Development and validation of a LC method for the separation and determination of the anticancer‐active FeIII(4‐methoxy‐salophene) using the new second‐generation monolith

LC method with the newly introduced second‐generation monolithic silica RP‐18e column has been developed for the separation of Fe^III(salophene) and four methoxy‐substituted Fe^III(salophene) complexes. The method has been validated for the quantitation of Fe^III(4‐OMe‐salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high‐resolution continuum‐source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first‐ and second‐generation HPLC monolithic columns have been compared for this purpose. The newly introduced second‐generation monolithic silica column Chromolith® HighResolution RP‐18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe^III(4‐OMe‐salophene) was found to be between 1 and 200 μg/mL. Detection and quantitation limits were 0.3 and 1 μg/mL, respectively.     

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Comparison of thermal‐ and photo‐polymerization of lauryl methacrylate monolithic columns for CEC

Lauryl methacrylate‐based (LMA) monolithic columns for CEC, prepared using either thermal initiation or by UV‐irradiation in the presence of AIBN have been compared. Thermal polymerization was carried out at 70°C for 20 h. For UV initiation, the effects of the time exposure to UV light and irradiation energy were investigated. For each initiation process, the influence of composition of porogenic solvent (1,4‐butanediol/1‐propanol ratio) on the physical and electrochromatographic properties of the resulting monoliths was also evaluated. Photochemically lauryl methacrylate stationary phases initiated showed higher permeabilities and better efficiencies than those prepared by thermal initiation. After optimization of polymerization mixture, photopolymerized columns provided a permeability of 4.25×10^?13 m² and a minimum plate height of 13.4 μm for a mixture of polycyclic aromatic hydrocarbons. Similar column‐to‐column and batch‐to‐batch reproducibilities, with RSD values below 11.6 and 11.0 % for the thermal‐ and UV‐initiated columns, respectively, were obtained.     

Adsorption behavior of large plasmids on the anion-exchange methacrylate monolithic columns

The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing "car-parking problem" phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.     

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

Polymethacrylate‐based monolithic capillary columns, prepared by γ‐radiation‐induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion‐pair reversed‐phase capillary liquid chromatography with high‐resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow‐split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high‐resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ‐ray‐induced monolithic column lies in the batch‐to‐batch reproducibility and long‐term high‐temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene‐divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.     

Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM^®) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM^® Disk with epoxy chemistry. After this, the immobilized CIM^® Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM^® Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap^® metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.     

Fabrication and Characterization of a Dye-Immobilized Yttria-Stabilized Zirconia Pellicular Adsorbent for Expanded Bed Adsorption Chromatography

This study deals with the fabrication and characterization of a pellicular adsorbent appropriate for the expanded bed adsorption (EBA) process. The synthesized adsorbent has an yttria-stabilized zirconia nucleus coated with agarose. Morphological analysis of the coated particles was performed by light-scattering microscopy and showed an average diameter of 197.54 and 202.25 µm, for the nucleus and coated particle, respectively. A screening for the reactive dyes reactive blue 19 (RB19), reactive blue 21 (RB21) and reactive orange 107 (RO107) was performed after immobilization onto the pellicular adsorbent by changing the pH, aiming at finding the binding capacity of these to adsorb bovine serum albumin (BSA). The reactive orange 107 was selected and it was more stable at pH 4.5. Study of the kinetics between BSA and the dye-immobilized particle showed that equilibrium is reached before 1 h. The adsorption isotherm of BSA onto RO107-immobilized adsorbent fitted the Langmuir model showing a q_m = 102.328 mg BSA/mL of adsorbent. The pellicular adsorbent also showed good expansion even at a high operating flow rate. Therefore, at a linear velocity as high as 2725 cm/h, a dynamic capacity of 15.7 mg of BSA/mL of adsorbent was obtained.     

