Clinica Chimica Acta 403 (2009) 126–130

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Clinica Chimica Acta

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Fully automatic method for the determination of fat soluble vitamins and vitamin Dmetabolites in serum

J.M. Mata-Granados a,b,⁎, J.M. Quesada Gómez a,c, M.D. Luque de Castro b

a SANYRES (GRUPO PRASA), E-14012, Córdoba, Spainb Department of Analytical Chemistry, Annex C-3, Campus of Rabanales, University of Córdoba, E-14071, Córdoba, Spainc Mineral Metabolism Unit, Hospital Reina Sofia of Córdoba, E-14004, Córdoba, Spain

⁎ Corresponding author. Department of I+D+i, SanSpain. Tel.: +34957218615.

E-mail address: jm.matagranados@gmail.com (J.M. M

0009-8981/$ – see front matter. Crown Copyright © 20doi:10.1016/j.cca.2009.01.029

a b s t r a c t

a r t i c l e i n f o

Article history:

Background: Fat soluble vi Received 9 October 2008Received in revised form 27 January 2009Accepted 30 January 2009Available online 6 February 2009

Keywords:Vitamin DVitamin EVitamin AOn-line SPEHPLC

tamins 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 for30 s and injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration, thenon-line transferred to a reversed-phase analytical column by a 15% methanol–acetonitrile mobile phase at1.0 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 α- and δ-tocopherols, respectively.Results: Detection limits were between 0.0015 and 0.26 µg/ml for the target compounds, the precision(expressed as relative standard deviation) between 0.83 and 3.6% for repeatability and between 1.8 and4.62% for within laboratory reproducibility. Recoveries between 97–100.2% and 95–99% were obtained forlow 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, anduser unattendance, making it a useful tool for high-throughput analysis in clinical and research laboratories.

Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction

Vitamins A (retinol) and E (a-tocopherol) are classified as non-enzymatic antioxidants [1]. Vitamin A “scavenges” singlet oxygen andsuperoxide anions. Apart from direct interaction with active oxygenspecies, vitamin A is thought to inhibit free radical synthesis viaincreasing the activity of detoxifying systems [2]. Moreover, hypervi-taminosis A is known to cause accelerated bone metabolism andspontaneous fractures in laboratory animals [3,4] and its high dietaryintake has been associated with osteoporosis [5].

Vitamin E protects unsaturated fatty acids located in both cell andorganelle membranes against endo- and exogenous free radicals andactive oxygen species, which are involved in the initiation and spreadof membrane damage caused by nonenzymatic lipid peroxidation[6,7]. Therefore, both vitamins are thought to play an important role indiseases such as atherosclerosis, diabetes mellitus and cancer.

Vitamin D2, which comes from yeasts and plants, and vitamin D3,which is found in oily fish and cod liver oil and is made in the skin,share essentially the same biological potency in humans [8–11]. Thedifferences between vitamin D2 and vitamin D3 are a double bond

yres group, E-14012, Córdoba,

ata-Granados).

09 Published by Elsevier B.V. All ri

betweenC22 andC23 and amethyl group on C24 for vitaminD2 [8–10].Once vitaminD2 or vitamin D3 enters the circulation, they are bound tothe vitamin D-binding protein and transported to the liver wherecytochromeP450-vitaminD-25-hydroxylase (CYP27A1) introduces anOH on carbon 25 to produce 25-hydroxyvitamin D [25(OH)D] [8–11].25(OH)D enters the circulation and is the major circulating form ofvitaminD. Because the hepatic vitamin D-25-hydroxylase is not tightlyregulated, an increase in the cutaneous production of vitamin D3 oringestion of vitamin D will result in an increase in circulating levels of25(OH)D [8–13]. Therefore, its measurement is used to determinewhether a patient is vitamin D deficient, vitamin D sufficient, orvitamin D intoxicated [8–11]. The main biological effect of vitamin D isto maintain the serum calcium within the normal range. It accom-plishes this by increasing the efficiency of intestinal absorption ofdietary calcium and by recruiting stem cells in the bone to becomemature osteoclasts, which, in turn, mobilize calcium stores from thebone into the circulation [8–12].

