Qualitative/quantitative strategy for the determination of glufosinate and metabolites in plants.

A simple method for the simultaneous determination of glufosinate and its metabolites in plants based on liquid chromatography-ultraviolet (LC-UV) absorption detection after derivatization with fluorenylmethoxycarbonyl chloride (FMOC-Cl) of some analytes to facilitate separation is reported here. Nonavailable standard metabolites were identified by LC-TOF/mass spectrometry (MS), which also confirmed all target analytes. Ultrasound-assisted extraction was used for sample preparation (power of 70 W and duty cycle of 0.7 s/s for 10 min) with subsequent evaporation of the extractant, reconstitution and filtration as the cleanup/concentration step prior to derivatization, and chromatographic separation and detection at 270 nm for underivatized analytes and 340 nm for those that were derivatized. The chromatographic analysis was completed in 40 min using a Luna® column (C18 phase). The analytical characteristics of the method were linear dynamic range of the calibration curves within 0.047-700 µg/mL with a regression coefficient (rc) of 0.999 for glufosinate, 0.077-700 µg/mL with a rc of 0.998 for N-acetyl-glufosinate, and 0.116-600 µg/mL with a rc of 0.998 for 3-(methylphosphinico)propanoic acid. The precision for the determination of glufosinate (studied at two levels, 0.1 and 5 µg/mL) was 2.7 and 6.0 % for repeatability and 4.7 and 7.2 % for within-laboratory reproducibility, respectively. Identification and confirmatory analysis of the presence of glufosinate and metabolites in the extracts from treated plants was carried out by LC-TOF/MS in high-resolution mode for the precursor ion. The method was validated by analyzing wheat (Triticum aestivum) samples (resistant and susceptible biotypes) treated with 300 g of glufosinate/ha following conventional agronomical practices.

Analysis of esterified and nonesterified fatty acids in serum from obese individuals after intake of breakfasts prepared with oils heated at frying temperature.

In this study, levels of esterified and nonesterified fatty acids (EFAs and NEFAs, respectively) were compared in obese individuals (body mass index between 30 and 47 kg m(-2)) in basal state and after intake of four different breakfasts prepared with oils heated at frying temperature. The target oils were three sunflower oils--pure, enriched with dimethylsiloxane (400 µg mL(-1)) as lipophilic oxidation inhibitor, and enriched with phenolic compounds (400 µg mL(-1)) as hydrophilic oxidation inhibitors--and virgin olive oil with a natural content of phenolic compounds of 400 µg mL(-1). The intake of breakfasts was randomized to avoid trends associated to this variability source. EFAs and NEFAs were subjected to a sequential derivatization step for independent gas chromatography-mass spectrometry analysis of both fractions of metabolites in human serum. Derivatization was assisted by ultrasonic energy to accelerate the reaction kinetics, as required for high-throughput analysis. Statistical analysis supported on univariate (multifactor ANOVA) and multivariate approaches (principal component analysis and partial least squares-discriminant analysis) allowed identification of the main variability sources and also discriminating between individuals after intake of each breakfast. Individuals' samples after intake of breakfasts prepared with virgin olive oil were clearly separated from those who ingested the remaining breakfasts. The main compounds contributing to discrimination were omega-3 and omega-6 EFAs with special emphasis on arachidonic acid and eicosapentaenoic acid. These two polyunsaturated fatty acids are the precursors of eicosanoid metabolites, which are of vital importance as they play important roles in inflammation and in the pathogenesis of vascular and malignant diseases as cancer.

Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to µ-LC-LIF determination.

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

Vitamin D3 levels in women and factors contributing to explain metabolic variations.

The elucidated metabolism of vitamin D3 in humans has been the support to explain the high involvement of this liposoluble vitamin in physiological functions. Clinical studies have associated levels of vitamin D3 metabolites with several disorders. Despite this knowledge, there is a controversy regarding the estimation of deficiency and the physiological and supraphysiological levels of vitamin D3 metabolites. The association between serum concentrations of vitamin D3 metabolites and several potentially influential factors (namely, age and anthropometric, seasonal, spatial and metabolic factors) is analyzed in this study. For this purpose, 558 women were recruited and interviewed in several Spanish provinces before blood sampling. Serum vitamin D3 and its metabolites were determined using an SPE-LC-MS/MS platform. The concentration range for vitamin D3 was 1.7-21.1 nmol/L and was influenced by body mass index (BMI), waist-to-hip ratio (WHR) and seasonal period. 25-hydroxyvitamin D3 levels were within 4.8-147.2 nmol/L and were related to WHR, season, latitude and calcium intake. The range of 24,25-dihydroxyvitamin D3, 0.3-15.0 nmol/L, was associated to BMI, WHR, season, latitude and calcium intake. Finally, energy intake influenced the vitamin D 25-hydroxylase through the 25-hydroxyvitamin D3/vitamin D3 ratio, which regulates the synthesis of the circulating form. According to these results, it is worth emphasizing the relevance of all these factors to explain the variability in serum levels of vitamin D3 and its metabolites. All these factors should be considered in future studies assessing the alteration of vitamin D3 metabolism.

