J. Biomedical Science and Engineering, 2010, 3, 454-458
doi: 10.4236/jbise.2010.35063 Published Online May 2010 (http://www.SciRP.org/journal/jbise/
Published Online May 2010 in SciRes. http://www.scirp.org/journal/jbise
Determination of isotretinoin in pharmaceutical formulations
by reversed-phase HPLC
Carla Aiolfi Guimarães1*, Farid Menaa2, Bouzid Menaa3*, Ivo Lebrun4, Joyce S. Quenca-Guillen1,
Aline Vivian Vatti Auada4, Lucildes P. Mercuri5, Paula Ferreira5, Maria Inês Rocha Miritello Santoro1
1Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil;
2Department of Dermatology, School of Medicine, University of Wuerzburg, Wuerzburg, Germany;
3Fluorotronics, Inc., 1425 Russ. Bvld, San Diego Technology Incubator, San Diego, USA;
4Department of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil;
5Department of Exact and Earth Sciences, Diadema, Federal University of São Paulo, São Paulo, Brazil.
Email: carlaaiolfi@usp.br; bouzid.menaa@gmail.com
Received 4 February 2010; revised 1 March 2010; accepted 2 March 2010.
The development of facile and rapid quantification of
biologically active biomolecules such as isotretitoin in
therapeutic drugs contained in many generic formu-
lations is necessary for determining their efficiency
and their quality to improve the human health care.
Isotretritoin finds its applications in the maintenance
of epithelial tissues. Different processes to date such
as normal phase HPLC, or gas chromatrography am-
ong others are able to separate and quantify isote-
troin. However, the extraction is quite complex and in
the case of HPLC, the analysis requires long reten-
tion times. In such context, an isocratic reversed-
phase high-performance liquid chromatography (HP-
LC) technique coupled with an UV-vis detector is
described here for easy separation and quantification
of 13-cis-retinoic (isotretinoin) from soft gelatin cap-
sule formulations. The isotretinoin was extracted
from three different commercial drug samples with
tetrahydrofuran (THF) solvent by a procedure that
can be completed in less than 10 minutes. Subsequent
separation and quantification were accomplished in
less than 5 minutes under isocratic reversed-phase
conditions on a Lichrospher RP18 column and a mo-
bile phase consisting of 0.01% TFA/acetonitrile
(15/85, v/v) at a flow rate of 1.0 mL/min. Isotretoin
was detected for the three samples via its UV-vis ab-
sorbance at 342 nm. The method was validated and
the results showed good linearity, precision and ac-
curacy for sensitive and selective quantitative deter-
mination of isotretinoin in the different pharmaceu-
tical formulations. We found that the average isot-
retinoin content in two of the three commercial pro-
ducts fell outside the 90-110% United States Pha-
rmacopeia specifications. Consequently, the facile
extraction and the precise method for the biomole-
cule quantification open up tremendous possibilities
in improving the quality control of drugs which can
exist as different generic brands.
Keywords: 13-Cis Retinoic Acid; Reversed-Phase
Chromatography; Isotretitoin Extraction; Isotretinoin
Quantification; Pharmaceuticals Formulation
Retinoic acid is a very potent biomolecule in promoting
growth and controlling differentiation and maintenance
of epithelial tissue for vitamin A deficient animals. In-
deed, all-trans-retinoic acid (tretinoin) appears to be ac-
tive form of vitamin A in all tissues except the retina,
and it is 10-100-fold more potent than retinol in various
systems in vitro. Isomerization of this compound in the
body yields to 13-cis-retinoic acid (isotretinoin), which
is nearly as potent as tretinoin in many of its actions on
epithelial tissues. Vitamin A and other retinoids have
found wide applications in the treatment of skin disor-
ders and may find important roles in cancer chemopre-
vention and therapy [1].
One of the most clinically useful vitamin A derivatives
is 13-cis-retinoic acid (isotretinoin). It was approved in
1982 to treat severe recalcitrant nodular acne and re-
mains the drug of choice for this therapeutic treatment.
