Re view 0 bersichtsbericht

Determination of vitamin E in different biological samples

Bestimmung von Vitamin E methanol and extraction with n-hexane [3, 5, 11 - 13, 17, 29,
in verschicdenen biologischen Proben dureh HPLC 31, 47, 60, 66] or heptane [7]. Zaspel and Scallany however
used acetone to extract the tocopherols from plasma; the
Zusammenfassung. Die Ubersicht behandelt folgende removal of water from the plasma by means of addition of
Aspekte: Isolierung des Vitamins aus biologischem Mate- anhydrous sodium sulfate, proved to be necessary to obtain
rial, Lebensmitteln und Pharmazeutica; instrumentelle tocopherol concentrations comparable to those found in the
Probleme (Injektion, reversed-phase und straight-phase n-hexane extracts [71]. Lehman and Martin added methanol
HPLC); Standardisierung; Nachweisgrenzen. to the sample; after mixing and centrifugation, an aliquot
was injected into the chromatograph without further clean-
Summary. The different methods for the determination of
up [37]. Since the 7-tocopherol level in plasma of clinically
vitamin E in all types of biological materials are reviewed.
normal cattle and swine is one order of magnitude lower
The following subjects are dealt with: isolation of the vit-
than that in human plasma, the impact of the determination
amin from biological samples, foodstuffs and pharmaceuti-
method is more critical. Nevertheless, the same determina-
cals; instrumental aspects (injection, reversed-phase HPLC,
tion procedure as for human plasma is used [41].
straight-phase HPLC); standardization; detection limits.
When tocopherols are to be determined in platelets,
erythrocytes or cell culture media, precipitation of the pro-
teins is accomplished by the addition of methanol or ethanol.
Thereafter an extraction with hexane [45], heptane [3] or a
Introduction chloroform solution [38] is carried out. Sometimes an in-
Vitamin E is a generic name for the biologically active ternal standard and an antioxidant are added [23, 30, 38].
members of the fat-soluble tocopherol- and tocotrienol- Some authors saponified the platelet or cell suspension
families. ~-Tocopherol has the highest vitamin E activity for before extraction [23, 30, 46].
men and animals, but is not always the most abundant form To determine tocopherol concentrations in tissues,
of the tocopheryl series [39]. homogenization followed by the extraction with an organic
Vitamin E has been determined by a wide variety of solvent is the most widely used method [26-28, 37, 62, 64,
methods that were reviewed recently [48]. For many years 65, 67, 71]. Westenberg freeze-dried the brain tissue before
biological tests were the only reliable method. However since extraction and made use of tocol as an internal standard
suitable separation and purification could be applied, [67]. Microsomes were analyzed by Hill and Burk. They
spectrophotometry was mostly used. The most widely saponified the proteins before heptane was added to extract
applied methods to purify extracts for tocopherol determina- the tocopherols [24]. Ascorbic acid, pyrogallol or butylated
tion are chromatographic ones: paper, thin-layer, column, hydroxytoluene may be added to prevent oxidation of the
gas-liquid and high-performance liquid chromatography tocopherols [24, 28, 62, 64, 65, 67].
(HPLC). The latter method allows a rapid, simple and
selective determination of tocopherols and tocotrienols in
2 Food
biological materials. This method requires relatively little
clean-up and offers at once the possibility for the determina- Most food samples to be analyzed for vitamin E are cleaned
tion of several fat-soluble vitamins in the same run on the up from interfering substances by saponification (alkaline
same column. digestion) and extraction. An exception exists for plant oil,
The purpose of this publication is to review the different where the sample is almost invariably dissolved in n-hexane
high-performance liquid chromatographic methods of vit- and, without further purification, injected into the HPLC
amin E analysis in all kinds of biological samples. column [1, 14, 21, 25, 55, 61]. Stancher and Zonta however
first saponified the oil before extraction [56],
For non-oil food the homogenization of the sample by
Isolation of vitamin E mixing, grinding etc. is necessary. Then alkaline saponifica-
1 Biological material tion of the test material followed by extraction with organic
Separation of plasma-tocopherols by HPLC is nearly always solvents is carried out [2, 44, 51, 54, 57, 69]. The saponifica-
preceded by deproteination of the plasma.with ethanol or tion is used to eliminate fats, liberate natural tocopherols
and hydrolyse added tocopheryl esters to free alcohols. Some
Offprint requests to: A. Deschuytere authors first extract the sample and then saponify the extract
Fresenius Z Anal Chem (1986) 324:! 4
9 Springer-Verlag 1986
[9, 10, 42]. McMurray and Blanchflower however stated that of hydrocarbon chains bonded to silica: methanol/water
the saponification of the extract leads to low recoveries and mixtures (methanol 8 5 - 1 0 0 % , water 1 5 - 0 % ) are most
is therefore not recommended [43]. Piironen et al. stated that often used as mobile phases [ 3 - 5 , 8, 11, 12, 1 5 - 2 0 , 22, 23,
the recovery is also dependent upon the amount of KOH 2 6 - 2 9 , 37, 38, 4 1 - 4 3 , 50, 5 2 - 5 4 , 60, 63, 67, 69-71].
