Anal. Chem. 1989, 6 1 , 1718-1723
1718
Determination of Serum Cholesterol by a Modification of the Isotope Dilution Mass Spectrometric Definitive Method Polly Ellerbe,* Stanley Meiselman, Lorna
T.Sniegoski, Michael J. Welch, and Edward White V
Center for Analytical Chemistry, National Institute of Standards and Technology (formerly National Bureau of Standards), Gaithersburg, Maryland 20899
An Isotope dlutlon mass spectrometrlc (ID/MS) method for cholesterol is descrlbed that uses capillary gas chromatography wHh c h ~ l e r t e r d - ~ ~as C the , labeled Internal standard. Labeled and unlabeled cholesterol are converted to the trlmethylsHyl ether. Combined capfflary column gas chromatography and electron m a c t mass spectrometry are used to obtaln the ak#ldance ratk d the urlsbekd and labeled [M"] Ions from the derlvatlve. Quantltatlon Is achleved by measurement of each sample between measurements of two standards whose unlabeled/labd.d ratlos bracket that of the sample. Seven pools were analyzed by thb method: standard reference materlal (SRM) 1951, which condsts of three frozen serum pools wHh low, medium, and high levels of cholesterol; SRM 1952, which condsts of three freeze-drled serum pools wlth low, medlum, and high lev- of cholmterok and SRM 909, a freeze-drled serum pool. The method is a modlflcatlon of our original deflnitlve method for cholesterol. The modifled method uses much better chromatographlc separatlons to assure spectfklty and a new method of Implementlng selected Ion monttorlng on a magnetic mass spectrometer to obtaln hlgh-precislon measurements of ion Intensity ratlos on narrow gas chromatographic peaks. The m o d W method has a coettlcknt of varlatbn (CV) d 0.22%, which Is an Improvement over the orlglnal method's CV of 0.36%. The measurements were found to be free of Interference. The high precidon and abemce of bias qualm this method as a candidate deflnltlve method.
Heart disease is the number one cause of death in the United States, and epidemiologic studies have shown a strong correlation between coronary heart disease and blood cholesterol levels. Therefore, a major effort, directed by the Cholesterol Education Panel of the National Institutes of Health, is under way to educate physicians and the general public on the importance of having one's blood cholesterol measured and of taking corrective action if the level is elevated. A wide variety of methods and instrumentation is used to measure serum cholesterol, and the results vary considerably. Proficiency testing programs of clinical laboratories have shown large discrepancies in cholesterol results among the laboratories (I). For the cholesterol education program to be effective, the measurements must be accurate enough to allow for proper diagnosis and long-term monitoring. An accuracy base in the form of serum-based materials with known concentrations of cholesterol can provide laboratories with an important component in assuring that patients' samples are accurately measured. To this end, a method of demonstrated accuracy and high precision, i.e., a definitive method, is necessary. Universal acceptance of the requirements for a method to be called definitive has not been achieved. The National Committee for Clinical Laboratory Standards has published guidelines for definitive methods ( Z ) , which define 0003-2700/89/0361-1718$01.50/0
a set of rules for the acceptance or rejection of a given method as definitive. Our group at the National Institute of Standards and Technology (NIST) (formerly National Bureau of Standards) has developed methods that are, according to those guidelines, definitive methods for cholesterol (3),glucose (41, uric acid (5,6),urea (7), and creatinine (8)in human serum. Other laboratories have published methods that they describe as definitive for cortisol (911),cholesterol (12), creatinine (13), glucose (14), and uric acid (15). The first definitive method developed at NIST was for cholesterol (3). Many modifications to the instrumentation have been made to improve precision since then (7,8), and capillary columns have replaced packed columns in the GC. The use of capillary columns leads to relatively narrow chromatographic peaks, which required the development of a new method for multiple ion monitoring (16) in order to achieve the required precision. When the need to measure cholesterol again arose, we saw the opportunity of using our modified instrumentation and modern capillary GC columns to improve the previous method. Isotope dilution mass spectrometry (ID/MS) has been the technique of choice for definitive methods since it does not depend on sample recovery and can be tested for the presence of bias and interferences. Our use of ID/MS is based on spiking a sample with a labeled version of the analyte as an internal standard, equilibrating, processing the sample, and the measuring the ratio of unlabeled to labeled analyte by using gas chromatography/mass spectrometry (GC/MS). Any loss of material after spiking does not affect the results since it is the ratio of unlabeled to labeled analyte that is measured. We tested for bias in sample preparation by preparing independent multiple sets of samples. The probability of