Food Chemistry 175 (2015) 100–105

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Food Chemistry

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Analytical Methods

Comparison of UPLC and HPLC methods for determination of vitamin C

http://dx.doi.org/10.1016/j.foodchem.2014.11.1040308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +48 61 8569320; fax: +48 61 8543993.E-mail address: a.gliszczynska-swiglo@ue.poznan.pl (A. Gliszczynska-Swigło).

Inga Klimczak, Anna Gliszczynska-Swigło ⇑The Poznan University of Economics, Faculty of Commodity Science, al. Niepodległosci 10, 61-875 Poznan, Poland

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 March 2013Received in revised form 25 October 2014Accepted 18 November 2014Available online 26 November 2014

Keywords:HPLCUPLCVitamin CFood analysisBeveragesPharmaceutical preparations

Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC)methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehy-droascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparationswere compared. Both methods are rapid: total time of analysis was 15 and 6 min for HPLC and UPLCmethods, respectively. The methods were validated in terms of linearity, instrument precision, limitsof detection (LOD) and quantification (LOQ), accuracy and recovery. Intra- and inter-day instrument pre-cisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% forUPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively(HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049 lg/mL forHPLC and 0.024 lg/mL for UPLC while LOQs were 0.149 and 0.073 lg/mL for HPLC and UPLC, respectively.These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in phar-maceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and con-sumes less eluent.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

There is an increasing need for fast and ultra-fast separationmethods with good efficiency and resolution. Ultra performanceliquid chromatography (UPLC) has become a widely used tech-nique, which takes full advantages of chromatographic principlesto perform separation, using short columns packed with smallerparticles (sub-2 lm). This has led to a shorter analysis time,improved peak efficiency (peak width), better resolution anddecreased use of solvents compared with conventional high-perfor-mance liquid chromatography (HPLC) (de Villiers et al., 2006;Guillarme, Nguyen, Rudaz, & Veuthey, 2007). Moreover, the UPLCsystem enables the detection of analytes at very low concentrationsbecause of the improved signal-to-noise ratio (Guillarme et al.,2007). The injection volume in UPLC can be significantly reducedwithout loss of sensitivity (Guillarme, Nguyen, Rudaz, & Veuthey,2008; Spacil, Nováková, & Solich, 2008). However, the use of shortcolumns (50–100 mm), packed with sub-2 lm particles in conven-tional liquid chromatography (LC), are limited by increased columnback-pressure (>40 MPa) which is not compatible with conven-tional instrumentation (de Villiers et al., 2006). The necessity ofusing instrumentation dedicated to UPLC is a limitation of this tech-nique (Guillarme et al., 2007).

Vitamin C is the most important water-soluble antioxidant.Both, ascorbic acid (AA) and its oxidation product, dehydroascorbicacid (DHAA), have vitamin C activity. Because humans cannot syn-thesise AA, its main sources in the diet are fruits, vegetables andtheir products as well as dietary supplements containing AA. Thereare significant variations in vitamin C content in food. For example,in fruits it may range from 2–10 mg/100 g in plums and apples upto 1000–1300 mg/100 g in rosehip and acerola, whereas in fruitjuices, it may range from 0–30 mg/L in apple juice to 280–860 mg/L in orange juice (Belitz, Grosch, & Schieberle, 2009;Davey et al., 2000). Supplements containing vitamins or dietaryminerals are included as a category of food in the Codex Alimentar-ius. Currently, AA is the most widely used vitamin/antioxidant sup-plement worldwide. It is available as a single compound or inmulti-compound preparations. It is also used in cosmetic and foodindustry as an antioxidant. Vitamin C in food can be lost, particu-larly during thermal processing or storage. Its loss (10–60%) isdue to both oxidation and leaching (Combs, 2008). Thus, additionof AA to fortify foods or restore vitamin C losses during processingor storage is common.

