Anal, Chem. 1986, 58, 1681-1685
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Determination of Serum Creatinine by Isotope Dilutian Mass Spectrometry as a Candidate Definitive Method Michael J. Welch,* Alex Cohen, Harry S. Hertz, Kwokei J. Ng, Robert Schaffer, Pieter Van Der Lijn, and Edward White V Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
An Isotope dllutlon mass spectrometrlc (IWMS) method for
serum creatlnlne Is described whlch uses ~reatlnlne-'~C,as the labeled Internal standard. Creatlnlne Is separated from creatlne and converted to the ethyl ester of N-(4,6-dlmethyl-2-pyrlmldlnyl)-Nmethylglyclne. Comblned capillary column gas chromatography and electron Impact mass spectrometry are used to obtaln the abundance ratio of the unlabeled and labeled [M COOC&]+ Ions from the derlvathe. Quantltatlon Is achieved by measurement of each sample between measurements of two standards whose unlabeled/labeied ratios bracket that of the sample. Four freeze-dried human serum pools lncludlng Natlonal Bureau of Standards Standard Reference Materlal 909 were analyzed wlth this method. The coefficients of varlatlon for a slngle measurement ranged from 0.15 to 0.27% for the four pools. The measurements were found to be free of Interference. The hlgh precision and absence of significant blas quaUfy this method as a candldate definltlve method.
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Serum creatinine concentrations are commonly measured in clinical laboratories in evaluating renal function. Clinical methods generally involve a version of the Jaffe method or an enzymatic procedure ( I ) . Because of significant discrepancies in results among the methods in use (Z), a need exists for a method of demonstrated accuracy and precision, Le., a definitive method, to provide an accuracy base against which the other methods can be judged. Universal acceptance of the requirements a method must meet to be considered definitive have not been achieved. The National Committee for Clinical Laboratory Standards has published guidelines for definitive methods (3),which, if adopted, should result in more uniform approaches to establishing methods as definitive. Isotope dilution mass spectrometry (ID/MS) has been the technique of choice for definitive methods for both organic and inorganic analytes. Organic analytes for which definitive methods have been described include cholesterol (4),glucose (5), uric acid (6, 7),urea (8), cortisol (9-11), and other steroid hormones (12). ID/MS methods have been reported for creatinine, but until recently none was described as a definitive method. Bjorkhem et al. (13)reported an ID/MS method that uses creatinine-15N2 as the labeled species. The creatinine is isolated by HPLC and converted into a bis(trifluoroacetate) of the (&hydroxy2-methy1)ethyl derivative. The peak heights of the [M - 69]+ ions from the labeled and unlabeled forms of the derivative were measured by GC/MS. The results had a coefficient of variation (CV) of 3%. The method was used as a reference method for the evaluation of some routine methods (14,15). Lawson et al. (16)described an ID/MS method that uses the same internal standard and HPLC for isolation of the creatinine but employs a direct insertion probe for introduction of underivatized creatinine into the mass spectrometer and measurement of the ratios of the molecular ions. Precision was good for the analysis of one quality control serum (CV 0003-2700/86/0358-1681$01 S O / O
= 0.44%), but evidence for the absence of systematic bias in the measurements was not provided. Since the ions monitored are relatively small (m/z 113 and 115) and the samples are measured without prior gas chromatographic separation, there is a chance for significant interference at one or both masses. Siekmann (17)has recently reported an ID/MS method that he proposes as a definitive method. The method utilizes ~ r e a t i n i n e - ' ~ C , ' ~asN ~ the internal standard, ion exchange chromatography for isolating creatinine, conversion of creatinine to its trimethylsilyl (Me&%) derivative, and capillary GC/MS for measurement of the intensity ratios of the ions at m/z 329 and 332. For 13 different control sera analyzed, the CV's ranged from 0.32 to 1.04% with an average of 0.77%. Measurements for demonstration of the absence of bias were not reported, nor were studies on column memory effects, a potential source of significant error for compounds with Me3& groups attached to nitrogen. We have developed an ID/MS method for serum creatinine (18) that fulfills the stringent requirements of a definitive method (3). This method utilizes addition of a known weight of creatinine-13C2to a known weight of serum. Creatinine is separated from creatine and other serum components by column chromatography with a weakly acidic resin (19). Separation from creatine is necessary because under the derivatization conditions used, both give the same derivative. The isolated creatinine is converted to the ethyl ester of N-(4,6-dimethyl-2-pyrimidinyl)-N-methylglycine by reaction with 2,4-pentanedione and ethanol in the presence of acetic acid. For measurement the derivative is injected into a gas chromatograph equipped with a nonpolar-supporbcoated open tubular (SCOT) column directly connected to a low-resolution, magnetic sector mass spectrometer. Isotope ratio measurements are made from the abundances of the [M - COOCzH5]+ ions at m / z 150 and 152. Standards are made by combining and derivatizing known amounts of unlabeled creatinine and creatinine-13Cz.Standards with isotope ratios slightly higher and slightly lower than that of each sample are measured immediately before and after the sample. Use of this measurement technique (bracketing) has been shown to produce results having high precision (4-6,8,10). On measuring the creatinine level in the same samples using different chromatographic conditions and different ionization techniques, we found no evidence of bias in the measurement process.
