The impact of prenatal mercury on neurobehavioral functioning longitudinally assessed from a young age to pre-adolescence in a Spanish birth cohort.

The objective is to investigate the relation between cord blood mercury concentrations and child neurobehavioural functioning assessed longitudinally during childhood until pre-adolescence. METHODS: The study involves mothers and their offspring engaged in the Spanish INMA birth cohort (n = 1147). Total mercury (THg) was determined in cord blood. Behavioural problems were assessed several times during childhood using the ADHD-DSM-IV at age 4, SDQ at ages 7 and 11, CPRS-R:S and the CBCL at ages 7, 9 and 11. Covariates were obtained through questionnaires during the whole period. Multivariate generalised negative binomial (MGNB) models or mixed-effects MGNB (for those tests with information at one or more time points, respectively) were used to investigate the relation between cord blood THg and the children's punctuations. Models were adjusted for prenatal fish intake. Effect modification by sex, prenatal and postnatal fish intake, prenatal fruit and vegetable intake, and maternal polychlorinated biphenyl concentrations (PCBs) was assessed by interaction terms. RESULTS: The geometric mean ± standard deviation of cord blood THg was 8.22 ± 2.19 µg/L. Despite adjusting for fish consumption, our results did not show any statistically significant relationship between prenatal Hg and the children's performance on behavioural tests conducted between the ages of 4 and 11. Upon assessing the impact of various factors, we observed no statistically significant interaction. CONCLUSION: Despite elevated prenatal THg exposure, no association was found with children's behavioural functioning assessed from early childhood to pre-adolescence. The nutrients in fish could offset the potential neurotoxic impact of Hg. Further birth cohort studies with longitudinal data are warranted.

Exposure to mercury among Spanish adolescents: Eleven years of follow-up.

The toxic effects of mercury exposure on human health are a public health concern. The most important source of this exposure is the consumption of fish and marine mammals. This study aims to describe hair mercury concentrations and their evolution from birth until eleven years of age in adolescents from the INMA (Environment and Childhood) birth cohort study, and to assess the association of hair mercury concentrations at eleven years of age with sociodemographic and dietary factors. The sample comprised 338 adolescents from the sub-cohort of Valencia (in eastern Spain). Total mercury (THg) was measured in hair samples collected at 4, 9 and 11 years old and in cord blood at birth. The equivalent of hair for cord-blood THg concentrations was calculated. Fish consumption and other characteristics at 11 years old were collected through questionnaires. Multivariate linear regression models were conducted to explore the association between THg concentrations, fish consumption and covariates. The geometric mean of hair THg concentrations at 11 years of age was 0.86 µg/g (95%CI: 0.78-0.94) and 45.2% of the participants presented concentrations above the equivalent RfD proposed by the US EPA (1 µg/g). Consumption of fish such as swordfish, canned tuna and other large oily fish was associated with higher levels of hair mercury at 11 years of age. Swordfish had the highest effect with an increase of 125% in hair mercury (95%CI: 61.2-214.9%) given a 100 g/week increase in its consumption, and, taking into account the frequency of consumption, canned tuna was the main contributor to Hg exposure among our population. The hair THg concentrations at 11 years of age represented a reduction of around 69% with respect to that estimated at childbirth. Even though THg exposure shows a sustained decreasing trend, it can still be considered elevated. INMA birth cohort studies provide a longitudinal assessment of mercury exposure in a vulnerable population, its associated factors and temporal trends, and this information could be used to adjust recommendations about this issue.

Pre and postnatal exposure to mercury and sexual development in 9-year-old children in Spain: The role of brain-derived neurotrophic factor.