Several analytical methods have been proposed for the determi-nation of vitamin C, including titrimetric (Suntornsuk, Gritsanapun,Nilkamhank, & Paochom, 2002), enzymatic (Shekhovtsova,Muginova, Luchinina, & Galimova, 2006), chemiluminometric(Pires, Marconi, Meneses, & Zagatto, 2006), spectrophotometric(Llamas, Di Nezio, & Fernández Band, 2011), fluorometric (AOAC,

I. Klimczak, A. Gliszczynska-Swigło / Food Chemistry 175 (2015) 100–105 101

2007), and amperometric (Wawrzyniak, Ryniecki, & Zembrzuski,2005) but separation techniques such as capillary electrophoresis(Dong et al., 2007), gas chromatography (Silva, 2005) and liquidchromatography (LC) (Nováková, Solich, & Solichová, 2008;Spínola, Mendes, Camara, & Castilho, 2012; Tarrago-Trani,Phillips, & Cotty, 2012), are regarded as more accurate. Amongchromatography techniques, HPLC methods are much more fre-quently used than gas chromatography.

Many chromatographic methods refer to the determination ofAA. Some of them are dedicated to the determination of AA andTAA content as the sum of AA and DHAA after reduction of DHAAto AA (Kału _zewicz et al., 2012; Spínola et al., 2012; Tarrago-Traniet al., 2012). This is essential for fruits and vegetables becausemany harvesting and post-harvest handling procedures promoteoxidation or degradation of AA (Hernandez, Lobo, & González,2006; Kału _zewicz et al., 2012; Lee & Kader, 2000). In many plantcrops, DHAA may represent up to 10% of total vitamin C and ittends to increase during storage (Lee & Kader, 2000). However, inthe case of some fruit juices (e.g. stored short-term orange juices),losses due to oxidation can be insignificant (Fernández-García,Butz, Bognàr, & Tauscher, 2001; Sánchez-Moreno, Plaza, deAncos, & Cano, 2003). For other types of juices, literature data inthis respect are limited (Brenes, Del Pozo-Insfran, & Talcott,2005; González-Molina, Moreno, & García-Viguera, 2009). There-fore, it is important to determine whether DHAA analysis is neces-sary for all type of juices.

Enhanced sensitivity and separation power of UPLC in compar-ison to conventional HPLC decreases the time and cost of analysis,and has become increasingly important in liquid chromatographyapplications. These include analysis of food and plant compounds,e.g. vitamins (de Brouwer et al., 2010; Hampel, York, & Allen, 2012;Spínola et al., 2012), phenolics (Gruz, Novak, & Strnad, 2008;Herrero et al., 2011) and alkaloids (Ortega et al., 2010; Yi et al.,2012). In the fields of food safety, UPLC is used for determinationof pesticides residues and their metabolites (Leandro, Hancock,Fussell, & Keely, 2006; Li et al., 2013), and heterocyclic aromaticamines (Barceló-Barrachina et al., 2006). Major applications ofUPLC in pharmaceutical analyses include quality control and sta-bility monitoring of products, drug discovery and development(Nováková & Vlcková, 2009; Wren & Tchelitcheff, 2006). Thereare few methods dedicated solely to vitamin C determination infoods (Spínola et al., 2012).

Due to the widespread application of vitamin C in food industry,and its losses during processing or storage, it is necessary to moni-tor levels of this compound in food and pharmaceutical prepara-tions using low-cost, fast and reliable analytical tools. Samplepreparation before analysis is crucial to obtain accurate results.According to literature data, 1–10% meta-phosphoric acid can beused to prepare samples for AA determination in beverages, fruitsand biological samples (final concentration from 0.2% to 5%)(Karlsen, Blomhoff, & Gundersen, 2005; Odriozola-Serrano,Hernández-Jover, & Martín-Belloso, 2007; Tiwari, O’Donnell,Muthukumarappan, & Cullen, 2009; Valdramidis, Cullen, Tiwari, &O’Donnell, 2010). The weakness of these methods is incompletedata on stability of AA during sample preparation and analysis.Therefore, the first aim of the present study was to determine thestability of AA in meta-phosphoric acid used for samplepreparation.

