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An isotope pattern deconvolution (IPD) quantification method has been applied for the determination of five substances (amphetamine, benzoylecgonine, cocaine, methamphetamine and MDMA) in wastewater for the application in wastewater-based epidemiology (WBE). A previously validated method that used a calibration curve for quantification was modified to apply IPD. The two approaches were compared in terms of analytical uncertainty in recovery studies of quality control samples, i.e. six wastewater samples from different geographical origins spiked at two concentration levels. Both methods were reliable as they passed (z-score < 2) in an interlaboratory exercise. After 60 individual determinations, IPD provided 11 results outside recovery limits (70-120%) while the previous method produced 31 adverse results. All mean values for IPD were accurate whereas 6 out of 10 results showed RSD values higher than 30% or recoveries outside limits when using the former method. Moreover, the calculated method bias for the latter doubles that of IPD, which, in turn, makes the combined uncertainty (u(c)) much higher. Consequently, a simple change of data treatment-IPD quantification methodology-resulted in a lower uncertainty of the estimated illicit drug concentration, one of the main steps contributing to the final uncertainty in the normalized daily drug consumption through WBE. The current study demonstrated that the employment of IPD can also be very interesting for future applications of WBE, especially when matrix effects are high, complicating accurate quantification. In addition, when a high number of samples and/or compounds need to be analysed, IPD is faster than calibration and, eventually, cost-effective when isotopically labelled internal standard is highly expensive.

A Liquid Chromatography coupled to tandem mass spectrometry (LC-MS/MS) based method have been developed for the determination of the main estrogen compounds -estrone (E1) and 17β-estradiol (E2)- in human serum. Two isotope dilution mass spectrometry (IDMS) quantification procedures have been used: a classical calibration curve-based method were compared to a recently developed isotope pattern deconvolution (IPD) method. IPD is based on isotopic abundance measurements and multiple linear regression. Validation was performed in terms of intra-assay repeatability (n = 5), inter-assay reproducibility (n = 9) and accuracy using spiked steroid-free serum at 5 concentration levels and 3 certified reference materials. Both methodologies meet EMEA requirements yielding recoveries between 79-106% and coefficient of variations of 1.7-8.3% along all experiments. Limits of quantification as low as 5 ng/L were achieved. 40 real samples were analysed for comparison purposes showing a great correlation between calibration and IPD concentration values. Real samples were also quantified by routine immunoassay analysis, which showed a significant proportional bias of 2.55 for E1 and good correlation for E2. While methods were considered suitable for routine or countercheck analysis within the context of hospital's needs, IPD has demonstrated to be faster and cost saving., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Vitamin D testing in analytical clinical laboratories has been experiencing a rapid increase of demand over the last years, as it plays a key role in several disorders. Due to the narrow ranges of medical significance regarding its concentration levels in human serum, accurate and precise determinations of vitamin D metabolites are required., Methods: We present an isotope dilution mass spectrometry quantification method for the re-certification of routine commercial standards used in method validation steps, isotope pattern deconvolution (IPD) based on LC-MS/MS., Results: IPD allowed to compensate for the observed biases of +4.7% for 25(OH)D3, -29% for 25(OH)D2 and -30% for 24,25(OH) 2 D 3 standard concentrations, respectively in an easy, cheap and straightforward way., Conclusions: Is has been observed that, in some cases, discrepancies may exist between stated purity or amount of routinely used commercial standards and actual values, which would lead to unwanted bias in the developed methodologies. The present correction has helped meeting the regulations established by international standardization programs, including Vitamin D Standardization Program (VDSP)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13 C 2 -testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g -1 . The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An isotope dilution mass spectrometry (IDMS) method for the determination of selected endogenous anabolic androgenic steroids (EAAS) in urine by UHPLC-MS/MS has been developed using the isotope pattern deconvolution (IPD) mathematical tool. The method has been successfully validated for testosterone, epitestosterone, androsterone and etiocholanolone, employing their respective deuterated analogs using two certified reference materials (CRM). Accuracy was evaluated as recovery of the certified values and ranged from 75% to 108%. Precision was assessed in intraday (n=5) and interday (n=4) experiments, with RSDs below 5% and 10% respectively. The method was also found suitable for real urine samples, with limits of detection (LOD) and quantification (LOQ) below the normal urinary levels. The developed method meets the requirements established by the World Anti-Doping Agency for the selected steroids for Athlete Biological Passport (ABP) measurements, except in the case of androsterone, which is currently under study., (Copyright © 2017. Published by Elsevier B.V.)

