Development and Application of HPLC/Post-Column Method for Analysis of Low Molecular Weight Organic Acids in Beers, Wines and Fruit Juices

A post-column reaction method, which involves use of the pH indicator BTB, was developed and applied to the analysis of low molecular weight organic acids in commercial beers, wines, and fruit juices. Organic acids identified in these beverages were formic, acetic, citric, pyruvic, tartaric, malic, succinic, lactic, and pyroglutamic. Amounts of total acids in samples ranged from 980.4 mg/L to 513.5 mg/L in beers, from 7,502.3 mg/L to 5,573.3 mg/L in wines, and from 11,162.8 mg/L (orange) to 2,995.8 mg/L (mango) in fruit juices. Citric acid was found in the greatest amount-ranging from 286.7 ± 4.5 mg mg/L to 139.0 ± 5.3 mg mg/L in beers. The greatest level of malic acid was in a wine with 4,248.4 mg/L. The method developed is applicable to determine amounts of organic acids, formic and acetic acids, in beverages quickly and accurately.


Introduction
Low molecular weight organic acids (LMWOAs), including formic, acetic, malic, citric, tartaric, lactic, succinic, and oxalic acid, are found in various beverages, such as beers, wines and fruit juices (citrus, apple, grape, and melon), as well as in some foods [1,2]. These acids contribute characteristic tastes to beverages.
For example, formic acid possesses pungent odor and sour taste in proper dilution. It is particularly adaptable to the pineapple flavor.
Acetic acid also has a pungent and stinging sour odor, but it gives a clean-sour and acid taste at dilute concentrations (lower than 1%) in water and been used in flavor compositions, such as butter, chocolate, grape strawberry and wine. Malic acid is present in plum, peach, apricot and related fruits and used for imitation fruit flavors, such as maple [3]. Citric acid is the major acid component of citrus species and possesses a clean acid taste in an aqueous solution [3,4]. In addition to the role of organic acids in taste and flavor of beverages, they also play an important role in quality of beverages, including beers [5], wines [6] and fruit juices [7]. A capillary electrophoresis (CE) with spectrophotometric method achieved successfully analysis of a limited number of LMWOAs [8].
This method has been widely used for LMWOAs analysis in foods and beverages [9]. However, the resolution and detection of LMWOAs formic and acetic, achieved better by chromatographic methods than by the spectrophotometric method [10]. In the case of the most commonly used gas chromatography (GC), a tedious derivatization of organic acids is required to prepare samples for analysis because they are highly soluble in water [11]. On the other hand, HPLC, which can take aqueous samples directly, has been widely used to analyze LMWOAs in water samples; such as wine, 122 beverage meads [15]. A HPLC interfaced to enzyme reactor analyzed oxalic acid in fruit and vegetable juices [16]. After the electronspray ionization system was advanced to interface between HPLC and mass spectrometers (LC/MS), LC/MS application for the analysis of water-soluble chemicals, including LMWOAs, developed significantly [17]. However, separation of LMWOAs by HPLC, formic acid and acetic acid, remains relatively difficult.
In the present study, a previously reported simple and accurate post-column reaction method for LMWOAs [18] was improved and successfully applied to analyze LMWOAs, including formic and acetic acids, in beers, wines, and fruit juices.

Beverage Samples
All commercial beverages-10 kinds of beers, 6 types of wines (5 red and 1 white), and 25 kinds of fruit juices-were bought from a local market in Seoul, Korea.

Preparation of Beer and Wine Samples
Beer and wine samples (2 mL each) were filtered with an Arcadis Syringe Filter with 0.45 mm PVDF membrane (Waters Co, Milford, MA).

Preparation of Fruit Juice Samples
Well-mixed juice samples were centrifuged at 10,000 rpm for 5min at 4 ºC to collect the supernatant. The supernatant was filtered with an Arcadis Syringe Filter with 0.45mm PVDF membrane (Waters Co, Milford, MA) and were analyzed with HPLC.

Results and Discussion
As mentioned above, analysis of LMWOAs is a difficult process.
There have been advances in HPLC/MS methods recently but some problems, such as insufficient resolution of formic acid and acetic acid, remain. In order to resolve these problems, we developed a post-column reaction method, which involved use of pH indicator BTB. This method and related theory were originally advanced nearly three decades ago [18]. 123 of detection (LOD) of acids were 25.0mg/L for citric, malic and quinin, lactic; 12.5mg/L for formic and acetic; and 50mg/L for pyroglutamic. The limits of quantitation (LOQ) of each acid were 75.0mg/L for citric, malic, quinin, and lactic; 30.0 mg/L for formic; 50.0 mg/L for acetic; and 150 mg/L for pyroglutamic.

Figure 2
Organic Acids Found in Beer Samples  Table 1 shows the results of LMWOAs analysis in beer samples.
There are three monoprotic acids (lactic, acetic and pyroglutamic),    Table 2 shows the results of LMWOAs analysis in wine samples.

Organic Acids Found in Wine Samples
The total amounts of acids were similar in all wine samples, ranging components [9]. Lactic acid and tartaric acid levels ranged from 3,784mg/L to 452mg/L and 1978mg/L 866mg/L. These results are consistent to the ones from the present study. As mentioned above, the quality of wines depends on, in part, acidity associated with the composition of acids [22]. The results in the present study demonstrate that the composition of acids in different brands of wines varies. Therefore, the analytical method developed can be useful in evaluating wine quality as well as a guide to improvement in the winemaking process.  One previous report demonstrated satisfactory analysis of organic acids in fruit juices (apple, peach, pear and apricot) using HPLC with an ion-exclusion column [23]. Another previous study reported the amounts of citric, malic, quinin and tartaric acids in apple, orange, cranberry, white/red grapes, and pomegrante juices analyzed using LC/MS/MS [24]. Effect of malic acid for the inactivation of common food pathogens on fresh-cut lettuce was reported, suggesting that malic acid possesses some biological activity [25]. The presence of formic and acetic acids was, however, not reported in these studies. On the other hand, the present study

Conclusion
The present study demonstrates that the method developed was useful to determine LMWOAs, formic acid and acetic acid, present in samples with complex matrices of beverages. Acids composition are important to evaluate quality of beverages. Therefore, the method developed in the present study would useful to analyze LMWOAs levels for evaluating quality of beverages. The CE method is also well-established method for LMWOAs analysis. Therefore, it is recommended to use both methods, the present method for formic acid and acetic acid.