A Drug Stability Study Using Surface-Enhanced Raman Scattering on Silver Nanoparticles

Round 1

Reviewer 1 Report

In the present manuscript, a SERS characterization of forced degradation study on Pemetrexed (PMT) was performed. The obtained results were interesting, although I cannot recommend it to be published in the current form. Thus, some changes and other studies should be performed before publication in the Applied Sciences journal.

1- The first paragraph of the introduction must be fundament with more references. Since there are several sentences in a row without any reference.

2- In the sentence “Jansen et al. [12] and Warner et al. [9] reported identified several PMT DPs through oxidation and hydrolysis.” The authors should eliminate the word “reported” or “identified”.

3- “Jansen et al. [12] and Warner et al. [9] reported identified several PMT DPs through oxidation and hydrolysis. Respaud et al. also mentioned only two potential DPs, des-glutamate and glutamic acid [13].” The authors must include in the sentences which were the methods that were used to identify the DPs.

4- The scale bar of the Figure 1b should be more explicit. The letters with the size should be bigger. Alternatively, the authors can include in the legend the size of the scale bar.

5- In overall, the paper is too simple. The obtained results should be complemented with other techniques to better understand which the DPs were obtained. For instance, the authors could complete the paper with HPLC.

Author Response

Response to Reviewer 1 Comments

We appreciate the time of you and we would like to thank you for your constructive comments. We have addressed all the comments in the revision. For each comment, we identify the section of the manuscript on which we made the pertinent changes.

Point 1: The first paragraph of the introduction must be fundament with more references. Since there are several sentences in a row without any reference.

Response 1: Thank you for your valuable comments. We agree with you. We added the following text to the beginning of the first paragraph and added references through the text.

“The drug safety and effectiveness are important concerns ​due to the potential adverse effects through their degradation products on patient health and environment [1,2,].  Investigation of possible molecular changes in a drug molecule over time without interference from excipients, impurities, and degradation products (DPs) is required [3].”

Point 2: In the sentence “Jansen et al. [12] and Warner et al. [9] reported identified several PMT DPs through oxidation and hydrolysis.” The authors should eliminate the word “reported” or “identified”.

Response 2: We corrected the sentence.

Point 3: “Jansen et al. [12] and Warner et al. [9] reported identified several PMT DPs through oxidation and hydrolysis. Respaud et al. also mentioned only two potential DPs, des-glutamate and glutamic acid [13].” The authors must include in the sentences which were the methods that were used to identify the DPs.

Response 3: The text is modified as following:

“Jansen et al. [18] and Warner et al. [15] identified several PMT DPs through oxidation and hydrolysis using HPLC and NMR. First, the DPs were separated a reversed-phase preparative HPLC method, then they were identified with NMR. In another study, Respaud et al. also identified only two potential DPs, des-glutamate and glutamic acid [19]. In their study, they employed a HPLC instrument equipped an ultraviolet and evaporative light scattering detector (HPLC-UV-ELSD) to determine the DPs of PMT.”

Point 4: The scale bar of the Figure 1b should be more explicit. The letters with the size should be bigger. Alternatively, the authors can include in the legend the size of the scale bar.

Response 4: A scale bar on the TEM image in Figure 2b is added. We also added a sentence as

“The colloidal suspension of AgNPs has a maximum absorbance at 430 nm as seen in Figure 2a. Figure 2b and 2c show a representative TEM and DLS plot with an average hydrodynamic size of 60 nm of AgNPs, respectively.”

Point 5: In overall, the paper is too simple. The obtained results should be complemented with other techniques to better understand which the DPs were obtained. For instance, the authors could complete the paper with HPLC.

Response 5: Thank you very much for bringing this up. As mentioned in the manuscript, HPLC is most common used technique for degradation studies of drugs. Our aim in here is to show how easy to investigate stability of a drug using SERS and influence of acidic and hydrolytic conditions on SERS activity. In the manuscript, the degradation studies of PMT using HPLC were briefly summarized. In addition, the SERS analysis results were confirmed with the previous HPLC study results. We believe it is not necessary to repeat already reported studies.

