s 70 (2007) 363–368www.elsevier.com/locate/jbbm

J. Biochem. Biophys. Method

Simple GC-FID method development and validation for determination ofα-tocopherol (vitamin E) in human plasma

Fatma Demirkaya, Yucel Kadioglu ⁎

Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240, Erzurum, Turkey

Received 23 December 2005; received in revised form 13 August 2006; accepted 13 August 2006

Abstract

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in humanplasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. Todetermine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol wasextracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used ascarrier gas at a flow-rate of 2 ml min−1. Calibration curves were linear over the concentration range 1–30 μg ml−1 (for standard solutions andsolutions without endogenous α-tocopherol in plasma) and 5–34 μg ml−1 (for solutions with endogenous α-tocopherol in plasma). Absoluterecovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. Thelimit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml−1 and 0.30 μg.ml−1, respectively.Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) wasbetter than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with thismethod.© 2006 Elsevier B.V. All rights reserved.

Keywords: α-tocopherol; GC-FID; Plasma

1. Introduction

In nature, vitamin E occurs in eight different forms (α-, β-, γ-and δ-tocopherol and α-, β-, γ- and δ-tocotrienols) with varyingbiologic activities [1]. The most biologically active form ofvitamin E is α-tocopherol (5,7,8 trimethyltocol) which is presentin lipoproteins (Fig. 1). It has been recognized as a classic freeradical scavenger with chain-breaking properties and is one of themost important of fat-soluble antioxidants in biological systems[2,3]. α-tocopherol protects against cardiovascular diseases [4,5]and it may play a key role in the prevention of some types ofcancer [6,7]. Several papers have also assessed the effects ofvitamin E administered with the aim of reducing the incidence ofsevere diseases, such as retrolental, fibroplasia, intraventricular

⁎ Corresponding author. Tel.: +90 442 2311539; fax: +90 442 2360962.E-mail addresses: yucelkadi@hotmail.com, yucel@atauni.edu.tr

(Y. Kadioglu).

0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jbbm.2006.08.006

hoemorrhage, bronchopulmonary dysplasia and hemolytic ane-mia [8,9]. In addition, reduced plasma α-tocopherol levels havealso been reported in the Smith-Lemli-Opitz syndrome [10]. Forthis reason, there is an enormous public interest in vitaminsupplements. Also an industrial pressure with limited control overmarketing and quality exist. If the intake of vitamin E is in-sufficient or if special dietary requirements exist multivitamins orvitamin preparations can be taken in order to prevent vitamindeficiency. Numerous such preparations often formulated areavailable on the market, But this vitamin has caused toxic effectswhen a person intakes it at high dose. Therefore, the determinationof vitamin levels in biological fluid is very important.

Further studies are required to clarify the roles of vitamin E, oneof the fat-soluble antioxidant vitamins and it is essential to havesimple and rapid analyticalmethods available for themeasurementof this substance in a routine manner. Several methods have beenreported for the determination of α-tocopherol including Volta-metric [11], Spectrometric [12] and Chromatographic [13–17]

Fig. 1. Chemical structure of α-tocopherol (vitamin E).

364 F. Demirkaya, Y. Kadioglu / J. Biochem. Biophys. Methods 70 (2007) 363–368

methods. An extensive survey of the literature revealed no GC-FID method for the determination of α-tocopherol in humanplasma that did not require the derivatization of α-tocopherol. Wepresented a rapid, simple, accurate, sensitive and precise analyticalmethod of determination of α-tocopherol with a simple samplepreparation without the derivatization in human plasma. Thedeveloped method was applied for the determination of concen-trations of endogenous α-tocopherol in blood of twelve healthyvolunteers who had no history of vitamin E drug usage.

2. Materials and methods

2.1. Chemicals

α-tocopherol was purchased from Sigma (St, Louis, Mo,USA). Ethanol was HPLC grade (Merck, Germany). Hexane,dichloromethane, other chemicals and solvents were analyticalgrade (Merck, Germany). All gases were supplied by Hava°(Ankara, Turkey).

2.2. Instrumentation and analytical conditions

Chromatographic analysis was carried out on an Agilent6890N Network gas chromatography system equipped with aflame ionization detector, an Agilent 7683 series autosampler, anAgilent chemstation. HP-5 column with 0.25 μm film thickness(30 m×0.320 mm I.D., USA) was used for separation. Splitlessinjection was used and the carrier gas was nitrogen at a flow-rateof 2 ml min−1. Nitrogen (25 ml min−1), hydrogen (40 ml min−1)and dry air (400 ml min−1) were used as auxiliary gases for theflame ionization detector. The injector and dedector temperatureswere 300 °C. The oven temperature was held at 150 °C for 1 minand then increased to 320 °C at a rate of 28 °C min−1, where thetemperature was held for 3 min.

