development of a liquid chromatographic time-of-flight mass spectrometric method for the...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2003; 17: 2797–2803

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1263

Development of a liquid chromatographic time-of-flight

mass spectrometric method for the determination of

unlabelled and deuterium-labelled a-tocopherol in blood

components

Wendy L. Hall1, Yvonne M. Jeanes1, Jonathan Pugh2 and John K. Lodge1*1Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK2Micromass UK Ltd., Atlas Park, Simonsway, Manchester M22 5PP, UK

Received 6 August 2003; Revised 10 October 2003; Accepted 10 October 2003

A method is described for the analysis of deuterated and undeuterated a-tocopherol in blood com-

ponents using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF)

mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuter-

ated (d3 or d6) a-tocopherol standards were found with negative ion mode electrospray ionisation.

Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure

standards were linear in the range tested (0–1.5mM, 0–15pmol injected). For quantification of d0

and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled

internal standard (d3-a-tocopherol) was employed. To counter matrix ion suppression effects, stan-

dard response curves were generated following identical solvent extraction procedures to those of

the samples. Within-day and between-day precision were determined for quantification of d0- and

d6-labelled a-tocopherol in each blood component and both averaged 3–10%. Accuracy was

assessed by comparison with a standard high-performance liquid chromatography (HPLC) method,

achieving good correlation (r2¼ 0.94), and by spiking with known concentrations of a-tocopherol

(98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected,

respectively. The assay was used to measure the appearance and disappearance of deuterium-

labelled a-tocopherol in human blood components following deuterium-labelled (d6) RRR-a-toco-

pheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small

sample volumes, was reproducible and robust, and was capable of high throughput when large

numbers of samples were generated. Copyright # 2003 John Wiley & Sons, Ltd.

Vitamin E is a term used to describe a number of tocopherols

(a-, b-, d-, and g-) and tocotrienols (a-,b-, d-, and g-) of which a-

tocopherol is the most biologically active comprising over

90% of vitamin E in the body.1,2 The most widely researched

function of a-tocopherol is that of the major lipophilic chain-

breaking antioxidant; however, recent evidence suggests

a-tocopherol to have further roles in cellular signalling which

are independent of its antioxidant action.3 These actions have

led to clinical trials involving vitamin E in coronary disease

prevention, some of which have shown vitamin E to have a

beneficial effect.3,4 Thus it is important to gain a greater

understanding of the absorption, transport and distribution

of vitamin E.

Deuterium-labelled a-tocopherol can be a useful tool with

which to measure the rate of absorption, transport and

uptake in various blood components and excretion as urinary

metabolites. Indeed, the use of deuterated tocopherols has

greatly increased the knowledge of vitamin E transport.2,5 For

example, such studies confirmed that vitamin E isoforms are

absorbed indiscriminately in the intestine but that the

naturally occurring RRR form of a-tocopherol is retained on

the plasma,6,7 and plasma recycling of a-tocopherol is in

itself rapid.8 As well as absorption and transport, deu-

terated tocopherols can be used for biokinetic analyses of

a-tocopherol which relate to its antioxidant function; for

example, using cigarette smokers as models of in vivo

oxidative stress.9,10

The use of stable-isotope-labelled compounds requires

mass spectrometry as a quantitation tool. Previous methods

for the analysis of deuterated vitamin E have required gas

chromatography/mass spectrometry (GC/MS).2,5,8–11 How-

ever, the disadvantage of GC/MS is the need for sample

clean-up and prior derivatisation of the samples which can

greatly increase experimental error, especially as the

trimethylsilyl derivatives are unstable and rapidly hydrolyse

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: J. K. Lodge, School of Biomedical and Mole-cular Sciences, University of Surrey, Guildford GU2 7XH, UK.E-mail: [email protected]/grant sponsors: British Heart Foundation; The RoyalSociety.

