There is an urgent need to search for new sorbents of pollutants presently delivered to the environment. Recently biochar has received much attention as a low-cost, highly effective heavy metal adsorbent. Biochar has been identified as an efficient material for cobalt (Co) immobilization from waters; however, little is known about the role of Co immobilization in soil. Hence, in this study, a batch experiment and a long-term incubation experiment with biochar application to multi-contaminated soil with distinct properties  were conducted to provide a brief explanation of the potential mechanisms of Co (II) sorption on wheat straw biochar and to describe additional processes that modify material efficiency for metal sorption in soil. The soil treatments with 5% (v/w) wheat straw biochar proved to be efficient in reducing Co mobility and bioavailability. The mechanism of these processes could be related to direct and indirect effects of biochar incorporation into soil. The FT-IR analysis confirmed that hydroxyl and carboxyl groups present on the biochar surface played a dominant role in Co (II) surface complexation. The combined effect of pH, metal complexation capacity, and the presence of Fe and Mn oxides added to wheat straw biochar resulted in an effective reduction of soluble Co (II), showing high efficiency of this material for cobalt sorption in contaminated soils.  In recent decades, industry’s reliance on cobalt as a material essential for enabling technological development has caused considerable growth in the use of cobalt and accidental release of this metal into the environment. Metal ore mining and the smelting process , alloys and chemicals containing cobalt (Co), sewage effluents, and urban and agricultural runoff (phosphate fertilizers and pesticides)  have bee2+, Mg2+, K+, Na+, P, NH4+, NO3−), and the total contents of trace elements  were determined to describe the properties of the material. The total surface area was determined using a BET  specific surface area analyzer Gemini VII 2390 Series . The cation exchange capacity and exchangeable cations  were determined according to the modified method described by Munera-Echeverri et al. [3) solution at a pH of 8.5 had a similar pH to WSBC. This extractant decreased calcium in solution , and this decrease enhanced the dissolution of Ca-phosphates. The nitrate content was analyzed according to the ISO 14256-1:2003 procedure on a UV-Vis Cary 60 . The pH values were measured at a ratio of 1:5 (w/v) in deionized water after the sample was shaken for 1 h at 130 rpm with a calibration check pH meter . The ash content was determined by weight loss after combustion at 750 °C for 6 h in a muffle furnace according to ASTM D7348-13 [3 was determined following the Scheibler method with a calcimeter [Biochar was produced from wheat straw (WSBC) at the pyrolysis temperature of 550 °C and time remaining in the reactor 60 s. The BET surface area, cation exchange capacity (CEC), pH in deionized water, CNHSO elemental composition, ash and carbonates content (% volume/dry weight), exchangeable cations and anions content  at afforested sites at different distances and locations from the copper smelter in SW Poland , expecting cobalt enrichment from smelter emissions. In the samples, pre-scanning studies for a wide range of heavy metals, including cobalt, were done on a microwave plasma-atomic emission spectrometer MP-AES 4200  after microwave sample digestion in 70% nitric acid (1:10 w/v ratio). The total concentration of cobalt remained in a wide range from 4.5 to 74 mg/kg, depending on the distance from the smelter and the soil type. Two soils with the highest Co concentrations, differing in texture, were chosen for the incubation experiment. Large soil samples, 15 kg each, were collected of Cutanic Luvisol and Fulvic Brunic Arenosol according to the FAO (Food and Agriculture Organization of the United Nations) guidelines  at a disAfter soils were incubation with 5% (w/v) wheat straw biochar, the (Community Bureau of Reference (BCR) sequential extraction procedure was applied to measure the following four fractions of cobalt in the tested soil: exchangeable and bound to carbonates (Fraction 1), reducible or bound to Fe and Mn-oxides (Fraction 2), oxidizable or bound to organic substances (Fraction 3), and residual (Fraction 4). Acetic acid, hydroxyl ammonium chloride, hydrogen peroxide plus ammonium acetate, and aqua regia stages of the sequential extraction procedure were applied to the soil samples, respectively ,44,45. Tt-tests were used to test for significant differences in cobalt fractions between biochar treated and untreated soils (p < 0.05). The obtained data were compiled using Microsoft Excel 2016 and Statistica Statsoft 13.3.The metal sorption batch experiments were performed in triplicate and the soil sorption experiments were performed in six replicates. The data are presented as the mean values with the relative standard deviation (RSD). Student’s The elemental composition of the tested wheat straw biochar is presented in 2, with the cation exchange capacity (CEC) of 63 cmolc/kg. The total ash content was 32.4% and the contribution of calcium carbonates was 3.07% (w/dw), however almost 90% of exchangeable cations in CEC was a K+, followed by Ca2+, Mg2+, and a very low content of Na+ and NH4+  groups. This change confirms that the O-H groups take part in Co (II) and Cu (II) complexation on wheat straw biochar surface. The carboxyl peak observed for a pure wheat straw biochar at 1624 cm−1 was shifted to much smaller values, i.e., 1583 cm−1 or 1570 cm−1 in the spectra of BC treated with salts , Cu (II), and mix Co (II) + Cu (II) biochars . From thth salts .−1, characteristic for C=O carboxylic group, can be explained by the interaction with Co (II) and Cu (II) ions with free carboxyl groups on the biochar surface and change to carboxylates, which indicates the important role of carboxyl groups on the biochar surface in metal binding. To provide more details about the type of the metal binding to carboxylic group on the biochar surface at 1624 and 1420 cm−1, a calculation of Δ according to Nakamoto [The decrease in the wavenumber of the peak 1624 cmNakamoto  was perf2O or OH groups. The results of the FT-IR analysis also showed that metal ions complexation could be related to an abundance of carbonates and polysaccharides such as moieties in biochar, since changes of peak at 1080 cm−1 related to Si–O, C–O, and S=O groups were observed in metal spiked biochar. Very low concentrations of sulfur in biochar , and bridging . The cal biochar  can limirmations .Wheat straw biochar was applied to soils to verify the hypothesis that application of this material can immobilized cobalt ions in soil. Copper fractionation for tested soils was described in the previous paper  and is nThe application of 5% (w/v) wheat straw biochar affected cobalt speciation in both tested soils. In sand + 5% WSBC treatment, a significant decrease in exchangeable cobalt Fraction F1 (F1) from 17.4% to 7.3% was determined. The observed reduction in Co content in F1 was mainly balanced by their increased content in Fraction 2 (F2), reducible or bound to Fe and Mn-oxides  by 9.4%.The application of SEM-EDX proved that the metal ions can be bound on the biochar surface. Examination of the SEM-EDX element map, 2/g, depending on pyrolysis (slow or fast), although, in most research, lower values can be found with good efficiencies for heavy metal removal [Biochar application can have a direct or indirect impact on cobalt immobilization/mobilization process in soil. Indirectly, biochar can affect soil sorption properties and pH, reducing the presence of metal in exchangeable and soluble forms in soil solution. The direct effect can be related to biochar properties such as sorption capacity, oxygen functional groups, and mineral components content  increasing or supporting soil sorption capacity for cobalt. All mentioned processes were considered in our study. The tested wheat straw biochar had high SSA as compared with other straw-derived biochars produced under similar conditions, as porosity of biochar significantly increases between 400–600 °C . Gul et  removal ,53. Cati removal . Wheat s removal . The whe removal ,54. Bioc removal , modifyi removal ,19,55. O removal . Wheat s removal . In othe removal ,58, simi removal ,60. As o removal . Similar removal . Sun et  removal  showed t removal , suggest removal . Zhang e removal  observed removal . These f2+ removal as compared with materials produced at high temperatures with a more aromatic structure. However, biochar applied to soil vs. solution undergoes many abiotic and biotic processes causing sorbent oxidation, called biochar aging [As oxygen-containing functional groups are predominant mechanisms of divalent cations the result of our study suggests that biochars produced at lower temperatures or oxidized during pretreatments  have betar aging , which car aging  and genear aging . Uchimiyar aging  describear aging  observedp > 0.05) in biochar treated soils. The Mn and Fe oxides and organometallic moieties such as Fe–O–C can be formed on the biochar surface during pyrolysis. Most of the Fe in biochar is present in crystalline phases ranging from zerovalent iron to ferric oxides [2 which could explain the high stability of Co forms after WSBC application to loam soil. Pan et al. [Findings of our study showed that wheat straw biochar can support soil sorption complex acting as a source of several mineral components, for example, Fe and Mn oxides ,70, silic oxides . The rolc oxides ,75,76,77c oxides . Kabata-c oxides  describen et al.  describeThe results of our study showed that wheat straw biochar has good removal efficiencies in single-metal systems determined in a batch experiment with cobalt salts in the solution. However, the capacities of biochar for cobalt sorption can be modified in multiple-metal systems due to the competition between the heavy metals present in soil and lower stability of the material due to cation exchange and the surface oxidation process that biochar undergoes under soil conditions. As biochar can affect soil properties, changing conditions of metal immobilization, and as soil conditions can have an impact on surface properties of biochar, predictions about biochar efficiency for metal sorption in soil are difficult and need further recognition. 2+, decreasing mobility and availability of this element in soil.The results of the experiments showed that wheat straw biochar is an efficient sorbent of CoThe dominant mechanisms of cobalt sorption on biochar surface are related to oxygen functional groups present on the biochar surface, mainly carboxylic and hydroxyl groups.Biochar can support soil sorption complex with mineral components such as Fe and Mn oxides which increase efficiency of cobalt immobilization.The efficiency of cobalt removal from soil can be modified by biochar oxidation and interactions with soil constituents which makes the process more complex as compared with cobalt removal from aqueous solutions.The results from this study suggest that application of biochar is a feasible strategy for remediation of cobalt contaminated soils, reducing health risks related to human exposure to Co from anthropogenic sources.Further and more complex studies are necessary to recognize the problems of cobalt soil pollution, potential risks, and remediation solutions, including biochar application.


