Nirav P. Chauhan*, Naazneen Surti, Manish P. Patel
Gujarat Technological University, Ahmedabad, Gujarat, India.
L. M. College of Pharmacy, Ahmedabad, Gujarat, India.
*Corresponding Author E-mail: niravpharma07@gmail.com
ABSTRACT:
Aim: The aim is to develop simple validated analytical method for analysis of Methoxy-Poly (Ethylene Glycol)-Succinimidyl Succinate (5000) by UV Spectroscopy and to study the forced degradation. Results and conclusion: The proposed method was successfully applied for the determination of Methoxy-Poly (Ethylene Glycol)-Succinimidyl Succinate in bulk. The results were demonstrated, that the procedure is accurate, precise and reproducible (relative standard deviation < 2%).
KEYWORDS: Linker, Methoxy-Poly (Ethylene Glycol)-Succinimidyl Succinate (5000).
INTRODUCTION:
PEGylation is the method involved with appending the strands of the polymer PEG to molecules, most normally peptides, proteins, and antibody, which can work on the wellbeing and effectiveness of numerous therapeutics.1,2,3 it produces adjustments in the physiochemical properties recalling changes for adaptation, electrostatic restricting, hydrophobicity and so on. These physical and synthetic changes increment essential upkeep of the remedial specialist. Likewise, it can impact the restricting preferring of the helpful moiety to the cell receptors and can modify the ingestion and circulation designs.4,5,6
Methoxy PEG Succinimidyl Succinate is a pegylated, amine and sulfhydryl reactive cross-linker.
This cross-linker is non-cleavable and water soluble.1,2 It contains an amine reactive methoxy and a sulfhydryl reactive Succinimidyl group linked with polyethylene glycol of different length.
The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the cross-linked molecules, thus reducing aggregation of labelled proteins in solution and minimizing steric hindrance.7,8,9
Nanoparticles of biodegradable polymer were formulated by solvent evaporation method followed by attachment of methoxy PEG Succinimidyl succinate under continuous shaking at controlled temperature for 24 hours. Liposome comprises method of formulation consisting of thin lipid film hydration with sonication and further attachment with methoxy PEG Succinimidyl succinate.10,11
The PEGylated formulation comprises few huge pharmacological benefits over the unmodified structure, like superior medication solvency, decreased measurement recurrence with possibly diminished harmfulness and without reduced adequacy, broadened circling life, expanded medication dependability, and improved insurance from photolytic debasement; PEGylated structures may likewise be qualified for patent protection also.12,13,14
As there is an unmet need for quantification of PEGylated nanoparticulate system and quantification of linker attached to nanoparticulate system so the aim was to develop and validate a simple, fast, and reliable ultraviolet (UV) spectroscopic method for the determination of MPEG-SS-5000 in bulk and attached to Nanoparticle.15,16
Chemicals reagents:
Methoxy PEG Succinimidyl Succinate was purchased from Laysan Bio Inc., Sodium Chloride, Potassium dihydrogen Phosphate, Disodium Hydrogen Phosphate and Distilled water were purchased from SRL chemical having analytical grade and Distilled water was purchased from Himedialabs.
List of Instruments:
Weighing balance, UV-Visible double beam spectrophotometer (UV-1800, Shimadzu, Japan) with 1cm matched quartz cells, Micropipette of variable volumes (Microlit, India) and Digital balance (Denver Instrument, Germany), pH meter were used
Preparation of Phosphate Buffer pH8:
Accurately weighed 2.38gram of disodium hydrogen phosphate, 0.19gram of potassium dihydrogen phosphate and 8gram of sodium chloride and dissolved in sufficient water to produce 1000ml. Adjust the pH if required.17,18
Preparation of stock solution:
100 mg of Methoxy PEG Succinimidyl Succinate was weighed and transferred in to 100ml volumetric flask, then dissolved in Phosphate Buffer Ph 7.4 and made up to the volume with the same solvent. This solution contains 1000μg/ml concentration. The standard stock solution was further diluted with Phosphate Buffer pH 7.4 to get the concentration of 100μg/ml to 600μg/ml and the solution was scanned between 200 and 400nm using the same solvent as blank. The spectrum was observed in that range and the λ max was found to be 260nm and was selected as analytical wavelength.
