STABILITY INDICATING ASSAY METHOD AND IMPURITY …shodhganga.inflibnet.ac.in/bitstream/10603/9714/12/13_chapter 5.pdf · 5. STABILITY INDICATING ASSAY AND IMPURITY PROFILING OF DROTAVERINE

Chapter 5: Stability indicating assay and impurity profiling of DRT

118

Chapter 5

STABILITY INDICATING ASSAY

METHOD AND IMPURITY PROFILING

OF DROTAVERINE HYDROCHLORIDE


119

5. STABILITY INDICATING ASSAY AND IMPURITY PROFILING OF

DROTAVERINE HYDROCHLORIDE (DRT)

A simple, sensitive, fast, and accurate RP-HPLC stability indicating assay method was described

for DRT. The method involves use of simple mobile phase and separation was achieved on

octadecyl stationary phase with isocratic mode. For studying the degradation behavior of DRT,

extensive stress degradation studies were carried out as per ICH guidelines and all degradation

products formed in the stress studies were separated with the developed stability indicating assay

method. DRT was found to be stable in thermal and solid state photolytic stress conditions and

susceptible for degradation in acid, alkaline, neutral, and oxidative and solution state photolysis

stress conditions. Total three degradation products were observed with maximum degradation in

alkaline and neutral stress conditions. Subsequently, the developed assay method was validated

and the results were within the range of acceptance criteria. Finally the applicability of the

method was proved when it was applied for the determination of DRT in its pharmaceutical

tablet formulations.

The simple and sensitive RP-HPLC method for the determination of related impurities of DRT

was developed. The developed method was selective and could separate all the impurities found

in DRT. The method was also validated as per ICH guidelines and was found to be reliable from

the results of all the validation parameters. The method was then extended for the detection and

quantification impurities in tablet formulations of DRT where it was found that the impurities

detected in API of DRT were also observed in each tablet formulation of DRT in higher amount

(above identification threshold) indicating the possible cause of degradation of DRT.

As both the impurities detected in DRT and in its all the formulations were same as the

degradation products DP-I and DP II, the same were targeted for their isolation and structural

characterization. For this, Prep-HPLC method was developed for the isolation and isolated

degradation products of DRT, which were further characterized by using spectroscopic

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

spectroscopic techniques and elemental analysis the structures of all isolated degradation

products were proposed. The postulated mechanism for the formation of all the degradation

products of DRT from parent drug also helps in knowing the intrinsic stability of DRT.


120

5.1. Chemicals and materials

Analytically pure (98.30 %) Drotaverine Hydrochloride (DRT) Active Pharmaceutical

Ingredient was procured from Troikaa Pharmaceutical Ltd., (Ahmedabad, India), with

Certificate of Analysis.

Methanol, acetonitrile, potassium dihydrogen phosphate, orthophosphoric acid, formic

acid, and ammonia used for mobile phase preparation were of HPLC grade, Merck,

Mumbai, India.

Hydrochloric acid, sodium hydroxide, and hydrogen peroxide (30 % w/v) used for stress

degradation studies were of analytical reagent grade, CDH Chemicals, Delhi, India.

Calibrated micropitte were used for purpose for measurement and transfer.

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

USA) was used throughout the study.

The description of three tablet formulations of DRT is given in Table 5.1

TABLE 5.1 Detail information about DRT tablet formulations

Sr.

No.

Name of brand and its

manufacturer

Label

claim

(mg)

Average

Weight (mg) a

Batch

No.

Manufa

cturing

date

Expiry

date

A

Doverin, Intas

Pharmaceuticals Ltd.

Ahmedabad, Gujarat

40 283.4 J003 03/2009 03/2011

B

Drotin, Martin and

Harris labs. Ltd.

Haridwar, Uttarakhand

40 219.5 TDR-

62 07/2010 03/2012

C

Beralgan, Aventis

Pharma Ltd. Ankleshwar,

Gujarat

40 229.4 029001 11/2009 11/2011

a Average weight of 20 Tablets

5.2. Equipments/ Instruments

Details of Equipments/Instrument are described in section 4.2.


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5.3. Identification of Drotaverine Hydrochloride (DRT)

The identification of procured sample of DRT 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)

5.3.1. Melting point determination

Determination of melting point of DRT was carried out using melting point apparatus using

open capillary method.

TABLE 5.2 Comparison of melting point of DRT with reported melting point

Drug Reported melting point [1] Observed melting point

DRT 208-210 (ºC) 206-208 (ºC)

5.3.2. UV spectroscopy

UV spectrum of methanolic solution of DRT (20 µg/mL) was scanned in the range of 200-600

nm on UV-VIS spectrophotometer.

FIGURE 5.1 UV-spectra of methanolic solution of DRT (20 µg/mL) showing λmax at 281 nm


122

TABLE 5.3 Comparison of reported λmax with obtained λmax of DRT

Drug Reported λmax [2] Obtained λmax

DRT 280 nm 281 nm

5.3.3. FT-IR Spectroscopy

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

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

Table 5.4.

FIGURE 5.2 FT-IR spectra of DRT

TABLE 5.4 Important frequencies of DRT obtained in FT-IR spectra

Sr.

No. Functional group

Theoretical frequency

(cm-1

) [3-4]

Observed

frequency (cm-1

)

1 Secondary Amines (-NH) Str. 3500-3100 3477.03

2 Aromaticity 3000-3150 3049.87

3 Methyl (-CH3) Str. 3000-2850 2986.27

4 Aromaticity(Benzene overtones) 1550-1650 1598.70

5 Amine (C-N) Str. 1350-1000 1148.40

6 C-O ethoxy 1000-1100 1041.37


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5.3.4. Mass spectroscopy

The MS and MS/MS study of DRT was performed and spectra are shown in Figure 5.3 and 5.4

The (M+1) peak

was obtained at 398.3 m/z which confirms molecular weight of DRT at 397.0.

Figure 5.4 represents daughter ions of DRT at different m/z. The fragmentation pattern of DRT

is proposed in Figure 5.5.

FIGURE 5.3 Full scan MS spectra of DRT

FIGURE 5.4 MS/MS spectra of DRT at molecular peak of 398.3


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O

O

NCH2H2C

H3C

H3C

H2C

O

O

CH2H2C

CH3

CH3

H

O

O

NCH2H2C

H3C

H3C

H2C

OH

O

H2C

CH3

H

DRT m/z 398

m/z 370

O

O

NCH2H2C

H3C

H3C

H2C

OH

OH

H

m/z 342

O

O

NCH2H2C

H3C

H3C

H2C OH

H

m/z 326

HO

HO

N

H2C OH

H

m/z 270

HO

HO

N

H2C

H

m/z 254

N-H

HOm/z 162

H

O

O

NH3CH2C

H3C

H2C OH

H

m/z 310

O

O

NCH2H2C

H3C

H3C

H2C

OH

OCH3

H

m/z 354

HO

OH2CH3C

N

H

m/z 190

FIGURE 5.5 Proposed fragmentation pattern of DRT from MS/MS spectroscopic studies

5.3.5. NMR spectroscopy

The 1H and

13C NMR spectra of DRT were recorded as described in section 4.2. 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.


