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www.JCE.DivCHED.org • Vol. 83 No. 2 February 2006 • Journal of Chemical Education 273

Over the past several years, we have made a concertedeffort to modernize our lower-division analytical chemistrycourse in two ways: (i) by incorporating more instrumentaltechniques and (ii) by using natural samples (that often existas complex mixtures) for analysis. Towards this end, we havetaken a cue from Barros et al. (1) and their use of a facile,aqueous reaction between α-dicarbonyl compounds in wineand beer and o-phenylenediamine. The result is a series ofquinoxaline compounds that demonstrate strong absorptionproperties and are exceedingly stable. We have used this ana-lytical system to teach the subtleties of assaying one or morecomponents of a complex mixture as well as a lesson in theadvantages or disadvantages of using instrumental methodsin the determination of analyte concentrations.

Three α-dicarbonyl compounds are commonly found inwine: glyoxal (1), methylglyoxal (2), and 2,3-butanedione (3).The last compound is thought to be responsible for the “but-tery” flavor of certain wines. Reaction of these α-dicarbonylcompounds with o-phenylenediamine yields the compoundsquinoxaline (4), 2-methylquinoxaline (5), and 2,3-dimethylquinoxaline (6) (Figure 1).

Methylglyoxal (MG), also known as pyruvaldehyde or2-oxopropanal, is present throughout the human body. Al-though harmless at low concentrations, methylglyoxal be-comes toxic when its concentration exceeds that of theSH-glutathione present, or approximately 1 mM (2).Methylglyoxal has also been identified as an important me-tabolite in the cellular and extracellular formation of advancedglycation end products, which are precursors of hyperglyce-mia and diabetes. Recent studies have shown that MG is anaturally occurring intermediate in a side-chain of the gly-colysis process. In particular, the formation of MG by thenonenzymatic or enzymatic elimination of phosphate fromdihydroxyacetone phosphate and glyceraldehyde-3-phosphateis thought to be the major source of MG in the body (3). Inspite of this study, the biosynthesis of the MG in animals is“still complicated, uncertain” (4), and very controversial. Theon-going debate stems largely from the fact that measure-ment of MG concentrations in biological tissues has not beenvery successful. Ingested MG is rapidly destroyed, which can-not be compensated by increasing the exposure to MG sincethe compound becomes toxic beyond certain levels (2).

While the quantitative analysis of MG in living tissueshas been unsuccessful, determination of this α-dicarbonylcompound in food and beverages has met with greater suc-cess (5). Particularly in wine, the measurement of MG andits derivatives has been accomplished with methods such high-performance liquid chromatography (HPLC) (1), gas chro-matography interfaced with a mass spectrometer (GC–MS)or a thermoionic detector (6). These techniques do not sepa-rate the α-dicarbonyls directly, but rather detect and separatethe quinoxaline derivatives formed by reactions with o-phe-nylenediamine. In this work, HPLC analyses were highly ef-fective in quantifying quinoxaline, methylquinoxaline, anddimethylquinoxaline derived from glyoxal, methylglyoxal, and2,3-butanedione present in authentic wine samples. Similarly,GC–MS was valuable in confirming the identities of thequinoxaline adducts. The results reported here are easily re-peatable and provide a robust set of experiments for studentsin analytical chemistry.

Experiment

The experiment is performed in groups of students, threestudents per group, four to five groups in a class, at least twodifferent wines per group. Groups of students rotate throughthe instruments and, with multiple instruments (at least twoof each), the experiment can be completed in a total of threeto four lab periods of three to four hours each. In the firstlab period, all of the derivatization reactions are performedand analysis of the authentic quinoxaline samples and winesamples may begin. The times required on each instrument

Assaying ααααα-Dicarbonyl Compounds in Wine: WA Complementary GC–MS, HPLC, and VisibleSpectrophotometric AnalysisTammy J. Dwyer* and Jeremiah D. FilloDepartment of Chemistry, University of San Diego, San Diego CA 92110; *tdwyer@sandiego.edu

Figure 1. α-Dicarbonyl compounds of interest and the correspond-ing products of their reactions with o-phenylenediamine.