Validation of method development:
Linearity:
A calibration curve was plotted between concentration and absorbance. Methoxy PEG Succinimidyl succinate was linear in the concentration range of 100-600µg/ml at 260nm. The linearity was repeated for six times and LOD and LOQ values were calculated.19,20,21
Precision:
The repeatability of the developed method was confirmed by the precision analysis. The intermediate precision of the method was confirmed by intraday and Interday analysis i.e. the analysis of methoxy PEG Succinimidyl succinate was repeated three time intraday and Interday. For this process 300μg/ml was used. The percentage SD and RSD were calculated.22,23,24
Accuracy:
Accuracy of the method was confirmed by the recovery studies. To the pre analyzed known quantity of Methoxy PEG Succinimidyl Succinate was added in aliquots comprising spiked level 80,100 and 120%. The amount of recovery was calculated. This procedure was repeated 3 times and the %RSD was calculated.25
Assay:
A quantity of powder equivalent to 10mg of Methoxy PEG Succinimidyl Succinate was taken in a 50ml volumetric flask dissolved and diluted up to the mark with the Phosphate Buffer pH7.4 and filtered using Whatmann filter paper No.40. From filtrate, the desired concentration (200µg/ml) was then analyzed using UV.26,27,28,29
RESULT AND DISCUSSION:
The λ max of methoxy PEG Succinimidyl Succinate was found to be 260nm and was selected as analytical wavelength. Methoxy PEG Succinimidyl Succinate in bulk was estimated at 260nm by using phosphate buffer pH 7.4. Linearity range was found to be 100−600μg/ml. The correlation coefficient was found to be 0.9998.
The linearity is shown in figure 1 and the concentration with the absorbance value are given in table 1
Figure: 1 Calibration of Methodxy PEG Succinimidyl Succinate
Table No: 1 Optical Characteristics of methoxy PEG Succinimidyl Succinate
|
Parameters |
Values |
|
λmax (nm) |
260 |
|
Beer’s law limit (µg/ ml) |
100-600 |
|
Correlation coefficient (r) |
0.9998 |
|
Regression equation (y=mx+c) |
Y = 0.0005x + 0.0185 |
|
Slope(m) |
0.0005 |
|
Intercept(c) |
0.0185 |
|
LOD (µg/ ml) |
2.98 |
|
LOQ (µg/ ml) |
9.04 |
The %RSD for intraday and inter-day was observed to be less than 2%. Hence the developed method was precise. The results are shown as per above table.
Table No: 2. Precision study of methoxy PEG Succinimidyl Succinate
|
Repeatability (300μg/ml) |
Intraday |
|||||||||
|
Samples |
Absorbance |
Concentration (μg/ml) |
Absorbance |
Mean |
SD |
RSD |
Mean RSD |
|||
|
Sample 1 |
Sample 2 |
Sample 3 |
||||||||
|
1 |
0.171 |
100 |
0.072 |
0.073 |
0.073 |
0.072666667 |
0.00058 |
0.79 |
0.44 |
|
|
2 |
0.171 |
300 |
0.171 |
0.172 |
0.172 |
0.171666667 |
0.000577 |
0.34 |
||
|
3 |
0.172 |
600 |
0.33 |
0.331 |
0.331 |
0.330666667 |
0.00058 |
0.17 |
||
|
4 |
0.172 |
Interday |
||||||||
|
5 |
0.172 |
Concentration (μg/ml) |
Absorbance |
Mean |
SD |
RSD |
Mean RSD |
|||
|
Sample 1 |
Sample 2 |
Sample 3 |
||||||||
|
6 |
0.173 |
|||||||||
|
Mean |
0.172 |
100 |
0.071 |
0.073 |
0.072 |
0.072 |
0.001000 |
1.39 |
0.91 |
|
|
SD |
0.000753 |
300 |
0.17 |
0.173 |
0.172 |
0.171666667 |
0.001528 |
0.89 |
||
|
%RSD |
0.44 |
600 |
0.331 |
0.33 |
0.333 |
0.331333333 |
0.001528 |
0.46 |
||
Table No: 3. LOD, LOQ and Ruggedness study of methoxy PEG Succinimidyl Succinate
