Determination of ascorbic acid in beers by high-performance liquid chromatography with electrochemical detection

Journal of Chromatography,

405 11987) 347-356

Elsevier Science Publishers B.V., Amsterdam -

Printed in The Netherlands

CHROM. 19 695

DETERMINATION OF ASCORBIC ACID IN BEERS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

N. MOLL* I Ail&e Chaptal, 54630 Richardmenil (France) and J. P. JOLY Laboratoire de Chimie Organique III (VA 486 [es Nancy Ceakx (France)

CNRS), lJnrversitk de Nancy I, B.P. 239,54506

Yanabeuvre

(First received February 17th, 1987; revised manuscript received April 22nd, 1987)

SUMMARY

A reversed-phase high-performance liquid chromatographic method with electrochemical detection was developed for the determination of ascorbic acid in beers at the nanogram level. The column was packed with Cl8 bonded silica, commercially available or prepared in the laboratory. The eluent was a citrate buffer solution with an ion-pairing reagent. Several fatty amines were tested as such reagents. Good recoveries indicated that no interfering substances were eluted together with ascorbic acid. The main advantages are the rapid and simple preparation of the samples and the specificity and sensitivity of the detector, which are superior to those of any other detection systems proposed for the determination of ascorbic acid in beers.

INTRODUCTION

The use of ascorbic acid (AA) as an antioxidant in beers was first proposed by Gray and Stone’ J. Following its introduction into American breweries, AA was used in various European countries other than F.R.G. A kinetic study of the oxidation of beer3 showed that it undergoes oxidation much faster if it contains increased amounts of an antioxidant such as AA. When all the antioxidant is consumed, the beer is further oxidized but much more slowly. The level of AA in a beer during storage gives information about the oxido-reduction level and the behaviour of the product towards oxidation. As far as the determination of AA in beer is concerned, the special interest for the brewer is its reducing power, that is, it is only the reduced AA that is significant. The first methods proposed utilized the reducing activity of AA towards coloured reagents such as 2,6-dichlorophenolindophenol (DCI), 2,2’-dipyridyl-Fe3+ and Nbromosuccinimide4. The most commonly used method for assaying AA was oxidative titration with DCI, a method recognized by the Association of Official Analytical 0021-9673/87/$03.50

0

1987 Elsevier Science Publishers B.V.

Asahipak GS-320 hydrophilic gel

FBondapak NH2

Alltech NH2

Partisil 10 SAX

pBondapakcarbohydrate

Zorbax NH*

* Determination of ascorbic acid alone. ** Simultaneous determination of ascorbic acid and dehydroascorbic

Fresh and processed fruit and vegetables* Fresh fruits and vegetables** Tropical root crops from the South Pacific Orange juice**

Urine samples, processed foods** Fruits and vegetables**

LiChrosorb NH*

acid

CHjCN-CH&OOH-HZ0 (87:2: 11) 0.05 M KH2P0&H,CN (25:75) CH&N-Hz0 (70:30) containing 0.01 M KH2P04 (PH 4.3) 0.1 M sodium acetate buffer at pH 4.25 0.05 M KHZP0,,-CH3CN (2575) (v/v) CH,CN-0.005 M KH2P04 at pH 4.6 (70:30) 0.015 M tartrate buffer solution at pH 3, containing 2 mM EDTA and 0.05% fi-thiodiglycol

Methanol-2 . 10m3 M ammonium salt at pH 5 (S&50) 2.5 mM KH2P04-CH3CN (50:50)

PBondapak Ci a

Selected foods and multivitamin products* Biological samples, foods and pharmaceutical vitamin preparations** Animal tissues**

LiChrosorb NH1

Eluent (v/v)

ng

0.05 pg

25 ng

0.01 pg

0.5 fig

Detection limit

DETECTION

Derivatization, fluorimetric monitoting: excitation 325 nm, emission, 400 nm

Post-column derivatization, 254 254

250

254

268

254

254

254

UV wavelength (run) for AA detection

WITH UV AND FLUORIMETRIC

Column

OF ASCORBIC ACID USING LIQUID CHROMATOGRAPHY

Substrate

DETERMINATION

TABLE I

15

14

13

12

11

10

8, 9

7

6

Ref.

