Dennis Bernieh

LC-HRMS analysis of dried blood spot samples for assessing adherence to cardiovascular medications

Research suggests that ~60% of patients prescribed cardiovascular drugs do not take their medication correctly. The analysis of dried blood samples (DBS) by liquid chromatography-high resolution mass spectrometry (LC-HRMS) for assessing medication adherence to candidate therapeutic drugs used in cardiovascular therapy was investigated. Specificity using this analytical method was based on the measurement at the accurate mass to charge ratio of the target analyte. To evaluate the method 8mm discs were punched from each DBS and extracted followed by subjecting to LC-HRMS analysis. Trials on 6 commonly UK used cardiovascular drugs are reported demonstrating the ability of the system to detect the target analytes during the 24 hour repeat prescription cycle. Samples from volunteers with confirmed adherence were used to validate the response from the system as were samples from volunteers receiving no medication. No false positives were observed and adherence assessment for bisoprolol, ramipril, amlodipine, valsartan, doxasozin and simvastatin was demonstrated using the LC-HRMS method. Furthermore examples of incorrect adherence were identified.

Quantitative LC–HRMS determination of selected cardiovascular drugs, in dried blood spots, as an indicator of adherence to medication

HighlightsThe first LC–HRMS method for the simultaneous quantification of the top ten UK prescribed cardiovascular drugs in dried blood spots developed.A micro‐analytical method for monitoring medication adherence.Validation showed good linearity, accuracy and precision for the ten target drugs.Drug recoveries, sample stability and matrix effects were investigated.The validated method successfully identified control volunteers who were known to be either adherent or non‐adherent. Abstract Dried blood spot (DBS) sampling was investigated as a means of obtaining micro‐volume blood samples for the quantitative analyses of ten commonly UK prescribed cardiovascular drugs as an indicator of medication adherence. An 8 mm disc was punched out from each DBS from calibration, quality control and volunteer samples and extracted using methanol containing the internal standard. Each extract was evaporated to dryness, the residue reconstituted in methanol:water (40:60 v/v) containing 0.1% formic acid and analysed by LC–HRMS. Chromatography was performed using gradient elution on a Zorbax Eclipse C18 HD 100 mm × 2.1 mm, 1.8 &mgr;m pore size column with the column oven temperature at 40 °C. Flow rate of the mobile phase was 0.6 ml/min with a run time of 2.5 min. Electrospray positive ionization was used for MS detection. Drug recoveries from spiked blood spots were 68% for simvastatin and ≥87% for all other target drugs. Compound specificity was obtained operating the MS with a 5 ppm mass window. The LC–HRMS method was validated, with results for accuracy and precision within acceptable limits; analytes were stable at room temperature for at least 10 weeks and different blood spot volumes and haematocrit values had no significant effect. The LC–HRMS assay was used to analyse DBS samples from volunteers, some of whom were prescribed one or more of the target drugs. In results from 37 volunteers the assay successfully identified volunteers who were known to be either adherent or nonadherent; confirmed the correct drug/drugs for multiple prescriptions; demonstrated no false positives from other cardiovascular drugs; revealed several examples of unsuspected non‐adherence. These results indicated that the developed assay was suitable for trials with patients.

Quantitative dried blood spot analyses: An aid to medicine optimization for heart disease patients

