Electrochemical study of the flavour enhancer maltol. Determination in foods by liquid chromatography with amperometric detection

ANALYTIC4 CHIMICA ACTA

ELSEVIER

Analytica Chimica Acta 327 (1996) 6.5-71

Electrochemical study of the flavour enhancer maltol. Determination in foods by liquid chromatography with amperometric detection M. Jestis Portela, Department of Analytical

Zurifie G6mez de Balugera, Ram6n J. Barrio*

Chemistry

M. Arhzazu

Goicolea,

Faculty of Pharmacy, University of Basque Country, 01006 Vitoria-Gasteiz,

Spain

Received 31 October 1995; revised 5 January 1996; accepted 21 January 1996

Abstract The electrochemical behaviour of malt01 on a glassy carbon electrode was studied. The results were applied to electrochemical detection in liquid chromatography (TX-ED). Liquid chromatography (LC) and oxidative amperometric detection with a glassy carbon electrode were used for the determination of malt01 in food samples. A mobile phase of methanol-acetonitrilecitrate/phosphate buffer, pH=2.4 (50:48:2) was used with the detector operated at E=lOOOmV vs. Ag/ AgCl reference electrode. Under optimal conditions detection and determination limits of 0.1 and 3.7 ng respectively were obtained. The method was used for the determination of malt01 in cakes. Conditions for clean-up of the sample were

established using solid phase extraction cartridges. Keywords: Liquid chromatography; Amperometry; Cakes; Malt01

1. Introduction Maltol, 3-hydroxy-2-methyl-4H-pyran-4-one, occurs naturally in certain conifers, but it is also formed when certain disaccharides are heated. Maltol, a potent flavour enhancer, is added to foods in minute amounts, at levels ranging from 50 to 200mg kg-’ [l]. Studies indicated that the average daily intake of malt01 per person in the US was estimated as 0.4 mg. This corresponds to about 0.01 mg kg-’ per day for a person who weighs 60 kg. The UN Joint FAO/WHO

* Corresponding author. Fax: 34 945 130756. SOOO3-2670/96/$15.000 1996 Elsevier Science B.V. AU rights reserved PZZ SOOO3-2670(96)00066-9

Export Committee on Food Additives concluded that up to 2mg kg-’ per day was an acceptable level of consumption for human beings [2]. At the recommended concentration malt01 does not have a flavour on its own but modifies or enhances the inherent flavours of the foods to which it is added. Determination of malt01 has been mainly performed by gas chromatography-mass spectrometry (GC-MS) [3,4]. In addition WNIS spectrometry has been used after extracting the coloured complex formed with Fe@) [S]. Electrochemical detection (ED) in a flow injection analysis system (FIA) has been used to determine malt01 after its reaction with a Cu(II)-phenantroline complex [6].

66

M.J. Port&

et al./Analytica

Several forms of electrochemical detection in liquid chromatography (LC-ED) are in use today. The most important method is direct-current (DC) amperometry. For analytes that can be oxidized or reduced, detection is usually sensitive and highly selective. Such detection offers fast response and a wide linear range. The major application of DC amperometry detection in LC is the determination of molecules containing phenol or cathechol functional groups [7]. These groups can be oxidized when they reach the surface of the glassy carbon electrode and the resulting current is measured. Some informative reviews [8-lo] and books [ll] have been published on this subject. Recently other substances with several oxidizable groups have been determined by this technique [ 121. In this work, the electrochemical oxidation of malt01 at a glassy carbon electrode has been investigated. The effect of the supporting electrolyte and pH on the oxidation curves has been examined and voltammograms have been obtained at various scan rates to elucidate the reaction mechanism. The results have been applied to the development of a rapid LC-ED method for the determination of malt01 in food samples.

