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Chapter 5: Stability indicating assay and impurity profiling of DRT
118
Chapter 5
STABILITY INDICATING ASSAY
METHOD AND IMPURITY PROFILING
OF DROTAVERINE HYDROCHLORIDE
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling 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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
121
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
123
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
124
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
125
FIGURE 5.6 1H NMR spectra of DRT
FIGURE 5.7 13
C NMR Spectra of DRT
Chapter 5: Stability indicating assay and impurity profiling 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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
127
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
128
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
133
products obtained after stress studies.
a b
a
Chapter 5: Stability indicating assay and impurity profiling of DRT
134
a
b
c
d
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
136
TABLE 5.7 Results from stress degradation study of DRT
NSD = No Significant Degradation
5.4.2.3. Method validation
1. System suitability
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
- -
Chapter 5: Stability indicating assay and impurity profiling of DRT
137
2. Linearity and range
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
8. Solution stability
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
143
5.5. Related Impurities Method for Drotaverine Hydrochloride by RP-HPLC
5.5.1. Experimental
5.5.1.1. Chromatographic conditions
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.
1. System suitability
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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. Results and discussion
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).
Chapter 5: Stability indicating assay and impurity profiling of DRT
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]
1. System suitability
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
148
2. Linearity and range
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
specification limit)
100.04
102.0 2.1 101.65
104.29
IV
(200 % wrt to
specification limit)
101.07 101.9 1.6
103.73
100.86
Mean 103.00
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
8. Solution stability
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
152
5.6. Isolation and characterization of Degradation Products (DPs) of DRT
5.6.1. Experimental
5.6.1.1. Chromatographic conditions
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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. Results and discussion
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
156
FIGURE 5.15 RP-HPLC chromatogram of isolated DPs of DRT (a) DP I and (b) DP II showing
chromatographic purity
a
b
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of DRT
162
FIGURE 5.20 1H NMR spectra of DP I (a) and DP II (b)
a
b
Chapter 5: Stability indicating assay and impurity profiling of DRT
163
FIGURE 5.21 13
C NMR spectra of DP I (a) and DP II (b)
b
a
Chapter 5: Stability indicating assay and impurity profiling of DRT
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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
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)
Chapter 5: Stability indicating assay and impurity profiling of DRT
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
Chapter 5: Stability indicating assay and impurity profiling of 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.
Chapter 5: Stability indicating assay and impurity profiling of DRT
170
5.7. References
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[7] Bakshi, M.; Singh, S. Development of validated stability indicating assay methods-
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[10] International Conference on Harmonization of Technical Requirements for
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[12] International Conference on Harmonization of Technical Requirements for
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Chapter 5: Stability indicating assay and impurity profiling of DRT
171
[13] International Conference on Harmonization of Technical Requirements for
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
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[15] Baertschi, S.W. Analytical methodologies for discovering and profiling degradation-
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