技術報告 Technical Report

Jpn. J. Hosp. Pharm. 技術報告〔19(1)99-105(1993) 〕

Simultaneous Determination of Inorganic Cations in Cardioplegic Solution by Photometric Ion Chromatography ― A. Comparison with Autoanalyzer Method

NOBUMASA HAYASE*† 1, SHIGETAKA AKUTSU† 1, SHUN- ICHIINAGAKI† 1,

and HIROMICHI HOSOKAWA† 2

Departmemt of Hospital Pharmacy† 1 and Clinical Laboratories† 2, Asahikawa Medical College Hospital

Received June 20, 1992 (Accepted September 30, 1992 )

A simultaneous determination of four inorganic cations in cardioplegic solution was examined by indirect photometric ion chromatography (PIC). Cardioplegic solution was diluted 100-fold with 2.5mM copper sulfate aqueous solution, treated with SEP-PEK C18and ACCELL PLUS QMA cartridges, and then injected into the chromatograph. Large amounts of sodium, potassium and magnesium and small amount of calcium were separated on a Zorbax SCX-300 column with 2.5mM copper sulfate solution as an eluent, and detected as negative absorption peaks by an ultraviolet (UV) photometric detector at 230 nm. Straight calibration curves for the cations were obtained from 0.05 to 1.5mM by using a peak height ratio method. The reproducibility of the data was good enough. When cardioplegic solution was diluted 4- fold with the eluent, the determination of calcium in sample solutions was sufficiently good, in spite of over-loadings of the other cations on column. The results obtained by the PIC method were more accurate than those of the autoanalyzer method.

Keywords- cardioplegic solution; inorganic cations; photometric ion chromatography; simultaneous determination; autoanalyzer

Introduction

Cardioplegic solution has been developed for clinical use of myocardial protection during aortic cross- clamping in the field of cardiac surgery 1). The aortic cross- clamping makes the heart ischemia during operation. It is believed that cardioplegic solution can protect the heart against ischemic damage, and against reperfusion injury following ischemia, because of depressing of the myocardial metabolism 2). Bretschneider et al. 3)and Hearse et al. 4)proposed the use and efficacy of cold chemical cardioplegia for the myocardial protection during coronary bypass surgery and heart arrest. The proposed solutions are now used as Bretschneider Solution and St. Thomas Hospital Solution, respectively. After that, various cardioplegic solutions have been developed to improve myocardial preservation, such as glucose-ins- ulin- potassium (GIK) solution 5). These solutions consist of inorganic anions, cations and some drugs.

†1,2 旭川市西神楽4線5号3-11;4-5-3-11 ,Nishikagura,Asahikawa,078 Japan 100 病院薬学 Vol. 19, No. 1 (1993)

However, the optimal concentration of each additive has not been given yet. It is very difficult to find

the optimal concentrations of the cations, because the effects of cations on the myocardium are variable with temperature and other additives 1). Furthermore, several cations interact with anions, for instance

calcium and magnesium ions react with carbonate and phosphate ions to form insoluble compounds

under the conditions of preparation or preservation 6). Therefore, it is important and necessary to

monitor the precise concentrations of the cations before clinical use for assessing the efficacy of car-

dioplegic solutions 7).

Ion selective electrode (ISE) methods have commonly been used in the determination of in-

organic cations and anions in biomedical materials or pharmaceutical preparations 8). Although this

method is rapid and convenient, it cannot determine several ions simultaneously. On the other hand, ion

chromatography (IC) 9) has become widely used for the determination of inorganic and organic ions.

However, IC method needs a conductivity detection.

Indirect photometric ion chromatography (PIC) based on the difference of the absorbance of sam-

ple and eluent (mobile phase) species has attracted much attention in the field of IC, since this method can be performed on a conventional high- performance liquid chromatographic (HPLC) system equipped

witha UV detector, and no derivatization procedures are required 10). Miyazaki et al. 11) developed a

simultaneous determination of inorganic cations by using PIC method. However, there is few applica-

tions of PIC to analyses of inorganic cations in pharmaceutical preparations. This paper, therefore,

deals with the application of this method to the simultaneous determination of calcium, sodium,

potassium and magnesium ions in cardioplegic solution. To elucidate the value of PIC method, the analytical results were compared with those of autoanalyzer method which consisted of ISE assay and

colorimetry.

