Stability of extemporaneously prepared sitagliptin phosphate solution.

Sitagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that is used orally in conjunction with diet and exercise to control sugar levels in type 2 Diabetes Mellitus patients. This study aimed to extemporaneously prepare SiP solution (1% w/v) using pure Sitagliptin phosphate (SiP) powder and assess its stability according to pharmaceutical regulatory guidelines. Four SiP solutions, coded T1, T2, T3, and T4, were extemporaneously prepared using pure SiP powder as a source of API. The most suitable one, in terms of general organoleptic properties, was selected for further investigations, including stability studies. For this last purpose, samples of the T4 solution were kept under two storage conditions, room temperature (25˚C and 60% Relative Humidity) and accelerated stability conditions (40˚C and 75% Relative Humidity). Assay, pH, organoleptic properties, related substances, and microbial contamination were evaluated for 4 consecutive weeks. A High-Performance Liquid Chromatography (HPLC) analytical method was developed and validated to be used for the analysis and quantification of SiP in selected solution formulation. The adopted formula had a pH on the average of 3 to 4. During the stability tests, all pH values remained constant. Furthermore, after 4 weeks of storage under both conditions, the SiP concentration was close to 100%. A stable SiP extemporaneous solution was successfully prepared using pure SiP powder. Patients with swallowing problems who use feeding tubes and are unable to take oral solid dosage forms may benefit from this research. Community pharmacists can prepare the solution using sitagliptin powder as the source of the active ingredient.

Introduction

Diabetes mellitus (DM) is a metabolic disorder characterized by a high blood sugar level resulting from defects in insulin secretion, insulin action, or both [1, 2]. Untreated hyperglycemia is associated with long-term complications including retinopathy with a potential loss of vision, nephropathy leading to renal failure, and peripheral neuropathy with risk of foot ulcers, amputations [3]. Diet and exercise can help some people to manage type 2 diabetes [4]. However, when lifestyle changes aren’t enough to lower blood sugar levels, the physician may prescribe suitable antidiabetic drugs. Some of these drugs are administered orally such as α-glucosidase inhibitors, Biguanides, DPP-4 inhibitors, Glucagon-like peptides, Sulfonylureas, and Meglitinides [5]. Gliptins are a group of the most common drugs used for lowering high blood glucose. SiP chemically is 3-amino-1-(3-trifluoromethyl)-6,8-dihydro-5H-(1,2,4) triazolo[4,3-a] pyrazin-7-yl]- 4-(2,4,5-trifluorophenyl) butane-1-phosphoric acid hydrate] (Fig 1) [6].

Fig 1

Chemical structure of SiP.

SiP is the most commonly prescribed gliptin to treat diabetes mellitus, alone or with metformin. SiP binds to the dipeptidyl peptidase 4 enzyme, inhibiting the breakdown of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP) [7]. These hormones are released by the gut after meals and target the pancreas by raising glucose-dependent insulin secretion and decreasing glucagon output from pancreatic α-cells [8]. SiP has many side effects including arthralgia, myopathy, pruritus, pancreatitis and it may increase hepatic enzymes level [9]. Unfortunately, SiP is available in markets only as a solid dosage form which is unsuitable for patients with swallowing problems. In fact, the prevalence of swallowing difficulties is one of the most common problems among pediatric and geriatric patients [10]. This problem will increase in elderly people as a result of many reasons including Alzheimer’s disease, carcinoma, and stroke. Patients with this problem will be less compliant and receive less than optimal treatment with tablets and capsules [11]. Patients having swallowing problems or underuse of feeding tubes may face serious challenges when they should be administered solid oral dosage forms. Accordingly, the community or hospital pharmacist should develop or prepare a new convenient oral liquid dosage form for those patients [12-15]

Therefore, the involved pharmacist should raise several questions to manage such cases, such as if the drug is available in a suitable liquid formulation and, if not, whether they can formulate a convenient oral liquid formulation of the drug or not [14, 15]

To the best of our knowledge, only glibenclamide and metformin are available as a liquid dosage form to treat diabetes mellitus. Unfortunately, SiP is only available as a solid dosage form. This study was conducted to prepare a SiP oral liquid dosage form and to evaluate its chemical and physical stability using bulk SiP powder.

Materials and methods

Materials

For this study, SiP powder, Sorbitol, Mannitol, peppermint flavor, cherry flavor powder, and strawberry flavor powder were obtained from Pharmacare -PLC Ramallah, Palestine.

Instruments and chromatographic conditions

The high-performance liquid chromatography (HPLC) system (Dionex, Thermo Scientific) consisted of an ultimate 3000 model pump (SN 8031808), autosampler (SN 8031972), column oven (SN 8031817), PDA detector (SN 8031310), and Chromeleon software V.6.8 (Dionex, Germany). Weights were measured using Oahu’s balance (RI097) (Switzerland), and pH was detected by using Mettler-Toledo PH meter (TYPE: MP225) (ID: RI015) (Columbus, US). A multichannel stirrer model (MS -52M) (Jeio-Tech, Korea) was used to confirm dissolving, and a stability chamber for accelerated storage conditions at 40 ˚C and 75% RH (RI 091) (Memmert, Germany) was used to evaluate the quality of the product at these harsh conditions. A UV-spectrophotometer (Jasco V 730, Japan) was used to check the suitable wavelength at which SIP showed the maximum absorbance.

Chromatographic conditions

To make a mobile phase ratio of 1:1, 500 ml of 0.1 M potassium dihydrogen phosphate buffer (13.6 g potassium dihydrogen phosphate dissolved in 1000 ml of distilled water) and 500 ml of methanol were combined. Phosphoric acid was used to change the pH to 4.5, after which the solution was degassed and filtered through a 0.45 membrane filter. The column oven temperature was 222°C, the run time was approximately 20 minutes, and the analyte retention time was approximately 6.3 min.