undetected interferences was reduced by measuring all samples with the use of a prominent ion from electron impact ionization and then selecting a representative subset of samples to be measured at other prominent ions, with other GC columns, and/or with another mode of ionization. Therefore, for an interfering species to be undetected, it would have to have the same retention time as that of the analyte on the different GC columns, the same ions at all the masses used for measurement in each method of ionization, and the same abundance ratios among these ions as that of either the labeled or unlabeled version of the analyte being measured. These sets of measurements are called the confirmatory measurements. Standard reference material (SRM) 909, freeze-dried human serum, is certified for only one level of cholesterol, and that level is low compared to normal human serum levels. The present work was done to certify two new human serum SRMs that each consist of three levels and cover a clinically significant range of cholesterol levels for humans. Our modified ID/MS method for serum total cholesterol fulfills the stringent requirements for a definitive method (2). The method is based on the addition of a known weight of ch~lesterol-'~C~ to a known weight of serum. The cholesterol esters are hydrolyzed, and the mixture of unlabeled and la@ 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
beled cholesterol is extracted and converted to their trimethylsilyl ethers. For measurement, the derivative is injected into a GC equipped with a nonpolar fused silica capillary column that has been directly inserted into the ion source of a magnetic sector mass spectrometer. The principal isotope ratio measurements are made from the abundances of the [M+'] ions at m j z 458 and 461. Standards are made by combining and derivatizing known amounts of pure unlabeled cholesterol and ch~lesterol-'~C~ Standards with weight ratios slightly higher and slightly lower than that of each sample are measured immediately before and after the sample. Use of this measurement technique, known as bracketing, has produced results of high precision (3). Confirmatory measurements made on cholesterol in the same samples using different ions, different chromatographic conditions, and different ionization techniques provided evidence that there was no bias in the measurement process.
ION SOURCE
1719
PRIEITEP CKT
GC
COMPUTER STRIP [WART RECORDER
1
Figure 1. Electric beam switching.
to the time necessary for the magnet to settle after switching between ions and due to the use of real-time data display. This dead time was too large to allow the number of measurement cycles necessary for good precision over the 5-20 s wide peaks of the cholesterol TMS derivative eluting from capillary columns. A method suitable for obtaining data on such nmow capillary EXPERIMENTAL SECTION column peaks has been developed by changing the method of switching between masses and abandoning the display of data Materials. Samples of standard reference material 909, a as it is acquired. The new means of switching masses eliminates freeze-dried human serum, were obtained from the Office of the dead time due to magnet settling (16). This is done by Standard Reference Materials (OSRM) at NIST. Other serum selecting the masses of interest by using the pair of deflection pools analyzed were SRM 1951, consisting of three levels of plates located after the magnet and before the collector slit, as cholesterol in frozen liquid sera, donated by the Centers for shown in Figure 1. This pair of plates was previously used only Disease Control (CDC), Atlanta, GA; and SRM 1952, consisting to wobble the ion beam across the collector slit, which allowed of three levels of cholesterol in freeze-dried sera, donated by the the analyst to observe the peak on a peak monitor or oscilloscope. College of American Pathologists (CAP), Skokie, IL. SRM 911a The ion beam passing between these plates is several mass units cholesterol (cholest-5-en-3-01(3p)) with a certified purity of 99.8 wide in the m a s region used for these measurements. The magnet f 0.1% was obtained from OSRM. Ch01est-5-en-25,26,27-~~C~-3-01 is set to the lower mass (or between the masses) of interest. Both (3p) with a measured isotopic purity of 99.3 atom % was obtained a square wave signal,which selects the mass, and a triangular wave from MSD Isotopes, St. Louis, MO. Other reagents were of ACS signal, which sweeps (28 ms/sweep) the selected ion beam across grade and used as is. the slit, are applied to the summing point of an amplifier that Serum Densities. Serum densities were measured according drives the deflection plates. The square wave voltage from a 16-bit to a procedure described previously (17). digital to analog converter (DAC) under computer control alSample Preparation and Derivatization. The procedures ternately switches the ion beams through the collector slit. The used for sample preparation have been previously described (3). triangular wave from a 12-bit DAC sweeps about 1 mass unit Briefly, frozen samples were allowed to thaw, and freeze-dried across the slit for a preset number of times per measurement cycle. samples were reconstituted by weight (Procedure A)(18). A Deflection plate voltages are set such that the unlabeled and weighed aliquot of an ethanol solution, containing a known labeled ion beams will alternately be switched to the collector slit. quantity of ch~lesterol-'~C~, freshly prepared for each set of The specific hardware used provides a mass range that is about samples, was added to a weighed aliquot of each serum sample. 