Early exposure to mercury has been related to endocrine disruption. Steroid hormones play a crucial role in neural cell migration, differentiation, etc., as well as protecting against several neurotoxic compounds. We investigate the relation between mercury exposure and children's sexual development, and we evaluate the possible influence of different brain-derived neurotrophic factor (BDNF) polymorphisms on this association. Our study sample comprised 412 9-year-old children participating in the INMA cohort (2004-2015). Mercury concentrations were measured at birth (cord blood) and at 4 and 9 years of age (hair). Sexual development was assessed by levels of sex steroid hormones (estradiol and testosterone) in saliva and the Tanner stages of sex development at 9 years (categorized as 1: prepuberty and >1: pubertal onset). Covariates and confounders were collected through questionnaires during pregnancy and childhood. Polymorphisms in the BDNF gene were genotyped in cord blood DNA. Multivariate linear regression analyses were performed between mercury levels and children's sexual development by sex. Effect modification by genetic polymorphisms and fish intake was assessed. We found marginally significant inverse associations between postnatal exposure to mercury (at 9 years) and testosterone levels (ß[95%CI] = -0.16[-0.33,0.001], and -0.20[-0.42,0.03], for boys and girls, respectively). Additionally, we found that prenatal mercury was negatively associated with Tanner stage >1 in boys. Finally, we found significant genetic interactions for some single nucleotide polymorphisms in the BDNF gene. In conclusion, pre and postnatal exposure to mercury seems to affect children's sexual development and BDNF may play a role in this association, but further research would be needed.

Pre and postnatal exposure to mercury and respiratory health in preschool children from the Spanish INMA Birth Cohort Study.

Effects of mercury on maturing immune system have been reported, however the association with respiratory and allergy problems during infancy remains unclear. The aim of this study is to evaluate the association between pre and postnatal mercury exposure and respiratory and allergy problems among preschool children and to examine the role of potential modifying factors. Study subjects were children participant in Spanish Childhood and Environment Project (INMA, 2003-2008). We measured total mercury levels in cord blood (n = 1868) and hair at 4 years of age (n = 1347). Respiratory outcomes (wheezing, severe wheezing, chestiness, persistent cough, eczema and otitis) were obtained by questionnaires administered to parents. Associations were investigated by logistic regression adjusted for socio-demographic and lifestyle-related variables in each cohort and subsequent meta-analysis. We tested effect modification by factors related to individual susceptibility, diet and co-exposure with other pollutants. The geometric mean of cord blood and hair total mercury was 8.20 µg/L and 0.97 µg/g, respectively. No statistically significant association between pre or postnatal mercury exposure and respiratory and allergy outcomes was found. Notwithstanding, lower maternal intake of fruits and vegetables increased the risk of some respiratory outcomes due to the prenatal exposure to mercury (pint < 0.05). Moreover, an inverse association between prenatal mercury exposure and some respiratory outcomes was observed among children with higher maternal exposure to organocholorine compounds or smoking (pint < 0.05). Also, sex and postnatal smoking exposure modulated mercury postnatal effects on persistent cough (pint < 0.05). In conclusion, no association between pre and postnatal mercury exposure and respiratory and allergy problems among the whole population at study was found. However, diet and other toxicants could modulate this relation, especially during prenatal period. More research on this topic is warranted due to the limited evidence.

Exposure to mercury among 9-year-old children and neurobehavioural function.

Mercury (Hg) is an environmental neurotoxicant whose main route of exposure in humans is the consumption of seafood. The aim of this study was to explore the relationship between Hg exposure at 9 years old and behaviour assessed at 9 and 11 years old. Study subjects were mother-child pairs participating in the INMA (Environment and Childhood) Project in Valencia (Spain). Total Hg (THg) was measured in hair samples from the children at 9 years old. Behaviour and emotions were assessed at 9 (n = 472) years and 11 (n = 385) years of age using the Child Behaviour Checklist test (CBCL) and the Conners Parents Rating Scale-Revised: Short Form (CPRS-R:S). Furthermore, the attention function was assessed by the Attention Network Test at 11 years old. Socio-demographic, lifestyle and dietary information was collected through questionnaires during pregnancy and childhood. Polymorphism in BDNF, APOE and GSTP1 were genotyped in cord blood DNA. Multivariable negative binomial regression models were built in order to study the association between THg concentrations and the scores obtained by the children at 9 and 11 years old. Effect modification by sex and genetic polymorphisms was assessed. The association between Hg levels and CBCL scores was positive (worse neurobehavioural development) for the CBCL internalizing and total problem scales (Incidence Rate Ratio [95% confidence interval] = 1.07 [1.01, 1.13] and 1.05 [0.99, 1.11], respectively). The association between Hg and the externalizing and total problems CBCL scales and the Attention Deficit Hyperactivity Disorder (ADHD) index of the CPRS-R:S was different according to sex, with boys obtaining worse scores with increasing Hg, compared to girls. Statistically significant interactions were also observed for genetic polymorphisms affecting the association between early exposure to Hg and both CBCL and CPRS-R:S scores. In conclusion, postnatal Hg exposure is associated with poorer neurobehavioural development in 9- and 11-year-old children. Sex and the presence of certain genetic polymorphisms modified this association.