Isotretinoin is the only treatment that affects all the ma-
jor factors involved in the pathogenesis of acne. Its ex-
hibits several activities, in particular a capacity to de-
crease sebaceous gland activity, to correct the keratinisa-
tion defect in acne and to reduce the population of the
bacterium Propionibacterium acnes [2].
Isotretinoin is a strong lipophilic molecule, almost in-
soluble in water and only partially soluble in oil. It is
labile upon heating, light and air. In such conditions,
these physicochemical properties impose considerable
C. A. Guimarães et al. / J. Biomedical Science and Engineering 3 (2010) 454-458 455
Copyright © 2010 SciRes. JBiSE
limitations upon formulation options. For instance, the
degradation of isotretinoin (in its solid state) in the
presence of air is an autocatalytic process that proceeds
rapidly and a simple dry powder formulation would not
have a sufficient shelf-life. A suspension in oil is more
stable [2]. Suspension in oil also minimizes chemical
degradation, as lipid preparations, especially those with
a low peroxide number, have been shown to enhance
stability [3]. Owing to the photolability and sensitivity to
heat and oxidation of the retinoids, their quantitative
determination in pharmaceutical is particularly important
for the quality control of finished products and for sta-
bility-indicating assays [1].
Indeed, for all pharmaceutical agents, one of the most
important therapeutic determinants is the amount of drug
the patient is exposed to. Previous studies comparing the
quality of a variety of generic with innovator products
have revealed important deficiencies in the amount of
active ingredient among generics [4,5]. Isotretinoin and
its trans-isomer, tretinoine, are known to be teratogens
that can cause fetal malformations during pregnancy for
intance [6,7]. It is possible for a patient to be treated
with a different isotretinoin-based generic products dur-
ing a course of therapy because of an assumption that
‘generic isotretinoins’ are essentially identical and bio-
equivalent to the innovator product. Under these circum-
stances, the pharmaceutical quality and consistency
among products become of paramount importance [2].
A number of previous methods for determination of
isotretinoin have been described in the literature. The
analytical method generally employed in the United
State Pharmacopoeia (USP) is a normal phase HPLC
system [8]. Isotretinoin has also been determined by gas
chromatography in soft and hard capsules formulations
[9]. A reversed phase HPLC method with fluorescence
detection has been described [1] for the determination of
retinoids in pharmaceutical dosage forms. Simultaneous
determination of tretinoin and isotretinoin in derma-
tological formulations has been also reported via normal
phase HPLC [10]. HPLC [1], Microcalorimetry [11], UV
radiation monitor [12], thin layer chromatography (TLC)
and preparative layer chromatography (PLC) [13], cap-
illary zone electrophoresis (CZE) and micellar elec-
trokinetic capillary chromatography (ME-KC) [14] have
been also employed to study retinoid degradation prod-
ucts in commercial preparation and raw materials.
Although the above methods are able to separate and
quantify isotretinoin, the extraction procedure is quite
complex and HPLC analysis involves long retention
times [10]. In that context, the aim of this study was to
develop a rapid method using an isocratic high perform-
ance liquid chromatography (HPLC) coupled to UV-vis
detector to determine and quantify the isotretinoin in soft
capsule formulations from three commercial samples.
The validation method with its precision and accuracy
was checked and the isotretinoin quantification was car-
ried out for three commercial products for which the
content of the biomolecule was compared to the United
States Pharmacopeia specifications.
2.1. Chemicals and Reagents
The content of isotretinoin was determined from the
concentrations of standard solutions. The standards for
13-cis-retinoic acid were from United States Pharm-
copeia reference standard (USP:RS). The three commer-
cial products were purchased from two different phar-
maceutical companies (their names have been omitted
for commercial reasons) named as Branded Original
Product A (Product A), Generic Product B (Product B)
and Generic Product C (Product C). All three commer-
cial soft-gel capsules contained 20 mg of isotretinoin.
Acetonitrile, trifluoroacetic acid (TFA), tetrahydrofuran
(THF) were HPLC grade and purchased from Merck
2.2. Laboratory Precaution
All experiments were performed using amber glass volu-
metric flasks under yellow light conditions to avoid
degradation of isotretinoin.
