DETERMINATION OF VITAMINS A, E, AND K AND UBIQUINONES IN PLASMA BY VERY HIGH SPEED LIQUID CHROMATOGRAPHY

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1 BUNSEKI KAGAKU Vol. 33, pp.e309-e314, 1984 The Japan Society for Analytical Chemistry, 1984 DETERMINATION OF VITAMINS A, E, AND K AND UBIQUINONES IN PLASMA BY VERY HIGH SPEED LIQUID CHROMATOGRAPHY Kouichi ABER, Kenichi KUSUBE, Keeko MUKASA, Yoshinobu ISHIGURO, Tadashi SATO, Seiji ISHIKAWA, and Haruhiko HOSHIDA Tsukuba Research Laboratories, Eisai Co., Ltd., 1-3 Tokodai 5-chome, Toyosato-machi, Tsukuba-gun, Ibaraki , Japan A quantitative method for analyzing vitamins A, E and K and ubiquinones in plasma, based on recently developed very high speed liquid chromatography, is described. Vitamins A, E, and K and ubiquinones are extracted from ƒêl of plasma with 5 ml of hexane after addition of 1 ml of water and 2 ml of ethanol containing each internal standard. Hexane extracts ( 4 ml ) are evaporated under a nitrogen stream at 40 Ž. The residue is dissolved in 100 ƒêl of isopropanol or hexane. The solution is subjected to the very high speed liquid chromatography. Total time required for sample preparation and analysis is some 10 min. The actual chromatographic separation is completed in about 5 min. The detection limits for vitamin A ( retinol ), vitamin E ( ƒ -tocopherol ), vitamin K1 and ubiquinone-10 obtained with a UV detector are 0.056, 0.28, 0.28 and 0.49 ng, respectively. This method offers significant advantages in terms of sensitivity and speed of sample treatment compared to conventional methods. Very high speed liquid chromatography ( VHSLC ) has recently been established by DiCesare et al1)-3). VHSLC represents a major advance in high performance liquid chromatographic technology which enables the separation of compounds in 1-5 min. This paper describes the application of VHSLC to the analysis of vitamins A, E and K and ubiquinones ( UQ ) in plasma, with emphasis on the practical aspects including rapid sample preparation and quantification procedures. EXPERIMENTAL Material Vitamin A ( retinol ) and (3-naphthyl myristate were obtained from Sigma Chemical Co., Ltd. ƒ -, ƒà- ƒá- and ƒâ-tocopherols were individually isolated from soy bean scum or wheat germ oil, and were purified according to the method of Kijima4). Tocol, eicosanyl naphthoate, UQ-7, UQ-8, UQ-9 and 2,3, 6-trimethy1-5- decaprenyl-1, 4- benzoquinone ( TQ-10 ) were synthesized according to the conventional methods5)6), and purified by column chromatography and recrystallization. Methanol, ethanol, hexane and isopropanol of analytical grade were used without further purification.

2 E310 BUNSEKI KAGAKU Vol. 33(1984) Appratus The VHSLC system was consisited of a Shimadzu LC-5A ( Shimadzu Corporation, Kyoto, Japan ) with a Rheodyne loop injector. The UV detector used was a Shimadzu SPD-2A detector. SHIM-PACK PC18-03/S0505 ( PC18 ) and SHIM-PACK ASI-03/S0505 ( ASI ) were used as VHSLC columns. These columns ( 50 mm ~ 4.6 mm I.D. ) have over 5000 theoretical plates per column. PC18 is packed with 3 Đm spherical C18 bonded phase particles and ASI, 3 pm spherical silica gel particles. The conventional columns used for comparison were Nucleosil C18 ( 5 Đm, 150 mm ~ 4.6 mm I.D. ) and Lichrospher ( 5 Đm, 250 mm ~ 4.6 mm I.D. ). VSHLC conditions for the determination of vitamins A, E and K and UQ are shown in Table 1. Table 1 VHSLC conditions for the determination of vitamins A, E and K and UQ Sample treatment Modified procedures, based on the previous methods7)-11), were used. Twenty to fifty of plasma was poured into a brown test tube, and then 1 ml of water, 2 ml of ethanol containing each suitable internal standard ( 5-naphtyl myristate, tocol, eicosanyl naphthoate, and TQ-10 for the determinations of vitamins A, E and K and UQ, respectively ) were added with constant mixing. To the mixture, 5 ml of hexane was added and then centrifuged at approximately 3000 rpm for 5 min. Four ml of the hexane layer was transferred to another brown tube and evaporated to dryness under a stream of dry nitrogen gas. The residue was dissolved in 100 Đl of isopropanol or hexane and the resulting solution was subjected to the VHSLC system. RESULTS Separation of vitamins A, E and K and UQ The separations of standards were excellent as shown in Fig. 1. All of them were eluted within 5 min. Minimum detectable quantities and calibration of compounds Since peaks elute very rapidly in VHSLC, quick response of detector is important. The SPD-2A UV detector has three response times : 0.25, 0.55 and 1.55 s. The relation of peak height of UQ 7-10 and TQ-10 to the response time was investigatea ( Table 2 ). To obtain the increased sensitivity of detection, the response time of the detector

