Re-certification of hydroxyvitamin D standards by isotope pattern deconvolution.

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)2D3 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).

Comparison of isotope pattern deconvolution and calibration curve quantification methods for the determination of estrone and 17ß-estradiol in human serum.

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 17ß-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.

Determination of selected endogenous anabolic androgenic steroids and ratios in urine by ultra high performance liquid chromatography tandem mass spectrometry and isotope pattern deconvolution.

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.

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry.

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.

Time to initiation of multidrug-resistant tuberculosis treatment and its relation with outcome in a high incidence district in Lima, Peru.

OBJECTIVE: To determine the time from diagnosis to start of multidrug resistant tuberculosis (MDR TB) treatment in Lima, Peru. METHODS: We studied new smear-positive TB adults that were started on MDR TB treatment or that were switched to it between June 2008 and December 2011. RESULTS: Time from the first positive smear to MDR-TB treatment was >30 days in 35% (13/37) of patients. Among the 27% (24/88) of patients that switched to MDR-TB treatment, time from the last dose of a drug-susceptible regimen was >30 days. CONCLUSION: Start of and switching to MDR TB treatment is still delayed.

Capabilities of microbore columns coupled to inductively coupled plasma mass spectrometry in speciation of arsenic and selenium.

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.

Secondary interactions, an unexpected problem emerged between hydroxyl containing analytes and fused silica capillaries in anion-exchange micro-liquid chromatography.

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.

Method optimization for the determination of four mercury species by micro-liquid chromatography-inductively coupled plasma mass spectrometry coupling in environmental water samples.

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.

Multielemental determination of arsenic, selenium and chromium(VI) species in water by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

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.

Simultaneous determination of arsenic species and chromium(VI) by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry.

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.

An assessment of heavy metals and boron contamination in workplace atmospheres from ceramic factories.

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.

Application of the Azomethine-H method to the determination of boron in workplace atmospheres from ceramic factories.

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.