Exposure to 4,4'-methylenediphenyl diisocyanate (MDI) during moulding of rigid polyurethane foam: determination of airborne MDI and urinary 4,4'-methylenedianiline (MDA)

Occupational exposure to 4,4'-methylenediphenyl diisocyanate (MDI) was measured during moulding of rigid polyurethane foam. The aim of the study was to find out whether an MDI-derived urinary amine metabolite could be detected in the urine of workers exposed to apparently low levels of MDI. Airborne MDI was sampled on 1-(2-methoxyphenyl)-piperazine (2MP)-impregnated glass fibre filters and determined by high-performance liquid chromatography (HPLC) using ultraviolet (UV) and electrochemical (EC) detection. The limit of detection of MDI was 3 ng ml-1 for a 20 microliters injection. The precision of sample preparation, expressed as relative standard deviation (RSD), was 1.3% with UV detection and 2.1% with EC detection at a concentration of 70 ng MDI ml-1 (n = 6). The 2MP-MDI derivative was stable at +4 degrees C up to eight weeks. The accuracy of the method was validated in an international quality control programme. Workers (n = 57) from three different factories participated in the study. Urinary 4,4'-methylenedianiline (MDA) metabolite was determined after acid hydrolysis as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry using chemical ionisation and monitoring negative ions. The limit of detection in urine was 0.2 nmol l-1. The precision of six analyses for a urine sample spiked to a concentration of 1 nmol l-1 was 29% (RSD). The MDI concentrations were below the limit of detection in most (64%) of the air samples collected in the worker's breathing zone. Still, detectable amounts of MDA were found in 97% of the urine samples. Monitoring of urinary MDA appears to be an appropriate method of assessing MDI exposure in work environments with low or undetectable MDI concentrations in the workplace air.     

Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

Identification and determination of sulphamethazine and N4-acetylsulphamethazine in meat by high-performance liquid chromatography with photodiode-array detection

A simple and selective method for the determination of sulphamethazine (SMT) and its metabolite, N4-acetylsulphamethazine (N4-AcSMT), in meat by high-performance liquid chromatography (HPLC) with photodiode-array detection was developed. The drugs were extracted from meat with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond-Elut C18 clean-up procedure. The HPLC separation was carried out on a Supersphere RP-18e column (125 X 4.0 mm I.D.) using 0.05 M sodium dihydrogenphosphate (pH 4.5)-acetonitrile (8:2) as the mobile phase at a flow-rate of 0.5 ml/min, and monitored with a photodiode-array detector. The recoveries of SMT and N4-AcSMT from meat fortified at 0.5 micrograms/g were 90.1-93.3 and 93.0-94.4%, respectively, with coefficients of variation of 1.9-3.2 and 1.5-2.7%. The limits of detection were 0.02 micrograms/g for each drug. SMT was found in ten samples of imported meat (12.5%) at levels ranging from 0.05 to 1.05 micrograms/g.     

Determination of thiobarbituric acid adduct of malondialdehyde using on-line microdialysis coupled with high-performance liquid chromatography.

An on-line analytical system for the continuous monitoring of malondialdehyde (MDA) was developed. This method involves the use of microdialysis perfusion, on-line derivatization and on-line HPLC analysis. This method gave a linear response for MDA concentrations and HPLC peak areas in the range from 0.051 microM to 2.43 microM. The intra-day (RSD = 1.6-10.5%) and inter-day (RSD = 1.1-9.3%) precisions were acceptable. The average in vitro probe recovery of MDA standard was 18.4 +/- 1.0%. The detection limit was 0.03 microM, corresponding to 0.6 pmol for an injection volume of 20 microl. This method was used for in vitro peroxidation investigations, which provided evidence for elevated MDA levels following the incubation of metal ions to a linoleic acid solution.     

