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On-line coupling of capillary electrophoresis with microdialysis for determining saccharides in dairy products and honey
Food Chemistry 316 (2020) 126362
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On-line coupling of capillary electrophoresis with microdialysis for determining saccharides in dairy products and honey
T
⁎
Petr Tůmaa, , Blanka Sommerováa, Václav Daněčekb a b
Charles University, Third Faculty of Medicine, Department of Hygiene, Ruská 87, 100 00 Prague 10, Czech Republic Charles University, Third Faculty of Medicine, Department of Biophysics, Ruská 87, 100 00 Prague 10, Czech Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Contactless conductivity detection Flow-gating interface Food analysis Polydimethylsiloxane Sequence analysis
Free sucrose, lactose, galactose, glucose and fructose were determined in yoghurts, milk and honey using on-line coupling of capillary electrophoresis with microdialysis. The dairy products were diluted 50-fold with 10 mmol/ L NaOH and sampled using laboratory-made microdialysis probes. The microdialysate was brought to the entrance of the electrophoretic capillary and the coupling consisted in a polydimethylsiloxane (PDMS) cross connector working in the flow-gating interface regime. The electrophoretic analysis was performed in 50 mmol/ L NaOH (pH 12.6) background electrolyte, where baseline separation of the five saccharides was achieved in 3.5 min. The LOQs varied in the range 2.3–7.3 mg/L, the number of separation plates varied between 176,000 plates/m for glucose to 326,000 plates/m for galactose and the relative standard deviation (RSD) for ten consecutive analyses of fruit yoghurt was 0.2% for the migration time and 4.4–7.6% for the peak area.
1. Introduction Microdialysis (MD) is a modern semi-invasive technique for continuous sampling of living tissue and organs, used primarily because of its minimal unpleasantness of sampling, with little influence on the ongoing biochemical and physiological processes (Brunner & Derendorf, 2006; Kennedy, 2013; Schultz & Kennedy, 2008; Sloan, Nandi, Linz, Aldrich, Audus, & Lunte, 2012; Zhang, Sun, Wang, Han, & Wang, 2012). Microdialysis sampling is performed using miniature probes, which are inserted into the studied tissue so that the microdialysis membrane is in direct contact with the tissue medium (Lee, Slaney, Hower, & Kennedy, 2013; Nandi & Lunte, 2009; Slaney et al., 2011). The actual probe most frequently consists of a 10–30 mm long microdialysis tube with a diameter of several tenths of a millimeter, connected to two tubes (Kennedy, 2013; Lee et al., 2013; Nandi & Lunte, 2009; Saylor & Lunte, 2015). The input tube brings the acceptor solution from the syringe pump to the probe, where it washes the inside of the microdialysis tube and is gradually enriched in the analytes that diffuse from the surrounding tissue through the microdialysis membrane into the flowing acceptor solution. This process produces a solution of the microdialysate flowing from the probe through the outlet tube, which is collected for subsequent analysis. Primarily small hydrophilic molecules diffuse through the microdialysis membrane and the efficiency of
the microdialysis sampling, measured as the ratio between the concentrations of the substance in the microdialysate and in the tissue, decreases exponentially with increasing flow rate. The flow rate varies between 0.5 and 10 µL/min and the microdialysis commonly has a yield of about 20–50% for small hydrophilic substances (Nandi & Lunte, 2009; Saylor & Lunte, 2015). Reduction of the flow rate is limited primarily by the very small volume of microdialysate obtained for subsequent instrumental analysis, which is critical especially in the offline arrangement, where the microdialysate must be further handled during its laboratory treatment (Tůma, Jaček, Fejfarová, & Polák, 2016; Tůma, Sommerová, & Šiklová, 2019). Consequently, it is much better to analyze the microdialysate flowing out of the probe in real time through on-line coupling between the microdialysis apparatus and a compatible microanalytical technique. A useful instrumental technique for these purposes consists in capillary (CE) (Guihen & O'Connor, 2009, 2010; Nandi & Lunte, 2009) or chip (Huynh, Fogarty, Martin, & Lunte, 2004; Nandi, Desaias, & Lunte, 2010; Saylor & Lunte, 2015) electrophoresis performed in capillaries and channels with a small inner diameter (ID) that is directly comparable with the ID of the tubes used in the microdialysis probe. In addition, CE is characterized by high separation efficiency, which is a necessary condition for the analysis of complicated clinical and food samples. Its short separation time is a vital factor in sequential analyses
Abbreviations: BGE, background electrolyte; C4D, contactless conductivity detection; DEI, deionised water; FGI, flow-gating interface; ID, inner diameter; MD, microdialysis; OD, outer diameter; PDMS, polydimethylsiloxane ⁎ Corresponding author. E-mail address: [email protected] (P. Tůma). https://doi.org/10.1016/j.foodchem.2020.126362 Received 17 July 2019; Received in revised form 24 October 2019; Accepted 4 February 2020 Available online 05 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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at short time intervals and it has the additional advantage of requiring small amounts of reagents, making it a suitable technique for green chemistry (Koel, Borissova, Vaher, & Kaljurand, 2011). It is important to develop a suitable interface between MD and CE, as this is not currently available commercially and individual laboratories make their own. The main function of the interface lies in separation of the continuous stream of collected microdialysate into discrete zones, which are injected into the capillary only at specified times. A potential technical solution consists in a cross-shaped flow-gating interface (FGI) in which the following meet in a single point: i) the entrance into the separation capillary, ii) the exit from the microdialysis probe, iii) the capillary bringing the gating solution (usually the background electrolyte for electrophoretic separation, BGE) and iv) the exit to waste (Gong, Zhang, & Maddukuri, 2018; Opekar & Tůma, 2017). In the normal FGI working regime, the microdialysate is deflected from the entrance into the separation capillary by a stream of gating solution and is fed to the waste. Only during injection is the flow of gating solution stopped by switching the valve to the OFF position so that the microdialysate fills the space in front of the entrance into the separation capillary and is injected into it. The valve is then switched to the ON position, renewing the flow of gating solution, deflecting the microdialysate from the entrance into the separation capillary and the actual CE separation takes place. This series of individual steps is constantly repeated, forming the basis of sequence analysis (Opekar & Tůma, 2017). This contribution is concerned with the development and construction of a coupling between a commercial Agilent CE instrument and microdialysis sampling of yoghurts, milk and honeys to determine the contents of free saccharides. The saccharides are separated in aqueous solutions of sodium hydroxide and detected by contactless conductivity detection (C4D). The axial construction of C4D consists of two tubular metal electrodes with a length of more than 2 mm that are placed around the outer capillary surface. The electrodes are separated by a detection gap of about one millimetre into which is inserted a shielding foil. The first electrode acts as a transmitter of an alternating signal with a frequency between hundreds of kHz and tens of MHz; the second electrode acts as the receiver. C4D represents a universal detection technique for CE and is employed particularly for the detection of minerals, amino acids, saccharides, low-molecular weight organic acids and other compounds with low absorbance in the UV region (Kubáň & Hauser, 2018, 2019; Opekar, Tůma, & Štulík, 2013; Šolínová & Kašička, 2006).
