determination of vitamins d by gas-liquid chromatography
TRANSCRIPT
THE JOURNAL OF VITAMINOLOGY 18, 165-171 (1972)
Determination of Vitamins D by Gas-Liquid
Chromatography
II. Rapid Assay for Vitamin Dz in the Presence
of Vitamins A and E1,2
KIYOSHI TSUKIDA AND KAYOKO SAIKI3
Kobe Women's College of Pharmacy, Higashinada-ku, Kobe
(Post No. 658)
(Received July 5, 1972)
In addition to our previous findings on GLC and mass spectroscopic determination of vitamins D, extensive studies on the effect of interfering substances have
presented a new simple GLC determination method of vitamin D2 in the presence of vitamins A and E. Thus, by employing TLC before applying a sample for
GLC evaluation, vitamin E and its related compounds which interfere the GLC
determination are eliminated completely and the greater part of vitamin A is also removed in the form of anhydrovitamin A simultaneously. Exhaustive conver
sion of vitamin A into its anhydro derivative and removal of unreacted vitamin A from vitamin D are not necessary. The proposed method is a simple, inexpen
sive, accurate, and time-saving quantification method of vitamin D2. Main advantages and limitation of the method are discussed in detail.
In a previous paper we presented the data concerning the basic conditions for GLC determination of vitamins D (1). It was also reported
(2) that coexistence of vitamin A could be allowed for GLC determination of vitamins D,
provided A/D value was 50 or less. Otherwise, vitamin A gave rise to cause a tailing phenomenon over vitamin D peaks (twin peaks, pyro and isopyro peaks) which have primarily well differentiated retention times from those of vitamin A and its related compounds. Furthermore, it should be stressed that the presence of vitamin E (a-tocopherol) gave a serious trouble
for the present purpose, though so-called degradation products of vitamins A and D did not
interfere an analysis except for a particular case. For elimination of vitamin E, several attempts employing a basic alumina (3) or an Amberlyst resin column (4) did not give any improvement on GLC analysis of vitamin D.
We now propose a simple and a rapid GLC determination of vitamin D2 in the presence of vitamins A and E by adopting TLC for eliminating vitamin E etc. and for suppressing A/D value below the tolerance limit.
PROPOSED DETERMINATION METHOD FOR VITAMIN D2
a) Saponification and benzene-washing
(Isolation of the unsaponifiable matter)
1 Determination of Vitamins D. III; Part II, Internat. J. Vit. Nutr. Res., 42 (2), in press.2 Following abbreviations are used: GLC
, gas-liquid chromatography; TLC, thin-layer chromatography; A/D, a content ratio of vitamin A to vitamin D; pyro, pyrocalcif erol; isopyro, isopyrocalcif erol; i, d., internal diameter; BHT, butylated hydroxytoluene; G. R., guaranteed reagent.
3 月田 潔,斎 木加代子
165
166 TSUKIDA AND SAIKI
Reagents:Benzene, G. R.Ethanol 99.5% v/v, G. R.2N KOH in ethanol, fresh0.5N and 1N KOH in water, fresh25% Sodium ascorbate in waterBHT, pure
Procedure:
Proceed according to the Mulder's method
(3) essentially. Weigh a sample containing not
less than 50ƒÊg of vitamin D2 into a saponifica
tion flask (200ml). Add BHT (10mg), dissolve
in ethanol (50ml), and then add 2N KOH in
ethanol (10ml) and 25% sodium ascorbate (1
ml) with swirling. Keep a sample solution at
80•‹(bath temperature) for 30min in a stream
of nitrogen and then cool immediately. Add
100.0ml of benzene pipetted accurately and mix
together. Pour the solution into a separatory
funnel without rinsing. Add 1N aqueous KOH
(40ml) and shake vigorously for 10 sec. After
standing for 5min, separate the layers and
discard the turbidz aqueous layer. Wash the
benzene layer similarly with 0.5N aqueous KOH
(40ml), and then repeatedly with water (40ml
portions) until the aqueous layer remains neutral
to phenolphthalein. Filter the benzene solution
through a phase separating paper (Whatman
1PS, siliconized) and store it in a well-stoppered
flask.
