Optimization of XCMS parameters for LC-MS metabolomics: an assessment of automated versus manual tuning and its effect on the final results.

INTRODUCTION: Several software packages containing diverse algorithms are available for processing Liquid Chromatography-Mass Spectrometry (LC-MS) chromatographic data and within these deconvolution packages different parameters settings can lead to different outcomes. XCMS is the most widely used peak picking and deconvolution software for metabolomics, but the parameter selection can be hard for inexpert users. To solve this issue, the automatic optimization tools such as Isotopologue Parameters Optimization (IPO) can be extremely helpful. OBJECTIVES: To evaluate the suitability of IPO as a tool for XCMS parameters optimization and compare the results with those manually obtained by an exhaustive examination of the LC-MS characteristics and performance. METHODS: Raw HPLC-TOF-MS data from two types of biological samples (liver and plasma) analysed in both positive and negative electrospray ionization modes from three groups of piglets were processed with XCMS using parameters optimized following two different approaches: IPO and Manual. The outcomes were compared to determine the advantages and disadvantages of using each method. RESULTS: IPO processing produced the higher number of repeatable (%RSD < 20) and significant features for all data sets and allowed the different piglet groups to be distinguished. Nevertheless, on multivariate level, similar clustering results were obtained by Principal Component Analysis (PCA) when applied to IPO and manual matrices. CONCLUSION: IPO is a useful optimization tool that helps in choosing the appropriate parameters. It works well on data with a good LC-MS performance but the lack of such adequate data can result in unrealistic parameter settings, which might require further investigation and manual tuning. On the contrary, manual selection criteria requires deeper knowledge on LC-MS, programming language and XCMS parameter interpretation, but allows a better fine-tuning of the parameters, and thus more robust deconvolution.

Comparison of liver and plasma metabolic profiles in piglets of different ages as animal models for paediatric population.

Liver plays an important role in drug metabolism, so studying the grade of maturation of this organ would help to develop more appropriate dosing regimens for paediatric populations. Nevertheless, considering the invasive nature of liver analyses there are obvious ethical boundaries, particularly in babies and children. In this work, we investigated the suitability of blood plasma as an alternative matrix to evaluate the biological age of liver. With this aim, we studied the correlation of plasma and liver metabolomic profiles obtained by HPLC-TOF-MS for piglets of different ages (newborns, neonates and infants). By means of Pearson correlation analysis we observed that 360 and 1784 pairs of metabolite features were significantly correlated in positive and negative ionization mode, respectively. Procrustes analysis was applied in order to assess the similarity of the clustering resulting from the data obtained from the two matrices and the two ionisation modes. The Procrustes distances were low for both ESI+ (0.3753) and ESI- (0.3673) and, hence, liver and plasma are expected to provide similar discriminatory information. Furthermore, we found that Multiblock Principal Component Analysis (MB-PCA) readily allowed us to combine the data obtained from both matrices and to better understand the clustering according to the three study groups. Considering all these results, we suggest that plasma can provide valuable insight into the maturation grade of liver in order to provide accurate dosing in paediatric population.

Efficacy of Protein Baits with Fipronil to Control Vespa velutina nigrithorax (Lepeletier, 1836) in Apiaries.

The yellow-legged hornet (Vespa velutina nigrithorax), outside its natural range, has become a major threat to domestic bees. Several control methods have been used to fight against V. velutina, but the results achieved are not satisfactory. The use of protein baits with biocides has shown to be an effective method to control invasive wasp populations, but they have not been used to control V. velutina. Thus, the efficacy of protein baits containing fipronil to reduce the presence of hornets in apiaries was evaluated in this study. After laboratory determination of the optimal efficacy of a protein bait at a 0.01% concentration of fipronil, field trials were conducted involving 222 beekeepers. The data reported by the 90 beekeepers who completed the requested questionnaire demonstrated that in the groups of apiaries with the highest pressure of hornets (groups with 10-30 and >30 hornets), there was a significant decrease in the presence of V. velutina, lasting at least two weeks. The reduction in the number of hornets was positively correlated with bait consumption, and bait consumption was positively correlated with the number of hornets present at the time of treatment. Although the method used has shown good efficacy and the concentration of fipronil used was very low; possible negative effects on the environment should also be evaluated.