Fig. 1. Scheme for on-line coupling of the microdialysis apparatus with the capillary electrophoresis instrument through a flow-gating interface made from a PDMS cross-connector.
entrances of this PDMS cross-connector (dimensions: height 15 mm, length 15 mm, thickness 3 mm). The inserted capillaries were firmly sealed in PDMS to avoid leaking of solutions and the connector had a minimal internal dead volume. PDMS connectors could be made by a more complicated procedure using lithographic techniques (Unger, Chou, Thorsen, Scherer, & Quake, 2000). The inlets and outlets of crossconnectors (Fig. 1A): i) To the upper inlet was connected a capillary with ID/OD 100/ 360 µm and length 3.0 cm, the other end of which was connected to the outlet of a three-way magnetically controlled valve (Science Instruments and Software, CR). The BGE with a flow rate of 83 µL/ min was pressed out of a 50 mL injection syringe using a linear piston pump (TJ-3A; MRC LTD, Israel) and was fed to the PDMS cross-connector through this inlet. When the valve was moved to the OFF position just before injecting the sample into the CE capillary, the BGE passed from the valve directly into the waste. ii) The microdialysate was fed to the right-hand inlet through a capillary with ID/OD 100/150 µm; the connection of the thinner capillary in the PDMS cross-connector was sealed by a drop of UVcured adhesive around the outer surface of the capillary 2 mm from its end. The actual laboratory-made MD probe was made from a 4 cm long microdialysation tube (ID/OD 200/216 µm, cut-off 13 kDa, Spectrum Labs, USA), into both of whose ends two fused silica ID/OD 75/150 µm capillaries were inserted to a depth of 1 mm and the connections were sealed with UV-cured adhesive. The inlet capillary was connected with the acceptor solution (10 mmol/ L NaOH) by a 5 mL injection syringe. A solution flow rate of 5 µL/ min was provided by a linear piston pump (Double NE-4500 OEM, New Era Pump Systems, U.S.A.). The MD probe was immersed in the sample and the outlet was brought to the PDMS connector. Several acceptor solutions, such as 10 mmol/L HCl, DEI and 10 mmol/L NaOH, were tested for microdialysis of saccharides with the highest recovery for an aqueous solution of NaOH, resulting from deprotonation of saccharides at high pH. The microdialysis flow rate was changed in the 1–10 µL/min range with an optimized value of 5 µL/min. The employed flow rate is closely related to the amount of sample that is injected into the separation capillary to achieve baseline separation of the saccharides. iii) A 15 mm stainless steel tube cut from an ID/OD 400/800 µm injection syringe (B/Braun, Melsungen, Germany) was inserted into the bottom outlet and the tube was mechanically connected to the grounding contact from the high-voltage source. The other end of the tube was connected using UV-cured adhesive to a 5 cm long capillary ID/OD 100/360 µm, which fed the solution from the PDMS cross-connector to the waste. iv) The injection end of the separation capillary was inserted into the
2. Experimental 2.1. Instrumentation for FGI In this work, the main FGI component was a cross-shaped polydimethylsiloxane (PDMS) connector with four entrances for close connection of the fused silica capillaries with an outer diameter (OD) of about 360 µm (Polymicro Technologies, Phoenix, U.S.A.). The connector was made in the laboratory by the casting technology described in general in ref. (Zhang & Gong, 2014) and specifically modified. Briefly, first a square frame with a 25 mm long outer edge and inner square opening with dimensions of 15 × 15 mm was cut from silicon foil with a thickness of 3 mm. Then 4 cm long pieces of fused silica capillary with OD 280 µm were inserted perpendicularly through the side walls of the frame, so that the capillaries formed a square cross that was fixed in the center by a drop of UV-cured adhesive (MANSON, CR). The frame with the capillaries was placed on a glass support and silicon rubber (RTV 615, ELCHEMCo, CR) mixed with a hardening component in a ratio of 10:1 v/v was poured into this prepared form; the mixture was then left to polymerize at 50 °C for 2 h. After hardening, the casting capillaries were first removed from the sides and then the polymer formed was cut out of the frame with a scalpel. Capillaries (OD 360–380 µm) could be inserted from the sides to any depth into the 2
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solutions were prepared using deionized water (DEI, 18.2 MΩ cm, Direct Q 3 UV, Millipore, Molsheim, France). Sucrose and D-lactose monohydrate were purchased from Sigma, Switzerland, D-fructose from Sigma, USA, D-galactose from Sigma, Italy, D-glucose anhydrous and NaOH from Fluka, Buchs, France, Na2HPO4 7H2O and Na3PO4 12 H2O from Aldrich, Steinheim, and LiOH from Aldrich, Milwaukee, USA. The 50 and 100 mmol/L aqueous solutions of NaOH were prepared by diluting carbonate-free 40% m/m NaOH with DEI (the carbonate-free NaOH solution was prepared from solid NaOH in the laboratory).
left inlet and its detection end was connected to the Agilent CE instrument. 2.2. Capillary electrophoresis Injection of the sample, flushing the capillary, application of the separation voltage, control of the experiment, including collection and processing of data were provided by the HP3D CE system (Agilent Technologies, Waldbronn, Germany). A 34.0 cm long fused silica capillary (ID 10 µm, OD 360 µm) with a length to C4D of 21 cm was inserted into the cassette with the incorporated C4D. The injection end of the capillary was led out of the CE instrument and connected with the PDMS cross-connector, where the part of capillary leading out of the cassette had a length of 15 cm. The other end remained in the CE instrument and was inserted in the electrophoretic vial, which could be pressurized or evacuated. From this end, the capillary was flushed with BGE between the individual analyses by the application of an overpressure of 920 mbar (92 kPa) or, on the other hand, the sample zone was injected into injection end of the capillary by the application of a vacuum of −50 mbar (−5 kPa). A high-voltage electrode was simultaneously placed in the output vial and the injection end was grounded; the micro-dialyzation part of the apparatus thus had zero potential versus ground and there was no danger of injury. The cassette including the incorporated C4D was thermostated at 25 °C. The axialtype C4D worked with a excitation sinusoidal signal with a frequency of 1 MHz and effective voltage value of 80 V (ADMET, CR) (Tůma, 2017). The tubular electrodes were 2.0 mm long and the detection gap equaled 1.0 mm. A shielding foil was inserted between the electrodes. A new capillary was flushed with 100 mmol/L NaOH for 20 min and the solution was left in the capillary for 30 min; then it was flushed with water for 10 min and finally with 50 mmol/L NaOH as BGE for 20 min. Between the individual analyses, the capillary was flushed with BGE from the end vial at a pressure of 920 mbar for 5 min or by electroosmosis through application of a voltage of 10 kV to the high-voltage electrode for 3 min. EOF was directed from the injection end towards the high-voltage electrode. Sample injection from the PDMS crossconnector into the capillary was hydrodynamic and was performed by application of a vacuum of −50 mbar (-5 kPa) to the end vial for 40 s, unless stated otherwise. The complete CE analysis consisted of a sequence of the following steps, which were controlled by Agilent ChemStation software, and synchronisation with an external three-way valve was provided through an A/D convertor (Interface 35900E, Agilent). The analysis was commenced by 5 min flushing of the capillary with BGE from the end vial; 15 s before the end of the flushing, the flow of gating solution to FGI was stopped by switching the valve to the OFF position. Then the microdialysate filled the space in the cross-connector in front of the entrance to the CE capillary, followed by hydrodynamic injection of the sample. The valve was switched to the ON position and the gating solution deflected the flow of the microdialysate away from the inlet into the capillary. A separation voltage of 10 kV with a ramp of 1 kV/s was applied 15 s after switching, see the time schedule (Table 1).