b) Acid treatment and TLC (Complete
elimination of vitamin E including its relating
compounds and simultaneous removal of the
greater part of vitamin A)
Reagents:
Acetone, G. R.0.1N HCl in methanol, fresh0.1N KOH in water, fresh
Reference solution containing vitamin D2 and previtamin D2, prepared by refluxing of vitamin D2 (crystals, Philips-Duphar) solution in isooctane (G. R.) for 30min, followed by evaporation to dryness in vacuo and by dissolving the residue in an appropriate volume of acetone (G. R.)
TLC:
Plate (250ƒÊ-thick, 25•~25cm), Kieselgel
HF254 Merck; use the plate within 24hr of its
preparation (activated at 120•‹for 1hr)
Developing solvent, benzene (G. R.)-ethy
lene dichloride (G. R.)-acetone (G. R.)=70:
30:0.5; when vitamin E is not included con
spicuously, another solvent system, e.g., benzene
(G. R.) acetone (G. R.)=9:1 can be employed
Procedure:
Add approximately equi-volume of 0.1N HCl
in methanol to the benzene solution described
above, and stand for 15min at room tempera
ture. Neutralize the solution immediately with
0.1N KOH aqueous solution, and wash repea
tedly with water until the aqueous layer remains
neutral to phenolphthalein. The acid-treated
benzene solution thus obtained is colored yellow
by conversion of the large portion of vitamin A
into anhydrovitamin A. Filter the benzene
solution through a phase separating paper, pipet
50ml of the filtrate, and evaporate in vacuo.
Dissolve the residue in acetone (1ml) and use
it for subsequent TLC as soon as possible.
Streak the acetone solution (0.2ml) (pref e
rably ca. 20-50ƒÊg vitamin D2) onto a thin
layer plate and also apply a single spot of the
reference solution containing vitamin D2 and
previtamin D2 on an extension of the basic
line. Develop the chromatogram for ca. 1hr
and examine the result quickly under ultraviolet
light (254nm). Encircle the corresponding area
at the same height as the spots of vitamin D2
and previtamin D2 ex the reference solution.
Scrape off this area of Kieselgel powder (vita
min D2+previtamin D2, concomitant vitamin
A is allowed) with a flat spatula. Extract the
substances with acetone, followed by washing
with the same solvent (20ml in total) and filter.
c) GLC determinationReagents:Internal standard solution, 7-dehydrochole
steryl acetate (crystals, pure) in acetone (G. R.)
Standard mixture of vitamin D2 and the internal standard, fresh; details are as described in the procedure
GLC:Apparatus, Gas Chromatograph GC-4AP
(Shimazu Seisakusho) equipped with a hydrogen flame ionization detector; Column, glass, 1.5m
GLC DETERMINATION OF VITAMIN D 167
long and 4mm i.d., packed with 1.5% OV
- 17 on Shimalite W (80-100 mesh, silanized);
Operational parameters, temperatures (column
250•‹, injector port 260•‹, and detector 300•‹),
flow rates (nitrogen [carrier gas] 60ml/min,
hydrogen [detector gas] 0.8kg/cm2, and air 1
kg/cm2)
Procedure:
Add the internal standard solution to the
sample solution described above (vitamin D2:
internal standard=2:1 is preferable). Apply
1-5ƒÊl of the acetone solution concentrated to
an appropriate volume (preferably 0.5-2ƒÊg vita
min D2 per an applied volume of the solution)
into the instrument by on-column injection using
a 10ƒÊl microsyringe. Estimate a peak area
(half-width•~peak height) ratio A (pyro or
isopyro/internal standard) from the recording
chart. From an independent GLC tracing,
determine the standard peak area ratio S bet
ween vitamin D2 (pyro or isopyro) and the
internal standard ex a newly prepared standard
mixture (vitamin D2: internal standard=2:1 by
weight, internal standard is the same concentra
tion solution when A value was estimated).
