Validation of an optimized method for the determination of iodine in human breast milk by inductively coupled plasma mass spectrometry (ICPMS) after tetramethylammonium hydroxide extraction

In this study a novel method to determine iodine concentrations in human breast milk was developed and validated. The iodine was analyzed by inductively coupled plasma mass spectrometry (ICPMS) following tetramethylammonium hydroxide (TMAH) extraction at 90 °C in disposable polypropylene tubes. While similar approaches have been used previously, this method adopted a shorter extraction time (1 h vs. 3 h) and used antimony (Sb) as the internal standard, which exhibited greater stability in breast milk and milk powder matrices compared to tellurium (Te). Method validation included: defining iodine linearity up to 200 μg L⁻¹; confirming recovery of iodine from NIST 1549 milk powder. A recovery of 94–98% was also achieved for the NIST 1549 milk powder and human breast milk samples spiked with sodium iodide and thyroxine (T4) solutions. The method quantitation limit (MQL) for human breast milk was 1.6 μg L⁻¹. The intra-assay and inter-assay coefficient of variation for the breast milk samples and NIST powder were <1% and <3.5%, respectively. NIST 1549 milk powder, human breast milk samples and calibration standards spiked with the internal standard were all stable for at least 2.5 months after extraction. The results of the validation process confirmed that this newly developed method provides greater accuracy and precision in the assessment of iodine concentrations in human breast milk than previous methods and therefore offers a more reliable approach for assessing iodine concentrations in human breast milk.     

Confirmatory analysis of firocoxib in bovine milk by rapid resolution liquid chromatography tandem mass spectrometry

A rapid method has been developed to analyse for firocoxib (FIRO) residue in bovine milk. Milk samples were extracted with acetonitrile and sample extracts were purified on Evolute? ABN solid phase extraction cartridges. Aliquots were analysed by rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS). The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCα) was 1.18 ng/mL and for the detection capability a (CCβ) value of 2.02 ng/mL was obtained. The measurement uncertainty of the method was 27%. Fortifying bovine milk samples (n = 18) in three separate assays, show the accuracy of the method to be between 96 and 105%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng/mL) was less than 11% respectively.     

Excess iodinuria in infants and its relation to the iodine in maternal milk

ObjectiveIodine is an essential micro nutrient, and a deficiency or excessive intake of this mineral is related to changes in thyroid function. In Brazil, both deficiency and excessive intake of iodine are common; however, excessive intakes have recently been observed. Thus, the objective of the present study was to assess the iodine concentration in maternal milk, taking into account the salt iodine concentration of the participating households and in the infants’ urine.MethodUrine samples from 33 infants (less than 6 months of age), maternal milk samples and samples of the kitchen salt used by the mothers were collected. The iodine levels in the urine and maternal milk were assessed by ICP-MS; the iodine levels in the salt were assessed by titration.ResultThe median iodinuria value in the infants was 293 μg/L; the mean iodine concentration was 206 μg/L in the maternal milk and 39.9 mg I/kg in the salt. There was a positive correlation between the iodine concentration in the maternal milk and the infant iodinuria value.ConclusionThe median infant iodinuria was elevated due to the high iodine concentration present in the maternal milk. High iodine values were caused by high salt iodine levels, which should be reduced.     

Endocrine responses and milk emission characteristics in high yielding Alpine dairy goats under once daily milking management

