STABILITY INDICATING ASSAY METHOD AND IMPURITY …shodhganga.inflibnet.ac.in/bitstream/10603/9714/11/12... · 2015. 12. 4. · Chapter 4: Stability indicating assay and impurity profiling

Chapter 4: Stability indicating assay and impurity profiling of MIL

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Chapter 4

STABILITY INDICATING ASSAY

METHOD AND IMPURITY PROFILING

OF MILNACIPRAN HYDROCHLORIDE


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4. STABILITY INDICATING ASSAY AND IMPURITY PROFILING OF

MILNACIPRAN HYDROCHLORIDE (MIL)

The simple, sensitive, and isocratic RP-HPLC method is described for the determination of

active content and related impurities of MIL. RP-HPLC method was developed with objective to

separate all the degradation products formed in forced degradation studies and related impurities

of MIL in API. Subsequently, exhaustive forced degradation studies were carried out as per the

ICH guidelines to study the degradation behavior of MIL. The developed RP-HPLC method

could separate all the degradation products formed in the stress studies and could also separate,

detect and quantify the related impurities present in the MIL. In the stress studies, it was found

that MIL was stable in thermal and photolytic stress degradation conditions and 8.6, 11.8, 4.2

and 28.5 % degradation was observed in acid, alkaline, neutral, and oxidative hydrolytic stress

conditions respectively. The developed and optimized method was further validated as per ICH

prescribed guidelines and the results were within the range of acceptance criteria. Subsequently,

the applicability of proposed method was proved by using the method for the estimation of active

content and related impurities of MIL in pharmaceutical capsule formulations.

Another stability indicating HPTLC method was also developed and validated for the

determination of MIL. The developed method could also separate degradation products formed

in the stress degradation conditions with sufficient difference in their RF values. The developed

method was also validated according to ICH guidelines. The method was successfully applied for

the estimation of MIL in different pharmaceutical capsule formulation. Both the developed

methods (RP-HPLC and HPTLC) were statistically compared for the assay results of MIL and

found that there was no significance difference between the results obtained. Thus, any method

can be used in routine analysis of MIL.

During the impurity analysis of MIL, one impurity (MIL-IMP) was obtained API and capsule

formulations of MIL in concentration of 0.08 and 0.12 % respectively. It was found that, MIL-

IMP was same as neutral degradation product obtained in stress degradation studies which was

further targeted for isolation and characterization. Isolation of MIL-IMP was carried out by

suitable prep-HPLC method and further characterization was done using different spectroscopic

techniques like UV, FT-IR, Mass, and NMR spectroscopy. From the results of all spectroscopic

studies, the structure of the isolated impurity was proposed as 1-phenyl-3-azabicyclo [3.1.0]


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hexan-2-one. The mechanism through which this impurity may have generated from MIL is also

proposed.

The stress degradation behavior of MIL in different stress conditions was evaluated with the help

of LC-MS/MS study. LC-MS analysis was used to find out molecular weight of each degradation

product. The proposed structures of degradation products were then confirmed from further

fragmentation pattern studies of MIL and its each degradation product with the help of MS/MS

studies. Finally, the probable mechanisms was proposed which also confirm the generation of

formed degradation products in different hydrolytic stress degradation conditions which helped

in prediction of degradation pathways of MIL.


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4.1. Chemicals and materials

Analytically pure (99.9 %) Milnacipran Hydrochloride (MIL) Active Pharmaceutical

Ingredient was procured from Torrent Pharmaceutical Ltd., (Ahmedabad, India), along

with Certificate of Analysis.

Methanol, acetonitrile, potassium dihydrogen phosphate, ammonium acetate,

orthophosphoric acid, glacial acetic acid, formic acid, triethylamine and ammonia used for

mobile phase preparation were of HPLC grade, Merck, Mumbai, India

Chloroform, ammonia, hydrochloric acid, sodium hydroxide and hydrogen peroxide (30 %

w/v) used for solvent preparation and stress degradation studies are of analytical reagent

grade, CDH Chemicals, Delhi, India.

De-ionized water prepared using Milli-Q plus purification system Millipore (Bradford,

USA) was used throughout the study.

The membrane filters 0.22 µm and syringe filters 0.45 µm used for mobile phase filtration

and sample filtration were supplied by Millpores Ltd. Bangalore.

All the glasswares including volumetric flask, pipette, measuring cylinder, beaker were of

Class A borosil glass.

Calibrated micropittes were used for purpose for measurement and transfer.

The description of two capsule formulations of MIL is given in Table 4.1

TABLE 4.1 Detail information about MIL capsule formulations

Sr.

No.

Name of brand and its

manufacturer

Label

claim

(mg)

Net

content

(mg)

Batch

No.

Manufac

turing

date

Expiry

date

A

Milnace 50; Torrent

Pharmaceuticals Ltd.

Ahmedabad, India

50 101.4 CC96

002 06/2008 06/2011

B

Milborn 50; Sun

Pharmaceutical Ltd. Baroda,

India

50 148.2 GK90

988 05/2008 05/2011


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4.2. Equipments/ Instruments

Following is the list of equipments and instruments used throughout the project work.

All the instruments were calibrated periodically as per in house SOP of Department of

Pharmaceutical Analysis, Institute of Pharmacy, Nirma University.

Melting point apparatus, T0603160; EIE Instruments Pvt Ltd., Ahmedabad India

Analytical balance, CX220, Citizen, USA.

Ultrasonicator, D-compact, EIE instrument Pvt. Ltd., Ahmedabad

Digital pH meter, PH-MV-TEMP. Meter, LTLUTRON, Taiwan

UV-visible spectrophotometer, Double beam, UV- 2450, Shimadzu, Japan

Fourier Transform Infrared spectrometer (FT-IR), JASCO FT/IR-6100 series (Jasco, Japan).

High precision water bath (for degradation assembly), EIE instrument Pvt. Ltd., Ahmedabad

India

Hot air oven, EIE 108, EIE Instruments Pvt. Ltd., Ahmedabad India

Temperature and Humidity chamber, EIE Instruments Pvt Ltd., Ahmedabad India

High Performance Liquid Chromatography (HPLC), consisted of Binary Pumps -Jasco PU-

2080 and Solvent Mixing Module-Jasco MX-2080, Rheodyne loop injector with 20 µL fixed

loop ,equipped with Photo Diode Array (PDA) Detector MD-2015 Plus (Jasco Japan), with

BORWIN Software for the data acquisition and data collection.

Preparative-High Performance Liquid Chromatography (Prep-HPLC), Shimadzu Prep-

HPLC system (Kyoto, Japan) was equipped with Binary pumps LC6AD/7A pump, PDA

detector SPD-M20A and Rheodyne loop injector (100 µL) with Shimadzu CLASS VP software.

A Phenomenex C18 semi preparative column (10 µm, 250 x 10 mm id; Torrance, USA) was

used for sample loading.


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Rotary Evaporator, Rota vapor R-200, Buchi Labortechnik, Switzerland was used for the

evaporation and concentration of eluents.

High Performance Thin Layer Chromatography, (Camag; Muttenz, Switerzerland) system

consists of Linomat V sample applicator fitted with 100 µL Applicator syringe (Hamilton,

Bonadauz, Switzerland). With Camag TLC scanner III operated in reflectance-absorbance

mode and controlled by WINCATS software; Visual detection of spots was carried out by

Camag UV cabinet with dual wavelength UV lamp (254 nm and 366 nm). The Camag Twin-

trough Chambers were used throughout.

Liquid Chromatography-Mass Spectroscopy, LC-MS/MS studies were carried out on a

system in which LC part consisted of Varian Prostar HPLC, comprising of an on-line degasser,

binary pumps (Prostar 210), with auto injector and PDA detector (Prostar 335). The MS system

consisted of Varian 500 with ion trap mass analyzer (Varian, USA). The data was collected and

processed using System Control software.

Mass spectroscopy, The MS system consisted of API 2000 Q-Trap Mass Spectrometer (Applied

Bio systems, PerkinElmer, Germany). The data was collected and processed using ANALYST

software.

NMR spectroscopy, The 1H and

13C NMR experiments were carried out at processional

frequencies of 300 MHz and 75 MHz, respectively in CDCl3 at 25 °C temperature on BRUKER

NMR (Bruker Ltd. Switzerland).

CHN-O analyzer, (Thermo, Finnigan) was used for elemental analysis equipped with thermal

conductivity detector and data was collected and processed with Egger software.


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4.3. Identification of Milnacipran Hydrochloride (MIL)

The identification of procured sample of MIL was carried out by following methods

1 Melting point determination

2 UV-VIS spectroscopy

3 FT-IR spectroscopy

4 Mass spectroscopy and

5 NMR Spectroscopy (1H and

13C)

4.3.1. Melting point determination

Determination of melting point of MIL was carried out using melting point apparatus using open

capillary method.

TABLE 4.2 Comparison of melting point of MIL with reported melting point

Drug Reported melting point [1] Observed melting point

MIL 179 -181 (ºC) 180 -182 (ºC)

4.3.2. UV spectroscopy

UV spectrum of MIL (20 µg/mL) in methanol was taken and scanned in the range of 200-400 nm

on UV spectrophotometer.

FIGURE. 4.1 UV-spectra of methanolic solution of MIL (20 µg/mL)


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TABLE 4.3 Comparison of reported λmax with obtained λmax of MIL

Drug Reported λmax [2] Obtained λmax

MIL 220 nm 220.0 nm

4.3.3. FT-IR Spectroscopy

FT-IR spectrum of MIL was recorded in diffused reflectance mode. Theoretical wave numbers

responsible for functional groups are compared with observed wave numbers and presented in

Table 4.4.

FIGURE 4.2 FT-IR spectra of MIL

TABLE 4.4 Important frequencies of MIL obtained in FT-IR spectra

Sr.

No. Functional group

Theoretical frequency

(cm-1

) [3-4]

Observed

frequency (cm-1

)

1 Amines (-NH2) Str. 3500-3100 3254.97, 3232.11

2 Methyl (-CH3) Str. 3000-2850 2810.74

3 Carbonyl Amide (R2-N-C=O) Str. 1680-1630 1613.18

4 Amine (C-N) Str. 1350-1000 1149.37

5 Monosubstituted Benzenes OPB 900-690 736.97


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4.3.4. Mass spectroscopy

The MS and MS/MS of MIL were performed on API 2000 Q-Trap Mass Spectrometer as

described in section 4.2. The analysis was performed in positive ionization mode with turbo ion

spray interface. The data was collected and processed using Analyst software. The parameters

for Ion Source, IS = 45,00 V, Declustering Potential, DP = 20 V, Entrance Potential, EP = 10 V

were set with nebulizer gas as air at a pressure of 25 psi and curtain gas as nitrogen at a pressure

of 35 psi. The MS and MS/MS spectra of MIL are shown in Figure 4.3 and 4.4 The (M+1) peak

was obtained at 247.3 m/z which confirms molecular weight of MIL at 246.0. Figure 4.4

represents daughter ions of MIL at different m/z. The fragmentation pattern of MIL is proposed

in Figure 4.5.

