Analyzing Alkyl Bromide Genotoxic Impurities in Febuxostat Based on Static Headspace Sampling and GC-ECD.

Herein, a sensitive and selective gas chromatography-electron capture detector (GC-ECD) method was developed and validated for the quantification of trace levels of five bromo-containing genotoxic impurities in Febuxostat active pharmaceutical ingredient (API) after headspace sampling (HS). Multivariate experimental designs for the optimization of static headspace parameters were conducted in two stages using fractional factorial design (FFD) and central composite design (CCD). The optimum headspace conditions were 5 min of extraction time and a 120 °C extraction temperature. Baseline separation on the analytes against halogenated solvents was carried out using an Agilent DB-624 (30 m × 0.32 mm I.D., 1.8 µm film thickness) stationary phase under isothermal conditions. The method was validated according to ICH guidelines in terms of specificity, linearity, the limits of detection and quantification, precision and accuracy. The linearity was assessed in the range of 5-150% with respect to the specification limit. The achieved LOD and LOQ values ranged between 0.003 and 0.009 and 0.01 and 0.03 µg mL-1, respectively. The accuracy of the method (expressed as relative recovery) was in the range of 81.5-118.2%, while the precision (repeatability, inter-day) was less than 9.9% in all cases. The validated analytical protocol has been successfully applied to the determination of the impurities in various Febuxostat API batch samples.

A floating 3D printed polypill formulation for the coadministration and sustained release of antihypertensive drugs.

Polypharmacy is a common issue, especially among elderly patients resulting in administration errors and patient inconvenience. Hypertension is a prevalent health condition that frequently leads to polypharmacy, as its treatment typically requires the co-administration of more than one different Active Pharmaceutical Ingredients (API's). To address these issues, floating hollow torus-shaped dosage forms were developed, aiming at providing prolonged gastric retention and sustained drug release. The dosage forms (polypills) containing three anti-hypertensive API's (diltiazem (DIL), propranolol (PRP) and hydrochlorothiazide (HCTZ)) were created via Fused Deposition Modelling 3D printing. A multitude of the dosage forms were loaded into a capsule and the resulting formulation achieved prolonged retention times over a 12-hour period in vitro, by leveraging both the buoyancy of the dosage forms, and the "cheerios effect" that facilitates the aggregation and retention of the dosage forms via a combination of surface tension and shape of the objects. Physicochemical characterization methods and imaging techniques were employed to investigate the properties and the internal and external structure of the dosage forms. Furthermore, an ex vivo porcine stomach model revealed substantial aggregation, adhesion and retention of the 3D printed dosage forms in porcine stomach. In vitro dissolution testing demonstrated almost complete first-order release of PRP and DIL (93.52 % and 99.9 %, respectively) and partial release of HCTZ (65.22 %) in the 12 h timeframe. Finally, a convolution-based single-stage approach was employed in order to predict the pharmacokinetic (PK) parameters of the API's of the formulation and the resemblance of their PK behavior with previously reported data.

Chemometric Optimization of SPE for the Determination of Multiclass Pesticides in Portable Water Samples by UHPLC-MS/MS.

This study aimed on the development of a SPE-UHPLC-MS/MS method for the simultaneous determination of pesticide residues in drinking water samples. A chemometric approach was applied to optimize the efficiency of the SPE pretreatment procedure. This study involved (i) the application of a Full Factorial Design for the screening of the significant factors, (ii) the application of a Central Composite Design for the determination of the optimal conditions and (iii) the evaluation and validation of the significance of the statistically proposed models. Oasis HLB cartridges were used for the extraction. The optimum sample volume was 300 mL and the elution solvent 3 mL of the mixture of methanol:ethylacetate 70:30 v/v. The method was validated according to the international guidelines. Recoveries were ranged from 63 to 116% and the detection limits were between 0.1 and 1.5 pg mL- 1. The validated method could be used in routine analysis for pesticides screening.

Utilization of a pH-switchable hydrophilicity solvent for the microextraction of clomipramine from human urine samples.