Ultrasphere ODS Altex

Nucleosil 7 Cs

Mixture of ascorbic acid derivatives

Beer samples

Onion extract

Partisil 10 SAX LiChrosorb RP-18

Tissues of marine animals

6 ng

w

0.50

0.95

0.80

2.5 ng

3ng

Detection limit

0.75

0.70

Acetate buffer (0.07 M, pH 4.75) Acetate bufkr (0.07 M, pH 5.25) 60 mA4 sodium acetate adjusted to pH 4.6 with 1 M acetic acid l/15 M phosphate buffer (pH 5.5), 0.028 M oxalic acid in water, pH 2.9 44 mM acetic acid, 16 mM sodium acetate and 1.8 mA4 1,5-dimethylhexylamine in ethanol-water (6.7:93.3) 5.0 g acetic acid in 1 I Hz0 adjusted to pH 5.5 with 1 M NaOH

Zipax SAX

Foodstuffs, pharmaceuticals, body fluids

Vydac SAX

Applied potential ( V) vs. Ag-AgCl

Elwnt

DETECTION

Column

OF ASCORBIC ACID BY HPLC WITH ELECTROCHEMICAL

Substrate

DETERMINATION

TABLE II

20

19

18

17

16

Ref.

2

b Ei

350

N. MOLL, J. P. JOLY

Chemists (AOAC)S. However, interference by other reducing substances is inevitable. With the development of high-performance liquid chromatography (HPLC), various methods for the determination of AA in biological samples with UV or fluorimetric detection have been proposed (Table I). The detection limit is not far below the microgram level, which may be too high for the detection of the trace levels present in some instances. With beers, detection by UV absorption is non-specific, as numerous substances absorb at the same wavelength as AA. A more sensitive and specific method that can be used is high-performance liquid chromatography with electrochemical detection (HPLC-ED), by which the detection limit reaches the nanogram level (Table II). However, there is little information in the literature1 s+16about the determination of AA in beers by HPLC-ED. This paper describes a simple, specific and accurate method for the determination of AA in its non-oxidized form in beers. The procedure utilizes HPLC-ED in the reversed-phase mode with complexing amines as ion-pairing reagents. After degassing, beer samples (5 ~1) are injected directly into the chromatograph. The good recoveries indicate that no interferences are present and the detection limit is about the nanogram level for 5 ,nl of beer (0.2 mg of AA per litre of beer). EXPERIMENTAL Reagents

Water for the preparation of the eluent and the stock and standards solutions of AA was freshly doubly distilled to prevent contamination by metal ions (iron and copper) which act as catalystsin AA oxidation. Just before use, oxygen and carbon dioxide were removed by bubbling with argon or degassing in an ultrasonic bath for 10 min. All chemicals were of analytical-reagent grade. Ascorbic Acid was obtained from Fluka and citric acid monohydrate (I), trisodium citrate dihydrate (II) and tetrasodium ethylenediaminetetraacetate trihydrate (EDTA) from Merck. Several organic amines were tested as ion-pairing reagents, including octylamine (III) and dodecylamine (IV) (Fluka), tridecylamine (V) and N,N-dimethyldodecylamine (VI) (Aldrich). N-Methyldodecylamine (VII) was prepared in the laboratory as follows2*. lXhloro-n-dodecane (Fluka) (1.27 mole) and 340 ml of an ethanolic monomethylamine solution (Prolabo) (2.73 moles) were heated in a reactor under pressure at 140°C. for 18 h. After cooling, 2 moles of 50% sodium hydroxide (Prolabo) (160 g) were added and that mixture was stirred at room temperature in the reactor for 3 h. The mixture was decanted and the aqueous phase extracted three times with 150 ml of diethyl ether. The organic phases were combined, dried over magnesium sulphate (Prolabo) and evaporated under reduced pressure. The residue was kept over potassium hydroxide for 48 h and distilled at 1 lo-120°C at 1 mmHg. N-Methyldodecylamine (152 g) was obtained as a pure product (yield 60%). Citrate &fleer solution (pH 4.4)

A 0.1 mole amount of I (21.01 g/l solution) and 0.1 mole of II (29.41 g/l solution) were dissolved separately in 1 1 of doubly distilled water and 280 ml of the

HPLC-ED

OF ASCORBIC

ACID IN BEERS

351

former solution and 220 ml of the latter were pipetted into a l-l graduated flask and diluted to volume with doubly distilled water. The buffer solution can be kept for several days in a refrigerator. Stock solution of ascorbic acid

For the preparation of a stock solution, 30 mg AA were weighed and dissolved in a small amount of oxygen-free doubly distilled water and the solution was transferred into a lOO-ml graduated flask and diluted to volume with doubly distilled water. The flask was stoppered and stored in a refrigerator until use. The stock solution must be prepared every day. Standard solutions of ascorbic acid

