## Mobile phase optimization method for steroids separation

**Applied Medical Informatics **
**Original Research **
**Vol. 18, No. 1, 2/2006, pp: 17-24 **
** **

Mobile Phase Optimization Method for Steroids Separation

**Carmen Elena Stoenoiu1, Sorana Daniela Bolboacă2, Lorentz Jäntschi1 **
1 Technical University of Cluj-Napoca, Cluj-Napoca, Romania

2 „Iuliu Haţieganu“University of Medicine and Pharmacy, Cluj-Napoca, Romania

**Abstract** *- The paper presented a mathematical model developed for optimization of mobile phase *

composition in thin layer chromatography applied on steroids separation. The proper solvents system

was experimental identify as being a mixture of chloroform, cyclohexan, and methyl-ethyl-cetone. An

original mathematical model was developed and used in order to identify the composition of mobile

phase. Starting with the mathematical model and with the optimization procedure a computer

program has been developed. The proposed model is able to optimize simultaneously many mobile

phases with respect of the shortest analysis time and of the selectivity of compounds. The efficiency of

the mathematical and optimization models is demonstrated on a sample of five androstane isomers.

**Key words: **Androstane Isomers, Thin-Layer Chromatography (TLC), Mobile Phase Optimization

**Introduction**
functions [4,5]; mineralocorticoids – maintain
The steroids hormones are natural or synthetic
blood volume and control renal excretion of
compounds with physiological activities derived
electrolytes [6]); sex steroids (androgens,
from cholesterol. With a single exception,
estrogens, and progestagens – produce sex
differences and support reproduction [7]);
cyclopentanophenanthrene skeleton and atomic
phytosterols (steroids that naturally occur in
numbering system as cholesterol (see Figure 1
plants [8]); and ergosterols (steroids that occur
[1], note that the carbon 18 and above can be
supplements [9]). Androstane is a steroid
hydrocarbon from which androgens are derived
and has the generic structure as is shown in

**Figure 1. Steroid nucleus **
**Figure 2. Androstane generic structure **
The term “steroids” has been introduced by
Callow R.K. & Young F. G. in 1936 [2] for the
techniques used for the separation of a mixture
group of compounds comprising the sterols, bile
of compounds by their distribution between two
phases, was introduced in 1901 by Mikhail
steroids include: anabolic steroids (class of
steroids that increase muscle and bone synthesis
technique used since 1963 [11] for separation
[3]), corticosteroids (glucocorticosteroids –
chemical compounds including of steroids. The
regulate aspects of metabolism and immune
method is still used in separation of compound
because of its advantages comparing with
steps under different conditions. The most
column chromatography [12]: (1) single use of
delicate problem in steroids separation is to
the layer simplifies sample preparation chouse the optimum mixture of solvents in order procedures; (2) simplicity of development by
dipping the plate into a mobile phase in a
chamber; (3) high sample through-put with low
previous investigated for identification of
operating cost because multiple samples can be
run simultaneously with standards on a single
experimental results were obtained by the
plate using a very low volume of solvent; (4)
solvent system with the following compounds:
selective and sensitive post-chromatographic
Chloroform – Cyclohexan - Methyl-Ethyl-
detection and identification; (5) visual Cetone. The obtained experimental data are observation and direct recording of the entire
presented in Table 1. The best results expressed
chromatogram including all components, the
as mobile phase composition and objective
origin, and the mobile phase front; and (6) the
function obtained by using Simplex, and Prisma
ability to repeat detection and quantification

**Table 1. Experimental data obtained for separation of androstane isomers **
5.51 6.10 6.09 2.45 3.32 0.50 0.31 0.19 0.26 0.64 7.76
7.43 7.92 7.91 6.14 6.48 0.51 0.21 0.19 0.31 0.32 8.84
2.05 2.97 2.76 0.15 0.28 0.27 0.21 0.22 0.23 0.15 9.64
3.45 5.03 4.59 0.58 1.33 0.54 0.31 0.29 0.21 0.35 8.91
0.53 0.96 0.75 0.15 0.20 0.41 0.31 0.22 0.25 0.25 9.04
6.23 0.85 6.89 4.00 4.82 0.56 0.31 0.24 0.29 0.51 8.85
6.44 6.83 6.80 5.64 4.10 0.52 0.21 0.11 0.21 0.82 8.55
0.92 1.89 2.47 0.24 0.26 0.43 0.32 0.21 0.22 0.22 8.77
0.00 0.00 0.00 0.00 0.00 0.46 0.32 0.15 0.24 0.21 8.41
8.26 8.42 8.34 7.29 7.00 0.31 0.16 0.15 0.21 0.47 8.93
I = amount of information; w = width of the compound spot; i = 1…4 (1 = 5α-androstane-3β-ol, 2 = 5α-
androstane-3α-ol, 3 = 5α-androstane-17β-ol, 4 = 5β-androstane-3α,17β-diol, 5 = 5β-androstane-3β,17β-diol)

