Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP-4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

General and selective isolation procedure for high-performance liquid chromatographic determination of anabolic steroids in tissues

A multi-residue method has been developed for the determination of anabolic steroids in animal tissue. The analytes are extracted from tissue with methanol and the extract is subjected to two solid-phase extractions, one using a non-specific adsorbing material, such as graphitized carbon black (Carbopack B), and the other Amberlite CG-400 I in the OH form. This procedure allowed the neutral anabolics (testosterone, trenbolone and progesterone) to be isolated and separated from the acidic type (phenolic group), such as diethylstilbestrol, oestradiol, zeranol/zearalenone and their respective metabolites. The determination was effected using high-performance liquid chromatography with different detectors (ultraviolet, fluorimetric and electrochemical). Several analytical parameters were studied: chromatographic conditions, recoveries, evaporation step, solvent flow-rate, cartridges reusability, interference of plastic cartridges. For all the anabolics investigated the recoveries were greater than 83.6%.     

A selective method for the determination of benzo[a]pyrene in soil using porous graphitic carbon liquid chromatography columns

A back-flushing procedure using porous graphitic carbon (PGC) HPLC columns has been used successfully for the cleanup of soil samples for the determination of benzo[a]pyrene in ppb levels by an ODS-fluorescence HPLC column. The procedure was tested on nine random soil samples taken from an industrial area of the Kingdom of Bahrain. The mean percent recovery from the PGC column was 96% and the average coefficient of variation for the whole method was 5.2%.     

Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.     

Reversed-phase high-performance liquid chromatographic determination of dexamethasone in bovine tissues.

A sensitive and selective high-performance liquid chromatographic procedure is described for the determination of the synthetic corticosteroid dexamethasone (DXM), in bovine muscle, kidney, liver and fat tissues, using methylprednisolone as the internal standard. Following extraction with ethyl acetate (muscle, kidney and liver) or diethyl ether (fat) and clean-up of the tissue extract, the drug residue was isolated using a C18 solid-phase extraction column. Separation of DXM was achieved by reversed-phase high-performance liquid chromatography with ultraviolet detection at 254 nm. By using this procedure, DXM levels as low as 0.01 mg kg-1 can be detected in muscle, kidney, liver and fat.     

Improved high-performance liquid chromatographic procedure for the determination of chlormethiazole levels following solid-phase extraction from plasma

An improved high-performance liquid chromatographic method for the determination of chlormethiazole levels in plasma is described. The drug is extracted from plasma using commercially available reversed-phase extraction columns; recovery values obtained using Sep-Pak C18 and Bond Elut C1, C2, C4, C6, C8, C18 columns are compared. Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of 0.025 M sodium acetate buffer, pH 4.5-acetonitrile (67:33) at a flow-rate of 1.6 ml/min in conjunction with a 15-cm Jones Chromatography Apex ODS column. The analytical column was protected by a Waters Assoc. Guard-Pak module containing a Guard-Pak CN insert. Using ultraviolet detection at 254 nm chlormethiazole levels in the region of 50 ng/ml can be measured with only 500 microliter of plasma.     

Normal-phase high-performance liquid chromatographic determination of spiramycin in eggs and chicken

A precise method is presented for determination of residual spiramycin (SP) in chicken eggs and tissues by high-performance liquid chromatography (HPLC). The sample preparation was performed by homogenizing with a mixture of acetonitrile and n-hexane (5:4, v/v) to minimize the fat amount followed by ultra-filtration using a MolCutII(R). The extracts containing SP were free from interfering compounds when examined by the normal-phase HPLC using a LiChrosorb(R) NH(2) column and a mobile phase of acetonitrile-water (85:15, v/v) with a photo-diode array detector. The average recoveries from spiked SP (0.1, 0.5 and 1.0 ppm) were in excess of 89.0% with coefficients of variation between 1.4 and 2.4%. The limit of detection was 0.1 ppm.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

Improved high-performance liquid chromatographic method for the determination of coenzyme Q10 in plasma.

Coenzyme (Co) Q10 was dissociated from lipoproteins in plasma by treatment with methanol and extraction with n-hexane. Subsequent clean-up on silica gel and C18 solid-phase extraction cartridges with complete recovery (99 +/- 1.2%) produced a clean extract. High-performance liquid chromatographic (HPLC) separation was performed on a C18 reversed-phase column. Three simple, rapid procedures are presented: HPLC with final UV (275 nm) detection, a microanalysis utilizing a three-electrode electrochemical detector and a microanalysis with column-switching HPLC and electrochemical detection. The methods correlate very well with classical ethanol-n-hexane extraction with UV detection. The identity and purity of the Co Q10 peak were investigated and the resulting methods were concluded to be suitable for total plasma Co Q10 determination. The average level in healthy subjects was 0.80 +/- 0.20 mg/l; the minimum detectable Co Q10 plasma level was 0.05 and 0.005 mg/l for UV and electrochemical detection, respectively. The methods were applied to many samples and the plasma Co Q10 reference values for healthy subjects, athletes, hyperthyroid, hypothyroid and hypercholesterolaemic patients are given.     

A highly sensitive high-performance liquid chromatographic procedure for the determination of isocyanates in air

Summary A sensitive high-performance liquid chromatographic procedure is described to analyse hexamethylene diisocyanate (HDI), 2,4- and 2,6-toluene diisocyanate (TDI), and 4,4-diphenylmethane diisocyanate (MDI) in air. The isocyanates are trapped on a sorbent coated with 1-(2-methoxyphenyl)piperazine (MPP). The resulting derivatives are separated using a column switching technique using either a diode array UV detector or an electrochemical detector. Working ranges are 1–5000 and 0.05–400 pmol for UV and EC detection, respectively. Virtually no breakthrough occurs if an air volume of up to 1500 l is sampled, and relative detection limits between 0.1 and 1 ng/m³ can be achieved. The procedure can be used to determine HDI and MDI in work place atmospheres and indoor air.Dedicated to Prof Dr. E. Lahmann on the occasion of this 65th birthday     

Improvement of extraction procedure for biogenic amines in foods and their high-performance liquid chromatographic determination.

A high-performance liquid chromatography method is described for the simultaneous determination of the biogenic amines tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine in cheese. The optimization of the procedure for the extraction of amines from the matrix is described. The separation of dansyl derivatives of the amines was achieved by reversed-phase liquid chromatography with gradient elution, followed by UV detection at 254 nm. The mobile phase was acetonitrile-0.01 M phosphate buffer (pH 7)-water. Under these conditions, rapid elution of the amines in less than 13 min was obtained. Validation of the method included calibration experiments, addition of standard amines for the determination of amine recoveries and repeatability tests.