Anal. Chem. 1988, 60, 2237-2239
2237
Isotope Dilution Liquid Chromatography/Atmospheric Pressure Ionization Mass Spectrometry for Determination of Serum Cholesterol Akiko Takatsu* a n d Sue0 Nishi National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305,J a p a n
The development of llquld chromatography/atmospherlc pressure lonlzatlon mass spectrometry (LC/APIMS) for the accurate determlnallon of serum cholesterol Is descrlbed. The method Incorporates stable Isotope dllutlon, and (M H H,O)+ Ions are monltored durlng LC/MS. Sallsfactory agreement between the analytlcal results and the certlfled value of the NBS SRM serum Is obtalned wHh the relatlve standard devlatlon of 0.6%. I t Is concluded that the,LC/ APIMS method would be accurate and precise In addltlon to belng more &pie and rapid compared wlth GWMS or off-line LWMS.
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Chromatography coupled with mass spectrometry is a powerful analytical methodology for analysis of biological samples. Combined liquid chromatograpy/mass spectrometry (LC/MS) is making an increasing contribution in place of gas chromatography/ma spectrometry (GC/MS), since it should enable us to determine the nonvolatile or thermally labile compounds without the need for prior derivatization. Isotope dilution mass spectrometry (IDMS) has been widely accepted as a reliable analytical method for highly accurate determinations. We have been developing the definitive method for determining human serum organic constituents, such as cholesterol or glucose, in clinical chemistry by using this technique (1,2).In principle, LC/MS should be equal to GC/MS with regard to precision and accuracy. While GC/MS has made a great contribution in this area, there have been very few examples of the use of LC/MS with the isotope dilution technique until now. Some investigations of IDMS with thermospray LC/MS have been carried out, but reported precisions are not very satisfactory compared with that of GC/MS (3-6). The atmospheric pressure ionization (API) technique has been recently developed for one of the practical interfaces of LC/MS for routine use (7). In this paper we describe the LC/APIMS method for the accurate determination of serum cholesterol, which is currently performed with GC/MS (8-14). We compare the method with the GC/MS and the off-line LC/MS methods, which we have already reported (1). EXPERIMENTAL SECTION Materials. Human serum reference material (NBS SRM 909) was obtained from the U.S.National Bureau of Standards (NBS). The lyophilized serum was reconstituted as described in the certificate. SRM cholesterol with certified purity of 99.8% (SRM 911a) was obtained from NBS. A stock standard solution of cholesterol (about 1.3mg/g) was prepared by dissolving an accurately weighed amount of cholesterol in a weighed amount of ethanol. [3,4‘%]Cholesterol (isotopic enrichment of 88%)was purchased from Commissariat a 1’Energie Atomique, France, and dissolved in ethanol. Calibration mixtures were prepared from the stock standard solution by adding the labeled cholesterol solution so that the ratios of natural to labeled cholesterol in the calibration mixtures were between 0.8 and 1.2 by weight. All other chemicals used
were of analytical reagent grade. Sample Preparation. The sample preparation procedure for LC/APIMS measurement of serum cholesterol was the same as that reported for the off-line LC/MS method (1). The serum sample and the labeled cholesterol solution, both of which contained about 0.5 mg of cholesterol, were mixed after weighing and hydrolyzed with ethanolic potassium hydroxide at 50 OC for 3 h. Hydrolyzed cholesterol was extracted with hexane and dissolved in methanol after evaporation of the hexane. Aliquots of the methanol solution, in which the concentration of cholesterol was about 1 mg/mL, were subjected to LC/MS measurements. LC/MS Measurement. For the chromatographic separation, a Hitachi 655A liquid chromatograph was used. Separation was performed on a YMC A-303 column (25 cm X 4.6 mm, 5 wm Cl&. Samples were eluted with methanol at a flow rate of 1.3 mL/min. A 20-rL aliquot of the calibration mixture or of the methanol solution from the serum sample was injected. A Hitachi M-80 LCAPI double-focusing mass spectrometer equipped with an API ion source was used. The LC effluents were introduced directly into the mass spectrometer with a Teflon tube and analyzed with APIMS under the following conditions for the ion source: neblizer temperature, 260 OC; drift voltage, 170 V. The isotope ratio measurement was performed with selected ion monitoring (SIM) at m/z 369 and 371 with a switching rate of 0.1 s/ion. Calculation. The peak intensity ratio was determined by measuring peak areas from the mass fragmentogram. The standard