Lipidomics: An omics discipline with a key role in nutrition.

The importance of lipidomics to unveil crucial aspects in the nutrition field is afforded in this article. With this aim, historical facts such as demonization of fats, enthronization of carbohydrates, and subsequent changes in the food pyramid are first discussed. After considering basic and analytical aspects of lipidomics and the upstream information this omics provides, its key role in personalized nutrition (PN), and the importance of lipids as nutrition biomarkers are critically discussed by appropriate examples. Pendent challenges to clarify the role of lipidomics in nutrition are overcome limiting factors, design of new lipidomics-based biomarkers, unveil mechanisms involved in lipidomics processes, and integrate lipidomics with other omics for a more complete and validated information useful in PN. The conclusions of this study also include the scant role of analytical chemists in the lipidomics-nutrition binomial, basically supported on analytical data.

Dry sweat as sample for metabolomics analysis.

Sweat is gaining popularity in clinical metabolomics as this biofluid is non-invasively sampled and its composition is modified by several pathologies. There is a lack of standardized strategies for collection of human sweat. Most studies have been carried out with fresh sweat collected after stimulation. A promising and simple alternative is sampling dry sweat by a solid support impregnated with a suited solvent. This research was aimed at comparing the metabolomics coverage provided by dry sweat collected by two solid supports (gauzes and filter papers) impregnated with different solvents. The dissolved dry sweat was analyzed by a dual approach: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among the tested sampling strategies, filter paper impregnated with 1:1 (v/v) ethanol‒phosphate buffer resulted the combination providing the highest metabolomics coverage (tentative identification of one hundred seventy-five compounds). Dry and fresh sweat were compared by using pools from the same individuals to evaluate compositional differences. Families of metabolites such as carnitines, sphingolipids and N-acyl-amino acids, among others, were exclusively identified in dry sweat. Comparison of both samples allowed concluding that dry sweat is better for analysis of low polar metabolites and fresh sweat is more suited for polar compounds. As most of the identified metabolites are involved in key biochemical pathways, this study opens interesting possibilities to the use of dry sweat as a source of metabolite markers for specific disorders. Sampling of dry sweat could provide a standardized approach for collection of this biofluid, thus overcoming the variability limitations of fresh sweat.

Ultrasound-enhanced enzymatic hydrolysis of conjugated female steroids as pretreatment for their analysis by LC-MS/MS in urine.

A fast, selective and sensitive method is here proposed for the analysis of female steroid hormones as conjugated forms (mainly, glucuronides and sulfates). The method has been applied to female urine samples to assess the metabolism of these compounds. The method implements an enzymatic hydrolysis (beta-glucuronidase with sulfatase activity) kinetically enhanced by ultrasonic energy in order to generate the free steroid forms. This enables a drastic shortening of the time required for this step as compared with conventional protocols (from 12-18 hours to 30 min). The reaction kinetics of the ultrasound-enhanced hydrolysis was characterized by comparison to that of the conventional protocol. After hydrolysis, the free steroid hormones were isolated and preconcentrated by automated solid-phase extraction and the eluate was subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The target analytes were confirmed and quantified by multiple reaction monitoring (MRM). The detection and quantification limits were within 0.06-0.8 ng mL(-1) and 0.19-2.69 ng mL(-1), respectively. The precision of the method, expressed as intra-day and inter-day variability, ranged between 2.1 and 5.2% and between 4.9 and 8.0%, respectively. A complementary study was carried out to assess the storage conditions of urine samples. This study is crucial in those applications involving metabolic processes as the integrity of the sample has to be preserved.

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%.

Evaluation of short-term storage prior to analysis of vitamin D3 and metabolites in human serum by liquid chromatography coupled to tandem mass spectrometry.

