Download 1. INTRODUCTION Tretinoin is the trans isomeric form of retinoic

1. INTRODUCTION
Tretinoin is the trans isomeric form of retinoic acid which is generally used for the
treatment of acute promyelocytic leukaemia, acne vulgaris, keratosis pilaris. It is
also used for the treatment of hair loss, ageing, etc. It increases the collagen
production that can reduce the appearance of stretch marks, which are indications
that they can slow skin ageing or reduce wrinkle formations. Topical Tretinoin,
known to be very susceptible to degradation under daylight by oxidation of the
conjugated double bonds which is neither retarded nor lessened by the presence of
antioxidant, is used for treating mild to moderate acne, fine wrinkles and
hyperpigmentation. Its chemical structure includes a functional acid group and a
side chain with conjugated double bonds, both susceptible to redox reactions. The
vitamin C redox system comprises L-ascorbic acid, monodehydroascorbic acid and
dehydroascorbic acid. Each of these substances has different physicochemical
properties but possess antioxidant capacity for both hydrophilic and lipophilic
substances. Tretinoin or all trans retinoic acid is easily oxidizable, thermally
unstable and it isomerizes fast when exposed to radiation. Tretinoin is an
endogenous retinoid metabolite of Vitamin A that binds to intracellular receptors
in the cytosol and nucleus, but cutaneous levels of tretinoin in excess of
physiologic concentrations occur following application of a tretinoin-containing
topical drug product. The structure of Tretinoin is shown in Figure 1.
Figure 1: STRUCTURE OF TRETINOIN
O
OH
112
Physical properties of Tretinoin:
Molecular formula
:
C20H28O2
Molecular mass
:
300.0
IUPAC name
:
(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6trimethylcyclohexen-1-yl)nona-2,4,6,
8-tetraenoic acid
Appearance
:
Yellow-to-light-orange crystalline powder
having a characteristic floral odour.
Nature
:
Slightly acidic
Pka
:
4.2
Solubility
:
It is soluble in dimethylsulfoxide, slightly
soluble in polyethylene glycol 400, octanol, and
100% ethanol. It is practically insoluble in
water, mineral oil and glycerin.
Available HPLC methods:
Literature survey on the various HPLC analytical methods available to determine
Tretinoin in various forms is given below:
Jiang XG et al1 proposed how Tretinoin and its active stereoisomer Isotretinoin
can be simultaneously determined by reversed-phase high pressure liquid
chromatographic method with a UV detector adjusted to 348 nm. Separation was
accomplished on YWG-C18 column by using a MeOH : NH4Ac buffer (pH 6.0)
85:15 (V:V), chlorpromazine being chosen as internal standard.
Tashtoush BM et al2 proposed a rapid method using an isocratic high-pressure
liquid chromatography and UV detection for determination of both all-trans
113
retinoic acid (tretinoin) and 13-cis retinoic acid (isotretinoin) in dermatological
preparations. Tretinoin and isotretinoin samples were extracted with acetonitrile
by a procedure that can be completed in less than 10 min. Subsequent separation
and quantification of amounts was accomplished in less than 15 min using
reversed-phase HPLC with isocratic elution with 0.01% trifluoroacetic
acid/acetonitrile (15:85 v/v).
A. Zarghi et al3 proposed how a new formulation of topical tretinoin–minoxidil
solution was prepared and the chemical stability of tretinoin was studied for 6
months at 4°C. A reversed phase high performance liquid chromatography method
was developed for determination of tretinoin using cyanocobalamine as an internal
standard. Tretinoin was shown to be stable for at least 6 months in refrigerated
storage conditions.
Michael B. Kril et al4 proposed a stability indicating reversed-phase highperformance liquid chromatographic method to quantify tretinoin in cream
formulations. Tretinoin cream samples were dissolved directly in tetrahydrofuran
and diluted for injection. Separation was accomplished on a 15 cm Nova-Pak C18
column using a tetrahydrofuran—phosphate buffer solvent system (42:58, v/v) and
1.0 ml/min flow-rate. The method was able to separate tretinoin from its
degradation products formed under stressing conditions.
Ye YR et al5 proposed a new HPLC method based on reverse phase separation
and photodiode-array detection for the simultaneous determination of tretinoin and
clindamycin phosphate, and their degradation products in topical formulations.