Formulation of SiP oral solution

A quality by testing (QbT) method was applied to guarantee the desired final quality of the SiP solution. Accordingly, several trials were carried out to achieve the most suitable formula that met the desired quality in terms of compliance of taste and dissolving behavior as shown in Table 1. Initially, the organoleptic properties of the suggested formulas were evaluated by the formulator himself. These tests were approved by the local ethics committee (Institutional Review Board [IRB] of An-Najah National University). After that, the formula that showed the most suitable taste, odor, and color was selected for stability studies according to the ICH guidelines for stability studies except analysis time intervals [16].

Table 1

Formulation of SiP oral solution.

Ingredients	T 1	T 2	T 3	T 4
Sitagliptin Phosphate Monohydrate	6.4 g	6.4 g	6.4 g	6.4 g
Liquid Sorbitol 70% w/v	140 ml	140 ml	140 ml	140 ml
Mannitol	14 g	14 g	14 g	14 g
Peppermint	2 ml	0	0	0
Cherry	2 g	3 g	3 g	0
Strawberry	0	0	2 g	4 g
Purified water up to 640 ml to produce 1% w/v concentration	640 ml	ml	640 ml	640 ml

The SiP solution (T4) was prepared to a final concentration of 10 mg/ml. Three samples were taken from the stored SIP solution for initial analysis and the remaining samples were stored at room temperature and in the stability chamber for stress conditions to analysis at 0, 7, 14, 21, and 28 days.

The T4 oral solution was made in triplicate using 6.4 g of the drug dissolved in 30 ml of distilled water. After that, 140 ml of 70% sorbitol w/v was applied to the previous mixture. Meanwhile, mannitol was continuously mixed into the mixture using a magnetic stirrer. The flavor (Strawberry) was then dissolved in 10 ml of water and added to the resulting mixture. Finally, filtered water was applied until the required amount was achieved, and the mixture was thoroughly mixed to produce a homogenized solution. The obtained solutions were poured into plastic bottles with a capacity of 100 ml. Three bottles were held at room temperature for stability testing, while the other three were put in an accelerated stability chamber (40°C, 75% RH).

Quality control of the oral solution

The obtained oral solutions were first examined for clarification, flavor, odor, and color. Chemical and physical stability, assay, pH, and microbial contamination were all studied further using the formula that provided the best results.

Chemical analysis

According to the validation portion, the amount of SiP in the obtained solution was investigated using a new validated HPLC analytical process. The HPLC experimental conditions were optimized on the Octadecyl silane C18 chemically bonded column (C18 150 X 4.6 porous silica column with 5 μ particle size or equivalent) that was purchased from ACE, (United Kingdom). The mobile phase was prepared by mixing 500 ml of 0.1M potassium dihydrogen phosphate buffer (13.6 g of potassium dihydrogen phosphate dissolved in 1000 ml distilled water) and 500 ml of methanol to make a mobile phase ratio of 1:1. Phosphoric acid was used to adjust the pH to 4.5, after that the solution was degassed and filtered through a 0.45 membrane filter. After being analyzed using a UV-spectrophotometer in the UV range (220–320), the maximum absorption was located at a wavelength of 260 nm (260 nm). The flow rate used was 0.5 ml/minute and the injection volume was 10 μl.

Analytical validation

The method was validated under the ICH guidelines [17, 18]. Parameters such as system suitability, selectivity, linearity, range, accuracy (recovery), and precision (repeatability) were all validated. For example, separated normal and placebo solutions were analyzed under the same conditions to determine the method’s specificity. The results were compared with that of a sample solution that had been analyzed on the same day to check if there is any interference between the SiP peak and any other peak in the chromatogram. To determine the accuracy of the method, three different concentrations were prepared at 60%, 100%, and 140% of the final assay concentration for SiP. Three samples were taken from each concentration. Two injections were given to each sample. For SiP, standard solutions were prepared with the same concentrations as the final assay concentration. The accuracy parameter (percent of recovery) was calculated. On the other hand, the precision parameter was determined on six sample- solutions with a final concentration of 0.1285 mg/ml for SiP. The sample and the standard solutions were prepared as directed in the specificity test. The precision parameter (coefficient of variation) was calculated. To assess the linearity of the method, five concentrations of the sample solutions were employed for constructing calibration curves covering a concentration ranging from 0.0771 to 0.1799 mg /ml. Concerning ruggedness and robustness, the same trial was used to prepare the sample solutions that were used in each part of the ruggedness and robustness parameter. Standard and sample solutions were prepared and analyzed by two different analysts, different flow rates, different % buffer in the mobile phase, different pH values for the mobile phase, different two instruments, different two columns, and filter versus centrifuge. Parameters of system suitability were determined by injecting six samples each one composed of 10.0 μl of the standard solution with a final concentration of 0.1285 mg/ml SiP. According to the obtained chromatograms, parameters such as injection precision, tailing factor and theoretical plates were calculated. This analytical procedure validation was conducted using three trials of the product and one trial of the placebo product that was prepared in the laboratory.

Preparation of stock, sample, and standard solutions

Diluent

Highly purified water was prepared by using a Millipore Milli-Q Plus water purification system.

Standard stock solution

Transfer 64.25 mg of Sitagliptin phosphate monohydrate RS/WS (accurately weighed) to 50 ml volumetric flask, add 40 ml of diluent, stir and sonicate to dissolve, dilute to volume using the same diluent, and mix.

Standard solution

Using a volumetric pipette, transfer 5 ml of standard stock solution to 50 ml volumetric flask, diluted to 50 ml using diluent, and mix well.

Sample solution

Using a volumetric pipette, transfer 5 ml of the reconstituted solution (64.25 mg of Sitagliptin phosphate monohydrate) to a 100 ml volumetric flask, add 75 ml of diluent, shake and sonicate for 30 min, leave to return to room temperature, dilute with diluent to the volume, and mix. After that, transfer 10 ml of the supernatant to a 50-ml volumetric flask, dilute with diluent to volume, and mix. Pass a portion of this solution through a nylon filter having a 0.45-μm, reject the first 10 ml, and use the filtrate as the Assay preparation.

Procedure. Inject 10 μl of the standard solution and sample solution directly into the HPLC column under the optimized chromatographic conditions. Suitability requirements were fixed for column efficiency to be not less than 800 theoretical plates from the SiP peak, tailing factor not more than 2.0, and relative standard deviation below 2.0% for the standard solution.