Each module contained a melt-extruded and molded formulation of 40% w/w metoprolol succinate in a PEG1500 and Kollidon® VA64 erodible hydrophilic matrix surrounded by polylactic acid and/or polyvinyl acetate as additional release rate-controlling polymers. Drug release testing confirmed the generation of predictable, combined drug release kinetics for dosage forms, independent of dose, based on a product’s constituent modules and enhanced product variety through a minimum of six dosage form release profiles from only three module variants. Based on these initial results, the potential of the reconfigurable modular product design concept is discussed for unified integration into a pharmaceutical mass customization/mass personalization context.Independent individualization of multiple product attributes, such as dose and drug release, is a crucial overarching requirement of pharmaceutical products for individualized therapy as is the unified integration of individualized product design with the processes and production that drive patient access to such therapy. Individualization intrinsically demands a marked increase in the number of product variants to suit smaller, more stratified patient populations. One established design strategy to provide enhanced product variety is product modularization. Despite existing customized and/or modular product design concepts, multifunctional individualization in an integrated manner is still strikingly absent in pharma. Consequently, this study aims to demonstrate multifunctional individualization through a modular product design capable of providing an increased variety of release profiles independent of dose and dosage form size. To further exhibit that increased product variety is attainable even with a low degree of product modularity, the modular design was based upon a fixed target dosage form size of approximately 200 mm The provision of individualized pharmaceutical therapy, which ultimately aims to elicit optimal therapeutic outcomes a priori from all patients, requires the integration of key patient attributes into the design and manufacture of pharmaceutical products ,7,8,9,10As target patient segments become progressively smaller, with each segment having unique needs from the pharmaceutical product, enabling simultaneous and independent individualization of multiple product features, i.e., multifunctional individualization, will demand a marked increase in the number of product variants. This encompasses, for example, simultaneous provision of individualized dosing ,13,14,15The aim of our study is to demonstrate multifunctional individualization by modular dosage forms through a product design concept capable of providing an increased variety of release profiles independent of the dose and size of the dosage form. In doing so, with a focus on the product platform and specifically on product modularity as an approach to provide affordable variety ,36, thisModular products have been defined previously as that which comprises building blocks or modules , wherebyNotably, Recently, Siiskonen et al. have developed a customized product concept for modular pharmaceutical products to address the requirement for enhanced product variety in the provision of individualized therapy . BuildinThe product was designed as a solid oral dosage form with a modular architecture. For the purpose of this study, a dosage form was assumed to comprise two out of three possible module variants, i.e., a low degree of product modularity. These three module variants were designed to provide unique drug release profiles but contain the same dose. A key feature for simultaneous and independent tailoring of the dose and drug release to an individual is the spatial separation of the dose- and release-controlling functionalities in the module variants. Reconfigurable assembly of these module variants into dosage forms, combining either two identical or two dissimilar module variants, was designed to increase variety in resulting in vitro drug release profiles on the dosage form level for a single API-containing drug product, without needing to increase the number of module variants. Importantly, it was only through one component  that variety in the release kinetics of the module variants was introduced whilst other components  remained fixed. Altogether, spatial separation of features of interest supports multifunctionality, whereas reconfigurable assembly of module variants into dosage forms and a balance between standardization and differentiation of submodular components promote affordable variety. These characteristics therefore underpin the suitability of this concept for tackling the product variety–volume complexity challenge.Our work proposes and constitutes, to the best of the authors’ knowledge, a first demonstration of a product concept that supports multifunctional individualization whilst also meeting the challenge from enhanced product variety management. Except for an early recommendation in our previous work , for theThe realization of the proposed concept is based on the hypothesis that, in the absence of interactions between module variants, module variants with independently controlled dose- and drug release-determining functionalities can contribute predictably and independently to the net dose and drug release functionalities of the dosage form they construct.Although co-development of product and manufacturing platforms in a mass customization context is crucial to support enhanced pharmaceutical product variety, especially at lower volumes, for the purpose of demonstrating our proposed concept, the product platform  is considered within the scope whereas manufacturing platforms, beyond their technical ability to prototype with the intended product performance, are considered out of scope. Since manufacturing networks are out of scope, scale-up and dedicated stability testing under varying conditions relevant to storage and transport are also beyond the scope of this study.® VA64) was supplied by BASF . Polyethylene glycol 1500 , sodium phosphate dibasic dihydrate  and sodium phosphate monobasic monohydrate  were purchased from Sigma-Aldrich Chemie GmbH . ZMorph polylactic acid (PLA) filament  was obtained from ZMorph S.A.  and Aquasolve™ polyvinyl acetate (PVA) filament  was purchased from Formfutura BV .Metoprolol succinate  was obtained from AstraZeneca . Vinylpyrrolidone-vinyl acetate copolymer  and the other was customized for each module variant (lid). The standardized water-insoluble PLA cup enclosed the bottom and sides of the core, allowing the surface area available for drug release to remain constant during dissolution. The presence and/or type of lid provided the module variants with their distinct drug release kinetics. Module variant 1 (MV1) with no lid was designed to provide rapid drug release, module variant 2 (MV2) with a water-soluble lid of predefined thickness was designed to generate a lag phase followed by rapid drug release from the core, and module variant 3 (MV3) with a water-insoluble lid with a central orifice was designed to provide slower initial drug release kinetics through the reduced area available for initial hydration of the core.® TinkerCAD™ . The generated ‘.stl’ file was subsequently imported into Simplify3D®  for control of printing parameters and subsequent printing. Critical cup and lid model dimensions are described in Fused deposition modeling (FDM) was used to fabricate the cup and lid submodular components. Digital models of the cup and lid were created as ‘.stl’ files using AutodeskA ZMorph VX multitool 3D printer , equipped with a single 0.3 mm diameter extrusion nozzle, was used to independently print the cup and lid submodular components from PLA and PVA filaments, respectively, for downstream assembly. The lids were designed with overhanging side walls and the cups and lids were all printed with matching 0.1 mm layer heights to facilitate interlocking and adhesion and prevent dislodging of the lids once the modules were assembled. Prior to the printing of each component, the build platform was leveled and heated to 60 °C to facilitate adhesion of the structures onto the platform. The set temperature for the nozzle was 200 °C, for both PLA cups and PVA lids.w/w MS:PEG1500:VA64 was prepared by HME. This drug concentration allowed a dose of approximately 25 mg MS (lower limit of marketed dose) to be incorporated into each dosage form. The incorporation of both low molecular weight PEG and VA64 as carriers facilitated the melt molding and melt extrusion processes, respectively, whilst promoting rapid drug release. MS powder, PEG1500, and VA64 powder were weighed in a weigh boat in a 2.5 g batch size and mixed with a spatula until homogeneous upon visual inspection. HME was performed using a 5 mL capacity Xplore micro compounder , affixed with conical mixing screws and a circular die, 1.5 mm in diameter. The physical mixtures were fed via a hopper into the barrel maintained at a constant temperature profile of 80 °C and a screw speed of 50 rpm during feeding and recirculation. This processing temperature was sufficiently low to allow consistent feeding into the barrel without particle bridging in the hopper and an appropriate material viscosity for consistent extrusion through the die and sufficiently high to operate within the motor load (torque) limit of the extruder at the chosen batch size. After complete feeding (˂ 1 min), mixtures were recirculated for 10 min to aid homogenization prior to extrusion through the die to obtain a cylindrical filament, which was allowed to cool at room temperature. Ejection was force-controlled at 100 N until a maximum screw speed of 400 rpm was reached. Filaments were stored in sealed plastic bags at room temperature prior to further processing.Hot melt extrusion (HME) was the first step in core fabrication and was selected in order to obtain a homogeneous distribution of the drug in a carrier via HME’s dispersive and distributive mixing. A formulation containing 40:20:40% Melt molding was the second step in core fabrication. Empty PLA cups fabricated by FDM were weighed in a Mettler MT5 analytical balance . Melt extruded filaments were cut with a blade and manually filled into each PLA cup. The cups with unmelted filaments were placed in an oven  at 100 °C for 15 min, the shortest duration required to achieve molding at this temperature. A higher temperature was required during melt molding than melt extrusion due to the absence of shear stress or applied force from rotating screws in the former process. This melt molding temperature allowed the molten formulation to fill the cup whilst the surrounding PLA cup maintained its structural integrity. The selection of a melt molding process for core fabrication allowed the core formulation to form a good seal with the inner bottom and sides of the surrounding PLA cup. In the absence of a lid, the exposed surface area for subsequent hydration and drug release would be identical in all module variants. The cups for MV1 and MV2 were filled completely in 2 sequential additions of 15 min each whereas the cups for MV3 were overfilled in 3 sequential additions of 15 min each to prevent formation of an air pocket beneath the MV3 lid orifice. The core-containing cups were weighed, and the mass of the cores calculated. Filled cups were stored in closed plastic well plates at room temperature prior to analysis.deg). Materials were weighed in open 100 µL aluminium crucibles and heated from 30 to 500 °C at 10 K/min under a nitrogen atmosphere set at a flow rate of 50 mL/min. Tdeg was reported as the first observed deflection (mass loss) from the initial baseline