Communication Electrochemistry, 88(6), 522–524 (2020)

Determination of Lactate by Sequential Injection AnalysisUsing a Fluoride Ion-selective Electrode Detector

Yuichi TAKAHASHI and Takashi MASADOME*

Department of Applied Chemistry, Faculty of Engineering, Shibaura Institute of Technology,Toyosu, Koto-ku, Tokyo 135-8548, Japan

* Corresponding author: masadome@sic.shibaura-it.ac.jp

ABSTRACT

Principle of determination of lactate using sequential injectionanalysis system

C3H6O3 + O2 C3H4O3 + H2O2 (1)

Lactateoxidase

Lactate Pyruvic acid

+ + H2O2 + 2 H2O + H+

+ F- (2)

4-Aminoantipyline(4-AAP)

4-Fluorophenol(4-FP)

Produced F- ion is detected with F- ISE

Horseradish Peroxidase(HRP)

A combination of sequential Injection Analysis (SIA) system and theion-selective electrode (ISE) detector is considered to be one of avery effective analytical method. Reaction of lactate with lactateoxidase produces pyruvic acid and hydrogen peroxide. When theproduced hydrogen peroxide is reacted with 4-aminoantipyrine (4-AAP) and 4-fluorophenol (4-FP) in the presence of horseradishperoxidase (HRP), F− ion is produced. The concentration of lactatecan be indirectly determined by measuring the F− ion by F− ISE.Based on this principle of detection of lactate, a new measurementmethod of lactate using an SIA with ISE detector was developed.

© The Author(s) 2020. Published by ECSJ. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY,http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any medium provided the original work is properly cited. [DOI:10.5796/electrochemistry.20-00097]. Uploading "PDF file created by publishers" to institutional repositories or public websites is not permitted by the copyright

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Keywords : Ion-selective Electrode, Lactate, Sequential Injection Analysis

1. Introduction

Blood lactate concentration is used as an important index for thedegree of fatigue of the body. Besides, when the blood lactateconcentration becomes high, the blood is acidified and becomeslactic acidosis, which can be diagnosed by measuring the bloodlactate concentration.

The optical methods using enzymes for lactate determination areused dominantly. For example, lactate is catalyzed by lactatedehydrogenase in the presence of NAD+ and NADH is produced.The concentration of NADH is measured spectrophotometrically(absorbance at 340 nm wavelength) and it corresponds to theconcentration of lactate.1 The optical methods require relativelylarge apparatus, high analytical skills and high cost. Furthermore,they are difficult to miniaturize and to apply to colored and turbidsamples.

Therefore, development of simpler analytical methods usingelectrochemical sensors for the determination of lactate isrequired.2–16 Among the electrochemical sensors, potentiometryusing ion-selective electrodes (ISEs) has very simple analyticalprocedures and low-cost apparatus and high selectivity to a primaryion, and applicable method to turbid and colored samples.17–20

Siddiqi reported a potentiometric determination method of glucoseusing F¹ ISE. In the method, F¹ ion generated by the reaction ofglucose, glucose oxidase and horseradish peroxidase (HRP) in thepresence of the fluoroaromatic compounds is detected by the F¹

ISE.17 An initial-rate potentiometric method for the determination ofuric acid using the F¹ ISE was reported based on the principle ofmonitoring the rate of fluoride ion produced from the reactions ofthe 4-fluorophenol with 4-aminoantipyrine (4-AAP), uricase, uricacid and HRP.18 These batchwise methods are very tedious and

time-consuming and requires much amounts of reagents and waste.Koncki et al. reported a new potentiometric flow injection analysis(FIA) of alkaline phosphatase (ALP) by detection with the F¹ ISE ofF¹ ion produced a reaction between ALP and monofluorophos-phate.19 This very rapid method uses smaller amount of reagent thanthe batchwise method. However, the FIA method still requiresrelatively large amounts of reagents due to continuous flowing ofreagents yet.

On the other hand, sequential injection analysis (SIA) method hasthe advantages of automating complicated analysis operations andreducing the amounts of reagents and waste, compared withconventional FIA methods.20–24 In the SIA method, a sample anda reagent of strictly determined small volume (microliter order) aresequentially injected into a holding coil using a personal computer-controlled syringe pump and a multi-port valve, and mixing andchemical reaction are performed by bidirectional flow and thereaction product are detected. Contrastingly, FIA is an instrumentalanalysis method in which a sample solution is introduced into theflow of a reagent solution to induce a chemical reaction and thereaction product is detected. In the FIA method, it is necessary toflow the reagent solution continuously. Therefore, the amounts ofreagents and waste in the SIA method is much less than those in theFIA method. In a clinical examination, Cl¹, Na+, and K+ ionsare measured by potentiometric ISE method. By measuring theconcentration of the hydrogen peroxide generated by the reactionof the substrate with various oxidases (oxidase system) usingpotentiometric F¹ ISE, clinical important samples of not only Cl¹,Na+, K+ but also lactate, uric acid and so on, could be determinedsimultaneously. Therefore, potentiometric ISE method may be moreversatile than the amperometric method2,4,5,7–10,12,13 in a clinicalexamination.