in the presence of moisture. Recently, a number of LC/MS

methods for the analysis of tocopherols have been estab-

lished.12–14 These methods involve either single14 or triple

quadrupole12,13 instruments. Until now, no time-of-flight

mass spectrometry (TOFMS) methods for vitamin E analysis,

or indeed analysis of biomolecules, have been available. TOF

mass spectrometers offer increased sensitivity for the gen-

eration of full mass range spectra compared with scanning

instruments such as quadrupoles. This offers the advantage

of allowing quantitation to be performed on any mass

observed in the acquired range along with simultaneous

exact mass confirmation of the analyte elemental composi-

tion. Additionally, the higher resolution associated with TOF

spectra confers greater specificity of analyte ion selection in

the presence of potential nominally isobaric matrix inter-

ferences. The aim of this work was to develop a rapid,

quantitative method for the determination of deuterium-

labelled and unlabelled a-tocopherol in blood components

using liquid chromatography (LC) TOFMS.

MATERIALS AND METHODS

TocopherolsRRR-[5,7-Methyl-(2H3)2]-a-tocopheryl acetate (d6) and all

racemic-[5-methyl-2H3]-a-tocopheryl acetate (d3) were kind

gifts from Cognis Nutrition and Health. Purities of the acetates

were 98.8% for both species. Isotopic purity was determined to

be >99.9%. d6-RRR-a-Tocopherol acetate was encapsulated

(150 mg) for human consumption. RRR-a-Tocopherol (d0;

purity� 99.0%) was purchased from Fluka (Poole, UK).

Reagents and solventsLC/MS grade methanol (LC/MS Chromasolv), butylated

hydroxytoluene (BHT), ascorbic acid and lithium perchlorate

were purchased from Sigma-Aldrich Chemical Co. (Poole,

UK). Sodium dodecyl sulphate (SDS) and potassium hydro-

xide (KOH) was from BDH, hexane (HPLC grade) was from

Fisher (Loughborough, UK), absolute ethanol from Hayman

Ltd. (Witham, UK), and phosphate-buffered saline (PBS)

tablets from Oxoid Ltd. (Basingstoke, UK).

Samples for analysisHuman plasma, erythrocytes, platelets and lymphocytes

were isolated from blood donated by healthy, normolipidae-

mic male and female volunteers, aged 23–54 years, at various

times following supplementation with a capsule containing

150 mg d6-RRR-a-tocopheryl acetate taken with a standard

breakfast. These procedures were approved by the Univer-

sity of Surrey Advisory Committee on Ethics.

Platelets were isolated from whole blood by first centrifu-

ging whole blood at 280 g for 14 min at 28C to obtain platelet-

rich plasma (PRP). The PRP was then centrifuged at 1120 g for

15 min at 28C to pellet the platelets. After removal of the

platelet-poor plasma the platelets were washed with Tris

buffer (15 mM Tris, 134 mM NaCl, 5 mM KCl, pH 7.4 with

HCl) before reconstitution in 1 mL of the Tris buffer. Samples

were then aliquoted and snap-frozen in liquid nitrogen and

stored at �808C prior to analysis.

Lymphocytes were isolated from whole blood using

Histopaque-1077 (Sigma-Aldrich) according to the manufac-

turer’s protocol. After washing the lymphocytes three times

with PBS, pH 7.4, the lymphocytes were reconstituted into 0.5

mL PBS, aliquoted, and snap-frozen in liquid nitrogen and

stored at �808C prior to analysis.

Erythrocytes were carefully removed from whole blood

after centrifugation at 1550 g for 10 min and washed three

times with saline. Haematocrit was then measured and the

cells aliquoted, snap-frozen in liquid nitrogen, and stored at

�808C prior to analysis.

Plasma was collected following platelet removal (platelet-

poor plasma). Plasma was then aliquoted, snap-frozen in

liquid nitrogen, and stored at �808C prior to analysis.