Postnatal exposure to mercury and neuropsychological development among preschooler children.

The objective of this study was to describe the postnatal exposure to Hg and to evaluate its association with neuropsychological development among preschool children. The study population are 4-5 years old children (n = 1252) participant in the Spanish INMA Project. Total Hg was measured in cord blood and in hair samples taken at 4 years of age (2008-2012). Neuropsychological development was assessed using the McCarthy Scales of Children's Abilities (MSCA). Information on covariates and possible confounders was obtained by questionnaires during pregnancy and childhood. Generalized additive and linear regression models were built in order to assess the relationship between MSCA scores and Hg exposure. We also evaluated the magnitude of the possible bias generated from measurement error in seafood intake estimate from questionnaire and Hg determination. The geometric mean of hair Hg was 0.98 µg/g [95% confidence interval (CI) 0.94, 1.03]. In the regression analysis, the association between Hg and the MSCA scores was positive for all the scales and statistically significant for the verbal (ß = 0.89; 95%CI 0.38, 1.39), memory (ß = 0.42; 95%CI 0.09, 0.76) and general cognitive scales (ß = 1.35; 95%CI 0.45, 2.25). However, these associations were clearly attenuated when we adjusted by the children's fish intake variables or when took into account theoretical scenarios of low precision in fish intake and Hg measurements. Hg levels in this Spanish population were high in comparison with other European countries; however, we did not observe adverse effects on child neuropsychological development associated with this postnatal exposure to Hg.

Exposure to mercury among 9-year-old Spanish children: Associated factors and trend throughout childhood.

Mercury is considered a neurotoxicant and human exposure occurs mainly from the consumption of marine species. We aimed to describe total mercury concentrations (THg) and associated factors in 9-year old children, as well as to explore the trend in THg from 4 to 9 years of age. The study population consisted of 9-year-old children participating in the INMA (Environment and Childhood) birth cohort study in Valencia, Spain (n = 405, 2013-2014). THg in hair samples was measured by atomic absorption spectrometry at the age of 4 and 9 years. Sociodemographic and dietary data was obtained through questionnaires. Multiple linear regression was used to explore the association between THg and covariates. The geometric mean (95% confidence interval) of hair THg at 9 years old was 0.89 µg/g (0.81, 0.98). Thirteen percent of children had THg above the equivalent to the Provisional Tolerable Weekly Intake proposed by the World Health Organization. THg were higher among children whose mothers had a healthy body mass index before pregnancy. Children with non-smoker mothers and worker fathers had also higher THg. Children's fish intake at 9 years-old was positively associated with THg, being swordfish, canned tuna and lean fish (i.e. hake, sea bream and sole) the most associated categories. Levels decreased by around 22% between 4 and 9 years old. Birth cohort studies, such as the INMA Project, allow the longitudinal evaluation of Hg exposure and the possible effects on children's health. This information can be used to formulate diet recommendations in vulnerable populations.

Bioanalytical chromatographic method validation according to current regulations, with a special focus on the non-well defined parameters limit of quantification, robustness and matrix effect.

Method validation is a mandatory step in bioanalysis, to evaluate the ability of developed methods in providing reliable results for their routine application. Even if some organisations have developed guidelines to define the different parameters to be included in method validation (FDA, EMA); there are still some ambiguous concepts in validation criteria and methodology that need to be clarified. The methodology to calculate fundamental parameters such as the limit of quantification has been defined in several ways without reaching a harmonised definition, which can lead to very different values depending on the applied criterion. Other parameters such as robustness or ruggedness are usually omitted and when defined there is not an established approach to evaluate them. Especially significant is the case of the matrix effect evaluation which is one of the most critical points to be studied in LC-MS methods but has been traditionally overlooked. Due to the increasing importance of bioanalysis this scenario is no longer acceptable and harmonised criteria involving all the concerned parties should be arisen. The objective of this review is thus to discuss and highlight several essential aspects of method validation, focused in bioanalysis. The overall validation process including common validation parameters (selectivity, linearity range, precision, accuracy, stability…) will be reviewed. Furthermore, the most controversial parameters (limit of quantification, robustness and matrix effect) will be carefully studied and the definitions and methodology proposed by the different regulatory bodies will be compared. This review aims to clarify the methodology to be followed in bioanalytical method validation, facilitating this time consuming step.

