Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol index

Accepted Manuscript Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol index Edyta Nalewajko-Sieliwoniuk, Magdalena Iwanowicz, Sławomir Kalinowski, Anatol Kojło PII:

S0003-2670(16)30121-0

DOI:

10.1016/j.aca.2016.01.033

Reference:

ACA 234373

To appear in:

Analytica Chimica Acta

Received Date: 30 September 2015 Revised Date:

22 December 2015

Accepted Date: 24 January 2016

Please cite this article as: E. Nalewajko-Sieliwoniuk, M. Iwanowicz, S. Kalinowski, A. Kojło, Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol index, Analytica Chimica Acta (2016), doi: 10.1016/ j.aca.2016.01.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

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Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol

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index

Edyta Nalewajko-Sieliwoniuka,*, Magdalena Iwanowicza, Sławomir Kalinowskib, Anatol Kojłoa

Department of Analytical Chemistry, Institute of Chemistry, University of Białystok, Hurtowa

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a

1, 15-399 Białystok, Poland

Department of Chemistry, University of Warmia and Mazury, 10-957 Olsztyn, Poland

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b

Abstract

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In this work, we present a novel chemiluminescence (CL) method based on direct-injection detector (DID) integrated with the multi-pumping flow system (MPFS) to chemiluminescence determination of the total polyphenol index. In this flow system, the sample and the reagents

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are injected directly into the cone-shaped detection cell placed in front of the photomultiplier window. Such construction of the detection chamber allows for fast measurement of the CL

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signal in stopped-flow conditions immediately after mixing the reagents. The proposed DIDCL-MPFS method is based on the chemiluminescence of nanocolloidal manganese(IV)hexametaphosphate-ethanol system. The application of ethanol as a sensitizer, eliminated the use of carcinogenic formaldehyde. Under the optimized experimental conditions, the chemiluminescence intensities are proportional to the concentration of gallic acid in the range from 5 to 350 ng mL-1. The DID-CL-MPFS method offers a number of advantages, including *

Corresponding author. Address: Institute of Chemistry, University of Bialystok, Hurtowa 1, 15-399 Bialystok, Poland. Tel.: +48 85 7457831; fax: +48 85 7388052. E-mail address: [email protected] (E. NalewajkoSieliwoniuk).

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ACCEPTED MANUSCRIPT low limit of detection (0.80 ng mL-1), high precision (RSD = 3.3%) and high sample throughput (144 samples h-1) as well as low consumption of reagents, energy and low waste generation. The proposed method has been successfully applied to determine the total polyphenol index (expressed as gallic acid equivalent) in a variety of plant-derived food

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samples (wine, tea, coffee, fruit and vegetable juices, herbs, spices).

Keywords: Direct-injection detector; Multi-pumping flow system; Polyphenolic antioxidants;

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Chemiluminescence; Nanocolloidal manganese(IV); Green Analytical Chemistry

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1. Introduction

Recent years have witnessed a surge of interest in the field of green analytical chemistry (GAC). GAC is focused on developing techniques and analytical methodologies that reduce

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or eliminate the use of toxic reagents, ensure lower amounts of generated residues that are less hazardous to human health and the environment [1]. Flow-based techniques facilitate development of greener analytical procedures mainly by the exploitation of new flow

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approaches and changes in system design. This minimizes reagent and sample consumption as well as waste generation without hindering analytical performance.

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One of the approaches which could be considered an evolution of the flow injection concept towards GAC is the multi-pumping flow system (MPFS) [1]. The MPFS is constructed from a set of solenoid-operated pulse micro-pumps, each dedicated to propelling a different solution [2]. Among other advantages, this strategy fulfills low energy requirements and is very effective in minimizing reagent consumption and waste generation (in contrast to classical flow injection analysis). A fixed volume of reagents is dispensed into the system only when necessary.

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ACCEPTED MANUSCRIPT In 2011, Koronkiewicz and Kalinowski [3] constructed an innovative direct-injection photometric detector (DID) integrated with the solenoid pulse-pump flow system. The idea of the sample and reagent injection directly into the detection chamber allowed for significant reduction in their consumption, minimized the waste generation and increased the sample

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throughput compared to the MPFS systems with a typical configuration (containing a confluence point and reaction coil) [4]. In 2015, the same authors presented a novel directinjection chemiluminescence detector (DID-CL) [5] with a cone-shaped reaction-detection

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chamber where a CL reaction takes place. The sample and the reagents are injected directly into the detection chamber in counter-current using solenoid micro-pumps. The proposed

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detector allows for fast and effective mixing of the injected solutions and measurement of the analytical signal in stopped-flow conditions from the moment of reagents mixing, which is crucial for rapid chemiluminescent reactions.

Polyphenolic compounds are the secondary metabolites found in plants. They are

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widespread constituents of foodstuffs of plant origin and are major natural antioxidants in our diet. The increasing interest in these substances is a consequence of their biological properties such as antioxidant, anti-allergic, anti-thrombotic, anti-bacterial and anti-inflammatory [6]. As

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a result of their potential beneficial effects on human health [7], inclusion of polyphenol-rich food products in our diet is recommended.

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The necessity for quality control of foods of plant origin, functional foods and dietary supplements containing polyphenols fosters the development of automatic, cost-effective, high-throughput and environmentally friendly methods based on flow analysis for determination of polyphenolic antioxidants. Among all assays commonly used for this purpose, the most popular are those based on spectrophotometric and electrochemical detection [8]. In recent years, flow chemiluminescence methods of determination of polyphenolic compounds and their antioxidant activity have received an increasing attention.

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ACCEPTED MANUSCRIPT This is mainly due to their great sensitivity and low limits of detection, which are several orders of magnitude lower in comparison with spectrophotometric assays. It gives possibility of high dilution of a sample, which considerably reduces the interference from matrix components of food samples.

