~ 25 ~

2. DEVELOPMENT AND VALIDATION OF NEW VISIBLE

SPECTROPHOTOMETRIC AND RP-HIGH PRESSURE LIQUID

CHROMATOGRAPHIC ASSAY METHODS OF MELPHALAN IN PURE

AND DOSAGE FORMS

2.1 DRUG PROFILE OF MELPHALAN

Melphalan is a phenylalanine derivative of nitrogen mustard and also known as

phenylalanine mustard, L-phenylalanine mustard, L-sarcolysin or L-PAM. Melphalan is a bi-

functional alkylating agent which is active against selective human neoplastic diseases

(Indian Pharmacopia, 1996; Sweetman, 2005 and Facon et al, 2007). It is known chemically

as 4-[bis(2-chloroethyl)amino]-L-phenylalanine (Fig. 2.01, P.25). The molecular formula is

C13H18Cl2N2O2 and the molecular weight is 305.20.

Fig. 2.01. Chemical structure of melphalan

It is available in tablet form (ALKERAN) for oral administration. Each film-coated

tablet contains 2 mg melphalan and the inactive ingredients viz., colloidal silicon dioxide,

crospovidone, hypromellose, microcrystalline cellulose, magnesium stearate macrogol, and

titanium dioxide.

Literature survey reveals that one LC-MS (Mirkou et al, 2009) and a few HPLC

methods (Brightman et al, 1999; Silvestro et al, 1991; Nath et al, 2008; Chang et al, 1978,

Rolf et al, 2003) have been reported for the estimation of melphalan. Existing analytical

methods reveal that relatively little attention was paid in developing economical methods,

~ 26 ~

therefore it made a need to develop sensitive and flexible analytical methods, which

prompted the author to choose melphalan for the investigation. In the present chapter the

author developed eight sensitive visible spectrophotometric methods and one RP-HPLC

method that are validated. These methods have been extended to pharmaceutical

formulations as they are simple, economical and sensitive.

~ 27 ~

2.2 VISIBLE SPECTROPHOTOMETRIC METHODS FOR

DETERMINATION OF MELPHALAN

Literature survey reveals that one LC-MS

(Mirkou et al, 2009) and a few HPLC

methods (Brightman et al, 1999; Silvestro et al, 1991; Nath et al, 2008; Chang et al, 1978,

Rolf et al, 2003) have been reported for the estimation of melphalan. Existing analytical

methods revealed that no attention was paid in developing visible spectrophotometric

methods by exploiting thoroughly the analytically functional groups present in melphalan.

This made the author to develop eight sensitive visible spectrophotometric assay methods for

melphalan in dosage forms. The developed visible spectrophotometric methods were

validated.

2.2.1. EXPERIMENTAL:

2.2.1. a. Instruments used

An Elico, UV - Visible digital spectrophotometer [SL – 159] with 1cm matched quartz

cells were used for the spectral and absorbance measurements. For pH measurements, an

Elico LI-120 digital pH meter was used.

2.2.1. b. Preparation of solutions and reagents:

All the reagents used in this assay were of analytical grade and the reagent solutions

were prepared using double distilled water.

i. Preparation of standard drug solutions:

The pharmaceutical grade pure sample of melphalan (99.28%) was procured from

Celon Laboratories Limited, Andhra Pradesh, India. A 1.0 mg/ml solution was prepared by

dissolving 100 mg of pure melphalan in 100 ml of methanol and this stock solution was

diluted step wise with distilled water to get the working standard solutions of concentration of

~ 28 ~

40µg/ml for methods M7, M9, M14a & M14b; 80µg/ml for methods M4 & M8; 100µg/ml for

methods M2 & M10 respectively.

ii. Preparation of tablet solution:

An accurately weighed portion of tablet (ALKERAN), content equivalent to about 100

mg of melphalan was transferred into a 100 ml volumetric flask. Added about 80ml of

methanol and shaken well for about 20 min. The contents were diluted with methanol up to

the mark and mixed thoroughly and filtered the solution. Then, the filtrate was evaporated to

dryness. The so obtained residue was used for the preparation of formulation solutions for

different methods as given under standard solutions preparations. These solutions were

analyzed as under procedures described for bulk solutions.

iii. Preparation of reagents:

METHOD M2:

Fe(III) solution (1.0% W/V): About 100 mg of anhydrous ferric chloride was accurately

weighed and dissolved in 100ml of distilled water.

PHEN (0.2% W/V): Prepared by dissolving 200 mg of o-phenanthroline in 100 ml of 0.1N

hydrochloric acid.

O-Phosphoric acid solution: Prepared by mixing 1.3 ml of o-phosphoric acid with 100 ml of

distilled water.

METHOD M4:

FC REAGENT (Loba - 2N): Folin Cio Calteu reagent was used as it is.

Na2CO3 (10%): Prepared by dissolving 10 gms of sodium carbonate in 100 ml of distilled

water.

METHOD M7:

Vanillin (0.4%W/V): Prepared by dissolving 400 mg of Vanillin in 100 ml of CH3OH.

H2SO4 (Merck): Used as it is.

~ 29 ~

METHOD M8:

NQS (0.5%W/V): Prepared by dissolving 500 mg of Naphtha quinone sulphate in 100 ml of

distilled water.

NaOH (20% W/V): Prepared by dissolving 20 gms of sodium hydroxide in 100 ml of

distilled water.

METHOD M9:

Ninhydrin (1.0% W/V): Prepared by dissolving 1.0 gm of ninhydrin in 100 ml of acetone.

METHOD M10:

CA (0.1% W/V): Prepared by dissolving 100 mg of p-chloranilic acid initially in 20 ml of

isopropyl alcohol followed by dilution to 100 ml with chloroform.

METHOD M14a:

MB (0.1% W/V): Prepared by dissolving 100 mg of Methylene Blue in 100 ml of distilled

water and subsequently washed with chloroform to remove chloroform soluble impurities.