used, the recovery being highest with the lowest addition Sometimes other mobile phases may be used [7, 24, 35, 36,
of KOH [51]. During these saponification and extraction 49, 56]. Some authors use two reversed-phase columns in
procedures antioxidants such as ascorbic acid and pyrogallol series [2, 56] or have the main column preceded by a guard
are often added to avoid oxidative losses. Some- column [3, 5, 7, 17, 27, 36, 53, 56, 71]. When several fat-
times the saponification step is omitted and only extraction soluble vitamins are to be determined simultaneously,
is carried out. This method results, however, in the same or changing the mobile phase by gradient elution may be neces-
lower recoveries than when the sample is saponified [51]. sary in order to obtain the desired separation within a rea-
Cohen and Lapointe used tetraethylenepentamine to pre- sonable length of time [2, 56, 69, 70]. It is usually not neces-
cipitate chlorophyll and other interfering elements in the sary to use gradient elution when a single vitamin or vitamins
sample, without altering the vitamin E concentration [54]. with similar chromatographic properties are to be separated,
If only the vitamin E quantity that is added to the food, although this method can sometimes be applied to increase
usually as e-tocopherylacetate, has to be determined, an selectivity [26].
extraction with an organic solvent is sufficient. Saponifica-
tion is not carried out and the vitamin is determined in its
3 Straight-phase HPLC
ester form [18, 36, 53, 62]. However, when the sum of the
natural and synthetic vitamin E content is to be determined, For separation of all the free tocopherols and tocotrienols,
saponification is necessary, c~-Tocopheryl acetate is trans- straight-phase HPLC is preferable, even though this tech-
formed into the corresponding alcohol and can be deter- nique is known to be more sensitive to retention variation
mined together with the endogene tocopherols of the sample [33]. Tocopheryl esters elute in reversed order and with
[59]. greater difficulty than the free tocopherols [19]. Silica
The HPLC determination of the tocopherol isomers in columns with mobile phases consisting of a single hy-
intravenous lipid emulsions is described by Gutcher et al.; drocarbon (n-hexane) or mixtures of a hydrocarbon with
they used a chloroform/methanol mixture for the extraction small quantities of a more polar solvent (alcohols,
of the vitamins [22]. ethers . . . . ) are most often used [1, 9, 13, 14, 19, 25, 31, 32,
The procedure used for the analysis of milk is very similar 34, 4 4 - 4 7 , 51, 55, 5 7 - 5 9 , 61-67]. Sometimes isooctane is
to the one used for plasma analysis i.e. deproteination of the used instead of hexane [10, 21] or a mixture of hexane/
sample by ethanol, followed by extraction with n-hexane dichloromethane/-isopropanol [9]. Westenberg used a
[15, 32]. Pickston first saponified the sample before extrac- column with a chemically bonded primary amine, the sepa-
tion [50]. ration efficiency of the different tocopherols was equal or
even superior to that obtained with other columns [67].
Speek et al. added butylated hydroxytoluene to the mobile
3 Pharmaceuticalproduets
phase to prevent the E vitamins from oxidative degradation
For the preparation of vitamin E containing tablets, gelatine [55]. Guard columns are also used to enhance the separation
capsules or other pharmaceutical formulations, ~-toco- of theisomers [9, 46, 51, 63 - 65].
pherol as free alcohol, acetate- or succinate-ester is used.
