Calibration and evaluation of routine methods by serum certified reference material for aldosterone measurement in blood.

We attempted to study the standardization of aldosterone measurement in blood. The serum certified reference material (serum CRM) was established by spiking healthy human serum with pure aldosterone. ID-LC/MS/MS as a reference measurement procedure was performed by using the serum CRM. LC-MS/MS as a comparison method (CM) was routinely used for clinical samples, and the values with and without calibration by the serum CRM were compared. The serum CRM demonstrated similar reactivity with peripheral blood plasma as clinical samples in routine methods (RM) of RIA, ELISA, and CLEIA. In comparison between RM and CM, the results in regression analysis indicated that the range of the correlation coefficient (r) was 0.913 - 0.991, the range of y intercept was 0.9 - 67.3 pg/mL and the range of slope was 0.869 - 1.174. The values by RM in 100 - 150 pg/mL for the diagnostic level, had a significant calibration effect, and the relative difference between calibrated value in RM and result by CM was within ±20%. Furthermore, the calibrated value using the serum CRM was 10,187 pg/mL, which corresponds to measured value of 14,000 pg/mL using RIA for the adrenal venous sampling. Measured values between plasma and serum as a sample for the aldosterone measurement from clinical samples showed no significant differences. In conclusion, we succeeded to prepare the certified reference material of aldosterone for RM. Then, we can accurately calculate corrected values by using our equation for four RMs of determination of aldosterone.

Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography-mass spectrometry for the measurement of progesterone in human serum.

Miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography-mass spectrometry (GC-MS) for the determination of progesterone in human serum sample is described. The detection limit and the quantification limit of progesterone in human serum sample are 0.5 and 2 ng mL(-1) (ppb), respectively. The calibration curve for progesterone is linear with a correlation coefficient of >0.999 in the range of 2-500 ng mL(-1). The average recoveries of progesterone in human serum samples spiked with 5, 50 and 200 ng mL(-1) progesterone are 97.4% (R.S.D.: 9.3%), 100.3% (R.S.D.: 4.7%) and 99.8% (R.S.D.: 3.4%), respectively. This simple, accurate, sensitive and selective analytical method can be applicable for the determination of progesterone in human serum samples.

Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography-mass spectrometry for determination of benzophenone and derivates in human urine sample.

The determination of benzophenones (BPs) in human urine sample by miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography-mass spectrometry (GC-MS) is described. As analytes, BP, its metabolites benzhydrol (BP-OH) and 2-hydroxybenzophenone (2OH-BP), and its derivatives 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-hydroxy-4-methoxy-4'-methylbenzophenone (BP-10) were selected. The detection limit and the quantification limit of BPs in human urine sample are 5-10 and 20-50 pg mL(-1), respectively. The calibration curve for BPs is linear with correlation coefficient higher than 0.99 in the range of 0.02-10 or 0.05-10 ng mL(-1). The average recoveries of BPs in human urine samples spiked with 0.5 and 5 ng mL(-1) BPs are 89.8-100.2% (RSD: 2.5-9.3%) and 89.3-99.9% (RSD: 2.9-3.7%), respectively. Ten human urine samples were analyzed using the present method. BP-OH and BP-3 were detected in all the samples within the range of 0.24-5.91 and 0.43-5.17 ng mL(-1), respectively. This simple, sensitive, and selective analytical method was successfully applied to the determination of trace amounts of BPs in human urine samples.

Characterization of scallop midgut gland certified reference material for quantification of diarrhetic shellfish toxins.

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ±â€¯0.061) mg/kg for OA and (0.45 ±â€¯0.11) mg/kg for DTX1.

Molecular basis of wax-based color change and UV reflection in dragonflies.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania, and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum. Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics.

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for trace analysis of triclosan in water sample.

A simple and highly sensitive method called stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS), which is used for the determination of trace amounts of 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan) in river water samples, is described. A stir bar coated with polydimethylsiloxane (PDMS) is added to a 10 mL water sample and stirring is carried out for 120 min at room temperature (25 degrees C) in a vial. Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 5 ng L(-1) (ppt). The method shows linearity over the calibration range (0.02-20 microg L(-1)) and the correlation coefficient is higher than 0.997 for triclosan standard solution. The recovery of triclosan in river water samples ranges from 91.9 to 108.3% (RSD: 4.0-7.0%). This simple, accurate, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in river water samples.