1.3% of the mass being monitored. The program controlling data The cholesterol esters were hydrolyzed by using KOH in ethanol, acquisition is started before elution of the GC peak of interest. and the cholesterol was extracted with hexane. The hexane layer One acquisition is a series of repeated cycles. was evaporated and the residue taken up in methanol for storage The following describes one measurement cycle. The square and handling. This procedure has been shown (3)to hydrolyze wave voltage is switched to select the mass of the unlabeled at least 99.9% of the cholesterol esters in serum. An aliquot of analyte, and acquisition of ion intensity data begins. During the methanol solution was dried, and bis(trimethylsily1)acetamide acquisition, the triangular wave applied to the deflection plate was added to form the trimethylsilyl (TMS) derivative of chosweeps the ion beam across the collector slit. Upon completion lesterol. of a preset number of sweeps (10 for cholesterol measurement), Lathosterol-Cholesterol Separation. Lathosterol (5adata acquisition is interrupted. The square wave is switched to cholest-7-en-38-01)was derivatized by following the procedure select the mass for the labeled analyte, and acquisition starts again above. Aliquots of the lathosterol derivative and the cholesterol for the same number of sweeps as set for the unlabeled analyte. derivative were mixed and examined by GC/MS using the When that preset number of sweeps is reached, acquisition is principal measurement conditions described below. interrupted again, and the next measurement cycle begins. Calibration Standards. Three independent sets, each conThe process is repeated continuously before and during the taining 10 or 11standards, were prepared. For each set, ethanolic elution of the GC peak of interest until the ion intensity signal stock solutions of SRM cholesterol and cholesterol-'3C3 were drops below a preset level, at which time data acquisition is ended. prepared by weight. Weighed portions of each solution were A base line is calculated and subtracted from the intensities combined to provide a series of standard mixtures whose unlameasured across the GC peak. The intensities for each mass are be1ed:labeled weight ratios ranged from 0.8 to 1.1. Portions of then summed across the peak, and an intensity ratio is calculated. these mixtures were derivatized as needed, following the procedure The ratios, number of cycles across the peak, and the sum of described above. intensities are then displayed. The total dead time per meaGC/MS Instrumentation. The instrumentation consisted surement cycle was reduced from 640 to 80 ms, allowing for enough of a gas chromatograph combined with a single focusing magnetic measurement cycles across chromatographic peaks only a few sector mass spectrometer controlled by a data acquisition system seconds wide to assure accurate and precise ratio measurements. designed for isotope ratio measurements (7). The instrumentation GC/MS Conditions. The principal measurements were made has been substantially modified to permit high-precision meaon the [M+'] ions at m / z 458 and 461 from electron impact (ET) surements to be made on narrow chromatographic peaks. Such on a nonpolar GC column. Confirmatory measurements were high-precision measurements cannot be made on unmodified made by using the [M - (TMS)OC,H,]+ ions at m/z 329 and 332 instrumentation. Measurement of the intensity ratio of unlabeled from E1 and the [M + NH4 - (TMS)OH]+ions at 386 and 389 to labeled analyte was made by selected ion monitoring. The from ammonia chemical ionization (CI) on the same nonpolar GC system as previously described (7)had serious limitations when column and by using the m / z 458/461 from E1 on a moderateapplied to the measurement of analytes eluting from capillary polarity column. For measurement under E1 conditions, the m a s columns. In each measurement cycle, there was dead time due
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
spectrometer was operated at 70 eV with an emission current of 0.5 mA and an ion source temperature of 200 "C. For measurement under ammonia CI conditions, the emission current was 1 mA, the source manifold pressure (ionization gauge) was 8 X Pa (6 X Torr), the analyzer pressure was 1 X lo4 Pa (8 x Torr), and the source temperature was 200 "C. For the principal measurements, the confirmatory measurements at m/z 329/332,and the confirmatory ammonia CI measurements, the GC was equipped with a 15 m long, 0.32 mm i.d., nonpolar (95% dimethyl-, 5% phenylmethylpolysiloxane) fused silica capillary column of 1-pm film thickness. An adjustable splitter was placed at the front of the column, and the end of the column was placed directly into the source. The splitter was set to give a vent to column ratio of 