2.3. Preparation of the standard and quality control solutions

A standard stock solution of α-tocopherol was prepared withethanol to a concentration of 400 μg ml−1 and stored in the darkunder refrigeration. Standard solutions at 1, 2, 4, 5, 7, 10, 20 and30 μg ml−1 concentrations of α-tocopherol were prepared bydiluting with ethanol appropriate volumes of stock solution. Thestandard solutions were prepared daily. Preparation of plasmaworking solutions; a suitable amount of standard solutions wasspiked in 0.5 ml plasma containing endogenous α-tocopheroland then extracted with liquid–liquid extraction method. Theconcentrations of extracted solutions containing endogenous α-

tocopherol were 5, 6, 8, 9, 11, 14, 24 and 34 μg ml−1. Thequality control solutions at 2, 15 and 25 μg ml−1 concentrationsof α-tocopherol were prepared in the same manner, spiked inplasma and extracted. The concentrations of quality controlsolutions of plasma were 6, 19 and 29 μg ml−1.

2.4. Extraction from plasma

0.5 ml of the plasma sample was added in 20 ml capacity glasstube. After adding ethanol (1 ml), the sample was shaken. 5 ml ofhexane and dichloromethane mixture (9:1) then was added. Thesolution was vortexed for 1 min. They were then centrifuged at3000 ×g for 7 min. The organic phase was transferred to anotherglass tube and evaporated to dryness at 40 °C under the aid of agentle stream of nitrogen. The residue was dissolved in ethanol.The solutions were filtered through phonomenex (0.45 μm poresize, diameter of 25 mm filter) and transferred to an autosamplervial for analysis. 2 μl of sample was injected into the GC-FIDsystem to obtain the quantity of α-tocopherol.

2.5. Subjects

The study was performed on twelve healthy volunteers(ten females and two males). Their ages ranged from 20 to32 (26.08±3.85) and their weights ranged from 52 to 80 kg(62.66±8.13) (Table 4) All the subjects were informed for thepurpose, protocol and risk of the study, They were in good healthas determined by a physical examination and clinical laboratorytest results. Subjects did not take medication containing α-tocopherol for at least two weeks prior to the entire study. Theirblood samples (3 ml) were collected in ETDA tubes in themorning (8:00 A.M.) and centrifuged at 2000 ×g for 10 min.These samples were stored at −20 °C until analysis.

3. Results

3.1. Specificity

The specificity of this method has been demonstrated by therepresentative chromatograms for standard α-tocopherol inhuman plasma shown in Figs. 2 and 3. The retention time of α-tocopherol in human plasma and standard solution is 8.4 min. Theclean-up of biological samples were achieved by developed liq-uid–liquid extraction procedure. No further matrix interferencewas noticed the exception of matrix interference of α-tocopheroldetermination in human plasma samples. Different temperatureprograms were investigated for the exception of matrix

Fig. 2. Chromatogram of obtained using the assay spiked procedure. A) Plasma containing endogenous α-tocopherol. B) Plasma spiked with 10 μg ml−1 α-tocopherol.C) Plasma spiked with 20 μg ml−1 α-tocopherol.

Fig. 3. A) Chromatogram of obtained calibration graph point using the assay spiked procedure. B) Calibration graphs (n=6). C) Plasma containing endogenousα-tocopherol.

365F. Demirkaya, Y. Kadioglu / J. Biochem. Biophys. Methods 70 (2007) 363–368

Table 1Linearity of α-tocopherol

Range(μg ml−1)

LRa Sa Sb R

Standard α-tocopherolsolutions

1–30 y=1.0797x−0.0495

0.0331 0.0491 0.9998

Without endogenousα-tocopherol in spikedhuman plasma

1–30 y=1.0553x+0.1127

0.2007 0.0315 0.9987

With endogenousα-tocopherol in spikedhuman plasma

5–34 y=1.0553x+0.2253

0.3204 0.0315 0.9987

aBased on six calibration curves: LR: linear regression, Sa: standard deviation ofintercept of regression line, Sb: standard deviation of slope of regression line, R:coefficient of correlation, y: peak-area, x: α-tocopherol concentration (μg ml−1).