Simultaneous analysis of benzophenone sunscreen compounds in water sample by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry.

A method for the simultaneous measurement of benzophenone (BP) sunscreen compounds, its derivatives 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), 2-hydroxy-4-methoxy-4'-methylbenzophenone (BP-10), 2-hydroxybenzophenone (2OH-BP), 3-hydroxybenzophenone (3OH-BP) and 4-hydroxybenzophenone (4OH-BP), in water samples was developed using stir bar sorptive extraction (SBSE) with in situ derivatization followed by thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). The detection limit is 0.5-2 ng L(-1) (ppt) for the seven BPs. The method shows good linearity and the correlation coefficients are equal to or higher than 0.990 for all the analyte. The average recoveries of BPs range from 102.0 to 128.1% (RSD<15.4%, n=6). Trace amounts of BPs in river water samples were determined by the present method.

Mercury speciation and analysis in drinking water by stir bar sorptive extraction with in situ propyl derivatization and thermal desorption-gas chromatography-mass spectrometry.

A method for mercury analysis and speciation in drinking water was developed, which involved stir bar sorptive extraction (SBSE) with in situ propyl derivatization and thermal desorption (TD)-GC-MS. Ten millilitre of tap water or bottled water was used. After a stir bar, pH adjustment agent and derivatization reagent were added, SBSE was performed. Then, the stir bar was subjected to TD-GC-MS. The detection limits were 0.01 ng mL(-1) (ethylmercury; EtHg), 0.02 ng mL(-1) (methylmercury; MeHg), and 0.2 ng mL(-1) (Hg(II) and diethylmercury (DiEtHg)). The method showed good linearity and correlation coefficients. The average recoveries of mercury species (n=5) in water samples spiked with 0.5, 2.0, and 6.0 ng mL(-1) mercury species were 93.1-131.1% (RSD<11.5%), 90.1-106.4% (RSD<7.8%), and 94.2-109.6% (RSD<8.8%), respectively. The method enables the precise determination of standards and can be applied to the determination of mercury species in water samples.

Miniaturized hollow fiber assisted liquid-phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for analysis of bisphenol A in human urine sample.

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml(-1) (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1-50 ng ml(-1). The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml(-1) BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.

Hollow-fiber-supported liquid phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for determination of chlorophenols in human urine samples.

A simple and highly sensitive method that involves hollow-fiber-supported liquid phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) was developed for the determination of chlorophenols (CPs) such as 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TrCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) in human urine samples. Human urine samples were enzymatically de-conjugated with beta-glucuronidase and sulfatase. After de-conjugation, HF-LPME with in situ derivatization was performed. After extraction, 2 microl of extract was carefully withdrawn into a syringe and injected into the GC-MS system. The limits of detection (S/N=3) and quantification (S/N>10) of CPs in the human urine samples are 0.1-0.2 ng ml(-1) and 0.5-1 ng ml(-1), respectively. The calibration curve for CPs is linear with a correlation coefficient of >0.99 in the range of 0.5-500 ng ml(-1) for DCP and TrCP, and of 1-500 ng ml(-1) for TeCP and PCP, respectively. The average recoveries of CPs (n=6) in human urine samples are 81.0-104.0% (R.S.D.: 1.9-6.6%) with correction using added surrogate standards. When the proposed method was applied to human urine samples, CPs were detected at sub-ng ml(-1) level.

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry.

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.

Determination of benzophenones in river-water samples using drop-based liquid phase microextraction coupled with gas chromatography/mass spectrometry.