EXPERIMENTAL SECTION Materials. Samples of Standard Reference Material (SRM) 909, a freeze-dried human serum, were obtained from the Office of Standard Reference Materials (OSRM), National Bureau of Standards (NBS). Other serum pooh analyzed were freeze-dried samples obtained from the Centers for Disease Control (CDC), Atlanta, GA. SRM 914 creatinine (2-amino-1,5-dihydro-lmethyl-4H-imidazol-4-one) with a certified purity of 99.8 i 0.1% was obtained from OSRM. C r e a t i ~ ~ i n e -(2-amino-1,5-di~~C~ hydro-l-(methyl-'3C)-4H-imidazol-4-one-5-'3C) with an isotopic purity of 95 atom % and creatinine-d3(Z-amino-1,5-dihydro-1(methyl-d3)-4H-imidazol-4-one) with an isotopic purity of 99.4 atom 70 were synthesized in this laboratory by a procedure described elsewhere (20). Creatinine-'*C (2-amin0-1,5-dihydro-l0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
methyl-4H-imidaz01-4-one-4-'~C) with an activity of 3.52 mCi/ mmol and creatine-14C (N-(aminoiminomethy1)-N-methylglycine-l-14C) with an activity of 9.0 mCi/mmol) were from commercial suppliers. 2,4-Pentanedione was distilled at 10 kPa (75 torr). The fraction boiling at 69 "C was collected and stored at -3 "C. Heptane was distilled. Other reagents were ACS grade and used as is. Sample Preparation. A fresh stock solution, consisting of approximately 6 mg of ~reatinine-'~C~ in 100 mL of water (both quantities accurately weighed), was prepared for each set of samples. The freeze-dried sera were reconstituted on a weight basis, as previously described (a), to correct for vial-vial variations in fill weight. Weighed aliquots of the reconstituted sera, containing 12-30 pg of creatinine, were combined with weighed aliquots from the aqueous solution of creatinine-I3C2such that the unlabeled/labeled creatinine ratio fell in the range of 0.184.26. The tubes containing the spiked serum were gently swirled, and the walls were washed down with water to bring the total volume to about 13 mL (not a critical quantity). The mixtures were allowed to equilibrate for about 24 h at room temperature and then passed through a weakly acidic ion exchange resin in the the H" form followed by 200 mL of water. Creatinine was quantitatively eluted with 100 mL of 1 M aqueous ammonia, and the effluent was freeze-dried. The residue was mixed with methanol and filtered, and the filtrate was concentrated. Ethanol was added and the solution concentrated again to eliminate methanol. The solution was transferred with ethanol rinses to a reaction tube for derivatization. Equilibration Study. To test for complete equilibration between labeled creatinine and endogenous creatinine in serum, a serum sample was spiked with an aqueous solution of creatinine-d,. After a brief mixing, the solution was divided into six aliquots, which were allowed to equilibrate for different periods (2, 4 , 8 , 22, 29, and 47 h) and were then processed as described above. Creatinine-Creatine Separation. Creatinine-14Cwas mixed with unlabeled creatinine in an aqueous solution and placed on the weak acid resin column. The elution of creatinine with water and then aqueous ammonia was followed by liquid scintillation counting. Creatine-14Cwas mixed with a serum sample and its elution was similarly followed. Calibration Standards. Four independent sets of standards were prepared. For each, aqueous stock solutions of SRM creatinine and creatinine-13C2were prepared by weight. Weighed portions of each solution were combined to provide a series of standard mixtures whose unlabeled/labeled creatinine weight ratios ranged from 0.18 to 0.26. Portions of these mixtures were derivatized as needed following the procedures described below. Derivatization. Creatinine, isolated from serum or from standard mixtures, was converted to the ethyl ester of N-(4,6dimethyl-2-pyrimidinyl)-N-methylglycineas follows. Glacial acetic acid (10 pL) and 2,4-pentanedione (50 WL)were added to a tube containing 50-115 pg of creatinine in ethanol. Ethanol was added to bring the total volume to 600 pL. The reaction tube was briefly shaken and placed in a heating block at 85 "C for 66-72 h. The reaction mixture was then concentrated under a stream of nitrogen at room temperature. The product was dissolved in heptane and transferred to a valved, septum-capped vial where the volume was adjusted to 150 FL for ID/MS measurements. GC/MS Conditions. The instrumentation consisted of a combined gas chromatograph, single-focusing magnetic sector mass spectrometer and a control and data acquisition system intended for isotope ratio measurements. The system and its use have been thoroughly described (8). For measurement under electron impact (EI) conditions, the mass spectrometer was operated at 72 eV with an emission current of 0.5 mA and an ion source temperature of 200-220 "C. The principal measurements were of the abundance5 of the [M - COOC2H,]+ions at m / z 150 and 152. Confirmatory measurements were made by using the [M + HI+ ions at m / z 224 and 226 from ammonia chemical ionization (CI) and the M+. ions at m / z 223 and 225 from EI. The conditions for measurements using ammonia CI were as follows: emission, 1 mA at 200 eV; source manifold pressure (Penning gauge), 5 x Pa (4 x torr); and analyzer pressure (ionization gauge), 7 X Pa ( 5 x torr). The source temperature control was critical for good precision in the CI mode and was maintained at 170 f 2 "C for