Clomipramine (CLP) is a tricyclic antidepressant drug, and its determination in biological samples is of high importance in clinical and forensic evaluations to assure appropriate drug concentrations. In the present study, benzoic acid was employed as a pH-switchable hydrophilicity solvent (SHS) for the microextraction of CLP from authentic human urine samples prior to its determination by high performance liquid chromatography-ultraviolet detection (HPLC-UV). The microextraction protocol was based on the phase transition of the SHS through pH alteration that resulted in its rapid dispersion and simultaneous phase separation. The obtained solid was collected in a syringe filter, dissolved in methanol, and analyzed. The main parameters that affected the efficiency of the microextraction procedure were studied and optimized to ensure high extraction efficiency for CLP and the analytical method was validated. Under optimum conditions, good linearity was observed between 0.05 and 5.0 µg mL-1. The limit of detection and limit of quantification were found to be 0.015 and 0.05 µg mL-1, respectively. The RSD values for intra-day repeatability and inter-day precision were 2.4-8.9 % and 1.7-9.1 %, respectively. The relative recovery values were within 90.0 and 110.0 % in all cases, demonstrating good method accuracy. The proposed SHS microextraction showed cost-efficiency, handling simplicity, and rapidity resulting in enhanced sample throughput. Moreover, the proposed method exhibited a green character and good applicability based on its evaluation by Green Analytical Procedure Index and Blue Applicability Grade Index.

Sol-gel Carbowax 20M-zwitterionic ionic liquid composite sorbent-based capsule phase microextraction device combined with HPLC/post-column derivatization for the determination of lanreotide, a human somatostatin analogue in urine.

In this research, a sol-gel Carbowax 20M-zwitterionic ionic liquid composite sorbent-based capsule phase microextraction (CPME) device was developed in combination with liquid chromatography-post column derivatization for the first ever reported determination of a somatostatin analogue - lanreotide in human urine. The sol-gel Carbowax 20M-zwitterionic ionic liquid composite sorbent was encapsulated in the lumen of a polypropylene capillary tube and characterized by FT-IR spectroscopy and SEM with energy dispersive X-ray spectroscopy (EDS). The main steps of the CPME workflow were optimized to obtain high extraction efficiency for the target analyte. After the separation of the analyte on a C8 stationary phase, the peptide was derivatized online with o-phthalaldehyde before the fluorescence detection. The main experimental parameters of CPME and the post-column procedures were systematically investigated and optimized. The method was validated in terms of selectivity, linearity, accuracy, precision, limits of detection (LOD), and limits of quantification (LOQ). The relative bias ranged between 88.8 and 115.6 % for the peptide, while the RSD values for repeatability and intermediate precision were less than 14.3 %. The achieved limit of detection (LOD) was 0.2 µΜ while the limit of quantitation (LOQ) was established as 0.9 µΜ. Finally, the sol-gel Carbowax 20M-zwitterionic ionic liquid composite sorbent-based microextraction capsules were found to be reusable for at least 20 extractions. The developed method presented adequate overall performance, and it could be applied in the analysis of selected peptide in human urine samples.

Fabricating a designer capsule phase microextraction platform based on sol-gel Carbowax 20M-zwitterionic ionic liquid composite sorbent for the extraction of lipid-lowering drugs from human urine samples.

