Determination of Water-Soluble Vitamins

in Pharmaceutical Preparations by Reversed-Phase

High-Performance Liquid Chromatography
with a Mobile Phase Containing Sodium
Dodecylsulphate and n-Propanol

I. Almagro / M. P.San Andres / S.Vera*

Departamento de Quimica Analitica, Facultad de Quimica, Universidad de Alcald, 28871 Alcald de Henares (Madrid), Spain;
E-Maih soledad.vera@uah.es

The use of mass spectrometry is becoming

KeyWords more widespread and can be combined
with several other separation techniques,
Column liquid chromatography for example gas chromatography or capil-
Water-solu ble vitamins lary electrophoresis. The subject has been
Pharmaceutical preparations treated thoroughly in the literature [1 4].
Analytical methods have been de-
scribed for some water-soluble vita-
Summary mins-thiamin (B1), pyridoxine (B6), ribo-
flavin (B2), nicotinic acid, nicotinamide,
A reversed-phase liquid chromatographic method with sodium dodecylsulphate-n-propanol- and folic acid (B9)-and the literature pro-
water as mobile phase has been used to separate and determine sixwater-soluble vitamins in vides guidance in conducting state-of-the-
l",,velve minutes. The analytical characteristics linear range, sensitivity, detection limits, and art separation analysis [5 12], primarily
precision were evaluated. The lowest detection limits were those of nicotinic acid (not usually by reversed-phase (RP) HPLC or re-
present in pharmaceutical products), 0.7 mg L 1, nicotinamicle, 1.3 mg L 1, and pyricloxine, versed-phase ion-pair (RP IP) HPLC [8
1.4 mg L 1.When the method was applied to the determination of the vitamins in pharmaceu- 12].
tical samples the values found agreed with those on the labels. Since Armstrong first effectively de-
monstrated the usefulness of replacing the
traditional organic modifiers used in re-
versed-phase liquid chromatography with
an aqueous micellar solution, so-called
Introduction (e) no single analytical approach can be micellar liquid chromatography (MLC)
used for simultaneous quantification of has been developed, and has been de-
The vitamins are essential organic sub- all the vitamins in a biological matrix. scribed in many publications [13 18]. Of-
stances in the metabolic processes that oc- ten the presence of an organic modifier,
cur in the nutrition of living bodies. Their The vitamins, depending on their solubi- e.g. a short- or medium-chain alcohol,
analysis is a major analytical challenge, lity, are classified as fat-soluble (vitamins was necessary to reduce retention times
because: A, D, E, and K) or water-soluble (B-com- and to improve the poor efficiency found
plex, C, folic acid, pantothenic acid, nico- when the mobile phase contains surfac-
(a) there is a need to quantify them in a tinic acid, and biotin). Historically, the B- tant only [18].
wide variety of biological matrices, which complex was measured by microbiologi- When the surfactant is dissolved in a
include both foods and body fluids; cal assay; nowadays several more power- polar solvent, e. g. water, as the main com-
(b) the concentrations present are usually ful techniques are available, including ponent, normal micelles are present.
very low, except in matrices which have high-performance liquid chromatography These structures also occur when the or-
been enriched; (HPLC) with detectors operating over a ganic modifier is present in small quanti-
(c) they can occur in several chemically di- continuous range of selective modes; nor- ties (< 15% v/v). Another type of aggre-
verse, but biologically non-convertible mal or synchronous fluorescence, radio- gate is also formed in systems in which the
forms; immunoassay (RIA), and enzyme-linked organic modifier, e.g. an alcohol, is the
(d) some are sensitive to heat, extremes of immunosorbent assay (ELISA), which dominant component. When the alcohol
pH, or degrading enzymes; and use specific protein-binding selectivity. chain is equal to or larger than six carbon

Original Chromatographia 2002, 55, February (No. 3/4) 185

0009-5893/00/02 185- 04 $ 03.00/0 9 2002 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
 4 Table I. Precision within-day and day-to-day.

I0.04 AU 10.04 AU Vitamin Within-day a Day-to-day b

a) b) tR (min) RSD% Area RSD% tR (min) RSD% Area RSD%
Thiamin 10.3 0.3 4058095 1.3 10.3 0.1 3782703 4.8
Riboflavin 2.47 0.3 4565045 1.5 2.44 1.0 4817611 5.2
Nicotinamide 7.43 0.05 1282709 2.4 7.41 0.3 1253476 3.7
Nicotinic acid 4.61 0.4 1463206 2.6 4.67 0.6 1462138 1.4
Pyridoxine 8.06 0.05 2578157 1.7 8.05 0.2 2584188 1.1
Folic Acid 7.07 0.04 1846982 2.6 7.05 0.3 1812195 3.7
n=5;bn=5.

Table II. Analytical figures of merit for determination of the vitamins.

Vitamin 2exp Linear range r Slope RSD% LOD Q.L.

