Development and validation of an LC‐MS/MS method for simultaneous quantitative analysis of free and conjugated bisphenol A in human urine

Bisphenol A (BPA) is an environmental endocrine‐disrupting chemicals that is widely used in common consumer products. There is an increasing concern regarding human exposure to BPA owing to the potential adverse effects associated with its estrogenic activity. For assessing environmental exposure to BPA, it is essential to have a sensitive, accurate and selective analytical method, especially one that can detect low BPA levels in complex sample matrices. In this study, we developed and validated an accurate, sensitive, and robust liquid chromatography–tandem mass spectrometry method for simultaneous quantification of free BPA and BPA β‐d ‐glucuronide (BPA‐gluc) concentrations in human urine with only a single injection. Calibration curves were linear over a concentration range of 1–100 ng/mL for BPA and 10–1000 ng/mL for BPA‐gluc. The levels of the analytes were determined quantitatively with HPLC/ESI‐MS/MS by using negative electrospray ionization in the select ion monitoring mode and a pentaflouraphenyl propyl column. The validated method was applied to the analysis of spot urine specimens collected from randomly selected healthy human subjects. Copyright © 2013 John Wiley & Sons, Ltd.     

Concentration of bisphenol A in thermal paper

Abstract

Bisphenol A (BPA) is widely used as a color developer in thermal paper. Thermal paper is ubiquitous in daily life due to its use in cash register receipts, so opportunities for human contact abound. For this study, 10 blank cash register receipts were obtained from businesses in suburban Boston. BPA was extracted and analysis of concentration was performed using gas chromatograph/flame ionization detector. In some receipts, BPA was not detected but in others it was as high as 19 mg for a 12-inch long receipt, which is in line with concentrations indicated in patents. This study is intended to highlight the potential for human exposure to BPA as well as the ease with which exposure may be reduced through the use of BPA-free thermal paper.     

Evaluation of two membrane‐based microextraction techniques for the determination of endocrine disruptors in aqueous samples by HPLC with diode array detection

In this study, the viability of two membrane‐based microextraction techniques for the determination of endocrine disruptors by high‐performance liquid chromatography with diode array detection was evaluated: hollow fiber microporous membrane liquid–liquid extraction and hollow‐fiber‐supported dispersive liquid–liquid microextraction. The extraction efficiencies obtained for methylparaben, ethylparaben, bisphenol A, benzophenone, and 2‐ethylhexyl‐4‐methoxycinnamate from aqueous matrices obtained using both approaches were compared and showed that hollow fiber microporous membrane liquid–liquid extraction exhibited higher extraction efficiency for most of the compounds studied. Therefore, a detailed optimization of the extraction procedure was carried out with this technique. The optimization of the extraction conditions and liquid desorption were performed by univariate analysis. The optimal conditions for the method were supported liquid membrane with 1‐octanol for 10 s, sample pH 7, addition of 15% w/v of NaCl, extraction time of 30 min, and liquid desorption in 150 μL of acetonitrile/methanol (50:50 v/v) for 5 min. The linear correlation coefficients were higher than 0.9936. The limits of detection were 0.5–4.6 μg/L and the limits of quantification were 2–16 μg/L. The analyte relative recoveries were 67–116%, and the relative standard deviations were less than 15.5%.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a novel stability indicating RP-HPLC method for the quantitative determination of marbofloxacin in its tablets

A fast, feasible, isocratic, stability indicating reverse phase-high performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantitative determination of marbofloxacin in marbofloxacin tablets. The method was developed using Zorbax SB C18 (150?mm?×?4.6?mm), 5-µm column thermostated at 30°C, mobile phase A (1?mL of trifluoroacetic acid in 1000?mL of water), mobile phase B (acetonitrile) in the ratio of 83:17?v/v at flow rate of 1.0?mL/min, and an injection volume of 10?µL. The analyte was monitored at a wavelength of 298?nm. The method was validated in accordance with the Food and Drug Administration (FDA) Veterinary International Conference on Harmonization guidelines. To demonstrate stability indicating ability of method, drug product was subjected to the stress condition of acidic, basic, humidity, thermal, oxidative, and photolytic degradation.     