10:1, and the GC was operated at a temperature of 270 "C with a helium flow rate of 3 mL min-I. The injection port and interface to the mass spectrometer were maintained at 250-270 "C. Under these conditions the retention time for the cholesterol derivative was about 6 min, and the GC column gave about 1000 theoretical plates per meter. Confirmatory measurements also were made at m/z 458/461using a 30 m long, 0.32 mm i.d., intermediate-polarity (50% dimethyl-, 50% phenylmethylpolysiloxane) fused silica column of 0.5-pm f i thickness. The column temperature was 280 "C, the flow rate was 3 mL min-', and the retention time of the cholesterol derivative under these conditions was about 8 min. Measurement Protocol. For the measurement of each sample, two standards were chosen: one whose ion intensity ratio was slightly lower than that of the sample and one whose ion intensity ratio was slightly higher. All the standards in the three sets prepared were considered as one group from which the proper standards for each sample could be chosen. Each standard and sample were measured twice in succession. The two observed intensity ratios were acceptable only if they agreed within 0.5%; if not, a third measurement was made, which had to agree with one of the other two, and the three were averaged. The average constituted one valid measurement. The calculated peak areas for each standard-sample-standard group had to be within a factor of 2 of each other, or the measurement was discarded. Each time a standard was used again in any given half-day, only a single measurement was made, as long as the new ion intensity ratio was within 0.5% of the previous value for that standard. Measurements were made in the following order: lower (or higher) weight-ratio standard, sample, higher (or lower) weight-ratio standard. Thus, each measurement of a sample was immediately bracketed both in time and ratio by measurements of standards. On a second day the order of standards was reversed and the measurement process repeated. 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. The weight ratios for each sample for both days had or a third day's measurement was made. to agree within 0.570, The third measurement had to agree with one of the other days' measurements within 0.5%, or all measurements were discarded. The average of the two (or three if necessary) valid measurements is the reported result. RESULTS AND DISCUSSION Instrumentation. The overall precision of the measurements reported here requires that the relative abundances of two ions be determined with a precision of about 0.1% for a sequence of standards and samples. This is accomplished by the combination of a GC/MS instrument specifically modified to produce high-precision results and a rigid measurement protocol which uses that instrument under optimal conditions. Both the protocol and earlier versions of the instrumentation have been previously described (3, 7). The two modes of multiple ion monitoring applicable to our magnetic sector mass spectrometer, accelerating voltage switching and magnetic switching, were extensively investigated. With accelerating voltage switching we have been unable to obtain precisions better than about 1%, but magnetic switching, combined with other instrumental improvements, gave satisfactory precision. Magnetic switching is, however, slow, and as a result does not permit the necessary
number of measurements (about 30 for each mass) to produce the required precision when applied to the relatively narrow gas chromatographic peaks eluting from capillary columns. One solution to this problem would be the use of a quadrupole mass spectrometer since these instruments can switch masses rapidly. However, tests on two unmodified commercial quadrupole GC/MS systems gave precisions that ranged from 0.5% to 3%. We therefore implemented a new method of multiple ion monitoring for magnetic sector instruments in which the ion beam is electricallyswitched between the masses of interest by electric deflection after mass separation (16). This method, like accelerating voltage switching, is intrinsically capable of much faster switching rates and lower dead times than magnetic switching. For the present measurements with the present hardware, the switching rate is about 4 timea faster than the magnetic switching rate, and the precision is at least as good as we found with peaks from packed GC columns and magnetic switching. The only disadvantage apparent so far, a mass range limited to about 1.3% of the mass to be measured, is the result of the specific hardware used to implement the method. Choice of Labeled Material. In our previous work on cholesterol wing packed columns (3),we had wed a cholesterol labeled with seven deuteriums as our labeled material (cholest-5-en-25,26,26,26,27,27,27-d7-3-ol (36)). This labeled material is not suitable for use on capillary columns as it is partially or completely resolved from unlabeled cholesterol, and for high-precision work, the labeled and unlabeled anal@ should coelute. We chose to use a carbon-13-labeled material (~holest-5-en-25,26,27-~~C~-3-01(3~)) as our labeled cholesterol. No separation of this material from unlabeled cholesterol was