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Determination of water-soluble vitamins in vitamin premixes, bioacitve dietary

supplements, and pharmaceutical preparations using high-efficiency liquid
chromatography with gradien...

Article in Moscow University Chemistry Bulletin · August 2010

DOI: 10.3103/S0027131410040103

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ISSN 00271314, Moscow University Chemistry Bulletin, 2010, Vol. 65, No. 4, pp. 260–268. © Allerton Press, Inc., 2010.
Original Russian Text © A.A. Bendryshev, E.B. Pashkova, A.V. Pirogov, O.A. Shpigun, 2010, published in Vestnik Moskovskogo Universiteta. Khimiya, 2010, No. 4, pp. 315–324.

Determination of WaterSoluble Vitamins in Vitamin Premixes,

Bioacitve Dietary Supplements, and Pharmaceutical Preparations
Using HighEfficiency Liquid Chromatography
with Gradient Elution
A. A. Bendryshev, E. B. Pashkova, A. V. Pirogov, and O. A. Shpigun
Division of Analytical Chemistry
email: zhab@mail.ru
Received September 7, 2009

Abstract—The influence of the pH and temperature of the mobile phase on the retention and separation of
14 vitamins and vitameric forms has been studied for four different stationary phases. The chromatographic
conditions allowing the separation of 12 watersoluble vitamins and vitameric forms have been proposed. It
has been established that the best separation of watersoluble vitamins can be achieved by employing gradient
elution. The mobile phase A, (0.6% phosphoric acid) pH 1.5–1.8; mobile phase B, acetonitrile. For the sep
aration of nicotinic and ascorbic acid it is preferable to use stationary phases Luna C18(2) and Gemini C18,
while for the separation of riboflavin and riboflavin5phosphate the Synergy Hydro RP and Zorbaz SBC18
stationary phases should be used. The selected conditions have been used for the determination of vitamins
in commercial samples of vitamin preparations. The results are in good accordance with the producer data.

Key words: watersoluble vitamins, HPLC, chromatography, pharmaceutical preparations, bioactive addi
tives, premixes.
DOI: 10.3103/S0027131410040103

INTRODUCTION simultaneously. In the majority of cases, reversephase

Water soluble vitamins include compounds with
very different structure and properties. Usually, this
group includes ascorbic acid (C), thiamine (B1), nic
otinamide (in different sources it is referred to as PP,
B3, or B5), pyridoxine (B6), pantothenic acid (called
B5 or B3 in different sources), folic acid (B9 or BC),
cyanocobalamin (B12), riboflavin (B2), biotin (H),
and, in some cases, rutin (P) [1, 2]. In addition to the
listed compounds, some of the vitamins can exist in
the form of some other substances (vitameric forms).
For example, PP is introduced into vitaminenriched
mixtures either as nicotinamide or as nicotinic acid.
Vitaminenriched mixtures are composed of sev
eral vitamins and there is a necessity for the determi
nation of the concentration of each of them both in
the initial mixture and in the food products, enriched
with such mixtures. Nowadays, according to the pro
cedures provided by the twelfth addition of the State
Pharmacopoeia of Russia, each vitamin is determined
separately, with the employed analytical methods usu
ally being old and timeconsuming (titration, thin
layer chromatography [3]). Highperformance liquid
chromatography (HPLC) is one of the most popular
methods for the determination of watersoluble vita
mins due to its ability to determine several compounds
HPLC is used [4–22]. It is difficult to perform the
simultaneous determination of all the watersoluble
vitamins in isocratic mode. Such a problem can be
solved using either ionpair reagents or gradient elu
tion. The conventional method used for the simulta
neous determination of several vitamins is ionpar
chromatography [5–9]. However, frequently complete
resolution of the peaks of different vitamins with each
other or with the matrix components is not achieved.
In this case, the ionpair variant provides few possibil
ities for the improvement of the resolution of the
neighboring peaks.
In our opinion, a more promising approach is gra
dient elution. Studies devoted to the determination of
watersoluble vitamins in different samples using gra
dient elution can be found [10–14]; however, there are
still many unclear points in this problem. In particular,
in different studies, the order of vitamin elution differs
even when similar mobile phases are used. When the
optimal conditions are selected, significantly differing
mobile and stationary phases are used and no universal
approach to the sample treatment is developed. Little
attention is paid to the separation of vitameric forms
and the stability of the vitamins and their forms in the
sample treatment and separation processes. In addi