|
LOD and LOQ |
Ruggedness |
|||||||||
|
Sample |
Intercept |
Slope |
Factor |
Level of Change |
Absorbance |
Mean |
SD |
%RSD |
||
|
Sample1 |
Sample 2 |
Sample 3 |
||||||||
|
1 |
0.0171 |
0.0009 |
Different Analyst |
Analyst-1 |
0.172 |
0.173 |
0.172 |
0.17 |
0.0006 |
0.34 |
|
2 |
0.0181 |
0.0009 |
||||||||
|
3 |
0.0185 |
0.0009 |
Analyst-2 |
0.17 |
0.172 |
0.172 |
0.17 |
0.0012 |
0.67 |
|
|
4 |
0.0191 |
0.00087 |
||||||||
|
5 |
0.0189 |
0.00089 |
|
|||||||
|
6 |
0.0192 |
0.0009 |
||||||||
|
Mean |
0.0185 |
0.0009 |
Different Instrument |
Instrument-1 |
0.173 |
0.171 |
0.17 |
0.17 |
0.00153 |
0.89 |
|
SD |
0.0008 |
|
||||||||
|
LOD |
2.98 |
Instrument-2 |
0.172 |
0.17 |
0.172 |
0.17 |
0.00115 |
0.67 |
||
|
LOQ |
9.04 |
|||||||||
The method was sensitive as LOD and LOQ were observed to be in sub-microgram level. The method was rugged as observed by the %RSD values which are less than 2%. The results of assay show that the amount of Methoxy PEG Succinimidyl Succinate was found between 99.70 to 101.42
The ruggedness of the developed method was confirmed by using different instruments and different analysts. The % RSD was calculated. The % RSD by using different analyst was found to be 0.94% and 0.86%. The results are within the limit. So it indicates the developed method was rugged. The results were shown as per above table
Table No: 4. Accuracy study of methoxy PEG Succinimidyl Succinate
|
Accuracy |
|||||||||||
|
Linker |
Spiked level |
Amount present in mixture (μg/ml) |
Amount added (μg/ml) |
Absorbance |
Amount Recovered (μg/ml) |
Average |
% Recovery |
Average % recovery |
SD |
% RSD |
|
|
|
80% |
100 |
80 |
0.14 |
181.12 |
179.22 |
100.62 |
99.57 |
0.96986 |
0.97 |
|
|
80% |
100 |
80 |
0.138 |
178.84 |
99.35 |
||||||
|
80% |
100 |
80 |
0.137 |
177.70 |
98.72 |
||||||
|
100% |
100 |
100 |
0.155 |
198.27 |
199.03 |
99.13 |
99.51 |
1.18952 |
1.20 |
||
|
100% |
100 |
100 |
0.158 |
201.70 |
100.85 |
||||||
|
100% |
100 |
100 |
0.154 |
197.12 |
98.56 |
||||||
|
120% |
100 |
120 |
0.173 |
218.84 |
219.22 |
99.47 |
99.65 |
1.31 |
1.31 |
||
|
120% |
100 |
120 |
0.171 |
216.55 |
98.43 |
||||||
|
120% |
100 |
120 |
0.176 |
222.27 |
101.03 |
||||||
The accuracy of the developed method was carried out by standard addition method. The known amount of pure Methoxy PEG Succinimidyl Succinate was added to the previously analyzed solution. Good recoveries at spiked level 80% were observed from 99.35% to 100.62%, Recoveries at spiked level 100% were observed from 99.13% to 100.85% and Recoveries at spiked level 120% were observed from 99.47% to 101.03%, concluding that the method was accurate. The results are shown as per above table.
Table No: 5 Assay of methoxy PEG Succinimidyl Succinate
|
Assay |
|||||||
|
Sample |
Absorbance |
linker recovered |
% linker calculated |
Avg linker recovered |
SD of linker recovered |
%RSD of linker recovered |
SD of % linker found |
|
1 |
0.156 |
199.41 |
99.70 |
199.79 |
1.72 |
0.86 |
0.86 |
|
2 |
0.159 |
202.84 |
101.42 |
||||
|
3 |
0.155 |
198.27 |
99.13 |
||||
|
4 |
0.157 |
200.55 |
100.28 |
||||
|
5 |
0.155 |
198.27 |
99.13 |
||||
|
6 |
0.156 |
199.41 |
99.70 |
||||
The results of assay show that the amount of Methoxy PEG Succinimidyl Succinate was between 99.70 to 101.42. The results were shown as per above table.