There are few methods for simultaneous determination of fat-soluble vitamins in biological fluids. Most of them consist of an ex-traction step prior to liquid chromatography analysis using diode arraydetection [14–17] or, less frequently, mass spectrometry (MS) [18–20].However, the reported methods are relatively time-consuming, expen-sive, and cumbersome, especially for vitamin Dmetabolites due to theirsensitivity to both temperature and light. For this reason, the

ghts reserved.

Table 1Prep steps of solid phase extraction.

Time Action Parameter Comment

0:00 Change cartridgeSample loop Load Load sample

0:01 Select solvent Methanol Activating cartridgeSet flow rate 4.0 ml/min

0:30 Select solvent 30% ACN 15 mM SDS Conditioning1:00 Set flow rate 1.0 ml/min Equilibration1:30 Sample loop Inject Load sample3:30 Select solvent 30% acetonitrile Washing interferences5:30 Set valve 1 Elute Eluting analytes with mobile phase

Start analysisSelect solvent NoneSet flow rate 0.0 ml/min

6:00 Set valve 1 PurgeSelect solvent Methanol Purge tubingSet flow rate 4.0 ml/min

7:00 Select solvent 30% acetonitrile Purge tubing8:00 Set flow rate 0.0 ml/min

Select solvent None15:00 End program

127J.M. Mata-Granados et al. / Clinica Chimica Acta 403 (2009) 126–130

development of automatic and sensitivemethods is required in order todetermine these fat-soluble compounds, of great interest in scientificareas such as the clinical field and metabolomics. In this research, amethod based on hyphenated solid phase extraction (Prospekt) withliquid chromatography and ultraviolet detection is developed todetermine vitamins A, D3 and D2, α-tocopherol, δ-tocopherol, 25OHD3

and 25OHD2 in human serum.

2. Materials and methods

2.1. Reagents and working solutions

25 Hydroxyvitamin D3 (25OHD3), 25 hydroxyvitamin D2 (25OHD2), vitamin A,vitamin D3, vitamin D2, delta tocopherol, alpha tocopherol and human serum albuminfrom Sigma (Sigma-Aldrich, St. Louis, MO, USA), methanol, acetonitrile and sodiumdodecyl sulfate (SDS) from Scharlab (Barcelona, Spain) were used.

Stock standard solutions were prepared by dissolving 1 mg of each analyte in 10 mlmethanol (for 25OHD2 and 25OHD3) or 10 mg (for vitamin D3, vitamin D2, δ-tocopheroland vitamin A) or 30 mg (for α-tocopherol) in 10 ml ethanol. The standard solutionswere prepared by dilution of the appropriate volume of stock solutions in ethanol. Astandard working solution containing each analyte at a concentration close to thereference healthy human value— 0.5, 0.02, 0.005, 0.02, 0.02, 0.1 and 5 µg/ml for vitaminA, 25OHD3, 25OHD2, vitamin D2, vitamin D3, δ-tocopherol and α-tocopherol,respectively — was used for optimisation of the chromatographic step. A 50 mMphosphate buffer solution pH 7.0–7.5 with 5 mg/dl of human serum albumin was usedto optimise the preconcentration step at analyte concentrations as above. A 30% (v/v)acetonitrile–water solution was used as washing solution and a 30% (v/v) acetonitrile–water 15 mM in SDS solutionwas used as loading solution and a 60% (v/v) acetonitrile–water 150 mM in SDS solution was used to dilute the samples. The initial mobile phasewas used as elution solution. Pure methanol as well as a 30% (v/v) acetonitrile–watersolutionwas used to purge the tubing of the Prospekt system at the end of the SPE steps.

2.2. Apparatus

The experimental setup used for the automatic determination of the target analytesin serum samples is shown in Fig. 1.