FIGURE 4.3 Full scan MS spectra of MIL


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FIGURE 4.4 MS/MS spectra of MIL at molecular peak of 247.3

CH2

NH2

C

O

N

CH2

CH2

MILm/z 247

H

CH3

CH3

C

O

N

H2C

CH2

CH3

CH3

CH2 CH2

m/z 230 m/z 202

CH2

NH

C

O

N

CH2

CH2

CH3

CH3

m/z 100

m/z 174

HNOC

CH2H3C

CH2

m/z 157

OC OC

CH2

m/z 129m/z 117

FIGURE 4.5 Proposed fragmentation pattern of MIL from MS/MS spectroscopic studies


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4.3.5. NMR spectroscopy

The 1H and

13C NMR experiments on MIL were carried out on NMR instrument as described in

section 4.2.15. The 1H and

13C chemical shifts were reported on the δ scale in ppm, relative to

tetra methyl silane (TMS) at δ 0.00 in 1H NMR and CDCl3 at 77.0 ppm in

13C NMR,

respectively.

FIGURE 4.6 1H NMR spectra of MIL


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FIGURE 4.7 13

C NMR Spectra of MIL


55

CH2

NH2

C

O

N

CH2

CH2

H3C

H3C

12

34

5

6

7

8

9

10

11

12

13

14

1516

1718

HCl

MIL

TABLE 4.5 1H NMR and

13C NMR spectral assignments for MIL

1H NMR

13C NMR

Position Chemical Shift

(ppm)

Coupling Constant

(Hz) Position

Chemical

Shift (ppm)

2 1.18 (t) 10.5 1 25.23

3a 3.67 (d) 10.5 2 34.54

3b 2.54 (t) 10.8 3 17.98

7,11 7.30 (m) -

8-10 7.20 (m) - 4 170.45

12 1.78 (m) - 6 138.23

13 8.77 (s) - 7,11 128.77

15,17 3.29 (m) - 8,10 125.60

16 0.88 (t) 6.9 9 127.0

18 1.18 (t) 6.9 12 42.80

15 39.40

16 12.11

17 41.80

18 12.85


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4.4. Stability Indicating Assay and Impurity Profiling of MIL by RP- HPLC

4.4.1. Experimental

4.4.1.1. Chromatographic conditions

Following chromatographic conditions were optimized and were kept constant throughout the

analysis.

Column: C18 PUROSPHERE STAR Hyber 250 × 4.5 mm i.d., with 5 µm particle size.

Mobile phase: Buffer: Acetonitrile (72:28 v/v).

Buffer preparation: 0.0125M potassium dihydrogen orthophosphate; add 0.3 % ammonia and

adjust the pH of buffer to 3.65 ± 0.02 with 1 M orthophosphoric acid.

Flow Rate: 1.0 mL/min; Detection wavelength: 220 nm; Injection volume: 20 µL.

4.4.1.2. Preparation of solutions

Standard and sample solutions: The standard stock solution 1 mg/mL was prepared by

dissolving accurately about 100 mg of MIL with methanol in 100 mL volumetric flask. The 20

µg/mL and 200 µg/mL concentration was prepared after diluting aliquots from the stock solution

with diluent (water: acetonitrile 50:50, v/v) for stability indicating assay and impurity study of

MIL, respectively.

Diluted standard solution: From the working standard stock solution (200 µg/mL) of MIL,

aliquots were diluted suitably with diluent to achieve the concentration of 0.2 µg/mL of MIL

which was used as diluted standard preparation.

Sample solution for assay and related impurities of MIL marketed capsule formulations:

Twenty capsules weighed and their net content was determined. Capsule powder equivalent to

about 50 mg MIL for each brand was accurately weighed and transferred to a 100 mL volumetric

flask with addition of about 80 mL of methanol. The mixture was sonicated for 20 min with

occasional shaking, and volume was made up to the mark with methanol. The above solutions

were centrifuged in centrifuge tubes at 2500 RPM in the Research Centrifuge for 15 min and

were filtered through 0.45 µm syringe filter. The first 10 mL of filtrate was rejected. Aliquots of

remaining filtrate was further diluted with diluent to obtained the solution having concentration

of 20 and 200 µg/mL for assay and related impurities method respectively.


57

4.4.1.3. Stress (forced) degradation studies [5-7]

The stress degradation studies were carried out as per ICH guidelines, by forcibly

degrading MIL under different stress conditions such as hydrolytic, oxidative, dry heat

(thermal) and light exposure.

The stress studies were carried out by preparing MIL solution of 2 mg/mL in respective stressors

as described in Table 4.6. A minimum of four samples were generated for every stress condition,

viz., Initial (zero time) sample containing the drug with stressor and the drug solution subjected

to stress treatment, the blank solutions stored under normal conditions, and the blanks subjected

to identical conditions. The detail of stress degradation conditions applied and optimized is given

in Table 4.6

TABLE 4.6 Optimized stress degradation studies conducted on MIL

Stress degradation conditions Stressor

Acid hydrolysis 1 N HCl, reflux at 100 °C for 4 h

Alkaline hydrolysis 0.1 N NaOH, reflux at 100 °C for 4 h

Neutral hydrolysis Water, reflux at 100 °C for 4 h

Oxidative degradation 3 % H2O

2 reflux at 100 °C for 4 h

Thermal degradation Drug powder kept in hot air oven at 120°C for 48 h

Photolytic degradation solution state aqueous solutions were exposed to direct sunlight

for 8 h in total two days

Photolytic degradation solid state drug powder was exposed to direct sunlight for 8 h

in total two days

Accelerated stability study Drug powder kept in temp. and humidity chamber at

40 ºC and 75 % RH for 1 month

Preparation of forced degraded samples

After exposure of MIL to all stress degradation conditions, the stress study samples were

prepared for RP-HPLC analysis. The hydrolytic and solution state photolytic samples were

suitably diluted with diluent to get concentration 20 µg/mL. Acidic and alkaline hydrolytic

stressed samples were appropriately neutralized with equimolar concentrations of NaOH and

HCl prior to injecting on HPLC. The methanolic stock solutions of thermal and accelerated

stability stress study samples were prepared to get the concentration of 2 mg/mL and were

suitably diluted

with diluent to get concentration 20 µg/mL. All the above samples were

analyzed on optimized RP-HPLC method as described in section 4.4.1.1. along with their

respective initial samples and blanks stored at 25 ºC as described above. All the samples were


58

allowed to run till the 2.5 times of the retention time of MIL. The response of MIL obtained in

every stress conditions were compared with the responses of respective initial samples and the

degradation of MIL was reported in terms of % degradation.

4.4.1.4. Method validation

The optimized stability indicating assay method and related impurities method for MIL were

validated as per ICH [8] and USP [9] recommended guidelines for following parameters.

1. System suitability

2. Linearity and range

3. LOD and LOQ

4. Specificity

5. Precision

A. Method precision (Repeatability)

B. Intermediate precision (Ruggedness)

6. Accuracy (Recovery)

7. Robustness

8. Solution stability


The system suitability test was performed to ensure that the complete testing system was suitable

for the intended application and it was performed by injecting five replicate injections of

standard preparation of MIL. The parameters measured were retention time, peak area,

theoretical plates, and asymmetry of MIL (standard solutions prepared as described in section

4.4.1.2.).


For establishment of linearity of MIL by stability indicating assay method, the calibration curve

was obtained at seven levels in the concentration range of 5-50 µg/mL for MIL respectively.

For related impurities method, linearity was determined over the range of LOQ to 200 % of the

specification limit. (LOQ is the reporting threshold as specified by ICH guidelines (i. e. 0.05 %).

The sample solutions for linearity of MIL were prepared by making the dilution given in Table


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4.7. Samples at each linearity level were analyzed in triplicate as described in section 4.4.1.1 and

the response was measured in the form of area under the curve of MIL.

TABLE 4.7 Linearity study of MIL (Unknown impurity)

Linearity level Volume (mL) taken

from standard stock a

Diluted with

diluent (mL)

Conc. of the solution

(µg/mL)

I (LOQ) 0.1 200 0.1 (0.05 %)

II 0.075 100 0.15

III 0.1 100 0.2

IV 0.15 100 0.3

V 0.175 100 0.35

VI 0.2 100 0.4 a Concentration of stock solution: 0.2 mg/mL of MIL (Standard weights 100.01 mg, 100.00 mg

and 99.99 mg)

3. Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were calculated from standard deviation of the response and the slope values of

the three linearity curves using the formula 3.3 α/S for LOD and 10 α/S for LOQ, where α is

standard deviation of response and S is mean of slope of three calibration curves.

Precision at LOQ: The theoretically obtained value of LOQ was verified by injecting six

replicates at this concentration (prepared by making dilutions from standard solutions) and

reporting the RSD value of mean area at LOQ concentration.

4. Specificity

Specificity is ability of an analytical method to measure the analyte free from interference due to

blanks (diluent and mobile phase) and degradation products formed in forced degradation studies

and was performed as described in section 4.4.1.3.

5. Precision

The precision of an analytical method is the closeness of agreement (degree of scatter) between

series of measurements obtained from multiple samplings of the same homogeneous sample

under the prescribed conditions.

A. Method precision (Repeatability)

For repeatability study, six sample sets were prepared by individually weighing accurately about

100 mg of MIL in different volumetric flasks to get concentration of 1.0 mg/mL and were further


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diluted with diluent individually to get concentration of 20 µg/mL. All the samples were

analyzed as described in section 4.4.1.1. The response obtained from each sample was

extrapolated to find out the mean assay value with RSD.

In related impurities method, the six sample sets were prepared having concentration of 200

µg/mL from above solutions and were analyzed as described in section 4.4.1.1. The responses of

impurities detected in each sample sets were measured and % of individual and total impurities

in each sample set was calculated. The mean of total impurities in six samples sets was found

with RSD.

B. Intermediate precision (Ruggedness)

The intermediate precision study was performed at three different levels i.e. intraday, interday,

and different analysts precision.

For intraday and interday precision studies, the samples were prepared and analyzed as described

in repeatability studies, three times at the interval of three hours on same day and on different

consecutive days, respectively. For intermediate precision by different analyst study, the whole

method precision experiment was performed by different analyst.

The results of intermediate precision studies in stability indicating assay method were reported

as mean assay of MIL and RSD of assay results obtained in each intermediate precision studies.

Similarly results of intermediate precision studies in related impurities method were reported as

the mean of total impurities and RSD of mean values of impurities.