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We present here a new measurement method for the rapid extraction and accurate quantification of technical nonylphenol (NP) and 4-t-octylphenol (OP) in complex matrix water samples by UHPLC-ESI-MS/MS. The extraction of both compounds is achieved in 30min by means of hollow fiber liquid phase microextraction (HF-LPME) using 1-octanol as acceptor phase, which provides an enrichment (preconcentration) factor of 800. On the other hand we have developed a quantification method based on isotope dilution mass spectrometry (IDMS) and singly (13)C1-labeled compounds. To this end the minimal labeled (13)C1-4-(3,6-dimethyl-3-heptyl)-phenol and (13)C1-t-octylphenol isomers were synthesized, which coelute with the natural compounds and allows the compensation of the matrix effect. The quantification was carried out by using isotope pattern deconvolution (IPD), which permits to obtain the concentration of both compounds without the need to build any calibration graph, reducing the total analysis time. The combination of both extraction and determination techniques have allowed to validate for the first time a HF-LPME methodology at the required levels by legislation achieving limits of quantification of 0.1ngmL(-1) and recoveries within 97-109%. Due to the low cost of HF-LPME and total time consumption, this methodology is ready for implementation in routine analytical laboratories., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work we have developed and validated an accurate and fast methodology for the determination of 4-nonylphenol (technical mixture) in complex matrix water samples by UHPLC-ESI-MS/MS. The procedure is based on isotope dilution mass spectrometry (IDMS) in combination with isotope pattern deconvolution (IPD), which provides the concentration of the analyte directly from the spiked sample without requiring any methodological calibration graph. To avoid any possible isotopic effect during the analytical procedure the in-house synthesized (13)C1-4-(3,6-dimethyl-3-heptyl)phenol was used as labeled compound. This proposed surrogate was able to compensate the matrix effect even from wastewater samples. A SPE pre-concentration step together with exhaustive efforts to avoid contamination were included to reach the signal-to-noise ratio necessary to detect the endogenous concentrations present in environmental samples. Calculations were performed acquiring only three transitions, achieving limits of detection lower than 100ng/g for all water matrix assayed. Recoveries within 83-108% and coefficients of variation ranging from 1.5% to 9% were obtained. On the contrary a considerable overestimation was obtained with the most usual classical calibration procedure using 4-n-nonylphenol as internal standard, demonstrating the suitability of the minimal labeling approach., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Isotope dilution mass spectrometry (IDMS) based on isotope pattern deconvolution (IPD) has been applied here to MS/MS (QqQ) in order to carry out the quantification and confirmation of organic compounds in complex matrix water samples without the use of a methodological IDMS calibration graph. In this alternative approach, the isotope composition of the spiked sample is measured after fragmentation by SRM and deconvoluted into its constituting components (molar fractions of natural abundance and labeled compound) by multiple linear regression (IPD). The procedure has been evaluated for the determination of the pharmaceutical diclofenac in effluent and influent urban wastewaters and fortified surface waters by UHPLC (ESI) MS/MS using diclofenac-d(4) as labeled compound. Calculations were performed acquiring a part and the whole fragment cluster ion, achieving in all cases recoveries within 90-110% and coefficients of variation below 5% for all water samples tested. In addition, potential false negatives arising from the presence of diclofenac-d(2) impurities in the labeled compound were avoided when the proposed approach was used instead of the most usual IDMS calibration procedure. The number of SRM transitions measured was minimized to three to make possible the application of this alternative technique in routine multi-residue analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We present here a new environmental measurement method for the rapid extraction and accurate quantification of Cr(VI) in solid samples. The quantitative extraction of Cr(VI) is achieved in 10 minutes by means of focused microwave assisted extraction using 50 mmol/L Ethylendiamintetraacetic acid (EDTA) at pH 10 as extractant. In addition, it enables the separation of Cr species by anion exchange chromatography using a mobile phase which is a 1:10 dilution of the extracting solution. Thus, neutralization or acidification steps which are prone to cause interconversion of Cr species are not needed. Another benefit of using EDTA is that it allows to measure Cr(III)-EDTA complex and Cr(VI) simultaneously in an alkaline extraction solution. The application of a 10 minutes focused microwave assisted extraction (5 min at 90 °C plus 5 min at 110 °C) has been shown to quantitatively extract all forms of hexavalent chromium from the standard reference materials (SRM) candidate NIST 2700 and NIST 2701. A double spike isotope dilution mass spectrometry (IDMS) procedure was employed to study chromium interconversion reactions. It was observed that the formation of a Cr(III)-EDTA complex avoided Cr(III) oxidation for these two reference materials. Thus, the use of a double spiking strategy for quantification is not required and a single spike IDMS procedure using isotopically enriched Cr(VI) provided accurate results.