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In our previous work [9], we investigated a number of chemiluminescence reactions with luminol, nanocolloidal Mn(IV), KMnO4 and Ce(IV) reagents in order to select the most sensitive and selective one for the determination of polyphenolic antioxidants. We found that

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these requirements were achieved when Mn(IV)-hexametaphosphate-formaldehyde CL system was used for polyphenols detection. This CL reaction was previously applied by us for

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the FI-CL [10] and HPLC-FI-CL [11] determination of polyphenolic compounds in extracts from Cirsium palustre (L.) and for the MCFA-CL [12] determination of the total polyphenol index/antioxidant activity of food products. However, the necessity of applying formaldehyde (carcinogenic by inhalation) in order to enhance the emission intensity to analytically useful

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levels caused this CL system imperfect for routine application. For many years formaldehyde had been the most effective enhancer in chemiluminescence reactions based on nanocolloidal manganese(IV). In 2014, Smith et al. [13] investigated a wide range of enhancers and they

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found that ethanol could be a safe and inexpensive alternative to formaldehyde without compromising signal intensity or sensitivity.

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The objective of this study was to employ the direct-injection chemiluminescence detector combined with the multi-pumping flow system in order to develop an improved automated flow-based method for determination of polyphenolic antioxidants. It is the first example of practical application of the DID-CL-MPFS for the analysis of real samples. Moreover, the aim of this study was to examine whether the addition of ethanol, as a more environmentally friendly reagent, can provide similar emission intensities to that of the formaldehyde enhancer

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ACCEPTED MANUSCRIPT for nanocolloidal Mn(IV)-based CL detection of polyphenols without affecting the analytical performance and selectivity of the method.

2.1.

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2. Materials and Methods

Reagents and solutions

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All the chemicals used in this work were of analytical grade, and all the solutions were prepared with purified water obtained from the Milli-Q Plus water purification system

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(Millipore S.A., Molsheim, France). Sodium hydroxide, potassium permanganate, formaldehyde, acetic acid, phosphoric acid, citric acid, ethanol, potassium chloride, sodium chloride, magnesium chloride, calcium chloride, manganese(II) chloride, sodium carbonate, oxalic acid, sodium tetraborate and sodium metabisulphite were supplied by POCH (Gliwice,

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Poland). Folin–Ciocalteu (F-C) reagent was supplied by Fluka (Steinheim, Germany). Xanthin, caffeine, theophylline, theobromine, proline, DL-malic acid, tartaric acid, ascorbic acid, glucose, fructose, sucrose, zinc chloride, iron(III) chloride, sodium formate, sodium

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hexametaphosphate, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were obtained from

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Sigma-Aldrich (Steinheim, Germany). Quercetin, rutin, (±)-catechin, epicatechin, epicatechin gallate, gallic acid and caffeic acid were supplied by Sigma-Aldrich (Steinheim, Germany). 1000 µg mL-1 stock solutions of gallic acid, caffeic acid, epicatechin and epicatechin gallate, 500 µg mL-1 stock solutions of quercetin and catechin and 10 µg mL-1 stock solution of rutin were prepared by dissolving an appropriate amount of each compound in water (Method 1) or ethanol (Method 2). All stock solutions were kept in the dark at a temperature of 4 °C. Working standard solutions of polyphenols were prepared daily by dilution of the stock solutions with 1 mol L-1 formaldehyde (Method 1) or 90 % ethanol (Method 2). 5

ACCEPTED MANUSCRIPT A transparent brown nanocolloidal manganese(IV) stock solution was prepared according to the procedure described in our previous work [9]. The CL reagent was prepared daily by dissolving 1.5 g sodium hexametaphosphate in 50 mL 1.7 × 10-3 mol L-1 stock

2.2. Sample pre-treatment of tea, coffee, herbs and spices

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solution of manganese(IV).

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Seven samples of different kinds of tea: Camellia sinensis (one white, two green and two black), two fruit teas (the first tea was a mixture of hibiscus, raspberry, blackberry,

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chokeberry, wild rose and elderberry; the second tea was a mixture of chokeberry, hibiscus, cranberry and apple), as well as seven samples of dried herbs and spices (Origanum majorana L., Origanum vulgare L., Ocimum basilicum L., Thymus vulgaris L., Artemisia dracunculus L., Rosmarinus officinalis L., Matricaria chamomilla) and two kinds of coffee Coffea Arabica

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(instant and ground roasted) were purchased from a local supermarket. The infusions of tea, coffee, herbs and spices were prepared as follows: 1 g of each sample was extracted with 100 mL of hot water at a temperature of 80°C (tea and coffee) or boiling water (herbs and spices)

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for 10 min. After this time, the extracts were cooled to a temperature of 25°C in a water bath

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and filtered through a filter paper (in case it was necessary). In order to estimate the total polyphenolic content in the investigated samples by the DID-CL-MPFS method they were diluted with 90% ethanol to fit the concentration of polyphenolic compounds to the concentration range of the calibrating curve of gallic acid. To reduce the consumption of ethanol, the initial dilutions were done with water. Then small volumes of water solutions (a few tens of microliters) were diluted with 90% ethanol. Infusions of green, black and white tea, as well as instant and ground roasted coffee were diluted 4000 times, extracts of fruit tea and Matricaria chamomilla were diluted 500 times, the remaining extracts of herbs and spices 6

ACCEPTED MANUSCRIPT were diluted 1500 times. In order to determine the total reducing capacity of the tested samples by the F-C method, they were diluted with water (except for extracts of fruit tea) to fit the concentration of polyphenols to the concentration range of the calibrating curve of gallic acid (infusions of instant coffee, white tea, green tea and all investigated herbs and

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spices were diluted 8 times, infusions of black tea and ground roasted coffee were diluted 4 times). Before the determination of the total antioxidant activity of food samples by the DPPH

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method, they were diluted 10 times with water.

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2.3. Sample pre-treatment of wine, fruit and vegetable juices

Five samples of vegetable and fruit juices (elderberry, blackcurrant, orange, carrot and tomato juice) and five grape wines of various types (red, rosé and white) were purchased from a commercial source. All investigated samples were kept in a dark place at a temperature of

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4°C. Prior to the analysis, vegetable juices were filtered through a filter paper. In order to estimate the total polyphenolic content by the DID-CL-MPFS method, all the samples were diluted with 90% ethanol to fit the concentration of polyphenolic compounds to the

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concentration range of the calibrating curve of gallic acid. To reduce the consumption of

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ethanol, the initial dilutions were done with water. Elderberry juice was diluted 12000 times, red wine was diluted 10000 times, blackcurrant juice was diluted 5000 times, tomato juice was diluted 3600 times, orange juice was diluted 2500 times, white and rosé wines were diluted 2000 times, carrot juice was diluted 1300 times. In order to determine the total reducing capacity of investigated beverages by the F-C method, they were diluted with water (red wine was diluted 20 times, rosé wine, elderberry and blackcurrant juices were diluted 8 times, white wine, orange, carrot and tomato juices were diluted 4 times). In order to estimate

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ACCEPTED MANUSCRIPT the total antioxidant activity of investigated samples by the DPPH method, all of them were diluted 10 times with water.