Before the extraction can be carried out, these products have
4 Detection
to be freed from the tablet coating, capsule wall or other
shell vehiculum [16, 19, 20, 34, 49, 69, 70]. Sometimes the If an electrochemical detector is used, the solubility of the
samples are saponificated [34]. supporting electrolyte is of prime importance. Since this
solubility is too low in the hydrophobic solvents used in
straight-phase HPLC, reversed-phase HPLC is to be applied
Instrumental aspects [7, 15, 29, 60]. However, straight-phase HPLC can be used
if the column eluate is mixed with an alcoholic electrolyte
1 Injection solution at the outlet of the column, before detection is
The extract is dried under reduced pressure or by using a performed [25]. Electrochemical detection of the tocopherols
gentle nitrogen stream, except when it is immediately in- is carried out at a potential of + 0.7 V [15, 29, 60] or + 1.0 V
jected onto the column [37, 64, 65]. The former method is [7] versus an Ag/AgC1 reference electrode.
mostly used in case of voluminous extracts from food Detection is more frequently accomplished by a fixed or
samples, the latter is used for plasma extracts. The residue variable wavelength spectrometric or a fluorimetric proce-
is then redissolved in a solvent miscible with the mobile dure. Although the possibility of interferences, and thus
phase or in the solvent used as the mobile phase. Further false results, is greater with UV detection [7], this detection
separation of the sample is performed on the HPLC column. method is still often used. The most commonly used
wavelengths are 254 nm [6, 16, 34, 67, 70] and a range of
wavelengths between 275 and 298 nm [ 2 - 9 , 1 1 - 1 3 , 1 7 -
2 Reversedphase HPLC 20, 22, 24, 2 6 - 2 8 , 31, 32, 3 4 - 3 6 , 41, 45, 46, 49, 50, 5 2 -
Non-aqueous reversed phase HPLC is most widely used 54, 58, 68, 71]. UV detection at 290 nm is reported to be
for practical determinations, however/% and 7-tocopherol more sensitive than at 254 nm [67].
coelute. This is no problem because sources of/%tocopherol Fluorescent detection is claimed to be more selective
are so limited that usually it will only be present in minor and provides lower detection limits [37, 41, 59, 71]. The
concentrations [40]. Reversed-phase columns are made up fluorescent intensity is greater with methanol than with
a hydrocarbon solvent. This implies that sample manipula- 6. Cavins JF, Inglett GE (1974) Cereal Chem 51:605-609
tions are minimized because tocopherol bound in tissues 7. Chou PP, Jaynes PK, Bailey JL (1985) Clin Chem 31(6):
is released by methanol [37]. The mostly used excitation 880-882
wavelengths vary between 290 and 296 nm, and the wave- 8. Cohen H, Lapointe M (1978) J Agric Food Chem 26(5): 1210-
1213
length of the cut-off emission filter is between 320 and
9. Cohen H, Lapointe M (1980) J Assoc Off Anal Chem
340 nm [1, 6, 10, 14, 21, 37, 38, 4 1 - 4 4 , 46, 51, 55, 61, 63, 63(6): 1254-1257
66, 71]. Jansson et al. however, used an emission wavelength 10. Cort WM, Vicente TS, Woysek EH, Williams BD (1983) J Agric
of 370 nm [31, 32]. H a t a m and Kayden carried out Food Chem 31(6):1330-1333
fluorimetric analysis at a short excitation wavelength 11. De Leenheer AP, De Bevere VO, Cruyl AA, Claeys AE (1978)
(205 nm), stating that this increases the selectivity twentyfold Clin Chem 24(4): 585- 590
in comparison with the usual wavelength [23]. 12. De Leenheer AP, De Bevere VO, DeRuyter MG, Claeys AE
(1979) J Chromatogr 162:408-413
13. De Leenheer AP, De Bevere VO, Claeys AE (1979) Clin Chem