This work demonstrates, for the first time, the applicability of multiple spiking isotope dilution analysis to molecular mass spectrometry exemplified by the speciation analysis of mercury using GC(EI)MS instrumentation. A double spike isotope dilution approach using isotopically enriched mercury isotopes has been applied for the determination of inorganic mercury Hg(II) and methylmercury (MeHg) in fish reference materials. The method is based on the application of isotope pattern deconvolution for the simultaneous determination of degradation-corrected concentrations of methylmercury and inorganic mercury. Mass isotopomer distributions are employed instead of isotope ratios to calculate the corrected concentrations of the Hg species as well as the extent of species degradation reactions. The isotope pattern deconvolution equations developed here allow the calculation of the different molar fractions directly from the GC(EI)MS mass isotopomer distribution pattern and take into account possible impurities present in the spike solutions employed. The procedure has been successfully validated with the analysis of two different certified reference materials (BCR-464 and DOLT-4) and with the comparison of the results obtained by GC(ICP)MS. For the tuna fish matrix (BCR-464), no interconversion reactions were observed at the optimized conditions of open focused microwave extraction at 70 degrees C during 8 min. However, significant demethylation was found under the same conditions in the case of the certified dogfish liver DOLT-4. Methylation and demethylation factors were confirmed by GC(ICP)MS. Transformation reactions have been found to depend on the sample matrix and on the derivatization reagent employed. Thus, it is not possible to recommend optimum extraction conditions suitable for all types of matrices demonstrating the need to apply multiple spiking methodologies for the determination of MeHg and Hg(II) in biological samples. Double spike isotope dilution analysis methodologies using widespread GC(EI)MS instrumentation are proposed here for the routine analysis of inorganic mercury and methylmercury in fish samples. The estimated method detection limits were below 10 ng g(-1) for both mercury species. Precision was evaluated for the concentrations present in the certified reference materials (CRMs) which vary from 0.1 to 5 microg g(-1), achieving values of coefficients of variation ranging from 7% to 2%. The concentrations obtained in both CRMs analyzed were in excellent agreement with the certified values, demonstrating the accuracy of the method at these concentration levels.

The performance of microHPLC-microconcentric nebulizer-inductively coupled plasma-mass spectrometry (ICP-MS) coupling for the simultaneous determination of As(III), As(V), monomethylarsenic acid (MMA), dimethylarsinic acid (DMA), selenite (SeIV) and selenate (SeVI) in water was evaluated. The accurate reduction of the off-column dead volume, specially the capillary of the micronebulizer, as well as the optimization of chromatographic conditions led to the claimed advantages expected for microbore columns: a significant diminution of sample and solvent consumption without sacrificing sensitivity and the overall resolution in faster analysis time (less than 5 min). Detection limits are in the range 0.03-0.04 microg L(-1) for arsenic species and 0.35 microg L(-1) for selenium species. The developed method was validated by analysing different spiked environmental water samples. Linearity, tested up to 50 microg L(-1), showed correlation coefficients above 0.999 and no matrix effect for high saline water samples. Good accuracy and repeatability was obtained for spiked influent and effluent water treatment plant.