Low toxicity deep eutectic solvent-based ferrofluid for the determination of UV filters in environmental waters by stir bar dispersive liquid microextraction.

In this work, a low toxicity deep eutectic solvent-based ferrofluid is presented for the first time as magnetic fluid to be used as an efficient solvent in liquid-based microextraction techniques. This ferrofluid is made of a hydrophobic deep eutectic solvent, composed by menthol and thymol in a 1:5 molar ratio as carrier solvent, and oleic acid-coated cobalt ferrite (CoFe2O4@oleic acid) magnetic nanoparticles. This material was characterized via magnetism measurement, scanning electron microscopy, infrared spectroscopy and density measurement. The determination of UV filters in environmental water samples was selected as model analytical application to test the extraction performance of this new ferrofluid by employing stir bar dispersive liquid microextraction, prior to liquid chromatography-tandem mass spectrometry analysis. The response surface methodology was used as a multivariate optimization method for extraction step. Under the optimized conditions, good analytical features were obtained, such as low limits of detection between 7 and 83 ng L-1, good repeatability (relative standard deviations, RSD (%) below 15%), enrichment factors between 46 and 101 and relative recoveries between 80 and 117%, proving the good extraction capability of this ferrofluid. Finally, the method was successfully applied to three environmental waters (beach and river waters), finding trace amounts of the target UV filters. The presented low toxicity deep eutectic solvent-based ferrofluid results to be a good alternative to conventional solvents used in liquid-phase microextraction techniques.

HPLC-(Q)-TOF-MS-Based Study of Plasma Metabolic Profile Differences Associated with Age in Pediatric Population Using an Animal Model.

A deep knowledge about the biological development of children is essential for appropriate drug administration and dosage in pediatrics. In this sense, the best approximation to study organ maturation is the analysis of tissue samples, but it requires invasive methods. For this reason, surrogate matrices should be explored. Among them, plasma emerges as a potential alternative since it represents a snapshot of global organ metabolism. In this work, plasma metabolic profiles from piglets of different ages (newborns, infants, and children) obtained by HPLC-(Q)-TOF-MS at positive and negative ionization modes were studied. Improved clustering within groups was achieved using multiblock principal component analysis compared to classical principal component analysis. Furthermore, the separation observed among groups was better resolved by using partial least squares-discriminant analysis, which was validated by bootstrapping and permutation testing. Thanks to univariate analysis, 13 metabolites in positive and 21 in negative ionization modes were found to be significant to discriminate the three groups of piglets. From these features, an acylcarnitine and eight glycerophospholipids were annotated and identified as metabolites of interest. The findings indicate that there is a relevant change with age in lipid metabolism in which lysophosphatidylcholines and lysophoshatidylethanolamines play an important role.

Aspergillus fumigatus Fumagillin Contributes to Host Cell Damage.

The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated.

Chromatographic methods for echinocandin antifungal drugs determination in bioanalysis.

The increase of fungal resistance to drugs, such as azole family, gave rise to the development of new antifungals. In this context, echinocandins emerged as a promising alternative for antifungal therapies. Following the commercialization of caspofungin in 2001, echinocandins became the first-line therapy for invasive candidiasis in different patient populations. The quantification of these drugs has gained importance since pharmacokinetic/pharmacodynamic and resistance studies are a paramount concern. This fact has led us to exhaustively examine the methodologies used for the analysis of echinocandins in biological fluids, which are mainly based on LC coupled to different detection techniques. In this review, we summarize the analytical methods for the quantification of echinocandins focusing on sample treatment, chromatographic separation and detection methods.

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications.

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.

Instability of calcium channel antagonists during sample preparation for LC-MS-MS analysis of serum samples.