125

FIGURE 5.6 1H NMR spectra of DRT

FIGURE 5.7 13

C NMR Spectra of DRT


126

O

O2

345

6

7 8

9

10

1

CH2

H2C

H3C

H3C

H2C

O

O

CH2

H2C

CH3

CH311

12

13

1415

16

17

18

19

21

22

23

24

25

20

N

TABLE 5.5 1H NMR and

13C NMR spectral assignments for DRT

Position 1H

δ (multiplicity, j)

13C

δ Position

1H

δ (multiplicity, j)

13C

δ

2 3.92 (t,7.8) 40.57 14 7.10 (d,1.8) 114.01

3 3.00 (t,8.0) 25.12 15 - 155.85

4 6.75 (s) 111.54 16 - 148.93

5 - 147.96 17 6.78 (s) 113.39

6 - 147.35 18 4.04 (m, 6.9) 64.24

7 7.33 (s) 113.93 19 1.40 (m, 6.9) 14.17

8 - 174.17 20 4.04 (m) 64.54

9 - 125.75 21 1.40 (m) 14.28

10 - 116.61 22 4.19 (q, 6.9) 64.88

11 4.54 (s) 37.74 23 1.48 (t, 6.9) 14.48

12 - 133.56 24 4.04 (m) 64.88

13 6.88 (dd,1.8) 121.04 25 1.40 (m) 14.48

δ= Chemical Shift (ppm), j = Coupling Constant (Hz)


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5.4 Stability Indicating Assay Method (SIAM) for DRT by RP-HPLC

5.4.1. Experimental

5.4.1.1. 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 (57:43, v/v).

Buffer preparation: 0.0125 M potassium dihydrogen orthophosphate; add 0.2 % ammonia and

adjust the pH of buffer to 4.0 ± 0.02 with 1 M orthophosphoric acid.

Flow rate: 0.8 mL/min; Detection wavelength: 240 nm; Injection volume: 20 µL.

5.4.1.2. Preparation of solutions

Standard solutions: The standard stock solution 1 mg/mL was prepared by dissolving accurately

about 100 mg of DRT with methanol in 100 mL volumetric flask. The aliquots of this stock

solution were diluted with diluent (water: acetonitrile, 50:50, v/v) to get concentration of 10

µg/mL.

Sample solution for assay of DRT in tablet formulations: DRT tablet powder equivalent to 100

mg DRT for each brand (Table 5.1) 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 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 the filtrate was rejected and

subsequent filtrate was further diluted with diluent to obtain the solution of 10 µg/mL.

5.4.1.3. Stress degradation studies [5-8]

The stress degradation studies were carried by forcibly degrading DRT under different

stress conditions such as hydrolytic, oxidative, dry heat (thermal), photolytic degradation and

accelerated stability testing.

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

stressors, and was used for stress studies under optimized conditions as given in Table 5.6. A

minimum of four samples were generated for every stressed condition, viz., Initial (zero time)


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sample containing the drug with stressor and the drug solution subjected to stress treatment, the

blank solutions stored under normal condition, and the blanks subjected to identical conditions.

TABLE 5.6 Optimized stress degradation studies conducted on DRT

Stress degradation conditions Stressor

Acid hydrolysis 1 N HCl, reflux at 100 °C for 24 h

Alkaline hydrolysis 0.1 N NaOH, reflux at 100 °C for 2h

Neutral hydrolysis water, reflux at 100 °C for 24 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 DRT to all above 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 10 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 with concentration of 2 mg/mL and were suitably

diluted with diluent to get concentration 10 µg/mL. All the above samples were analyzed on

optimized RP-HPLC method as described in section 5.4.1.1. along with their respective initial

samples and blanks as described above. All the samples were allowed to run till the 2.5 times of

the retention time of DRT. The response of DRT obtained in every stress conditions were

compared with the responses of respective initial samples and the degradation of DRT was

reported in terms of % degradation.

5.4.1.4. Method validation [9-10]

The optimized stability indicating assay method for DRT was validated for following

parameters.


129

1. System suitability

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 (10 µg/mL). The parameters measured were retention time, peak area,

theoretical plates, and asymmetry of DRT.

2. Linearity and range

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

seven levels in the concentration range of 2-25 µg/mL for DRT. The solutions (20 µL) were

analyzed in triplicate as described in section 5.4.1.1. Peak area and concentrations were

subjected to least square regression analysis to calculate calibration equation and correlation

coefficient.

3. 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 5.4.1.3.

4. Precision

A. Method precision

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

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

individually to get concentration of 10 µg/mL. All the samples were analyzed as described in

section 5.4.1.1. The response obtained from each sample was extrapolated to find out the mean

assay value with RSD.

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, 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 were reported as mean assay of DRT and RSD of

assay results obtained in each intermediate precision studies.


130

5. Accuracy

Accuracy of stability indicating assay method for DRT was performed by recovery studies. Most

widely used synthetic mixture of tablets excipients (i. e. lactose, starch, magnesium stearate and

talc) were prepared (placebo) in the ratio of their permitted concentration in formulation of

tablets.

Known amounts of DRT corresponding to 80-120 % of the label claim (40 mg) were added to

placebo mixtures at three different levels in triplicate. For level I, II and III approximately 32, 40

and 48 mg of DRT (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, 0.1 mL from each flask were taken and diluted to 10 mL with diluent and the resulting

solutions were analyzed as described in section 5.4.1.1.

6. Robustness

Deliberate changes in the following parameters which affects % assay of DRT and system

suitability parameters were studied.

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

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.

7. Solution stability

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.

5.4.1.5. Method application to pharmaceutical formulations of DRT

The stability indicating assay method was used for the quantification of DRT in three different

brands of pharmaceutical tablet dosage forms of DRT. The description of tablets formulations of

DRT is given in Table 5.1.


131

The sample solutions of various marketed tablet formulations of DRT were prepared (as

described in section 5.4.1.2.) and analyzed as described in section 5.4.1.1. The percentage assay

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

that of samples.

