534
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
Determination of Individual Ubiquinone Homologues by Mass Spectrometry and High Performance Liquid Chromatography Sukehiro Imabayashi, Tetsuya Nakamura, Yoshio Sawa, Jiro Hasegawa, Kenya Sakaguchi, Takeshi Fujita, Yutaka Mori, and Kiyoshi Kawabe Eisai Research Laboratories, Eisai Co., Ltd.. Koishikawa 4, Bunkyo-ku, Tokyo 112, Japan
Ubiquinone homologues in various animal tissues (liver, kidney, heart, and spleen) were determined by direct inlet selected ion monitoring. Samples were prepared by solvent extraction, after treating with lipase to eliminate substances that might interfere with the analysis. The detection limit by direct inlet selected ion monitoring for ubiquinone-10 was 0.1 ng. The same samples were also subjected to high performance liquid chromatography and results of this latter analysis were in good agreement with results by direct inlet selected ion monitoring.
T h e biological significance of ubiquinone as a redox carrier in the respiratory chain has been well established ( I ) . Further, from a physiological standpoint, it was reported that localized deficiencies of ubiquinone occurred in t h e gingiva of patients with periodontal diseases ( 2 ) ,in human heart disease ( 3 ) ,and in leucocytes of patients with essential hypertension ( 4 ) . T h e test was performed by measuring the variation of specific activity of succinate-dehydrogenase ubiquinone reductase in tissues ( 5 ) . However, the quantitative analysis of ubiquinone has not been described in these cases. This may be due partly t o the fact t h a t a practical and reliable procedure for analysis of ubiquinone homologues in biological samples had not been developed. However, a number of studies have been carried out on the quantitative analysis of ubiquinone homologues (i.e., ubiquinone-n, with n representing the number of isoprenoid side chains, hereafter, simply as Q-n) widely occurring in animals a n d plants. A survey of t h e occurrence of ubiquinone in various vertebrates has been reported (6). Ramasarma (7) described a comprehensive review of t h e analytical d a t a in animal tissues, plants, and microorganisms. As a conventional method for the determination of total Q-n,spectrophotometric which is based o n the difference in absorbancy analysis (8), of t h e oxidized and reduced ubiquinone a t 275 n m or a has been used. Since each of these modified Craven test (8), methods utilizes the quinone nuclei as the analytical principle, they do n o t give satisfactory results for determination of individual homologues of ubiquinone. Casey e t al. (9) developed a method using reverse-phase paper chromatography. It consists of direct estimation of individual spots developed o n t h e paper by densitometry. T h e Q-n concentration (in human blood) is also being analyzed ( I O ) by electron capture gas chromatography. T o develop a sensitive a n d simple method, t h e present authors a t t e m p t e d t o determine individual ubiquinone homologues in various tissues of animals a n d human blood by direct inlet selected ion monitoring (DI-SIM) (11, 12),which recently drew attention as a means of microanalysis, for the determination of individual ubiquinone homologues in animal tissues and human plasma. Additionally, determination was m a d e by high performance liquid chromatography ( H P L C ) (13, 1 4 ) to compare t h e results of both methods. For the pretreatment of samples, in place of a conventional strong alkali saponification method, a milder enzymic hydrolysis using lipase was employed. 0003-2700/79/0351-0534$01.00/0
EXPERIMENTAL