**Table 2. Mobile phase composition and corresponding values for objective function according **
**with applied method **
No. Method Chloroform : Cyclohexan : Methyl-Ethyl- Fob
Starting with the experience obtained in

*Mathematical Model and Optimization *
optimization of the mobile phase in High-
Performance Thin-Layer Chromatography of a
The quantitative measure of a chromatographic
sample of benzodiazepines [14,15], the aim of
parameter put in a mixture of three solvents
the present research was to develop and to
depends on the composition of mobile phase.
The dependence equation could be one with six
or with seven parameters (Eq.(1) and Eq.(2)):

**Material and Method **
*1,x2,x3) = a1x1 + a2x2 + a3x3 + a4x1x2 *
A sample of five androstane was the material of
the present study. The following isomers were

*M7(x1,x2,x3) = a1x1 + a2x2 + a3x3 + a4x1x2 *(2)
included into analysis: 5α-androstane-3β-ol, 5α-
androstane-3α-ol, 5α-androstane-17β-ol, 5β-
androstane-3α,17β-diol, 5β-androstane-3β,17β-

*1, x2, x3* are molar fraction of the three

*1 + x2 + x3 = 1)*,

*M6* and

*M7* are
estimators and then predictors of choused
chromatographic parameter, and

*a1, a2, a3, a4, *
chromatographic parameter and then used in

*a5, a6, a7* are coefficients first determined based

**Figure 3. Androstane isomers include into analysis **
The following chromatographic parameters
of the separation compounds for the eluent

*e*.
were modeled starting from Eq.(1) and Eq.(2):
By application of one of the above describe equations (Eq.(3)-Eq.(5)) on a series of

*p*
*jFj(Sm(e),Inf(e,m),RSA(e),RRP(e)) *
ore more than one rows (one row for each
where

*Fob* is an objective function which
characterized the separation with the eluent

*e* in
report with selection of coefficients

*aj*,

* 1 ≤ j ≤ 4*,

* *
parameter that is modeled by using Eq.(1) or

*Fj *are functions that contain an expression of
Eq.(2). The optimization model has a unique
four parameters,

*aj *are coefficients choused
solution for

*p ≥ 6* (Eq.(1)), and for

*p ≥ 7*
arbitrary or through of a defined ponderate
mathematical relation of the

*Fj* functions and
A system can be build for each row from Mob
respectively of the number of equidistant
matrix with

*p* linear equations (where p = 6
intervals

*m*,

*RSA *is the mean of separation
resolution by using the eluent

*e*, and

*RRP* is the

*ob(j) = a1x1 + a2x2 + a3x3 + a4x1x2 + . *
resolutions used in separation with the eluent

*e*.
where

*xi* are the molar fractions of each solvent (

*i = 1, 2, 3*) that enter into the composition of

*RS(i,j,e) = 2·(l(i)-l(j))/(w(i)+w(j)) *
the

*ej* eluent (

*j = 1, 2, …, p*).
where

*i*,

*j* are two separation compounds,

*w(i)*
To the above describe system (Eq.(6)) the least
and

*w(j)* are the width of the compound’s spots,
squared method was applied for construction of
and

*RS* is the matrix of calculated resolution for
the system with unique solution (