The next aim of this study was to validate and compare UPLCand HPLC methods for AA and TAA determination. The validatedHPLC and UPLC methods were applied to different fruit juicesand vitamin supplements. Moreover, the concentration of AA andTAA was determined in different fruit juices stored for 24 and48 h after opening to verify the necessity of determination of bothAA and DHAA to assess the concentration of vitamin C in this kindof product.

2. Materials and methods

2.1. Materials

Cartons or bottles of commercial fruit beverages (juices anddrinks) were purchased from local markets. Vitamin C pharmaceu-tical preparations were bought in local pharmacy. Bottles of fruitjuices (three for each type of juice originating from two differentbatches; n = 6) were stored after opening for 24 and 48 h in thecold (4–6 �C) to assess the effect of time on the concentration ofDHAA. Meta-phosphoric acid and AA (min 99.7%) were purchasedfrom Merck (Darmstadt, Germany), DL-dithiothreitol (DTT; min99%) was from Sigma–Aldrich (Steinheim, Germany). Deionized-doubly distilled water was filtered through a 0.45 lm filter forHPLC (Millipore, Bedford, MA, USA). Methanol (Chempur, PiekarySlaskie, Polska) was HPLC grade. All other chemicals were reagentgrade.

2.2. Sample preparation

The vitamin C extraction method was adapted from Ross (1994).Beverage sample (0.5 mL) and 10% meta-phosphoric acid (0.5 mL)were mixed using a vortex (5 min) (final concentration of meta-phosphoric acid was 5%), centrifuged at 8500g for 10 min, andinjected onto the HPLC or UPLC column to determine AA content(Gliszczynska-Swigło & Tyrakowska, 2003). Before injection ontoUPLC column, the samples of fruit juices or drinks were diluted1:50 or 1:10, respectively, using 10% meta-phosphoric acid.

The sum of AA and DHAA (vitamin C content; TAA) was deter-mined after reduction of DHAA to AA using DTT. The juice sample(0.2 mL) and 5% DTT (0.2 mL) were mixed and diluted to 2 mL with10% meta-phosphoric acid and injected onto HPLC or UPLC columnto determine vitamin C (Gliszczynska-Swigło et al., 2006).

Vitamin C tablets were dissolved in 25 mL 10% meta-phosphoricacid and treated as fruit beverages samples. Before injection ontoHPLC or UPLC column, the samples of tablets were diluted 1:50or 1:150, respectively, using 10% meta-phosphoric acid. Threeindependent extractions were carried out for all samples. The onlydifference between sample preparation for HPLC and UPLC was themagnitude of sample dilution before injection.

2.3. Chromatographic determination of AA

The vitamin C was determined using Waters 600 high-perfor-mance liquid chromatograph (Waters Corp., Milford, MA, USA)equipped with LiChrospher C18 (250 � 4.0 mm, 5 lm, Merck KGaA,Germany) fitted with the same guard column. A gradient of mobilephase composed of methanol (solvent A) and 5 mmol/L KH2PO4, pH2.65 (solvent B) was used according to the following program: lin-ear increment starting with 5–22%A in 6 min and the return to theinitial conditions within the next 9 min with the flow rate of0.8 mL/min. The eluate was detected using a Waters 996 photodi-ode array detector set at 245 nm (Gliszczynska-Swigło et al.,2006). The injection volume was 20 lL.

