SIMULTANEOUS ESTIMATION OF METHOTREXATE AND TRETINOIN BY REVERSE PHASE –HPLC METHOD

SIMULTANEOUS ESTIMATION OF METHOTREXATE AND

TRETINOIN BY REVERSE PHASE –HPLC METHOD

Aditya S., Arun P.*, Neelesh D., Neeraj K., Shailendra P., Shivani T. and Stuti P.

Shri Ram Group of Institution Faculty of Pharmacy, Jabalpur M.P.

ABSTRACT

A simple fast, precise and accurate liquid chromatographic method was

developed for simultaneous estimation of Methotrexate(MTX) and

tretioin(ATX) combined dosage form by RP-HPLC (Reverse Phase

High Performance Liquid Chromatography) method. Chromatography

was out on a column -18 and ACN:Buffer (85:15 v/v) of resultant pH

3.0, as mobile phase, at a flow rate of 1.2ml/min and measured at

340nmth. The calibration curves linearity range were 1-6ug/ml for

methorexate and 1-16 ug/ml for tretioin, regression coefficient values

are 0.9989 and 0.9999 respectively. The average retention time of

Methotrexate and trition were found to be 1.95 and 8.98 min

respectively. The method was reproducible, with good resolution and sharp peak of

Methotrexate and tretioin. There results of the analysis have been validated statistically.

KEYWORDS: METHOTREXATE, TRETIONIN, RP-HPLC, ASSAY, UV DETECTION.

INTRODUCTION

Analytical chemistry plays vital role in development of science, which involves separation,

identification and determination of the relative amounts of components in a sample of matter.

Drug should be available in such a form that quality including bioavailability, adequate

plasma concentration, desired duration and onset of action, accurate dose, safety, efficacy and

stability on storage of product, can be assured during the storage of the product.

Volume 6, Issue 10, 535-548. Research Article ISSN 2277– 7105

*Corresponding Author

Arun P.

Shri Ram Group of

Institution Faculty of

Pharmacy, Jabalpur M.P. Article Received on 06 July 2017,

Revised on 26 July 2017, Accepted on 16 August 2017

DRUG PROFILE

Table for Drug Profile of MTX.

Common Name Methotrexate (MTX)

IUPAC Name (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl) (methyl)amino)benzamido)pentanedioic acid Chemical Formula C20H22N8O5

Mol. Mass 454.44 g/mol

Physical Parameters MTX is a bright yellow-orange, odorless powder.

Solubility

MTX is practically insoluble in water, chloroform and ether. It is freely soluble in dilute solutions of alkaline and carbonates; it is slightly soluble in dilute HCL and alcohol.

Uses

Intake of MTX is dangerous, so is useful only in the following conditions :

MTX is used to treat only life-threatening cancer.  It is ingested in severe psoriasis.

 It also works in rheumatoid arthritis.

 It is also applicable in psoriatic arthritis, mycosis fungoides, and a variety of solid tumors.

Adverse Effects

Dry cough, shortness of breath; diarrhea, vomiting, white patches or sores inside your mouth or on your lips; blood in your urine or stools; fever, chills, body aches, sore throat and headache with a severe blistering, peeling and red skin rash, nausea, stomach pain, low fever, loss of appetite, dark urine, clay-colored stools, jaundice (yellowing of the skin or eyes).

Pharmacokinetics

1. Bioavailability--17–90% 2. Protein binding—70% 3.Metabolism—Hepatic

4. Half Life—3–15 hours (dose dependent) 5. Excretion—Renal (48–100%).

Therapeutic Cons

Pregnancy cat : D(AU) X(US)

Legal Status : Prescription Only (S4)(AU), POM(UK). Routes : Oral, iv, im, sc, intrathecal.

Table for Drug Profile of ATRA.

Common Name Tretinoin (all-trans retinoic acid or ATRA)

IUPAC Name 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)–nona-2,4,6,8-tetraenoic acid

Chemical Formula C20H28O2

Mol. Mass 300.4412 g/mol

Chemical Structure

Physical Parameters Tretinoin is a slightly yellow powder.

chloride.