5.4.2. Results and discussion

The presence of isoquinoline ring with two diethoxy groups makes the drug liable to loss of ethyl

groups leading to formation of alcoholic products. The resonating double bond present around

methylidine makes the drug to susceptible for the attack of nucleophilic agents which leading to

hydroxylation which may lead to formation keto structure.

5.4.2.1. Method development and optimization[11]

Non-polar stationary phase was tried for bringing the retention of the drug as the drug non-polar

in nature. Several modifications in the mobile phase composition were tried in order to bring about

proper good peak shape of drug and selectivity between the degradation products. The modifications

included the changing type and strength of buffer used for bringing ionization of drug, pH of buffer,

type, and ratio of the organic modifier, and flow rate. From the different mobile phases tried

mobile phase consisting mixture of phosphate buffer and acetonitrile (57: 43, v/v) was found to

be satisfactory when separation was carried on C18 stationary phase. Ammonia (0.2 %) was

added in the buffer preparation as peak reagent and the pH of the buffer was set to 4.0 ± 0.02,

which resulted in good peak with acceptable asymmetry and theoretical plates for DRT as shown

in system suitability parameters (Table 5.8). The RP-HPLC chromatogram obtained from

developed RP-HPLC method for DRT is shown in Figure 5.8.


132

FIGURE 5.8 RP-HPLC chromatogram of DRT (10 µg/mL) for stability indicating assay method

5.4.2.2. Stress degradation behavior of DRT

From the results of stress degradation studies of DRT it was observed that three major

degradation products (designated as DP I, DP II and DP III) were seen in acid, alkaline, neutral

and oxidative hydrolysis (Figure 5.9 a, b, c, d), however the % of degradation observed in these

hydrolytic conditions were different i.e. 9.9, 26.74, 24.13 and 12.44 % respectively (Table 5.8).

Two degradation products i.e. DP I and DP II were observed in solution state photolytic

degradation with 3.98 % degradation of DRT. The results of forced degradation study shows that

DP I, DP II and DP III are hydrolytic degradation products of DRT formed due to neutral

hydrolysis and their % of formation was enhanced in presence of alkaline condition. Amongst all

the major degradation products of DRT, DP III was formed only under refluxed condition as it

was not seen in photolytic degradation.

From the Table 5.8, it can been concluded that, DRT is stable in dry heat/thermal and solid state

photolytic stress studies as there was no change in peak area of stress samples compared to initial

peak area of DRT. DRT is however susceptible for hydrolysis in all the hydrolytic conditions

with order of degradation as alkaline ˃ neutral ˃ oxidative ˃ acidic ˃ solution state photolytic

degradation.

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


133

products obtained after stress studies.

a b

a


134

a

b

c

d


135

FIGURE 5.9 RP-HPLC chromatograms of DRT after a. Acid b. alkaline c. Neutral d. Oxidative

and e. Solution state photolytic treatment (I, II, and III are the major Degradation Products (DPs)

of DRT)

d

e


136

TABLE 5.7 Results from stress degradation study of DRT

NSD = No Significant Degradation

5.4.2.3. Method validation


The system suitability parameters were evaluated for the developed method by calculating the

RSD values of retention time, peak area, asymmetry, and theoretical plates of five standard

replicates (Table 5.8). The values are within the acceptable range indicating that the system is

suitable for the intended analysis.

TABLE 5.8 System suitability parameters of developed method for DRT

Parameters Observation RSD

Rt (min) 4.74 0.44

Peak area 663752 0.40

Theoretical plates 12656 0.97

Asymmetry 1.29 0.55 a Mean of five replicates

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 643889 579904 9.93

9.61 (0.998),

18.09 (0.997),

24.51 (0.998)

90.1

Alkaline

hydrolysis 639009 480901 26.74

10.71 (0.995),

18.20 (0.996),

24.51 (0.998),

75.3

Neutral hydrolysis 665787 505098 24.13

9.94 (0.996),

18.09 (0.998),

24.53 (0.999),

75.9

Oxidative

hydrolysis 658890 576908 12.44

10.06 (0.997),

18.61 (0.995),

24.77 (0.996),

87.6

Photolytic solution

state 665909 639347 3.98

9.97 (0.996),

18.10 (0.994), 96.0

Thermal/Dry Heat 671123 672233 NSD - -

Photolytic solid

state 671123 675667 NSD

- -

Accelerated

stability 671123 665098 NSD

- -


137


The correlation coefficient values obtained in linearity study for developed RP-HPLC method

(Figure 5.10) which confirms the good linearity of the method over the range studied (Table 5.9).

TABLE 5.9 Linearity of DRT by developed RP-HPLC method

Linearity Level Conc. of DRT ( µg/mL) Response observed (AUC) a RSD

I 2 117040 13.5

II 5 319994 6.6

III 7.5 484530 3.6

IV 10 661921 1.0

V 15 1007565 0.7

VI 20 1335476 3.6

a Mean of three replicates

FIGURE 5.10 Calibration curve of developed RP-HPLC method for DRT

3. Specificity

The specificity was evaluated from the forced degradation studies as described in section 5.4.2.2.

where Figure 5.9 a, b, c, d and e shows, DRT peak well separated from all the degradation

products formed during the different stress conditions with sufficient resolution (≥ 2). The peak


138

purity for DRT and all the degradation products of DRT were more than 0.999 indicating purity

of each separated peak and absence of interference due to other co eluting peaks. Thus specificity

study ensures the selectivity of the developed analytical method which is able to separate and

quantify DRT in presence of different degradation products.

4. Precision

The results (Table 5.10) of all the precision studies (Repeatability, intraday, interday and

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

criteria (98-102 %, ≤ 2 respectively) which proves the good precision of developed method.

TABLE 5.10 Precision study for DRT by SIAM

Precision study Observation

Mean Assay a RSD

Repeatability 99.23 0.77

Intraday b 98.89 0.57

Interday c 99.13 0.70

Different analyst d 99.19 0.74

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

6. Accuracy

The recovery for DRT was between 98.2 and 100.8 % with RSD of 1.0 % (Table 5.11), indicates

that the developed method was accurate for the determination of DRT in pharmaceutical

formulations.