Microbial contamination testing

This test was conducted according to the pharmacopeial specification and guidelines [19]. Precisely, to prepare the culture media, 28 g of nutrient agar dehydrated powder was dissolved using 1000 ml of distilled water. The obtained solution was brought to boiling and kept under vigorous mixing. Then, the solution was sterilized by placing it in the autoclave for 15 min at 125°C. The sterilized solution was then poured into sterilized Petri dishes. Then, the Petri dishes were placed for 24 h in the refrigerator. After that, 0.1 ml of each syrup was placed on the petri dish and incubated for 48 h at 37°C, and then by visual inspection using the colony counter we detected the presence of Escherichia coli, total aerobic microbial count, or total combined yeast and mold counts.

Results

SiP powder was used to make a T4 oral solution. The pH, assay, related substances, organoleptic properties (Table 2), and microbial contamination of this oral solution were all tested (Table 3). Also, T4 was subjected to further stability studies as reported in Table 2. This formula showed good chemical, physical, and microbial stability during the whole period of study (4 weeks). Indeed, the drug concentration remained unchanged despite being stored under room or accelerated conditions for one month. In addition, all other stability parameters remained stable as per day 0.

Table 2

Quality control tests for solutions from pure powder.

Q.C.	0 Day		7 Days		14 Days		21 Days		28 Days
	25˚C	40˚C	25 ˚C	40 ˚C	25 ˚C	40 ˚C	25 ˚C	40 ˚C	25 ˚C	40 ˚C
Assay (%w/v)	98.5	N/A	100.2	97.47	100.7	98.8	101.7	99.8	102.1	100.2
	±0.3		±0.36	±0.3	±0.3	±0.4	±0.3	±0.3	±0.3	±0.3
Related substances (%w/v): Unspecified impurities
	0.04	0.04	0.05	0.07	0.07	0.07	0.07	0.08	0.07	0.08
Total impurities	0.04	0.04	0.05	0.07	0.07	0.07	0.07	0.08	0.07	0.08
pH	3	3.1	3.1	3.5	3.2	3.6	3.3	3.7	3.5	4
Organoleptic Properties	Complies		Complies		Complies		Complies		Complies

Table 3

Microbial limit tests after 28 days of storage at room temperature.

Microbiology	Results	Limits
Escherichia coli bacteria	Negative/ml	Absent
Total combined yeast and molds count	< 10 cfu/ml	not more than 101 cfu/ml
Total aerobic microbial count	< 10 cfu/ml	not more than 102 cfu/ml

Regarding the microbial stability of SiP, no signs of contamination were observed as summarized in Table 3.

Stress degradation studies showed that alkaline degradation is the major degradation pathway of SiP. Actually, the forced degradation analysis aimed to see how well the analytical method could classify and differentiate degradation products from unbroken SiP. The results showed that the HPLC approach used can distinguish the main degradation product (fumarate adduct) with a relative retention time of 1.25 min (Fig 3).

Fig 3

HPLC Chromatogram of SiP and its degradation products substance (A and B before and after magnification.

Since all validation parameters were within the acceptable ranges as stated, the used analytical method was found to be accurate for the assay of SiP in the prepared solutions (Table 4). Moreover, the parameters of system suitability were assessed and they were within the acceptance criteria as summarized in Table 5. The related substances test was carried out according to the European Pharmacopeia (EP 9.0) method of analysis [20]. The approach was precise since isolated normal and placebo solutions were tested under the same conditions and the results were compared to those of a reference solution analyzed the same day, with no interference between the SiP peak and any other peak in the chromatogram. The retention time obtained for SiP was about 5. 48 min as reported in Fig 2. Additionally, stress testing was performed under different stress conditions, including degradation by 0.1N HCl, 0.1N NaOH, H2O2, light, and heat. At all the conditions, there was no interference between the SiP and any other degrades and the purity of the SiP was 0.999 as reported in Table 6. The method also was accurate since the analyzed three different concentrations (60%, 100%, and 140%) of SiP solutions showed the percent of recovery of 99.43%, 101.29%, and 101.02%, respectively. On the other hand, the calculated precision parameter showed a coefficient of variation equal to 0.48%. To determine the method’s linearity, a linear calibration curve was created using five SiP concentrations ranging from 0.0771 to 0.1799 mg/ml. The obtained calibration curve showed a correlation coefficient close to 1 and a Y-intercept equal to 11904.150 and a limit of detection equal to 77.1 μg/ml, which ensures that the method is linear within this range. Standard and sample solutions were prepared and evaluated by two separate analysts, with different flow rates, percent buffer in the mobile phase, pH values for the mobile phase, different two instruments, different two columns, and filter versus centrifuge, and no major variations were identified. No significant differences were observed due to these changes as the coefficient of variance for all of them was below the recommended limit of 2% (Tables 4 and 7). The system suitability was determined by injecting 10.0 μl of the standard solution and finding the results within the recommended range. The relative standard deviation on six replicate injections was found to be 0.63%, tailing factor 1.9±0.2%, and the column efficiency expressed by several theoretical plates for the six replicate injections and was 4222.

Table 4

Summary results of the validation of the assay of SiP solution.

Parameter	Statistical Measure	Result	Limits
Specificity	No interference between SiP chromatogram and other chromatograms	Complies	No interference between the active material peak and any other peaks.
Accuracy	For 60%	99.43	98.00–102.00
	For 100%	101.29
	For 140%	101.02
Linearity	• Correlation Coefficient	0.99930	• Min 0.9950
	• Y-Intercept	11904.150	• ±2.0% of the average area
Precision	Coefficient of Variation (RSD)	0.48%	Maximum of 2.0%

Table 5

Summary results of system suitability.

Parameter	Results	Limit
Injection Precision (RSD)	0.63	< 2.0% (for six injections).
Tailing Factor	1.2	< 2.0
Number of Theoretical Plates	4222	> 2000
Capacity Factor	7.5	> 2

Fig 2

HPLC chromatograms of SiP sample (A) and reference standard (B).

Table 6

Summary results of SiP stressed conditions.

