Search

Benzo[c]fluorene (BcFE) concentrations in benzene/ethanol extracts of airborne particulates were determined by high-performance liquid chromatography (HPLC) with fluorescence detection. HPLC conditions were as follows: columns, two ZORBAX Eclipse PAH (4.6 i.d. × 250 mm, 3.5 μm) and one Inertsil ODS-P (4.6 i.d. × 250 mm, 5 μm) in series; mobile phase, acetonitrile-water (98:2, v/v), 0.3 mL/min; detection wavelengths, excitation 309 nm, emission 354 nm. Particulate-phase BcFE concentrations in the atmosphere varied seasonally (winter > summer). The concentrations were 11000 ± 6100 pg m(-3) (winter) and 40 ± 12 pg m(-3) (summer) in Beijing, China, and 13 ± 5.0 pg m(-3) (winter) and 2.7 ± 0.52 pg m(-3) (summer), in Kanazawa, Japan. In both cities, the particulate-phase BcFE concentration in the atmosphere was lower than that of benzo[a]pyrene (BaP) by a factor of 0.03 - 0.43. However, the mutagenic contribution of particulate-phase BcFE in the atmosphere in winter calculated from the mutagenicity relative potency factor was greater than that of BaP.

A convenient HPLC method for the quantitative determination of EDTA in foods was developed. EDTA in food samples was easily extracted with water by ultrasonication. After converting to Fe(III) complex in the presence of Fe(III) ions, EDTA was separated on a reversed-phase C30 column and detected with ultraviolet detection (260 nm). Citrate and malate, which are present in many foods, also formed Fe(III) complexes but they did not interfere the chromatographic detection of EDTA. The method allowed determination of EDTA in foods at concentrations as low as 0.01 mmol/kg. Good recoveries (95.2-101%) were obtained by the standard addition method on four samples with high repeatability (RSD, 0.8-3.4%). The method was successfully applied to the analysis of EDTA in carbonated drinks, jellies, canned beans, canned corn and food supplements., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chelating agent EDTA is widely used, and as a result is showing up widely in the aquatic environment. Here we describe a preconcentration procedure for measuring EDTA concentration in sea water samples by HPLC. The procedure consists of forming an Fe(III) complex followed by solid-phase extraction using an activated carbon cartridge. After the preconcentration, EDTA was quantified by HPLC with ultraviolet detection (260 nm). The enrichment permitted the determination of EDTA at concentrations as low as 1 nM. Good recoveries were obtained for both brackish and full-strength sea water with high repeatability (RSD<6%). The method was applied to sea water samples taken from near the mouth of the Oyabe River in Japan., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An on‑line concentration and fluorescence determination HPLC for polycyclic aromatic hydrocarbons (PAHs) in seawater was proposed. An online concentration column packed with octadecyl polyvinyl alcohol polymer, a pump and a column switching valve were introduced in the conventional HPLC with a fluorescence detector. Only 1.0-100 mL seawater sample was introduced into the concentration column at 1.0 mL min(-1) without any other pretreatment except filtration. Then the trapped PAHs totally flew into the separation column and eluted separately to be detected fluorogenically. The proposed method had good linearity with correlation coefficients (r) ranged from 0.951 to 0.998, and limits of detection ranged from 0.002 to 0.50 ng L(-1) for 15 PAHs as 100 mL seawater was loaded. The sensitivity of the method was 10 to 100 times higher than those reported by other works. The proposed method was applied to the determination of PAHs in the seawater samples collected in the Japan Sea with satisfactory results and to check the present benzo[a]pyrene concentration at the beaches in Noto peninsula, Japan polluted with C-heavy oil spilled from the tanker in 1997.