Relevant secondary interactions (hydrogen-bond type), additional to the main anion-exchange mechanism, were found when a method for As, Se and Cr speciation was developed based on microLC-inductively coupled plasma mass spectrometry (ICP-MS) coupling. In order to get the claimed analytical performance characteristics of the microbore columns, microLC systems are equipped with very narrow bore fused silica capillaries. When a mobile phase of NH(4)NO(3) at pH 8.7 was used, a notable tailing was observed for As(III), As(V), MMA and Se(IV), species containing hydroxyl groups in its chemical structure at this pH value. However, additional interactions appeared neither when the fused silica capillaries of the capillary LC system were substituted for polyetheretherketone (PEEK) nor operating at pH below 8.5. A mechanism to explain the additional interaction observed is proposed and tested in this work. It seems that high pH values produce a partial hydrolysis of the siloxane groups of the fused silica capillaries. Under these conditions, degradation products of silica, containing ionized silanol groups, reach the column and interact with the anion-exchange resin. Then, ionized silanol groups, retained on the column, can interact with the hydroxyl moiety of the aforementioned analytes leading to severe peak tailing and broadening. Different strategies were evaluated to solve the problem. The addition of a salt containing hydroxyl groups in the mobile phase such as hydrogen phosphate, the diminution of the pH and the use of PEEK capillaries in the microHPLC system demonstrated to be suitable. Finally, two alternative microHPLC-ICP-MS separations, based on a gradient elution of NH(4)NO(3) at pH 8.0 and NH(4)NO(3)/NH(4)H(2)PO(4) at pH 8.7, were optimized and compared. Results showed better peak shapes for some species when hydrogen phosphate was added to the mobile phase.

A method based on the coupling microHPLC-microneb-ICPMS has been developed for Hg(II), MeHg+, EtHg+ and PhHg+ species. Gradient elution using methanol and l-cysteine at pH 3.0 allowed the chromatographic separation of all species in less than 13 min (total analysis time 15 min). The direct coupling of microLC to ICPMS through a Micromist nebulizer permits the analysis of environmental water without sample pretreatment and derivatization steps. Nebulizer type, organic modifier and column length were the main parameters tested. The methanol content and pH of the mobile phase greatly affected the retention time and sensitivity of the method. Key factors to obtain high signal to noise ratio, at concentrations below 1 microg L(-1), were found to be the nebulization step and traces of Hg present in the complexing agent. A detailed optimization of carrier and make up gas flow rates have enabled the nebulization of the methanol gradient elution with good mass transport efficiency, low organic solvent loading into the plasma and excellent precision. The performance of the microHPLC-microneb-ICPMS method developed was evaluated on a surface water sample filtered (0.22 microm) and spiked with 0.5 microg L(-1) (as Hg) of each species. Precision (R.S.D., n=6) for all species of Hg varied from 0.5 to 2.1%. Detection limit, defined as three times the standard deviation (n=6), ranged from 8 ng L(-1) for EtHg+ to 32 ng L(-1) for PhHg+ and was noticeably lower than those reported in previous LC-based methods. Accuracy was suitable with recoveries ranging from 85 to 100% when tested at two levels (0.5 and 10 microg L(-1)) in groundwater samples. Recovery was matrix affected when water samples of high salinity (depurated wastewater and seawater) were used.

