AJPCR_25783_RA <1>

Online - 2455-3891
Vol 11, Issue 7, 2018 Print - 0974-2441
Research Article
1 SIMULTANEOUS ESTIMATION AND FORCED DEGRADATION STUDIES OF AMILORIDE 1
2 2
3 HYDROCHLORIDE AND FUROSEMIDE IN A PHARMACEUTICAL DOSAGE FORM USING 3
4 REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD 4
5 5
6 6
7 7
8 JAVED S SHAIKH, NUTAN N RAO* 8
9 Department of Quality Assurance, Oriental College of Pharmacy, Sanpada, Navi Mumbai, Maharashtra - 400 705, India. 9
10 Email: nutan.rao@ocp.edu.in 10
11 11
12 Received: 09 March 2018, Revised and Accepted: 05 April 2018 12
13 ABSTRACT 13
14 14
15 Objective: The present study describes the stability indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for 15
16 simultaneous estimation of amiloride hydrochloride and furosemide in pharmaceutical dosage forms. 16
17 17
18 Methods: The proposed RP-HPLC method was developed using Shimadzu LC-2030 HPLC system equipped with UV detector, and chromatographic 18
19 separation was carried on Shim-pack C18 (250 mm×4.6 mm, 5 µ) column at a flow rate of 1 ml/min and the runtime was 4min. The mobile phase 19
20 consisted of water and acetonitrile in the ratio of 35:65, and elements were scanned using a UV detector at 281 nm. 20
21 21
22 Results: The retention time of amiloride hydrochloride and furosemide was found to be 1.92 min and 3.14min, respectively. Linearity was found to 22
23 be 12–28 ppm for amiloride hydrochloride and 96–224 ppm for furosemide, respectively. Limit of detection and limit of quantification for amiloride 23
24 hydrochloride were 0.381 ppm and 1.156 ppm and for furosemide were 2.00 ppm and 6.068 ppm, respectively. 24
25 25
26 Conclusion: The stability indicating method was developed by subjecting the drugs to stress conditions such as acid and base hydrolysis, oxidation, 26
27 humidity, photolytic, and thermal degradation, and the degraded products formed were resolved successfully from the samples. 27
28 28
29 Keywords: Amiloride hydrochloride, Furosemide, Reverse-phase high-performance liquid chromatography, Degradation, Validation, ICH. 29
30 30
31 © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. 31
32 org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i7.25783 32
33 33
34 34
35 INTRODUCTION Chem Products, Dombivili, Maharashtra, India. Commercial tablet 35
36 dosage form, Frumil, was purchased from local markets. The HPLC grade 36
37 Amiloride hydrochloride is a potassium-sparing diuretic. It is chemically 3, 37
acetonitrile and water were purchased from Thomas Baker. Analytical
AQ1
38 5-diamino-N-(diaminomethylene)-6-chloropyrazinecarboxamidemonohydr 38
grade orthophosphoric acid (OPA), hydrochloric acid, sodium hydroxide,
39 ochloridedihydrate [1,2] (Fig. 1). It works by inhibiting sodium reabsorption 39
and hydrogen peroxide were purchased from S.D. Fine Chemicals.
40 in renal epithelial cells by binding to sodium channels. Inhibition of sodium 40
41 reabsorption creates a negative voltage in the luminal membranes of principal Instrument 41
42 cells, situated at the distal convoluted tubule and collecting duct. This negative 42
The chromatographic separation was carried out by Shimadzu LC-
43 voltage decreases the potassium and hydrogen ion secretion [2,3]. It is used in 43
2030 HPLC system equipped with UV detector and autosampler. The
44 conjunction with diuretics to spare potassium loss. 44
LabSolution software was used for signal monitoring and processing.
45 Photolytic degradation was done in UV chamber, and hot air oven was 45
46 Furosemide is a loop diuretic. It is chemically 4-chloro-2- furfurylamino-5- 46
employed for thermal degradation.
47 sulphamoyl benzoic acid (Fig. 1). Furosemide inhibits water reabsorption 47
48 in the nephron by blocking sodium and chloride reabsorption in the 48
Selection of wavelength
49 ascending limb of the loop of Henle [4]. Furosemide helps to maintain 49
AQ2
Both the drugs were scanned by UV individually, in a wavelength range of
50 potassium and minimize the risk of alkalosis, in the treatment of edema 50
200–400 nm and maxima for each drug was measured. The corresponding
51 associated with hepatic cirrhosis and congestive heart failure [2,5]. 51
UV spectrum graphs of the drugs such as amiloride hydrochloride and
52 hydrochlorothiazide are shown in Fig. 2. The detection wavelength was 52
53 The thorough literature survey reveals that few analytical methods such 53
selected from the overlay UV spectrum and was found to be 281 nm.