Iprodione is an agricultural fungicide that is difficult to detect in foods by HPLC because it coelutes with natural compounds in the food. We previously showed that food matrix could be degraded with titanium dioxide powder (TP). Here we describe an improved method for detection of iprodione using silica gel supported titanium dioxide (SGT). To synthesize SGT, titania-sol was mixed with diethanolamine, 2-propanol, and titanium tetraisopropoxide. After titania-sol was infiltrated into the silica gel (particle diameter 4 mm), the mixture was dried and then heated. Crude basil extract containing iprodione was mixed with SGT in a quartz vial, and the vial was irradiated with a UV light to selectively decompose the matrix interfering with the iprodione determination. In HPLC chromatograms of the treated solution, the interference peak decreased 35 times faster with SGT than with TP. When SGT (11 g) was added to the extract (20 mL) of dry basil (2 g), black light irradiation for 30 min was enough to quantify iprodione. The recovery rate of iprodione was 99.1%. Thus, the photocatalytic cleanup method using SGT is effective for analyzing residual iprodione in dry basil.

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2-100 ng x mL(-1) was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng x g(-1) and 0.39 ng x g(-1), respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

A simple and sensitive method for measuring airborne particle-associated benz[a]anthracene-7,12-quinone (BaAQ) based on two-dimensional high-performance liquid chromatography (HPLC) with fluorescence detection was established. The system involves an ODS column for sample clean up, a 6-port switching valve, an ODS column for trapping the fraction containing BaAQ, an ODS column for sample separation, and a Pt-Rh catalytic column for reduction of BaAQ to a corresponding fluorescent compound. The accuracy of the assay, as applied to airborne particulate sample extracts spiked with known amounts of BaAQ was 94-108%. The detection limit was 97 fmol per injection (signal-to-noise ratio=3), and the calibration range was from 1 to 100 pmol with excellent proportionality (R(2)=0.9995). BaAQ in airborne particles collected in Osaka, Japan every 3 h was successfully measured using the established analytical method with simple sample preparation steps: ultrasonic extraction in organic solvent and concentration under reduced pressure and/or nitrogen stream.

To measure the actual exposure of a person to 1-nitropyrene (1-NP) in airborne particulate matter, it is considered more accurate to collect air samples with a portable air sampler than to sample at a fixed location. However, because the portable samplers can sample only small volumes, a sensitive method is needed to analyze the compounds that are collected on a filter. Here we describe a high-performance liquid chromatographic (HPLC) method with fluorescence detection that is sensitive and precise enough for use with portable air samplers. The developed column-switching system successfully removed the interfering substances in the samples with only a simple pretreatment. To improve the precision of the measurement, deuterated 1-NP was used as an internal standard, and it eluted immediately prior to 1-NP with sufficient resolution (R(s), 1.50). The detection limit was 0.32 fmol/injection, and the calibration range was from 2 to 100 fmol. The proposed method was applied to determining 1-NP in fine airborne particulate matter (PM(2.5)) at two sites with low pollution levels. 1-NP was detected in all samples at concentrations in the low fmol/m(3) range. The proposed method has enough sensitivity and precision to determine 1-NP in the limited air volume of the portable sampler.

Anion-exchange solid-phase extraction accompanied with high-performance liquid chromatography has been developed for the determination of six kinds of aminopolycarboxylic acids (APCAs) in river water [N-(2-hydroxyethyl)ethylenediaminetriacetate (HEDTA), ethylenediaminetetraacetate (EDTA), 1,3-propanediaminetetraacetate (PDTA), diethylenetriaminepentaacetate (DTPA), 1,2-propanediaminetetraacetate (MeEDTA), and O,O'-bis(2-aminoethyl)ethyleneglycoltetraacetate (GEDTA)]. The enrichment of APCAs using an anion-exchange cartridge was successfully done by the removal of anions, which competed with APCAs in anion-exchange processes. Barium chloride solution was added to river water and the mixture was passed through On Guard II Ag and H cartridges and then a Bond Elut Jr.SAX cartridge to enrich APCAs. After elution, APCAs were analyzed on two reversed phase C30 columns connected in series and detected with ultraviolet detection. The enrichment using solid-phase extraction permitted the determination of APCAs in river water at concentrations as low as 1nM. Good recoveries (83-111%) were obtained for each APCA by the standard addition method on three river water samples with high accuracy (RSD 1.8-9.5%). Applying this method, two kinds of APCAs, EDTA and DTPA, were determined in samples from the Oyabe and Senbo Rivers in Japan.

