Chapter 5 Results and Discussion -...

Chapter 5 Results and Discussion

Department of Pharmaceutics Jamia Hamdard

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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.



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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



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Fig. 4: IR spectra of aceclofenac

Fig. 5: DSC thermogram of aceclofenac



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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



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Fig. 7: IR spectra of paracetamol

Fig. 8: DSC thermogram of paracetamol



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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



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Fig. 10: IR spectra of telmisartan

Fig. 11: DSC thermogram of telmisartan



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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



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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.



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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



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



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Fig. 16: TOF-MS/MS spectra of aceclofenac



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Fig. 17: TOF-MS spectra of diclofenac



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Fig. 18: TOF-MS/MS spectra of diclofenac



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Fig. 19: TOF-MS spectra of paracetamol



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Fig. 20: TOF-MS/MS spectra of paracetamol



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Fig. 21: TOF-MS spectra of para-aminophenol



87

Fig. 22: TOF-MS/MS spectra of para-aminophenol



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Fig. 23: Proposed MS/MS fragmentation mechanism of aceclofenac



89

Fig. 24: Proposed MS/MS fragmentation mechanism of diclofenac



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Fig. 25: Proposed MS/MS fragmentation mechanism of paracetamol

Fig. 26: Proposed MS/MS fragmentation mechanism of para-aminophenol



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Fig. 27: UPLC-TOF-MS/MS chromatogram of aceclofenac (1ng/mL)

Fig. 28: UPLC-TOF-MS/MS chromatogram of diclofenac (1ng/mL)



92

Fig. 29: UPLC-TOF-MS/MS chromatogram of paracetamol (1ng/mL)

Fig. 30: UPLC-TOF-MS/MS chromatogram of para-aminophenol (1ng/mL)



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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



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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


PEAK

ARE

A

Fig. 33: Linearity curve of diclofenac



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y = 6.743x + 26.224R2 = 0.9999

0

1000

2000

3000

4000

5000

6000

7000

8000

0 200 400 600 800 1000 1200


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


PEA

K AR

EA

Fig. 35: Linearity curve para-aminophenol



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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



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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



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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



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Fig. 36 TOF-MS spectra of aceclofenac after acid hydrolysis (1 N HCl, 25°C, 1 h)



100

Fig. 37 TOF-MS spectra of aceclofenac after alkali hydrolysis (1 N NaOH, 25°C, 1 h)



101

Fig. 38 TOF-MS spectra of paracetamol after acid hydrolysis

(1 N HCl, 25°C, 1 h)



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Fig. 39 TOF-MS spectra of paracetamol after alkali hydrolysis

(1 N NaOH, 25°C, 1 h)



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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.



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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)



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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.



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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



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.



108

Fig. 45: TOF-MS spectra of telmisartan



109

Fig. 46: TOF-MS/MS spectra of telmisartan



110

Fig. 47: TOF-MS spectra of hydrochlorothiazide



111

Fig. 48: TOF-MS/MS spectra of hydrochlorothiazide



112

Fig. 49: TOF-MS spectra of DSA



113

Fig. 50: TOF-MS/MS spectra of DSA



114

Fig. 51: Proposed MS/MS fragmentation mechanism of telmisartan



115

Fig. 52: Proposed MS/MS fragmentation of hydrochlorothiazide



116

Fig. 53: Proposed MS/MS fragmentation of DSA



117

Fig. 54: UPLC-TOF-MS/MS chromatogram of telmisartan (1ng/mL)

Fig. 55: UPLC-TOF-MS/MS chromatogram of hydrochlorothiazide (1ng/mL)



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)



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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


PEAK

ARE

A

Fig. 58: Linearity curve of telmisartan



120

y = 11.649x + 129.93R2 = 0.9998

0

2000

4000

6000

8000

10000

12000

14000

0 200 400 600 800 1000 1200


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.



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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



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



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



124

Fig. 61: TOF-MS spectra of telmisartan after acid hydrolysis (1 HCl, 25°C, 1 h)



125

Fig. 62: TOF-MS spectra of telmisartan after alkali hydrolysis (1 NaOH, 25°C, 1 h)



126

Fig. 63: TOF mass spectra of hydrochlorothiazide after acid hydrolysis

(1 N HCl, 25°C, 1 h)



127

Fig. 64: TOF mass spectra of hydrochlorothiazide after alkali hydrolysis

(1 N NaOH, 25°C, 1 h)



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



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)



130

Fig. 66: TOF-MS spectra of telmisartan and hydrochlorothiazide combined tablets

after 12 months of long term stability conditions



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

Chapter 5 Results and Discussion -...

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