260
 DETERMINATION OF WATERSOLUBLE VITAMINS IN VITAMIN PREMIXES
tion, the detection conditions are significantly varied
[1, 2].
The aim of the present study is to create a method
for the determination of watersoluble vitamins using
the HPLC method in the gradient elution mode and to
optimize the condition for their separation.
EXPERIMENTAL
Chromatographic system. Liquid chromatograph
Agilent 1100 was used for the study (Agilent, U.S.)
consisting of a gradient pump, degassing agent of the
mobile phase autosampler, column thermostat, and
diode matrix detector. For the separation, the follow
ing chromatographic columns were used: Synergy 4u
hydroRP (250 × 4.6 mm) (Phenomenex, U.S.),
Gemini 5u C18 (250 × 4.6 mm) (Phenomenex, U.S.),
Zorbax SBC18 (250 × 4.6 mm) (Agilent, U.S.). For
the study, all the columns were equipped with precol
umns Security Guard C18 (Phenomenex, U.S.). The
samples were injected with an autosampler. The vol
ume of the injected sample was 20 µl. The Chemsta
tion software (Agilent, U.S.) was used for the registra
tion of the chromatograms.
Reagents and solvents. The following reagents were
used for the study: thiamine hydrochloride (analytical
grade), thiamine phosphate chloride (analytical
grade), nicotinamide (analytical grade), ascorbic acid
(analytical grade), pyridoxine (analytical grade), pyri
doxal (analytical grade), pyridoxamine (analytical
grade), pantothenic acid (analytical grade), folic acid
(98%), riboflavin5phosphate (analytical grade),
nicotinic acid (analytical grade), biotin (analytical
grade), phosphoric acid (85%) (SigmaAldrich,
U.S.), acetonitrile (HPLCgradient grade, Pancreac,
Spain), sodium hydrocarbonate (analytical grade,
Labtekh, Russia), sodium dihydrophosphate (analyti
cal grade, Labtekh, Russia), and sodium hydroxide
(analytical grade, Labtekh, Russia).
The preparation of vitamin solutions. For all the
vitamins except riboflavin, biotin and folic acid, the
standard solutions were prepared as follows: a
weighted portion of the vitamin (approximately 0.1 g)
was placed in a 100 ml volumetric flask, then 20 ml of
deionized water and 1 ml of phosphoric acid were
added, mixed, 59 ml of deionized water was added
and, with periodical vigorous shaking, the solution was
subjected to ultrasound until complete dissolution; the
water temperature in the ultrasound bath was con
trolled and did not exceed 25°C. The resultant solu
tion was brought to the mark of the deionized water
and thoroughly mixed. The addition of phosphoric
acid was necessary in order to improve the stability of
the vitamins. To prepare standard solutions of biotin
and folic acid, a weighted portion of the vitamin was
placed in a 100 ml flask; then, 10 ml of deionized water
and 2 ml of 200 mM sodium hydrocarbonate solution
were added; the solution was mixed and diluted with
60 ml of deionized water; with periodical vigorous
MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65
261
shaking, the solution was subjected to ultrasound until
complete dissolution; the temperature in the ultra
sound bath did not exceed 25°C. The resultant solu
tion was brought to the mark of the deionized water
and thoroughly mixed. Due to the low solubility of
riboflavin, a weighted portion of this vitamin (approx
imately 0.03 g) was placed into a 1000 ml flask, where
200 ml of deionized water and 15 ml of phosphoric
acid were added, mixed, and then diluted with 700 ml
of deionized water; it was then periodically shaken vig
orously and subjected to ultrasound until complete
dissolution, with the temperature in the ultrasound
bath not exceeding 25°C. The resultant solution was
brought to the mark of the deionized water and thor
oughly mixed. All the solutions were kept at 4°C in a
dark place. The solution of ascorbic acid was stable for
a day, while the solutions of the other vitamins were
stable for two weeks. The calibration curves of the vita
mins were prepared daily. The concentration of the
calibration solutions was chosen so that the concen
trations of different vitamins and their ratios were
close to the concentrations and the ratios of the vita
mins in the tested samples. The required amount of
the stock solution and 0.1 ml of phosphoric acid was
added to a 25 ml volumetric flask, the solution was
brought to the mark of the deionized water and thor
oughly mixed. The calibration solutions were kept at
4°C.
RESULTS AND DISCUSSION
To chose the optimal conditions for the determina
tion of vitamins, the influence of the chromatographic
parameters on their retention needs to be studied. The
factors which have the greatest influence on the reten
tion of the compounds are the nature of the stationary
phase, the pH of the mobile phase, the content of ace
tonitrile in the eluent, and the profile of the gradient
elution.
The influence of pH of the mobile phase. Since
many vitamins have an acidic or basic nature, the pH
of the eluent has a great influence on their retention.
In reversephase HPLC, the compound in nondisso
ciated form is retained stronger than in the dissociated
form. The values of the pKa and pKb of the vitamins
are significantly different from each other and, there
fore, the retention time and the selectivity can be sig
nificantly altered by changing the pH of the mobile
phase. For this experiment, the pH of the mobile
phase was varied in the range from 1.5 to 7.5. When
studying the influence of pH, the range of the stability
of the employed columns and the instability of some
vitamins in a basic medium was taken into account. In
the pH range from 2.5 to 7.5, eluent A was a 50 mM
NaH2PO4 buffer solution. The required pH value was
provided by the addition of NaOH or phosphoric acid.
Eluent B was acetonitrile.
For the study of the influence of the pH of the
mobile phase on the vitamin retention time, gradient
No. 4 2010
262 BENDRYSHEV et al.