The chromatographic separation was performed with a ProStar system (Varian,Palo Alto, California, USA), consisting of a pump (ProStar 240, solvent delivery mixture),

Fig. 1. Schematic diagram of the SPE-HPLC arrangement for automatic sampleextraction–individual separation determination. In the extraction mode (A), a freshextraction cartridge is placed in the loop on the switching valve (1). The cartridge iswashed and conditioned with solvent from the solvent delivery unit (SDU). An aliquotof the sample is led to the SPE cartridge by the loading solvent from the SDU. Theanalytes are adsorbed and a washing solution is circulated through the cartridge toremove potential interferences. In the elution mode (B), the valve is switched to flushthe analytes to the analytical column with the LC mobile phase. After the analytes haveleft the cartridge, the valve is switched again to the extraction mode (A), and theextraction cycle repeated while the chromatographic step takes place.

a diode array detector (ProStar 330) and an autosampler (ProStar 410) furnished with acolumn oven and 0.6 ml sample loop. A Prospekt system (Spark Holland, Emmen, TheNetherlands), comprising a unit for SPE cartridge exchange and a solvent delivery unit(SDU) for SPE solvent delivery was hyphenated to the chromatograph for fullyautomatic analysis. The overall system was controlled and the data acquired andprocessed by using Star Chromatography Workstation version 6.0 software. Peek tubeof 0.5 and 0.25 mm i.d. (VICI, Houston, Texas, USA) was used to connect all valves of theProspekt unit and this unit with the analytical column, respectively. A cartridge packedwith Bond Elut C8 (Varian) as sorbent material 10×2 mm was used for SPE. Theanalytical column was a 5 µm Inertsil ODS-2 column, 150×4.6 mm i.d. (GL Sciences).

2.3. Preparation of serum samples

Venous blood was collected into a Vacutainer® tube (Becton Dickinson). The tubewas not opened to ambient air and placed in ice or kept refrigerated until processing.Blood samples were processed within 2 h after collection and centrifuged at 4000 g for10min to extract serum, which was collected, placed in a plastic ware tube and stored ator below−80 °C until analysis. Appropriate volumes (reported in Sections 3.5 and 3.6)of standard solutions of the analytes were added in the validation step.

2.4. Proposed method

A volume of 0.5 ml of serum is supplemented with 0.5 ml 60% (v/v) acetonitrile–water solution 150 mM in SDS and vortexed for 30 s in a 1.8 ml injection vial, thenplaced in the autosampler tray, which is thus ready for analysis. 0.6 ml of the content ofthe injection vial is injected into the system following the sequence of automaticoperations described in Table 1. Basically, the sample preparation process starts withpreparation of the cartridge by activation of its stationary phase with methanol,conditioning and equilibration with a 30% (v/v) acetonitrile–water solution 15 mM inSDS; then, the sample is loaded into the cartridge with a 30% (v/v) acetonitrile–watersolution 15 mM in SDS (flow pumped by the SDU). Under these conditions, the targetcompounds are retained in the cartridge and a 30% (v/v) acetonitrile–water solutionwas used as cleaning solution. The chromatographic step starts by switching valve 1 andputting the cartridge into contact with the initial chromatographicmobile phase, whichis 85:15 acetonitrile–methanol at 1.0 ml/min. At minute 5, a linear gradient is pro-grammed to increase the flow rate at 1.5 ml/min in 4 min. The chromatographic step isdeveloped at room temperature.

A last step consists of purging the tubing of the Prospekt system. The chroma-tographic and detection steps overlap with the SPE step of the next sample in order toimprove the analysis frequency.

A photodiode array detector was used for detection of the target analytes. The threewavelengths selected (265 nm for vitamin D compounds, 292 nm for tocopherols and325 nm for vitamin A); correspond to those of maxima absorption of these compounds.The software contains a peak purity method to assess the purity of each peak using thecompounds spectra.

3. Results and discussion

The optimisation sequence followed in this study consisted of twosteps: the first, focused on the best separation–determination of thecompounds using HPLC and, the second, on the most efficientpreconcentration–cleanup of the target compounds by the automaticSPE system. High repeatability and reproducibility of the injections

128 J.M. Mata-Granados et al. / Clinica Chimica Acta 403 (2009) 126–130

were observed after optimisation; therefore, the use of an internalstandard was unnecessary. A standard solution and a serum poolspiked with the analytes, respectively, were used for optimisation ofthese steps.