Vitamin D has been widely determined in clinical trials to elucidate its biochemical involvement in a great number of pathologies. The analysis of vitamin D and its hydroxymetabolites in biofluids such as serum or plasma is a challenging task due to limitations associated to the low concentrations of some metabolites (typically, dihydroxymetabolites), methodological interferences, and the low stability of the compounds. Among these limitations, efforts have been targeted at optimizing instrumental improvements to develop more sensitive and selective methods, while the stability of vitamin D and metabolites has not been exhaustively evaluated. In this research, several aspects regarding the short-term storage conditions of human serum have been studied to evaluate their influence on the determination of vitamin D3 and metabolites. An experimental plan has been applied to assess the influence of two relevant parameters: the storage temperature for a period of two months and the number of freeze-thaw cycles. The storage temperature affected in a different manner to vitamin D3 and its metabolites, being vitamin D3 and 1,25-dihydroxyvitamin D3 the two analytes more affected by this parameter. Concerning the freeze-thaw cycles, this variable must be limited to two cycles owing to its significant influence on the variability for quantitation of dihydroxymetabolites in human serum. Finally, lyophilization was also tested to check if serum concentrations of vitamin D3 and its metabolites were affected by this preprocessing step. The results revealed that only vitamin D3 experienced a decrease in serum concentration after two months, which does not constitute a real problem as vitamin D3 is not currently a crucial parameter to be determined in clinical trials due to its scant biological activity.

Determination of primary fatty acid amides in different biological fluids by LC-MS/MS in MRM mode with synthetic deuterated standards: Influence of biofluid matrix on sample preparation.

The recent growing interest in primary fatty acid amides (PFAMs) is due to the broad range of physiological effects they exhibit as bioindicator of pathological states. These bioactive lipids are usually in biological samples at the nanomolar level, making their detection and identification a challenging task. A method for quantitative analysis of seven main PFAMs (lauramide, myristamide, linoleamide, palmitamide, oleamide, stearamide and behenamide) in four human biofluids -namely, urine, plasma, saliva and sweat- is here reported. Two sample preparation procedures were compared to test their efficiency in each biofluid: solid-phase extraction (SPE) and protein precipitation. The latter was the best for plasma and urine, while the analysis of saliva and sweat required an SPE step for subsequent suited determination of PFAMs. Detection of the seven metabolites was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Quantitative analysis was supported on the use of stable isotopically labeled internal standards (SIL-ISs) in the calibration method, which required the synthesis of each IS from the precursor deuterated fatty acids. Detection limits for the target analytes were within 0.3-3 ng mL-1. The method was applied to a small cohort of male and female volunteers (n = 6) to estimate the relative concentration profiles in the different biofluids. The analytical features of the method supported its applicability in clinical studies aimed at elucidating the role of PFAMs metabolism.

Ultrasonic enhancement of leaching and in situ derivatization of haloacetic acids in vegetable foods prior to gas chromatography-electron capture detection.

A continuous ultrasound-assisted approach to enhance the extraction of nine haloacetic acids (HAAs) from vegetables with in situ derivatization to methyl esters for their gas chromatography (GC) analysis is presented. The optimization of simultaneous extraction (using acidic methanol as extractant) and derivatization enabled the completion of both steps in 15 min. Ultrasound assistance has proved to enhance both linked steps, which results in a considerable shortening of the overall analysis time (i.e. 552.1 and 552.2 EPA methods for analysis of these compounds in drinking water require 1 and 2 h, respectively, only for derivatization). After sample preparation, the esterified HAAs were isolated by liquid-liquid extraction with n-hexane and analysed by GC-electron capture detection. The proposed method is an interesting alternative to present methods for the determination of HAAs in vegetable foods. This is an area unjustifiably forgotten by reference laboratory organisms as proved by the absence of official methods for analysis of the target compounds in these samples. The proposed method can be applied to the analysis of HAAs in any solid sample after optimization of the main variables involved in the extraction-derivatization step.

Automated solid-phase extraction for concentration and clean-up of female steroid hormones prior to liquid chromatography-electrospray ionization-tandem mass spectrometry: an approach to lipidomics.

