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.     

Determination of bisphenol A in river water and body fluid samples by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry

A new method, based on stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water, urine, plasma, and saliva samples. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volumes and extraction time are investigated. Then, the stir bar is subjected to TD followed by GC-MS. The detection limits of BPA in river water, urine, plasma, and saliva samples are 1-5, 20, 100, and 20pgml(-1) (ppt), respectively. Calibration for BPA was shown to be linear with a correlation coefficient of >0.99. The average recoveries of BPA in all samples are higher than 95% (R.S.D. < 10%) with correction using an added surrogate standard, 13C12-bisphenol A. This simple, accurate, sensitive, and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.     

The analysis of estrone and 17beta-estradiol by stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry: application to urine samples after oral administration of conjugated equine estrogens

The development of a sensitive and solvent-free method for the measurement of estrone (E(1)) and 17beta-estradiol (17beta-E(2)) in human urine samples is described. The deconjugated estrogens were derivatized in situ with acetic acid anhydride and the derivatives were extracted directly from the aqueous samples using stir bar sorptive extraction (SBSE). The compounds containing a secondary alcohol function are further derivatized by headspace acylation prior to thermal desorption and gas chromatography/mass spectrometry (GC/MS). A number of experimental parameters, including salt addition, temperature and time, were optimized to increase the recovery of E(1) and 17beta-E(2) by SBSE. The derivatization reactions were also optimized to obtain the highest yields of the acylated estrogens. Detection limits of 0.02 and 0.03 ng mL(-1) were obtained for E(1) and 17beta-E(2), respectively. The method was applied to determine the effect of conjugated equine estrogen intake on the excretion of E(1) and 17beta-E(2) in human urine samples. Increased levels of the endogenous estrogens were detected after administering a standard dose of Premarin to a female volunteer. Routine monitoring of estrogen levels is recommended to avoid a high urinary excretion of E(1) and 17beta-E(2), nowadays enlisted as endocrine disrupting chemicals (EDCs), during hormone replacement therapy.     

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.     

Stir bar sorptive extraction and liquid chromatography with UV detection for determination of antidepressants in plasma samples

A sensitive and reproducible stir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) method is described for the determination of sertraline, mirtazapine, fluoxetine, citalopram, paroxetine, imipramine, nortriptyline, amitriptyne, and desipramine in plasma samples. Important factors in the optimization of SBSE efficiency are discussed, such as extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions: solvents, modes (magnetic stir, ultrasonic), time, and number of desorption steps. The SBSE/LC-UV method showed to be linear in a concentration ranging from the limit of quantification (LOQ) to 1000.0 ng mL(-1). The LOQ values ranged from 10.0 ng mL(-1) to 40.0 ng mL(-1). The inter-day precision of the SBSE/LC-UV method presented coefficient of the variation lower than 15%. Based on figures of the merit results, the SBSE/LC-UV methodology showed to be adequate to the antidepressants analyses from therapeutic to toxic therapeutic levels. In order to evaluate the proposed method for clinical use, the SBSE/LC-UV method was applied to the analysis of plasma samples from elderly depressed patients.     

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.     

Solvent-enhanced microwave-assisted derivatization following solid-phase extraction combined with gas chromatography-mass spectrometry for determination of amphetamines in urine

An approach using microwave-assisted derivatization (MAD) following solid-phase extraction (SPE) combined with gas chromatography-mass spectrometry (GC-MS) was developed to determine amphetamines in urine samples. The parameters affecting the derivatization efficiency - including microwave power and irradiation time - were investigated. Besides, solvent is thought critically important to MAD. Derivatization performance was studied using various solvents and compared with the performance obtained without solvent. Derivatization efficiency was clearly found to be enhanced by the presence of solvent. The highest derivatization efficiencies were obtained in ethyl acetate (EA) under microwave power of 250W for 1min. Calibration curves for all amphetamines were linear over a range from 1 to 1000ng/mL, with correlation coefficients above 0.9992. The intra-day and inter-day precision were less than 15%. The applicability of the method was tested by analyzing amphetamine-abusing subjects urine samples. Accordingly, the solvent-enhanced MAD-GC-MS method appears to be adequate for determining amphetamines in urine.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography-mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97-99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1-3% for samples prepared concurrently and 2-11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.     