METHOD M14b:

MV (0.1% W/V): Prepared by dissolving 100 mg of Methylene Violet in 100 ml of distilled

water and subsequently washed with chloroform to remove chloroform soluble impurities

2.2.2. RECOMMENDED PROCEDURES:

After systematic and detailed study of the various parameters involved, the following

procedures were recommended for the assay of melphalan in bulk samples and

pharmaceutical formulations.

METHOD M2: Aliquots of standard melphalan solution (100 μg/ml) containing 5.0 to 25.0

μg (0.5 – 2.5 ml) were transferred into a series of 10 ml volumetric flasks and 1.0 ml of

0.003M ferric chloride was added to each flask. Then 1.0 ml of o-PHEN solution was added

to all flasks and the volumes in all volumetric flasks were equalized with water. The contents

in each flask were gently boiled for 30 min and then cooled to room temperature. To the

~ 30 ~

above flasks 2.0 ml of OPA was added and the final volume of all volumetric flasks was

brought to 10 ml with water. The absorbance of the colored product was measured at 510 nm

against corresponding reagent blanks and the amount of melphalan was assayed from

corresponding calibration graph drawn (Fig. 2.03, P.40).

METHOD M4: Into a series of 10 ml volumetric flasks, standard solution (80 μg/ml) of

melphalan in the concentration range of 4.0 – 20.0μg (0.5 – 2.5 ml) were transferred. Then

3.0 ml of sodium hydroxide solution and 1.0 ml of FC reagent were successively added and

kept aside for 5 min. The volume was made up to 10 ml with water. The absorbance was

measured at 735 nm against reagent blank. The amount of melphalan was deduced from its

Beer-Lambert’s plot (Fig. 2.06, P. 41).

METHOD M7: Into a series of 10 ml calibrated tubes, aliquots (0.5 – 2.5 ml, 40 g/ml) of

standard melphalan solution, 2.0 ml of vanillin and 3.0 ml of conc. sulphuric acid were added

successively and the total volume in each flask was brought to 10 ml by the addition of

methanol. The contents were heated by placing in heating water bath (maintained at 500C)

for 15 min. Then the flasks were colored and made up to the mark with methanol and the

absorbance was measured at 560nm against a reagent blank. The concentration of drug

present in dosage sample was deduced from Beer-Lambert plot (Fig. 2.09, P.42).

METHOD M8: Aliquots of standard melphalan solution (0.5 – 2.5 ml, 80 g/ml) were

transferred into a series of 10 ml of calibrated tubes containing 1.0 ml of 0.02N NaOH and

1.0 ml of NQS solution and the contents in each tube were heated at 500C for 5 min and

cooled for 2 min in ice water. The absorbance of each solution was measured after 5 min at

480 nm against a reagent blank prepared similarly. The amount of melphalan was calculated

from its calibration graph (Fig. 2.12, P.43).

~ 31 ~

METHOD M9: Aliquots of standard melphalan solution (0.5 – 2.5 ml, 40 g/ml) was

transferred into a series of 10 ml calibrated tubes containing to each flask, 2.0 ml of 2%

ninhydrin solution was added and diluted to volume with acetone. The solutions were heated

on a boiling water bath for ten minutes. After cooling the solutions to room temperature, they

were made up to mark with acetone. The absorbance of each solution was measured at 580

nm against the reagent blank. The content of the unknown was computed either from

calibration curve (Fig. 2.15, P.44).

METHOD M10: Into a series of 10 ml calibrated tubes containing aliquots of standard

melphalan solution (0.5 – 2.5 ml, 100 g/ml), 2.0ml of p-chloranilic acid was added and kept

aside for 30 min at laboratory temperature and made up to the mark with chloroform. The

absorbance of the colored solutons was measured at 525 nm against the reagent blank. The

amount of the drug was calculated from Beer’s law plot (Fig. 2.18, P.45).

METHOD M14a & M14b: Aliquots of standard drug solution (0.5 – 2.5 ml, 40 g/ml) and 1.0

ml of pH 9.8 buffer solution were placed separately in a series of 125 ml separating funnels.

A volume of 1.0 ml of MB (M14a) / MV (M14b) was added. In each funnel, the total volume of

aqueous phase was adjusted to 10 ml with distilled water. Then 10 ml of chloroform was

added in each separating funnel, the contents were shaken for 2 min and allowed to separate.

The organic layer was collected through cotton plug and the absorbance was measured at 615

nm (M14a) and at 620 nm (M14b) against reagent blank. Both the colored species were stable

for 2 hours. The amount of drug in a sample was obtained from the Beer’s Lambert plot (Fig.

2.21, P.46 for M14a; Fig. 2.24, P.47 for M14b).

~ 32 ~

2.2.3. Results and Discussion:

a. Optimization of Experimental conditions for the proposed methods:

The developed spectrophotometric methods are direct and are based on the

determination of the colored product generated. Preliminary experiments were carried out in

developing procedures for the new methods (M2, M4, M7, M8, M9, M10, M14a and M14b) by

varying parameters one at a time, keeping the others fixed and then observing the effect

produced on the absorbance of the colored species. The following experiments were

conducted for this purpose and the conditions so developed were incorporated in

recommended procedures.

METHOD M2: In developing the procedure, studies were carried out on the effect of volume

of Fe (III) and o-phenanthroline solution, volume and pH of the buffer solution, order of

addition of reagents, nature of solvents for final dilution, heating time and temperature. The

optimum conditions are incorporated in Table. 2.01 (P.48).

METHOD M4: The optimum conditions in this method were developed based on the studies

of the effects of various parameters viz., nature and volume of base (sodium carbonate),

volume of 2N FC reagent solution for maximum color development, incubation time, the

order of addition of reagents and the stability of the colored species formed after the final

dilution and the studies were incorporated in Table. 2.02 (P.49).