54 as RP-HPLC and UV methods are reported for simultaneous estimation 54
55 of amiloride hydrochloride and furosemide in pharmaceutical dosage 55
Chromatographic conditions
56 forms [3,6,7]. Thus, the present investigation was held out to acquire 56
The chromatographic separation of analytes was carried out using
57 new, simple, accurate, rapid, and cost-effective stability indicating 57
ShimadzuRP-HPLC system with Shim-pack GIST C18 (250 × 4.6 mm,
58 RP-HPLC method for the simultaneous estimation of amiloride 58
5 µ) column. The mobile phase of a mixture of water and acetonitrile
59 hydrochloride and furosemide in pharmaceutical dosage form. The 59
was in the ratio of 35:65 and column temperature was maintained at
60 suggested method was applied successfully to split up the degraded 60
25°C. The analytes were detected at 281 nm using UV detector. The
61 products from the samples. 61
runtime was set at 4 min at a flow rate of 1 ml/min.
62 62
63 METHODS 63
Preparation of standard stock solution
64 Reagents and chemicals Standard stock solutions of amiloride hydrochloride and furosemide 64
65 Amiloride hydrochloride and furosemide standards were provided by were prepared separately by dissolving 100 mg of amiloride 65
66 Alkem Laboratories, Navi Mumbai, Maharashtra, India, and Yarrow hydrochloride and 100 mg of furosemide in 100 ml volumetric flasks 66
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Asian J Pharm Clin Res, Vol 11, Issue 7, 2018, 1-7

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10 Fig. 1: Structure of amiloride hydrochloride and furosemide 10
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Fig. 2: Overlain spectra of amiloride hydrochloride and furosemide
31 31
32 32
33 with water:acetonitrile (35:65) as diluent and sonicated for 10 min. dish and kept in a humidity chamber at 95% relative humidity, at 25°C 33
34 From the above solution, 0.2 ml of amiloride hydrochloride and 1.6 for 120 h. A photolytic degradation study was carried out by placing the 34
35 ml of furosemide were transferred separately to 10 ml volumetric tablets in a Petri dish in a photolytic chamber for 7 days. 35
36 flasks, and 1  ml 5% OPA was added as a supporter and sonicated for 36
37 5 min and made up the volume with diluent to get 20ppm of amiloride RESULTS AND DISCUSSION 37
38 hydrochloride and 160ppm of furosemide standard stock solution. 38
Method development
39 39
Preparation of sample solution A series of tests was taken with different columns such as Inertsil
40 40
Ten tablets (Frumil tablets: 5 mg amiloride hydrochloride and 40 mg ODS and Shim-pack C18 column with different mobile phases to
41 41
furosemide) were weighed and the average weight of each tablet was produce a suitable RP-HPLC method for estimation of amiloride
42 42
calculated; then, the weight equivalent to 1 tablet was transferred into hydrochloride and furosemide in tablet dosage form, and finally, a
43 43
44 a 100 ml clean dry volumetric flask, add 30  ml of diluent, sonicated for typical chromatogram was obtained with water and acetonitrile in 44
45 25 min, and make up to the final volume with diluent and filtered. 2  ml the ratio of 35:65. The chromatographic separation was performed on 45
46 of the filtered solution was pipetted out into a 10 ml volumetric flask Shim-Pack C18 (250 × 4.6 mm, 5 µ) column by injecting 20 μL, and the 46
47 and 1  ml 5% of the OPA was added as a supporter and sonicated for analytes were detected with UV detector at 281 nm. The retention time 47
48 5 min, and volume made up to 10 ml with diluent. of amiloride hydrochloride and furosemide was found to be 1.92 min 48
49 and 3.14 min, respectively. Forced degradation studies for amiloride 49
50 Method validation hydrochloride and hydrochlorothiazide in tablet dosage form were also 50
51 The method validation was done according to the ICH guidelines with above carried out using the developed method, and the degraded compounds 51
52 developed RP-HPLC method for simultaneous estimation of amiloride were effectively resolved. The optimized conditions were given in 52
