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APPLICATION NOTE

Liquid Chromatography

Author: Wilhad M. Reuter PerkinElmer, Inc. Shelton, CT

Analysis of Organic in Fruit Juices by Introduction In food, organic acids, such as malic, HPLC and UV Detection ascorbic and citric acids, originate from natural biochemical processes or are added as , acidulants and/ or stabilizers. Organic acids contribute to the sensory properties of foods and beverages by providing both and aroma. In particular, citric is widely used in soft drinks to provide the sour taste. The monitoring of these organic acids is essential for quality control during the processing of juices and related products, as well as for evaluating juice authenticity and purity. In addition, the use of organic acids in foods and beverages is regulated in many countries, though regulations vary widely.1 This application note presents a simple and robust HPLC method for the analysis of organic acids typically found in store-bought fruit juices. The method conditions and performance data, including repeatability and linearity, are provided. Experimental Table 1. HPLC Method Parameters. Hardware/Software HPLC Conditions PerkinElmer Brownlee Validated Aqueous C18, 5 µm, A PerkinElmer Altus™ HPLC system was used, including the A-10 Column: 4.6 x 250-mm (Part # N9303549) Solvent/Sample Manager, A-10 column heater and A-10 UV Mobile Phase: Isocratic; 25-mM K-phosphate buffer; pH 2.4 detector (PerkinElmer, Shelton, CT, USA). A PerkinElmer Analysis Time: 8.0 min.; wash/equilibration time = 6.0 min ™ Brownlee Validated Aqueous C18 5 µm, 4.6 x 250-mm column Flow Rate: 1.5 mL/min. (~3000 psi; 200 bar) was used for all analyses (PerkinElmer, Shelton, CT, USA). All Oven Temp.: 30 ºC instrument control, analysis, and data processing was performed UV Detection: Wavelength: 210 nm via Waters® Empower® 3 Chromatography Data Software (CDS). Injection Volume: 20 µL Sampling (Data) Rate: 5 pts./sec Method Parameters The HPLC method parameters are shown in Table 1. For calibration purposes, five levels were prepared via serial dilution of the stock standard, using water as diluent. Solvents, Standards and Samples The water, used for both solvent and diluent, was HPLC-grade. Commercial fruit juices, including grape juice, juice, For buffering the mobile phase and adjusting the pH to 2.4, both apple juice and , were obtained from a local monobasic potassium phosphate and phosphoric acid were used, store. These were diluted 9:1 with HPLC-grade water, filtered obtained from Sigma Aldrich®, Inc (Allentown, PA). and then injected. An standard kit (part # 47264) was obtained from The buffered mobile phase and all standards and samples were Sigma Aldrich®, Inc. Of the included organic acids, the following first filtered through 0.45 µm filters before injection. were used for this application: oxalic acid, , quinic acid, , ascorbic acid, shikimic acid, citric acid, and Results and Discussion . Additionally, acetic acid was added as a standard, Figure 1 shows the chromatogram of a standard mixture of the which was also obtained from Sigma Aldrich®, Inc. 10 organic acids, all separated in under eight minutes. A stock standard was prepared containing the 10 organic acids As shown in Figure 2, the chromatographic repeatability of indicated above. Individual concentrations varied depending on 10 replicates of the standard mix demonstrates exceptional the individual organic acid’s UV absorptivity and expected highest reproducibility. The average retention time (RT) %RSD for all concentration in fruit juices. The concentration, in ppm, of each 10 analytes was 0.08 %. organic acid in the stock standard solution is presented in Table 2.

Table 2. Concentrations (ppm) of organic acids in the stock standard solution. Oxalic Tartaric Quinic Malic Ascorbic Shikimic Acetic Citric Succinic Fumaric 141 543 1002 640 160 21 1005 962 608 81

0.12 Fumaric

0.10

0.08 Oxalic AU 0.06

0.04 Tartaric Ascorbic

0.02 Shikimic Acetic Malic Quinic Citric Succinic

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Minutes

Figure 1. Chromatogram of organic acid standard mix; UV at 210 nm.

2 absorbicabsorbic malicmalic

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

6 Minutes1.0x1.0x10 10 6

500000 5 Figure 2. Overlay500000 of 10 replicates of organic acid standard mix. 8.0x8.0x10 10 5

6.0x10 5 400000400000 6.0x10 5 ea ea

Ar 4.0x10 5

Ar 5 Figure300000 3300000 presents examples of the linearity plots for four of the organic 4.0xacids.10 Theseabsorbic four, as well as the other six organic acids, all 2

ea 5 resultedea in R values > 0.999. 2.0x10 5 Ar 200000 malic 2.0x10 Ar 200000 absorbic

0.0 0.0 100000100000 malic

1.0x10 65 -2.0-2x1.00x10 5 0 0 0.000.00 20.0200 .00 40.0400 .00 60.0600 .00 80.0800 .00 100.00100.00 120.00120.00 140.00140.00 160.00160.00 Conc. (ppm) 500000 8.0x10 5 Conc. (ppm) 1.0x10 6 -10000-100000 0 0.000.00Malic 100.00Acid100.00 200.00200.00 300.00300.00 400.00400.00 500.00500.00 600.00600.00 Ascorbic Acid 5 400000 ConcCo. (pncpm. (p)pm) 6.0x10 500000 8.0x10 5 ea

