Analytical QbD for the optimization of a multimode HPLC method for the investigation of hydrochlorothiazide, diltiazem and propranolol release from 3D printed formulation.

Since 3D printing technology is an emerging field in pharmaceutical technology, the present study aimed at the development of a mixed-mode liquid chromatographic method for the separation and determination of hydrochlorothiazide, diltiazem, and propranolol to investigate their in-vitro release performance from 3D printed tablets. Due to the unique properties of the mixed-mode stationary phase, the three drugs were separated in less than 8 min under isocratic elution. Method development was accomplished following the Analytical Quality by Design principles and was evaluated using risk assessment and multivariate analysis. The influences of critical method parameters on critical method attributes (were screened using a 2-level fractional factorial design and subsequently optimized through a central composite design. The method operable design region was approved by the establishment of a robust zone using Monte Carlo simulation and capability analysis. The validation of the HPLC method was performed based on the total error concept. The relative bias was varied between ─ 11.6 % and 10.5 % and the RSD values for repeatability and intermediate precision were below 4.4 % in all cases. The limits of detection (LOD) ranged between 0.17 - 0.90 µg/mL and were adequate for the specific application. The developed method was successfully applied to the analysis of the studied drugs in in-vitro drug release samples obtained from 3D-printed tablets combining the above-mentioned active pharmaceutical ingredients (APIs).

O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements.

Herein, a new, direct paper-based fluorimetric method is described for the quantitative determination of glutathione (GSH) molecules in nutritional supplements. Briefly, the proposed analytical method is based on the fluorescence emission resulting from the direct and selective chemical reaction of GSH molecules with the derivatization reagent that is o-phthalaldehyde (OPA) in acidic conditions at room temperature. The intensity of the emitted fluorescence on the surface of the analytical paper devices after irradiation with a lamp at 365 nm is proportional to the concentration of GSH and is measured using a smartphone as the detector. This methodology, which is suitable for measurements in laboratories with limited resources, does not require specialized instrumentation or trained personnel. The protocol governing the proposed method is simple and easily applicable. Essentially, the chemical analyst should adjust the value of pH on the surface of the paper by adding a minimal amount of buffer solution; then, after adding a few microliters of the derivatization reagent, wait for the surface of the paper to dry and, finally, add the analyte. Subsequently, the irradiation of the sensor and the measurement of the emitted fluorescence can be recorded with a mobile phone. In the present study, several parameters affecting the chemical reaction and the emitted fluorescence were optimized, the effect of interfering compounds that may be present in dietary supplements was examined, and the stability of these paper sensors under different storage conditions was evaluated. Additionally, the chemical stability of these paper devices in various maintenance conditions was studied, with satisfactory results. The detection limit calculated as 3.3 S/N was 20.5 µmol L-1, while the precision of the method was satisfactory, ranging from 3.1% (intra-day) to 7.3% (inter-day). Finally, the method was successfully applied to three different samples of dietary supplements.

High-throughput and green optical sensing of thiocyanate in human saliva based on microplates and an overhead book scanner as detector.

An instrumental-free, high-throughput assay has been developed for the quantification of thiocyanate in human saliva. The proposed green method is based on the rapid reaction of the analyte with Fe(III) under acidic pH in a microplates format to form a colored complex that is captured as an image by an overhead book scanner. Optimization included the effects of the amount concentration of Fe(III), acidity and reaction time / complex stability using a total volume of 300 µL per well. Validation towards the matrix effect was focused on the specific application and was performed using both artificial and human saliva. The linearity of the developed assay was up to 500 µM thiocyanate offering a lower limit of quantification (LLOQ) of 30 µM. The green potentials were evaluated by both the Green Analytical Procedure (GAPI) and Blue Applicability Grade (BAGI) indexes. The thiocyanate content in the saliva of non-smoking volunteers ranged between 750 and 1350 µΜ, while elevated concentrations were verified in smoking individuals (1860-3080 µΜ). Statistical agreement with a corroborative method was assessed using the Bland-Altman plot.

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.

High performance liquid chromatography coupled with post - Column derivatization methods in food analysis: Chemistries and applications in the last two decades.

High performance liquid chromatography coupled with post-column derivatization is used for increasing the sensitivity and selectivity of the desirable analytes after the chromatographic separation. The transformation of the analytes can be conducted through the addition of a suitable reagent in the eluted stream or the ultraviolet irradiation of the eluted analytes, forming detectable derivatives for ultraviolet or fluorescence detectors. This review focuses on the developed methods using high performance liquid chromatography coupled with post-column derivatization for the determination of substances in food samples during the last two decades. The significance of the determination of each analyte in foods and the existing guidelines in each case are discussed. Preparation of the samples and the analytical methods are commented. For each analyte, official methods and commercially available systems and reagents are mentioned, as well.