UPLC determination of vitamin C was done using Acquity™ultra high performance liquid chromatograph equipped withACQUITY UPLC BEH C18 (100 � 2.1 mm; 1.7 lm; Waters, Milford,MA, USA) fitted with guard column. A gradient of mobile phasecomposed of methanol (solvent A) and 5 mmol/L KH2PO4, pH2.65 (solvent B) was used according to the following program: lin-ear increment starting with 5–15%A in 1 min, from 15% to 35% forthe next 1 min and the return to the initial conditions within thenext 4 min with the flow rate of 0.2 mL/min. The eluate wasdetected using a Waters Acquity™ photodiode array detector setat 245 nm. The injection volume was 5 lL.

102 I. Klimczak, A. Gliszczynska-Swigło / Food Chemistry 175 (2015) 100–105

AA was identified by comparing its retention time and ultravi-olet spectrum with that of the AA standard. UV-spectrum was alsoused to confirm the purity of AA separated from other possiblecompounds present in extract. Quantification of AA or vitamin C(as the sum of AA and DHAA) was done using the external standardmethod with AA as a standard. For each extract, at least two injec-tions were made.

The content of DHAA was determined indirectly by subtractionof AA from TAA.

2.4. Method validation

Validation of HPLC and UPLC methods included linearity, instru-ment precision and sensitivity. Quantification of AA was performedusing an external standard method. The eight-point (25–300 lg/mLfor HPLC and 5–50 lg/mL for UPLC) calibration curves (n = 4; gener-ated during a 6-month period) were prepared with the standardsolutions of AA in 10% meta-phosphoric acid at concentrationsspanning those present in samples.

Instrument precision was checked from six consecutive injec-tions of fruit juice and vitamin C tablet extracts prepared asdescribed in Section 2.2.

The LOD and LOQ were calculated from the calibration curve atconcentrations from 0.1 to 3 lg/mL (HPLC) or 0.05–3 lg/mL(UPLC). The following equation was used:

LOD ¼ 3:3�rb

LOQ ¼ 10�rb ;

where r is the standard deviation of y-intercepts and b is the slopeof the calibration curve.

The efficiency of the extraction method was determined byspiking samples at two concentration levels of AA (50% and 100%of the AA level in the product). Spiked and unspiked samples weretreated in the same way throughout the whole procedure. Threeindependent extractions were carried out for each product andAA level. Each sample was injected onto the HPLC or UPLC columnstwice.

2.5. Statistical analysis

Statistical analyses were performed using the Statistica 9.0(StatSoft, Inc., 2000) program. Results on the effect of storage timeon the AA and TAA concentrations in juice samples were assessedusing ANOVA. Differences between fresh and stored samples wereevaluated by the Dunnett’s test. The differences between HPLC andUPLC results as well as between AA and TAA concentrations instored fruit beverages were evaluated by the Student’s t test. Thedifferences at 5% level were considered as significant.

3. Results and discussion

3.1. Stability of AA in meta-phosphoric acid

The results of our study show that the stability of AA in juicesamples using 2.5% meta-phosphoric acid (final concentration ofmeta-phosphoric acid was 1.25%) was significantly less than with10% meta-phosphoric acid (meta-phosphoric acid final concentra-tion of 5%). The concentration of AA in juice samples (n = 6)decreased from 252.7 to 232.2 mg/L (8%) in 1 h. In the same time,the concentration of AA in tablet samples dissolved in 2.5% meta-phosphoric acid (n = 6) decreased from 62.5 to 46.5 mg/tablet(26%). Juice or tablet samples dissolved in 10% meta-phosphoricacid were more stable. However, after 24 h, at least 97.3% of AAwas still present in juice or tablet samples. Based on these results,10% meta-phosphoric acid was used for sample preparations (final

concentration of meta-phosphoric acid was 5% or 10% for juicesand tablets, respectively).

3.2. Analytical characteristics

In this paper, HPLC and UPLC methods for vitamin C determina-tion were compared. AA and DTT were eluted from an HPLC systemin 4.01 and 13.06 min, respectively. Using UPLC system, retentiontimes of AA and DTT were 1.56 and 3.30 min, respectively(Fig. 1). Using UPLC method, the total time of analysis was 2.5times shorter than with HPLC and solvent consumption decreasedfrom 12 to 1.2 mL/min.