Ar 5 300000 4.0x10 400000 6.0x10 5 ea 2.0xea 10 5 Ar

200000 Ar 5 300000 4.0x10

0.0 100000ea 2.0x10 5

Ar 200000

-2.0x10 5 0 0.0 100000 shikimic 2 0.00 20.00 40shikimic.00 60.00 80.00 100.00 120.00 140.002 160.00 shikimic R = 0.99986 shikimic Conc. (ppm) R = 0.99991 -100000 -2.0x10 5 0 0.00 100.00 200.00 300.00 400.00 500.00 600.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 Conc. (ppm) Conc. (ppm) -100000 0.00 100.00 200.00 300.00 400.00 500.00 600.00 Conc. (ppm)

6 1.0x1.0x10 10 6

800000800000 Shikimic Acid 5 Citric Acid 8.0x8.0x10 10 5

600000600000 5 shikimic 6.0x6.0x10 10 5 shikimic

400000 ea ea 400000 ea 5 ea Ar 4.0x10 5 Ar Ar 4.0x10 Ar shikimic shikimic 200000 5 200000 2.0x2.0x10 10 5

0 0.0 0.0 0 R2 = 0.99989 R2 = 0.99987 1.0x10 6 5 -2.0-2x1.00x10 5 800000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.000.00 200.00200.00 400.00400.00 600.00600.00 800.00800.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 5 Conc. (ppm) 8.0x10 ConcCo. (pncpm. (p)pm) Conc. (ppm) 1.0x10 6

600000800000 6.0x10 5 8.0x10 5

400000600000 ea ea 5 Figure 3. Linearity plots for malic, ascorbic, shikimic and citric acids; UV at 210 nm. Ar 4.0x10 5 Ar 6.0x10

200000400000 ea 5 ea 2.0x10 5

Ar 4.0x10 Ar

0.0 2000000 2.0x10 5

-2.0x10 5 0 0.0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.00 200.00 400.00 600.00 800.00 Conc. (ppm) Conc. (ppm) -2.0x10 5

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.00 200.00 400.00 600.00 800.00 Conc. (ppm) Conc. (ppm)

3 Four fruit juices were subsequently analyzed, with the resulting chromatograms shown in Figures 4 and 5. As an example of the chromatographic overlap of sample and standard, Figure 4b shows the grape juice sample overlaid upon the stock standard mix, both normalized to the highest peak.

0.080 Grape Juice

0.070 a

0.060 Malic 0.050 Tartaric

0.040 AU Oxalic

0.030

0.020 Succinic Fumaric

0.010 Shikimic Citric

0.000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

b Malic Fumaric Oxalic Tartaric Shikimic Succinic Citric

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

Figure 4. Chromatogram of grape juice sample (a) and same sample overlaid with standard mix (b); UV at 210 nm.

4 0.100 0.090 Citric

0.080 Oxalic

0.070

0.060

0.050 AU Ascorbic

0.040

0.030 Malic

0.020

0.010 Shikimic Fumaric

0.000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

0.110

0.100 Apple Juice

0.090 Ascorbic

0.080

0.070

0.060 Malic

AU 0.050

0.040

0.030

0.020

0.010 Shikimic Fumaric Citric 0.000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

Cranberry Juice 0.050

0.040 Shikimic Citric Malic

0.030 Quinic AU

0.020

0.010 Ascorbic

0.000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

Figure 5. Chromatograms of orange, apple and cranberry juice samples, with identified organic acids; UV at 210 nm.

5 The individual organic acid content for each of the four juices Conclusion is presented in Table 3. Comparing the four juices, they varied This work demonstrated the effective chromatographic considerably from one another in the proportion of the organic acids separation and quantitation of 10 commonly found organic they contained. Overall, both citric and malic acids predominated in acids in fruit juices using a PerkinElmer Altus HPLC system all four juices, with small amounts of shikimic acid in each. with A-10 UV detector. The results exhibited exceptional Interestingly, grape juice was the only juice that showed repeatability and linearity over the tested concentration ranges. significant amounts of tartaric and succinic acids, while none of the juices showed any detectable level of acetic acid. Oxalic acid Four different fruit juices were effectively analyzed, with each was detected at a significant level in the orange juice, but it showing various amounts and combinations of the 10 organic was not quantifiable or detected in the other three juices due acids. The chromatographic separation was achieved in under to significant matrix interference in the early-eluting region. eight minutes and, in general, quantitation of the organic acids in all four juices was easily achieved.

Table 3. Resulting averaged organic acids content for four analyzed fruit juices. All References samples were run in triplicate. Grape Juice Orange Juice Apple Juice Cranberry 1. Production and Packaging of Non-Carbonated Fruit Juices Organic Acid (ppm) (ppm) (ppm) Juice (ppm) and Fruit Beverages (2nd edition), Philip R. Ashurst (editor), Oxalic NQ 15.2 ND ND Springer Science and Business Media LLC, 1999. Tartaric 135.0 NQ ND ND Quinic ND NQ ND 311.7 Malic 327.4 177.5 365.1 190.2 Ascorbic ND 39.4 92.1 9.6 Shikimic 1.8 1.6 1.4 6.8 Acetic ND ND ND ND Citric 34.9 761.5 27.2 303.8 Succinic 178.2 ND ND ND Fumaric 2.8 2.4 2.6 ND ND = Not detected NQ = Not quantifiable

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