The method has been shown to be stability indicating, accurate, and precise for
two different formulation vehicles. Separation was achieved on a reverse phase
C18 column (Lichrospher, RP18, 5 microm, 25 cm x 4.6 mm ID, Phenomenex,
USA) using a simple gradient with aqueous-acetonitrile and aqueous-methanol
mobile phases.
114
Wang Y et al6 reported a liquid chromatography-mass spectrometry method for
simultaneous determination of retinol and 9-cis, 13-cis, and all-trans retinoic acid
(ATRA) in rat prostate. Mass spectrometric signal responses for ATRA were
compared using positive ion atmospheric-pressure chemical ionization (APCI) and
electrospray, as well as positive ion and negative ion APCI. Ventral prostate tissue
samples were homogenized and extracted following simple protein precipitation
without derivatization. Baseline separation of 9-cis, 13-cis, and ATRA standards
was obtained by using non-porous silica C18 column. Selected ion monitoring of
the ions m/z 301 and m/z 269 was carried out for mass spectrometric quantitative
analysis. The ion of m/z 301 corresponded to the protonated molecule of ATRA,
whereas the ion of m/z 269 corresponded to loss of water or acetic acid from the
protonated molecule of retinol or the internal standard retinyl acetate respectively.
Klvanova J et al7 established a rapid and simple method for determination of Alltrans retinoic acid (ATRA), 13-cis retinoic acid (13CRA) and all-trans retinal
(ATRAL) by HPLC. They separated ATRA, 13CRA and ATRAL by simple
isocratic normal phase HPLC. Both retinoic acid isomers and ATRAL were eluted
within 13 min and all components were well resolved.
Gundersen TE et al8 proposed a fully automated isocratic high-performance
liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cisretinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-transretinoic acid, using on-line solid-phase extraction and a column switching
technique allowing clean-up and pre-concentration in a single step. A 500microliter sample of serum was diluted with 750 microliters of a solution
containing 20% acetonitrile and the internal standard 9, 10-dimethylanthracene.
About 1000 microliters of this mixture was injected on a 20 x 4.6 mm I.D. poly
ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak
C18, 37-53 microns, 300 A particles. Proteins and very polar compounds were
washed out to waste, from the pre-column, with 0.05% TFA-acetonitrile (8.5:1.5,
115
v/v). Components retained on the pre-column were backflushed to the analytical
column for separation and detection at 360 nm. Baseline separation was achieved
using a single 250 x 4.6 mm I.D. Suplex pKb-100 column and a mobile phase
containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium
acetate-glacial acetic acid. A total time of analysis of less than 30 min, including
sample preparation, was achieved.
Yokoyama H et al9 established a high performance liquid chromatography system
that allowed simultaneous quantification of various retinoids. They applied the
retinoids to a high performance liquid chromatography system with a silica gel
absorption column. Samples were separated by the system with a binary multistep
gradient with two kinds of solvent that contained n-Hexane, 2-propanol, and
glacial acetic acid in different ratios. Each retinoid was detected at a wavelength of
350 nm. This condition allowed separation of 13-cis-retinoic acid, 9-cis-retinoic
acid, all-trans-retinoic acid, 13-cis-retinol, all-trans-retinol, all-trans-4-oxo-retinoic
acid, and 13-cis-4-oxo-retinoic acid as distinct single peaks. Each retinoid was
also analyzed separately and its retention time determined.
Beatrice Disdier et al10 described a gradient reversed-phase high-performance
liquid chromatographic technique for the easy separation and quantification of
some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and
their corresponding 4-oxometabolites, in plasma. The method involved a diethyl
ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13cis-acitretin as internal standards. A Nova-Pak C18 steel cartridge column was
used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5%
tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min.
Detection was by absorbance at 350 nm.
M Brisaert et al11 investigated the degradation of a tretinoin lotion placed in front
of a xenon lamp. Analysis was performed with HPLC. The tretinoin lotion was
degraded to about 20% of its initial concentration within 30 min. Incorporation of
116
tretinoin in β-cyclodextrin or in some surfactants (Brij®s) did not have any effect
on the photodegradation of tretinoin. Neither could a UV-B sunscreen retard the
photodegradation of tretinoin while a UV-A sunscreen had very little effect.
Irradiation with selected wavelengths revealed that 420 nm seemed to be the most
harmful wavelength for the degradation of tretinoin and not the wavelength of
maximum absorption (350 nm) as expected. Then the addition of the yellow
colourants chrysoin and fast yellow, absorbing in the region of 420 nm, was tested.
