Population pharmacokinetic modelling to characterize the effect of chronic kidney disease on tenofovir exposure after tenofovir alafenamide administration.

BACKGROUND: Tenofovir alafenamide is gradually replacing tenofovir disoproxil fumarate, both prodrugs of tenofovir, in HIV prevention and treatment. There is thus an interest in describing tenofovir pharmacokinetics (PK) and its variability in people living with HIV (PLWH) under tenofovir alafenamide in a real-life setting. OBJECTIVES: To characterize the usual range of tenofovir exposure in PLWH receiving tenofovir alafenamide, while assessing the impact of chronic kidney disease (CKD). METHODS: We conducted a population PK analysis (NONMEM®) on 877 tenofovir and 100 tenofovir alafenamide concentrations measured in 569 PLWH. Model-based simulations allowed prediction of tenofovir trough concentrations (Cmin) in patients having various levels of renal function. RESULTS: Tenofovir PK was best described using a one-compartment model with linear absorption and elimination. Creatinine clearance (CLCR, estimated according to Cockcroft and Gault), age, ethnicity and potent P-glycoprotein inhibitors were statistically significantly associated with tenofovir clearance. However, only CLCR appeared clinically relevant. Model-based simulations revealed 294% and 515% increases of median tenofovir Cmin in patients with CLCR of 15-29 mL/min (CKD stage 3), and less than 15 mL/min (stage 4), respectively, compared with normal renal function (CLCR = 90-149 mL/min). Conversely, patients with augmented renal function (CLCR > 149 mL/min) had a 36% decrease of median tenofovir Cmin. CONCLUSIONS: Kidney function markedly affects circulating tenofovir exposure after tenofovir alafenamide administration in PLWH. However, considering its rapid uptake into target cells, we suggest only a cautious increase of tenofovir alafenamide dosage intervals to 2 or 3 days only in case of moderate or severe CKD, respectively.

Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Development and validation of a liquid chromatography coupled to tandem mass spectrometry method for the monitoring of temsavir plasma concentrations in people living with HIV.

A majority of people living with HIV (PLWH) now have access to HIV treatment with high antiviral potency and favorable tolerability profile. However, in some treatment experienced PLWH viral strains resistant to major current classes of antiretrovirals have emerged, usually due to periods with continued virus replication in the presence of failing drug regimens and thus selection pressure. In such context, new treatment options are therefore needed. Fostemsavir (RUKOBIA®) is the prodrug of temsavir, a first-in-class oral attachment inhibitor approved for the treatment of heavily treatment-experienced adults with multidrug-resistant HIV-1 infection. In this case RUKOBIA® is part of a complex regimen of antiretroviral drugs, often in addition to other drugs for chronic co-morbidities (e.g., heart disease, diabetes mellitus, hepatic and renal impairment, etc). In such a multi-drug regimen context, therapeutic drug monitoring (TDM) of temsavir can be necessary to exclude or adjust for relevant drug-drug interactions. A highly selective assay by liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) was therefore developed for the quantification of temsavir in human plasma. A convenient sample preparation using protein precipitation with acetonitrile followed by supernatant dilution was carried out. Temsavir and fostemsavir were separated in less than 2 min using a multi-step UPLC gradient, thus ensuring adequate quantification of temsavir. The assay for the quantification of temsavir was extensively validated over the large range of clinically relevant concentrations from 1 to 10,000 ng/mL, in accordance with international bioanalytical method guidelines. The method achieves excellent performance in terms of trueness (99.7 - 105.3%), repeatability and intermediate precision (both from 1.6% to 5.8%). This LC-MS/MS method is now part of the routine analyses of the Laboratory of the Service of Clinical Pharmacology of Lausanne (CHUV), Switzerland, as an integrated part of our general TDM Service for antiretrovirals.

Optimized LC-MS/MS quantification of tuberculosis drug candidate macozinone (PBTZ169), its dearomatized Meisenheimer Complex and other metabolites, in human plasma and urine.

Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals. Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using ß-expectation tolerance intervals for known and newer analytes. The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.

Development and validation of a multiplex UHPLC-MS/MS assay with stable isotopic internal standards for the monitoring of the plasma concentrations of the antiretroviral drugs bictegravir, cabotegravir, doravirine, and rilpivirine in people living with HIV.

