Analysis of eight free progestogens in eggs by matrix solid-phase dispersion extraction and very high pressure liquid chromatography with tandem mass spectrometry

A rapid method to identify and quantify unconjugated progestogens in eggs is presented. Samples were prepared based on matrix solid-phase dispersion (MSPD) using C18 as dispersant, followed by a clean-up step with graphitized carbon black (GCB) solid-phase extraction (SPE) cartridges. The analytes were separated by very high pressure LC on a BEH C18 column for a period of 5 min. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was operated in the positive time-scheduled multi-reaction monitoring mode. Recovery studies were performed at two fortification levels. Average recoveries of the target compounds varied from 83.8% to 111.2% and relative standard deviations ranged from 10.5% to 23.7%. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.2-2.0 microg kg(-1) and 0.6-5.0 microg kg(-1), respectively. Investigation of real samples indicated that the range of progesterone in eggs was 9.9-40.0 microg kg(-1).     

Determination of 4-nonylphenol, nonylphenol monoethoxylate, nonylphenol diethoxylate and other alkylphenols in fish and shellfish by high-performance liquid chromatography with fluorescence detection

A simple and highly sensitive method is described for the HPLC determination of 4-nonylphenol (NP), 4-nonylphenol mono- (NP1EO) and diethoxylates (NP2EO) in fish and shellfish together with bisphenol A (BPA), 4-tert.-butylphenol (BP) and 4-tert.-octylphenol (OP). The NP, NP1EO, NP2EO and other alkylphenols in the samples are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR clean-up the sample extract is analyzed by HPLC with a fluorescence detection. Recoveries in Japanese smelt, carp and corbicura are 81.8–84.3% for NP, 83.5–84.3% for NP1EO, 90.5–96.2% for NP2EO, 70.7–72.9% for BPA, 71.0–73.4% for BP and 77.1–83.2% for OP spiked at 0.5 μg each chemical per 5 g of the fish and shellfish samples. The detection limits are 2 ng/g for NP, NP1EO and NP2EO, and 1ng/g for BPA, BP and OP.     

Optimization of a matrix solid-phase dispersion method for the determination analysis of carbendazim residue in plant material

The objective of this paper was to prove that matrix solid-phase dispersion (MSPD) coupled with high performance liquid chromatography (HPLC) and column switching could be used for the determination and quantification of carbendazim residue in plant samples. By comparing results obtained after optimization of the extraction conditions on an acidic silica gel column, it was determined that sorption and retention of carbendazim were achieved via specific interactions. The method of standard additions was used for quantitative analysis. Its performance was evaluated and validated: the detection limit (UV-Vis detection at lambda=279 nm) was 0.02 microg/g, the relative standard deviations (R.S.D.) were between 2.7 and 4.1% and the recoveries were ranging from 84.3 to 90.7% at the 0.04, 0.08 and 0.1 microg/g fortification levels. The method was successfully tested on cereal samples, and the results obtained with the present off-line MSPD-HPLC procedure were found to compare well with those obtained with procedure involving LLE.     

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography--mass spectrometry for measurement of phenolic xenoestrogens in human urine samples

A high-sensitivity analytical method that uses stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) for the simultaneous measurement of trace amounts of phenolic xenoestrogens (PXs), such as 2,4-dichlorophenol (DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol (OP), 4-nonylphenol technical isomers (NP), pentachlorophenol (PCP) and bisphenol A (BPA), in human urine samples was developed. The urine sample (1 ml) was de-conjugated by adding beta-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of acetonitrile. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC-MS. The detection limits of DCP, BP, OP, NP, PCP and BPA in the urine samples were 20, 10, 10, 50, 20 and 20 pg ml-1 (ppt), respectively. The calibration curves for PXs were linear and had correlation coefficients higher than 0.99. The average recoveries of those analytes in the urine samples were higher than 95% (RSD: <10%, n=6) with correction using the added surrogate standards. This simple, accurate, sensitive and selective method can be used in the determination of PXs in human urine samples.     