in the weight vs. temperature curve in the absence of water loss. Glass transition temperatures (Tg) and/or melting points (Tm) of the raw materials and melt extruded filaments were probed between room temperature and below Tdeg of the drug, which represents the range of storage and processing conditions for the materials. Tg of raw materials below room temperature (MS and PEG1500) were obtained from existing literature. These thermal events were measured by differential scanning calorimetry (DSC) in a DSC 2 STARe system instrument . The samples were weighed in 40 µL aluminium crucibles, which were sealed by lids with a pinhole for subsequent analysis. The instrument was run in a heat-cool-heat cycle at 10 K/min under a 50 mL/min nitrogen atmosphere from 25 to 170 °C. STARe software  was used for instrument control and subsequent analysis of thermograms. Tg values were determined at the midpoint of the Tg range and Tm at the peak of the melting endotherm. Furthermore, the percentage crystallinity of MS in the melt extruded filament was calculated by normalizing the melting enthalpy of MS in the melt extruded filament to the melting enthalpy of the pure MS powder  and to a nominal MS content of 40% w/w in the filament. All samples were prepared in triplicate for analysis.Thermogravimetric analysis (TGA) using a TGA/DSC 3+ STARe system instrument  was performed on raw materials to determine the onset of thermal degradation . The assembled module variants comprising the cup, core, and affixed lid (for MV2 and MV3) were analyzed intact using a high-resolution SkyScan 1272 instrument , with a 50 kV, 181 µA X-ray source and a charge-coupled device (CCD) detector fiber-optically coupled to a scintillator. SkyScan 1272 control software  was used for instrument operation. Samples were imaged in batches of 4 at a pixel size of 19.8 µm or smaller. Three-dimensional imaging was achieved by rotation through 180° with steps of 0.5° averaging 2 frames per position, with 3 × 3 binning, resulting in a resolution of 1344 pixels × 896 pixels and a total acquisition time of 23 min per batch. Image reconstruction was performed from a batch of 4 objects using NRecon  at a resolution of 19.8 µm, adjusting misalignment, reducing ring artifacts, and applying beam hardening correction (10%). During reconstruction, the subscans were fused in the z-direction and both x/y shift correction and rotation correction were applied. DataViewer  was used for data analysis. Core diameters for all module variants, lid thicknesses for MV2, and orifice sizes for MV3 were obtained from DataViewer. The orifice diameter was measured for a 0.4 mm orifice size although final drug release tests were conducted on MV3 with a 0.8 mm orifice size. A digital caliper set at 0.8 mm was used to verify that 0.8 mm orifices were consistently printed.In vitro drug release testing of MS from the module variants, both alone and in combinations of two module variants (to represent the dosage form variants), was carried out in duplicate using a USP 2 dissolution apparatus  with a suspended stainless-steel stationary basket (mesh size 8), which housed the sample. To prevent floating during dissolution, the module variants were glued to the metal flap supplied with the stationary basket using ethyl cyanoacrylate adhesive. The USP 2 apparatus was operated at 37 ± 1 °C, 50 rpm with 900 mL pH 6.8 phosphate buffer (I = 0.1 M) and was equipped with an autosampler  programmed to withdraw 1.5 mL sample aliquots at predefined time intervals without media replacement. Withdrawn aliquots were analyzed by ultra-performance liquid chromatography coupled with ultraviolet spectroscopy (UPLC-UV) to quantify MS concentration. For all drug release tests involving MV3, to eliminate the accumulation of air from the dissolution medium at the orifice of MV3, the dissolution medium was stirred for 2 h prior to commencement of the drug release test and the lid of the dissolution vessel was tapped gently during dissolution at 0 min and 15 min, respectively.® Scientifica, Usmate, Italy). At predefined time intervals, 1 mL aliquots were withdrawn. UV absorbance spectrophotometry was used to quantify MS absorbance at 275 nm and MS concentration was calculated from a calibration curve (concentration range 2–100 µg/mL). As an indication of homogeneity, MS content was also determined along the length of the melt extruded filament by dissolving 5 mm long filament sections (n = 10) weighing approximately 5 mg in 10 mL phosphate buffer and quantifying MS via UV absorbance as described above.Prior to USP 2 dissolution of individual module variants and dosage form variants, preliminary investigations of drug release kinetics from cores only, MV1, and MV3, were performed in duplicate in 250 mL glass beakers containing 100 mL pH 6.8 phosphate buffer (I = 0.1 M), which maintained sink conditions  for MS dissolution. The purpose of this preliminary release test was to guide selection of MV3 orifice size based on the orifice size that would provide distinct drug release kinetics compared to MV1 and which could therefore be used to demonstrate reconfigurability. Samples were suspended in the medium using a stainless steel stationary basket analogous to that described in the USP 2 dissolution setup. This smaller scale dissolution test was operated at room temperature with 500 rpm stirring with an Arex magnetic stirrer  featuring a binary solvent manager, column manager, and sample manager. The UPLC system was equipped with a reversed phase Acquity UPLC BEH C18 column  and an Acquity UPLC PDA detector operated at 275 nm . MS calibration standards were prepared ranging in concentration from 0.25 to 20 µg/mL for construction of a calibration curve and subsequent quantification of MS concentrations in samples. The % relative standard deviation (RSD) for all standard curves were below 5%. Of the standard or sample 10 uL was injected into a mobile phase pumped at 1 mL/min through the column, maintained at 40 °C, in an isocratic gradient of 75% water containing 0.03% trifluoroacetic acid and 25% acetonitrile containing 0.03% trifluoroacetic acid. Empower 3 Chromatography Data Software  was used for both instrument control and data analysis.Functional realization of the product concept requires accurate and precise translation of the dose- and drug release-determining design features into the physical construct. w/w. With a % RSD of 3.2% in filament sections weighing approximately 5 mg, the filament was deemed homogeneous on an approximately 6-fold smaller length scale than that required for core fabrication. With this homogeneous distribution of MS in melt extruded filaments, the dose for MV1, MV2, and MV3 depended on reproducibility of both FDM-printed PLA cups and melt molded cores. FDM dispensing precision of the PLA for the cups was high with a 1.1% RSD in mass. In contrast, the somewhat lower precision in melt molded core mass of 3.6% and 4.2% RSD for normal filled cores (MV1 and MV2) and overfilled cores (MV3), respectively, reflects the reliance on manual sectioning and filling of extruded filaments into the PLA cup prior to melt molding, compared to the automated dispensing during FDM to generate the PLA cups. Regardless, core masses were within an acceptable 5% RSD in all cases, which, together with the filament drug content homogeneity, was expected to translate to acceptable precision in dose.MS content measured along the length of the melt extruded filament was 39.4% ± 1.3% The drug release rate-determining functionality was designed as the presence and/or type of lid in each module variant given that the fastest drug release was provided by the core. This translated to core diameter precision (2.8% RSD), lid thickness precision (6.4% RSD), and orifice diameter precision (2.2% RSD) for MV1, MV2, and MV3. The dimensional data in w/w MS: 20% w/w PEG1500: 40% w/w VA64).The cores are the dose-controlling features of all module variants and were melt molded from melt extruded drug-containing filaments. m, midpoint). In the melt extruded filament comprising 40% w/w MS: 20% w/w PEG1500, and 40% w/w VA64, melting endotherms were visible in the region of PEG1500 and MS. The melting endotherm corresponding to MS was observed in the melt extruded filament at a Tm of 130 °C. Comparing the melting enthalpies of pure crystalline MS with that of MS in the filament revealed a reduction in the melting enthalpy from 178.7 to 68.1 J/g . The melting enthalpy of pure crystalline MS is in agreement with that reported in the literature [1/2 [1/2 [1/2 [g of MS and PEG1500 below the temperature range of interest in this study was reported as 2.2 °C [g of VA64 was determined as approximately 111 °C determined from heat cycle 2. Previous literature corroborates the presence of a characteristic broad endothermic peak between 25 and 100 °C due to water loss in heat cycle 1 and therefore Tg determination of VA64 from heat cycle 2 [Pure crystalline MS displayed a melting endotherm at 139 °C , measured as the gradient of a linear fit between 10% and 60% drug release [MV1 and MV3 exhibited faster release kinetics under USP 2 test conditions relative to their performance in preliminary drug release testing, which could be attributed to an increase in temperature from room temperature (preliminary tests) to 37 °C (USP 2) as well as a change in the agitation method from a magnetic stirrer to paddle apparatus. This temperature difference also contributed to a reduced solubility of dissolved gases and formation of air bubbles in the USP 2 dissolution media, which was not evident during preliminary tests. Air bubbles, originating from the medium, immobilized periodically on the surface of MV1 and MV2, could temporarily reduce surface area available for dissolution, contributing to the observed variability in individual release rates under USP 2 testing conditions. This argument is supported by the lack of variability in individual release rates from MV3 due to the air bubble removal protocol described in the methods section applied to all MV3-containing dissolution tests.The solid lines represent minimum and maximum drug release kinetics for the dosage form variants (black) and module variants (grey). Three unique release profiles are shown for the dosage form variants comprising two identical module variants, analogous to that of the single module variants. The dose released (mg) corresponds to the complete drug release (100%) in all cases. Furthermore, Sources of variability for dosage forms constructed from MV1 and MV2 are as described for The dose released (mg) corresponds to the complete drug release (100%) in all cases. As with the combination of identical module variants in Independently achieve predictable, combined dosing and predictable, combined drug release kinetics for dosage forms based on their constituent modules and,Contribute to the enhanced product variety necessary for the provision of individualized therapy.This study demonstrated how enhanced product variety for multifunctional individualization can be realized using reconfigurable modularization. These study outcomes were underpinned by key differences between our proposed modular pharmaceutical product concept and conventional modular pharmaceutical product concepts, as summarized in In both the conventional and proposed modular product concepts, assembly may be physical assembly  ,52,81, vBeyond the demonstration of enhanced drug release flexibility independent of dose and the increase in total variety of release profiles, this concept provides additional opportunities for expanding flexibility while preserving affordability. (i) Increase the total range of dosage form drug