The widespread use of highly active antiretroviral treatments has dramatically changed the prognosis of people living with HIV (PLWH). However, such treatments have to be taken lifelong raising issues regarding the maintenance of both therapeutic effectiveness and long-term tolerability. Recently approved or investigational antiretroviral drugs present considerable advantages, allowing once daily oral dosage along with activity against resistant variants (eg, bictegravir and doravirine) and also parenteral intramuscular administration that facilitates treatment adherence (eg, long-acting injectable formulations such as cabotegravir and rilpivirine). Still, there remains a risk of insufficient or exaggerated circulating exposure due to absorption issues, abnormal elimination, drug-drug interactions, and others. In this context, a multiplex ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) bioassay has been developed for the monitoring of plasma levels of bictegravir, cabotegravir, doravirine, and rilpivirine in PLWH. A simple and convenient protein precipitation was performed followed by direct injection of the supernatant into the UHPLC-MS/MS system. The four analytes were eluted in less than 3 minutes using a reversed-phase chromatography method coupled with triple quadrupole mass spectrometry detection. This bioassay was fully validated following international guidelines and achieved good performances in terms of trueness (94.7%-107.5%), repeatability (2.6%-11%), and intermediate precision (3.0%-11.2%) over the clinically relevant concentration ranges (from 30 to 9000 ng/mL for bictegravir, cabotegravir, and doravirine and from 10 to 1800 ng/mL for rilpivirine). This sensitive, accurate, and rapid UHPLC-MS/MS assay is currently applied in our laboratory for routine therapeutic drug monitoring of the oral drugs bictegravir and doravirine and is also intended to be applied for the monitoring of cabotegravir/rilpivirine levels in plasma from PLWH receiving once monthly or every 2-month intramuscular injection of these long-acting antiretroviral drugs.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.

Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma.

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 µm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

Applicability of supercritical fluid chromatography - mass spectrometry to metabolomics. I - Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances.

The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between -6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO2 was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand). To avoid system overpressure at very high organic modifier proportion, columns of 100 × 3 mm I.D. packed with sub-3 µm superficially porous particles were selected. In terms of organic modifier, a mixture of 95% MeOH and 5% water was selected, with 50 mM ammonium formate and 1 mM ammonium fluoride, to afford good solubility of analytes in the mobile phase, limited retention for the most hydrophilic metabolites and suitable peak shapes of ionizable species. A sample diluent containing 50%ACN/50% water was employed as injection solvent. These conditions were applied to a representative set of metabolites belonging to nucleosides, nucleotides, small organic acids, small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances, quaternary ammonium metabolites, phosphate-based substances, carbohydrates and amino acids. Among all these metabolites, 65% of the compounds were adequately analyzed with excellent peak shape, 23% provided distorted peak shapes, while only 12% were not detected (mostly metabolites having several phosphate or several carboxylic acid groups).

What are the current solutions for interfacing supercritical fluid chromatography and mass spectrometry?

Mass spectrometry (MS) is considered today as one of the most popular detection methods, due to its high selectivity and sensitivity. In particular, this detector has become the gold standard for the analysis of complex mixtures such as biological samples. The first successful SFC-MS hyphenation was reported in the 80's, and since then, several ionization sources, mass analyzers and interfacing technologies have been combined. Due to the specific physicochemical properties and compressibility of the SFC mobile phase, directing the column effluent into the ionization source is more challenging than in LC. Therefore, some specific interfaces have to be employed in SFC-MS, to i) avoid (or at least limit) analytes precipitation due to CO2 decompression, when the SFC mobile phase is not anymore under backpressure control, ii) achieve adequate ionization yield, even with a low proportion of MeOH in the mobile phase and iii) preserve the chromatographic integrity (i.e. maintaining retention, selectivity, and efficiency). The goal of this review is to describe the various SFC-MS interfaces and highlight the most favorable ones in terms of reliability, flexibility, sensitivity and user-friendliness.

Systematic evaluation of matrix effects in supercritical fluid chromatography versus liquid chromatography coupled to mass spectrometry for biological samples.

Matrix effects (ME) is acknowledged as being one of the major drawbacks of quantitative bioanalytical methods, involving the use of liquid chromatography coupled to mass spectrometry (LC-MS). In the present study, the incidence of ME in SFC-MS/MS and LC-MS/MS in the positive mode electrospray ionization (ESI+) was systematically compared for the analysis of urine and plasma samples using two representative sets of 40 doping agents and 38 pharmaceutical compounds, respectively. Three different SFC stationary phase chemistries were employed, to highlight the importance of the column in terms of selectivity. Biological samples were prepared using two different sample treatments, including a non-selective sample clean-up procedure (dilute and shoot (DS) and protein precipitation (PP) for urine and plasma samples, respectively) and a selective sample preparation, namely solid phase extraction (SPE) for both matrices. The lower susceptibility to ME in SFC vs. reversed phase LC (RPLC) was verified in all the experiments performed on urine, and especially when a simple DS procedure was applied. Also, with the latter, the performance strongly varied according to the selected SFC stationary phase, whereas the results were quite similar with the three SFC columns, in the case of SPE clean-up. The same trend was observed with plasma samples. Indeed, with the PP procedure, the occurrence of ME was different on the three SFC columns, and only the 2-picolylamine stationary phase chemistry displayed lower incidence of ME compared to LC-MS/MS. On the contrary, when a SPE clean-up was carried out, the results were similar to the urine samples, with higher performance of SFC vs. LC and limited discrepancies between the three SFC columns. The type of ME observed in LC-MS/MS was generally a signal enhancement and an ion suppression for urine and plasma samples, respectively. In the case of SFC-MS/MS, the type of ME randomly varied according to the analyzed matrix, selected column and sample treatment.

