Huijgen, H.J.

Kok, W.Th.

Sanders, G.T.B.

van Ingen, H.E.

English

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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)UvA-DARE (Digital Academic Repository)Analytical evaluation of kone microlyte determination of ionized magnesium.van Ingen, H.E.; Huijgen, H.J.; Kok, W.Th.; Sanders, G.T.B.Publication date1994Published inClinical chemistryLink to publicationCitation for published version (APA):van Ingen, H. E., Huijgen, H. J., Kok, W. T., & Sanders, G. T. B. (1994). Analytical evaluationof kone microlyte determination of ionized magnesium. Clinical chemistry, 40, 52.General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an opencontent license (like Creative Commons).Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, pleaselet the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the materialinaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letterto: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. Youwill be contacted as soon as possible.Download date:09 Mar 2023CLIN. CHEM. 40/1, 52-55 (1994) #{149}Automation and Analytical Techniques52 CLINICALCHEMISTRY, Vol. 40, No. 1, 1994Analytical Evaluation of Kone Microlyte Determination of Ionized MagnesiumHuub E. van Ingen,”2 Henk J. Hutjgen,”4 Wim Th. Kok,3 and Gerard T. B. Sanders1We performed an analytical evaluation of a commerciallyavailable instrument for determining ionized magnesiumthrough use of a neutral carrier, liquid-membrane-basedion-selective electrode. Reproducibility (CV 2-4%), linear-ity (0.30-2.50 mmol/L), lower limit of detection (0.30mmol/L), and absence of interference from Ca2 indicateadequate performance for measuring ionized magnesiumin plasma or serum samples in the normal to high-con-centration range. Sodium in excess of 150 mmol/L causeda negative bias, which can be explained by ionicstrength-induced changes in activity coefficients. The use of hep-arm as an anticoagulant must be restricted to concentra-tions <15 units/mL because of the bindingof magnesiumto heparin. The mean ± SD concentration of ionizedmagnesium and its fraction of total magnesium in 76healthy volunteers were 0.56 ± 0.05 mmol/L and 0.65 ±0.04, respectively.IndexIngTerms: reference values/ion-se!ectwe electrodeIon-selective electrodes (ISE) have been developed fordetermining W, Na, K, C1, ionized Ca2 (iCa2),and Li in clinical samples (1-3). Until recently, noISE was available that reliably measured ionized Mg2(iMg) in plasma, serum, or blood. Efforts have beenconcentrated on the use of neutral ionophore-basedmembranes, composed of ionophore/poly(vinylchloride)(PVC)/plasticizer (1/33/66, by wt). To improve selectiv-ity, researchers inserted into the membranes an organicanion in an equiinolar ratio to the neutral ionophore (4,5). Problems with such systems have mainly beencaused by insufficient selectivity against Na, Ca2,and H, as well as the limited lifetime of the electrode.Two experimental systems to measure iMg in se-rum have been described. Rouilly et al. (6) used a cellassembly of the kind Hg.Hg2C12, KC1 (saturated)IKC1 (3mol/L)samplelImembraneMgCl2 0.7 mmol/L, CaC12 1.2mmol/L, KC1 4 mmol/L, NaCl 140 mmol/L, AgCl:Ag at21#{176}C.The membrane consisted of ionophore ETH 5282[(N’ ,N”,N”’-imino-di-6, l-hexandiyl)-tris(N-heptyl-N-‘Academic Medical Centre, Dept. of Clinical Chemistry, B1-239, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.2Present address: Department of Clinical Chemistry, Dr. Danielden Hoed Cancer Center, Groene Hilledijk 301, 3075 BA Rotten-dam, The Netherlands.3Laboratory for Analytical Chemistry, University of Amster-dam, The Netherlands.