Current Pharmaceutical Analysis - Volume 10, Issue 3, 2014

Tyrosine kinase inhibitors (TKIs) are a class of targeted drugs with antiangiogenic and antitumor activities. Due to inter-individual metabolic variability, an accurate therapeutic drug monitoring (TDM) represents a key element for the patient treatment. Here a fast and easily accessible method for the quantification of 3 TKIs (with one active metabolite) in human plasma after extraction is described. Sample pre-treatments were performed by solid phase extraction (Oasis^® MCX μElution technology). Chromatographic separation was performed on a Waters Acquity UPLC^® system with diode array detection (DAD) using a gradient of ammonium formate-acetonitrile on BEH C18 2.1x50 mm column. The analytical methods were validated by using the accuracy profiles approach (β-expectation set at 95%). The methods were successfully validated for sunitinib (10 – 250 ng/mL), N-desethyl sunitinib (15 – 250 ng/mL), axitinib (15 – 250 ng/mL) and pazopanib (20 – 200 μg/mL). The first concentration levels validated were considered as limit of quantification (LOQ). The validated method will be used in a clinical research study to determine TKI plasma levels and in this way help physicians to optimize the posology in order to achieve the best therapeutic response for their patients.

We developed and validated a rapid hydrophilic interaction chromatography (HILIC) method using a core-shell column in a conventional HPLC to assess the dissolution profile of rivastigmine hydrogen tartrate (RHT, 6 mg) from hydrophilic matrix controlled-release tablets. The dissolution profile was determined from 10-360 min in a USP dissolution apparatus II using water at 37 °C at 50 rpm. The chromatographic separation was performed over 5 min using a Kinetex^® (100 x 4.6 mm, 2.6 μm) HILIC column and an isocratic mobile phase consisting of 80:20 acetonitrile:10 mM pH 5.8 ammonium acetate buffer at 30 ºC. The flow rate was 2.0 mL min-1 and the detection wavelength was 217 nm. The method was linear over a range of 1-30 μg mL^-1. The intra- and inter-day precision was < 4.0% RSD for the assay and < 15% RSD for the dissolution test. The methods exhibited good recovery (> 90% in the assay and > 95.0% in the dissolution test). The separation method tolerated small variations in flow and temperature. These techniques may be used as a rapid and accurate assay of RHT in hydrophilic matrixes, and to assess the dissolution profile of the drug in modified pharmaceutical dosage form.

Protein phosphorylation is one of the most important post-translational modifications involved in the regulation of many cellular processes and highly efficient analytical tools are required to enrich, purify and characterize phosphoproteins. However, there are only a few analytical approaches involving phosphoprotein enrichment. The presented study describes the introduction of trivalent cerium, holmium and thulium cations for the selective co-precipitation of phosphorylated proteins with an excess of phosphate anions. Rare earth metal ions are known to be hard acceptors with a strong preference for oxygen-containing anions such as phosphates with which they form very tight ionic bonds. The high selectivity of this method can be explained by the high number of free coordination sites of rare earthmetal ions, with eight and nine being the most common coordination numbers. This could be shown for the specific precipitation of caseins from standards, bovine milk, spiked HeLa cell lysate, and human saliva samples using matrix-assisted laser desorption/ ionization mass spectrometry. Phosphoproteins were specifically precipitated by CeCl3, HoCl3 and TmCl3 in the presence of phosphate. Separation from non-phosphorylated proteins was carried out using an enhanced protocol and yielded stable pellets. Stringent washing steps were performed to remove interacting proteins and tryptic digestion on-pellet allowed the recovery of phosphopeptides. Quantification was carried out by colorimetric detection using the BCA assay which showed that the amount of applied precipitant is highly dependent on the precipitation recovery. More than 95% protein recovery could be achieved for the applied rare earth metal ions.

A new simple and highly sensitive HPLC-UV method for the analysis of paclitaxel in Taxol^® and Abraxane^® has been developed which also allows the quantification in biological samples of pharmacokimnetic studies with a very easy sample preparation. The proposed method has advantages over previously reported methods, such as an increase in sensitivity up to 20 times, good accuracy in terms of recovery that allows to work without addition of internal standard and a simple sample preparation in one step. The analysis was carried out using a Zorbax Eclipse XDB-C18 (4.6 × 150 mm, 3.5 μm) with a column guard. The mobile phase consisted of acetonitrile: water (50: 50) and the flow rate was 1 mL/min, the detection wavelength was 227 nm, the injection volume 5 μL and the analysis was performed at room temperature. The method was validated according ICH guidelines and FDA guidelines for validation of bioanalytical methods. Linearity was evaluated in the range from 0.005 – 50.0 μg/mL, with a regression coefficient (R2) of 0.9967. LOD and LOQ were 0.001 and 0.005 μg/mL respectively.

