Anal. Chem. 1984, 56,713-719
DOE-Pittsburgh Energy Technology Center under Interagency Agreement No. DE-AI22-82PC53141. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental proce-
713
dure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
Determination of Serum Urea by Isotope Dilution Mass Spectrometry as a Candidate Definitive Method Michael J. Welch,* Alex Cohen, Harry 5.Hertz, Fillmer C. Ruegg, Robert Schaffer, Lorna T. Sniegoski, and Edward White V
Center for Analytical Chemistry, National Bureau of Standards, Washington, D.C. 20234
An isotope dilution mass spectrometric (ID/MS) method for serum urea Is descrlbed. The method utlllzes urea-“O as the labeled Internal standard and Involves Isolation of urea from serum, converslon to 6-methyC2,4-bls[(trlmethylsilyl)oxy]pyrlmidine, capillary column gas chromatography for sample Introduction, and measurement of the abundance ratio of the (M 15)’ Ions from the labeled and unlabeled derlvatlve. Quantltation is achieved by measurement of each sample between measurements of two standards whose unlabeled/ labeled ratios bracket that of the sample. Results are of high precislon, wlth coetflclents of varlatlon for a single measurement of 0.17% for NBS Standard Reference Material 909, a freeze-drled human serum pool, and 0.19% overall for five frozen serum pools, and have been shown to be free of measurement Interferences. The method is therefore of Sufficient accuracy and preclslon to be considered a “deflnltlve” method.
coefficients of variation were less than 5%. In this paper we describe an ID/MS method for serum urea of sufficient accuracy and precision to be considered a definitive method. We add a known weight of urea-180 to a known weight of serum such that the measured ratio of unlabeled to labeled urea is approximately 1:1. After an equilibration period, the serum is freeze-dried. The urea is extracted with methanol, the methanol is evaporated, and the urea is sublimed. The urea is then converted to 6-methyluracil by using modifications of published syntheses (13,14). For gas chromatography, the derivative is converted to its trimethylsilyl ether, 6-methyl-2,4-bis[(trimethylsilyl)oxy] pyrimidine. For our ID/MS measurements, samples are bracketed with standards, a calibration technique shown to provide excellent precision (2,3). Ion abundance ratios are measured for both samples and standards by using the (M - 15)’ ions at m / z 255 and 257. The quantity of unlabeled urea in the sample is then found by linear interpolation. As tests for measurement bias, randomly selected samples are measured again by using two other pairs of ions.
The measurement of serum urea levels is an important clinical test for kidney function as well as for protein metabolism. As part of a program for standardization of clinical methods, we have undertaken for serum urea development of a “definitive” method ( I ) , i.e., a method of demonstrated high accuracy and precision, to provide an accuracy base to which reference and routine methods can be compared. Isotope dilution mass spectrometry (ID/MS) is a technique which has been shown to be capable of meeting the rigorous requirements of a definitive method (2-8). Although ID/MS has been employed in the determination of serum urea, none of the reported methods can be considered a definitive method. Bjorkhem et al. (9, 10) reported an ID/MS method utilizing ureaJ5N2 as the labeled analogue and derivatization of the urea to a dimethylated 5,5-diallylbarbituric acid. The coefficient of variation (CV) for a single measurement by this method was 3.6%, which the authors judged to be sufficiently precise for use as a reference method for their laboratory. Two ID/MS urea methods intended for use in studies of metabolism have been reported. Nissim et al. (11) employed urea-16N2as the labeled analogue and converted the urea to its trifluoroacetyl derivative. While the method was satisfactory for its intended purpose, its precision (CV 7%) does not meet the requirements of a definitive or a reference method. Tserng and Kalhan (12)also used urea-16N2but converted urea to the trifluoroacetyl or trimethylsilyl derivatives of 2-hydroxypyrimidine. Their method was applied to enrichments of as low as 0.1%. With higher enrichment,
EXPERIMENTAL SECTION Serum. Both freeze-dried and frozen sera were studied. The freeze-dried serum was Human Serum Standard Reference Material (SRM) 909 from the National Bureau of Standards. The serum was reconstituted in the vials in which it was supplied by addition of 10 mL of the diluent water supplied with the serum. Each vial with ita contents was weighed before and after adding the water, and later, the clean empty vial was weighed again so that the weights of freeze-dried serum and added water were known. After addition of the water, the mixture was gently swirled periodically to dissolve the serum and ensure homogeneity. The frozen sera were of bovine origin and were five different pools supplied by the Centers for Disease Control (CDC),Altanta,GA. This serum was stored at -20 O C until samples were needed, at which time the serum was allowed to thaw at room temperature. Urea. The unlabeled urea used as the primary standard was SRM 912 with a purity of 99.7 h 0.1%. Urea-180was synthesized as described below. Urea-13C,16N2 was commercially available and had an isotopic purity of 90 atom % 13C and 95 atom % lSN. Synthesis of Urea-180. The synthesis of urea-180 from cyanamide and H2180(97 atom % l80)followed published procedures (15, 16) with the following modifications. Hydrogen chloride gas was used in place of hydrochloric acid. After the crude product was extracted into ethanol and acetone, the solution was treated with aqueous silver nitrate to remove any remaining cyanamide and then with aqueous sodium chloride to remove any excess silver. The urea-180 was twice sublimed at 100 “C and 15-27 Pa (0.11-0.20 torr) with a dry ice cooled condenser and recrystallized from acetone-ethanol. The yield of product (90 atom % l80)was 0.8 g, or 28%, mp 134.0-134.3 O C .