best results. For the principal E1 and for CI measurements the gas chromatograph was equipped with a 15-m nonpolar (poly(dimethylsiloxane)) SCOT column with an adjustable splitter at the front of the column and a fused silica transfer line to the mass spectrometer (8). The splitter was set to a vent-to-column ratio of 6:1, and the GC was operated at 130 OC with a helium flow rate of 3 mL/min. The injection port and interface to the mass spectrometer were maintained at 170-200 "C. Under these conditions the retention time for the creatinine derivative was 25 min. Confirmatory measurements in the E1 mode were done with a moderate polarity (50%poly(phenylmethylsi1oxane)) fused silica column operated at 133 "C and directly inserted into the ion source. Measurement Protocol. For the measurement of each sample, two standards were chosen: one whose ion abundance ratio was slightly higher than the sample and another whose ratio was slightly lower. The sample was measured between measurements of these standards. This was done by injecting one standard, followed 5.0 min later by the sample and 5.0 min later yet by the second standard. The solvent front from the third injection eluted approximately 5 min before the creatinine derivative from the first injection. The 5.0-min interval was chosen because at this interval no impurities from any one injection coelute with the creatinine derivative from another injection. The ion source filament and accelerating voltage were turned on after the last solvent front eluted. The ratio was measured for each peak as previously described (8). The quantity of analyte in the sample was calculated by linear interpolation of the measured ratio of the sample between the measured ratios of the standards, whose weight ratios were known ( 4 , 5 , 8). The order of injection was reversed for a second measurement, done on a separate day. The two calculated results for the sample were required to agree within 0.5%. If they agreed, as in almost all cases they did, they were averaged and their mean was the reported result. If not, a third measurement was made. If the third agreed within 0.5% of one of the other two (true for the remaining cases), the three values were averaged. A difference in peak height of more than a factor of 2 within an individual standard-sample-standard group served as grounds for data rejection, as has been discussed previously (8).
RESULTS AND DISCUSSION Choice of Labeled Material. AB had been found previthe choice of labeled material was critical ously with urea (8), to achievement of high precision. In our initial work with creatinine-d3 as the labeled material, we were unable to find conditions that would give reproducible ratios. We synthesized creatinine-13C2 for use in this ID/MS method in order to eliminate possible isotope effects from deuterium. Although there is not a primary isotope effect in the derivatization reaction with either labeled material, the likelihood of measurable isotope effects are much less with I3C than with deuterium. With creatinine-13C2as the labeled material, we found that our results were highly reproducible. Equilibration Study. The addition of an isotopically labeled material to a serum matrix may not immediately result in a complete equilibration of the labeled form with the endogenous form. The time required for complete equilibration depends upon the analyte and the matrix. Our ID/MS results for glucose and uric acid were found to be dependent upon equilibration times (5, 6). We studied the equilibration of endogenous creatinine with creatinine-d3and found it to be complete in 2 h. For convenience, 24 h equilibration times were used. C r e a t i n i n d r e a t i n e Separation and Interconversion. The presence in serum of creatine, the open ring form of creatinine, is a possible source of bias in our method, since creatine not removed from the sample will form the same derivative as creatinine. Experiments with an aqueous solution of creatinine-14C on the weak acid resin demonstrated that with the water washes 99% of the creatinine is retained. The retained creatinine is completely eluted with aqueous ammonia. The small amount of radioactive material that
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
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Table I. Test of Consistency of Two Independent Sets of Standard Mixtures (SRM Creatinine Creatinine-"C2)
+
Cii3 1003
bracketed std"
by
weight ratios (unlabeled/ labeled) mean measured by ID/MS weighed-in
B
c
0.24965 0.23705 0.2196 0.25765 0.22825 0.216 25
fl" I
50
IS0
100
200
m/z
Figure 1. Electron impact mass spectrum and structure of the ethyl ester of N-(4,6dimethyl-2-pyrimidinyl)-N-methylglyclne.