A sol-gel Carbowax 20 M/3-[(3-Cholamidopropyl) dimethyl ammonio]-1-propanesulfonate composite sorbent-based capsule phase microextraction device has been fabricated and characterized for the determination of four statins (pravastatin, rosuvastatin, pitavastatin, and atorvastatin) in human urine. The presence of ionizable carboxyl functional groups in statins requires pH adjustment of the sample matrix to ensure that the target molecules are in their protonated form (pH should be 2 units below their pKa values) which not only is cumbersome but also risks unintended contamination of the sample. This challenge was addressed by introducing zwitterionic ionic liquid in addition to neutral, polar Carbowax 20 M polymer in the sol-gel-derived composite sorbent. As such, the composite zwitterionic multi-modal sorbent can simultaneously extract neutral, cationic, and anionic species. This particular attribute of the composite sorbent eliminates the necessity of the matrix pH adjustment and consequently simplifies the overall sample preparation workflow. Various experimental parameters such as the sample amount, extraction time, salt addition, stirring rate, and elution solvent type that may affect the extraction performance of the statins were investigated using a central composite design and the one-parameter-at-a-time approach. The analytes and the internal standard were separated on a C18 column with gradient elution using phosphate buffer (20 mM, pH 3) and acetonitrile as mobile phase. The analytes were detected at 237 nm. The method was validated, and linearity was observed in the range 0.10-2.0 µg mL-1 for all compounds. The method precision was better 9.9% and 10.4% for intra-day and inter-day, respectively, while the relative recoveries were acceptable, ranging between 83.4 and 116% in all cases. Method greenness was assessed using the ComplexGAPI index. Finally, the method's applicability was demonstrated in the determination of the statins in authentic human urine after oral administration of pitavastatin and rosuvastatin-containing tablets.

Smartphone-Based High-Throughput Fluorimetric Assay for Histidine Quantification in Human Urine Using 96-Well Plates.

A high-throughput fluorimetric assay for histidine was developed, using a 96-well plates platform. The analyte reacts selectively with o-phthalaldehyde under mild alkaline conditions to form a stable derivative. Instrumental-free detection was carried out using a smartphone after illumination under UV light (365 nm). The method was proved to be linear up to 100 µM histidine, with an LLOQ (lower limit of quantification) of 10 µM. The assay was only prone to interference from glutathione and histamine that exist in the urine samples at levels that are orders of magnitude lower compared to histidine. Human urine samples were analyzed following minimum treatment and were found to contain histidine in the range of 280 to 1540 µM. The results were in good agreement with an HPLC corroborative method.

A review of bioanalytical applications of microextraction techniques combined with derivatization.

Microextraction techniques have attracted the attention of many researchers working in the field of bioanalysis due to their unique advantages, mainly in downsizing the scale of sample preparation steps. In parallel, analytical derivatization offers a powerful combination in terms of additional sensitivity, selectivity and compatibility with modern separation techniques. The aim of this review is to discuss the most recent advances in bioanalytical sample preparation based on the combination of microextraction and analytical derivatization. Both innovative fundamental reports and analyte-targeted applications are included and discussed. Dispersive liquid-liquid extraction and solid-phase microextraction are the most common techniques that typically combined with derivatization, while the development of novel and greener protocols is receiving substantial consideration in the field of analytical chemistry.

In vitro and in silico computational methods for assessing vaginal permeability.

OBJECTIVE: Vaginal administration is an important alternative to the oral route for both topical and systemic use. Therefore, the development of reliable in silico methods for the study of drugs permeability is becoming popular in order to avoid time-consuming and costly experiments. METHODS: In the current study, Franz cells and appropriate HPLC or ESI-Q/MS analytical methods were used to experimentally measure the apparent permeability coefficient (Papp) of 108 compounds (drugs and non-drugs). Papp values were then correlate with 75 molecular descriptors (physicochemical, structural, and pharmacokinetic) by developing two Quantitative Structure Permeability Relationship (QSPR) models, a Partial Least Square (PLS) and a Support Vector Machine (SVM). Both were validated by internal, external and cross-validation. RESULTS: Based on the calculated statistical parameters (PLS model A: R2 = 0.673 and Q2 = 0.594, PLS model B: R2 = 0.902 and Q2 = 0.631, SVM: R2 = 0.708 and Q2 = 0.758). SVM presents higher predictability while PLS adequately interprets the theory of permeability. CONCLUSIONS: The most important parameters for vaginal permeability were found to be the relative PSA, logP, logD, water solubility and fraction unbound (FU). Respectively, the combination of both models could be a useful tool for understanding and predicting the vaginal permeability of drug candidates.