release profiles: the release rate and mechanism is defined by the lid in our proposed product concept, either through the absence of the lid (MV1) or through the lid material and design (MV2 and MV3). Alternative lid materials or designs could therefore translate into alternative release kinetics or mechanisms for each module variant allowing either slower or faster kinetics than those demonstrated in this study. Modification of only the lid allows the balance between standardization and differentiation in the submodular components and the total number of module variants to remain fixed. This balance is one already established approach to simultaneously promote affordability (through standardization) and variety (through differentiation) during mass customization ,36. In a(iia) Increase the total number of dosage form release profiles within a given range (through fine-tuning): Current academic research on individualized release predominantly utilizes a fine-tuning approach as a means to improve drug release flexibility, whereby modification of one release-determining structural feature allows tuning of drug release iteratively, on a continuous scale, and which confines variety in release profiles to variants of the same release profile shape or mechanism. Examples include modifying shell thickness in core–shell designs to prolong or shorten lag times ,87, modi3  to remain within the currently recommended dimensions of marketed pharmaceutical tablets [3 each. Multifunctionality requires that the tailoring of product features such as the dose and drug release be performed independently of each other and of dosage form size [(iib) Increase the total number of dosage form drug release profiles within a given range (through reconfiguration): Reconfiguration has been highlighted as a critical design feature of the proposed modular product concept to increase the number of dosage form variants without increasing the number of module variants that are designed and fabricated. Additionally, the dosage form was constrained in size to enhance patient acceptability. Our modular design was based upon a fixed target dosage form size of approximately 200 mm tablets . Since t(i) and (ii) Modify both the range and number of dosage form release profiles through combined approaches: The total range of release kinetics selected in this study across the three module variants allowed demonstration of predictable, combined “monophasic” drug release and predictable, combined “biphasic” drug release. Since our results confirm that it is possible to construct dosage form release profiles predictably from their constituent module release profiles, tailoring the time gap between the release kinetics of one module variant and the next so that they are either more similar or less similar to each other can either achieve an overlap or reduce the overlap between the combined release profiles on a dosage form level. Tailoring the gap between module variant release kinetics together with modifying the number of modules that are reconfigured into a final dosage form can therefore result in multiphasic release profiles, if desired, for certain APIs and/or therapeutic indications.(iii) Simultaneously tailor dose and drug release: Although this study demonstrated additive dosing from individual modules to final dosage forms, the delivered dose from the dosage form remained fixed  between dosage form variants. Identical doses within individual module variants, if desired, could have been achieved through overfilling of MV1 and MV2 cores to match overfilling of MV3 cores. Alternatively, achieving scalable individualized dosing with modular dosage form design concepts at a fixed dosage form size was already addressed in our previous work . As descRegarding individualized drug release, medical and biological knowledge gaps in defining target individual in vivo release profiles remain. Optimization of the product and its subsequent translation to in vivo performance was beyond the scope of this study. Nevertheless, our in vitro demonstration of an increased variety of predictable drug release kinetics provides a pre-emptive step towards realizing the overarching goals of individualized therapy, once these knowledge gaps are filled. Furthermore, although independent tailoring of dose and drug release through modularization was highlighted in this study, future explorations of modular product concepts for individualization will need to be extended to encompass additional product attributes such as the appearance, choice of API, composition, and sensory properties of a dosage form.Robust functional realization and industrialization of our product concept and future product concepts with their individualized attributes will rely, in part, on processing technologies’ capabilities to fabricate parts reproducibly. Appropriate precision in FDM, HME, and melt molding was achieved in this study for adequate prototyping of the product concept. However, to expand potential utility of the concept and improve robustness of the module variants, alternative or modified formulations and processing strategies  can be exploited. These can, for example, reduce core porosity and the contribution of air to orifice obstruction and variable release kinetics, achieve complete amorphization in the filament for drugs with poor aqueous solubility, or facilitate automation for increased throughput during scale-up and industrialization of the concept. The ability of the selected processes to achieve high precision in dose-controlling, release-controlling, and size-controlling structural features will remain critical.Beyond product performance, unified integration with process and production will be required to promote access to affordable individualized therapy. Whilst initial explorations of product designs for individualization in a mass customization context have been performed ,94, to tThrough the provision of an increased variety of drug release profiles independent of the dose and size of the dosage form, a product concept for multifunctional individualization has been demonstrated. To the best of our knowledge this is the first demonstration that fulfills a crucial overarching requirement of pharmaceutical products for individualization, i.e., simultaneous and independent individualization of multiple product features in the context of a unified product–process–production approach for the provision of affordable variety.The attainment of predictable, combined dosing and drug release with respect to the module variants from which a dosage form is constructed highlights the role of reconfigurable modularization in achieving predetermined dose and drug release accurately and predictably. In this regard, this study provides a first indication of the potential to reverse engineer drug release kinetics for a dosage form, from its composite module variants, based on a predetermined target release profile.Furthermore, through reconfiguration during the final assembly stage (module variant to drug product variant) to manage product variety, this product concept is primed to translate already established key mass customization principles  to the pharmaceutical value chain. Although the road to realization still requires optimization for robust performance, extended applicability to different APIs, and translation into clinical applicability, the dynamic product design presented here provides numerous additional opportunities for expanding flexibility, which remain to be harnessed prior to realization of affordable individualized therapy.


We evaluated the use of some neutrophil products, namely; autologous rabbit antimicrobial neutrophil extract (rANE), heterologous porcine antimicrobial neutrophil extract (pANE), neutrophil degranulation products (DGP) and neutrophil microvesicles (MVs) for stimulation of monocyte-derived macrophages (MDMs) to improve healing. Two animal models were evaluated; the rabbit model for autologous osteochondral transplantation (OT) with application of rabbit ANE, DGP or MVs for MDMs stimulation, and the ovine model of the insertion of a Ti implant with the use of porcine ANE, and ovine DGP or MVs for MDMs stimulation. Macrophage activity was assessed on the basis of free radical generation and arginase activity. We estimated that DGP acted in a pro-inflammatory way both on rabbit and ovine MDMs. On the other hand, MVs acted as anti-inflammatory stimulator on MDMs in both experiments. The response to ANE depended on origin of extract (autologous or heterologous). Macrophages from rabbits before and after OT stimulated with autologous extract generated lower amount of NO and superoxide, especially after transplantation. In the ovine model of Ti implant insertion, heterologous ANE evoked increased macrophage pro-inflammatory activity. Our study revealed that these neutrophil products could regulate activity of macrophages, polarizing them into pro-or anti-inflammatory phenotypes that could enhance bone and osteochondral tissue healing. Inflammation as a response of an organism to local injury involves leukocytes migration to damaged sites to fight bacterial infection and to ensure tissue repair. However, non-resolving inflammation is the cause of many inflammatory disorders such as arthritis and implant rejection ,2. GenerNeutrophils as important effectors of the innate immunity, are involved in many immune mechanisms. As is well known, they are the first line of defense against bacterial and fungal infections and they release some antimicrobial factors, such as reactive oxygen species (ROS) and proteolytic enzymes, capable of, among other things, eliminating the invading pathogens. Except that, neutrophils are participants of coordination of the general inflammatory response. When neutrophil function is insufficient as in excessive bacterial infection their activity should be enhanced. On the other hand, some disorders are involved in excessive neutrophil activation, in which case attenuation of the neutrophil response is needed ,5.There is a continuing debate over the role of neutrophils in bone repair, some authors reported their negative influence on this process. Neutrophils could promote tissue injury by release of some enzymes such as elastase, and free radicals generation and, furthermore, the neutrophil-induced inflammatory response could exacerbate the already existing tissue damage . Other aAntimicrobial peptides (AMP) apart from their direct microbicidal role are known as compounds with immunomodulatory function. Some of them, e.g., human cathelicidin LL-37 suppress pro-inflammatory activation of macrophages, and promote angiogenesis and wound healing . PreviouImplanted biomaterials can significantly influence the immune response and macrophage polarization, either enhancing or reducing inflammation depending on their physicochemical properties . TitaniuKnowledge about the mechanisms involved in degeneration of the cartilage surrounding osteochondral defects which leads to progression of osteoarthritis (OA) is still incomplete . MacrophTo assess the potential of neutrophil-derived products for modifying of the macrophage response we prepared two animal models; the rabbit model of autologous osteochondral graft and the ovine model of insertion of Ti implant into the tibial growth plate. Two different orthopaedic models and two different surgical procedures were considered for assessment of repair process in two different tissues of musculoskeletal system. In the rabbit model we assessed the in vitro response of rabbit monocyte-derived macrophages (MDMs) to autologous rabbit antimicrobial neutrophil extract (rANE), rabbit neutrophil degranulation products (DGP) or rabbit neutrophil microvesicles (MVs). In the ovine model, MDMs were treated in vitro with heterologous porcine neutrophil extract (pANE), ovine DGP or ovine neutrophil MVs.For the experiment, eight New Zealand white rabbits, male, 6 months old, weighing 3.5–4.0 kg were used. The experiment was conducted in accordance with the International Council for Laboratory Animal Science Guidelines for the care and the use of laboratory animals and was approved by the Local Ethics Committee Number II in Lublin (52/2017).TM, Piramal Healthcare Ltd., West Drayton, UK) was used. The autologous osteochondral transplantation (OT) was performed with the use of specially designed instrumentation . The procedure included the collection of full thickness grafts and transplantation from one knee joint into the other in the same rabbit  then isolated neutrophil suspension was homogenized and released neutrophil granules were collected by centrifugation at 25,000× g, 40 min, 4 °C and stirred overnight in 10% acetic acid at 4 °C. The extract containing the antimicrobial peptides (ANE) was separated from the granules , lyophilized and stored at −20 °C. The lyophilized portions were resuspended in the culture medium to a concentration of 20 μg/mL immediately before further experiments  ethylendiamine dihydrochloride 1% sulphanilamide and 2.5% H3PO4) were mixed and incubated at room temperature for 10 min and then absorbance was measured. Values obtained were converted into micromoles (µM) of NaNO2 based on a standard curve . Superoxide anion generation was measured colorimetrically (absorbance read at 545 nm) after incubating neutrophils with 0.1 mg/mL nitroblue tetrazolium  solution in phosphate-buffered saline (PBS) at room temperature for 10 min [Nitric oxide (NO) generation was determined on the basis of nitrite measurement as the stable product of NO in culture medium. Briefly, 100 µL of the culture supernatant and 100 µL of Griess reagent (0.1% r 10 min .7) was incubated with 1 μg/mL fMLP for 20 min at 37 °C 5% CO2. After incubation neutrophils were centrifuged . Obtained supernatant was centrifuged again 20,000× g 30 min, 4 °C and pellet was resuspended in PBS at the original incubation volume. Protein concentration was determined by the Lowry’s method using BSA as standard [The suspension of isolated neutrophils , at 37 °C and 5% CO2 for 24 h in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% bovine calf serum . Cultures were microscopically evaluated (Olympus CK-40) and when cells differentiated into macrophages we started the experiment. Then, the cultures without additional stimulation were control groups (BCS), whereas other were stimulated with DGP, neutrophil MVs or ANE. Measurements of MDMs activity were conducted after 24 and 72 h of incubation at 37 °C with 5% CO2. The functional analysis of MDMs cultures after different stimulation was conducted on the basis superoxide and NO generation and assessment of arginase activity in vitro, as described previously [Peripheral blood mononuclear cells (PBMCs) were isolated as described previously  and culteviously ,24.The study was performed in a group of 8 sheep, females, BCP local breed, 4 months old, about 20 kg body weight, derived from the Bezek Experimental Farm, University of Life Sciences, Lublin. All animals were fed, housed and cared according to the rules concerning the protection of animals. The experiment was approved by the local Ethics Committee No II in Lublin (No 84/2019). For the surgery, animals were anaesthetized with xylazine (0.1 mg/kg intramuscularly) and butorphanol (0.1 mg/kg). Additionally, for local anaesthesia 2% lignocaine was used. The proximal tibia was exposed with standard surgical approach. Then a titanium plate was implanted directly into the proximal growth plate of the tibia .6 cells/mL. DGP preparation was conducted similarly as in rabbit model and cell-free supernatant was collected and stored at −20 °C until later [2. Enzyme release, NO level and superoxide anion generation were measured as in a previously described rabbit model.Porcine ANE was prepared as previously described . The solil later . Culture2 as described in the rabbit model.Isolation of blood derived monocytes, culture and functional assessment were conducted similarly as for preparation and evaluation of rabbit MDMs. Blood for preparation of neutrophil-derived products was collected 7 days before insertion of the Ti implant. At that time hematological tests were also conducted. Monocytes for MDMs cultures were isolated 7 days before Ti implant insertion and 2 h after implantation. These cells were stimulated with DGP, neutrophil MVs or pANE or left without additional stimulation and marked as BCS MDMs (control). Functional analyses of MDMs were conducted in two measurements; after 24 h and 72 h of incubation at 37 °C with 5% COp < 0.05 was set as statistical significance.All in vitro experiments were performed at least three times, and the quantitative data were expressed as mean ± standard deviation. Data obtained were analyzed with Statistica 13.1 . Significance was calculated using a one-way analysis of variance (ANOVA) followed by a Tukey’s test and Isolated neutrophils were used for preparation of neutrophil-derived autologous products. Activity of neutrophils culture used for preparation of DGP was assessed on the basis of enzyme release and NO or superoxide generation and results were summarized in p < 0.05) in measurement after 72 h incubation in comparison with results in measurement after 24 h incubation was observed (p < 0.05) higher in cultures stimulated with DGP in measurement after 72 h in group after OT (p < 0.05) in both cultures before and after OT compared to the BCS groups  superoxide production in all studied groups and measurements  the differences were insignificant (p < 0.05) (We estimated that after stimulation with DGP MDMs cultures generated significantly higher amounts of NO in comparison with BSC MDMs groups in measurement after 24 h incubation at 37 °C and 5% COnificant a. In culnificant b. Argina < 0.05) c. The ra < 0.05) d.Addition of MV-enriched pellet into the macrophage cultures induced decrease of NO generation before and after implantation after 24 h of incubation, whereas after 72 h incubation, a significant decrease of NO generation was noted only in the group after implantation a. StimulOur study revealed that autologous or heterologous neutrophil-derived products could modulate the activity of macrophages, polarizing them into pro- or anti-inflammatory phenotypes. This response may be crucial for repair process in osteochondral tissues or bone regeneration. Previous reports underlined that neutrophils are not only directly microbicidal cells but are also regulators of macrophage function ,15,16,26First we evaluated in vitro activity of degranulation products of isolated rabbit and ovine neutrophils. Obtained results indicated that stimulated neutrophils released elastase, MPO from primary granules, ALP from secondary granules, and generated superoxide and NO. As estimated previously proteins from degranulated neutrophils stimulated monocytes and may alter macrophage function ,16,17,262, whereas the response in the ovine model was more pronounced after 24 h of incubation in both MDMs cultures before and after implantation of the Ti plate into the tibia. According to Hussen et al. bovine DGP do not polarize monocyte–derived macrophages but differentiated them towards a mixed phenotype with enhanced antimicrobial functions [We estimated that in both our models DGP acted as pro-inflammatory stimulator of macrophages. This study revealed that both rabbit and ovine DGP enhanced NO and superoxide production by MDMs in vitro. This response in the rabbit model was noted in both groups before and after osteochondral transplantation after 72 h MDMs incubation at 37 °C and 5% COunctions . In our unctions ,27,28.In the next step of the experiment we estimated that after stimulation with DGP arginase activity decreased in both rabbit MDMs cultures before and after OT in comparison with BCS stimulated macrophages. In the ovine model of Ti implant insertion, decrease of arginase activity in response to DGP was noted only in the group of MDMs obtained after implantation. These results were consistent with the research of Campbell et al.  which inFor better functional analysis of interactions between NO generation and arginase activity in MDMs cultures we calculated urea/nitrite ratio, which was previously introduced by Geelhaar-Karsch et al. . In our Another stimulator used in our study was neutrophil MVs-enriched pellet. Extracellular microvesicles are cell-derived membrane fragments of granules generated by most eukaryotic cells. These structures can transfer biologically active molecules participating in cell adhesion, chemotaxis and angiogenesis . In the In the rabbit model we observed decreased arginase activity in MDMs cultures obtained before and after OT and stimulated with MVs-enriched pellets in comparison with BCS MDMs. On the other hand, in our ovine model, increase of arginase activity in both MDMs cultures before and after implantation was observed. Rhys et al. estimated that MVs alone did not affect macrophage phenotype, suggesting that they inhibit classical activation rather than imparting alternative activation. According to these authors, potential therapeutic application of neutrophil MVs could be considered during treatment of arthritis .Depending on the specific biochemical conditions, the NO/L-ornithine imbalance may be protective or harmful to the target cells or tissues; thus, understanding of the main regulatory mechanisms of the arginase/NOS interplay is crucial for the development of successful diagnosis and treatment . In the In our experiment, macrophage response to antimicrobial extract depended on the origin of extract (rabbit or porcine ANE), animal species (rabbit or sheep) and the status of animal models . We estimated that macrophages from rabbits before and after osteochondral transplantation stimulated with autologous extract generated a lower amount of NO, especially after transplantation both in cultures incubated for 24 and 72 h. Superoxide generation and arginase activity were also diminished in all studied groups of MDMs. The results obtained were consistent with our previous findings . In the We revealed that some neutrophil-derived products, namely; autologous ANE and MVs can cause an anti-inflammatory response of macrophages, whereas others, such as DGP or heterologous ANE, can cause pro-inflammatory activity of these cells. The macrophage responses to neutrophil-derived products during two different tissue repair processes were similar, thus the results obtained may be applicable in different species and different repair process models. These results gives rise to consideration of the use of some natural neutrophil-derived products in different disorders of the musculoskeletal system, however, further research is needed. The importance of this finding lies in the potential of developing innovative therapeutic approaches based on autologous and heterologous neutrophil-derived products for modulation of the macrophage response. To summarize, precise and time-optimized regulation of inflammation is crucial to normal bone osteochondral tissue repair. Understanding of the complex and often contradictory role of inflammation in the musculoskeletal system is needed to develop novel and effective therapies for a variety of pathological conditions. In this context, modulation of macrophage polarization that enhances bone and osteochondral tissue healing represents a new potential strategy for treatment.


ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM ( Knowledge of molecular mechanisms that protect oocytes against polyspermia is important both in basic research and in the future of human medicine, as well as to improve the efficiency of in vitro production (IVP) in animals. It is known that there are two mechanisms that prevent more than one sperm from entering the oocyte . The firIt is generally accepted that frequent abnormal development of in-vitro-produced porcine embryos and early embryonic mortality are often caused by polyspermic penetration. Previous studies described the relationship of higher occurrence of polyspermic penetration with lower meiotic competence, which is characteristic of oocytes of prepubertal gilts ,11. MachDue to the requirements for large numbers of oocytes, prepubertal gilts represent the prevailing source of experimental porcine oocytes. Oocytes of prepubertal gilts before the onset of oestrus do not reach the optimal developmental competence, which results in higher polyspermy rate and significantly lower blastocyst yield than in adult sows, despite there being no differences in nuclear maturation ,14. The Knowledge of the mechanism of oocyte development during folliculogenesis and its effect on developmental potential of the oocyte is an essential prerequisite for improvement of in vitro fertilization (IVF) and in vitro production (IVP) systems. Low monospermic penetration rate and high rate of polyspermy are still the main unsolved problems in porcine embryo production . AlthougThe function of CGs was long believed to start and finish by exocytosis of their contents at the moment of oocyte penetration. However, in some recent studies, authors reported that some CGs undergo exocytosis prior to fertilization, and some cortical granules components function beyond the time of fertilization in regulation of embryonic cleavage and preimplantation development . The process of the oocyte acquiring maturity is highly advanced and requires the involvement of many genes that directly or indirectly influence the achievement of MII stage. The molecular background during the reorganization of intracellular structures in the maturing oocyte has not been well known. Further research is needed to establish the thorough mechanisms regulating these processes.The objective of the study was to further understand the expression of genes related to morphogenesis before and after in vitro maturation of swine oocytes to better define gene pathways involved in maturation. Furthermore, the aim was to better understand the distribution and density of CGs in porcine oocytes in regard to meiotic competency for IVM porcine oocytes.All chemicals used in the present study were purchased from Sigma-Aldrich Chemicals Co.  unless otherwise stated.Ovaries from adult cycling sows were obtained from an experimental slaughterhouse and transported to the laboratory within 2 h at 31–33 °C. The average age of the animals was 40 months and they were derived from crossbred Landrace X Czech Large White. The ovaries of each donor were evaluated and only those with proper morphologic status (presence of ovarian follicles of different sizes) were used. Ovaries were assessed according to morphology of follicles and presence of corpora lutea.Oocytes with higher meiotic competence from medium follicles (MF)—5–9 mm and with lower meiotic competence from follicles smaller than 5 mm (SF)—were collected from ovaries by follicle aspiration and cutting of the ovarian cortex, respectively. Oocytes from larger follicles (≥10 mm) were not included in the study. All healthy cumulus–oocyte complexes with dark, evenly granulated ooplasm and at least two cumulus layers were used for the experiments. 2 for 90 min. Then, the oocytes were transferred to DPBSm and washed twice. During the washing procedure, oocytes were examined under inverted microscope and classified as blue stained (BCB+) or colorless (BCB−). Only BCB+ oocytes were used in molecular analyses (“Before IVM” group) or IVM followed by second BCB test and molecular analyses (“After IVM” group).The Brilliant Cresyl Blue (BCB) staining assay was performed for assessment of porcine oocytes’ quality and maturity and for selection of oocytes for later tests. The enzyme glucose-6-phosphate converts the BCB stain from blue to colourless. In oocytes that completed the growth, activity of the enzyme decreases, and the stain cannot be reduced, resulting in blue oocytes (BCB+). Oocytes were rinsed twice with modified Dulbecco PBS (DPBS)  supplemented with 50 μg/mL streptomycin, 50 IU/mL penicillin , 0.4% BSA (w/v), 0.34 mM pyruvate, and 5.5 mM glucose (DPBSm). They were then treated with 13 μM BCB  diluted in DPBSm at 38.5 °C and 5% CO−1 penicillin, 50 µg mL−1 streptomycin, 10% BFS , and gonadotropins  in a four-well multidish  for 44 h at 39 °C in a humidified atmosphere of 5% CO2. The mean maturation rate was 85%. Only oocytes with verified MII stage represented matured oocytes in the experiment. Oocytes in germinal vesicle stage and mature oocytes were manually denuded of cumulus cells in IVM culture medium with 0.1% (w/v) hyaluronidase (Sigma Aldrich). After washing, the oocytes were fixed in 3.7% paraformaldehyde for 60 min at room temperature. They were washed in PBS and mounted on glass slides, avoiding oocyte compression, using Vectashield medium  containing 1 μM of DNA dye  specific for dyeing of chromatin. The slides were stored below 0 °C until examination. The oocytes were examined with the use of a laser scanning confocal microscope  equipped with argon and helium–neon lasers. The 532 nm excitation band and 550–590 nm detector were used for detection of chromatin. The 40× Leica HCX PL APO CS objective, pinhole, offsets, gain, and Acousto-Optical Beam Splitter (AOBS) were adapted.Isolated cumulus–oocyte complexes that were not used for examination at germinal vesicle (GV) stage were matured in groups of 25–30 in 500 µL of M199 medium , supplemented with 0.20 mM sodium pyruvate, 0.57 mM cysteamine, 50 IU mLcgd). The Index of CGs distribution represents the ratio between mean intensity signal in the peripheral part of oocyte and the mean intensity signal in the central part of oocyte. In matured oocytes (n = 197) all CGs were localized in the peripheral area. The density of CGs from each optical section, scanned at the apical pole of each oocyte, was counted at three different areas on each optical section. Number of CGs/100 µm2 in each oocyte was counted.The methods for staining CGs were based on those reported by Yoshida et al.  and Wangcgd and density of cortical granules were statistically analyzed by the independent samples t-test using STATISTICA CZ, version 10 software . Differences with p < 0.05 were considered as statistically significant.Microphotographs of CGs distribution from confocal microscopy were saved and processed using the NIS-ELEMENTS AR 3.0 software . The values of IFor histological purposes, ovaries from five animals were collected. Immediately after collection, samples were fixed in the Bouin’s solution for 48 h. Consequently, ovaries were dehydrated with increasing concentrations of ethyl alcohol and xylen and then embedded in paraffin blocks. Subsequently, they were cut into 3–4 μm thick sections with a semiautomatic rotary microtome . All ovary sections were stained with a routine hematoxylin and eosin (H&E) staining method following the protocol: deparaffinization and rehydration, staining with H&E, and dehydration. Histological sections were evaluated by light microscope and selected pictures were taken with a use of high-resolution scanning technique and Mirax Midi BF/FL microscope scanner .® . The obtained amount of total mRNA was measured using the optical density at 260 nm, and the purity of RNA from the absorption ratio 260/280 nm (above 1.8) . RNA quality was determined with the use of Bioanalyzer 2100 . Obtained RNA integrity (RIN) ranged from 8.5 to 10 with an average of 9.2 . RNA was diluted to a 100 ng/L concentration with an OD260/OD280 ratio of 1.8/2.0. Each sample was separated and secured for further analysis; only 100 ng was used for microarray assays. The remaining RNA was used for RT-qPCR analysis.The oocyte samples were divided into two groups. The first group contained pooled oocytes (SF and MF) before IVM, the second group included pooled oocytes (SF and MF) after IVM. Each group was combined into three independent samples representing separate experimental groups. Total RNA was extracted from the samples with RNeasy MinElute Kit Cleanup  and TRI Reagent® WT Expression Kit, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA was used for biotin labeling and fragmentation by Affymetrix GeneChip® WT Terminal Labeling and Hybridization . Biotin-labeled fragments of cDNA (5.5 μg) were hybridized to Affymetrix® Porcine Gene 1.1 ST Array Strip (48 °C/20 h). Then, microarrays were washed and stained according to the technical protocol, using Affymetrix GeneAtlas Fluidics Station . The array strips were scanned employing Imaging Station of GeneAtlas System . The preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Operating Software . Quality of gene expression data was checked according to quality control criteria provided by the software. Obtained CEL files were imported into downstream data analysis software [All the experiments were carried out in triplicate ,24. Totasoftware ,26,27.http://www.bioconductor.org/), based on the statistical R  programming language. For background correction, normalization and summation of raw data, the Robust Multiarray Averaging (RMA) algorithm implemented in “affy” package of BioConductor  was applied. Biological annotation was taken from BioConductor “oligo” package where annotated data frame object was merged with normalized data set, leading to a complete gene data table. Statistical significance of analyzed genes was performed by moderated t-statistics from the empirical Bayes method. Obtained p values were corrected for multiple comparisons using Benjamini and Hochberg’s false discovery rate. The selection of significantly changed gene expression was based on p value beneath 0.05 and expression fold higher than |2|. All analyses were performed using BioConductor software . Gene symbols for up- or downregulated genes from each of the compared groups were loaded to DAVID by “RDAVIDWebService” BioConductor package . For further analysis, we chose the enriched GO terms that had at least five genes and p-value (Benjamini) lower than 0.05. The enriched GO terms were