2.3. HPLC Instrumentation and Conditions
The HPLC system consisting of Agilent model 1100
(Agilent, USA) connected to a UV/Visible absorbance
detector (agilent) was used for all separation. Chroma-
tographic separations were performed using reversed-
phase chromatographic using a HPLC column [Lichro-
spher RP18 (5 µm, 125 × 4 mm i.d., Merck, USA)].
Isotretinoin was detected by the UV-vis absorbance at
342 nm [8] with the sensitivity set at 0.1 aborbance unit
full scale (Aufs).
The mobile phase used was composed of 0.01% tri-
fluoroacetic acid and acetonitrile (15/85, v/v) at a flow
rate of 1.0 mL/min. The injection volume was 20 µL and
the column was maintained at 40ºC.
2.4. Standard Preparation Procedures
Stock solutions were prepared by dissolving the appro-
priate amounts of isotretinoin in acetonitrile. A set of
working standard solutions was produced by diluting
aliquots of the stock solutions to give the desired con-
centrations of the analytes. For the method linearity as-
sessment, the concentration range was 5.0 to 30.0 µg/mL
(the concentrations were determined using the calibr-
ation graphs. The standard solutions were stable for at
least 3 days at 4ºC).
2.5. Extraction of Soft-Gel Capsules
Isotretinoin capsules were opened carefully using a
sharp blade and the content of these capsules was ex-
456 C. A. Guimarães et al. / J. Biomedical Science and Engineering 3 (2010) 454-458
Copyright © 2010 SciRes.
tracted directly with THF solvent. The solution contai-
ning isotretinoin was shaked for 5 min. 1 mL of aliquot
for the resulting solution was diluted in acetonitrile to
obtain the final solution concentration (20 µg/mL) that
was analyzed by HPLC in comparison with the appro-
priate standard solution.
2.6. Quantification of Isotretinoin
A standard curve was constructed by injecting samples
containing isotretinoin standard at concentrations rang-
ing from 5.0 to 30.0 µg/mL. The peak area was deter-
mined and plotted versus the concentration of isotre-
tinoin. For the recovery studies, known volumes of iso-
tretinoin standard solutions were analyzed, and the
absolute recovery was calculated by comparing the peak
area obtained from isotretinoin in the commercial cap-
sule with the peak area of samples derived from the
standard solutions.
The objective of this work was to develop a rapid me-
thod using an isocratic high performance liquid chro-
matography (HPLC) system to determine quantitatively
the isotretinoin in hard gelatin capsule formulations. In
order to validate an efficient method for analysis of
drugs in pharmaceutical formulations, preliminary tests
were performed with the objective to select adequate and
optimum conditions. Parameters such as detection wave-
length, ideal mobile phase and their proportions, con-
centration of the standard solutions, and flow rate were
exhaustively tested and the quantitative determination of
isotretoin in the gel capsules was made possible under
the ideal conditions described in this paper. The pro-
posed method is simple and do not involve laborious
time-consuming sample preparation.
3.1. Chromatography
The conditions for a rapid and simple HPLC separation
with UV detection were developed using an isocratic elu-
tion with a mobile phase composed of 0.01% trifluo-
roacetic acid and acetonitrile (15/85, v/v). These condi-
tions gave well resolved sharp peaks (Figure 1) for
Absorbance (mAU)
Elution time (min)
Figure 1. Chromatogram of samples. (a) isotretinoin standard (20 µg/mL); (b)
Product A (20 µg/mL); (c) Product B (20 µg/mL); (d) Product C (20 µg/mL).
Conditions: Lichrospher RP18 column (5 µm, 125 × 4 mm i.d., Merck), mobile
phase 0.01% TFA /acetonitrile (15/85, v/v), flow rate 1.0 mL/min, UV detection at
342 nm and ambient temperature (24 ± 2ºC).