Dilution of 2, 4, 6, 8, 10 and 12 ml of the stock solution to 100 ml with oxygen-free doubly distilled water gave standard solutions containing 6, 12, 18, 24, 30 and 36 mg/l of AA, respectively. As dilute solutions of AA are unstable, it is recommended, for reproducible results, that the standard solutions are prepared just prior to the analysis and that the dilution of the stock solution is repeated for each point of the calibration graph (five replicates each). A volume of 5 ,ul of each sample was injected immediately into the HPLC column without prior filtration (to avoid oxygen intake). Beer samples

A bottle of beer was opened and 10-20 ml of beer were used to rinse a lOO-ml beaker, then 50 ml of the sample were poured into the beaker and degassed in an ultrasonic bath for 10 min. Volumes of 5 ~1 of beer were injected immediately into the HPLC column. Three replicate determinations were conducted for each beer sample. HPLC

apparatus and procedures

The chromatographic system (Waters Assoc.) included a Model 6000 A pump, a Model U6K injector and a Model 460 electrochemical detector equipped with a glassy carbon working electrode. The potential of the detector was set at +0.60 V vs. Ag-AgCl. Chromatographic data were recorded with a Model 4290 integrator (Spectra-Physics). The pre-column (25 x 3.9 mm I.D.) was packed with 30 pm Rsil ClsHL (RSL, Eke, Belgium). Two analytical columns were used: a 300 x 3.9 mm I.D. PBondapak Cl8 (Waters Assoc.) and a 300 x 3.9 mm I.D. column packed with a Cl* stationary phase prepared in our laboratory in the following way. LiChrospher Si 300 silica, 10 pm (Merck) was first conditioned for 16 h in a closed receptacle maintained at 20°C and 65% relative humidity. The conditioned silica (3 g) was dispersed in 30 ml of anhydrous toluene and 3 mmole (1.225 g) of octadecyltrichlorosilane (Aldrich) were added. The mixture was refluxed for 4 h under a slight pressure of dry nitrogen without agitation. After cooling, the silica was filtered off and rinsed successively with anhydrous toluene, 96% ethanol, distilled water, 96% ethanol and diethyl ether. Drying of the bonded silica was conducted at 50°C and 10 mmHg for 14 h. A 3.64g amount of silica was obtained, corresponding to a weight increase of 21.3%. Results

352

N. MOLL, J. P. JOLY

of elemental analysis gave C = 15.14% and H = 3.43%. The surface coverage (N) was calculated using the equationz3 N (~mol/m2) =

lo6 PC 1200 nc - PC (AA-

1 1). s

= 3.67

where PC is the percentage of carbon in the bonded phase, nc is the number of carbon atoms in the bonded silane molecule, M is the molecular weight of the bonded silane molecule and S is the specific surface area of the unbonded silica in m’/g. The result indicates that all the silanol sites were bonded. IR spectroscopy (silica pellet, 15 mg, without KBr)24 gave CH, 2855 and 2925 ctr-‘. Packing of the column. A 1.4-g amount of bonded silica was dispersed in tolueneeisopropanoll96% ethanol (1: 1: 1, v/v/v) by ultrasonication according to the classical slurry method 25. It was then percolated through the column with 100 ml of n-hexane at 450 bar. Eluent for HPLC. The eluent consisted of citrate buffer solution (PH 4.4) EDTA (5 s lop4 M) to complex with metal ions and each of the different amines (1 mM). The mixture was vigorously homogenized and filtered through a 0.45-pm membrane filter. The eluent can be stored for several days in a refrigerator. Before use, it must be degassed for 15 min in an ultrasonic bath and kept under a slight argon pressure during the HPLC. The flow-rate of the eluent was 1 ml/min. RESULTS