METHOD M7: The effect of parameters, such as nature and strength of acid, concentration

and volume of vanillin, order of addition of reagents, solvent for final dilution were studied

and the optimum conditions chosen for this procedure is reported in Table. 2.03 (P.50).

~ 33 ~

METHOD M8: In developing this method a systematic study of the effects of various

parameters were under taken by varying one parameter at a time and controlling all other

fixed. The effects of various parameters such as volume and strengths of NQS & NaOH,

solvent for final dilution, time on the stability and intensity of colored species were studied.

The optimum conditions are incorporated in Table. 2.04 (P.51).

METHOD M9: The conditions were fixed based on the study of the effects of various

parameters such as volume of ninhydrin, nature and conc. of reducing agent, pH and volume

of the buffer, order of addition of the reagents, heating time and temperature, solvent for final

dilution and stability of the colored products after final dilution. The optimum conditions

were established for these method developments are presented in Table. 2.05 (P.52).

METHOD M10: The optimum conditions in this method were developed based on the study

of the effects of various parameters such as strength and volume of reagent, solvents used for

final dilution in the formation and stability of the colored species. The results are

incorporated in Table. 2.06 (P.53).

METHOD M14a & M14b: The optimum conditions in these methods were fixed based on the

study of the effects of various parameters such as type and concentration of acid used in

buffer, concentration of dye - MB (M14a), or MV (M14b), choice of organic solvent, shaking

time, temperature, intensity and stability of the colored species in organic phase. The results

are incorporated in Table. 2.07 (P.54).

b. Spectral Characteristics:

After establishing optimized conditions in each developed method, the colored products

were scanned on a spectrophotometer in the visible region against similar reagent blank. The

absorbances for each developed method were recorded and the plotted absorption spectra

~ 34 ~

were graphically represented in Fig. 2.02, P.40 for M2, Fig. 2.05, P.41 for M4, Fig. 2.08,

P.42 for M7, Fig. 2.11, P.43 for M8, Fig. 2.14, P.44 for M9, Fig. 2.17, P.45 for M10, Fig.

2.20 & Fig. 2.23, P.46 & 47 for M14a and M14b.

c. Optical Characteristics:

Standard solutions containing of melphalan in each linearity level were prepared and

were analyzed at their maximum absorption (max) for each developed method. Calibration

graphs were obtained by plotting absorbance versus the concentration of drug by least

squares regression analysis. Beer’s law plots and Ringbom plots of melphalan for each

developed method were recorded graphically (Fig. 2.04 to 2.25, P.40 – 47) and their results

(i.e. slope, intercept, Beer’s law limits, correlation, molar absorptivity, Sandell’s sensitivity,

optimum photometric range, LOD and LOQ) were calculated and are reported (Table. 2.08,

P.55).

d. Precision:

The precision for each developed method was ascertained from the absorbance values

obtained by actual determination of six replicates of a fixed amount of melphalan. The

%RSD (percent relative standard deviation) and percent range of error (at 0.05 and 0.01

confidence limits) were calculated for the proposed methods and are represented in Table.

2.09 (P.56).

e. Accuracy:

The accuracy for each proposed method was determined by taking different amounts of

bulk samples of melphalan within the Beer’s law limits and was expressed as the percentage

of analyte recovered by the assay. The accuracy results (percent error) for each developed

methods were reported in Table. 2.08 (P.55).

~ 35 ~

f. Interference studies:

Under optimum conditions, interference studies were conducted to study the effect of

wide range of excipients and other active ingredients usually present in the formulations of

melphalan with the proposed methods (M2, M4, M7, M8, M9, M10, M14a & M14b). These

studies reported that the common excipients usually present in dosage forms of melphalan did

not interfered in the proposed methods.

g. Analysis of formulations:

Commercial formulations containing melphalan (ALKERAN) were successfully

analyzed by the proposed methods. The values obtained by the proposed are summarized in

Table. 2.09 (P.56).

f. Chemistry of the colored species:

Melphalan possess different functional moieties such as α-amino acid and aromatic

3o- nitrogen of varied reactivity. Eight proposed methods are based on the reactivity of

presence of several substituent’s i.e. reducing behaviour [Fe III/o-PHEN (M2), FC (M4)],

condensation reaction (due to the presence of amine) [VANILLINE (M7)], substitution [NQS

(M8)], charge transfer interaction (due to the presence of tertiary nitrogen) [PCA (M10)],

condensation [NH (M9)] and neutral ion association complex (due to complex formation

between negatively charged carboxylic acid on the drug and basic dye i.e., protonated amino

group in the dye, which can be extractable in to chloroform) [MB (M14a), MV (M14b)].

The chemistry of the colored species formed in each proposed method for the assay of

melphalan has been presented in respective schemes given below.

METHOD M2: Melphelan when treated with an oxidant [Fe(III)], it undergoes oxidation,

giving products of oxidation (inclusive of reduced form of oxidant, Fe (II) from Fe (III),

~ 36 ~

besides un reacted oxidant). The reduced form of Fe III (i.e., Fe II) has a tendency to give

colored complex on treatment with o-PHEN (Scheme. 2.01, P.36).

N

N

NN

N N

N

N

N

N

O - PHEN

3Fe +2

Fe +2

Fe +3 (Excess)

Fe +2 + Unreacted Fe +3

Unreacted Fe +3 + O- Phosphoric Acid Fe +3 OPA Complex

Coloured Complex

Melphalan

Scheme.2.01: Reaction of melphalan with Fe(III)/O-PHEN

METHOD M4: The reaction was based on the reduction reaction of FC reagent by the

unshared pair of electrons in melphalan. It probably can reduce tungstate/molybdate in

presence of sodium carbonate solution, thereby producing reduced species having

characteristic blue color complex that is read spectrophotometrically.

METHOD M7: This visible spectrophotometric method is based on condensation reaction of

aromatic aldehyde (vanillin) with the primary amine of melphalan forming a color complex

(Scheme. 2.02, P.36).