‘Author for correspondence. Fax Int + 31-205664440.6Nonstandard abbreviations: ISE, ion-selective electrode; iCa2,ionized calcium; iMg, ionized magnesium; PVC, poly(vinylchlo-ride); KTpC1B, potassium tetrakis(p-chlorophenyl)borate; AAS,atomic absorption spectrometry; and TES, 2-{[tris(hydroxy-methyl)methyllamino)ethanesulfonic acid.Received March 29, 1993; accepted September 7, 1993.methylmalonamide)]/PVC/o-nitrophenyloctyl etherplasticizer (by wt, 1/33/65), and potassium tetrakis(p-chlorophenyl)borate (KTpC1B) in a molar ratio of 1.5relative to the ionophore. This system had two majordrawbacks: Interference from calcium necessitated cal-ibration of the electrode with solutions containing aniCa2 concentration identical to that in the sample to bemeasured, and the measurements were performed in anopen system, which led to rather large pH shifts byevaporation of CO2.Maj-Zurawska and Lewenstam (7) used a more ele-gant approach by incorporating a Mg-selective elec-trode in the Microlyte 6 analyzer (Kone Instruments,Espoo, Finland), making possible the simultaneousmeasurement of Na, K, iCa2, and pH. The mem-brane composition of the Mg2 ISE was ionophore ETH5220 [N,N’-octamethylenebis(N’,N’-dioctylmalondiam-ide)IIPVC/chloroparaffin/o-nitrophenylphenyl ether (1/33/32.5/32.5, by wt) and KTpC1B in a molar ratio of 0.70relative to the ionophore. Measurement conditions wereconsiderably improved over the aforementioned methodby increasing the temperature to 37#{176}Cand using aclosed system of flow-through electrodes. However, theauthors stifi reported considerable interference fromCa2, for which no clear solution has been presented.Recently, Kone introduced an iMg determinationon their Microlyte 6 ion analyzer that allows measure-ment of iMg without interference from Ca2 in thesetting of a routine clinical chemistry laboratory. Wehave performed an extensive analytical evaluation ofthis instrument, including the determination of a lim-ited set of reference values.Materials and MethodsThe Microlyte 6 ion analyzer was used with the fol-lowing electrodes installed: Ag/AgC1 reference elec-trode, Ca2, Na, K, C1, pH, and Mg2. The Mg-selective membrane was composed of ETH 5220dispersed in a PVC matrix, with chloroparaffln and o-ni-trophenylphenyl ether as plasticizers and KTpCIB asadditive.To calibrate the instrument for all ions except Mg2,we used two aqueous standard solutions, followed bymeasurement of a third aqueous standard with an in-termediate value as a linearity check. The Mg2 ISE iscalibrated with standard solutions 1 (Ca2 1.25 mmoJJL,Mg2 0.60 mmol/L) and 2 (Ca2 0.75 mmol/L, Me 0.30mmolJL). The third calibration solution has a Mg2 con-centration identical to calibration solution 2 (0.30mmol/L) but Ca2 at 1.75 mmol/L. This procedure en-ables calculation of the selectivity coefficient KPtCa,which is stored subsequently in the instrument andused for correction of iMg values for iCa2, and neces-Ca, mmoIIL0.781.351.8540 sara-0.47-0.72-0.70CLINICALCHEMISTRY, Vol. 40, No. 1, 1994 53sitates simultaneous implementation of the Mg2 andCa2 electrodes. Each measurement takes 2 min, fol-lowed by a one-point calibration, which also takes 2mm. Thus 15 samples can be measured in an hour. Theinstrument uses 150 L of sample; measurement tem-perature is 3 7#{176}C.Total Mg2 was measured by atomic absorption spec-trometry (A.AS) (PE 2100; Perkin-Elnier, Gouda, TheNetherlands).Selectivity coefficients were determined by the fixedinterference method (8). Electromotive force valuesneeded for this purpose were measured directly by dis-connecting the electrodes from the instrument and con-necting them with a Century SS-1 pH/mV meter (Beck-man Instruments, Fullerton, CA). Interference fromCa2, Na, and Li was also evaluated by addition ofthese ions as their chloride salts to a solution containingNaCI 140 mmol/L, KC1 4.5 mmol/L, MgCl2 0.6 mmollL,and 2-{[tris(hydroxymethyl)methyl]amino}ethanesul-fonic acid (TES) 5.0 mmol/L, pH 7.4.Response times are defined as the length of timeelapsing between the instant the concentration of iMgchanges and the first instant at which the electrodepotential becomes equal to its steady-state value within1 mV (8). We measured these times with the sameexperimental system as was used for the determinationof the selectivity coefficients. The electrode was equili-brated with an electrolyte solution (composition identi-cal to the solution used for the interference studies),after which serum samples with different iMg24 concen-trations were introduced.The lower limit of detection was determined as thej2 concentration at the intersection of the extrapo-lated two linear segments of the calibration curve (8).This curve was determined by addition of MgC12 to asolution containing NaCI 140 mmolJL, KC1 4.5 mmol/L,CaC12 1.25 mmol/L, and TES 5.0 mmolJL, pH 7.4.Intraassay reproducibifity was determined with bothcommercial control materials (from human origin) andpatients’ samples. Commercial control samples wereused for calculating day-to-day reproducibility.Linearity was determined either by mixing standardsolutions or by mixing pooled patients’ samples to whichMgC12 had been added.Blood samples were drawn into either plain or hep-arm-containing tubes with an evacuated blood-collect-ing system (Venoject; Terumo Europe N.y., Leuven,Belgium). All procedures followed were in accordancewith the rules laid down in the Helsinki Declaration of1975, as revised in 1983.All chemicals used were of analytical-reagent gradefrom E. Merck B.V. (Amsterdam, The Netherlands).Sodium heparin (Thromboliquine#{174}, sodium salt) was ob-tained from Organon Teknika B.V. (Boxtel, The Neth-erlands). Lithium heparin for addition experiments waspooled from Venoject tubes.ResultsThe response times of the Mg-selective electrodewere always <30 s. Thus, a measurement time of 120 sallows reading of the potential practically at steadystate.The selectivity coefficient K#{176}qgcawas determinedwith three concentrations of the interfering ion. Beth arelatively new electrode and one at the end of its lifetimewere used. Results are shown in Table 1. The selectivitycoefficient KPOtMg,Na was determined at two sodium con-centrations (125 and 150 mmo]/L); was -2.9and -3.1, respectively. These selectivity coefficients areequal to or smaller than earlier reported values (7).Interference from Ca2, Na, and Li was determinedby the addition of standards to buffered solutions (Fig.1). The addition of Na resulted in a negative bias,whereas added Ca2 and Li produced no significantinterference.Within-calibration CVs, measured with assays of con-trol serum, were 3.9% (iMg 0.48 mmol/L, n = 10) and1.3% (iMg 0.92 mmol/L, n = 10). Assays of patients’samples gave within-calibration CVs of 2.2% (iMg0.50 mmoJiL, n = 10) and 2.6% (iMg 0.81 mmolfL, n =10). Day-to-day reproducibffity, calculated as CV, was3.5% (iMg 0.57 mmol/L, n = 36) and 2.9% (iMg 1.05mmol/L, n = 35).The lower limit of detection was 0.30 mmolfL iMg.The linear range for assay of serum samples and di-lute electrolyte solutions extended from 0.30 to 2.50mmol/L iMg. The comparison between Mg2 in diluteelectrolyte solutions determined by AAS and by ISE wasanalyzed by the regression method of Passing and Bab-bk, which resulted in ISE = 0.966AAS + 0.029 mmol/L(range: 0.30-2.50 mmollL). Slope and intercept were notsignificantly different from 1 and 0, respectively.The lifetime of the electrode was limited by a gradualdecrease of the calibration slope, resulting in decreasingsensitivity. Determination of KPOtMg,Ca after measure-ment of 1000 serum samples showed a slight change inCa2 selectivity (Table 1). However, stable values ofcontrol samples proved that the instrument still appliedan appropriate correction factor. After measurement of-1000 serum samples, the electrode should be replacedbecause of the decrease in sensitivity.The influence of heparin on iMg2 and iCa2 mea-surements was determined by collecting blood from twohealthy volunteers into different amounts of sodium andlithium heparin. As shown in Fig. 2, a lithium heparinconcentration of 15 units/mL induced