Dosage Cream or Gel : 0.01%, 0.025%, 0.04%, 0.05%, 0.1%. Cream:

0.02%, 0.025%, 0.05%, 0.1%.

Uses

1. It is commonly used to treat Acne Vulgaris and Keratosis Pilaris.

2. It is also used to treat Acute Promyelocytic Leukemia (APML).

3. It is also used in the treatment of Photoaging.

4. Topical tretinoin is also used to treat and reduce the appearance of Stretch Marks by increasing collagen production in the dermis.

Contraindications

Combining other topical acne medications (for example, salicylic acid) with tretinoin may lead to excessive skin irritation.

Use of abrasive soaps or cleansers, astringents, skin waxes and other products that irritate the skin may add to tretinoin induced skin irritation.

Adverse Effects

Redness, Scaling, Itching, And Burning of skin.

It is aTeratogen. Dyspepsia, Fever, Weight gain, Peripheral Edema in patients with Acute Promyelocytic Leukemia.

Pharmacokinetics 1. Protein binding—> 95% 2. Half Life—0.5-2 hours

Therapeutic Cons

Pregnancy cat. : C(topical), D(oral)(USA)

Legal Status : Prescription Only (S4)(AU) Routes : Topical, oral.

EXPERIMENTAL PROFILE

Types of Chemical Analysis

Qualitative Method

When a completely unknown sample is taken for analysis, presence of one or more component in the sample and prediction of structural features of the compound, are done by qualitative method.[4,5]

Quantitative Method

This method deals with the determination of quantity i.e. content of each component or

specified component of the sample, whose chemical behavior is known. Quantitative analysis

can be classified into two categories

• Classical Method of Analysis. • Instrumental Method of Analysis.

A. Classical Method of Analysis

In these methods volume and mass are used as means of detection i.e. Gravimetry, titrimetry

B. Instrumental Method of Analysis

These methods are based on the measurement of specific and nonspecific physical properties

of a substance by using instrument.

Table for Type of Chemical Analysis.

Sr. No. METHOD PRINICIPLE APPLICATION

I Qualitative Analysis Chemical behaviour of the

Substance.

Detection of Presence or absences of Substance.

II (A)

QuantitativeAnalysis Classical Methods

1. Titrimetric Methods

Determination of volume of known concentration which is required to react quantitatively with a measured volume of solution of the substance to be measured.

(a) Acidimetry and

Alkylimetry Neutralization reaction. Alkaline and acidic substances.

(b) Redox Titration

Change of oxidation among the the reaction of ion to form precipitates.

Ionic substance.

(c) Precipitation titration Complex formation of ion. Complexing agent.

(2) Gravimetric Method

Producing and weighing of compound or element in pure form after chemical treatment.

Analysis of standards.

(B) Instrumental Method[6]

1. Spectroscopic Method Interaction of electromagnetic radiation with matter.

(a) Ultraviolet and Visible

Spectroscopy

Excitation of valence

electrons. Quantitative analysis.

(b) Infra-red Spectroscopy Excitation of molecular

vibration.

Structure determination and identification of organic and inorganic compounds.

(d) Atomic Absorption

Spectroscopy

Absorption of atomic

resonance line. Trace and quantitative analysis for metals.

(e) Atomic Emission

Spectroscopy.

Light emission from excited electronic stases of atoms.

Quantitative analysis for all elements emitting in UV – VIS spectrum.

(f) X-Ray Diffraction Diffraction of x- rays from

crystal planes

Identification of solids compounds. Crystalline structure.

(g) X-ray Fluorescence Re-emission of x-ray from

excited atoms.

Quantitative analysis of analysis of elements especially non metals.

(h) Fluorometry and

Phosporimetry.

Emission of light energy by

electron. Inorganic and organic substance.

(i ) Nephelometry and

Turbidimetry

Intensity of transmitted light as a function of concentration of the dispersed phase.

In water treatment plant in sewage work, in beverage bottling industry etc.

(j) Mass Spectroscopy

Ionisation of molecule, and conversion of molecule into fragment ions.