2. Experimental 2.1. Chemicals and reagents Malt01 and Prazosin were supplied by Sigma (St. Louis, MO, USA). Methanol, acetonitrile of HPLC grade were obtained from BDH (Poole, Dorset, UK) and citric acid monohydrate and disodium hydrogen phosphate used in the supporting electrolyte from Merck (Darmstadt, Germany). The supporting electrolyte used in the mobile phase was prepared by mixing 0.02 mol 1-l citric acid and 0.05 mall-’ disodium hydrogen phosphate to yield the desired pH. The resulting buffer solution was mixed with methanol and acetonitrile to obtain the desired concentrations of eluent. 2.2. Instrumentation 2.2.1. Cyclic voltammetry Voltammetric curves were obtained by linear sweep with a Metrohm (Herisau, Switzerland) E-

Chimica Acta 327 (1996) 65-71

506 polarecord and a Metrohm E-613 scanner coupled with a Yokogama (Tokyo, Japan) 3022 A4 X-Y recorder. A glassy carbon Metrohm Mod. 6.1204.000 electrode was used as the working electrode, a platinum electrode as the auxiliary electrode and an Ag/AgCl (3 mol 1-l KCl) electrode as reference in a Metrohm 663 VA stand. 2.2.2. Chronoamperometry Coulometric measurements on malt01 stock solution were performed with a three-electrode system. The working electrode was formed by connecting two glassy carbon electrodes in series in order to increase the electrodic surface. The cathode was separated from the main compartment by means of an agaragar salt bridge. The cell system was connected to a PARC (Princeton, NJ) potentiostat/galvanostat Model 263A controlled by a data station EG and G PARC Model 270/250 (Princeton, New Jersey, USA) Research Electrochemistry Software 4-23. 2.2.3. Liquid chromatography The LC system consisted of a Waters Model 510 pump (Waters, Milford MA, USA), a model U6K 25 ul loop injector (Waters Association), a Knauer degasser on-line and a 25 cmx4.1 mm i.d. 10 urn Spherisorb Cis column (Tracer, Barcelona, Spain). A Princeton Applied Research (Princeton, New Jersey, USA) Mod. 400 electrochemical detector equipped with a thin-layer cell having a glassy carbon electrode was used as the amperometric detector. The electrochemical data were processed by means of a Metrohm 714 IC-Metrodata chromatography workstation. 2.3. Procedures 2.3.1. Preparation of the food samples After crumbling 50 mg of the cake, the sample was diluted in a small volume of deionized water. The mixture was shaken vigorously for 5 min and filtered through a Millipore GVWPG4700 0.22pm filter (Milford, MA, USA). The solution was then passed through an activated Bond-Elut Cis cartridge (Varian, Harbor City, CA, USA), where malt01 was collected. The cartridge was rinsed with water and the malt01 was eluted with 3 ml of mobile phase (methanolacetonitrile-citrate/phosphate buffer 50:48:2). The

M.J. Portela et al./Analytica

67

Chimica Acta 327 (19%) 65-71

whole extraction process was controlled with an Analytichem International (Harbor City, CA, USA) Vat Elut 10 vacuum system. A solution 1.43x lop3 mall-1 of the internal standard prazosin was prepared. A 1OOul aliquot of this solution was added to the obtained solution of the extraction procedure and diluted to 10 ml. An aliquot of this solution (25 ul) was injected into the chromatograph.

3. Results

and discussion

The cyclic voltammogram of Malt01 on a glassy carbon electrode is shown in Fig. 1. The compound exhibited a broad irreversible oxidation wave at a potential of 95OmV This peak is probably due to the oxidation of the enolic group. The mechanism is discussed below, but this enolic group is the only oxidizable group present in the molecule. It was found that the value of Ep shifted to more negative potentials with increasing pH (Fig. 2). Values of the electrochemical transfer coefficient, CYIZ (where (Y is the transfer coefficient and n is the number of electrons involved) were calculated at each pH value, according to the following equation: ofi = 0.048/E,

I

0.6 PA

- Epj2,

where Epn is the potential in volts that corresponds to iJ2. These values vary between 0.64 and 0.55 for pH values between 2.17 and 7.77. The number of hydrogen ions involved in the electrode reaction, m, (Table 1) was calculated from the plot Ep vs. pH according to the expression AE,/ApH

= (O.O59/an)m.