Author Response File: Author Response.docx

Reviewer 2 Report

1. Silver nanoparticles have been used in SERS for over a decade, what is your innovation?
2. Silver nanoparticles are easily oxidized on the substrate, which can affect its SERS effect. How did you overcome this problem?
3. The scale bar of TEM is missing.
4. The variation of SERS at different times and under different conditions is given in Figures 4-6. This information is the focus of this paper. I suggest that the authors summarize the peak of change (possibly in a table), which would be more intuitive.
5. In the discussion of SERS peaks, the authors should cite reasonable references to support the chemical bonds corresponding to the different peaks.
6. The authors need to update the reference.None of the literature is post-2020.

Author Response

Response to Reviewer 2 Comments

We appreciate the time of reviewers and we would like to thank them for their constructive comments. We have addressed all the comments in the revision. For each comment, we identify the section of the manuscript on which we made the pertinent changes.

Point 1: Silver nanoparticles have been used in SERS for over a decade, what is your innovation?

Response 1: We agree with you, the innovation is not the application of SERS for drug stability study. We wanted to emphasize the importance of the influence of the experimental condition used for the forced degradation on a SERS experiment performed with the same goal. The surface properties such as charge, dissolution and aggregation status of the AgNPs after the treatment of drug molecules under forced degradation conditions such as HCl and H₂O₂ exposure influence the SERS outcome.

Point 2. Silver nanoparticles are easily oxidized on the substrate, which can affect its SERS effect. How did you overcome this problem?

Response 2: Thank you very much. This is a valid point and we are aware of this since we have been using AgNPs as SERS substrates for years. It is indeed one of the points of this study to show how experimental conditions affect the SERS experiment for such an application. The oxidation rate of AgNPs can be varied. The reason is the particle size, as well as the particle size distribution, can have a significant effect on the oxidation rate (Hua Qi et al 2010 Nanotechnology 21 215706). Because, the size of NPs increases surface energy of NPs decreases (Journal of Colloid and Interface Science 303 (2006) 415–418). In this study, the oxidation of AgNPs concentrated 32 times at ambient conditions may have an effect on enhancement factor (EF). However, we showed that citrate reduced AgNPs of 50 nm size have a very high SERS activity in our previous studies [J. Phys. Chem. C 2008, 112, 10338–10343]. From our experience, the AgNPs as they are synthesized and used as SERS substrates behave good and they show good SERS activity.

The main point here is that AgNPs can be oxidized in the presence of H₂O₂ (Anal. Chem. 2011, 83, 5873–5880), which is one of the forced degradation conditions. In our previous study, we showed that H₂O₂ reacted with AgNPs if they remained unreduced. We discussed this point in the manuscript in details.

Point 3. The scale bar of TEM is missing.

Response 3: The scale bar is added.

Point 4. The variation of SERS at different times and under different conditions is given in Figures 4-6. This information is the focus of this paper. I suggest that the authors summarize the peak of change (possibly in a table), which would be more intuitive.

Response 4: Thank you for valuable suggestion. We added the following table in the “supporting material”.

Table S1. Tentative SERS band assignments [1-5]

 Point 5. In the discussion of SERS peaks, the authors should cite reasonable references to support the chemical bonds corresponding to the different peaks.

Response 5: We have cited the related literatures for assignment of the bands. In addition, we have added several new sentences in the “Result and Discussion” section as below.

“In Table S1, the assignment of each band observed on the SERS spectra is provided.”

“After 30 h exposure, new bands start to appear at 640, 1051, 1100, 1253, 1437, 1615 cm^-1 can be assigned HCH bending, CH in plane bending, NH₂ rocking, CH₂ symmetric deformation and scissoring of NH₂, respectively [29, 36].”

“Galla et al. reported that when PMT exposed 3% H₂O₂ for 30 min, 17.15% of PMT degraded [20]”.

Point 6. The authors need to update the reference. None of the literature is post-2020.

Response 6: The reference list is updated.