Structure determination and identification of organic compound. Analysis of trace volatile in non volatiles.

(k) Nuclear Magnetic

Resonance Spectroscopy

Reorientation of magnetic nuclei in a magnetic field.

Structure determination and identification of organic compound and molecular

conformation.

(l)

Electron Spin Resonance Spectroscopy

Reorientation of magnetic electrons in a magnetic field.

4. Hyphenated Method[8]

(a) GC-Mass Spectroscopy

Combine separation efficiency of GC with sensitivity and

specificity of mass spectroscopy.

Identification and analysis of mixtures.

(b) GC-IR Spectroscopy

Combine separation efficiency of GC with specificity of IR.

Separation and structural determination of mixtures.

(c) HPLC-Mass Spectroscopy

Combine separation efficiency of HPLC with sensitivity and specificity of mass spectroscopy.

Analysis and identification of mixtures.

3. Chromatographic method[7]

(a) Gas Chromatography (GC)

Partitioning between vapour phase and solid/liquid.

Multi-component and qualitative analysis of volatile organics.

(b) Liquid

Chromatography(HPLC)

Partitioning between liquid and solid phase

Separation technique for ionic materials. Used for

multicomponent analysis.

(c) Size-Exclusion Chromatography

Solute size dependent partitioning between packing pore volume and interstitial volume.

Separation according to hydrodynamic volume, Determination of polymer molecular weight distribution.

(d) High-Performance Thin Layer Chromatography

Partitioning between liquid

solution and solid. Multi-component analysis.

(e) Paper Chromatography Partitioning between liquid

solution and paper. Separation of amino acids.

2. Electrochemical Method:

(a) Potentiometry

Difference in electrode potential depends on concentration of ionic species in solution

PH determination, fluride determination.

(b) Voltametry and Polarography

Voltage current time Relationship during electrolysis.

Metals, anions and many organic species.

(c) Stripping Method

Electrodeposition of ion onto an electrode surface area and electrochemically stripped from electrode back into solution.

Determination of metals in tap water.

(d) Amperometry Method

Difference in voltage is a function of

volume of the titrating reagent. Trace metal analysis.

(e) Coulometry Method

The extent of chemical reaction of an electrode is directly proportional to the quantity of electricity passing through electrode.

Reagents which are difficult to analyze by ordinary chemical means like nitrates.

(f) Electrogravimetry Method

The amount of chemical action is proportional to quantity of electricity which passes through the electrode.

Metal elements.

(g) Conductance

5. Miscellaneous Method:

(a) Thermal Analysis

Change in physical and/or chemical property of substances as a function of temperature.

Determination of purity and thermal stability.

(b) Refractrometry Method

Refractive index is function of concentration.

Determination of composition of binary liquid or gaseous mixtures.

(c) Polarimetry Method

Substance solution cause

rotation of plane polarized light.

Quantitative analysis of optically active compounds.

(d) Radiometric Methods

Counting of radioactive species

produced by substance. Trace and ultra trace element analysis.

(e)

Biological and Microbiological Method

Analysis of microbial inhibitions.

Biological assay of elements microbiological assay of antibiotic and vitamins.

The instrumental methods are more accurate, precise, sensitive, selective and less time

consuming than classical methods.

Chromatography

Chromatography is ―a separation process that is achieved by distribution of substance

between two phases, stationery phase and mobile phase. Chromatography is probably the

most power full and versatile analytical technique available to modern chemist. Its power

arises from its capacity to determine quantitatively many individual components present in

the mixture in one, single analytical procedure.

Table for Choice of Method.

Substance of similar chemical type Partition Chromatography

Substance of different chemical type Adsorption Chromatography

Gases and volatile Substance Gas Chromatography

Ionic and Inorganic Substance Ion Exchange Chromatography, Paper or

Thin Layer Chromatography

Ionic form non-ionic Substance Ion Exchange or Gel Chromatography

Biological materials, compounds of high

molecular weight Gel Chromatography

High Performance Liquid Chromatography

Early in the development of liquid chromatography, scientist realized that increase in column

efficiency could be brought about by decreasing the partial size of packaging. The technology

for producing and using pickings with particle diameter as smalls 3 to10µm was developed.

