METHODS OF ANALYSIS AND QUALITY CONTROL

METHOD OF ANALYSIS OF PALMITOLEIC ACID IN SE A

BUCKTHORN OIL

M. P. Mogilevskaya, L. D. Ageeva, UDC 615.356 : [615.322 : 582.866].012.07

Yu. A. Koshelev, and M. Ts. Yanotovskii

Currently, sea buckthorn oil is evaluated on the total carotenoid (not l e s s than 1.8 g / l i t e r ) content, and
this value is accepted for control of o f f - t h e - s h e l f p r e p a r a t i o n s . It is known that the carotenoid concentration
v a r i e s significantly depending upon the v a r i e t y of the plant and growth conditions and upon the influence of t e m -
p e r a t u r e , light, and storage t i m e of the b e r r i e s . It has also been r e p o r t e d [1] that about 77% of the biologically
active substances in this oil are fatty acids. Because of this, it is possible to hypothesize that the fatty acid
composition of s e a buckthorn oil might be a useful c h a r a c t e r i s t i c for control of the production p r o c e s s .
Medicinal p r e p a r a t i o n s of sea buckthorn oil are obtained by e x t r a c t i o n of dried crushed b e r r i e s with sun-
flower oil. By use of g a s - liquid chromatography it was established [2] that s e a buckthorn oil differs f r o m
sunflower oil only by the p r e s e n c e of palmitoleie acid. Palmitoleic acid o c c u r s in whale, seal, cod, and other
m a r i n e animal oils, but is found only in negligible quantities in vegetable oils (about 1% in cotton, soybean,
olive, and s e v e r a l other oils).
F i g u r e I shows a chromatographic c o m p a r i s o n of the fatty acids found in sunflower (a) and in sea buckthorn
(b) oil obtained by extraction with ethyl ether. F r o m the e h r o m a t o g r a m it can be seen that sunflower and sea
buckthorn contain principally the same fatty acids, with the exception of palmitoleic acid. The concentration of
palmitoleic acid in relation to the total of all the fatty acids was 40-47% and was variable depending upon plant
v a r i e t y (Table 17. These data showthe possibility of adopting the quantitative determination of palmitoleic
acid as an index in control of the production p r o c e s s for p r e p a r a t i o n of sea buckthorn oil.
In e a r l i e r methods the palmitoleic acid concentration was determined with r e f e r e n c e to the total fatty
acid composition and these methods w e r e probably not reliable owing to the significantly variable quantities of
fatty acids found in sea buckthoru, as in sunflower oil.
A g a s - liquid c h r o m a t o g r a p h i c method has been developed for the determination of the absolute quantity
of fatty acids, specifically palmitoleie acid, in sea buckthorn oil and in i n t e r m e d i a t e production products and
p r e p a r a t i o n s of it. Myristic acid was used as an internal standard because p r e l i m i n a r y analysis of the fatty
acid composition of a l a r g e number of lots of sunflower and sea buckthorn oil showed that m y r i s t i c acid was
e i t h e r absent o r o c c u r r e d in some lots of oil in quantities of l e s s than 0.5%. Because of the deficiency of pal-
mitoleic acid in some of the samples to be analyzed, palmitic acid was used for calibration as the sensitivity
of the flame-ionization detector is proportional to the c a r b o n atom concentration and the coefficient for c o r -
recting the o b s e r v e d r e a c t i o n (plateau peak) depends only upon the m o l e c u l a r weight and the concentration of
c a r b o n in the component [3] .*

EXPERIMENTAL
Sample P r e p a r a t i o n . The fatty acid m e t h y l e s t e r s are p r e p a r e d by suspending about 1000 ~g of the sub-
stance to be analyzed in about 500 pg of a c c u r a t e l y weighed m y r i s t i c acid. Then 6-7 drops of methyl alcohol
and 1-2 drops of acetyl chloride are added. The methanolysis is c a r r i e d out in a r e i n f o r c e d r e a c t i o n v e s s e l
on a hot plate f o r 1 h. The residual methanol is removed, s e v e r a l drops of hexane (0.1-0.2 ml) are added
and 1 # l i t e r of the solution is analyzed by gas-liquid c h r o m a t o g r a p h y .
Analysis Conditions. The analysis was conducted on a Chrom-4 c h r o m a t o g r a p h with a flame-ionization
detector and a glass column. The stationary phase was 10~polyethyleneglycol succinate on Chromaton N-
AW-HMDS (0.160-0.200 ram). Column length was 2.4 m; t e m p e r a t u r e , 180~ vaporization t e m p e r a t u r e , 250~
and sensitivity 1 : 100.
Calibration Curve, A calibration c u r v e is obtained using a c c u r a t e l y weighed r e f e r e n c e palmitic acid and

*Not given in Russian original L i t e r a t u r e Cited s e c t i o n - Consultants Bureau.