Calculate a quantity of vitamin D2 as follows:
A: S=x:w
x: Vitamin D2 (weight) in a final sample solution applied for GLC
w: Vitamin D2 (weight) in a final solution of a standard mixture applied for GLC
RESULTS AND DISCUSSION
a) GLC of Vitamin D2 and Interfering Substances
Since chemical methods heretofore proposed for microdetermination of vitamin D commonly suffer primarily from lack of specificity, it has been a serious problem how to separate the vitamin in a pure state free from numerous interfering substances. Though it is simple and one of the most representative separation techniques, TLC can not differentiate vitamin D from tachysterol. On the contrary, TLC does separate previtamin D from vitamin D and unfortunately this phenomenon gives rise to trouble how to deal with a thereto-equilibrium
which exists between these two compounds. It is quite impossible to separate minute amount of vitamin D quantitatively on a limited plate from large excess of interfering substances. It is for this reason that purification of vitamin D still relies upon an employment of multi-columns
(adsorption, partition, or gel filtration) in spite of its great disadvantages, viz., insecure reproducibility of a column condition and time-consuming operation. Among recent techniques of instrumental analysis, infrared and nuclear magnetic resonance spectroscopies are not always adequate for microdetermination at present time. Considering every qualifications for a routine assay involving f utural prospects, GLC technique would be most practical and would have unlimited possibilities (1), though mass spectroscopy is the most direct determination method of all (5).
On GLC determination of vitamin D, several papers have been published successively since the work of Ziff er et al. appeared in 1960 (6). Basic studies on an analytical form (unmodified vitamin D), stationary phase of the column (1.5
% OV-17), internal standard (7-dehydrocholesteryl acetate), and operational parameters were already reported by present authors, excellent calibration curves of vitamins D between peakarea ratio and weight ratio (vitamin D/internal standard) had being obtained (1, 2). Since a GLC separation tracing itself reflects a determination value of a definite ingredient immediately, tailing phenomenon as well as overlap with other peaks caused by concomitants would then be urgent problem for present purpose, even though there exist many problems concerning multiplicity of pre-treatment depending upon a form of a sample for analysis (3, 4, 7).
Vitamin A and its related compounds on GLC Under the operated condition, vitamin A (possibly as degradation products) possessing a smaller retention time was eluted rapidly from the column and up to 50 times the weight of vitamin A could be allowed as a concomitant for the present (Table 1). With increasing
quantities over this tentative tolerance limit, vitamin A did induce tailing phenomenon over vitamin D peaks region (Fig. 1). Similar observations were also obtained on the effects of vitamin A related compounds (anhydrovita-
168 TSUKIDA AND SAIKI
TABLE 1GLC determination of vitamin D2 in the presence of vitamin A*
* Vitamin D2 is determined by GLC using the
pyro -and the isopyro-D2 peaks. The ratio of total vitamin D2 to the internal standard is 2:1.
FIG. 1 Gas chromatographic behavior of a mixture of vitamins A acetate and D2
a: pyro thermal cyclization product. b: isopyro thermal cyclization product. Weight ratio of vitamin D2 to vitamin A acetate: A, 1:1; B, 1:50; C, 1:100
min A etc.) and of vitamin A degradation products prepared artificially by irradiation, oxidation (8), or heating of the original vitamin. However, when a sample was treated according to the procedure b) described above, even 500 times the weight of concomitant vitamin A and its related compounds did not interfere with an analysis at all.
Vitamin D related compounds on GLC Interfering effect of so-called vitamin D degrada
tion products obtained by irradiation (white or ultraviolet light) or by prolonged standing at room temperature of vitamin D in crystals or in various solution was investigated. Among these compounds, only lumisterol and a product
(only end absorption in its ultraviolet spectrum) isolated from ultraviolet irradiated vitamin D in hexane solution were not separated quantitatively from vitamin D2 (pyro peak). In this case it is
possible, however, to estimate vitamin D based on isopyro peak instead of pyro peak unless the vitamin content is extremely small.