Determination of fenbendazole and its metabolites in trout by a high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for the simultaneous determination of fenbendazole (FBZ), fenbendazole sulfoxide (FBZ-SO) and fenbendazole sulfone (FBZ-SO2) in trout muscle and skin tissues. The compounds were extracted with acetonitrile and the extract was concentrated and cleaned up by solid phase extraction on C18 and CN cartridges. The extract was analysed by reversed-phase gradient HPLC on a C18 column followed by ultraviolet detection at 290 nm. The method's detection limits were 4.0 micrograms kg-1 for FBZ, 4.5 micrograms kg-1 for FBZ-SO and 3.8 micrograms kg-1 for FBZ-SO2. The mean recovery in muscle was 88% for FBZ, 94% for FBZ-SO and 92% for FBZ-SO2 at a level of 5-150 micrograms kg-1. The corresponding mean recoveries in skin tissue were 88, 81 and 86% at a level of 10-100 micrograms kg-1. The mean relative repeatability standard deviation was 9.2% at a level of 5 micrograms kg-1, 5.9% at a level of 10-100 micrograms kg-1 and 2.3% at a level of 150 micrograms kg-1. The method was applied to trout given feed containing FBZ in an aquaculture pilot plant. The three compounds FBZ, FBZ-SO and FBZ-SO2 were all detected in muscle and skin tissues shortly after administration. The concentrations were generally highest in skin tissue.     

Determination of hexahydrophthalic anhydride in air using gas chromatography.

Two methods for the determination of hexahydrophthalic anhydride (HHPA) in air were developed. In a solid sorbent method, HHPA was sampled in Amberlite XAD-2 tubes, eluted in toluene and analysed by gas chromatography with flame ionization detection. The sampling rates were 0.2 and 1.0 l/min. At 15 micrograms/m3 (relative humidity less than 2%) and 27 micrograms/m3 (relative humidity 70%) no breakthrough was observed. However, at 160 micrograms/m3 (relative humidity less than 2%), 6% breakthrough was found. The sampling efficiency of the sampling rates 0.2 and 1.0 l/min did not differ. In a bubbler method, HHPA was sampled in bubblers filled with 0.1 M sodium hydroxide solution. The sodium salt of hexahydrophthalic acid was formed. No breakthrough was observed using a sampling rate of 1.0 l/min. The samples were stable during storage for eight weeks in a refrigerator. The HHP acid was esterified with methanol-boron trifluoride and analysed by gas chromatography-flame ionization detection. Apparatus for the generation of standard atmospheres of HHPA, in the range of 10-3000 micrograms/m3, was developed using the diffusion principle. For the solid sorbent method the precision (coefficient of variation) of the overall method was 2-7%, and for the bubbler method 3-19% (range 15-160 micrograms HHPA/m3; relative humidity = less than 2-70%). A comparison between the two methods was performed using the standard atmosphere. The concentrations found by the solid sorbent method were 86-98% of those found by the bubbler method (range 15-160 micrograms HHPA per m3; relative humidity = less than 2-70%). In work environment air, 93% was found using the solid sorbent method relative to the bubbler method at a mean concentration of 330 micrograms/m3 (coefficient of variation = 39%; range 200-540 micrograms/m3). For both methods, concentrations greater than 3 micrograms/m3 could be quantified at 60 min sampling with a sampling rate of 1.0 l/min.     

Multiresidue chromatographic method for the determination of macrolide residues in muscle by high-performance liquid chromatography with UV detection.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.     

Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphamonomethoxine (SMMX), sulphadimethoxine (SDMX), sulphisozole (SIZ), nalidixic acid (NA), oxolinic acid (OXA), piromidic acid (PMA), furazolidone (FZ) and sodium nifurstyrenate (NFSA) in cultured fish was developed. The drugs were extracted with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (150 x 4.6 mm I.D.) using 5 mM aqueous oxalic acid-acetonitrile (55:45) as the mobile phase with detection at 265 nm (0.04 a.u.f.s.). The calibration graphs were rectilinear from 1 to 20 ng for OXA, from 2 to 50 ng for SMMX, SDMX, SIZ, NA, PMA and FZ and from 5 to 100 ng for NFSA. The recoveries of each drug added to fish were 65.0-89.5%. The detection limits were 0.02 micrograms/g for OXA, 0.05 micrograms/g for SMMX, SDMX, SIZ, NA, PMA and FZ and 0.1 micrograms/g for NFSA.     