*MMCP*); the
separation of the compound

*i* by the compound

* *
solution is obtained by applying the following
where

*i* is one of the separation compounds,

*e* is the eluent used as mobile phase,

*l(i)* is the
where

*(k,0)* *= 1, 2, …, 6* for Eq.(1) and

*(k,0) = *
coordinate at which the

*e* eluent had migrated,

*1, 2, …, 7 *for Eq.(2),

*A(k)* are the series of terms

*l(e)* is the coordinate at which the eluent had
known from Eq.(6),

*M2* is the mean calculated
migrated, and

*RF* is the series of retention factor
for the product of Mob series and

*A(k)*, and

*MMCP* is the extended matrix of system of the
optimum point was identified, this being the
linear equations which is used in determination
optimum composition of mobile phase (

*x1*,

*x2*,
The Gaussian method was applied to found the
solution for Eq.(7). The found solutions for the

*A0 = (a01, a02, …, a06)* - for Eq.(1)

*A0 = (a01, a02, …, a07)* - for Eq.(2)

* *
The

*A0* coefficients are used in prediction of the
chromatographic parameter of interest by using
integrated into a program dedicated to the
one of the equations Eq.(1) or Eq.(2). For
optimization of the mobile phase composition.
example if

*Y* is the choused chromatographic
The program is freely available from the
parameter, the Mob matrix (the predictor of

*Y*),
as well as the estimator of

*Y*, had more than one
http://vl.academicdirect.org/molecular_dynamic
row. If

*z* is the number of Mob matrix rows (and
implicit the number of predictors) then the
The generic equations that proved to obtained
estimator of choused chromatographic performances in optimization of the mobile parameter

*Ŷ* is:
phase, according with the chromatographic
parameter of interest and composition of
The optimum can be obtained by application of
a maximization or minimization function (as is
for example the characterization of a separation
The values of the retention factor obtained
experimentally and by mathematical model,

*o = opt(Ŷ), where opt = "max" or opt = *
experimental and optimized retention factor,
associated 95% confidence interval, the squared
Moving through all domains of possible values
correlation coefficient are presented in Table 5.
for the composition of the mobile phase, the

**Table 3. Results of optimization procedure **
∆rf = a1x1+a2x2+a3x3+a4x1x2+a5x1x3+a6x2x3+a7x1x2x3
a1x1+a2x2+a3x3+a4x1x2+a5x1x3+a6x2x3+a7x1x2x3
Objective function (Fob) Fob = a1x1+a2x2+a3x3+a4x1x2+a5x1x3+a6x2x3+a7x1x2x3

**Table 4. Experimental and optimized retention factor for androstane isomers **
Exp. = retention factor obtained from experimental data;
MathM. = retention factor obtained by mathematical model

**Table 5. Results of correlation between **
**experimental and optimized values for **
Cyclohexan – Methyl-Ethyl-Cetone = 60 : 0 :

**retention factor **
36) are presented in Table 6. The correlation
coefficient obtained between experimental and
5α-androstane 3β-ol 0.9934 [0.9714-0.9985] 0.9869
optimized values, associated 95% confidence
5α-androstane 3α-ol 0.9648 [0.8536-0.9918] 0.9307
intervals and squared correlation coefficients are
95% CIr = 95% confidence intervals for correlation
The graphical representation of the retention factor estimated through optimization versus
retention factor experimentally obtained for 5β-

**Figure 5. The plot of the retention factor **
androstane-3β,17β-diol compound is presented

**obtained through optimization procedure **
The squared correlation coefficient between
objective function calculated based the experimental data and objective function
calculated by using the mathematical model for
the optimum mobile phase (Chloroform – Cyclohexan – Methyl-Ethyl-Cetone = 60 : 0 :
36) was equal with 0.8921 (95%CIr = [0.7769 –
0.9871]). Applying the mathematical model on experimental data presented in Table 1, the
diagrams of the objective function obtained for presented in Figure 6 was obtained (the
composition of the used optimum mobile phase
was Chloroform – Cyclohexan – Methyl-Ethyl-Cetone = 60 : 0 : 36).