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2.4. Apparatus

The multi-pumping flow system (Fig. 1) consisted of the solenoid micro-pumps (P1– P4) (Cole-Parmer, USA) of a nominal volume of 20 µL (product no. P/N73120-10) and 50 µL

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(product no. P/N73120-22), the flow lines of PTFE tubing (0.8 mm i.d.) and the direct-

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injection chemiluminescence detector. The pumps with smaller volume (P1 and P2) were used for injecting the sample and the CL reagent solutions, the bigger one (P4) for propelling the carrier solution (water). The construction of the direct-injection chemiluminescence detector had been discussed in detail by Koronkiewicz and Kalinowski in their last work [5]. It was built using one block of Teflon. Inside this block, a cone-shaped reaction-detection chamber

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was drilled (with a total volume of 280 µL) and closed with a transparent glass window placed in front of the photomultiplier tube (operated at 1200 V or 1250 V). There are two

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inlets into the detector chamber at its bottom and one outlet at the top of the chamber (for an easier escape for potential air bubbles). One of the inlets was used for injecting the mixture of

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the sample and the CL reagent while the second one was utilised for cleaning the detector chamber using a carrier solution. The photomultiplier and solenoid micro-pumps were PCcontrolled by software developed in the Delphi programming language [14]. The main window of the program used in this work is presented in Fig. 2b. Absorption

spectra

were

monitored

spectrophotometer (Hitachi, Japan).

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using

a

model

U-3900H

UV-Vis

ACCEPTED MANUSCRIPT 2.5. Flow manifold

The configuration of the DID-CL-MPFS system used in this work is depicted in Fig. 1. The solutions contained respectively: polyphenolic compounds (as a sample) in 1 mol L-1

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formaldehyde (Method 1) or 90 % ethanol (Method 2) and the mixture of Mn(IV) and sodium hexametaphosphate in 6 mol L-1 phosphoric acid (as the CL reagent).

The working sequence of the solenoid micro-pumps in a single measurement cycle is

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presented in Table 1 and Fig. 2a. In step 1, the sample line was primed with the sample solution. In order to do this, pump P1 was alternately switched ON (for 0.2 s) or OFF (for 0.35

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s) six times in total. In step 2, the detection chamber was washed with water. Pump P4 was switched ON/OFF alternately six times for time intervals of 0.2 s (ON) and 0.5 s (OFF). Next, the baseline was recorded under the stopped-flow conditions (step 3). After that time, the solutions of the sample and the CL reagent were injected into the detection chamber using the

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solenoid micro-pumps P1 and P2 (step 4) which were alternately switched ON (for 0.2 s) or OFF (for 0.35 s) nine times in total. In step 5, the CL signal was recorded under the stoppedflow conditions. In order to wash the detection chamber before the next measurement cycle,

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ten repetitions of the water injection using pump P4 were performed (step 6). Pump P4 was switched ON/OFF alternately for time intervals of 0.2 s (ON) and 0.5 s (OFF). Pump P3 was

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used only at the end of the day for removing (with water) the mixture of the sample and the CL reagent from a ‘blind space’ between the confluence point and the inlet to the detector chamber [5].

The determination of polyphenols was based on the net CL intensity which was calculated according to the formula: ∆I = IS−I0, where IS is an emission from the CL reaction in the presence of polyphenols (solutions of polyphenols were prepared in 1 mol L-1

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ACCEPTED MANUSCRIPT formaldehyde (Method 1) or in 90 % ethanol (Method 2)) and I0 is a blank (CL signal registered for 1 mol L-1 formaldehyde (Method 1) or for 90 % ethanol (Method 2)).

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2.6. Folin-Ciocalteu (F-C) method

The procedure described by Djeridane et al. [15] was employed for the determination of the total reducing capacity of the plant-derived food samples with the F-C reagent. The

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gallic acid solutions were used to prepare a calibrating curve in the concentration range of 5 – 300 µg mL-1 and the results were expressed as mg of gallic acid equivalent per litre of a

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sample.

2.7. DPPH free radical scavenging assay

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In order to determine the radical scavenging activity of the tested food samples against the DPPH radical, a spectrophotometric method proposed by Miliauskas et al. [16] was applied. The decrease in absorbance of the DPPH radical solution was measured at 515 nm.

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The scavenging activity of investigated samples was calculated according to the formula:

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% Inhibition = [(AB – AA)/AB] × 100,

where: AB – absorption of blank sample (t = 0); AA – absorption of the plant-derived food sample (t = 15 min).

2.8. Statistical analysis

The software Microsoft Office Excel 2007 (Microsoft Corp, USA) was used for preparing the data analysis. The results were presented as mean ± standard deviation. Outliers

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ACCEPTED MANUSCRIPT were eliminated from the dataset using the Q-test. Correlations between the results obtained by the chemiluminescence method (DID-CL-MPFS) and the spectrophotometric assays (F-C and DPPH) were determined on the basis of a linear regression and a correlation coefficient

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(r).

3. Results and Discussion

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3.1. Development of the multi-pumping flow system with direct-injection chemiluminescence

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detector

The proposed method is based on the enhancing effect of polyphenols on chemiluminescence of nanocolloidal manganese(IV). In our previous studies, we had used sodium hexametaphosphate and formaldehyde in order to effectively enhance the CL of

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Mn(IV) to an analytically useful level [9-12]. In the literature, we found that ethanol could be a safe and inexpensive alternative to formaldehyde enhancer [13]; we decided to check

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whether it could also be applied for polyphenols determination.

