Chapter 5 Results and Discussion -...
Transcript of Chapter 5 Results and Discussion -...
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
70
The aim of the present work was to develop UPLC/Q-TOF-MS/MS method for the simultaneous
estimation of drugs along with their degadation products in fixed-dose combination tablets. In
the present research work two different and most widely used fixed-dose combination tablets
containing aceclofenac and paracetamol; and telmisartan and hydrochlorothiazide were selected
for study. The tablets containing aceclofenac and paracetamol are used for acute painful
condition in adults for relief from various diseases related with pain and inflammation, such as
acute pain in osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, low back pain, dental
pain, fracture, painful pharyngitis and tonsillitis. The other fixed-dose combination tablets
containing telmisartan and hydrochlorothiazide are used worldwide for the treatment of mild to
moderate hypertension.
The widespread use of these pharmaceutical combination products has stimulated the
development of analytical methods for simultaneous determination of both the components
present in it. The literature survey revealed that simultaneous determinations of these drugs in
tablets have been reported by UV, HPLC and HPTLC. But these methods are still only for the
determination of drugs without demonstrating its separation from their major degradation
products. Moreover these methods suffer from relatively low sensitivity, specificity, and long
analysis time. Hence in the present work analytical methods was developed for simultaneous
determination of these drugs along with their known degradation products.
The ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry was chosen
to provide for required fast, high resolution separations having the necessary sensitivity and
associated advantages over the other analytical techniques. UPLC is a novel chromatographic
technique utilizing high linear velocities, which is based on concept using columns with smaller
packing (1.7-1.8 µm porous particles) and operated under high pressure (up to 15000 psi). This is
an extremely powerful approach which dramatically improves peak resolution, sensitivity and
speed of analysis. In addition to UPLC, the use of orthogonal quadrupole time-of-flight mass
spectrometry (Q-TOF-MS) with low and high collision energy full scans acquisition
simultaneously performed, allows the generation of mass information with higher accuracy and
precision, which is ultimately helpful in structure elucidation and identification of drugs and their
degradation products.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
71
Physicochemical Characterization and Identification of drugs The drugs were characterized according to IP 1996, USP 2000 and BP 2008 using UV, IR, DSC
and mass spectrometry.
ACECLOFENAC Aceclofenac is white crystalline powder. It is soluble in methanol and insoluble in water. The
absorption maximum (λmax) was found to be 276 nm. This was in accordance with reported value
(275 nm, BP, 2008). The UV spectrum of aceclofenac is given in Fig. 3. The IR spectrum was
found to exhibit peaks similar to those reported in the literature (BP, 2008). The IR spectrum of
aceclofenac is given in Fig. 4. DSC thermogram of aceclofenac sample was obtained in the
temperature range of 50 to 300°C as shown in Fig. 5. The sample showed sharp endothermic
peak at 155.51°C. The result is within the limit given in BP, 2008. The mass spectrum of
aceclofenac was similar with the reported methods in literature (Celiz et. al., 2009).
Fig. 3: UV spectra of aceclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
72
Fig. 4: IR spectra of aceclofenac
Fig. 5: DSC thermogram of aceclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
73
PARACETAMOL Paracetamol is white crystalline powder. It is soluble in methanol and insoluble in water. The
absorption maximum (λmax) was found to be 249 nm which was in accordance to monographs (IP
1996, USP 2000). The UV scan is shown in Fig. 6. The IR spectrum was found to exhibit peaks
similar to those reprted in the literature (IP 1996). The IR spectrum is shown in Fig. 7. The
melting point of paracetamol was found to be 172°C which was in accordance to monograph
given in IP 1996. DSC thermogram of paracetamol given in Fig. 8. The mass spectrum of
paracetamol was similar with the reported methods in literature (Chen, X., et. al., 2005; Hu, L.,
et. al., 2009; Wang, A. et. al., 2008).
Fig. 6: UV spectra of paracetamol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
74
Fig. 7: IR spectra of paracetamol
Fig. 8: DSC thermogram of paracetamol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
75
TELMISARTAN Telmisartan is white crystalline powder. It is soluble in methanol and insoluble in water. The
absorption maximum (λmax) was found to be 295 nm which was in accordance to monographs
(BP, 2008). The UV scan is shown in Fig. 9. The IR spectrum was found to exhibit peaks similar
to those reported in the literature (BP, 2008). The IR spectrum is shown in Fig. 10. The melting
point of telmisartan was found to be 271.41°C which was in accordance to monograph given in
BP, 2008. DSC thermogram of telmisartan given in Fig. 11. The mass spectrum of telmisartan
was similar with the reported methods in literature (Yan, T. et al., 2008; Shah, R.P. et. al., 2010).