TABLE 5.11 Accuracy study of DRT by SIAM

Level % Recovery Mean Recovery RSD

I

(80 % wrt to LC)

99.0 99.0 0.81 98.2

99.8

II

(100 % wrt LC)

98.3 99.83 1.33 100.6

100.6

III

(120 % wrt LC)

100.8 99.60 1.04 99.0

99.0

Mean 99.5


139

7. Robustness

The results of robustness studies are summarized in Table 5.12. In any condition assay value of

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

TABLE 5.12 Robustness study of DRT by SIAM

Robustness condition Observation

System suitability %

Assay

% difference

in assay b RSD

a Rt T A

- 5% Acetonitrile (Buffer:

Acetonitrile 59:41 v/v) 0.82 5.12 10998 1.25 99.77 + 0.54

+ 5% Acetonitrile (Buffer:

Acetonitrile 54:46 v/v) 0.27 4.32 9991 1.30 99.09 - 0.14

- 0.05 Changed pH of buffer of

mobile phase - 3.95 0.94 4.80 11109 1.27 99.67 + 0.44

+ 0.05 Changed pH of buffer of

mobile phase - 4.05 0.74 4.73 12998 1.29 98.94 - 0.29

- 10% Change in flow rate - 0.72

mL/min 0.92 5.10 11909 1.29 98.25 - 0.98

+ 10% Change in flow rate - 0.88

mL/min 1.42 4.35 9564 1.31 99.93 + 0.71

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 5.13), it was found that the % difference of

impurities is more than 2 % when compared with initial showing standard and sample solution

stability of DRT is upto 24 hr.

TABLE 5.13 Solution stability study of DRT by SIAM

Interval

Observation

% Assay % Difference

STD* Sample* STD Sample

Initial 100 99.46 - -

12 h 98.99 98.66 - 1.01 - 0.80

24 h 98.26 98.18 - 1.74 - 1.28

48 h 95.92 95.98 - 4.08 - 3.48

* Result are from duplicate injection of same solution

5.4.2.4. Method application


140

Figure 5.11 a, b and c represents the chromatograms of DRT in three different tablet brands. The

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

satisfactory, accurate, and precise for estimation of DRT without interference of excipients, as

indicated by the good recovery and acceptable standard deviation (SD) values (Table 5.15).

(a)

(b)


141

FIGURE 5.11 Representative RP-HPLC chromatograms of DRT (10 µg/mL) in Brand A (a);

Brand B (b) and Brand C (c)

TABLE 5.14 Summary of results for DRT assay in marketed tablet dosage forms

Formulation Amount of drug recovered a (mg) ± SD

b % Assay ± SD

b

A 39.5 ± 0.20 98.8 ± 0.51

B 39.2 ± 0.20 98.0 ± 0.50

C 39.7 ± 0.24 99.3 ± 0.40 a Label claim = 40 mg;

b n = 3

5.4.3. Conclusion

The developed stability indicating RP-HPLC method for the determination of DRT was found to

be simple, selective, sensitive, and economical.

The results from the stress degradation studies shows that DRT is susceptible for degradation in

all the hydrolytic degradation conditions and maximum degradation was observed in alkaline and

neutral stress degradation conditions. The developed stability indicating assay method was

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

applicability of the method was proved when it was applied for the estimation of DRT in

pharmaceutical tablets formulations of DRT.

(c)


142

The two of the three degradation products of DRT (DP I and DP II) were found in all the

hydrolytic and in solution state photolytic stress degradation conditions where DP III was

obtained only in refluxed conditions of stress. Hence it was concluded that the alkaline and

neutral conditions are required for the formation of DP I and DP II however DP III is formed

only in harsher conditions i.e. refluxing DRT at higher temperatures.

It is further required to do LC-MS/MS study for characterization of degradation products and

elucidation of degradation pathway of DRT.


143

5.5. Related Impurities Method for Drotaverine Hydrochloride by RP-HPLC

5.5.1. Experimental


The optimized method for stability indicating assay of DRT as described in section 5.4. was

applied for detection and quantification of related impurities in DRT with same chromatographic

conditions as described in section 5.4.1.1.

5.5.1.2. Preparation of solutions

Diluted standard preparation: The diluted standard solution of DRT with concentration of 0.5

µg/mL was prepared from the standard stock preparation of DRT as described in section 5.4.1.2.

Preparation of sample solution: The standard stock solution described in section 5.4.1.2. was

further diluted with diluent to get the concentration of 100 µg/mL.

Sample solution for related impurities of DRT in tablet formulations: The same procedure as

described in section 5.4.1.2. for assay of DRT from marketed formulations was followed for

related impurities detection with only change in sample concentration 100 µg/mL of DRT.

5.5.1.3. Method validation

The optimized related impurities method for DRT was validated for following validation

parameters.


The system suitability test was performed by injecting five replicate injections of diluted

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

theoretical plates, and asymmetry of DRT.

2. Linearity and Range

The 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 DRT were prepared by making the dilution given in Table 5.1. Samples at each


144

linearity level were analyzed in triplicate as described in section 5.4.1.1 and the response was

measured in the form of area under the curve of DRT.

TABLE 5.15 Linearity study of DRT (Unknown impurity)

Linearity

level Volume (mL) taken

from standard stock a

Diluted with

diluent (mL)

Conc. of the solution

(µg/mL)

I (LOQ) 0.05 100 (0.05 %) 0.05

II 0.125 100 0.125

III 0.25 100 0.25

IV 0.5 100 0.5

V 0.75 100 0.75

VI 1 100 1.0

a Stock solution: 0.1 mg/mL of DRT

3. LOD and LOQ

LOD, LOQ, and Precision at LOQ for DRT by related impurities method was determined as

described in section 4.4.1.4.

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 5.4.1.3.

5. Precision

A. Method Precision

For repeatability study, the six sample sets of DRT were prepared having concentration of 100

µg/mL and were analyzed as described in section 5.4.1.1. 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

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

and different analysts precision.


145

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.

6. Accuracy

The accuracy of the method for unknown impurity was studied with respect to recovery of DRT.

The accuracy of unknown impurity with respect to DRT was determined over the range of LOQ

to 200 % of the specification limit of impurity (LOQ being 0.05 µg/mL to 1.0 µg/mL) at IV

levels.

The procedure as described in accuracy study of section 5.4.1.4. was followed in accuracy study

for unknown impurity. The placebo mixture was prepared and standard stock solution of DRT

(0.1 mg/mL), was spiked (0.05, 0.25, 0.5, and 1.0 mL) at different levels in 100 mL volumetric

flasks in triplicate containing constant weights of placebo, followed by addition of 80 mL of

diluent and the solution was then 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 5.4.1.1.

7. Robustness

The robustness was studied by making the deliberate changes in chromatographic parameters and

procedure as described in section 5.4.1.4.

8. Solution Stability

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

standard solution and sample solution) 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

and comparing the results with the freshly prepared diluted standard solutions analyzed

simultaneously as described in section 5.4.1.1.


146

5.5.1.4. Method application to pharmaceutical tablet formulations of DRT

The developed and validated related impurities method was successfully applied for the

estimation of impurities in three different brands of tablet formulations of DRT. The description

of tablet formulations of DRT is given in Table 5.1.