These colourants did indeed retard the photo-degradation of tretinoin more or less
depending on the concentration of the dye. Finally we only had to select a
concentration that was still effective but that did not colour the skin.
M.G Brisaert et al12 proposed a HPLC method on a reverse-phase column to
analyze the dermatological preparations that seemed to be the most suitable one
and also the sample preparation for this method was relatively simple. The
decomposition of tretinoin in preparations which were exposed to radiation was
very fast. 10% decomposition was noted between less than 1 h and 181 h,
depending on the formulation of the preparation. The tretinoin stability was also
influenced by temperature, depending on the ingredients of the dermatological
preparations. The adjuvant Brij 35 S, used as solubilizing agent, had a very bad
influence on the chemical stability of tretinoin.
S. Strohschein et al13 compared two different types of RP stationary phases in
their ability to separate cis/trans isomers of retinoic acid (tretinoin) by LC-NMR
coupling. Only by recording of 1H NMR spectra, the structural identification of the
separated compounds was possible, since their absorption coefficients are very
similar and their mass is identical, and therefore identification by UV-Vis is not
unambiguous and identification with LC-MS fails due to identical fragmentation
patterns. A commonly used C18 phase and a recently developed C30 phase have
been used for the separation of a mixture of thermal isomerized retinoic acids.
Three isomers could be separated and identified with the separation on a C18
117
column, whereas five cis/trans isomers could be identified by the use of a C30
column.
The list of available brands of this drug is shown in Table 1.1.
Table 1.1: List of brand names of Tretinoin
S.No
Brand name
Formulation
Available
Manufacturer
strength (w/w)
1
Comedolytic
Cream
0.025%
Fem Care
2
Eudyna
Cream
0.05%
Zydus Cadila
3
Pinoin
Ointment
0.03%
East West
4
Pinoin
Ointment
0.05%
East West
5
Retino-A
Cream
0.025%
J&J (Ethnor)
6
Retino-A
Cream
0.05%
J&J (Ethnor)
7
Retinol
Cream
0.025%
Psycorem
118
2. EXPERIMENTAL
2.1. Instrumentation
A Shimadzu electronic balance (AX-200) was used to weigh the drug and then for
wavelength checking UV-2306 spectrophotometer was used. An isocratic
Shimadzu HPLC model (VP series) instrument with Inertsil ODS C18 column
(250 mm x 4.6 mm, 5µm) was used to develop a High Pressure Liquid
Chromatographic method for the quantitative estimation. The instrument was
equipped with a LC 20AT pump for solvent delivery and variable wavelength
programmable SPD-10AVP detector. Degassing of the mobile phase was done
using a Loba ultrasonic bath sonicator. A 20µL Rheodyne inject port (7725i) was
used for injecting the samples. Data was analyzed by using PEAK software.
2.2. Chemicals and Solvents
Methanol, acetonitrile and orthophosphoric acid of HPLC grade were purchased
from E.Merck, Mumbai, India. Tretinoin, as a pharmaceutical form, in the brand
name of Pinoin was purchased from the local market.
2.3. The Mobile phase
The mobile phase containing acetonitrile, methanol and 0.1% orthophosphoric
acid in the ratio of 75:05:20 (v/v/v) was used for the elution.
2.4. Standard solution of the drug
Initially a stock solution was prepared by dissolving 10 mg of the drug in the
solvent, made upto 100 ml in a volumetric flask and appropriate dilutions were
done using the solvent chosen. A standard solution of 10 ppm was obtained by this
process for subsequent analysis.
119
2.5. Sample solution
The ointment form of Tretinoin (Pinoin) equivalent to 10 mg of the drug was
dissolved in 5 ml of the mobile phase taken in 10 ml volumetric flask. After
dissolution the solution was filtered through Ultipor Nylon 6, 6 membrane sample
filter paper and the filtrate was adjusted to the mark with the same solvent to
obtain a concentration of 10 ppm.
3. METHOD DEVELOPMENT
Development of a suitable RP HPLC method involves selection of the appropriate
wavelength, solvent, stationary and mobile phases. In order to establish these
requirements, a systematic study on the effect of various factors involved was
undertaken by varying each of them keeping all other conditions constant as
follows:
3.1. Detection of wavelength
The
wavelength
of
maximum
absorbance
was
recorded
on
an
UV
spectrophotometer using a solution of the drug and found to be 236 nm.