Experimental

1. Reagents. Sodium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate and

caesium sulfate (guaranteed reagent, Kanto Chemical Co., Inc.) were used as standard compounds. All

other reagents were also of guaranteed grade. Each solution of the standard compounds, and the eluent

(copper sulfate solution) were prepared with distilled deionized water. The eluent was filtered through a 0.22- um membrane (TYPE GS, Millipore Co.) before use. Calibration standards containing cations from

0.05 to 1.5mM were prepared by mixing each solution. SEP- PAK C18 cartridges (360mg of C18 sorbent,

9•~13mm) and ACCELL PLUS QMA cartridges (360mg of trimethylammonium exchangers, 9•~13

mm) were purchased from Waters Millipore Co. and activated by passing 2mL of methanol followed by

5mL of distilled water before use.

2. Sample Preparations. Cardioplegic solution was prepared by dissolving 7.03g sodium

chloride, 1.789g potassium chloride, 0.163g potassium dihydrogenphosphate, 0.176g calcium chloride

dihydrate, 0.32g magnesium sulfate heptahydrate, 2.033g magnesium chloride hexahydrate, and

1.982g glucose in 1000mL distilled water. For the determinations of sodium, potassium and

magnesium, 1mL of cardioplegic solution and 1mL of 50mM caesium sulfate (as internal standard)

solution were pipetted into a 100mL volumetric flask, and then diluted with 2.5mM copper sulfate solu-

tion which was the eluent of PIC (100- fold diluted cardioplegic solution). The sample solution for the

analysis of calcium was similarly prepared by pipetting 25mL of cardioplegic solution and 1mL of 50 病院薬学 Vol. 19, No. 1 (1993) 101

mM caesium sulfate solution into a 100 mL volumetric flask and diluted (4- fold diluted cardioplegic solution). Ten mL of the sample solution ran through SEP- PAK C18 and ACCELL PLUS QMA cartridges to eliminate organic and anionic matters. Fifty pL of the effluent were injected into the chromatograph mentioned below. 3. Appratus and Conditions of PIC. The chromatographic system consisted of a HITACHI 635A pump, a Rheodyne 7125 injector (loop of 100 pL), a HITACHI 655A- 21 Variable Wavelength UV Monitor and a HITACHI 056 recorder. The cation- exchange separating column was a Zorbax SCX- 300 (4.6 mm i.d. x 25 cm, Shimadzu). The eluent was 2.5 mM copper sulfate solution and was kept at a flow rate of 1.2 mL / min. The detection wavelength was 230 nm (sensitivity of 0.08 a. u. f. s.). The entire course of the study was performed at ambient temperature. 4. Autoanalyzer Method. Cardioplegic solution was introduced directly into a BECKMAN ASTRA- 4 automated stat-routine analyzer system. The system included a glass electrode and a valinomycin membrane electrode for the determination of sodium and potassium contents, respectively. The selectivitycoefficients of both electrodeswere Kricatic,= pot 0.003 NaKand K pot 0.001,k pot KNa=where K potvalue ij is de- finedas a interferenceratio of j-ion againsti- ion electrode). The calciumcontents were determinedby colorimetrywith o- cresolphthalein complexone 13). Two concentrationlevels, that is, 100 and 150 mM of sodiumsulfate, 20 and 30 mM of potassiumsulfate, and 2 and 5 mM of calciumsulfate were used for calibrations.However, magnesium contents were not determinedby the system.

