Is supercritical fluid chromatography hyphenated to mass spectrometry suitable for the quality control of vitamin D3 oily formulations?

Nowadays, many efforts are devoted to improve analytical methods regarding efficiency, analysis time and greenness. In this context, Supercritical Fluid Chromatography (SFC) is often regarded as a good alternative over Normal Phase Liquid Chromatography (NPLC). Indeed, modern SFC separations are fast, efficient with suitable quantitative performances. Moreover, the hyphenation of SFC to mass spectrometry (MS) provides additional gains in specificity and sensitivity. The present work aims at the determination of vitamin D3 by SFC-MS for routine Quality Control (QC) of medicines specifically. Based on the chromatographic parameters previously defined in SFC-UV by Design of Experiments (DoE) and Design Space methodology, the method was adapted to work under isopycnic conditions ensuring a baseline separation of the compounds. Afterwards, the response provided by the MS detector was optimized by means of DoE methodology associated to desirability functions. Using these optimal MS parameters, quantitative performances of the SFC-MS method were challenged by means of total error approach method validation. The resulting accuracy profile demonstrated the full validity of the SFC-MS method. It was indeed possible to meet the specification established by the European Medicines Agency (EMA) (i.e. 95.0 - 105.0% of the API content) for a dosing range corresponding to at least 70.0-130.0% of the API content. These results highlight the possibility to use SFC-MS for the QC of medicine and obviously support the switch to greener analytical methods.

Optimization and validation of a fast supercritical fluid chromatography method for the quantitative determination of vitamin D3 and its related impurities.

In the uprising context of green analytical chemistry, Supercritical Fluid Chromatography (SFC) is often suggested as an alternative to Normal Phase Liquid Chromatography. Indeed, SFC provides fast, efficient and green separations. In this report, the quantitative performances of SFC were challenged on a real-life case study: the Quality Control (QC) of vitamin D3. A rapid and green SFC method was optimized thanks to the Design of Experiments-Design Space (DoE-DS) methodology. It provided robust and high quality separation of the compounds within a 2min timeframe, using a gradient of ethanol as co-solvent of the carbon dioxide. The analytical method was fully validated according to the total error approach, demonstrating the compliance of the method to the specifications of U.S. Pharmacopeia (USP: 97.0-103.0%) and European Pharmacopeia (EP: 97.0-102.0%) for an interval of [50-150%] of the target concentration. In order to allow quantification of impurities using vitamin D3 as an external standard in SFC-UV, correction factors were determined and verified during method validation. Thus, accurate quantification of impurities was demonstrated at the specified levels (0.1 and 1.0% of the main compound) for a 70.0-130.0% dosing range. This work demonstrates the validity of an SFC method for the QC of vitamin D3 raw material and its application to real samples. Therefore, it supports the switch to a greener and faster separative technique as an alternative to NPLC in the pharmaceutical industry.

Monitoring of anatabine release by methyl jasmonate elicited BY-2 cells using surface-enhanced Raman scattering.

A new application of surface-enhanced Raman scattering (SERS) in the field of plant material analysis is proposed in this study. The aim was to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 (BY-2) cells. Gold nanoparticles (AuNps) were used as SERS substrate. The first step was to study the SERS activity of anatabine in a complex matrix comprising the culture medium and BY-2 cells. The second step was the calibration. This one was successfully performed directly in the culture medium in order to take into account the matrix effect, by spiking the medium with different concentrations of anatabine, leading to solutions ranging from 250 to 5000µgL(-1). A univariate analysis was performed, the intensity of a band situated at 1028cm(-1), related to anatabine, was plotted against the anatabine concentration. A linear relationship was observed with a R(2) of 0.9951. During the monitoring study, after the MeJa elicitation, samples were collected from the culture medium containing BY-2 cells at 0, 24h, 48h, 72h and 96h and were analysed using SERS. Finally, the amount of anatabine released in the culture medium was determined using the response function, reaching a plateau after 72h of 82µg of anatabine released/g of fresh weight (FW) MeJa elicited BY-2 cells.

A simple calibration approach based on film-casting for confocal Raman microscopy to support the development of a hot-melt extrusion process.

When developing a new formulation, the development, calibration and validation steps of analytical methods based on vibrational spectroscopy are time-consuming. For each new formulation, real samples must be produced and a "reference method" must be used in order to determine the Active Pharmaceutical Ingredient (API) content of each sample. To circumvent this issue, the paper presents a simple approach based on the film-casting technique used as a calibration tool in the framework of hot-melt extrusion process. Confocal Raman microscopic method was successfully validated for the determination of itraconazole content in film-casting samples. Then, hot-melt extrusion was carried out to produce real samples in order to confront the results obtained with confocal Raman microscopy and Ultra High Performance Liquid Chromatography (UHPLC). The agreement between both methods was demonstrated using a comparison study based on the Bland and Altman's plot.