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, HCHO or ethanol,H3PO 4  3 )6    → Mn(II)* + polyphenolox Mn(IV) + polyphenol (NaPO

Therefore,

two

Mn(II)* → Mn(II) + hν

multi-pumping

flow

systems

with

the

direct-injection

chemiluminescence detector based on Mn(IV)-hexametaphosphate-formaldehyde (Method 1) and Mn(IV)-hexametaphosphate-ethanol (Method 2) CL systems were optimized in order to obtain the highest analytical signals. The measurements were conducted in the DID-CLMPFS manifold presented in Fig. 1. Univariate searches were performed on the selection of an appropriate: volume of the sample and the CL reagent, concentration of reagents and photomultiplier voltage. The studied ranges and the optimal values of these parameters are 11

ACCEPTED MANUSCRIPT summarized in Table 2. For the optimization studies, we selected gallic acid, known to be a strong antioxidant. Moreover, this compound is the most frequently applied as a standard in the studies related to the determination of the total polyphenolic content and total antioxidant

50 ng mL-1 (Method 1) and 100 ng mL-1 (Method 2).

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3.1.1. Influence of the sample and the CL reagent volume

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activity of food samples. The concentration of gallic acid used in the optimization studies was

The first optimized parameter was the volume of the sample and the CL reagent. These

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solutions were injected using pumps P1 and P2, which delivered a volume of 20 µL per stroke (Fig. 1). As depicted in Fig. 2, the switching pattern of the solenoid micro-pumps demonstrates that pumps P1 and P2 were switched ON/OFF at the same time. The injections were repeated between 4 and 11 times, corresponding to a total volume of the sample and the

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CL reagent of 160 – 440 µL. It was observed that, in spite the total volume of the reactiondetection chamber of 280 µL, the CL signal increased with the increasing volume of injected mixture of the sample and the CL reagent up to 360 µL (corresponding to 9 simultaneous

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injections of both solutions). For 400 µL, a light decrease in the photocurrent was observed for both methods. Therefore, 9 was chosen to be the number of injections, corresponding to

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360 µL of liquid (180 µL of the sample solution and 180 µL of the CL reagent solution).

3.1.2. Effect of the reagents concentration

The effect of the concentration of nanocolloidal Mn(IV) (prepared in 6 mol L-1 phosphoric acid) on the CL signal of gallic acid was studied varying its concentration (from 2 × 10-4 to 1.7 × 10-3 mol L-1), while maintaining the other parameters as constant. In both

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ACCEPTED MANUSCRIPT methods, a significant increase in analytical signal height was observed with the increase of Mn(IV) concentration up to 1.7 × 10-3 mol L-1. Higher concentrations of Mn(IV) were not investigated due to poor solubility of manganese dioxide. Therefore, the concentration of 1.7 × 10-3 mol L-1 was selected for further studies.

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The enhancing effect of the concentration of sodium hexametaphosphate on analytical signal height was examined in the range from 0 to 5% (w/v). In both methods, a light increase in the photocurrent occurred for sodium hexametaphosphate concentration of up to 3%, while

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for higher concentrations, the CL signal decreased around 20% (Method 1) or remained constant (Method 2). Considering these results, a 3% sodium hexametaphosphate solution was

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chosen as optimal for both methods. Furthermore, the stability of the CL reagent (obtained by dissolving sodium hexametaphosphate in a 1.7 × 10-3 mol L-1 solution of manganese(IV)) was investigated for the period of a work day. The stability studies were performed by injecting the standard solution of gallic acid 3 times every 30 min over a period of 7 hours, without

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replenishing the reagent vessel. The results are presented in Electronic Supplementary Material (Fig. S1). The chemiluminescence intensity remained almost constant for about 270 min (RSD = 4.6%, n = 10). After that time, it started to increase and the RSD of 15

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measurements performed during 420 min was 9.8%. Therefore, we recommend using this CL reagent for about 4.5 hours from the moment sodium hexametaphosphate is mixed with

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manganese(IV).

The next optimized parameter was the concentration of formaldehyde and ethanol

enhancers. In Method 1, formaldehyde was used as a solvent for preparing the working solutions of polyphenolic compounds. The effect of its concentration was studied within the range of 0.1 − 4 mol L-1. The highest analytical signals were obtained for 1 mol L-1 formaldehyde and this concentration was used in further experiments. In Method 2, ethanol was used as a solvent for preparing the working solutions of polyphenols and, at the same

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ACCEPTED MANUSCRIPT time, as an enhancer of Mn(IV)-based chemiluminescence. The influence of ethanol concentration on the analytical signals of gallic acid was tested over a range of 0 to 100%. It was observed that with the increase in ethanol concentration from 0 to 90%, the CL signal of gallic acid increased by 91%. For higher concentrations of ethanol, about 20% decrease in

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photocurrent was observed. Therefore, 90% ethanol was used in subsequent experiments. As the final step, the influence of photomultiplier voltage on analytical signals of gallic acid was investigated in the range of 1100 – 1300 V. The maximum value of ∆I was

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obtained when the voltage of the photomultiplier was 1200 V (Method 1) or 1250 V (Method

3.2. Analytical characteristic

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2). Therefore, those values were selected for further studies.

After optimizing the experimental conditions, analytical parameters of the DID-CL-

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MPFS methods based on Mn(IV)-hexametaphosphate-formaldehyde (Method 1) and Mn(IV)hexametaphosphate-ethanol (Method 2) CL systems were evaluated. The linear ranges, the calibration graphs, the correlation coefficients (r) and the limits of detection (LOD) obtained

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for standard solutions of seven polyphenols known as strong antioxidants (gallic acid,

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epicatechin, caffeic acid, epicatechin gallate, catechin, quercetin and rutin) are presented in Table 3. As it can be seen from the results presented in Table 3, the linearity ranges obtained in Method 2 were 1.3 – 2.9 times longer than those obtained in Method 1. The limits of detection calculated as 3Sb/S, where Sb is the residual standard deviation and S is the slope of the calibration graph, were between 0.8 ng mL-1 (for gallic acid) and 17 ng mL-1 (for rutin). Both methods exhibited the highest sensitivity for the detection of gallic acid. Therefore, this compound was selected as a standard for the determination of the total polyphenol index in the tested plant-derived food samples. The within-day repeatability of the Method 1,

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ACCEPTED MANUSCRIPT expressed as a relative standard deviation of 20 subsequent measurements of 100 ng mL-1 gallic acid, was 3.4%. In the case of the Method 2, the value of RSD was comparable and equal to 3.3% for 100 ng mL-1 of gallic acid. The between-days reproducibility of the Method 1 and Method 2, expressed as RSD of the slopes of the calibration graphs of gallic acid

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registered on three different days (n=6), was 4.9% and 4.3%, respectively. The total time of a programmed cycle necessary for obtaining a CL peak was 25 s (Table 1). Therefore, the

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sampling rate obtained for both methods was 144 determinations per hour.

