International Journal of Life Sciences 9 (5) : 2015; 41 - 44 Founded 2007 An Independent, Open Access, Peer Reviewed, Non-Profit Journal International Journal of InternationalInternational JournalJournal ofof LifeLife SciencesSciences ISSN 2091-0525 website: http://nepjol.info/index.php/IJLS/index ifeSciences LCopyright © International Journal of Life Sciences

Research Article

Determination of Organic Acids in Fruit juices by UPLC

Fereshteh Khosravi1*, Nahid Rastakhiz1, Behzad Iranmanesh2, Seyyed Sina Seyyed Jafari Olia3

1Department of Chemistry, Kerman Branch, Islamic Azad University, Kerman, Iran. 2Department of medicine, shahid beheshti University,Tehran, Iran. 3Department of Civil Engineering, Islamic Azad University, Kerman Branch.

Article Information ABSTRACT Key words: A reversed phase UPLC method for separation and quantification of organic acids (oxalic, citric, Organic acids; tartaric, malic and ascorbic and lactic acids) in fruit juices was developed. The chromatographic UPLC; Fruit separation was performed with a Surveyor Thermo Electron system at 10°C by using a potassium dihydrogen orthophosphate buffer (pH3.1) as mobile phase, an Hypersil Gold a Q Analytical Column and diode array detection at λ=254 nm for ascorbic acid and λ=214 nm for the other organic acids. Organic acid profiles of seven species of fruits: sweet orange, white and red apple, lemon, lime, white and pink grape fruit were established. Species significantly affect the organic acid distribution of fruit juices. In all juices, the most abundant organic acid was citric acid, ranging from 5.22to 62.42g/l. Fruit juices are good sources of ascorbic acid (0.137-0.625g/l). The average ascorbic acid was the highest in lemon juice followed by sweet orange juice, sweetie and white grapefruit.

INTRODUCTION organic acids. The organic acid composition of fruits is also of interest due to its impact on the sensory properties. Citrus fruits, one of the important fruit crop Organic acids are naturally found in vegetables and fruits groups, are consumed mostly as fresh or as juice because and may be formed during processes like fermentation or of their nutritional value and special flavor. Consumption may be added into food during the manufacturing of citrus juice is found to be beneficial in preventing process. Organic acids can be separated from food by coronary diseases and chronic asthma (Abd-Ghafar et al., water or solvent extraction, vapour distillation, or 2010). Citrus fruit extracts are also found to have precipitation with lead or other elements (Joslyn 1970). antioxidant, anti-inflammatory, anti-tumor, anti-fungal Today, the most common way used for the extraction of and blood clot inhibition activities (Abeysinghe et al., organic acids present in fruits and vegetables is the 2007). These health benefits of citrus fruit have mainly solvent extraction. The maturation stage and been attributed to the presence of bioactive compounds, geographical origin affect the amounts and types of such as ferrulic acid, hydrocinnamic acid, cyanidin organic acids found in different kinds of fruits (Cámara et glucoside, hisperidine, vitamin C, carotenoid and al. 1994). The identification and quantitative analysis of naringin content (Abeysinghe et al., 2007; Xu et al., 2008). major organic acids in fruits is considered very important for food and beverage technology and quality evaluation Citrus fruits are classified as acid fruits, since their (Hasib et al., 2002). Organic acids are a useful index of soluble solids are composed mainly of organic acids and authenticity in fruit products, since they have lower sugars, which are used as the main index of maturity and susceptibility to change during processing and storage one of the major analytical measures of flavor quality. The than other components of fruits (Camara et al., 1994). main acids of citrus fruits are citric and malic acids with Accurate knowledge of organic acid levels (and ratios) trace amounts of tartaric, benzoic, oxalic and succinic might be useful for determining the percentage juice and acids reported (Karadeniz, 2004). also for detecting misbranding and/or adulteration in fruit juices, since each fruit has a unique pattern of Organic acid accumulation in the vacuole of cells of citrus

*Corresponding author Email address: [email protected]

Copyright reserved © International Journal of Life Sciences DOI:dx.doi.org/10.3126/ijls.v9i5.12690 fruits is a developmentally regulated process, the degree determinations were made in isocratic conditions, at and timing of which varies greatly among species and ambient tempareture, using a mobile phase made of 0.2 varieties and is highly susceptible to agroclimate (Canel % acetonitrile and 50 mM phosphate solution (dissolve et al., 1995). 6.8 g potassium dihydrogen phosphate in 900 ml water; the pH value should be adjusted to pH =2.8 with phosphoric acid and then filled to 1000 ml with water) MATERIALS AND METHODS filtered through a polyamide membrane (0.2 μm) and degassed in a vacuum. The flow rate of the mobile phase Fresh fruits of sweet orange, lemon, lime and grapefruit, was 1.2 ml/min for all the chromatographic separations. white and red apple varieties of commercial ripened The separation column was balanced with mobile phase stages were purchased from the local markets. Healthy until the baseline was stabilized. Sample injections were fruits were selected randomly for uniformity of shape made at this point. The volume injected was 5 μl for either and color. prepared sample or standard solution.