OH

O CH3

CHO

HOOC CH H2C

NH2

N

Cl

Cl

OH

O CH3

CH

HOOC CH H2C

N

N

Cl

Cl

- H2O

+

Vanillin

Melphalan

Coloured product

Scheme 2.02: Reaction of melphalan with Vanillin

~ 37 ~

METHOD M8:

This method was based on the condensation of melphalan with NQS in an alkaline

medium to form an orange-colored product (Scheme. 2.03, P.37).

HOOC CH H2C

NH2

N

Cl

Cl

HOOC CH H2C

N

N

Cl

Cl

O

O

SO3 Na

- NaHSO3

O

OH

+

NQS

Melphalan

Coloured product

Alkaline Medium, 500 C

Scheme 2.03: Reaction of melphalan with NQS

METHOD M9: In the present method, the NH2 group of melphalan combines with ninhydrin

molecule forming amino derivative, which is capable of condensing with another ninhydrin

molecule to give a colored complex (diketohydrindylindene-diketohydrindamine –

Ruhemenn’s purple).

METHOD M10: A dononr-acceptor complex formation takes place between melphalan and

PCA (Scheme 2.01, P.37).

.

O O

OHCl

HO Cl

PCA

Acceptor

Melphalan Donor -Acceptor complex

Donor

Scheme 2.04: Reaction of melphalan with PCA

~ 38 ~

METHOD M14a& M14b: The carboxylate anion (negative charge) of melphalan is

expected to attract the oppositely charged part of the dye (positive charge of methylene

blue / Methylene violet) and behave as single unit being held together by electrostatic

attraction (Scheme.2.05, P.39).

~ 39 ~

Scheme 2.05: Reaction of melphalan with MB & MV

~ 40 ~

Fig.2.02. Absorption spectra of melphalan for Method M2

Fig.2.03. Beer’s law plot of melphalan Fig.2.04. Ringbom plot of melphalan

for Method M2 for Method M2

~ 41 ~

Fig.2.05. Absorption spectra of melphalan for Method M4

Fig.2.06. Beer’s law plot of melphalan Fig.2.07. Ringbom plot of melphalan

for Method M4 for Method M4

~ 42 ~

Fig.2.08. Absorption spectra of melphalan for Method M7

Fig.2.09. Beer’s law plot of melphalan Fig.2.10. Ringbom plot of melphalan

for Method M7 for Method M7

~ 43 ~

Fig.2.11. Absorption spectra of melphalan for Method M8

Fig.2.12. Beer’s law plot of melphalan Fig.2.13. Ringbom plot of melphalan

for Method M8 for Method M8

~ 44 ~

Fig.2.14. Absorption spectra of melphalan for Method M9

Fig.2.15. Beer’s law plot of melphalan Fig.2.16. Ringbom plot of melphalan

for Method M9 for Method M9

~ 45 ~

Fig.2.17. Absorption spectra of melphalan for Method M10

Fig.2.18. Beer’s law plot of melphalan Fig.2.19. Ringbom plot of melphalan

for Method M10 for Method M10

~ 46 ~

Fig.2.20. Absorption spectra of melphalan for Method M14a

Fig.2.21. Beer’s law plot of melphalan Fig.2.22. Ringbom plot of melphalan

for Method M14a for Method M14a

~ 47 ~

Fig.2.23. Absorption spectra of melphalan for Method M14b

Fig.2.24. Beer’s law plot of melphalan Fig.2.25. Ringbom plot of melphalan

for Method M14b for Method M14b

~ 48 ~

Table.2.01. Optimum Conditions Established in Method M2 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE

REMARKS

max (nm) 500 – 520 510 ----

Effect of volume of Fe(III) solution 0.5 – 1.5 ml 1.0 ml

Variation of volume below and above of this range gave

erratic results

Effect of volume of PHEN 0.5 – 1.5 ml 1.0 ml

Variation of volume below and above of this range gave

erratic results

Effect of heating time 25 – 40 min 30 min Below 25 min the colored complex was not completely

formed.

Effect of volume of O-phosphoric acid 1.0 – 3.0 ml 2.0 ml A minimum of 1.0 ml of o-phosphoric acid was required.

Nature of solvent for final dilution Distilled Water Distilled Water -------

Stability of the colored species after

final dilution. ----- 60min

The absorbance of the colored product decreased slowly after

60 min.

~ 49 ~

Table.2.02. Optimum Conditions Established in Method M4 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) 730 – 745 735 ----

Vol. of 2N FC reagent on color

development 1.0 – 2.0 ml 1.0 ml

Beyond upper and lower limits, low absorbance value or

precipitation was observed.

Effect of Vol. of NaOH solution on

color development 2.5 – 4.0 ml 3.0 ml

Maximum color development was achieved only in the

concentration range mentioned.

Effect of the order of addition of

reagents on color development Melphalan, NaOH, FC Melphalan, NaOH, FC The mentioned order of addition was required.

Keeping time prior to final dilution for

constant absorbance values 10 – 20 min 15 min

For getting maximum absorbance 5min, was necessary for

maximum color development.

Solvent for final dilution Distilled Water Distilled Water Distilled water has been found to be the best solvent.

Stability period after final dilution ---- 50 min ----

~ 50 ~

Table.2.03. Optimum Conditions Established in Method M7 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) 550 – 570 560 ----

Volume of vanillin

1.5 – 3.0 ml

2.0 ml

2.0 ml of Vanillin was necessary for covering broad range of

Beer’s law limits.

Effect of vol. of conc. H2SO4 on color

development

2.0 – 4.0 ml 3.0 ml Less than 3.0 ml of conc. H2SO4 results in low absorbance

values and greater than 3.0 ml results in instability of the

colored product.

Effect of the order of addition reagents

on color development.

Melphalan, Vanillin,

Conc. H2SO4

Melphalan, Vanillin,

Conc. H2SO4

If the order of addition is changed, low absorbance values

resulted.

Effect of temperature and time 40–500C, 10–20 min 50

0C,15 min. Above 50

0C methanol evaporates.