This study was done to verify if the lack of increase of milk fat content observed in Alpine dairy goats under once daily milking (ODM) compared to twice daily milking (TDM), results from disturbances of animals and/or milk ejection reflex. In this respect, we determined the milk yield and composition in the same 12 multiparous Alpine dairy goats when they were managed first under TDM (period 1: P1) and then under ODM (period 2: P2). Furthermore, oxytocin (OT) and cortisol (CORT) releases and milk emission kinetic of these goats were measured at morning milking 2 and 4 times in P1 and P2, respectively.ODM compared to TDM, caused 18 and 23% reductions, respectively in daily milk yield and milk fat content without milk protein content modification.Although baseline concentration of blood OT was lower under ODM than TDM management (7.0 pg/mL vs 17.8 ± 2.0 pg/mL, respectively), ODM did not modify the total amount of OT released during milking (15,484 pg/mL/32 min vs 18,996 ± 2865.3 pg/mL/32 min, respectively), the peak concentration of OT (57.2 pg/mL vs 73.6 ± 10.5 pg/mL, respectively) or the time to reach it (3.0 min vs 2.0 ± 0.5 min, respectively) by comparison to TDM.ODM compared to TDM, never modified the baseline concentrations of CORT (6.1 ng/mL vs 7.1 ± 1.1 ng/mL, respectively), the total amount of CORT released (11,727 ng/mL/32 min vs 10,073 ± 1522.2 ng/mL/32 min, respectively), the peak concentrations of CORT (16.1 ng/mL vs 15.1 ± 2.1 ng/mL, respectively) and the time to reach it (13.1 min vs 11.0 ± 1.5 min).During ODM, milk flow latency was reduced (−61%) while the mean flow rate was increased (+28%) by comparison to TDM. ODM compared to TDM management, did not modify the maximum flow rate (1.6 L/min vs 1.6 ± 0.2 L/min, respectively) or the time to reach this maximum (103.0 s vs 95.0 ± 10.0 s, respectively). The total milking duration at morning milking was not different (250.0 s vs 221.0 ± 22.0 s, respectively) although the morning milk yield was significantly higher under ODM management (2.63 kg/d vs 1.87 ± 0.1 kg/d, respectively).This results show that milk emission is improved under ODM management in Alpine goats without inhibition or disturbance of neuro-hypophyseal OT release pattern and lack of activation of the hypothalamic–pituitary–adrenal axis and plasma cortisol increase, disproving our initial hypotheses of a disturbance of animals and/or incomplete milk ejection to explain the low fat content of milk under ODM in this breed.     

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C₁₈ column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.     

Concentrations of preptin,salusins and hepcidins in plasma and milk of lactating women with or without gestational diabetes mellitus

This study was undertaken to ascertain whether human milk contains preptin, salusin-alpha (salusin-α) and -beta (salusin-β) and pro-hepcidin and hepcidin-25, and whether there are relationships between plasma and milk preptin, salusin-α and -β and pro-hepcidin and hepcidin-25 concentrations in lactating mothers with and without gestational diabetes mellitus (GDM). Blood was obtained from non-lactating women (n = 12), non-diabetic lactating women (n = 12), and GDM lactating women (n = 12). Colostrum, transitional milk, and mature milk samples were collected just before suckling from healthy and GDM lactating women. Peptides concentrations were determined by ELISA and EIA. Mammary gland tissues were screened immunohistochemically for these peptides. Women with GDM had significantly higher plasma and colostum preptin concentrations than healthy lactating women during the colostral and transitional milk period. Salusin-alpha and -beta levels in milk and plasma were lower in women with GDM. Salusin-α and -β were significantly lower in both plasma and colostrums of GDM than of healthy lactating women. Women with GDM had significantly higher colostum prohepcidin and hepcidin-25 concentrations than healthy lactating women during the colostral period. Plasma prohepcidin was also higher in women with GDM than in healthy lactating women during the colostral period, but plasma prohepcidin and hepcidin-25 levels decreased during mature milk period. Transitional milk pro-hepcidin and hepcidin-25 levels in women with GDM were higher than in healthy lactating women. All these results revealed that the mammary gland produces those peptides, which were present in milk at levels correlating with plasma concentrations.     

Saliva as a medium for aldosterone measurement in repeated sampling studies

BackgroundSaliva is a readily available biological fluid, making it convenient in diagnosis of diseases and in multi-sampling protocols. Several salivary steroids give a useful index of free plasma levels. Increased incidence of primary aldosteronism (PA) in approximately 10% of the hypertensive population has increased interest in the mineralocorticoid aldosterone.MethodsA biotinylated-aldosterone tracer and a commercially available antibody are used in a time-resolved fluorescence immunoassay (TR-FIA) to measure salivary aldosterone (SA). Saliva was collected in various multi-sampling protocols: Investigation of diurnal rhythm in healthy and PA patients, ACTH stimulation test and posture test in healthy subjects.ResultsMethod validation showed a sensitivity of 19 ng/L and intra-/inter-assay precision between 7.2–10.1% and 8.7–15.7%, respectively. SA correlated significantly (y = 0.2995x ± 0.01, r² = 0.60) to plasma aldosterone measured by a commercial radioimmunoassay. SA (median; 95%CI) was at 111 (95–127) ng/L in PA (n = 84) and 50 (44–56) ng/L in healthy subjects (n = 60). After change in posture, aldosterone increased in both, saliva (57 (47–63) ng/L to 95 (84–117) ng/L) and plasma (26 (26–41) ng/L to 135 (110–181) ng/L). Peak levels were reached after 1 h, and were higher in females than in males.ConclusionsSA correlates well to plasma aldosterone and mirrors responses during conditions of stress. SA is significantly higher in PA, and the diurnal rhythm seen in the healthy is blunted in PA. We additionally found gender-dependent differential responses to posture, with higher increases in females. Measurement of aldosterone in saliva presents a useful and convenient method for application in multi-sampling studies.     