detected. Interferences. Lathosterol is a possible interference because it forms a TMS derivative of identical molecular weight and very similar structure to that of the cholesterol derivative. It did not interfere in our original cholesterol method, but it has interfered in measurements elsewhere (19). A mixture of cholesterol-TMS and lathosterol-TMS was examined to see if the two derivatives were resolved. There was 24 s from the computer-calculated cutoff of the cholesterol chromatographic peak to the start of the lathosterol peak when the principal measurement conditions were used. Thus, lathosterol does not interfere with cholesterol in this method. Other likely steroid impurities have different molecular weights and different retention times and should not interfere. The confirmatory measurements described below were run to test for such potential interferences. Memory Effects. We tested the derivative for memory effects resulting from material remaining in the injector, on the column, or in the source, although none were expected. If a memory effect were present, injections of a sample or standard of one unlabeldlabeled ratio would affect the ratio measured for subsequent injections of sample or standard. We injected sequentially the unlabeled cholesterol derivative, the labeled derivative, and the unlabeled derivative and measured each ratio. The ratios for the injections of the unlabeled material were not significantly different. Even when the ratio differences between consecutive measurements were the most extreme, and any memory effect should be most evident, no memory effect was observed. Standards Cross-Check. The accuracy of results for serum samples is limited by the accuracy of the standard mixtures used for calibration. For definitive methods we prepare a t least two independent sets of standards and test each standard by bracketing with standards from another set. The weight ratio determined by the ID/MS measurements is then compared with the gravimetric weight ratio for that standard. The agreement between these values is an indi-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
Table I. Test of Consistency of Two Independently Prepared Sets of Standard Mixtures (SRM 911a Cholesterol and Cholesterol-"CI)
std"
bracketed b y
29 26 23 32 31 24
4, 5 10, 6 6, 1 6, 1 1, 2 1, 2
weight ratios (unlabe1ed:labeled) mean measd by ID/MS weighed in 0.8330 0.8672 0.9037 0.9139 0.9354 0.9663
0.8341 0.8686 0.9025 0.9130 0.9344 0.9673
-0.13 -0.07 +0.13 +0.10 +0.10 -0.10
mean diff 'Standards
23-32
Table 111. Cholesterol in SRM 1951" (mg dL-I) pool
diff, %
70 72 73
meanb set 1 set 2 set 3
pool CV of 1 mean measmt,' %
209.9 242.1 282.1
210.4 242.3 282.0
1.
Table 11. Cholesterol in Human Serum SRM 909" (mmol L-I 9') sample
set 1
concentrationb set 2
1 2 3 4 5 6
4.332 4.333 4.329 4.332 4.335 4.330
4.341 4.342 4.338 4.331 4.322 4.331
4.338 4.336 4.332 4.329 4.342 4.327
mean
4.332
cv, %
0.05
4.334 0.18
4.334 0.13
set 3
Statistical Summary overall mean = 4.333 CV of single measurementC= 0.23% re1 std error of mean = 0.032% "Three independently prepared sets. Three vials per set and two samples per vial. bThe mean of day 1 and day 2 valid measurements. CV of a single measurement is calculated as (s2(sets) + s2(vials)+ s2(aliquots)+ s2(measurement))1/2. cation of the bias between sets. For cholesterol, three independent sets were prepared and tested by bracketing. The three seta were combined into one group, which was used for all cholesterol measurements. The results of the measurements comparing sets 1 and 3, with set 3 as "samples", are shown in Table I. The mean difference between set 1 and set 2 was 4 . 0 1 % and between set 2 and set 3 was -0.01% (results not shown). Serum Results. The results of the principal measurements for cholesterol in SRM 909 are shown in Table 11. Three seta of six samples each (two samples from each of three vials) were independently prepared and analyzed. These results indicate the excellent within-set and between-set precision obtainable with this method. The coefficient of variation (CV) for a single measurement was 0.23%, while the relative standard error of the mean was 0.032%. When the original definitive method was last used to analyze SRM 909, the result for cholesterol was 4.341 mmol L-'g-', which is 0.2% higher than the mean with the modified method. However, a t that time we had observed a small, but real, decrease in cholesterol concentration of approximately 0.1% per year, as measured with the original definitive method. Therefore, most of the difference is probably attributable to the time difference of about a year in storage. The results in Table I1 are in millimoles of cholesterol per liter of reconstituted serum per gram of dry serum. These concentrations are based on reconstitution of each vial of freeze-dried serum by addition of 10.00 mL of water at 22 "C. Measurements of other analytes in this SRM have shown that
210.6 242.5 281.7
210.7 242.2 282.0
0.36 0.25 0.23
re1 SE,d % 0.010 0.066 0.066
"Three independent sets, three vials of each pool per set, one sample per vial. bMean of three valid results. 'CV of a single measurement. dRel SE is relative standard error. Table IV. Cholesterol in SRM