A simple HPLC method for the determination of adulteration in apple juice was developed. The method is based on the detection of D-malate, derived from racemic malic acid, which is added as an acidulant. A variable-wavelength optical rotation detector was used to determine the enantiomeric excess (ee). Using anion-exchange chromatography with a phosphate buffer eluent and UV (210 nm) detection, the limit of detection for L-malate was 2 microg. With an injection of 13.4 microg of malate, the standard deviation of the ee calibration curve was 2.5%. Several apple juice samples were analyzed according to the proposed procedure, and the results agreed with those obtained using enzymatic kits for food analysis.

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1h at 120 degrees C and the recovery for biocytin was 65.7+/-2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, "natto", rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.

A sensitive and variable-wavelength optical rotatory (OR) detector for high-performance liquid chromatography is presented. This design is entirely different from that of conventional OR detectors consisting of a crossed polarizer pair. By placing a polarizing prism and a retardation plate into a commercial circular dichroism (CD) detector, the OR signal was obtained. The Mueller matrix approach was used to prove the principle of the OR signal appearance. Sugars and 4-androstene-3,17-dione were chosen as test compounds. The limit of detection was below 0.5 microg of injected sucrose at 260 nm, which was superior to that obtained with a conventional OR detector. For 4-androstene-3,17-dione, which is CD active, and shows a large anomalous OR dispersion curve, our detector gave a large OR signal with approximately half the intensity of the CD signal at 340 nm.

Using a fast reversible reaction of aminopolycarboxylic acids (APCAs) into Fe(III)-APCA complexes in the presence of Fe(III) ions, seven kinds of APCAs [nitrilotriacetate (NTA), N-(2-hydroxyethyl)ethylenediamine-triacetate (HEDTA), ethylenediamine-tetraacetate (EDTA), 1,3-propanediamine-tetraacetate (PDTA), diethylenetriamine-pentaacetate (DTPA), 1,2-diaminopropane-tetraacetate (MeEDTA), and O,O'-bis(2-aminoethyl)ethyleneglycol-tetraacetate (GEDTA)] in cosmetics and synthetic detergents were separated on two reversed-phase C30 columns connected in series and detected with ultraviolet detection. Simple pretreatment, consisted of thousand times dilution of samples and addition of 100 microl of the Fe(III) solution containing 10 mM Fe(III) chloride and 0.5 M sulfuric acid to 10 ml of diluted samples, permitted the determination of APCAs in cosmetics and synthetic detergents at concentration level of 0.1 mM, except 0.3 mM for GEDTA. APCAs except GEDTA could be detected at concentration level of 0.03 mM and GEDTA could be detected at concentration level of 0.09 mM. Good recoveries (95-110%) were obtained for each APCA by the standard addition method on two diluted samples with high accuracy (RSD 0.2-9.1%). Three APCAs (EDTA, HEDTA and NTA) were detected in various concentrations in cosmetics and synthetic detergents and the other APCAs were not detected in any of the samples. This method requires no tedious pretreatment and takes only 15 min for one analysis, so it is useful for determination of APCAs.

1-Nitropyrene (1-NP) is enriched in diesel exhaust particulate matter (DPM) relative to other sources of particulate matter (PM), and has been proposed as a marker for DPM. However, in ambient air, 1-NP concentrations are typically in the low pg/m(3) range. Therefore, collection of large volume air samples coupled with extensive sample clean-up procedures has been required to achieve adequate detection limits to measure 1-NP in ambient samples. We report here an improved LC-MS/MS method suitable for the detection and quantification of 1-NP in low volume ambient PM samples. The method involves ultrasonic extraction of ambient PM in organic solvent, concentration of the sample under reduced pressure, and two-dimensional HPLC analysis of the extract. 1-NP is isolated on the first HPLC column, then converted to 1-aminopyrene (1-AP) via online reduction in a column packed with a Pt/Rh catalyst. The 1-AP containing fraction from the first column is refocused on a trapping column, then eluted through a second HPLC column prior to MS/MS detection. Deuterated (d(9)) 1-NP (1-dNP) is added to each sample prior to extraction as an internal standard for quantification of 1-NP. The accuracy and precision of the assay, as applied to ambient particulate standard reference materials are 110+/-5.7% for SRM 1650b, 116+/-7.1% for SRM 2975, 108+/-5.8% for SRM 1649a, and 53+/-9.2% for SRM 1648. The analytical limit of detection was 152 fg on column, and analytical limit of quantitation 221 fg on column. To our knowledge, the sensitivity of this method is comparable with GC-NICI-MS methods while having the advantage of considerably less extensive sample preparation. This method is an approximately 10-fold improvement in sensitivity over HPLC methods utilizing fluorescence and/or chemiluminescence detection.