2.4. Treatment of the food samples The milk and yoghurts were purchased in a local retail outlet and the honey was obtained from a local beekeeper; specifically: semiskimmed milk (Olma, CR), Florian blueberry-flavored full-fat yoghurt (Olma, CR), plain full-fat yoghurt (Choceňská mlékárna, CR) and flower and honey-dew honey (from the Dívčí Kopy area, CR). 10 mmol/L NaOH was added to a 1.0 g sample of the tested yoghurt or milk to a final volume of 50.0 mL. The sample was enclosed in a screw-top plastic bottle and placed in an ultrasound bath (Sonorex Super RK106, Bandelin Electronic, Berlin, D) for 15 min. Then 10.0 mL of the suspended sample was transferred to a 12 mL glass vessel, 50 µL of 1.0 mol/L LiOH were added as an internal standard (IS) and a MD probe was inserted into the sample. The IS was used only to monitor the separation process; the peak area of the analyte was not corrected to the peak area of the IS for quantification. MD sampling was performed in a suspension stirred by a magnetic stirrer (MiniMag, Benchmark Scientific, U.S.A.). For the quantitative determination, the suspension was spiked with an aqueous solution of the individual saccharide standards. For the qualitative measurement, the MD probe was immersed directly in the untreated yoghurt, milk or honey. 2.5. Treatment and evaluation of the results All the CE analyses of the model samples were carried out in five consecutive runs and the plots represent the means ± the standard deviations. The Origin 7.0 program (OriginLab Corporation, Northampton, U.S.A.) was used to evaluate and statistically treat the experimental data. The number of theoretical plates was calculated from the formula, N = 5.54 (tM/w1/2)2/Lef, where tM is the migration time, w1/2 is the peak width at half-height and Lef is the effective length of separation capillary. The peak resolution, R, was computed from the relationship, R = 2(tM2 − tM1)/(w1 + w2), where tM is the migration time of the tested analyte and w is the peak width at the baseline. The LOD and LOQ values were determined from the peak height as the average concentrations corresponding to a signal/noise ratio of 3 or 10 respectively; the background C4D noise is 2.0 µV. Agilent ChemStation software was used for data collection from the C4D and subsequent data analysis, including precise measurement of migration times and peak areas. 3. Results and discussion 3.1. Electrophoretic separation of saccharides
2.3. Chemicals Based on the recommendation in ref. (Carvalho, da Silva, & do Lago, 2003; Duarte et al., 2019), alkaline BGEs based on an aqueous Na2HPO4/Na3PO4 mixture at pH > 12 mixed in various ratios was first tested for separation of the saccharides in combination with C4D. In phosphate BGEs, the C4D recording contains the system peaks that overlap directly with the saccharide peaks; the baseline is wavy and the electropherograms cannot be used for quantification of most of the saccharides. Consequently, further separation was performed in a carbonate-free aqueous solution of 50 mmol/L NaOH optimized for CE/ C4D determination of saccharides in foods, beverages and microdialyzates, based on our previous studies (Tůma et al., 2019; Tůma,
All the chemicals employed were of analytical purity and the Table 1 Time events during the process of sample injection in FGI. Time (s)
Event
0 15 55 70
Switch the valve to the OFF position Start sample injection by applying pressure Stop sample injection and switch the valve to the ON position Switch the high voltage on and start separation
3
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Table 2 Migration time (tM), number of theoretical plates (N) per meter, length of the separation capillary, given in thousands, electrophoretic resolution between two neighboring peaks (R, for sucrose given relative to the EOF zone) and RSD for the migration time and peak area for ten consecutive CE/MD analyses of fruit yoghurt. tM (s)
Sucrose Lactose Galactose Glucose Fructose
171.4 188.2 193.2 198.3 206.5
± ± ± ± ±
0.3 0.4 0.4 0.4 0.2
N/1000 (m−1)
R
280 252 326 176 177
4.3 5.5 1.6 1.5 1.9
± ± ± ± ±
10 7 20 5 4
± ± ± ± ±
0.1 0.1 0.0 0.0 0.0
RSD – time (%)
RSD – area (%)
0.2 0.2 0.2 0.2 0.2
5.9 4.7 7.6 5.7 4.4
suspension of the dairy product with standard additions of the individual saccharides. The CE analysis must be performed with a delay of at least 3 min from the spiking time, which is necessary to establish an equilibrium on the microdialysis membrane and thus to transport the microdialysate from the MD probe to the PDMS interface. The dependence of the peak area on the concentration for the tested concentration range can be described by a linear regression dependence with coefficient of determination (r2) greater than 0.999 for all the saccharides (Table 3). The slopes for the peak areas were used to calculate the saccharide concentrations in the dairy products. The LODs vary in the range 0.7–2.2 mg/L (2.9–6.9 µM). These are sufficiently low values for determination of the saccharides in a 50-fold diluted dairy product. The CE/MD technique enables determination of the whole saccharide profile in fruit and plain yoghurts and milk (Table 4). The determined concentrations are in good agreement with the value of the total saccharide content given on the labels of the individual products. Quantification performed by direct spiking of an aqueous suspension of the dairy product, which is subsequently sampled by microdialysis, successfully eliminates the effect of the matrix and yields relevant results. The actual analysis then corresponds to the predominant content of sucrose in fruit yoghurts, which is not generally known.
Fig. 2. CE/MD analysis of 50-fold diluted dairy products. A – complete electropherogram of fruit yoghurt with designation of the IS and EOF zones, B – detail of the separation of saccharides in fruit yoghurt, C – detail of the separation in plain yoghurt, D – detail of the separation in milk. Peak identification: 1 - sucrose, 2 – lactose, 3 – galactose, 4 – glucose, 5 – fructose. CE conditions: capillary length 34 cm, length to C4D 21 cm, ID/OD 10/360 µm, BGE 50 mmol/L NaOH, pH 12.6; hydrodynamic sample injection by a pressure of 50 mbar for 40 s, voltage/current 10 kV/ 3.0 µA, C4D with carrier frequency of 1 MHz and amplitude of 80 V.
Málková, Samcová, & Štulík, 2011; Vochyanová, Opekar, Tůma, & Štulík, 2012). Although this is not a classical buffer, it yielded the best results of all the tested solutions. At pH 12.6, saccharides migrate as anions against the direction of the fast EOF, which draws them into the detector. On the electropherogram, the peak of the Li+ ions used as IS appears first, followed by the EOF zone with electroneutral substances and then the peaks of the saccharides separated down to the baseline in the order of the slowest migrating sucrose, through lactose, galactose, glucose to the fastest migrating fructose (Fig. 2). The saccharide zones appear in C4D as negative peaks because the BGE co-ions are OH− ions, which have greater mobility than the saccharide anions. The employed BGE exhibits high electrical conductivity and must thus be combined with capillaries with small ID (Tůma, Samcová, & Štulík, 2011), here a 10 µm capillary, and simultaneously it is necessary to perform the separation at lower electric field intensities, here only 10 kV/34 cm. Otherwise, large amounts of Joule heat are released, manifested in waviness of the C4D baseline and it becomes difficult to evaluate the individual saccharide peaks. The migration times (tM), numbers of separation plates (N), chromatographic resolutions (R) and repeatabilities of the migration times and peak areas (RSD) for ten consecutive CE/MD analyses of fruit yoghurt are summarized in Table 2. Baseline separation of five saccharides is achieved in 3.5 min, with the lowest resolution value of two neighboring peaks (galactose/glucose) equal to 1.5. The number of theoretical plates is high and varies in the range 176,000–326,000 plates/m; the highest N value was found for galactose, which is present in the lowest quantitative amount in the yoghurt sample, see Fig. 2. This demonstrates that the highly efficient CE separation can be performed even under conditions when a larger part of the effective separation path is not thermostated, that sharp zones of the sample are injected into the separation capillary and also that uncontrolled mixing of the sample with the BGE does not occur in the PDMS connector.
3.3. Intra-day repeatability and inter-day reproducibility Intra-day repeatability was determined for ten consecutive CE/MD analyses of 50-fold diluted fruit yoghurt. The obtained RSD values for the migration time were excellent and attained a value of 0.2% for all the saccharides. RSDs for the peak area were in the range 4.4–7.6%, where the worst values were obtained for the minority peak of galactose. These RSDs for the peak area are slightly elevated compared to the normally cited values of 2–4% for commercial CE instruments but fully comparable with the RSD-area values for a laboratory-made instrument (Opekar & Tůma, 2016), Table 2. Inter-day reproducibility was tested for one fruit yoghurt, which was repeatedly processed over three successive days and analyzed 10 times each day. The obtained RSD values varied in the range 1.0–1.5% for the migration times and 6.5–10.0% for the peak areas. Table 3 Linear parameters of the calibration dependence for standard additions of saccharides to the microdialysate samples.