N

N

N

N

N

N

4 5 6

NH2

NH2

HC

C

O

O

HH3C

CC

O

O

CH3H3C

CC

O

O

H

1 2 3

CH3

CH3

CH3

In the Laboratory

274 Journal of Chemical Education • Vol. 83 No. 2 February 2006 • www.JCE.DivCHED.org

Figure 5. HPLC traces for different wine samples following reac-tion with o-phenylenediamine: (A) Chardonnay, (B) SauvignonBlanc, (C) Cabernet Sauvignon, and (D) Merlot. The retention timesfor the reaction products (determined using authentic samples) are5.2 min (quinoxaline), 5.7 min (2-methylquinoxaline), and 6.1 min(2,3-dimethylquinoxaline).

Figure 4. Standard curves for authentic samples of the reaction prod-ucts quinoxaline, 2-methylquinoxaline, and 2,3-dimethylquinoxalinedetermined using HPLC.

2-methylquinoxaline

2,3-dimethylquinoxaline

quinoxaline

Concentration / (10�5 mol/L)

Pea

k A

rea

60000

50000

40000

30000

20000

10000

00.0 0.5 1.0 1.5 2.0

Figure 3. Gas chromatogram of a wine sample (San Andre’sChardonnay; top trace) showing the presence of methylglyoxal (fol-lowing reaction with o-phenylenediamine via the detection ofmethylquinoxaline). The single ion monitoring (SIM) mode gives riseto the “glitch” in the center of the chromatogram. The mass spec-trum of 2-methylquinoxaline (bottom trace) displays a clean frag-mentation pattern for the aromatic compound.

Figure 2. Visible spectra of several wine samples following reactionwith o-phenylenediamine. The wines shown here are Sauvignon blanc(Amber Hill) and Chardonnay (Sutter Home and Charles Shaw).

Sauvignon Blanc (AH)

Chardonnay (SH)

Chardonnay (CS)

Wavelength / nm

Abs

orba

nce

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0250 300 350 400 450 500 550 600 650 700

In the Laboratory

www.JCE.DivCHED.org • Vol. 83 No. 2 February 2006 • Journal of Chemical Education 275

(given optimum conditions) are: UV–vis, approximately 0.5hour (five wavelength scans); GC–MS, approximately 3 hours(five injections at 30 minutes each); HPLC, approximately2.0 hours (includes equilibration times and five injections at10 minutes each). At the conclusion of the experiment, priorto students submitting written lab reports, a “class dialogue”is held to discuss the complementarity of the techniques andresults.

Preparation of Quinoxaline CompoundsIn a typical reaction, 500 µL of wine are combined with

1.5 mg of o-phenylenediamine in an Eppendorf tube. Thereaction proceeds to completion at room temperature in twohours or if the mixture is warmed to 60 �C for 30 minutes.

Visible SpectrophotometryAuthentic samples of quinoxaline, 2-methylquinoxaline,

and 2,3-dimethylquinoxaline can be purchased from Aldrichand used without further purification. The λmax value for eachcompound is measured in aqueous solution and in metha-nol using a spectrophotometer (in our case a Thermo-Spectronic UNICAM UV500). In either solvent, theabsorption spectra of the various quinoxalines are similar withλmax = 315 nm. Since the results for the three compounds 4,5, and 6 (following derivatization of wine samples with o-phenylenediamine) are overlapping and indistinguishable(Figure 2), students discover that this method proves unsuit-able for quantitative analysis.