The drug degradation studies conducted by SERS technique;

• Yilmaz, H.; Cobandede, Z.; Yilmaz, D.; Cinkilic, A.; Culha, M.; Demiralay, E.C. Monitoring forced degradation of drugs using silica coated AgNPs with surface-enhanced Raman scattering. Talanta 2020, 214, 120828.
• El-Zahry, M.R.; Lendl, B. Structure elucidation and degradation kinetic study of Ofloxacin using surface enhanced Raman spectroscopy, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2018, 193, 63–70.
• El-Zahry, M.R.; Refaat, I.H.; Mohamed, H.A.; Rosenberg, E.; Lendl, B. Utility of surface enhanced Raman spectroscopy (SERS) for elucidation and simultaneous determination of some penicillins and penicilloic acid using hydroxylamine silver nanoparticles, Talanta 2015, 144, 710–716

The pemetrexed degradation studies;

• Stolarczyk, E.U.; Stolarczyk, K.; Łaszcz, M.; Kubiszewski, M.; Leś, A.; Michalak O. Pemetrexed conjugated with gold nanoparticles – Synthesis, characterization and a study of noncovalent interactions, European Journal of Pharmaceutical Sciences 2017,109,13-20.
• Roland JWM, Robin C, Rob J. van K, Robert J, Ethan den B, Lindemans J, Theo Luider M., A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multitargeted anti-folate pemetrexed in plasma from lung cancer patients. Anal Bioanal Chem, 2010; 398(7-8): 2943–2948. DOI: 10.1007/s00216-010-4192-8.
• Meesters RJW, Cornelissen R, Klaveren RJV, Jonge R, Boer E, Lindemans J, Luider TM, A new ultrafast and high throughput mass spectrometric approach for the therapeutic drug monitoring of the multitargeted antifolate pemetrexed in plasma from lung cancer patients. Anal BioanalChem, 2010; 398, 2943-2948
• Rivory LP, Clarke SJ, Boyer, M, Bishop JF, Highly sensitive analysis of the antifolate pemetrexed sodium: A new cancer agent in human plasma and urine by High Performance Liquid Chromatography. J Chromatography B: Bio Med SciAppl, 2001; 765, 135-140
• Li Wang, Yan Sun S, Jian J, Chuan Li, Determination of pemetrexed in Sprague-Dawley rats by LC/MS/MS and study on its pharmacokinetics. Asian J Pharmaco dynamics and Pharmacokinetics, 2006; 6(3):240-246.
• Kolmer, E.W. J.; Teulen, M. J. A.; Boosman, R. J.; Rouw, N.; Burgers, J.A.; Heine, R. Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support micro dosing studies. Biomedical Chromatography. 2022, 36:e5277.
• Adyanth, M.N.M.K.; Ravi, T.C.K.W.P. Determination of related substances in pemetrexed disodium (Form-IV) in bulk drug samples by HPLC, Pharm. Technol. 2014, 38 (2).
• Narenderan, S.T.; Ramesh, J.; Babu, B.; Meyyanathan, S.N.A. stability-indicating LC–MS/MS method optimization for Pemetrexed through design of experiments: Identification and characterization of major oxidative degradation product. Journal of Pharmaceutical and Biomedical Analysis 2020, 183, 113150
• Jansen, P.J.; Smith, W.K.; Baertschi, S.W.; Dorman, D.E.; Kemp, C.A.; McCune K.A. Determination of the degradation chemistry of the antitumor agent pemetrexed disodium, J. Pharm. Sci. 2016, 105 (11) 3256–3268.
• Respaud, R.; Tournamille, J.F.; Croix, C.; Laborie, H.; Elfakir, C.; Viaud-Massuard. M.C. Development of an ion-pairing reversed-phase liquid chromatography method using a double detection analysis (UV and evaporative light scattering detection to monitor the stability of Alimta®-pemetrexed preparations: identification and quantification of l-glutamic acid as a potential degradation product, J. Pharm. Biomed. Anal. 2011, 54 (2), 411–416.
• Galla, V.K.; Archana, V.; Jinka, R. A new rapid Stability indicating RP-PDA-UPLC method for the estimation of Assay of Pemetrexed disodium-An anti-Lung cancer drug from lyophilized parenteral formulation, J. Appl. Pharm. Sci. 2017, 7 (10) 131–137.
• Hemchand, S.; Babu, R.R.C.; Annapurna, M.M. A new validated stability-indicating gradient RP-HPLC method for the determination of pemetrexed disodium and its process related substances, J. Drug Deliv. Ther. 2019, 9 (3-s) 588–610.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors attended most of the topics excepted point 5. I can accept this form of the manuscript. 

Reviewer 2 Report

This version can be accepted.