**Figure 4. Optimized versus experimental **
**retention factor for 5**β

**-androstane-3**β

**,17**β

**-**
experimental data the graphical representation of the retention factor at optimum mobile phase (Chloroform – Cyclohexan – Methyl-Ethyl-Cetone = 60 : 13 : 27) the graphical
representation presented in Figure 5 was obtained. The spots of dark color indicate the optimum mobile phase obtained by the
mathematical model (Chloroform – Cyclohexan

**Figure 6. The diagram of the objective **
**function (Cyclohexan & Methyl-Ethyl-**
The values of the resolution for separation
obtained from the experimental data and respectively by the mathematical model for the
phase is increased. In the region where the spots
diagram as darkest spots (Cyclohexan – Methyl-
are darkest, the composition of the optimum
Ethyl-Cetone = 0 : 36), the color becoming
mobile phase can be established according with
lighter as the distance from the optimum mobile
the value of the objective function (Fob).

**Table 6. Experimental and optimized separation resolution for androstane isomers **
1.3611 3.8333 3.7176 1.1944 12.3678 1.0685 2.5867 0.0000 0.6809
5α-androstane-3β-ol & MathM. 1.5995 2.8743 1.0491 4.3299 1.7656 11.7357 1.3874 2.2069 1.5223 0.0000
-1.5132 2.7842 -0.6123 -0.5712 0.6321 -0.3189 0.3798 -1.5223 0.6809
1.3714 2.8980 2.7470 0.6984 1.6500 1.1429 4.8438 0.0000 0.3478
5α-androstane-3β-ol & MathM. 2.0585 1.0231 1.1217 3.1298 0.9909 1.5426 1.6400 4.5318 0.8971 0.4453
0.3483 1.7763 -0.3828 -0.2925 0.1074 -0.4971
3.1463 7.6000 7.6533 1.1515 5.2471 2.1918 2.0923 0.0000 3.7308
MathM. 9.1778 4.6253 3.5548 5.0261 2.7285 5.2772 2.6190 3.2725 1.9742 2.6115
-1.1252 -1.479 4.0452 2.6272 -1.577 -0.0301 -0.4272 -1.1802 -1.9742 1.1193
2.2892 8.4286 4.7640 1.0000 2.6355 3.4925 2.0308 0.0000 3.2308
MathM. 5.0588 3.3510 3.4511 3.0638 2.5547 2.4993 4.2739 2.6682 2.4726 2.3214
-1.2167 -1.0618 4.9775 1.7002 -1.5547 0.1362 -0.7814 -0.6374 -2.4726 0.9094
0.0500 0.9767 1.4667 0.7925 21.9636 0.1875 2.1887 0.0000 0.5161
5α-androstane-3α-ol & MathM. 0.0000 3.5802 0.0000 4.7737 0.6087 20.3429 0.0000 0.6302 1.1904 0.0000
-3.307 0.1838 1.6207 0.1875 1.5585 -1.1904 0.5161
6.8462 12.8182 17.1154 2.8929 10.5000 5.6667 6.1111 0.0000 6.1081
MathM. 15.2315 8.5199 6.2142 10.8550 5.8671 11.1565 6.4194 8.9897 3.0710 4.5436
6.604 6.2604 -2.9742 -0.6565 -0.7527 -2.8786 -3.071 1.5645
5.4340 14.9444 11.2121 2.7143 9.6829 5.3010 6.0370 0.0000 4.5079
MathM. 8.3549 6.2852 5.7482 8.0072 5.5387 9.6940 6.9897 7.1844 4.5026 3.3839
-2.5023 -0.8512 9.1962 3.2049 -2.8244 -0.0111 -1.6887 -1.1474 -4.5026 1.124
7.0800 11.6000 16.0400 2.5532 10.9057 7.2500 10.3721 0.0000 5.8333
MathM. 17.6358 9.1133 6.3593 12.5177 4.6293 10.9451 7.7706 11.9674 2.5576 4.3175
-1.458 -2.0333 5.2407 3.5223 -2.0761 -0.0394 -0.5206 -1.5953 -2.5576 1.5158
5.6078 13.4054 10.1875 2.3404 5.5200 5.8065 10.2791 0.0000 4.3226
MathM. 8.7378 5.6239 5.4412 8.6127 4.4771 5.4223 7.4977 10.6272 3.9262 3.7798
-2.0631 -0.0161 7.9642 1.5748 -2.1367 0.0977 -1.6912 -0.3481 -3.9262 0.5428
1.0794 0.6842 2.6786 0.2000 2.0500 2.9903 0.0909 0.0000 0.8529
-diol & 5β-androstane MathM. 2.0475 1.7197 0.8932 1.3022 0.4973 2.2265 2.7229 0.8321 0.0000 0.4648-3β,17β-diol
-0.209 1.3764 -0.2973 -0.1765 0.2674 -0.7412 0
Exp. = experimental resolution of separation; MathM. = resolution of separation obtained by mathematical model