Simultaneous determination of malondialdehyde and ofloxacin in plasma using an isocratic high-performance liquid chromatography/fluorescence detection system

This study establishes the applicability of using high-performance liquid chromatography (HPLC) with fluorescence detection for the simultaneous determination of malondialdehyde (MDA) and ofloxacin (OFL). The MDA and OFL were separated through a reverse-phase C18 column (250 mm × 4.6 mm) at a flow rate of 1.0 mL min⁻¹ and then detected using a fluorescence detector (excitation: 532 nm; emission: 553 nm). The separation conditions were optimized by varying the concentration and pH of the phosphate buffer and the percentage of organic solvent; the optimal mobile phase was a mixture of 50 mM phosphate buffer (adjusted to pH 5.8 with potassium hydroxide) and methanol (45:55, v/v). The retention times of MDA and OFL were 3.6 and 5.9 min, respectively, with detection limits (at a signal-to-noise ratio of 3) of 0.015 and 4.0 μM, respectively. This method afforded linear responses between the MDA and OFL concentrations and the HPLC peak areas within the ranges 0.15-2.43 μM and 0.06-1.0 mM, respectively. The precisions of the determinations of MDA and OFL, measured in terms of relative standard deviations, were 1.6-5.0% and 1.9-3.6%, respectively, for intra-day assays and 1.0-4.3% and 0.3-1.8%, respectively, for inter-day assays. The average recoveries of MDA and OFL spiked in plasma were 100.4% and 98.8%, respectively. To the best of our knowledge, this paper describes the first practical analytical approach toward simultaneously monitoring the levels of MDA and OFL in plasma. The OFL-induced oxidative stress measured using this method indicated that OFL treatment did not markedly increase the level of MDA.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Reversed-phase high-performance liquid chromatographic separation and electrochemical detection of retinol and its isomers

Baseline separation of the isomers of retinol using reversed-phase high-performance liquid chromatography (HPLC) in less than 30 min is presented. A new approach to the detection of retinol using electrochemical detection is developed. The oxidative electrochemistry of retinol is studied at a glassy-carbon electrode using coulometry, ultraviolet-visible spectrophotometry and HPLC. Amperometric detection in HPLC for retinol provided a linear response from 0 to 1.5 micrograms/ml and a detection limit of 4.1 ng/ml. Electrochemical detection was compared to ultraviolet-visible absorbance detection for the determination of retinol in human serum extracts. Good agreement is found for the results obtained with the two detectors.     

Characterization of polyarylamide fibers by inverse gas chromatography

We determined a group of estrogenic compounds by solid-phase microextraction (SPME) coupled to high-performance liquid chromatography (HPLC) with both ultraviolet (UV) and electrochemical detection (ED). A modified liquid chromatograph was used. Polyacrylate fibers (85 microns) were used to extract the analytes from the aqueous samples. Dynamic and static modes of desorption were compared and the variables affecting both absorption and desorption processes in SPME-HPLC were optimized. Static desorption gave the best recoveries and peak shapes. The performance of the SPME-HPLC-UV-ED method was checked with river water and wastewater. The method enabled estrogenic compounds to be determined at low-microgram l-1 levels in real water samples. Limits of detection were between 0.3 and 1.1 micrograms l-1 using UV detection and between 0.06 and 0.08 microgram l-1 using ED. beta-Estradiol was found in samples from a wastewater treatment plant at concentrations between 1.9 and 2.2 micrograms l-1.     

Ultrasensitive detection of malondialdehyde with surface-enhanced Raman spectroscopy

Malondialdehyde (MDA) is a biomarker of lipid peroxidation that has been widely associated with food rancidity as well as many human diseases. Most current MDA detection methods involve MDA reaction with thiobarbituric acid (TBA), followed by UV-visible and/or fluorescence detection of high-performance liquid chromatography (HPLC)-separated TBA-MDA. Herein, we report the first proof-of-concept study of surface-enhanced Raman detection of a TBA-MDA adduct using silver nanoparticles as the SERS substrate and the 632.8 nm HeNe laser as a Raman excitation source. Current SERS detection limit of TBA-MDA is 0.45 nM, ~100 times higher than the 36 nM fluorescence sensitivity recently reported with the HPLC-purified TBA-MDA. Molecular specificity of the SERS technique was studied by comparing the SERS spectrum of TBA-MDA with those acquired with TBA adducts of other TBA-reactive compounds (TBARCs) that includes formaldehyde, acetaldehyde, butyraldehyde, trans-2-hexenal, and pyrimidine. Compared to TBA and TBA adducts with those TBARCs, the SERS activity of TBA-MDA adduct is significantly higher. The possibility of direct SERS detection of TBA-MDA in a reaction mixture (without HPLC separation) has also been investigated.     