Sample preparation and vitamin E extractionTo ensure that signals fell into the dynamic range of the instru-

ment, and to reduce matrix ion suppression effects (discussed

later), aliquots of blood components (100mL) were diluted

with PBS prior to extraction, and the final dried extracts

were reconstituted into a relatively large volume of ethanol

(500mL). It was found that plasma and erythrocyte aliquots

required diluting 1:5 and 1:10 with PBS, respectively, while

platelet and lymphocyte aliquots did not require dilution.

Total vitamin E was extracted from the blood components

by a combination of SDS, ethanol and hexane, in the presence

of BHT (10mL of a 1 mg/mL solution in ethanol), as described

previously.15 For LC/TOFMS analysis an internal standard

(d3-labelled a-tocopherol, 50 pmol) was added to the aliquot

prior to extraction. Lymphocyte and platelet aliquots were

sonicated for 3 min prior to extraction to obtain a homo-

genous solution. In separate experiments plasma samples

underwent saponification to extract vitamin E. The method

used was that of Podda et al.16 using KOH and ascorbic acid.

Following extraction and hexane evaporation, extracts

were reconstituted into 500 mL of ethanol ready for

LC/TOFMS analysis. All samples were prepared and

analysed in duplicate.

Standard preparationStandard stock solutions of d0-, d3- and d6-a-tocopherol were

prepared in ethanol. The hexadeuterated RRR-[5,7-methyl-

(2H3)2]-a-tocopheryl acetate (d6) and trideuterated all race-

mic-[5-methyl-2H3]-a-tocopheryl acetate (d3) were first

hydrolysed using KOH as described.16 The concentrations

of the stock solutions were then determined using the molar

extinction coefficient (e292¼ 327016) and diluted accordingly.

For quantitation, standards underwent the same extraction

procedure as that of the blood components (described above).

Aliquots of d0 and d6 were added to PBS (100mL) containing

BHT (10 mL of 1 mg/mL solution in ethanol). A range of d0

and d6 concentrations (0–400 pmol) with a constant concen-

tration of internal standard (d3, 50 pmol) was used.

Following extraction and evaporation, extracted standards

were reconstituted in 500mL ethanol. Final concentrations of

standard curves therefore typically ranged from 0–2 mM (for

the analysis of plasma and erythrocytes) or 0–0.8 mM (for the

analysis of platelets and lymphocytes), containing 0.1 mM

internal standard, and consisted of at least five calibration

points. Standards were also analysed in duplicate.

Liquid chromatographyThe HPLC system used was a Waters Alliance system

(Waters Corporation, Milford, MA, USA). The Waters 2695

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2797–2803

2798 W. L. Hall et al.

separations module comprises a solvent delivery system,

online degasser, peltier-cooled autosampler (set at 48C), con-

troller, and column oven (set at 308C). The mobile phase con-

sisted of 100% methanol (LC/MS grade) with a total run time

of 3.5 min. The tocopherols were separated on a reverse-

phase Waters Symmetry1 column (2.1� 50 mm, C18,

3.5 mm particle size) at a flow rate of 0.3 mL/min. a-Tocopher-

ols eluted at 2.2 min.

Mass spectrometrySamples were analysed using a Micromass LCTTM (Micro-

mass UK Ltd., Manchester, UK) orthogonal acceleration

TOF mass spectrometer, with negative ion mode electro-

spray ionisation (ESI). The LCT consists of an orthogonal

ion extraction (Z-Spray) source equipped with an electro-

spray inlet. The ions are transferred via a dual hexapole to

a pusher region where the ions are deflected into the analy-

ser flight tube at a rate of 20 kHz (50 ms flight time). Ion arri-

val times are recorded using a multichannel plate detector

(MCP) linked to a 3.6 GHz time-to-digital converter

(TDC). The individual 50 ms digitised spectra are summed

over a defined interval (1 s) to produce the full mass range

spectra. To optimise conditions the instrument was tuned

on a 10 mM solution of a-tocopherol in ethanol continuously

infused into the ion source. Optimum ionisation parameters

were found to be: negative ion electrospray ionisation

(ESI–); desolvation gas temperature, 2508C; source tem-

perature, 1208C; capillary voltage, 2800 V; sample cone vol-

tage, �35 V; extraction cone voltage 3 V; RF lens, 350.