A method for determination of free and glucuronide-conjugated female steroid hormones in urine at the pgmL(-1) level is here presented. For this purpose, a dual approach with or without beta-glucuronidase hydrolysis has been developed to succeed in this analysis. The target analytes were two progestogens - progesterone and pregnenolone - and three endogenous estrogens - estradiol, estriol and estrone. Separation and detection were carried out by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS-MS) with a triple quadrupole (qQq) mass detector. The determination step was optimized by multiple reaction monitoring for highly selective identification and sensitive quantification of female hormones in a complex sample such as human urine. As these compounds are present in urine at very low concentration (ngmL(-1) level), a preconcentration and clean-up step by solid-phase extraction was automatically carried out prior to the chromatographic step in order to improve the sensitivity of the method. This sample pretreatment was performed using a lab-on-valve (LOV) manifold which provided preconcentration factors ranging from 59.1 to 72.3 for 10mL urine. The detection and quantification limits were in the ranges 1.8-18pg and 6-61pg on-column, respectively, with precision values from 1.93 to 10.99%, expressed as relative standard deviation. These results enable to conclude the suitability of the LOV-LC-qQq approach for determination of the lipidomic profiling of the main female steroid hormones in a difficult matrix as human urine. The method can be potentially applied to the clinical and other metabolomic areas.

Static-dynamic sequential superheated liquid extraction of phenols and fatty acids from alperujo.

Superheated liquids of different polarity have been used for sequential extraction of fatty acids and phenols from alperujo. Multivariate methodology has been used to optimise the static-dynamic extraction. Forty-two minutes are required to complete extraction (20 mg/kg of fatty acids and up to 2,200 mg/kg of hydroxytyrosol in the raw material used). The efficacy of the extraction has been demonstrated and compared with that of conventional methods (Folch and stirring-based methods for fatty acids and phenols, respectively), which needed 4.5 and 24 h for the extraction of fatty acids and phenols, respectively. The non-polar and polar extracts were injected into GC-MS and HPLC-MS-MS equipment, respectively, for individual separation-quantification of the target compounds. The simplicity of the experimental setup and the low costs of the raw material make the proposed method advisable when extraction of both fractions is required.

Liquid-liquid extraction for the enrichment of edible oils with phenols from olive leaf extracts.

A liquid-liquid extraction method to enrich edible oils--olive, sunflower, and soy oils--with phenols from olive leaf extracts is proposed. After microwave assistance to remove the phenols from three varieties of olive leaves, concentrations in the extracts between 12921 and 5173 mg/L of oleuropein, between 488 and 192 mg/L of apigenin-7-glucoside, between 444 and 219 mg/L of luteolin-7-glucoside, and between 501 and 213 mg/L of verbascoside were obtained, which clearly depended on the target variety. After optimization of the liquid-liquid extraction step, the concentrations in oils were 442, 162, and 164 mg/L of oleuropein, respectively, which were also enriched in apigenin-7-glucoside (between 8 and 15 mg/L, depending of the oil), lutelin-7-glucoside (between 11 and 12 mg/L), and verbascoside (between 11 and 13 mg/L). The oil-extract distribution factor of these compounds was also calculated for all olive leaf varieties and edible oils using different extracts concentrations and also different oil-extract volume ratios. Thus, a door is open to enrichment of any oil with olive phenols at preset concentrations using extracts preconcentrated as required and taking into account the distribution factor of the target compounds between the oil and the extracts.

The analytical process to search for metabolomics biomarkers.

The scant number of available metabolomics biomarkers does not reflect the extent of the research in this field. Looking for the reasons of failure, the authors, as analytical chemists, critically discuss each of the steps in the analytical process that requires improvements. They find insufficient information about how the experimental part is developed. After revising the steps from sampling to obtainment of the analytical sample (from typical samples such as blood and urine to others less common such as sweat or saliva), the need for data and metadata for either reproduction of a given study or for taking the study as starting point after biomarker discovery is criticized. The separation and analysis steps are also revised as does data treatment. After the sources of errors from the analytical process are overcome, subsequent steps in the implementation of biomarkers to reach the final aim of clinical adoption should be supported as required.

Study of sample preparation for determination of endocannabinoids and analogous compounds in human serum by LC-MS/MS in MRM mode.

Endocannabinoids are lipids with a key role in physiological processes such as the immune response or the metabolism. This involvement explains their association to pathologies such as cancer, obesity or multiple sclerosis. The determination of endocannabinoids constitutes a challenge for clinical laboratories due to the variety of biological matrices and the wide range of concentrations at which they can be found. This research deals with the comparison of three sample preparation strategies (viz., on-line SPE, off-line SPE for interferents removal, and protein precipitation) for subsequent LC-MS/MS analysis of 14 endocannabinoids and analogous compounds in serum. As a result, the on-line coupling between SPE and LC-MS/MS is proposed as the best approach for this determination. The proposed method allows full automation of the overall process, shortening of the analysis time, and avoidance of errors associated with sample preparation steps. The improvement in sensitivity and selectivity thus achieved allows obtaining quantification limits at the pg mL-1 level, which makes possible the application of the method for clinical studies.

