Determination Spectrometry. of Serum Glucose by Isotope Dilution Mass. National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305

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1 ANALYTICAL SCIENCES OCTOBER 1988, VOL Determination Spectrometry of Serum Glucose by Isotope Dilution Mass Akiko TAKATSU and Sueo NISHI National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305 An accurate and precise method for the determination of serum glucose by isotope dilution mass spectrometry was studied for use in establishing the accuracy of glucose methods in clinical laboratories. After adding uniformly labeled [13C6} glucose to the serum, glucose was separated from the serum matrix and converted into a-d-glucofranose cyclic 1,2:3,5-bis(n-butylboronate)-6-acetate (glucose-bba). Measurements were performed with combined capillary gas chromatography-mass spectrometry. The ratios of the peak areas at m/z 297 and 303 for samples and the calibration mixtures were used for quantification. Analytical results of the SRM serum showed satisfactory agreement with the certified value and the relative standard deviation of the replicates was within 0.5% for all of the serum samples tested. Keywords Isotope dilution mass spectrometry, gas clinical chemistry, definitive method chromatography/mass spectrometry, serum glucose, Glucose determination is one of the most important and most frequently performed procedures in clinical laboratories. The accuracy of any measurement is essential in clinical chemistry. In order to evaluate the accuracy of routine methods, a "reference method" of known accuracy and a method of higher accuracy and precision, i.e. a "definitive method" are needed.' There is therefore an increasing need for the establishment of both definitive and reference methods. Isotope dilution mass spectrometry (IDMS) has been considered as being a highly accurate and precise method which can be used as a definitive method. Glucose determination by IDMS was demonstrated by Sweeley et al. using a trimethylsilyl derivative.2 As a reference method for a serum glucose determination, Bjorkhem et al. reported the GC-MS method using an 0-methyloxime-penta-trimethylsilyl derivative.3 The definitive method proposed by the US National Bureau of Standards (NBS) included a condensation reaction for 1,2:5,6-di-0-isopropylidene a-n-glucofranose, commonly known as diacetone glucose, introduced to a mass spectrometer via a gas chromatograph equipped with a packed column4, initially a direct insertion probes They also studied the capillary GC-MS procedure with an a-d-glucofuranose cyclic 1,2 : 3,5- bis(n-butylboronate)-6-acetate (glucose BBA) derivative and compared the results.4 In Canada Pelletier et al. performed the capillary GC-MS method with a methyloxime-penta-trimethylsilyl derivative6 and proposed this as a definitive method.' Recently in Japan the Japan Society of Clinical Chemistry studied an interlaboratory comparison program in order to establish a glucose reference method and to examine the methods that are in routine use in clinical laboratories. Through that program we have been able to develop a highly specific IDMS method that can provide target values. We had already reported a similar approach for serum cholesterol.8 For a serum glucose determination we examined various glucose derivatives and the conditions for GC-MS and chose the method with a glucose-bba derivative based on that reported by NBS.4 Some modifications of the procedure were required in order to perform the method in our laboratory. In the present paper we describe an IDMS method for a serum glucose determination and evaluate the accuracy and the precision Experimental of our method. Serum samples Vials of pooled human sera were supplied by the Japan Society of Clinical Chemistry, which were prepared for the interlaboratry comparison program among the clinical laboratories. Human serum reference material (NBS SRM 909) was obtained from the National Bureau of Standards (Gaithersburg, MD, USA). The lyophilized serum was reconstituted, as described in the certificate. We determined its specific gravity by weighing the reconstituted serum in a calibrated small glass tube. Chemicals SRM glucose with a certified purity of 99.9% (SRM