Typically ions in the m/z range 350–500 Da were collected

from 1.5–3.5 min following injection, and acquired at 4000

FWHM resolution. Spectra were acquired at a rate of 1 spec-

trum/s (0.95 s average, 0.05 s delay). The target masses of

the ions of interest were used to identify each tocopherol

species by producing an extracted mass chromatogram

from the total ion chromatogram (TIC) over a 0.1 Da mass

window, thereby utilising the specificity gained by acquir-

ing spectra at high resolution. The identified peaks were

then integrated and amounts calculated from response

curves generated from a range of standards using the stan-

dard quantitation processing function with Mass Lynx ver-

sion 4.0 software. Ions generated by deuterium-labelled and

unlabelled a-tocopherol were: m/z 429.3742 for d0-RRR-a-

tocopherol, m/z 432.3973 for d3-all rac-a-tocopherol, and

m/z 435.4116 for d6-RRR-a-tocopherol.

HPLC/electrochemical detection (ECD)The same LC system to that described above was used but

incorporating a Bioanalytical Systems (BAS) electrochemical

detector with a glassy carbon electrode operating at an

applied potential of þ0.5 V. Tocopherols were separated on

a Waters Spherisorb ODS-2 column (4.6� 250 mm, 5mm par-

ticle size) using a mobile phase consisting of 99% methanol,

1% water and 0.1% lithium perchlorate at a flow rate of

1.5 mL/min. Tocopherols were quantitated using an internal

standard (d-tocopherol, 0.5 mM final concentration) with

responses calculated from a range of pure a-, g- and d-

tocopherol standards in ethanol. Data were analysed using

Waters MilleniumR32 software.

StatisticsData are expressed as means� standard deviations. Within-

day and between-day precision are shown as coefficients of

variance (standard deviation/mean� 100¼CV%). In addi-

tion to using the Pearson correlation coefficient to assess the

association between HPLC/ECD and TOFMS, Bland-Altman

plots17,18 were made to compare the difference between

methods. Bland-Altman tests are sensitive to detecting bias

between two methods estimating the same variable.17,18 Since

there was a large range in concentrations tested (plasma

and platelet unlabelled a-tocopherol), the between-method

variation was larger as concentrations increased. Therefore,

the percentage difference ((HPLC/ECD�TOFMS/(HPLC/

ECDþTOFMS/2))� 100) was used rather than the differ-

ence between methods.

RESULTS AND DISCUSSION

Method developmentAs this is the first method to employ LC/TOFMS to analyse

tocopherols, the appropriate method of ionisation was first

established. Ionisation was tested on both positive and nega-

tive ion mode electrospray (ESI) and atmospheric pressure

chemical ionisation (APCI) by tuning on a 10mM solution of

d0-RRR-a-tocopherol constantly infused into the spectro-

meter and observing the TIC following a 1-s acquisition.

Negative ion ESI was found to give the optimum response

(data not shown); hence this was used in subsequent method

development.

Typical TICs and spectra over the mass range of interest for

the tocopherol species following an injection of 0.5 pmol of

either d0, d3 or d6 standard are shown in Fig. 1. TOF ESI–

produces an isotopically resolved single ion of exact mass

(Fig. 1(B)). This mass for d0 (429.3742) is nominally isobaric

with the [M–H]� calculated mass of a-tocopherol (429.3733).