366 F. Demirkaya, Y. Kadioglu / J. Biochem. Biophys. Methods 70 (2007) 363–368

interference. At the end of this investigation, the best tempera-ture program was selected for a good resolution. The tem-perature of the GC oven was as follows; initial temperature150 °C, hold 1 min, final temperature 320 °C, hold 3 min, ramprate 28 °C min−1. When the ramp rate was more or less than28 °C min−1, the good resolution of the peaks (analyte peak andmatrix interference peaks) was not obtained. The plasmasamples received from different hospitals have also been testedand showed no significant interference at the retention times ofcompounds of interest. It can be seen that endogenous α-tocopherol was present in the human plasma samples. Weestimated that endogenous α-tocopherol of plasma samples inour study has a concentration of approximately 4 μg ml−1 by thestandard curve according to the procedure of the standardaddition method and is within the range reported by literature[18].

3.2. Stability

The vitamin E study is unstable and especially sensitive tolight, heat, oxygen and peroxide [19]. Stock solutions of α-tocopherol (400 μg ml−1) were prepared in absolute ethanol andkept in the dark at 4 °C. The stability of the analyte stocksolution was tested during 14 days and the results indicated thatthe stock solution was stable within this period. Also, theworking solutions for the spiking plasma sample were found tobe stable after 14 days with no significant change in con-centration when stored at −20 °C.

Table 2Precision and accuracy of α-tocopherol

Added(μg ml−1)

Within-day

Found±SDa Precision %RSDb Accurac

Plasma poolsd

6 6.16±0.2128 3.45 −2.6619 20.04±0.2633 1.31 −5.4729 26.81±0.1953 0.72 7.55aSD: Standard deviation of six replicate determinations. bRSD: Relative standard deAverage of six replicate determinations. cAccuracy: (%relative error) (found−addeddPlasma volume (0.5 ml).

3.3. Linearity

The linearity of the peak area response versus concentration forthe standard α-tocopherol samples and samples without theendogenous α-tocopherol in spiked plasma were studiedapproximately from 1 to 30 μg ml−1. The calibration curveswere obtained with eight concentrations (1, 2, 4, 5, 7, 10, 20 and30 μg ml−1) of the analyte solution in spiked plasma sample aftersubtracting the basal concentration of the endogenous α-tocopherol in plasma. The linearity of samples with theendogenous α-tocopherol in spiked plasma was studied approx-imately from 5 to 34 μg ml−1 because the endogenous α-tocopherol in human plasma has a concentration of approximately4 μg ml−1. The linear regression equations obtained by using theleast-squares method (n=6) and correlation coefficients arepresented in Table 1.

3.4. Precision and accuracy

Assay precision was determined by repeatability (within-day)and intermediate precision (between-day). Within-day was eval-uated by assaying samples, at same concentration and during thesame day. The between-day was studied by comparing the assayon different days (6 days). The accuracy of this analytic methodwas evaluated by checking at three different concentration of α-tocopherol. Three quality control solutions (2, 15, 25 μg ml−1)were prepared, spiked in plasma and extracted. The quality con-trol solutions extracted from plasma has 6, 19, 29 μg ml−1

concentrations. The within-day relative standard deviation (RSD)was b3.50% for human plasma (n=6) and the between-dayrelative standard deviation (RSD) was b4% for human plasma(n=6). Precision studies of GC-FID method showed acceptableRSD values and relative errors for accuracy were b8%. Theseresults were given in Table 2.

3.5. Limit of detection and quantification

LOD and LOQ were determined by an empirical method thatconsisted of analyzing a series of standard solutions containingdecreasing amounts of α-tocopherol. The LOQ defined as thelowest concentration of measured value of standard solutionswas 0.35 μg ml−1. LOQ for plasma was 4.35 μg ml−1. TheLOD for determination of standard α-tocopherol solutions wasapproximately 0.30 μg ml−1 being comparable to those reported

Between-day

yc Found±SDa Precision %RSDb Accuracyc

6.21±0.2366 3.80 −3.5019.76±0.5317 2.69 −4.0027.03±0.8167 3.02 6.79

rivation.) / added×100.

Table 3Recovery of α-tocopherol (n=6)

Added(μgml−1)

Found concentrationstandard α-tocopherolsample (μg ml−1)

Found concentration (μgml−1) human plasmacontaining 4 μg ml−1

endogenous α-tocopherol

%Recovery

1 1.0367 0.8705 83.962 2.1000 2.1616 102.934 4.1841 4.1361 98.855 4.9427 4.5794 92.647 7.0165 7.0558 100.5610 9.6233 9.8018 101.8515 15.5097 16.0426 103.4320 20.1057 20.8920 103.9125 25.8861 22.8089 88.1130 30.0502 29.5051 98.18

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in literature [20–22]. Both accuracy and precision of this valuewere well within the proposed criteria (RSD %: 20%).