.
Fig. 9: UV spectra of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
76
Fig. 10: IR spectra of telmisartan
Fig. 11: DSC thermogram of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
77
HYDROCHLOROTHIAZIDE Hydrochlorothiazide is white crystalline powder. It is soluble in methanol and slightly soluble in
water. The absorption maximum (λmax) was found to be 271 nm which was in accordance to
monographs (IP, 1996). The UV scan is shown in Fig. 12. The IR spectrum was found to exhibit
peaks similar to those reported in the literature (IP, 1996). The IR spectrum is shown in Fig. 13.
The melting point of hydrochlorothiazide was found to be 275.16°C which was in accordance to
monograph given in IP, 1996. DSC thermogram of hydrochlorothiazide given in Fig. 14. The
mass spectrum of hydrochlorothiazide was similar with the reported methods in literature (Fang,
W. et. al. 2005; Liu, F. et. al., 2008; Yan, T et al., 2008).
.
Fig. 12: UV spectra of hydrochlorothiazide
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
78
Fig. 13: IR spectra of hydrochlorothiazide
Fig. 14: DSC thermogram of hydrochlorothiazide
. Conclusion: On the basis of UV, IR, DSC and mass spectrometric analysis, it was confirmed
that the obtained samples of aceclofenac, paracetamol, telmisartan, and hydrochlorothiazide was
authentic and pure.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
79
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION
Development of UPLC-QTOF-MS method for simultaneous estimation
of aceclofenac and paracetamol and their degradation products All the compounds have strong responses in the positive ionization mode and they form
protonated molecules in the full scan mass spectra. Therefore, the positive ions, [M+H]+ at m/z
354.07 for aceclofenac, m/z 296.23 for diclofenac, m/z 152.07 for paracetamol, and m/z 110.07
for para-aminophenol were selected as the precursor ions as shown in Fig. 15, 17, 19 and 21
respectively. Moreover under the selected MS/MS conditions the precursor ions were
fragmented to major product ions at m/z 215.07 for aceclofenac, m/z 214.06 for diclofenac, m/z
110.06 for paracetamol, and m/z 65.04 for para-aminophenol, as shown in Fig. 16, 18, 20, and
22, respectively. Quantitation was done on the basis of major product ions. The product ion
spectra of aceclofenac and diclofenac suggested that the fragmentation of molecules occurs
from carboxylic group and loss of carbon dioxide results in the formation of one common
product ion, which was identified as C6H3Cl2NHC7H5+ at m/z 250.05, and 252.04 shows the
isotopic pattern of two chlorine atoms. This product ion is further fragmented in to another
product ion, C6H4ClNC7H5+ with higher intensity at m/z 215.07 for aceclofenac and 214.06 for
diclofenac as shown in Fig. 23 and 24. The product ion spectra of paracetamol was due to the
fragmentation of molecule from acetamide group and loss of neutral molecule, namely ketene
(CH2=C=O) results in the formation of major product ion at m/z 110.06, whereas for para-
aminophenol, it was due to the formation of C6H5O+ (phenoxy cation) at m/z 93.05, which in
turn converted in to most intense peak of C5H5+ (cyclopentadienium) at m/z 65.04 as shown in
Fig. 25 and 26.
Different mobile phase containing acetonitrile-water or methanol-water along with modifiers
such as ammonium acetate and or formic acid were tested to obtain the best chromatographic
conditions. Under the conditions used, acetonitrile was chosen as the organic solvent because it
resulted in sharp symmetrical peaks with requisite sensitivity when compared with methanol.
Ammonium acetate was found to have the ability to promote ionization of analytes, improve the
peak symmetry and easily miscible with organic solvent, hence it was selected as suitable buffer
for the mobile phase. Using isocratic mobile phase composition of acetonitrile-2mM
ammonium acetate (40:60, v/v) at a flow rate of 0.25 mL/min gave good peak shapes with short
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
80
separation times. The retention time was found to be 1.50 min for aceclofenac, 1.05 min for
diclofenac, 0.61 min for paracetamol, and 1.61 min for para-aminophenol with the total
chromatographic run time of 2 min for each compound, as shown in Fig. 27, 28, 29, 30 and 31
respectively which turned out to be much shorter analysis time when compared with previously
reported methods.