Obtaining exact mass greatly increases the confidence in the

correct assignment of the compound of interest. For example,

a nominal mass of 429.3 has the possibility of thousands of

possible empirical formulae, while a mass within�5 ppm can

only result from a very limited number of elemental

compositions. In this case the closest match corresponds to

C29H49O2, which is the formula for a-tocopherol. The clear

and monoisotopically resolved spectrum obtained by ESI-

TOF contrasts with the spectrum obtained in previous

methods employing either single14 or triple12,13 quadrupole

mass spectrometers. In these methods, APCIþ MS/MS,12

APCIþ MS,14 and ESþ MS/MS13 produced a cluster of ions

for a-tocopherol in them/z range 423–432, the expected target

protonated ion not being the predominant species. The

presence of these additional ions has been attributed both to

the oxidation of a-tocopherol, and to the protonation

followed by dehydrogenation of a-tocopherol, during ionisa-

tion12 and represents novel ionisation characteristics. How-

ever, this appears to occur only in quadrupole spectrometers

since only the expected target ions were observed during all

ionisation methods with TOFMS.

The principle of quantitation from TOF full ‘scan’ spectra

by integrating extracted mass chromatograms over a narrow

(0.1 Da) window allows greater specificity than typically

observed in selected ion recording (SIR) experiments

LC/TOFMS analysis of tocopherols in blood components 2799


employed on single quadrupole mass spectrometers,

although the overall sensitivity and linear dynamic range

are generally lower. The optimum specificity and selectivity

and linear dynamic range are usually obtained from multiple

reaction monitoring experiments performed on triple quad-

rupole instruments where quantitation is performed on the

most abundant fragment ions generated from the selected

precursor ion. However, in this example where the target

concentrations are relatively high and the dynamic range less

than three orders of magnitude, the benefits of exact mass and

high-resolution full spectrum acquisition conferred by TOF

data outweigh the limitations. With a-tocopherol the main

fragment observed during quadrupole MS was found to be at

m/z 165,12–14 which corresponds to the loss of the phytyl

chain. TOFMS does not produce product ions if the cone

voltage is kept relatively low. However, in-source fragmen-

tation can be induced by increasing the sampling cone

voltage. This was assessed in our experiments. a-Tocopherol

fragmentation occurred when the cone voltage was increased

to �70 mV. Among others, a fragment at m/z 163.3 was

observed (data not shown) which corresponds to an ion 1 Da

lower in mass than the major fragment ion observed in

previous methods.12–14

Typical peak area versus concentration curves for d0-, d3-

and d6-a-tocopherol standards demonstrated a linear

response in the range 0–1.5 mM (0–15 pmol injected). Con-

centrations in excess of 5 mM a-tocopherol (150 pmol injected)

resulted in non-linearity. TOF instruments generally have a

relatively low dynamic range, which is offset by the increased

sensitivity of these machines, so increasing concentrations of

analyte will eventually saturate the detector and produce a

non-linear response.

Quantification of a-tocopherolAn internal standard, d3-labelled all racemic a-tocopherol

(50 pmol added, 0.1 mM final concentration in extract),

was used for the quantification of unlabelled (d0) and d6-

labelled a-tocopherol in biological samples. The d3-labelled

a-tocopherol was added to the blood components prior to

extraction thereby accounting for any losses in the extraction

procedure.

First, we established the concentration range of tocopher-

ols that did not exceed the dynamic range of the detector. By

increasing concentrations of either d0 or d6 (0–10mM) and

maintaining a constant concentration of internal standard d3

(0.1mM), we observed saturation of the detector at concentra-

tions of d0 and d6 in excess of 5mM (as described above),

which also resulted in suppression of the internal standard

signal. Thus a maximum concentration of 4mM was deemed

appropriate. Therefore, biological samples, such as plasma,

had to be diluted prior to extraction, or reconstituted in larger

volumes, in order not to exceed the dynamic range of the

instrument. This has the added benefit of requiring very low

volumes of sample for quantitation. Indeed<10mL of plasma

can be used (standard HPLC methods generally use 100 mL).