a nonsignificantincrease in iMg; the other curves showed a monoto-nous decrease with increasing heparin concentration. Acomparison of iMg2 measured in blood collected simul-taneously into plain serum tubes and lithium heparinTable 1. Calcium selectivity coefficients determined bythe fixed interference method.Iogl#{176}’,,, after measurement of1000 eera-0.44-0.55-0.59tubes (15 units/mL lithium heparin) showed signifi-cantly higher iMg2 values in lithium heparin plasma(paired t-test, n = 39; average difference 0.02 1, t = 6.85,SD = 0.019, probability of equality P <0.0005). As ex-pected, iCa2 values detected in the lithium heparintubes were slightly lower than in the serum samples.Reference values for iMg and the fraction of iMgdetermined in 76 healthy volunteers (33 men, 43___________________________ women, median age 32 years, range 18-60) were 0.56 ±1.4 i.e 1.8 2 0.05 mmol/L and 0.65 ± 0.04 (mean ± SD), respectively.2# (jiIJL)Discussion0.8. The precision of the determination of iMg2 was0.7 slightly less than reported for systems measuring iCa2,which are in the range 1.0-1.5% (9, 10). Given that the1 0.8 biological CV for j2 is unknown, we were not able to+I o.s --- - #{149}‘ calculate whether an analytical CV of 2-3% contributessignificantly to the tot l variation.0.4 Because no reference system is available for the mea-0.3- surement of iMg, we could only estimate the accuracyof the method by comparing values for iMg measured010 in aqueous solutions with values measured by AAS. TheU + (iiin*) results of the linearity experiments did not indicate biasin the linear range, which therefore suggests adequateaccuracy.Linearity in the normal to high range of Mg2 con-centration (0.50-2.50 mmol/L) was excellent. The lowerlimit of the linear range (0.30 mmol/L) indicates a slightdivergence from AAS values. If we assume that thedistribution of Mg in ionized and bound fractions doesnot change in hypomagnesemia, a value of 0.30 mmolJLfor iMg corresponds to a total Mg2 of 0.46 mmoIiL.Magnesium values <0.46 mmol/L are seen in -2% of-- 100 0 0 180 100N& (mmol/L) the Mg2 requests in our laboratory, which makes thislimitation a source of concern.Fig. 1. interference from Ca2 Na and U on detection of iCa2-corrected iMg, determined by addition of the interfering ions The apparently negative interference from Na ionstheir chloride salts to a buffered electrolyte solution; curves are can be explained by the influence of the ionic strength.drawn through the means of tiiplicates. The activity coefficients for Mg2 in the solution used,1.101.000.800.700.400-l0a2,LlHepiCa,NaH.p#{149}i15I I30 80 1 180- -Fig. 2. Influence of sodium and lithium heparin on iMg and iCa2, expressed as fractions of the simultaneously measured serum value ofthese ions.Each data point represents the mean of duplicate determinationsin plasmasamples obtained from two healthy volunteers. The dashed lines show the 2 SDconfidence Interval of the lMg2 serum value.54 CLINICALCHEMISTRY, Vol. 40, No. 1, 19941’o.s0.40.30.2- 0.8-j0.50.40.3IICLINICAL CHEMISTRY, Vol. 40, No. 1, 1994 55calculated from the Debye-Huckel equation, are 0.325and 0.311 at NaC1 concentrations of 140 and 160mmol/L, respectively. That is, the increase in ionicstrength decreases the Mg2 activity by 4%. Becausethe ionic strength of plasma with an increased concen-tration of Na will also be increased, this situationrepresents a phenomenon that is active in vivo. Correc-tion for this negative bias is thus not necessary if onewishes to report a physiologically relevant activity-de-rived value, which is one of the advantages of ISE mea-surements (11).The gradual decrease in calibration slope was thelimiting factor for the lifetime of the electrode. Whencompared with the number of Mg requests in our ownlaboratory (10-20 per day), a lifetime of 1000 samples isacceptable.In our view, the recommended sample type for thedetermination of iMg