CONCLUSION:
In this study a simple, precise, accurate and sensitive UV-spectroscopy method was developed for the analysis of methoxy PEG Succinimidyl Succinate in bulk. The correlation coefficient (γ) value of the proposed method was close to 1.0, which indicates that the concentration used for plotting calibration curve were obeying Beer’s law strictly. Statistical validation shows that the method was reproducible and accurate. Also the various parameters were calculated such as standard deviation and percentage relative standard deviation. The values complies all the limits as per ICH guidelines.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank Gujarat Technological University for Research support and National fellowship for Person with Disability for their kind support.
REFERENCES:
1. Menon Rudolph S, Zalipsky S, Seltzer R. Covalent attachment of polyethylene glycol to proteins and evaluation. Biotechnology and Applied Biochemistry. 1992;15(1):100–14.doi.org/10.1111/j.1470-8744.1992.tb00198.x
2. Bentley MD, Roberts MJ, Harris JM. Protein PEGylation and its chemistry. Advanced Drug Delivery Reviews. 2002 Jun 17;54(4):459–76.doi.org/10.1016/S0169-409X(02)00022-4
3. Hsu H-J, Han Y, Cheong M, Král P, Hong S. Dendritic PEG outer shells enhance serum stability of polymeric micelles. Nanomedicine. 2018 Aug;14(6):1879–89.DOI: 10.1016/j.nano.2018.05.010
4. Calvo P, Gouritin B, Angelo JD, Noel J, Georgin D, Fattal E, et al. Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery. Pharm Res. 2001; 18(8):1157–66. Doi: 10.1023/a: 1010931127745.
5. Sánchez-López E, Ettcheto M, Egea MA, Espina M, Calpena AC, Folch J, et al. New potential strategies for Alzheimer’s disease prevention: pegylated biodegradable dexibuprofen nanospheres administration to APPswe/PS1dE9. Nanomed Nanotechnol Biol Med. 2016;13:1171–82.doi.org/10.1016/j.nano.2016.12.003
6. Jain A, Chasoo G, Singh SK, Saxena AK, Jain SK. Transferrin-appended PEGylated nanoparticles for temozolomide delivery to brain: in vitro characterisation. Journal of Microencapsulation. 2011 Feb 1;28(1):21–8.doi.org/10.3109/02652048.2010.522257
7. Zhang X, Chen G, Zhang T, Ma Z, Wu B. Effects of PEGylated lipid nanoparticles on the oral absorption of one BCS II drug: a mechanistic investigation. IJN. 2014 Nov 26;9(1):5503–14.
8. Zhao S, Wang W, Huang Y, Fu Y, Cheng Y. Paclitaxel loaded human serum albumin nanoparticles stabilized with intermolecular disulfide bonds. Med Chem Commun. 2014 Oct 23;5(11):1658–63.
9. Pang X, Yang P, Wang L, Cao J, Cheng Y, Sheng D Human serum albumin nanoparticulate system with encapsulation of gefitinib for enhanced anti-tumor effects in non-small cell lung cancer. Journal of Drug Delivery Science and Technology. 2019 Aug 1;52:997–1007.
10. Wan X, Zheng X, Pang X, Zhang Z, Jing T, Xu W The potential use of lapatinib-loaded human serum albumin nanoparticles in the treatment of triple-negative breast cancer. International Journal of Pharmaceutics. 2015 Apr 30;484(1):16–28.
11. Cho H-Y, Lee CK, Lee Y-B. Preparation and Evaluation of PEGylated and Folate-PEGylated Liposomes Containing Paclitaxel for Lymphatic Delivery. Journal of Nanomaterials. 2015 Jan 20;2015:e471283. doi.org/10.1155/2015/471283
12. Wei M, Guo X, Tu L, Zou Q, Li Q, Tang C, et al. Lactoferrin-modified PEGylated liposomes loaded with doxorubicin for targeting delivery to hepatocellular carcinoma. IJN. 2015 Aug 12;10(1):5123–37.doi.org/10.2147/IJN.S87011
13. Ghaferi M, Asadollahzadeh MJ, Akbarzadeh A, Ebrahimi Shahmabadi H, Alavi SE. Enhanced Efficacy of PEGylated Liposomal Cisplatin: In Vitro and In Vivo Evaluation. International Journal of Molecular Sciences. 2020 Jan;21(2):559. doi.org/10.3390/ijms21020559
14. Yani MV, Anjani IAW, Narayana IGS, Wihandani DM, Supadmanaba IGP. Combination of Cisplatin-Withaferin Based on PEGylated Liposome Nanoparticles as Alternative Therapy for Ovarian Cancer. Journal of Medicine and Health.2020,2(5):1129
15. Taneja N, Singh KK. Rational design of polysorbate 80 stabilized human serum albumin nanoparticles tailored for high drug loading and entrapment of irinotecan. International Journal of Pharmaceutics. 2018 Jan 30;536(1):82–94.DOI: 10.1016/j.ijpharm.2017.11.024
16. Almandoz MC, Sancho MI, Duchowicz PR, Blanco SE. UV–Vis spectroscopic study and DFT calculation on the solvent effect of trimethoprim in neat solvents and aqueous mixtures. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2014 Aug 14;129:52–60. doi.org/10.1016/j.saa.2014.02.191
17. Yu M, Ding Z, Jiang F, Ding X, Sun J, Chen S, et al. Analysis of binding interaction between pegylated puerarin and bovine serum albumin by spectroscopic methods and dynamic light scattering. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2011 Dec 1;83(1):453–60.