subjected to the hierarchical clusterization algorithm and presented as heat maps. Functional annotation clustering of differentially expressed genes was performed using DAVID   [Subsequently, we analyzed the relationship between the genes belonging to chosen GO terms with the GOplot 1.0.2 package  . The GoPhttps://string-db.org/). A list of gene names was used as query for interaction prediction. Searching criteria were based on co-occurrences of genes/proteins in scientific texts (text mining), coexpression, and experimentally observed interactions. The results of such analysis generated a gene–protein interaction network where the intensity of the edges reflects the strength of the interaction score. Besides interaction prediction, STRING also allowed us to perform functional enrichments of GO terms based on previously uploaded gene sets.Interactions between differentially expressed genes/proteins belonging to an ontology group were investigated by STRING10 software  . The Reactome FIViz  app is designed to find pathways and network patterns related to cancer and other types of diseases. This app accesses the pathways stored in the Reactome database, allowing pathway enrichment analysis for a set of genes, visualizing hit pathways using manually laid-out pathway diagrams directly in Cytoscape 3.6.0 software , and then investigating functional relationships among genes in hit pathways. The app can also access the Reactome Functional Interaction (FI) network, a highly reliable, manually curated, pathway-based protein functional interaction network covering over 60% of human proteins.Finally, the functional interaction between genes that belong to the chosen GO BP terms were investigated by REACTOME FIViz application to the Cytoscape 3.6.0 software  into cDNA. RT-qPCR was performed in a real-time LightCycler PCR detection system  using SYBRR Green I as a detection dye, with target cDNA quantified using a relative quantification method . The relcgd) in MF and SF oocytes regardless to the size of follicles are presented in cgd values between oocytes with higher and lower meiotic competence derived from medium and small follicles, respectively, was statistically significant (p < 0.05). Icgd in oocytes with lower meiotic competence (1.48 ± 0.31) shows a tendency of central orientation of CGs in the cytoplasm of oocytes derived from SF (cgd = 2.22 ± 0.49) than their concentration in the central part, indicating movement of CGs towards the cortical area during the acquisition of meiotic competence.The values of the Index of CGs distribution (I from SF . On the p ˂ 0.05) between these experimental groups , 12,258 porcine transcripts were analyzed. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. This set of genes consisted of 419 different transcripts. Subsequently, the genes were used for identification of significantly enriched GO BP terms. Whole transcriptome profiling by Affymetrix microarray allowed us to analyze the gene expression changes in freshly isolated oocytes before the in vitro procedure (“before IVM”) compared to after in vitro maturation (“after IVM”), as pooled samples from SF and MF. Using AffymetrixDAVID software was used for extraction of the genes belonging to “cellular component morphogenesis”, “negative regulation of cell differentiation”, “morphogenesis of a branching structure”, “regulation of cellular component size”, and “branching morphogenesis of a tube” Gene Ontology Biological Process terms (GO BP). We found 39 genes belonging to the ontological group GO BP terms described above. From the results obtained during the analysis of microarrays, 33 genes were significantly represented in downregulated, and only six were upregulated after in vitro maturation. These sets of genes were subjected to the hierarchical clusterization procedure and are presented as heat maps . p values, and LogFC are shown in Sets of the differentially expressed genes belonging to “cellular component morphogenesis”, “negative regulation of cell differentiation”, “morphogenesis of a branching structure”, “regulation of cellular component size”, and “branching morphogenesis of a tube” GO BP terms with their official gene symbols, fold changes in expression, corrected The enrichment of each GO BP term and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway were calculated as Z-score and are shown on the circle diagram in Moreover, in the Gene Ontology database, genes that formed one particular GO group can also belong to other different GO term categories. For this reason, we explored the gene intersections between the selected GO BP terms. The relation between those GO BP terms was presented as a circle plot  as well https://cytoscape.org/) are shown in A STRING-generated interaction network was created with differentially expressed genes belonging to the “cellular component morphogenesis”, “negative regulation of cell differentiation”, “morphogenesis of a branching structure”, “regulation of cellular component size”, and “branching morphogenesis of a tube” ontology groups. The intensity of the edges reflects the strength of interaction score . FinallyThe results obtained from the expression microarrays were subjected to quantitative verification using the RT-qPCR technique. The direction of changes in the expression of 37 of 39 genes has been confirmed . Such diThe presented research, both at the molecular level of gene expression and distribution of cortical granules, was aimed at identifying processes leading to full maturity by the oocyte. In vitro maturation of oocytes is still difficult, which translates into a decrease in IVF process efficiency.The transfer of CGs is associated with meiotic maturation of oocytes and is often considered a determinant of maturity. Therefore, it is very important to know the mechanisms regulating the dynamics of CGs. We have evaluated the distribution of CGs in immature oocytes, both from small and medium follicles. There is a tendency towards central CGs orientation in SF oocytes with lower meiotic competence. On the contrary, in MF oocytes, the peripheral CGs concentration was twice as high as the central concentration , Table 12 of the cortical ooplasm surface decreases during the IVM process. CGs appear to be a dynamic structure, dependent on many factors, and are characterized by a complex distribution during the development of oocytes. The molecular mechanism of formation and releasing of cortical granules are not well known. Many studies emphasize that the fate of the embryo is determined at a very early stage of development, before embryo genome activation (EGA), and even before fertilization. It is also suggested that all mechanical actions on cells are reflected at the molecular level [Additionally, the average value of peripheral density of CGs in MF was lower than in SF . Therefoar level .The microarray study was concentrated on the analysis of genes belonging to “cellular component morphogenesis”, “negative regulation of cell differentiation”, “morphogenesis of a branching structure”, “branching morphogenesis of a tube”, and “regulation of cellular component size” ontological groups by conducting in vitro culture of porcine oocytes in primary culture. The chosen groups refer to the organization of cell structure and its morphogenesis or component size, which may be of key importance for the proper development of oocytes and its usefulness for IVF processes. Analysis of microarrays was performed before and after in vitro maturation of swine oocytes, which enabled us to evaluate molecular markers during IVM. In general, downregulation of the expression of the genes after IVM was observed, except for the group “cellular component morphogenesis”, which, in part, showed upregulation after IVM .The gene expression profile in in-vitro-maturing porcine oocytes showed an increased expression in relation to the state before IVM. All upregulated genes belonged exclusively to the “cellular component morphogenesis” group, associated with the creation of new cells and their components. The gene that is involved in the organization of cytoskeleton, especially microtubules and actin filaments—ARHGEF2 (Rho/Rac guanine nucleotide exchange factor 2) —is upregThe second analyzed gene is closely related to the assembly of the microtubules. The MAP1B gene (microtubule associated protein 1B) plays a predominant role in the development of the nervous system and neural flexibility , althougFurthermore, it was demonstrated that the transport of CGs from the central part of the cell to the membrane is actin-dependent ,6. It isA recently published study on oocytes (OCs) from pigs showed no progression of meiosis and poor distribution of CGs after exposure of OCs to BFA (brefeldin A—a beta-lactam antibiotic) . The indThe next gene, CXCL12 (C-X-C motif chemokine ligand 12), is an extremely important gene that exhibited an increase in expression after IVM of swine oocytes. CXCL12 is also known as SDF-1  and is a CXC group chemokine with a wide range of effects, including hematopoiesis and embryo development . Due to In contrast, increased expression of DAB2 (DAB adaptor protein 2) gene was shown. DAB2 is involved in regulation processes such as cellular adhesion , which iThe FN1 gene (fibronectin) was upregulated in our current studies and is also strongly associated with cellular adhesion processes. Fibronectin is a major component of the extracellular matrix, also involved in the migration of cells associated with the basement membrane from connective tissue . The expHowever, the SOX9 gene (SRY-box transcription factor 9) is associated with the processes related to sex differentiation—its activation of transcription and gonadal development ,62. It aThe remainder of the analyzed genes showed downregulation after IVM of swine oocytes in vitro culture. A large group of genes was closely or indirectly related to oocytes, ovarian follicles, or the reproductive system in general. The first gene that belonged only to the “cellular component morphogenesis” of the ontological group was GJA1 (gap junction protein Alpha 1), one of the key factors in oocyte maturation ,67. GJA1The second gene that may function as a potential marker of oocytes maturation is MAP3K1 (mitogen-activated protein kinase kinase kinase 1). It was shown that this gene plays a role in follicular atresia, which was associated with inhibition of MAP3K1 expression in porcine GCs, indicating its antiapoptotic effect . PreviouThe MMP14  gene is responsible for a wide range of functions associated with angiogenesis, cell