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This article not subject to U.S. Copyright. Published 1984 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Other Reagents. Only ACS reagent-grade chemicals were used. Methanol, ethanol, and acetone were redistilled. Calibration Standards. Mixtures of known amounts (2.5-3 mg each) of SRM urea and urea-'*O were prepared such that the weight ratios (unlabeled/labeled) were in the range of 0.78-0.97. Two sets of standard mixtures were prepared independently from separate standard solutions of labeled and unlabeled urea. The individual standard mixtures were prepared with weighed aliquots of these solutions and then concentrated to dryness and stored at -4 "C. As needed, portions of each mixture was derivatized by the procedure described below. Sample Preparation. For each sample a weighed aliquot of serum containing about 0.83 mg of urea was added to a flask. A weighed aliquot of a standard solution of urea-'*O was added such that the unlabeled/labeled ratio fell within the range of the calibration standards. The inner walls of each flask were washed with water and the mixture (about 60 mL total volume) was swirled. The mixtures were allowed to equilibrate at room temperature for 22-24 h and then freeze-dried at 190-280 Pa (1.4-2.1 torr). The residue was mixed with methanol and centrifuged, and the supernatant transferred to a sublimation flask. Sublimation of each sample was accomplished at 100 "C for 45 min at 50-90 Pa (0.4-0.7 torr). The sublimate was dissolved in methanol, placed in a tared vial, dried, and weighed. Derivatization. Urea was dissolved in a known amount of methanol and an aliquot containing about 100 pg of total urea, whether from serum or a standard mixture, was transferred to a 2-mL ampule. After the solvent was allowed to evaporate, 2.5 p L of diketene and 25 pL of glacial acetic acid were added. The ampule was flame sealed and placed in a heating block at 82-84 "C for 46-48 h. After the reaction mixture cooled, it was transferred with methanol to a conical vial fitted with a Teflonvalved cap. The methanol was evaporated and 50 pL of bis(trimethylsily1)acetamide (BSA) was added to complete the derivatization. The derivative was stored in the excess BSA for the ID/MS measurements. As a measure of the total losses in sample preparation, ion abundances of samples were compared with ion abundances for solutions of known concentration of the derivative. Yields averaged 85%. Instrumentation. A Varian MAT Model CH 7A, single-focusing, magnetic sector mass spectrometer equipped with a combined electron impact/chemical ionization ion source and interfaced to a gas chromatograph was used for the ID/MS measurements. The gas chromatograph was modified for use with capillary columns by the installation of pressure control, an adjustable splitter, and a direct connection from the column to the mass spectrometer. The connec,tion was a fused silica line, the GC end of which was positioned a few centimeters into the end of the column. The opening in the fused silica was restricted with a tapered piece of very fine glass to keep the end of the GC column near atmospheric pressure, The fused silica line ran inside the line of sight quartz tube through a heated interface to within a few centimeters of the ion source. The mass spectrometer and the system used for multiple ion selection and data acquisition have been extensively modified from that described in our previous ID/MS work (2, 3). The mass spectrometer was modified by installation of a 270 L/s turbomolecular pump for the ion source region. Ionization gauges were installed in the ion source and analyzer regions. A microcomputer with 64 kbytes of memory and two 8-in. floppy disk drives was installed to perform several functions as illustrated in Figure 1. It drives a 16-bit digital-to-analog converter (DAC) to control the switching of the magnetic field between the selected m/z values for the isotope ratio measurements. The computer generates a triangular wave which is sent to the beam deflection plates to sweep the selected ion signal rapidly back and forth across the mass spectrometer detector slit. The frequency (maximum rate 35.65 Hz) of the wave is software controlled while the amplitude (k150 V), which sets the mass range covered, is controlled manually. The ion intensity signal from the electron multiplier goes through a solid-statepreamplifier which replaces the vacuum tube preamplifier originally supplied with the system. The signal is then sent to the amplifier (provided with the instrument) and then to the computer, via a 12 bit analog-to-digital converter (ADC). The computer acquired and
Rwam& Par meter File Btorage
G.C.