Stress solution	Assay after one day	Degradation of the main peak	Interference with the main peak	Purity check for the main peak	UV spectrum for the main peak
H2O kept at 60 ˚C	99.6%	No	No	0.999	Similar
0.1 N NaOH	86.2%	Partial	No	0.999	Similar
3% H2O2	97.6%	No	No	0.999	Similar
0.1 N HCl	96.7%	No	No	0.999	Similar
At 120˚C	97.8%	No	No	0.999	Similar
In UV Chamber	99.1%	No	No	0.999	Similar

Initial result for the stock solution was = 99.3%.

Retention time for the main peak was = 5.48 min.

Table 7

Summary ruggedness and robustness results of the assay validation of SiP solution.

Ruggedness and Robustness	Coefficient of Variation. (RSD)	Different analyst	Different time analysis	Different Flow rate	Different % buffer in the mobile phase	Different pH for mobile phase	Different instruments	Different Columns	Filter vs centrifuge	Max 2.0%
	Coefficient of Variation. (RSD)	0.84	0.10	0.41	0.10	0.54	0.53	0.61	0.69

Once the appropriate formula was assessed for solubility and organoleptic properties, it was stored at two storage conditions, room temperature (25±3˚C) and accelerated conditions (40˚C/75% RH), to assess its stability. Throughout the stability analysis, the solution exhibited chemical, physical, and microbial stability (4 weeks). In fact, there was no appreciable change in pH which remained within the accepted range (3–4) till the end of the study period and the mean % of remained SiP was close to 97% ± 0.2 at room temperature. Also, until the end of the fourth week of storage, bottles stored under stability conditions of 40°C/75% RH displayed similar stability. Furthermore, there were no visible signs of organoleptic changes such as odor, color, or crystal formation in the solutions. Furthermore, the solutions showed no signs of microbial contamination and no signs of microbial development, as claimed in Table 3.

Discussion

The pharmacist faces difficulties in community and hospital pharmacy practice with the preparation of liquid dosage forms that are not commercially available for pediatric and geriatric patients who have trouble swallowing tablets or capsules, as well as patients who may obtain medicines through nasogastric or gastrostomy tubes [21]. To the best of our knowledge, SiP is not available in any liquid oral dosage forms that could not be suitable for patients with swallowing difficulties. In fact, during the Covid-19 epidemic where geriatric patients with diabetes are considered highly vulnerable to ending up in the ICU, and having a suitable liquid dosage form for this important drug may be of great help to the physicians. At the beginning of the development of the oral liquid formulation, it was noted that the SiP bulk powder was freely soluble in water. As a result, an oral solution of this drug can be conveniently prepared without compromising absorption, as is the case for water-insoluble drugs. Furthermore, SiP has an oral bioavailability of about 87%, and its pharmacokinetic parameters are unaffected by food intake. Besides, it reaches maximum plasma concentration within 3 h [22]. Accordingly, there is no need for either dissolution or bioequivalence studies (as in capsules, tablets, and suspensions) to demonstrate that the administered dose is bioequivalent to the brand tablets. On the other hand, a strong bitter taste of the solution was detected. As a result, several attempts were made to disguise or mitigate this unfavorable bitterness. At the end of our attempts, we successfully developed a new extemporaneous SiP oral liquid formula with suitable organoleptic properties. Also, a new analytical method was developed and validated to assess the quality and stability of the obtained SiP extemporaneous solution using pure SiP powder [23]. Based on validation parameters such as specificity, device linearity, suitability, accuracy, range, precision, and ruggedness, the current validation study aims to ensure that the established SiP solution assay method is successful and reproducible. The validated HPLC method showed that SiP was eluted after 5.45 min of the run while the main related substance was eluted 6.467 (Fig 2A and 2B). A freshly prepared solution of SiP was tested using this HPLC method and correlated with the SiP reference standard. In fact, both retention time and the shape of the peaks were comparable between the reference standard solutions and the sample (Fig 2).

The stress degradation studies showed that alkaline degradation is the major degradation pathway of SiP (Fig 3) [24, 25]. As a result, it’s critical to build an HPLC system that can identify degradation products. As a consequence, the approach can be used to investigate the stability of SiP oral solution. After that, the selected formula was assessed for its assay, pH, microbial contamination, and organoleptic properties including color, smell, taste, and turbidity during the whole period of the study (one month) to estimate the expiry date of the obtained formula (Tables 2 and 3). At the end of the 28 days, more than 97% of the initial concentration of SiP was detected in the solutions which were stored in a selected stability condition. Moreover, no observable changes in color, odor or apparent microbial growth were observed in any sample during the stability study’s period. Besides, no apparent change in the mean pH was observed in any of the tested samples. Since the SiP liquid dosage form is not commercially available, community and hospital pharmacists may benefit from this knowledge because they can prepare such a formula on a prescription using pure SiP powder. This could be very useful for a wide range of patients who need oral hypoglycemic drugs in a liquid dosage form.

Conclusion

Using pure SiP powder, an extemporaneous SiP solution was successfully prepared. In terms of stability and organoleptic properties, the used formula was of high quality. The community pharmacist can extemporaneously prepare the solution using this active ingredient from the sponsor of the generic. Scientific details and instructions on how to compound commercial immediate-release tablets into oral solution, as well as information on the solution’s expiration date, should be provided in pharmaceutical manufacturers’ product inserts.

28 Oct 2020

PONE-D-20-27192

Stability of extemporaneously prepared Sitagliptin Phosphate Solution

PLOS ONE

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The above manuscript describes a method to evaluate the Stability of Extemporaneously Prepared Sitagliptin Phosphate Solution for patients with swallowing difficulties or need feeding tubes to take medicines. The research topic is interesting; however, it should be noted out that the manuscript exhibits many shortcomings and errors. Some are summarized below:

Abstract: Line 31, Replace the word “champers” with “chambers”

Abstract: Line 34, Replace the word “validates” with “validated”

Introduction: Line 55, Delete the word “phosphate”

Introduction: Line 59, The word “Mellitus” should be written as mellitus

Materials and Methods: Line 82-85, Repetition of the word “Pharmacare PLC”

Materials and Methods: Line 93, Incubator was used as stability chamber for stability studies. How temperature and humidity can be varied simultaneously in an incubator? ICH guideline was not followed while carrying out stability studies.