Standardization 25(3):425- 428
14. de Lumen BO, Fiad S (1982) J Agric Food Chem 30:50-53
Internal standards are to be used for quantification because 15. Deschuytere A, Deelstra H (1985) Proc Eur Food Chem
the importance of errors during sample handling is reduced I I I : 4 4 - 50
and therefore the precision of the method is improved. The 16. Dolan JW, Orant JR, Tanaka N, Giese RW, Karger BL (1978)
internal standards are added before saponification and ex- J Chromatogr Sci 16:616-622
traction. The most c o m m o n used internal standard is tocol 17. Driskell WJ, Neese JW, Bryant CC, Bashor MM (1982) J
[11 - 1 3 , 29, 31, 32, 38, 46, 67]. ~-Tocopheryl acetate is used Chromatogr 231:439-444
in samples, such as blood fractions [3, 5, 46, 47, 71] or food 18. Eriksen S (1980) J Assoc Off Anal Chem 63(5) : 1154-1157
19. Eriksson T, Sorensen B (1977) Acta Pharm Suec 14:475-484
[67], that do not contain added vitamin E. Other internal 20. Eriksson M, Eriksson T, Sorensen B (1978) Acta Pharm Suec
standards used for tocopheryl quantification are tert-butyl- 15:274-281
hydroxyanisole in oils [21], cholesterolphenyl acetate in food 21. Gertz C, Hermann K (1982) Z Lebensm Unters Forsch
[2] or 3-tocopherol in human plasma [7, 60] or milk [15] 174: 390- 394
because of its low concentration in these samples. When 22. Outcher GR, Lax AA, Farrell PM (1984) J Parent Entomol
several vitamins are quantified in the same analysis, retinyl Nutr 8(3) :269-273
acetate [17] or naphthalene [45] can be used. 23. Hatam LJ, Kayden HJ (1979) J Lipid Res 20:639-645
24. Hill KE, Burk RF (1984) Biochem Pharmacol 33(7):1065-
1068
25. Hiroshima O, Ikenoya S, Ohmae M, Kawabe K (1981) Chem
Detection limits Pharm Bull 29(2): 451 - 455
The minimum detectable quantities vary with the kind of 26. Howell SK, Wang YM (1982) J Chromatogr 227:174-180
27. Hung SSO, Cho YC, Slinger SJ (1980) J Assoc Off Anal Chem
detector used. For electrochemical and fluorescent detec- 63(4): 889- 893
tion, the limit is lower (0.001 - 0 . 0 1 ~g injected) than for UV 28. Hunt DF, Organisciak DT, Wang HM, Wu RLC (1984) Curr
detection ( 0 . 0 5 - 0.1 gg injected). Eye Res 3(11): 1281 -- 1288
The recovery is very much dependent u p o n the extraction 29. Ikenoya S, Abe K, Tsuda T, Yamano Y, Hiroshima O, Ohmae
and/or saponification method used [1, 43, 51, 67, 71]. F o r M, Kawabe K (1979) Chem Pharm Bull 27(5):1237-1244
plasma and erythrocytes with an appropriate extraction 30. Ishibashi K, Abe K, Ohmae M, Katsui G (1977) Vitamins (in
procedure, the recovery is usually almost quantitative. For Japanese) 51:415-422
food- and feedstuffs and tissues it varies between 80 and 31. Jansson L, Nilsson B, Lindgren R (1980) J Chromatogr
181:242-247
100%.
32. Jansson L, Akesson B, Holmberg L (1981) Am J Clin Nutr
The coefficient of variation differs with the kind of 34:8-13
tocopherol that is being determined. In plasma it varies 33. Karger BL, Giese RW (1978) Anal Chem 50:1048
between 1.5 and 6% for c~- and 7-tocopherol. For/~-toco- 34. Kountourellis JE, Raptouli A, Botsoglou NA, Georgarakis M
pherol the variation coefficient is significantly higher [7.0 to (1985) Pharmazie 40(H3): 193 - 194
22.0%), possibly because of its low concentration in plasma 35. Landen WO Jr (1981) J Chromatogr 211:155-159
[31]. For foodstuffs the variation coefficient is situated in 36. Landen WO Jr (1982) J Assoc Off Anal Chem 65(4):810-816
the range between 1.5 and 6.0%. In certain cases however, 37. Lehmann J, Martin HL (1982) Clin Chem 28(8):1784-1787
