Mélisande Bernard

Liquid chromatography with tandem mass spectrometry for the simultaneous identification and quantification of cardiovascular drugs applied to the detection of substandard and falsified drugs

The counterfeiting of pharmaceuticals has been detected since about 1990 and has alarmingly continued to pick up steam. We have been recently involved in an evaluation program of some of the most commonly prescribed cardiovascular drugs in Africa, for analysing an important number of tablets or capsules obtained from different places in seven African countries. A reversed-phase high-performance liquid chromatography with tandem mass spectrometry method was developed and validated to simultaneously control the identity and the quantity of acenocoumarol, amlodipine, atenolol, captopril, furosemide, hydrochlorothiazide and simvastatin in tablets. Their separation was performed on a Kinetex® C(18) (100 mm × 2.1 mm inside diameter, 2.6 μm) column using a gradient elution of 20 mM ammonium formate buffer and acetonitrile (90:10 10:90 v/v) at a flow rate of 0.5 mL/min. The analytes were detected using electrospray ionisation tandem mass spectrometry in both positive and negative modes with multiple reaction monitoring. Tandem mass spectrometry fragmentation patterns of captopril, furosemide and acenocoumarol, up to now not detailed in the literature, were also studied to assist in the selection of the most relevant transitions towards the objectives. The developed method was validated as per International Conference on Harmonisation guidelines with respect to specificity, linearity, trueness, precision, limits of detection and quantification. It has been successfully applied to the control of oral forms of seven cardiovascular drugs collected in African countries.

Identification of the major degradation pathways of ticagrelor

Ticagrelor is a direct-acting and reversible P2Y12-adenosine diphosphate (ADP) receptor blocker used as antiplatelet drug. Forced degradation under various stress conditions was carried out. The degradation products have been detected and identified by high-pressure liquid chromatography multistage mass spectrometry (LC-MS(n)) along with high-resolution mass spectrometry. C18 XTerra MS column combined with a linear gradient mobile phase composed of a mixture of 10 mM acetate ammonium/acetonitrile was shown suitable for drug and impurity determinations and validated as a stability indicating method. Structural elucidation of the degradation products relied on MS(n) studies and accurate mass measurements giving access to elemental compositions. Up to nine degradation products resulting from oxidation/auto-oxidation, S-dealkylation and N-dealkylation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics was also studied in order to assess the molecules shelf-life and to identify the most important degradation factors.

A comprehensive study of apixaban's degradation pathways under stress conditions using liquid chromatography coupled to multistage mass spectrometry

Apixaban is a novel anticoagulant drug acting as a direct, selective and reversible inhibitor of the coagulation factor Xa. Forced degradation under stress conditions were carried out in order to establish its stability profile. The drug was shown to be stable under photolytic, thermolytic and oxidative conditions, while under hydrolytic conditions, up to seven degradation products were generated for about 15% of drug degradation. The degradation products have been detected by linear gradient reversed phase high-performance liquid chromatography coupled with a photo diode array and with electrospray ionization tandem mass spectrometry. A combination of multistage mass spectrometry and high-resolution mass spectrometry (HR-MS) allowed the structural elucidation. The product ions of the degradation products were compared to those of the apixaban protonated ion so as to assign the most structures possible. This required a study in depth of the drugs fragmentation pattern, which has not been reported so far. In view of the products formed, it appears that hydrolysis of the oxopiperidine moiety of apixaban occurred in acidic medium, whereas that of the tetrahydro-oxo-pyridine moiety would further happen under alkaline conditions. Asides from characterization, the LC method was shown to indicate stability and validated as per the criteria described by the ICH guidelines.

Characterization of the surface physico-chemistry of plasticized PVC used in blood bag and infusion tubing

Commercial infusion tubing and blood storage devices (tubing, blood and platelets bags) made of plasticized PVC were analyzed by spectroscopic, chromatographic and microscopic techniques in order to identify and quantify the additives added to the polymer (lubricants, thermal stabilizers, plasticizers) and to put into evidence their blooming onto the surface of the devices. For all the samples, deposits were observed on the surface but with different kinds of morphologies. Ethylene bis amide lubricant and metallic stearate stabilizers were implicated in the formation of these layers. In contact with aqueous media, these insoluble deposits were damaged, suggesting a possible particulate contamination of the infused solutions.

Identification of dabigatran etexilate major degradation pathways by liquid chromatography coupled to multi stage high-resolution mass spectrometry

Dabigatran etexilate (DABET) is an oral direct thrombin inhibitor that has been approved for the prevention of blood clot formation. As the active pharmaceutical ingredient (API) may undergo degradation, leading to drug activity loss or to the occurrence of adverse effects associated with degradation products, thorough knowledge of the APIs stability profile is required. Since very few studies have been reported on the drug stability profile, a study related to DABETs behaviour under stress conditions was carried out in order to identify its major degradation pathways. DABET was subjected to hydrolytic (acidic and alkaline), oxidative, photolytic and thermal stress, as per ICH-specified conditions. Up to ten degradation products along with dabigatran, the active metabolite of DABET, were formed and detected by reverse phase liquid chromatography in gradient mode (LC) coupled to UV and mass spectrometry (UV-MS). Structures were determined by elemental composition determination and study of the fragmentation patterns, using high-resolution mass spectrometry in multistage mode (HR-MSn). Under hydrolytic stress conditions, O-dealkylation may occur and formation of benzimidic acid derivatives was also observed. DABET was shown to be much less susceptible to photolysis and oxidative stress, even if N-dealkylation was highlighted. In view of the structures identified, various degradation pathways of DABET have been proposed.