eluted in the water wash may have been from impurities or the result of creatinine-14Cconverting to creatine-14C. Since the labeled atoms in creatinine-13C2are not at the ring opening position, there can be no primary isotope effect in the conversion of unlabeled and %,-labeled creatinine to creatine, and consequently no significant effect upon the unlabeled/ labeled ratio. Creatine-14Cwas mixed with serum and placed on the resin to follow the elution of creatine and determine if it converts to creatinine either in the serum or on the resin. Creatine was completely eluted (>99.9%) with the water washes, demonstrating that there is no conversion of creatine to creatinine under our experimental conditions. The two experiments demonstrate that creatinine can be isolated free of creatine. Thus, there is no bias resulting from serum creatine in this method. Choice of Derivative. In order to introduce creatinine into the mass spectrometer through a gas chromatograph, it is necessary to convert it into a suitable derivative. The structure of the derivative chosen for this work, the ethyl ester of N(4,6-dimethyl-2-pyrimidinyl)-N-methylglycine, is shown in Figure 1. We adapted a procedure used for the derivatization of arginine in peptides (21,22)to form this derivative, which has several advantages. It can be formed in consistently good yields. Typical yields of creatinine, through the entire procedure, ranged from 70 to 75%. It has satisfactory gas chromatographic properties, including the absence of labile substituents likely to lead to chromatographic memory effects. The E1 mass spectrum has most of the ion intensity concentrated at two masses, both of which contain the labeled atoms. Memory Effects. We tested the derivative for column memory effects, although none were expected. If a memory effect is present, injections of a sample or standard of one unlabeled/labeled ratio will affect the ratio measured for subsequent injections of other samples or standards. We injected the unlabeled creatinine derivative, followed at 5-min intervals by the creatinine-I3C2derivative and the unlabeled derivative again and measured the ratios. The ratios for the two injections of the unlabeled material were not significantly different. The lack of effect on the measured ratios, even where the ratio differences between consecutive measurements were the most extreme and memory effects should be most evident, provides strong evidence for the absence of column memory effects. Electron Impact Mass Spectra. The electron impact mass spectrum for the creatinine derivative, the ethyl ester of N-(4,6-dimethyl-2-pyrimidinyl)-N-methylglycine is shown in Figure 1. The base peak, at m / t 150, results from the loss of the carbethoxy group. This ion and the corresponding ion for the labeled creatinine derivative at m/z 152 were chosen for the ratio measurements because of their intensity and the
0.2679 0.2493 0.2370 0.2193 0.2582 0.2284 0.2166 0.2083
difference, %
+0.14 +0.02 +0.14 -0.21 -0.06 -0.16
"Standards A-D were from one set, E-H from the other. bNot bracketed. absence of interference at these masses. Samples and standards were also free of interferences at m / z 223 and 225, the molecular ions for the unlabeled and labeled creatinine derivative, respectively. The intensities for these ions were considerably less, but were sufficient for confirmatory ratio measurements. Chemical Ionization Spectra. Isobutane, methane, and ammonia were tested as reagent gases. The base peak for the creatinine derivative was [M H]+ with all three reagent gases, but significant amounts of [M - H]+ were also found. Since [M - H]+ for the labeled derivative has the same mass as [M H]+ for the unlabeled, it would contribute to the intensity measured for the unlabeled form. If the relative abundance of [M - H]+ were constant under varying C1 conditions, a nonlinear interpolation could be applied (23). However, with isobutane as the reagent gas, the ion abundance ratios were very dependent upon sample size and source temperature. The relative abundance of the [M - H]+ ion was found to depend upon the reagent gases in the following order: NH3