Fig. 15: TOF-MS spectra of aceclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
81
Fig. 16: TOF-MS/MS spectra of aceclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
82
Fig. 17: TOF-MS spectra of diclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
83
Fig. 18: TOF-MS/MS spectra of diclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
84
Fig. 19: TOF-MS spectra of paracetamol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
85
Fig. 20: TOF-MS/MS spectra of paracetamol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
86
Fig. 21: TOF-MS spectra of para-aminophenol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
87
Fig. 22: TOF-MS/MS spectra of para-aminophenol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
88
Fig. 23: Proposed MS/MS fragmentation mechanism of aceclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
89
Fig. 24: Proposed MS/MS fragmentation mechanism of diclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
90
Fig. 25: Proposed MS/MS fragmentation mechanism of paracetamol
Fig. 26: Proposed MS/MS fragmentation mechanism of para-aminophenol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
91
Fig. 27: UPLC-TOF-MS/MS chromatogram of aceclofenac (1ng/mL)
Fig. 28: UPLC-TOF-MS/MS chromatogram of diclofenac (1ng/mL)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
92
Fig. 29: UPLC-TOF-MS/MS chromatogram of paracetamol (1ng/mL)
Fig. 30: UPLC-TOF-MS/MS chromatogram of para-aminophenol (1ng/mL)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
93
Fig. 31: UPLC-TOF-MS/MS chromatogram of aceclofenac and paracetamol (Mixed
standards 1 ng/mL each)
The linear calibration plot was obtained over the concentration range of 1-1000 ng/mL for
aceclofenac and paracetamol, 0.01-1 ng/mL for diclofenac and 0.01-5 ng/mL para-
aminophenol. For all the compounds the correlation coefficient was more than 0.999. The
results exhibited that an excellent correlation existed between the peak area and concentration
ranges as stated for all the compounds. The linearity data are summarized in Table 7.
Table 7: Results of linearity data, LOD and LOQ of ACF, DCF,
PCM, & PAP
Parameters ACF DCF PCM PAP
Linear range (ng/mL) 1-1000 0.01-100 1-1000 0.01-10
Slope 5.3103 7.620 6.743 11.897
Intercept 22.224 35.225 26.224 1.2007
Correlation coefficienta 0.9996 0.9996 0.9999 0.9998
LOD (ng/mL) 0.01 0.002 0.02 0.001
LOQ (ng/mL) 1 0.01 1 0.01
aAverage of six replicates
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
94
Linearity graph are given in Fig. 32, 33, 34, 35. The results of LOD and LOQ are presented in
Table 7. The obtained results indicated that higher sensitivity of the method, which was
comparable with earlier reported methods.
y = 5.3103x + 22.224R2 = 0.9996
0
1000
2000
3000
4000
5000
6000
0 200 400 600 800 1000 1200
CONCENTRATION (ng/mL)
PEAK
AR
EA
Fig. 32: Linearity curve of aceclofenac
y = 7.62x + 35.225R2 = 0.9996
0100200300400500600700800900
0 20 40 60 80 100 120
CONCENTRATION (ng/mL)
PEAK
ARE
A
Fig. 33: Linearity curve of diclofenac
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
95
y = 6.743x + 26.224R2 = 0.9999
0
1000
2000
3000
4000
5000
6000
7000
8000
0 200 400 600 800 1000 1200
CONCENTRATION (ng/mL)
PEA
K AR
EA
Fig. 34: Linearity curve of paracetamol
y = 11.897x + 1.2007R2 = 0.9998
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12
CONCENTRATION (ng/mL)
PEA
K AR
EA
Fig. 35: Linearity curve para-aminophenol
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
96
The low values of RSD less than 2% were obtained for all the compounds suggested that an
excellent precision of the method. The results of precision are presented in Table 8.
Table 8: Results obtained from precision of ACF, DCF, PCM, & PAP
aMean of six replicates (n = 6) After spiking 50, 100, and 150% levels of standards to pre-analyzed tablet sample, the
recoveries of all the compounds was found to be in the range 99-101% with RSD less than 2%,
indicated that accuracy of the method was adequate. The results are presented in Table 9.
Table 9: Results obtained from recovery studies of ACF, DCF, PCM, & PAP
Analyte Conc. added (ng/mL) Conc. found (ng/mL) Recovery(%)a RSD (%)
50% Level of test conc. ACF DCF PCM PAP
50 0.1 250 1.25
49.98
0.1002 248.24
1.248
99.96 100.2 99.29 99.84
0.98 1.24 0.92 1.46
100% Level of test conc. ACF DCF PCM PAP
100 0.2 500 2.5
99.96 0.199
500.12 2.485
99.96 99.50 100.02 99.40
1.42 1.25 1.22 1.38
150% Level of test conc. ACF
DCF PCM PAP
150 0.3 750 3.75
150.15 0.2992 749.95
3.745
100.10 99.73 99.99 99.86
0.96 1.22 0.98 1.16
aMean of six replicates (n = 6)
Precision ACF DCF PCM PAP Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Intraday
Interday
Different analyst
99.98
99.92
100.12
1.44
1.65
1.82
99.46
100.22
100.32
1.75
1.88
0.92
100.18
99.96
99.98
1.24
1.46
0.96
100.05
99.94
100.12
1.11
1.62
1.35
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
97
The reliability of the method during normal usage was checked by determination of robustness.