LC-MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma.

A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150mm×4.6mm, 3µm) column using a gradient elution mode with a run time of 15min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68-106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.

Validation of a fast liquid chromatography-UV method for the analysis of drugs used in combined cardiovascular therapy in human plasma.

Ultra-performance liquid chromatography (UPLC) was investigated as a faster alternative to high-performance liquid chromatography (HPLC) for the simultaneous analysis of drugs usually prescribed in cardiovascular therapy. Upon a previously developed and validated solid phase extraction (SPE)-HPLC-photodiode array (PDA)-fluorescence (FLR) method, separation of chlorthalidone (CLTD; diuretic), valsartan and its metabolite (VAL and VAL-M1 respectively; angiotensin II receptor antagonist drugs) and fluvastatin (FLUV; statin) was performed in human plasma using an RP C18 column (50mmx2.1mm, 1.7microm, Waters Acquity UPLC (BEH)) and a tunable UV-vis (TUV) detector. After method transfer, different system variables were modulated to study the evolution of responses of the analytes and the endogenous interferences. The improved method was fully validated and the results were compared with its precursor HPLC method relating to analysis time, efficiency and sensitivity. The studied compounds were separated in less than 8min and the method showed good linearity (20-3000microg/L for chlorthalidone, 110-1100microg/L for valsartan-M1, 67-1900microg/L for valsartan and 48-1100microg/L for fluvastatin), precision and accuracy. The proposed method was found to be reproducible (RSD<10%), accurate (RE<15%), robust and suitable for quantitative analysis of the studied drugs in plasma obtained from patients under combined cardiovascular treatment.

Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma.

This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm x 3.9 mm, 3 microm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31-3000 microg/l for chlorthalidone, 20-1000 microg/l for valsartan-M1, 10-5000 microg/l for valsartan and 14-1000 microg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78-91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment.

Separation and quantitation of several angiotensin II receptor antagonist drugs in human urine by a SPE-HPLC-DAD method.

In this work, an SPE-HPLC method coupled to photodiode array detection was validated in human urine matrix, in order to monitor four antihypertensive angiotensin II receptor antagonist drugs in patients under cardiovascular treatment. For that purpose, experimental design was used. Quantitation was accomplished by the internal standard method. The obtained LOQs were 95, 113, 125, and 85 ng/mL for eprosartan, telmisartan, irbesartan, and valsartan, respectively. The intraday and interday precision and accuracy at four concentration levels in the working range (LOQ-15 microg/mL) were always lower than 11% RSD and 8% relative error. The urine samples proved to be stable during 4 h at room temperature, after three thaw-freeze cycles, and for 2 months at -20 degrees C. No interferences from other endogenous compounds or co-administered drugs were found. The method has been successfully applied to monitor the renal elimination of eprosartan and valsartan during 24 h.

Biovalidation of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite valeryl-4-hydroxy-valsartan in human plasma.

A simple and fast method for the simultaneous determination of the antihypertensive drug Valsartan and its metabolite in human plasma has been validated. The proposed method deals with SPE, followed by an HPLC separation coupled with fluorimetric and photometric detection. The optimization of the SPE-HPLC method was achieved by an experimental design. The separation was performed on an RP C18 Atlantis 100 mmx3.9 mm column. The mobile phase consisted of a mixture of ACN 0.025% TFA and phosphate buffer (5 mM, pH = 2.5) 0.025% TFA and was delivered in gradient mode at a flow rate of 1.30 mL/min. The eluent was monitored with a fluorescence detector at 234 and 378 nm excitation and emission wavelengths, respectively, and at 254 nm using a photometric detector. The full analytical validation was performed according to the Food and Drug Administration (FDA) 'guidance for industry: bioanalytical method validation' and the recoveries obtained for Valsartan and its metabolite ranged from 94.6 to 108.8%. The validated method was successfully applied to 12 plasma samples obtained from patients under antihypertensive treatment with Valsartan.

Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples.

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.