1,4-Dihydropyridines calcium channel antagonists (1,4-DHP CCAs) are photolabile and the products of their photodecomposition have no pharmaceutical activity. In our previous work we have presented a screening procedure for eleven 1,4-DHPs in plasma by LC-MS-MS using multiple reaction motoring. The laboratory process includes preparation and storage of stock solutions, plasma storage, solid-phase extraction, reconstitution of extracts and storage time in an autosampler for LC-MS-MS analysis. Prior to validation of the analytical procedure, we have tested the stability of these compounds by exposure to light. Methanolic solutions have been exposed to laboratory and UV light and the stability of the compounds in plasma was tested by exposure of spiked plasma samples to laboratory light at room temperature. Stability during freeze-thaw cycles and stability during 2 month storage at -20 degrees C have been tested as well. Products of photodecomposition have been identified after forced degradation and the degree of degradation has been quantified using LC-UV-DAD and LC-MS-MS, respectively. A 96% degradation after only 2h has been observed when solutions of nifedipine or nisoldipine were exposed to laboratory light in clear glass vials. In plasma samples degradation was 25% in only 2h for both compounds. The main degradation product was produced by oxidation of the dihydropyridinic ring resulting in the pyridine analogue that has been described as the first metabolite in the metabolic pathway. Only minor degradation was found for the other tested compounds after 2h light exposure in methanolic solutions. Furthermore, lercanidipine and nicardipine were also degradated by esterhydrolysis. Several additional minor degradation products were found for the other tested 1,4-DHPs, however, some of them could not be identified. Preconditions for storage and handling of plasma samples prior to and during analysis for 1,4-DHP CCAs are suggested in order to avoid photodecomposition of the analytes.

Improvement of the chromatographic separation of several 1,4-dihydropyridines calcium channel antagonist drugs by experimental design.

A high-performance liquid chromatographic method with diode array detection has been developed and optimized for the separation of five calcium channel blockers belonging to the 1,4-dihydropyridine subgroup (nifedipine and related drugs). The possibility of the simultaneous drug analysis allows a decrease of time during the assay as well as a saving of reagents and solvents. In this work, the effect of four experimental parameters (organic modifier percentage, pH value, concentration of the buffer in the mobile phase, and column temperature) on the chromatographic resolution are investigated by experimental design in order to optimize the chromatographic separation of five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine, and lercanidipine). Fractional factorial design, central composite design, and finally the Multisimplex program are used to establish the optimal conditions in terms of resolution and minimum analysis time. Optimal separation of the five compounds under study is achieved in less than 12 min using a Sulpecosil LC-ABZ+Plus C18 column, a composition of mobile phase of acetonitrile-10mM acetic acid acetate buffer pH 5 (72:28, v/v) at a flow rate of 1 mL/min, a column temperature of 30 degrees C +/- 0.1 degrees C, and a detection wavelength of 238 nm.

Cardiovascular drug determination in bioanalysis: an update.

The great impact of cardiovascular diseases in human health has led to the development of a huge number of drugs and therapies to improve the treatment of these diseases. Cardiovascular drug analysis in biological fluids constitutes an important challenge for analytical scientists. There is a clear need for reliable methods to carry out both qualitative and quantitative analysis in a short time of analysis. Different problems such as drug monitoring, analysis of metabolites, study of drugs interactions, drugs residues or degradation products, chiral separation, and screening and confirmation of drugs of abuse in doping control must be solved. New trends in sample preparation, instrumental and column technology advances in LC and innovations in MS are described in this work.

Simultaneous determination of five 1,4-dihydropyridines in pharmaceutical formulations by high-performance liquid chromatography-amperometric detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of five 1,4-dihydropyridines: amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine. These drugs are widely used in the treatment of hypertension, angina pectoris and the therapy of cerebrovascular spasms of various origins. The chromatographic separation was performed on a Supelcosil LC ABZ + Plus C18 column with a mobile phase consisting of acetonitrile-10 mM acetate buffer (72:28, v/v) at a flow rate of 1 ml/min. The temperature was set at 30 +/- 0.2 degrees C. The amperometric detector, equipped with a glassy carbon electrode was operated at +1100 mV versus Ag/AgCl in the direct current mode. Under these chromatographic conditions, the drugs eluted in less than 12 min. The method showed to be linear over the range 4.5-15 microg/ml with a within-day and day-to-day repeatabilities in terms of R.S.D. lower than 15%, an accuracy greater than 98% and detection limits varying from 90 ng/ml (amlodipine) to 1.55 microg/ml (nitrendipine). The method was successfully applied to commercially available pharmaceuticals with relative errors lower than 5%. The validity of the method was examined comparing the results obtained with those of HPLC with photometric detection.

Square wave voltammetric determination of the angiotensin-converting enzyme inhibitors cilazapril, quinapril and ramipril in pharmaceutical formulations.