Preparation of juice sample Chemicals The citrus fruit juice was extracted by cutting the fruit in Citric acid, malic acid, tartaric acid, oxalic acid, ascorbic half and careful hand-squeezing to obtain the juice. The acid and lactic acid were purchased from Merck and juice was passed through a strainer to remove pulp and Sigma-Aldrich. Acetonitrile, potassium dihydrogen seeds. The freshly squeezed juice was centrifuged at orthophosphate and phosphoric acid were of analytical 3000 rpm for 10 min and twice, the supernatant was purity or for chromatographic use. The water used was diluted 1:5. The dilutions were membrane filtered (0.20 ultrapure, Basic TWF. μm) before injection. Two samples were analyzed in duplicate. RESULTS AND DISCUSSION Preparation of juice sample The citrus fruit juice was extracted by cutting the fruit in half and careful hand- In Fig. 1 the chromatogram of ascorbic acid in the squeezing to obtain the juice. The juice was passed standard solution and real samples was given. The through a strainer to remove pulp and seeds. The freshly linearity of the method was evaluated according to area squeezed juice was centrifuged at 3000 g for 10 min and response. Selected wavelength for ascorbic acid, the supernatant was diluted 1:50 for citric acid retention time, concentration ranges of linear response, determination and 1:5 for the other acids. The dilutions correlation coefficients and detection of limit were were membrane filtered (0.45 µm) before injection. Two summarised in Tab. 1. samples were analyzed in duplicate. The detection limit (LOD) could be defined as the Organic acid standards smallest peak detected with a signal height three times A mixed standard stock solution was prepared that of the baseline, while the limit of quantification containing 1000 mg/l citric acid, 2000 mg/l malic acid, (LOQ) referred to the lowest level of analyte which could 300 mg/l oxalic and ascorbic acid respectively, 700 mg/l be determined with an acceptable degree of confidence. tartaric acid and 400 mg/l lactic acid. The stock solution and the corresponding dilutions was made in ultrapure In the present work, detection limits were estimated water and stored in dark places between the according to the hypothesis that a peak, to be detected, experiments, at low temperature (+4°C). should have a signal-to-noise ratio >3. The reproducibility of test peak area and retention time was Analysis by UPLC tested with the help of Chrom Quest software, which The organic acids in the samples test solution were calculated the relative standard deviations (RSD) for the separated by reversed phase chromatography on a 150 retention time of the analytes for all levels of the mm×4.6 mm i.d., 5 μm particle ZORBAX Eclipse XDB-C18 calibration graphs and for a peak area at each calibration analytical column, of which was detected by absorbance level. Precision of areas was <2% RSD while precision of and quantified with external calibration graph. For the retention times was <0.5% RSD. simultaneous detection of the seven analytes, the detector was set at λ=254 nm for ascorbic acid and λ=214 The relative standard deviations (RSD) for the retention nm for the other organic acids. This setting was chosen time were between 0.047% for oxalic acid and 0.209% since ascorbic acid has its maximum optical absorbance for citric acid therefore, in standard solutions, the close to 254 nm. developed HPLC method provided stable retention times. The RSD values for peak areas were between 0.028% and The UPLC analysis was performed with a Agilent 1200 1.530% indicating the stability of the method in terms of series system. Integration, data storage and processing peak area. The detection limit (LOD) could be defined as were performed by Chemstation software. The the smallest peak detected with a signal height three

2 times that of the baseline, while the limit of quantification Fig. 1. Chromatograms of the a) ascorbic acid standard (LOQ) referred to the lowest level of analyte which could solution 30mg/l b) grape fruit c) orange d) lemon be determined with an acceptable degree of confidence.

Precision was tested on ten replicated analyses of independent preparations of sweet orange juice. The RSD value was % indicating that the method was precise with a high degree of repeatability. The recovery of organic acids from citrus fruits ranged from 97.2 to 103.6% . The amount organic acids found in citrus juices were shown in Tab. 2.

Tab. 1. Retention times, concentration ranges of lineal response, equations of the calibration graphs and correlation coefficients for standard org anic acids λ,nm RT(min) Concentration Correlation Det ection range (mg/l) coefficient limit(mg/l) (r2) Oxalic acid 254 2.01 0.2-300 0.9998 0.1 Tartaric acid 254 2.41 1-700 0.9993 0.5

Malic acid 254 3.09 10-1000 0.9992 10 Lactic acid 254 3.45 25-500 0.9992 20 Ascorbic acid 254 3.2 0.5-200 0.999 0.5 Citric acid 254 6.25 16-1000 0.9998 8

Malic acid 254 3.09 10-1000 0.9992 10 Lactic acid 254 3.45 25-500 0.9992 20 Ascorbic acid 214 3.2 0.5-200 0.999 0.5 Citric acid 254 6.25 16-1000 0.9998 8