The linearity of the plot ip vs. d/2 in the range of scan rates from 10 to 800 mv s-l and the observation of the graph i&dn vs. v (where c is the concentration and v the scan rate), which clearly does not exhibit any slope, indicate that the oxidation process is diffusion controlled (Fig. 3). At the same time the number of electrons, n, was determined by controlled potential coulometry: after an electrolysis of the blank (citric-phosphate buffer, pH=2.7) at a potential of 1OOOmV vs. Ag/AgCl electrode, a 5 x lop4 mol 1-l solution of malt01 was

F IV) Fig. 1. Cyclic voltammogram on a glassy carbon electrode for 5 x lop4 mol 1-l of maltol. Scan rate: 100 mV s-‘; citric-phosphate buffer (pH=2.1)-methanol-acetonitrile 50:48:2.

electrolysed for 26000s. The malt01 concentration that still remains in solution, calculated by linear scan voltammetry, was 4.83x 10P4mol 1-l. With these data, the number of electrons transferred per

68

M.J. Portela et ai./Analytica 1000

1

0

0

900

0

=

800

--

700

--

0

0

.% w”

0 0 0

800

500

--

0

-l 0

2

4

8

8

Chimica Acta 327 (1996) 65-71 Table 1 Number of hydrogen malt01

EPi2

(Yn

m

2.17 2.91 4.00 4.51 4.92 6.00 6.62 7.03 7.77

950 919 850 825 800 725 700 657 600

875 837 775 738 725 650 612 580 525

0.64 0.59 0.64 0.55 0.64 0.64 0.55 0.62 0.64

0.68 0.62 0.68 0.58 0.68 0.68 0.58 0.65 0.68

10 7000

molecule of malt01 oxidized was found to be 1.02. The same result was obtained on the whole pH range studied: n=1.04 at pH=3.48 and n=O.98 at pH=5.30. These results allow to suggest the following mechanism (Scheme 1):

The

electrochemical

reaction

of

Ep (mV)

Fig. 2. Effect of the pH on the peak potential of maltol. Scan rate: lOOmVs_‘; citric-phosphate buffer-methanol-acetonitrile 50:48:2.

1.

reaction

PH

PH

Scheme

ions taking part in the oxidation

(al

6000

. . .

5000 z C

4000

-P

3000 i

mechanism. .

These results are in accordance with others found in the literature about the oxidation of compounds containing an enolic group [ 13,141.

.

0

l

l

l

4

3.1. Liquid chromatography 0

Previous data obtained in Section 3 showed that hydrogen ions take part in the oxidation reaction of maltol. That is why the parameters oxidation potential and pH must be studied together. The hydrodynamic voltammograms were obtained by injecting 25 ul volume of 7.9x low6 mol 1-l standard solution and varying the potential between 850 and 105OmV and the pH between 3.0 and 6.0 (Fig. 4). In Fig. 4 it can be observed that the highest chromato-

.

200

400

600

600

1000

1200

v (mV/s)

Fig. 3. Plot of (a) $ vs. vln and (b) i&v’” malt01 of 5x10-4mo11-‘.

vs. v for a solution of

graphic peak height (h,) was obtained at a potential value of 1000 mV and at pH=3.0, within a flow rate of l.Omlmin-‘. On the other hand, the highest signalto-noise was obtained at lOOOmV, then this potential was selected for the analytical work.

M.J. Portela et alJAnalytica

Table 2 Effect of the methanol and acetonitile contents on the capacity factor and resolution for 25ng of malt01 injected. Flow rate: 1 ml min.’

hp(nA) 7 6 5 4 3 2 1 0 800

900

1000

1100

EOW Fig. 4. Hydrodynamic injected. H pH =3.0, Flow rate: 1 ml min-‘.

69

Chimica Acta 327 (19%) 65-71

0

voltammograms pH =3.8, 0

Buffer %

MeOH %

60 52 51 50 50 50 49 48 41 45

40 48 48 50 48 45 48 50 48 45

AcN % 0 0

1 0 2 5 5 2 5 10

R

RS

0.88 0.60 0.65 0.47 0.69 0.49 0.44 0.44 0.38 0.45

1.61 0.89 1.00 0.59 1.17 0.63 0.52 0.52 0.41 0.54

for 25ng of malt01 pH =5.02, 0 pH =5.96.