Materials and Methods: Line 95, “Three samples were taken for initial analysis”, however, in Abstract 4 types of samples were mentioned (eg. T1, T2, T3 and T4). Which version is correct (3 or 4)?

Materials and Methods: Line 100, Table 1, Procedure for preparing 1 % solution of Sitagliptin Phosphate solution was not correct.

Materials and Methods: Line 102, “Five gram” precisely of the drug was dissolved in 30 mL distilled water. Whereas, in Table 1, 6.4 g was mentioned.

Materials and Methods: There was no mention of mobile phase ratio in the entire manuscript, which is primary requirement for method to reproduce.

Materials and Methods: The method sample preparation was not satisfactory

Materials and Methods: Authors have not mentioned procedure for performing validation parameters, therefore, it is not possible to judge whether ICH guidelines were followed or not.

Results: Line 185, The pH was mentioned as “3 ± 0.15”, whereas, in Table 2, pH was in the range of 3-4.

Results: Presentation of data and statistical analysis are not as per regulatory guidelines. Data’s presented in Table 2, 4 and 5 are not as per ICH guidelines

Results: Chromatograms presented at the end of the manuscript are not visible properly and does not reflect the result or outcome of the study.

Reviewer #2: Research article should be recommended after such correction. Author doing good research work in the area of pharmaceutical formulation development and for the same he/she also develop a good HPLC metho.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comment Sheet.docx

Click here for additional data file.

3 Jan 2021

Response Letter

Date: 15-11-2020

Girish Sailor

Academic Editor

Subject: Revision of manuscript entitled " Stability of extemporaneously prepared Sitagliptin Phosphate Solution "

Submission number: PONE-D-20-27192

Below you can find The answer to the inquiries raised by the Editorial office (Journal requirement) and reviewers.

I. Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

• Done

2.Thank you for stating the following in the Acknowledgments Section of your manuscript:

[The authors would like to acknowledge An-Najah National University for its support]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

I am sorry for this inconvenient, I would like to state that I have no funding for this project, it is only a research effort between my institution and Pharmacare laboratories only for academic purposes and there are no any commercial benefits for any of the authors or institutions.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

[The author(s) received no specific funding for this work]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Thank you very much and I am sorry for this inconvenient. Also, I would like to state that I have no funding for this project, it is only a research effort between my institution and Pharmacare laboratories only for academic and public issues and there are no any commercial benefits for any of the authors or institutions.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Done

4.Thank you for stating the following in the Competing Interests section:

[The authors have declared that no competing interests exist].

We note that one or more of the authors are employed by a commercial company: Pharmacare PLC

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

• These authors at Pharmacare company played a pure academic rule in this manuscript such as carrying analytical experiments.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

• An-Najah National University provide support in the form of salaries to the following authors:

o AN Z and

o N J

• Pharmacare PLC provide support in the form of salaries to the following authors:

o Y Z

o A K

o M G

• No salary from any for the following authors (they were students at the time of conducting the project:

o T A S

o S S

o La O

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

• Not applicable

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

• Not applicable

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

• Not applicable

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

• Done

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

I declare that no author has competing of interest in this manuscript.

5.We noticed you have some minor occurrence of overlapping text with the following previous publication, which needs to be addressed:

- https://insights.ovid.com/eujhph/201705000/01735171-201705000-00005

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

• Done

Answer to reviewers’ comments

II. Reviewers comments

Reviewer 1

Reviewer #1: The above manuscript describes a method to evaluate the Stability of Extemporaneously Prepared Sitagliptin Phosphate Solution for patients with swallowing difficulties or need feeding tubes to take medicines. The research topic is interesting; however, it should be noted out that the manuscript exhibits many shortcomings and errors. Some are summarized below:

I would like to thank the reviewer for his comments that I appreciate and I believe they will raise the quality of the manuscript. The whole manuscript is now revised and all his/her comments are now answered appropriately.

Abstract: Line 31, Replace the word “champers” with “chambers”

Done

Abstract: Line 34, Replace the word “validates” with “validated”

Done

Introduction: Line 55, Delete the word “phosphate”

Done

Introduction: Line 59, The word “Mellitus” should be written as mellitus

Done

Materials and Methods: Line 82-85, Repetition of the word “Pharmacare PLC”

Done

Materials and Methods: Line 93, Incubator was used as stability chamber for stability studies. How temperature and humidity can be varied simultaneously in an incubator? ICH guideline was not followed while carrying out stability studies.

I agree with you. Actually, it was a typing mistake. Now the sentence is revised accordingly.

Materials and Methods: Line 95, “Three samples were taken for initial analysis”, however, in Abstract 4 types of samples were mentioned (eg. T1, T2, T3 and T4). Which version is correct (3 or 4)?

Actually, we developed 4 different formulas (T1, T2, T3, AND T4). T4 was selected for further analysis. Analysis was repeated on 3 samples which were taken from T4. Now the stamen is revised to clarify this issue.

Materials and Methods: Line 100, Table 1, Procedure for preparing 1 % solution of Sitagliptin Phosphate solution was not correct.

The procedure is now revised and corrected accordingly

Materials and Methods: Line 102, “Five gram” precisely of the drug was dissolved in 30 mL distilled water. Whereas, in Table 1, 6.4 g was mentioned.

Thank you very much for this notification. I agree with you and the procedure is now revised accordingly.

Materials and Methods: There was no mention of mobile phase ratio in the entire manuscript, which is primary requirement for method to reproduce.

Now the mobile phase ratio is included

Materials and Methods: The method sample preparation was not satisfactory

Now the sample preparation is revised and changed appropriately.

Materials and Methods: Authors have not mentioned procedure for performing validation parameters, therefore, it is not possible to judge whether ICH guidelines were followed or not.

The procedure is now included

Results: Line 185, The pH was mentioned as “3 ± 0.15”, whereas, in Table 2, pH was in the range of 3-4.

Thank you again for this notification. Now the numbers are adjusted accordingly.