Standards. All-trans standard samples (Ql0,Q9,and Qe)were prepared by condensing a boric acid ester of 2,3-dimethoxy-5methyl-1,4-benzohydroquinone with decaprenol (or isodecaprenol), solanesol, octaprenol, respectively, and oxidizing the resultant borate with AgzO (15). DI-SIM internal standard 2,3-dimethoxy-~-methy1-6-3’,7’,11’,15’,19’,23’,27’,31’,35’,39’-decameth tetracontanyl benzoquinone (hereafter referred to as IS-1)was prepared by catalytic reduction of Qlo with a Pd-C catalyst, followed by oxidation with PbOz (10). HPLC internal standard 2,3,5-trimethyl-6-decaprenyl-1,4-benzoquinone (16) (hereafter referred to as IS-2) was prepared by condensing a boric acid ester of 2,3,5-trimethyl-1,4-benzohydroquinone with decaprenol (or isodecaprenol), and oxidizing the resultant borate with Ag,O. H u m a n s a n d Animals. Humans. Ten healthy males (28 to 43 years of age, weighing 58 to 79 kg) were used. Mice. Five- to six-week-old female ICR/JCL mice were purchased from CLEA Japan Inc., Tokyo. They were maintained on a NMF (Oriental Yeast Co., Tokyo) pellet diet for two weeks and mice weighing about 22 g were used in the present study. Guinea Pigs. Healthy three-month-old males and females were maintained for two weeks on an ORC-4 diet (Oriental Yeast Co., Tokyo). The weights were about 250 g when used. Beagles. Twenty-four- to thirty-eight-month-old males and females were maintained on a CD-1 diet (CLEA Japan Inc., Tokyo). The weights were about 10 kg. Rats. Fifteen-week-old male and female Sprague-Dawley rats were maintained for three weeks on a CE-2 diet (CLEA Japan Inc., Tokyo). Pretreatment of the Samples. One to two mL of plasma was placed in a Pyrex glass test tube and 2.0 gg of IS-1 was added. To the mixture 2 mL of 1% pancreatin (Sigma Chemical Company) solution and 0.2 mL of 4% sodium taurocholate (BDH Chemicals Ltd.) solution were added. Subsequently, the solution was incubated at 37 “C for 2 h. Then 5 mL of ether was added and the mixture was sufficiently shaken and centrifuged. The ether phase was collected in a Pyrex glass test tube. This extraction procedure was repeated three times. The sample was evaporated to dryness under reduced pressure. The resultant residue was dissolved in 0.2-0.5 mL of ethyl acetate. Sample pretreatment in a quantitative analysis of Q-n in tissues was as follows. The rats were decapitated, the liver removed, and a portion of the liver (about 0.5 to 2 g of wet liver) was homogenized in a Potter Elvehjem homogenizer. The homogenate thus obtained was treated in the same manner as pretreatment of the plasma sample mentioned above. Extraction with ether was repeated three times to ensure complete extraction of IS and Q-n. The homogenized tissues were kept in a freezer and an aliquot of the homogenate was used for determination. The determination errors in one day and between days were examined. Such errors in the DI-SIM were within &5%. Following the present experimental procedure, it was observed that the relative recovery ratio of Q-n and IS-1 from the plasma and organs were almost 100%. Direct Inlet Selected Ion Monitoring. The instruments used were a JEOL JMS--D100 and JMS-D300, both with a magnetic sector. For JMS-D300 equipment, the computer system (JMA-2000, JEOL, Tokyo) was attached to ascertain the contamination of interfering substances. This computer system is specially designed t o detect minute amounts of interfering substances in biological samples. Measuring conditions were as follows, ionizing current, 300 PA; ionizing voltage, 25 eV; chamber 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979
535
Table I. Relative Intensities of Parent Ion Peaks of Ubiquinone Homologues and Internal Standards
ionizing voltage QTl
m/ e
7 0 eV
30 eV
Q, Q, Q,,
728 796 86 4 882 832
60 31 22
64 33 24
IS-1 IS-2
looa
looa
26
28
' Arbitrarily assigned.
subject 1 2 3
,
4 m L
Craven test, Pg/mL
1 . 3 5 I 0.11' 1 . 2 6 = 0.09 1 . 0 4 :? 0.11
1 . 4 1 k 0.36 1.15 t 0 . 2 3 1 . 0 3 2 0.17
DI-SIM
-Ai___
IS-I
Table 11. Comparison of Human Plasma Level Determined b y DI-SIM or Craven Test 0
,
I
I
I
I
!
4
2
I
r-
10
8
6 rnin
Figure 1. Direct inlet selected ion monitoring (DI-SIM) of standard Qg,
and the internal standard (IS-1). Measuring conditions: ionizing current, 300 PA; ionizing voltage, 25 e V ; chamber temperature, 200 "C: sample temperature, from 150 to 270 OC. Measuring mass fragment: Q9, mle796 [M 21': Qlo, mle864 [M 21'; IS-1, m / e Qlo,
' Mean t SEM for three determinations for a sample.