C. A. Guimarães et al. / J. Biomedical Science and Engineering 3 (2010) 454-458 457
Copyright © 2010 SciRes. JBiSE
The precision of proposed method was evaluated thro-
ugh intra-day repeatability of responses after replicate (n
= 10) injection of sample solutions (20.0 µg/mL). The
precision is expressed as the relative standard deviation
(RSD) amongst responses. The standard deviation
the isotretinoin commercial formulations but also for the
isotretinoin standards. The retention time was of ap-
proximately 3.8 min. The shorter elution time makes the
method especially useful for routine analysis of isotreti-
noin in pharmaceutical formulations. The validation ex-
periments as described in the following section were
completed to determine if the method could achieve the
reproducibility and accuracy required for analysis of
3.2. Method Validation
3.2.1. Method Validation
The quantification of the chromatogram was performed
using the peak area of isotretinoin standards for known
content of isotretinoin and as references for the determi-
nation of the content for the commercial samples to which
we determined the peak areas of their respective chro-
matograms. Five standard solutions ranging from 5.0 to
30.0 µg/mL in concentration (three replicates each),
were injected into the HPLC system. The calibration
graph was obtained by plotting the peak areas of the
standard solutions against the theoretical standard con-
centrations. The linearity was evaluated by linear least-
squares regression analysis. The correlation coefficient
obtain with linear regression of curve was 0.997. Statis-
tical analysis indicated excellent linearity as shown in
Table 1.
3.2.2. Accuracy
To evaluate the accuracy of the proposed method, re-
covery tests were carried out with all samples. The
measurements were performed by adding known amo-
unts of standard solutions to sample followed by analysis
using the proposed method. The recovery values ob-
tained were 101.2, 99.4 and 99.3% (why don’t you have
100%) for the three soft gelatin capsule formulations,
confirming the accuracy of the proposed method. The
percentage of recovery results are presented in Table 1.
3.2.3. Precision
amongst replicate responses and relative standard devia-
tion values (RSD) were less than 1.0%, indicating preci-
sion of the method. The statistical data results obtained
in the analysis of commercially available samples are
shown in Table 2.
According to the results obtained only one formula-
tion (Product A (branded named)), was found to be good
agreement with the claimed content of the drugs. Indeed,
the average percentage of isotretinoin content (Table 3)
for the Product A was evaluated to 105 ± 0.05% which
fits with United States Pharmacopeia (USP) specifica-
tions. In contrast, the content for the generic products
formulations Products (Product B and Product C), fell
outside the United States Pharmacopeia (USP) specifica-
tions. The content percentage of isotretinoin was found
to be 128.5 ± 0.08 and 115.5 ± 0.04 in Product B and
Product C, respectively. The USP XXX [8] monograph
for isotretinoin capsules states that the capsules must
contain not less than 90.0% and not more than 110% of
the labeled amounts of isotretinoin. The results and the
accuracy of the method shows that it is necessary to con-
trol the content of the active biomolecule for drug safety,
but also that the precision of the method and rapid acqui-
sition of the data can be a plus for pharmaceutical indus-
tries for the quality control of their products.
Table 1. Statistical results of linear regression analysis in the
determination of isotretinoin by proposed method.
Statistical parametrs Results
Intercept of curve (b) –328.09
Slope of curve (a) 138.86
Linear correlation coefficient (r) 0.997
Table 2. Recovery data of standard solutions added to the
samples analyzed by using the proposed HPLC method.
*Avarage of three determination
Table 3. Statistical data obtained in the analysis of samples using the proposed method.
Sample Declared theoretical
concentration (µg/mL)
Found experimental
concentration (µg/mL)Content (%) RSD (*) (%) Content (%)
Confidence interval (**)
Product A 20.0 21.0 105.0 0.43 105.0 ± 0.05
Product B 20.0 25.7 128.5 0.70 128.5 ± 0.08
Product C 20.0 23.1 115.5 0.30 115.5 ± 0.04
(*) Average of ten determination (**) 95.0% confidence level.