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3.3. Selectivity studies

The effect of twenty four chemical species naturally existing in plant-derived food products was evaluated by spiking the standard solutions of gallic acid (100 ng mL-1) with increasing amounts of these substances. The concentration ratio of the interfering compound

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to gallic acid was considered acceptable if the relative error of determination was lower than ± 5%. As it can be seen from the results presented in Table 4, Method 1 exhibited a slightly better selectivity to some of the investigated interfering compounds compared with Method 2.

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In Method 1, the most important effect on the analytical signal of gallic acid was observed for iron(III) chloride, manganese(II) chloride and ascorbic acid. In Method 2, in addition to the

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substances listed above, an interfering effect was also observed for caffeine, oxalic acid, sodium tetraborate and sodium metabisulphite. Prior to the analysis, the samples were diluted at least 1:500 (up to 1:12000) times (v/v) (as described in Section 2.2 and Section 2.3). Comparing the tolerable concentrations of the tested compounds (Table 4) with their contents in real samples, it can be concluded that, after an appropriate dilution of the sample, none of them cause an interfering effect. Therefore, both Mn(IV)-based methods could be used for the

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ACCEPTED MANUSCRIPT selective determination of polyphenolic compounds without any additional steps for masking or removing substances naturally present in the analysed samples.

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3.4. Method application

Both presented methods exhibited similar analytical characteristics and both allow for a selective determination of polyphenols in the presence of matrix compounds. Therefore,

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taking into account that ethanol, in comparison to formaldehyde, is safe and environmentally friendly reagent, we chose Method 2 for the determination of the total polyphenol index in 26

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plant-derived food products (tea, coffee, herb and spice infusions, wines, fruit and vegetable juices). The samples were prepared according to the procedures described in Section 2.2 and Section 2.3. Each sample was analyzed in triplicate. The results were expressed as milligrams of gallic acid equivalent per litre of a sample. As it can be seen from the results presented in

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Table 5, the highest contents of polyphenolic antioxidants were found in: elderberry juice (2713 ± 39 mg L−1), red wine (1090 ± 51 and 1149 ± 58 mg L−1), green, black and white tea infusions (from 352 ± 13 to 778 ± 38 mg L−1), instant coffee (718 ± 16 mg L−1) and

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blackcurrant juice (454 ± 17 mg L−1). The lowest total polyphenol index was determined in: fruit teas (60.9 ± 2.2 and 100 ± 4 mg L−1), extracts of Matricaria chamomilla (54.1 ± 2.8 mg

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L−1) and Rosmarinus officinalis L. (89.4 ± 4.0 mg L−1). The next step was to check the correlation between the results obtained by the DID-

CL-MPFS method with those obtained by spectrophotometric F-C and DPPH methods (described in Section 2.6. and Section 2.7.). The F-C method used to be recommended for the determination of the total phenolic content. Nowadays, due to its low selectivity to polyphenols, is rather suggested for the determination of the total reducing capacity [18]. Nevertheless, owing to the fact that in the majority of plant-derived food products

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ACCEPTED MANUSCRIPT polyphenols are the most abundant antioxidants, the F-C method gives a good approximation of the total content of polyphenolic compounds in almost all cases [19]. The results of spectrophotometric assays are summarized in Table 5. In the case of all investigated types of samples, the data obtained by the DID-CL-MPFS method correlates highly with those

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obtained by the F-C and DPPH methods (r ≥ 0.950). Linear correlations of the results obtained by DID-CL-MPFS and spectrophotometric assays are shown in Electronic Supplementary Material (Fig. S2). A positive correlation suggests that the DID-CL-MPFS

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method could be applied not only for the determination of the total polyphenol index but also

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for antioxidant/reducing activity of plant-derived food products.

3.6. Comparison of the DID-CL-MPFS system with other flow systems of the new generation with CL detection

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A critical comparison of the DID-CL-MPFS system with other automated flow systems of the new generation for the CL determination of polyphenolic antioxidants was carried out. The main parameters usually taken into account to evaluate an analytical

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procedure are presented in Table 6. The proposed method affords the advantages of a significantly lower limit of detection, lower waste generation per determination and higher

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sample throughput than those observed in the references cited [12, 20-25]. Such a low limit of detection and the associated with this need of high dilution of the samples (as described in Section 2.2 and Section 2.3) resulted in an effective dilution of the interfering matrix components. Upon analyzing the selectivity of the DID-CL-MPFS method, it can be concluded that the comparison is also very favorable to the proposed procedure. None of the individually investigated twenty four chemical species naturally existing in different foodstuffs of plant origin caused the interfering effect. Therefore, the proposed method as

17

ACCEPTED MANUSCRIPT well as the previously developed MCFA-CL method [12] could be used for the determination of the total polyphenol index/antioxidant activity of diverse plant-derived food products (such as wine, tea, coffee, fruit and vegetable juices, herbs, spices, extracts of olive oil). However, the DID-CL-MPFS method is an example of a faster and ‘greener’ flow-based method. It

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offers 2.4 times higher sample throughput and 5.5-fold lower waste generation per assay in comparison with the MCFA-CL method.

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4. Conclusions

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An automated and environmentally friendly flow method based on the direct-injection chemiluminescence detector integrated with the multi-pumping flow system to determination of the total polyphenol index was developed. The main advantages of the DID-CL-MPFS method are: simplicity, low cost, portability (small weight and size of the flow system),

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selectivity to polyphenols, low consumption of reagents and energy. Moreover, our newly developed DID-CL-MPFS method is superior to previously described automated flow systems of the new generation for the CL determination of polyphenolic antioxidants due to

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its lower limit of detection (minimization of matrix effects), lower waste generation and higher sample throughput. Application of ethanol as a sensitizer allowed for elimination the

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use of toxic formaldehyde. The DID-CL-MPFS method fulfills the criteria of green analytical chemistry and could be applied for the selective determination of the total polyphenolic content and their antioxidant/reducing activity in a big variety of food products of plant origin.