The retention times and peak area of each compound did not change significantly when mobile
phase composition, flow rate, injection volume and column temperature were deliberately
modified. Thus, the method was found to be robust with respect to variability in
chromatographic conditions. The results and the experimental range of the variables evaluated
in the robustness assessment are presented in Table 10.
Table 10: Chromatographic conditions and range investigated during robustness testing of
ACF, DCF, PCM, & PAP
aMean of six replicates (n = 6); bOptimized value
UPLC coupled with quadrupole time-of-flight mass detection showed high specificity because only the ions derived from the analytes of interest were monitored. The comparison of the chromatograms of the blank and sample solutions indicated that no interferences were detected from mobile phase components and excipients of the formulation. The specificity was also determined by forced degradation studies. Under stress testing degradation was observed when the aceclofenac and paracetamol individual standard solutions were subjected to acidic and alkaline hydrolysis as seen from the significant drop in assay values and appearance of degradation peaks in the chromatograms. After acidic and alkaline hydrolysis, aceclofenac was
Variables Range
ACF DCF PCM PAP
Recovery (%)a
RSD (%)
Recovery (%)a
RSD (%)
Recovery (%)a
RSD (%)
Recovery (%)a
RSD (%)
Flow rate (mL/min)
0.20 0.25b
0.30
99.64 99.98 99.96
0.981.241.38
99.75 100.05 99.84
1.26 1.32 1.18
99.98 100.15 99.65
1.38 1.15 0.66
99.46 99.94 99.92
0.901.12
1.76 Acetonitrile (%)
35 40b
45
99.92 100.24 99.88
1.341.460.94
99.97 100.10 99.66
0.85 1.42 1.36
99.62 99.92 99.78
0.54 0.86
1.12
99.90 99.96 99.84
1.351.26
1.58 Injection volume (µL)
5 10b
20
99.48 100.24 99.95
1.120.961.22
99.64 99.90 99.75
1.72 1.66 1.52
99.45 99.98 99.66
1.12 0.96
1.22
99.90 99.95 99.34
1.420.85
1.11 Column temperature (°C)
35 40b
45
99.66 100.35 100.12
1.451.681.72
99.46 100.11 99.65
0.85 1.48 1.82
99.75 99.96 99.92
1.64 1.24
1.52
99.46 100.04 99.95
1.140.78
1.28
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
98
degraded into diclofenac with the appearance of peaks at m/z 296 and paracetamol was degraded into para-aminophenol with the appearance of intense peaks at m/z 110 in their mass spectrum. The percentage of diclofenac from degraded sample of aceclofenac in acidic and alkaline conditions after 1 h was found to be 5.12% and 3.26%, respectively which was calculated by area (%) with respect to the initial area of aceclofenac peaks. The percentage of para-aminophenol from degraded sample of paracetamol in acidic and alkaline conditions after 1 h was 4.64% and 2.32%, respectively with respect to the initial area of paracetamol peaks. This was further confirmed by co-injection of reference standard solution of diclofenac and para-aminophenol, the obtained chromatograms were found similar with that of reference standard of each compound, indicating that there was no co-elution of unknown degradation peak at the retention times of respective compounds. No degradation was observed when both the drugs were subjected to oxidative and photolytic stress conditions. The results of forced degradation studies are presented in Table 11. TOF-MS spectra of aceclofenac after acid and alkali hydrolysis are shown in Fig. 36 and 37. TOF-MS spectra of paracetamol after acid and alkali hydrolysis are shown in Fig. 38 and 39. Proposed degradation mechanisms of aceclofenac and paracetamol are presnted in Fig. 40 and 41. Table 11: Results obtained from forced degradation studies of ACF & PCM
Stress conditions ACF PCM
Assay
(%)a
Major degradation
productsb
Assay
(%)a
Major degradation
productsb
No degradation (Control) 99.99 100.02
Acid hydrolysis
(1 N HCl, 25°C, 1h)
94.62 DCF (5.12%) 95.22 PAP (4.64%)
Alkali hydrolysis
(1 N NaOH, 25°C, 1h)
96.46 DCF (3.26%) 97.15 PAP (2.32%)
Oxidation
(3% H2O2, 25°C, 1h)
99.95 99.97
Photolytic
(UV light at 254 nm, 24 h)
99.98 99.99
aMean of three replicates (n = 3); bPeak area (%) against respective standards
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
99
Fig. 36 TOF-MS spectra of aceclofenac after acid hydrolysis (1 N HCl, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
100
Fig. 37 TOF-MS spectra of aceclofenac after alkali hydrolysis (1 N NaOH, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
101
Fig. 38 TOF-MS spectra of paracetamol after acid hydrolysis
(1 N HCl, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
102
Fig. 39 TOF-MS spectra of paracetamol after alkali hydrolysis
(1 N NaOH, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
103
