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La determinación de metabolitos de vitamina D es muy importante en el metabolismo óseo, en enfermedades coronarias, cáncer, inmunología innata, etc. Desafortunadamente, la variabilidad entre los métodos para la determinación de los metabolitos de la vitamina D limita la capacidad de los clínicos para monitorizar el estado, la suplementación y la toxicidad de vitamina D. En este trabajo se presenta un método automático para la determinación de los metabolitos más importantes de la vitamina D. Se inyectan 0,2 ml de suero en una plataforma XLC-MS/MS (eXtraction Liquid Chromatography-tandem Mass Spectrometry) para ser limpiados y preconcentrados mediante extracción en fase sólida (SPE). Los analitos retenidos en el cartucho de SPE son eluídos directamente por la fase móvil cromatográfica que contiene un 10% de agua en metanol con 5 mM de formato amónico como agente ionizante a un flujo de 0,3 ml/min para la separación de los analitos y posterior detección mediante espectrometría de masas (MS/MS) triple cuádrupolo. Los límites de detección oscilaron entre 3,5 y 8,2 pg/ml. Los coeficientes de variación oscilaron entre un 1,5 y 2,3% intra-ensayo en un mismo día y 2,5-3,9% inter-ensayo realizado durante una semana. La recuperación osciló entre 97 y 99,7% para todos los analitos. El tiempo total de análisis fue de 20 minutos. Por tanto, el método propuesto es robusto, barato y adecuado para su uso en laboratorios clínicos y de investigación.

There is controversial information about the impact of vitamin A on bone. Some epidemiological studies show that excessive intake of vitamin A, or an excess of serum vitamin A, has related with adverse impact on bone mass; however, other studies did not find these links, and some authors have proposed that this vitamin might promote a better bone health. The present work aims to contribute to clarify the real role of vitamin A in bone tissue. For this purpose, a cross-sectional study of 154 osteoporotic non-treated postmenopausal women (> 65 years old) was carried out. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. We assessed concentrations of serum retinol, osteocalcin, parathyroid hormone, alkaline phosphatase, calcium, and phosphorus. We also studied demographic and anthropometric parameters. Spearman's correlations between retinol levels and other variables found negative correlations with BMD in both lumbar spine (R = - 0.162, P < 0.01) and femoral neck (R = - 0.182, P < 0.01), as well as alkaline phosphatase (R = - 0.110; P < 0.05) and phosphorus (R = - 0.110; P < 0.05). A positive correlation between retinol and fertile window was observed (R = 0.158; P < 0.01). After multivariable adjustment, we still found a negative correlation between serum retinol and BMD, both at the lumbar spine (R = - 0.210; P < 0.01) and at the femoral neck (R = - 0.324, P < 0.001). It is concluded that elevated serum-retinol levels are associated with an increased risk of low bone mass and thus with osteoporotic fractures. Therefore, osteoporosis-risk assessment should include quantification of serum metabolite of vitamin A.