We have developed a method to determine nitrocatecholamines (nitroCAs: nitronorepinephrine (nitroNE), nitroepinephrine (nitroE), and nitrodopamine (nitroDA)) in rat brain using high-performance liquid chromatography-peroxyoxalate chemiluminescence (POCL) reaction detection. This method consists of in-line extraction of amines, including nitroCAs, followed by separation on an ODS column, reduction of nitroCAs with a reducer column, derivatization with ethylenediamine, and finally the POCL reaction detection. Use of the reducer column increased the sensitivity of detection of nitroCAs by 45-172%. The limits of detection (signal-to-noise ratio of 3) were 75fmol for nitroNE, 125fmol for nitroE, and 150fmol for nitroDA. By using this method, not only nitroNE, but nitroE and nitroDA were determined in rat brain. The concentrations of nitroNE and nitroE in rat brain were 9.2+/-2.0 and 13.5+/-2.5 pmol/g tissue, respectively (n=5).

A method is presented for determining monohydroxy polycyclic aromatic hydrocarbons (OHPAHs) having 2-, 3- and 4-rings in human urine by using high-performance liquid chromatography with fluorescence detection. A urine sample containing conjugates of OHPAHs was hydrolysed in the presence of beta-glucuronidase/aryl sulfatase and the solution was cleaned up with a solid-phase extraction (C(18) and silica). Eight OHPAHs, namely 1- and 2-hydroxynaphthalenes, 2-hydroxyfluorene, 2-, 3- and 4-hydroxyphenanthrenes, 3-hydroxyfluoranthene and 1-hydroxypyrene, were separated and 1- and 9-hydroxyphenanthrenes co-eluted on an alkylamide-type reversed-phase column with fluorimetric detection. The urinary concentrations of OHPAHs were quantified by using deuterated 1-hydoxypyrene as an internal standard. The method showed good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r (2) ranged from 0.996 to 0.999). The limits of detection (S/N=3) were in the range from 2.3 fmol to 2.2 pmol per injection. This method was successfully applied to urine samples from non-smoking taxi drivers, traffic policemen and rural villagers of Chiang Mai, Thailand. The results showed higher urinary concentrations of OHPAHs in rural villagers, consistent with higher respiratory exposure to PAHs.

Current on-line solid-phase extraction methods combined with HPLC for shortening the clean-up operation are not suitable for simultaneously detecting compounds that have a wide variety of hydrophobicities. To solve these problems, we designed a new on-line sample preparation system. The system consists of an eluting pump, a mixing TEE connector, a 10-port 2-position valve and a solid-phase extraction precolumn. The eluate from the precolumn is diluted with a weak solvent from the HPLC at the TEE connection to load low hydrophobic compounds onto the analytical column. The proposed on-line sample preparation system was successfully applied to the simultaneous analysis of 21 pesticides in river water using LC/TOF-MS. In this method, the recoveries from river water samples were 67 to 126% (mean 83%), the reproducibility (CV%) was in the range from 1.1 to 11% (mean 5.6%), the calibration curve was linear in the range from 1 ppb to 500 ppb (r > 0.999) and the detection limits (S/N = 3) were in the range from 0.0034 ppb (daimuron) to 3.3 ppb (oxine-copper).