Sucrose Lactose Galactose Glucose Fructose
3.2. Quantification of saccharides in diluted dairy products The saccharides were quantified by direct spiking of an aqueous 4
Range (g/L)
Slope – area (mV.s.L/g)
r2
Slope – height (mV.L/g)
LOD (mg/ L)
LOQ (mg/ L)
0.01–1.2 0.01–0.75 0.01–0.15 0.01–0.75 0.01–0.75
12.6 ± 0.2 9.0 ± 0.2 25.2 ± 0.4 25.3 ± 0.3 25.6 ± 0.4
0.9996 0.9999 0.9993 0.9998 0.9994
6.0 2.8 8.8 6.1 4.9
1.0 2.2 0.7 1.0 1.2
3.3 7.3 2.3 3.3 4.0
± ± ± ± ±
0.1 0.0 0.4 0.1 0.1
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processes occurring in living tissues and organs at a molecular level, was used for the first time to determine saccharides in dairy products. The microdialysis apparatus was connected on-line with CE analysis using a PDMS cross-connector, where this CE/MD combination works in the flow-gating interface regime. The developed instrumentation facilitates the determination of the entire profile of saccharides in dairy products with minimum requirements on laboratory treatment of complicated food matrices and with a detection limit at the micromolar concentration level. The analytical technique is useful for rapid monitoring of food quality, monitoring changes during food processing and detection of falsification.
Table 4 Determination of the content of saccharides in yoghurt and milk in g per 100 g of product.
Sucrose Lactose Galactose Glucose Fructose Total Declared
Fruit yoghurt (g/ 100 g)
Plain yoghurt (g/ 100 g)
Milk (g/ 100 g)
5.83 3.11 0.33 1.89 1.95 13.1 13.9
– 2.85 ± 0.0 0.83 ± 0.0 – – 3.7 3.8
– 4.36 ± 0.0 – – – 4.4 4.6
± ± ± ± ±
0.0 0.0 0.0 0.0 0.0
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Fig. 3. Direct CE/MD analysis of saccharides in undiluted and untreated dairy products and honeys. A – fruit yoghurt, B – plain yoghurt, C – milk, D – flower honey, E – honey-dew honey. Peak identification: 1 - sucrose, 2 – lactose, 3 – galactose, 4 – glucose, 5 – fructose. The CE conditions are same as in Fig. 2 with the exception of the sample injection: 50 mbar for 1 s.
3.4. Qualitative analysis of saccharides in undiluted dairy products and honey In further tests, the MD probe was immersed directly in undiluted yoghurt, milk or honey. In these analyses, it was necessary to reduce the amount of sample injected into the capillary to an impulse corresponding to the application of 50 mbar for 1 s, to avoid exceeding the separation capacity of the capillary; all the other experimental parameters remained unchanged. Direct CE/MD analysis of undiluted foods provides information on their qualitative composition, from which it follows that the saccharide profile of fruit yoghurt consists of: sucrose, lactose, galactose, glucose and fructose; plain yogurt: lactose, galactose; milk: lactose; and flower and honey-dew honey: glucose and fructose (Fig. 3). This analytical procedure is suitable for rapid screening of saccharides in foods and detection of possible falsification. It follows from the described examples that plain yoghurt, milk and both kinds of honey contain only naturally occurring saccharides. In contrast, in addition to lactose and galactose derived from milk, fruit yoghurt also contains added glucose and fructose from fruit and, in addition, a large amount of sucrose. This procedure is not suitable for quantification as it does not enable spiking of an undiluted sample because of the very slow diffusion of saccharides in solid matrices. The developed CE/MD method was also successfully tested for the qualitative analysis of saccharides in apple and orange juice, red wine and Coca Cola. The results for these samples were equally satisfactory, confirming that. 4. Conclusions Microdialysis sampling of an object, familiar from monitoring 5
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from diabetic foot. Analytica Chimica Acta, 942, 139–145. https://doi.org/10.1016/j. aca.2016.09.008. Tůma, P., Málková, K., Samcová, E., & Štulík, K. (2011). Rapid monitoring of mono- and disaccharides in drinks, foodstuffs and foodstuff additives by capillary electrophoresis with contactless conductivity detection. Analytica Chimica Acta, 698(1–2), 1–5. https://doi.org/10.1016/j.aca.2011.04.055. Tůma, P., Samcová, E., & Štulík, K. (2011). Contactless conductivity detection in capillary electrophoresis employing capillaries with very low inner diameters. Electroanalysis, 23(8), 1870–1874. https://doi.org/10.1002/elan.201100264. Tůma, P., Sommerová, B., & Šiklová, M. (2019). Monitoring of adipose tissue metabolism using microdialysis and capillary electrophoresis with contactless conductivity detection. Talanta, 192, 380–386. https://doi.org/10.1016/j.talanta.2018.09.076. Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., & Quake, S. R. (2000). Monolithic microfabricated valves and pumps by multilayer soft lithography. Science, 288(5463), 113–116. https://doi.org/10.1126/science.288.5463.113. Vochyanová, B., Opekar, F., Tůma, P., & Štulík, K. (2012). Rapid determinations of saccharides in high-energy drinks by short-capillary electrophoresis with contactless conductivity detection. Analytical and Bioanalytical Chemistry, 404(5), 1549–1554. https://doi.org/10.1007/s00216-012-6242-x. Zhang, A. H., Sun, H., Wang, P., Han, Y., & Wang, X. J. (2012). Modern analytical techniques in metabolomics analysis. Analyst, 137(2), 293–300. https://doi.org/10. 1039/c1an15605e. Zhang, Q. Y., & Gong, M. J. (2014). Prototyping of poly(dimethylsiloxane) interfaces for flow gating, reagent mixing, and tubing connection in capillary electrophoresis. Journal of Chromatography A, 1324, 231–237. https://doi.org/10.1016/j.chroma. 2013.11.043.
3390/s130302786. Saylor, R. A., & Lunte, S. M. (2015). A review of microdialysis coupled to microchip electrophoresis for monitoring biological events. Journal of Chromatography A, 1382, 48–64. https://doi.org/10.1016/j.chroma.2014.12.086. Schultz, K. N., & Kennedy, R. T. (2008). Time-Resolved Microdialysis for In Vivo Neurochemical Measurements and Other Applications. In Annual Review of Analytical Chemistry, vol. 1 (pp. 627-661). Palo Alto: Annual Reviews. https://doi. org/10.1146/annurev.anchem.1.031207.113047. Slaney, T. R., Nie, J., Hershey, N. D., Thwar, P. K., Linderman, J., Burns, M. A., & Kennedy, R. T. (2011). Push-pull perfusion sampling with segmented flow for high temporal and spatial resolution in vivo chemical monitoring. Analytical Chemistry, 83(13), 5207–5213. https://doi.org/10.1021/ac2003938. Sloan, C. D. K., Nandi, P., Linz, T. H., Aldrich, J. V., Audus, K. L., & Lunte, S. M. (2012). Analytical and Biological Methods for Probing the Blood-Brain Barrier. In R. G. Cooks & E. S. Yeung (Eds.), Annual Review of Analytical Chemistry, vol. 5 (pp. 505-531). Palo Alto: Annual Reviews. https://doi.org/10.1146/annurev-anchem-062011143002. Šolínová, V., & Kašička, V. (2006). Recent applications of conductivity detection in capillary and chip electrophoresis. Journal of Separation Science, 29(12), 1743–1762. https://doi.org/10.1002/jssc.200600167. Tůma, P. (2017). Frequency-tuned contactless conductivity detector for the electrophoretic separation of clinical samples in capillaries with very small internal dimensions. Journal of Separation Science, 40(4), 940–947. https://doi.org/10.1002/ jssc.201601213. Tůma, P., Jaček, M., Fejfarová, V., & Polák, J. (2016). Electrophoretic stacking for sensitive determination of antibiotic ceftazidime in human blood and microdialysates
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On-line coupling of capillary electrophoresis with microdialysis for determining saccharides in dairy products and honey
T
⁎
Petr Tůmaa, , Blanka Sommerováa, Václav Daněčekb a b
Charles University, Third Faculty of Medicine, Department of Hygiene, Ruská 87, 100 00 Prague 10, Czech Republic Charles University, Third Faculty of Medicine, Department of Biophysics, Ruská 87, 100 00 Prague 10, Czech Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Contactless conductivity detection Flow-gating interface Food analysis Polydimethylsiloxane Sequence analysis
Free sucrose, lactose, galactose, glucose and fructose were determined in yoghurts, milk and honey using on-line coupling of capillary electrophoresis with microdialysis. The dairy products were diluted 50-fold with 10 mmol/ L NaOH and sampled using laboratory-made microdialysis probes. The microdialysate was brought to the entrance of the electrophoretic capillary and the coupling consisted in a polydimethylsiloxane (PDMS) cross connector working in the flow-gating interface regime. The electrophoretic analysis was performed in 50 mmol/ L NaOH (pH 12.6) background electrolyte, where baseline separation of the five saccharides was achieved in 3.5 min. The LOQs varied in the range 2.3–7.3 mg/L, the number of separation plates varied between 176,000 plates/m for glucose to 326,000 plates/m for galactose and the relative standard deviation (RSD) for ten consecutive analyses of fruit yoghurt was 0.2% for the migration time and 4.4–7.6% for the peak area.
