Development, Optimisation, Validation and Inter-Laboratory Verification of a Reversed Phase HPLC Method for Quantification of Human Recombinant Insulin

Iyire, Affiong; Russell, Craig; Dennison, Tom; Rajoli, Rajith K. R.; Saleem, Imran; Rahman, Ayesha; Mohammed, Afzal-Ur-Rahman

doi:10.24297/jbt.v7i1.7192

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…The method was precise with repeatability giving 1.1% RSD; mean % recovery ranging from 78.38 to 103.93%; and limits of detection and quantification at 0.158 μg/mL and 0.528 μg/mL respectively. All but one of the mean recovery RSD% values were under the acceptable 15% upper limit for pharmaceutical analysis (Iyire et al 2018 ). Recovery at the lowest concentration in the range gave the lowest recovery, informing the use of the indirect method for quantifying the amount of pilocarpine hydrochloride entrapped in the niosomes.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of sonication on stability-indicating properties of optimized pilocarpine hydrochloride-loaded niosomes in ocular drug delivery

2021

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Niosomes are increasingly explored for enhancing drug penetration and retention in ocular tissues for both posterior and anterior eye delivery. They have been employed in encapsulating both hydrophilic and hydrophobic drugs, but their use is still plagued with challenges of stability and poor entrapment efficiency particularly with hydrophilic drugs. As a result, focus is on understanding the parameters that affect their stability and their optimization for improved results. Pilocarpine hydrochloride (HCl), a hydrophilic drug is used in the management of intraocular pressure in glaucoma. We aimed at optimizing pilocarpine HCl niosomes and evaluating the effect of sonication on its stability-indicating properties such as particle size, polydispersity index (PDI), zeta potential and entrapment efficiency. Pilocarpine niosomes were prepared by ether injection method. Composition concentrations were varied and the effects of these variations on niosomal properties were evaluated. The effects of sonication on niosomes were determined by sonicating optimized drug-loaded formulations for 30 min and 60 min. Tween 60 was confirmed to be more suitable over Span 60 for encapsulating hydrophilic drugs, resulting in the highest entrapment efficiency (EE) and better polydispersity and particle size indices. Optimum sonication duration as a process variable was determined to be 30 min which increased EE from 24.5% to 42% and zeta potential from (−)14.39 ± 8.55 mV to (−)18.92 ± 7.53 mV. In addition to selecting the appropriate surfactants and varying product composition concentrations, optimizing sonication parameters can be used to fine-tune niosomal properties to those most desirable for extended eye retainment and maintenance of long term stability.

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Section: Methodsmentioning

confidence: 99%

Evaluation of sonication on stability-indicating properties of optimized pilocarpine hydrochloride-loaded niosomes in ocular drug delivery

2021

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“…The British Pharmacopoeia, 2016 and The United States Pharmacopeial, 2019 both recommend reversephase high-performance liquid chromatography (RP-HPLC) for human insulin analysis, as do Moses A et al, 2019. However, according to several studies, this method is time-consuming (Iyire et al, 2018), as well as has an unsatisfactory peak, resolution and tailing (Najjar et al, 2014). Therefore, previous studies have contributed to the development and validation of human insulin assays that are both isocratic and gradient (Iyire et al, 2018;Moslemi et al, 2003;Moussa et al, 2010;Najjar et al, 2014;Ravi et al, 2007;Zuben et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…However, according to several studies, this method is time-consuming (Iyire et al, 2018), as well as has an unsatisfactory peak, resolution and tailing (Najjar et al, 2014). Therefore, previous studies have contributed to the development and validation of human insulin assays that are both isocratic and gradient (Iyire et al, 2018;Moslemi et al, 2003;Moussa et al, 2010;Najjar et al, 2014;Ravi et al, 2007;Zuben et al, 2020). Although glycerol is a common excipient in pharmaceutical products containing human insulin, no study has described nor investigated its effect together with meta-cresol and A-21 desamido insulin.…”

Section: Introductionmentioning

confidence: 99%

Development of Analysis and Validation of Human Insulin-Containing Matrices Using Reverse Phase-Liquid Chromatography