Metabolomics analysis of human sweat collected after moderate exercise.

Sweat is a promising biofluid scarcely used in clinical analysis despite its non-invasive sampling. A more frequent clinical use of sweat requires to know its whole composition, especially concerning to non-polar compounds, and the development of analytical strategies for its characterization. The aim of the present study was to compare different sample preparation strategies to maximize the detection of metabolites in sweat from humans collected after practicing moderate exercise. Special emphasis was put on non-polar compounds as they have received scant attention in previous studies dealing with this biofluid. Sample preparation by liquid-liquid extraction (LLE) using extractants with different polarity index was compared to deproteination. Then, derivatization by methoxymation with subsequent silylation was compared to direct analysis of sweat extracts to check the influence of derivatization on the subsequent determination of volatile organic compounds (VOCs). 135 compounds were tentatively identified by combining spectral and retention time information after analysis by gas chromatography coupled to mass spectrometry in high resolution mode (GC-TOF/MS). Lipids, VOCs, benzenoids and other interesting metabolites such as alkaloids and ethanolamines were identified. Among the tested protocols, methyoxiamination plus silylation after LLE with dichloromethane was the best option to obtain a representative snapshot of sweat metabolome collected from different body parts after moderate exercise. Passive and active sweat pools from a cohort of volunteers (n = 6) were compared to detect compositional differences which can be explained by the sampling process and sweating induction. As most of the identified compounds are metabolites involved in key biochemical pathways, this study opens new opportunities to extend the applicability of human sweat as a source of metabolite biomarkers of pathologies or specific processes such as dehydration or nutritional unbalance.

In-column micro-high-performance liquid chromatographic concentration-separation prior to ultraviolet detection for the determination of chlorophenols in water samples.

Two methods for the determination of all chlorophenols (CPs) plus phenol have been developed, both based on multiple sample injection - 2 or 12 injections - for in-column concentration prior to total content determination (TCD) of CPs and individual separation-quantification (ISQ) of each CP in drinking-water by micro-liquid chromatography-ultraviolet absorption spectrometry (microLC-UV). A Zorbax SB C18 capillary column (150 mm x 0.5mm; 5 microm particle size) and a monitoring wavelength of 210 nm were used in both methods. The limits of detection (LODs) and quantification (LOQs) are 9 and 36 ng/l, respectively, for the TCD method, and range between 3-13 ng/l and 8-21 ng/l, respectively, for the ISQ method, with linear dynamic ranges from LOQs to 2.00 microg/l. The precision, expressed as relative standard deviation (RSD), ranges between 2.25 and 6.91% for repeatability and between 3.08 and 8.06% for within laboratory reproducibility for both methods, and the errors, also expressed as RSD for all compounds, range between 0.57 and 9.35%. The time necessary for the TCD and the ISQ methods are 20 and 92 min, respectively. The accuracy of the methods and potential matrix effects were studied by using spiked water samples and recoveries between 90.9 and 111.1% were obtained. Then, the ISQ method was applied to different samples including tap, mineral and spring water.

Ultrasound-assisted extraction and silylation prior to gas chromatography-mass spectrometry for the characterization of the triterpenic fraction in olive leaves.

One of the most important fractions of bioactive compounds isolated from plants is that formed by triterpenic compounds, which have proved to be anti-bacterial, antifungal, anti-inflammatory, cytotoxic and anti-tumour. A method for leaching and determination of the main triterpenic compounds (oleanolic acid, ursolic acid, uvaol, erythrodiol) in olive leaves is here presented. Quantitative leaching was obtained with ethanol as leachant and ultrasonic assistance for 20 min, a very short time as compared to conventional procedures by maceration, which usually requires at least 5 h. After isolation, an aliquot of the ethanolic leachate was silylated to derivatize the analytes prior to gas chromatography-mass spectrometry analysis. Silylation reaction was also assisted with ultrasound in order to accelerate the derivatization step, which only required 5 min--a dramatic shortening in comparison to conventional silylation of terpenic compounds with derivatization times ranging from 30 min to 3 h. The proposed method has demonstrated to be useful for isolation and characterization of the triterpenic fraction in plants; the capability of ultrasound to assist sample preparation (acceleration of leaching and derivatization) has also been proved.

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.