Determination of aromatic amines in aqueous extracts of polyurethane foam using hydrophilic interaction liquid chromatography and mass spectrometry

A method is presented for the determination of aromatic amines in aqueous extracts of polyurethane (PUR) foam. The method is based on the extraction of PUR foam using aqueous acetic acid (0.1%, w/v) followed by determination of extracted aromatic amines using hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS) with positive electrospray ionisation. The injections of volumes up to 5 μL of aqueous solutions were made possible by on-column focusing with partially filled loop injections. The fragmentation patterns for 2,4- and 2,6-toluene diamine (TDA) and 4,4′-methylene dianiline (MDA) were clarified by performing a hydrogen-deuterium exchange study.TDA and MDA were determined using trideuterated 2,4- and 2,6-TDA and dideuterated 4,4′-MDA as internal standards. Linear calibration graphs were obtained over the range 0.025-0.5 μg mL⁻¹ with correlation coefficients >0.996 and the instrumental detection limit for each compound was <50 fmol. The stability of the amines was influenced by the matrix, so their concentrations decreased over time.Agreement was observed between the results of analyses of PUR foam extracts by HILIC-MS/MS and results obtained by ethyl chloroformate derivatisation and reversed phase (RP) liquid chromatography-mass spectrometry (LC-MS/MS).TDA was observed to be unstable in extracts of foam but not in pure solutions.     

Analysis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine by ion chromatography and pulsed amperometric detection

A novel high-performance liquid chromatography (HPLC) method is presented for the detection and trace level determination of the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV). The tripeptide, an intermediate in penicillin production, is derived from fungal fermentation. The technique relies on ion-exchange separation of the tripeptide on an anion-exchange column followed by detection by reduction on a gold electrode using pulsed amperometry. The sensitivity of direct determination of ACV is increased by employing pulsed amperometric detection (PAD) over direct ultraviolet detection. Choice of the working electrode and optimisation of electrode potentials was based on cyclic voltammograms recorded for the tripeptide in the mobile phase. A linear regression equation was obtained over the range 0-100 micrograms ml-1. The detection limit in fermentation broths was found to be 0.1 micrograms ml-1 whereas in buffer the detection limit was found to be 10 ng ml-1. A good correlation coefficient was observed when ACV concentrations determined by ion chromatography-PAD were compared with measurements obtained by pre-column derivatisation with fluoromethylorthochloroformate followed by HPLC separation on a reversed-phase C18 silica column with UV detection. The procedure has been applied to the measurement of natural levels of ACV in fermentation broths of selected strains of Aspergillus nidulans and Penicillin chrysogenum.     

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.     

High-performance liquid chromatography with fluorescence detection for the simultaneous determination of 3,4-methylenedioxymethamphetamine, methamphetamine and their metabolites in human hair using DIB-Cl as a label

This paper describes a highly sensitive HPLC method for the simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in human hair samples. The amphetamines investigated were derivatized with the fluorescent reagent, DIB-Cl to yield highly fluorescent DIB-derivatives, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 nm and 430 nm, respectively. The separation was achieved on an ODS column with an isocratic mobile phase composed of acetonitrile-methanol-water (30:40:30, v/v/v). The limits of detection for the four compounds obtained by the proposed method ranged from 11 to 200 pg/mg. The method was successfully applied to the determination of MDMA and MDA in hair samples obtained from MDMA abuser.     

Determination of malondialdehyde in traditional fish products by HPLC

The oxidation state of traditional fish products was measured by determining the malondialdehyde (MDA) level by HPLC and the results were compared to those given by a spectrophotometric method. The procedure involves oxidation of the products by incubation at 40 degrees C for 3 d. Samples were steam distilled in a Kjeldahl distillation apparatus and the MDA was determined in the aqueous distillates by HPLC, using a micro-Bondapak C18 column, with mixed mobile phase of 1% acetic acid-acetonitrile (85 + 15; v/v). A total time of 2 min was necessary to assay each distillate and only MDA was detected. MDA can be determined at a level of 1.5 x 10(-8) mol l-1. The highest rate of oxidation of the samples, as shown by the changes in the TBA test and MDA concentration determined by HPLC, was observed in smoked fish and the lowest in dried-salted fish.     

Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.