Acknowledgement

18

ACCEPTED MANUSCRIPT The equipment used in this work was partly supported by EU funds via the project with contract number POPW.01.03.00-20-034/09. References

analysis. A review, Anal. Chim. Acta 714 (2012) 8–19.

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[1] W.R. Melchert, B.F. Reis, F.R.P. Rocha, Green chemistry and the evolution of flow

[2] J.L.F.C. Lima, J.L.M. Santos, A.C.B. Dias, M.F.T. Ribeiro, E.A.G. Zagatto, Multi-

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pumping flow systems: an automation tool, Talanta 64 (2004) 1091-1098.

[3] S. Koronkiewicz, S. Kalinowski, A novel direct-injection photometric detector integrated

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with solenoid pulse-pump flow system, Talanta 86 (2011) 436–441.

[4] S. Koronkiewicz, S. Kalinowski, Application of direct-injection detector integrated with

the multi-pumping flow system to photometric stopped-flow determination of total iron, Talanta 96 (2012) 68–74.

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[5] S. Koronkiewicz, S. Kalinowski, Direct-injection chemiluminescence detector. Properties and potential applications in flow analysis, Talanta 133 (2015) 112–119. [6] E. Hurtado-Fernández, M. Gómez-Romero, A. Carrasco-Pancorbo, A. Fernández-

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Gutiérrez, Application and potential of capillary electroseparation methods to determine antioxidant phenolic compounds from plant food material, J. Pharm. Biomed. Anal. 53 (2010)

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1130–1160.

[7] P.M. Kris-Etherton, K.D. Hecker, A. Bonanome, S.M. Coval, A.E. Binkoski, K.F. Hilpert, A.E. Griel, T.D. Etherton, Bioactive compounds in foods: Their role in the prevention of cardiovascular disease and cancer, Am. J. Med. 113(9B) (2002) 71S–88S. [8] L.M. Magalhães, M. Santos, M.A. Segundo, S. Reis, J.L.F.C. Lima, Flow injection based methods for fast screening of antioxidant capacity, Talanta 77 (2009) 1559–1566.

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ACCEPTED MANUSCRIPT [9] E. Nalewajko-Sieliwoniuk, J. Malejko, M. Święczkowska, A. Kowalewska, A study on the selection of chemiluminescence system for the flow injection determination of the total polyphenol index of plant-derived foods, Food Chem. 176 (2015) 175–183. [10] J. Malejko, E. Nalewajko-Sieliwoniuk, J. Nazaruk, J. Siniło, A. Kojło, Determination of

chemiluminescence detection, Food Chem. 152 (2014) 155-161.

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the total polyphenolic content in Cirsium palustre (L.) leaves extracts with manganese(IV)

[11] E. Nalewajko-Sieliwoniuk, J. Malejko, M. Mozolewska, E. Wołyniec, J. Nazaruk,

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Talanta, Determination of polyphenolic compounds in Cirsium palustre (L.) extracts by high

38–44.

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performance liquid chromatography with chemiluminescence detection, Talanta 133 (2015)

[12] E. Nalewajko-Sieliwoniuk, J. Malejko, A. Pawlukiewicz, A. Kojło, A novel multicommuted flow method with nanocolloidal manganese(IV)-based chemiluminescence detection for the determination of the total polyphenol index, Food Anal. Methods, DOI:

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10.1007/s12161-015-0274-8

[13] Z.M. Smith, J.M. Terry, N.W. Barnett, P.S. Francis, Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection, Talanta

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130 (2014) 221–225.

[14] 〈http://pracownicy.uwm.edu.pl/kalinow〉 8th of September 2015

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[15] A. Djeridane, M. Yousfi, B. Nadjemi, D. Boutassouna, P. Stocker, N. Vidal, Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds, Food Chem. 97 (2006) 654–660.

[16] G. Miliauskas, P.R. Venskutonis, T.A. van Beek, Screening of radical scavenging activity of some medicinal and aromatic plant extracts, Food Chem. 85 (2004) 231–237. [17] D.C. Montgomery, G.C. Runger, Applied statistics and probability for engineers, John Wiley Sons Inc, New York, 2003.

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ACCEPTED MANUSCRIPT [18] D. Huang, B. Ou, R.L. Prior, The chemistry behind antioxidant capacity assays, J. Agric. Food Chem. 53 (2005) 1841–1856. [19] J.D. Everette, Q.M. Bryant, A.M. Green, Y.A. Abbey, G.W. Wangila, R.B. Walker, Thorough study of reactivity of various compound classes toward the Folin−Ciocalteu

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reagent, J. Agric. Food Chem. 58 (2010) 8139–8144.

[20] S.R.P. Meneses, K.L. Marques, C.K. Pires, J.L.M. Santos, E. Fernandes, J.L.F.C. Lima, E.A.G. Zagatto, Evaluation of the total antioxidant capacity by using a multipumping flow

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system with chemiluminescent detection, Anal. Biochem. 345 (2005) 90–95.

[21] M. Wada, M. Kira, H. Kido, R. Ikeda, N. Kuroda, T. Nishigaki, K. Nakashima, Semi-

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micro flow injection analysis method for evaluation of quenching effect of health foods or food additive antioxidants on peroxynitrite, Luminescence 26 (2011) 191–195. [22] A.R. Araujo, F. Maya, M.L. Saraiva, J.L. Lima, J.M. Estela, V. Cerda, Flow system for the automatic screening of the effect of phenolic compounds on the luminol-hydrogen

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peroxide-peroxidise chemiluminescence system, Luminescence 26 (2011) 571–578. [23] M.F. Andrade, S.G.F. Assis, A.P.S. Paim, B.F. Reis, Multicommuted flow analysis procedure for total polyphenols determination in wines employing chemiluminescence

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detection, Food Anal. Methods 7 (2014) 967–976. [24] E. Fassoula, A. Economou, A. Calokerinos, Development and validation of a sequential-

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injection method with chemiluminescence detection for the high throughput assay of the total antioxidant capacity of wines, Talanta 85 (2011) 1412–1418. [25] D. Karampelas, A. Economou, A. Calokerinos, A novel hybrid flow-injection/sequentialinjection methodology for the rapid evaluation of the total antioxidant capacity of wines using inhibition of the alkaline luminol-potassium permanganate chemiluminescent reaction, Microchem. J. 118 (2015) 223–230.