In this study, a fast, simple and highly sensitive method that employs liquid phase microextraction (LPME)-GC/MS was developed to analyze trace benzophenones (BPs) in river-water samples. The tip of a 10-microl microsyringe filled with toluene (3 microl) was inserted into 2 ml of a river-water sample, and fixed at 5 mm below the water surface of the sample. A toluene droplet was made on the tip of the syringe, and extraction was conducted while agitating at 500 rpm for 15 min. After extraction, 2.0 microl of the extract was put into the syringe again, and directly introduced to GC/MS. The limits of detection (S/N = 3) and quantification (S/N >10) of BPs were 10 and 50 pg ml(-1), respectively. The results of a recovery test ranged over 93.3 - 101.1% (RSD, less than 10%; n = 6). The results of BPs determinations in the river-water samples showed that BPs (ND - 68.9 pg ml(-1)) were detected.

Interlaboratory comparison of liquid chromatography-tandem mass spectrometry quantification of diarrhetic shellfish toxins in scallop midgut glands.

An interlaboratory comparison (ILC) was organized as a measure of the analytical competency in the liquid chromatography-tandem mass spectrometry quantification of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in scallop midgut gland samples. The test sample was prepared using boiled midgut glands of naturally contaminated scallops with DTX1 and its esters by spiking with OA, and homogeneity and stability of this test sample was assessed to be appropriate. Twenty laboratories participated in the ILC based on the Japanese official testing method; they submitted two sets of analytical concentrations of target analytes along with the details of their analytical protocols. For assessing these data, assigned values were established from another ILC where ten participants quantified the target analytes by the standard addition method. The mean analytical results of the former ILC showed good agreement with the assigned values, and the corresponding relative reproducibility standard deviations met the criterion of CODEX STAN 292. Meanwhile, the results of more than half of the participants were out of the uncertainty range of the assigned values; these participants were encouraged to investigate their protocols to improve their analytical capability.

Dual derivatization-stir bar sorptive extraction--thermal desorption--gas chromatography-mass spectrometry for determination of 17beta-estradiol in water sample.

A novel method for the trace analysis of 17beta-estradiol (E2) in river water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ acylation (first derivatization) and thermal desorption (TD) with quartz wool assisted (QWA) in tube silylation (second derivatization), followed by gas chromatography-mass spectrometry (GC-MS), and is called the "dual derivatization method." The optimum conditions for SBSE with in situ acylation, such as the volume of acetic acid anhydride and the extraction time, were investigated. In addition, the optimum conditions for TD with QWA in tube silylation, such as the volume of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the TD temperature and hold time, were investigated as well. The detection limit (S/N = 3) and the quantitation limit (S/N>10) of E2 in the river water sample were 0.5 and 2 pg ml(-1) (ppt), respectively, by the dual derivatization method. In addition, the detection limit was 0.1 pg ml(-1) by using dual derivatization method with multi-shot mode. The calibration curve for E2 was linear in the range of 0.002-10 ng ml(-1) with correlation coefficients >0.999. The average recoveries of E2 (n = 6) at the concentrations of 0.05 and 1.0 ng ml(-1) from the river water sample were 93.1 (RSD: 1.4%) and 98.4% (RSD: 0.8%), respectively, with correction using the added surrogate standard, 17beta-estradiol-(13)C(4). This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of E2 in water samples.

Liquid phase microextraction with in situ derivatization for measurement of bisphenol A in river water sample by gas chromatography-mass spectrometry.

A new method that involves liquid phase microextraction (LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water samples. The LPME conditions, such as the type of extraction solvent and the extraction time, are investigated. Then, the extract is directly injected into GC-MS. The detection limit and the quantification limit of BPA in river water sample are 2 and 10pgml(-1) (ppt), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 10-10,000pgml(-1). The average recoveries of BPA in river water samples spiked with 100 and 1000pgml(-1) BPA are 104.1 (RSD: 8.9%) and 98.3 (RSD: 3.2%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C(12). This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.

Novel stir bar sorptive extraction methods for environmental and biomedical analysis.

Stir bar sorptive extraction (SBSE) is sample preparation technique that involves the extraction and enrichment of organic compounds from a liquid sample. The technique is based on the principle of sorptive extraction. A large amount of extraction phase is coated on a stir bar. An analyte is extracted into the extraction phase, based on its octanol-water partitioning coefficient and the phase ratio. Recently, various methods involving SBSE were developed in order to further facilitate analysis and improve sensitivity. In this review, we focused on the novel methods that involve SBSE with in situ derivatization, SBSE with in situ de-conjugation, thermal desorption (TD) in the multi-shot mode and TD with in tube derivatization method. Those methods were applied successfully to the trace analysis of environmental and biological samples and extremely low detection limits were achieved.