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6. Accuracy (Recovery study)

Accuracy of stability indicating assay method and related impurities method was performed by

recovery studies. Most widely used synthetic mixture of capsule excipients (i. e. dicalcium

phosphate, lactose and microcrystalline cellulose) were prepared (placebo) in the ratio of their

permitted concentration in formulation of capsules.

For stability indicating assay method, known amounts of MIL corresponding to 80-120 % of the

label claim (50 mg) were added to placebo mixtures at three different levels in triplicate. For

level I, II and III accurately about 40, 50 and 60 mg of MIL (which correspond to 80, 100 and

120 % of the label claim) was weighed and mixed with constant weight of placebo in 100 mL

volumetric flask, about 80 mL methanol was added and the flasks were sonicated for 15 min and

volumes were made upto the mark with methanol. All the solutions were filtered through

whatman filter paper 41. From the above filtrate, 0.2 mL from each flask were taken and diluted

to 10 mL with diluent and the resulting solutions were analyzed as described in section 4.4.1.1.

In related impurities method, the accuracy of the method for unknown impurity was studied with

respect to recovery of MIL. The accuracy of unknown impurity in respect to MIL was

determined over the range of LOQ to 200 % of the specification limit (LOQ being 0.1 µg/mL to

0.4 µg/mL) at four levels.

From the methanolic standard stock solution of MIL (0.2 mg/mL), solutions at different levels

were spiked (0.05, 0.075, 0.1, and 0.2 mL) with constant weight of placebo, in 100 mL

volumetric flasks. About 80 mL of diluent was added and was sonicated for about 10 min and

volume was made upto the mark with diluent. All the solutions were filtered through whatman

filter paper 41 and the resulting solutions were analyzed as described in section 4.4.1.1.

7. Robustness

The robustness of an analytical procedure refers to its ability to remain unaffected by small and

deliberate variations in method parameter and provides an indication of its reliability for the

routine analysis. Deliberate changes in the following parameters which affects % assay of MIL

and system suitability parameters were studied of stability indicating assay and related

impurities method respectively.

i. Change in % organic phase of mobile phase by ± 5.0 %


62

ii. Change in pH of buffer of mobile phase by ± 0.05 of set pH

iii. Change in the flow rate of the mobile phase by ± 10 % of the original flow rate.


The solution stability was also carried out to check the stability of both the solutions (standard

and sample) till 48 h when stored at ambient temperature in laboratory. It was performed by

doing the analysis of both the solutions at 0, 12, 24, and at 48 h intervals and comparing the

results with the freshly prepared standard solutions analyzed simultaneously.

4.4.1.5. Method application to pharmaceutical formulations of MIL

The stability indicating assay method was used for the quantification of MIL and related

impurities method was used for detection and quantification of related impurities of MIL in two

different brands of pharmaceutical capsule dosage forms of MIL.

The sample solutions of various marketed capsule formulations of MIL were (prepared as

described in section 4.4.1.2.) analyzed as described in section 4.4.1.1. The percentage assay of

MIL was calculated from responses of the standard solution with the same concentration as that

of samples. The impurities detected above 0.05 % were taken in consideration and the % of each

individual impurity and total impurities were found out.

4.4.2. Results and discussion

MIL is carboxamide moiety having polar groups. The amide functional group of MIL is more

liable to hydrolysis to give acid and amine. However the presence of tertiary nitrogen makes the

drug more prone to formation of N-oxide of parent drug.

4.4.2.1. Method development and optimization [10-11]

The nature of stationary phase for separation was selected on the basis chemistry of drug.

Various columns with different stationary phases were tried. From the different trials, column

with C18 stationary phase was selected which gave proper retention, good theoretical plates, and

resolution. Further elution was also optimized using buffered mobile phase, since MIL is basic

drug having polar groups. Phosphate buffer with 0.0125 M gave good ionization of drug where

the MIL was eluted at appropriate time from column. Further the percentage of organic phase

(acetonitrile) was also optimized as its concentration affected elution of MIL and its DPs. From

the different mobile phases tried mobile phase consisting of phosphate buffer: acetonitrile (72:


63

28, v/v) was found to be satisfactory. Since MIL contains two amino groups in structure tailing

was obtained and a tailing in peak of MIL, was avoided by adding 0.3 % v/v of triethylamine and

finally pH of buffer was optimized to 3.65, where the drug gave symmetric and sharp peak for

MIL at 1.0 mL/min flow rate with good theoretical plates and acceptable tailing factor. Under the

chosen experimental conditions, the chromatogram showed a single peak at Rt 5.68 min having

theoretical plates 14,107 with asymmetry of 1.29 (Figure 4.8 a ) which is most appropriate for

the assay determination. At optimized chromatographic conditions, all DPs obtained in stress

degradation conditions were well separated from MIL as well as from each other.

For related impurities method, higher concentration of sample solution was injected and two

additional peaks at Rt 8.6 and 11.3 min were obtained. The amounts of both the impurities were

determined as 0.08 % and 0.03 % respectively, related to area of parent peak (Figure 4.8 b). The

chromatogram also shows that detected impurities were well separated from MIL and from

individual impurities with good resolution (more than 2).


64

FIGURE 4.8 RP-HPLC chromatograms of MIL (a) 20 µg/mL for stability indicating assay

method and (b) 200 µg/mL for related impurities method

4.4.2.2. Stress degradation behavior of MIL [12]

The stress degradation studies were performed to prove the stability indicating power of the

developed RP-HPLC method and as the part of specificity study of validation parameter. Table

4.8 shows the % of degradation obtained in each condition and the retention times of major

degradation products obtained in stress study.

From the results of stress degradation studies of MIL, it was observed that MIL was stable in dry

heat/thermal and photolytic stress studies as the response of MIL in terms of peak area was

nearly same as was obtained in zero time samples of MIL in these conditions.

However MIL was susceptible for degradation in all the hydrolytic conditions which was

confirmed from the additional peaks obtained in the respective chromatograms and also the

decrease in area of MIL in each hydrolytic condition when compared with areas of their zero

samples.

The mass balance was calculated, from the responses obtained MIL and all the degradation

products obtained after stress studies.


65

TABLE 4.8 Results from stress degradation study of MIL

NSD= No Significant Degradation

Stress degradation

condition

Initial

peak

area

Total Peak

area

after stress

Appr.

Degradation

observed (%)

Rt. (min) of

major DPs and

peak purity

% Mass

balance

achieved

Acid hydrolysis 999120 913456 8.6 2.78 (0.995),

8.69 (0.998) 91.4

Alkaline hydrolysis 978889 861285 11.8

2.69 (0.996),

7.23 (0.998),

8.69 (0.995)

88.0

Neutral hydrolysis 1010099 969055 4.2 8.68 (0.997) 95.9

Oxidative

hydrolysis 998820 722087 28.5

3.36 (0.996),

6.05 (0.995),

8.68 (0.998)

72.3

Photolytic solution

state 1010099 1010055 NSD - -

Thermal/Dry Heat 1010133 1010035 NSD - -

Photolytic solid state 1010133 1013032 NSD - -

Accelerated stability 1010133 1010965 NSD - -


66


67

FIGURE 4.9 Representative RP-HPLC chromatograms of MIL (20 µg/mL) in acid (a), alkaline

(b), neutral (c) and oxidative (d) hydrolytic conditions. (I, II, III, IV, and V are the Degradation

Products (DPs) of MIL)


68

As seen in Table 4.8, amongst the hydrolytic degradation the order of degradation behavior is

oxidative ˃ alkaline ˃ acidic ˃ neutral.

Three, two and one degradation products were formed under alkaline (I, IV and V), acidic (I and

V), and neutral (V) hydrolysis conditions respectively (Figure 4.9 a. b. c.). One common

degradation product V was obtained in all the hydrolyzed samples of MIL irrespective of pH of

solution. Degradation product I is the other common degradation product obtained in acidic and

alkaline hydrolytic degradation conditions. Total three degradation products (II, III, and V) were

observed in oxidative hydrolytic condition (Figure 4.9 d). The maximum degradation in peroxide

degradation is justified by the acidic pH of hydrogen peroxide which makes the amide moiety of

MIL susceptible to hydrolytic attack leading to formation of degradation products II, III, and V.

From the results of stress degradation studies it was found that the Rt of degradation product (V)

formed in all the hydrolytic stress conditions was same with one of the impurity detected at Rt

8.6 min in MIL in related impurities method.

4.4.2.3. Method validation [13-14]


The system suitability for stability indicating assay method and related impurities method was

evaluated by calculating the RSD values of retention time, peak area, asymmetry, and theoretical

plates of five standard replicates. The experimental results (Table 4.9) showed that the values are

within the acceptable range indicating that the system is suitable for the intended analysis.

TABLE 4.9 Results from system suitability parameters in two methods

Parameters Stability indicating assay method Related impurities method

Observation a RSD Observation

a RSD

Rt (min) 5.68 0.21 5.66 0.19

Peak area 1003825 0.59 9741 3.69

Theoretical plates 14133 0.46 9523 0.78

Asymmetry 1.29 0.96 1.29 0.55 a

Mean of five replicates


For evaluation of linearity in stability indicating assay and related impurities method, peak area

and concentrations were subjected to least square regression analysis to calculate calibration

equation and correlation coefficient.


69

The correlation coefficient values obtained in both stability indication and related impurity

method (Figure 4.10 and Figure 4.11) confirms the good linearity of the method over the range

studied (Table 4.10).

TABLE 4.10 Results from linearity study of MIL by two methods

Stability indicating assay method Related impurities method

Linearity

level

MIL Conc.

(µg/mL)

Mean response a

and RSD

observed

Linearity

Level

MIL Conc.

(µg/mL)

Mean response a

and RSD

observed

I 5 249002; 2.2 I (LOQ) 0.1 3356; 6.8

II 10 488823; 1.1 II 0.15 6612; 1.4

III 15 755137; 0.1 III 0.2 10011; 2.8

IV 20 1003825; 0.2 IV 0.3 15542; 1.9

V 30 1539458; 0.2 V 0.35 18879; 0.1

VI 40 2039356; 2.4 VI 0.4 20012; 0.6

VII 50 2679905; 2.2 a

Mean of three replicates

3. LOD and LOQ

From the triplicate results of linearity study, SD and slope value was found to be 213 and 57166

respectively which is further used to calculate LOD and LOQ values. LOD value was found to

be 0.01 µg/mL and LOQ was 0.04 µg/mL. The RSD value of theoretically calculated LOQ

preparation was found to be 8.18 with mean area 1973.

FIGURE 4.10 Calibration curve of developed RP-HPLC method for MIL (SIAM)


70

FIGURE 4.11 Calibration curve of developed RP-HPLC method for MIL (Related

impurities method)

4. Specificity

The specificity was evaluated from the forced degradation studies as described in section 4.4.2.2.

where Figure 4.9 a, b, c, d. shows, MIL peak is well separated from all the degradation products

formed during the different stress conditions with sufficient resolution (i.e. ˃ 2). In the stress

degradation studies, the Peak Purity values (obtained by PDA detector) of all degradation

products of MIL were more than 0.999, ensuring purity of degradation peaks with no merging.