Stability period after final dilution ---- 45 min --

~ 51 ~

Table.2.04. Optimum Conditions Established in Method M8 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) 480 – 490 480 ---

Effect of NQS on color development 0.5 – 1.5 ml 1.0 ml The use of less than 0.5 ml NQS resulted in a decrease in

absorbance, greater than 1.0 ml resulted in cloudiness.

Effect of NaOH,0.02N on the absorbance

of the final colored species 0.5 – 1.5 ml 1.0 ml

Less than 0.5 ml and greater than 1.0 ml was found to

disturb Beer’s law obeyance in a broad range.

Solvent for final dilution Distilled water Distilled water Other than water miscible solvent did not enhance the

intensity of final colored solution.

Stability period after final dilution ---- 45 min Stable up to 45 min.

~ 52 ~

Table.2.05. Optimum Conditions Established in Method M9 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) 570 – 590 580 ----

Volume of Ninhydrin in acetone

required 1.5 – 2.5 ml 2.0 ml

2.0 ml of Ninhydrin was found to be necessary for color

product formation.

Time and temperature for maximum

color development

10 – 15 min in boiling

water bath

10 min in boiling

water bath

Heating in water bath for 10 min was required to obtain better

results in sensitivity & reproducibility.

Solvent for final dilution Acetone Acetone No advantage was observed with usage of other water

miscible solvents instead of Acetone.

Stability of the colored species ---- 60 min The absorbance of the colored product decreased slowly with

time after 1 hour.

~ 53 ~

Table.2.06. Optimum Conditions Established in Method M10 for Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) 520 – 530 525nm ----

Effect of volume of CA reagent 1.5 – 2.5 ml 2.0 ml Beyond 2.0 ml absorbance values increased.

Waiting time for the maximum color

development at laboratory temperature 25 – 35 min 30 min

Waiting time 30 min was necessary for the maximum color

development.

Solvent for final dilution Chloroform Chloroform Chloroform was found to be suitable for final dilution to give

better absorbance values.

Stability of colored species after final

dilution --- 50 min

After 50 min, the intensity of the colored product was found to

decrease slowly with time.

~ 54 ~

Table.2.07. Optimum Conditions Established for Methods M14a & M14b For Melphalan Determination

PARAMETER OPTIMUM RANGE CONDITIONS IN

PROCEDURE REMARKS

max (nm) M14a

M14b

610 – 620

610 – 630

615

620

--

Effect of buffer on color development 9.0 – 10.0 pH-9.8 Variations of the pH less than 9.0 and greater than 10.0

resulted in low absorbance values

Volume of buffer required for maximum

intensity of color (ml) 0.5 – 1.5 ml 1.0 ml

Optimum volume of 1.0 ml of buffer was sufficient for

maximum color development

Effect of volume of dye

MB (M14a)

MV(M14b)

0.1 – 1.0 ml

0.1 – 1.0 ml

0.5 ml

0.5 ml

0.5 ml of MB for M14a and MV for M14b dye was necessary

for covering the broad range of Beer’s law limits

Choice of organic solvent for extraction of

colored complex Chloroform Chloroform

Chloroform was preferred for its selective extraction of the

colored drug-dye complex from the aqueous phase.

Effect of shaking time (min) 1 – 5 min 2 min Constant absorbance values were obtained for the shaking

period of 1-5 min.

Stability of the colored species ----- 60 min

The colored species after separation from organic phase was

stable for 60 min, after wards the absorbance gradually

decreases.

~ 55 ~

Table.2.08. Results of Optical, Regression Parameters, Precision And Accuracy of the Proposed Methods for Melphalan

* Average of six determinations

PARAMETER M2 M4 M7 M8 M9 M10 M14a M14b

max (nm) 510 735 560 480 580 525 615 620

Beer’s law limits (g/mL) 5.0 – 25.0 4.0 – 20.0 2.0 – 10.0 4.0 – 20.0 2.0 – 10.0 5.0 – 25.0 2.0 – 10.0 2.0 – 10.0

Molar absorptivity (1 mol-1.cm-1) 3.699 x 103 4.589 x 103 1.350 x 103 8.320 x 103 1.246 x 104 4.862 x 103 7.164 x 103 1.201 x 104

Sandell’s sensitivity (g.cm-2/0.001 absorbance unit) 0.18767 0.161729 0.07875 0.1087735 0.083092 0.15555 0.120187 0.085144

Optimum photometric range (g/mL) 6.0 – 23.0 5.5 – 18.0 3.0 –8.0 5.0 – 18.0 2.5 – 9.0 5.5 – 22.5 3.0 – 9.0 3.5 – 8.5

Regression equation (Y=a+bc) ; slope (b) 0.0178 0.0150 0.0447 0.0276 0.0400 0.0159 0.0253 0.0402

Intercept (a) 0.0062 0.0012 0.0020 0.0014 0.0021 0.0029 0.0030 0.00112

Correlation coefficient (r) 0.9993 0.9999 0.9997 1.000 0.9994 0.9998 0.9993 0.9992

Standard deviation on intercept (Sa) 0.00219 0.00114 0.00681 0.00132 0.00451 0.00382 0.00344 0.00478

Standard deviation on slope (Sb) 0.000132 0.0000109 0.000102 0.00010 0.000690 0.000230 0.000518 0.000721

Standard error on estimation (Se) 0.00208 0.00109 0.00649 0.00649 0.00430 0.00364 0.00328 0.00456

LOD(g/mL) 0.377 0.230 0.4600 0.144 0.236 0.729 0.407 0.364

LOQ(g/mL) 1.23 0.76 1.52 0.478 1.12 2.40 1.35 1.18

Relative standard deviation (%)* 0.679 0.771 0.514 0.840 0.791 1.151 1.067 1.165

% Range of error (confidence limits)

0.05 level 0.567 0.645 0.430 0.703 0.661 0.962 0.892 0.974

0.01 level 0.840 0.954 0.363 1.040 0.978 1.424 1.320 1.441

~ 56 ~

Table.2.09. Assay and Recovery of Melphalan in Commercial Dosage Forms [ALKERAN]

METHOD PHARMACEUTICAL

FORMULATION

LABELED

AMOUNT (mg)

AMOUNT FOUND*

Mean S.D(mg)

% RECOVERY BY

PROPOSED METHOD

M2

ALKERAN

2.0

1.99+0.173 100.00

M4 1.99+0.153 100.00

M7 1.98+0.227 99.49

M8 1.98+0.192 99.49

M9 1.98+0.181 99.49

M10 1.99+0.201 100.00

M14a 1.98+0.124 99.49

M14b 1.99+0.147 100.00

* Average of six determinations.