Fig. 40: Proposed degradation mechanism of aceclofenac
Fig. 41: Proposed degradation mechanism of paracetamol
The validated method was applied to the determination of aceclofenac and paracetamol in commercially available tablets containing 100 mg of aceclofenac and 500 mg of paracetamol. The aceclofenac content from tablets was 98.96-99.98% with RSD 1.46% and paracetamol content was 98.25-100.12% with RSD 1.75%. The low values of RSD indicated that method was suitable for routine analysis of aceclofenac and paracetamol in tablets without any interference from excipients. The low analysis time of 2 min allowed a rapid determination of the drugs, which is an important advantage for routine quality control analysis.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
104
The method was successfully employed in stability study of marketed tablets. The results
obtained after 12 months of long term and 6 months of accelerated stability study showed that
tablets were chemically as well as physically stable. The content of aceclofenac and
paracetamol in tablets did not change significantly during storage under 12 months of long
term and 6 months of accelerated conditions compared to the initial values (ANOVA, P
>0.05). The limits of diclofenac and para-aminophenol in tablets at different stages of long
term and accelerated storage conditions were within the Pharmacopoeial limit (not more than
0.1% with respect to peak area of aceclofenac and paracetamol, respectively). Except slight
increase in amounts (0.12%) of degradation products in 12 months. The results of stability
studies are shown in Table 12. The TOF-MS spectra of aceclofenac and paracetamol combined
tablets after 12 months of long term stability conditions is shown in Fig. 42. The method could
be applied for routine quality control analysis of aceclofenac and paracetamol, and to monitor
the level of their degradation products, diclofenac and para-aminophenol in bulk drugs and in
pharmaceutical formulations during stability studies.
Table 12: Results obtained from stability studies of ACF & PCM tablets Storage conditions
ACF
(%)a
DCF
(%)a
PCM
(%)a
PAP
(%)a
Initial point
3 months
30°C/65% RH
40°C/75% RH
6 months
30°C/65% RH
40°C/75% RH
9 months
30°C/65% RH
12 months
30°C/65% RH
100.02
99.96
99.26
98.92
98.92
98.64
98.42
0.02
0.03
0.06
0.06
0.08
0.10
0.11
100.42
99.94
99.12
98.98
98.08
98.45
98.12
0.04
0.06
0.08
0.08
0.10
0.11
0.12 aAverage from three determinations via peak area (%), (n = 3)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
105
Fig. 42 TOF-MS spectra of aceclofenac and paracetamol combined tablets after 12 months
of long term stability conditions
The stability data were evaluated according to the guidelines ICH Q1E. After extrapolation, the
95% lower confidence limit for the mean regression curve intersected the lower acceptance
criteria for assay at 24 months for aceclofenac and 27 months for paracetamol, as shown in Fig.
38 and 39. As per the guidelines the shelf life (expiry dates) should be based on the stability of
the least stable active. Because of the shelf life of aceclofenac (24 months) was found less than
shelf life of paracetamol (27 months), the expiry date of combination tablet product was
proposed as 24 months.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
106
Fig. 43: Shelf life determination of aceclofenac (a) Mean regression line, (b) 95% Lower
confidence limit of mean, (c) Lower acceptance criteria for assay
Fig. 44: Shelf life determination of paracetamol (a) Mean regression line, (b) 95% Lower
confidence limit of mean, (c) Lower acceptance criteria for assay
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
107
Development and validation of UPLC-QTOF-MS method for
simultaneous estimation of telmisartan and hydrochlorothiazide and
their degradation products
All the compounds have strong responses in the negative ionization mode and they form protonated molecules in the full scan mass spectra. Therefore, the negative ions, [M-H]- at m/z 513.08 for telmisartan, m/z 295.86 for hydrochlorothiazide, and m/z 283.88 for DSA were selected as the precursor ions, as shown in Fig. 45, 47, and 49. Moreover under the selected MS/MS conditions the precursor ions were fragmented to major product ions at m/z 513.18→469.13 for telmisartan, 295.91→204.94 for hydrochlorothiazide, and 283.95→169.00 for DSA, as shown in Fig. 46, 48, and 50, respectively. Quantitation was done on the basis of major product ions. The product ion spectra of telmisartan was suggested that the fragmentation of molecules occurs from carboxylic group and loss of carbon dioxide results in the formation of one major product