The sample solutions for related impurities of DRT in different tablet brands (prepared as

described in section 5.4.1.2.) were analyzed as described in section 5.4.1.1. The impurities

detected above 0.05 % were taken in consideration and the % of each individual impurity and

total impurities were calculated.


5.5.2.1. Development and optimization of related impurities method[11]

The method of related impurities of DRT was developed with the aim to detect and quantify all

the impurity at low concentration of reporting threshold as specified by ICH guidelines (0.05 %).

With keeping sensitivity and selectivity in mind the optimized conditions of stability indicating

assay method of DRT was applied for the detection and quantification of impurities of DRT.

Figure 5.12 shows good peak shape of DRT at 100 µg/mL concentration. The system suitability

parameters are mentioned in Table 5.16. At this concentration of DRT, two additional peaks

were also seen at Rt 9.9 (designated as IMP I) and at 18.2 min (designated as IMP II)

respectively (Figure 5.12). The peak area of IMP II was more (0.48 %) as compared to peak area

IMP I (0.12 %). Both the detected impurities were found to be well separated from each other

and from parent drug with good resolution (˃ 2).


147

FIGURE 5.12 RP-HPLC chromatogram of DRT (100 µg/mL) for related impurities method

showing IMP I at Rt 9.99 and IMP II at 18.24 mins respectively.

5.5.2.2. Method validation [12-13]


The system suitability was evaluated by calculating the RSD values of retention time, peak area,

asymmetry, and theoretical plates of five standard replicates. The experimental results (Table

5.16) showed that the values are within the acceptable range indicating that the system is suitable

for the intended analysis.

TABLE 5.16 System suitability parameters for DRT diluted standard preparation (Unknown

impurity)

Parameters Observation a RSD

Rt (min) 4.74 0.21

Peak area 43642 4.19

Theoretical plates 13217 0.88

Asymmetry 1.29 1.15 a Mean of five replicates


148


The correlation coefficient value obtained in related impurities method (Figure 5.13) confirms

the good linearity of the method over the range studied.

FIGURE 5.13 Plot of linearity curve for DRT by the developed method

3. LOD and LOQ

From the triplicate results of linearity study, SD and slope value was found to be 435.6 and

79469 respectively which is further used to calculate LOD and LOQ values. LOD value was

found to be 0.02 µg/mL and LOQ was 0.05 µg/mL. The RSD value of theoretically calculated

LOQ preparation was found to be 5.5 with mean area 6806.

4. Specificity

The developed RP-HPLC method for determination of impurities of DRT was specific as proved

from the results of the stress degradation studies. The peak purity for all the observed

degradation products of DRT were more than 0.999 indicating purity of each separated peak and

absence of interference due to other co eluting peaks.

5. Precision


149

The results of all the precision studies (Table 5.17) obtained in related impurities method

(repeatability, intraday, interday and different analyst), shows that RSD values are within the

acceptance criteria which proves the good precision of developed method.

TABLE 5.17 Precision study DRT by related impurity method (Unknown impurity)

Presicion study Mean of total Impurities (%) a RSD

Repeatability a 0.60 1.51

Intraday b 0.60 2.8

Interday c 0.62 4.17

Different analyst d 0.62 5.24

a n= 6

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

c Mean value of day I and day

II observations; d

Mean value of analyst I and analyst II observations

6. Accuracy

The mean recovery at LOQ level is 102.8 % with 3.2 % RSD which is within the acceptance

criteria. Similarly, the recovery range at level II, III, and IV between 100.04 to 106.11 % which

also is within the acceptance criteria (Table 5.18).

TABLE 5.18 Accuracy study of DRT by related impurity method (Unknown impurity)

Level % Recovery Mean Recovery RSD

I

LOQ (0.05 %)

104.96

102.8 3.2 104.56

99.01

II

(50 % wrt to

specification limit)

106.11

105.3 0.9 105.48

104.33

III

(100 % wrt to


100.04

102.0 2.1 101.65

104.29

IV

(200 % wrt to


101.07 101.9 1.6

103.73

100.86

Mean 103.00


150

7. Robustness

The results of robustness studies are summarized in Table 5.19. The assay value of sample is not

deviating more than 2.0 % indicating that the method is robust in nature.

TABLE 5.19 Robustness study of DRT by related impurities method

Robustness condition

Observation

System suitability % Total

impurities

Absolute

difference % RSD a Rt T A

- 5% Acetonitrile (Buffer:

Acetonitrile 59:41 v/v) 4.12 5.10 9998 1.23 0.58 -0.02

+ 5% Acetonitrile (Buffer:

Acetonitrile 55 : 45 v/v) 3.88 4.30 8891 1.27 0.57 -0.03

- 0.05 Changed pH of buffer of

mobile phase - 3.95 5.66 4.76 10109 1.34 0.61 +0.01

+ 0.05 Changed pH of buffer of

mobile phase - 4.05 4.38 4.74 9998 1.30 0.60 0.0

- 10% Change in flow rate - 0.72

mL/min 2.35 5.13 9909 1.34 0.62 +0.02

+ 10% Change in flow rate - 0.88

mL/min 4.77 4.39 8881 1.29 0.60 0.0

a from five values of standard area;

b % difference compared from the method precision result; T=

Theoretical plates; A = Asymmetry


There was no significant difference in the % assay of diluted standard preparation from initial

and also the not much deviation in % of total impurities found in samples indicates that standard

and samples solutions are stable at ambient temperature for 24 h (Table 5.20)

TABLE 5.20 Solution stability study of DRT by related impurity method

Interval Observation

% Assay of STD* Total Impurities (%)* Absolute difference

STD Sample

Initial 100.0 0.65 - -

12 h 98.97 0.95 - 1.03 + 0.30

24 h 98.22 1.44 - 1.78 + 0.79

48 h 95.88 2.45 - 4.22 + 1.80

* Result are from duplicate injection of same solution


151

5.5.2.3. Method application

The proposed method of related impurities was applied for the determination of impurities in

tablet formulations of DRT in three different brands.

The impurities detected in DRT at Rt 9.9 min and 18.2 min as described in section 5.5.2.1 were

also found in all the tablet formulations of DRT in different amount. The results from impurity

analysis of DRT tablet formulations are summarized in Table 5.21.

TABLE 5.21 Summary of results for related impurities for DRT in marketed tablet dosage forms

Formulation % of impurities Found

a

Total impurities a (%)

IMP I IMP II

A 0.11 1.21 1.32

B 0.15 2.33 2.48

C 0.19 0.48 0.67 a Mean value of three determinations

From Table 5.21, it was observed that IMP I and IMP II found in all the three formulations above

the identification threshold specified by ICH guidelines, thus it is very much essential to

characterize these two impurities found in DRT formulations.