Validation of analytical procedures using HPLC‐ELSD to determine six sesquiterpene lactones in Eremanthus species

Eremanthus species display sesquiterpene lactones with therapeutic potential. We are proposing the development of a new analytical method that has been completely validated to qualify Eremanthus species and its main lactones in raw material using HPLC‐ELSD. For the sample preparation, 10.0 mg of powdered Eremanthus leaves was extracted with a 5 mL MeOH/H₂O (9:1 v/v) solution containing scopoletin at 140 µg/mL as the internal standard. For the separation of eight compounds, six of which were lactones, one internal (IS) and one secondary standard were performed utilizing monolithic columns with a nonlinear gradient. The selectivity, stability, precision and matrix effects parameters showed values of RSD of <10%. The six lactones and scopoletin (IS) were recovered with a proportion between 74 and 90% with accuracy represented by error at ?25.41%. The linear dynamic range was obtained between 10.0 and 310.0 µg/mL for all compounds with r² > 0.9987. The limits of detection and quantitation ranged from 2.00 to 6.79 µg/mL and from 6.00 to 20.40 µg/mL, respectively. Assessing the robustness study, this method can be used in inter‐laboratory studies. Using the HPLC‐ELSD method, six sesquiterpene lactones including 4β,5‐dihydro‐2′,3′‐dihydroxy‐15‐deoxy‐goyazensolide, goyazensolide, 4β,5‐dihydro‐2′,3′‐epoxy‐15‐deoxy‐goyazensolide, centratherin, 4β,5‐dihydro‐15‐deoxy‐goyazensolide and lychnofolide, were detected and quantified from distinct Eremanthus species, which were collected in different regions. Copyright © 2015 John Wiley & Sons, Ltd.     

Stability of mycophenolate mofetil in polypropylene 5% dextrose infusion bags and chemical compatibility associated with the use of the Equashield® closed‐system transfer device

Stability studies are necessary in healthcare settings as they facilitate fast, cost‐effective and efficient work related to batch manufacturing and availability of supplies. We studied the stability of 1–10 mg/mL mycophenolate mofetil (MMF) in polypropylene 5% dextrose infusion bags prepared from Cellcept^® and with a generic brand name (Micofenolato de Mofetilo Accord) at different storage temperatures. To ensure chemical compatibility during preparation, we also tested MMF sorption to the Equashield^® closed‐system drug transfer device used in this step. For this, a validated stability‐indicating high‐performance liquid chromatography method was developed for the quantification and identification of MMF in the infusion bags. The analytical selectivity of the assay was determined by subjecting an MMF sample to extreme values of pH, oxidative stress and heat conditions to force degradation. Protected from light, 1–10 mg/mL MMF in infusion polypropylene bags prepared from reconstituted Cellcept^® 500 mg or Accord 500 mg in 5% dextrose was stable for at least 35 days when stored at 2–8°C or between ?15 and ?25°C, and for 14 days when stored at 25°C. MMF loss owing to chemical sorption to the Equashield^® closed‐system drug transfer device set was negligible.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Determination of chlorpheniramine in human plasma by HPLC‐ESI‐MS/MS: application to a dexchlorpheniramine comparative bioavailability study

In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether–dichloromethane, 80:20, v/v) and analyzed by HPLC‐ESI‐MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH₄OH on a Gemini Phenomenex C₈ 5 μm column (50 × 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05–10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra‐batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter‐batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine^®, Schering‐Plough). The study was conducted using an open, randomized, two‐period crossover design with a 2 week washout interval. Since the 90% confidence interval for C_max and AUC ratios were all within the 80–125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption. Copyright © 2009 John Wiley & Sons, Ltd.     

Potential of long capillary monolithic columns for the analysis of protein digests

The gain in separation efficiency for protein digests using long monolithic columns has been evaluated for a LC‐MS system with capillary monolithic columns of different lengths (150 and 750 mm). A mixture of BSA, α‐casein and β‐casein tryptic digests was used as a test sample. Peak capacity and productivity (peak capacity per unit time) were determined from base peak chromatograms and MS/MS data were used for protein identification by MASCOT database searching. Peak capacity and protein identification scores were higher for the long column. Analyses with similar gradient slope for the two columns produced ratios of the peak capacities that were slightly higher than the expected value of the square root of the column length ratio. Peak capacity ratios varied from 2.7 to 4.0 for four different gradient slopes, while protein identification scores were 2–4 times higher for the long column. Similar values were obtained for the productivity of both columns and the highest productivity was obtained at gradient times of 45 and 75 min for the short and long column, respectively. The use of long monolithic columns improves peptide separation and increases reliability of protein identification for complex digests, especially if longer gradients are chosen.     