$.@A) The effect of the buffer molar@ on the height of the peak was examined at pH=3.3 using phosphatecitrate buffer ranging from 0.02 to O.lOmoll-‘. It was observed that iP decreases when the ionic strength increases. That is why the minimum value of the buffer molarity that guarantees a stable pH value, 0.02 mol-‘, was selected Two organic modifiers, methanol and acetonitrile, were used to optimize the time of analysis, the peak width and the resolution. Table 2 shows the influence of the CHsCN and CHsOH contents in the mobile phase on the capacity factor, 2, and the resolution R,. The optimum separation conditions are R,>l, k’>l and a retention time, ta. which is as short as possible because at high values of fa the internal standard prazosin peak becomes broader. Acceptable results were obtained by a mobile phase containing citrate buffer-methanol-acetonitrile 60:40:0 or 5 1:48: 1. However, these conditions resulted in an unacceptably wide prazosin peak. A composition of 50:48:2 was used yielding a successful separation of malt01 and prazosin and an acceptable separation of the injection peak from the malt01 peaks. The mobile phase flow rate was 1.Oml min-’ and retention times obtained for malt01 and internal standard prazosin were 1.92 and 3.53 rnin, respectively. The detector used in this paper is based on the principle of a “thin-layer” cell. Because the mass transport to the electrode is controlled by both

n

4 -n

3 --

n

2 --

n

n

n

W 1 --

7 094

OS

0.8

1

flow rate (ml/min.) Fig. 5. Effect of the flow rate on the detector response for 25 ng of malt01 injected. pH=3.1 and E=lOOOmV.

diffusion and convection, the dependence of the detector response on flow rate must be studied. The results (Fig. 5) indicate that the response is optimum for a flow rate of 1.Oml rnin-‘, although if the system pressure increases flow rate of 0.8 or 0.9mlmK’ could be used with no loss of signal. 3.2. Calibration,

recovery and precission

The determination of the malt01 was based on the linear dependence of the relationship “area,alt,,l/ on the concentration of the injected ae%ra2osin” flavour enhancer (in ng). A linear response was

M.J. Portela et al./Adytica

70

Chimica Acta 327 (1996) 65-71

b

a

I

I

1 nA

1 nA

-

2 Fig. 6. (a) Chromatogram

4

6

8

tImin)

of a blank cake sample. (b) Chromatogram

observed from 3 to 40ng of malt01 injected. The linear calibration plot corresponds to the equation Arntito~/ Apmzosin= -0.2790 (r = 0.9988).

0’

+ 0.1525 [Maltol]

At the applied potential of +l .O V and for a signalto-noise ratio of 3:1, the detection limit was found to be 0.1 ng of malt01 injected. Typical chromatograms obtained for a blank sample and a malt01 spiked cake sample containing 195 mg kg-’ are shown in Fig. 6. The peaks for malt01 and the internal standard prazosin were identified on the basis of the retention time compared with that of the standard compound and by co-chromatography of the standard and the spiked cake sample under the optimum conditions selected before. The recovery of malt01 was determined by spiking 50mg fractions of maltol-free cake sample with 0.006 and 0.01 mg of malt01 (47.5 and 80 pl of a solution of 126.11 mg 1-l of maltol, respectively). These were then extracted using the proposed method. The same amounts of malt01 were diluted in the mobile phase and injected directly without

2

.4

6

0

t(n

1)

of a malt01 spiked cake sample. For conditions

see text.

extraction. At low concentration the recovery was 98.14&1.95% (mean f RSD, n=5 at the 95% confidence level). At higher concentrations the recovery was 99.88f0.94%. In order to assess the precision of the method ten 50mg maltol-free cake samples were spiked with malt01 to produce a concentration of 200 mg kg-‘. This level represents the typical amount of malt01 added to foods. The samples were analysed by the proposed method. The relative standard deviation (RSD) was 0.83%. The within-day precision of the method was established by repeated determinations every two days (n=lO) on a cake sample (200mg kg-‘), the RSD was 0.67%. In conclusion, the oxidation of malt01 could be carried out using a glassy carbon electrode and the electrochemical reaction mechanism is proposed. The results were used to obtain a method for malt01 determination in foods by LC with amperometrical detection. The main advantages of the proposed method of analysis are the simplicity and low cost of the extraction procedure. The results of the statistical analysis indicated that recovery and accuracy are satisfactory.

M.J. Portela et alJAnalytica

Acknowledgements This work was supported by from the Education, University ment of Basque Government EA177/93 research project of University UPV/EHU.

Chimica Acta 327 (19%) 65-71

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