5.5.3. Conclusion

The developed related impurities method for DRT is simple, involves use of simple mobile phase

with isocratic elution and easy extraction procedure. The method could detect, separate, and

quantify all the found impurities in DRT API with sufficient resolution. The reliability of the

method was proved form the acceptable results of all the validation parameters.

It was observed that both the impurities (IMP I and IMP II) detected in DRT and its tablets

formulation are degradation impurities of DRT since their Rt. was same as degradation products

(DP I and DP II) of stress studies of DRT and also gave same PDA spectra in the range of 200-

800 nm.

Further, it is necessary to elucidate the structures of both the Degradation Products (DPs) of DRT

as they are quantified above the identification threshold as prescribed by ICH guidelines.


152

5.6. Isolation and characterization of Degradation Products (DPs) of DRT

5.6.1. Experimental


For the isolation of DPs of DRT, method was developed and optimized on analytical and then

transferred to Prep-HPLC.

Chromatographic conditions (analytical HPLC)

Column: C18 column (Inertsil ODS 3V, 250 x 4.6 mm id 5µm).

Mobile phase: Water (pH of water adjusted to 3.60 ± 0.02 with formic acid after addition of 0.2

% ammonia as peak reagent): Acetonitrile (55:45, v/v).

Flow rate: 0.8 mL/min; Detection wavelength: 240 nm; Injection volume: 20 µL.

5.6.1.2. Solution preparation

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

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

degradation as described in section 5.4.2.2. Since major degradation products were found in

neutral conditions of degradation, neutral degradation was considered as a source for the

generation of degradtion products of DRT. The prepared aqueous solution of DRT was then

stressed by refluxing at 100 °C for 110-115 h to generate all the degradation products in

maximum ammount.

5.6.1.3. Isolation of DPs of DRT by Prep-HPLC[14-16]

The Prep-HPLC method developed as described in section 5.6.1.1. was scaled up for column

(Semi-Prep-HPLC column), Flow rate (6.0 mL/min), and injection volume (1.0 mL) but was

modified for gradient programming for reducing run time on Prep-HPLC system. Table 5.22

depicted gradient program used for Prep-HPLC method in which solution A and B represents

mobile phase and acetonitrile respectively.


153

Table 5.22 Gradient programming of mobile phase for Prep-HPLC method

Time (min) Solution A Solution B

0.01 100 0

7.00 100 0

7.01 75 25

9.00 45 55

12.00 75 25

13.00 100 0

The neutral degraded sample solution of DRT prepared as described in section 5.6.1.2. was

loaded on Prep-HPLC and eluents containing targeted DPs were collected and concentrated by

evaporating acetonitrile portion of eluents at room temperature under high vacuum on rota

evaporator. The concentrated aqueous layers were further dehydrated with solid sodium sulphate

(approximately 1 g) and further extracted with chloroform (50 mL each time) thrice for each DP

of DRT. The collected combined chloroform layers were then evaporated individually in rota

evaporator to get solid masses of DPs of DRT. Before characterization of isolated DPs using

different spectroscopic techniques, chromatographic purity of each DP was checked using

developed RP-HPLC method as described in section 5.6.1.1.

5.6.1.4. Characterization of isolated DPs by spectroscopic techniques

The isolated and purified DPs (designated as DP I and DP II) were further analyzed by different

spectroscopic techniques like UV, FT-IR, Mass and NMR spectroscopy for characterization and

structural elucidation.

5.6.1.4.1 UV Spectroscopy

The standard solution (20 µg/mL) of DP I and DP II were prepared individually in methanol and

used to do analysis in UV-VIS region from 200-800 nm to determine their ƛ max.

5.6.1.4.2 FT-IR spectroscopy

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

spectra of DP I and DP II were recorded in the range of wave number 400-4000 cm-1

and

compared with spectra of DRT recorded as described in section 5.3.3.

5.6.1.4.3 Mass spectroscopy

The MS and MS/MS experiments were performed on a Varian MS system as described in

section 4.2. The analysis was performed in positive ionization mode with Electrospray interface.


154

The mass to charge (m/z) ratio was recorded in the range of 50-800 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.

5.6.1.4.4 NMR spectroscopy

The 1H and

13C NMR studies on DP I and DP II were carried out on instrument as described in

section 4.2.

5.6.1.5. Elemental Analysis

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

nitrogen in isolated DPs on CHN-O element analyzer as described in section 4.2.


5.6.2.1. Method development and optimization

The isolation and purification of all the DPs of DRT, Prep-HPLC method was developed on

analytical HPLC. Method development was initiated using neutral degraded sample of DRT

where all targeted degradation products (DP I, DP II, and DP III) were formed in sufficient

quantity (Figure 5.9 c). Satisfactory separation between all the DPs of DRT was achieved when

mobile phase described in section 5.6.1.1. was used where DP I, DP II and DP III eluted at

Rt.7.8, 25.8 and 30.8 mins, respectively (Figure 5.14) which were well separated from DRT peak

(Rt 5.66). Sufficient resolution was obtained between DRT and all the DPs which would be

helpful for isolation of DPs in pure form.


155

FIGURE 5.14 RP-HPLC chromatogram (10 µg/mL) of neutral treated DRT showing well

resolved DPs of DRT

5.6.2.2. Isolation and purification of DPs

The eluent fractions were collected containing DP I, DP II and DP III and processed as described

in section 5.6.1.3. to get solid mass of DP I (yield 70 mg) and DP II (yield 50 mg). However DP

III was not obtained in sufficient quantity for further characterization. DP I and DP II showed

melting point 216-218, and 212-214 °C respectively.

5.6.2.3. Chromatographic purity of isolated DP-I and DP-II

Before carrying out the spectroscopic experiments, the purity of isolated DPs was carried out as

described in section 5.6.1.1. The chromatographic purity of isolated DP I and DP II was found to

be 95.4 and 99.1% respectively. Figure 5.15 represents the RP-HPLC chromatograms of isolated

DP I (a) and DP II (b) showing the chromatographic purity.


156

FIGURE 5.15 RP-HPLC chromatogram of isolated DPs of DRT (a) DP I and (b) DP II showing

chromatographic purity

a

b


157

5.6.2.4. Spectroscopic characterization of isolated DPs

5.6.2.4.1 UV spectroscopic analysis

The results from UV spectroscopic analysis of both the DPs of DRT are depicted in Table 5.23.

The UV spectra of both the DPs (Figure 5.16 a, and b) suggests the probable presence of parent

chemical moiety in structure of both the DPs as both have very near UV absorption maxima as

that was observed for DRT (281nm).