Results: Presentation of data and statistical analysis are not as per regulatory guidelines. Data’s presented in Table 2, 4 and 5 are not as per ICH guidelines

Done

Results: Chromatograms presented at the end of the manuscript are not visible properly and does not reflect the result or outcome of the study.

Better resolution chromatogram are now included

Comment sheet

Que.1: How you have selected a different formulation ingredients and ratio?

Thank you for your comment. Actually, we followed the quality by testing method which is now reported in the methodology.

Que.2: Have you taken UV Spectrum for selection of wavelength in SiP solution? Is there same or different?

Yes, they are same. Now we included this in the methodology and instrument sections.

Que.3: Which guideline follow for the evaluation of stability studies of SiP solution?

We followed the ICH guidelines for stability testing of pharmaceutical products and now this statement is included in the methodology.

Que.4: How you have evaluated organoleptic properties of SiP oral liquid formulation?

The organoleptic properties were evaluated by the formulator himself during the development phase. This was approved by the local ethics committee (Institutional Review Board [IRB] of An-Najah National University). This statement is now included in the methodology

Que.5: Have you develop HPLC method or cite any other research article method? Describe it.

The HPLC method was developed and validated by the team according to the ICH guidelines.

Reviewer #2: Research article should be recommended after such correction. Author doing good research work in the area of pharmaceutical formulation development and for the same he/she also develop a good HPLC method.

I would like to thank the reviewer for this positive comment and for the efforts that he/she did to improve the quality of the manuscript. We tried our best to answer the comments and to modify the manuscript accordingly. We believe that the manuscript is now much better and much closer to the final acceptance.

Best Regards

Submitted filename: Response Letter 15-11-2020.docx

Click here for additional data file.

25 Feb 2021

PONE-D-20-27192R1

Stability of extemporaneously prepared Sitagliptin Phosphate Solution

PLOS ONE

Dear Dr. Zaid,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

As per the reviewer's comment, the manuscript need major revision before consider for publication.

==============================

Please submit your revised manuscript by Apr 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Girish Sailor

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: After going through the revised manuscript, these are my observations:

Major problems:

#1. As pointed out by the previous reviewer, the stability study was conducted in an incubator. In the revised manuscript, it is changed as stability chamber. This change of equipment seems to be surreal.

#2. The chromatograms looks like edited figures. Authors need to send original chromatogram for review.

#3. There is contradiction in the statements given under abstract section and in the methodology that, in the abstract authors state that four SiP solutions coded T1, T2, T3 and T4 were prepared by the authors, while in the methodology, authors state that the formulae were prepared and evaluated by a separate formulator. These statements are contradicting and confusing to the reader.

#4. As pointed out by the previous reviewer, the analytical method validation section is not satisfactory even after revision with respect to the procedure and the results reported.

#5. The usage of English language throughout the manuscript is to the standard mandated for a research article and need major editing.

Other observations:

#1. Line 2: Change Diabetic Mellitus to Diabetes mellitus.

#2. Line 32: Change stability chambers to stability conditions.

#3. Authors have mentioned in the abstract, 'The obtained solution (T4)........' T4 is one among the four solutions prepared and has not been obtained separately. Need to change the sentence accordingly.

#4. Line 42: “This study provides a solution….” should be revised meaningfully.

#5. The chemical name “3-amino-1-(3-trifluoromethyl)-6,8-dihydro-5H-(1,2,4) triazolo[4,3-a] pyrazin-7-y]- 4-(2,4,5-trifluorophenyl) butane-1-phosphoric acid hydrate]” Should be corrected by changing pyrazine-7-y to pyrazine-7-yl.

#6. Line 85: remove the semicolon.

#7. Under the heading ‘Instruments and Chromatographic conditions”, chromatographic conditions are not mentioned.

#8. Procedure for preparation of the formulae should be re-written in the sentence format, and not as bulleted points.

#9. Under the heading “ Quality control of the oral solution” authors state that the best formula was selected based on the organoleptic properties by the formulator (who is different from the author). If so, why authors are again unnecessarily describing about other formulae. Discussion on T, T2, T3, and T4 become unnecessary since authors have not formulated but have only carried out the stability studies.

#10. Under the heading, “Chemical analysis”, authors state that the amount of SiP in obtained solutions were investigated. If authors have already selected T4 for further evaluation based on organoleptic properties, which are the other solutions???

#11. Line 134: Column dimensions should be written in a standard format. What authors mean by “Pours” ????

#12. Line 135: Word “optimum” is not necessary.

#13. There is inconsistency in writing mL or ml. Please follow the journal format.

#14. Paragraph describing HPLC method development need revision. Mobile phase ratio should be represented in a generally used format. Column oven temperature is an important parameter, but is not reported. Total run time and Rt of analyte also needs mention.

#15. Line 142: Weights were measured…. This part should be placed in materials section.

#16. Analytical method validation section needs revision in terms of correct procedure and proper usage of English language. Why precision study is done on a concentration of 0.1285 mg/mL? How the range was calculated? What is the LLOQ and LOQ of the method?

#17. The title “Preparation of Stock, Sample and Standard solutions” are confusing. Is there any separate stock solution and standard solution? What is the concentration of standard stock solution? What would be the expected concentration in the final diluted sample solution should be mentioned.

# 18. In Table 2, related substances are specified as decimal points. What it represents? There is inconsistency in the usage of capital letters. For example: Complies.

#19. There is inconsistency in the text for “Table”

#20. Table 4 is incomplete in many respects. Results are not represented as required by the guideline. Authors have provided the results of validation in 3 tables. This should be comprehended and made a single concise Table.

#21. There is inconsistency in representing the retention time. Some place it is represented with 3 significant figures and in another place in two.

#21. Number of theoretical plates is represented as 4222 in Table 5, while in the text it is mentioned as 930 ±1

#22. Procedure for forced degradation study should be mentioned in the text and the chromatogram for the stability indicating assay method should be cited in the results.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10 Apr 2021

Dear Editor,

Many thanks for providing us with the opportunity to revise our manuscript. We would also like to thank yourself and the reviewers for the time and expertise in providing feedback.