Aging may enhance both oxidative stress and bone-marrow mesenchymal stem-cell (MSC) differentiation into adipocytes. That reduces osteoblastogenesis, thus favoring bone-mass loss and fracture, representing an important worldwide health-issue, mainly in countries with aging populations. Intake of antioxidant products may help to retain bone-mass density. Interestingly, a novel olive-pomace physical treatment to generate olive oil also yields by-products rich in functional antioxidants. Thus, diet of postmenopausal women was supplemented for two months with one of such by-products (distillate 6; D6), being rich in squalene. After treatment, serum from such women showed reduced both lipidic peroxidation and oxidized low-density lipoprotein (LDL). Besides, vitamin E and coenzyme Q10 levels increased. Furthermore, culture medium containing 10% of such serum both increased osteoblastogenesis and reduced adipogenesis in human MSC from bone marrow. Therefore, highly antioxidant by-products like D6 may represent a relevant source for development of functional products, for both prevention and treatment of degenerative pathologies associated with aging, like osteoporosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the relationship between vitamin E status and osteoporosis in early postmenopausal women. Anthropometric data, osteoporosis risk factors, vitamin E serum levels, bone mineral density (BMD) and other serum parameters which may influence bone mineral density in postmenopausal women were analyzed in a cross-sectional study. The association between osteoporosis and age, age of menopause, body mass index, osteocalcin, calcium, vitamin D, vitamin E (measured as 25 hydroxyvitamin D and as α-tocopherol:lipids ratio, respectively), bone alkaline phosphatase, smoking status, leisure physical activity and alcohol intake were modeled by a multivariate logistic regression and multi-linear regression analysis in 232 early postmenopausal women. A lower vitamin E:lipid ratio was associated with osteoporosis in multivariate logistic regression. In a multivariate linear model with BMD of the lumbar spine as a dependent variable, the vitamin E:lipid ratio was clearly related with BMD of the lumbar spine (F ratio = 6.30, p = 0.002). BMD of the lumbar spine was significantly higher in the highest tertile of the vitamin E:lipid ratio than in the lowest tertile. The mean vitamin E:lipid ratio was significantly lower in osteoporotic postmenopausal women (T score ≤-2.5) (3.0 ± 0.6 μmol/mmol) than normal (neither osteoporotic nor osteopenic) postmenopausal women (T score >-1) (3.5 ± 0.7 μmol/mmol) using multivariable-adjusted BMD. These findings highlight that vitamin E may increase BMD in healthy postmenopausal women.

The effect of breakfast intake of fried oils containing natural antioxidants or a synthetic autooxidation inhibitor on the metabolism of essential fatty acids focused on obese individuals. Serum levels of eicosanoids were compared in individuals before and after intake of different breakfasts. Univariate descriptive analysis was used to characterise the cohort selected for this study and multivariate analysis to reveal statistical differences of normalised eicosanoids concentrations (determined by solid-phase extraction coupled to LC-MS/MS) depending on the edible oil used for breakfast preparation. The results showed that the intake of breakfast prepared with pure sunflower oil subjected to deep frying causes an effect over the eicosanoids profile that enables discrimination versus the rest of individuals. The effect was a significant increase in the concentration of hydroxyoctadecadienoic acid (HODE) metabolites, indicative markers of the intake of fried oils. The concentration of HODE metabolites was lower when the oil contained either natural antioxidants from olive-oil pomace or a synthetic autooxidation inhibitor as dimethylsiloxane. The comparison of the effect of fried sunflower oils with fried extra virgin olive oil shows the benefits associated to the consumption of the latter., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlabelled: Postmenopausal women who were vitamin D deficient and had high serum levels of retinol had an eight times higher risk of having osteoporosis. A high retinol level together with vitamin D deficiency/insufficiency is an additional risk factor for osteoporosis., Purpose: The aim of this study was to evaluate the association between vitamin D deficiency/insufficiency and excess of vitamin A intake as an osteoporosis risk factor in healthy postmenopausal women, Design: The design is a cross-sectional study of 232 healthy postmenopausal women., Methods: Bone mass was evaluated by dual energy X-ray absorptiometry. Serum calcium, albumin phosphorus, creatinine, total high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, and triglycerides analyzed by standard methods and retinol and 25-hydroxyvitamin D [25(OH)D] measured by an online solid-phase extraction coupled with high-pressure liquid chromatography-ultraviolet detection., Results: Prevalence of vitamin D deficiency [25(OH)D < 20 ng/mL] was 70.1 %; 14.3 % had a 25(OH)D < 10 ng/mL, and 23.6 % had insufficiency [25(OH)D 21-29 ng/mL]. Prevalence of high serum levels of retinol (>80 μg/dL) was 36.4 %. Among subjects with 25(OH)D <20 ng/mL (n = 152), 60.4 % (n = 92) had serum levels of retinol > 80 μg/dL. Bone density measurements revealed that the risk of osteoporosis was ~8 times higher in women with the highest retinol levels, as compared with women with the lowest retinol levels. In women with 25(OH)D < 20 ng/mL, the risk for osteoporosis increased substantially in women who had the highest blood levels of retinol compared to the women with lowest retinol levels., Conclusions: Higher retinol levels together with vitamin D deficiency could be a significant additional risk factor for osteoporosis, underscoring the need for improved physician and public education regarding optimization of vitamin D status in postmenopausal women and developing policies to avoid high serum levels of vitamin A.