All-Union S c i e n t i f i c - R e s e a r c h Vitamins Institute, Moscow. T r a n s l a t e d f r o m K h i m i k o - F a r m a t s e v t i c h e s k i i
Zhurnal, Vol. 12, No. 3, pp. 143-146 March, 1978. Original article submitted April 4, 1977.

0091-150X/78/1203-0409507.50 9 1979 Plenum Publishing Corporation 409

 3
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,7 a 8 rain 2 4 6 rain

Fig. 1. G a s - l i q u i d c h r o m a t o g r a p h y of the m e t h y l e s t e r s of s u n -
flower (a) and s e a b u c k t h o r n (b) oil e x t r a c t e d b y organic s o l v e n t s .
1) P a l m i t i c ; 2) s t e a r i c ; 3) oleic; 4) linoleic; 5) p a l m i t o l e i c .

TABLE 1. The Absolute Concentration of

P a l m i t o l e i c Acid in Some V a r i e t i e s of Sea
Buckthorn B e r r i e s
Absolute concentration
Variety of palmitoleic acid, %

New Alia 31,4~ 1.5

Grant Katutn 3t,4-- 1,8
Vitamin 24,9~2,4
Oil 28,2~ 1,6
I

the internal s t a n d a r d m y r i s t i c a c i d with m a s s r a t i o s gi/gST of 0.1 to 1.0, w h e r e gl i s t h e weight of p a l m i t i e

acid and gST is the weight of m y r i s t i c acid. The r e l a t i v e c a l i b r a t i o n coefficient of the s u b s t a n c e d e t e r m i n e d
in r e l a t i o n to the s t a n d a r d is calculated f r o m the equation:
K = ~I"SsT
gST"$1 "
The c o n c e n t r a t i o n of p a l m i t o l e i c acid (C, in %) in p r e p a r a t i o n s and i n t e r m e d i a t e production products is c a l -
culated using the f o r m u l a :

c = K"SpzST, 100.
5ST'g p
w h e r e K is the r e l a t i v e c a l i b r a t i o n coefficient, Sp is the a r e a under the p e a k for p a l m i t o l e i e acid m e t h y l e s t e r ,
SST is the a r e a u n d e r the peak for m y r i s t i c acid methyl e s t e r , gST is t h e w e i g h t of m y r i s t i c acid, and g p is
the weight of s e a buckthorn off.
A t y p i c a l e h r o m a t o g r a m is shown in Fig. 2. The s e n s i t i v i t y of the p r o p o s e d method has a v a r i a t i o n coef-
ficient of 5.5 relative%.
The method was also used f o r c o n s t r u c t i o n of a kinetic c u r v e of the diffusion p r o c e s s . The p u r p o s e of
t h i s study was to d e t e r m i n e the c o r r e l a t i o n between p e r c e n t c o n c e n t r a t i o n of p a l m i t o l e i c acid and c a r o t e n o i d s
during c o n s e c u t i v e e x t r a c t i o n s of s e a buckthorn oil f r o m the pulp. Samples w e r e r e m o v e d during the diffusion
p r o c e s s and w e r e analyzed f o r p a l m i t o l e i c acid and total c a r o t e n o i d s (Fig. 3). The diffusion c u r v e shows a
c o r r e l a t i o n between the c o n c e n t r a t i o n of p a l m i t o l e i c acid and of c a r o t e n o i d s during the e x t r a c t i o n p r o c e s s .
B e c a u s e of this , the absolute c o n c e n t r a t i o n of p a l m i t o l e i c acid m u s t be a second p a r a m e t e r by which to follow
the technological p r o c e s s for producing s e a buckthorn oil, as well as, a c h a r a c t e r i s t i c of the quality of the
finished product. Use of g a s - liquid c h r o m a t o g r a p h y for the d e t e r m i n a t i o n of s e a buckthorn oil through its
p a l m i t o l e i c acid content m a k e s p o s s i b l e production control and also the determin'ation of its concentration in
different p r e p a r a t i o n s .

410
 g/liter [
i z, z~ I
%
10
9 ao F J~
9 1,8k

',< 1I
;,~ /I
,.4 2//
2

c.,pf~ 1
\ 2_ 4 min 10 2 4 6" 8 10 12 14

Fig. 2 Fig. 3
Fig. 2. Gas-liquid c h r o m a t o g r a m of a simulated mixture of
the methyl e s t e r s of m y r i s t i c (1) and palmitoleic (2) acid.
Fig. 3. Variation in the concentration of carotenoids (1) and
absolute concentration of palmitoleic acid (2). Abscissa) No.
of diffuser.

LITERATURE CITED
Io N. A. Shugam, "Study of biologically active substances", Author' s A b s t r a c t of Candidate' s Dissertation,
Moscow (1969).
2. B. N. Tyutyunnikov, C h e m i s t r y of Fats [in Russian], Moscow (1966), p. 22.

411