Investigating the applicability of polar fabric phase sorptive extraction for the HPLC quantitation of salivary vitamin B12 following administration of sublingual tablets and oral sprays.

In this study, a simple and rapid fabric phase sorptive extraction (FPSE) protocol combined with high performance liquid chromatography-ultraviolet detection (HPLC-UV) was developed for the monitoring of salivary vitamin B12 levels. Different sol-gel coated cellulose and polyester membranes were evaluated and sol-gel Carbowax 20 M coated polyester membranes were chosen for the selective extraction of the target analyte from saliva samples. Face-centered central composite design (FC-CCD) was employed for the investigation and optimization of sample volume, extraction time and stirring rate, while the other experimental factors were investigated using the classical one-factor-at-a- time" (OFAT) method. Validation of the FPSE-HPLC-UV method was conducted according to the FDA guidelines for bioanalytical methodologies. The lower limit of quantification for vitamin B12 was 0.10 µg mL-1 and the linear range was 0.10-10.0 µg mL-1. The relative recoveries for intra-day and inter-day studies were 87.5-113.8% and 88.2-119.2%, respectively. The relative standard deviation was better than 8.2% in all cases, demonstrating good method precision. The sol-gel Carbowax 20 M coated FPSE membranes were found to be reusable for up to 25 times. Finally, the proposed scheme was successfully employed for the quantitation of salivary vitamin B12 at different time points following the administration of sublingual tablets and oral sprays.

Development and evaluation of pulsed - Post column derivatization in liquid chromatography as a concept to minimize reagent consumption.

In the current article, we propose an alternative approach to reduce the consumption of the reagents in liquid chromatography coupled to on-line post column derivatization. In our proposal post column reagents do not flow continuously but they are instead introduced as well-defined pulses (at microliter levels) that are merged on-line with the eluted analytes through precise tuning (Pulsed-Post Column Derivatization, Pulsed-PCD). The profiles of the pulses in terms of time and flow rate were investigated "visually" using caffeine as model compound (at 274 nm). The robustness of the procedure was evaluated by Monte Carlo simulations and was verified taking into account the precisions of typically used propulsion systems. As a proof of concept, we selected the determination of histidine in human urine after separation by cation exchange chromatography and Pulsed-PCD derivatization with o-phthalaldehyde. The consumption of the derivatizing reagent was downscaled to the microliter level per run, while the analytical results were within the expected ranges (110 - 1520 µmol L-1) and with good agreement with the corroborative method based on classic HPLC-PCD.

Determination of Free Histidine in Complex Hair Care Products with Minimum Sample Preparation Using Cation-Exchange Chromatography and Post Column Derivatization.

In this communication, we describe the first analytical method for the determination of free histidine in hair care products (shampoos and conditioners). Cation-exchange chromatography combined with postcolumn derivatization and fluorimetric detection enabled the accurate (recovery: 83.5-114.8%) and precise (2.4-5.6% RSD) determination of free histidine without matrix interferences at concentration levels down to 1.5 mg kg-1. Real commercially available samples were found to contain the amino acid at levels ranging between 70 and 535 mg kg-1.

Analytical quality by design approach for the determination of imidazole in sildenafil API and its formulations using zwitterionic HILIC stationary phase.

Herein, the development of a HILIC method for the determination of imidazole (Imp E) in sildenafil citrate API and its final formulations is reported. The main goal of this study was to develop a robust, application-specific HPLC method according to the Analytical Quality by Design principles for the analysis of the above impurity. After the risk assessment study, the high-risk method parameters were sequentially screened and optimized by using 2-level fractional factorial and Box-Behnken designs. The mathematical models were combined with the Monte-Carlo simulations to identify the Method Operable Design Region. The method was thoroughly validated between 25 % and 150 % of the target concentration limit of the imidazole using the total-error concept. The relative bias varied between 1.6 % and 5.6 % and the RSD values were lower than 5.8 % for repeatability and intermediate precision. The limit of detection and the lower limit of quantification were satisfactory and found to be 0.025 and 0.125 µg mL-1 imidazole, respectively. The applicability of the proposed approach has been demonstrated in the analysis of several sildenafil citrate API batches and final products.