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Amphetamine and methamphetamine determination in urine by reversed-phase high-performance liquid chromatography with simultaneous sample clean-up and derivatization with naphthoquinone 4-sulphonate on solid-phase cartridges

A liquid-solid procedure is proposed for sample clean-up and derivatization of amphetamine and methamphetamine in urine samples. The reagent was 1,2-naphthoquinone 4-sulphonate, and a commercial C₁₈ packing cartridge was used. The samples derivatized at room temperature were chromatographed on a 5-μm Hypersil ODS (250×4 mm I.D.) with an elution gradient of acetonitrile-water containing propylamine. Under these conditions, the amines were eluted with short retention times. The procedure was used to determine amphetamine, or methamphetamine with its metabolite amphetamine,in spiked urine samples. The detection limit (at a signal-to-noise ratio of 3) for amphetamine (0.1 μg/ml) was similar to that obtained with liquid-liquid derivatization and to those obtained with immobilized reagents on a polymeric solid support. The detection limit for methamphetamine (0.4 μg/ml) was higher than with the liquid-liquid procedure because of the lower reactivity on the cartridge. The precision and accuracy of the method were also studied.     

A sensitive method for quantitation of beta-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) in human urine by isotope dilution liquid chromatography-positive ion-electrospray-tandem mass spectrometry

A method for measurement of an important biological marker, 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) of sulfur mustard agent HD [bis-(2-chloroethyl)sulfide] in human urine, to quantify HD exposure, is presented. It employs TiCl3 reduction of beta-lyase metabolites to SBMTE, and automated solid-phase extraction sample preparation, followed by isotope dilution liquid chromatography-positive ion-electrospray ionization-tandem mass spectrometry with 7.5 min/sample cycle time, to achieve SBMTE quantitation of up to 200 samples/24h a day. Percent relative standard deviations over the calibration range varied from 12.0% at 0.1 ng/mL to 0.9% at 100 ng/mL, and the limit of detection from a 0.5 mL sample was below the lowest level calibration standard of 0.1 ng/mL.     

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: a cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.     

Simultaneous determination of 5-hydroxyindoles and catechols from urine using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

To make analytes amenable for fluorescence (FL) detection, polymer monolith microextraction (PMME) coupled to high-performance liquid chromatography with FL detection was developed for the simultaneous determination of catechols and 5-hydroxyindoleamines (5-HIAs) from urine samples. In this method, a two-step pre-column derivatization method was employed to derivatize the analytes and a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was used as the extraction medium for PMME. The conditions for the derivatization and subsequent extraction of 5-HIAs and catechols derivatives were optimized. Using our optimum conditions, the detection limit of the target analytes were 0.11–21 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations less than 12% and a recovery of higher than 82%. In this study, we show how our proposed method can be used as a rapid sensitive technique for the determination of catechols and 5-HIAs from urine samples.     

Improvement and validation the method using dispersive liquid-liquid microextraction with in situ derivatization followed by gas chromatography-mass spectrometry for determination of tricyclic antidepressants in human urine samples

A simple, rapid and sensitive method termed dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC/MS) was developed for the determination of tricyclic antidepressants (TCAs) in human urine sample. An appropriate mixture of methanol (disperser solvent), carbon tetrachloride (extraction solvent), and acetic anhydride (derivatization reagent) was injected rapidly into human urine sample. After extraction, the sedimented phase was analyzed by GC/MS. The calibration curves obtained with human urine were linear with a correlation coefficient of over 0.99 in the range of 2.0/5.0-100 ng mL(-1). Under the optimum conditions (carbon tetrachloride: 10 μL, methanol: 150 μL), the detection limits and the quantification limits of the tricyclic antidepressants were 0.5-2.0 ng mL(-1) and 2.0-5.0 ng mL(-1), respectively. The average recoveries of TCAs were 88.2-104.3%. Moreover, the inter- and intra-day precision and accuracy was acceptable at all concentrations. The results showed that DLLME is applicable to the determination of trace amounts of TCAs in human urine sample.     

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography–mass spectrometry (GC–MS) analysis. A one-step derivatization using 100 μL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations of all targeted compounds showed good intra- and inter-day (within 7 days) precision (<10 %), and good stability (<20 %) within 4 days at room temperature (23–25 °C), or 7 days when stored at ?20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistical analysis of the concentrations of these targeted metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.     

Picomolar concentrations of morphine in human urine determined by dansyl derivatization and liquid chromatography-mass spectrometry

Morphine is present in varying amounts as an endogenous product in human urine. Derivatization of morphine contained in urine with dansyl chloride yields a known product, which can be quantified by liquid chromatography mass spectrometry with high selectivity and sensitivity. Urine samples of 51 healthy individuals were spiked with stable-isotope labeled morphine, hydrolyzed and subjected to solid phase extraction followed by derivatization of morphine with dansyl chloride. The dansyl derivatives of naturally occurring morphine and deuterated internal standard were then detected by liquid chromatography-triple quadrupole mass spectrometry. Using the [N-CD(3)]-labeled internal standard and solid-phase extraction, a limit of detection of 35 fmol/ml (10 pg/ml) and a limit of quantification of 87.5 fmol/ml (25 pg/ml) was determined for morphine in human urine. This new LC-MS/MS method allowed the detection of endogenous morphine in human urine of 51 volunteers with an average value of 156.4 fmol/ml (44.7 ng/ml).