3.2. Choice of stationary phase
An Inertsil ODS C-18 5µm column having 250 x 4.6mm internal diameter was
chosen for the method development after a number of trials using different
octadecyl columns of various types and configurations from different
manufacturers were performed. It gave the expected separation with good
chromatographic peak shapes.
3.3. Selection of the Mobile phase
As selection of stationary and mobile phases depends upon the nature of the
sample and properties of the molecule a number of solvents were analyzed, mixed
in various proportions and tested under isocratic conditions with varied flow rates
to separate the drug on the ODS C-18 column with various combinations. An ideal
120
separation was achieved with mobile phase containing acetonitrile, methanol and
0.1% orthophosphoric acid in the ratio of 75:05:20 (v/v/v). This was finally
selected as it gave a well defined chromatographic peak with better resolution,
base line separation and low tailing factor.
3.4. Flow rate
An effective flow rate is one that is minimum with a short run time which can
minimize the usage of solvents. The optimum flow rate of 1.0 ml/min was attained
by varying it between 0.5–1.5 ml/min. This was ideal for the successful elution of
the analyte.
3.5. Optimized chromatographic conditions
Optimization of mobile phase was performed based on chromatographic
separation, peak shape and peak area obtained. The composition, pH and flow rate
of the mobile phase were changed to optimize the separation conditions. Based on
the above proceedings, the Chromatographic conditions thus optimized are shown
in Table 1.2.
These optimized conditions were maintained for the determination of Tretinoin in
bulk and pharmaceutical forms. When blank solution containing only the mobile
phase without the drug was injected, no peak was obtained. The chromatograms of
standard, blank, tablet sample are shown in Figure 2, 3 and 4 respectively.
121
Table 1.2: Optimized chromatographic conditions for estimation of Tretinoin
S.No
Parameter
Condition
1
Mobile phase
Acetonitrile : Methanol : 0.1% OPA (75:05:20)
2
Pump mode
Isocratic
3
Mobile phase pH
4.2
4
Diluent
Mobile phase
5
Column
Inertsil ODS C-18, 5µm, 250 x 4.6mm
6
Column Temp
Ambient
7
Wavelength
236 nm
8
Injection Volume
20 µL
9
Flow rate
1.0 ml/min
10
Run time
12 min
11
Retention Time
7.005 min
122
Figure 2: Chromatogram of standard solution
Figure 3: Chromatogram of blank (No Peak)
123
Figure 4: Chromatogram of formulation
4. RESULTS AND DISCUSSION
The experimental method developed above was employed for its
subsequent validation and determination of Tretinoin in bulk and pharmaceutical
forms. The following results were obtained correspondingly.
Validation of a proposed analytical method to determine the assay should
meet the requirements for the intended analytical application as per ICH
guidelines14. The typical analytical parameters used in validation of the assay
include Precision, Accuracy, Linearity, Robustness, Limit of detection, Limit of
Quantification, Selectivity or Specificity.
124
4.1 Linearity
Linearity is the method's ability to obtain peak area results that are proportional to
the concentration of the analyte within a given range. Linearity was performed by
preparing standard solutions of Tretinoin at different concentration levels
including working concentration mentioned in experimental condition i.e. 10 ppm.
Twenty micro liters of each concentration was injected in duplicate into the HPLC
system. The peak responses were read at 236 nm and the corresponding
chromatograms were recorded. From these chromatograms, the mean peak areas
were calculated and linearity plots of concentration over the mean peak areas were
constructed individually. The calibration plot is shown in Figure 5. The
regressions of the plots were computed by least square regression method.
Linearity results obtained are presented in Table 1.3.
Figure 5: Calibration plot for Tretinoin
The results obtained indicate a linear relationship between peak response and
concentration of Tretinoin in the range of 2-10 ppm.
125
Table 1.3: Linearity studies of Tretinoin
Level
Concentration of Tretinoin (in ppm)
Mean peak area
1
2
98053.2
2
4
193934.7
3
6
291203.9
4
8
382250.6
5
10
476133.5
Range:
Slope
47223.82
2 to 10
Intercept
4972.23
Correlation coefficient
0.9999
ppm
4.2 Precision
Precision is the degree of reproducibility of an analytical method under normal
operational conditions. Precision is determined by using the method to assay a
sample for a sufficient number of times to obtain statistically valid results.
Precision of the method was performed as Intraday precision and Inter day
precision. The precision is then expressed as the relative standard deviation.