TABLE 5.23 Results from the UV spectral analysis of DPs of DRT

Compound Observed absorption maxima (ƛ max)

DP I 250, 285, 360,

DP II 228, 283, 328

FIGURE 5.16 UV spectra of (20 µg/mL) DP I (a) and DP II (b) in methanol

a

b


158

5.6.2.4.2 FT-IR spectroscopy

Figure 5.17 shows FT-IR spectra of DP I and DP II obtained in diffused reflectance mode with

characteristic frequencies observed reported in Table 5.24.

FIGURE 5.17 FT-IR spectra of DP I (a) and DP II (b)

a

b


159

TABLE 5.24 Important frequencies of DPs obtained in FT-IR spectra

Compound Wave number (cm-1

)

DP I 3587, 3093,3014, 2806, 1592, 1139, 1043

DP II 3079, 2994, 2849, 1655, 1590, 1516, 1139,1041,

5.6.2.4.3 Mass spectroscopy

The MS of DP I and DP II exhibited molecular ion at m/z (M+1) 414 and 412 amu respectively

(Figure 5.18). Further MS/MS studies on both the DPs was also carried out to study their

fragmentation pattern and to predict the structures of both the DPs (Figure 5.19). Table 5.25

depicts the summarized results of mass spectroscopic studies performed.

a


160

FIGURE 5.18 Mass spectra of DP I (a) and DP II (b) showing m/z value 414.2, 412.2 amu

respectively

b

a


161

FIGURE 5.19 MS/MS of DP I (a) and DP (II)

Table 5.25 shows the summarized results of Mass spectroscopic experiments carried on DP I and

DP II

TABLE 5.25 Summary of results from the mass spectroscopic analysis

Compound Observed parent ions (m+1) and major daughter ions

DP I 414 , (m+1),

412, 396, 384,370, 367, 357, 354,310, 234, 262, 218, 190, 162

DP II 412 (m+1), 396, 384, 370, 367, 354, 353, 340, 326, 324, 262, 253 234, 218, 190,162

5.6.2.4.4 NMR spectroscopy

For the further confirmation of proposed structures of DP I, and DP II, NMR spectroscopic

experiments were carried out. The NMR experiments could not be done on DP III because of its

low recovery. Figure 5.20 (a and b) shows the 1H NMR of DP I and DP II and Figure 5.21. (a

and b) shows 13

C NMR of DP I and DP II. The results from the NMR (1H and

13C) spectral data

for DRT and DP I and DP II are compiled in Table 5.26.

b


162

FIGURE 5.20 1H NMR spectra of DP I (a) and DP II (b)

a

b


163

FIGURE 5.21 13

C NMR spectra of DP I (a) and DP II (b)

b

a


164

TABLE 5.26 NMR (1H and

13C) spectral assignments for DRT, DP I and DP II

Position

1H δ, (multiplicity, j)

13C δ ( ppm)

DRT DP I DP II DRT DP I DP II

2 3.92 (t,7.8) 3.94 (t,7.5) 3.91 (t,7.8) 40.57 42.21 47.19

3 3.00 (t,8.0) 2.91 (t,7.8) 2.79 (t,7.7) 25.12 25.54 25.42

4 6.75 (s) 7.18 (s) 6.73 (s) 111.54 111.09 111.15

5 - - - 147.96 147.56 148.54

6 - - - 147.35 147.11 147.09

7 7.33 (s) 6.77 (s) 6.90 (s) 113.93 113.57 111.91

8 - - - 174.17 166.21 164.90

9 - - - 125.75 121.66 128.28

10 - - - 116.61 120.98 119.46

11 4.54, s 6.69, s - 37.74 170.93 192.90

12 - - - 133.56 133.11 131.01

13 6.88 (dd,1.8) 7.72 (dd,1.5) 7.55 (dd,1.8) 121.04 124.37 126.44

14 7.10 (d,1.8) 7.59 (d,1.5) 7.65 (d, 1.5) 114.01 114.08 112.87

15 - - - 155.85 153.30 153.86

16 - - - 148.93 148.09 151.53

17 6.78 (s) 6.90 (d,8.4) 6.85 (d,8.7) 113.39 111.57 111.96

18 4.04 (m) 4.03 (m) 4.01 (m) 64.24 64.57 64.51

19 1.40 (m) 1.40 (m) 1.40 (m) 14.17 14. 63 14.10

20 4.04 (m) 4.03 (m) 4.01 (m) 64.54 64.50 64.51

21 1.40 (m) 1.40 (m) 1.40 (m) 14.28 14.63 14.10

22 4.19 (q,6.9) 4.16 (m) 4.18 (m) 64.88 64.88 64.89

23 1.48 (t,6.9) 1.49 (m) 1.48 (t,7.1) 14.48 14.71 14.64

24 4.04 (m) 4.03 (m) 4.01 (m) 64.88 64.88 64.89

25 1.40 (m) 1.40 (m) 1.40 (m) 14.48 14.71 14.64

26 - 4.28 (s) - - - -

*Refer structures for numbering (Figure 5.27 a. and b.); δ= chemical shift; j= coupling

constant in Hz

5.6.2.4.5 Elemental Analysis of DP I and DP II

Table 5.27 depicts the results from the elemental analysis of DP I and DP II, which supports the

proposed structures.


165

TABLE 5.27 Results from the elemental analysis of DP I and DP II

Element estimated (%) DP I DP II

Carbon 69.38 69.81

Hydrogen 7.21 7.32

Nitrogen 3.15 3.22

5.6.2.6. Structural elucidation of DPs of DRT

5.6.2.6.1. Structural elucidation of DP I

The spectral data of DP I was compared with that of DRT. As observed in Figure 5.22.a. the

mass spectrum of DP I exhibited molecular ion [M+H] + at 414, which is 16 Da more than that of

DRT (Figure 5. 3. a). The 1H NMR spectrum showed all signals corresponding to DRT with

small shift in the δ ppm values, additionally a singlet was observed at 4.28 ppm. Compared to

DRT, signal obtained due to proton at position 11 was observed at downfield position of 6.69

ppm suggesting substitution at this carbon with electronegative group (Table 5.26). In 13

C NMR

also, the signal obtained due same numbered carbon at 37.74 ppm was not observed but

downfield signal at 170.93 ppm near to chemical shift value of hydroxyl group was obtained for

DP I supporting 1H NMR results. This assumption was further supported by the results of FT-IR

analysis of DRT and DP I where the characteristic broad peak of hydroxyl group was seen at

wave number 3587 cm-1

(Figure 5.24 a), confirming the hydroxylation of the parent drug. The

other characteristic frequencies observed at 1042, 1509, 2985, and 3049 at cm-1

, due to ethoxy

group (-O-C-), amine (-C-N), aliphatic alkanes and aromatic alkanes respectively, obtained for