GC–MSThe retention times and masses of the standard

quinoxaline samples are measured using a GC–MS (in ourcase a Hewlett-Packard 5890A�5970). For the reaction be-tween α-dicarbonyl compounds in various wines and o-phe-nylenediamine, 1 µL of the reaction mixture is injecteddirectly onto the GC–MS. A temperature gradient was es-tablished whereby the initial column temperature was 60 �Cfollowed by heating at 2��min to 120 �C (over 30 min), then

sdnuopmoCenilaxoniuQrofstluseRSM–CG.1elbaT

dnuopmoC noitneteRnim/emiT (/ssaM m )z/

enilaxoniuQ 8.81 67,031enilaxoniuqlyhteM-2 8.32 67,711,441

enilaxoniuqlyhtemiD-3,2 3.03 67,711,851

heating at 15��min to a final temperature of 230 �C, and hold-ing there for 5 min. We used splitless mode injection andsingle ion monitoring (SIM) to enhance the sensitivity of ourinstrument. In SIM, the mass selective detector was turnedon for only a short time, corresponding to the expected re-tention times plus or minus 45 seconds. A typical result for2-methylquinoxaline derivatized from the methylglyoxal in thewine sample is shown in Figure 3 and representative reten-tion times for the quinoxaline reaction products are presentedin Table 1.

HPLCLiquid chromatography is performed using a reverse-

phase C18 column (we used a Waters 610E HPLC and aHiggins Analytical CLIPEUS 5-µm column). The eluent con-sists of 50:50 acetonitrile:sodium acetate buffer (0.040 M,pH 4) and the absorbance is monitored at 315 nm. Stocksolutions of authentic quinoxaline compounds (using the elu-ent as solvent) are used to construct standard curves span-ning the concentration range 5.0 × 10�7 M to 2.0 × 10�5 M(Figure 4). Typical chromatograms of wine samples in whichall three glyoxals are present in varying amounts and are con-verted into quinoxalines prior to analysis are shown in Fig-ure 5. Under these conditions, the retention times are 5.21± 0.01 min for quinoxaline, 5.61 ± 0.06 min for 2-methylquinoxaline, and 5.99 ± 0.20 min for 2,3-dimethylquinoxaline. The concentrations of α-dicarbonylcompounds 4, 5, and 6 determined in the various winesamples are shown in Table 2.

selpmaSeniWmorfdevireDsenilaxoniuQrofstluseRCLPH.2elbaT

yteiraV dnarB /]enilaxoniuQ[01( −6 )L/lom

/]enilaxoniuqlyhteM[01( −6 )L/lom

/]enilaxoniuqlyhtemiD[01( −6 )L/lom

nongivuaStenrebaC wahSselrahC )60.0(34.4 )50.0(43.2 )03.0(64.9nongivuaStenrebaC emoHrettuS — — )5.0(8.51nongivuaStenrebaC kcuhCkcuBowT — — )91.0(36.5

tolreM sirroM.W.J — — )4.0(6.31yannodrahC reitsuG&notraB — )50.0(91.1 )12.0(08.2yannodrahC wahSselrahC — )40.0(46.7 )02.0(35.3yannodrahC s'erdnAnaS )60.0(34.3 — )262.0(26.0yannodrahC emoHrettuS — — )91.0(61.4oigirGtoniP elloCled )80.0(54.1 )50.0(45.1 )42.0(43.1

cnalBnongivuaS lliHrebmA )60.0(13.3 )50.0(47.1 )42.0(73.1gnilseiRetihW tioneBuaetahC — — —

lednafniZetihW emoHrettuS — )500.0(757.0 )91.0(41.5

N ETO : .sesehtnerapninevigseitniatrecnuetulosbA

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276 Journal of Chemical Education • Vol. 83 No. 2 February 2006 • www.JCE.DivCHED.org

Hazards

o-Phenylenediamine is a toxic substance and cancer sus-pect agent. In case of contact with eyes or skin, rinse imme-diately with plenty of water. Acetonitrile and methanol arepoisonous substances and are harmful if inhaled or absorbedthrough skin. Compounds 4–6 are irritating to eyes, respira-tory system, and skin.

Discussion

We have used the formation of quinoxaline compoundsfrom the reaction of o-phenylenediamine with glyoxal,methylglyoxal, and 2,3-butanedione present in wine samplesto enhance our teaching of various analytical chemistrythemes. Students learn the value of (i) derivatization of ana-lytes to facilitate their detection; (ii) separation prior to quan-titation of analyte concentrations when their structures arevery similar; and (iii) the complementarity of techniques forcorroborating results.