3.6. Recovery

The analytical recovery of α-tocopherol from human plasmawas assessed by direct comparison of concentrations of α-tocopherol samples (without endogenous α-tocopherol in plasma)obtained after the whole extraction procedure by using six rep-licate at ten concentrations levels (1,2, 4, 5, 7, 10, 15, 20, 25 and30 μg ml−1) in the calibration graph versus standardα-tocopherolsolutions. α-tocopherol was extracted from blood with 5 ml of themixture of hexane and dichloromethane (9:1). This solvent mix-ture gave an excellent recovery. Analytical recoveries of α-to-copherol from plasma were 83% to 104%, as shown Table 3. Nointerfering peaks were detected in the plasma samples containingendogenous α-tocopherol in the analysis.

4. Discussion

In the present study, we report a highly selective GC-basedseparation combined a flame ionization detection that enabled us

Fig. 4. Chromatogram of plasma samples obta

to quantify the α-tocopherol without derivatization in humanplasma. Avariety of indices have been used to assess vitamin E tobe an antioxidant [2,3]. Currently, the most frequently used directmeasure is the plasma vitamin E concentration. Because theplasma of vitamin E can be affected by many physiological andpathologic conditions as well as certain drugs. The usefulness ofvitamin E (α-tocopherol) as an antioxidant has been questioned[3–10]. Some studies suggested that α-tocopherol concentrationin plasmamight be preferred, as antioxidant, although limited dataare available.

Today, HPLC andGC is considered to be the method of choicefor the determination of α-tocopherol in plasma. GC method isvery common that the previously reported methods usually re-quire a derivatization step. Here, we reported that our proceduredoes not require derivatization step. In the present study, thesamples were collected from twelve healthy volunteers and the α-tocopherol in plasma was extracted with a mixture of hexane anddichloromethane (9:1). The analytical recovery of this methodwas very good.

An important aspect of the implementing a new assay inroutine clinical practice is that it should be thoroughly evaluatedbefore introduction for routine use. In the present study, theevaluation of precision and accuracy for α-tocopherol wascalculated from the samples analyzed six replicate on one dayand six days. Furthermore, the linearity, precision, accuracy,stability of the assay for α-tocopherol in plasma were assessedbased on results of the analysis of eight samples with inter-related concentrations and LOD and LOQ values of standard α-tocopherol was determined. We suggest that this method will beuseful for studies concerning the metabolism of α-tocopheroland also particularly routine quantitation of α-tocopherol inhuman plasma.

5. Simplified description of the method and its applications

Plasma samples of twelve healthy volunteers were preparedaccording to extraction procedure and analyzed in order to checkthe applicability of the method in real clinical samples. Typical

ined from the twelve healthy volunteers.

Table 4Characteristics of healthy volunteers and α-tocopherol concentrations in plasmasamples obtained from the twelve healthy volunteers obtained using the assayprocedure

Healthyvolunteer

Sex Age(years)

Weight(kg)

Height(cm)

α-tocopherolconcentration(μg ml−1)

HB F 25 56 164 5.77SK F 23 60 162 10.97LE F 20 52 167 9.07BA F 28 58 155 10.18AO F 21 64 161 8.42AG F 23 66 164 6.12BO F 29 59 157 7.98CC F 31 63 157 10.06FA F 26 61 160 5.26SY F 26 57 154 8.54VK M 29 76 181 4.77YO M 32 80 184 5.68Mean±SDa – 26.08±3.85 62.66±8.13 163.83±9.56 7.73±2.14

aSD: Standard deviation of six replicate determinations.

368 F. Demirkaya, Y. Kadioglu / J. Biochem. Biophys. Methods 70 (2007) 363–368

chromatograms of plasma samples obtained with twelve healthyvolunteers are present in Fig. 4. Characteristics of healthy vol-unteers and concentrations of analyte found after analysis aresummarized in Table 4.

The present study describes the novel GC-FID method fordetermination of α-tocopherol (vitamin E) without derivatizationin human plasma. The proposed method is simpler than HPLCmethods and more reliable than spectrometric methods and easilyapplied to the analysis of large numbers of human plasma. Alsoone-step liquid–liquid extraction procedure used in human plasmais fairly rapid and advantageous.

In conclusion, this assay method for the determination of α-tocopherol from human plasma was completely validated byusing sensitivity, stability, specificity, linearity, accuracy andprecision parameters. We successfully applied this method to thedetermination of α-tocopherol concentration in blood of twelvehealthy volunteers. The GC-FID method has high recovery andexcellent reproducibility. For these reasons, it can be used fordetermining plasma of α-tocopherol in routine measurement aswell as in pharmacokinetic studies for clinical use.

Acknowledgements

The authors are grateful to the Directorship of the Universityof Atatürk for the financial support of this work.

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