53 hydrochloride and furosemide. Several parameters were evaluated such Table 1 and Fig. 3. 53
54 as system suitability, precision, accuracy, linearity, robustness, limit of 54
55 detection (LOD), and limit of quantification (LOQ) [8,9]. System suitability 55
56 System suitability was performed to verify the acceptability of the 56
57 Forced degradation studies resolution and repeatability of the system. System suitability was 57
58 The ICH degradation was attempted under various stress conditions performed by injecting six replicate injections of the standard solution 58
59 such as acid, alkaline, oxidation, thermal, humidity, and photolytic (100%), and parameters such as peak area, USP tailing, theoretical 59
60 conditions to evaluate the interference of degradation impurities. These plates, retention time, and peak asymmetry were evaluated. The % 60
61 studies help to know the inherent stability characteristic of the active relative standard deviation (RSD) was determined and reported within 61
62 molecules in drug product and the possible degradation products. the limits [10]. The results are shown in Table 2. 62
63 63
64 Acid, base, and oxidation degradations were performed by adding 5  ml Accuracy 64
65 of 1 N HCl, 5  ml of 1 N NaOH, and 5  ml of 30% peroxide solution (H2O2), The accuracy of the proposed method was evaluated by calculating the 65
66 respectively, to the sample solutions, and these samples were kept at room recovery studies of the test drug at three different concentration levels 66
67 temperature for 3 h. Thermal degradation was performed by keeping the (80%, 100%, and 120%) by the standard addition method [11]. A known 67
68 tablets in a Petri dish and then placed them in an oven at 105°C for 48 h. amount of amiloride hydrochloride and furosemide was added to the 68
69 Humidity degradation was performed by placing the tablets in a Petri prequantified sample solution, and three replicates of each concentration 69

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14 Fig. 3: Chromatogram of standard amiloride hydrochloride and furosemide 14
15 15
16 16
Table 1: Optimized chromatographic condition Robustness
17 17
Robustness of the proposed method has been evaluated by small
18 Parameter Optimized condition 18
deliberate changes in the system parameters such as flow rate,
19 19
Column Shim‑Pack C18 (250×4.6 mm, 5 µ) column wavelength, and temperature [14]. It was found that none of the above
20 20
Mobile phase Water and acetonitrile in the ratio of 35:65 parameters caused an alteration in the peak area, retention time, and
21 21
Flow rate 1 ml/min USP tailing by small changes such as ±0.2 ml change in flow rate, ±2 nm
22 22
Wavelength UV detector at 281 nm wavelength, and ±2°C changes in temperature. The % RSD was found
23 Injection volume 20 µL 23
to be within the limits, and the method was found to be robust. The
24 Temperature 25°C 24
robustness results are shown in Table 5.
25 Retention time Amiloride hydrochloride 1.921 min and 25
26 furosemide 3.146 min 26
Assay of marketed formulation
27 27
Analysis of marketed formulation (Frumil tablets: 5 mg amiloride
28 28
hydrochloride and 40 mg furosemide) was purchased from local
29 Table 2: System suitability parameters 29
markets. Ten tablets were weighed and average weight of each tablet
30 30
was calculated; then, the weight equivalent to 1 tablet was transferred
31 Parameters Amiloride Furosemide 31
into a 100 ml clean dry volumetric flask, add 30  ml of diluent, sonicated
32 hydrochloride 32
for 25 min, and make up to the final volume with diluent and filtered.
33 Retention time (min) 1.921 3.146 33
2  ml of the filtered solution was pipetted out into a 10 ml volumetric
34 USP plate count 2876 6454 34
flask, and 1  ml 5% of OPA is added as a supporter and made up to 10 ml
35 USP tailing 1.243 1.191 35
with diluent. From the resulting solution, 20 μL was injected into HPLC
36 36
system and peak areas were recorded. The % assay of the marketed
37 37
formulation was found to be 99.55% for amiloride hydrochloride and
38 were injected into developing chromatographic conditions. The percentage 38
99.9% for furosemide as shown in Table 6.