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H(2)O(2) CL system. The detection limits (signal-to-noise ratio=3) were in the range from 5fmol (6-OHNP and 8-OHNP) to 12fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair. Fifteen kinds of PAHs classified as priority pollutants by the US EPA were quantified with four perdeuterated PAHs as internal standards. After 50 mg hair samples were washed with n-hexane to remove external contamination of PAHs, the samples were digested in 2.5 M sodium hydroxide. The digests were extracted with n-hexane and then analyzed by HPLC. Eleven kinds of PAHs were identified in hair samples of 20 subjects, and 10 kinds of PAHs were eventually quantified using the internal standards. For anthracene, chrysene and benzo[k]fluoranthene, significant differences were observed between smokers and non-smokers. Although benzo[b]fluoranthene, dibenz[a,h]anthracene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were observed in the particulates of indoor and outdoor air, they were not detected in all hair samples. The analysis of PAHs in human hair should be useful as a new biomarker to evaluate the exposure to PAHs.

An automatic HPLC system for analyzing nitropolycyclic aromatic hydrocarbons (NPAHs, nitroarenes) in airborne particulates was previously described (Anal. Chim. Acta, 2001, 445, 20). Some problems with this system were that it generated a peak originating from an ascorbic acid solution that elutes at a retention time close to that of 1,6-dinitropyrene (DNP), and that it was able to analyze only 1,3-, 1,6-, 1,8-DNPs and 1-nitropyrene (I-NP). Here, we describe an improved system that effectively removes the interfering peak by introducing an ODS column just after the pump for the ascorbic acid solution, and which is capable of analyzing several additional compounds (2-, 4-NPs, 2-nitrofluorene. 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitroperylene and 6-nitrobenzo[a]pyrene etc.). The improved sensitivities were achieved by concentrating the compounds in a benzene-ethanol extract from airborne particulates, by increasing the loading time of the sample solution from 20 to 38 min, and by increasing the flow rate of an ascorbic acid solution from 1.3 to 1.8 mL/min.

Changes in the chemical structures of morphine and codeine in the presence of hydrogen peroxide, a major component of hair dye and decolorant treatments, were examined with high-performance liquid chromatography/mass spectrometry (LC/MS). A mixture of morphine and hydrogen peroxide solution, after incubation at 39 degrees C for 24 h, produced two reaction products (hydroxymorphines). When codeine was used in place of morphine, one reaction product (hydroxycodeine) was produced, in which the benzene ring was hydroxylated.

A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant p450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by beta-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91-103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.

A liquid chromatographic (LC) method has been developed for the separation of 11 monohydroxybenzo[a]pyrenes (OH BaPs) positional isomers, and for their detection using electrospray ionization mass spectrometry (ESI-MS). All OH BaPs isomers were separated on an octadecylsilyl (C(18))-bonded amorphous organosilica column utilizing gradient elution with acetonitrile-water and triethylamine (TEA) at pH 11.0 and determined by MS, except 2- and 8-OH BaPs which were coeluted. The lower detection limits were in the range from 1.6 micro g/L for 12-OH BaP to 12 micro g/L for 5-OH BaP without any sample enrichment. The relative standard deviations of area response were in the range from 1.8% (9-OH BaP) to 4.9% (12-OH BaP) except for 9.4% (7-OH BaP). The developed method was successfully applied to incubation mixtures of BaP and CYP1A1/epoxide hydrolase. This method identified 1-, 3- and 9-OH BaPs as the major metabolites, and 2- (and/or 8-) and 12-OH BaPs as the minor metabolites in the incubation mixture., (Copyright 2002 John Wiley & Sons, Ltd.)

The change of chemical structure of cocaine in the presence of hydrogen peroxide, a main component of hair dye and decolorant treatments, was studied. High-performance liquid chromatography/mass spectrometry (LC/MS) was used for the separation and identification of cocaine derivatives. After a mixture of cocaine and hydrogen peroxide solutions was incubated at 39 degrees C (this temperature is commonly used when the hair is treated with hair dye or decolorant) for 24 h, six reaction products were detected by LC/MS. Two of them were ecgonine methyl ester and benzoylecgonine, which are metabolites of cocaine. The other reaction products were assumed to be ortho-, meta- and para-hydroxycocaines and dihydroxycocaine, in each of which the benzene ring was hydroxylated by the reaction. These five reaction products (except for dihydroxycocaine) were found immediately after mixing cocaine and hydrogen peroxide. Therefore, the above reaction products might be present in the hair of cocaine users that had treated their hair with hair dye or decolorant., (Copyright 2002 John Wiley & Sons, Ltd.)