Phototransformation patterns of the antiplatelet drug tirofiban in aqueous solution, relevant to drug delivery and storage

Tirofiban is a synthetic, nonpeptidic fibrinogen receptor antagonist used as an antiplatelet drug for intravenous delivery. As the active pharmaceutical ingredient may undergo light exposure during manufacturing, storage and/or delivery, there is a need to acquire an extensive knowledge of its major photochemical-degradation pathways. Thus, photochemical-degradation of tirofiban under simulated light irradiation in aqueous solution and in the absence of photosensitizers or photocatalysts, has been investigated in terms of mechanisms. The structural characterization of the photochemical products was carried out with using performance liquid chromatography-multistage high-resolution mass spectrometry along with on-line hydrogen/deuterium exchange. The identification of the twelve detected photochemical products suggested that the photo-transformation of tirofiban occurred via multiple reaction pathways, initiated either by electron or hydrogen atom transfer. These included the photo-oxidation of the piperidine moiety without impacting the secondary amine, the hydroxylation of the methylene group activated by the aromatic ring, the oxidation of the alkyl-sulfonamide group and also the decarboxylative oxidation of the molecule. Hydroxylated compounds, geminal and vicinal-diol compounds, were highlighted suggesting that most of the photoproducts are more hydrophilic than the drug. Understanding the main photo-degradation routes is a good basis to work out efficient measures so as to mitigate or avoid tirofiban instability.

Photodegradation of aqueous argatroban investigated by LC/MSn: Photoproducts, transformation processes and potential implications

Argatroban (ARGA), used as intravenous anticoagulant drug, has been reported to photodegrade under light exposure, requiring specific precautions at handling, storage and administration. Thus, for the first time, aqueous ARGA photodegradation under aerobic conditions has been described in terms of photoproducts, phototransformation processes and potential implications. ARGA significant photoproducts were successfully separated and characterized by gradient reversed-phase liquid chromatography coupled with high-resolution multistage mass spectrometry (LC/HR-MSn). Hitherto still not available in literature, ARGA in-depth fragmentation study was conducted so as to thoroughly sort out the main mechanisms specific to the molecule and therefore, to propose a fragmentation pattern relevant to the identification of ARGA related substances. Thereafter, in view of the structural characteristics of the photoproducts formed, ARGA photodegradation pathways could be worked out, showing that whether by direct photolysis or through photosensitization, the methyltetrahydroquinoline nitrogen and that of guanidine group would be mainly involved in photolysis initiation reactions, through one-electron oxidation along with proton loss. Desulfonation, cyclisation affording compounds of diazinane type, and/or rearrangements with transfer of the methyltetrahydroquinoline group toward the guanidine function were observed accordingly. Having a good insight into ARGA photodegradation pathways allows for consistent measures in view of mitigating or avoiding the drug decay and the related potential effects.

Biocompatibility assessment of cyclic olefin copolymers: Impact of two additives on cytotoxicity, oxidative stress, inflammatory reactions, and hemocompatibility

This work reports the biocompatibility evaluation of cyclic olefin copolymers (COC) as candidates for implantable medical devices. The focus was to establish the influence of two major additives (antioxidant and lubricant) on the overall biocompatibility. The cytotoxicity was evaluated according to ISO 10993-5 guidelines using L929 fibroblasts, HUVEC, and THP-1-derived macrophages. Oxidative stress (ROS, GSH/GSSG, and SOD analysis) and pro-inflammatory cytokines (Il-6 and TNF-α secretion) were quantified using THP-1 cells in direct contact with films. Hemocompatibility was assessed through haemolysis testing, dynamic blood coagulation, platelet adhesion, and activation (membranous P-selectin expression). Results show that the different types of COC have successfully passed the in vitro biocompatibility tests. The presence of antioxidant induces however a slight decrease in ROS production in correlation with a high SOD activity and a modification in blood coagulation profile probably linked to antioxidant recrystallization phenomenon on the surface of COC. The lubricant presence reduced haemolysis, fibrinogen adhesion, and platelet activation. Surface nanotopography of COC highlights different types of needles and globules according to the present additive. Those primary results indicate that COC are promising biomaterial. However, additives influenced some biological parameters pointing out the necessity of a global approach of risk analysis for biocompatibility evaluation.

Degradation pathways study of the natriuretic and β-adrenoceptor antagonist tienoxolol using liquid chromatography–electrospray ionization multistage mass spectrometry

Tienoxolol is a pharmacologically active molecule designed with the functional groups ketothiophene, alkyl benzoate and arylpropanolamine so as to combine a diuretic and a β-adrenoreceptor antagonist into a single molecule. Its degradation products generated in several stress media have been determined by high-pressure liquid chromatography (HPLC) coupled to a hybrid mass spectrometer with a triple quadrupole-linear trap. A Polaris(®) column with a C18-A stationary phase and a linear gradient mobile phase composed of a mixture of trifluoroacetic acid 1% (v/v) and acetonitrile allowed for optimal separation. Structural elucidation of the degradation products has been based on MS/MS techniques, by comparing their fragmentation patterns to the precursors data. Up to seven degradation products of the active ingredient, resulting from hydrolysis, oxidation, dehydration and transamidation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics have been studied to assess the molecules shelf life and to identify the most important degradation factor.