curve was obtained by plotting the masa ratio vs relative peak area between the m/z 369 and 371 ions from the measurement of the calibration mixtures. With this curve the mass ratio of the sample was calculated from the peak intensity ratio obtained by the sample measurement. The cholesterol concentration of the sample was calculated from the equation C = (RM)L/ W (W is the weight of the serum sample, L is the weight of the labeled cholesterol added to the sample, and RM is the calculated mass ratio). RESULTS AND DISCUSSION LC/MS Conditions. LC conditions were chosen in the view of following points. Good Separation. Other sterols or compounds that might interfere with the cholesterol determination with APIMS should be separated with LC. In the labeled material, chemical impurities that gave similar mass spectra to cholesterol existed. Separation from these compounds was confirmed with UV detection before APIMS measurement. Good Sensitivity. Since in APIMS a solvent acts as a reagent in the chemical ionization at atmospheric pressure, sensitivity as well as spectrum pattern might change with the solvent. In the previous study we had used acetonitrile/2propanol as the mobile phase for LC, and good separation was obtained (1). But in the APIMS, the intensity of the dehydrated ion (M + H - H20)+peak was greater with methanol than with acetonitrile/2-propaol, while the mass spectra of cholesterol with both solvents were similar, being characterized by the abundant dehydrated ion and no other fragment ions. This is explained by the smaller proton affinity of methanol. So it should be preferable to use methanol as the mobile phase. The API mass spectrum of natural cholesterol with methanol is shown in Figure 1.
0003-2700/88/0360-2237$01.50/00 1988 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2238
I
(M+H-H20)'
369
t
'
.
"l
371
I
I
I
1
. , L I
,
1.
d
./.
L
I
L
Figure 1. A P I mass spectrum of natural cholesterol.
Table I. Retention Times of Sterols retention sterols (systematic name) ergocalciferol
time, min 8.2
(9,10-secoergosta-5,7,10( 19),22-tetraen-3@-01) desmosterol (cholesta-5,24-dien-3@-01) ergosterol (ergosta-5,7,22-trien-3@-01)
epicoprostanol (56-cholestan-3a-01) 7-dehydrocholesterol (choleeta-5,7-dien-3j3-01) lathosterol (5a-cholest-7-en-3@-01) cholesterol (5-cholest-en-3@-01) stigmasterol (stigmasta-5,22-dien-3@-01) campesterol (ergost-5-en-3@-01) cholestanol (5a-cholestan-3~-01) 8-sitosterol (stiemast-5-en-38-01)
11.0 11.1 11.3 12.0
13.4 13.8
14.5 15.0 15.5 16.4
Shorter Chromatographic Run. For the quantitative analysis it is essential to avoid a change of instrumental conditions between the measurements of calibration mixtures and of the samples. So, a shorter chromatographic run should be preferable to minimize the instrumental drift during the measurements. When the YMC A-303 column was used, separation was satisfactory with methanol. The entire chromatographic run was about 16 min at a flow rate of 1.3 mL/min. Table I summarizes the retention times of sterols that may be present in serum. It is demonstrated that the cholesterol peak is free from interference by any of these sterols. Isotope ratio measurement was performed by monitoring m/z 369 and 371, corresponding to the (M H - H20)+ions of unlabeled and labeled cholesterol, respectively. The mass fragmentogram of cholesterol in human serum with labeled cholesterol added is shown in Figure 2. The small peak before cholesterol at m / z 371 was due to an impurity in the labeled material. Calibration of Isotope Ratio Measurement. To calculate the mass ratio of the sample from the measured peak intensity (peak area) ratio, the relation between the peak intensity ratio and mass ratio for calibration mixtures is necessary. If R12 is defined as RI2 [(Rx 1)(Ry - RI)]/[(Ry + 1)(RI - Rx)] (1) where RI, Rx, and Ry are the measured peak intensity ratios for the sample spiked with labeled material, pure unlabeled sample, and pure labeled internal standard, respectively, the mass ratio is linear with R12 (15-17). Since in our experiment we used the [3,4-1Y!]cholesterol with an isotopic purity of 88% as an internal standard, the mass of which differed only 2 amu from that of the natural one, both natural and labeled cholesterol contributed to both of the signals measured for the peak intensity ratio (Rx # a;Ry # 0). Thus, the relation between the peak intensity ratio and mass ratio was expected to be nonlinear. However, under similar conditions satisfactory results have often been obtained by using linear interpolation in the narrow mass ratio range (14,17). And our regression equation was RI = 1.1587(RM) + 0.1301, with the
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Figure 2. Mass fragmentogram of serum cholesterol with labeled cholesterol added.