1 --+
ION SOURCE
PRINTER Permanent Recorda
CRT Real Time Display Of Data Magnet Stability Signal
Ion intensity Signal
Figure 1. Diagram of the Isotope dilution gas chromatograph/mass spectrometer and the associated control and data acquisltlon system.
stores the intensity signal for each monitored mass. As the intensity signals are acquired they are displayed numerically on a CRT and are also used to drive a strip chart recorder for monitoring the gas chromatographic peak shape and retention time. Simultaneously,the signal sent to the beam deflection plates is used to drive the time base of an oscilloscope where the shape and position of the mass peak can be monitored. After data acquisition is completed, the stored ion intensities are corrected for background and summed and the ratio of the summed intensities at the two masses is calculated in accordance with the protocol described later. The data reduction program is identical with that used for previous work (2,3).The data and results are printed to provide a permanent record. The operating programs, data reduction programs, and parameter files are stored on the disks. Each parameter file consists of magnetic field settings for a pair of ions to be monitored and the number of triangular wave sweeps to be generated for each dwell time. The magnetic field settings can be adjusted from the keyboard prior to data acquisition with the adjusted settings replacing the old settings in the parameter file. During a run the magnetic field can be adjusted manually to keep the observed peak centered within the mass window set by the amplitude of the triangular wave. GC/MS Procedure. The gas chromatograph was equipped with a 30-m 0.5 mm i.d. SE-30 SCOT column operated at 125 "C with a flow of helium of 3 mL/min. The adjustable splitter was set to a split ratio of 6:l vent to column. The injection port and the interface to the mass spectrometerwere maintained at 170-200 "C. Under these conditions, the retention time for the trimethylsilyl derivative of 6-methyluracil was 25 min. Typically, 6-8 pg of derivative in about 1 pL of BSA was injected and the resulting peaks were 70-80 s wide at the base. For measurement under electron impact (EI) conditions, the mass spectrometerwas operated at 72 eV with an emission current of 0.5 mA and a source temperature of 200-220 "C. The principal measurements were made in the E1 mode monitoring the (M 15)' ions at m / z 255 and 257. The doubly charged ions, [M 2(CH3)]*+,at m/z 120 and 121 were used for confirmatory measurements in the E1 mode. For isobutane chemical ionization (CI), the mass spectrometer was operated at 170 eV with an emission current of 1 mA and a source temperature of 180-190 "C. The Pa (4 X lod source manifold Penning gauge pressure was 5 X torr) with the analyzer pressure at 7 X lo-' Pa (5 X lo-' torr). Confirmatory measurements, in the CI mode, were made by monitoring the (M + 1)+ ions at m / z 271 and 273. For all of the measurements,the dwell time for each mass was 700 ms (25 sweeps at 28.05 ms/sweep). The magnetic field took approximately 300 ms to stabilize after each switch, making the total time approximately 2 s for one complete cycle including both masses. Thus typical GC peaks had 35-40 measurement cycles across them. Injections of standards and sample were made in groups of three as described in the measurement protocol section. Shortly after the last solvent peak in each group eluted, the amplifier output was zeroed and the accelerating voltage and emission current were turned on. These remained on until the last GC peak in the group eluted. Measurement Protocol, After a preliminary ratio measurement of the ion abundances of a sample is made, two calibration standards are chosen: one whose ratio is slightly higher
ANALYTICAL CHEMISTRY, VOL. 56,
than that of the sample and another whose ratio is slightly lower. The sample is measured between these standards. This is accomplished by injecting one standard, followed at 5.5-min intervds by the sample and then the second standard. The solvent front from the third injection elutes approximately 5 min before the urea derivative from the first injection. The 5.5-min interval was chosen because at this interval no impurities from one injection coelute with the urea derivative from other injections. Data for the calculation of abundance ratios are acquired for each peak in the following manner: the computer acquires and stores all of the intensity data separately for each monitored mass, finds the peak maximum for the lower mass as the data are acquired, and terminates data acquisition when the intensity has fallen to 1%of the peak maximum. The data are then processed and abundance ratios calculated according to the following procedure. The intensity data for the lower mass are examined to find the cycle, N , in which the GC peak began. The median intensity of the seven preceding cycles ( N - 7 to N - 1)is found and used as the base line for the lower mass data. For the higher mass data the base line is offset one cycle earlier, i.e., the median intensity for cycles N - 8 to N - 2 is found. The intensities acquired for each mass are corrected by subtracting the corresponding base line. These corrected intensities are summed from the start of