**Table 7. Results of correlation between experimental and optimized values for separation **
**resolution **
5α-androstane-3β-ol & 5α-androstane-3α-ol 0.9365 [0.7477 – 0.9851]
5α-androstane-3β-ol & 5α-androstane-17β-ol 0.8602 [0.5029 – 0.9663]
5α-androstane-3β-ol & 5β-androstane-3α,17β-diol 0.7262 [0.1778 – 0.9303]
5α-androstane-3β-ol & 5β-androstane-3β,17β-diol 0.3879 [0.3198 – 0.8177]
5α-androstane-3α-ol & 5α-androstane-17β-ol 0.9632 [0.8475 – 0.9915]
5α-androstane-3α-ol & 5β-androstane-3α,17β-diol 0.7021 [0.1298 – 0.9234]
5α-androstane-3α-ol & 5β-androstane-3β,17β-diol 0.4362 [0.2666 – 0.8361]
5α-androstane-17β-ol & 5β-androstane-3α,17β-diol 0.8682 [0.5261 – 0.9684]
5α-androstane-17β-ol & 5β-androstane-3β,17β-diol 0.5850 [0.0706 – 0.8876]
5β-androstane-3α,17β-diol & 5β-androstane-3β,17β-diol 0.8346 [0.4319 – 0.9598]
r = correlation coefficient; 95% CIr = 95% confidence intervals for correlation coefficient;

**Discussion**
The proposed mathematical model for minimum value obtained from experimental optimization of mobile phase of androstane
isomers has been developed, the aim of the
retention factor (experiment number 6), and
from 0.0000 to 21.9636 for the resolution of
As it can be observed from the Results chapter
three parameters were optimized: the retention
experimental and optimized value differ from
factor, the separation resolution and the
one experiment to other. For example, for the
objective function. Analyzing the obtained
first experiment with one exception (separation
results (Table 3) two observation can be done.
resolution between 5α-androstane-3α-ol and
First observation refers the generic dependence
5α-androstane-17β-ol), the value obtained
equation used: for all three parameters the best
though optimization vas greater comparing with
results were obtained with the seven parameters
value obtained experimentally. The greater
equation (Eq.(2)). The second observation refers
difference in separation resolution of 2.5023
the optimum mobile phase which is identical for
was observed for 5α-androstane-3α-ol and 5β-
separation resolution and objective function
androstane-3β,17β-diol compounds (first
experiment, Table 6). At the opposite sides are
Cetone = 64 : 0 : 36, see Table 3), and are very
closed to the optimum mobile phase obtained
experiments, with one exception the value
for the retention factor that is of 60 : 13 : 27.
obtained through optimization was smaller
Analyzing the data from Table 4 it can be
observed that the differences between retention
experimentally. The greatest difference was of
factor obtained from experimental data and the
9.1962 for the experiment number 3, resolution
value obtained through optimization were very
separation between 5α-androstane-3α-ol and
small, this sustaining the abilities of the
5β-androstane-3β,17β-diol, and of 1.5645 for
optimization procedure. The minimum value of
experiment number ten (separation resolution
the absolute difference between these values is
of 0.0000 (see the results from the Table 4, the
androstane-3α,17β-diol). Analyzing the
experiment number 9) to 0.2390 (see the results
performances of the optimization procedure,
regarding the resolution separation, the best
androstane-3α-ol isomer. Note that the value
performances were obtained for 5α-androstane-
obtained through optimization was less than the
3α-ol and 5α-androstane-17β-ol compounds (r
value obtained from experimental data (see
= 0.9632, see Table 7). Thus, for the separation
resolution the mathematical model had not the
The performances of the proposed optimization
same stability as for the retention factor (the
model can be analyzed thought the correlation
value of the correlation coefficients vary from
coefficient and associated squared value. The
0.3879 to 0.9632, with two values less than 0.5,
closer the value of correlation coefficient is to -
three values between 0.5-0.75, and five values
1 or +1 the better the optimization procedure is.
Looking at the correlation coefficients presented
Some performance was obtained in optimization
in Table 5 it can be observed that in four cases
of objective function, but this performance is
out of five the value is greater than 0.99. The
less comparing with the performances obtained
exception is observed for the compound 5α-
for optimization of the retention factor.
androstane 3α-ol for each the correlation
The assessment of the proposed mathematical
coefficient is of 0.9648, being considered that
model in optimization of the mobile phase of
there is a strong relationship between the androstane isomers by thin layer
experimental value and value obtained through
chromatography with three solvents can be done
by analyzing the obtained results and of
The performances obtained by using of the
advantages and disadvantages. The greatest
proposed mathematical model for the separation
advantage of the mathematical model results
resolution parameter are not so good comparing
from its faster ability in obtaining the optimum
with those obtained for retention factor (see
composition of the mobile phase. Looking at the
Table 6). This difference can be explained by
obtained composition of the optimum mobile
the difference between the distributions of data:
phase it can be observed that the optimization of
the retention factor, of the separation resolution
8. Gül MK, Şeker M. Comparative analysis of
and of the objective function lead to very
similar compositions of mobile phases (see
(Brassica napus L.) and olive (Olea europaea L.)
Table 3). This observation can be explained by
the moderately polar character of the androstane
isomers and shown us that the small variations
9. Perera CO, Jasinghe VJ, Ng FL, Mujumdar
in composition of the optimum mobile phase do
AS. The effect of moisture content on the
conversion of ergosterol to vitamin D in shiitake
As a concluding remark it can be say that the
proposed mathematical model proved to assure
10. Senchenkova EM. In: Gillispie (Ed.).
accurate results on analysis of the separation of
Dictionary of scientific biography; Charles
androstane isomers. But, more researches are
Scribner Sons: New York, 1976;13:486-488.
necessary to be done in order to analyze the
11. Dyer WG, Gould JP, Maistrellis NA, Peng
stability and validity of the proposed model. If
TC, Ofner P. Thin-layer chromatography of
the mathematical model will prove its stability
and validity could become a useful method in
standardized procedure. Steroids 1963;1:271-
separation of androstane isomers from drugs,
12. Sherma J. Thin-Layer Chromatography of
Pesticides – A Review of Applications for