Table 1. Gradient modes (the flow rate was 0.8 ml/min)

Gradient 1 2 3 4 5 6 7 8 9 10 11
mode
Time, min Acetontrile (eluent B) concentration, %

0 0 0 0 0 5 5 3 0 0 0 0
2 – – – 5 – – – – – – –
5 – – – – – – 3 – – – –
6 0 – – – – – – 0 – – –
7 – – 20 20 20 5 – – – – –
8 – – – – – 20 – – – – –
10 – 20 25 – – – 20 – – – –
15 18 – – – – – – – – – –
28 18 20 25 20 20 20 20 18 – – –
29 50 50 50 50 50 50 50 50 – – –
30 15 15 15 15 15 15 15 15 – – –
31 0 0 0 0 0 0 0 0 – – –
40 – – – – – – – – 30 20 10
41 0 0 0 0 0 0 0 0 0 0 0

profile no. 1, indicated in Table 1, was used (the flow
rate was 0.8 ml/min). At the first step of the gradient
elution, no acetonitrile was present in the mobile
phase, providing the best retention and separation of
poorly retained compounds.
The general principles of the influence of the pH of
the mobile phase on the vitamin retention times were
the same for all the considered stationary phases.
However, the stationary phase can significantly influ
ence the order of the elution of the vitamins and the
selectivity of the separation of some of the vitamin
pairs. A more detailed discussion of the influence of
the stationary phase will be given below. The depen
dence of the retention time of watersoluble vitamins
on the pH of the column for the column Luna C18(2)
is provided in Fig. 1. Let us consider the influence of
the pH on the retention time of the vitamins for the
column Luna C18(2) in more detail.
For nicotinic acid, the growth of the retention time
from 5.7 to ~10 min is observed along with an increase
of the pH from 1.5 to 7.5. This phenomenon can be
explained in the following way: this is an amphoteric
compound due to the presence of a carboxyl group and
the basic nitrogen atom in the pyridine ring in its
structure (pKa = 2.07, pKb = 4.9). As a result, at high
pH values, the carboxyl group is completely dissoci

Retention time, min

(a) (b)
16 1
2 24 1
3 2
12 4
3
5 20 4
8 5
6
6
16
4 7
0 12
1 2 3 4 5 6 7 8 0 2 4 6 8
pH

Fig. 1. Dependence of the retention time of the vitamins on the pH of the mobile phase: (a) ((1) nicotinamide, (2) thiamine,
(3) pyridoxine, (4) pyridoxal, (5) nicotinic acid, (6) pyridoxamine, (7) ascorbic acid); (b) ((1) biotin, (2) riboflavin, (3) ribofla
vine5phosphate, (4) cyanocobalamin, (5) folic acid, (6) pantothenic acid (column Luna C18(2), 250'4.6 mm).

MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65 No. 4 2010

 DETERMINATION OF WATERSOLUBLE VITAMINS IN VITAMIN PREMIXES
ated, with the pyridine nitrogen atom remaining
deprotonated. At low pH values the situation is the
opposite: the nicotinic acid is in the form of a proto
nated pyridine base. At medium pH values, the nico
tinic acid exists as a mixture of the indicated forms.
The alteration of the retention time of nicotinic acid
along with an increase of the pH from 1.5 to 3.5 is,
apparently, associated with an increase of the share of
the form with the dissociated carboxyl group. At pH >
3.5, no additional dissociation on the carboxyl group
occurs (as it is completely dissociated), while the
extent of nitrogen protonation decreases along with an
increase of the pH; i.e., the retention time in this pH
range depends on the share of the forms with proto
nated nitrogen in the pyridine ring.
The retention time of nicotinamide (pKa = 3.3)
increases from 4.5 to about 16 min along with an
increase of the pH from 1.5 to 3.5. For a further
increase of the pH, the retention time grows somewhat
more. At low pH values, nicotinamide is completely in
the protonated from and the endcaping groups of the
adsorbent are nondissociated; and at high pH values, a
dissociation of the groups of the stationary phase
occurs, while the fraction of the protonated nicotina
mide in the mixture is reduced. As the dependences
show, the deprotonation of the charged nitrogen atom
of the pyridine ring of the vitamins combined with the
ionization of the chemically attached hydroxyl groups
of the adsorbent leads to the improvement of the sorp
tion and the retention time grows. The retention time
of ascorbic acid decreases from 6.5 to 3.4 min along
with an increase of the pH. Ascorbic acid is a dihydric
one (pKa1 = 4.18, pKa2 = 11.6) and dissociates at rela
tively low pH values. The ability of vitamin C towards
such dissociation definitely influences its retention
time on the stationary phases. Since the dissociation of
vitamin C by the second stage is negligible, it may be
concluded that no ascorbic acid in the form of a dian
ion is present in the mixture and, consequently, it does
not influence the sorption of the vitamin on the whole.
The retention is apparently explained by the presence
of the noncharged and anionic form of vitamin C and
by the ionization of the adsorbent groups as well.
Pantothenic acid has pKa = 4.41 and, as a result, it
becomes anionic along with an increase in the pH of a
medium. Its retention time deceases from 1 to 13 min
as a result of an increase of the pH from 1.5 to 7.5,
respectively. The retention time of folic acid (pKa =
2.3; 8.3) is somewhat higher than that of pantothenic
acid, but the tendencies of the change of this parame
ter are generally the same. The retention time is 19 and
15 min at pH 1.5 and 7.5, respectively.
The retention time of biotin is constant in the pH
range from 1.5 to 4 (23 min); however, as a result of the
further increase of the pH of the mobile phase, its
sharp reduction to 16 min is observed; pKa of the car
boxyl group of biotin is about 4.5 and at high pH values
biotin is present entirely in anionic form, which is
retained on the column less strongly.
MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65
263
The retention time of riboflavin (pKa = 10.2)
hardly changes when the pH of the eluent is varied.
The riboflavin molecule does not contain groups that
are capable of dissociation in the considered pH range.
For riboflavin5phosphate, some decrease in the
retention time is observed (for less than a minute). For
cyanocobalamin, the retention time hardly depends
on the pH value.
The thiamine molecule includes a positively
charged thioanisole fragment and as a result is a highly
polar organic compound. At pH < 3.5, thiamine has a
low retention time (about 3.8 min). When the pH
increases (>3.5), the retention time of thiamine
sharply increase to 14.3 min and hardly changes along
with the further growth of the pH. Such behavior
seems to be associated with the fact that at pH > 3.5,
thiamine becomes a free base, i.e., the deprotonation
of the nitrogen atom in the pyrimidine ring occurs and
this fragment of the molecules becomes uncharged,
creating the possibility for the interaction between
C18groups of the adsorbent and the thiamine mole
cule.
Thiamine monophospate behaves similarly to thia
mine and has a close retention time in the pH range
from 1.5 to 5.5 (3.5 min at pH 1.7). We did not succeed
in separating these compounds completely in this pH
range. When the pH is increased to 5.5, the retention
time of thiamine monophosphate increases to 14 min.
Pyridoxine is a derivative of pyridine and its mole
cule contains a nitrogen atom of the pyridine ring,
which has strong basic properties. For pyridoxine, a
relatively small increase of the retention time was
observed with the increase of the pH from 12 to
15 min. Pyridoxamine is poorly retained in the sta
tionary phase at pH 1.5 (3.1 min). With the increase of
the pH to 7, the retention time of pyridoxamine
increases to 6 min. If the pH is further increased, the
sharp growth of the retention time to 13 min at pH 8 is
observed. When the pH increases from 1.5 to 5, the
retention time for pyridoxal grows from 7 to 14 min,
and then becomes almost constant.
The Influence of the Stationary Phase
According to the data in the literature data [1, 2],
reverse C18 phases are most frequently used for the
separation of watersoluble vitamins. However, C18
columns of different producers can have significantly
different properties due to the differences of the initial
silica and polymers, synthetic procedures, attach
ment, and endcaping density. Such differences can
significantly influence the retention of the com
pounds. Therefore, in order to find the optimal sepa
ration conditions, we have considered several types of
columns. The stationary phases used for the study
included Synergi Hydro RP C18, Gemini C18, Luna
C18(2), and Zorbax SBC18. Unfortunately, com
plete information about the columns is not always pro
vided by the producers, but nevertheless their main
No. 4 2010
264 BENDRYSHEV et al.