the GC peak (cycles N and N - 1for the lower and higher masses, respectively) to the point where the intensity of the lower mass has fallen to 2% of the peak maximum (cycles N + W, and N - 1+ W for the lower and higher masses, respectively)where W is the number of cycles across the peak. The resulting sum for the lower mass is then divided by the sum for the higher mass to give an ion abundance ratio. The ratios obtained from the 2% cutoff data are used for calculation but the results are not significantly affected if other cutoffs are used as long as the same cutoff point is used for both standards and samples. The quantity of analyte in the sample is calculated by linear interpolation of the ion abundance ratio of the sample between the ion abundance ratios of the standards, whose weight ratios are known (2, 17). Each sample is measured as described above and then remeasured on a separate day with the order of injection reversed. If the results of the two runs agree to within 0.5%, they are averaged and the mean is the reported result; if not, a third run is made. If the third agrees within 0.5% of one of the other two, the mean of the three runs is the reported result. If it does not, the three values are discarded and the system is studied to find and correct the cause of the nonreproducibility before measurements are resumed. For these urea measurements no values were discarded. Data are also rejected when the intensity of any GC peak in a group of three differs by more than a factor of 2 from either of the other peaks. When this happens the data are discarded and the measurements are repeated, using adjusted quantities as necessary.
RESULTS AND DISCUSSION For a method to be considered “definitive”, it must be demonstrated that the method produces results of high precision and that no evidence for significant bias can be found. Demonstration of the precision of a method is straightforward, but to prove that the results are unbiased is not possible, when the “true” answer is unknown. It is, however, possible to provide evidence for the absence of significant bias in wellcontrolled measurement systems which allow individual sources of systematic error to be evaluated. The combination of isotope dilution, gas chromatography/mass spectrometry, and the technique of bracketing each sample by calibration standards when conducted under a strict measurement protocol provides one approach to achieving a well-controlled measurement system. The advantages of this approach have been previously described (2). We will discuss the instrumentation and special techniques used, some aspects of the chemistry, the precision obtainable, and evidence that biases are probably very small for our ID/MS serum urea method. Possible sources of bias and imprecision are also discussed.
Instrumentation and Special Techniques for High Precision. We modified commercial instrumentation and
NO. 4, APRIL 1984 715
used special techniques to significantly improve the precision commonly attained in organic isotope dilution mass spectrometry. Figure 1is a schematic representation of the gas chromatograph/mass spectrometer and the associated control and data acquisition system used in this work. In this section we describe and discuss the instrument modifications, special techniques used, and the rationale for each. For the vacuum system of the mass spectrometer, replacement of the oil diffusion pump and cold trap on the ion source with a turbomolecular pump resulted in lower operating pressures and the elimination of small, repetitive pressure pulses and their effects. Ionization gauges installed on the source and analyzer sections enabled us to more accurately monitor operating conditions. Switching between selected ions on magnetic sector instruments is frequently accomplished by varying the accelerating voltage. Our experience has been that better precision is obtained when the ion source conditions are kept constant and the magnetic field is switched; however, this is a slower process, since the magnetic field must stabilize before data acquisition is begun. On our instrument and under the conditions used, the time for magnet switching and settling is approximately 30% of each 2-9 measurement cycle. The triangular wave signal sent to the beam deflection plates sweeps the ion beam rapidly back and forth across the detector slit. The amplitude of the triangular wave is chosen such that the signal reaching the detector covers a range of about one mass unit centered about the mass of interest. As a result, small changes in the ion beam position (which occur as a consequence of changing instrumental conditions such as drift of the magnetic field or ion source pressure changes) cause only the portion of base line measured around the mass peak to be different. Consequently, changes in beam position and irregularities in the shape of the mass peak have no observable effect on the precision. Our previous technique for beam sweeping (2,3) employed continuous sweeping, with measurement dwell times beginning and ending a t random points in the sweep. This resulted in poorer precision when slow sweep rates were used. Our present system sweeps the beam only during the measurement dwell times and accumulates data for a preset integral