Measurement of bisphenol A and bisphenol B levels in human blood sera from healthy and endometriotic women

A sensitive HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) in human blood serum. The detection limits of the method were 0.18 and 0.20 ng/mL for BPA and BPB, respectively. A single‐step liquid–liquid extraction was used for the pre‐treatment of serum samples. The recoveries of BPA and BPB spiked to sera were 85.6 and 87.7%, respectively. The analyses of sera from both healthy and endometriotic women emphasized the absence of bisphenols in all the control cases (11 women), whereas BPA was found in 30 sera (51.7%) and BPB was found in 16 sera (27.6%) in the group of 58 patients with endometriosis; in nine of such sera BPA and BPB were present simultaneously. Only relatively to the sera quantitated, BPA concentrations ranged from 0.79 to 7.12 ng/mL (mean concentration 2.91 ± 1.74 ng/mL), whereas BPB concentrations ranged from 0.88 to 11.94 ng/mL (mean concentration 5.15 ± 4.16 ng/mL). Therefore, the presence of at least one of the two bisphenols was verified in a percentage as high as 63.8% in the sera from endometriotic women, suggesting the existence of a relationship between endometriosis and BPA and/or BPB exposure. Indeed, it is well known that bisphenols can work as xenoestrogens, owing to their structural similarity to natural and synthetic estrogens (e.g. estradiol and dietilstilbestrol). However, further studies are necessary to confirm this hypothesis and to assess the actual dose at which exposures to bisphenols are able to increase the sensitivity of the endometriotic cells to estradiol. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated HPLC method for the determination of fluconazole in human plasma

A high-performance liquid chromatographic assay with UV detection was developed for the determination of fluconazole in human plasma. The method utilized solid-phase extraction for sample clean-up. The separation was performed on a C18 column by isocratic elution with a mobile phase of 10 mM acetate buffer at pH 5.0 and methanol and UV detection at 210 nm. Validation was performed according to the current recommendations of the USFDA bioanalytical method validation guidance. The method proved to be specific, accurate, precise and linear between 200 and 10,000 ng/mL with correlation coefficients greater than 0.999. The coefficient of variation was within 11% and relative deviation was less than 10%.     

Determination of Phenolic Endocrine Disruptors in Cosmetics by High-Performance Liquid Chromatography Mass Spectrometry

An analytical method for the simultaneous determination of bisphenol A, 4-t-octylphenol, 4-n-octylphenol, and 4-n-nonylphenol in cosmetic samples has been developed. These compounds have toxic effects on human health as they have shown to produce endocrine disrupting properties. Therefore, their presence in cosmetics should be avoided according to the current European Regulations on cosmetic products. The method is based on high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) detection. Standard addition calibration was used to avoid matrix effects. The limits of detection values ranged between 7 and 15?ng?mL^?1 (threefold of the residual standard deviation of regression lines). The proposed method was validated, and good recovery (90–106%) and repeatability values (2.7–8.2%) were obtained. Finally, the method was successfully applied to ten commercially available cosmetic samples. The good analytical features of the proposed method make it useful to carry out the quality control of cosmetic products and raw materials to assure the safety of users.     

QbD-driven development and validation of HPLC method for determination of Bisphenol A and Bis-sulphone in environmental samples