Results and Discussion

1. PIC Conditions. The choices of column and eluent were considered to permit simultaneous determination of both monovalent and divalent cations in cardioplegic solution. It is generally observed that a styrene- divinylbenzene copolymer based cation exchange column retains divalent cations longer than a silica-based resin column. Therefore, there are some problems to determine both monovalent and divalent cations simultaneously with a styrene- divinylbenzene copolymer resin column. The Zorbax SCX- 300 is a conventional silica-based cation exchange column for HPLC whose low ion-exchange capacity permits the simultaneous determination of those in PIC with copper sulfate solution. Figure 1 shows a typical PIC chromatogram of a mixture of five cations, namely, sodium, potassium, magnesium, calcium and caesium. The eluent concentration and the flow-rate were adjusted to 2.5 mM and 1.2 mL / min, respectively, to give acceptable resolution and retention time. The detection wavelength was set at 230 nm, because it was critical point to observe a deep trough of the eluent and a stable baseline in the chromatogram. Negative peaks in the elutions detected by an ultraviolet (UV) monitor were altered to positive peaks by changing signal polarity in a recorder. These conditions for PIC were in similar to those in the literature). Under the described chromatographic conditions, the five cations including caesium as an internal standard were separately determined within 25 min. Capacity factors (k') were as follows: sodium, 1.9; potassium, 2.7; caesium, 4.0; magnesium, 15.9; and calcium, 21.3. 2. Pretreatment of Sample Solution. It is important for IC to eliminate additives from sample specimines. Indeed, it was observed that when the sample solutions were injected directly into the chromatograph, a water-dip became wide and noise peaks appeared around the peak of caesium to make the accuracy of the determination worse. To prevent these interferences, the sample solutions 102 病院薬学 Vol. 19, No. 1 (1993)

Fig. 1. A Chromatogram for a Mixture of Five Cations The experimental conditions are the same as in the text. Thepeaks of chromatogram represent cation standards containing sodium(1 mM), potassium (1 mM), caesium (1 mM), magnesium (0.5 mM) and calcium (0.5 mM). were pretreated with SEP-PAK C18and ACCELL PLUS QMA cartridges before injection into the chromatograph. 3. Detection Limits and Calibration Curves. Straight calibration curves for the four cations were obtained from 0.05 to 1.5 mM by using a peak height ratio method. The correlation coefficients (r) and equations of the regression analysis for calibration curves were as follows: sodium, r= 0.995, y= 1.45x + 0.32; potassium, r= 0.999, y= 1.46x- 0.05; magnesium, r= 0.999, y= 0.95x; calcium, r= 0.998, y = 0.45x+ 0.01, where y is the peak height ratio and x is the concentration of cation in the calibration standard. The high value of intercept for the sodium calibration curve may be caused by con- taminations from reagents, glass apparatus, filters and pretreatment cartridges, but its distinct origin 病院薬学 Vol. 19, No. 1 (1993) 103

A B

Retention time (min.)

Fig. 2. Typical Chromatograms for (A) 100- Fold and (B) 4-Fold Diluted Cardioplegic Solution The experimental conditions are the same as in the text.

Table 1. Comparison of the Results Obtained for Four Cations in Cardioplegic Solution Using the PIC and Autoanalyzer Methods

Each value represents the mean •}standard deviation (mM) of 10 determinations. *1 Theoretical values were calculated from formula of cardioplegic solution. *2The results were obtained from 100 -fold diluted cardioplegic solution with the eluent of PIC. *3The result was obtained from 4-fold diluted cardioplegic solution with the eluent of PIC. 104 病院薬学 Vol. 19, No. 1 (1993)

was not attributed. The limits of detection were 20 ƒÊM for sodium and potassium , and 15 ƒÊM for magnecium and calcium, respectively . These values were based on a signal- to- noise ratio of 3 in the chromatograph. The precision of the PIC method for the determinations of cations was assessed by repeated analyses (10 times) of standard mixture containing known amounts (0 .5 or 1 mM) of four cations. The coefficient of variation (CV%) ranged from 1 .01 to 2.14% within-day determinations.