Vitamin E and its related compounds on GLC Vitamin E and most of its related compounds give serious obstacles for GLC determination of vitamin D. In most types of vitamin drugs, for example, vitamin E is usually contained in a larger amount compared with other vitamins and the peak of vitamin E or its acetate overlaps completely on the pyro or on the isopyro peak, respectively (Table 2). Thus, elimination of vitamin E and its related compounds should now be projected as one of the most important pre-treatment for GLC determination of vitamin D. Separation of vitamin E by employing an appropriate column treatment such as basic alumina or Amberlyst resin column did not give any improvement at a level of GLC analysis, viz., the former column
produced new minor peaks (isomers or a degradation product ?) ex vitamin D and the latter column could not suppress an elution of vitamin
E degradation product (and vitamin E*l) responsible for overlapping on the vitamin D peaks region (Fig. 2). However, when a sample is treated according to the procedure b) described
TABLE 2
Relative retention data on 1.5 on OV-17 column
* Vitamin D2 and D3 give twin peaks, the larger
pyro peak being eluted earlier.
*1 Over 10 times the weight of vitamin E could not be suppressed .
GLC DETERMINATION OF VITAMIN D 169
FIG. 2 Gas chromatographic behaviors of vita
mins D and E
•c•c vitamin D2, -a-tocopherol (a) or ƒ¿-toco
pheryl acetate (b)
A: genuine samples, B: after saponifying and
passing through a basic alumina column, C: after
saponifying and passing through an Amberlyst A-26
column
above, these interfering substances can be eliminated completely.
b) Acid Treatment and TLC
For complete elimination of vitamin E and its related compounds, TLC separation was investigated. Concerning vitamin A, it was
FIG. 3 TLC chromatograms of vitamins A, D, and E g, genuine; s, after saponification; a, after acid treatment Fluorescence: 1B, light blue; Y, yellow; V, violet; RB, reddish brown* Vitamin A acetate: D:E acetate=140:1:160 by weight
practically impossible to separate vitamin D
from excess amount of vitamin A quantitatively,
and hence present TLC should aim at a restrain
of A/D value below 50 or less. This was done
by converting the greater part of vitamin A
into anhydrovitamin A in advance, followed by
TLC separation. When large excess of vita
mins A and E exist, the system of benzene
ethylene dichloride acetone (70:30:0.5) is pre
ferable. Since this developing agent is not
projected for separation of vitamins A and D,
but for complete elimination of vitamin E and
its related compounds as well as anhydrovitamin
A, it is possible to employ another solvent
system, e,g., benzene acetone (9:1), provided
vitamin E is not contained significantly. On a
TLC plate, a rather complicated chromatogram
ex vitamin E was observed and BHT was
found at a front with the solvent. Preferable
amount of vitamin D on a TLC plate would
be 20-50ƒÊg. Typical TLC pattern of vitamins
D, E, and A (genuine, after saponification, and
after acid treatment) were illustrated in Fig. 3
and GLC recoveries of vitamin D both after
acid treatment and after TLC were given in
Table 3.
c) GLC Determination of Vitamin D on a
Laboratory-blended Sample
Surprisingly, little information have been
available on the determination of vitamin D in
the presence of vitamins A and E except for a
few methods employing multi-columns (3, 4) or TLC (7). A GLC determination of vitamin D2 (as the trimethylsilyl ether) in multiple-vitamin tablets, using a 3% OV-210 column was reported by Edlund et al., but the same authors concluded that this method could not be applicable to those containing vitamin E (9)*2. Recently, Feeter et al. (10) separated vitamins D and tocopherols in the form of the propionate on GLC using 2% SE52 column. Even apart from a few problems such as preparation and conditioning of the column or reaction rate and
yield of esterification etc., difficulty will
*2 It may be applicable with much better result than our present method to those containing vitamin E,
only if vitamin E is eliminated according to our present procedure.