1. Introduction Microdialysis (MD) is a modern semi-invasive technique for continuous sampling of living tissue and organs, used primarily because of its minimal unpleasantness of sampling, with little influence on the ongoing biochemical and physiological processes (Brunner & Derendorf, 2006; Kennedy, 2013; Schultz & Kennedy, 2008; Sloan, Nandi, Linz, Aldrich, Audus, & Lunte, 2012; Zhang, Sun, Wang, Han, & Wang, 2012). Microdialysis sampling is performed using miniature probes, which are inserted into the studied tissue so that the microdialysis membrane is in direct contact with the tissue medium (Lee, Slaney, Hower, & Kennedy, 2013; Nandi & Lunte, 2009; Slaney et al., 2011). The actual probe most frequently consists of a 10–30 mm long microdialysis tube with a diameter of several tenths of a millimeter, connected to two tubes (Kennedy, 2013; Lee et al., 2013; Nandi & Lunte, 2009; Saylor & Lunte, 2015). The input tube brings the acceptor solution from the syringe pump to the probe, where it washes the inside of the microdialysis tube and is gradually enriched in the analytes that diffuse from the surrounding tissue through the microdialysis membrane into the flowing acceptor solution. This process produces a solution of the microdialysate flowing from the probe through the outlet tube, which is collected for subsequent analysis. Primarily small hydrophilic molecules diffuse through the microdialysis membrane and the efficiency of
the microdialysis sampling, measured as the ratio between the concentrations of the substance in the microdialysate and in the tissue, decreases exponentially with increasing flow rate. The flow rate varies between 0.5 and 10 µL/min and the microdialysis commonly has a yield of about 20–50% for small hydrophilic substances (Nandi & Lunte, 2009; Saylor & Lunte, 2015). Reduction of the flow rate is limited primarily by the very small volume of microdialysate obtained for subsequent instrumental analysis, which is critical especially in the offline arrangement, where the microdialysate must be further handled during its laboratory treatment (Tůma, Jaček, Fejfarová, & Polák, 2016; Tůma, Sommerová, & Šiklová, 2019). Consequently, it is much better to analyze the microdialysate flowing out of the probe in real time through on-line coupling between the microdialysis apparatus and a compatible microanalytical technique. A useful instrumental technique for these purposes consists in capillary (CE) (Guihen & O'Connor, 2009, 2010; Nandi & Lunte, 2009) or chip (Huynh, Fogarty, Martin, & Lunte, 2004; Nandi, Desaias, & Lunte, 2010; Saylor & Lunte, 2015) electrophoresis performed in capillaries and channels with a small inner diameter (ID) that is directly comparable with the ID of the tubes used in the microdialysis probe. In addition, CE is characterized by high separation efficiency, which is a necessary condition for the analysis of complicated clinical and food samples. Its short separation time is a vital factor in sequential analyses
Abbreviations: BGE, background electrolyte; C4D, contactless conductivity detection; DEI, deionised water; FGI, flow-gating interface; ID, inner diameter; MD, microdialysis; OD, outer diameter; PDMS, polydimethylsiloxane ⁎ Corresponding author. E-mail address: [email protected] (P. Tůma). https://doi.org/10.1016/j.foodchem.2020.126362 Received 17 July 2019; Received in revised form 24 October 2019; Accepted 4 February 2020 Available online 05 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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at short time intervals and it has the additional advantage of requiring small amounts of reagents, making it a suitable technique for green chemistry (Koel, Borissova, Vaher, & Kaljurand, 2011). It is important to develop a suitable interface between MD and CE, as this is not currently available commercially and individual laboratories make their own. The main function of the interface lies in separation of the continuous stream of collected microdialysate into discrete zones, which are injected into the capillary only at specified times. A potential technical solution consists in a cross-shaped flow-gating interface (FGI) in which the following meet in a single point: i) the entrance into the separation capillary, ii) the exit from the microdialysis probe, iii) the capillary bringing the gating solution (usually the background electrolyte for electrophoretic separation, BGE) and iv) the exit to waste (Gong, Zhang, & Maddukuri, 2018; Opekar & Tůma, 2017). In the normal FGI working regime, the microdialysate is deflected from the entrance into the separation capillary by a stream of gating solution and is fed to the waste. Only during injection is the flow of gating solution stopped by switching the valve to the OFF position so that the microdialysate fills the space in front of the entrance into the separation capillary and is injected into it. The valve is then switched to the ON position, renewing the flow of gating solution, deflecting the microdialysate from the entrance into the separation capillary and the actual CE separation takes place. This series of individual steps is constantly repeated, forming the basis of sequence analysis (Opekar & Tůma, 2017). This contribution is concerned with the development and construction of a coupling between a commercial Agilent CE instrument and microdialysis sampling of yoghurts, milk and honeys to determine the contents of free saccharides. The saccharides are separated in aqueous solutions of sodium hydroxide and detected by contactless conductivity detection (C4D). The axial construction of C4D consists of two tubular metal electrodes with a length of more than 2 mm that are placed around the outer capillary surface. The electrodes are separated by a detection gap of about one millimetre into which is inserted a shielding foil. The first electrode acts as a transmitter of an alternating signal with a frequency between hundreds of kHz and tens of MHz; the second electrode acts as the receiver. C4D represents a universal detection technique for CE and is employed particularly for the detection of minerals, amino acids, saccharides, low-molecular weight organic acids and other compounds with low absorbance in the UV region (Kubáň & Hauser, 2018, 2019; Opekar, Tůma, & Štulík, 2013; Šolínová & Kašička, 2006).