correlation coefficient r > 0.999, which shows linearity was satisfactory,although the high value of the intercept explained by the presence of an unlabeled impurity in the labeled material was obtained. To estimate the error introduced by using the linear interpolation instead of nonlinear one, mathematical analysis is undertaken. To simplify this consideration, we assume that only two standards are used for calibration and estimate the difference between the calculated mass ratios, using linear and nonlinear calibration. Let RM denote the mass ratio of the sample with the internal standard and let RI denote the peak intensity ratio. If one assumes a linear relation between RM and RI, i.e. RM = A(R1) + B, where A and B are constants, then one obtains
RMs =
[(RIs - RIL)/(RIH - RIL)I(RMH- RML) + RML (2)
where the subscript S refers to the sample mixture and H and L refer to the calibration standards having higher and lower mass ratios, respectively. The nonlinear equation is expressed as RM = A'(RI2) + B', using R12 as defined in eq (1). Then one obtains RMs' = [(RIZs - R I ~ L ) / ( R I ~-HR I ~ L ) ] ( R M H - RML)
+
RML
= [@Is - RIL)(RIH- RX)]/[(RIH - RIL)(RIs Rx)](RMH - RML) RML
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(3) F is defined as the difference between the calculated mass ratio using two calibration procedures. F = RMs - RMs' = [(RIs - RIL)(RIH- RIS)][(RMH- RML)/ (RIH - RIL)I/(Rx - RIs) (4) Since in our case the calculated peak intensity at the m / z 371 of natural cholesterol resulting from natural isotopic abundance is about 4.7%, Rx - RIS was around 20. The value of (RMH - RML)/(RIH- RIL), which is given by the slope of our regression equation, is about 1, and simple mathematics gives (RIH - RIs)(RIs - RIL) < [(RIH - RIs) + (RIs - RIL)I2/4 = (RIH - RIL)'/4 = (RMH- RML)2/4 Since our experiment was performed between mass ratio
ANALYTICAL CHEMISTRY, VOL. 60, NO.
Table 11. Analytical Results of Cholesterol in Human Serum (NBS SRM 909) by IDMS (LC/APIMS) Method ((mmol/L)/e) samDle r e d i c a t e
measmt 1
2
1
2
4.403 4.372 4.387
4.369 4.409 4.389
average average o f averages (RSD) certified concn (difference)
3
4
5
4.356 4.340 4.409 4.418 4.365 4.347 4.414 4.361 4.344 4.379 0.027 (0.62%) 4.359 f 0.017 (+0.46%)
values of 0.8 and 1.2, the maximum value of RMH- RML was 0.4. So,
(RIH - RIs)(RIs - RIL) < (RMH - RML)2/4 < 0.04 By substituting these values into eq 4, we reach the conclusion that F will be less than 0.002 (about 0.2%). Compared with the measurement precision of about 0.6%,this difference is not very significant. IDMS Results. Analytical results for the human serum reference material (NBS SRM 909) are shown in Table 11. Five replicates of the vial were prepared independently and analyzed. The isotope ratio measurement was repeated two times for each sample. Because the concentration was based on the weighed mass in our method, we measure the specific gravity to calculate the concentration per liter of the reconstituted serum. Excellent agreement (within 0.5%) between the analytical results obtained by this method and the certified value of NBS showed that the accuracy was satisfactory. The relative standard deviation (RSD) was 0.6 % ,which was almost the same as the reproducibility of the isotope ratio measurement with APIMS. The reported RSDs with GC/MS had ranged from 0.23% to 2.4%, and the reproducibility of this LC/APIMS method is better than some of those (8-11) and worse than others (12-14). Since in LC/APIMS no derivatization was required, sample