A novel multi-beam polarized photometric detector (PPD) for high-performance liquid chromatography (HPLC) is described. By pairing a polarizing prism with a thin quartz plate as a retarder, many linear polarized beams are produced at every 1/2 wavelength of the plate, and the polarizing axes of the adjacent beams intersect each other. The addition of another prism inclining its polarizing axis by pi/4 against the first one enables the simultaneous measurement of optical rotations based on the PPD at many wavelengths. The combination of these optics with a photo-diode array detector can be used to construct a modulated type polarimeter. This detector is designed to measure the optical rotation of an analyte at its absorption band. The spline function connecting the points at 1/4 wavelengths of the plate was used as a baseline to extract the PPD waves. The use of the similarity factor as a noise filter gave high sensitivity. Application of the proposed technique to an analyte carrying the Cotton absorption band provided good results.

The effect of hydrogen peroxide, a main component of hair dye and decolorant treatments, on methamphetamine (MA) was studied. Two analytical methods, thin-layer chromatography (TLC) and high-performance liquid chromatography/mass spectrometry (LC/MS), were used for the separation and identification of MA derivatives. Mixtures of MA solutions and hydrogen peroxide that had been incubated at 39 degrees C for 24 h were shown to contain para-hydroxy MA by TLC and para-, meta- and ortho-hydroxy MAs by LC/MS. In addition, MA N-oxide and N-hydroxy MA were found in MA/hydrogen peroxide mixtures immediately after mixing. Therefore, we concluded that MA changed to MA N-oxide and N-hydroxy MA before changing to para-, meta- and ortho-hydroxy MAs.

Nitropolycyclic aromatic hydrocarbons (NPAHs), which are known to be carcinogenic and/or mutagenic, are considered to be one of the air pollutants that cause lung cancer. In the last two decades, a number of sensitive and selective methods have been developed for the determination of NPAHs and related compounds in environmental and biological samples. This paper describes the state of the art of the methods and applications., (Copyright 2000 John Wiley & Sons, Ltd.)

A high-performance liquid chromatographic method has been developed for the determination of saccharides in sake, an alcoholic beverage brewed from rice. Saccharides in sake were separated on a normal phase (carbamoyl bonded silica) column using a linear gradient elution of water in acetonitrile. Seven saccharides, glucose, maltose, isomaltose, maltotriose, panose, isomaltotriose and ethyl alpha-D-glucoside, were determined by a polarized photometric detector. Unidentified peaks suggesting saccharides with polymerization degrees over 4 were also observed. The proposed method did not require any sample clean-up treatment. As an application, saccharide compositions in various kinds of sake were compared., (Copyright 2000 John Wiley & Sons, Ltd.)

Black hairs that had been removed from a methamphetamine (MA) addict were treated with permanent wave, dye or decolorant liquids, and MA and amphetamine (AP) were quantified by a high-performance liquid chromatography/chemiluminescence detection method. The concentrations of MA and AP in the hair decreased significantly in all cases. Both MA and AP were stable in the permanent wave treatments, but not stable in the dye or decolorant treatments. As possible reasons for the decrease, the elution of MA and AP from hair in the permanent wave treatment, and the degradation of MA and AP in the dye or decolorant treatments might be considered. These results suggested that treatments of hair with permanent wave, dye or decolorant liquids interfered with determination of MA and AP in hair.

HPLC/circular dichroism detection using a conventional spectrophotometric detector has been established. A split-type flow cell developed for a polarized photometric detector, which is a non-modulated polarimeter, was inserted into this detector. When two rays of circularly polarized light with opposite rotational senses to each other were incident in both cells by combining a polarizer and a 1/4 wave retarder in front of the cell assembly, the output from this spectrophotometric detector became solely the signal based on circular dichroism. Selective detection of a chiral analyte, in which the ratio of the difference in the absorbance between two rays mentioned above and the absorbance by normal rays in over 1/1000, is feasible using the proposed instrument.

