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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

OH

O

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Physical properties of Tretinoin:

Molecular formula : C20H28O2

Molecular mass : 300.0

IUPAC name : (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-

trimethylcyclohexen-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

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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 high-

performance 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.

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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 All-

trans 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-cis-

retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-

retinoic acid, using on-line solid-phase extraction and a column switching

technique allowing clean-up and pre-concentration in a single step. A 500-

microliter 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,

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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 13-

cis-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

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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

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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

strength (w/w)

Manufacturer

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

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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.

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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

121

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.

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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

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Figure 2: Chromatogram of standard solution

Figure 3: Chromatogram of blank (No Peak)

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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.

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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.

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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:

2 to 10

ppm

Slope

Intercept

Correlation coefficient

47223.82

4972.23

0.9999

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

127

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 Conc. (in ppm) Injection No. Peak Area %RSD

Tretinoin

10

1 475708.6

0.587

2 473984.3

3 476291.6

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.

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Table 1.5: Interday Precision Results for Tretinoin

Sample Conc.(in ppm) Injection No. Peak Area %RSD

Tretinoin

10

1 476279.1

0.805

2 467379.2

3 471014.0

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.

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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 pre-

analyzed 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 % RSD

50 6 291229 100.0089

0.23 100 8 380659 99.58378

150 10 474564 99.67047

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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

65:05:30

473268

474622

99.39828

99.68265

Wavelength 238

234

473152

474564

99.37391

99.67047

pH 4

4.4

477354

478167

100.2564

100.4272

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.

131

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.

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Table 1.10: Formulation results

Brand Dose

(gm)

Sample

Conc.

Standard

area

Sample

area

Amount

found

% assay

Pinoin 1 10 ppm 98053.2 1165.2 9.5 ppm 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.

133

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 normal-

phase 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 on-

line solid-phase extraction and column switching. Determination of 9-cis-

retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-all-trans-

retinoicacid 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 by high performance liquid

134

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-oxo-

metabolites 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.