The angiotensin-converting enzyme inhibitors cilazapril, quinapril and ramipril are reduced at a hanging mercury drop electrode in the pH range 3.5-13 using Britton-Robinson buffers as supporting electrolyte and KCl as ionic medium. Square wave voltammetry has proved to be the most suitable electroanalytical technique for the quantitative voltammetric determination of these antihypertensive drugs. Optimisation of the chemical and instrumental variables was carried out. Analyses were performed in 0.02 M borate buffer at pH 9.5 and 0.5 M KCl as ionic medium, using a pulse amplitude of 50 mV and a frequency of 150 Hz. A linear relationship between peak current and concentration was found in the interval 0.5-8 microg/ml for cilazapril and up to 6 microg/ml for quinapril and ramipril, allowing the direct determination of their pharmaceutical formulations alone or mixed with hydrochlorothiazide. Good accuracy and repeatativity were obtained.

Bioanalytical chromatographic method validation according to current regulations, with a special focus on the non-well defined parameters limit of quantification, robustness and matrix effect.

Method validation is a mandatory step in bioanalysis, to evaluate the ability of developed methods in providing reliable results for their routine application. Even if some organisations have developed guidelines to define the different parameters to be included in method validation (FDA, EMA); there are still some ambiguous concepts in validation criteria and methodology that need to be clarified. The methodology to calculate fundamental parameters such as the limit of quantification has been defined in several ways without reaching a harmonised definition, which can lead to very different values depending on the applied criterion. Other parameters such as robustness or ruggedness are usually omitted and when defined there is not an established approach to evaluate them. Especially significant is the case of the matrix effect evaluation which is one of the most critical points to be studied in LC-MS methods but has been traditionally overlooked. Due to the increasing importance of bioanalysis this scenario is no longer acceptable and harmonised criteria involving all the concerned parties should be arisen. The objective of this review is thus to discuss and highlight several essential aspects of method validation, focused in bioanalysis. The overall validation process including common validation parameters (selectivity, linearity range, precision, accuracy, stability…) will be reviewed. Furthermore, the most controversial parameters (limit of quantification, robustness and matrix effect) will be carefully studied and the definitions and methodology proposed by the different regulatory bodies will be compared. This review aims to clarify the methodology to be followed in bioanalytical method validation, facilitating this time consuming step.

Quantitative determination of the calcium channel antagonists amlodipine, lercanidipine, nitrendipine, felodipine, and lacidipine in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometric method has been developed and validated for the quantification of the five 1,4-dihydropyridine calcium channel antagonists amlodipine, lercanidipine, nitrendipine, felodipine, and lacidipine in human plasma. Sample preparation involved solid-phase extraction on RP-C18 cartridges with good recovery for all the compounds. Sample analysis was performed on a Luna RP-C18 analytical column (15 mm x 2 mm ID, 3.0 microm) with a Sciex API 365 triple quadrupole mass spectrometer with turboionspray source and multiple reaction monitoring. The method is sensitive with a limit of detection below 1 ng/mL for each drug in plasma, with good linearity (r(2) > 0.998), over the therapeutic concentration range (1 to 40 ng/mL). All the validation data, such as accuracy, precision, and interday repeatability, were within the required limits. The method can be used for pharmacokinetic studies and therapeutic drug monitoring of the compounds in humans.

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists.

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.

Separation of the high-ceiling diuretic Torasemide and its metabolites by capillary zone electrophoresis with diode-array detection.

A capillary zone electrophoretic method was developed for the separation of the high-ceiling loop diuretic Torasemide and three of its metabolites (M1, M3 and M5) using an experimental design approach. Two different experimental designs were employed to optimize the developed method: (i) a fractional factorial design examining six factors at two levels (2(6-2)) and (ii) a central composite design examining two factors at two levels (2(2)+2x2+p). The factors studied were: pH, buffer concentration, proportion of boric acid in the mixed boric acid:potassium dihydrogen phosphate background electrolyte, temperature, applied voltage and percentage of organic modifier. Resolution between peaks was established as response. Separation of the four studied compounds was achieved in less than 8 min, using an electrolyte of 20 mM boric acid:potassium dihydrogen phosphate (75:25 v/v) with 15% MeOH adjusted to pH 9.7 with KOH, at a potential of 28 kV. Detection wavelength and temperature were 206 nm and 35 degrees C, respectively.