Analytical characteristics of HPLC and UPLC methods included:linearity, precision and sensitivity (Table 1). Quantification of AAwas performed using an external standard method. The curve forAA in HPLC method was linear to atleast 300 lg/mL. In UPLCmethod, lower concentrations must be used: a maximum concen-tration cannot be higher than 50 lg/mL. The RSD values of thestandard curve slopes obtained with HPLC and UPLC systems were2.78%, 1.82%, respectively. Linear correlation coefficients (r) forboth standard curves were 0.999.

Instrument precision was checked from six consecutive injec-tions of AA extracts from fruit juice and pharmaceutical prepara-tion. In the case of fruit juice, the intra-day (the daily) and inter-day (day-to-day) RSDs obtained using HPLC were 2.2% and 2.4%,respectively, whereas with UPLC – 1.7% and 1.9%, respectively.For vitamin C tablets, the intra- and inter-day RSDs obtained usingHPLC were 0.4% and 0.5%, respectively; with UPLC system – 0.1%and 0.3%, respectively (Table 1). Precision of AA determination inpharmaceutical preparation using both methods was higher thanobtained for fruit juice. It is probably the effect of less complexmatrix of tablet than fruit juice.

The LOD and the LOQ for AA under HPLC conditions were0.049 lg/mL and 0.149 lg/mL, respectively. These results are muchlower than those reported by Sánchez-Mata, Camara-Hurtado,Diez-Marquês, and Torija-Isasa (2000), Hernandez et al. (2006)and Vidovic et al. (2008) but slightly higher than those reportedby Valente, Albuquerque, Sanches-Silva, and Costa (2011). In UPLCmethod, LOD and LOQ were 0.024 and 0.073 lg/mL, respectively(Table 1). These results are very similar to those obtained bySpínola et al. (2012) but better than those reported by Zhang,Chen, Liao, and Ren (2009) as well as by Talekar, Vora, Gawade,and Gopala (2013). Moreover, the extraction solvent used in thepresent study was simpler than that described by Spínola et al.(2012) and guaranteed stability of sample within at least 24 h.The proposed UPLC method can be an alternative for existingmethods for vitamin C determinations in juices and pharmaceuti-cal preparations.

The efficiency of the extraction was determined based on therecovery of AA from spiked juice and tablet samples. It was foundthat the recovery of AA was not lower than 98.9% (Table 2).

3.3. Quantification of AA in fruit beverages and vitamin C tablets

The validated HPLC and UPLC methods were applied to differentfruit juices and vitamin supplements. The content of vitamin C intested products is reported in Table 3. DHAA was not present inany sample (the results obtained for samples prepared with andwithout DTT were not statistically different). The results obtainedusing HPLC and UPLC methods were very similar although, forsome products, the results obtained using UPLC were slightlyhigher than those obtained by HPLC. It may be due to the highersensitivity of UPLC than HPLC.

The changes in AA, TAA and DHAA concentrations in differentopened fruit juices stored for 24 and 48 h under refrigeration con-ditions are presented in Table 4. DHAA was not observed in any

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Juice sample Juice sample with DTT

A24

5 [a.u

.]

Retention time [min]

AA

DTT

A

0 1 2 3 4

DTT

AAB

A24

5 [j.u

.]

Retention time [min]

Fig. 1. Typical HPLC (A) and UPLC (B) chromatograms of ascorbic acid separated from fruit juice and pharmaceutical preparation.

Table 1Method validation parameters for determination of AA in juices and vitamin C tablets.

