Ann Van Eeckhaut

Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects.

Liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry is currently the method of choice for the quantitative determination of drugs in biological matrices. The advantages of this technique include high specificity, sensitivity and throughput. However, co-eluting matrix components, which are not observed in the chromatogram, can have a detrimental effect on the analysis, since they can cause ion suppression or enhancement of the analyte. The evaluation of matrix effects on the quantitative analysis of drugs in biological fluids is an important and sometimes overlooked aspect of assay validation. In this review, the influence of matrix effects on bioanalytical LC-MS/MS methods is discussed and illustrated with some examples. In addition, possible solutions to reduce or eliminate matrix effects are highlighted. A literature overview of validated LC-MS/MS methods published from January till June 2008 is also included. Although matrix effects are investigated in most papers, there is no consensus on how matrix effects should be evaluated during method validation. In addition, the definition of specificity should be changed for LC-MS/MS based methods.

The absolute quantification of endogenous levels of brain neuropeptides in vivo using LC–MS/MS

Neuropeptides seem to play an important role when the CNS is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano-LC-MS/MS methods. A number of LC-MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. The optimized and validated methods can be used to investigate the in vivo neuropeptide release during pathological conditions, in this way initiating new and immense challenges for the development of new drugs.

Strategies to reduce aspecific adsorption of peptides and proteins in liquid chromatography-mass spectrometry based bioanalyses: an overview.

In the drug-discovery setting, the development of new peptide and protein-based biopharmaceuticals attracts increased attention from the pharmaceutical industry and consequently demands the development of high-throughput LC-MS methods. Regulatory guidelines require bioanalytical methods to be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity and stability, but also in terms of carryover. Carryover results from the aspecific adsorption of analyte(s) to parts of the analytical system and thus introduces bias in both identification and quantification assays. Moreover, nonspecific binding occurs at the surface of materials used during sample preparation, such as pipette tips, sample tubes and LC-vials. Hence, linearity, sensitivity and repeatability of the analyses are negatively affected. Due to the great diversity in physicochemical properties of biomolecules, there is no general approach available to minimize adsorption phenomena. Therefore, we aim to present different strategies which can be generically applied to reduce nonspecific binding of peptides and proteins. In the first part of this review, a systematic approach is proposed to guide the reader through the different solvents which can be used to dissolve the analyte of interest. Indeed, proper solubilization is one of the most important factors for a successful analysis. In addition, alternative approaches are described to improve analyte recovery from the sample vial. The second part focuses on strategies to efficiently reduce adsorption at components of the autosampler, column and mass spectrometer. Thereby carryover is reduced while maintaining a sufficiently wide dynamic range of the assay.

Pressor and Renal Hemodynamic Effects of the Novel Angiotensin A Peptide Are Angiotensin II Type 1A Receptor Dependent

Recently, a new derivative of angiotensin (Ang) II, called “Ang A,” has been discovered to be present in plasma of healthy humans and, in increased concentrations, in end-stage renal failure patients. The objectives of the study were to investigate the blood pressure and renal hemodynamic responses to Ang A in normotensive and hypertensive rats and in genetically modified mice and the binding properties of Ang A to Ang II type 1 (AT1) or Ang II type 2 (AT2) receptors. Intravenous and intrarenal administration of Ang A induced dose-dependent pressor and renal vasoconstrictor responses in normotensive rats, which were blocked by the AT1 receptor antagonist candesartan but were not altered by the AT2 receptor ligands PD123319, CGP42112A, or compound 21. Similar responses were observed after intravenous administration in spontaneously hypertensive rats. Deletion of AT1a receptors in mice almost completely abolished the pressor and renal vasoconstrictor responses to Ang A, indicating that its effects are mediated via AT1a receptors. Ang A was less potent than Ang II in vivo. The in vitro study demonstrated that Ang A is a full agonist for AT1 receptors, with similar affinity for AT1 and AT2 receptors as Ang II. Overall, the responses to Ang A and Ang II were similar. Ang A has no physiological role to modulate the pressor and renal hemodynamic effects of Ang II.

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.

Development of a validated capillary electrophoresis method for enantiomeric purity testing of dexchlorpheniramine maleate

A capillary zone electrophoresis method has been developed for the detection of 0.1% of (R)-levochlorpheniramine maleate in samples of (S)-dexchlorpheniramine maleate. Using 1.5 mM carboxymethyl-beta-cyclodextrin in an acidic background electrolyte, resolution values of more than 10 were obtained. Under these conditions the R-enantiomer is migrating in front of the bulk S-enantiomer. The assay was validated for linearity (2-10 microg/ml; R2 = 0.9992), selectivity [(RS)-pheniramine maleate and (RS)-brompheniramine maleate], limit of detection (0.25 microg/ml), limit of quantification (0.75 microg/ml), analytical precision (intra- and inter-day variability), repeatability of the method (RSD = 5.0%) and accuracy. In samples of dexchlorpheniramine maleate from two different manufacturers, concentrations of, respectively, 0.15% and 1.95% (m/m) of levochlorpheniramine maleate were detected. The method was compared to the HPLC method described in the European Pharmacopoeia III monograph.

Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT2 receptor agonist, to the striatum in rats

A microbore liquid chromatography method coupled to UV detection was developed and validated in order to monitor the passage of compound 21 (C21), a non-peptide angiotensin II type 2 receptor agonist, to the striatum of rats. For this purpose, sampling from the striatum was performed using the in vivo microdialysis technique. Separations were performed on a C₁₈ microbore (1mm i.d.) column using gradient elution. The retention time for C21 was found to be 6.3 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient ≥ 0.999. The limit of detection and the limit of quantification were 3 and 10 ng/ml respectively. The intra-day and the inter-day precision (RSD%) ranged between 0.5 and 4.6% with an average recovery of 101.5±10.0% (mean±SD, n=15). In vivo experiments were performed on rats to measure the concentration of C21 in striatal dialysates after intraperitoneal (10 or 50mg/kg) or intravenous injection (10 mg/kg or 20 mg/kg) of C21 and suggest minimal passage of the compound to the striatum.

Development and evaluation of a linear regression method for the prediction of maximal chiral separation of basic drug racemates by cyclodextrin-mediated capillary zone electrophoresis

An important step in method development of chiral separations with neutral cyclodextrins (CDs) as chiral selectors is the estimation of the CD concentration that gives the highest degree of separation. From the equation [S]opt=1/(K1K2)(1/2) this optimal CD concentration can be calculated if any knowledge is available about the binding constants K1 and K2 of both enantiomer complexes. These values can be obtained by measuring the effective velocities of each enantiomer as a function of the selector concentration and fitting these profiles by non-linear least-square regression. An alternative approach has been developed which makes it possible to predict the optimal CD concentration from a few experiments performed at low CD concentrations. The model is developed using some antimycotic imidazole derivatives (econazole, miconazole and isoconazole) as test substances and hydroxypropyl-beta-CD as chiral selector. The results obtained by this method are in good agreement with those from non-linear least-square regression.

Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection

Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column (1 mm internal diameter), conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM (mean ± SD, n = 30). The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.

An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates

AIM An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. RESULTS Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. CONCLUSION The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.