A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry.

Fatty acids are a well-established class of compounds targeted as biosignatures for future missions to look for evidence of life on ocean worlds such as Europa and Enceladus. In order to establish their abiotic or biotic origin, we need to separate and quantify fatty acids to determine their relative abundances within a sample. In this study, we demonstrate the high potential of capillary electrophoresis coupled to mass spectrometry (CE-MS) for the efficient separation and sensitive detection of a wide variety of fatty acids. Three derivatization strategies were evaluated to allow the detection of fatty acids by positive ionization mode MS. Furthermore, CE-MS conditions were optimized to provide maximum separation efficiencies and detection sensitivities for the analysis of saturated and unsaturated fatty acids with even- and odd-numbered carbon chain lengths. Optimum separation and detection were obtained using a background electrolyte of 2 M acetic acid in 45% acetonitrile, after derivatization of the fatty acids with 2-picolylamine or N,N-diethylethylenediamine. The limits of detection for the derivatized fatty acids using the optimized method ranged from 25 to 250 nM. The optimized method was also used for the analysis of fatty acids in cell cultures and natural samples. Two distinctive biosignatures were obtained for the microorganisms Halobacillus halophilus and Pseudoalteromonas haloplanktis. In addition, multiple fatty acids were detected in a natural sample from Mono Lake, California.

Detection of Biosignatures by Capillary Electrophoresis Mass Spectrometry in the Presence of Salts Relevant to Ocean Worlds Missions.

Capillary electrophoresis (CE) is a promising liquid-based technique for in situ chemical analysis on ocean worlds that allows the detection of a wide range of organic molecules relevant to the search for life. CE coupled with mass spectrometry (MS) is particularly valuable as it also enables the discovery of unknown compounds. Here we demonstrate that CE coupled to MS via electrospray ionization (ESI) can readily analyze samples containing up to half the saturation levels of salts relevant to ocean worlds when using 5 M acetic acid as the separation media. A mixture containing amino acids, peptides, nucleobases, and nucleosides was analyzed in the presence of two salts, NaCl and MgSO4, based on their relevance to Europa and Enceladus. We demonstrate here CE-MS limits of detection for these organics ranging from 0.05 to 1 µM (8 to 89 ppb) in the absence of salts. More importantly, we demonstrate here for the first time that organics in the low micromolar range (1-50 µM) are detected by CE-MS in the presence of 3 M NaCl without desalting, preconcentration, or derivatization. This demonstration highlights how CE-MS is uniquely suited for organic analysis on future missions to ocean worlds.

Autonomous CE Mass-Spectra Examination for the Ocean Worlds Life Surveyor.

Ocean worlds such as Europa and Enceladus are high priority targets in the search for past or extant life beyond Earth. Evidence of life may be preserved in samples of surface ice by processes such as deposition from active plumes, hydrofracturing, or thermal convection. Terrestrial life produces unique distributions of organic molecules that translate into recognizable biosignatures. Identification and quantification of these organic compounds can be achieved by separation science such as capillary electrophoresis coupled to mass spectrometry (CE-MS). However, the data generated by such an instrument can be multiple orders of magnitude larger than what can be transmitted back to Earth during an ocean world's mission. This requires onboard science data analysis capabilities that summarize and prioritize CE-MS observations with limited computational resources. In response, the autonomous capillary electrophoresis mass-spectra examination (ACME) onboard science autonomy system was created for application to the ocean world's life surveyor (OWLS) instrument suite. ACME is able to compress raw mass spectra by two to three orders of magnitude while preserving most of its scientifically relevant information content. This summarization is achieved by the extraction of raw data surrounding autonomously identified ion peaks and the detection and parameterization of unique background regions. Prioritization of the summarized observations is then enabled by providing estimates of scientific utility, including presence of key target compounds, and the uniqueness of an observation relative to previous observations.

Targeted metabolomics of whole blood using volumetric absorptive microsampling.

Volumetric absorptive microsampling (VAMS) enables the collection of small and accurate quantities of biological fluids. Therefore, this sampling technique is of great interest for volume-limited samples or serial collection of samples. In this study, we examined the potential of VAMS for targeted mass spectrometry (MS)-based metabolomics. The targeted analysis of 36 major metabolites from only 10 µL of whole blood was optimized. A design of experiments was carried out to maximize the extraction of metabolites. Moreover, critical steps in sample preparation and sample analysis were studied and characterized, such as the addition of internal standards to tips of VAMS devices before sample collection. A reversed-phase UHPLC-MS/MS method was used to analyze organic acids, whereas hydrophilic interaction chromatography (HILIC)-MS/MS was selected for the determination of amino acids. Overall, the optimum extraction solvent was acetonitrile-water in a proportion of 60:40 (v/v), providing good recoveries and resulting in the detection of all target metabolites in whole blood with good repeatability (less than 15% RSD on peak area). Furthermore, the stability of the analytes in dried whole blood, which is of critical importance in metabolomics studies, was investigated. The amino and organic acids were stable for at least 4 days when stored at room temperature. This is in contrast to the instability of these compounds in wet blood, thereby showing the great potential of VAMS in metabolomics studies.

Capillary electrophoresis-mass spectrometry for targeted and untargeted analysis of the sub-5 kDa urine metabolome of patients with prostate or bladder cancer: A feasibility study.