The automated method developed for the determination of carotenoids uses 200 μL of serum, which was mixed with 400 μL of tetrahydrofuran, vortexed for 1 min, settled for 10 min, centrifuged for 6 min and the supernatant injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration. A 10% water-acetonitrile mobile phase at 1.5 mL min(-1) eluted the retained compounds and transferred them on-line to a reversed-phase analytical column for individual separation of the target analytes. Visible detection was performed at 450 and 460 nm. The detection limits for the target analytes were between 3 and 30 ng mL(-1); the precision (expressed as relative standard deviation) ranged between 2.83 and 5.06% for repeatability and between 3.80 and 7.40% for within laboratory reproducibility. The total analysis time was 18 min. The proposed method is reliable, robust, and has an excellent potential for high-throughput use in both clinical and research laboratories., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Standard operating procedures (SOPs) are of paramount importance in the analytical field to ensure the reproducibility of the results obtained among laboratories. SOPs gain special interest when the aim is the analysis of potentially unstable compounds. An SOP for analysis of lipid hydroperoxides (HpETEs) is here reported after optimization of the critical steps to be considered in their analysis in human serum from sampling to final analysis. The method is based on automated hyphenation between solid-phase extraction (SPE) and liquid chromatography-mass spectrometry (LC-MS). The developed research involves: (i) optimization of the SPE and LC-MS steps with a proper synchronization; (ii) validation of the method-viz. accuracy study (estimated as 86.4% as minimum value), evaluation of sensitivity and precision, which ranged from 2.5 to 7.0 ng/mL (0.25-0.70 ng on column) as quantification limit and precision below 13.2%), and robustness study (reusability of the cartridge for 5 times without affecting the accuracy and precision of the method); (iii) stability study, involving freeze-thaw stability, short-term and long-term stability and stock solution stability tests. The results thus obtained allow minimizing both random and systematic variation of the metabolic profiles of the target compounds by correct application of the established protocol., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A selective and sensitive, fully automated platform for verification and quantitative determination of target peptides in biofluids is proposed and then validated by development of a method for analysis of cathelicidin in human serum. The method is based on the on-line coupling of solid-phase extraction (SPE) and tandem mass spectrometry with direct infusion. Mass spectrometry analysis was carried out by multiple reaction monitoring using three transitions (one for quantitative analysis and two for qualitative analysis), all them confirmed by in silico fragmentation of the target peptide. Samples were prepared in the SPE workstation on a polymeric divinylbenzene resin by preconcentration, deproteinization, and cleanup, removing salts and interferences after direct injection of human serum. The analytical process required 12 min. The limits of detection and quantitation were 2.5 and 8.25 μg/L, respectively (0.20 and 0.66 pg on column). Repeatability and within-laboratory reproducibility were 2.4% and 2.7%, respectively. A dual-cartridge configuration was used to test recovery of cathelicidin in serum, resulting in 80%. Because quantitative retention in the cartridge was assessed, determination of cathelicidin was validated without using synthetic peptides labeled with stable isotopes. The hyphenated system allows full automation, thereby improving reproducibility and accuracy, as demanded by clinical analysis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prostanoids are potent biologically active lipid molecules demanding for analysis methods combining precision, sensitivity and high-throughput for pharmacological and clinical applications. The present research describes the development and validation of an on-line automated method based on solid-phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for the quantification of prostanoids in human serum. This approach overcomes the main limitation of previous methods involving manual protocols, such as analyte losses, metabolites degradation and time-consuming protocols, are minimized. Human serum (100 μL) was directly injected into an automatic solid-phase extraction workstation for cleanup and preconcentration of the target metabolites. The eluate was on-line transferred to a reversed-phase analytical column for chromatographic separation prior to mass spectrometry detection in selected reaction monitoring mode. The detection limits for the target analytes ranged from 2.3 to 63.3 pg on column. The precision (expressed as relative standard deviation) was within 3.30 and 6.15% for repeatability and from 4.16 to 11.11% for within-laboratory reproducibility. Accuracy was evaluated with spiked and non-spiked serum samples to estimate concentration differences that could be affected by matrix effects or inefficient SPE performance. Accuracy, estimated as recovery factor, was from 87.7 to 100% for the target compounds. The proposed method is reliable and has an excellent potential for high-throughput use in both clinical and research laboratories by minimizing analyst intervention., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Inflammation is a complex cascade process involved in the pathogenesis of a number of diseases or generated as response to external or internal stimuli. Current research is focused on the development of assays for fast identification and quantitation of inflammation biomarkers. Eicosanoids are the oxidation metabolites of polyunsaturated fatty acids (mainly 20-carbon fatty acids) that play a regulation role in inflammation and, therefore, they have proved to be involved in different pathological states such as cancer, atherosclerosis, arthritis and cardiovascular or immunological diseases. Eicosanoids can be metabolized by different oxygenase enzymes to prostanoids such as prostaglandins and thromboxanes or hydroxyl fatty acids such as hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids. A high-throughput automated approach is here presented for direct eicosanoid analysis in biofluids such as human serum and cells culture media. The approach is based on a hyphenated system composed by a solid-phase extraction workstation (Prospekt 2 unit) on-line coupled to a liquid chromatograph-triple quadrupole-tandem mass spectrometer. The detection limits for the target analytes ranged from 0.009 to 204 pg on-column, with precision between 2.65% and 7.33%, expressed as relative standard deviation. Accuracy studies with a dual-cartridge configuration resulted in recoveries between 78.6% and 100%, which validated internally the proposed approach ensuring highly efficient cleanup of proteins and salts. The method is reliable, robust and endowed with a great potential for implementation in clinical and routine laboratories. Analysis of culture media of stem cells stimulated with arachidonic acid was carried out to evaluate its incidence on the eicosanoid profile of the exometabolome.