170 TSUKIDA AND SAIKI
TABLE 3GLC recoveries of vitamin D2
*1 A ratio of the pyro or the isopyro peak area to the internal standard peak area .
Total vitamin D2 to the internal standard is 1:1 by weight.
*2 Apply the acetone solution in the instrument.
*3 Vitamin D2 (50ƒÊg) is developed on a plate, eluted with acetone. The internal
standard is added and the solution is concentrated prior to its GLC analysis.
*4 After anhydration, neutralization, and washing, the internal standard is added to
the benzene solution. Evaporate the solvent and the residue is dissolved in
acetone.
*5 Saponificationacid treatment TLCGLC; Vitamin A:D:E in a sample=140:
1:160 (by weight).
be encountered at once when a sample is contaminated with a large excess of vitamins A and E, and qualification of trioctanoin for an internal standard is also questionable.
Our method indicates that overall recoveries of vitamin D in a laboratory-blended sample
(vitamin A:D:E=140:1:160 by weight) are satisfactory and sufficiently accurate (Fig. 4 and Table 3). Four to five hours are required for an overall analysis. Practical applicability of our method for determination of vitamin D in commercial multiple-vitamin drugs was confirmed in some pertinent experiments.
d) Scope and Limitation
Main characteristics (Table 4) and advantages of our present method could be summarized as follows:
1. Unmodified vitamin D2 is adopted as an
FIG. 4 GLC determination of vitamin D2 in a laboratory-blended mixture (vitamin A:D:E=140:1:160 by weight)
a:pyro, b:isopyro, c:internal standard
analytical form for GLC determination.2. By employing TLC, vitamin E and its
related compounds are eliminated completely
and the greater part of vitamin A is also
GLC DETERMINATION OF VITAMIN D 171
TABLE 4Determination of vitamin D2 in the presence o f vitamins A and E
removed in the form of anhydrovitamin A simultaneously. Complete conversion of vitamin
A into its anhydro derivative and separation of vitamin D from unreacted vitamin A are not
necessary. Even a sample possessing a high
A/D value can be analyzed without any trouble.
3. Tedious pre-treatment using multi-colu
mns can be omitted. It is a simple, inexpensive,
accurate, and time-saving quantification method of vitamin D2.
4. Consideration on previtamin D and
tachysterol are not necessary.
5. Involving futural prospects such as ad
vanced automation of analysis, it possesses the
qualification for an excellent routine assay.It should be also pointed out as a minor
disadvantage of this method that TLC procedure has still time-consuming factor in some sense
and scraping of Kieselgel powder may cause
fluctuating data in analysis, though our scraping
procedure itself is not necessary to be so strict as in general case.
REFERENCES
1. Tsukida, K., and Saiki, K., J. Vitaminol., 16, 293 (1970).
2. Tsukida, K., and Saiki, K., presented at the 126th Meeting of Fat-Soluble Vitamins Research Committee (Jul., 1970).
3. Mulder, F. J., de Vries, E. J., and Keuning, K. J., Pharm. Weekblad, 100, 1457 (1965).
4. Ueda, F., Makino, T., Kazama, A., and Wata nabe, K., Vitamins, 44, 31(1971); J. Vitaminol., 17, 142 (1971).
5. Tsukida, K., and Saiki, K., Internat. J. Vit. Nutr. Res., 42, (2) (1972), in press.
6. Ziff er, H., Vanden Heuvel, W. J. A., Haahti, E. O. A., and Horning, E. C., J. Am, Chem. Soc.,
82, 6411 (1960).7. Bolliger, H. R., and Konig, A., Z. Analyt.
Chem., 214, 1 (1965).8. Kobayashi, T., Vitamins, 32, 482 (1965).9. Edlund, D. O., and Anfinsen, J. R., J, Ass. Off. Analyt. Chem., 53, 287 (1970).
10. Feeter, D. K., Jacobs, M. F., and Rawlings, H. W., J. Pharm. Sci., 60, 915 (1971).