Effect of sterilization process on the formation of mono(2-ethylhexyl)phthalate from di(2-ethylhexyl)phthalate.

The risk assessment of di(2-ethylhexyl)phthalate (DEHP) migrating from polyvinyl chloride (PVC) medical devices is an important issue. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags is hydrolyzed by esterase into mono(2-ethylhexyl)phthalate (MEHP). However, MEHP is thought to be even more toxic than the parent compound. Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The limits of quantification (LOQs) of DEHP and MEHP were 2.5 and 0.75 ng/ml, respectively. In this study, the effect of sterilization process on the levels of DEHP and MEHP migration was investigated. The level of migration of DEHP from gamma(gamma)-ray sterilized PVC sheet was low compared with that of the unsterilized control. By contrast, the level of MEHP migration from the gamma-ray sterilized PVC sheet was high compared with that of the unsterilized control. In addition, a high content of MEHP was found in the gamma-ray sterilized PVC sheet.

Stir bar sorptive extraction with in situ de-conjugation and thermal desorption gas chromatography-mass spectrometry for measurement of 4-nonylphenol glucuronide in human urine sample.

4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.

Evaluation and analysis of exposure levels of di(2-ethylhexyl) phthalate from blood bags.

BACKGROUND: The US FDA and The Ministry of Health, Labor and Welfare of Japan have indicated that the risk assessment of di(2-ethylhexyl) phthalate (DEHP) released from polyvinyl chloride (PVC) medical devices requires immediate attention. In particular, the analysis of the exposure to DEHP from blood bags is very important for medical treatment. However, human exposure to DEHP via blood transfusion remains poorly understood. We evaluated DEHP and mono(2-ethylhexyl) phthalate (MEHP) levels, migration patterns, and metabolism in blood products for the detailed assessment of exposure to DEHP. METHODS: A method that is based on column-switching liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with on-line extraction was used for the direct analysis of DEHP and MEHP in the blood products. From the Japanese Red Cross Society, 78 blood products (red blood cell concentrate: n=18, irradiated red blood cell concentrate: n=18, whole blood: n=18, blood platelet: n=18, and frozen plasma: n=6) were sampled in January 2003 for use in this study. RESULTS: The detection levels of DEHP and MEHP ranged from 1.8 to 83.2 microg/ml and from 0.1 to 9.7 microg/ml, respectively. The levels of MEHP and DEHP in the blood products were increased with increasing storage time. In addition, whole blood products in PVC bags had the highest DEHP levels compared to the other blood products. Our results indicate that the maximum level of human exposure to DEHP released from blood bags is 0.7 mg/kg weight/time. CONCLUSION: This first quantitative evidence may be useful for the risk assessment of DEHP released from blood bags.

Stir bar sorptive extraction and trace analysis of alkylphenols in water samples by thermal desorption with in tube silylation and gas chromatography-mass spectrometry.

A novel method called thermal desorption (TD) with in tube silylation followed by gas chromatography-mass spectrometry (GC-MS), which is used for the determination of trace amounts of alkylphenols (APs) in river water samples, is described. APs are extracted from river water samples and concentrated by the stir bar sorptive extraction (SBSE) technique. The stir bar coated with polydimethylsiloxane (PDMS) is added to 2.0 ml water sample and stirring is carried out for 60 min at room temperature (25 degrees C) in the vial. Then, the PDMS stir bar is subjected to TD with in tube silylation followed by GC-MS. The detection limit is of the sub pg ml(-1) (ppt) level. The method shows good linearity and the correlation coefficients are higher than 0.99 for all analytes. The average recoveries of APs are higher than 90% (R.S.D.: 3.6-14.8%, n=6). This simple and sensitive analytical method may be used in the determination of trace amounts of APs in river water samples.