39 recovery result of amiloride hydrochloride and furosemide was found to 39
40 be within limits of 100–101%, indicating that the developed method was Forced degradation studies 40
41 found to be accurate. The percentage recovery results are shown in Table 3. Forced degradation studies of the drug formulation were carried out 41
42 42
by treating the drug samples under stress-induced conditions such
43 Precision as acid and base hydrolysis, oxidation, humidity, and photo- and
43
44 The precision of an analytical procedure may be defined as the closeness 44
thermal-degradation to evaluate the ability of the proposed
45 of agreement between a series of measurements obtained from multiple 45
method to separate amiloride hydrochloride and furosemide from
46 sampling of the same homogeneous sample under the prescribed conditions. 46
its degradation products as shown in Figs. 5-11. The % assay of
47 The method precision and system precision studies were carried out by 47
amiloride hydrochloride and furosemide with respect to untreated
48 injecting six replicates of both standard and test solutions with the same 48
sample and % assay results obtained from treating the samples
49 concentration [12]. The % RSD was calculated from the chromatogram and 49
with various stress conditions had a difference which was within
50 the results obtained were within the limits of 2%, and the proposed method 50
the acceptable limits. The results of stress studies are shown in
51 was found to be precise. The precision data are given in Table 4. 51
Table 7.
52 52
53 Linearity Acid degradation 53
54 The linearity of the method was determined at different concentration Acid (1 N hydrochloric acid) degradation study showed 1.3% and 1.9% 54
55 levels ranging from 12 to 28 ppm of amiloride hydrochloride and from degradation for amiloride hydrochloride and furosemide, respectively. 55
56 96 to 224 ppm of furosemide. All the concentrations were prepared and However, no degradation product was observed in the chromatogram 56
57 injected into the system. The linearity curve was constructed by plotting (Fig. 6). 57
58 peak area versus concentration of the analyte. From the results obtained, 58
59 the proposed method was found to be linear. The regression coefficient Alkali degradation 59
60 (r2) was found to be 0.999 and 0.999 for amiloride hydrochloride and The degradation in base (1 N sodium hydroxide) was found to be 1.1% 60
61 furosemide, respectively, and the result is shown in Fig. 4. and 3.9% for amiloride hydrochloride and furosemide, respectively. 61
62 However, no degradation product was observed in the chromatogram 62
63 LOD and LOQ (Fig. 7). 63
64 In the present study, the LOD and LOQ of amiloride hydrochloride and 64
65 furosemide were evaluated based on the standard calibration curve Oxidative degradation 65
66 method [13]. LOD is performed to know the lowest concentration Oxidative degradation study in 30% hydrogen peroxide gave 66
67 level of the analyte that gives a measurable response. LOD and LOQ around 2.2% and 6.9% for amiloride hydrochloride and furosemide, 67
68 for amiloride hydrochloride are 0.381 ppm and 1.156 ppm and for respectively. However, no degradation product was observed in the 68
69 furosemide are 2.00 ppm and 6.068 ppm, respectively. chromatogram (Fig. 8). 69

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35 Fig. 4: Linearity graph of (a) amiloride hydrochloride and (b) furosemide 35
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37 Table 3: Percentage recovery results of amiloride hydrochloride and furosemide 37
38 38
39 Spiked (%) Percentage recovery Mean percentage %RSD 39
40 recovery 40
Amiloride Furosemide Amiloride Furosemide
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hydrochloride hydrochloride
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43 80 99.7 100 100.1 0.74 0.50 43
44 100.8 99.6 44
45 101.1 100.6 45
100 100.2 100 100.1 0.47 0.06
46 46
100 99.9
47 47
100.9 100
48 120 101 99.7 100.7 0.23 0.61 48
49 101.4 100.7 49
50 101 100.8 50
51 51
RSD: Relative standard deviation
52 52
53 Photolytic degradation 53
54 Table 4: Results of method precision for amiloride AQ3
54
In photolytic  UV degradation, the drug degraded was 0.3% and 0.65%
55 hydrochloride and furosemide for amiloride hydrochloride and furosemide, respectively. However, no 55
56 degradation product was observed in the chromatogram (Fig. 9). 56
57 % Assay 57
58 S. No. Amiloride hydrochloride Furosemide Thermal degradation 58
59 In thermal degradation, there was no degradation peak observed in 59
60 1 101.1 100.9 60
2 101.7 100.2 the chromatogram, and degradation was 1.2% and 2.7% for amiloride
61 hydrochloride and furosemide, respectively (Fig. 10). 61
3 101 100.6
62 62
4 101.6 100.5
63 5 100.9 99.7 Humidity degradation 63
64 6 101.5 100.2 In humidity degradation, the drug degraded was 0.6% and 11.5% 64
65 Mean±SD ±0.35 ±0.41 for amiloride hydrochloride and furosemide, respectively. However, 65
66 %RSD 0.35 0.41 no degradation product was observed in the chromatogram 66
67 67
SD: Standard deviation, RSD: Relative standard deviation (Fig. 11).