Tab. 2. Organic acids content(gr/l) of fruit juices

Fruit Ox alic Tartaric Malic Lactic Citric Ascorbic aci d acid acid acid acid acid Sweet orange 0.119 0.409 1.156 1.789 12.989 0.526

Red apple 0.268 0.237 0.871 1.229 12.998 0.009 White apple 0.125 0.113 0.397 0.632 7.102 0.004 Lime 0.131 0.011 6.122 0.612 62.422 0.442 Lemon 0.084 0.086 0.989 1.322 85.256 0.911

Pink grapefruit 0.147 0.106 2.819 0.696 20.723 0.622 White grapefruit 0.117 0.156 1.802 0.725 23.022 0.741 The amounts of each organic acid found in fruit juices were shown in Tab. 2. It was clear that the impact of species was significant on organic acid distribution of Precision was tested on six replicated analyses of fruit juices. As indicated by previous researchers (Cunha independent preparations of sweet orange juice. The RSD et al., 2002; Karadeniz, 2004), citric acid is the major values ranged from 0.154 to 3.18% indicating that the organic acid found in fruit juices (5.22-62.42g/l), method was precise with a high degree of repeatability, followed by malic acid and lactic acid. In general, oxalic, especially for citric, ascorbic, lactic and oxalic acids (RSD tartaric and ascorbic acids were present in minor <2%). The recovery of organic acids from citrus juices quantities in citrus juices. For sweet orange, lemon, white ranged from 95.8 and 102.1% confirming the accuracy of grapefruit and red grape fruit, white apple and red apple the separation and analysis conditions. juices, lactic was found to be in higher concentrations than malic acid. The malic acid content of lime (6.122g/l) The developed method was applied to establish the was significantly higher than that of the other fruit juices organic acid profiles of ten commercially-available (0.387 -2.819 g/l) while the tartaric acid content was the species of citrus fruits and the samples were eluted from lowest (0.011 g/l). Higher amounts of tartaric acid were the system within 20 minutes. found in sweet orange (0.409 g/l).

3 CONCLUSION REFERENCES

Abeysinghe DC, Li X, Sun CD, Zhang WS, Zhou CH and Chen KS (2007). Bioactive This work is a contribution to the development of a rapid compounds and antioxidant capacities in different edible tissues of citrus fruit of four species. Food Chem. 104:1338-1344. and precise HPLC procedure for quantitative Abd-Ghafar MF, Prasad KN, Weng KK and Ismail A. (2010). Flavonoid, hesperidine, total determination of organic acids in fruit juices under phenolic contents and antioxidant activities from Citrus species. Afr. J. Biotechnol. 9(3):326-330. reverse phase conditions. Oxalic, tartaric, malic, lactic, Aktas AH, Şen S, Yılmazer M and Cubuk E (2005). Determination of carboxylic acids in citric and ascorbic acids have been determined apple juice by RP HPLC, Iran. J. Chem. Chem. Eng. 24(1):1-6. Buchberger WW. (2000). Detection techniques in ion analysis: what are our choices?. J. simultaneously and eluted from the column within 20 Chromatogr. A 884(1-2):3-22. Camara MM, Diez C, Torija ME and Cano MP. (1994). HPLC determination of organic minutes. Considering the easiness and conciseness of acids in pineapple juices and nectars. Eur. Food Res. Technol. 198:52-56. sample preparation, the proposed analytical procedure Canel C, Bailey-Serres JN and Roose ML (1995). Pummelo fruit transcript homologous to ripening-induced genes. Plant Physiol. 108:1323-1324. could be considered as an efficient, accurate and rapid Casella IG and Gatta M. (2001). Determination of electroactive organic acids by anion- method of organic acid determination. exchange chromatography using a copper modified electrode. J. Chromatogr. A 912(2):223-233. Cunha SC, Fernandes JO and. Ferriera IMLVO (2002). HPLC/UV determination of organic The method could successfully used to quantify organic acids in fr. Hasib A, Jaouad A, Mahrouz M and Khouili M (2002). HPLC determination of organic acids in natural and commercial fruit juices. Citric acid acids in Moroccan apricot. Cienc. Tecnol. Aliment. 3:207-211. Karadeniz F. (2004). Main organic acid distribution of authentic citrus juices in Turkey. was the main acid in all fruit juices followed either by Turk. J. Agric. For. 28:267-271. malic or lactic acid varying within fruit species. Linget C, Netter C, Heems D and Vérette E (1998). Online dialysis with HPLC for the automated preparation and analysis of amino acids, sugars and organic acids in grape juice and wines. Analusis 26(1):35-39. It was observed that the organic acids present in citrus Xu G, Liu D, Chen J, Ye X, Ma Yand Shi J (2008). Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chem. 106:545-551. juices were species, cultivar and horticultural practice dependent and could be considered as an active © International Journal of Life Sciences NOTE: Authors/contributors are responsible for originality, contents, correct parameter for authenticity determination. references, and ethical issues.

Submit your next manuscript to IJLS with a - 1. Convenient online submission, 2. Rapid editorial review followed by peer review, 3. Immediate publication on acceptance. 4