While spectrophotometry is determined to be minimallyuseful in this experiment, the GC–MS method proved to beideal to confirm that the compounds analyzed in the winesamples were indeed the glyoxals based on the formation ofquinoxalines; however, it was less useful for quantitation ow-ing to the poor signal-to-noise of our older instrument. Wedid achieve quite acceptable separation of the quinoxalinesby GC–MS that was essential to identify each of the analytecomponents. HPLC was the superior method for quantita-tion. The GC–MS results were in agreement with the HPLCresults regarding the presence or absence of the glyoxals inthe wine samples.

There are several trends that emerge from the data pre-sented in Table 2. In general, our results suggest that red winevarieties have higher concentrations of 2,3-butanedione (com-pared to white wines) and perhaps this correlates with theirtaste. Our results also suggest that white wine varieties tendto have significant, measurable concentrations of at least twoand often all three of the α-dicarbonyl compounds glyoxal,methylglyoxal, and 2,3-butanedione while red wines do not.Finally, it appears that wines typically characterized as “dry”(red wines and Chardonnay) tend to contain higher concen-trations of α-dicarbonyl compounds than “sweet” wines suchas a Riesling.

Given that wine is such a complex mixture of substances,there is very little in the literature attributing quantities ofα-dicarbonyl compounds to elements of aroma or flavor.

Nonetheless, one can find broad ranges for levels of glyoxal,2-methylglyoxal, and 2,3-butanedione in different wine va-rieties. Glyoxal is present in a variety of beverages includingtea, beer, and wine, with concentrations ranging from 6.2 ×10�6 to 7.6 × 10�5 M in white wines and 2.7–3.8 × 10�5 M inred wines (1, 6, 7). 2-Methylglyoxal is also found in a vari-ety of beverages with 1.5 × 10�6 to 4.0 × 10�5 M present inwhite wines and 9.0 × 10�6 to 4.0 × 10�5 M in red wines (1,6, 8). Finally, 2,3-butanedione is associated with a butteryaroma and is found at levels of 1.0–5.8 × 10�5 M in whitewines and 2.3 × 10�6 to 3.3 × 10�5 M in red wines (1, 6, 9).The results of the present studies are certainly consistent withthese ranges. One possible extension of these studies, if onehad access to samples from a winery, would be to determinehow the concentrations of the α-dicarbonyl compoundschange during the stages of the wine-making and fermenta-tion process.

WSupplemental Material

Instructions for the students and notes for the instruc-tor are available in this issue of JCE Online.

Literature Cited

1. Barros, A.; Rodrigues, J. A.; Almeida P. J.; Oliva-Teles, M. T.J. Liq. Chrom. Rel. Technol. 1999, 22, 2061–2069.

2. Szent-Gyorgyi, A. Proc. Natl. Acad. Sci. USA 1977, 74, 2847.3. McLellan, A. C.; Phillips, S. A.; Thornalley, P. J. Anal. Biochem.

1992, 206, 17.4. Ohmori, S.; Mori, M.; Shiraha, K.; Kawase, M. In Enzymol-

ogy and Molecular Biology of Carbonyl Metabolism 2; Weiner,H., Flynn, T. G., Eds.; A. R. Liss: New York, 1989; p 398.

5. Hayashi, T.; Shibamoto, T. J. Agric. Food Chem. 1985, 33,1090–1093.

6. De Revel, G.; Pripis-Nicolau, L.; Barbe, J. C.; Bertrand, A. J.Sci. Food Agric. 2000, 80, 102–108.

7. Kielhorn, J.; Pohlenz-Michel, C.; Schmidt, S.; Mangelsdorf,I. Concise International Chemical Assessment Document 57,Glyoxal; Inter-Organization Programme for the Sound Man-agement of Chemicals (World Health Organization): Geneva,Switzerland, 2004.

8. Hayashi, T.; Shibamoto, T. J. Agric. Food. Chem. 1985, 33,1090–1093.

9. Martineau, B.; Acree, T.; Henick-Kling, T. Food Res. Intern.1995, 28, 139–143.