Thus specificity study ensures the selectivity of the developed analytical method which is able to

separate and quantify MIL in presence of different degradation products.

5. Precision

The results (Table 4.11) of all the precision studies obtained in stability indicating assay method

(Repeatability, intraday, interday and different analysts), shows that the mean assay values and

RSD values are within the acceptance criteria (98-102 %, ≤ 2 respectively) as specified by ICH

guidelines which proves the good precision of developed method.


71

TABLE 4.11 Precision study of MIL by the two methods

Precision study

Observation


Mean Assay a RSD

Mean of total

impurities (%) a

RSD

Repeatability a 100.31 0.95 0.08 5.95

Intraday b 100.19 1.03 0.08 6.8

Interday c 100.64 0.71 0.08 7.8

Different analyst d 99.75 1.5 0.08 6.0

a n= 6

b Mean value of initial, 3 h, 6 h interval observations;

c Mean value of day I and day II

observations; d

Mean value of analyst I and analyst II observations

Precision results in related impurities methods are also within the acceptance criteria confirming

the reproducibility of the method.

6. Accuracy

The accuracy in both the methods was calculated as the percentage of the drug recovered and

also expressed as the RSD between the measured mean concentrations and added concentration.

In stability indicating assay method, the recovery for MIL was between 99.4 and 101.6 % with

RSD of 1.0 % (Table 4.12), indicating that the developed method was accurate for the

determination of MIL in pharmaceutical formulations.

The mean recovery in related impurities method at LOQ level is 86.5 % which is within the

acceptance criteria (i. e. between 80-120 %). Similarly the recovery range at level II, III and IV

between 95.9 to 106.6 % (Table 4.13), which is also within the acceptance criteria (90-110 %).

TABLE 4.12 Accuracy study of MIL by SIAM

Level % Recovery Mean Recovery RSD

I

(80 % wrt to LC)

99.0

99.17 0.29 99.0

99.5

II

(100 % wrt LC)

100.7

100.80 0.75 101.6

100.1

III

(120 % wrt LC)

99.4

100.40 0.95 101.3

100.5

Mean 100.1


72

TABLE 4.13 Accuracy study of MIL by related impurities method

Level % Recovery Mean Recovery RSD

I

LOQ (0.05 %

86.51

86.5 2 88.29

84.82

II

(75 %)

99.54

100.5 1.2 101.85

100.07

III

(100 %)

101.09

104.8 3 106.6

106.60

IV

(200 %)

101.24

100.4 4.1 95.91

104.04

Mean 98.05

7. Robustness

The results of robustness studies are summarized in Table 4.14 and 4.15 for stability indicating

assay and related impurities method respectively. In any condition assay value of sample is not

deviating more than 2.0 % indicating that both the methods are robust in nature.

TABLE 4.14 Robustness study of MIL by SIAM

Robustness condition

Observation

System suitability %

Assay

% difference

in assay b RSD

a Rt T A

- 5 % Acetonitrile (Buffer:

Acetonitrile 70.6:29.4 v/v) 0.53 6.18 15771 1.29 101.9 + 1.68

+ 5% Acetonitrile (Buffer:

Acetonitrile 73.4:26.6 v/v) 0.93 5.32 11542 1.31 99.5 - 0.71

+ 0.05 Changed pH of buffer of

mobile phase -3.70 0.75 5.68 14413 1.29 99.5 - 0.68

- 0.05 Changed pH of buffer of

mobile phase -3.60 0.22 5.66 13399 1.30 100.6 + 0.39

+ 10% Change in flow rate -1.1

mL/min 0.36 5.32 13390 1.28 100.5 + 0. 29

- 10% Change in flow rate -0.9

mL/min 0.54 5.91 14401 1.34 100.6 + 0.39

a from five values of standard area;

b % difference compared from the method precision result;

T = Theoretical plates; A= Asymmetry


73

TABLE 4.15 Robustness study of MIL by related impurities method


Observation

System suitability % Total

impurities

Absolute

difference* % RSDa Rt. T A

- 5 % Acetonitrile (Buffer:

Acetonitrile 70.6:29.4 v/v) 1.67 6.09 9898 1.29 0.08 0.0

+ 5 % Acetonitrile (Buffer:

Acetonitrile 73.4:26.6 v/v) 4.72 5.48 7779 1.28 0.08 0.0

+ 0.05 Changed pH of buffer of

mobile phase – 3.70 0.71 5.44 7899 1.28 0.08 0.0

- 0.05 Changed pH of buffer of

mobile phase – 3.60 4.39 5.55 7884 1.31 0.09 + 0.01

+ 10 % Change in flow rate – 1.1

mL/min 3.58 5.48 7779 1.28 0.08 0.0

- 10 % Change in flow rate – 0.9

mL/min 3.40 6.12 8766 1.31 0.07 - 0.01

a from five values of standard area;

b % Difference compared from the method precision result;

T= Theoretical plates; A = Asymmetry


From the results of the solution stability study (Table 4.16), it was found that the assay value

difference of standard and sample is less than that specified by the acceptance criteria of ICH ( ≤

2 %) indicating stability of sample and solution at ambient temperature for 48 h. Also it was

found that the difference of total impurities from initial value was not more than 0.05 % absolute

or 10 % of value.

TABLE 4.16 Solution stability of MIL by two methods

Interval

Observation


% Assay % Difference %

Assay of

STD*

% Total

Impurities*

Absolute

difference

STD* Sample* STD Sample STD Sample

Initial 100.0 99.6 - - 100.0 0.08 - -

12 h 99.9 100.5 - 0.1 + 0.9 100.1 0.08 + 0.1 0.0

24 h 100.0 99.4 0.0 - 0.2 99.7 0.09 - 0.3 + 0.01

48 h 99.5 100.1 - 0.05 + 0.05 99.6 0.09 - 0.3 + 0.01

* Result are from duplicate injection of same solution


74

4.4.2.4. Method application

Figure 4.12 a and b represents the chromatograms of MIL in two marketed formulations. The

assay results obtained by the applied stability indicating assay method were found to be

satisfactory, accurate, and precise for estimation of MIL from its pharmaceutical capsule

formulation without interference of excipients, as indicated by the good recovery and acceptable

standard deviation (SD) values (Table 4.17).

TABLE 4.17 Summary of results for assay and related impurities for MIL in marketed capsule

dosage forms

Formulation Amount of drug

recovered a (mg) ± SD

b

% Assay

± SD b

% of MIL-IMP

% of total

impurities ± SD b

A 49.7; 0.28 99.4; 0.55 0.12 0.12; 0.008

B 50.5; 0.40 101.0; 0.80 0.10 0.10; 0.005 a Label claim = 50 mg;

b n = 3


75

FIGURE 4.12 Representative RP-HPLC chromatograms (20 µg/mL) of MIL in Brand A (a) and

Brand B (b)


76

FIGURE 4.13 Representative RP-HPLC chromatogram of MIL (200 µg/mL) Brand A (a) and

Brand B (b)

a

b


77

In related impurities method, one common impurity detected at Rt 8.6 min designated as MIL-

IMP (which was also detected in MIL having concentration 0.08 %). Two additional impurities

(Rt 7.0 and at 11.3 min) were also detected in Brand A (Figure 4.13) at concentration level of

0.03 and 0.02 % respectively. However these detected impurities were not taken into further

consideration because they were below the reporting threshold as specified by ICH guidelines.

The common impurity at Rt 8.6 min found in both the formulation, was above the identification

threshold, and hence needs to be identified for structural elucidation.

4.4.3. Conclusion

The developed stability indicating assay method and related impurities method for the

determination of MIL and its related impurities were found to be simple, sensitive fast and

economical. The methods were reliable as the results from all the validation parameters produced

satisfactory results in both the methods and can be further applied for the estimation of active

content of MIL and related impurities in pharmaceutical formulations of MIL as proved from the

statistical results of method application.

From stress degradation study, it was found that MIL is susceptible for degradation in all the

hydrolytic conditions with maximum degradation in oxidative hydrolytic condition. The

common degradation product was obtained at Rt 8.6 min in all the hydrolytic degradation

conditions. This was the only degradation product in neutral hydrolysis of MIL. Hence it was

concluded that the common degradation product is the neutral degradation product formed in

aqueous condition of stress studies. Further LC-MS/MS studies can be performed for knowing

the degradation behavior, by targeting all the formed degradation products of MIL in stress

studies.

Common impurity was found in both MIL and its capsule formulations in the concentration

range of 0.08-0.12 % respectively, this impurity was also found as common degradation product

in all the hydrolytic degradation conditions. Since the impurity found in both the formulations is

above the ICH recommended identification threshold further studies needs to be extended for its

isolation and structural elucidation by different spectroscopic techniques. The studies will be

helpful for prediction of origin of impurity from MIL.


78

4.5. High Performance Thin Layer Chromatography (HPTLC) Stability

Indicating Assay Method (SIAM) for Milnacipran Hydrochloride

4.5.1. Experimental

4.5.1.1. Chromatographic procedure [15-16]

Chromatography was performed on 10 × 10 cm aluminum TLC plates precoated with 250 µm

layers of silica gel (E. Merck, Darmstadt, Germany; supplied by Merck India, Mumbai India).

Samples were applied in the form of bands, under a continuous flow of nitrogen, by means of a

Camag (Muttenz, Switerzerland) Linomat V sample applicator fitted with 100 µL Applicator

syringe (Hamilton, Bonadauz, Switzerland). A constant application rate of 0.1 µL per second

was used and the distance between the adjacent bands was also optimized. The plates were then

conditioned for 20 min in a presaturated twin-trough glass chamber (10 x 10 cm2). The spotted

plate was then placed in mobile phase (chloroform: methanol: ammonia, 6.4:2.5:0.2; v/v/v) and

ascending development was performed to a distance of around 80 mm from the point of

application at ambient temperature. Subsequent to the development, plates were dried in a

current of air with the help of an air dryer and spots was visualized in Camag UV cabinet with

dual wavelength UV lamp (254 nm and 366 nm) and densitometric scanning was performed at

220 nm with Camag TLC scanner III operated in reflectance-absorbance mode and controlled by

WinCats software. The slit dimensions (4 × 0.2 mm) were also optimized and kept constant

throughout the analysis.

4.5.1.2. Preparation of standard solutions

Standard solutions A stock solution of MIL was prepared by dissolving accurately about 100 mg

of MIL with 100 mL methanol. Aliquots of this solution were suitability diluted with methanol

to get working standard solutions of MIL having concentration of 0.1 mg/mL.

Solutions of forced degradation studies: All the stock solutions of stress degradation studies

were prepared as described in section 4.4.1.1. and 4.4.1.3. Methanol was used as diluent to get

working solution having concentration 0.1 mg/mL.