Vortex‐assisted liquid–liquid micro‐extraction and high‐performance liquid chromatography for a higher sensitivity methyl methacrylate determination in biological matrices

A vortex‐assisted liquid–liquid micro‐extraction coupled with high‐performance liquid chromatography, with UV–vis, is proposed to pre‐concentrate methyl methacrylate and to improve separation in biological matrices. The use of 1‐octanol as extracting phase, its volume, the need for a dispersant agent, the agitation conditions and the cooling time before phase separation were evaluated. In optimum conditions, enrichment factors of 20 (±0.5) and enrichment recovery of 99% were obtained. The straightforward association of this extraction process with the HPLC method, previously regulated by the International Organization for Standardization, afforded a detection limit of 122 ng/mL and a quantification limit of 370 ng/mL. The within‐batch precision, relative standard deviation, was 3% for a sample with 1.49 µg/mL and 4% for a sample with 13.4 µg/mL. The results showed a between batch‐precision of 21% for experiments performed on five different days, for a sample with a concentration of 1.10 µg/mL in methyl methacrylate. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of aripiprazole and its active metabolite,dehydroaripiprazole, in plasma by capillary electrophoresis combining on‐column field‐amplified sample injection and application in schizophrenia

A sensitive high‐performance CZE combining on‐column field‐amplified sample injection (FASI) has been developed for simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in human plasma. A sample pretreatment by means of liquid–liquid extraction (LLE) (diethyl ether) with subsequent quantitation by FASI‐CZE was used. The separation of aripiprazole and dehydroaripiprazole was performed using a BGE containing 150 mM phosphate buffer (pH 3.5) with 40% methanol and 0.02% PVA as a dynamic coating to reduce interaction of analytes with the capillary wall. Before sample loading, a methanol plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (10 kV, 30 s) to introduce sample cations and the applied voltage was 20 kV with on‐column detection at 214 nm. Several parameters affecting the separation and sensitivity of the drug and its active metabolite were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer. The linear ranges of the method for test drug and its active metabolite, in plasma using amlodipine as an internal standard, were over the range 5.0–100.0 ng/mL. One female volunteer (25 years old) was orally administered a single dose of 10 mg aripiprazole (Abilify^®, Otsuka) and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied to monitor the concentration of aripiprazole and dehydroaripiprazole in plasma collected after oral administration of 20 or 30 mg aripiprazole (Abilify^®, Otsuka) daily at steady state in one schizophrenic patient.     

Investigation on Adsorption Dynamics of Protein/Tween‐20 Mixture at the Surface of Solution by Surface Pressure Measurement

The dynamic surface tension (γ_t) and apparent diffusion coefficient (D) of nonionic surfactant Tween‐20 in the presence and absence of bovine serum albumin (BSA) have been investigated via the measurements of surface pressure (π) at different time (t). The curves of γ_t～t are obtained from π～t isotherms. Results show that the γ_t～t relationships of Tween‐20 solution with or without BSA accord with the Ward‐Tordai equation in the region of initial adsorption. D value obtained from the γ_t～t^1/2 curves shows that the diffusion of Tween‐20 slows down with the increase of the concentration of Tween‐20 (c _Tween‐20). And D value of Tween‐20 in the presence of BSA is almost the same as that of the system without BSA when c _Tween‐20 is constant, suggesting that the interaction between Tween‐20 and BSA is weak.     

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates

The aim of this work was to devise a one‐step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild‐type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)–IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K_D of 4.53 × 10⁻⁷ m was obtained from batch isotherm measurements. The combination of tailor‐made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one‐step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial‐Zn(II) and EPI‐30–IDA–Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A‐Sepharose CL‐4B. This MAb preparation revealed on SDS–PAGE two protein bands with M_r of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a gas chromatography-mass spectrometric method for the determination of sildenafil and desmethyl-sildenafil in whole blood