Development of a LC-MS/MS method for the determination of isomeric glutamyl peptides in food ingredients.

A liquid chromatography with tandem mass spectrometry method was developed for the determination of 27 glutamyl di- and tripeptides in food ingredients. Such compounds are of importance for the food industry, as they can modulate the perception of basic tastes (sweet, salty, and umami). Due to their high polarity, the hydrophilic interaction chromatography mode was selected to have sufficient retention on the column and the best separation was obtained on an amide hybrid silica stationary phase packed with 1.7 µm particles. Thorough optimization of the mobile phase was performed as the start-composition had to be free of ammonium to avoid on-column cis-trans isomerization of the first eluting proline dipeptide. A baseline separation was achieved for all α and γ isomers whereas only a partial resolution was obtained for γ-Glu-Leu and γ-Glu-Ile, for which only the position of a methyl group differs. A fast sample preparation, based on successive dilutions, was performed before injection into the liquid chromatography with tandem mass spectrometry system. The developed method was then applied for the semi-quantification of glutamyl di- and tri-peptides in four different food ingredients. The methodology will further support the optimization of production processes to select the conditions for which the peptide concentrations would be the highest.

A systematic investigation of sample diluents in modern supercritical fluid chromatography.

This paper focuses on the possibility to inject large volumes (up to 10µL) in ultra-high performance supercritical fluid chromatography (UHPSFC) under generic gradient conditions. Several injection and method parameters have been individually evaluated (i.e. analyte concentration, injection volume, initial percentage of co-solvent in the gradient, nature of the weak needle wash solvent, nature of the sample diluent, nature of the column and of the analyte). The most critical parameters were further investigated using in a multivariate approach. The overall results suggested that several aprotic solvents including methyl tert-butyl ether (MTBE), dichloromethane, acetonitrile or cyclopentyl methyl ether (CPME) were well adapted for the injection of large volume in UHPSFC, while MeOH was generally the worst alternative. However, the nature of the stationary phase also had a strong impact and some of these diluents did not perform equally on each column. This was due to the existence of a competition in the adsorption of the analyte and the diluent on the stationary phase. This observation introduced the idea that the sample diluent should not only be chosen according to the analyte but also to the column chemistry to limit the interactions between the diluent and the ligands. Other important characteristics of the "ideal" SFC sample diluent were finally highlighted. Aprotic solvents with low viscosity are preferable to avoid strong solvent effects and viscous fingering, respectively. In the end, the authors suggest that the choice of the sample diluent should be part of the method development, as a function of the analyte and the selected stationary phase.

Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid chromatography.

In the last years, supercritical fluid chromatography has largely been acknowledged as a singular and performing technique in the field of separation sciences. Recent studies highlighted the interest of SFC for the quality control of pharmaceuticals, especially in the case of the determination of the active pharmaceutical ingredient (API). Nevertheless, quality control requires also the determination of impurities. The objectives of the present work were to (i) demonstrate the interest of SFC as a reference technique for the determination of impurities in salbutamol sulfate API and (ii) to propose an alternative to a reference HPLC method from the European Pharmacopeia (EP) involving ion-pairing reagent. Firstly, a screening was carried out to select the most adequate and selective stationary phase. Secondly, in the context of robust optimization strategy, the method was developed using design space methodology. The separation of salbutamol sulfate and related impurities was achieved in 7min, which is seven times faster than the LC-UV method proposed by European Pharmacopeia (total run time of 50min). Finally, full validation using accuracy profile approach was successfully achieved for the determination of impurities B, D, F and G in salbutamol sulfate raw material. The validated dosing range covered 50 to 150% of the targeted concentration (corresponding to 0.3% concentration level), LODs close to 0.5µg/mL were estimated. The SFC method proposed in this study could be presented as a suitable fast alternative to EP LC method for the quantitative determination of salbutamol impurities.