This work aims to develop and validate a UPLC stability indicating method for quantitative analysis of Caspofungin, grounded in statistical analysis able to estimate measurement uncertainty, and determine drug degradation kinetics. The validation was performed as recommended in ICH guidelines. The chromatographic method was conducted applying the following conditions: 220 nm wavelength, 10 μL injection volume, 25 °C oven temperature, 0.6 mL/minute flow, and gradient elution involving mobile phases A and B, composed of 0.1 % solution of triethylamine pH adjusted to 2.5 with the aid of phosphoric acid and acetonitrile, respectively. The validation of the UPLC method demonstrated the selectivity, robustness, linearity, precision, accuracy and the ability to indicate caspofungin stability. Statistical analysis demonstrated the method is linear in the concentration range between 20 and 80 μg/mL (y= 30908912.1196x – 11246.7143; r= 0.9997), precise (repeatability: 1.09%, intermediate precision: 1.79%) and accurate (recovery range: 95.61–101.36%). According to these results, the uncertainties associated with accuracy and precision were the most significant, contributing to 57% overall uncertainty. On the other hand, repeatability of sample and standard peak areas were almost insignificant (less than 5% of overall uncertainty). The analysis of measurement uncertainty proved to be useful for validating the results and a very useful tool in routine analysis. The degradation kinetics were calculated using the UPLC method, which is a validated and well-established technique, capable of indicating thermal stability (80 °C) of caspofungin in solution. The drug kinetics of degradation showed a first order reaction (y = - 0.0078 x + 2.0168, r = 0.9993) and half-life (t1/2) estimated at 3.9-minute analysis under the conditions tested, indicating that caspofungin is strongly sensitive to heat stress condition.

The present work proposes a simple, accurate and low-cost method employing simultaneous detection (conductometric and potentiometric) during a precipitation titration using silver nitrate as titrant for diltiazem hydrochloride (DTZ) determination. Both methods were based on the chemical reaction between chloride ions coming from diltiazem hydrochloride molecule and Ag(I) ions, yielding the precipitate AgCl(s). The conductance of the solution and the electric potential of the cell (using an Ag-wire electrode) were measured as a function of the volume of titrant. Under optimized experimental conditions the method was applied for DTZ determination in pharmaceuticals and the results obtained are in close agreement with those obtained using an official method proposed by the British Pharmacopoeia.

In this work, the development of flow system that is simple, precise and accurate for determining fluoride in mouthwash is described. The determination of fluoride was based on the on-line formation of a Fe(III)–salicylate complex, which is consumed in the presence of fluoride, generating the colourless hexafluoroferrate(III) complex ion, [FeF6]³-. The proposed procedure exhibits a linear concentration range of 8.0 × 10^-5–1.2 × 10^-3 mol L^-1, a detection limit of 3.5 x 10^-5 mol L^-1, a higher sampling frequency of 44 h^-1 and relative standard deviations below 0.2%. The interference of the red colour of the samples was removed using waste-seed specimens of Mabea fistulifera Mart. as a biosorbent material.

Nitazoxanide, 2- [(5-nitro-1, 3-thiazol-2-yl) carbamoyl] phenyl] ethanoate (NTZ) is a new broad-spectrum, antiparasitic drug. The acid, alkaline and hydrogen peroxide oxidative degradation were studied in order to analyze nitazoxanide in the presence of its degradation products using high performance liquid chromatographic assay. The liquid chromatographic separation was achieved using two columns namely the solid core Poroshell 120 EC-C18 (4.6x 100mm, i.d. particle size, 2.7μm) and the fluorinated Pursuit 5PFP (4.6x150mm, i.d. particle size, 5μm), and a mobile phase composed of acetonitrile: water (90:10 v/v, pH 2.5 using glacial acetic acid and triethylamine) delivered at a flow rate 0.3mL min-1. The analyte(s) were detected using a UV detector set at 320 nm. The linearity of the proposed method was in the range of 20-200 μg mL^-1 (r=0.9996) and (r=0.9998) using Poroshell 120 EC-C18 and Pursuit 5PFP columns, respectively. The proposed methods were validated in terms of accuracy, selectivity, sensitivity and precision for the analysis of nitazoxanide in bulk form and in pharmaceutical formulations. The degradation products produced from the forced stress tests studies did not interfere with the analysis of nitazoxanide. This indicates that the proposed methods are stability- indicating assays and are suitable for the determination of nitazoxanide in quality control laboratories.

Proteomic approaches posses a huge potential in understanding diseases such as cancer. These analyses rely on the extraction of proteins from clinical specimens and their subsequent enzymatic digestion in peptides prior to mass spectrometric analysis. The characterisation of chosen subproteomes – like phosphoproteome – which are rich reservoirs of potential biomarkers for disease, requires much more sophisticated procedures due to the additional phosphoproteomic sample preparation steps. The addition of sophisticated analytical procedures is mandatory but make the parallel processing of clinical samples prone to plenty of errors and, thus compromise the reproducibility needed for confident comparative high-throughput studies. The presented review focuses on relevant developments in the phosphoproteomic field that could, in the near future, facilitate the rapid discovery of tumour biomarkers or new drug targets for cancer treatment. In terms of methodological advances, the enrichment of phosphoproteins and mass spectrometry detection of peptides will be especially highlighted. The discussion part will also contribute to new areas of interest for scientists that are investigating phosphoproteins.