Lead is a nonphysiological metal that has been implicated in toxic processes that affect several organ systems in humans and other animals. Although the brain generally has stronger protective mechanisms against toxic substances than other organs have, exposure to lead results in several neurophysiological and behavioral symptoms. The administration of a single injection (i.p.) of lead acetate in mice is a model of acute Pb2 + toxicity. In the present study, this model was used to explore the magnitude of the effect of different doses, time intervals and mice strains on several biobehavioral parameters. We investigated the effects of acute lead acetate administration on body and brain weight, brain lead acetate accumulation and specially, spontaneous locomotion and brain catalase activity. Lead acetate was injected i.p. in outbred (Swiss or CD1) and inbred (BALB/c, C57BL/J6 or DBA/2) mice at doses of 0, 50, 100, 150 or 200 mg/kg. At different time intervals following this acute treatment, several biochemical, physiological and behavioral responses were recorded. Results indicated that acute lead acetate has deleterious dose-dependent effects on brain and body weight. The effect on body weight in the present study was transient, although lead acetate was detected in neural tissues for several days after administration. Spontaneous locomotor activity only was reduced up until 24 hours. The effect of lead on body weight was strain-dependent, with Swiss mice showing greater resistance compared to the other strains. Total brain catalase activity in lead-pretreated Swiss mice showed a significant induction. This enzymatic upregulation could provide a protective mechanism for oxidative stress in these mice.

A new method for the simultaneous chromatographic separation and determination of arsenite, arsenate, mono-methylarsonic acid, dimethylarsinic acid, selenite, selenate and hexavalent chromium in water is presented. Speciation was achieved by on-line coupling of anion-exchange LC and inductively coupled plasma mass spectrometry (ICP-MS). Optimisation of the chromatographic conditions led to baseline separation of the seven species in 14 min using gradient elution with NH4NO3 20 mM, pH 8.7-NH4NO3 60 mM, pH 8.7 as mobile phase. Detection limits are in the range 40-60 ng l(-1) for arsenic species, around 130 ng l(-1) for Cr(VI), and higher for Se(IV) and Se(VI) (1.2 and 1.4 microg l(-1) respectively). The method showed good accuracy and repeatability, and no interference of chloride on 75As, 77Se or 53Cr was observed. The developed method was applied to the analysis of several environmental surface water samples.

The simultaneous determination of As(III), As(V), monomethylarsenic acid (MMA), dimethylarsinic acid (DMA) and Cr(VI) in fresh water has been carried out by coupling an anion-exchange column to an inductively coupled plasma-mass spectrometer. Optimisation of chromatographic conditions led to baseline separation of signals from the five species in approximately 9 min using gradient elution. Detection limits were 0.02-0.05 microg As l(-1) and 5.5 microg Cr l(-1). Repeatability was 2-3% for arsenic species and higher, i.e., 8%, for Cr(VI) due to the higher background for this species. Arsenic species and hexavalent chromium stability in surface water samples was evaluated, and storage conditions were set to 1 day at 4 degrees C in polyethylene flasks (without acidification) in order to avoid As(III)-As(V) conversions. The method was applied to the analysis of surface water.

The occupational exposure in workplace atmospheres of ceramic factories to some metals (Ba, Cd, Co, Cr, Cu, Ni, Zn) and boron has been studied. During 1994 samples were collected at several ceramic factories from the Comunitat Valenciana (Spain) in a random fashion to avoid correlation problems regarding data analysis; factories were sampled to provide a representative cross-section. Powdered samples were treated with concentrated nitric acid according to the National Institute for Occupational Safety and Health (NIOSH) digestion procedure for the analysis of metals in air samples. Heavy metals were analyzed by AAS and boron by UV/Visible spectrophotometry using the Azomethine-H method. Concentrations found for Cd, Cr, Cu, Ni and Zn were considered very low in the environment of this industrial sector. However, Co, Ba and B were detected in several samples with concentrations above the threshold limit value (TLV), referred to airborne concentrations of substances in workplace atmospheres. Principal component analysis (PCA) has also been carried out in order to classify the workplaces studied and their potential hazard to the workers.

The determination of boron in airborne particulates from ceramic factories' workplaces has been investigated. Samples containing raw or fritted material powder from ceramic factories have been digested in Erlenmeyer flasks using nitric acid as reagent. Analysis was performed by the Azomethine-H method. Recoveries for different raw materials were quantitative, whereas for fritted materials they widely depend on their composition. Relative standard deviations for all materials tested were lower than 8%. The accuracy of the procedure was tested by the addition of known amounts of boron to different types of materials. Standard addition calibrations proved that there was no influence of the matrix. In workplace atmospheres of ceramic industries boron was usually present, in the environment of enamel factories with levels higher than the recommended exposure value.