Liquid chromatographic determination and depletion profile of oxytetracycline in milk after repeated intramuscular administration in sheep

A simple, rapid and specific ion-pair liquid chromatographic method for the routine determination of the marker residue of oxytetracycline in sheep milk, at levels as low as 20 microg/kg, has been developed. Milk samples were acidified and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulphate and concentrated into diluted phosphoric acid. Separation was carried out isocratically on a Nucleosil C(18) column using a mobile phase that contained both positively and negatively charged pairing ions. The in-house validated method gave overall recoveries and overall relative standard deviations better than 86% and 4.6%, respectively. The method was successfully applied to study the depletion of oxytetracycline in sheep milk and to estimate the withdrawal period after intramuscular administration of a commercial oxytetracycline formulation.     

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples

Alkylphenols, 4-nonylphenol (NP) and 4-tert-octylphenol (OP), in human urine and plasma samples were analyzed using stir bar sorptive extraction (SBSE) in combination with thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The method involved correction by stable isotopically labeled surrogate standards, 4-(1-methyl)octylphenol-d5 (m-OP-d5) and deuterium 4-tert-octylphenol (OP-d). A biological sample was extracted for 60 min at room temperature (25 degrees C) using a stir bar coated with a 500 microm thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was analyzed by TD-GC-MS in the selected ion monitoring (SIM) mode without any derivatization step. The average recoveries in human urine and plasma samples spiked with NP and OP at levels of 0.5 and 10 ng ml-1 were between 95.8 and 99.8% with correction using the added surrogate standards. The limits of quantitation were 0.2 ng ml-1 for NP and 0.02 ng ml-1 for OP. We measured the background levels of NP and OP in five human urine and three human plasma samples from healthy volunteers. NP and OP were not detected in all human urine samples (N.D. < 0.2 ng ml-1 for NP, and N.D. < 0.02 ng ml-1 for OP). However, 0.2-0.3 ng ml-1 for NP and 0.1-0.2 ng ml-1 for OP in human plasma samples were observed by this method.     

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Measurement of Phenolic Environmental Estrogens in Women with Uterine Leiomyoma

Objectives

To investigate the effect of phenolic environmental estrogens on uterine leiomyoma from the perspective of clinical epidemiology.

Methods

Urine and blood samples were collected from Han women with uterine leiomyoma and women without uterine leiomyoma, living in Nanjing, China, between September 2011 and February 2013. A total of 156 urine samples and 214 blood samples were collected from the uterine leiomyoma group and 106 urine samples and 126 blood plasma samples from the control group. Bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP) concentrations were determined by solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Results

Phenolic environmental estrogens in the uterine leiomyoma and control groups were compared based on: gravida>3 and gravida ≤ 3. In participants with gravida>3, urine OP concentration was significantly (P<0.05) higher in the uterine leiomyoma group than in the control group. In participants with gravida ≤ 3, urine NP concentration was significantly (P<0.05) higher in the uterine leiomyoma group compared to controls. Despite obstetric history, urine BPA mean exposure concentration was significantly (P<0.05) different between uterine leiomyoma group and control group. The urine BPA concentration was not significantly (P>0.05) different between gravida>3 and gravida ≤ 3 patients. There was no significant (P>0.05) difference in plasma concentrations of BPA, OP and NP between the leiomyoma group and control group. Mean exposure concentration and range of distribution of BPA, OP and NP plasma concentration differed between the uterine leiomyoma and control group.

Conclusion

Exposure level of phenolic environmental estrogens in human was related with leiomyoma tumorigenesis.     

Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.     

Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection

A high-performance liquid chromatographic (HPLC) assay for the determination of nicotine and cotinine in human milk was developed using an extraction by liquid-liquid partition combined with back extraction into acid, and followed by reverse-phase chromatography with UV detection of analytes. The assay was linear up to 500 microg/l for both nicotine and cotinine. Intra- and inter-day relative standard deviations (R.S.D.) were <10% (25-500 microg/l) for both nicotine and cotinine. Limits of quantitation (LOQ) were 10 and 12 microg/l for nicotine and cotinine, respectively, while the limits of detection (LOD) were 8 and 10 microg/l for nicotine and cotinine, respectively. The mean recoveries were 79-93% (range 25-500 microg/l) for nicotine and 78-89% (range 25-500 microg/l) for cotinine. The amount of fat in the milk did not affect the recovery. We found that this method was sensitive and reliable in measuring nicotine and cotinine concentrations in milk from a nursing mother who participated in a trial of the nicotine patch for smoking cessation.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Validation of an HPLC-UV method according to the European Union Decision 2002/657/EC for the simultaneous determination of 10 quinolones in chicken muscle and egg yolk

Herein two different methods are proposed for the determination of 10 quinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken muscle and egg yolk. Two different HPLC systems were used comparatively and the respective methods were fully validated. The analytes were initially extracted from chicken muscle and egg yolk and purified by a solid phase extraction using LiChrolut RP-18 cartridges. Recoveries varied between 96.6 and 102.8% for chicken muscle and 96.4-102.8% for egg yolk. HPLC separation was performed at 25 degrees C using an ODS-3 PerfectSilTarget (250 mmx4 mm) 5 microm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of 0.1% trifluoroacetic acid (TFA)-ACN-CH3OH, delivered by a gradient program, different for each method. In both cases caffeine was used as internal standard at the concentration of 7.5 ng/microL. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed methods were validated according to the criteria of Commission Decision 2002/657/EC. The LODs for chicken muscle varied between 5.0 and 12.0 microg/kg and for egg yolk was 8.0 microg/kg for all examined analytes.     

Measuring environmental phenols and chlorinated organic chemicals in breast milk using automated on-line column-switching-high performance liquid chromatography-isotope dilution tandem mass spectrometry

Breast milk is one possible route of exposure to environmental chemicals, including phenols and chlorinated organic chemicals for breast-fed infants. We developed a highly sensitive method of analyzing breast milk for triclocarban (3,4,4'-trichlorocarbanilide) and eight phenolic compounds: bisphenol A (BPA), 4-tert-octylphenol (4-tOP), ortho-phenylphenol (OPP), 2,4-dichlorophenol, 2,5-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, and 2-hydroxy-4-metoxybenzophenone (BP-3). The method includes adding a solution containing a stable isotope of each chemical, enzymatic hydrolysis of the conjugated chemicals in the milk, and on-line solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. It can also be used to measure the free (unconjugated) species by omitting the enzymatic deconjugation step. The method, validated using pooled breast milk samples, has inter-day coefficient of variations ranging from 4.8 to 18.9% for most analytes, and spiked recoveries generally about 100%. Detection limits for most analytes are below 1 ng/mL in 100 microL of breast milk. We tested the usefulness of the method by measuring concentrations of these nine compounds in 20 breast milk samples. BPA, OPP, and BP-3 were detected in more than 60% of the samples tested. The free species of these compounds appear to be most prevalent in milk.     

Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography-tandem mass spectrometry

A reliable and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) confirmation method has been developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in chicken muscle. Samples were extracted with basic ethyl acetate, defatted with hexane, and cleaned up on Oasis MCX cartridges. LC separation was achieved on a XTerra C(18) column with gradient elution using a mobile phase composed of acetonitrile and water at a flow rate of 0.20 mL/min. The analysis was carried out on a triple-quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via electrospray interface operated in the positive and negative ionization modes, with deuterated chloramphenicol-d5 (d(5)-CAP) as the internal standard. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for each analyte with one precursor ion and two product ions. Limits of detection (LODs) were 0.1 microg/kg for CAP, 0.2 microg/kg for FF and 1 microg/kg for TAP and FFA in chicken muscle. Linear calibration curves were obtained over concentration ranges of 0.3-20 microg/kg for CAP, 0.5-20 microg/kg for FF and 3-100 microg/kg for TAP and FFA in tissues. Mean recoveries of the 4 analytes ranged from 95.1% to 107.3%, with the corresponding intra- and inter-day variation (relative standard deviation, R.S.D.) less than 10.9% and 10.6%, respectively. The decision limit (CCalpha) and detection capability (CCbeta) of the method were also reported.     