preparation was simpler than in GC/MS. It was also a rapid method compared with off-line LC/MS. We had reported an RSD of 0.7% for off-line LC/MS and 0.5% for GC/MS (1). Since in the off-line LC/MS method sample-sample variance in the collection at an LC or in the introduction into a mass spectrometer was significant, we expected better precision with on-line LC/MS. As expected, the present results were better than those obtained with off-line LC/MS and closer to those with the GC/MS. There has been no example of determination of cholesterol with thermospray LC/MS, but for other compounds relatively
20, OCTOBER 15, 1988
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large RSDs were reported. Results of sorbitol analysis show an RSD of 2.76% at a 1:l ratio of labeled to unlabeled compound (5). Recently Gaskell et al. performed thermospray LC/MS for the determination of serum cortisol. They compared the method with GC/MS and reported an RSD of 7%, which was inferior to that with GC/MS (6). They thought that this large RSD was attributable to the poorer sensitivity or instability of the mass spectrometer. In the case of LC/ APIMS, stable operation was possible for a long time and sensitivity was satisfactory. For serum cholesterol determination the materials are sufficiently abundant so that ultimate sensitivity is not an issue, but the accuracy and the precision of the measurement should be important. For that purpose this method is quite satisfactory. Future studies will be aimed at developing a definitive method for clinical chemistry. LC/APIMS could be expected to be used for highly precise analysis in place of GC/MS. ACKNOWLEDGMENT We thank Y. Katoh and Y. Numajiri for technical assistance and Naka Works, Hitachi, Ltd., for use of the LC/APIMS. LITERATURE CITED (1) Takatsu, A.; Nishi, S. C l h . Chem. (Wlflston-Salem, N . C . ) 1987, 33, 1113-11 17. (2) Takatsu, A.; Nlshl, S., submitted for publicatlon in Anal. Sci. (3) Liberato, D. J.; Yergey, A. L.; Weintraub. S. T. Bkmed. Envkon. Mass Spectrom. 1988, 13, 171-174. (4) Yergey, A. L.; Esteban, N. V.; Llberato, D. J. Biomed. Envkon. Mass Spectrom. 1987, 14, 623-625. (5) Esteban, N. V.; Llberato, D. J.; Sldbury, J. B.; Yergey, A. L. Anal. C h M . 1987, 59, 1674-1677. (6) Gaskell, S. J.; Rollins, K.; Smith, R. W.; Parker, C. E. Biomed. Envkon. Mass Spectrom. 1987, 14, 717-722. (7) Sakairi, M.; Kambara, H. Anal. Chem. 1988, 6 0 , 774-780. (6) Bjorkhem, I.; Blomstrand, R.; Svensson, L. C l h . Chim. Acta 1974, 54, 165-193. (9) Siekmann, L.; Huskes, K. P.; Breuer, H. Fresenius' Z. Anal. Chem. 1978, 279. 145-146. ( I O ) Gambert, P.; Lallemant, C.; Archambauit, A,; Maume, 8. F.; Padieu, P. J . ChrOmtOgr. 1979, 162, 1-6. (11) Wolthers, B. G.; Hindriks, F. R.; Muskiet, F. A. J.; Groen, A. Clln. Chim. Acta 1980, 103. 305-315. (12) Cohen, A.; Hertz, H. S.; Mandel, J.; Paule, R. C.; Schaffer, R.; Sniegoski, L. T.; Sun, T.; Welch, M. J.; White, E. V. Clh. Chem. (WlnstonSalem, N . C . ) 1980, 2 6 , 854-860. (13) Freudenthal. J.; Derks, H. J. 0.M.; Gramberg, L. G.; ten Hove, 0.J.; Klaassen, R. Blomed. Mass Spectrom. 1981, 8 . 5-9. (14) Pelletier, 0.; Wright, L. A.; Breckenridge, W. C. Clln. Chem. (WlnstonSalem, N . C . ) 1987, 33, 1403-1411. (15) Plckup, J. F.; MaPherson, K. Anal. Chem. 1978, 48. 1885-1890. (16) Colby, B. N.; McCaman, R. W. B/omed. Mass Spectrom. 1976, 6 , 225-230. (17) Yap, W. T.; Schaffer, R.; Hertz, H. S.; White. E. V.; Welch, M. J. Biomed. Mass Spectrom. 1983, 10, 262-264.
RECEIVED for review March 1,1988. Accepted July 20,1988.