We think that all comments raised by the reviewers are legitimate and requires consideration. We would like to profoundly thank them for their constructive comments which have greatly improved the manuscript.

Please find below our response to reviewers comments. We have considered carefully all of the comments and have amended the manuscript as appropriate. The amended text is highlighted in red font throughout the manuscript. We have provided a detailed response to each of the comments.

Review Comments to the Author

Reviewer #3: After going through the revised manuscript, these are my observations:

Major problems:

#1. As pointed out by the previous reviewer, the stability study was conducted in an incubator. In the revised manuscript, it is changed as stability chamber. This change of equipment seems to be surreal.

I agree with respected reviewer actually the correct name is stability chamber and I apologized for this mistake.

#2. The chromatograms looks like edited figures. Authors need to send original chromatogram for review.

Thank you for this important correction and now we attached the original chromatograms.

#3. There is contradiction in the statements given under abstract section and in the methodology that, in the abstract authors state that four SiP solutions coded T1, T2, T3 and T4 were prepared by the authors, while in the methodology, authors state that the formulae were prepared and evaluated by a separate formulator. These statements are contradicting and confusing to the reader.

We went throughout the whole MS and we could not find anything related to this comment even though the formulator is one authors.

#4. As pointed out by the previous reviewer, the analytical method validation section is not satisfactory even after revision with respect to the procedure and the results reported.

Actually, the method was validated according to the international guidelines that followed and used by all respected pharmaceutical industry around the world (for example the ICH guidelines (https://www.ich.org/pdf.ICH/s7dep4.pdf)).

#5. The usage of English language throughout the manuscript is to the standard mandated for a research article and need major editing.

The language was revised by one of the authors who is a native speaker.

Other observations:

#1. Line 2: Change Diabetic Mellitus to Diabetes mellitus.

Done

#2. Line 32: Change stability chambers to stability conditions.

Done

#3. Authors have mentioned in the abstract, 'The obtained solution (T4)........' T4 is one among the four solutions prepared and has not been obtained separately. Need to change the sentence accordingly.

Done

#4. Line 42: “This study provides a solution….” should be revised meaningfully.

Done

#5. The chemical name “3-amino-1-(3-trifluoromethyl)-6,8-dihydro-5H-(1,2,4) triazolo[4,3-a] pyrazin-7-y]- 4-(2,4,5-trifluorophenyl) butane-1-phosphoric acid hydrate]” Should be corrected by changing pyrazine-7-y to pyrazine-7-yl.

Thank you

#6. Line 85: remove the semicolon.

Done

#7. Under the heading ‘Instruments and Chromatographic conditions”, chromatographic conditions are not mentioned.

Done

#8. Procedure for preparation of the formulae should be re-written in the sentence format, and not as bulleted points.

Done

#9. Under the heading “ Quality control of the oral solution” authors state that the best formula was selected based on the organoleptic properties by the formulator (who is different from the author).

Actually, as we mentioned earlier in this letter the formulator is one of the authors which is stated in authors contributions section.

If so, why authors are again unnecessarily describing about other formulae. Discussion on T, T2, T3, and T4 become unnecessary since authors have not formulated but have only carried out the stability studies.

We corrected as requested thank you again

#10. Under the heading, “Chemical analysis”, authors state that the amount of SiP in obtained solutions were investigated. If authors have already selected T4 for further evaluation based on organoleptic properties, which are the other solutions???

Corrected as requested.

#11. Line 134: Column dimensions should be written in a standard format. What authors mean by “Pours” ????

Porous corrected it was typos mistake.

#12. Line 135: Word “optimum” is not necessary.

Corrected

#13. There is inconsistency in writing mL or ml. Please follow the journal format.

Done

#14. Paragraph describing HPLC method development need revision. Mobile phase ratio should be represented in a generally used format. Column oven temperature is an important parameter, but is not reported. Total run time and Rt of analyte also needs mention.

Done in the chromatographic conditions section.

#15. Line 142: Weights were measured…. This part should be placed in materials section.

Done as requested.

#16. Analytical method validation section needs revision in terms of correct procedure and proper usage of English language. Why precision study is done on a concentration of 0.1285 mg/mL? How the range was calculated? What is the LLOQ and LOQ of the method?

Because this is the concentration that was used in the preparation of standard and sample in the method of analysis.

Range 0.0771 to 0.1799 mg /ml = 0.1285*60% to 0.1285*140% which means from 60% to140% of the used concentration.

LLOQ and LOQ of the method?

The detection limit for sitagliptin phosphate was: 0.95 µg/ml.

The quantitation limit for sitagliptin phosphate was: 1.973 µg/ml.

The detection limit for Impurity A was: 0.493 µg/ml.

The quantitation limit for Impurity A was: 1.34 µg/ml.

The detection limit for Impurity B was: 0.814 µg/ml.

The quantitation limit for Impurity B was: 1.34 µg/ml.

#17. The title “Preparation of Stock, Sample and Standard solutions” are confusing. Is there any separate stock solution and standard solution? What is the concentration of standard stock solution? What would be the expected concentration in the final diluted sample solution should be mentioned.

Standard stock solution: Transfer 64.25 mg of Sitagliptin phosphate monohydrate RS/WS (accurately weighed) to 50 ml volumetric flask, add 40 ml of diluents, stir and sonicate to dissolve, dilute to volume using diluents, and mix.

Standard solution: Transfer 5 ml of standard stock solution to 50 ml volumetric flask, complete to volume using diluent and mix.

# 18. In Table 2, related substances are specified as decimal points. What it represents? There is inconsistency in the usage of capital letters. For example: Complies.

Done

#19. There is inconsistency in the text for “Table”

Done

#20. Table 4 is incomplete in many respects. Results are not represented as required by the guideline. Authors have provided the results of validation in 3 tables. This should be comprehended and made a single concise Table.

Done

#21. There is inconsistency in representing the retention time. Some place it is represented with 3 significant figures and in another place in two.

Done

#21. Number of theoretical plates is represented as 4222 in Table 5, while in the text it is mentioned as 930 ±1

Corrected as 4222

#22. Procedure for forced degradation study should be mentioned in the text and the chromatogram for the stability indicating assay method should be cited in the results.