Therefore, sample dilution into the dynamic range of the

instrument is an important and necessary prerequisite for

quantitation with TOFMS.

During initial spiking and recovery experiments with

increasing concentrations of d6-labelled a-tocopherol into

blood components, we noticed that quantitating using

calibration curves prepared in ethanol resulted in calculated

amounts much smaller than that added. This was first

attributed to matrix ion suppression effects due to the

biological matrix as previously noted.13 However, further

Figure 1. Typical LC/TOFMS extracted mass chromatograms (A) and mass spectra (B) of a-tocopherolsused in the method. The structures of the species d0 (RRR-a-tocopherol, unlabelled), d3 (all rac-a-5(CD3)-tocopherol) and d6 (RRR-a-5, 7-(CD3)2-tocopherol) are also shown. Tocopherols elute at 2.2 min.

The mass spectra shown in (B) demonstrate the presence of a single main species with accurate mass.



experiments revealed this to be mainly due to matrix effects

arising from the extraction procedure itself. To counter this,

standards underwent identical extraction to those of the

biological samples. Thus, in order to quantitate d0- and d6-

labelled a-tocopherol in samples, calibration curves were

generated using increasing concentrations of d0 and d6 and a

constant concentration of internal standard, then treated in

the same way as samples (solvent extraction, as described in

the previous section). The same concentration of internal

standard was added to unknown samples. A response curve

is generated to determine amounts from the relationship:

Response ¼ Areaunknown

� ðInternal standardconcentration= Internal standardareaÞ

(where unknown is either d0 or d6 in the standard or

unknown sample).

A typical response curve for d0- and d6-a-tocopherol is

shown in Fig. 2. The curve is non-linear and fits a polynomial

(coefficient of determination >0.995).

Typical TICs and extracted mass chromatograms of the

tocopherol species in representative plasma and platelet

extracts taken from an individual 3 h following ingestion of

d6-labelledRRR-a-tocopheryl acetate are shown in Fig. 3. The

ion pattern of the peak eluting at 2.2 min (not shown) showed

the presence of the three tocopherol species (d0, d3 and d6)

and no other ions in the range, comparable with mass spectra

of pure standards (Fig. 1).

It has been suggested previously that saponification

of biological samples may be required in the analysis of

a-tocopherol using LC/MS without multiple quadrupole

instruments, in order to remove potential interfering species

Figure 2. Representative area response curve used for the

determination of d0- and d6-labelled a-tocopherol in blood

components using d3-labelled a-tocopherol as an internal

standard. This concentration range was used for determina-

tion of d0 and d6 in plasma. The coefficient of determination

for each species was >0.995. The inset shows an expanded

region from 0–0.3mMwith response for both d0 and d6 on the

y-axis.

Figure 3. Typical LC/TOFMS mass chromatograms of vitamin E extracts from plasma and

platelets taken 3 h following supplementation of an individual with 150mg d6-labelled RRR-a-tocopheryl acetate. The total ion chromatograms (TICs) demonstrate the elution of the tocopherol

peak at 2.2min. Extracted mass chromatograms of the tocopherol species d0, d3 and d6 are shown

over a mass window of 0.1Da.



such as phospholipids and their oxidation products with m/z

similar to that of a-tocopherol.12 To investigate any potential

interfering compounds with the TOFMS method, a number

of plasma samples (n¼ 8) underwent extraction by saponi-

fication16 and this analysis was compared with those

extracted using the solvent (ethanol and hexane) method

(n¼ 8) performed in parallel. The spectrum of the tocopherol

peak eluting at 2.2 min was similar for both methods (data not

shown) and the d0 and d6 concentrations determined were

also similar (solvent: [d0] mM 20.8� 1.5, [d6] mM 8.0� 4; cf.

saponification: [d0] mM 21.3� 2, [d6] mM 8.3� 0.5). These

observations, coupled with the exact mass measurement of

a-tocopherol (which substantially increases confidence as

described above), suggest that both methods of vitamin E

extraction can be used in the analysis of tocopherols using

TOFMS.