3.1. Optimisation of the chromatographic separation and detection steps

Theexperimental set up formobile phase optimisationwas theProStarsystem furnished with a 100 μL sample loop. The composition of themobile phase and the column temperature were studied by a univariateapproach. The response variables used for optimisation of this step were:(a) Peak area, (b) the resolution expressed as (tr2−tr1)/tr1, where tr is theretention time; and (c) retention factor expressed as (tr−t0)/t0,where t0is the dead volume time. The first of these responses should be as narrowas possible and the resolution should be as high as possible in order toachieve thebest resolution in the shortest timewithmaximumsensitivity.The ideal retention factor is between 2 and 10.

Individual direct injections of standard solutions of the analyteswere used to collect the spectrum of each analyte at the maximumabsorption wavelength to improve both the selectivity and sensitivityof the method. The optimum mobile phase and detection parametersare detailed under “proposed method”.

Fig. 2. Chromatogram from a sample with concentrations of the target analytes close to thetext). Concentrations: vitamin A (tr=3.733): 0.100 µg/ml, vitamin D3 (tr=11.053): 5 ng(tr=4.344): 2.5 ng/ml, δ-tocopherol (tr=10.055): 530 ng/ml, α-tocopherol (tr=12.879

3.2. Optimisation of the automatic solid-phase extraction step

The optimisation of on-line sample preparation was aimed atobtaining maximum extraction efficiency and cleanup.

The SPE sorbent, sample volume, loading solvent (type, volumeand flow rate) and elution flow rate were studied by a univariateapproach because the high number of non-continuous variables (i.e.SPE sorbent, loading solvent, washing solvent) made the use of thetypical multivariate design difficult. Spiked serum was used for thestudy of the SPE sorbent and other variables of this step.

A hyphenated SPE system (dual SPE cartridge configuration) [21]was used for optimisation of this step. The response variable used forthe optimisation of this step was the sorption ratio of each compoundbetween cartridge 1 and cartridge 2, which ensures optimum reten-tion when a single cartridge is used.

3.2.1. SPE sorbentSix types of SPE sorbents — namely, Hysphere C8(EC), Hysphere

C18(EC), Hysphere C8, Bond Elut C18-OH, Bond Elut C18 and Bond ElutC8 — were tested as first cartridge in order to select the best one interms of preconcentration and cleanup. Hysphere C18(EC) wasselected as cartridge 2 due to its generic retention behaviour and

ir LOQs. Monitoring wavelengths: 265 nm (A), 325 nm (B), 292 nm (C) (for details, see/ml, vitamin D2 (tr=10.536)=2.5 ng/ml, 25OHD3 (tr=4.127): 5 ng/ml, 25OHD2

): 1 µg/ml.

Table 3Repeatability (Sr), within-laboratory reproducibility (Swr) and recovery for each analyteat three concentration levels.

Analyte Repeteabilitya Reproducibility a Recovery b

(low level)Recoveryb

(mediumlevel)

Recovery b

(highlevel)

Vitamin A 1.06 2.4 97.0 96.5 95.025OHD3 0.83 1.8 100.2 99.5 99.825OHD2 0.92 1.86 99.8 99.3 99.2Vitamin D2 1.9 3.12 98.9 99.0 98.2Vitamin D3 1.85 3.23 99.1 99.4 99.9δ-tocopherol 2.3 4.51 98.3 98.4 98.1α-tocopherol 3.6 4.62 97.8 98.1 97.2

a Expressed as percent of relative standard deviation.b Expressed as percent of [final concentration− initial concentration]/added

concentration.

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the information obtained from the literature. The best results wereobtained for Bond Elut C8 cartridge.

3.2.2. Loading solventSeveral aqueous mixtures of methanol and acetonitrile were

assayed as loading solvent in previous studies. Acetonitrile wasselected as organic solvent in the loading aqueous solution.Concentrations of acetonitrile in water from 10 to 50% were studiedand the best results were obtained with 30% of acetonitrile, whichprovided recoveries close to 100% for vitamin A, 25OHD3 and25OHD2, but was decreased to 60% for vitamin E. Loading solutionswith acetonitrile above 30% decreased vitamin A recovery, increasedthe back pressure in the system and yielded progressively dirtyextracts, as shown in the undesirable peaks that appeared at thebeginning of the chromatogram in Fig. 2. In order to increase vitaminE recovery and avoid protein precipitation SDS was added to theloading solution at concentrations always higher than the criticalmicellar concentration. Concentrations of SDS higher than 15 mMpromoted a decrease in the recovery of vitamin A and 25OHD3, butincreased that of vitamin E. A 15 mM concentration of SDS in theloading solution was used as a compromise solution, at which therecovery of vitamin E was 70%.