A (AML) and atorvastatin (ATV) were subjected to acidic hydrolysis and their degradation products were separated and their structures were confirmed. A simple stability indicating isocratic reversed phase high performance liquid chromatographic method was developed and validated for the simultaneous determination of AML and ATV in presence of their hydrolytic degradation products. The proposed RP-HPLC method utilizes Agilent Zorbax® ODS 5 um, 4.6 x 250 mm column, at ambient temperature, mobile phase consisting of acetonitrile:methanol:phosphate buffer solution (45: 30: 25 by volume), pH adjusted to 2.5±0.1 with orthophosphoric acid, at a flow rate of 1.0 mL/min and UV detection at 254 nm. The proposed method was valid in the range of 0.8–30 μg/mL (r=0.9999) for AML and 0.4-30 μg/mL (r=1.0000) for ATV. The degradation products did not interfere with the determination of both drugs and the assay can thus be considered stability-indicating. The proposed method was validated according to ICH guidelines and successfully applied to the estimation of AML and ATV in bulk powder and pharmaceutical dosage forms.Introduction: Over 355 million prescriptions were dispensed for cardiovascular diseases in the UK in 2013. Half of these, costing the NHS £2.3 billion, were wasted because patients do not take their medicines as prescribed. A method using dried blood spot (DBS) sample collection followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated for quantification of eleven commonly UK prescribed cardiovascular drugs: amlodipine, atenolol, atorvastatin, bisoprolol, diltiazem, doxazosin, lisinopril, losartan, ramipril, simvastatin and valsartan. Thus medication efficiency, adherence or drug/drug interactions can be assessed from reference pharmacokinetic data.B (2-bromo-2-nitropropane-1,3-diol) is an effective preservative employed in cosmetic formulations. However, under specific conditions, bronopol can decompose releasing low levels of formaldehyde and nitrites. These last compounds can react with any secondary amines or amides to produce significant levels of carcinogenic nitrosamines. The production of nitrosamines in cosmetic products has to be avoided. For this reason, although the use of bronopol is authorized in the current European Regulation, the maximum allowed concentration of bronopol in cosmetic products is 0.1%. Nevertheless, there is still no official method of analysis for the determination of this compound. The aim of this work is to propose a fast and simple analytical method to determine bronopol in cosmetic products. The proposed method is based on a vortex-assisted emulsification extraction, as one-step solution extraction process to prepare cosmetic samples, followed by liquid chromatography with spectrophotometric detection at 250 nm. The best results were achieved by using a C18 column at the following optimized conditions: Temperature, 40oC; Flow rate, 0.5 mL min-1; mobile phase, ethanol and a 1% acetic acid aqueous solution were used to perform the separation by elution gradient. Satisfactory results were obtained for the analysis of 19 cosmetic samples including creams, shampoos and bath gels, with good recoveries and repeatability. Limits of detection and quantification were at the low Bg mL−1 levels. These good analytical features, as well as its environmentally-friendly characteristics, make the presented method suitable for the determination of bronopol in cosmetic products.T growing social concern about health and beauty has encouraged in recent years a remarkable increase in the use of cosmetic products. Because of this widespread use, it is necessary to carry out adequate quality controls to ensure not only the effectiveness but also the safety in users. In this respect, European legislation on cosmetic products includes a list of compounds whose use in cosmetic products is banned. A clear example of banned compounds that can be found in cosmetic at trace levels are the so-called N-nitrosamines, since mutagenic, carcinogenic and teratogenic activity has been attributed to these compounds. Their presence in cosmetic products it could be attributed to unintentional causes. Thus, these compounds are formed with relative ease from an amine (either secondary or tertiary) and a nitrosating agent such as nitrite or oxides of nitrogen. Among N-nitrosamines found in cosmetics, N-Nitroso-Diethanolamine (NDELA) is undoubtedly the most typical one. Thus, the development of reliably analytical methods is highly encouraged. The aim of this work is to develop an analytical method for the determination of NDELA in cosmetic products, by exploiting the high potential of the Reversed-Phase Dispersive Liquid-Liquid Micro-Extraction (RP-DLLME) as cleanup and enrichment technique for highly polar and water-soluble compounds as NDELA. In contrast to conventional DLLME, in this approach a small volume of water is dispersed into a bulk organic solution containing the analytes as extraction solvent. The main parameters involved in RP-DLLME were optimized and the method was successfully validated in terms of linearity, precision and accuracy.A hybrid development strategy of Quality by design (QbD) and one factor at time (OFAT) approaches was used to develop a stability indicating HPLC method for quantitative determination of cefditoren pivoxil (CTP) in bulk powder and pharmaceutical formulations. A forced degradation studies were performed under acid, alkaline, thermal and photolytic stress conditions. Chromatographic separation achieved in less than 10 min. using a RP C-18 column, mobile phase [methanol: acetate buffer pH 4.5 (55:45, v/v)], flow rate 1.5 mL min-1 and UV detection at 225 nm. Optimization of column, pH, and wavelength implemented according to OFAT approach, while elution temperature and methanol content in the mobile phase considering QbD approach. The method was validated to meet official requirements including specificity, linearity, precision, accuracy and robustness. The drug response was linear (r=0.9999) in range of 89-672 μg mL-1, the limit of detection (LOD) and limit of quantitation (LOQ) were 5.31 μg mL-1 and 16.1 μg mL-1, respectively. The intraand inter-day precisions were 0.11%, 0.44% respectively. The proposed method was successfully applied for the determination of CTP in bulk and tablets with acceptable accuracy and precisions. The obtained