Preparation of sample solutions for Assay of MIL in marketed formulations: The extraction

procedure for MIL from capsule formulations is same as described in section 4.4.1.2. However


79

the solutions for working concentration (0.1 mg/mL) were prepared in methanol and 20 µL of

these solutions were analyzed as described in section 4.5.1.1.

4.5.1.3. Stress degradation studies

The stress degradation studies were carried out as per ICH guidelines, by forcibly degrading MIL

under different stress conditions such as hydrolytic, oxidative, thermal, and photolytic

degradation as described in the section 4.4.1.3. The optimized stress degradation conditions

which showed significant degradation as described Table 4.9 were studied in HPTLC. For the

evaluation of degradation behavior of MIL by HPTLC, the stress study samples along with their

initial samples were spotted (20 µL, 2000 ng/spot) on the activated chromatoplates and were

analyzed as in section 4.5.1.1.


To prove the reliability and reproducibility, the developed method was validated for following

validation parameters.


For establishment of linearity of MIL by proposed method, the calibration curve was obtained at

seven levels in the concentration range of 500-6000 ng/spot. For this the different increasing

amounts of MIL working standard (0.1 mg/mL) was spotted three times on individual plates and

analyzed as described in section 4.5.1.1. (Table 4.18).

TABLE 4.18 Preparation of different linearity levels of MIL

Linearity Level Volume Applied (µL) Concentration (ng/spot)

I 5 500

II 10 1000

III 20 2000

IV 30 3000

V 40 4000

VI 50 5000

VII 60 6000

2. Specificity (Interference from excipients and degradation products)

The specificity of the method was ascertained by analyzing MIL in presence of excipients of

MIL capsule formulation.


80

Similarly specificity of the method was also proved by forcibly degrading MIL in different stress

conditions and subsequently analyzing MIL in presence of degradation products. The peak purity

of MIL and each degradation product obtained was assured by comparing the spectra at three

different levels, that is, peak start (s), peak apex (m) and peak end (e) positions.

3. Precision

A. Method precision

For repeatability study, six sample sets were prepared by individually weighing MIL in six

different volumetric flasks to get concentration of 1.0 mg/mL and were further diluted with

methanol individually to get concentration of 0.1 mg/mL. Standard having same concentration

(0.1 mg/mL) was also prepared in the similar manner as that of sample. The standard (5

replicates) and six samples sets were spotted on (20 µL, 2000 ng/spot) previously activated plate

and were analyzed as described in section 4.5.1.1. The mean assay of six samples sets was found

and RSD was reported.

B. Intermediate precision

The intermediate precision study was performed at three different levels i.e. intraday, interday,

and different analysts precision.

For intraday and interday precision studies, the procedure described in repeatability study of

precision parameter was repeated three times at the interval of three hours on same day and on

different consecutive days, respectively. For intermediate precision by different analyst study,

the whole method precision experiment was performed by different analyst.

The results of intermediate precision studies was reported as mean assay of MIL and RSD of

assay results obtained in each intermediate precision studies.

4. Accuracy

The accuracy of the method was performed by recovery studies as described in accuracy study of

section 4.4.1.4.

The working solution in this HPTLC method however was prepare in methanol and resulting

solutions were spotted on (20 µL, 2000 ng/spot) previously activated plate and were analyzed as

described in 4.5.1.1. The responses obtained after scanning each spots were measured.


81

5. Robustness

The Robustness of the method was studied by making deliberate changes in the following

parameters and subsequently change in % assay of MIL was observed.

i. Change in plate activation time by ± 20%

ii. Change in chamber saturation time by ± 20%

iii. Change in total volume of mobile phase by ± 10%

iv. Change in development distance of mobile phase by ± 10%


The solution stability was also carried out to check the stability of both the solutions (standard

and sample) till 48 h when stored at ambient temperature in laboratory. It was performed by

doing the analysis of both the solutions at 0, 12, 24 and at 48 h intervals and comparing the

results with the freshly prepared standard solutions analyzed simultaneously as described in

section 4.5.1.1.


The proposed developed and validated HPTLC method was successfully extended for the

estimation of MIL in two different brands of pharmaceutical capsule formulations. The

description of two capsule formulations is given in Table 4.3.

4.5.1.6. Method comparison

The assay results obtained in the developed HPTLC method for MIL were statistically compared

with the assay results obtained RP-HPLC method by paired t-test at 5 % level of significance.

4.5.2. Results and Discussion

4.5.2.1. Method development and optimization of chromatographic conditions

Selection of best solvent system is the critical step in HPTLC method development. From the

different solvent systems tried, the mobile phase consisting of chloroform and methanol in ratio

of 6.4:2.5 v/v gave good separation between MIL and its degradation products with optimum RF

value for MIL; however tailing of MIL peak was observed which was avoided by addition of 0.2

mL ammonia in mobile phase. The optimized mobile phase was chloroform: methanol:


82

ammonia, 6.4:2.5:0.2; v/v/v which gave symmetric, well resolved peak of MIL with RF 0.45

(Figure 4.14). The mobile phase selected gave best band for MIL with appropriate RF value.

Well defined band was obtained when the chamber was saturated with mobile phase for 20 min

at ambient temperature. Reproducible responses were obtained at optimized slit dimensions of 4

× 0.2 mm. For quantitive purpose the densitometric scanning was carried at wavelength 220 nm

where MIL and its degradation products exhibit sufficient UV absorption and estimation of MIL

was achieved without hampering sensitivity.

4.5.2.2. Degradation behavior of MIL

The representative chromatograms for the hydrolyzed degraded samples of MIL are shown in

Figure 4.15 (a, b, c and d). From the results of stress degradation study (Table 4.19), MIL was

found to be stable in thermal, photolytic degradation, and accelerated stability conditions. The

observed order of degradation in hydrolytic degradation was oxidative ˃ basic ˃ acidic ˃ neutral.

TABLE 4.19 Results from stress degradation study of MIL by HPTLC method

NSD=No significant degradation

One of the degradation product of MIL at RF 0.80 ± 0.01 (represented as D5) obtained in all

hydrolytic and in oxidative degradation conditions, was the only degradation product peak

obtained in neutral hydrolysis, it indicates that MIL hydrolyzed in presence of water during

acidic, basic hydrolysis which was also confirmed by scanning and comparing the spectra of

degradation spot with RF 0.80 ± 0.01 obtained in these conditions (Figure 4.16). However the

effect of acidic pH of hydrogen peroxide leads to formation maximum degradation products.

Similarly the degradation product formed in acidic and basic hydrolysis at RF 0.04 ± 0.01 and

0.05 ± 0.01 respectively (represented as D1) may be same since both the spots displayed similar

spectras (Figure 4.17).

Stress degradation

condition

Initial

peak area

Peak area

after stress

Appr. degradation

observed (%)

Rf values of

major DPs

Acid hydrolysis 6577.44 5512.88 16 0.04, 0.81

Alkaline hydrolysis 6299.09 4094.12 35 0.05, 0.23,0.80

Neutral hydrolysis 6844.90 6133.12 10 0.81

Oxidative hydrolysis 7013.88 4909.09 30 0.06, 0.24, 0.80.

Thermal/Dry heat 6899.23 6842.09 NSD -

Photolytic solid state 6899.23 6890.33 NSD -

Accelerated stability 6899.23 6870.76 NSD -

Photolytic solution state 6844.51 6804.88 NSD -


83

FIGURE 4.14 HPTLC chromatogram of MIL (2000 ng/spot) showing RF 0.45

FIGURE 4.15 HPTLC chromatogram of MIL a. Acid b. Alkaline c. Neutral and d. Oxidative

degraded. D1, D2, D3, D4, and D5 are the degradation products of MIL

a b

c d


84

FIGURE 4.16 Overlayed UV spectra of band at RF 0.80 ± 0.01 (D5) in acid degraded

(a, with peak purity 0.99945), alkaline degraded (b with peak purity 0.99965), neutral

degraded (c with peak purity 0.99915) and oxidative degraded MIL (d with peak purity

0.99908) MIL samples respectively.

FIGURE 4.17 Overlayed UV spectra of band at RF 0.04 ± 0.01 (D1) in acid degraded

(a, with peak purity 0.99933), alkaline degraded (b with peak purity 0.99945) MIL

samples respectively


85



For evaluation of linearity, observed peak area and concentrations were subjected to least square

regression analysis to calculate calibration equation and correlation coefficient. The observed

linearity confirming adherence of the system to Beer’s law. The regression analysis equation was

y = 2.753x +1322.283 with correlation coefficient (r) was 0.9966 (Figure 4.18).

FIGURE 4.18 Calibration plot of MIL for linearity study by the developed method


86

2. Specificity

For the evaluation of specificity of the method, spots obtained at RF value 0.45 corresponding to

MIL from API as well as marketed formulations were scanned in UV region from 200 to 700 nm

And overlay spectra are given in Figure 4.19. Correlation of spectra was found to be more 0.999

indicates specificity of method.

To check purity of each peak obtained due to MIL and its degradation products was scanned at

three different positions of peaks and obtained peak purity values were more than 0.999 for each

peak. The results indicate that method is specific for MIL since there is no interference to MIL

peak from excipients as well as degradation products.

FIGURE 4.19 Spectra comparison of MIL (a) Bulk (b) Capsule formulation A and (c) Capsule

formulation B

3. Precision

The results (Table 4.20) of all the precision studies (Method precision, intraday, interday and

different analysts), shows that the mean assay values and RSD values are within the acceptance

criteria (98-102 % and ≤ 2 respectively) as specified by ICH guidelines which proves the good

precision of developed method.


87

TABLE 4.20 Precision study of MIL by the developed method

Precision study Observation

Mean Assay a RSD

Repeatability 99.5 1.17

Intraday b 99.7 1.06

Interday c 100.2 1.16

Different analyst d 99.8 0.91

a n= 6;

b Mean value of initial, 3 h, 6 h interval observations;

c Mean value of day I and day II

observations; d

Mean value of analyst I and analyst II observations

4. Accuracy

The recovery for MIL was between 98.2 and 101.4 % with RSD of 1.1 % (Table 4.21),

indicating that the developed method was accurate for the determination of MIL in

pharmaceutical formulations.

TABLE 4.21 Accuracy study of MIL by the developed method

Level % Recovery Mean recovery RSD

I

(80 % wrt to LC)

101.4

100.3 1.78 98.2

101.2

II

(100 % wrt LC)

98.2

99.2 1.00 100.2

99.2

III

(120 % wrt LC)

100.3

99.8 0.50 99.7

99.3

Mean 99.7

5. Robustness

The results of robustness studies are summarized in Table 4.22. In any condition assay value of

sample is not deviating more than 2.0 % indicating that method is robust in nature.