1
2 10
3 5 14
8 9 11
4 6 7 12
13

Luna C18 Synergi Hydro

4 5 7 8 14
3 10
13
2 6 9
12
1 11
0 1 2 3 4 5 6 7 8 9
k

Fig. 2. The selectivity rows for the stationary phases Luna C18(2) and Synergi Hydro RP. The pH of the mobile phase was 1.7.
Notation: (1) pyridoxamine, (2) thiamine phosphate, (3) thiamine, (4) nicotinamide, (5) ascorbic acid, (6) nicotinic acid,
(7) pyridoxal, (8) pyridoxine, (9) pantothenic acid, (10) folic acid, (11) cyanocobalamin, (12) riboflavine5phosphate,
(13) riboflavin, (14) biotin.

features are known; for example, the column Synergi
Hydro RP has a hydrophilic endcaping, which pro
vides better retention and separation of hydrophilic
compounds. For the synthesis of the column Gemini
C18, a hybrid silica gelpolymer matrix is used instead
of silica gel, which allows the column to operate both
in a strongly basic and strongly acid pH region, and
excludes the interaction between the compounds and
silanol groups. The column Luna C18(2) is a column
with increased attachment density of the functional
groups, which provides a stronger shielding of the
adsorbent grain and an aggregate increase of the col
umn capacity and, consequently, the increase of the
retention time and some improvement of the selectiv
ity. Due to a special endcaping, Zorbax SBC18 can
operate at low pH values. The presence of the endcap
ing can lead to the alteration of the selectivity and the
capacity.
The results of the experiments allow us to divide the
considered stationary phases into two groups. The first
group includes Luna C18(2) and Gemini C18, and the
second one includes Synergi Hydro RP and Zorbax
SBC18. The difference between these groups is
clearly observed at pH 1.5–1.8. The selectivity row for
the stationary phases Luna C18(2) and Synergy Hydro
RP are given in Fig. 2.
When the vitamins are separated using the station
ary phases Luna C18(2) and Gemini C18 at pH 1.5–
1.8, nicotinic acid is eluted before ascorbic acid. The
peaks of these compounds are clearly resolved with
each other and the nicotinamide peak. The peaks of
thiamine, nicotinamide, and other polar compounds
are symmetric; the resolution is sufficient for the
simultaneous qualitative determination of thiamine,
nicotinamide, and nicotinic and ascorbic acid. The
values of the retention times for the pairs riboflavin
riboflavin5phosphate and thiaminethiamine phos
phate almost coincide and do not allow the separation
of these compounds. In addition, the retention time of
cyanocobalamin is also very close to that of riboflavin
5phosphate. It is to be noted that the retention time
of the compounds on the Gemini C18 column is
somewhat lower than on the other columns. When the
separation is made using the columns Synergi Hydro
and Zorbax SB C18 at pH 1.5–1.8, ascorbic acid is
eluted before nicotinic acid, with the peaks not being
resolved to the baseline. The peaks of nicotinamide
and thiamine are somewhat unsymmetrical and there
is no pH value that allows achieving simultaneous
complete separation of the peaks of thiamine, nicoti
namide, and ascorbic and nicotinic acids.
The differences in the selectivity and the elution
order of the vitamins in different phases is, obviously,
first of all, associated with the presence of hydrophilic
endcaping in the stationary phases of the second
group, which provides additional interaction with the
polar compounds leading to somewhat better selectiv
ity in the pairs riboflavinriboflavin5phosphate and
thiamine–thiamine phosphate, as well as to an
increase in the retention time of nicotinic acid and
some asymmetry of the peaks of thiamine and nicoti
namide.
Unfortunately, complete separation of all the con
sidered vitamins and vitameric forms cannot be
achieved at any pH or by using any of the stationary
phases. However, in real medical preparations, pre
mixes, bioactive dietary supplements, and vitamin
enriched products, only one form of the vitamins are
present and, therefore, in the majority of cases, the
appropriate conditions for the separation of the vita

MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65 No. 4 2010

 DETERMINATION OF WATERSOLUBLE VITAMINS IN VITAMIN PREMIXES
mins present in the sample can be selected. Satisfac
tory separation of the majority of the vitamins can be
contained at different pH values. However, the best
separation is provided at pH 1.5–1.9. In addition, the
majority of vitamins are more stable in the acid
medium. If there is a necessity of the complete separa
tion of nicotinic and ascorbic acids, it is reasonable to
use the phases of the first group, and if it is the neces
sary to separate the pair riboflavin–riboflavin5phos
phate, the stationary phases of the second group are to
be used.
The Influence of the Gradient Elution Profile
One of the main conditions for the achievement of
the most efficient separation of the substances is the
selection of the optimal gradient mode. Using differ
ent gradient profiles, fine resolution of the chromato
graphic peaks of the substances with close retention
times can be obtained and the total time required for
the analysis can be decreased as well.
In order to vary the conditions of the gradient elu
tion, the initial concentration of acetonitrile, the
duration of the isocratic region at the beginning, the
rate of growth of the acetonitrile concentration, the
final concentration of acetonitrile, and the duration of
the elution were changed. At the end of the each chro
matographic determination, the concentration of ace
tonitrile was increased up to 50% for a short time in
order to elute the strongly adsorbed admixtures from
the column. After each gradient elution, the chro
matographic system was equilibrated under the initial
conditions for 6 min. The considered gradient profiles
are given in Table 1.
For the selection of the optimal conditions for the
gradient elution, a standard mixture of vitamins was
used as the test mixture. In all these experiments, the
components of the mobile phase were 0.6% phospho
ric acid with pH 1.76 and acetonitrile. In some of the
studies and procedures [1, 2] dedicated to the determi
nation of vitamins, the initial period of the gradient
elution is implemented at a nonzero concentration of
acetonitrile. Obviously, this is associated with the loss
of the properties of some columns in 100% aqueous
media due to the socalled collapse of the C18 groups.
For the selection of the optimal separation conditions,
the initial concentration of acetonitrile in the mobile
phase was varied. It was established that the best sepa
ration of the poorly adsorbed compounds can be
achieved at the zero concentration of acetonitrile. An
increase of the acetonitrile concentration leads to the
reduction of the retention time and the resolution
between the peaks of the neighboring compounds
deteriorates. The phenomenon of the collapse of the
C18 groups was not observed for the employed col
umns.
The best gradient mode for the separation of water
soluble vitamins appeared to be gradient no. 1. Its
application provided not only good resolution and
MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65
265
symmetric shapes of the peaks of the analytes but also
a rather uniform selectivity of the vitamin determina
tion in the range between and 25 min.
The Influence of Temperature
The choice and the provision of the column opti
mal temperature is very important for the successful
separation and determination of the analytes. The
sorption process on the employed stationary phase is
known to be exothermic and therefore the change of
the column temperature more or less influences the
equilibrium of this process. It results in the change of
the retention time of the analytes and the efficiency of
their chromatographic peaks.
To study the influence of temperature, we have
conducted a series of experiments at values of the col
umn thermostat temperature ranging from 20 to 60°C
inclusively, with a 10°C step. The upper limit was cho
sen after taking into consideration the stability of the
stationary phase. All the experiments were conducted
using gradient mode no. 1.
Just as expected, the reduction of the retention
time of the compounds was observed along with an
increase of the temperature. However, no significant
reduction of the retention time was achieved and the
maximal change with the transition from 20 to 60°C
was 1.5–2 min for all the vitamins. The reduction of
the retention time for different vitamins happens
almost simultaneously and as a result the variation of
the temperature of the chromatographic column does
not allow improving the separation. In addition, it
appeared that the increase in temperature leads to the
growth of the baseline fluctuation. For these reasons,
all further experiments were conducted at 20°C.
Therefore, the optimal conditions for the separa
tion of the vitamins are as follows: gradient profile no.
1, eluent A is 0.6% phosphoric acid (pH 1.70); eluent
B is acetonitrile; and the temperature of the column is
20°C. For the separation of nicotinic and ascorbic
acid, the columns Luna C18(2) or Gemini C18 should
be used, and if there is a necessity for the separation of
the riboflavin–riboflavin5phosphate and thiamine–
thiaminephosphate pairs, the stationary phases Syn
ergi Hydro C18 or Zorbax SBC18 should be used.
The chromatogram of the model vitamin mixture, reg
istered in the selected conditions, is presented in
Fig. 3.
The Selection of the Detection Conditions
The absorption spectra of each of the vitamins were
registered using the diode matrix detector. The values
of the maxima of vitamin absorption at pH 1.7 are pro
vided in Table 2. Qualitative determination of each of
the vitamins should be performed using the wave
lengths providing the best detection limits and suffi
cient resolution between the peaks of the determined
vitamin and the neighboring peaks. For a number of
No. 4 2010
266 BENDRYSHEV et al.