Liquid chromatography and supercritical fluid chromatography as alternative techniques to gas chromatography for the rapid screening of anabolic agents in urine.

This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS and UHPSFC-MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC-MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid-liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC-MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC-MS/MS and only 14% for GC-MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC-MS/MS with 76% of the analytes displaying relative matrix effect between -20 and 20%, while the GC-MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents.

Fast and sensitive supercritical fluid chromatography - tandem mass spectrometry multi-class screening method for the determination of doping agents in urine.

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48-68% of the compounds and higher than 50% for 83-87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.

Evaluation of innovative stationary phase ligand chemistries and analytical conditions for the analysis of basic drugs by supercritical fluid chromatography.

Similar to reversed phase liquid chromatography, basic compounds can be highly challenging to analyze by supercritical fluid chromatography (SFC), as they tend to exhibit poor peak shape, especially those with high pKa values. In this study, three new stationary phase ligand chemistries available in sub -2 µm particle sizes, namely 2-picolylamine (2-PIC), 1-aminoanthracene (1-AA) and diethylamine (DEA), were tested in SFC conditions for the analysis of basic drugs. Due to the basic properties of these ligands, it is expected that the repulsive forces may improve peak shape of basic substances, similarly to the widely used 2-ethypyridine (2-EP) phase. However, among the 38 tested basic drugs, less of 10% displayed Gaussian peaks (asymmetry between 0.8 and 1.4) using pure CO2/methanol on these phases. The addition of 10mM ammonium formate as mobile phase additive, drastically improved peak shapes and increased this proportion to 67% on 2-PIC. Introducing the additive in the injection solvent rather than in the organic modifier, gave acceptable results for 2-PIC only, with 31% of Gaussian peaks with an average asymmetry of 1.89 for the 38 selected basic drugs. These columns were also compared to hybrid silica (BEH), DIOL and 2-EP stationary phases, commonly employed in SFC. These phases commonly exhibit alternative retention and selectivity. In the end, the two most interesting ligands used as complementary columns were 2-PIC and BEH, as they provided suitable peak shapes for the basic drugs and almost orthogonal selectivities.

Supercritical fluid chromatography in pharmaceutical analysis.

In the last few years, there has been a resurgence of supercritical fluid chromatography (SFC), which has been stimulated by the introduction of a new generation of instruments and columns from the main providers of chromatographic instrumentation, that are strongly committed to advancing the technology. The known limitations of SFC, such as weak UV sensitivity, limited reliability and poor quantitative performance have been mostly tackled with these advanced instruments. In addition, due to the obvious benefits of SFC in terms of kinetic performance and its complementarity to LC, advanced packed-column SFC represents today an additional strategy in the toolbox of the analytical scientist, which may be particularly interesting in pharmaceutical analysis. In the present review, the instrumentation and experimental conditions (i.e. stationary phase chemistry and dimensions, mobile phase nature, pressure and temperature) to perform "advanced SFC" are discussed. The applicability of SFC in pharmaceutical analysis, including the determination of drugs in formulations and biofluids is critically discussed.

Possibilities of retention modeling and computer assisted method development in supercritical fluid chromatography.

The multi-modal retention mechanism in supercritical fluid chromatography (SFC) results in a non-linear dependency of log(k) on the fraction of organic solvent φ and log(φ). In the present study, the possibility of retention modeling for method development purposes in SFC was investigated, considering several non-linear isocratic relationships. Therefore, both isocratic and gradient runs were performed, involving different column chemistries and analytes possessing diverse physico-chemical properties. The isocratic retention data of these compounds could be described accurately using the non-linear retention models typically used in HILIC and reversed-phase LC. The interconversion between isocratic and gradient retention data was found to be less straightforward than in RPLC and HILIC because of pressure effects. The possibility of gradient predictions using gradient scouting runs to estimate the retention parameters was investigated as well, showing that predictions for other gradients with the same starting conditions were acceptable (always below 5%), whereas prediction errors for gradients with a different starting condition were found to be highly dependent on the compound. The second part of the study consisted of the gradient optimization of two pharmaceutical mixtures (one involving atorvastatin and four related impurities, and one involving a 16 components mixture including eight drugs and their main phase I metabolites). This could be done via individual retention modeling based on gradient scouting runs. The best linear gradient was found via a grid search and the best multi-segment gradient via the previously published one-segment-per-component search. The latter improved the resolution between the critical pairs for both mixtures, while still giving accurate prediction errors (using the same starting concentrations as the gradient scouting runs used to build the model). The optimized separations were found in less than 3 h and 8 h of analysis time (including equilibration times), respectively.