Limits of quantification and detection (LOQ andLOD)LOD and LOQ were calculated using pure standards and

blood components spiked with varying concentrations of

d6-a-tocopherol prior to extraction. Values for LOQ and

LOD are presented in Table 1. The LOD was determined

from the amount injected which produced a peak with a sig-

nal/noise ratio of <5:1. This was found to be in the region of

5 fmol injected for standards, but 2-3-fold higher in blood

components, presumably because of either matrix effects or

losses during the extraction process. The LOQ was deter-

mined from the lowest point in the standard response curve

in which a reliable amount can be obtained. The values

between 50 and 100 fmol are approximately 3-fold higher

than the LOD for each blood component. The LOQ and

LOD are similar for plasma and platelets, but were higher

in erythrocytes presumably due to matrix ion suppression.

It should be noted though that these values for LOD and

LOQ using TOFMS represent an overestimate since: (i) they

were performed on diluted samples and (ii) the MCP gain

and TDC threshold can be altered to vary sensitivity if

required. Thus the LOQ is potentially lower. However, in

our experiments, these conditions were sufficient to detect

d6-labelled a-tocopherol in all blood components at various

times following 150 mg d6-RRR-a-tocopheryl acetate supple-

mentation in humans.

Performance of the TOFMS methodPerformance of the new LC/TOFMS method was evaluated

by accuracy and precision. To assess quantitative accuracy

the TOFMS method was compared with a HPLC/ECD meth-

od.16 Although HPLC/ECD is an established method it can-

not be considered a gold standard. However, comparison

of these two methods would indicate the accuracy of

LC/TOFMS to some extent. Non-deuterated samples

were obtained from volunteers taking part in a separate a-

tocopherol supplementation study. Plasma and platelet sam-

ples (n¼ 13) were used so that a wide range of a-tocopherol

values could be compared and from different matrices. d3-a-

Tocopherol was used as an internal standard for the TOFMS

method, while d-tocopherol was used as an internal standard

for the HPLC/ECD method. The use of different internal

standards required separate extraction for the two methods,

which may have added another source of error to the varia-

tion resulting from the method of detection. The LC/TOFMS

method correlated closely with HPLC/ECD (r2¼ 0.939). The

% differences between HPLC/ECD and TOFMS determined

with Bland-Altman plots17,18 showed that 9 out of 13 data

points were scattered randomly between the upper and

lower 20% range (data not shown). The upper and lower lim-

its of agreement (�2 SD) were 41% different due to two out-

lying data points. The mean percentage difference between

methods was �6%.

Plasma was also spiked with increasing amounts of d6-

labelled a-tocopherol (20–300 pmol added, 0.2–3 mM final

Table 1. Limit of detection (LOD) and limit of quantification

(LOQ) of a-tocopherol in blood components analysed by LC/

TOFMS

Standard Plasma RBCs Platelets

LODa 5 15 30 15S/Nb 5:1 3:1 3:1 3:1LOQa,c 50 100 50

a LOD and LOQ values in fmol injected.b Signal-to-noise ratio at LOD.c LOQ values extrapolated from lowest point in standard curve.

Table 2. Precision of LC/TOFMS method in the

determination of unlabelled (d0) and deuterium-labelled

(d6) a-tocopherol. Values shown are coefficients of

variation (CV) following a number of determinations either

within- or between-day

Variation(CV %) Iona Curveb Standardc Plasmad rbcsd Plateletsd

Within-daye d0 0.4 5 3 4 4d6 0.4 6 3 7 4

Between-dayf d0 0.4 4 7 5 7d6 0.4 4 10 6 3

a Variation in analysis of either d0 or d6, with an approximately 10-fold difference in concentration between d0 and d6.b Variation in the coefficient of determination of the standardresponse curve used for determination of either d0 or d6 amounts.Two concentrations of internal standard (d3) were tested (final con-centration 0.1 or 0.5 mM) and gave similar results.c Based on peak areas of pure standards in ethanol (0.5 pmol injec-tions).d Based on quantification of either d0 or d6 using d3 as internal stan-dard as described.e Based on n¼ 8 determinations of the same sample during the courseof a day (except curve; n¼ 3).f Based on n¼ 8 determinations of the same sample over the course of4 weeks.