4.2.1. Intraday precision
The Intraday precision was studied by preparing and injecting six replicate
standard solutions of Tretinoin (10 ppm) using the proposed method. The percent
126
relative standard deviation (% RSD) was calculated for the peak areas and it was
found to be 0.587%, which is well within the acceptance criteria of not more than
2.0%. Results of intraday system precision studies are shown in Table 1.4.
Table 1.4: Intraday Precision Results for Tretinoin
Sample
Tretinoin
Conc. (in ppm)
Injection No.
Peak Area
1
475708.6
2
473984.3
3
476291.6
10
%RSD
0.587
4
471487.7
5
470678.9
6
469564.2
4.2.2. Interday precision
The interday precision was studied by preparing and injecting six replicates of
standard solutions of Tretinoin (10 ppm) on two different days over a period of
one week. The percent relative standard deviation (% RSD) was calculated and it
was found to be 0.696%, which is well within the acceptance criteria of not more
than 2.0%. Results of interday system precision studies are shown in Table 1.5.
127
Table 1.5: Interday Precision Results for Tretinoin
Sample
Conc.(in ppm)
Tretinoin
Injection No.
Peak Area
1
476279.1
2
467379.2
3
471014.0
10
%RSD
0.805
4
473599.9
5
474272.9
6
467027.8
4.3. Selectivity
Selectivity of an analytical method is its ability to measure accurately an analyte in
the presence of possible interference creatable substances such as synthetic
precursors, excipients, etc. The selectivity of method was confirmed by comparing
the chromatograms of blank, standard and tablet sample. It was found that there is
no interference due to excipients in the tablet formulation and also found good
correlation between the retention times of standard and sample. The results are
shown in Table 1.6.
128
Table 1.6: Selectivity Study
Name of the solution
Retention Time (in min)
Blank
No peak
Standard
7.005
Sample
7.167
4.4. Accuracy
Accuracy of an analytical method is the extent to which test results are close to
their true value. It is measured from the result of a quantitative determination of a
well characterized known sample. The amount measured is compared to the
known amount. The accuracy of the method was determined by standard addition
method. A known amount of standard drug was added to the fixed amount of preanalyzed tablet solution. Peak area was compared before and after the addition of
the drug. The standard addition method was performed at 50%, 100% and 150%
level of 4 ppm. The solutions were analyzed at each level as per the proposed
method. The percent recovery and % RSD was calculated and results are presented
in Table 1.7. This indicates that the proposed method was accurate.
Table 1.7: Accuracy results
% Level
Conc. (in ppm)
Area
% Recovery
50
6
291229
100.0089
100
8
380659
99.58378
150
10
474564
99.67047
129
% RSD
0.23
4.5. Robustness
Robustness of analytical method is a measure of its capacity to remain unaffected
by small but deliberate variations in method parameters and provides an indication
of its reliability during normal usage. This was carried out by varying two
parameters from the optimized chromatographic conditions. The results are shown
in Table 1.8.
Table 1.8: Robustness results
Parameter changed
Change
Area
% Recovery
Standard
-
476133
-
Mobile phase
85:05:10
473268
99.39828
65:05:30
474622
99.68265
238
473152
99.37391
234
474564
99.67047
4
477354
100.2564
4.4
478167
100.4272
Wavelength
pH
4.6. Limit of detection and Limit of quantification
Limit of detection (LOD) is the lowest concentration of analyte in a sample that
can be detected but not necessarily quantified. In chromatography the detection
limit is the injected amount that results in a peak height of at least twice or three
times as high as the baseline noise level.
Limit of quantification (LOQ) is the minimum injected amount that gives precise
measurements. In chromatography it typically requires peak heights of 10 to 20
times higher than baseline noise at precision of <10-15% RSD between results.
130
The sample was dissolved by using the mobile phase and injected until the peak
disappeared. After 0.025 ppm dilution, peak was not observed clearly. So it
confirms that 0.025 ppm is the Limit of Detection and Limit of Quantification was
found to be 0.0824 ppm. The LOD and LOQ of Tretinoin are given in Table 1.9.
Table 1.9: Limit of Detection and Limit of Quantification for Tretinoin
Parameter
Measured volume
Limit of Quantification
0.025 ppm
Limit of Detection
0.0824 ppm
4.7. Formulation:
The validated method was applied for the assay of commercial ointment
containing Tretinoin. The formulation of Tretinoin equivalent to 10 mg of drug
was taken in 10 ml of volumetric flask containing 5 ml of mobile phase and was
shaken to dissolve the drug and then filtered through Ultipor N66 Nylon 6,6
membrane sample filter paper. Volume of the filtrate was adjusted to the mark
with the same solvent to obtain concentration of 10 ppm. An aliquot of this
solution was injected into HPLC system. Peak area of Tretinoin was measured and
compared against the peak area of the standard solution. The proposed method
was able to estimate Tretinoin in the ointment formulation with an accuracy of
95 %. The results presented good agreement with the labeled content as shown in
Table 1.10.