Fig. 1. Scheme for on-line coupling of the microdialysis apparatus with the capillary electrophoresis instrument through a flow-gating interface made from a PDMS cross-connector.
entrances of this PDMS cross-connector (dimensions: height 15 mm, length 15 mm, thickness 3 mm). The inserted capillaries were firmly sealed in PDMS to avoid leaking of solutions and the connector had a minimal internal dead volume. PDMS connectors could be made by a more complicated procedure using lithographic techniques (Unger, Chou, Thorsen, Scherer, & Quake, 2000). The inlets and outlets of crossconnectors (Fig. 1A): i) To the upper inlet was connected a capillary with ID/OD 100/ 360 µm and length 3.0 cm, the other end of which was connected to the outlet of a three-way magnetically controlled valve (Science Instruments and Software, CR). The BGE with a flow rate of 83 µL/ min was pressed out of a 50 mL injection syringe using a linear piston pump (TJ-3A; MRC LTD, Israel) and was fed to the PDMS cross-connector through this inlet. When the valve was moved to the OFF position just before injecting the sample into the CE capillary, the BGE passed from the valve directly into the waste. ii) The microdialysate was fed to the right-hand inlet through a capillary with ID/OD 100/150 µm; the connection of the thinner capillary in the PDMS cross-connector was sealed by a drop of UVcured adhesive around the outer surface of the capillary 2 mm from its end. The actual laboratory-made MD probe was made from a 4 cm long microdialysation tube (ID/OD 200/216 µm, cut-off 13 kDa, Spectrum Labs, USA), into both of whose ends two fused silica ID/OD 75/150 µm capillaries were inserted to a depth of 1 mm and the connections were sealed with UV-cured adhesive. The inlet capillary was connected with the acceptor solution (10 mmol/ L NaOH) by a 5 mL injection syringe. A solution flow rate of 5 µL/ min was provided by a linear piston pump (Double NE-4500 OEM, New Era Pump Systems, U.S.A.). The MD probe was immersed in the sample and the outlet was brought to the PDMS connector. Several acceptor solutions, such as 10 mmol/L HCl, DEI and 10 mmol/L NaOH, were tested for microdialysis of saccharides with the highest recovery for an aqueous solution of NaOH, resulting from deprotonation of saccharides at high pH. The microdialysis flow rate was changed in the 1–10 µL/min range with an optimized value of 5 µL/min. The employed flow rate is closely related to the amount of sample that is injected into the separation capillary to achieve baseline separation of the saccharides. iii) A 15 mm stainless steel tube cut from an ID/OD 400/800 µm injection syringe (B/Braun, Melsungen, Germany) was inserted into the bottom outlet and the tube was mechanically connected to the grounding contact from the high-voltage source. The other end of the tube was connected using UV-cured adhesive to a 5 cm long capillary ID/OD 100/360 µm, which fed the solution from the PDMS cross-connector to the waste. iv) The injection end of the separation capillary was inserted into the
2. Experimental 2.1. Instrumentation for FGI In this work, the main FGI component was a cross-shaped polydimethylsiloxane (PDMS) connector with four entrances for close connection of the fused silica capillaries with an outer diameter (OD) of about 360 µm (Polymicro Technologies, Phoenix, U.S.A.). The connector was made in the laboratory by the casting technology described in general in ref. (Zhang & Gong, 2014) and specifically modified. Briefly, first a square frame with a 25 mm long outer edge and inner square opening with dimensions of 15 × 15 mm was cut from silicon foil with a thickness of 3 mm. Then 4 cm long pieces of fused silica capillary with OD 280 µm were inserted perpendicularly through the side walls of the frame, so that the capillaries formed a square cross that was fixed in the center by a drop of UV-cured adhesive (MANSON, CR). The frame with the capillaries was placed on a glass support and silicon rubber (RTV 615, ELCHEMCo, CR) mixed with a hardening component in a ratio of 10:1 v/v was poured into this prepared form; the mixture was then left to polymerize at 50 °C for 2 h. After hardening, the casting capillaries were first removed from the sides and then the polymer formed was cut out of the frame with a scalpel. Capillaries (OD 360–380 µm) could be inserted from the sides to any depth into the 2
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solutions were prepared using deionized water (DEI, 18.2 MΩ cm, Direct Q 3 UV, Millipore, Molsheim, France). Sucrose and D-lactose monohydrate were purchased from Sigma, Switzerland, D-fructose from Sigma, USA, D-galactose from Sigma, Italy, D-glucose anhydrous and NaOH from Fluka, Buchs, France, Na2HPO4 7H2O and Na3PO4 12 H2O from Aldrich, Steinheim, and LiOH from Aldrich, Milwaukee, USA. The 50 and 100 mmol/L aqueous solutions of NaOH were prepared by diluting carbonate-free 40% m/m NaOH with DEI (the carbonate-free NaOH solution was prepared from solid NaOH in the laboratory).
left inlet and its detection end was connected to the Agilent CE instrument. 2.2. Capillary electrophoresis Injection of the sample, flushing the capillary, application of the separation voltage, control of the experiment, including collection and processing of data were provided by the HP3D CE system (Agilent Technologies, Waldbronn, Germany). A 34.0 cm long fused silica capillary (ID 10 µm, OD 360 µm) with a length to C4D of 21 cm was inserted into the cassette with the incorporated C4D. The injection end of the capillary was led out of the CE instrument and connected with the PDMS cross-connector, where the part of capillary leading out of the cassette had a length of 15 cm. The other end remained in the CE instrument and was inserted in the electrophoretic vial, which could be pressurized or evacuated. From this end, the capillary was flushed with BGE between the individual analyses by the application of an overpressure of 920 mbar (92 kPa) or, on the other hand, the sample zone was injected into injection end of the capillary by the application of a vacuum of −50 mbar (−5 kPa). A high-voltage electrode was simultaneously placed in the output vial and the injection end was grounded; the micro-dialyzation part of the apparatus thus had zero potential versus ground and there was no danger of injury. The cassette including the incorporated C4D was thermostated at 25 °C. The axialtype C4D worked with a excitation sinusoidal signal with a frequency of 1 MHz and effective voltage value of 80 V (ADMET, CR) (Tůma, 2017). The tubular electrodes were 2.0 mm long and the detection gap equaled 1.0 mm. A shielding foil was inserted between the electrodes. A new capillary was flushed with 100 mmol/L NaOH for 20 min and the solution was left in the capillary for 30 min; then it was flushed with water for 10 min and finally with 50 mmol/L NaOH as BGE for 20 min. Between the individual analyses, the capillary was flushed with BGE from the end vial at a pressure of 920 mbar for 5 min or by electroosmosis through application of a voltage of 10 kV to the high-voltage electrode for 3 min. EOF was directed from the injection end towards the high-voltage electrode. Sample injection from the PDMS crossconnector into the capillary was hydrodynamic and was performed by application of a vacuum of −50 mbar (-5 kPa) to the end vial for 40 s, unless stated otherwise. The complete CE analysis consisted of a sequence of the following steps, which were controlled by Agilent ChemStation software, and synchronisation with an external three-way valve was provided through an A/D convertor (Interface 35900E, Agilent). The analysis was commenced by 5 min flushing of the capillary with BGE from the end vial; 15 s before the end of the flushing, the flow of gating solution to FGI was stopped by switching the valve to the OFF position. Then the microdialysate filled the space in the cross-connector in front of the entrance to the CE capillary, followed by hydrodynamic injection of the sample. The valve was switched to the ON position and the gating solution deflected the flow of the microdialysate away from the inlet into the capillary. A separation voltage of 10 kV with a ramp of 1 kV/s was applied 15 s after switching, see the time schedule (Table 1).