Biodiesel is an alternative fuel for diesel engines produced through transesterification of oleaginous feedstocks. To analyze the influence of the fatty-acid composition on biodiesel optimization, transesterification of several vegetable oils has been studied. Reactions were carried out in flasks filled with vegetable oils, heated to the reaction temperature and stirred at 1100 rpm. The reactions started when the methanol and potassium hydroxide solutions were added to the flasks. Concentration of catalyst, amount of methanol, reaction temperature and time were optimized using a factorial design and a surface response design. Also, a kinetics study was carried out to optimize the reaction time. Results showed that reaction parameters optimal values depend on the oil chemical and physical properties. It can be concluded from this field trial that the effect of both catalyst concentration and reaction time over the transesterification yield is greatly influenced by the saturation degree and fatty-acid chain length., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objectives: Association between vitamin D deficiency and excess of vitamin A as a potential risk factor of osteoporosis and fracture has been evaluated., Design and Methods: 232 healthy postmenopausal women were studied. Serum parameters were analyzed by standard methods and fat-soluble vitamins by an own HPLC method. QUS measurement of the calcaneal bone was carried out by Sahara., Results: 124 patients were considered non-osteoporotic and 101 (44.9%) were osteoporotic. The prevalence of high serum levels of retinol was 36.4% and vitamin D deficiency was 70.1%. 60.4% of women with vitamin D deficiency have high serum levels of retinol. In the whole population, the increased risk of osteoporosis was up to three times higher for the highest retinol quintile, as compared with the lowest retinol quintile. Whereas in women with vitamin D deficiency the risk of osteoporosis increased was up 5 times higher than women in the lowest quintile of retinol., Conclusions: Our results show that high retinol levels together with vitamin D deficiency are hitherto an overlooked risk factor for osteoporosis., (Copyright (c) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.

Vitamin D deficiency is recognized as one of the most common chronic medical conditions in the world. Vitamin deficiency has been associated with increased mortality. The aim of the study here presented was to evaluate the vitamin D endocrine system (VDES) status in healthy blood donors and critically ill patients baseline and in response to treatment during a week with two doses of 1.5 mg of 25-hydroxyvitamin D3 and 2 microg calcitriol (1,25(OH)2D3) IV on alternate days, by monitoring levels in serum of major vitamin D metabolites in critically ill patients. Group 1: healthy blood donors (control group) (n=92), and group 2: critically ill subjects from an intensive care unit (ICU) (n=33). Critically ill patients were divided into three groups: group A (n=12) is the control group; group B (n=11), administration PO 1,5 mg of 25(OH)D3, in days 0 and 4 of treatment; and group C (n=11), administration IV of 2 microg 1,25(OH)2D3 on alternate days. Baseline serum levels of vitamin D2 and 25(OH)D2 were not detected. Vitamin D3 (9.8 vs 26.0 nM) (p<0.05), 25(OH)D3 (13.3 vs 52.3 nM) (p<0.001), and 1,25(OH)2D3 (53.8 vs 120.5 pM) (p<0.01) serum levels were significantly lower in critically ill subjects than in healthy donors. After treatment in group B: 25OHD3 increased to 46.0+/-16.5 ng/ml (p<0.0001) (22.2%<75 nM, 11.1% <50 nM). 1,25(OH)2D3 increased to 121.8+/-61.8 pM<0.01 whereas were slightly decreased in the other groups during the study. 24,25(OH)2D3 serum levels were increased in patients treated with calcitriol 8.5+/-5.3 vs 24.8+/-16.3 nM (p<0.05) while the levels kept stable in group A patients. In summary, critically ill patients have a severe vitamin D deficiency, which can be easily corrected by administration of high doses of 25OHD (PO). The VDES functional deficiency could be probably also corrected through administration of calcitriol (IV). Both treatments could produce an improvement in the general health and probably a reduction in overall mortality risk of the critically ill patients., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A method for the determination of fatty acids in serum based on GC-MS (micro-SIS detection mode) has been developed and the separation and cis/trans isomers have been identified. A prior two-step extraction/derivatization procedure accelerated by ultrasound allows individual determination of esterified (EFAs) and non-esterified fatty acids (NEFAs), and shortening of the derivatization steps to 5 min for EFAs and 15 min for NEFAs. The total analysis time for 39 fatty acids was 61 min. The minimum LOD and LOQ values were 0.002 and 0.006 microg/ml, respectively. The proposed method was validated for EFAs and NEFAs using two different methods and the results show no statistical differences between the proposed method and those used as reference. The proposed derivatization-extraction methodology is suitable for fatty-acid analysis of human serum, and can be applied to nutritional and epidemiological studies.

Background: Fat soluble vitamins and vitamin D metabolites are key compounds in bone metabolism. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor vitamin D status, supplementation, and toxicity., Method: 0.5 ml serum was mixed with 0.5 ml 60% acetonitrile 150 mM sodium dodecyl sulfate, vortexed for 30 s and injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration, then on-line transferred to a reversed-phase analytical column by a 15% methanol-acetonitrile mobile phase at 1.0 ml/min for individual separation of the target analytes. Ultraviolet detection was performed at 265 nm, 325 nm and 292 for vitamin D metabolites, vitamin A and alpha- and delta-tocopherols, respectively., Results: Detection limits were between 0.0015 and 0.26 microg/ml for the target compounds, the precision (expressed as relative standard deviation) between 0.83 and 3.6% for repeatability and between 1.8 and 4.62% for within laboratory reproducibility. Recoveries between 97-100.2% and 95-99% were obtained for low and high concentrations of the target analytes in serum. The total analysis time was 20 min., Conclusions: The on-line coupling of SPE-HPLC endows the proposed method with reliability, robustness, and user unattendance, making it a useful tool for high-throughput analysis in clinical and research laboratories.

A screening test based on laser-induced fluorescence (LIF) and a method for individual identification - quantitation of aflatoxins (AFs) in olive leaves and drupes, based on chromatographic separation and triple-quad mass-spectrometry detection with electrospray ionization in positive mode, is here reported. The sensitivity and selectivity of both methods are enhanced by a preconcentration-cleanup step developed by a Prospekt station. The analysis frequency is at least 3.5 samples/h. The screening test makes able to detect the target analytes at concentrations of 0.7microg/kg without "false negatives". The LC-MS/MS method provides limits of detection (LOD) and quantification (LOQ) ranging between 0.01-0.03 and 0.03-0.11microg/kg, respectively. The linear dynamic range is between LOQ-50microg/kg. The between-day precision, expressed as relative standard deviation, ranges between 0.97-2.86% and the within laboratory reproducibility, also expressed as RSD, between 1.63% and 4.84%.

Objective: Simultaneous assessment of the status of lipid-soluble vitamins; retinol, alpha-tocopherol, 25 hydroxyvitamin D(3) and 24,25 dihydroxyvitamin D(3) in serum of blood donors, paradigm of a healthy population., Patients and Methods: Serum samples were supplied by the Regional Blood Donors Center in Cordoba from 215 healthy Spanish individuals (166 males and 99 females). Target analytes were determined using liquid-liquid extraction and separation-detection by HPLC., Results: The method was validated using standard reference material (SRM 968c, NIST). Standard errors were 1.4%, 2.1% and 1.8% for 25OHD(3), vitamin A and vitamin E, respectively. The ranges thus assessed were as follows: 17.1+/-8.0 nmol/L, for 24,25(OH)(2)D(3), 40.3+/-34.6 nmol/L for 25OHD(3), 2.57+/-0.7 micromol/L for retinol and 22.13+/-8.30 micromol/L for alpha-tocopherol. Females showed lower serum levels of retinol (p<0.01), alpha-tocopherol (p<0.01) and 25OHD(3) (p=0.028). A total of 10.4% subjects showed vitamin E deficiency, 85.4% had normal levels and 4.2% had high levels of vitamin E. 65.6% of the target subjects showed normal levels of retinol, and 1.6% had moderate or severe vitamin A deficiency. High levels of vitamin A were found in 32.8% of the subjects. Fourteen percent of the healthy subjects showed severe vitamin D deficiency (serum levels of 25OHD(3) <25 nmol/L), 50.8% had vitamin D(3) insufficiency (25OHD(3) from 25 to 50 nmol/L), 17.6% of the subjects had suboptimal 25OHD(3) serum levels (25OHD(3) from 50 to 75 nmol/L), only 16.8% had an adequate status of 25OHD(3) and 0.8% had high levels of vitamin D (25OHD(3)>200 nmol/L). Among subjects with vitamin D below 50 nmol/L, 49.38% had high levels of retinol (over 2.4 mumol/L). This association is considered a risk factor for osteoporosis and fracture., Conclusions: The reported data of high prevalence of lipid-soluble vitamin values outside the physiological range have important repercussions on public health. These data also uphold the need for simultaneous measurement of fat-soluble vitamins as a valuable tool in clinical practice as well as in epidemiological studies for awareness and correction.

A method for the rapid and simultaneous determination of ubiquinone-10 (coenzyme Q10, CoQ(10)) and the reduced form ubiquinol-10 (CoQ(10)H(2)) in human serum by LC-MS-MS with electrospray ionization (ESI) in the positive mode is here proposed. High selective identification and sensitive quantitation of both analytes have been carried out by monitoring the transition from the corresponding precursor ion to the product ion. Prior to the chromatographic analysis, serum samples (100 microl) were subject to a conventional pre-treatment based on protein precipitation, liquid-liquid extraction, evaporation to dryness and reconstitution with 95:5 methanol/hexane (v/v). The overall method has enabled to achieve low detection limits--5.49 and 15.8 ng/ml for CoQ(10) and CoQ(10)H(2), respectively--which were estimated with serum. The accuracy and potential matrix effects have been studied with spiked serum resulting recoveries between 92.82 and 106.97%. The proposed method has been applied to serum samples from healthy middle-age women, in which the CoQ(10)H(2)/CoQ(10) ratio has been used as marker of oxidative stress.

Automatic SPE has been coupled on-line to CE by a transfer tube and the replenishment system of the CE instrument. The approach allows the target analytes (viz. creatinine, creatine, xanthine, hypoxanthine, uric acid, p-aminohippuric acid and ascorbic acid in urine samples) to be removed from the sample matrix, cleaned up, preconcentrated and injected into the capillary. The detection limits range between 0.14 and 4.50 microg/mL, the quantification limits between 0.45 and 15.0 microg/mL, and linear dynamic ranges - which include the reference healthy human values - from the quantification limits to 1332 microg/mL. The precision, expressed as RSD, ranges between 0.38 and 2.22% for repeatability and between 1.79 and 7.61% for within-laboratory reproducibility. The errors, expressed as RSD for all compounds, range between 0.20 and 6.90%. The time for automatic SPE and that necessary for the individual separation-detection of the target analytes are 13 and 12 min, respectively; the analysis frequency is 5 h(-1). The accuracy of the method and potential matrix effects were studied by using spiked samples and recoveries between 96.00 and 103.07 % were obtained. The proposed method was applied to samples from healthy young students.

A new fully automated method for the determination of metabolites of Vitamin D(3) and Vitamins A and E has been developed. A robotic station for liquid-liquid extraction, connected on line with an automatic system for solid-phase extraction (Prospekt) and a liquid chromatograph were used and the complexity of the overall method was overcome by full automation. The eluate from the chromatograph was monitored by a photodiode-array detector at three wavelengths, namely, 265 nm for Vitamin D(3) metabolites, 291 nm for Vitamin E and 325 nm for Vitamin A-which are the maximum absorption wavelengths for the analytes. The time required per sample analysis was 35 min because of the overlapping development of the steps. The linearity obtained for serum samples (standard addition method) gives correlation coefficients (r(2)) ranging between 0.996 and 0.989, with standard deviation of the slope between 4.0 and 4.9%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10.1% in all cases-both expressed as relative standard deviation-for low concentrations of the analytes, namely, 3 ng/ml for 24,25(OH)(2) dihydroxyvitamin D(3), 10 ng/ml for 25(OH) hydroxyvitamin D(3), 100 ng/ml for Vitamin A and 2 microg/ml for Vitamin E. The method has been validated using a CRM (NIST, SRM968c).

A new fully automated high-performance liquid chromatography (HPLC) method using 1 ml of serum has been developed for the determination of retinol (Vitamin A), alpha-tocopherol (Vitamin E), 25-hydroxyvitamin D(3) and 24 R,25-hydroxyvitamin D(3). The eluate was monitored with a photodiode-array detector at three wavelengths-namely: 265 nm for Vitamin D(3), 291 nm for Vitamin E and 325 nm for Vitamin A. The detection limits were equal to or lower than 1 ng ml(-1) for all vitamins. The linearity obtained with serum samples (standard addition method) gives correlation coefficients (r(2)) ranging between 0.999 and 0.996 in all cases, with standard deviation of the slope between 3.2 and 1.6%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10% in all cases. The most outstanding features of the present method are its ease of use, its rapidity and fully automation, which enables its use for routine analysis. The time required per sample was 30 min, because the overlapped development of the steps. This method was used for the determination of normality range of these vitamins in healthy people in the 18-80-year-old interval.

An almost automated method for the determination of hydroxymetabolites of vitamin D(3) (cholecalciferol) in human serum is reported. The method consists of three steps: 1) a batch liquid-liquid extraction step with 2-propanol and hexane, and drying of the extract and reconstitution with phosphate buffer. 2) A cleanup and preconcentration step based on solid-phase extraction using Prospekt equipment, with CN group cartridges and elution with the chromatographic mobile phase. 3) A chromatographic step for individual separation of the target analytes starting with a 90:10 methanol-water mixture, then a linear gradient to obtain 100% methanol; followed by photometric detection. The method provides a linear range between 1.0 and 100 ng mL(-1) for 24,25-(OH)(2) vitamin D(3) and for 25-(OH)(2) vitamin D(3), and between 1.5 and 100 ng mL(-1) for 1,25-(OH) vitamin D(3), with correlation coefficients ranging between 0.993 and 0.987, repeatability between 1.9% and 4.8% and within-laboratory reproducibility between 2.8% and 8.8%.