88

TABLE4.22 Robustness study of MIL by developed method


Observation

System

suitability %

Assay

%

difference

in assay* RSDa RF

Change in plate activation time by - 20 % (24 min) 0.60 0.42 98.0 - 1.5

Change in plate activation time by + 20 % (36 min) 0.29 0.46 99.0 - 0.5

Change in volume of mobile phase by - 10 % (8.2 mL) 0.66 0.44 99.1 - 0.4

Change in volume of mobile phase by + 10 % (10 mL) 0.27 0.46 100.4 + 0.9

Change in chamber saturation time by - 20 % (16 min) 0.82 0.41 98.8 - 0.7

Change in chamber saturation time by + 20 % (24 min) 0.14 0.46 98.8 - 0.7

Change in development distance by - 10 % (72 mm) 0.65 0.44 98.4 - 1.1

Change in development distance by - 10 % (88 mm) 0.48 0.47 98.6 - 0.9 a

From three values of standard area; b % difference compared from the method precision result


Results from solution stability study (Table 4.23) shows that the assay value difference of

standard and sample is less than that specified by the acceptance criteria of ICH ( ≤ 2 %)

indicating stability of both the solutions of MIL.

TABLE 4.23 Solution stability of MIL by developed method

Interval

Observation

% Assay % Difference

STD* Sample* STD Sample

Initial 100.0 99.05 - -

12 h 100.11 99.66 + 0.1 + 0.61

24 h 99.98 99.12 - 0.02 + 0.07

48 h 99.44 99.01 - 0.6 - 0.04

* Result are from duplicate injection of same solution


The developed and validated stability indicating HPTLC method was successfully applied for

quantitation of MIL in two marketed formulations. The assay results are given in Table 4.24.

TABLE 4.24 Summary of results for assay for MIL in marketed capsule dosage forms

Formulation Amount of drug recovered a (mg) ± SD

b % Assay ± SD

b

A 49.6; 0.12 99.1; 0.24

B 50.4; 0.2 100.8; 0.3 a

Label claim = 50 mg; b n = 3


89

4.5.2.5. Method comparison

As reported method is not available for quantification of MIL from marketed formulation,

herewith both RP-HPLC and HPTLC methods were compared statistically. The results obtained

by paired t-test at 5 % level of significance are summarized in Table 4.25. The t cal value is less

than t tab value indicates that there is no significant difference between the assay results obtained

by the two methods (Table 4.25).

TABLE 4.25 Statistical comparison between assay results of MIL formulations by two

analytical methods

Statistical parameter

MIL pharmaceutical capsule formulation

A B

HPTLC HPLC HPTLC HPLC

Mean (n = 3) 99.13 99.40 100.76 101.03

Variance 0.05 0.30 0.12 0.61

P value 0.49 0.61

t stat (tcal) - 0.78 - 0.56

t Critical (tcrit) 3.18 3.18

tcal< tcrit yes Yes

Null hypothesis pass pass

4.5.3. Conclusion

This developed and validated stability indicating HPTLC method is specific, precise and

accurate and able to separate the drug from its all the degradation products. The method was

successfully applied for determination of MIL in its pharmaceutical capsule formulations which

suggest good reliability of the method as no significant difference in assay results were obtained

when developed method was compared with RP-HPLC method. The developed HPTLC method

can be conveniently used for routine quality control analysis of MIL in industries for batch

release.


90

4.6. Isolation and characterization of impurity of Milnacipran Hydrochloride

4.6.1. Experimental

4.6.1.1. Chromatographic conditions [17-20]

Following chromatographic conditions for Prep-HPLC were optimized and were kept constant

throughout for the isolation of MIL-IMP in pure form.

Chromatographic conditions

Column: C18 Phenomenax semi preparative column (10 µm, 250 x 10 mm id).

Mobile phase: Buffer: Acetonitrile (72:28; v/v).

Buffer preparation: 0.0125 M ammonium acetate; add 0.3 % ammonia and adjust the pH of

buffer to 3.65 ± 0.02 with glacial acetic acid.

Flow rate: 6.0 mL/min; Detection wavelength: 220 nm; Injection volume: 1 mL.

4.6.1.2. Solution preparation

The solution for sample loading on Prep-HPLC was prepared by dissolving 1 g of MIL in 100

mL distilled water (1 % solution); the prepared solution was then subjected to the stress

degradation as described in section 4.4.1.3. However the duration of refluxing was increased

from 4 h to 72 h to achieve maximum degradation of MIL.

4.6.1.3. Isolation of MIL-Impurity (MIL-IMP)

The neutral degraded sample solution of MIL prepared as described in section 4.6.1.2. was

loaded on Prep-HPLC with chromatographic conditions described in section 4.6.1.1.and eluents

containing targeted impurity were collected and concentrated by evaporating acetonitrile portion

of eluents at room temperature under high vacuum on Rota evaporator. The concentrated

aqueous layer was further dehydrated with solid sodium sulphate (approximately 1 g) and further

extracted with chloroform (50 mL each time) thrice. The collected combined chloroform layer

was evaporated in Rota evaporator to get solid mass of impurity (MIL-IMP). Before

characterization of isolated impurity using different spectroscopic techniques, chromatographic

purity of the isolated MIL-IMP was checked using developed RP-HPLC method.


91

4.6.1.4. Characterization of isolated MIL-IMP by spectroscopic techniques

The isolated and purified MIL-IMP was further analyzed by different spectroscopic techniques

like UV, FT-IR, Mass, and NMR spectroscopy for characterization and structural elucidation.

4.6.1.4.1 UV Spectroscopy

The standard solution (20 µg/mL) of MIL-IMP was prepared in methanol and scanned in whole

UV-VIS range 200-800 nm for determination of its maximum absorbing wavelength.

4.6.1.4.2 FT-IR spectroscopy

The FT-IR spectroscopic analysis was performed by diffused reflectance technique. The FT-IR

spectra of MIL and MIL-IMP were recorded in the range of wave number 400-4000 cm-1

and

compared with spectra of MIL recorded as described in section 4.3.3.

4.6.1.4.3 Mass spectroscopy

The MS and MS/MS experiments on MIL-IMP were performed on API 2000 Q-Trap Mass

Spectrometer as described in section 4.2.

4.6.1.4.4 NMR spectroscopy

The 1H and

13C NMR experiments on MIL-IMP were carried out at processional frequencies of

300 MHz and 75 MHz, as described in section 4.2.

4.6.1.1. Elemental Analysis

The elemental analysis was carried out to determine the amount of carbon, hydrogen and

nitrogen present in isolated MIL-IMP on CHN-O Element Analyzer as described in section 4.2.

4.6.2. Results and discussion

4.6.2.1. Method development and optimization

For the isolation of MIL-IMP in pure form Prep-HPLC method was developed. Here the mobile

phase tried for the development of Prep-HPLC method was with volatile buffer i.e. with

ammonium acetate keeping the buffer strength its pH and concentration of organic phase of

mobile phase same. The advantage of using volatile buffer is ease of extraction of MIL-IMP

from eluents collected from Prep-HPLC, since ammonium acetate buffer gets easily vaporized

on Rota evaporator even at room temperature. The pH was adjusted with glacial acetic acid after

addition of ammonia as a peak reagent. In this method the MIL and MIL-IMP eluted at Rt 6.6

and 9.3 min respectively (Figure 4.20).


92

FIGURE 4.20 Representative Prep-HPLC chromatogram of neutral treated solution of MIL

4.6.2.2. Isolation and purification of MIL-IMP

The eluent fractions were collected between 8.5 min to 9.8 min to recover MIL-IMP in pure

form. Off-white crystals of MIL-IMP were obtained with yield of 120 mg, having melting point

90-92 °C.

4.6.2.3. Chromatographic purity of isolated MIL-IMP

The purity of isolated MIL-IMP was checked using analytical RP-HPLC method as described in

section 4.4.1.2. and was found to be 99.2 % (Figure 4.21). Spiked solution of MIL with MIL-

IMP was also analyzed and found that the response of MIL-IMP in API was increased (Figure

4.22).


93

FIGURE 4.21 RP-HPLC Chromatogram of isolated MIL-IMP showing single peak (Rt 8.6 min)

FIGURE 4.22 RP-HPLC chromatogram of MIL spiked with MIL-IMP (Rt 5.6 min for MIL; Rt

8.6 min for MIL-IMP)


94

4.6.2.4. Characterization of isolated MIL-IMP by spectroscopic techniques

4.6.2.4.1 UV spectroscopic analysis

The UV spectra of MIL-IMP shows the absorption maxima at 217.0 nm which was very near to

that of MIL (220.0 nm) suggesting that the MIL-IMP structure may contain the same basic

moiety (Figure 4.24).

FIGURE 4.23 UV spectra of MIL-IMP showing λ max at 217 nm

4.6.2.4.2 FT-IR spectroscopy

Figure 4.24 shows the FT-IR spectra of MIL-IMP in diffused reflectance mode with

characteristic frequencies observed are reported in Table 4.26.


95

FIGURE 4.24 FT-IR spectra of MIL-IMP

4.6.2.4.3 Mass spectroscopy

The MS of MIL-IMP exhibited molecular ion at m/z (M+1) 174 amu (Figure 4.25). The MS/MS

study of MIL-IMP (Figure 4.26) was also carried out to study fragmentation pattern.

FIGURE 4.25 Mass spectra of MIL-IMP showing m/z value 174 amu


96

FIGURE 4.26 MS/MS spectra of MIL-IMP at molecular peak of 174

The spectroscopic results of MIL-IMP and MIL are summarized in Table 4.26.

TABLE 4.26 Summarized results of spectroscopic data of MIL and MIL-IMP

Compound

Observation from spectroscopic experiments

ƛ max

(nm)

IR

(Wave number, cm-1

)

Mass

(m+1) and major daughter ions

MIL 220 3253.3, 3232.1, 3154.5 ,

1616.0, 1455.9, 1149.0

247.2 (M+1), 230.2, 174.2, 157.1,

131.1, 100.1

MIL-IMP 217 3212.8, 3077.8,

1675.8,1482.9, 1199.5

174.2 (m+1), 146.2, 131, 103.1,

96.1, 91.1, 78.1

4.6.2.4.4 NMR spectroscopy

For the further confirmation of structure of MIL-IMP, the NMR spectroscopic experiments were

carried out. Figure 4.27 and 4.28 shows the 1H NMR and

13C NMR spectra of MIL-IMP

respectively. The NMR (1H and

13C) spectral data for MIL and MIL-IMP is compiled in Table

4.27.


97

FIGURE 4.27 1H NMR Spectra of MIL-IMP

FIGURE 4.28 13

C NMR Spectra of MIL-IMP


98

4.6.2.1. Elemental Analysis

The results from the elemental analysis of MIL-IMP are depicted in Table 4.27, which further

supports the proposed structure.