458 C. A. Guimarães et al. / J. Biomedical Science and Engineering 3 (2010) 454-458
Copyright © 2010 SciRes. JBiSE
The proposed HPLC methods for quantitative determi-
nation showed good linearity, precision and accuracy for
sensitive and selective quantitative determination of iso-
tretinoin in pharmaceutical formulations. This method is
not time-consuming and is easy to perform in any labo-
ratory. We ultimately show, according the United States
Pharmacopeia specifications that the average amount of
isotretinoin in 2 of the 3 commercial products ranged
outside the 90-110%. Safe use of isotretinoin, particu-
larly with respect to teratogenicity, remains a central
issue for therapeutics development. Therefore, it is im-
portant, that future research determines a comparative
study with pharmaceutical quality of isotretinoin prod-
ucts on patient exposure to isotretinoin by comparative
bioequivalence tests.
The authors acknowledge the CNPq (National Counsel of Techno-
logical and Scientific Development) CAPES (Foundation to Higher
Level People Improvement) and FAPESP (Research Support Founda-
tion in the State of São Paulo) for the financial support.
[1] Gatti, R., Gioia, M.G. and Cavrini, V. (2000) Analysis
and stability study of retinoids in pharmaceuticals by
fluorescence. Journal of Pharmaceutical and Biomedical
Analysis, 23(1), 147-159.
[2] Taylor, P.W. and Keenan, M.H.J. (2006) Pharmaceutical
quality of generic isotretinoin products, compared with
Roaccutane. Current Medical Research and Opinion,
22(3), 603-615.
[3] Ioele, G., Cione, E., Risoli, A., Genchi G. and Ragno, G.
(2005) Accelerated photostability study of tretinoin and
isotretinoin in liposomes formulations. International Jour-
nal of Pharmaceutics, 60(1-2), 251-260.
[4] Wetterich, U. and Mutschler, E. (1995) Quality of cefo-
taxine sodium preparations. Arzneimittel-Forschung Drug
Research, 45(1), 74-80.
[5] Lambert, P.A. and Conway, B.R. (2003) Pharmaceutical
quality of ceftriaxone generic products compared with
Rocephin. Journal Chemotherapy, 15(4), 357-368.
[6] Lammer, E.J., Chen, D.T., Hoar, R.M., Agnish, N.D.,
Benke, P.J. and Braun, J.T. (1985) Retinoic acid em-
bryopathy. New England Journal of Medicine, 313(14),
[7] Willhite, C.C., Wier, P.J. and Berry, D.L. (1989) Dose
response and structure-activity considerations in retinoid-
induced dysmor phogênesis. Critical Reviews in Toxicology,
20(2), 689-695.
[8] (2007) United States pharmacopeia. XXX, Pharma-
copeia Convention, Maryland, 2428-2429.
[9] Lima, E.M., Diniz, D.G. and Antoniosi-Filho, N.R. (2005)
Development of a gas chromatography method for the
determination of isotretinoin and its degradation products
in pharmaceuticals. Journal of Pharmaceutical of Bio-
medical Analysis, 38(4), 678-685.
[10] Tashtoush, M.B. Jacobson, E.L. and Jacobson, M.K.
(2007) A rapid HPLC method for simultaneous deter-
mination of tretinoin and isotretinoin in dermatological
formulations. Journal of Pharmaceutical and Biomedical
Analysis, 43(3), 859-864.
[11] Tan, X., Meltzer, N. and Lindenbaum, S. (1992) Solid-
state stability studies of 13-cis-retinoic acid and all-
transretinoic acid using microcalorimetry and HPLC
analysis. Pharmaceutical Research, 9(9), 1203-1208.
[12] Allwood, M.C. and Plane, J.H. (1984) The degradation of
vitamin A exposed to ultraviolet radiation. International
Journal of Pharmaceutics, 19(2), 207-213.
[13] Crank, G. and Pardijanto, M.S. (1995) Photooxidations
and photosensitized oxidations of vitamin A and its
palmitate ester. Journal of Photochemistry and Photobio-
logy A: Chemistry, 85(1-2), 93-100.
[14] Bempong, D.K., Honigberg, I.L. and Meltzer, N.M.
(1993) Separation of 13-cis and all-trans retinoic acid
and their photodegradable products using capillary zone
electrophoresis and micellar electrokinetic chromato-
graphy (MEC). Journal of Pharmaceutical and Biomedi-
cal Analysis, 11(9), 829-833.