Application of an innovative design space optimization strategy to the development of LC methods for the simultaneous screening of antibiotics to combat poor quality medicines.

The poor quality of medicines is a crucial problem of public health. Therefore, it is important to have analytical tools to attend decisions of the legal authorities while combating this offense. In this context, the main objective of this study was to develop generic methods able to trace, screen and determine several antibiotics and common associated molecules by mean of liquid chromatographic techniques. For that purpose, an innovative Design Space optimization strategy was applied, targeting 16 antibiotics and 3 beta-lactamase inhibitors. The robustness of the developed method allowed using its use in an environment where operational factors such as temperature are not easy to control and eased its transfer to Ultra High Performance Liquid Chromatography. To demonstrate its ability to quantify the targeted molecules, the developed and transferred method was fully validated for two active ingredients commonly used in association, sulbactam and ceftriaxone, using the accuracy profile as decision tool. Based on this successful step, the method was then used for the quantitative determination of these two active ingredients in three pharmaceutical brands marketed in the Democratic Republic of Congo. Two out of the three pharmaceutical products did not comply with the specifications.

Application of an innovative design space optimization strategy to the development of liquid chromatographic methods to combat potentially counterfeit nonsteroidal anti-inflammatory drugs.

In the context of the battle against counterfeit medicines, an innovative methodology has been used to develop rapid and specific high performance liquid chromatographic methods to detect and determine 18 non-steroidal anti-inflammatory drugs, 5 pharmaceutical conservatives, paracetamol, chlorzoxazone, caffeine and salicylic acid. These molecules are commonly encountered alone or in combination on the market. Regrettably, a significant proportion of these consumed medicines are counterfeit or substandard, with a strong negative impact in countries of Central Africa. In this context, an innovative design space optimization strategy was successfully applied to the development of LC screening methods allowing the detection of substandard or counterfeit medicines. Using the results of a unique experimental design, the design spaces of 5 potentially relevant HPLC methods have been developed, and transferred to an ultra high performance liquid chromatographic system to evaluate the robustness of the predicted DS while providing rapid methods of analysis. Moreover, one of the methods has been fully validated using the accuracy profile as decision tool, and was then used for the quantitative determination of three active ingredients and one impurity in a common and widely used pharmaceutical formulation. The method was applied to 5 pharmaceuticals sold in the Democratic Republic of Congo. None of these pharmaceuticals was found compliant to the European Medicines Agency specifications.

Comparison of the quantitative performances and measurement uncertainty estimates obtained during method validation versus routine applications of a novel hydrophilic interaction chromatography method for the determination of cidofovir in human plasma.

Method validation is essential to ensure that an analytical method is fit for its intended purpose. Additionally, it is advisable to estimate measurement uncertainty in order to allow a correct interpretation of the results generated by analytical methods. Measurement uncertainty can be efficiently estimated during method validation as a top-down approach. However, method validation predictions of the quantitative performances of the assay and estimations of measurement uncertainty may be far away from the real performances obtained during the routine application of this assay. In this work, the predictions of the quantitative performances and measurement uncertainty estimations obtained from a method validation are compared to those obtained during routine applications of a bioanalytical method. For that purpose, a new hydrophilic interaction chromatography (HILIC) method was used. This method was developed for the determination of cidofovir, an antiviral drug, in human plasma. Cidofovir (CDV) is a highly polar molecule presenting three ionizable functions. Therefore, it is an interesting candidate for determination by HILIC mode. CDV is an acyclic cytidine monophosphate analog that has a broad antiviral spectrum and is currently undergoing evaluation in clinical trials as a topical agent for treatment of papillomavirus infections. The analytical conditions were optimized by means of design of experiments approach in order to obtain robust analytical conditions. These ones were absolutely necessary to enable the comparisons mentioned above. After a sample clean-up by means of solid phase extraction, the chromatographic analysis was performed on bare silica stationary phase using a mixture of acetonitrile-ammonium hydrogen carbonate (pH 7.0; 20mM) (72:28, v/v) as mobile phase. This newly developed bioanalytical method was then fully validated according to FDA (Food and Drug Administration) requirements using a total error approach that guaranteed that each future result will fall within acceptance limits of ±30% with a probability of 95% over a concentration range of 92.7-1020ng/mL. A routine application of the cidofovir determination in two pre-clinical trials demonstrated that the prediction made during the pre-study validation was consistent by retrospective analysis of the quality control (QC) samples. Finally, comparison of the measurement uncertainty estimations calculated from the method validation with those obtained from the routine application of the method was performed, stressing that the estimations obtained during method validation underestimated those obtained from routine applications and that the magnitude of this underestimation was function of the cidofovir concentration. Finally, this new HILIC method is reliable, easily applicable to routine analysis and transposable at low cost in other laboratories.