(ram) (mgL 1) (areamg 1L) forslope a (mgL 1) (mgL 1)
Thiamine 245 6.72 100 0.9996 3.05 • 104 2.3 3.8 12.6
Riboflavin 270 0.77 155 0.9998 7.58 • 104 7.2 2.8 9.1
Nicotinamide 264 1.46 75 0.9998 3.17 • 104 3.6 1.3 4.3
Nicotinic acid 264 0.45 21 0.9995 2.71 • 104 5.3 0.7 2.2
Pyridoxine 290 1.02 60 0.9997 4.21 X 104 5.2 1.4 4.6
5 10 Time (min) 5 10 Time (min) Folicacid 264 1.84 90 0.9998 2.11 • 104 3.9 2.0 6.5
Figure 1. High-performance liquid chromato- //=7.
grams. (a) Standard mixture of vitamins. (b)
Sample 2.1 = riboflavin, 2 = nicotinic acid, 3 =
folic acid, 4 = nicotinamide, 5 = pyridoxine,
6 = thiamine.

atoms, the aggregates formed are reverse Experimental at r o o m temperature, on a LiChrosorb

micelles; if short-chain alcohols are pre-
sent it is not possible to distinguish these
reverse aggregates perfectly [19].
In R P H P L C the isotropic phase ob-
tained by use of large percentages of
short-chain alcohols does not cause pro-
blems and reduces the retention times of
analytes. In previous work we have used
mobile phases containing a surfactant, so-
dium dodecylsulphate (SDS) or hexade-
cyltrimethylammonium bromide (CTAB),
and high percentages of a short-chain al-
cohol, n-propanol or ethanol, for isocratic
reversed-phase separation and determina-
tion of polycyclic aromatic hydrocarbons
[20], antioxidants [21, 22] and inorganic
complexes [23 26], with very good re-
sults.
This paper describes the simultaneous
chromatographic determination of six
water-soluble vitamins, thiamine (B1),
pyridoxine (B6), riboflavin (B2), folic acid
(B9), nicotinic acid, and nicotinamide, by
reversed-phase H P L C with SDS-n-propa-
nol-water as mobile phase. Pharmaceuti-
cal preparations which contain vitamins
are interesting samples for analysis, be-
cause of their complex composition; for
this reason the proposed chromato-
graphic method was applied to such sam-
ples.
Reagents
All reagents were analytical-reagent
grade. Riboflavin (B2), nicotinic acid, ni-
cotinamide, thiamine (B1), pyridoxine
(B6), and folic acid (B9) were from Fluka
(Madrid, Spain). The surfactant sodium
dodecyl sulphate (SDS), and phosphoric
acid, were from Merck (Darmstadt, Ger-
many) and n-propanol was from Sharlab
(Barcelona, Spain). The vitamins were dis-
solved in the mobile phase, by diluting an
appropriate quantity of their stock solu-
tions with water or phosphoric acid.
These solutions were sonicated and stored
in the dark. The ultra pure water used was
obtained from a Millipore Milli-Q system
(Milford, USA). Before use all mobile
phases were filtered (0.451xm) and de-
gassed in an ultrasonic bath.
Equipment
Liquid chromatography was performed
with a Perkin-Elmer model 250 binary
pump, an Applied Biosystems model
785A programmable absorbance detec-
tor, and a Rheodyne injection valve, injec-
tion volume 20 IxL, all connected to a 486
personal computer equipped with Perkin-
Elmer T u r b o c h r o m 4.0 software for data
acquisition. Compounds were separated,
RP-18 column ( 1 5 0 m m • 3 . 9 m m i.d.,
particle size 10 ixm) from Sugelabor (Ma-
drid, Spain). The column effluent was
monitored at the wavelengths of maxi-
m u m absorbance of each vitamin.
Analytical Characteristics
of the Chromatographic Method
The method of calibration, relating peak
area to the quantity of analyte injected,
was used to determine the analytical char-
acteristics linear range, sensitivity, detec-
tion, and quantification limits. The range
of concentrations injected, for each vita-
min, in mg L 1, was 0.76 168 for ribofla-
vin, 0.45 45 for nicotinic acid, 0.73 66.5
for nicotinamide, 0.61 90 for pyridoxine,
0.67 84 for thiamin, and 0.92 100 for fo-
lic acid. The sensitivity was defined as the
slope of the calibration graph. The detec-
tion limit, LOD, was determined from the
calibration curve, as the amount of ana-
lyte that yields a signal equal to intercept
plus three times the statistic Sy/x, which es-
timates the random errors in the y-direc-
tion [27]. In the same way, the quantifica-
tion limit, QL, was calculated as the
amount of analyte that yields a signal
equal to intercept plus ten times the statis-
tic Sy/x.