38. Lehmann J, Martin HL (1983) Clin Chem 29:1840-1842
the coefficient of variation of the less abundant isomers may
39. Machlin LJ (1980) Vitamin E: A comprehensive treatise, Vol 1.
be somewhat higher [55]. M Decker Inc. New York
40. MacLaughlin PJ, Weihrauch JL (1979) J Am Diet Assoc
Acknowledgement. A. Deschuytere is a research assistant at the 75: 647- 665
Belgian National Fund for Scientific Research. 41. MacMurray CH, Blanchflower WJ (1979) J Chromatogr
178 : 525-- 531
42. MacMurray CH, Blanchflower WJ (1979) J Chromatogr
References 1 7 6 : 4 8 8 - 492
43. MacMurray CH, Blanchflower WJ Rice DA (1980) J Assoc Off
1. Abe K, Yuguchi Y, Katsui G (1975) J Nutr Sci Vitaminol Anal Chem 63(6):1258-1261
21 : 183 -- 188 44. Manz U, Philipp K (1981) Int J Vit Nutr Res 51:342-348
2. Barnett SA, Frick LW, Baine HM (1980) Anal Chem 52: 45. Matsuo M, Tahara Y (1977) Chem Pharm Bull 25(12):3381 -
610-614 3384
3. Bieri JG, Tolliver TJ, Catignani GL (1979) Am J Clin Nutr 32: 46. Mino M, Kitagawa M, Nakagawa S (1985) Am J Ctin Nutr
2143 -2149 41:631-638
4. Burns DT, Mackay C (1980) J Chromatogr 200 : 300 - 304 47. Nilsson B, Johansson B, Jansson L, Holmberg L (1978) J
5. Catignani GL, Bieri JC (1983) Clin Chem 29(4):708-712 Chromatogr 145:169 -- 172
48. Parrish DB (1980) CRC Crit Rev Food Sci Nutr 161 - 1 8 7 61. Van Niekerk PJ (1973) Anal Biochem 52:533-537
49. Pellerin F, Dumitrescu D (1980) Talanta 27:243-251 62. Vatassery GT, Hagen DF (1977) Anal Biochem 79:129-134
50. Pickston L (1978) N Z J Sci 21:383-385 63. Vatassery GT, Maynard VR, Hagen DF (1978) J Chromatogr
51. Piironen V, Varo P, Syvaoja E-L, Salminen K, Koivistoinen P 161:299-302
(1984) Int J Vit Nutr Res 53:35-40 64. Vatassery GT, Angerhofer CK, Peterson FJ (1984) J
52. Riickemann H, Ranfft K (1978) Z Lebensm Unters Forsch Neurochem 42(2): 554- 558
166:151-152 65. Vatassery GT, Angerhofer CK, Knox CA, Deshmukh DS
53. Shaikh B, Huang HS, Zielinski WL (1977) J Assoc Off Anal (1984) Biochim Biophys Acta 792 : 1 1 8 - 1 2 2
Chem 60(1) : 137 - 139 66. Vuilleumier J-P, Keller HE, Gysel D, Hunziker F (1983) Int J
54. S6derjhelm P, Andersson B (1978) J Sci Food Agric 29:697- Vit Nutr Res 53 : 265
702 67. Westerberg E, Friberg M, Akesson B (1981) J Liquid
55. Speek AJ, Schrijver J, Schreurs WHP (1985) J Food Sci Chromatogr 4(1): 109-121
50:121-124 68. Widicus WA, Kirk JR (1979) J Assoc Off Anal Chem
56. Stancher B, Zonta F (1983) J Chromatogr 256:93-100 62(3): 637-641
57. Stancher B, Zonta F (1983) Riv It Sost Gr LX:371-375 69. Wiggins RA (1976) Proc Anal Div Chem Soc 133-137
58. Tangney CC, Driskell JA, MacNair HM (1979) J Chromatogr 70. Williams RC, Schmit JA, Henry RA (1972) J Chromatogr Sci
172:513-515 10:494 - 501
59. Thompson JN, Hatina G (1979) J Liquid Chromatogr 71. Zaspel BJ, Csallany AS (1983) Anal Biochem 130:146-150
2(3): 327 - 344
60. Vandewoude M, Claeys M, De Leeuw I (1984) J Chromatogr
311:176-182 Received October 14, 1985