Electrochemistry Received: August 4, 2020

Accepted: August 18, 2020

Published online: September 15, 2020

The Electrochemical Society of Japan https://doi.org/10.5796/electrochemistry.20-00097

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A combination of the SIA system and the ISE detector isconsidered to be a very effective analytical method for determininglactate. Reaction of lactate with lactate oxidase produces pyruvicacid and hydrogen peroxide. When the produced hydrogen peroxideis reacted with 4-aminoantipyrine (4-AAP) and 4-fluorophenol(4-FP) in the presence of horseradish peroxidase (HRP), F¹ ion isproduced. The concentration of lactate can be indirectly determinedby measuring the F¹ ion by ISE. The purpose of this study is todevelop a new measurement method of lactate using SIA with ISEdetector based on the principle of detection of lactate.

2. Experimental

2.1 ChemicalsLactic acid, 4-AAP and HRP of reagent grade from Wako Pure

Chemicals Co. were used. 4-FP of reagent grade was obtained fromTokyo Chemical Industries Co. Lactate oxidase (106 units/mg) wasobtained from Toyobo Co. All other chemicals of reagent gradewere used with no further purification. Water (Mili-Q) was used inall experiments.

2.2 SIA system for the determination of lactateFigure 1 shows an SIA system for the determination of lactate.

All instruments were connected by teflon tubing with an innerdiameter of 0.5mm. First, a carrier solution (0.15M Tris-HCl,pH 8.0) of 300µL at a flow rate of 70 µL s¹1 was aspirated from thesyringe pump (SP) and sent to an ISE detector from 1 port of the six-way valve (SV) to clean the inside of the tube. Next, lactate (samplesolution) at a flow rate of 29 µL s¹1 was aspirated from 3 port of SVto clean teflon tubing and was discarded into waste. Thereafter, areagent solution (100µL) consisting of 3.0mM 4-AAP, 30.0mM 4-FP, 0.10 units/mL HRP and 10 unit/mL lactate oxidase, and lactate(100 µL) as a sample solution from 6 and 3 port of SV wereaspirated into a holding coil (HC) at a flow rate of 29 µL s¹1,respectively. The solution in the HC was sent to a reaction vessel(RV) from 2-port of SV, and the operation of returning the solutionin the RV to HC through SV was repeated four times and finally, theflow was stopped for 5min at RV in order to react reagent andsample solutions. The resulted reaction product (F¹ ion) wasaspirated into the HC and sent from 1-port of SV to the F¹ ISEdetector (FLC 12, TOA-DKK Co.) by the carrier solution (300 µL)at a flow rate of 29 µL s¹1 and the flow and the continuous flow ofthe carrier solution from the peristaltic pump (PP) to stabilize thepotential of the ISE detector were combined at the confluence point,and the combined solution was passed to the ISE detector. The

length of the teflon tubing between the confluence point and the ISEdetector was 10 cm. The potential difference between F¹ ISE (TOA-DKK Co.) and reference electrode (TOA-DKK) in the ISE detectorwas measured as a peak-shaped signal with a potentiometer (COM-20R, TOA-DKK). The above protocols were automaticallycontrolled by a personal computer.

3. Results and Discussion

The detection sensitivity for lactate depends on the concen-trations of HRP, hydrogen peroxide, lactate oxidase, as well as thereaction time between lactate and reagent solution. At first, the effectof the concentration of hydrogen peroxide on the sensitivity of HRPwas examined. For hydrogen peroxide in the concentration rangefrom 1.0 to 1.5 and 2.0mM, as the concentration of hydrogenperoxide increases, the detection sensitivity of HRP slightlyincreases. From the result, the concentration of hydrogen peroxidewas determined to be 2.0mM. Next, the effect of the concentrationof HRP on the sensitivity of hydrogen peroxide was examined. ForHRP in the concentration range from 0.005 to 0.1 units/mL, asthe concentration of HRP increases, the detection sensitivity forhydrogen peroxide increases. From the result, the concentration ofHRP (0.1 units/mL) was used in the next experiment. The effect oftime of stopped-flow at RV on the determination of lactate wasexamined. The detection sensitivity for lactate increases as the timeof stopped-flow increases in the range from 0 to 5min. In case thattime of stopped-flow is 5 and 10min, detection sensitivity for lactateis almost the same. From the result, the time of stopped-flow at RVwas determined to be 5min. The effect of concentration of lactateoxidase on the detection sensitivity of lactate was also examined. Asthe concentration of lactate oxidase increases in the range of 1.0–10.0 units/mL, the detection sensitivity for lactate increases. Fromthe result, the concentration of lactate oxidase was determined to be10 units/mL.