TABLE 4.27 Results from the elemental analysis of MIL-IMP

Element estimated (%) Observation

Carbon 75.31

Hydrogen 7.02

Nitrogen 7.83

4.6.2.6. Structural elucidation of MIL-IMP

The spectral data of MIL-IMP was compared with that of MIL. The ESI mass spectrum of MIL-

IMP exhibited molecular ion [M+H] + at m/z 174 which is 73 Da less than that of MIL (Fig.

4.25.). The stretching frequencies at 2940 and 2904 cm-1 observed in FT-IR spectra due to two

ethyl groups of MIL were absent in MIL-IMP. In NMR spectra also, the chemical shifts for two

ethyl groups in MIL were not seen for MIL-IMP (Table 4.28). These observations confirmed

absence of the diethyl amino group in MIL-IMP which may be the reason for its low absorptivity

value compared with MIL. Odd number molecular weight of MIL-IMP (173) suggests the

presence of single nitrogen in structure. Further appearance of single less intense peak at

frequency 3234 cm-1

in FT-IR spectra of MIL-IMP indicates possible presence of secondary

amine in structure as doublet was seen at 3253 and 3232 cm-1

due to primary amino group for

MIL. The shift of carbonyl stretching frequency to higher frequency (from 1616 of MIL to 1679

cm-1

), near the frequency of cyclic amides indicates the presence of cyclic amide group in the

structure which was further confirmed by 13

C NMR spectral data where the chemical shift due to

carbonyl carbon (C6) in MIL-IMP was found at more deshielded value of 178.32 ppm compared

to MIL (C4,170.45 ppm), this was supported by observation of chemical shift of N-H (1H)

proton obtained at more deshielded value near to chemical shift obtained due to lactam. The

results obtained from the elemental analysis of MIL-IMP, Mass and NMR spectral study

confirms the molecular formula of MIL-IMP as C11H11NO with proposed structure of 1-phenyl-

3-azabicyclo [3.1.0] hexan-2-one (Figure 4.29).


99

TABLE 4.28 NMR spectral assignments for MIL and MIL-IMP

MIL MIL-IMP 1H

13C

1H

13C

Position* δ (ppm) Multiplicity, j Position* δ (ppm) Position* δ (ppm) Multiplicity j Position* δ (ppm)

2 1.18 t, 10.5 1 25.23 1 6.51 s 2 43.12

3a 3.67 d, 10.5 2 34.54 2a 3.63 dd 3 19.27

3b 2.54 t, 10.8 3 17.98

2b 3.38 d 10.5 4 33.40

7,11 7.30 m 3 2.22 m 5 22.77

8-10 7.20 m 4 170.45 5a 1.54 q, 4.5 6 178.32

12 1.78 m 6 138.23 5b 1.15 t, 4.5 7 136.04

13 8.77 s 7,11 128.77 8,12 7.41 m 8,12 128.76

15,17 3.29 m 8,10 125.60 9,11 7.33 m 11,9 128.41

16 0.88 t, 6.9 9 127.0 10 7.25 M 10 127.12

18 1.18 t, 6.9 12 42.80

15 39.40

16 12.11

17 41.80

18 12.85

*Refer structures for numbering (Figure 4.29)


100

C

H H

H

O

H

H

N

H

1

2

3 4

5

6

7

89

10

1112

a

b

CH2

NH2

C

O

N

CH2

CH2

H3C

H3C

12

34

5

6

7

89

10

11

12

13

14

1516

1718

HCl

MIL MIL-IMP

a b

aH

bH

FIGURE 4.29 Chemical structures of MIL and MIL-IMP

Further interpretation of 1H NMR spectra of MIL and MIL-IMP revealed that the different

splitting pattern with different chemical shifts values for 3 Ha and 3Hb were obtained for MIL as

they are diastereotopic protons. In proposed structure of MIL-IMP, the two protons at 5H are

also diastereotopic wherein 5Ha interacts with proton of 3 H giving quartet however this type of

interaction may not be possible for 5Hb proton due to cyclopropane ring strain resulting into

triplet. This probability is also observed in case of protons of 2 H in MIL-IMP wherein 2Ha

gives deshielded doublet-doublet splitting pattern because of its coupling to 3H and 1H protons

but since the 2 Hb proton is above the plane of ring it may not interact with proton at 3H, but will

interact with 1 H giving doublet. The structure of MIL-IMP was finally confirmed by proposing

fragmentation pattern of MIL-IMP where obtained m/z values of daughter ions in MS/MS

spectrum (Figure 4.30) were in accordance with the proposed fragmentation of MIL-IMP.

4.6.2.7. Origin of MIL-IMP [21]

It is postulated that MIL-IMP is the degradation impurity which may arise during hydrolysis of

MIL. The mechanism of formation of MIL-IMP may be intramolecular nucleophilic reaction.

The amino group of MIL in hydrolytic condition attacks on carbonyl carbon, and a cyclic

intermediate is formed, followed by its protonation which induces the removal of secondary

amine leading to final formation of MIL-IMP. The plausible mechanism for formation of MIL-

IMP from MIL is outlined in Figure 4.31.


101

CH2

NH

OC

MIL-IMP m/z 174

CH2

NH2

m/z 146

CH3

m/z 131

CH2

NH

OC

m/z 117

m/z 96

CH2

m/z 129m/z 91m/z 78

H

FIGURE 4.30 The proposed MS/MS fragmentation pattern for the MIL-IMP

C

O

N

CH2

NH2

MIL

C N

CH2 N O

HH

C N

CH2 NH O

H

H-O-H

C N

CH2 NH O

H

HN-dealkylation

OH

C

CH2 NH

O

Water hydrolysis

R

R

R

R R

R

R

R

(R= -CH2-CH3)

NH2

R

R

MIL-IMP(1-phenyl-3-aza-bicyclo-

-[3.1.0]hexan-2-one)

FIGURE 4.31 Plausible mechanism for origin of MIL-IMP


102

4.6.3. Conclusion

The presences of impurities above defined levels are highly undesirable in APIs as well as in

pharmaceutical formulations. The impurity of MIL detected and quantified above identification

threshold is isolated by Prep-HPLC. Characterization is carried out by various spectroscopic

techniques like IR, Mass, and NMR spectroscopy. The structure of MIL-IMP is confirmed as 1-

phenyl-3-azabicyclo [3.1.0] hexan-2-one which may develop due to water hydrolysis during

manufacturing process of API as well as during formulation development.


103

4.7. LC-ESI-MS/MS study on degradation behavior of Milnacipran

Hydrochloride

4.7.1. Experimental

4.7.1.1. Chromatographic conditions

The RP-HPLC method described in section 4.4.1.1. was modified and optimized for LC-MS

study for evaluation of degradation products of MIL. For this following chromatographic

conditions were optimized and kept constant for LC-MS method.

Column: Inertsil ODS 3V column (5 µm, 4.6 x 250 mm id).

Mobile phase: Buffer: Acetonitrile (70:30, v/v).

Buffer preparation: Ammonium acetate 0.010 M with pH 3.60 ± 0.05 (adjusted with formic acid

after addition of 0.1 % ammonia as modifier).

Flow rate: 1.0 mL/min, Detection wavelength: 220 nm; Injection volume: 20 µL.

4.7.1.2. Preparation of samples for LC-MS study

The standard and stress degradation samples stock were prepared as described in section 4.4.1.2.

and 4.4.1.3. The stock solutions of standard, hydrolytic and oxidative stress samples were

suitably diluted with diluent (water: acetonitrile, 50:50, v/v) to get final concentration of 20

µg/mL. Standard and sample solutions (20 µL) were after filtration through 0.45 µm syringe

filter on LC-MS using autosampler.

4.7.1.3. Characterization of degradation products from LC-MS/MS studies [21-22]

The drug and the stress drug samples obtained in the degradation studies as described in section

4.4.1.3 were subjected to LC-ESI-MS/MS studies. LC-MS/MS studies were carried out on a

system as described in section 4.2. The mass spectrometer was run in positive ionization mode

with Electrospray ionization interface and mass to charge (m/z) ratio was recorded in the range of

100-300 m/z. The parameters for capillary and Rf voltage were 80 V, with nebulizer gas as air at

a pressure of 35 psi and curtain gas as nitrogen at a pressure of 10 psi.

Initially the parent ion (m+1) values were obtained using LC-MS studies according to the

retention time of parent drug and its all DPs (designated as I-V). Subsequently the MS/MS

studies were performed for MIL and all the DPs for obtaining their daughter ions. The

fragmentation pattern of MIL and each DP was studied in detail.


104

4.7.2. Results and Discussion

4.7.2.1. LC-MS/MS method development and optimization

For the development of selective and specific LC-MS method for MIL stress studies, various

organic phases were tried with water. However, poor peak shape was obtained for MIL.

Ammonium acetate buffer with low buffer strength was selected as it is compatible with LC-MS

analysis. It showed good separation of MIL as well as its degradation products but the problem

of tailing of the peaks was persistent. Ammonia (at concentration of 0.1 % v/v) was added as a

modifier to sharpen the peak as the drug contains two amino groups. The pH of the buffer of

mobile phase was adjusted to 3.60 with the help of glacial acetic acid and optimized mobile

phase was ammonium acetate buffer (pH 3.6; 0.010 M): acetonitrile (70:30, v/v). Under the

chosen experimental conditions, MIL eluted at Rt of 6.9 mins (Fig. 4.30.) and all the degradation

products formed in stress studies were well resolved from the parent drug as well as from other

which is essential for their easy characterization (Figure 4.33 a. b. c and d.).

FIGURE 4.32 Representative chromatogram of MIL (20 µg/mL) in LC-MS method


105

FIGURE 4.33 Representative chromatograms of MIL after a. Acid b. Alkaline d. Neutral and e.

Oxidative hydrolysis (I,II,III,IV,V are the degradation products of MIL) in LC-MS method


106

The chromatograms of stress degradation studies of MIL obtained with LC-MS method (Figure

4.33) suggest that the elution pattern and degradation behavior of MIL was same as observed in

RP HPLC method as described in section 4.4.2.2.

4.7.2.2. Characterization of degradation products by LC-MS study

All the degradation products (I-V) observed in the chromatograms (Figure 4.33) were targeted in

LC-MS for their characterization. The experimental masses (M+1) obtained with LC-MS studies

are represented in Figure 4.34. The obtained M+1 values of each DP in positive ESI mode were

compared to the molecular weights of the proposed structures of degradation products of MIL

(Figure 4.35). Further structures of all the DPs of MIL (I-V) were elucidated with the help of

MS/MS fragmentation pattern individually (Figure 4.36). The observed m/z values of molecular

ion peak and major fragments of MIL and its degradation products are given in Table 4.29.