HPLC UPLC

Standard linearity Range (lg/mL) 0–300 0–50r 0.999 0.999

Instrument precision(n = 6)

Intra-day

Juice Mean (mg/L) ± SD 248.2 ± 5.4 255.9 ± 4.3RSD (%) 2.2 1.7

Vitamin C tablet Mean (mg/tablet) 67.6 ± 0.3 67.7 ± 0.1RSD (%) 0.4 0.1

Instrument precision(n = 6)

Inter-day

Juice Mean (mg/L) ± SD 245.5 ± 5.8 253.5 ± 4.9RSD (%) 2.4 1.9

Vitamin C tablet Mean (mg/tablet) ± SD

65.8 ± 0.3 66.2 ± 0.2

RSD (%) 0.5 0.3

LimitsLOD (lg/mL) 0.049 0.024LOQ (lg/mL) 0.149 0.073

Table 3Comparison of vitamin C content in analysed samples as determined by HPLC andUPLC methods.

Sample Vitamin C content ± SD (%RSD) (mg/L or mg/tablet)

HPLC UPLC

Fruit juices and beveragesOrange juice 224.3 ± 0.9a (0.4) 230.9 ± 1.8b (0.8)White grapefruit juice 1 255.6 ± 3.6a (1.4) 267.2 ± 2.4b (0.9)Red grapefruit juice 2 467.1 ± 1.5a (0.3) 473.7 ± 1.4b (0.3)Blackcurrant nectar 209.6 ± 2.7a (1.3) 208.2 ± 2.7a (1.3)Multifruit drink 1* 86.6 ± 1.3a (1.5) 84.7 ± 0.3a (0.4)Multifruit drink 2* 87.6 ± 0.6a (0.7) 88.7 ± 0.6a (0.7)Ice Tea drink* 100.0 ± 0.4a (0.4) 97.5 ± 0.9b (0.9)

Vitamin preparationsMultivitamin preparation 64.4 ± 0.3a (0.5) 68.7 ± 1.6b (2.3)Vitamin C 65.8 ± 0.6a (0.9) 67.7 ± 0.1b (0.1)Vitamin C + rutin* 100.0 ± 0.1a (0.1) 99.7 ± 0.4a (0.4)

Values are mean ± SD (at least three independent extractions were carried out forthree products (n = 9) and at least two injections into UPLC or HPLC column weredone for each sample).a,b Means in each row are significantly different at a = 0.05.

* In accordance with producer declaration.

I. Klimczak, A. Gliszczynska-Swigło / Food Chemistry 175 (2015) 100–105 103

fresh juice (the results obtained for samples prepared with andwithout DTT were not statistically different). The decrease in AAof 0–2.9% and 4.1–6.4% in comparison to fresh juice was observedfor juices stored for 24 and 48 h, respectively. The concentration ofDHAA was from 0% to 5.4% depending on storage time and a kind ofjuice. It was reported that phenolic compounds protect AA fromdegradation but this protection depends on the type of compound(Miller & Rice-Evans, 1997; Özkan, Kırca, & Cemeroglu, 2004).Moreover, it was found that apple juice had the lowest protective

Table 2Recovery of AA from selected fruit beverages and pharmaceutical preparation.

Product AA content ± SD (mg/L) or (mg/tablet)

HPLC UPLC

Grapefruit juice 468.3 ± 0.8 454.2 ± 0.5

Blackcurrant nectar 208.1 ± 07 209.2 ± 0.7

Vitamin C supplement 100.1 ± 0.1 99.8 ± 0.2

a Percent of AA content in product.b Recovery mean ± SD (n = 3 in each level).

effect on AA in fortified fruit juices comparing to orange and black-currant juices. Apple juice phenolic acids were recognised as lessefficient protectors of AA than flavonoids of orange and blackcur-rant juices (Miller & Rice-Evans, 1997). These results may explainhigher losses of AA in apple juices than in multifruit juices ana-lysed in the present study. It was observed that oxidation processwas only partly responsible for the loss of AA because the concen-tration of DHAA did not compensated the decrease of AA in freshjuice. This was especially apparent for multifruit juices, in which

AA spiked levela (%) Recoveryb (%)

HPLC UPLC

50 99.8 ± 0.3 99.9 ± 0.2100 100.2 ± 0.2 100.0 ± 0.1

50 99.0 ± 0.7 99.3 ± 0.4100 98.9 ± 0.6 99.5 ± 0.5

50 99.9 ± 0.3 99.9 ± 0.2100 99.8 ± 0.4 99.8 ± 0.3

Table 4The changes in vitamin C content in fruit juices (mg/L) during short-term storage under refrigeration conditions.