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1 Table 5: Results of robustness 1

2 2
3 S. No. Parameter Amiloride hydrochloride Furosemide 3
4 RT NTP TF RT NTP TF 4
5 5
6 1 Flow rate 0.8  ml 2.313 3357 1.220 3.663 8027 1.178 6
7 Flow rate 1.2  ml 1.643 2397 1.270 2.531 5872 1.197 7
2 Temperature 23°C 1.927 2881 1.249 3.158 6488 1.193
8 8
Temperature 27°C 1.922 2896 1.240 3.147 6511 1.192
9 3 Wavelength 279 nm 1.923 2846 1.237 3.141 6337 1.196 9
10 Wavelength 283 nm 1.921 2885 1.239 3.145 6721 1.192 10
11 11
RT: Retention time, NTP: Number of theoretical plates, TF: Tailing factor
12 12
13 Table 6: Percentage content of marketed formulation 13
14 14
15 Tablet Drug Amount taken Amount found % Assay 15
16 16
FRUMIL (amiloride hydrochloride Amiloride hydrochloride 20 ppm 19.91 ppm 99.55
17 17
5 mg and furosemide 40 mg) Furosemide 160 ppm 159.84 ppm 99.90
18 18
19 19
20 Table 7: Forced degradation studies of amiloride hydrochloride and furosemide 20
21 21
22 Stress condition Amiloride hydrochloride Furosemide 22
23 23
24 % Assay % Difference w.r.t control % Assay % Difference w.r.t control 24
25 Control 99.7 NA 99.8 NA 25
26 Acid degradation 98.4 1.3 97.9 1.9 26
27 Base degradation 98.6 1.1 95.9 3.9 27
28 Oxidative degradation 97.5 2.2 92.9 6.9 28
29 Photolytic degradation 99.4 0.3 99.3 0.5 29
Thermal degradation 98.5 1.2 97.1 2.7
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Humidity degradation 99.1 0.6 88.3 11.5
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46 Fig. 5: Chromatogram of untreated tablet 46
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60 Fig. 6: Chromatogram of acid degradation 60
61 61
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63 CONCLUSION furosemide. The stress testing studies revealed that the method was 63
64 successfully employed to resolve the degraded products from the 64
Stability indicating RP-HPLC method has been developed and for
65 simultaneous estimation of amiloride hydrochloride and furosemide in sample. The proposed method was proved to be selective, accurate, 65
66 tablet dosage form. The validated method was successfully implemented precise, and rapid, and it can be used for the routine analysis of the 66
67 for the stress testing and analysis of amiloride hydrochloride and amiloride hydrochloride and furosemide in the formulation. 67
68 68
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Fig. 7: Chromatogram of base degradation
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28 Fig. 8: Chromatogram of oxidative degradation 28
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42 Fig. 9: Chromatogram of photolytic degradation 42
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Fig. 10: Chromatogram of thermal degradation
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Fig.11: Chromatogram of humidity degradation

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1 ACKNOWLEDGMENT furosemide in pure and pharmaceutical dosage form. Eur J Anal Chem 1
2 2017;12:385- 94. 2
The authors are thankful to our Principal Dr. Mrs. Sudha Rathod, 6. Kopuri G, Anusha K, Rao DS. Development of a new HPLC method for
3 3
Oriental College of Pharmacy Sanpada, Navi Mumbai, for providing simultaneous estimation of amiloride and hydrochlorothiazide. J Chem
4 4
a platform and facility to conduct research work. The authors are Pharm Sci 2015;8:661-3.
5 7. Narasimham YS, Barhate VD. Development and validation of stability 5
6 thankful to Alkem Laboratories for providing a gift sample of amiloride 6
indicating UPLC method for the simultaneous determination of beta-
7 hydrochloride. 7
blockers and diuretic drugs in pharmaceutical dosage forms. J Chem
8 Metrol 2010;4:1-20. 8
9 AUTHORS’ CONTRIBUTION 8. Tengli AR, Gurupadayya BM. Method development and validation of a 9
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All the authors have contributed equally.
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CONFLICTS OF INTEREST 9. Devanna N, Dudekula B, Ramachandraiah C. Development and
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validation for the simultaneous estimation of dolutegravir and
14 The authors confirm that this paper has no conflict of interests. 14
lamivudine in drug product by RP-HPLC. Int J Res Pharm Nano Sci
15 2017;6:173-80. 15
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AQ1: Kindly check the term.
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AQ2: Kindly review the sentence. 36
37 AQ3: Kindly check the edit made. 37
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