882
temperature, 200 "C; probe temperature programmed from 150 to 270 "C; measuring SIM: Q9, m / e 796 [M + 2]+; QLo,m / e 864 [M + 2]+; IS-1, m / e 882 [MI+; IS-2, m / e 832 [ M + 2 ] + . The appearance of parent ion peaks of ubiquinone homologues and internal standards under two ionizing voltage conditions is shown in Table I. The extract was dissolved in 1 to 5 pL of ethyl acetate, the presumed content of Qlo being 0.1-~100ng. At this point, the solution was placed in a glass capillary, which was heated with a drier to evaporate the solvent. A standard sample for a calibration curve was prepared by adding cholesterol in the amount of 1 to 5%. The cholesterol was added only in the standard samples to stabilize evaporation of ubiquinone homologues and to obtain a typical S1M pattern. In such a case, high reproducibility was easily obtained and a linear calibration curve was obtained. High P e r f o r m a n c e Liquid Chromatography. Chromatography was performed on a Shimadzu LC-1 pumping system, Shimadzu SPD-1 (scanning UV detector) and UVD-1 (254-nm U V detector). The column was a Shimadzu-Du Pont "permaphase ODs" (1m X 2.1 mm, particle size: 30 Gm). A solution of degassed spectroquality methanol (HPLC grade, Wako Chemicals, Osaka) and distilled water was used as the gradient-elution mobile phase programmed for a linear 1 7 ~per minute increase in methanol concentration from a starting mixture of 80/20 ( v / v ) methanol/water taken to 100% methanol. The measuring temperature was 45 "C. A 1000-psi head was developed at a flow rate of 1 mL/min. Column effluent was monitored at 254 nm (UL'D-1)
+
[MI+
+
and 275 nm (SPD-1). The purpose of using two differential UV detectors was to investigate interfering substances in more detail.
RESULT AND DISCUSSION T h e SIM spectrum of a standard sample is shown in Figure 1 and t h e chromatogram of a standard sample of HPLC analysis is shown in Figure 2 . T h e analysis of Qlo in human plasma was carried out by DI-SIM and Craven test (I 7 ) . T h e results obtained by t h e two methods are shown in Table 11. T h e range for the 10 individuals by DI-SIM was from 0.28 to 1.63 Fg/mL and t h e average was 0.65 @Lg/mL. Analysis of DI-SIM and HPLC on individual animal tissues is shown in Table 111. Results of analytical values by DI-SIM, Craven test, and HPLC were almost in agreement (Tables I1 a n d 111). For pretreatment of samples, a conventional method of alkali saponification could not entirely eliminate substances that interferred with the analysis (such as triglyceride.-like compounds) by the DI-SIM. Treatment with lipase removed this difficulty. T h e effectiveness of the lipase pretreatment was clearly shown on t h e SIM pattern of extracts of tissue samples. Using DI-SIM and H PLC analysis, measurements were made for t h e various animal tissues. In all cases, using
__-- _____
c I _
Table 111. Occurrence of Ubiquinone-9 and -10in Animal Tissues* HFLC
DI-SIM
animal mouse
rat guinea pig rabbit beagle d o g mongrel d o g
tissue liver kidney heart spleen thymus liver kidney liver kidney heart liver k idney liver kidney liver kidney
@gig wet tissue (mean + S E M ) . -__ ______ a
Q,
Q9
8 7 . 5 2 18.4 ( 6 ) b 3 2 1 . 5 I2 6 . 1 ( 6 ) 151.0 - 1 8 . 6 ( 6 ) 24.5 - 4.6 (6) 7 7 . 5 -: 27.0 ( 5 ) 138.5 f 1 8 . 7 ( 3 ) 1 0 3 . 2 i- 22.9 ( 3 )
6 . 7 I2 . 4 ( 4 ) 2 9 . 1 i 6.5 ( 3 ) 19.1 t 2 . 9 ( 5 ) N.D.C 28.7 T 11.0 ( 5 ) 77.5 230.7 220.5
+
7 . 5 (18)
i
10.5 (18)
t
8 . 6 (18)
Q, 83.5 8.3 ( 4 ) 2 9 8 . 0 . 35.1 ( 4 )
5.6 2 . 3 ( 4 ) 2 6 7 - 4 . 1 (4) +
169.2 - 85.1 ( 1 5 ) 1 2 8 2 f 47.2 (1.5) 4.5 * 2.0 ( 1 8 )
trace trace
____ Q l ?
79.7 224.5 227.8 77.8 147.7 + 138.3 2 177.7 : 142.4 213.5 i +
I
+
145.7 t 1 4 . 8 ( 5 ) 185.4 * 13.6 ( 5 )
trace trace
13.8 = 3 . 2 ( 4 )
trace
Figures in the parentheses represent number of animals.
_______
+
7 . 9 (18)
10.0 ( 1 8 ) 8.4( 1 8 ) 10.6 ( 4 ) 1 2 . 8 (4) 13.9 ( 5 )
11.0( 5 ) 29.7 [ A ) 33.9 (4)
N . D . : n n t detectable. - .-__
___
536
ANALYTICAL CHEMISTRY, VOL. 51, NO. 4, APRIL 1979 IS-2
I I
In studying the physiological role of ubiquinone in tissues or blood, i t is necessary t o determine the concentration of individual homologues in t h e mixture in a quantitative manner. Although this can be accomplished by several methods (8, 9, 17), these procedures require large amounts of samples. However, our method is a practical recommendation for quantitative analysis of individual ubiquinone higher t h a n Qs homologues, which are widely distributed in small amounts in the animal a n d vegetable kingdom.