A simple and green liquid chromatography method for the determination of ibuprofen in milk-containing simulated gastrointestinal media for monitoring the dissolution studies of three-dimensional-printed formulations.

A fast and green ultra-high-performance liquid chromatographic method was developed for the determination of ibuprofen in milk-containing simulated gastrointestinal media to monitor the dissolution of three-dimensional printed formulations. To remove interfering compounds, protein precipitation using methanol as a precipitation reagent was performed. The separation of the target analyte was performed on a C18 column using a mobile phase consisting of 0.05% v/v aqueous phosphoric acid solution: methanol, 25:75% v/v. Method validation was conducted using the total error concept. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results will be included in the defined bias limits. The relative bias ranged between ─1.1 and +3.2% for all analytes, while the relative standard deviation values for repeatability and intermediate precision were less than 2.8% and 3.9%, respectively. The achieved limit of detection was 0.01 µg/ml and the lower limit of quantitation was established as 2 µg/ml. The proposed method was simple, and it required reduced organic solvent consumption following the requirements of Green Analytical Chemistry. The method was successfully employed for the determination of ibuprofen in real biorelevant media obtained from dissolution studies.

Designing an "all-in-one" microextraction capsule device for the liquid chromatographic-fluorescence determination of doxorubicin and its metabolites in rat plasma.

In this study, an "all-in-one" microextraction device was designed and fabricated for the extraction of doxorubicin and its two metabolites from rat plasma prior to their determination by high performance liquid chromatography coupled to fluorescence detector. A sol-gel-based sorbent was synthesized in situ and incorporated within two conjoined porous polypropylene tubes together with a cylindrical magnetic bar in order to avoid the need of an external stirring bar. Among other sorbents investigated, the moderately polar sol-gel poly(tetrahydrofuran) was found to be advantageous due to its high affinity toward the target analytes. Systematic investigation of the critical parameters affecting the adsorption and the desorption step was carried out. Due to the "built-in" filtration mechanism of the porous microextraction capsules, the isolation of the analytes was performed directly in the plasma matrix without any previous sample pretreatment (i.e., protein precipitation, centrifugation, etc.). The proposed method was validated in terms of linearity, accuracy, precision, specificity, sensitivity, and stability according to the FDA guidelines. The limits of detection ranged between 1 - 2 ng mL-1 while the lower limits of quantitation of the analytes were calculated as 10 ng mL-1. The accuracy (% relative error) was found within -9.7 - 15.3% under both intra- and inter-day conditions. The precision was better than 13.4% in all cases. ComplexGAPI index was employed to present the green attributes of the developed protocol from the preparation of the microextraction device to the final determination of the analytes. Finally, the applicability of the fabricated stand-alone extraction device was demonstrated in the analysis of the target analytes in rat plasma after intravenous administration of doxorubicin in order to assess its pharmacokinetic profile.

Determination of bisphosphonate active pharmaceutical ingredients in pharmaceuticals and biological materials: An updated review.

Although almost 60 years have passed since their first application, bisphosphonates are still in use as medicine against osteoporosis. Due to their chemical structures and properties, these compounds have attracted the attention of many scientists. From an analytical point of view, various analytical methods have been published in the last decade for the determination of these drugs involving separation techniques (HPLC, GC, CE), electrochemical, sensors, spectrophotometry, IR, etc. The present article is a continuation of the 2008 review article of the authors on the analysis of bisphosphonates (C.K. Zacharis, P.D. Tzanavaras, JPBA 48 (2008) 483-496) and it focuses on bioanalytical and pharmaceutical QC applications on the analysis of this class of pharmaceutically active compounds presenting a critical discussion on advantages/disadvantages, figures of merit and analytical features of techniques and methods on this topic.