~ 57 ~

2.2.5. CONCLUSIONS:

Eight new simple, sensitive and economical visible spectrophotometric methods

were developed for the determination of melphalan in pure and dosage formulations

using Fe(III)-PHEN, FC, Vaniline, NQS, Ninhydrin, PCA, MB and MV as reagents.

The developed visible spectrophotometric methods for the determination of melphalan

were simple, sensitive and economical.

Melphalan possess different functional moieties such as α-amino acid and

aromatic 3o- nitrogen of varied reactivity. Eight proposed methods are based on the

reactivity of presence of several substituent’s i.e. reducing behaviour [Fe III/o-PHEN

(M2), FC (M4)], condensation reaction (due to the presence of amine) [VANILLINE

(M7)], substitution [NQS (M8)], charge transfer interaction (due to the presence of

tertiary nitrogen) [PCA (M10)], condensation [NH (M9)] and neutral ion association

complex (due to complex formation between negatively charged carboxylic acid on the

drug and basic dye i.e., protonated amino group in the dye, which can be extractable

in to chloroform) [MB (M14a), MV (M14b)]. The chemistry of the colored species

formed in each proposed method for the assay of melphalan has been presented in

respective schemes

The λmax of each proposed method for melphalan analysis was determined

by taking scans of the drug sample solutions in the visible region and was found to be

that only one peak was observed for each proposed method with maximum absorption,

that in turn was used for the development of calibration plots. The optical regression

parameters of each developed methods were calculated and were represented in the

corresponding table of the current section. All the calibration curves of the proposed

methods showed a good linear relationship between the absorbance and concentration

~ 58 ~

and coefficient correlation was higher than 0.9999. The obtained results of recovery

studies (%R.S.D) demonstrate the validity and accuracy of the proposed methods.

Finally, it is concluded that “the proposed methods developed in the present

study can be used as alternative methods and can be successfully applied to the

determination of melphalan in pharmaceutical preparations without any interference

from the excipients”.

~ 59 ~

2.3. RP-HIGH PRESSURE LIQUID CHROMATOGRAPHIC

METHOD FOR DETERMINATION OF MELPHALAN

Literature survey reveals that few HPLC methods (Brightman et al, 1999;

Silvestro et al, 1991; Nath et al, 2008; Chang et al, 1978, Rolf et al, 2003) and one LC-

MS (Mirkou et al, 2009) method have been reported for the estimation of melphalan in

pharmaceutical formulations and biological samples. The present section describes the

development of a simple RP-HPLC method using C18 column for determination of

melphalan in pure and tablet forms. The method was validated as per ICH guidelines

(2005).

2.3.1. EXPERIMENTAL:

2.3.1.a. Instrumentation:

The developed and validated method for determination of melphalan was

performed on an isocratic HPLC system (PEAK) consisting of isocratic liquid pump,

LC 8200 variable wavelength UV detector with Millennium® version 32 software on a

Dell computer. The analytical column used to achieve chromatographic separation was

a stainless steel ODS, C18 RP-Column (4.6mmx250mm) purchased from Waters

Corporation (Bedford, MA, USA) protected by a guard column of the same material.

2.3.1.b. Materials:

The pharmaceutical grade pure sample of melphalan (99.28%) was procured from

Celon Laboratories Limited, Andhra Pradesh, India. Acetonitrile solvent of HPLC

grade was obtained from E-Merck Ltd, Mumbai, India. Orthophosphoric acid of AR

~ 60 ~

grade was procured from Qualigens Fine chemicals, Mumbai, India. The HPLC grade

water was obtained from a Milli-Q RO water purification system.

2.3.1.c. Preparation of stock and working solutions:

An accurately weighed sample of 10 mg of melphalan was dissolved in 100 ml

methanol to give standard stock solution of 100 μg/ml. A series of working standard

solutions (2.0 μg/ml – 14 μg/ml) were obtained by diluting the stock solutions with

mobile phase (acetonitrile, water and 1% ortho phosphoric acid in the ratio of 70:27:3

v/v/v). All the volumetric flasks containing melphalan were wrapped with aluminium

foil and stored in the dark.

2.3.2. RESULTS AND DISCUSSION:

2.3.2. a. Method Development and Chromatographic conditions:

The development of HPLC methods for the determination of drugs has received

great attention in analytical research because of their importance in quality control

(ICH, 2005). Parameters such as detection of flow rate, wavelength, ideal mobile phase,

and concentration of the standard solution were studied.

Solvent and column selection:

Amongst popular and important solvents useful for separations of

pharmaceuticals, acetonitrile is one of the solvent of choice because of its

distinguishing properties viz., minimal chemical reactivity, low acidity and a

reasonably low boiling point. Further it is considered to be ideal for RP-HPLC

application in view of its (a) miscibility with water to form water mixtures with broad

range of polarities (b) low viscosity yielding low pressure drop (c) low UV absorption

cut-off (Kaljurand and Koel, 2011). Hence, in the present study, for the selection of

mobile phase, combination of acetonitrile with other solvent (water) and a modifier

~ 61 ~

(phosphoric acid) was tested. In the present study, ODS C18 analytical column was

chosen as the stationary phase due to excellent selectivity and low cost (Peng Zhang

and Xiu-Wei Yang, 2010).

