Blood represents a very important biological fluid and has been the target of continuous and exte... more Blood represents a very important biological fluid and has been the target of continuous and extensive research for diagnostic, or health and drug monitoring reasons. Recently, metabonomics/metabolomics have emerged as a new and promising 'omics' platform that shows potential in biomarker discovery, especially in areas such as disease diagnosis, assessment of drug efficacy or toxicity. Blood is collected in various establishments in conditions that are not standardized. Next, the samples are prepared and analyzed using different methodologies or tools. When targeted analysis of key molecules (e.g., a drug or its metabolite[s]) is the aim, enforcement of certain measures or additional analyses may correct and harmonize these discrepancies. In omics fields such as those performed by holistic analytical approaches, no such rules or tools are available. As a result, comparison or correlation of results or data fusion becomes impractical. However, it becomes evident that such obstacles should be overcome in the near future to allow for large-scale studies that involve the assaying of samples from hundreds of individuals. In this case the effect of sample handling and preparation becomes very serious, in order to avoid wasting months of work from experts and expensive instrument time. The present review aims to cover the different methodologies applied to the pretreatment of blood prior to LC-MS metabolomic/metabonomic studies. The article tries to critically compare the methods and highlight issues that need to be addressed.
The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the com... more The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.
A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of... more A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.
The analysis of amino acids presents significant challenges to contemporary analytical separation... more The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed-phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous-organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.
Journal of Liquid Chromatography & Related Technologies, 2000
ABSTRACT A rapid, accurate, and sensitive method has been developed for the quantitative determin... more ABSTRACT A rapid, accurate, and sensitive method has been developed for the quantitative determination of iodoamino acids, namely thyroxine (3,5,3′,5′-tetra-iodothyronine, (T4) and 3,5,3′-tri-iodothyronine (T3). These compounds are essential indicators in the clinical diagnosis of thyroid gland diseases.An Inertsil ODS-3, 150 × 4.0 mm, 5 μm analytical column was used with a mixture of CH3OH-H2O with 2% acetic acid, at a volume ratio 65:35, with a flow rate 1 mL/min. Detection was performed with a variable wavelength UV-visible detector at 240 nm, resulting in detection limits of 1 ng and 2 ng for T3 and T4, respectively, per 20 μL injection.For the quantitative determination, anthraquinone was used as internal standard at a concentration of 1.0 ng/μL. A rectilinear relationship was observed up to 28 and 40 ng/μL for T3 and T4, respectively.Analysis time was approximately 10 min (retention time of internal standard) while the two compounds are eluted within 5 min. The statistical evaluation of the method was examined, performing intra-day (n = 8) and inter-day calibration (n = 8) and was found to be satisfactory with high accuracy and precision results.The method was applied to the analysis of the iodothyronines in biological fluids, blood serum and urine, after solid phase extraction for sample clean-up and analyte retention, using diol cartridges. Percentage recovery of iodothyronines in spiked samples ranged from 79.90 to 103.15 for T3 and from 83.65 to 106.15 for T4, over the range of 0.5–3 ng/μL.No interferences were observed from endogenous compounds of human serum and urine.
Blood represents a very important biological fluid and has been the target of continuous and exte... more Blood represents a very important biological fluid and has been the target of continuous and extensive research for diagnostic, or health and drug monitoring reasons. Recently, metabonomics/metabolomics have emerged as a new and promising 'omics' platform that shows potential in biomarker discovery, especially in areas such as disease diagnosis, assessment of drug efficacy or toxicity. Blood is collected in various establishments in conditions that are not standardized. Next, the samples are prepared and analyzed using different methodologies or tools. When targeted analysis of key molecules (e.g., a drug or its metabolite[s]) is the aim, enforcement of certain measures or additional analyses may correct and harmonize these discrepancies. In omics fields such as those performed by holistic analytical approaches, no such rules or tools are available. As a result, comparison or correlation of results or data fusion becomes impractical. However, it becomes evident that such obstacles should be overcome in the near future to allow for large-scale studies that involve the assaying of samples from hundreds of individuals. In this case the effect of sample handling and preparation becomes very serious, in order to avoid wasting months of work from experts and expensive instrument time. The present review aims to cover the different methodologies applied to the pretreatment of blood prior to LC-MS metabolomic/metabonomic studies. The article tries to critically compare the methods and highlight issues that need to be addressed.
The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the com... more The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.
A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of... more A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.
The analysis of amino acids presents significant challenges to contemporary analytical separation... more The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed-phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous-organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.
Journal of Liquid Chromatography & Related Technologies, 2000
ABSTRACT A rapid, accurate, and sensitive method has been developed for the quantitative determin... more ABSTRACT A rapid, accurate, and sensitive method has been developed for the quantitative determination of iodoamino acids, namely thyroxine (3,5,3′,5′-tetra-iodothyronine, (T4) and 3,5,3′-tri-iodothyronine (T3). These compounds are essential indicators in the clinical diagnosis of thyroid gland diseases.An Inertsil ODS-3, 150 × 4.0 mm, 5 μm analytical column was used with a mixture of CH3OH-H2O with 2% acetic acid, at a volume ratio 65:35, with a flow rate 1 mL/min. Detection was performed with a variable wavelength UV-visible detector at 240 nm, resulting in detection limits of 1 ng and 2 ng for T3 and T4, respectively, per 20 μL injection.For the quantitative determination, anthraquinone was used as internal standard at a concentration of 1.0 ng/μL. A rectilinear relationship was observed up to 28 and 40 ng/μL for T3 and T4, respectively.Analysis time was approximately 10 min (retention time of internal standard) while the two compounds are eluted within 5 min. The statistical evaluation of the method was examined, performing intra-day (n = 8) and inter-day calibration (n = 8) and was found to be satisfactory with high accuracy and precision results.The method was applied to the analysis of the iodothyronines in biological fluids, blood serum and urine, after solid phase extraction for sample clean-up and analyte retention, using diol cartridges. Percentage recovery of iodothyronines in spiked samples ranged from 79.90 to 103.15 for T3 and from 83.65 to 106.15 for T4, over the range of 0.5–3 ng/μL.No interferences were observed from endogenous compounds of human serum and urine.
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Papers by H. Gika