Targeted and untargeted analyses of the sub-5 kDa urine metabolome of genitourinary cancer patients (prostate and/or bladder) were performed without chemical derivatization using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). For targeted analysis, endogenous levels of sarcosine and 5 other amino acid metabolites implicated in the progression of prostate cancer were quantified in four patients and in a pooled urine sample from healthy volunteers. An untargeted analysis (m/z 50 to 850) of patient urine was performed using the same CE-ESI-MS system identifying over 400 distinct molecular features per patient. All patient urine samples were collected at prostatectomy/cystectomy via catheter. Patient urine samples were filtered by centrifugation, with endogenous sarcosine enriched by solid-phase extraction, and the processed samples loaded onto CE-ESI-MS for analysis. Diagnostic information, digital pathological slides, and tissue samples were collected and stored in a comprehensive biobanking database. The introduction of urine sample collection into the surgery workflow was facile and is a promising strategy for addressing the translational research challenge of moving smoothly from "chromatogram to nomogram".

Volumetric absorptive microsampling: Current advances and applications.

Recently, volumetric absorptive microsampling (VAMS) has been introduced for the sampling of biological fluids, and more particularly whole blood, on a porous hydrophilic tip. VAMS enables the collection of small, accurate and precise blood volumes (10 or 20µL) regardless of the hematocrit. After drying, the samples can be stored or directly analyzed. The stability of various compounds in dried samples supported on VAMS tips varies from one day to a few months at room temperature, and increases at lower temperatures. The complete tip is used during a simple and straightforward sample preparation. Compounds can be extracted with a variety of solvents, and thereafter directly analyzed. A design of experiments is recommended to determine the optimal extraction conditions for a reproducible recovery. The recovery of compounds might be influenced by the hematocrit. In the last two years, various pharmacokinetic and therapeutic drug monitoring studies have been conducted with VAMS. This review covers the general aspects related with the use of VAMS and its applicability is demonstrated through examples.

Comparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling.

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC-MS/MS methods In this study, we present the development of a nanofluidic-LC-MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC-MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC-MS/MS method. A limit of quantitation of 50pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC-MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry.

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20µl of whole blood from tested subjects. In this study, we present the development and validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof-of-concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

Bioavailability enhancement of itraconazole-based solid dispersions produced by hot melt extrusion in the framework of the Three Rs rule.

Solid dispersion formulations made of itraconazole (ITZ) and Soluplus® (polyethylene glycol, polyvinyl acetate and polyvinylcaprolactame-based graft copolymer abbreviated SOL) were produced using hot melt extrusion. Since ITZ possesses a water solubility of less than 1ng/mL, the aim of this work was to enhance the aqueous solubility of ITZ, and thereby improve its bioavailability. The three formulations consisted of a simple SOL/ITZ amorphous solid dispersion (ASD), an optimized SOL/ITZ/AcDiSol® (super-disintegrant) ASD and an equimolar inclusion complex of ITZ in hydroxypropyl-ß-cyclodextrin (substitution degree=0.63, CD) with SOL. The three formulations were compared in vitro and in vivo to the marketed product Sporanox®. The in vitro enhancement of dissolution rate was evaluated using a biphasic dissolution test. In vitro dissolution results showed that all three formulations had a higher percentage of ITZ released than Sporanox® with the following ranking: SOL/ITZ/CD>SOL/ITZ/AcDiSol®>SOL/ITZ>Sporanox®. The bioavailability of these four formulations was evaluated in rats. The bioanalytical method was optimized so that only 10µL of blood was withdrawn from the rats using specific volumetric absorptive microsampling devices. This enabled to keep the same rats during the whole study, which was in accordance with the Three Rs rules (reduction, refinement and replacement). Furthermore, this technique allowed the suppression of inter-individual variability. Higher Cmax and AUC were obtained after the administration of all three formulations compared to the levels after the use of Sporanox® as follows: SOL/ITZ/AcDiSol®>SOL/ITZ/CD>SOL/ITZ>Sporanox®. The inversion in the ranking between SOL/ITZ/CD and SOL/ITZ/AcDiSol® made impossible the establishment of an in vitro-vivo correlation. Indeed, very different release rates were obtained in vitro and in vivo for the two optimized formulations. These results suggest that ITZ would be protected inside the core of the SOL micelles even during the absorption step at the intestine, while some agents present in the intestinal fluids could displace ITZ from the hydrophobic cavity of CD by competition.

Sampling only ten microliters of whole blood for the quantification of poorly soluble drugs: Itraconazole as case study.

Nowadays in animal studies, it is important to comply with the so-called Three Rs rule by replacing or reducing the number of tested animals. Volumetric absorptive microsampling (VAMS) can be used to collect small quantities (10 or 20µL) of whole blood, thereby limiting the amount of animals needed. In this study, a quantitative method was developed and subsequently validated for the poorly soluble drug itraconazole (ITZ) using VAMS and ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS). A proof of concept study showed that the optimized method is applicable to test the bioavailability of drug formulations containing ITZ. Using VAMS, smaller blood volumes can be taken per sampling point (10-20µL instead of the conventional 0.2-0.5mL) avoiding the sacrifice of animals. Moreover, the same rats can be used to compare different drug formulations which strengthens the validity of the results. In long-term bioavailability studies, it is necessary to guarantee the stability of the tested drugs supported on VAMS devices. In this study, we show that ITZ was only stable for 24h after collection with VAMS, but for at least two weeks by the storage of extracted samples at -80°C.