Significance of orthophosphoric acid:

Melphalan has two ionisable groups viz., carboxylic and amino groups, and

hence, in RP-HPLC, the retention time of melphalan can be changed by modifying pH

of the mobile phase. The degree of ionization of melphalan is dictated by pH of the

mobile phase and in addition, the operating pH of the mobile phase should be

maintained below 7.5 to sustain the stability of silica based stationary phase. Below pH

7.5, the amino group in melphalan will be in cationic form, whereas, the carboxylic

acid group will be neutralized. In the present case, the pH of the medium is maintained

acidic by the addition of orthophosphoric acid in order to suppress the ionization of

carboxylic group of melphalan. The degree of ionization and hence change in retention

behavior of drug takes place by varying the concentration of H3PO4 in the mobile

phase. The ion exchange interaction between positively charged amino group on

melphalan and negatively charged silanol groups on column will result in ‘mixed mode

retention’. It is a very well known fact that, mixed mode retention increases retention

time as well as broadens peak (Prathap et al, 2013). Such a mixed mode retention effect

is abolished in RP-HPLC by the addition of H3PO4 which causes ion suppression or

maintain silanol groups in unionized form. This yields narrow and symmetrical peak.

In the column, cleavage of siloxane linkage and silica dissolution can be observed

when the pH of mobile phase is below 2.0 and above 8.0 respectively. Hence, it is

essential to maintain the pH of the mobile phase in the range of 2.0 to 8.0 (Snyder et al,

2001; Nledner et al, 2008).

~ 62 ~

Absorption maximum for melphalan:

UV detection for melphalan in other methods was performed mostly in the

range of 250 – 262 nm (Silvestro et al, 1991; Nath et al, 2008; Chang et al, 1978;

Fedric Pinguet et al, 2010; Rolf et al, 2003), whereas in this method it was at a higher

wavelength, i.e., 275 nm which can be explained based on the higher polarity of the

mobile phase. It is well known that in presence of a polar solvent, the more polar *

orbital will be highly stabilized than the orbital leading to a net decrease in the

transition energy, which result in an increase in transition wavelength or a

bathochromic shift / red shift (Kemp, 1991).

Chromatographic conditions:

The mobile phase was filtered by passing through a 0.45 μm membrane filter

(Millipore, Bedford, MA, USA). Chromatographic analysis was carried out at ambient

temperature. The injection volume was 20 μl. Several tests were performed in order to

get satisfactory separation-resolution of melphalan in different mobile phases with

acetonitrile, water in various ratios (20:80, 40:60, 50:50, 60:40, 80:20) by using C18

column. But, the compounds were separated isocratically with a mobile phase

consisting of acetonitrile, water and 1% ortho phosphoric acid in the ratio of 70:27:3

(v/v/v). The effluent was monitored spectrophotometrically at a wavelength of 275nm.

The retention of melphalan on analytical column was evaluated at a flow rate of 1.0

ml/min. The typical chromatogram of sample solution of melphalan is shown in

Fig.2.26, P.63. The optimized chromatographic conditions for the determination of

melphalan are represented in Table.2.10, P.63. The developed method had a short

retention time (4.57 minutes) compared to 8 – 15 min reported by earlier authors

(Silvestro et al, 1991; Brightman et al, 1999; Rolf et al, 2003 and Nath et al, 2008).

~ 63 ~

Fig.2.26. HPLC chromatogram of melphalan

Table.2.10. Optimized Chromatographic Conditions

for the Determination of Melphalan

Elution Isocratic

Mobile phase Acetonitrile: water : 1 % ortho phosphoric acid (

70:27:3 v/v/v)

API Concentration 10 µg/ml

Column ODS C-18 RP ( 4.6 mm i.d x 250 mm)

Flow rate 1 min/ ml

Detection UV at 275 nm

Injection volume 20 micro liters

Temperature Ambient

Retention time 4.57 minutes

Run time 10 minutes

Area 768173.3 mAU

pH 4.8

Theoretical plates 3978

Pressure 20-25 Mpa

Tailing factor 1.17

~ 64 ~

2.3. b. METHOD VALIDATION:

i. Linearity:

The linearity for HPLC method was determined by determining response factor at

seven concentration levels ranging from 2.0–14.0μg/ml for melphalan (Fig.2.27-2.33,

P.64–66). The calibration curve was constructed by plotting response factor against

concentration of melphalan. The calibration curve showed good correlation between

concentration and peak area with a regression coefficient (r2) of 0.9985. The regression

equation for the calibration curve (Fig.2.34, P.67) was Y = 766518X + 25066.37 for

melphalan, where Y represents the peak area of analyte and X represents analyte

concentration. The results revealed that significant correlation exists between response

factor and concentration of drug within the concentration range indicated on Y-axis

(Table.2.11, P.67).

Fig.2.27. Linearity chromatogram of melphalan (2.0µg/ml)

~ 65 ~

Fig.2.28. Linearity chromatogram of melphalan (4.0µg/ml)

Fig.2.29. Linearity chromatogram of melphalan (6.0µg/ml)

Fig.2.30. Linearity chromatogram of melphalan (8.0µg/ml)

~ 66 ~

Fig.2.31. Linearity chromatogram of melphalan (10.0µg/ml)

Fig.2.32. Linearity chromatogram of melphalan (12.0µg/ml)

Fig.2.33. Linearity chromatogram of melphalan (14.0µg/ml)

~ 67 ~

Fig.2.34. Calibration curve of melphalan

ii. Precision:

The precision of the method was demonstrated by interday and intraday

variation studies. In the intraday studies, six repeated injections of standard solution

were made and the response factor of drug peaks was measured, and then % RSD

values were calculated. For inter-day variation studies, six repeated injections of

standard solutions were made for three consecutive days and response factor of drug

peaks was measured, and % RSD was calculated (Table.2.12, P.68). From the data

obtained, the developed RP-HPLC method was found to be precise. The percentages of

Table.2.11. Calibration of the RP-HPLC

for the estimation of Melphalan

Concentration in µg/ml Area (mAU)

0 0 2 168687.1

4 335787.0

6 502576.6

8 643699.3

10 782350.3

12 914253.3

14 1120622.0

Regression equation

Slope (a)

Intercept (b)

Correlation coefficient

Y = a X + b

76651.98

25066.37

0.9985

~ 68 ~

RSD for precision of the proposed method were found to be in the ranges 0.128 –

0.289, which was very much within the allowable limit of 2% (Isabel et al, 2004).

