycoxidation protein status in uremic patients.

Development and validation of a high-performance liquid chromatographic method for the determination of desmosines in tissues.

The development and the validation of a general strategy for the simple and accurate analysis of desmosines (isodesmosine and desmosine) in tissues coupled with the determination of collagen (as hydroxyproline) is described. The method is based on simplified sample (i.e., lung) pretreatment which involves, in a PTFE screw-capped Pyrex tube, homogenization, collagen extraction with hot 5% trichloroacetic acid and hydrolysis of the elastin-containing residue with 6 M hydrochloric acid, followed by cellulose minicolumn purification of desmosines from the hydrolysates, dansyl chloride pre-column derivatization of the purified desmosines and reversed-phase high-performance liquid chromatographic (HPLC) analysis of the dansyl derivatives using a Spherisorb ODS-2 column, an on-column enrichment sample device and a linear gradient of organic modifier (acetonitrile) in phosphate buffer. The simple sample pretreatment, the optimized chromatographic conditions and the short HPLC analysis time (less than 15 min) allow the accurate and rapid determination of desmosine and isodesmosine, thus permitting the determination of elastin in several kinds of tissues with a minimum of sample manipulation.

Development and use of carbon adsorbents in the liquid chromatographic separation of isomers

Abstract Porous carbonaceous sorbents, prepared by a replicate method on a silica template, were examined under the liquid chromatographic conditions. The carbons were obtained by pyrolysis of two different organic precursors (a phenol—formaldehyde resin and saccharose) at 600°C. Columns packed with 15–20-μm particles were obtained by means of high-viscosity slurry techniques. The separation of some pairs of E/Z diastereomers is described and discussed.