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.     

Revised reference values for selenium intake

The German, Austrian and Swiss nutrition societies are the joint editors of the ‘reference values for nutrient intake’. They have revised the reference values for the intake of selenium and published them in February 2015. The saturation of selenoprotein P (SePP) in plasma is used as a criterion for the derivation of reference values for selenium intake in adults. For persons from selenium-deficient regions (China) SePP saturation was achieved with a daily intake of 49 μg of selenium. When using the reference body weights the D-A-CH reference values are based upon, the resulting estimated value for selenium intake is 70 μg/day for men and 60 μg/day for women. The estimated value for selenium intake for children and adolescents is extrapolated using the estimated value for adults in relation to body weight. For infants aged 0 to under 4 months the estimated value of 10 μg/day was derived from the basis of selenium intake via breast milk. For infants aged 4 to under 12 months this estimated value was used and taking into account the differences regarding body weight an estimated value of 15 μg/day was derived. For lactating women compared to non-lactating women a higher reference value of 75 μg/day is indicated due to the release of selenium with breast milk. The additional selenium requirement for pregnant women is negligible, so that no increased reference value is indicated.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.     

Comparison of accuracy of ultrasonography,progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Evaluación de los hábitos alimentarios relacionados con la ingesta de yodo,el estado nutricional de yodo y disfunción tiroidea en cuatro poblaciones no seleccionadas (proyecto Tirobus)

IntroductionMost of the studies on urinary iodine levels in Spain in the last decade have reported a significant improvement. A survey was undertaken together with an information campaign on the thyroid gland, the importance of iodine intake and hypothyroidism in four Spanish cities. The goals of the survey were to obtain information on consumption of iodine-containing foods, to measure urinary iodine levels and to evaluate the prevalence of thyroid dysfunction.Materials and methodsA non-preselected population attending the information campaign centers located in Barcelona, La Coruña, Malaga and Madrid was studied. A questionnaire on fish, milk and iodized salt consumption was administered. Urinary iodine levels (Pino's method) and thyrotropin (TSH) concentrations (Whatman 903^® dry paper method) were measured.ResultsA total of 872 questionnaires were completed (Madrid 40%; La Coruña 27%; Malaga 19%; and Barcelona 14%). The mean age was 51 years (SD 16); 81% were women. A total of 60.6% of interviewees reported they consumed iodized salt, 90.8% reported daily milk intake and 29.3% reported fish consumption ≥3 times per week. The mean urinary iodine concentration was 143.2 μg/L. The prevalence of high TSH levels (>4 mUI/L) was 1.3% and that of low TSH levels (<0.4 mUI/mL) was 1.2%.ConclusionsAccording to the World Health Organization criteria, the median urinary iodine concentration, both overall or by city, is indicative of optimal iodine intake. In addition to iodized salt intake, consumption of products such as milk and fish has probably contributed to these positive results. The prevalences of undiagnosed hyperthyroidism and hypothyroidism detected in this study were similar to those found in other studies.     

An approach for manganese biomonitoring using a manganese carrier switch in serum from transferrin to citrate at slightly elevated manganese concentration