21

ACCEPTED MANUSCRIPT Table 1. Working sequence of the solenoid micro-pumps in a single measurement cycle Description

P1

P2

P4

Time [s]

1

Priming the sample line

ON/OFF a

OFF

OFF

3.3

2

Cleaning the chamber with water

OFF

OFF

ON/OFF b

3.7

3

Stopped-flow for recording the baseline

OFF

OFF

OFF

1.8

4

Inserting the sample and reagent

ON/OFF c

ON/OFF c

OFF

4.6

5

Stopped-flow for recording the CL signal

OFF

OFF

OFF

4.6

6

Cleaning the chamber with water

OFF

ON/OFF d

7.0

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Step

OFF

The pump P1 was alternately switched ON/OFF six times in total.

b

The pump P4 was alternately switched ON/OFF six times in total.

c

The pumps P1 and P2 were alternately switched ON/OFF nine times in total.

d

The pump P4 was alternately switched ON/OFF ten times in total.

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a

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ACCEPTED MANUSCRIPT Table 2. The studied parameters of Mn(IV)-based chemiluminescence systems for polyphenols detection Optimized parameter

Studied range

Optimal value

Mn(IV)-sodium hexametaphosphate-formaldehyde based method (Method 1) 2×10-4 - 1.7×10-3

1.7×10-3

Concentration of sodium hexametaphosphate [%]

1-5

3

Concentration of formaldehyde [mol L-1]

0.1 - 4

Volume of the sample and the CL reagent [µL]

160 - 440

360

Photomultiplier voltage [V]

1100 - 1300

1200

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Concentration of Mn(IV) [mol L-1]

1

Mn(IV)-sodium hexametaphosphate-ethanol based method (Method 2) Concentration of Mn(IV) [mol L-1]

1.7×10-3

Concentration of sodium hexametaphosphate [%]

1-5

3

Concentration of ethanol [%]

0 - 100

90

Volume of the sample and the CL reagent [µL]

160 - 440

360

Photomultiplier voltage [V]

1100 - 1300

1250

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2×10-4 - 1.7×10-3

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ACCEPTED MANUSCRIPT Table 3. Analytical parameters of Mn(IV)-sodium hexametaphosphate-formaldehyde and Mn(IV)-sodium hexametaphosphate-ethanol based DID-CL-MPFS methods Linearity range [ng mL-1]

Correlation LOD coefficient [ng mL-1] (r)

Calibration equation y = (a±SD)x + (b±SD) (n=3)

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Mn(IV)-sodium hexametaphosphate-formaldehyde based method (Method 1) 5 - 150

y=(0.620±0.020)x+(3.62±1.62)

epicatechin

5-250

y=(0.554±0.024)x+(2.67±0.40)

caffeic acid

10-250

y=(0.386±0.015)x+(0.604±0.262)

epicatechin gallate

5-250

y=(0.379±0.010)x+(0.480±0.071)

catechin

10-250

quercetin rutin

0.998

1.03

0.999

1.48

0.999

2.66

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gallic acid

1.70

y=(0.356±0.012)x+(0.967±0.494)

0.999

1.82

10-350

y=(0.352±0.011)x+(1.29±0.63)

0.999

2.38

25-500

y=(0.153±0.003)x+(1.67±1.17)

0.999

6.73

0.998

0.80

y=(0.560±0.018)x+(4.57±1.02)

0.999

2.05

y=(0.349±0.014)x+(2.81±1.60)

0.999

1.31

y=(0.324±0.016)x+(0.798±0.353)

0.999

0.84

y=(0.320±0.004)x+(4.02±1.54)

0.999

1.40

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0.998

Mn(IV)-sodium hexametaphosphate-ethanol based method (Method 2)

epicatechin

10-350

caffeic acid

5-500

epicatechin gallate

5-700

catechin

5-700

y=(0.566±0.008)x+(3.07±0.51)

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5-350

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gallic acid

quercetin

50-500

y=(0.288±0.008)x+(9.35±4.96)

0.999

10.5

rutin

250-1000

y=(0.124±0.003)x+(24.0±0.28)

0.999

17.0

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ACCEPTED MANUSCRIPT Table 4. The tolerable concentration ratio of the studied matrix substances to analyte in the determination of gallic acid (100 µg L-1) by Mn(IV)-sodium hexametaphosphateformaldehyde and Mn(IV)-sodium hexametaphosphate-ethanol based DID-CL-MPFS

The tolerable concentration ratio

10000 5000

8000 5000

4000

40000

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NaCl CaCl2·6 H2O

–

3000

Glucose Acetic acid Sucrose Fructose ZnCl2

3000 3000 2500 2000

KCl Malic acid Na2S2O5

1500 1300 1000

Tartaric acid

900

10

30 3000 900 500

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500 Proline Na2B4O7·10H2O 400

Mn(IV)-sodium hexametaphosphateethanol (Method 2)

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Ethanol Citric acid

Mn(IV)-sodium hexametaphosphate-formaldehyde (Method 1) 500000

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Interferent

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methods (relative error of determination ± 5%)

4000 300 0.3

8000 1100 5

300

300

Theophylline Xanthin Oxalic acid

Theobromine

250 30 10 10

250 30 0.4 150

Caffeine

9

7

Ascorbic acid MnCl2·4 H2O

5

0.09

0.1

0.3

FeCl3

0.06

0.1

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MgCl2

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ACCEPTED MANUSCRIPT Table 5. The total polyphenol index determined by the DID-CL-MPFS method, the total reducing capacity determined by the F-C method and antioxidant activity determined by the DPPH method in different plant-derived food products DID-CL-MPFS method

a

-1

a

(mg gallic acid L )

(mg gallic acid L-1)

Tea and coffee infusions

DPPH inhibition a

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Type of sample

F-C method

(%)