Determination of triazines in infant nutrient cereal-based foods by pressurized microwave-assisted extraction coupled with high-performance liquid chromatography-mass spectrometry

A method for determining triazine herbicides in infant nutrient cereal-based foods by pressurized microwave-assisted extraction (PMAE), coupled with high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS), is described. The key parameters of PMAE, including extraction solvent, extraction time and temperature, were optimized. The isolation of the target compounds from the matrix was found to be efficient when 2 g of nutrient cereal samples was extracted with 20 mL of methanol for 10 min at 105 degrees C. Final determination was accomplished by HPLC-ESI/MS. The recoveries from 66.2 to 88.6% were obtained for three compounds at fortification levels (5-500 microg kg(-1)) with relative standard deviations (R.S.D.) 相似文献   

Simultaneous determination of three residual barbiturates in pork using accelerated solvent extraction and gas chromatography-mass spectrometry

A new method was developed for the rapid extraction and unequivocal determination of barbital, amobarbital and phenobarbital residues in pork. The isolation of the analytes from pork samples was accomplished by utilizing an accelerated solvent extractor ASE 300. The procedure was automatically carried out in series for fat removing and extraction, respectively with n-hexane and acetonitrile pressurized constantly at 10.3 MPa for 30 min. After evaporation, the extracts were cleaned up on a C(18) solid phase extraction (SPE) cartridge and the barbiturates were eluted with hexane-ethyl acetate (7:3), evaporated on a rotary evaporator and derivatized with CH(3)I. The methylated barbiturates were separated on a HP-5MS capillary column and detected with a mass detector. Electron impact ion source (EI) operating in time program-selected ion monitoring mode (SIM) was used for identification and external standard method was used for quantification. Good linearity was obtained in the range from 0.5 microg/kg to 25 microg/kg. Average recoveries of the three barbiturates spiked in pork ranged from 84.0% to 103.0%, with relative standard deviations from 1.6% to 12%. The limit of detection (LOD) was 0.5 microg/kg for the three barbiturates (S/N>or=3). The quantification limit (LOQ) was 1 microg/kg for the three barbiturates (S/N>or=10).     

Matrix solid-phase dispersion extraction coupled with HPLC-diode array detection method for the analysis of sesquiterpene lactones in root of Saussurea lappa C.B.Clarke

We developed a reliable and effective method to determine costunolide and dehydrocostuslactone in the root of Saussurea lappa C. B.Clarke using matrix solid-phase dispersion (MSPD) extraction, HPLC separation and diode array detection (DAD). Several extraction parameters for the MSPD were optimized. Florisil was chosen as dispersing adsorbent with methanol as elution solvent. The ratio of Florisil to sample was selected to be 4:1 and no additional clean-up steps were needed. Linearities (r>0.9995) were determined to be in the range of 22.5-360.0 μg/mL for costunolide and 25.0-400.0 μg/mL for dehydrocostuslactone. Intra- and inter-day precisions were also determined with a relative standard deviation (RSD) less than 3.2%. The limits of detection were found to be 0.122 μg/mL for costunolide and 0.135 μg/mL for dehydrocostuslactone. The recoveries were in the range of 92.5-99.8% with relative standard deviations ranged from 1.2% to 3.5%. The proposed MSPD method required shorter time and lower solvent volume than maceration-ultrasonic and Soxhlet extraction methods.     

A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.     

GC-MS analysis of bisphenol A in human placental and fetal liver samples

A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography-mass spectrometry (GC-MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings.