Oskar Gonzalez

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.

LC-MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma

A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150mm×4.6mm, 3μm) column using a gradient elution mode with a run time of 15min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68-106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.

Development of an LC-MS/MS method for the quantitation of 55 compounds prescribed in combined cardiovascular therapy

This paper reports an LC-MS/MS method with positive electrospray ionization for the screening of commonly prescribed cardiovascular drugs in human plasma, including compounds with antihypertensive (57), antidiabetic (12), hypolipemiant (5), anticoagulant (2) and platelet anti-aggregation (2) effects. Sample treatment consisted of a simple protein precipitation with MeOH/0.1 M ZnSO₄ (4:1, v/v) solution after the addition of internal standard, followed by evaporation and reconstitution. Analytes separation was performed on a Polar-RP column (150 m x 2 mm, 4 μm) using a gradient elution of 15 min. The MS system was operated in MRM mode, monitoring one quantitation and one confirmation transition for each analyte. The recovery of the protein precipitation step ranged from 50 to 70% for most of the compounds, while some were considerably affected by matrix effects. Since several analytes fulfilled the linearity, accuracy and precision values required by the ICH guidelines, the method proved to be suitable for their quantitative analysis. The limits of quantitation varied from 0.38 to 9.1 μg/L and the limits of detection from 0.12 to 5.34 μg/L. The method showed to be suitable for the detection of plasma samples of patients under cardiovascular treatment with the studied drugs, and for 55 compounds reliable quantitative results could be obtained.

Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma.

This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm x 3.9 mm, 3 microm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31-3000 microg/l for chlorthalidone, 20-1000 microg/l for valsartan-M1, 10-5000 microg/l for valsartan and 14-1000 microg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78-91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment.

Validation of a fast liquid chromatography-UV method for the analysis of drugs used in combined cardiovascular therapy in human plasma.

Ultra-performance liquid chromatography (UPLC) was investigated as a faster alternative to high-performance liquid chromatography (HPLC) for the simultaneous analysis of drugs usually prescribed in cardiovascular therapy. Upon a previously developed and validated solid phase extraction (SPE)-HPLC-photodiode array (PDA)-fluorescence (FLR) method, separation of chlorthalidone (CLTD; diuretic), valsartan and its metabolite (VAL and VAL-M1 respectively; angiotensin II receptor antagonist drugs) and fluvastatin (FLUV; statin) was performed in human plasma using an RP C18 column (50mmx2.1mm, 1.7microm, Waters Acquity UPLC (BEH)) and a tunable UV-vis (TUV) detector. After method transfer, different system variables were modulated to study the evolution of responses of the analytes and the endogenous interferences. The improved method was fully validated and the results were compared with its precursor HPLC method relating to analysis time, efficiency and sensitivity. The studied compounds were separated in less than 8min and the method showed good linearity (20-3000microg/L for chlorthalidone, 110-1100microg/L for valsartan-M1, 67-1900microg/L for valsartan and 48-1100microg/L for fluvastatin), precision and accuracy. The proposed method was found to be reproducible (RSD<10%), accurate (RE<15%), robust and suitable for quantitative analysis of the studied drugs in plasma obtained from patients under combined cardiovascular treatment.

Performance Modeling of Fresnel‐Based CPV Systems: Effects of Deformations under Real Operation Conditions

Getting a lower energy cost has always been a challenge for concentrated photovoltaic. The FK concentrator enhances the performance (efficiency, acceptance angle and manufacturing tolerances) of the conventional CPV system based on a Fresnel primary stage and a secondary lens, while keeping its simplicity and potentially low‐cost manufacturing. At the same time F‐XTP (Fresnel lens+reflective prism), at the first glance has better cost potential but significantly higher sensitivity to manufacturing errors. This work presents comparison of these two approaches applied to two main technologies of Fresnel lens production (PMMA and Silicone on Glass) and effect of standard deformations that occur under real operation conditions.

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.

Quantitative determination of dobutamine in newborn pig plasma samples by HPLC–MS/MS

HighlightsIon‐pairing formation with HFBA and extraction with EtOAc was the optimal pig plasma sample treatment for the determination of dobutamine.A simple, selective and sensitive HPLC‐MS/MS method was developed and fully validated for the quantification of dobutamine in pig plasma samples. Abstract A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry (HPLC/MS‐MS) was performed as a method for the determination of dobutamine hydrochloride (DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine (TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for the treatment of plasma samples. The separation of dobutamine and internal standard was done using a Kinetex F5 (50 × 2.1 mm, 2.6 &mgr;m, 100 Å) analytical column. The mobile phase was a mixture of acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow rate was 0.25 mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in ESI+, using a cone voltage (CV) of 25 V and a collision energy (CE) of 25 eV. The weighted calibration curve (1/x2) was found to be linear over the concentration range of 1–100 ng/mL (r2 > 0.999). The limit of quantification (LLOQ) of the method was 1 ng/mL. The values of selectivity, carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.

Metabolomic analysis for the study of maturation in pediatrics: Effect of confounding factors in a pilot study

A pilot study for the investigation of the maturation grade of children has been carried out using plasma samples already analyzed in a previous pharmacokinetic study. By using a meticulous data treatment, possible confounding factors that may hinder the obtained results were identified. By doing so, it was possible to obtain enough evidence to support the feasibility of performing a larger study eluding some unwanted variability and minimizing not only the number of subjects involved but also the time and money spent on the study. In the pilot study the metabolic profiles obtained using UHPLC‐TOF‐MS technique of plasma samples from 14 newborn piglets (<5 days) were compared with the plasma profiles of 16 infant piglets (8 weeks). The type of anaesthesia administered, gender, vein or artery of blood extraction and time of sampling were studied as possible confounding factors. Unsupervised analysis by principal component analysis (PCA) clearly differentiated between neonates and children. During the data treatment and the statistical analysis, the effect of confounding factors such as the anaesthetic regimen was identified and removed, while the effect of the rest of studied factors was not considered relevant, and the discrimination between the two groups based on the age was maintained. This allowed extracting relevant conclusions for a future study design while avoiding the unnecessary sacrifice of animals. Furthermore, the results obtained demonstrate the utility of metabolomics in the discovery of novel putative plasma biomarkers such as carnitines that can be correlated with the maturation state of paediatric patients.