2.4. Treatment of the food samples The milk and yoghurts were purchased in a local retail outlet and the honey was obtained from a local beekeeper; specifically: semiskimmed milk (Olma, CR), Florian blueberry-flavored full-fat yoghurt (Olma, CR), plain full-fat yoghurt (Choceňská mlékárna, CR) and flower and honey-dew honey (from the Dívčí Kopy area, CR). 10 mmol/L NaOH was added to a 1.0 g sample of the tested yoghurt or milk to a final volume of 50.0 mL. The sample was enclosed in a screw-top plastic bottle and placed in an ultrasound bath (Sonorex Super RK106, Bandelin Electronic, Berlin, D) for 15 min. Then 10.0 mL of the suspended sample was transferred to a 12 mL glass vessel, 50 µL of 1.0 mol/L LiOH were added as an internal standard (IS) and a MD probe was inserted into the sample. The IS was used only to monitor the separation process; the peak area of the analyte was not corrected to the peak area of the IS for quantification. MD sampling was performed in a suspension stirred by a magnetic stirrer (MiniMag, Benchmark Scientific, U.S.A.). For the quantitative determination, the suspension was spiked with an aqueous solution of the individual saccharide standards. For the qualitative measurement, the MD probe was immersed directly in the untreated yoghurt, milk or honey. 2.5. Treatment and evaluation of the results All the CE analyses of the model samples were carried out in five consecutive runs and the plots represent the means ± the standard deviations. The Origin 7.0 program (OriginLab Corporation, Northampton, U.S.A.) was used to evaluate and statistically treat the experimental data. The number of theoretical plates was calculated from the formula, N = 5.54 (tM/w1/2)2/Lef, where tM is the migration time, w1/2 is the peak width at half-height and Lef is the effective length of separation capillary. The peak resolution, R, was computed from the relationship, R = 2(tM2 − tM1)/(w1 + w2), where tM is the migration time of the tested analyte and w is the peak width at the baseline. The LOD and LOQ values were determined from the peak height as the average concentrations corresponding to a signal/noise ratio of 3 or 10 respectively; the background C4D noise is 2.0 µV. Agilent ChemStation software was used for data collection from the C4D and subsequent data analysis, including precise measurement of migration times and peak areas. 3. Results and discussion 3.1. Electrophoretic separation of saccharides
2.3. Chemicals Based on the recommendation in ref. (Carvalho, da Silva, & do Lago, 2003; Duarte et al., 2019), alkaline BGEs based on an aqueous Na2HPO4/Na3PO4 mixture at pH > 12 mixed in various ratios was first tested for separation of the saccharides in combination with C4D. In phosphate BGEs, the C4D recording contains the system peaks that overlap directly with the saccharide peaks; the baseline is wavy and the electropherograms cannot be used for quantification of most of the saccharides. Consequently, further separation was performed in a carbonate-free aqueous solution of 50 mmol/L NaOH optimized for CE/ C4D determination of saccharides in foods, beverages and microdialyzates, based on our previous studies (Tůma et al., 2019; Tůma,
All the chemicals employed were of analytical purity and the Table 1 Time events during the process of sample injection in FGI. Time (s)
Event
0 15 55 70
Switch the valve to the OFF position Start sample injection by applying pressure Stop sample injection and switch the valve to the ON position Switch the high voltage on and start separation
3
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Table 2 Migration time (tM), number of theoretical plates (N) per meter, length of the separation capillary, given in thousands, electrophoretic resolution between two neighboring peaks (R, for sucrose given relative to the EOF zone) and RSD for the migration time and peak area for ten consecutive CE/MD analyses of fruit yoghurt. tM (s)
Sucrose Lactose Galactose Glucose Fructose
171.4 188.2 193.2 198.3 206.5
± ± ± ± ±
0.3 0.4 0.4 0.4 0.2
N/1000 (m−1)
R
280 252 326 176 177
4.3 5.5 1.6 1.5 1.9
± ± ± ± ±
10 7 20 5 4
± ± ± ± ±
0.1 0.1 0.0 0.0 0.0
RSD – time (%)
RSD – area (%)
0.2 0.2 0.2 0.2 0.2
5.9 4.7 7.6 5.7 4.4
suspension of the dairy product with standard additions of the individual saccharides. The CE analysis must be performed with a delay of at least 3 min from the spiking time, which is necessary to establish an equilibrium on the microdialysis membrane and thus to transport the microdialysate from the MD probe to the PDMS interface. The dependence of the peak area on the concentration for the tested concentration range can be described by a linear regression dependence with coefficient of determination (r2) greater than 0.999 for all the saccharides (Table 3). The slopes for the peak areas were used to calculate the saccharide concentrations in the dairy products. The LODs vary in the range 0.7–2.2 mg/L (2.9–6.9 µM). These are sufficiently low values for determination of the saccharides in a 50-fold diluted dairy product. The CE/MD technique enables determination of the whole saccharide profile in fruit and plain yoghurts and milk (Table 4). The determined concentrations are in good agreement with the value of the total saccharide content given on the labels of the individual products. Quantification performed by direct spiking of an aqueous suspension of the dairy product, which is subsequently sampled by microdialysis, successfully eliminates the effect of the matrix and yields relevant results. The actual analysis then corresponds to the predominant content of sucrose in fruit yoghurts, which is not generally known.
Fig. 2. CE/MD analysis of 50-fold diluted dairy products. A – complete electropherogram of fruit yoghurt with designation of the IS and EOF zones, B – detail of the separation of saccharides in fruit yoghurt, C – detail of the separation in plain yoghurt, D – detail of the separation in milk. Peak identification: 1 - sucrose, 2 – lactose, 3 – galactose, 4 – glucose, 5 – fructose. CE conditions: capillary length 34 cm, length to C4D 21 cm, ID/OD 10/360 µm, BGE 50 mmol/L NaOH, pH 12.6; hydrodynamic sample injection by a pressure of 50 mbar for 40 s, voltage/current 10 kV/ 3.0 µA, C4D with carrier frequency of 1 MHz and amplitude of 80 V.
Málková, Samcová, & Štulík, 2011; Vochyanová, Opekar, Tůma, & Štulík, 2012). Although this is not a classical buffer, it yielded the best results of all the tested solutions. At pH 12.6, saccharides migrate as anions against the direction of the fast EOF, which draws them into the detector. On the electropherogram, the peak of the Li+ ions used as IS appears first, followed by the EOF zone with electroneutral substances and then the peaks of the saccharides separated down to the baseline in the order of the slowest migrating sucrose, through lactose, galactose, glucose to the fastest migrating fructose (Fig. 2). The saccharide zones appear in C4D as negative peaks because the BGE co-ions are OH− ions, which have greater mobility than the saccharide anions. The employed BGE exhibits high electrical conductivity and must thus be combined with capillaries with small ID (Tůma, Samcová, & Štulík, 2011), here a 10 µm capillary, and simultaneously it is necessary to perform the separation at lower electric field intensities, here only 10 kV/34 cm. Otherwise, large amounts of Joule heat are released, manifested in waviness of the C4D baseline and it becomes difficult to evaluate the individual saccharide peaks. The migration times (tM), numbers of separation plates (N), chromatographic resolutions (R) and repeatabilities of the migration times and peak areas (RSD) for ten consecutive CE/MD analyses of fruit yoghurt are summarized in Table 2. Baseline separation of five saccharides is achieved in 3.5 min, with the lowest resolution value of two neighboring peaks (galactose/glucose) equal to 1.5. The number of theoretical plates is high and varies in the range 176,000–326,000 plates/m; the highest N value was found for galactose, which is present in the lowest quantitative amount in the yoghurt sample, see Fig. 2. This demonstrates that the highly efficient CE separation can be performed even under conditions when a larger part of the effective separation path is not thermostated, that sharp zones of the sample are injected into the separation capillary and also that uncontrolled mixing of the sample with the BGE does not occur in the PDMS connector.
3.3. Intra-day repeatability and inter-day reproducibility Intra-day repeatability was determined for ten consecutive CE/MD analyses of 50-fold diluted fruit yoghurt. The obtained RSD values for the migration time were excellent and attained a value of 0.2% for all the saccharides. RSDs for the peak area were in the range 4.4–7.6%, where the worst values were obtained for the minority peak of galactose. These RSDs for the peak area are slightly elevated compared to the normally cited values of 2–4% for commercial CE instruments but fully comparable with the RSD-area values for a laboratory-made instrument (Opekar & Tůma, 2016), Table 2. Inter-day reproducibility was tested for one fruit yoghurt, which was repeatedly processed over three successive days and analyzed 10 times each day. The obtained RSD values varied in the range 1.0–1.5% for the migration times and 6.5–10.0% for the peak areas. Table 3 Linear parameters of the calibration dependence for standard additions of saccharides to the microdialysate samples.