Development and Validation of an HPLC-UV Method for the Dissolution Studies of 3D-Printed Paracetamol Formulations in Milk-Containing Simulated Gastrointestinal Media.

Herein, a simple and rapid HPLC method for the determination of paracetamol milk-containing biorelevant media is proposed. The separation of the analyte from the milk-containing biorelevant media was accomplished isocratically using a mobile phase containing 25 mM phosphate buffer (pH = 3.0) and methanol, 80:20, v/v at a flow rate of 1 mL min-1. Following a protein precipitation-based sample clean-up, a thorough investigation of the effect of the precipitation reagent (methanol, acetonitrile, 10% v/v trifluoroacetic acid solution) on the analyte recovery was performed. The matrix effect was assessed in each biorelevant medium by comparing the slopes of the calibration curves of aqueous and matrix-matched calibration curves. The method was comprehensively validated using the accuracy profiles. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results will be included in the defined bias limits. The relative bias ranged between -4.5 and +3.9% for all analytes, while the RSD values for repeatability and intermediate precision were less than 2.7% and 3.0%, respectively. The achieved limit of detection (LOD) was 0.02 µg mL-1 and the lower limits of quantitation (LLOQ) were established as 10 µg mL-1, which corresponded to 2% of the highest expected concentration of paracetamol. The proposed scheme was utilized for the determination of paracetamol in dissolution studies of its 3D-printed formulation in milk-containing biorelevant media.

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core-shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box-Behnken experimental design, and the method was validated using the total error concept. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L-1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of -2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.

Single-Step Hydrolysis and Derivatization of Homocysteine Thiolactone Using Zone Fluidics: Simultaneous Analysis of Mixtures with Homocysteine Following Separation by Fluorosurfactant-Modified Gold Nanoparticles.

Herein, we report a new automated flow method based on zone fluidics for the simultaneous determination of homocysteine and homocysteine thiolactone using fluorimetric detection (λext = 370 nm/λem = 480 nm). Homocysteine thiolactone is hydrolyzed on-line in alkaline medium (1 mol L−1 NaOH) to yield homocysteine, followed by reaction with o-phthalaldehyde in a single step. Derivatization is rapid without the need of elevated temperatures and stopped-flow steps, while specificity is achieved through a unique reaction mechanism in the absence of nucleophilic compounds. Mixtures of the analytes can be analyzed quantitatively after specific separation with fluorosurfactant-capped gold nanoparticles that are selectively aggregated by homocysteine, leaving the thiolactone analogue in solution. As low as 100 nmol L−1 of the analyte(s) can be quantified in aqueous solutions, while concentrations > 2 µmol L−1 can be analyzed in artificial and real urine matrix following 20-fold dilution. The percent recoveries ranged between 87 and 119%.

Cereal-Based 3D Printed Dosage Forms for Drug Administration During Breakfast in Pediatric Patients within a Hospital Setting.

In an effort to combine a child-friendly dosage form for medication administration in hospitalized pediatric patients and a user-friendly automated process for its preparation by health-care providers, the current study proposes a method for drug administration with breakfast using semi-solid extrusion 3D printing. Cereal was used as the platform carrier of the hydrophobic ibuprofen and the hydrophilic paracetamol to develop the drug loaded cereal ink. Rheological analysis was performed to identify the cereal ink with optimum viscosity for extrusion printing. Drug distribution and crystallinity within the printed cereal were assessed with confocal Raman microscopy and thermal and X-ray diffraction analysis, respectively, indicating molecular dispersion of both drugs within the cereal. High cereal porosity was associated with a higher milk absorption capacity and a decrease in their flexural force upon immersion in milk. Dissolution studies were performed in biorelevant media under fasted and fed state conditions and in the presence of full-fat and low-fat milk showing dissolution enhancement of the poorly soluble ibuprofen in the presence of the higher fat content milk. Concealing drug administration under the auspice of this essential daily eating habit is expected to facilitate overcoming adherence barriers to medication intake by pediatric patients within a hospital setting.