Corrected as requested.

Once again, we would like to thank yourself and the reviewers for the time and expertise in providing feedback. We look forward to hearing from you soon.

4 May 2021

PONE-D-20-27192R2

Stability of extemporaneously prepared Sitagliptin Phosphate Solution

PLOS ONE

Dear Dr. Zaid,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Based on the reviewer comment,the manuscript still need some major revision.

==============================

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

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Reviewer #4: Yes

Reviewer #5: Partly

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Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #4: Yes

Reviewer #5: No

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Reviewer #4: Yes

Reviewer #5: No

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Reviewer #4: (No Response)

Reviewer #5: Dear author,

its a wild thought to produce a liquid oral preparation for the Sitagliptin. it could have been a better research article if you do your literature review in a proper way. I appreciate your effort with a strong disagreement. you never think about the hypoglycemic effect of the sorbitol and mannitol. hypoglycemia is a major side effect of OHGAs and Insulin. if mannitol and sorbitol has Hypoglycemic effect then how you can incorporate these polyhydroxy artificial sweeteners with a hypoglycemic drug. please read the reviewer comments attached.

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Reviewer #4: No

Reviewer #5: No

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Submitted filename: PLOS review.docx

Click here for additional data file.

7 Aug 2021

Dear Editor,

Many thanks for providing us with the opportunity to revise our manuscript. We would also like to thank yourself and the reviewer for the time and expertise in providing feedback.

We think that all comments raised by the reviewer are legitimate and requires consideration. We would like to profoundly thank hiim for his constructive comments which have greatly improved the manuscript.

Please find below our response to reviewer comments. We have considered carefully all of the comments and have amended the manuscript as appropriate. The amended text is highlighted in red font throughout the manuscript.We have provided a detailed response to each of the comments.

Thanking you,

Yours faithfully

Comments: -

1. Sitagliptin is a dipeptidyl peptidase enzyme inhibitor which produce hypoglycemic along with diet and exercise to control sugar level in type 2 Diabetic Patients.

• But according to America diabetic Association best therapy for type 2DM in patient with feeding tubes or patients with swallowing difficulty is insulin therapy is good.

• Corrected as requested

2. Is there any references regarding the Assay of sitagliptin tablets or sitagliptin phosphate powder?

No reference was found as well as we used a new method.

3. Please provide information regarding Diluents used to prepare the stock solution.

We provided information regarding Diluents as requested.

4. Preparation of stock, sample and standard solutions for HPLC are not clear.

Thank you for this suggestion and now we clearly explain the preparation of stock, sample and standard solutions for HPLC.

5. The sample T4 has a final concentration of 1%w/v. can you achieve 1% w/v if you dissolve 6.4 gm of SiP in 500 ml of water.

Yes since we used sitagliptin in its salt form and not as free base.

6. Is there any hypoglycemic effect has possessed by the sorbitol and mannitol? If it is there how you can suggest such a formulation. Please justify your suggestion.

Actually, these two are polare excipients and have very limited impact on sugar blood level.

https://jums.ac.ir/dorsapax/Data/sub_7/file/Handbook%20of%20pharmaceutical%20excipients.pdf

https://www.mayoclinic.org/diseases-conditions/diabetes/expert-answers/artificial-sweeteners/faq-20058038

Ohrem HL, Schornick E, Kalivoda A, Ognibene R. Why is mannitol becoming more and more popular as a pharmaceutical excipient in solid dosage forms?. Pharmaceutical development and technology. 2014 May 1;19(3):257-62.

7. Each 100 ml of SiP formulation contain 2.8 gm of sorbitol, which higher than the concentration of active pharmaceutical ingredient SiP (1.28 gm in 100ml). Can you justify this with above comment no.5.

Sorbitol is an artificial sweetner and its concentration is within the recommended level reported in the handbook of pharmaceutical excipients

https://jums.ac.ir/dorsapax/Data/sub_7/file/Handbook%20of%20pharmaceutical%20excipients.pdf

8. Regarding the sample solution preparation, author has to mention how you prepared the sample solution from the formulation (T4) you made.

Done as requested.

9. How you read the colony forming units after the microbial contamination test.

By visual inspection using the colony counter as reported in the revised manuscript

10. How you did the assay of SiP T4 formulation? If you did the assay after the validation or be

It is now reported in the revised manuscript

11. There is a study (IJPSR, 2013; Vol. 4(9): 3494-3503- http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.414.784&rep=rep1&type=pdf ) related with degradation products of Sitagliptin. Are you correlated your study with the previous study? Please include your comments in the discussion.

The comments are now reported in the discussion

12. Please take some more time to rewrite the methods, report, discussion and conclusion. It would be a better paper. I strongly suggest you to rewrite these portions.

All the required sections are rechecked and revised thoroughly as suggested.

Submitted filename: Response to the reviewer comments.docx

Click here for additional data file.

26 Oct 2021

Submitted filename: To PLOS.docx

Click here for additional data file.

30 Oct 2021

Dear Editor,

Thank you for giving us a chance to revise our manuscript. Many thanks for you and respected reviewers for their efforts in improving our manuscript.

Reviewer comments

1. The author made changes according to previous comments.

Thank you a lot

2. Some references not following the PLOS guidelines for referencing. The author should recheck the Reference style.

Answer: Thank you and now we corrected references as shown in the revised MS and we followed Plos One style.

3. In table no.1, “purified water up to 500 ml” shall be rewritten as purified water up to 640 ml to produce 1% w/v concentration.

Answer: Thank you for this suggestion and for all your efforts in reviewing our manuscript and now we corrected Table 1 as you recommended

Best Wishes

Submitted filename: Response Letter.docx

Click here for additional data file.

17 Dec 2021

Stability of extemporaneously prepared Sitagliptin Phosphate Solution

PONE-D-20-27192R4

Dear Dr. Zaid,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Girish Sailor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Dec 2021

PONE-D-20-27192R4

Stability of extemporaneously prepared sitagliptin phosphate solution

Dear Dr. Zaid:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Girish Sailor

Academic Editor

PLOS ONE