This technology required sophisticated instruments operating at high pressure, which

contrasted markedly with the simple glass columns of classic gravity-flow liquid

INSTRUMENTATION

The Essential Feature of Modern High Performance Liquid Chromatography In normal phase

LLC, the stationary phase is polar and the mobile phase is non-polar. In reversed phase LLC,

the polarity of these phases is reversed, i.e., stationary phase non-polar and mobile phase is

polar (RP – HPLC).

Fig: 1.1 Block diagram of a complete HPLC. The item connected by Dashed lines are

necessary only for gradient elution.

RP-High Performance Liquid Chromatography

Reverse phase chromatography refers to the use of a polar eluent with a non polar stationary

phase in contrast to normal phase chromatography, where a polar stationary phase is

employed with a non-polar mobile phase.

Reverse Phase Chromatography is widely used due to the following advantages

Many compounds such as biologically active substances have limited solubility in non polar

solvents that are employed in normal phase chromatography.

Ionic or highly polar compounds have high heats of adsorption on straight silica or

alumina columns and therefore can elute as a tailing peaks.

Column deactivation from polar modifiers is a problem in liquid solid chromatography which

Ionic compounds can be chromatographed via ion exchange chromatography. This mode of

chromatography is tedious because precise control of variables such as pH and ionic strength

is required for reproducible chromatography.

Step in HPLC Method Development

The steps involved in HPLC method development are summarized in following the series of

step.[10]

Fig-For Steps in HPLC Method Development.

Analytical Method Validation

The purpose of method validation is to demonstrate that the established method is ―fit for the

purpose‖. This means that the method, as used by the laboratory generating the data, will

provide data that meets the criteria set in the planning phase. There is not a single accepted

procedure for conducting a method validation. The ultimate objective of the method

validation is to provide evidence that the method does what it is intended to do, accurately,

reliably and reproducibly. The validation parameters as per ICH guidelines are:

2. Precision

3. Specificity

4. Limit of Detection

5. Limit of Quantitation

6. Linearity

7. Ruggedness

8. Robustness

9. System Suitability Testing.

Method Validation

On the basis of fixed parameters the method of estimation was validated for following

parameters.

1. Accuracy (Recovery Studies)

Recovery studies were carried out at 80%, 100% and 120% level. The results for recovery

studies showed that results were within acceptable limits, above 99% and below 101%.

Table for Results of recovery study of MTX-ATRA marketed formulation.

Precision

Table forData of Method Repeatability of MTX and ATRA Formulation.

Drug Label

claim

Amount Found*

(%)

Standard Deviation*

% Co- efficient of Variation*

RSD*

MTX (mg/tab) 7.5 99.64 0.0787 0.0311 0.019

ATRA (%gel) 0.1 98.12 0.098 0.0477 0.041

Replicate

Amount taken (µg/ml)

Amount added at

(µg/ml) % Recovery

MTX ATRA % MTX ATRA MTX ATRA

1 4 8

80%

3.2 6.4 99.50 99.80

2 4 8 3.2 6.4 100.10 100.40

3 4 8 3.2 6.4 100.00 99.60

1 4 8

100%

4 8 99.50 99.70

2 4 8 4 8 100.20 99.90

3 4 8 4 8 100.30 99.70

1 4 8

120%

4.8 9.6 99.00 100.30

2 4 8 4.8 9.6 100.00 100.00

3 4 8 4.8 9.6 99.50 99.50

Mean 99.79 99.87

S.D 0.431 0.307

RSD 0.004 0.003

Denotes average of six determinations

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Sample solution was subjected to Limit of Detection (LOD) and Limit of Quantitation (LOQ)

studies, results are given in Table.

Table for LOD and LOQ of MTX and ATRA Formulation.

Drug LOD (μg/mL) LOQ (μg/mL)

MTX 0.6 0.8

ATRA 0.5 0.7

System Suitability / Repeatability

Table for HPLC System suitability of MTX and ATRA Formulation.