ion, at m/z 469.5, as shown in Fig. 51. The product ion spectra of hydrochlorothiazide was suggested that the compound is fragmented by the loss of neutral molecule HCN, resulted in the formation of one intermediate product ion, at m/z 268.90. This product ion is further fragmented in to another product ion with higher intensity at m/z 204.93, as shown in Fig. 52. The product ion spectra of DSA was due to the loss of SO2 and NH3 molecule, results in the formation of one intermediate product ion at m/z 204.93. This product ion is further fragmented in to another product ion with higher intensity at m/z 169.00, as shown in Fig. 53. Different mobile phase containing acetonitrile-water or methanol-water along with modifiers such as ammonium acetate and or formic acid were tested to obtain the best chromatographic conditions. Under the conditions used, acetonitrile was chosen as the organic solvent because it resulted in sharp symmetrical peaks with requisite sensitivity when compared with methanol. Ammonium acetate was found to have the ability to promote ionization of analytes, improve the peak symmetry and easily miscible with organic solvent, hence it was selected as suitable buffer for the mobile phase. Using isocratic mobile phase composition of acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.2 mL/min gave good peak shapes with short separation times. The retention time was found to be 2.25 min for telmisartan, 1.22 min for hydrochlorothiazide, and 0.95 min for DSA with the total chromatographic run time of 3 min for each compound, as shown in Fig. 54, 55, 56, and 57, respectively.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
108
Fig. 45: TOF-MS spectra of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
109
Fig. 46: TOF-MS/MS spectra of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
110
Fig. 47: TOF-MS spectra of hydrochlorothiazide
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
111
Fig. 48: TOF-MS/MS spectra of hydrochlorothiazide
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
112
Fig. 49: TOF-MS spectra of DSA
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
113
Fig. 50: TOF-MS/MS spectra of DSA
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
114
Fig. 51: Proposed MS/MS fragmentation mechanism of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
115
Fig. 52: Proposed MS/MS fragmentation of hydrochlorothiazide
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
116
Fig. 53: Proposed MS/MS fragmentation of DSA
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
117
Fig. 54: UPLC-TOF-MS/MS chromatogram of telmisartan (1ng/mL)
Fig. 55: UPLC-TOF-MS/MS chromatogram of hydrochlorothiazide (1ng/mL)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
118
Fig. 56: UPLC-TOF-MS/MS chromatogram of telmisartan and hydrochlorothiazide
(Mixed standards 1ng/mL each)
Fig. 57: UPLC-TOF-MS/MS chromatogram of DSA (1ng/mL)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
119
The linear calibration plot was obtained over the concentration range of 1-1000 ng/mL for
telmisartan and hydrochlorothiazide, and 0.01-2 ng/mL DSA. For all the compounds the
correlation coefficient was more than 0.999. The results exhibited that an excellent correlation
existed between the peak area and concentration ranges as stated for all the compounds. The
linearity data are summarized in Table 13.
Table 13: Results of linearity data, LOD and LOQ of
TEL, HCTZ, & DSA
Parameters TEL HCTZ DSA
Linear range (ng/mL) 1-1000 1-1000 0.01-2
Slope 12.502 11.649 60.41
Intercept 247.41 129.93 0.1095
Correlation coefficienta 0.9998 0.9998 0.9996
LOD (ng/mL) 0.01 0.02 0.001
LOQ (ng/mL) 1 1 0.01
aMean of six replicates
y = 12.502x + 247.41R2 = 0.9998
0
2000
4000
6000
8000
10000
12000
14000
0 200 400 600 800 1000 1200
CONCENTRATION (ng/mL)
PEAK
ARE
A
Fig. 58: Linearity curve of telmisartan
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
120
y = 11.649x + 129.93R2 = 0.9998
0
2000
4000
6000
8000
10000
12000
14000
0 200 400 600 800 1000 1200
CONCENTRATION (ng/mL)
PEA
K A
REA
Fig. 59: Linearity curve of hydrochlorothiazide
y = 60.41x + 0.1095R2 = 0.9996
0
10
20
30
40
50
60
70
0 0.2 0.4 0.6 0.8 1 1.2
CONCENTRATION (ng/ML)
PEA
K AR
EA
Fig. 60: Linearity curve of DSA
Linearity graphs are shown in Fig. 58, 59, and 60. The results of LOD and LOQ are presented
in Table 13. The obtained results indicated that higher sensitivity of the method.
The low values of RSD less than 2% were obtained for all the compounds by evaluation of
intraday, interday and different analysts precision suggested an excellent precision of the
method. The results of precision are presented in Table 14.