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.

Stimulants that are controlled by the Stimulant Drug Control Law of Japan are methamphetamine (MA) and amphetamine (AP). MA is used by most stimulant addicts, and AP is detected as its main metabolite. We have developed a high-performance liquid chromatography method with chemiluminescence detection (CL-HPLC), for determining trace levels of MA and its metabolites in a single human hair sample, in which bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide are the postcolumn reagents. After washing a single hair sample with water and methanol, it was cut into pieces, extracted with a mixed solution of methanol and hydrochloric acid for 1 h under ultra-sonication and allowed to stand at room temperature overnight. Then the organic phase was evaporated to dryness. To the residues, 0.1 mL of carbonate buffer and 0.1 mL of dansyl chloride solution were added and the solution was heated at 45 degrees C for 1 h. An aliquot of the reaction mixture was then subjected to HPLC. MA and AP were chemiluminogenically detected as their dansyl derivatives from a sample of only a single hair. The detection limit was about 2 pg in an injected volume (20 microliters), and about 20 pg in a single hair sample. This detection limit was smaller than that by the gas chromatography/mass spectrometry (selective ion monitoring) method. Our method was useful as a screening test for stimulant users.

Various metal powders were batch screened as possible on-line reducers for nitropolycyclic aromatic hydrocarbons (NPAHs), in particular 1,3-dinitropyrene (1,3-DNP, 1,8-DNP and 1-nitropyrene (1-NP) and it metabolites. Zinc was found to be an excellent reducer in chemiluminescent compatible mobile phases without any associated disposal problems. Zinc reducer column parameters, namely pH, buffer concentration, packing composition (zinc:glass beads or zinc:silica gel) and column length were all optimized for the HPLC determination system with chemiluminescence detection, named NPAH analyser. The system consisted of three pumps, sample injector, reducer column packed with zinc:glass beads switching valve with a cutting loop, separation column (ODS), chemiluminescence detector and integrator. The mobile phase for the separation column and chemiluminescence reagent solution were respectively acetonitrile:imidazole-HClO44 and acetonitrile containing bis(2,4,6-trichlorophenyl)oxalate and H2O2. When the reducer column was introduced before the separation column, 1,3-, 1,6- 1,8-DNPs and 1-NP in benzene:ethanol extracts from airborne particulates were separately determined with detection limits of 2-50 fmol, respectively, in a 100 microL sample. When the reducer column was introduced just after the separation column, 1-NP, 1-nitrosopyrene and 1-aminopyrene in the incubation mixture of 1-NP and the Salmonella typhimurium YG1021 strain were separately determined with detection limits as low as sub fmol levels. 1,8-DNP and its metabolite were also separately determined in the incubation mixture of 1,8-DNP and the S. typhimurium TA98 strain.

Determinations of both the free and bound form of N-acetyl-neuraminic acid (NANA) in several human body fluids, such as serum, cerebrospinal fluid (CSF), saliva, urine, amniotic fluid, and milk were carried out by HPLC with fluorimetric detection. The method utilized 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) as a fluorimetric derivatizing reagent. Free-form NANA was obtained from the body fluids after ultrafiltration with Microcon 10 (YM-10 cellulose membrane, filtration limit M(r) = 10,000, Amicon). The DMB derivative of NANA was separated isocratically by a Nucleosil 5C18 column with a mixture of 0.1 M sodium phosphate buffer (pH 2.0)-methanol (75:25, v/v). A gradient elution system was used for urine analysis. Analysis times were 10-30 min. Recoveries of free NANA by ultrafiltration were satisfactory: 95.66 +/- 1.80% for serum and 97.27 +/- 1.55% for CSF, respectively. The high sensitivity and specificity render this method applicable to all the body fluids tested. Although a physiological role for free NANA has not yet been elucidated, the method presented promises to contribute to the basic understanding of the NANA metabolism.