ACKNOWLEDGMENT
0
4
12
8
16
20
min
Figure 2. HPLC of standard Q-n. Measuring conditions: column, Permaphase ODS 1 m X 2.1 mm i.d.; mobile phase, MeOH (HPLC grade)-water (pH 5.0-7.0); gradient, 80120-100/0 (MeOH/H,O); flow rate, 1 mL/min (70 kg/cm2); temperature, 45 O C ; measuring wavelengths, 254 nm (UVD-1) and 275 nm (SPD-1)
either one or the other of t h e two methods, consistently reproducible results were obtained (Table 111). In order t o be sure of the results of the H P L C analysis, two types of detectors (SPD-1 and UVD-1) were attached t o the H P L C apparatus a n d t h e measurements were made a t two different wavelengths (275 and 254 nm). The analytical results obtained were always the same for the two wavelengths. This fact means that the analytical data obtained are not disturbed by contaminants. I n t h e DI-SIM analysis, a check was also made by mass chromatography (JEOL model JMS D-300 with computer system). By use of a mass chromatography analysis, it was possible to confirm t h e adequacy of the established quantitative analytical procedure with DI-SIM. A comparative study of t h e DI-SIM method and Craven test method was made using normal human plasma as the sample (Table 11). T h e results obtained showed good compatibility of t h e ubiquinone concentration. Theoretically, in t h e DI-SIM analysis, the number of simultaneous determinations of individual ubiquinone homologues is permitted in accordance with the channel number of t h e multi-ion detector in the apparatus.
T h e technical assistance of Miwako Kuroki and Kazuko Kinoshita are gratefully acknowledged. T h e authors are deeply indebted to Junzo Tsutsumi, R. K. Garg, Tomio Takamatsu, a n d Kimio Hamamura, Eisai Research Laboratories, for their cooperation. Thanks are also due t o Atsushi Yamagishi, Executive Director of the Board a n d Director of Research Division, for encouragement and support of this study.
LITERATURE CITED (1) J. W. DePierre and L. Ernster, Ann. Rev. Biochem., 46, 201-262 (1977). (2) R. Nakamura, G. P. Littarru, K. Folkers, and E. G. Wilkinson, Proc. Nafl. Acad. Sci. U . S . A . , 71, 1456-1460 (1974). (3) G. P. Littarru, L. Ho and K. Folkers, Int. J . Vitam. Nutr. Res., 42, 413-434 (1972). (4) T. Yarnagami, Y. Iwamoto, and K. Folkers, Int. J . Vifam. Nutr. Res., 44, 404-414 (1974). (5) R. Nakamura, G. P. Littarru, and K. Folkers, I n f . J . Vitam. Nufr. Res., 43, 526-536 (1973). (6) A. T. Diplock and G. A. D. Haslewood, B~ochem.J . , 104, 1004-1010 (19671. (7) i.~Fiamasarma.Adv. Lipid Res., 6, 107-180 (1968). (8) F. L. Crane and Rita Barr, "Methods in Enzymology", D. B. McCormick and L. D. Wright, Ed., Academic Press, New York, 1971, Vol. 18, Part C, pp 137-181. (9) A. C. Casey, P. Myers, and A. Lee, J . Chromtcgr., 72, 365-371 (1972). (10) H. Morirnoto, I. Imada, T. Arnano, M. Toyoda, and Y. Ashida. Biochem. Med.. 7, 169-177 (1973). (11) C. G. Hamrnar and R. Hessiing, Anal. Chem., 43, 298-306 (1971). (12) W. Snedden and R. B. Parker, Anal. Chem., 43, 1651-1656 (1971). (13) P. L. Donnahey and F. W. Hemming, Biochem. SOC. Trans., 3, 775-776 (19751 - . - I \
(14) I. A. Tavares, N. J. Johnson, and F. W. Hemming, Biochem. Soc. Trans., 5. 1771-1773 (19771. (15) S.Kijima. I . Yamatsu, K. Harnamura, N. Minaml, Y. Yamagishi and Y. Inai, U.S. Patent 3,998,858 (1976); Chem. Abstr., 86, 1 7 1 6 6 4 ~(1977). (16) J. Green and D. McHale, "Biochemistry of Quinones", R. A. Morton, Ed., Academic Press, London and New York, 1965, pp 261-285. (17) E. Redalieu, I. M. Nilsson, T. M. Farley. K. Folkers, and F. R. Koniuszy, Anal. Biochem., 23, 132-140 (1968).
RECEIVED for review April 12, 1978. Accepted December 15, 1978.