Ambarsari

Suryadi²,

Yanuar³

2022

jppipa, pendidikan ipa, fisika, biologi, kimia

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Licensed drugs containing human insulin are used as a routine therapy to maintain blood glucose levels in patients with diabetes mellitus. Meanwhile, production and storage factors can reduce the potency of these drugs, and this is important for patients. Therefore, a fast and reliable analytical method is needed to ensure quality, as well as safety and effectiveness without being affected by the matrices content, such as m-cresol and glycerol. The study was developed with the high-performance liquid chromatography using a reverse-phase column ReliantÔ C-18 (4.6 x 150 mm, 5 mm), 40°C column temperature, 215 nm UV-Vis detector, 1 mL/min flow rate, and 20 mL injection volume, while, ethylparaben was used as an internal standard. Furthermore, the separation was conducted with solution A and B containing Na2SO4 pH 2.3 and Na2SO4 pH 2.3 in acetonitrile (55:45, v/v) in a ratio of 38:62, v/v, while validation was performed according to ICH guideline. The human insulin retention time was 5.04±0.009 minutes, with linearity of 9.74-146.03 mg/mL (r = 0.9997). Accuracy and precision were 100.71±1.11% and 0.64%, while LOD and LOQ were 0.193 mg/mL and 0.643 mg/mL respectively. Human insulin remained stable at 23°C for 48 hours as demonstrated by ANOVA, a0.05. Based on the results, this method has the potential to separate human insulin from A-21 desamido insulin as a degradant and matrices, as well as allowing for testing many products in a short time.

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“…Dalam monografi USP, insulin human dapat dikuantisasi menggunakan metode Kromatografi Cair Kinerja Tinggi (KCKT) fase terbalik. Beberapa publikasi juga melaporkan baik secara KCKT sistem isokratik maupun gradien [6][7][8][9][10] . Karena telah tersedia metode secara kimia, maka dilakukan pengembangan metode secara immunoassay yang lebih selektif dan peka 11 yaitu enzymelinked immunosorbent assay (ELISA) 12 .…”

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Production of Anti-Recombinant Human Insulin Antibody and Validation by Indirect ELISA

Ambarsari,

Suryadi,

Yanuar

2023

IJPST

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Human insulin potential has become an interest and is important in maintaining the success of therapyin patients with the availability of chemical-based analytical methods, however, only a few have beenusing immunoassays. This study aimed to produce IgG polyclonal antibodies from rabbits immunizedwith 1 mg/mL rhINS subcutaneously and validated by indirect ELISA. Antibody was precipitated andfractioned on a HiTrap® Protein A HP column before being quantified with a UV spectrophotometerat λ 280 nm. The characterization was conducted by Dot Blot test on a BCIP-NBT substrate, as well asSDS-PAGE and Western Blot with polyacrylamide gel concentrations of 7.5% and 17.5%. Validationwas performed using solutions containing glycerol and m-cresol as matrices spiked with rhINS. Thelinearity test in the rhINS concentration range of 80.11-200.28 μg/mL (r = 0.99) showed the linearresult. The accuracy and precision obtained an average of 99.11%±5.01 and 3.91%, while the LODand LOQ were 22.05 μg/mL and 73.51 μg/mL, respectively. Human insulin was stable at 2-8oC for 24hours (α: 0.05, ANOVA). In conclusion, in-house produced IgG polyclonal antibodies and goat anti-IgG peroxidase conjugate can be used for routine testing of human insulin.

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Development, Optimisation, Validation and Inter-Laboratory Verification of a Reversed Phase HPLC Method for Quantification of Human Recombinant Insulin

Cited by 5 publications

References 15 publications

Evaluation of sonication on stability-indicating properties of optimized pilocarpine hydrochloride-loaded niosomes in ocular drug delivery

Evaluation of sonication on stability-indicating properties of optimized pilocarpine hydrochloride-loaded niosomes in ocular drug delivery

Development of Analysis and Validation of Human Insulin-Containing Matrices Using Reverse Phase-Liquid Chromatography

Production of Anti-Recombinant Human Insulin Antibody and Validation by Indirect ELISA

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