Vitamin D insufficiency together with high serum levels of vitamin A increases the risk for osteoporosis in postmenopausal women.

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.

On-line coupling of automatic solid-phase extraction and HPLC for determination of carotenoids in serum.

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.

Standard operation protocol for analysis of lipid hydroperoxides in human serum using a fully automated method based on solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring.

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.

Automated method for targeting analysis of prostanoids in human serum by on-line solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring.

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.

Influence of vegetable oils fatty-acid composition on biodiesel optimization.

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.

Vitamin D deficiency and high serum levels of vitamin A increase the risk of osteoporosis evaluated by Quantitative Ultrasound Measurements (QUS) in postmenopausal Spanish women.

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.

Automated determination of folate catabolites in human biofluids (urine, breast milk and serum) by on-line SPE­HILIC­MS/MS.

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 chromatography­tandem mass spectrometry (HILIC­MS/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.

Evaluation of vitamin D endocrine system (VDES) status and response to treatment of patients in intensive care units (ICUs) using an on-line SPE-LC-MS/MS method.

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.

Fast, sensitive and highly discriminant gas chromatography-mass spectrometry method for profiling analysis of fatty acids in serum.

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.

Fully automatic method for the determination of fat soluble vitamins and vitamin D metabolites in serum.

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.

High through-put aflatoxin determination in plant material by automated solid-phase extraction on-line coupled to laser-induced fluorescence screening and determination by liquid chromatography-triple quadrupole mass spectrometry.

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%.

Inappropriate serum levels of retinol, alpha-tocopherol, 25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults: simultaneous assessment by HPLC.

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.

Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme Q10) in human serum by liquid chromatography tandem mass spectrometry to evaluate the oxidative stress.

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.

Automated method for the determination of fat-soluble vitamins in serum.

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.

Fully automated method for the determination of 24,25(OH)2 and 25(OH) D3 hydroxyvitamins, and vitamins A and E in human serum by HPLC.

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).

Automated method for the determination of vitamin D3 hydroxymetabolites in serum.

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%.