**Acknowledgements**
2002–2004. Acta Chromatogr. 2005;15:5-30.
13. Cimpoiu C, Hosu A, Hodisan S. Analysis of
UEFISCSU Romania through project some steroids by thin-layer chromatography ET108/2006.

**References **
14. Cimpoiu C, Jäntschi L, Hodisan T. A New
1. Steriod Skeleton [online]. Wikipaedia The
Method for Mobile Phase Optimization in High-
Free Encyclopedia Wikipaedia 21 October
2005 [cited 2006 January]. Available fro: URL:
(HPTLC), J. Planar. Chromatogr. - Mod. TLC
http://en.wikipedia.org/wiki/Image:Steroid-
15. Cimpoiu C, Jäntschi L, Hodisan T. A New
2. Callow RK, Young FG. Proceedings of the
Mathematical Model for the Optimization of the
Royal Society of London. Series A, Mobile Phase Composition in HPTLC and the Mathematical and Physical Sciences. 1936;
Chromatogr. Related Technol. 1999;22:1429-
3. Kuhn CM. Anabolic steroids. Recent. Prog.
Horm. Res. 2002;57:411-434. 4. Pierik M, Rutgeerts P, Vlietinck R, Vermeire S. Pharmacogenetics in inflammatory bowel disease. World J. Gastroenterol. 2006;12:3657-3667. 5. Wanner A, Horvath G, Brieva J.L, Kumar SD, Mendes ES. Nongenomic actions of glucocorticosteroids on the airway vasculature in asthma. Proc. Am. Thorac. Soc. 2004;1:235-238. 6. Gormley K, Dong Y, Sagnella GA. Regulation of the epithelial sodium channel by accessory proteins. Biochem J. 2003;371:1-14. 7. Kudwa AE, Bodo C, Gustafsson JA, Rissman EF. A previously uncharacterized role for estrogen receptor beta: defeminization of male brain and behavior. Proc. Natl. Acad. Sci. U S A. 2005;102:4608-4612.

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