3 E311 was set at 0.25 s. The calibration curves thus obtained were linear through the origin. The minimum detectable quantities of these materials ( S/N = 5 ) were approximately ten times smaller than those obtained by the conventional HPLC ( Table 3 ). A E Fig. 1 Separation of vitamins A, E and K and UQ standards Table 2 Effect of response time on peak height of UQ

4 E312 BUNSEKI KAGAKU Vol. 33(1984) Table 3 Comparison of the minimum detectable quantities (S/N=5) of VHSLC with those of HPLC Separation of vitamins A, E and K and UQ in plasma Figure 2 shows typical chromatograms of vitamins and UQ obtained with rat plasma. As shown in Table 4, the recoveries of them from rat plasma were satisfactory. The coefficient of variation ( n = 8 ) was ca. 2 % in all cases. A E K UQ Fig. 2 Separation of vitamins A, E and K and UQ inrat plasma

5 E313 Table 4 Recoveries of vitamins A,E and K and UQ from plasma Comparison of VHSLC and HPLC Table 5 summarizes the contents of vitamins A, E and K and UQ in rat plasma determined by the present VHSLC method and the previous HPLC methods7)9)10)11). They were agreed well each other. Table 5 Comparison of contents of vitamins A, E and K and UQ in rat plasma obtained by VHSLC and those by HPLC DISCUSSION HPLC has been generally used for the separation of vitamins A, E and K and UQ7)- 11) How ever, this procedure has the disadvatages of requiring rather large quantities of samples because of low sensitivity. In case of infant research, HPLC methods are not practical as they need as much as ml of plasma or serum. In addition, the total analysis time of HPLC method is usually min per sample and more rapid method has been desired. Therefore, we developed in the present investigation a more sensitive and rapid VHSLC method for vitamins A, E and K and UQ in plasma. DiCesare et al1)-3) already reported that the theoretical plates of their column ( 100 mm x 4.6 mm I.D. ) were over and many separation are completed in 1 or 2 min. In contrast, we used SHIM-PACK columns whose theoretical plates were over 5000

6 E314 BUNSEK I KAGAKU Vol. 33(1984) for the separation of vitamins A, E and K and UQ, obtaining excellent separation of them without extraordinary increase of column pressure. As the detector, fluorometers were not suitable since their response times were very slow ; the efficiency on the eluting peak reduced less than 10 %. Therefore, we used the SPD-2A UV detector which response quickly. VHSLC simply means performing modern liquid chromatography in extremely short time. The VHSLC method which we reported here, seems to be more rapid and sensitive than the conventional methods for determining vitamins A, E, and K and UQ. The authors express their deep gratitude to Dr. H. Nakamura, University of Tokyo, Dr. T. Nakamura, Mr. Y. Sawa, Miss Y. Hatori, Eisai Co., Ltd., Dr. H. Higashikuze, Shimadzu Corporation, and Mr. S. Ozawa, Takeda Chemical Co., Ltd., for their valuable discussion and assistances. REFERENCES 1) J.L. DiCesare, M.W. Dong, J.G. Atwood : J. Chromatogr., 217, 369 (1981). 2) J.L. DiCesare, M.W. Dong, L.S. Ettre : Chromatographia, 14, 257 (1981). 3) M.W. Dong, J.L. DiCesare : J. Chromatogr. Sci., 20, 49 (1982) 4) S. Kijima : 'Extraction and purification of natural vitamin E' in S. Shimazono and G. Katsui ( Eds. ) Vitamin E, pp.55 (Asakura,Tokyo) (1973). 5) Y. Yamano, S. Ikenoya, M. Anze, M. Ohmae, K. Kawabe : Yakugaku Zasshi, 98, 774 (1977). 6) R. Rueg, U. Gloor, R.L. Goel, G. Ryster, O. Wiss, O. Isler : Hely. Chin. Acta, 43, 2616 (1960). 7) K. Abe, K. Ishibashi, M. Ohmae, K. Kawabe, G. Katsui : Vitamins(Japan), 51, 275 (1977). 8) K. Abe, G. Katsui : Vitamins(Japan), 49, 259 (1975). 9) K. Abe, O. Hiroshima, K. Ishibashi, M. Ohmae, G. Katsui : Yakugaku Zasshi, 99, 192 (1979) 10) K. Abe, K. Katayama, S. Ikenoya, K. Takada, T. Yuzuriha, K. Hamamura, T. Namura, K. Yasuda, S. Yoshida, B.D. Watson, K. Kogure : 'Level of coenzyme Q in tissue' in K. Folkers and Y. Yamamura ( Eds. ) Biomedical and Clinical Aspects of Coenzyme Q, Vol. 3. Elsevier, Amsterdam, 1981, pp ) K.Abe, M. ohmae, K.Kawabe, G. Katsui : Vitamins(Japan), 53, 385 (1979) Keyword phrases very high speed liquid chromatography of vitamins and ubiquinones ; vitamins A, E and K in rat plasma ; UV detection ; pretreatment of plasma ; response time of UV detector ; separation of fat-soluble vitamins and ubiquinones. ( Received February 20, 1984 )