Table 3. Concentrations of unlabelled (d0) a-tocopherol inblood components determined by LC/TOFMS

Blood componenta-Tocopherol determined

by LC/TOFMS (n¼ 6)

Plasma (mmol/L) 27.1� 8.6Erythrocytes (mmol/mL packed cellhaematocrit)

5.2� 0.4

Platelets (mmol/g protein) 2.0� 0.6Lymphocytes (mmol/g protein) 1.7� 0.8



concentration) and, following extraction, the d6 concentra-

tion was determined by the LC/TOFMS method. The

accuracy of this quantitation was found to be 98� 8%

(n¼ 12).

To assess precision, plasma, erythrocyte and platelet

samples were extracted and analysed either on a number of

occasions during a single day, or a number of times on

separate days over a 4-week period. Table 2 shows the

precision of the LC/TOFMS method based on the coefficient

of variation from within-day and between-day quantitation.

Good precision was found with CVs ranging from 3–10%.

Higher CVs for between-day variation were observed for

erythrocytes, and in general CVs were higher for d6 than d0,

presumably because of the smaller amounts.

Application of the LC/TOFMS method tobiological samplesIn order to apply the LC/TOFMS method to biological

samples, d0- and d6-labelled a-tocopherol were analysed in

blood components taken from a range of individuals various

times following ingestion of a capsule containing 150 mg

d6-labelled RRR-a-tocopheryl acetate. Table 3 shows

baseline concentrations of d0-a-tocopherol in plasma, ery-

throcytes, platelets and lymphocytes in a range of individuals

(n¼ 6), determined by LC/TOFMS. Values are similar to

published values for a-tocopherol in blood components.19,20

Profiles of d6-labelled a-tocopherol in blood components in a

single individual are shown in Fig. 4. Following ingestion of

d6-labelled RRR-a-tocopheryl acetate, there is a rapid ap-

pearance of d6-a-tocopherol in the plasma (peak at 9 h) and

erythrocytes (peak at 24 h), followed by a decrease in d6 con-

centration. In contrast, platelet and lymphocyte d6-a-

tocopherol increases gradually over the time period. Similar

a-tocopherol turnover curves in plasma8,11 and erythro-

cytes21 following deuteratedRRR-a-tocopheryl acetate inges-

tion have been observed. No such data exists for platelets and

lymphocytes.

CONCLUSIONS

This study demonstrates that LC/TOFMS can be applied in

the analysis of labelled and unlabelled tocopherols in blood

components. The method is rapid, sensitive, accurate and

reproducible.

AcknowledgementsWe are grateful to Dr. Christine Gartner and Dr. James Clark

of Cognis Nutrition and Health (Dusseldorf, Germany and

LaGrange, IL, USA, respectively) for the gift of the deuterated

(d3 and d6) tocopheryl acetates, and to the Pharmacy Depart-

ment of St. Thomas’ Hospital for encapsulation. We are grate-

ful to the British Heart Foundation and The Royal Society

for funding and to the Medical Research Council for sup-

porting YMJ.

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Figure 4. Concentration of d6-labelled a-tocopherol deter-mined by LC/TOFMS in blood components in a single

individual following ingestion of a capsule containing 150

mg d6-labelled RRR-a-tocopheryl acetate.



development of a liquid chromatographic time-of-flight mass spectrometric method for the...

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