After high-dose-short-term exposure (usually from occupational exposure) and even more under low-dose long term exposure (mainly environmental) manganese (Mn) biomonitoring is still problematic since these exposure scenarios are not necessarily reflected by a significant increase of total Mn in blood or serum. Usually, Mn concentrations of exposed and unexposed persons overlap and individual differentiation is often not possible. In this paper Mn speciation on a large sample size (n = 180) was used in order to be able to differentiate between highly Mn-exposed or low or unexposed individuals at low total Mn concentration in serum (Mn(S)). The whole sample set consisted of three subsets from Munich, Emilia Romagna region in Italy and from Sweden. It turned out that also at low total Mn(S) concentrations a change in major Mn carriers in serum takes place from Mn-transferrin (Mn-Tf(S)) towards Mn-citrate (Mn-Cit(S)) with high statistical significance (p < 0.000002). This carrier switch from Mn-Tf(S) to Mn-Cit(S) was observed between Mn(S) concentrations of 1.5 μg/L to ca. 1.7 μg/L. Parallel to this carrier change, for sample donors from Munich where serum and cerebrospinal fluid were available, the concentration of Mn beyond neural barriers – analysed as Mn in cerebrospinal fluid (Mn(C)) – positively correlates to Mn-Cit(S) when Mn(S) concentration was above 1.7 μg/L. The correlation between Mn-Cit(S) and Mn(C) reflects the facilitated Mn transport through neural barrier by means of Mn-citrate. Regional differences in switch points from Mn-Tf(S) to Mn-Cit(S) were observed for the three sample subsets. It is currently unknown whether these differences are due to differences in location, occupation, health status or other aspects. Based on our results, Mn-Cit(S) determination was considered as a potential means for estimating the Mn load in brain and CSF, i.e., it could be used as a biomarker for Mn beyond neural barrier. For a simpler Mn-Cit(S) determination than size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS), ultrafiltration (UF) of serum samples was tested for suitability, the latter possibly being a preferred choice for routine occupational medicine laboratories. Our results revealed that UF could be an alternative if methodical prerequisites and limitations are carefully considered. These prerequisites were determined to be a thorough cleaning procedure at a minimum Mn(S) concentration >1.5 μg/L, as at lower concentrations a wide scattering of the measured concentrations in comparison to the standardized SEC-ICP-MS results were observed.     

Effect of dietary conjugated linoleic acid on serum levels of N2O5 and l-citrulline in goat kids

Forty new born male kids were allotted into two groups for the evaluation of the effects of dietary conjugated linoleic acid (CLA) on nitric oxide (NO) and l-citrulline levels in serum. The control group received no supplement CLA and the CLA group received 20 g/kg milk DM of CLA from birth to 1-month old. The kids were fed colostrum for 2 days and milk replacer from days 3 to 29. Blood samples (n = 40) were taken at 1, 8, 15, 22, and 29 days of age. N₂O₅ (nitrite + nitrate) concentration in serum ranged from 21.93 to 26.15 and 34.90 to 40.59 μmol in control and CLA kids. The l-citrulline values ranged from 0.30 to 0.40 and 15.54 to 19.81 μmol in control and CLA kids.     

Antioxidant activity of blueberry fruit is impaired by association with milk

The antioxidant properties of dietary phenolics are believed to be reduced in vivo because of their affinity for proteins. In this study we assessed the bioavailability of phenolics and the in vivo plasma antioxidant capacity after the consumption of blueberries (Vaccinium corymbosum L.) with and without milk. In a crossover design, 11 healthy human volunteers consumed either (a) 200 g of blueberries plus 200 ml of water or (b) 200 g of blueberries plus 200 ml of whole milk. Venous samples were collected at baseline and at 1, 2, and 5 h postconsumption. Ingestion of blueberries increased plasma levels of reducing and chain-breaking potential (+ 6.1%, p < 0.001; + 11.1%, p < 0.05) and enhanced plasma concentrations of caffeic and ferulic acid. When blueberries and milk were ingested there was no increase in plasma antioxidant capacity. There was a reduction in the peak plasma concentrations of caffeic and ferulic acid (? 49.7%, p < 0.001, and ? 19.8%, p < 0.05, respectively) as well as the overall absorption (AUC) of caffeic acid (p < 0.001). The ingestion of blueberries in association with milk, thus, impairs the in vivo antioxidant properties of blueberries and reduces the absorption of caffeic acid.     