results demonstrated that the proposed method has a great value for application in quality control labs and stability studies for CTP.C shell technology is gaining importance in HPLC analyses due to economic, fast and reproducible nature. Core shell columns are called as new generation columns. Various brand names are available with Sunshell, Halo, Ascentis Express, Poroshell 120, Kinetex, Accucore and Nucleoshell trade names. These columns provide ultra fast HPLC separations for a variety of phramaceuticals with moderate sample loading, capacity and low back pressure. Chemistries of these columns are C8, C18, RP Amide, Hilic, PFP, Phenyl, and RP-aqua as well WP C 18. Silica particles are of 2.6 and 1.6 μm as total and inner solid core diameters with 0.5 μm thickness of outer porous layer; having 90 A pore sizes and 150 m2/g surface areas. The proposed lecture will high light the importance of new generation columns with special emphasis on their textures and chemistries, separations, optimization and comparison (inter and intra stationary phases) and economy. Besides, future perspectives will also be discussed.N (NIL) is used to treat a type of blood cancer called Philadelphia chromosome positive chronic myeloid leukemia. Carbamazepine (CBZ) is used to treat seizures, nerve pain and bipolar disorder. The important drug interactions may occur when used in combination of carbamazepine with Nilotinib. Nilotinib is metabolized by CYP3A4. CBZ shows a strong CYP3A4 inducer feature. A simple, rapid and accurate high performance liquid chromatography method for the simultaneous determination of NIL and CBZ in their tablets were developed and validated. The developed method was applied to in vitro drug interaction studies in different pH environments, simulating empty and full stomach juice (pH 1 and 4), intestinal juice (pH 9), and blood pH (7.4) at 37oC. The method employs an isocratic elution using water and acetonitrile (30:70 v/v) and an RP-C18 HPLC column. Flow-rate was maintained at 1 mL/min, and the eluent monitored using a UV photo diode array detector at 281 nm. The calibration curves were linear over the concentration ranges of 0.0125-5 μg/mL for both drugs. For the interaction studies, in the presence of NIL, availability of CBZ was increased in all interaction medium. The increases of availability of CBZ in different pH medium from most to least were pH 7.4 > pH 4 > pH 9 > pH 1.2 > pH 6.8. For the application of NIL with CBZ, the availability of NIL increased mostly in pH 4 and moderate in pH 1.2 and pH 6.8. The availability of NIL showed decrease in pH 7.4 and 9.F therapeutic to lifestyle medicines, the counterfeiting of medicines has been on the rise in recent times. Estimates indicate that about 10% of medicines worldwide are counterfeits with much higher figures in developing countries. Currently, the rapid screening of medicines is a challenge leaving many patients at risk. This study considered the potential use of Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) for rapid quantitative analysis of tablet formulations. ATR-FTIR requires minimal sample preparation as it only requires crushing the tablet before analysis unlike the conventional methods where time-consuming solvent extraction of the Active Pharmaceutical Ingredient (API) is necessary. Reference spectra for pure API and excipients were recorded as part of a reference library for identification purposes. Preliminary studies were carried out with tablets having a single API (Paracetamol). API could be identified down to 5% w/w of the tablet. Tablet samples with multiple APIs were also identified. For quantitative analysis, IR spectra of standard mixtures of Paracetamol in excipient were recorded and used in calibration. Paracetamol tablets from Europe, Africa and Southeast Asia were then quantified based on calibration data. Quantification data for selected characteristic peak areas for each API/excipient mixture was linear. Results were in the expected range with conventional UV analysis confirming data obtained. Therefore, ATR-FTIR can be applied in the rapid identification and quantification of tablet formulations. The faster sampling time, simplicity and portability of ATR-FTIR makes it valuable in the authentication of medicines especially in developing countries where facilities are not readily available.O useful information about the chemical composition of samples containing multiple components still is a great challenge for analysts. One approach to the analysis of very complex samples of which there are no standards is by obtaining fingerprints, for which several chromatographic techniques are useful. In this work, we use high performance liquid chromatography. A chromatographic fingerprint is just a chromatogram that shows multiple peaks of different heights corresponding to the components of the samples. Samples with similar fingerprints possess similar nature and probably a common origin. Therefore, fingerprints show potential interest to determine the identity, authenticity and consistency between batches of medicinal herbs. An ideal assessment for fingerprint quality should include the number of visible peaks and their resolution. In this work, a strategy is reported to measure the level of information through the concept of peak prominence, developed in our laboratory. Peak measurements are ranked and the results are used to summarize the information level in the fingerprint, obtaining a numerical evaluation. Fractional factorial designs were performed to measure the impact of several extraction conditions (e.g. solvent nature and composition, temperature, time of extraction, time and temperature of storage, etc.), by processing the chromatograms of the extracts. The gradient program was optimized to maximize the chemical information. The methodology was applied to green tea samples.Various types of monoclonal antibody (mAb) products including intact mAbs, mAb fragments, engineered variants, and antibody-drug conjugates (ADCs) are being developed for the treatment of cancer and other diseases due to their excellent biocompatibility and high selectivity. The proliferation of monoclonal antibody therapeutics and their susceptibility to various biochemical modifications has highlighted the importance of characterizing these highly heterogeneous products for their safety and efficacy.