131
Table 1.10: Formulation results
Brand
Pinoin
Dose
Sample
Standard
Sample
Amount
(gm)
Conc.
area
area
found
1
10 ppm
98053.2
1165.2
9.5 ppm
% assay
95
5. CONCLUSION
The statistical evaluation of the proposed method revealed its good
linearity, reproducibility and its validation for different parameters made us to the
conclude that the current RP-HPLC method can successfully used for rapid and
reliable determination of Tretinoin in ointment formulation and also in bulk drugs.
Its chromatographic run time of 12 minutes allows the analysis of a large number
of samples in short period of time, making it suitable for the routine analysis of
Tretinoin and also quantification of Tretinoin in pharmaceutical dosage forms.
132
REFERENCES:
1. Jiang XG, Xi NZ., Zhongguo Yao Li Xue Bao. 1994, 15(5), 458-461.
2. Tashtoush BM, Jacobson EL, Jacobson MK., J Pharm Biomed Anal. 2007, 19,
43(3), 859-864.
3. A. Zarghi, M. Jenabi and A. J. Ebrahimian, HPLC determination of the
stability of tretinoin in tretinoin–minoxidil solution, Pharmaceutica Acta
Helvetiae 1998, 73(3), 163-165.
4. Michael B. Kril, Karen A. Burke, James E. DiNunzio and R. Rao Gadde,
Determination
of
tretinoin
in
creams
by
high-performance
liquid
chromatography, Journal of Chromatography A 1990, 522, 227-234.
5. Ye YR, Bektic E, Buchta R, Houlden R, Hunt B, Simultaneous determination
of tretinoin and clindamycin phosphate and their degradation products in
topical formulations by reverse phase HPLC, J Sep Sci. 2004, 27(1-2), 71-77.
6. Wang Y, Chang WY, Prins GS, van Breemen RB, Simultaneous
determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate
using liquid chromatography-mass spectrometry, J Mass Spectrom. 2001,
36(8), 882-888.
7. Klvanova J, Brtko J, Selected retinoids: determination by isocratic normalphase HPLC, Endocr Regul. 2002, 36(3), 133-141.
8. Gundersen TE, Lundanes E, Blomhoff R., Quantitative high-performance
liquid chromatographic determination of retinoids in human serum using online solid-phase extraction and column switching. Determination of 9-cisretinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-all-transretinoicacid and 4-oxo-13-cis-retinoic acid, J Chromatogr B Biomed Sci Appl.
1997, 691(1), 43-58.
9. Yokoyama H, Matsumoto M, Shiraishi H, Ishii H., Simultaneous
quantification
of
various
retinoids
133
by
high
performance
liquid
chromatography: its relevance to alcohol research, Alcohol Clin Exp Res.
2000, 24(4S), 26S-29S.
10. Beatrice Disdier, Hot Bun, Jacques Catalin, Alain Durand, Simultaneous
determination of all-trans-, 13-cis-, 9-cis-retinoic acid and their 4-oxometabolites in plasma by high-performance liquid chromatography, Journal of
Chromatography B: Biomedical Sciences and Applications 1996, 683(2), 143–
154.
11. M Brisaert, J Plaizier-Vercammen, Investigation on the photostability of a
tretinoin lotion and stabilization with additives, International Journal of
Pharmaceutics 2000, 199(1), 49–57.
12. M.G Brisaert, I Everaerts, J.A Plaizier-Vercammen, Chemical stability of
tretinoin in dermatological preparations, Pharmaceutica Acta Helvetiae 1995,
70(2), 161–166.
13. S. Strohschein, G. Schlotterbeck, J. Richter, M. Pursch, L. H. Tseng, H.
Handel, K. Albert, Comparison of the separation of cis/trans isomers of
tretinoin with different stationary phases by liquid chromatography-nuclear
magnetic resonance coupling, Journal of Chromatography A 1997, 765(2),
207–214.
14. Validation of compedial Assays-Guidelines' Pharmacopeial Convention,
Rockvilie, MD, 1985.
134