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
121
Table 14: Results obtained from precision of TEL, HCTZ, & DSA
aMe aMean of six replicates (n = 6) After spiking 50, 100, and 150% levels of standards to pre-analyzed tablet sample, the
recoveries of all the compounds was found to be in the range 99-101% with RSD less than 2%,
indicated that accuracy of the method was adequate. The results are presented in Table 15.
Table 15: Results obtained from recovery studies of TEL, HCTZ, & DSA
Analyte Conc. added (ng/mL) Conc. Found (ng/mL) Recovery(%)a RSD (%)
50% Level of test conc.
TEL
HCTZ
DSA
250
100
0.05
249.96
100.05
0.049
99.98
100.05
99.00
0.92
1.12
0.96
100% Level of test conc.
TEL
HCTZ
DSA
500
100
0.1
499.92
100.02
0.099
99.98
100.02
99.00
1.22
1.35
1.62 150% Level of test conc.
TEL
HCTZ
DSA
750
150
0.15
750.01
149.92
0.149
100.13
99.94
99.33
0.95
1.15
0.97 aMean of six replicates (n = 6)
The reliability of the method during normal usage was checked by determination of robustness.
The retention time and peak area of each compound did not change significantly when mobile
phase composition, flow rate, injection volume and column temperature were deliberately
Precision TEL HCTZ DSA Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Intraday
Interday
Different analyst
99.94
99.96
100.15
1.45
1.64
1.62
100.11
99.95
99.96
1.26
1.47
0.95
100.25
99.96
99.92
1.11
1.46
0.76
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
122
modified. Thus, the method was found to be robust with respect to variability in
chromatographic conditions. The results and the experimental range of the variables evaluated
in the robustness assessment are presented in Table 16.
Table 16: Chromatographic conditions and range investigated during
robustness testing of TEL, HCTZ, & DSA
aMean of six replicates (n = 6); bOptimized value
The specificity was determined by forced degradation studies. After acidic and alkaline
hydrolysis, telmisartan was degraded into unknown degradation product with the appearance
of peaks at m/z 162.82 and m/z 188.92, respectively. Whereas it was found stable under
oxidative and photolytic stress conditions. The percentage of telmisartan under degradation in
acidic and alkaline conditions after 1 h was found to be 94.62% and 96.46%, respectively
which was calculated by area (%) with respect to the initial area of telmisartan peaks. After
acidic and alkaline hydrolysis, hydrochlorothiazide was degraded into DSA. The percentage of
hydrochlorothiazide under degradation in acidic and alkaline conditions after 1 h was found to
Variables Range
TEL HCTZ DSA
Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Recovery
(%)a
RSD
(%)
Flow rate
(mL/min)
0.15
0.20b
0.25
99.66
99.92
99.96
0.97
1.25
1.33
99.72
100.11
99.64
1.35
1.12
1.17
99.97
100.15
99.67
0.92
1.10
1.76
Acetonitrile
(%)
45
50b
55
99.92
100.27
99.62
1.15
1.45
0.97
99.97
100.10
99.66
0.75
1.42
1.36
99.62
99.92
99.75
0.64
1.16
1.42
Injection
volume (µL)
5
10b
20
99.47
100.22
99.96
1.12
0.96
1.62
99.64
99.90
99.75
1.76
1.62
1.32
99.45
99.97
99.66
1.22
0.96
0.92
Column
temperature
(°C)
35
40b
45
99.66
100.35
100.12
1.45
1.68
1.72
99.46
100.11
99.65
0.95
1.47
1.22
99.75
99.96
99.92
1.64
1.24
1.52
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
123
be 93.22% and 92.25%, respectively which was calculated by area (%) with respect to the
initial area of hydrochlorothizide peaks. The percentage of DSA from degraded sample of
hydrochlorothiazide in acidic and alkaline conditions after 1 h was 6.32% and 8.64%,
respectively with respect to the initial area of hydrochlorothiazide peaks. This was further
confirmed by co-injection of reference standard solution of DSA, the obtained chromatograms
were found similar with that of reference standard of each compound, indicating that there was
no co-elution of unknown degradation peak at the retention times of respective compounds. No
degradation was observed when both the drugs were subjected to oxidative and photolytic
stress conditions. The results of forced degradation studies are presented in Table 17. TOF-MS
spectra of telmisartan after acid and alkali hydrolysis are given in Fig. 61 and 62. TOF-MS
spectra of hydrochlrothiazide after acid and alkali hydrolysis are given in Fig. 63 and 64.
Proposed degradation mechanism of hydrochlorothiazide is given in Fig. 65.