The volume and flow rate of the loading solvent were also studied.As guideline, the initial load volume should be at least twice the sumof the loop, transport tubing and cartridge volume. The load volumewas studied between 2 and 8 ml and the flow rate between 0.5 and4 ml/min. Flow rates above 1 ml/min provided low recoveries of theanalytes because the load solvent–sample mixture was not efficient;therefore, 1 ml/min was chosen as loading flow rate. The best loadvolume (the minimum providing clean extracts) was 2 ml.

3.2.3. Diluting solventThe samples were diluted to improve the load in the sample loop

and the recovery of several analytes. Percentages of acetonitrile from10 to 80% were studied and the best results were obtained for 60%. Inorder to increase vitamin E recovery the concentration of SDS in thediluting solvent was studied. In this case, the recoveries of vitamin A,25OHD3 and 25OHD2 were not affected for SDS concentrations higherthan 15 mM. Therefore, concentrations up to 200 mM were studied.The recovery of vitamin E was close to 98% for 150 mM SDS andleveled off for higher concentrations.

3.2.4. Washing solventThe composition of the washing solvent was studied using several

acetonitrile–water mixtures. The best results were provided by 30%acetonitrile as higher concentrations decreased the recovery of vitaminA (recoveryof 75% for50%acetonitrile). SDSwasnot used in thewashingsolvent as it promoted broadband chromatographic peaks. Washingvolumes between 1 and 6 ml and flow rates between 1 and 4 ml/minwere assayed; the optimumvalueswere 3mland1ml/min, respectively(higher flow rates promoted analytes elution).

Table 2Equations, linear ranges and figures of merit.

Analyte Equation Linearrange

Coefficientof regression(R2)

Limit ofdetection(µg/ml)

Limit ofquantification(µg/ml)

Vitamin A y=−39492+10858500C 0.042–1.0 0.9998 0.012 0.04225OHD3 y=1527+3119240C 0.007–0.2 0.9994 0.0021 0.00725OHD2 y=3193+5215930C 0.003–0.1 0.9973 0.0015 0.0031Vitamin D2 y=−4148+3223480C 0.012–0.1 0.9934 0.0017 0.0042Vitamin D3 y=2430+2166920C 0.021–0.2 0.9949 0.0025 0.082δ-tocopherol y=−11829+140548C 0.56–10 0.9920 0.18 0.56α-tocopherol y=−40187+327814C 0.86–20 0.9997 0.26 0.86

3.3. Calibration curves and linearity

Calibration plots were run for the target analytes using the peakarea at the monitoring wavelengths as a function of the standardconcentration of each compound. Slope, intercept, regression coeffi-cient, standard deviation of residuals and standard deviations of theslope and intercept of all analytes are listed in Table 2.

3.4. Analytical sensitivity and detection limits

The limit of detection (LOD) for each analyte was calculated as 3σdivided by the slope of the calibration equations at low concentration(where σ is the standard deviation of the estimate) [22]. The LODs arebetween 0.0015 and 0.26 mg/l for all analytes. The limits of quantifica-tion, expressed as the concentration of analyte which gives a signal 10σthe mean blank signal, are within 0.003 and 0.86 mg/l for all analytesunder study (see Table 2).

3.5. Evaluation of the precision of the method

Within-laboratory reproducibility and repeatability were evalu-ated in a single experimental set-up with duplicates by experimentscarried out with a serum pool. Two measurements of this sample perday were carried out on 7 days [23].

The results obtained are listed in Table 3. The repeatability,expressed as relative standard deviation (RSD), ranged between0.83 and 3.6% for all target analytes; the within laboratory reprodu-cibility, also expressed as RSD, ranged between 1.8 and 4.62%.