Table.2.12. Precision Data for the Determination of Melphalan

DAY PRECESSION AREA MEAN* % R.S.D.

Day-1 944509.0 0.289

Day-2 946797.7 0.128

Day-3 945650.9 0.206

*All the values are the averages of six determinations

iii. Sensitivity:

The Limit of Detection (LOD) was determined as lowest concentration giving

response and Limit of Quantification (LOQ) was determined as the lowest

concentration analyzed with accuracy method. These were determined by injecting

progressively low concentrations of the standard solutions using the developed RP-

HPLC method. The Limit of Detection (LOD) and the Limit of Quantification (LOQ)

for melphalan was found to be 0.5 µg/ml and 1.5 µg/ml respectively.

iv. Recovery studies:

Recovery studies were carried out by adding different amounts (50%, 100%, and

150%) of bulk samples of melphalan to 4 μg/ml so that overall concentration will be

within the linearity range. The accuracy was expressed in terms of percent recovery.

Their chromatographs are represented in Figs.2.35-2.37, P.69. The mean of percentage

recovery value was 98.65 (Table 2.13, P.69). The results were given in Table 3.2.4.

The statistical analysis of data obtained for the estimation of melphalan indicates a high

level of accuracy for the proposed method.

~ 69 ~

Fig.2.35. Accuracy chromatograms for melphalan (50%)

Fig.2.36. Accuracy chromatograms for melphalan (100%)

Fig. 2.37. Accuracy chromatograms for melphalan (150%)

Table.2.13. Recovery studies of the proposed HPLC method

for the Determination of Melphalan

AMOUNT

TAKEN

µg/ml

AMOUNT ADDED

µg/ml

TOTAL AMOUNT

µg/ml

AMOUNT

FOUND (µg/ml)* % RECOVERY MEAN

4 2 6 5.775 96.25%

98.65% 4 4 8 7.9132 98.91%

4 6 10 10.088 100.8%

* All the values are the averages of three determinations

~ 70 ~

v. Ruggedness & Robustness:

Ruggedness test was determined between two analysts, instruments and columns.

Robustness of the method was determined by small deliberate changes in flow rate,

mobile phase pH and mobile phase ratio. The content of the drug was not adversely

affected by these changes as evident from the low value of relative standard deviation

indicating that the method was rugged and robust.

vi. Estimation of melphalan in Tablet Dosage Form:

An RP-HPLC chromatogram with good shape and resolution was obtained for the

contents of melphalan tablet (ALKERAN Tablet) by following the above developed

method (Fig.2.38, P.71). For analysis of dosage formulations, average weight of ten

ALKERAN tablets was determined. The tablets were ground and mixed well. The

powder of the sample equivalent to 10 mg of melphalan was accurately weighed and

transferred into a 10 ml volumetric flask. About 7 ml of methanol was added, sonicated

to dissolve it completely and made the volume up to the mark with diluent. Mixed well

and filtered through Whatmann filter paper. An aliquot equivalent to 100 μg of the

sample was pipetted into a 10 ml volumetric flask and made up to the mark with the

mobile phase after filtration. From the absorbance value, the drug content per tablet

was calculated (on an average weight basis) and the results were tabulated (Table.2.14,

P.71). Good recovery values of drugs show that the proposed method can be

successfully applied to the determination of melphalan in pharmaceutical formulations

without any interference from common excipients.

~ 71 ~

Item 3, 4 are excipients that did not interfere with melphalan

Fig.2.38. Validation chromatogram of commercial formulations [Melphalan]

Table.2.14.Results of analysis of Melphalan Recovery studies

PHARMACEUTICAL

FORMULATION

AMOUNT OF

MELPHALAN (mg) % RECOVERY

LABELLED FOUND

ALKERAN 5.0 4.97 99.4 %

All the values are the averages of three determinations

~ 72 ~

2.3.5. CONCLUSIONS:

The results of optimization studies shows that the mixture of acetonitrile: water:

1% ortho phosphoric acid (70:27:3 v/v/v proportions) gave satisfactory results. The pH

of mobile phase was 4.8, the optimized flow rate was found to be 1.0 ml/min and the

retention time was found to be 4.57 minutes with the total runtime of 10 minutes. The

system suitability parameters such as tailing factor and percentage relative standard

deviation (%RSD) were found to be well within the acceptable limits.

A calibration curve was constructed and found to be linear within the range of 2

to 14 µg/ml. The correlation coefficient of calibration curve was 0.9995 for Melphalan

which indicates a very good correlation between the concentration and area under the

curve. In precision studies, percentage of relative standard deviation (%RSD) was

found to be less than 2% for within day and day to day variations which proves that the

method is precise. The recovery studies were performed by standard addition method in

which 50%, 100% and 150% of known amounts of standard melphalan were added to

pre-analyzed sample and were subjected to the proposed HPLC method. The results

showed good recovery ranging from 96.25 to 100.8% with a mean value of 98.65%.

The LOD and LOQ were found to be 0.5 µg/ml and 1.5 µg/ml respectively.

The method was found to be accurate, reproducible, specific and rapid, no

interfering peaks were observed at the elution times of drug. The method may be

applied for the estimation of melphalan in the bulk and in the pharmaceutical

formulation without interference and with good selectivity and sensitivity than many of

the reported methods.

~ 73 ~

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