AND DISCUSSION

The organic amines used as ion-pairing reagents in HPLC-ED are generally primary amines and we tested three (IIIIV). IV and V were abandoned, because they are solids with poor solubilities in the HPLC eluent. More substituted amines such as secondary (VII) and tertiary (VI) amines should give greater complexation in HPLC separation. They have the advantage of being liquids likely to be readily soluble in the eluent. Both VI and VII gave the best results with regard to the resolution of the peaks and the retention time of AA. The results presented below were obtained with the amine VII synthesized in our laboratory (see Experimental). An example of the HPLC profiles obtained for a French pale lager beer is shown Fig. 1A. Ascorbic acid is eluted at 4.5 min. Other peaks can be observed on the chromatogram, which can be attributed to other reducing substances in beers, and these are attributed to other reducing substances in beers, and these are discussed in the next section. Allowing for the presence of such compounds on the chromatograms, a beer analysis can be conducted once every 20 min. Coefficients of variation were calculated for AA in both water and beer over the concentration range 6-36 mg/l: The resulting values were 3.553.7% for AA in water (five determinations each) and 2.5% for AA in beer (three determinations each). Owing to the instability of AA after opening of the bottle, not more than three determinations could be carried out for a repeatability test. The recovery of added AA averaged 96.1%. The detection limit for a signal-to-noise ratio of 2: 1 was 0.9 ng (0.20 mg/l) with a 5-,~l injection volume.

HPLC-ED

OF ASCORBIC

(A)

ACID

IN BEERS

353

(B)

a

AA

AA

i :-

: : TIME 4min )

I\

Fig. 1. Chromatograms of ascorbic acid (AA) in a French lager beer (14.5” Plato), (A) 1 day after bottling (30.2 mg/l) and (B) 3 weeks after bottling (20.1 mg/l). Column, 300 x 3.9 mm I.D., packed with a Cts stationary phase prepared in the laboratory; mobile phase, citrate buffer (pH 4.4)-l mM N-methyldodecylamine (VII)-5 10m4 M EDTA at a flow-rate of I ml/min; volume injected, 5 ~1; applied potential, 0.6 V YS. Ag-AgCl.

The response of the detector decreases slowly with time because of the deposit of sample material on the electrode surface. To prevent erroneous determinations of AA, a freshly prepared standard was injected after every three beer samples. The determination of the calibration graph allowed the validity of the method to be verified, but in practice the concentration of AA in beer should preferably be determined with repeated analyses of a standard. Other reducing substances in beer

We tested several other beers for AA using the described method. Figure 2 shows the chromatograms of two export Bavarian pale beers (A) and (C), an export German pale beer (B) and an export Dutch pale beer (D). Following the Bavarian purity law of 1516, German beers are not treated with antioxidants, as is confirmed by the chromatograms, on which no peak due to AA could be detected. Export German beers are no longer treated with AA, which was not the case for some beers about 10 years ago (up to 70 mg/l of AA were found) 26. If these beers are not treated with antioxidants, they contain more natural reducing substances from the raw material or brewing process, including reductones with ene-diol functions, reducing sugars, melanoidins, tannins and polyphenols. A classification of these compounds was given by De Clerck and van Cauwenbergez7 using the ITT (indicator time test). Ascorbic acid and reductones were described as the most reactive reducing products. Electrochemical detection based on the same electron transfer properties is the most specific detection method available for evaluating electroactive compounds. The peaks observed on the chromatograms in Fig. 2 represent an equilibrium between numerous redox systems whose

354

N. MOLL, J. P. JOLY

L ID1 (B)

(A! t

0

4

T 00

4

6

12

16

6

12

t6

20

20

TIME 4min ) Fig. 2. HPLC profiles of (A) a Bavarian double bock beer, (B) a German pale strong beer, (C) a Bavarian pale beer and (D) a Dutch pale beer. All four beers were export beers. Chromatographic conditions as in Fig. 1.

standard potentials are unknown. The set potential of + 0.60 V is probably not the most suitable for their detection and there exist other reducing substances that are not detected at this potential. Taking into account the complexity of such a medium, it is not possible to attribute any of these peaks to known substances. Two observations were made: catechin added as an internal standard to beer A (Fig. 1) increased the peak eluted at 15 min, and pure synthesized Amadori compoundP have no reducing power. It can be seen in Fig. 3 that aqueous solutions of proline-fructose (A) and proline-maltulose (C) gave no detector response. The same solutions were

0

4

6

tPt620

0

4

6

12

16

20

(min ) Fig, 3, Chromatograms of aqueous solutions of pure Amadori compounds, proline-fructose and proline-maltulose, (A) and (C), respectively, just after dissolution and (B) and (D), respectively, after 1 h reflux. Chromatographic conditions as in Fig. 1. TIME

HPLC-ED

OF ASCORFW

ACID IN BEERS

355

AA (mg.lmi )

t

\7 b

131

30-Q

20

q

0

\(1)

D--f

1

‘;]_,. ‘,

,

‘

FrgT*R:;