3.6. Evaluation of the accuracy of the method

The accuracy of the method and potential matrix effects werestudied by analysing spiked samples. The recovery of the analytes wascalculated as [final concentration− initial concentration]/addedconcentration; evaluated by determining a sample repeatedly fivetimes in the same day under identical conditions, and at threeconcentration levels (low, medium and high concentration). Mediumlevel is close to the reference levels in a healthy population, high level

Table 4Profile of the target analytes in the population under study (expressed asmean±standarddeviation).

Parameter Values Values ref[24]

Values ref[25]

Values ref[26]

Values ref[27]

Age (n=80) 43±1525OHD3 (ng/ml) 18.8±6.7 17.2±8.3 19.7±9.5Vitamin A (mg/l) 0.63±0.09 0.575 0.52±0.13Vitamin D3 (ng/ml) 20.3±3.1δ-tocopherol (mg/l) 1.2±0.4α-tocopherol (mg/l) 8.4±1.4 11.8 12.6±2.4

130 J.M. Mata-Granados et al. / Clinica Chimica Acta 403 (2009) 126–130

is twice of medium level and low level is 20% of the high level.Medium level is: 20 ng/mL for 25OHD3 and vitamin D3, 0.5, 1.0 and8.4 mg/L for vitamin A, δ and α-tocopherol, respectively. The resultsobtained are listed in Table 3. The interferences were evaluated bycomparison of the UV spectra of the pure compound — saved in thecreated spectra library — and the UV spectra of the peak of the samecompound in the serum samples using the peak purity option of thePolyView 2000 software. Interferences were not found; therefore, themethod is selective for these compounds in serum. The performance ofthemethodwasperiodically checked for serum25OHD3 and 25OHD2 byparticipation in the Vitamin D External Quality Assurance Survey(DEQAS), promoted by Charing Cross Hospital (London, UK).

3.7. Comparison with other methods

The proposed method is fast, cheap and minimises samplehandling. These characteristics endow themethodwith high precisionand accuracy, which make the use of an internal standard unneces-sary, thus lowering the analysis cost. This aspect and its high samplethroughput make the method suitable for routine analysis.

Various HPLC methods have been described in the past for mea-surement of fat-soluble vitamins. All reported methods to determinevitamins A and E include a manual extraction step [14–20], in whichthe serum sample was mixed with ethanol to precipitate proteins andthen extracted with hexane. After centrifugation, the hexane phasewas evaporated to dryness under a nitrogen stream and the residuewas dissolved in methanol or in an acetonitrile–methanol mixture.This procedure is relatively tedious and complex, the reason whythese methods usually were costly, time-consuming, and with lowprecision and accuracy; so an expert worker and/or internal standardwere necessary to improve precision.

Despite that the use of a UV detector provides lower sensitivity andselectivity than a mass detector, the former is cheaper and moreavailable for routine analysis.

3.8. Application of the method

In order to demonstrate the applicability of the proposed method,it was used to determine the target analytes in serum from eighty“healthy” volunteers. A typical chromatogram of the diluted samplewith concentrations next to LOQ appears in Fig. 2. Demographicprofile and mean±standard deviation of each analyte in the samplesare shown in Table 4. The results obtained are similar to thoseprovided by other authors for healthy people in Spain.

4. Conclusions

Automation of sample preparation by the use of on-line SPE withautomatic valve switching and cartridge exchange has proved anexcellent approach for unattended analysis of the target compoundsin human serum samples. Minimising human intervention makes themethod easy to apply and improves reproducibility and accuracy. Inaddition to full automation, the proposed approach provides a closedsystem for maximum protection against degradation and highprecision because the number of sample transfer steps is significantlyreduced.

The method was designed to provide a fast chromatographicseparation of vitamins A, D3 and D2, α-tocopherol, δ-tocopherol andvitamin D metabolites (25OHD3, 25OHD2) between themselves andfrom interfering substances in serum. The on-line SPE is fast and simplewith no loss of analytes thanks to the use of an automatic closed system,which also provides good sensitivity, precision and accuracy. Further-more, a sample can be automatically prepared while running the chro-

matographic step of the previous one, so that continuous analysis can bedone, providing a high-throughput assessment of analytes in humanserum.

Acknowledgements

Investigators are grateful to Sanyres for financial support and PAIgroups CTS-413 and FQM-227 (Junta de Andalucía, Spain).

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