Table 17: Results obtained from forced degradation studies of TEL & HCTZ
Stress conditions TEL HCTZ
Assay (%)a Major degradation
productsb
Assay (%)a Major degradation
productsb
No degradation (Control) 100.10 100.02
Acid hydrolysis
(1 N HCl, 25°C, 1h)
94.62 m/z 162.81
93.22 DSA
(6.32%)
Alkali hydrolysis
(1 N NaOH, 25°C, 1h)
96.46 m/z 188.92
92.25 DSA
(8.64%)
Oxidation
(3% H2O2, 25°C, 1h)
99.95 99.97
Photolytic
(UV light at 254nm, 24 h)
99.97 99.99
aMean of three replicates (n = 3); bPeak area (%) against respective standard
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
124
Fig. 61: TOF-MS spectra of telmisartan after acid hydrolysis (1 HCl, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
125
Fig. 62: TOF-MS spectra of telmisartan after alkali hydrolysis (1 NaOH, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
126
Fig. 63: TOF mass spectra of hydrochlorothiazide after acid hydrolysis
(1 N HCl, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
127
Fig. 64: TOF mass spectra of hydrochlorothiazide after alkali hydrolysis
(1 N NaOH, 25°C, 1 h)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
128
Fig. 65: Proposed degradation mechanism of hydrochlorothiazide
The validated method was applied to the determination of telmisartan and hydrochlorothiazide
in commercially available tablets containing 40 mg of telmisartan and 12.5 mg of
hydrochlorothiazide. The content of both the drugs in tablets was found to be 98-100% with
RSD less than 2%, indicated that method was suitable for routine analysis of drugs in tablets
without any interference from excipients. The low analysis time of 3 min allowed a rapid
determination of the drugs, which is an important advantage for routine quality control
analysis.
The method was successfully employed in stability study of marketed tablets. Tablets were
stored in stability chamber and monitored to physical and chemical stability. The contents of
drugs were determined by applying the developed method at different stages of stability studies.
The results obtained after 12 months of long term and 6 months of accelerated stability study
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
129
showed that tablets were chemically as well as physically stable. The content of telmisartan and
hydrochlorothiazide in tablets did not change significantly during storage under 12 months of
long term and 6 months of accelerated conditions compared to the initial values (ANOVA, P
>0.05). Although the degradation products were observed in the stress testing of both the drugs
as pure form, but the tablets did not show any major degradation either at long term or at
accelerated storage condition except DSA from hydrochlorothiazide. The percentages of DSA
in tablets at different stages of long term and accelerated storage conditions were within the
Pharmacopoeial limit (not more than 0.1% with respect to peak area of hydrochlorothiazide).
Except slight increase (0.11%) in 12 months. The results of stability studies are presented in the
Table 18. TOF-MS spectra of telmisartan and hydrochlorothiazide combined tablets after 12
months of long term stability conditions is shown in Fig. 66. Hence it is suggested that the
method could be applied for routine quality control analysis of telmisartan and
hydrochlorothiazide, and to monitor the level of their degradation products in bulk drugs and in
pharmaceutical formulations during stability studies.
Table 18: Results from stability studies of TEL & HCTZ tablets Storage conditions
TEL
(%)a
HCTZ
(%)a
DSA
(%)a
Initial point
3 months
30°C/65% RH
40°C/75% RH
6 months
30°C/65% RH
40°C/75% RH
9 months
30°C/65% RH
12 months
30°C/65% RH
100.05
99.94
99.22
98.90
98.72
98.34
98.02
100.12
99.94
99.11
98.96
98.64
98.42
98.12
0.02
0.05
0.06
0.07
0.10
0.09
0.11
aAverage from three determinations via peak area (%), (n = 3)
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
130
Fig. 66: TOF-MS spectra of telmisartan and hydrochlorothiazide combined tablets
after 12 months of long term stability conditions
Chapter 5 Results and Discussion
Department of Pharmaceutics Jamia Hamdard
131
The stability data were evaluated as per the ICH Q1E guidelines. The graphs were plotted between assay (%) of drugs and time (months). After extrapolation, the 95% lower confidence limit for the mean regression curve intersected the lower acceptance criteria for assay at 26 months for telmisartan and 25 months for hydrochlorothiazide, as shown in Fig. 67 and 68. As per the guidelines the shelf life (expiry dates) should be based on the stability of the least stable active. Because of the shelf life of hydrochlorothiazide (25 months) was found less than shelf life of telmisartan (26 months), the expiry date of combination tablet product was proposed as 24 months.
Fig. 67: Shelf life determination of telmisartan (a) Mean regression line,
(b) 95% Lower confidence limit of mean, (c) Lower acceptance criteria for assay
Fig. 68: Shelf life determination of hydrochlorothiazide (a) Mean regression line,
(b) 95% Lower confidence limit of mean, (c) Lower acceptance criteria for assay