ABSTRACT

A large number of hazardous chemicals have entered the environment due to the rapid growth of urbanisation and industrial development and are exerting harmful effects on wildlife as well as on human health. Plastic materials are one of the most leading causes for this contamination which are widely used in daily activities of human beings, i.e. disposal purpose, food packaging, bottles, containers, cups, grocery bags, etc. These materials contain Bisphenol A (BPA) and Bis-sulphone (BIS) which have been recognised as potential endocrine disruptors. In the present study, a selective and reliable high-performance liquid chromatography (HPLC) based method was developed with the mobile phase of 10 mM ammonium acetate buffer-acetonitrile (58:42 v/v, pH: 5) using quality by design (QbD) approach and the method was validated for the simultaneous assessment of BPA and BIS. The method was observed with a good linearity range of 50–500 ng/mL with an r² value of 0.998 and 0.999 for BPA and BIS, respectively. The developed and validated method was applied for the estimation of endocrine-disrupting chemicals in sewage water and soil samples. The results showed a considerable amount of BPA and BIS in the samples. This preliminary data explored the presence of BPA and BIS in these environmental samples that give the primary awareness of the effluence of BPA and BIS in the environment.     

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.     

Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend^®) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min⁻¹. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and p^H; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and p^H have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control.     

纳米银增强化学发光法测定塑料中的双酚A

研究发现在碱性介质中,纳米银对luminol-KMnO4化学发光体系的发光信号具有强烈的增敏作用,而双酚A对该体系具有明显的抑制作用,据此结合流动注射技术,建立了流动注射化学发光分析法测定塑料中双酚A的方法。该方法测定双酚A的线性范围为8.0×10-11～1.0×10-8g.mL-1(0.9926)和1.0×10-8～8.0×10-8g.mL-1(0.9916),检出限(3σ)为6.5×10-11g.mL-1。对5.0×10-10g.mL-1和5.0×10-8g.mL-1双酚A平行测定11次,其相对标准偏差为1.4%和1.0%。对反应机理进行了讨论。该方法简单、快速、灵敏度高,用于塑料制品中双酚A的测定,回收率为97.0%～105.0%。     

Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.     

Accurate and sensitive determination of selected hormones,endocrine disruptors,and pesticides by gas chromatography–mass spectrometry after the multivariate optimization of switchable solvent liquid‐phase microextraction

Switchable solvent liquid‐phase microextraction was combined with gas chromatography and mass spectrometry to improve the sensitivity and accuracy for the determination of selected endocrine disruptors, pesticides, and hormones. The extraction method was used to complement gas chromatography with mass spectrometry by preconcentrating analytes for trace determinations. A Box–Behnken experimental design was used to evaluate the main variables and their interaction effects, and optimum parameters were selected based on the model of experimented results. Application of optimum extraction conditions to mixed standard solutions yielded limits of detection and quantitation values between 0.20–13 and 0.90–46 ng/mL, respectively. The accuracy and the applicability of the developed method was checked in tap water and two different wastewater samples by spiked recovery tests. The percent recoveries recorded for the analytes were between 91 and 110%, and percent relative standard deviation values were all below 10%. The results indicate that the method can be used for the accurate and sensitive determination of these analytes in the presented matrixes.     

Development of an HPLC method to analyze and prepare elsinochrome C and hypocrellin A in the submerged fermentation broth of Shiria sp. SUPER-H168

A rapid and sensitive analytical method based on reverse-phase high-performance liquid chromatography was first developed to simultaneously determine elsinochrome C (EC) and hypocrellin A (HA) in the submerged fermentation. The mobile phase consisted of acetonitrile-water 60:40 (v/v) with a flow-rate of 1 mL/min. The calibration curves were as follows: y = 37,625x + 249,775 for EC, y = 30,813x + 556,409 for HA and linear at the investigated concentration. The correlation coefficients (R(2) ) were 0.9989 and 0.9998 respectively for EC and HA. The limits of detection and quantification were 175 and 585 μg/L for EC and 205 and 610 μg/L for HA. The precisions of concentration and retention times were less than 2.5 and 0.3%. The recovery of the method was greater than 95.0%. The methodology was applied to analyze simultaneously EC and HA concentrations in a submerged fermentation, and was adequate for analysis of biosynthesis of perylenequinones. The method was also amplified to separate and purify EC and HA using a semi-preparative C(18) column. In addition, elsinochrome C was first identified in the submerged fermentation broth of Shiraia sp. SUPER-H168.     

Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

A novel method for the simultaneous determination of six benzodiazepines (BZDs) and four tricyclic antidepressants (TCAs) in biological fluids by HPLC with UV detection at 240 nm has been developed. After a deproteinization step biological fluids were analyzed by direct injection. SPE on Nexus cartridges was also applied. Since two compounds, namely imipramine and diazepam, were coeluting, a sequential SPE protocol has been developed. BZDs were eluted by a mixture of methanol/ACN(1:1), followed by the elution of TCAs with methanol. Separation was performed on a Kromasil C8 column (250 x 64 mm(2) id, 5 microm) using a mobile phase of 0.05 MCH3COONH4/ACN/methanol (initial composition 55:15:30 v/v/v) at a flow rate of 1.0 mL/min delivered by a gradient program within 15 min. Colchicine was used as the internal standard (4 ng/microL). The method was linear for all analytes up to 20 ng/lL, with coefficients of regression between 0.996 and 0.99996. LODs and LOQs were 0.08-1.17 and 0.28-3.91 ng/lL, respectively. Recovery was in the range of 92.8-108.7% for within-day and 91.9-109.9% for between-day assays, with RSD values lower than 10.0% for all matrices.     

Development and validation of an analytical method for determination of endocrine disruptor, 2,4-D, in paddy field water

The acidic herbicides are an important class of chemical compounds that are used to control a variety of weeds that threaten many crops. Owing to their low microbial activity levels, the acidic herbicides exhibit a residual activity remaining for periods of up to several months in soils and water. The principal objective of this study was to develop an analytical method based on liquid–liquid and solid‐phase extraction followed by HPLC, for the determination of 2,4‐D in paddy field water. The residues were verified via tandem mass spectrometry (MS/MS) in negative‐ion electrospray ionization (ESI) mode. Linearity was good over a concentration range of 1–100 µg/L with a correlation coefficient (r²) of 0.999. The mean recovery rates of triplicate results ranged from 85.2 to 90.85%. The limits of detection and quantitation were 0.4 and 1.0 µg/L, respectively. The method proposed herein was applied to field samples acquired from Hampyung and Sunchang counties, Republic of Korea. The analyte was detected at a concentration range of 6.8–12.8 and 3.55–24.0 µg/L, respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of an RP-HPLC-UV method for the determination of BOL-303225-A, a new coumarin-based anti-inflammatory drug, in rat plasma

A simple and rapid high-performance liquid chromatographic method was developed and validated for the analysis in rat plasma of BOL-303225-A, a new coumarin-based anti-inflammatory drug. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid (35.5:64.5 v/v) as mobile phase. The UV detector was set at 324 nm. The method proved to be linear (r(2) > 0.99) and precise (RSD < 7%) over the concentration range 29-940 ng/mL, and was suitable for the support of pharmacokinetic studies in rats.     

Development and validation of an HPLC method for the determination of process-related impurities in pridinol mesylate, employing experimental designs

A simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound pridinol mesylate, has been developed and validated. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C₁₈ column with a mobile phase containing 50 mM potassium phosphate buffer (pH 6.4), MeOH and 2-propanol (20:69:11, v/v/v), delivered at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 245 nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r² > 0.99) over the range 0.05-2.5% (5-250% with regards to the 1% specification limit for both process-related impurities); it is also precise, regarding repeatability (RSD ≤ 1.5% for all of the analytes) and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1-102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.     

Development of solid-phase extraction method and its application for determination of hydrochlorothiazide in human plasma using HPLC

A high-performance liquid chromatographic method was developed, validated and applied for the determination of hydrochlorothiazide in human plasma. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of hydrochlorothiazide and internal standard were investigated. The method involves solid-phase extraction on RP-select B cartridges followed by isocratic reversed-phase chromatography on a Hibar Lichrospher 100 RP-8 column with UV detection at 230 nm. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. Limit of quantification was 10 ng mL(-1). The method has been implemented to monitor hydrochlorothiazide levels in patient samples.