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.

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.

Developments in on-line, post separation sample manipulation in the last 22 years: Pharmaceutical and biomedical applications.

On-line post separation sample manipulation is a powerful approach increasing the sensitivity and selectivity in chemical analysis. Post separation sample manipulation includes the treatment of the analytes after their separation through a suitable separation technique, mainly liquid chromatography and capillary electrophoresis. Typically, post separation approaches include either the addition of a reagent/solvent to derivatize the analyte/enhance the sensitivity, pH change, or the conversion of the analyte through a photochemical/electrochemical system (reagent-free systems). This review focuses on the developed methods using post-column manipulation of sample with pharmaceuticals and biomedical applications, covering the period from 2000 to midle-2023. Chemistries combined with fluorescence, UV-vis and mass spectrometric detection are discussed employing both liquid chromatography and electrophoretic techniques for separation. Noteworthy instrumental modifications are also discussed.

A Novel Equipment-Free Paper-Based Fluorometric Method for the Analytical Determination of Quinine in Soft Drink Samples.

A simple, equipment-free, direct fluorometric method, employing paper-based analytical devices (PADs) as sensors, for the selective determination of quinine (QN) is described herein. The suggested analytical method exploits the fluorescence emission of QN without any chemical reaction after the appropriate pH adjustment with nitric acid, at room temperature, on the surface of a paper device with the application of a UV lamp at 365 nm. The devices crafted had a low cost and were manufactured with chromatographic paper and wax barriers, and the analytical protocol followed was extremely easy for the analyst and required no laboratory instrumentation. According to the methodology, the user must place the sample on the detection area of the paper and read with a smartphone the fluorescence emitted by the QN molecules. Many chemical parameters were optimized, and a study of interfering ions present in soft drink samples was carried out. Additionally, the chemical stability of these paper devices was considered in various maintenance conditions with good results. The detection limit calculated as 3.3 S/N was 3.6 mg L-1, and the precision of the method was satisfactory, being from 3.1% (intra-day) to 8.8% (inter-day). Soft drink samples were successfully analyzed and compared with a fluorescence method.

Naphthalene-2,3-dicarboxaldehyde as a pulsed-post column derivatization reagent; comparison with two alternative o-phthalaldehyde based chemistries for the determination of histamine.

In the present study, naphthalene-2,3-dicarboxaldehyde (NDA) was used in on-line post column derivatization (PCD) coupled to liquid chromatography under the new concept of Pulsed-PCD. In Pulsed-PCD, the reagents are introduced into the flowing stream of the mobile phase under precise timing overlapping the eluted analyte. The consumption of the reagents is minimized to a few microliters, resulting in a significant advantage, that is the use of expensive reagents in PCD. For this reason, NDA-CN chemistry was used for the determination of histamine in food samples, such as eggplant and spinach. Two additional methods were developed based on the reaction of histamine with o-phthalaldehyde (OPA), namely the classic OPA - nucleophilic compound reaction and the specific OPA - histamine reaction in alkaline medium. The chromatographic conditions and the Pulsed-PCD conditions were investigated, while the analytical figures of merit were satisfactory. In all three methods, a pulse of 50 µL was used (OPA/NDA + Buffer), reducing dramatically the consumption of PCD reagents.

Pulsed-post column derivatization coupled to green liquid chromatography for the determination of glutathione and cysteine based on thioacrylates formation.

In the present work, we developed a method for the determination of thiols (cysteine and glutathione) in yeast samples under the new concept of Pulsed-post column derivatization (Pulsed-PCD). For the chromatographic separation of the analytes, 100% aqueous mobile phase was used and the eluted compounds reacted on-line with the injected pulses (100 µL) of the derivatizing reagent (ethyl propiolate + Britton-Robinson buffer). Spectrophotometric detection of the derivatives was carried out at 285 nm. The Pulsed-PCD configuration, the selection of the analytical column and the pulsed-PCD reaction conditions were investigated. The method was validated for the determination of endogenous content of the analytes in dry and fresh yeasts, with LODs of 3.0 µmol L-1. The percent recovery ranged between 85.2 and 114.4% in all cases and the results were compared with a corroborative method based on classical PCD. The analytical greenness of the proposed method was evaluated using two tools; Analytical Eco-Scale and Green Analytical Procedure Index (GAPI). The greenness score of the HPLC-Pulsed-PCD method (score = 77) was compared with that of the corroborative HILIC-PCD method (score = 71) and was found to be greener in terms of the amount of chemicals used and the produced wastes.

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.

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.

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.