778 ± 38

1098 ± 53

460 ± 32

639 ± 90

33.5 ± 1.3

543 ± 28

815 ± 46

42.7 ± 2.7

352 ± 13

722 ± 26

35.8 ± 1.8

584 ± 19

32.6 ± 1.0

1386 ± 61

49.4 ± 2.9

466 ± 15

17.0 ± 0.5

100 ± 4

221 ± 9

9.61 ± 0.30

60.9 ± 2.2

150 ± 3

5.43 ± 0.50

289 ± 11

814 ± 7

29.6 ± 1.1

Origanum vulgare L. (dried)

257 ± 12

685 ± 26

28.1 ± 1.4

Ocimum basilicum L. (dried)

255 ± 12

580 ± 16

26.7 ± 1.0

Thymus vulgaris L. (dried) 246 ± 13

587 ± 5

22.4 ± 1.2

Artemisia dracunculus L. (dried)

147 ± 8

368 ± 9

12.7 ± 0.4

Rosmarinus officinalis L. (dried)

89.4 ± 4.0

174 ± 8

4.51 ± 0.05

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Green teas

356 ± 15

Coffea Arabica (instant coffee)

718 ± 16

Coffea Arabica (ground roasted coffee)

191 ± 9

Fruit teas

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Herb and spice infusions

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White tea

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Black teas

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Origanum majorana L. (dried)

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60.3 ± 2.9

ACCEPTED MANUSCRIPT Matricaria chamomilla (dried)

54.1 ± 2.8

181 ± 9

1.25 ± 0.09

1149 ± 58

4043 ± 93

83.6 ±1.8

1090 ± 51

3575 ± 27

80.7 ±1.3

141 ± 6

570 ± 19

161 ± 5

74.0 ± 3.71

119 ± 4

67.6 ± 3.44

Elderberry juice

2713 ± 39

2540 ± 59

Blackcurrant juice

454 ± 17

Orange juice

308 ± 16

Tomato juice

153 ± 6

Carrot juice

133 ± 6

Wines

Rosé wine (semi-dry)

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White wine (semi-dry)

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mean of three determinations ± SD

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Fruit and vegetable juices

a

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Red wine (dry)

27

7.55 ± 0.21

5.64 ± 0.07

5.53 ±0.36

79.8 ± 0.8

1059 ± 11

36.6 ± 2.0

734 ± 31

16.9 ± 0.6

128 ± 6

6.59 ± 0.11

439 ± 21

14.9± 0.3

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Table 6. Comparison of the analytical features achieved by the proposed DID-CL-MPFS method and other automated flow-based methods of the

CL detection

Linear range [mg L-1]

Limit of RSDh Sample Waste volume generation detection [%] [µL] (per assay) [mg L-1] [mL]

Sample throughput [h−1]

MPFS

Luminol-H2O2 and lucygeninH2O2

up to 0.016a

–

0.22.0

48

–

(160 and 70)i

0.7-10a 0.7-40b 10-60c

0.5 0.6 3.9

2.110

5

–

–

5.2

50

6.7

6.6

3.4

33

0.17

5.3

MPFS

Luminol-ClO-

10-100d

SIA

Luminol-H2O2- 0.17-34d Co(II)

FI-SI

LuminolKMnO4

0.15-2.6d 0.05

MCFAg

Mn(IV)-

0.005-

0.0015

Type of sample

Ref.

Farmaceutical formulations, tea extracts

–

[20]

30

Noni juices

–

[21]

12

Wine/grape seeds

Substances such as ascorbic acid, β-carotene and ferrous ion should be removed before the analysis.

[22]

180i

Wine

The effect of 8 wine constituents was studied. Copper and SO32− might cause interferences.

[23]

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Selectivity

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1.1i

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MSFIA- Luminol-H2O2- 1-100d SIAf horseradish peroxidase

75

1.9

60

Wine

The effect of 3 wine [24] constituents was studied. Their influence was negligible.

4.9

100

1.7

71

Wine

The effect of 4 wine [25] constituents was studied. Their influence was negligible.

2.4

1785

7.1

60

Wine, tea, coffee,

The effect of 24 chemical

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SMFIAe LuminolONOO-

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Flow method

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new generation with CL detection for determination of polyphenolic compounds

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[12]

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sodium 0.15d hexametaphosp hateformaldehyde

0.0008

3.3

180

1.3 (0.86 i)

144 (200i)

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Mn(IV)0.0050.35d sodium hexametaphosp hate-ethanol

for ascorbic acid was used as a standard compound,

b

Trolox was used as a standard compound,

c

ascorbyl palmitate was used as a standard compound,

d

gallic acid was used as a standard compound,

e

SMFIA- semi-micro flow injection analysis,

f

MSFIA-SIA - the combination of multisyringe flow injection analysis and sequential injection analysis,

g

MCFA - multicommuted flow analysis,

h

RSD - relative standard deviation,

i

calculated for the same type of sample (without taking into account the need for priming the sample line).

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a

29

species was studied. No interfering effect was observed.

Wine, tea, coffee, The effect of 24 chemical fruit and vegetable species was studied. No juices, herbs, spices interfering effect was observed.

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DIDMPFS

fruit and vegetable juices, herbs, spices, extracts of olive oil

This work

ACCEPTED MANUSCRIPT Figure captions

Fig. 1 Schematic diagram of the multi-pumping flow system with a direct-injection

content. P1, P2, P3 and P4 are the solenoid micro-pumps

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chemiluminescence detector (DID-CL-MPFS) for the determination of the total polyphenolic

Fig. 2 (a) The switching sequence of the solenoid micro-pumps in a single measurement

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cycle. Step 1 – priming the sample line, step 2 – cleaning the chamber with water, step 3 – stopped-flow for recording the baseline, step 4 – injection of the sample and CL reagent into

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the reaction-detection chamber, step 5 – stopped-flow for recording the CL signal, step 6 –

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cleaning the chamber with water; (b) an example of the CL signals

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

a)

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Fig. 2.

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b)

ACCEPTED MANUSCRIPT Highlights

A DID-CL-MPFS method for determination of the total polyphenol index is proposed.

•

Direct-injection detector enables fast measurement of CL in stop-flow conditions.

•

The method is based on the enhancement of manganese(IV)-based CL reaction.

•

Ethanol was used as a ‘green’ alternative to formaldehyde enhancer.

•

The method ensures minimal consumption of reagents and waste generation.

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•