4. Determination of Inorganic Cations in Cardioplegic Solution. Using the method described above, inorganic cations in 10 samples of 100- fold diluted cardioplegic solution were determined. Figure 2 A shows an example chromatogram for those. As the baseline was stable for 30 min after injection, successive injections were possible at 30 min intervals. In this case, it was able to determine the four cations simultaneously. However, the values of calcium were not enough accurate

(CV%= 15.1) because the peak of calcium was a barely detectable change in chromatogram. The calcium content in cardioplegic solution is one of serious factors for the arrest and cardioprotection in cardiovascular surgery 14). When 4- fold diluted cardioplegic solution was analysed by the same method, satisfactory analyses for calcium have been achieved. The chromatogram is shown in Figure 2 B. The baseline was stable, and the values of calcium were enough accurate (CV%= 8.8) in spite of

overloadings of the other cations on column, namely, out of full-scale in the recorder. Our data for car-

dioplegic solution were listed in Table 1.

5. Comparison of the PIC Method with the Autoanalyzer Method. Clinical analyzer

systems based on ISE or colorimetries are available for analysis of sodium, potassium, magnesium,

calcium and pH in body fluids. These instruments have advantages of small size consumption, short analysis time, and minimum operation cost. Table 1 shows a comparison of the results obtained for four

cations in cardioplegic solution by the PIC and autoanalyzer methods. The concentrations determined by the PIC method were in good agreement with those of theoretical values, but all the values obtained by the autoanalyzer method were higher than those of the PIC method or theoretical ones. It is con-

sidered that monovalent ions of sodium and potassium, and divalent ions of magnesium and calcium may affect each other in analyses of autoanalyzer method, because ISE selectivities or colorimetric reac- tions may not have enough specificity. In general, it is pointed out that the values determined by ISE

method show a tendency to overestimate because of interference by coexisting ions and compounds in

the sample 15). As PIC method determine the contents of cations separately, such disadvantage as au-

toanalyzer method has must be eliminated.

Conclusion

The PIC method using a conventional HPLC system seems very suitable and practical for the

simultaneous determination of inorganic cations in cardioplegic solution. Moreover, the results ob-

tained by the PIC method were more accurate than those by the automated routine analyzer system.

Acknowledgement The authors thank Dr. Kazuo Ichihara (Department of Pharmacology) and Dr.

Yasuyuki Fujita for helpful suggestions on the manuscripts.

References

1) M. Masuda and K. Tokunaga, Tanpakusitu Kakusan Kouso, 35, 1946- 1953 (1990). 2) K. Kinosita and T. Ehara, J. Mol. Cell Cardiol., 16, 405- 419 (1984). 病院薬学 Vol. 19, No. 1 (1993) 105

3) H. J. Bretschneider, G. Hubner, D. Knoll, B. Lohr, H. Nordbeck and P. G. Spieckermann, J. Cardiovasc. Surg., 16, 241- 260 (1975). 4) P. Junge, D. J. Hearse and M. V. Braimbridge, J. Thoracic. Cardiovasc. Surg., 73, 848- 855 (1977). 5) D. M. Lolly, J. F. Ray, W. 0. Myers, G. Sheldon and R. D.Sautter, Ann. Thorac. Surg., 26, 515- 524 (1978). 6) R. Aeba and T. Inoue, Rinshou Mizu Denkaishitu, 6, 15- 21 (1986- 7). 7)F. Yamamoto, M. V. Braimbrige and D. J. Hearse, J. Thoracic. Cardiovasc. Surg., 87, 908- 912 (1984). 8) J. Koryta, Anal. Chim. Acta., 183, 1- 46 (1986). 9) H. Small, T. S. Stevense and W. C. Bauman, Anal. Chem., 47, 1801- 1809 (1975). 10) H. Small and T. E. Miller, Anal. Chem., 54, 462- 469 (1982). 11) M. Miyazaki, K. Hayakawa and S. Choi, J. Chromatogr., 323, 443- 446 (1985). 12) V. P. Y. Gadzekpo and G. D. Christian, Anal. Chim. Acta., 164, 279- 282 (1984). 13) J. Gitelman, Anal. Biochem. , 18, 521- 531 (1967). 14) J. Amano, M. Sunamori and A. Suzuki, Adv. Myocardiol , 3, 33 -39 (1982). 15) M. Saito, Meth Clin. Appl. ISE., 8, 219- 225 (1987).

技 術 報 告 Technical Report

技術報告 Technical Report