2 488 ANALYTICAL SCIENCES OCTOBER 1988, VOL ) was obtained from NBS. A stock standard solution of glucose (about 2 mg/ g) was prepared by dissolving an accurately weighed amount of SRM glucose in a weighed amount of water. Uniformly labeled [13C6] glucose (98.8 atom% 13C) was purchased from MSD Isotopes (Division of Merck Frosst Canada Inc., Montreal, Canada) and dissolved in water. All other chemicals used were of analytical reagent grade. Calibration mixtures Calibration mixtures were prepared from the stock standard solution by adding a labeled glucose solution so that the ratios of natural to labeled glucose in the calibration mixtures were between 0.8 and 1.2 by weight. After being freeze dried, it was derivatized by the same procedure as used for the serum sample. Sample preparation A sample preparation procedure was similar to that reported by NBS4, but simplified. We derivatized the deproteinized serum directly and analyzed by GC-MS without any further purification, such as deionization with the ion exchange column or removing the pyridine. Briefly, our procedure was as follows. An equilibrated mixture of serum sample and the labeled glucose solution, each of which contained about 1 mg of glucose and was accurately weighed, was deproteinized with ethanol and the supernatant was finally freeze dried. A solution of n-butylboronic acid in pyridine was added and the solution was heated at 85 C for 1 h. After cooling, acetic anhydride was added. The reaction mixture was subjected to GC-MS analysis. Preparation of other derivatives A penta-trimethylsilyl derivative was formed by a reaction of dried glucose with N-trimethylsilylimidazole. For preparing aldononitrile acetate, glucose was heated with hydroxylamine hydrochloride in pyridine followed with acetic anhydride.9 Diacetone glucose was prepared by condensing glucose with acetone in the presence of sulfuric acid; the product was extracted with benzene.10 GC-MS procedure A Hewlett Packard 5830A gas chromatograph and a JEOL JMS D-300 mass spectrometer with a JMA 2000 data system were used. A fused silica capillary column coated with SE-54 (0.3 mm i.d. X 50 m L., Hewlett Packard) was coupled directly to the ion source of the mass spectrometer. The temperatures of the injector, column, and interface to the mass spectrometer were maintained at 250, 210 and 250 C, respectively. The split ratio of the vent to column was set to about 10:1. The ionization energy was 70 ev. Isotope ratio measurements were made by selected ion monitoring at m / z 297 and 303 with a switching rate of 0.1 s / ion. Calculation The peak intensity ratio was determined by measuring the peak areas from the mass fragmentogram. The calculation of the glucose concentration from the peak intensity ratio was similar to that of cholesterol determination.8 Each day the relation between the peak intensity ratio and the mass ratio was constructed from measurements of the calibration mixtures. Using this relation, the mass ratio of the sample was calculated from the peak intensity ratio obtained by a sample measurement. The glucose concentration of the sample was calculated using following equation: C=RMXL/ W, where W is the weight of the serum sample, L is the weight of the labeled glucose added to the sample, and RM is the calculated mass ratio. Results and Discussion Choice of derivative Since for a determination of glucose by gas chromatography it is necessary to convert glucose into a more volatile form, several derivatives of glucose for gas chromatographic analysis have been reported. We examined four of them, i.e. penta-trimethylsilyl derivative, aldononitrile acetate, diacetone glucose, and glucose-bba derivative. The mass spectrum of pentatrimethylsilyl derivative showed many fragment ions and no ion around the molecular ion. In IDMS, a larger difference between the measured mass for unlabeled and labeled glucose is preferable so that both natural and labeled material do not contribute to both of the signals measured for the peak intensity ratio. If the compound contains silicon, this is more severe because of the natural isotopic abundance contribution of silicon. Thus, judging from the mass spectrum, this derivative was not suitable for the IDMS method. Moreover, a- and f3- anomers were separated on the column and it was difficult to confirm that the same ratio of the anomers was obtained in the labeled glucose as in the unlabeled glucose. These problems are similar in the case of the methyloxime-penta-trimethylsilyl derivative, reported by Bjorkhem et al.3, or Pelletier et al.6'', since that derivative contains silicon and has syn- and anti- isomers. Aldononitrile acetate did not have sensitive fragment ions at the high-mass region (above m/z 200). In the preparation of diacetone glucose an elaborate clean-up procedure was required before any GC-MS measurement, since complicated by-products were formed during the reaction. When a solution of D-glucose was treated with n- butylboronic acid followed by acetic anhydride, a rapid and quantitative conversion to the glucose-bba took place. The preparation of the glucose-bba derivative was easy and its mass spectrum was suitable for an

3 ANALYTICAL SCIENCES OCTOBER 1988, VOL Fig. 1 Mass spectra of glucose-bba derivatives of (A) unlabeled glucose and (B) uniformly labeled [13C6] glucose. IDMS determination. We therefore decided to use the glucose-bba derivative for our IDMS measurements. The mass spectra of this derivative are shown in Fig. 1. The abundant ion in the high-mass region is that at m / z 297 for natural glucose (m/z 303 for labeled glucose), formed by an expulsion of a butyl radical from the molecular ion." Labeled internal standard In IDMS, possible isotope effects should be eliminated in order to obtain reproducible results. We used 13C glucose instead of deuterium labeled glucose, because isotope effects from deuterium, such as the separation of labeled material from natural one on the GC column, have often been reported and discussed.2 From a mass spectrometric measurement the unlabeled glucose content in the labeled glucose we used was estimated to be less than 1%. Equilibration When an isotopically labeled glucose is added to the serum, a complete equilibration of the labeled form with the endogenous form may not immediately occur, because serum glucose might interact with the serum matrix. NBS reported that the equilibration was dependent on time; an equilibration time of 20 h was necessary to establish complete equilibration.4 With our experiment the analytical results of 2 h equilibration samples were clearly lower than those of 6- and 20-h samples. We concluded that equilibration was necessary and in our measurement about 20 h equilibration time (overnight) was used for convenience. Since glucose is known to decompose little by little at room temperature, and we did not find the reason why a preservation reagent should not be used, 0.1 ml of a 0.23 mol/ 1 solution of sodium azide in water was added at equilibration.