Domoic acid exposure and associated clinical signs and histopathology in Pacific harbor seals (Phoca vitulina richardii)

Domoic acid (DA) is a potent neurotoxin that has caused strandings and mortality of seabirds and marine mammals off the California coast. Pacific harbor seals (Phoca vitulina richardii) are an abundant, nearshore species in California; however, DA exposure and toxicosis have not been documented for harbor seals in this region. To investigate DA exposure in harbor seals, samples were collected from free-ranging and stranded seals off California to assess exposure, clinical signs of toxicosis, and brain lesions in harbor seals exposed to DA. Domoic acid was detected in 65% (17/26) of urine samples collected from apparently healthy free-ranging seals, with concentrations of 0.4–11.7 ng/ml. Domoic acid also was detected in feces (2.4–2887 ng/g), stomach contents (1.4 ng/g; stranded only), milk (2.2 ng/ml; stranded only), amniotic fluid (9.7 ng/ml; free-ranging only), fetal meconium (14.6–39.8 ng/g), and fetal urine (2.0–10.2 ng/ml). Clinical signs indicative of DA toxicosis were observed in two live-stranded seals, and included disorientation, seizures, and uncoordinated movements. Histopathology revealed the presence of brain lesions consistent with DA toxicosis in two live-stranded seals, and one free-ranging seal that died during capture. Results indicated that harbor seals were exposed to DA, exhibited clinical signs and histological lesions associated with DA exposure, and that pups were exposed to DA in utero and during lactation via milk. Future investigation is required to determine the magnitude of impact that DA has on the health and mortality of harbor seals.     

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp³-bradykinin, des-Arg⁹-bradykinin and fragments of fibrinogen α-chain (Fib-α_[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH_4[666–687]) and complement component 4a (C4a_{[1337–1350]}). Ile¹³-ITIH_4[666–687], d20-C4a_{[1337–1350]} and Sar-D-Phe⁸-des-Arg⁹-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp³-bradykinin (4–200 ng/ml), des-Arg⁹-bradykinin (2–100 ng/ml), Fib-α_[605–629] (120–3000 ng/ml), ITIH_4[666–687] (0.4–10 ng/ml) and C4a_{[1337–1350]} (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.     

Assessment of salivary cotinine concentration among general non-smokers population: Before and after Spanish smoking legislations

BackgroundIn Spain, two smoke-free laws have been passed (Law 28/2005 and Law 42/2010).This study evaluates the association between Spanish smoking legislations and the second-hand smoke (SHS) exposure in an adult non-smoking population cohort in Barcelona (Spain).MethodsThis is a longitudinal study, before and after the implementation of two national smoking bans, in a representative sample of adults (≥16 years old) from Barcelona (Spain) surveyed in 2004–2005 and followed up in 2013–2014 (n = 736). We only analyzed non-smokers (n = 397). We obtained 9 ml of saliva sample for analysis of cotinine, a biomarker of recent tobacco exposure. We calculated geometric means of salivary cotinine concentration and their geometric standard deviation. We used linear mixed effect models, with individuals as random effects, to model the percentage change in salivary cotinine concentration and their 95% confidence intervals.ResultsThe percentage of participants with saliva samples with measurable concentrations of cotinine fell from 92.4% to 64.2% after both Spanish smoking legislations. The geometric mean of salivary cotinine concentration significantly decreased 88% (from 0.98 ng/mL to 0.12 ng/mL, p < 0.001) after the implementation of the two Spanish smoke-free legislations. The decrease of the GM salivary cotinine concentration was statistically significant independently of the sociodemographic variables.ConclusionThere was a large reduction in the salivary cotinine concentration among adult non-smokers and higher cotinine concentrations among those declaring exposure to SHS at home after both legislations. Moreover, after both Spanish smoke-free laws salivary cotinine concentration was homogenized according to sociodemographic variables.     

Determination of iodine in human milk and infant formulas

The aim of this study was to develop a method to determine iodine in human milk and infant formulas using ICP-MS. The milk samples were digested using an alkaline digestion (5% NH₃, 45 W, 2 min and 30 s), and the method was validated using a certified reference material (CRM) BCR CRM151. On the other hand the milk was separated in three fractions, whey, fat and caseins using ultracentrifugation (15 min, 4 °C, 50,000 rpm) and the iodine was determined in the different fractions. About 27 samples of different infant formulas and 14 samples of human milk have been studied. In the human milk the values found were between 144±93.2 μg kg⁻¹, whereas in the infant formulas the values were 53.3±19.5. For both types of samples the bigger amount of iodine is in the whey fraction, between 80% and 90%, whereas in the fat there is about a 2% of the total iodine and in the casein fraction the levels are between 5% and 10% depending on the type of sample.     

Development of a sensitive and selective method for the quantitative analysis of cortisol,cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.