186 Chromatographia 2002, 55, February (No. 3/4) Original

 Determination of Vitamins
in Pharmaceutical Preparations

Pharmaceutical preparations were pur-

~-~ ~ ~ chased from a pharmacy. Tablets were
io ~/) ~/) . io . io weighed, crushed, and dissolved in water
O containing a small quantity of phosphoric
.OO~m O . .O'
c~
, -~-~ ~: o44 , ~d , -~ acid. After filtration (0.45 ixm) an aliquot
,-~ 9 ~ - - ' 1 0 ~ ~,-~x~
was dissolved in the mobile phase and in-
~ ~ ~'~
~ . ~ ;~ ~ j ected into the chromatographic system.
~-~
_~-=
N.~ o o .~ O O N.~ O O N.~ O r

Results a n d Discussion

To optimize the composition of the mo-

bile phase, standards of the vitamins were
chromatographed separately in isocratic
mode, using different quantities of surfac-
tant and n-propanol, to determine the re-
I ---0 ---0 '-N I
tention time of each vitamin. A standard
mixture of vitamins was then analysed
and the chromatographic conditions were
adjusted to obtain the optimum peak
~ - ~
shape, efficiency, analysis time, selectivity,
~'~- ~ ~
O ~ -~ and resolution. On the basis of this study
-~ ~,.~ ~;--~ .~_o .~_o .~_o
chromatography was performed with a
mobile phase gradient prepared by mixing
0.03 M SDS-10% v/v PrOH-0.01 M H3PO4
(solution A) and 0.03 M SDS-60% v/v
PrOH-0.01 M H3PO4 (solution B). Elution
was started with 100% A and the composi-
tion was changed 1% A over a period of 5
min; the mobile phase flow rate was 1 mL
o min 1.
~e Figure la shows the separation of a
standard mixture of the six water-soluble
. ~ a2~ vitamins. The separation was achieved 12
min, quicker than separations achieved by
O
~ - RP HPLC or RP IP HPLC [8 12]. Most
of the vitamins in the mixture were sepa-
rated to baseline and eluted as sharp
ga peaks. For thiamin (peak 6) the peak
Y O .~
shape and the nature of the baseline neces-
sitated careful integration, checking the
precision of the retention time, and the va-
.~.~
ga, lue of the peak area.
%-~ The precision (within-day and day-to-
day), expressed as RSD%, obtained by
II
O
measurement of the retention time and
Z peak area for each vitamin, are listed in
O
Table I. The within-day precision was al-
O
ways < 3%; day-to-day values were accep-
table, because they did not exceed 5% ex-
cept for riboflavin, i. e. despite the appar-
O
ent problems with thiamin, RSD values
within-day and day-to-day were accepta-
io ble-there were no problems with reprodu-
O
cibility.
Table II shows the analytical figures of
merit for determination of the vitamins; it
also includes the experimental wavelength

Original Chromatographia 2002, 55, February (No. 3/4) 187

and the regression coefficients of the cali-
bration plots. There was good linear cor-
relation between peak area and the con-
centrations of the vitamins; the values of
the regression coefficients were always
> 0.999.
To determine whether small changes
affected the analytical procedure, besides
evaluating the precision to give the values
in Table I, a test of ruggedness was per-
formed to determine the RSD% of the
slopes of the calibration plots over a peri-
od of several days. It is apparent that the
values of RSD% were < 5% except for vi-
tamin B2, riboflavin, for which the value
was 7.2%.
The detection limits were small; the
lowest values were obtained for nicotinic
acid (0.7mgL 1), nicotinamide (1.3mg
L 1), and pyridoxine (1.4mg L 1).
A chromatogram obtained from one of
the pharmaceutical samples (sample 2) is
presented in Figure lb. Table III shows
the results obtained from five samples and
lists the amounts declared on the labels
and those found by the chromatographic
method; agreement was very good. It
should also be emphasized that Table III
shows that good results were obtained
from analysis of samples of diverse com-
position containing many compounds.
Conclusions
The proposed method, based on chroma-
tographic separation by use of mobile
phases containing SDS and high percen-
tages ofn-propanol, was used with success
for the determination of six water-soluble
vitamins in pharmaceutical preparations.
The advantages of the method are:
(a) The proposed procedure enables sim-
ple and quick analysis in one injection
18 8
with a total analysis time of 12 min, less
than that required by reversed-phase or
reversed-phase ion-pair HPLC.
(b) The analytical figures of merit are very
good; the high sensitivity and low detec-
tion limits should make the method widely
applicable. The low values of RSD% ob-
tained for the slopes of different calibra-
tion graphs show the ruggedness is good.
(c) The method is applicable to a wide
range of concentrations in pharmaceutical
preparations of different composition.
Acknowledgements
Financial support from the University of
Alcal~i (No. E025/2000) is gratefully ac-
knowledged.
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Received: Apr 4, 2001
Revised manuscripts
received: Jul 13 and Sep 17, 2001
Accepted: Sep 25, 2001
Original