4 490 ANALYTICAL SCIENCES OCTOBER 1988, VOL. 4 Chromatographic separation In order to avoid a lengthy chromatographic run, we examined the gas chromatographic conditions and found that with the use of an SE-54 fused silica capillary column the glucose-bba was eluted in approximately 12 min. In human serum a small amount of other hexoses existed; if they were not separated from glucose, they might have contributed to errors. We derivatized these hexoses in order to analyze by GC-MS, and confirmed that other hexoses were clearly separated from glucose. Chromatogram is shown in Fig. 2. Figure 3 shows mass fragmentograms of the glucose derivative of the serum sample with labeled glucose added. Other hexoses or compounds were hardly detected in the measurements. Calibration and calculation To minimize the effect of instrumental drift, a bracketing procedure in which each sample was measured between calibration mixtures of similar ratios, has often been adopted for IDMS.4,6,7 With the system we used, however, since the random measurement error was larger than the instrumental drift, it was Fig. 2 Reconstructed ion chromatogram of BBA derivatives of glucose and other hexoses. Peaks; a) fructose, b) mannose+ galactose, c) glucose. preferable to repeat the measurements and to use the mean results for the calculation. We therefore used the calibration-curve method. The regression equation was R,=0.978XRM+0.047, where R, and RM are the peak intensity ratio and the mass ratio, respectively, with a correlation coefficient r> This showed that the linearity was satisfactory. Analytical results of serum samples Analytical results for the human serum reference material (NBS SRM 909) are shown in Table 1. Four replicates of the vial were prepared independently and analyzed. An isotope ratio measurement was repeated two or three times for each sample. Because the concentration was based on the weighed mass in our method, we measured the specific gravity in order to calculate the concentration per liter of the reconstituted serum. The results obtained by this method agreed with the certified value of NBS. The analytical results of three human serum pools by this method are shown in Table 2. To determine the precision of the whole procedure, a set of separate preparations was made from the same serum pool. The isotope ratio measurement was repeated two or three times for each sample. A relative standard deviation of the replicates (RSDX) of within 0.5% was obtained for measurements of all of the serum samples. Error analysis The accuracy of the measurement for glucose in serum is naturally based on the purity of the unlabeled standard. The SRM that we used was certified to be 99.9% pure glucose and the uncertainty of the glucose content was 0.1%. Errors in preparing the standard solution could contribute to errors in the determination. We prepared the stock standard solution based on measurements of the mass and error of weighing (0.1%). We added a known amount of labeled glucose and unlabeled glucose to the SRM serum and determined Table 1 Analytical results of glucose in human serum (NBS SRM 909) by IDMS method (mmol/l per gram) Fig. I Mass fragmentograms of BBA derivatives glucose with labeled [13C6] glucose added. of serum

5 ANALYTICAL SCIENCES OCTOBER 1988, VOL Table 2 Analytical results of serum glucose by IDMS method (mg glucose/g serum) Table 3 Analysis of variance the amount of glucose by this method. Analytical recoveries were calculated by dividing the obtained amount by the amount of spiked unlabeled glucose and the expected endogenous glucose in the SRM serum, which was calculated from the measured concentration of the SRM serum. Analytical recoveries were and 100.0%, respectively. An analysis of the variance12 on the data of Tables 1 and 2 demonstrated that the random errors in mass spectrometric measurements mainly contributed to the overall precision (Table 3). The overall precision of measurements ranged between 0.4 and 1.8%, but the precision of replicates was excellent. After an improvement of the mass spectrometric analysis this method would fulfill the requirement of a RSD less than 0.5%, suggested for a definitive method.13 The accuracy should be improved by pooling the results obtained by several laboratories as well as different methods. References 1. N. W. Tietz, Clin. Chem., 25, 833 (1979). 2. C. C. Sweeley, W. H. Elliott, I. Fries and R. Ryhage, Anal. Chem., 38,1549 (1966). 3. I. Bjorkhem, R. Blomstrand, 0. Falk and G. Ohman, Clin. Chim. Acta, 72, 353 (1976). 4. E. White V, M. J. Welch, T. Sun, L. Sniegoski, R. Schaffer, H. S. Hertz and A. Cohen, Biomed. Mass Spectrom., 9, 395 (1982). 5. R. Schaffer, Pure Appl. Chem., 45, 75 (1976) Pelletier and S. Cadieux, Biomed. Mass Spectrom.,10, 130 (1983) Pelletier and C. Arratoon, Clin. Chem., 33, 1397 (1987). 8. A. Takatsu and S. Nishi, Clin. Chem., 33,1113 (1987). 9. G. 0. Guerrant and C. W. Moss, Anal. Chem., 56, 633 (1984). 10. D. J. Bell, J. Chem. Soc., 1935, J. Wiecko and W. R. Sherman, J. Am. Chem. Soc., 98, 7631 (1976). 12. R. F. Hirsh, Anal Chem., 49, 691A (1977). 13. A. Cohen, H. S. Hertz, J. Mandel, R. C. Paule, R. Schaffer, L. T. Sniegoski, T. Sun, M. J. Welch and E. White V, Clin. Chem., 26, 854 (1980). (Received April 20, 1988) (Accepted August 9, 1988)