_

0 8 16 12 2 4 6 Fig. 4. Ascorbic acid content vs. storage time for a French pale lager beer (I lo5 Plato) from three’different bottlings, (l), (2) and (3).

refluxed for 1 h, which resulted in partial thermal degradation of the Amadori compounds into reductones whose electrochemical response could be observed in (B) and (D), respectively. There is therefore a presumption that many such systems contribute to the general phenomena observed in beers. Storage

experiments

Results of storage experiments for three packagings of the same French beer are presented Fig, 4. Each point represents the mean value of three replicate determinations. The rate of decrease of the AA content differs from one batch to another and, in the same batch, from one bottle to another. However, the determination of AA during the storage related to oxygen measurements is of great interest for the brewer in predicting the stability of the product. CONCLUSION

We believe that the HPLC-ED approach affords convenience of sample preparation, specificity and sensitivity superior to those of methods proposed previously for AA-in beers. The detection of other electroactive components could give an indication of the total reducing power and be a useful tool throughout the malting and brewing process. The method gives rapidly, during storage, information on the oxido-reduction level of beers and can be related to other measurements for the prediction of the stability of the beer. REFERENCES 1 P. P. Gray and I. Stone, U.S. Pat., 2 159985, 1939. 2 P. P. Gray and I. Stone, U.S. Pat., 2159986, 1939. 3 .I. C. Andre and M. Moll, J. Am. Sot. Brew. Chem., in press.

356

N. MOLL, .I. P. JOLY

4 N. Moll and M. Moll, in G. Charalambous (Editor), The ShelfLife of Foods and Beverages, Elsevier, Amsterdam, 1986, pp. 97-140. 5 AOAC, Oficial Methods of Analysis of the Association of Oficial Analytical Chemists, Association of Official Analytical Chemists, Washington, DC, 13th xl., 1980, p. 746. 6 S. P. Sood, L. E. Sartori, D. P. Wittmer and W. G. Haney, Anal. Chem., 48 (1976) 796-798. 7 R. C. Rose and D. L. Nahrwold, And. Biochem., 114 (1981) 14&145. 8 N. Arakawa, M. Otsuka, T. Kurata and C. Inagaki, J. N&r. Sci. VitaminoZ., 27 (1981) 1-7. 9 M. Otsuka, T. Kuraia, E. Suzuki, N. Arakawa and C. Inagaki, J. Nutr. Ski., Vitaminol., 27 (1981) 9-15. 10 L. W. Doner and K. B. Hicks, Anal. Biochem., 115 (1981) 225-230. I1 R. B. H. Wills, P. Wilmalasiri and H. Greenfield, J. Agric. Food Chem., 32 (1984) 836-838. 12 A. Rizzolo, E. Porni and A. Polesello, Food Chem., 14 (1984) 189199. 13 B. Kacem, M. R. Marshall, R. F. Matthews and J. F. Gregory, J. Agric. Food Chem., 34 (1986) 271-274. 14 J. H. Bradbury and U. Singh, J. Food Sci., 51 (1986) 975-987. 15 T. Seki, Y. Yamaguchi, K. Noguchi and Y. Yanagihara, J. Chromatogr., 332 (1985) 283-286. 16 L. A. Pachla and P. T. Kissinger, Anal. Chem., 48 (1976) 364367. 17 R. S. Carr and J. M. Neff, Anal. Gem., 52 (1980) 2428-2430. 18 D. Sybilska, K. Duszczyk and M. Przasnyski, J. Chromatogr., 298 (1984) 352-355. 19 C. S. Tsao and M. Young, J. Chromatogr., 330 (1985) 408411. 20 B. Luckas, Fresenius’ 2. Anal. Chem., 323 (1986) 496497. 21 E. J. Knudson and K. J. Siebert, personal communication. 22 0. Westphal and D. Jerchel, Chem. Ber., 73 (1940) 1002~1011. 23 L. C. Sander and S. A. Wise, Anal. Chem., 56 (1984) 504510. 24 R. E. Majors and M. J. Hopper, J. Chromatogr. Sci., 12 (1974) 767-778. 25 J. J. Kirkland, J. Chromatogr. Sci., 9 (1971) 206&211. 26 N. Moll, Thesis, University of Nancy I (1977). 27 J. de Clerck and H. van Cauwenberge, BUN.Assoc. EC. Brass. Lotdvain, 52, No. 1, 2, 3 (1956) 87 pp. 28 N. Moll and B. Gross, J. Chromatogr., 206 (1981) 186-192.