is serum, a matrix that containsno potentially interfering anticoagulants. Second bestwould be plasma heparinized with sodium or lithiumheparin (maximal concentration 15 units/mL), whichwould enable measurement of iMg in the same sam-ple type recommended for analysis of iCa2 (12).We were not able to discern the cause of the positivebias (compared with serum) introduced when measuringiMg2 in plasma heparinized with lithium heparin, 15units/mL. Lithium interference was excluded by addi-tion experiments. iCa2 behaved as expected, with bothsodium and lithium heparin causing a negative bias incomparison with serum. A possible explanation for thepositive bias is the presence of preservatives in the lith-ium heparin blood-collection tubes. We realize thatcellular metabolism may continue in the plain tubesduring serum preparation (13); stifi, serum is our pref-erence.During the evaluation period, the Microlyte systemfunctioned without serious problems. User-friendliness,speed, calibration frequency, and shelf life of calibrantsall proved to be sufficient for “routine” analysis of iMgin a clinical chemistry laboratory.We conclude that the Microlyte 6 is a reliable andaccurate system for the determination of iMg. Its non-linearity in the lower range, however, is a potentiallimitation to the assessment of the iMg status in pa-tients with severe hypomagnesemia.We thank Kone Instruments Finland for providing us with theMicrolyte system and for their support during the evaluation.References1. Oesch U, Animann D, Simon W. Ion-selective membrane elec-trodes for clinical use. Cliii Chem 1986;32:1448-59.2. Hirst AD, Stevens JF. Electrodes in clinical chemistry. AnnCliii Biochem 1985;22:460-88.3. Koryta J. Theory and applications of ion-selective electrodes.Anal Chim Acta 1988;206:l-48.4. Otto M, Thomas JDR. Model studies on multiple channelanalysis of free magnesium, calcium, sodium and potassium atphysiological concentration levels with ion-selective electrodes.Anal Chem 1985;57:2647-51.5. MOUer M, Rouilly M, Rusterholz B, Maj-Zurawska M, Hu Z,Simon W. Magnesium selective electrodes for blood serum studiesand water hardness measurement. Mikrochim Acta 1988;3:283-90.6. Rouilly M, Rusterholz B, Spichiger UE, Simon W. Neutralionophore-based selective electrode for assaying the activity ofmagnesium in undiluted blood serum. Cliii Chem 1990;36:466-9.7. Maj-Zurawska M, Lewenstam A. Fully automated potentiomet-nc determination of ionized magnesium in blood serum. AnalChim Acta 1990;236:331.-5.8. Guilbault GO, Durst NA, Frant MS. Freiser H, Hansen EH,Light TS, et al. Recommendations for nomendature of ion.selec-tive electrodes. Pure Appl Chem 1976;48:129-32.9. Brauman J, Delvigne CH, Decornnck I, Wifiems D. Factorsaffecting the determination of ionized calcium in blood. Scand JClin Lab Invest 1983;43(Suppl 165):27-31.10. Bower GN Jr, Brassard C, Sena F’S. Measurement of ionizedcalcium in serum with ion-selective electrodes: a mature technologythat can meet the daily service needs. Cliii Chem 1986;32:1437-47.11. Siggaard-Andersen 0, Thode J, Fogh-Andersen N. What is“ionized calcium”? Scand J Clin Lab Invest 1983;43(Suppl 165):11-6.12. Bounk ABTJ, Buckley BM, Falch-Christiansen T, CovingtonAK Maas AHJ, MUller-Plathe 0, et al. IFCC recommendation onsampling, transport and storage for the determination of theconcentration of ionized calcium in whole blood, plasma andserum. Eur J Clin Chem Cliii Biochem 1991;29:767-72.13. Toffaletti J, Ernst P, Hunt P, Abranis B. Dry electrolyte.balanced heparinized syringes evaluated for determining ionizedcalcium and other electrolytes in whole blood. Cliii Chem 1991;37:1730-3.

Analytical evaluation of kone microlyte determination of ionized magnesium.

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Analytical evaluation of kone microlyte determination of ionized magnesium.

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