Sucrose Lactose Galactose Glucose Fructose
3.2. Quantification of saccharides in diluted dairy products The saccharides were quantified by direct spiking of an aqueous 4
Range (g/L)
Slope – area (mV.s.L/g)
r2
Slope – height (mV.L/g)
LOD (mg/ L)
LOQ (mg/ L)
0.01–1.2 0.01–0.75 0.01–0.15 0.01–0.75 0.01–0.75
12.6 ± 0.2 9.0 ± 0.2 25.2 ± 0.4 25.3 ± 0.3 25.6 ± 0.4
0.9996 0.9999 0.9993 0.9998 0.9994
6.0 2.8 8.8 6.1 4.9
1.0 2.2 0.7 1.0 1.2
3.3 7.3 2.3 3.3 4.0
± ± ± ± ±
0.1 0.0 0.4 0.1 0.1
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processes occurring in living tissues and organs at a molecular level, was used for the first time to determine saccharides in dairy products. The microdialysis apparatus was connected on-line with CE analysis using a PDMS cross-connector, where this CE/MD combination works in the flow-gating interface regime. The developed instrumentation facilitates the determination of the entire profile of saccharides in dairy products with minimum requirements on laboratory treatment of complicated food matrices and with a detection limit at the micromolar concentration level. The analytical technique is useful for rapid monitoring of food quality, monitoring changes during food processing and detection of falsification.
Table 4 Determination of the content of saccharides in yoghurt and milk in g per 100 g of product.
Sucrose Lactose Galactose Glucose Fructose Total Declared
Fruit yoghurt (g/ 100 g)
Plain yoghurt (g/ 100 g)
Milk (g/ 100 g)
5.83 3.11 0.33 1.89 1.95 13.1 13.9
– 2.85 ± 0.0 0.83 ± 0.0 – – 3.7 3.8
– 4.36 ± 0.0 – – – 4.4 4.6
± ± ± ± ±
0.0 0.0 0.0 0.0 0.0
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement Financial support from the Grant Agency of the Czech Republic, Grant No. 18-04902S, is gratefully acknowledged. References Brunner, M., & Derendorf, H. (2006). Clinical microdialysis: Current applications and potential use in drug development. TrAC Trends in Analytical Chemistry, 25(7), 674–680. https://doi.org/10.1016/j.trac.2006.05.004. Carvalho, A. Z., da Silva, J. A. F., & do Lago, C. L. (2003). Determination of mono- and disaccharides by capillary electrophoresis with contactless conductivity detection. Electrophoresis, 24(12–13), 2138–2143. https://doi.org/10.1002/elps.200305408. Duarte, G. F., Lobo, E. O., Medeiros, I., da Silua, J. A. F., do Lago, C. L., & Coltro, W. K. T. (2019). Separation of carbohydrates on electrophoresis microchips with controlled electrolysis. Electrophoresis, 40(5), 693–698. https://doi.org/10.1002/elps. 201800354. Gong, M. J., Zhang, N., & Maddukuri, N. (2018). Flow-gated capillary electrophoresis: A powerful technique for rapid and efficient chemical separation. Analytical Methods, 10(26), 3131–3143. https://doi.org/10.1039/c8ay00979a. Guihen, E., & O'Connor, W. T. (2009). Current separation and detection methods in microdialysis the drive towards sensitivity and speed. Electrophoresis, 30(12), 2062–2075. https://doi.org/10.1002/elps.200900039. Guihen, E., & O'Connor, W. T. (2010). Capillary and microchip electrophoresis in microdialysis: Recent applications. Electrophoresis, 31(1), 55–64. https://doi.org/10. 1002/elps.200900467. Huynh, B. H., Fogarty, B. A., Martin, R. S., & Lunte, S. M. (2004). On-line coupling of microdialysis sampling with microchip-based capillary electrophoresis. Analytical Chemistry, 76(21), 6440–6447. https://doi.org/10.1021/ac049365i. Kennedy, R. T. (2013). Emerging trends in in vivo neurochemical monitoring by microdialysis. Current Opinion in Chemical Biology, 17(5), 860–867. https://doi.org/10. 1016/j.cbpa.2013.06.012. Koel, M., Borissova, M., Vaher, M., & Kaljurand, M. (2011). Developments in the application of Green Chemistry principles to food analysis Capillary electrophoresis for the analysis of ingredients in food products. Agro Food Industry Hi-Tech, 22(5), 27–29. https://www.researchgate.net/publication/287915711. Kubáň, P., & Hauser, P. C. (2018). 20th anniversary of axial capacitively coupled contactless conductivity detection in capillary electrophoresis. TrAC Trends in Analytical Chemistry, 102, 311–321. https://doi.org/10.1016/j.trac.2018.03.007. Kubáň, P., & Hauser, P. C. (2019). Contactless conductivity detection for analytical techniques: Developments from 2016 to 2018. Electrophoresis, 40(1), 124–139. https://doi.org/10.1002/elps.201800248. Lee, W. H., Slaney, T. R., Hower, R. W., & Kennedy, R. T. (2013). Microfabricated Sampling Probes for in Vivo Monitoring of Neurotransmitters. Analytical Chemistry, 85(8), 3828–3831. https://doi.org/10.1021/ac400579x. Nandi, P., Desaias, D. P., & Lunte, S. M. (2010). Development of a PDMS-based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples. Electrophoresis, 31(8), 1414–1422. https://doi.org/10.1002/elps. 200900612. Nandi, P., & Lunte, S. M. (2009). Recent trends in microdialysis sampling integrated with conventional and microanalytical systems for monitoring biological events: A review. Analytica Chimica Acta, 651(1), 1–14. https://doi.org/10.1016/j.aca.2009.07.064. Opekar, F., & Tůma, P. (2016). Dual-channel capillary electrophoresis for simultaneous determination of cations and anions. Journal of Chromatography A, 1446, 158–163. https://doi.org/10.1016/j.chroma.2016.04.015. Opekar, F., & Tůma, P. (2017). Hydrodynamic sample injection into short electrophoretic capillary in systems with a flow-gating interface. Journal of Chromatography A, 1480, 93–98. https://doi.org/10.1016/j.chroma.2016.12.029. Opekar, F., Tůma, P., & Štulík, K. (2013). Contactless impedance sensors and their application to flow measurements. Sensors, 13(3), 2786–2801. https://doi.org/10.
Fig. 3. Direct CE/MD analysis of saccharides in undiluted and untreated dairy products and honeys. A – fruit yoghurt, B – plain yoghurt, C – milk, D – flower honey, E – honey-dew honey. Peak identification: 1 - sucrose, 2 – lactose, 3 – galactose, 4 – glucose, 5 – fructose. The CE conditions are same as in Fig. 2 with the exception of the sample injection: 50 mbar for 1 s.
3.4. Qualitative analysis of saccharides in undiluted dairy products and honey In further tests, the MD probe was immersed directly in undiluted yoghurt, milk or honey. In these analyses, it was necessary to reduce the amount of sample injected into the capillary to an impulse corresponding to the application of 50 mbar for 1 s, to avoid exceeding the separation capacity of the capillary; all the other experimental parameters remained unchanged. Direct CE/MD analysis of undiluted foods provides information on their qualitative composition, from which it follows that the saccharide profile of fruit yoghurt consists of: sucrose, lactose, galactose, glucose and fructose; plain yogurt: lactose, galactose; milk: lactose; and flower and honey-dew honey: glucose and fructose (Fig. 3). This analytical procedure is suitable for rapid screening of saccharides in foods and detection of possible falsification. It follows from the described examples that plain yoghurt, milk and both kinds of honey contain only naturally occurring saccharides. In contrast, in addition to lactose and galactose derived from milk, fruit yoghurt also contains added glucose and fructose from fruit and, in addition, a large amount of sucrose. This procedure is not suitable for quantification as it does not enable spiking of an undiluted sample because of the very slow diffusion of saccharides in solid matrices. The developed CE/MD method was also successfully tested for the qualitative analysis of saccharides in apple and orange juice, red wine and Coca Cola. The results for these samples were equally satisfactory, confirming that. 4. Conclusions Microdialysis sampling of an object, familiar from monitoring 5
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