Figure 2 shows the typical calibration peaks for lactate. A linearrelationship between peak heights and logarithmic concentrations oflactate with a slope of 53.8mVdecade¹1 in a concentration rangefrom 0.05 to 0.5mM was observed. The coefficient of variation forthe peak height of 0.1mM lactate was 7.0% (n = 12). The samplingrate for 0.1mM lactate was 5 samples h¹1. Effect of coexisting ionson the determination of lactate was also examined. Coexisting ofMgCl2 (10mM), CaCl2 (10mM), NaCl (200mM), KCl (10mM),Fe(NO3)3, Cu(NO3)2, glucose (10mM), Galactose (10mM), crea-tinine (1mM), creatine (0.1mM), cysteine (0.02mM), dopamine(0.1 µM), urea (10mM) and uric acid (0.5mM) did not interfere

Figure 1. Schematic diagram of the SIA system for the determination of lactate. SV: six-port selection valve, Holding coil: i.d.0.5mm © 480 cm, PP: peristaltic pump. All instruments were connected by teflon tubing with an inner diameter of 0.5mm.

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with the determination of 0.1mM lactate. However, coexisting of20 µM Bilirubin interfered with determination of 0.1mM lactatewith a negative error of ca. 40%, compared with 0.1mM lactatecontaining no Bilirubin. The serious negative error was removed byadding 0.05mM K4[Fe (CN)6] to the reagent solution used in Fig. 2.The mechanism of removal of interference by Bilirubin is thatferrocyanide is oxidized to ferricyanide by hydrogen peroxide in thepresence of peroxidase; this, in turn, oxidizes the chromogen (4-FP)and the latter oxidation perhaps involves a phenol (as opposed to 4-FP) intermediate, which does not react with bilirubin.25

The coexistence of 0.2mM ascorbic acid did not interfere withthe determination of 0.1mM lactate, but the coexistence of 0.5mMor more ascorbic acid for the determination of 0.1mM lactateshowed with a negative error of ca. 60%, compared with 0.1mMlactate containing no ascorbic acid. This interference was removedby firstly reacting 5 units/mL ascorbate oxidase solution as a reagentsolution with lactate and then by reacting the resulting product withthe reagent solution used in Fig. 2.

4. Conclusions

An SIA system using an F¹ ISE detector was developed forthe measurement of lactate. The developed SIA system has theadvantage of using fewer reagents, less sample volume, and lesscomplicated procedures than other determination methods for thedetermination of lactate. The sample throughput of the proposedSIA method for lactate was 5 samples h¹1 and may be lower thanthat of the other electrochemical sensors.3,5,7 The sample throughputof the proposed SIA method for lactate may be improved byincreasing of flow rate of the carrier solution. The sensitivity of theproposed SIA method for lactate is the same as that of otherelectrochemical methods or lower than that of them.2,4,5,7,8,10–12 Thereason may be as follows: Since the enzymatic reaction of thismethod is not a system in which lactate, which is the sample, isrecycled, but is a system in which it is oxidized and consumed by anoxidase, F¹ ion generated depends on the initial amount of lactatemolecules contained in the sample solution.

However, problems such as the peeling of an immobilizedenzyme, decrease in detection sensitivity of the sensor, and shortlifetime in electrochemical sensors does not occur because the F¹

ISE detector is used for at least 6 months in case 20 samples per day

are measured, keeping the electrochemical performance such as thedetection sensitivity for lactate.

Determination of lactate in biological samples such as a serum bythe present SIA system is examined in progress.

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Figure 2. Typical calibration peaks and calibration curve for lactate obtained by the SIA system. Reagent: 3mM 4-AAP, 30mM 4-FP,0.1 units/mL HRP and lactate oxidase: 10 units/mL. Stopped-flow time at reaction vessel: 5min. The inset shows a calibration curve forlactate.

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