31 research outputs found
Simple quantification of phytosterols and tocopherols using fast chromatography -Tandem mass spectrometry.
Tocopherols and Phytosterols are highly abundant compounds in waste products resulting from Canola Oil production. They have significant antioxidant and cholesterol lowering properties, respectively. Since Canola is a major crop product in Canada, effective extraction of these metabolites has economical impact. Hence, there is a need for the development of a fast and easy quantification method of these active metabolites.
Our analytical strategy relied on the use of fast chromatography -Tandem mass spectrometry (FC-MS/MS). A guard column was used to achieve fast separation and the method is compared to use of conventional C18 column. The mobile phase consisted of acetonitrile:methanol (99:1) with 0.1% acetic acid. The compounds were ionized in the positive ion mode using atmospheric pressure chemical ionization (APCI). The following parameters were employed: source temperature 380°C, curtain gas 40 psi, nebulizer current 2.5 µA and ion source gas 1 30 psi. 5α-Cholestan-3β-ol and Rac-tocol were used as internal standards for phytosterols and tocopherols, respectively.
Four phytosterols and four tocopherols, namely Stigmasterol, β-Sitosterol, Brassicasterol, Campesterol, Alpha-tocopherol, Gamma-tocopherol, Beta-tocopherol and Delta-tocopherol were determined using FC-Multiple-Reaction-Monitoring (MRM). The run time was 2 minutes only, compare to 6.5 minutes with the column injection. Beta-tocopherol and Gamma-tocopherol couldn’t be resolved on the guard column nor on the C18 column. The FC-MS/MS methods addressed the issue of cross talks among the target analytes. For example, stigmasterol and β-sitosterol precursor ions observed as [M+H-H20]+ has the same m/z values for the ion designated as [M+H-4H]+ for campesterol and brassicasterol. In fact, such interferences prevented the full removal of the column (i.e. loop injection). Calibration curves were established and a good linearity was achieved (0.25-10 µg/ml) with R2 of 0.996 and 0.997 for tocopherols and phytosterols, respectively.
In conclusion, a fast and simple FC-MS/MS method for the simultaneous quantitation of phytosterols and tocopherols was successfully developed
Insulin aggregation assessment by size-exclusion chromatography and capillary gel electrophoresis
Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may lead to complications in insulin therapy. For that reason, they are important parameters to investigate for insulin quality control.
The United States and European Pharmacopoeias currently use a SEC method with acidic mobile phase for the quantification of aggregates in insulin formulations. However, changes in aggregates assessment have been reported when the mobile phase composition differs from the sample dissolution medium. [1]
To investigate the impact of mobile phase on human insulin aggregates analysis, the aggregated human insulin samples were analyzed by SEC using neutral (nSEC) or acidic mobile phases (aSEC). The aggregated samples were obtained by dissolving human insulin in acidic media, followed by agitation for 8, 16, 24, 32, 40 and 48 hours respectively.
During SEC method development, the impact of arginine and acetonitrile addition to the mobile phase was pointed out.
Additionally, an orthogonal capillary-gel electrophoresis method (CGE) for the assessment of insulin aggregates was developed. The optimal pH 8.1 CGE buffer was formulated without SDS in order to preserve the non-covalent aggregates.
After the optimization of the methods, human insulin and aggregated samples were analyzed using nSEC, aSEC and CGE.
A similar increase of dimers percentage with incubation time was noticed by both nSEC and CGE, while no significant increase of dimers content was observed by aSEC. However, an insulin polymeric complex was detectable for some samples with aSEC.
The three methods were used to analyze an insulin formulation and a similar tendency was observed.
The results obtained emphasize the importance of mobile phase choice in SEC. The good correlation between dimers percentage obtained by nSEC and CGE suggests that these technics most probably enable the detection of the species initially present in the sample and do not change the composition of the sample during analysis.
The developed CGE method is a fast and reliable tool for the study of the complex process of insulin aggregation. Furthermore, the CGE method could be easily applied to other proteins since it proved to be highly reproducible and has the advantages of low sample consumption, and no expensive column or organic solvents are required
Analyse des concentrations de quatre type d'insulines différentes ( humaine 100 IU/ml, aspart 100 IU/ml, lispro 200 IU/ml et glargine 300 IU/ml) provenant de trois sources différentes (pharmacie hospitalière , officine de ville , et échantillons procurés au diabétologue par les représentants de l’industrie ).
Development of a Fast Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Simultaneous Determination of Phytosterols and Tocopherols
Study of insulin aggregation by SEC and CGE
Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient but only the monomeric form is active once dissociated in the bloodstream. Insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolded proteins results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. Moreover, during its production, insulin is often subjected to extreme conditions making lack of aggregates an important parameter to be controlled during its quality control.
United States and European Pharmacopoeias use both size exclusion chromatography (SEC) to assess the level of covalent high molecular weight species. This technique is reproducible, and easy to use but shows many drawbacks including possible changes in the aggregates composition by dilution into the HPLC system or adsorption of sample onto the stationary phase. For those reasons other techniques have been considered in the literature for studying aggregation of insulin. Optical microscopy, electron microscopy, dynamic light scattering, turbidimetry, Fourier Transform infrared spectroscopy, Raman spectroscopy, thioflavin T fluorescence and circular dichroism spectroscopy are some of them. In any cases, the use of orthogonal techniques is essential to assess the relevance of the results.
Results: In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by SEC and capillary electrophoresis (CE). CE shows many advantages in terms of sample and solvent consumption and enables analysis of samples under their native form. We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method
Analytical techniques currently used in the pharmaceutical industry for the quality control of RNA-based therapeutics and ongoing developments.
peer reviewedThe number of RNA-based therapeutics has significantly grown in number on the market over the last 20 years. This number is expected to further increase in the coming years as many RNA therapeutics are being tested in late clinical trials stages. The first part of this paper considers the mechanism of action, the synthesis and the potential impurities resulting from synthesis as well as the strategies used to increase RNA-based therapeutics efficacy. In the second part of this review, the tests that are usually performed in the pharmaceutical industry for the quality testing of antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) and messenger RNAs (mRNAs) will be described. In the last part, the remaining challenges and the ongoing developments to meet them are discussed
Follow-up of Insulin Aggregation productions in the context of formulation quality control
Application of cyclodextrins in capillary electrophoresis to improve stereo- and chemo selectivity of phosphorothioate oligonucleotides
peer reviewe
Quality control and aggregation follow-up of insulin formulations
The prevalence of diabetes is increasing every year making insulin formulations widely prescribed medicines. Fast and efficient methods to assess the quality of such biopharmaceutical products are thus required, more particularly methods to assess the content of API and potential aggregates.
We started our study on insulin aggregation using the European Pharmacopeia method. Since methods to assess aggregate content are often contradictory, we also developed original and orthogonal methods using size-exclusion chromatography (SEC) and capillary gel electrophoresis (CGE). It was demonstrated that methods under neutral conditions (SEC and CGE) yield to similar aggregate content contrary to pharmacopeia SEC method that works under acidic conditions. Ion-mobility Q-TOF mass spectrometer was also used to confirm the presence and the identity of insulin dimers. Then, we applied the three methods to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. We also used the CGE method to study the stability of human insulin under different storage conditions.
Finally, we used UHPLC and mass spectrometry to quantify insulin formulation from different supply chains. We demonstrated that all the analyzed formulations had a potency between 95.0 % and 105.0 % of the potency stated on the label. This was useful to dispel doubts regarding issues in insulin cold chain supply recently described in the litterature