Absorbance, absorbance units × 10−3

5
250

200

150 4 11

100

3 6
50 2
10 12
1 8 9
7
0
5 10 15 20 25 30 35
Time, min

Fig. 3. Chromatogram of the standard solution of vitamins; the column was Luna C18 (2) 250 × 4.6 mm; and gradient no.1 (see
the text) was used. Spectrophotometric detection: 0–3.2 min 292 nm, 3.2–7 min 254 nm, 7–13 min 292 nm, 13–18 min 200 nm,
18–20 min 297 nm, 20–21 min 2360 nm, 21–22.5 min 2790 nm, 2324 min 200 nm. The vitamins were (1) pyridoxamine,
(2) thiamine, (3) nicotinamide, (4) nicotinic acid, (5) ascorbic acid, (6) pyridoxal, (7) pyridoxine, (8) pantothenic acid, (9) folic
acid, (10) cyanocobalamin, (11) riboflavin, (12) biotin.

real samples, the optimal wavelengths could not coin on the obtained chromatograms was made using the
cide with the absorption maxima. It is associated with preliminarily created spectrum library. The detection
the absorption of the components of the matrix of the limits, the linearity ranges, and the wavelengths
tested sample. When optimizing the conditions of the employed for the detection are provided in Table 2.
determination, the identification of the vitamin peaks
The Analysis of Real Samples
Table 2. The detection wavelengths and analytical parame Under the proposed conditions, we analyzed the
ters for determining the vitamin
vitamin preparations Undevit, bioactive dietary sup
Wave Detection Linear plement Alfavit, and vitamin premix GSvit 12. For
Vitamin length, limit, range, the extraction of the vitamins from the tested samples,
nm mg/l mg/l 1% solution of phosphoric acid was used (pH 1.5). The
extraction of the vitamins from the tested samples was
Thiamine 244 0.01 0.01–100 performed using an ultrasound bath. A precisely
Thiamine phosphate 244 0.02 0.02–100 weighted portion of the tested samples was placed in a
Nicotinamide 244 0.03 0.03–300 250 ml glass flask, washed with the extraction agent,
Nicotinic acid 244 0.2 0.2–200 and placed in an ultrasound bath. The weight of the
portion was calculated so that the resultant concentra
Ascorbic acid 254 0.03 0.03–200 tions of all the determined vitamins were within the
Pyridoxine 292 0.02 0.02–100 analytical range. In order to analyze the preparations
Pyridoxal 292 0.1 0.1–100 in the form of tablets, 10–20 tablets should be taken,
Pyridoxamine 292 0.3 0.3–100 weighed, and then carefully ground in a porcelain
mortar. For further calculations, the content of the
Pantothenic acid 200 0.2 0.2–200 vitamins is referred to as the mass equivalent to one
Folic acid 297 0.006 0.006–100 tablet. The concentrations of the vitamins measured
Cyanocobalamin 360 0.2 0.2–100 and the stated by the producer in the tested prepara
Riboflavin phosphate 270 0.2 0.2–50 tions are given in Table 3. It confirms that the obtained
results are in accordance with the producer data.
Riboflavin 270 0.005 0.005–10 Unfortunately, we did not manage to determine the
Biotin 200 0.2 0.2–100 concentration of cyanocobalamine and biotin in the

MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65 No. 4 2010

 DETERMINATION OF WATERSOLUBLE VITAMINS IN VITAMIN PREMIXES 267

Absorbance, absorbance units × 10−3

3
700

10 6

500 2

18 7
300
5
1 4
100
6

0 5 10 15 20 Time, min

Fig. 4. Chromatogram of the extract of the Undevit pill. The column was Synergi Hydro 250 × 4.6 mm; gradient no. 1 was used
(see the text). Spectrophotometric detection – see Fig. 3. Vitamins: (1) thiamine, (2) nicotinamide, (3) ascorbic acid, (4) pyri
doxine, (5) pantothenic acid, (6) folic acid, (7) riboflavin.

preparations, as the content of these vitamins
appeared to be below the detection limit of the detec
tor used. The chromatogram of the extract of Undevit
pills is given in Fig. 4.
CONCLUSIONS
The influence of pH, temperature, and the station
ary phase on the retention of vitamins was studied. It
was established that the differences in the retention of
vitamins on different stationary phases are apparently
associated with the presence of different endcaping
groups and different attachment density of the
C18groups. The best separation of the vitamins and
the vitameric forms was achieved at the mobile phase
pH 1.5–1.8. The influence of the temperature on the
retention time of the vitamins appeared to be negligi
ble. The chromatographic conditions were proposed
so that they would allow the separation of 12 water
soluble vitamins and vitameric forms: eluent A was
0.6% phosphoric acid, pH 1.7; eluent B was acetoni
trile; the gradient profile was the following: 0–6 min
was 0% B, 6–15 min was from 0 to 18% B, 15–28 min
was 18%B, 28–29 min was from 18 to 50% B, 29–
30 min was from 50 to 15%B, 30–31 min was from 15
to 0% B; the eluent flow rate was 0.8 ml/min; and the
column temperature was 20°C. For the separation of
nicotinic and ascorbic acid, the columns Luna C18(2)
or Gemini C18 should be used, and if there is a neces
sity for separating the riboflavin–riboflavine5phos
phate and thiamine–thiaminephosphate pairs, the
stationary phases Synergi Hydro C18 or Zorbax SB

Table 3. The determination of the concentration of vitamins in some real samples (n = 3, p = 0.95)
Tested samples
Undevit Alfavit Premix GSvit 12
Vitamin
Producer’s Found, Producer’s Found, Producer’s Found,
data, mg/pill mg/pill data, mg/pill mg/tablet data, mg/pill mg/ 100 g
Thiamine 2 1.9 ± 0.1 1.5 1.4 ± 0.1 0.46–0.54 0.49 ± 0.02
Nicotinamide 20 19 ± 1 20 20 ± 1 3.7–4.3 4.0 ± 0.2
Ascorbic acid 75 73 ± 3 80 78 ± 3 56–64 62 ± 2
Pyridoxine 3 3.1 ± 0.1 1 1.0 ± 0.05 0.54–0.66 0.61 ± 0.03
Pantothenic acid 3 3.0 ± 0.1 5 5.0 ± 0.2 2.8–3.2 3.2 ± 0.2
Folic acid 0.07 0.071 ± 0.003 0.2 0.20 ± 0.01 0.045–0.055 0.045 ± 0.003
Riboflavin 2 2.0 ± 0.1 1.7 1.6 ± 0.1 0.74–0.86 0.81 ± 0.02

MOSCOW UNIVERSITY CHEMISTRY BULLETIN Vol. 65 No. 4 2010

268 BENDRYSHEV et al.
C18 are to be employed. The selected conditions were
used for the determination of vitamins in commercial
samples of vitamin preparations.
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