A high performance liquid chromatographic (HPLC) method with fluorescence detection after on-line reduction for the determination of 1-nitropyrene (NP), 1-nitrosopyrene (NSP), 1-aminopyrene (AP) and N-acetylaminopyrene (AAP) has been developed. The reduction efficiency of NP and NSP on a zinc column was found to be higher than that of an electrochemical reducer. Using a HPLC system equipped with a zinc column (4.0 mm i.d. x 10 mm) and an imidazole/HClO4 (pH 6.8): acetonitrile mobile phase, detection limits (S/N = 3) of 20-30 fmol for NP, NSP and AP and 350 fmol for AAP were obtained. NP, NSP and AP were determined in the incubation mixture of NP and Salmonella typhimurium, YG1021, by this method. Time course studies showed that a large ratio of NP was metabolized in the pre-incubation step of the Ames test.

An improved high-performance liquid chromatographic assay method for biotinidase activity was developed using the fluorimetric substrate biotinyl-6-aminoquinoline, which was found to be more specific than the biotinyl-4-aminobenzoate previously used. The new method measures the intensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm; emission 340 nm) for 4-aminobenzoate. The analysis of fluorescence in the visible spectrum reduced considerably the number of interfering peaks compared with analysis in the ultraviolet region. This method also made it possible to measure the biotinidase activity directly in samples usually difficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzyme reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, respectively, were demonstrated. Michaelis constants for biotinyl-6-aminoquinoline were measured at various stages of partial purification. Because the solubility of these synthetic substrates in the aqueous phosphate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the characterization of the kinetics of biotinidase.

A universal peroxyoxalate-chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual-head short-stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole-nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO-hydrogen peroxide. These two solutions were delivered by the two pump-heads. After the pH of the column eluate was adjusted to the optimum range (6.5-7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0-8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on-line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by some modifications of the postcolumn solutions.

A dual-head short-stroke pump has two advantages in the post-column peroxyoxalate chemiluminescence (PO-CL) detection system. The first is to increase the mixing efficiency of solutions. The second is to increase the stability of the PO-CL reaction by keeping the aryl oxalate and hydrogen peroxide solutions separate. The detection sensitivities for six polycyclic aromatic hydrocarbons (PAHs) increased in the present system by using bis(2,4-dinitrophenyl) oxalate (DNPO) or bis(2,3,4,5,6-pentafluorophenyl) oxalate (PFPO) instead of such popular aryl oxalates as bis(2,4,6-trichlorophenyl) oxalate (TCPO) and bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO). Both DNPO and PFPO increased the sensitivities by factors of 4.1-10.2 and 3.5-8.1, respectively. In addition, DNPO was more stable than PFPO in acetonitrile. These results suggest that DNPO is the most useful aryl oxalate for the sensitive PO-CL detection of PAHs.

A high-performance liquid chromatographic method for the determination of trace levels of methamphetamine, amphetamine and piperidine in human urine is reported. The three compounds, extracted into diethyl ether from alkaline urine, were derivatized with dansyl chloride, then separated on a reversed-phase column and detected by chemiluminescence after reaction with bis(2,4,6-trichlorophenyl) oxalate and hydrogen peroxide. The corresponding peaks obtained from human urine were identified as the dansyl derivatives by mass spectrometry. Methamphetamine levels as low as 2 x 10(-10) M in urine were determined. The sensitivity of the method is higher than that of Simon's reagent test and gas chromatography.

A convenient and reliable reversed-phase liquid chromatographic assay for four kinds of carboxylase, i.e. pyruvate carboxylase, acetyl-coenzyme A carboxylase, propionyl-coenzyme A carboxylase, and 3-methylcrotonyl-coenzyme A carboxylase, was developed. These are all biotin enzymes, which contain covalently bound biotin as a co-factor (co-enzyme). This chromatographic method, which is free from interferences, gives a stoichiometric and precise assay for these enzymes. It is non-radioisotopic, which makes it suitable for clinical laboratories. The assay time required for one sample is ca. 30 min. The method was satisfactorily applied to guinea-pig kidney homogenates after a simple pretreatment with acid.

A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection. By the proposed method, nitropyrene, 1,3-, and 1,8-dinitropyrenes were detected in sooty emissions of cars after simple purifications.

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5-7 and pH 3-4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflammatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.