number of sweeps, thus reducing the effect of sweep rate on precision. The accurate measurement of narrow peaks eluting from a capillary column requires excellent linearity of response from the signal processing system. The intensity of the signal changes rapidly, typically 3 orders of magnitude in 10-15 s on the steep sides of peaks. Replacement of the vacuum tube preamplifier supplied with the instrument with a modern solid-state electrometer led to improved linearity of response and, consequently, improved precision of measurement. The measurement protocol that we have adopted is designed to ensure that samples and standards are measured under nearly identical conditions. Mass spectrometric ion abundance ratio measurement can, and usually do, vary with time due to instrumental drift, Quantitation is therefore based upon ion abundance ratios for samples and standards measured close together in time. The protocol also requires that ion abundances from the sample and bracketing standards agree within a factor of 2, to reduce the possible effects from ion source pressure changes or from any nonlinear responses in ion detection and ion abundance signal processing. Remeasurement of the samples on a second day, with the order of standards reversed, serves as a test for bias resulting from column memory effects and gives us a measure of how well we can do replicate measurements on a single sample. The final precision and accuracy which can be achieved also depend critically on the handling of the sample and labeled material. For the lyophilized serum used in these experiments,
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 4, APRIL 1984
Table I. Test for Consistency of Two Sets of Standard Mixtures (SRM Urea std
bracketed ID/MS measd wt ratios (unlabeled/labeled) by run 1 run 2 run 3
range,
+
Urea-I80)
%
mean
weighed-in ratio
diff, %
0.89 0.26 0.41 0.21
0.847 07 0.856 49 0.916 27 0.952 24
0.786 68 0.850 14 0.857 25 0.916 87 0.951 99
-0.36 -0.09 -0.07 + 0.03
Set I A
B C D E
NB a G, H G,H J, J,
K K
0.850 26 0.855 37 0.918 b 1 0.951 1 9
0.842 70 0.857 58 0.916 51 0.953 23
0.848 24 0.85 6 53 0.914 30 0.952 30
av -0.12
F G H J K L
A, B A, B
c, D c, D
0.81278 0.84097 0.873 45 0.911 42
0.812 68 0.840 17 0.873 14 0.911 48
Set I1 0.812 91 0.838 73 0.873 25 0.910 58
NB' NBa
0.03 0.27 0.04 0.10
0.812 79 0.839 96 0.873 28 0.911 16
0.811 73 0.839 06 0.872 06 0.910 85 0.955 67 0.968 72
+ 0.13 +0.11 +0.14 t 0.03 av +0.10
' NB, not bracketed. the weight of dry serum can vary a few percent from vial to vial. Therefore, the serum was reconstituted by weight; that is, both the quantity of water and the dry serum were weighed. In general the required accuracy in the measurement of small aliquots can best be obtained gravimetrically, and therefore in the procedure where a quantity of material must be accurately known, the measurement is made gravimetrically rather than volumetrically. Use of Urea-13C,1SN2 as the Labeled Analogue. The choice of labeled urea is a critical factor in the success of the method. We investigated the use of urea-13C,16N2as the labeled material before choosing ureaJ80. Under the most carefully controlled conditions we were able to devise, three samples from the same vial of reconstituted serum that was used for the samples in Table I1 were spiked with urea-13C,15N2 and bracketed by standards prepared with ~ r e a - ~ ~ C , ' The ~N~. mean result was 7.260 mg of urea/g of dry serum with a CV for the mean of 0.17%. This uncertainty was mostly due to sample-sample differences. The mean concentration was 0.34% higher than the mean obtained by using urea-lsO as the isotope diluent. Confirmatory measurements at the alternate ions gave similar results, leaving the concentration difference between the labeled materials the same. In general, studies that used ~ r e a - l ~ C , ~consistently ~N, gave poorer precision within sets and much poorer precision between sets than we found with use of urea-180. For example, two earlier sets with urea-13C,15N2had means which differed by 1.29% and CV's for single measurements of 1.69 and 0.35%. On investigation, we found that changes in derivatization conditions affected the measured ratios enough to account for the poor precision observed between sets. In the derivatization to 6-methyluracil, nitrogen atoms from urea are directly involved in the cyclization reaction, thus a primary isotope effect may occur and be large enough to affect the relative amounts of labeled to unlabeled derivative that form. Also variations in freeze-drying conditions used when isolating the urea had some effect on the measured ratio. When urea-ls0 was used as the labeled material under carefully controlled conditions, problems with precision became much less significant. A disadvantage associated with the use of urea-180 is that there are contributions from the labeled material at the ion monitored for the unlabeled, and vice versa. These contributions cause the relationship between weight ratios and ion abundance ratios to be nonlinear. However, it has been shown by Yap et al. that, over the small weight ratio ranges covered by bracketing, and with the relatively small cross contributions, the errors caused by using linear interpolation in our
(In,
analyses are negligible (