DRT were also observed for DP I with small shift suggesting the retention of parent structure of

DRT and only hydroxylation has been taken place in generation of DP I (Table 5.24). Based on

these spectral data the molecular formula of DP I was proposed as C24 H31NO5 which was finally

confirmed from the elemental analysis results (Table 5.27) where the % of carbon, hydrogen and

nitrogen practically observed were 69.38, 7.21, and 3.15 % respectively. Finally the structure of

DP I was characterized as (6,7-diethoxy-3, 4-dihydroisoquinolin-1-yl) (3, 4-diethoxyphenyl)

methanol (Figure 5.22.a.) from the MS/MS study of DP I where the daughter ions obtained were

corresponding to the proposed fragmentation pattern of DP I (Figure 5.23)


166

O

O

N

2

345

6

7 8

9

10

1

CH2

H2C

H3C

H3C

CH

O

O

CH2

H2C

CH3

CH311

12

13

1415

16

17

18

19

21

22

23

24

25

20

HO26

O

O

N

2

345

6

7 8

9

10

1

CH2

H2C

H3C

H3C

C

O

O

CH2

H2C

CH3

CH311

12

13

1415

16

17

18

19

21

22

23

24

25

20

O

FIGURE 5.22 Chemical structures of DP I (a) and DP II (b)

5.6.2.6.2. Structural elucidation of DP II

The mass spectrum of DP II exhibited molecular ion [M+H] + at 412, (Figure 5.22 b.) which is

14 Da more than that of DRT. Similar to DP I, the 1H NMR spectrum of DP II also showed all

signals corresponding to DRT with small shift in the δ ppm values but signal at 4.54 ppm due to

proton of position 11 was not observed suggesting loss of additional hydrogen from DP I. Even

more downfield signal at 192.90 ppm was obtained in 13

C NMR spectrum suggesting more

electronegative (possible carbonyl) group. The downfield signal obtained at chemical shift value

of 47.19 ppm in DP II compared to DP I at 42.21 and DRT at 40.57 confirmed the substitution

with electronegative group at this position. The observation of characteristic FT-IR peak at wave

number 1655 cm-1

near to frequency of carbonyl groups confirmed the presence of carbonyl

group in structure of DP II with observation of all the remaining frequencies of ethoxy group (-

O-C-), amine (-C-N), aliphatic alkanes, and aromatic alkanes. Based on the above spectral

results, the molecular formula of the DP II was proposed as C24 h29NO5 which was also confirmed

from the elemental analysis report with practically observed values of % carbon; hydrogen and

nitrogen were 69.81, 7.32 and 3.22 % respectively. Finally the structure of DP II was

characterized as (6,7-diethoxy-3,4-dihydroisoquinolin-1-yl)(3,4-diethoxyphenyl) methadone

(Figure 5.22 b.) from the MS-MS study of DP II where the daughter ions obtained were

corresponding to the proposed fragmentation pattern of DP II (Figure 5.23)

(a) (b)


167

DP-I 414

O

O

O

O

H2C

H2C

H2C

H2CH3C

H3C

H3C

H3C

N

OH

O

O

O

O

H2C

H2C

H2C

H2CH3C

H3C

H3C

H3CDRT398

NH

OH

O

O

O

O

H2C

H2C

H2C

H2CH3C

H3C

H3C

H3C

N

O

HO

O

O

O

H2C

H2C

H2CH3C

H3C

H3C

N

O

384DP-II 412

N

O

235

O

O

O

H2C

H2C

H2CH3C

H3C

H3C

N

O

367

O

O

H2C

H2C

H3C

H3C

N

218

HO

OH2CH3C

N

218

HH

HH

H H

HH

N-H

HO

162

H

FIGURE 5.23 Proposed fragmentation pattern of DP I and DP II

5.6.2.7. Mechanism of Formation of DP I and DP II [17]

The stress degradation studies helped to propose the possible mechanism through which DP I

and DP II may have been formed from DRT.

As seen earlier, both degradation products are formed in neutral and alkaline degradation

conditions in maximum amounts compared to other conditions. Hence it is postulated that in

neutral and alkaline medium the DRT gets hydroxylated to DP I which upon further oxidation

leads to formation of DP II. The hydroxylated DP I may be an intermediate in the formation of

DP II which is may be formed through the ketoenoltautomerization mechanism (Figure 5.24)


168

O

O

NHCH2H2C

H3C

H3C

HC

O

O

CH2H2C

CH3

CH3

DRT

OH

H2O,NaOH, H2O2

O

O

NHCH2H2C

H3C

H3C

HC

O

O

CH2H2C

CH3

CH3

OH

O

O

NCH2H2C

H3C

H3C

HC

O

O

CH2H2C

CH3

CH3

DP-I

OH

O

O

NCH2H2C

H3C

H3C

C

O

O

CH2H2C

CH3

CH3

- H2

Oxidation/Keto-Enolization

DP-II

O

.

FIGURE 5.24 Plausible mechanism for the formation of DP I and DP II from DRT


169

5.6.3. Conclusion

The impurities detected above identification threshold (and also found as degradation products)

in the impurity analysis of DRT were targeted for the isolation and characterization by

spectroscopic techniques. Suitable Prep-HPLC method was developed and optimized for the

isolation which was also compatible with LC-MS method. All the DPs were isolated in pure

form and characterized using spectroscopic techniques and from the results of spectroscopic data

the structures of DP I and DP II could be proposed and finally were confirmed by MS/MS

fragmentation studies. Mechanism for formation of both the degradation products (DP I and DP

II) were postulated in given set of conditions.


170

5.7. References

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

Research Laboratories, USA, 2006, 14th

ed. p. 585.

[2] Vivek, S. R.; Santosh, V. G.; Upasana, P.P.; Mahima, S. Simultaneous determination of

drotaverine hydrochloride and aceclofenac in tablet dosage form by spectrophotometry.

Eurasian J. Anal. Chem. 2009, 4, 184‐190.

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

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

[6] 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.

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

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

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

and Francis Group, FL, USA, 2005.

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

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

[10] International Conference on Harmonization of Technical Requirements for

Registrations of 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.

[11] Rao, N. 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.


Registrations of Pharmaceuticals for Human Use Impurities in New Drug

Substances. Geneva; Q3A (R2); 2006.


171


Registrations of Pharmaceuticals for Human Use Impurities in New Drug Products.

Geneva; Q3B (R2); 2006.

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

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

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

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

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

Aamsterdam, Netherlands, 2006.

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

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

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