Product Storage (h) AA1 TAA DHAA2

Multifruit juice 1 0 392.3 ± 3.9a 394.2 ± 2.7a 024 381.1 ± 4.8A,a

(2.9%)389.2 ± 1.5A,b

(1.3%)8.1 ± 3.7(2.1%)

48 376.1 ± 1.5A,a

(4.1%)386.9 ± 2.0A,b

(1.9%)10.8 ± 2.2(2.8%)

Multifruit juice 2 0 435.8 ± 4.0a 433.1 ± 2.1a 024 428.4 ± 1.4a 429.4 ± 1.7a 048 414.4 ± 2.3A,a

(4.9%)426.2 ± 2.9A,b

(1.6%)11.8 ± 2.9(2.7%)

Apple juice 1* 0 254.6 ± 6.7a 251.2 ± 2.2a 024 247.2 ± 1.5a 245.8 ± 3.1a 048 238.3 ± 4.9A,a

(6.4%)251.9 ± 1.1b 13.6 ± 3.5

(5.4%)

Apple juice 2* 0 360.3 ± 1.6a 357.5 ± 2.5a 024 354.2 ± 2.9A,a

(1.7%)360.3 ± 3.9a 0

48 340.8 ± 1.5A,a

(5.4%)353.0 ± 2.2b 12.2 ± 2.3

(3.4%)

Values are mean ± SD (three juices from two different batches were analysed; at least three independent extractions were carried out for each bottle of product (n = 18) and atleast two injections into column were done for each sample).

A Means in the column for AA or TAA are significantly different from appropriate fresh sample (Dunnett’s test, a = 0.05).a,b Means in each row are significantly different (Student’s t test, a = 0.05).

1 In parenthesis – the loss of AA in comparison with fresh juice.2 Calculated as the difference between TAA and AA; in parenthesis – the percentage of TAA.* Enriched with ascorbic acid (according to the producer declaration).

104 I. Klimczak, A. Gliszczynska-Swigło / Food Chemistry 175 (2015) 100–105

probably not only oxidation but also degradation of AA occurred.The changes in the concentration of AA in apple juices were mainlydue to the oxidation thus the decrease in TAA during storage wasnot observed. Determination of vitamin C in fruit juices storedfor at least 24 h under refrigeration may require determination ofTAA because oxidation of AA may occur during storage.

4. Conclusions

Although many methods have been developed for determina-tion of AA or TAA, both HPLC and UPLC methods proposed in thisstudy for determination of AA or TAA (vitamin C) in fruit beveragesand pharmaceutical preparations can be recommended as rapid,precise and sensitive. Extraction of AA and DHAA from tested prod-ucts using 10% meta-phosphoric acid guaranteed determination ofvitamin C within 24 h without its essential loss. For fresh commer-cial beverages as well as for pharmaceutical preparations, it is notnecessary to determine both AA and DHAA. Determination of vita-min C in stored fruit juices may require determination of TAAbecause oxidation of AA may occur during storage. Both methodscan be useful in the routine qualitative and quantitative analysisof vitamin C in beverages and pharmaceutical preparations. How-ever, UPLC method is faster, more sensitive, consumes less eluentand it is more eco-friendly than the conventional HPLC method.In consequence, UPLC is cheaper than HPLC because a higher num-ber of analyses per unit of time can be performed and consumptionof eluent is much lower.

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