107

FIGURE 4.34 Representative LC-MS spectra of MIL (a) and its DP I (b), II (c), III (d), IV (e)

and V (f)


108

FIGURE 4.35 Proposed structures of DPs (I-V) of MIL

TABLE 4.29 Observed molecular ion and daughter ions of MIL and DPs

Peak (RRT) Observed m/z value Major fragments obtained in MS/MS studies

I (0.38) 192 174, 161, 148, 147, 134, 103

II (0.55) 263 247, 230, 202, 174, 157, 117,100

MIL (1) 247 230, 202, 174, 157, 117,100

III (1.04) 190 174, 162, 131, 129, 117, 96,

IV (1.33) 230 202, 174, 157, 129,117, 100

V (1.42) 174 131, 129, 117, 96,


109

FIGURE 4.36 Representative MS/MS spectra of MIL (a) and its DP I (b), II (c), III (d), IV (e)

and V (f)


110

4.7.2.3. Fragmentation studies of MIL and its DPs

For the confirmation of proposed structures of DPs of MIL, generalized fragmentation pattern

(Figure 4.37, 4.38 and 4.39) for MIL and each DP was outlined with the help of major fragments

observed in MS/MS studies which helped in confirmation of proposed structures of DPs.

CH2

NH2

C

O

N

CH2

CH2

MILm/z 247

H

CH3

CH3

CH2

NH2

C

O

N

H2C

CH2

H

CH3

CH3

O

C

O

N

H2C

CH2

H

CH3

CH3

CH2

IIm/z 263

CH2

IVm/z 230

m/z 202

CH2

HN

C

O

N

CH2

CH2

CH3

CH3

m/z 100

m/z 174

HNOC

CH2H3C

CH2

m/z 157

OCOC

CH2

m/z 129m/z 117

FIGURE 4.37 Proposed fragmentation pattern for MIL and its DPs II, and IV


111

CH2

NH2

Im/z 192

H

CH2

NH2

m/z 174

m/z 161 m/z 134m/z 148

HOOC

HOOC

OC

CH2

NH2

m/z 147

HOOCHOOC

m/z 103

FIGURE 4.38 Proposed fragmentation pattern for DP I

CH2

NHH

OC

Vm/z 174

CH2

NH

H

OC

IIIm/z 190

OCH3

m/z 131

CH2

NH

OC

m/z 117

m/z 96

CH2

NH2O

m/z 162

CH2

m/z 129

FIGURE 4.39 Proposed fragmentation pattern for DPs III and V


112

4.7.2.4. Mechanism of formation of DPs of MIL [21]

From the structures of degradation products, their origin of generation in each hydrolytic

condition can be proposed. This study helps in further confirmation of degradation products of

MIL.

As described in chapter 4.6. DP V is the common degradation product formed in all the

hydrolytic conditions due to presence of water. The mechanism for formation of lactum DP from

MIL is described in section 4.6.2.6.

DP-I with experimental mass of 192 is commonly found during acidic and alkaline hydrolytic

conditions but it is formed through different mechanism in acidic and alkaline environment. The

mechanism for acid catalyzed hydrolysis involves water acting as nucleophile which attacks on

activated carbonyl carbon. It results into a formation of positively charged intermediate which

gets neutralized by using a proton. Another protonation results into loss of dialkylamine with the

formation of acid (I). The mechanism for acidic degradation for MIL is outlined in Figure 4.40.

C

O

Acid hydrolysis

N

R

R

CH2

NH2

MIL

OH H

C

OH

N

R

R

CH2

NH2

C

OH

N

R

R

CH2

NH2

OH

H

H

C

O

NH

R

R

CH2

NH2

OHC

CH2

NH2

O

OH

H

+NH2

(R= -CH2-CH3)

H

R

R

I2-(aminomethyl)-1-phenyl-

-cyclopropanecarboxylic acid

FIGURE 4.40 Proposed mechanism for acid hydrolysis of MIL

Under the alkaline condition, hydroxylate ion acts as the nucleophile and attack on carbonyl

carbon. The resulting carboxylate anion looses the charged leaving group and form carboxylic

acid derivative (I). In basic hydrolysis free amine group gets liberated from drug molecule and


113

gives deamination product (IV) with m/z value 230. The mechanism for basic degradation for

MIL is outlined in Figure 4.41.

CAlkaline hydrolysis

N

R

R

CH2

NH2

MIL

C N

R

R

CH2

NH2

C

CH2

NH2

O

OH

+

O

OH

C

O

N

R

R

CH2

(R= -CH2-CH3)

O

H

OH

NH2

R

R

I

IV

2-(aminomethyl)-1-phenyl--cyclopropanecarboxylic acid

N,N-diethyl-2-methylene-1-phenyl--cyclopropanecarboxamide

FIGURE 4.41 Proposed mechanism for alkaline hydrolysis of MIL

The DP II may be formed due to the N-oxide formation of MIL whose m/z value matches with

the theoretically calculated mass value of proposed structure. In presence of water of 3 % H2O2,

MIL degraded to the DP V and it is further oxidized to form N-oxide of DP V to give DP III at

experimental mass of 190. The mechanism for formation of DP II and III respectively is given in

Figure 4.42.

.

C

O

N

CH2

NH2

MIL

C

CH2 NH

O

Oxidative hydrolysis

R

R

(R= -CH2-CH3)

C

O

N

CH2

NH2

R

R

Through water hydrolysis

Oxidative hydrolysis

C

CH2 N

O

O

O

II

V III

N-Oxide of MIL

1-phenyl-3-aza-bicyclo--[3.1.0]hexan-2-one

N-Oxide of 1-phenyl-3-aza-bicyclo--[3.1.0]hexan-2-one

FIGURE 4.42 Proposed mechanism for oxidative hydrolysis of MIL


114

4.7.2.5. Degradation pathways of MIL

Based on the degradation studies and the results of mass fragmentation data of degradation

products of MIL the most probable degradation pathway is proposed in Figure 4.43. The finding

of the study inferred that common degradation product (I) of acid and alkaline hydrolysis is

generated through different mechanism.

FIGURE 4.43 Proposed pathway for the formation of DPs of MIL

4.7.3. Conclusion

In this study, the intrinsic stability of MIL is established. The degradation products formed in

different stress conditions were characterized through mass fragmentation studies. The molecular

masses were established by recording LC-MS scans ESI mode. A more comprehensive

degradation pathway of the drug has been outlined. Lactum degradation product of MIL was also

found as impurity in MIL active pharmaceutical ingredient and its marketed formulations. It is

hoped that characterization of the unknown degradation products of the drug will be helpful to

pharmaceutical industries in setting up their limits.


115

4.8. References

[1] The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals. Merck Research

Laboratories, USA, 14th

ed. 2006, p.1069.

[2] Lakshmi, M. V.; Rao, S. N.; Ravi Kumar, B. N. V. Development and validation of RP-

HPLC method for the estimation of milnacipran tablet dosage forms. International Journal

of Research in Pharmacy and Chemistry 2010, 1, 40-43.

[3] Silverstein, R.M.; Bassler, G.C.; Morrill, T.C. Spectrometric Identification of Organic

Compounds, John Wiley and Sons, Inc, New York, 1991.

[4] Pavia, D. L.; Lampman, G. M.; Kriz, G.S. Introduction to Spectroscopy, 3rd

ed. Thomson

Learning Inc, USA, 2001.

[5] Alsante, K.M.; Roland, B.; Yoshiko, T. The role of degradants profiling in active

pharmaceutical ingredients and drug products. Adv. Drug Deliver. Rev. 2007, 59, 29-37.

[6] Singh, S.; Bakshi, M. Guidance on conduct of stress tests to determine inherent stability of

drugs. Pharm. Tech. On-line 2000, 24, 1-14.

[7] Bakshi, M.; Singh, S. Development of validated stability indicating assay methods-critical

review. J. Pharm. Biomed. Anal. 2002, 28 (6), 1011-1040.

[8] International Conference on Harmonization of Technical Requirements for Registrations of

Pharmaceuticals for Human Use. Validation of Analytical Procedures: Text and

Methodology. Geneva; Q2 (R1); 2005.

[9] USP, United States Pharmacopoeia, 31st ed. NF 26. United States Pharmacopeia

Convention, Asian edition, Rockville, 2008, p.1225.

[10] Baertschi, S.W. Pharmaceutical Stress Testing, Predicting Drug Degradation. Taylor and

Francis Group, FL, USA, 2005.

[11] Nageswara Rao, R.; Nagaraju, V. An overview of the recent trends in development of HPLC

methods for determination of impurities in drugs. J. Pharm. Biomed. Anal. 2003, 33, 335-

377.

[12] Baertschi, S.W. Pharmaceutical Stress Testing, Predicting Drug Degradation. Taylor and

Francis Group, FL, USA, 2005.

[13] Kazakevich, Y.; Lobrutto, R. HPLC for Pharmaceutical Scientist. John Wiley and Sons,

Inc., Hoboken, New Jersey, USA, 2007.


116


Pharmaceuticals for Human Use. Specifications: Test Procedures and Acceptance Criteria

for New Drug Substances and New Drug Products:

Chemical Substances Specification for Test Procedure Guidelines. Geneva; Q6A; 1996.

[15] Sethi, P.D. HPTLC Quantitative Analysis of Pharmaceutical Formulations. CBS publishers

and distributors, New Delhi, India, 1996.

[16] Stahl, E. Thin-Layer Chromatography A Laboratory Handbook. Springer international

edition, berline, Heidelberg, 2005.

[17] Ahuja, S.; Alsante, K.M. Handbook of Isolation and Characterization of Impurities in

Pharmaceuticals. 1st ed. Volume 5, Academic Press, United Kingdom, 2003.

[18] Baertschi, S.W. Analytical methodologies for discovering and profiling degradation-related

impurities. Trend. Anal. Chem. 2006, 25, (8), 758-767.

[19] Gorog, S.; Babjak, M.; Balogh, G.; Brlik, J.; Csehi, A.; Dravecz, F. Drug impurity profiling

strategies. Talanta 1977, 44, 1517-1526.

[20] Wellings, D. A. A Practical Handbook of Preperative HPLC. Elsevier publication,

Aamsterdam, Netherlands, 2006.

[21] March, J. Advanced Organic Chemistry, Reactions, Mechanisms and Structures. John Wiley

and Sons Wiley India (P) New Delhi, India, 2005.

[22] Niessen, W. M.A. Liquid Chromatography-Mass Spectrometry. Chromatographic science

series, Taylor and Francis Group, FL, USA, 2006.

[23] Watson, J.T.; Sparkman, O.D. Introduction to Mass Spectrometry. John Wiley and Sons,

Ltd. Sussex, England, 2007.

[24] International Conference on Harmonization of Technical Requirements for Registrations

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(R2); 2006.


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STABILITY INDICATING ASSAY METHOD AND IMPURITY …shodhganga.inflibnet.ac.in/bitstream/10603/9714/11/12... · 2015. 12. 4. · Chapter 4: Stability indicating assay and impurity profiling