Injection No

Standard Response MTX

2(µg/mL)

ATRA 4(µg/mL)

1. 1115973 452765

2. 1115750 452701

3. 1115130 452720

4. 1115127 452750

5. 1115247 452738

Average 1115342 452736

SD 420.9301 27

%RSD 0.25 0.012

Robustness

Table for HPLC Robustness of MTX-ATRA Formulation

Effect on AUC

S.No Sys Suitability Flow (-10%) Flow (+10%) pH= 2.97 pH= 3.03

MTX ATRA MTX ATRA MTX ATRA MTX ATRA MTX ATRA

1. 5973 2765 5965 2760 5950 2768 5971 2765 5978 2767

2. 5945 2770 5955 2740 5945 2779 5949 2754 5940 2770

3. 5956 2748 5950 2750 5940 2741 5950 2749 5965 2742

Mean 5957 2761 5959 2750 5947 2766 5956 2755 5953 2759

SD 13.1 11.5 12.3 10.5 10.4 14.5 9.5 9.2 15.4 12.6

Effect on Retention time S.

No

Rt Flow (-10%) Flow (+10%) pH= 2.97 pH= 3.03

MTX ATRA MTX ATRA MTX ATRA MTX ATRA MTX ATRA

1. 1.95 8.98 1.93 8.97 1.94 8.95 1.96 8.99 1.94 8.95

2. 1.93 8.97 1.95 8.95 1.92 8.92 1.94 8.95 1.92 8.96

3. 1.94 8.98 1.92 8.96 1.94 8.93 1.92 8.94 1.96 8.97

Mean 1.94 8.98 1.93 8.96 1.93 8.93 1.94 8.96 1.94 8.96

System Suitability Parameters

Table for System Suitability Test Parameters.

System Suitability Parameter Proposed Method

MTX ATRA

Retention Time (tR) 1.95 8.98

Capacity Factor (k) 0.95 7.98

Therotical plate Number (N) 2562 1535

Tailing Factor (T) 0.58 0.85

Resolution (R) 6.8

RESULTS AND DISCUSSION

RP-HPLC method for the Simultaneous estimation of MTX and ATRA

Determination of solubility of both drugs in different solvents.

Table for Result of Solubility of Drugs in Different Solvents.

Keywords: (+++), Freely Soluble; (++), Soluble; (+), sparingly soluble; (-), insoluble.

Linearity and Calibration curve For MTX

The Linearity range of MTX 1–6µg/ml in (Table No-7.2) with correlation coefficient 0.9989.

Table for Standard Calibration data of MTX.

Sr. No. Concentration(μg/ml) *Area Under Curve(AUC)

1 0 0

2 1 657109

3 2 1115973

4 3 1809860

5 4 2416707

6 5 2990067

7 6 3600123

Denotes average of three determinations.

Solvents Solubility

MTX ATRA

0.1 N NaOH +++ +++

Water + ++

Acetonitrile ++ +++

Methanol ++ ++

Chloroform - +

Fig. for Standard Curve for MTX Pure.

For ATRA

The Linearity range of ATRA 0–16 µg/ml in (Table) with correlation coefficient 0.9998.

Table for Standard Calibration data of ATRA.

Sr. No. Concentration(μg/ml) *Area Under Curve(AUC)

1 0 0

2 1 114123

3 2 226783

4 4 452765

5 8 913467

6 16 1825342

*Denotes average of three determinations.

DISCUSSION AND CONCLUSION

Methotrexate and Tretinoin both drugs are most commonly used drugs. Literature survey

reveals that Tretinoin and MTX are given in combination for effective management of

leukemia. The combination has shown great success rates in clinical practice. A simple,

rapid, accurate and precise Reverse Phase High Performance Liquid Chromatographic and

UV methods were developed and validated for Simultaneous estimation of Methotrexate and

Tretinoin in bulk and in pharmaceutical dosage form.

ACKNOWLEDGMENT

The authors would like to thank the personnel of medicinal chemistry research lab, Shri Ram

Group of Pharmacy for their sincere assistance to conduct this research.

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