Sildenafil (SDL) is a phosphodiesterase type 5 inhibitor and it is approved for the treatment of erectile dysfunction and pulmonary hypertension. SDL is extensively metabolized to its pharmacologically active metabolite, desmethyl‐sildenafil (DSDL). A sensitive and specific GC/MS method for the determination of SDL and DSDL in whole blood was developed and validated to support therapeutic drug monitoring of SDL patients. The combination of solid‐phase extraction with derivatization using BSTFA with 1% TMCS in acetonitrile efficiently reduced matrix effect and improved sensitivity of the method. In this assay, protriptyline was used as internal standard for both analytes. The LODs were 1.50 and 5.00 ng/mL for SDL and DSDL, respectively, whereas the respective LOQs were 5.00 and 15.0 ng/mL. The calibration curves were linear up to 500.0 ng/mL (SDL: R² 0.992, DSDL: R² 0.990). Absolute recovery values for both analytes ranged from 83.1 to 93.2%. Within‐ and between‐batch accuracy was less than 11.8 and 10.2%, respectively, whereas within‐ and between‐batch precision was less than 8.1 and 10.8%, correspondingly. The developed method is suitable for the determination of SDL and DSDL concentrations in blood samples obtained from patients under Viagra^® treatment, for pharmacokinetic studies or for the investigation of related forensic cases.     

Expanded Bed Chromatography as a Tool for Nanoparticulate Separation: Kinetic Study and Adsorption of Protein Nanoparticles

Expanded bed adsorption was investigated together with its suitability for the practical recovery of nanoparticulate mimics of products such as plasmid DNA and viruses as putative gene therapy vectors. The study assessed the binding of protein nanoparticles fabricated from bovine serum albumin (BSA) with average size of 80 nm as a model system and viral size/charge mimic to the streamline DEAE adsorbent in the expanded bed column chromatography. The adsorption kinetics and adsorption mechanism for the BSA nanoparticles on the adsorbent were studied. In batch adsorption studies, the factors nanoparticle concentration, contact time and adsorbent amount, affecting adsorption isotherms were investigated. Subsequently the data were regressed against the Lagergren equation, which represents a first-order kinetics equation and also against a pseudo-second-order kinetics equation. The results demonstrated that the adsorption process followed a Langmuir isotherm equation. The kinetics of the adsorption process followed a pseudo-second-order kinetics model with a rate constant value of 0.025 g mg^?1 min^?1. The dynamic binding capacity of the BSA nanoparticles on an expanded bed was calculated. The recovery of the nanoparticles was more than 85%.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

Preparation of a novel sorptive stir bar based on vinylpyrrolidone‐ethylene glycol dimethacrylate monolithic polymer for the simultaneous extraction of diazepam and nordazepam from human plasma

A new monolithic coating based on vinylpyrrolidone‐ethylene glycol dimethacrylate polymer was introduced for stir bar sorptive extraction. The polymerization step was performed using different contents of monomer, cross‐linker and porogenic solvent, and the best formulation was selected. The quality of the prepared vinylpyrrolidone‐ethylene glycol dimethacrylate stir bars was satisfactory, demonstrating good repeatability within batch (relative standard deviation < 3.5%) and acceptable reproducibility between batches (relative standard deviation < 6.0%). The prepared stir bar was utilized in combination with ultrasound‐assisted liquid desorption, followed by high‐performance liquid chromatography with ultraviolet detection for the simultaneous determination of diazepam and nordazepam in human plasma samples. To optimize the extraction step, a three‐level, four‐factor, three‐block Box–Behnken design was applied. Under the optimum conditions, the analytical performance of the proposed method displayed excellent linear dynamic ranges for diazepam (36–1200 ng/mL) and nordazepam (25–1200 ng/mL), with correlation coefficients of 0.9986 and 0.9968 and detection limits of 12 and 10 ng/mL, respectively. The intra‐ and interday recovery ranged from 93 to 106%, and the relative standard deviations were less than 6%. Finally, the proposed method was successfully applied to the analysis of diazepam and nordazepam at their therapeutic levels in human plasma. The novelty of this study is the improved polarity of the stir bar coating and its application for the simultaneous extraction of diazepam and its active metabolite, nordazepam in human plasma sample. The method was more rapid than previously reported stir bar sorptive extraction techniques based on monolithic coatings, and exhibited lower detection limits in comparison with similar methods for the determination of diazepam and nordazepam in biological fluids.