18. S S, K R, T AS. Analytical Method Development and Validation of Caffeine in Tablet Dosage Form By Using UV- Spectroscopy. 1. 2013 Jul 10;3(4):82–6.
19. Sri KV, Jain GV, Maduri M. UV Spectrophotometric Method for the Estimation of Rilpivirine in Bulk and Pharmaceutical Formulations. Research Journal of Pharmacy and Technology. 2014 Apr 28;7(4):463–6. doi.org/10.5958/0974-360X
20. Andressa TS, Gabriela R B, Isadora DM, Lisiane B, Donadel MM, Clésio SP. UV spectrophotometric method for quantitative determination of Bilastine using experimental design for robustness. Drug Analytical Research. 2017; 01(2): 38-43.doi.org/10.22456/2527-2616.79221
21. M Sashikala, S Mohan et .al; Validated UV-Visible Spectroscopic Analytical method development and stability studies on Oseltamivir. Research Journal of Pharmacy and Technology. 2020 Sep 4;13(9):4323–8. doi.org/10.5958/0974-360X.2020.00764.7
22. Lavanya Latha Reddy K., Sowjanya T., Bandhavi P., Raveendranath T., R.B. Desireddy, Naga Sowjanya G. Development and Validation of UV Spectrophotometric Method for Estimation of Amlodipine Besylate in Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2012 Oct 28;5(10):1320–3.
23. Manfed E. Wolff. Burger’s Medicinal Chemistry and Drug Discovery: Therapeutic Agent. John Willy and Sons, INC, New York.1997; Vol 5, 5th Ed.
24. Mastanamma SK, Reehana SK, L P, Kiran R. UV Spectrophotometric Method for the Determination of Sirolimus in Bulk and its Dosage Form. Research Journal of Pharmacy and Technology. 2019 Apr 30;12(4):1655–8. doi.org/10.5958/0974-360X.2019.00277.4
25. Sumithra M, Yuvanesh P, Mistry A. Analytical method development and validation of ambroxol hydrochloride by UV spectroscopy and forced degradation study and detection of stability. Research Journal of Pharmacy and Technology. 2016 Jul 28;9(7):794–800.doi.org/10.5958/0974-360X.2016.00152.9
26. Tegeli V, Birajdar A, Matole V. UV Spectrophotometric Method Development and Validation of Darunavir in bulk and Solid Dosage Form. Research Journal of Pharmacy and Technology. 2021 Jun 29;14(6):3262–4.doi.org/10.52711/0974-360X.2021.00567
27. Prof. Sushil D. Patil, Tejashri Dugaje, Dr. Sanjay J. Kshirsagar. Development and Validation of UV Spectrophotometric Method for Estimation of Cyproheptadine hydrochloride. Asian Journal of Research in Chemistry. 2019; 12(2):112-115.
28. Attia K. A. M. et al. spectrophotometric determination of Acotiamide hydrochloride tri-hydrate in the presence of its degradative product. The Journal of Biological and Chemical Luminescence. 2018; 1: 1-6
29. Vijayraj S. et al. Analytical process of drug by ultraviolet (UV) spectroscopy: A review. International Journal of Pharmaceutical Research and Analysis. 2012; 2 (2): 72-78.
Received on 13.04.2022 Modified on 16.07.2022
Accepted on 19.10.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(7):3275-3279.
DOI: 10.52711/0974-360X.2023.00539