migration, and apoptosis . There aThe next gene that showed a decrease in expression after IVM is inhibit subunit beta A (INHBA). It is believed that it plays an important role in reproduction and development through the coding of activins from TGF-beta superfamily ,85 and iAmong many genes influencing the morphology and maturity of oocytes, the extracellular component of the matrix, laminin subunit beta 2 (LAMB2), is probably an important factor. It is a noncollagen component of the basement membrane in the ovarian follicle. Studies on bovine ovarian follicles showed that the expression of this gene in GCs cells was different depending on the location of these cells in the follicle, which also resulted in their hormonal activity . LAMB2 dThe SMAD4 (Mothers against decapentaplegic homolog 4) gene is also strongly associated with ovarian follicle development and GCs function through its association with the TGF-beta signal pathway, moderating apoptosis ,91. In aDownregulation of IHH, ITGB1, and CD9 connected with folliculogenesis or oogenesis was revealed after IVM. IHH , whose expression in the mucous membrane of the murine uterus was caused by the addition of progesterone , and itsThe expression of the FST gene (follistatin) is observed in many tissues of the body, and its product is also called activin-binding protein. FST plays an important role in the regulation of activin and other members of the TGF-beta superfamily ,98, whicA decrease in expression of a large number of genes, which until now were not directly or indirectly related to reproductive processes of the maturation of oocytes or folliculogenesis, was revealed in the current study. The genes exhibiting a decrease in expression were involved in the broadly understood angiogenesis and development of the cardiovascular system, although the mechanisms involved in the building of the corpus luteum after ovulation are often based on the construction of vessels and remodeling of the resulting hemorrhagic corpus luteum. VEGFA , which may have a role in oocyte maturation, , CYR61  , CTNNA2 The last subgroup of genes that are characterized by many different functions with a wide range of mechanisms, but are usually not related to ovaries. The CDK6 (cyclin dependent kinase 6), ZCCHC11 , WWTR1 (WW domain containing transcription regulator 1), UBE2B (ubiquitin conjugating enzyme E2 B), NOTCH2 (notch receptor 2), SPTA1 , TPM1 (tropomyosin 1), which may be linked to oocyte maturation , and CAPThe present study was based on estimation of CGs distribution in the cytoplasm of oocytes acquiring meiotic competence and determination of their gene expression profile. The above changes are caused by many factors and should be further studied. During the maturation of oocytes, the density of CGs in the cytoplasm decreases. It is also worth noting that the distribution of CGs during oocyte maturation becomes more peripheral and probably occurs in concert with cytoskeleton filaments inside the oocyte. The described ARHGEF2 and MAP1B genes, whose increased expression was observed during this study, are closely related to the development of the cytoskeleton, and may therefore constitute an important new marker for the acquisition of meiotic competence of the oocyte. Among the analyzed ontological groups of genes, the increased expression group only concerned genes from the “cellular component morphogenesis” group, which includes the previously mentioned ARHGEF2 and MAP1B genes. Significant changes in the expression level of the examined genes before and after IVM of porcine oocytes may indicate their potential use as markers of competence acquisition by the oocyte. However, it should be noted that our results may serve as a preliminary study and a reference point for subsequent studies. Additionally, the advantage of the study is the research on a pig model, whose genetic homology to humans is very high. Therefore, the obtained results could be used to improve methods of acquiring oocytes of the best competence of both animals and, after validation in humans, in carrying out in vitro fertilization.


Gyps fulvus) nestling samples, wherein enrofloxacin and tolfenamic acid were most commonly detected . To our knowledge, this is the first study including NSAIDs in the exposure assessment of different classes of veterinary pharmaceuticals in live avian scavengers.Pharmaceuticals are still considered emerging pollutants affecting both aquatic and terrestrial ecosystems. Scavenging bird species may be exposed to veterinary drugs when they feed on livestock carcasses provided at supplementary feeding stations, as these are often stocked with ailing and/or recently medicated animals. Because those animals may be a source of several different pharmaceutical compounds, analytical methods to evaluate residue levels and exposure potential should enable detection and quantification of as many different compounds as possible, preferably from small sample volumes. Four different extraction methods were tested to conduct HPLC-MS-TOF analysis of some of the most common veterinary drugs used in livestock in Spain. The method deemed most viable was a simple extraction, using methanol and 100 µL of plasma, that allowed quantification of seven antibiotics  and five nonsteroidal anti-inflammatory drugs (NSAIDs) . The method was then applied to analysis of 29 Eurasian griffon vulture ( The use of pharmaceuticals leads to the constant release of bioactive substances into the environment. Although most of them are rapidly metabolized and degraded, there is a continuous input to the environment through different pathways, while other active substances are more persistent and even bioaccumulative . As suchGyps vulture population across South Asia during the 1990s [Withdrawal periods, time that must elapse after the administration of a veterinary drug to livestock until its residues in animal products for human consumption are below the maximum residue limits (MRLs), are established according to the legally binding food safety standards to avoid risks to human health . Howeverhe 1990s , known ahe 1990s ,15,16,17he 1990s  and othehe 1990s , ketoprohe 1990s  and flunhe 1990s ).For an adequate risk assessment of pharmaceuticals in avian scavengers, it should be taken into account that, considering their feeding habit, there is a real possibility that those species feeding on carcasses at SFSs are exposed to mixtures of pharmaceuticals. Among them, antibiotics and NSAIDs are some of the most commonly used in livestock . TherefoGyps fulvus) nestlings were analyzed. The Spanish vulture population represents a stronghold for this species in Europe, and it can be used as sentinel of pharma-pollution for other endangered species. This evaluation is paramount to avoid population crashes due to mortality directly caused by particular compounds, such as NSAIDs [The aim of this study was to validate a multiresidual analytical technique to detect, in small plasma volumes, veterinary pharmaceuticals (seven antibiotics and five NSAIDs) commonly used in the treatment of livestock. To check the validity of the analytical method, plasma samples from Eurasian griffon vulture  from the Laboratory Animals section from University of Murcia, with authorization code CEEA 177/2015 was used for validation of the technique. These hens were only fed with nonmedicated feed, and they never were treated with pharmaceuticals. Blood was collected by puncturing the brachial vein with a 23 G needle and 5 mL syringe, using heparin as anticoagulant. To obtain plasma, blood samples were centrifuged at 3000 × g for 10 min. To check the validity of the technique, we used plasma of nestling Eurasian griffon vultures (n = 29) sampled from a breeding population in the Central Mountains range and associated canyons in Castilla y León, Central Spain, where a large and increasing vulture population nests on cliffs [g for 10 min) in order to obtain plasma . Ethican cliffs , see lin plasma  in methanol were purchased from Cromlab . Ciprofloxacin-d8 hydrochloride hydrate (ref 32982) was purchased from Sigma Aldrich  and used as internal standard (IS). Methanol and acetonitrile residue quality (>99.9% purity) was obtained from Lab-Scan (Poland). For sample acidification, hydrochloric acid  37% diluted in double-distilled and deionised water until reaching a final concentration of 10% was used. Magnesium sulphate (MgSO4), sodium chloride (NaCl), sodium citrate dibasic sesquihydrate (NaCitrate dibasic sesquihydrate), sodium citrate tribasic dehydrate (NaCitrate tribasic dehydrate), PSA bonded silica  and C18 (Discovery DSC-18: octadecylsilane 18% C) were purchased from Supelco .A mix of certified reference standards at 100 µg mL−1 was made with an appropriate amount of methanol. This mix was used to spike the hen plasma samples for method validation.A standard mix containing every antibiotic and NSAID at 2000 ng mLTo detect and quantify the pharmaceuticals, an HPLC system consisting of vacuum degasser, autosampler and binary pump  was used. Standards or samples (40 µL) were thermostated at 4 °C and injected onto a reversed phase rapid resolution Waters Sunfire C18  HPLC column at a flow rate of 0.8 mL/min. The column was equilibrated at 40 °C. In the case of positive ionization, solvents A (MilliQ water with 0.01% formic acid) and B (acetonitrile) were used for the compound separation. For negative ionization analysis, solvents A (MilliQ water with 5 mM ammonium acetate) and B (acetonitrile) were used. In both conditions, the elution program consisted on a linear gradient from 0% to 45% solvent B for 15 min; a linear gradient from 45% to 95% solvent B for 12 min; 95% solvent B, maintained for 3 min; initial condition (0% solvent B), applied for 3 min before the next injection. The total run time was 33 min. The HPLC system was connected to a time-of-flight mass spectrometer Agilent 6220 accurate mass TOF  equipped with an electrospray interface operating in the positive and negative ionization mode, using the following operation parameters: nebulizer gas pressure, 60 psi; drying gas flow, 12 L/min at 350 °C in both conditions. The capillary spray, fragmentor and octopole voltages were 3500, 180 and 250 V, respectively, in positive ionization, and 3500, 180 and 250 V, respectively, in negative ionization. Profile data in the 100–1100 m/z range were acquired for MS scans in 4 GHz extended high-resolution mode with 2 spectra/s, 500 ms/spectrum and 9652 transients/spectrum. Reference masses at 121.0509 and 922.0098 were used for mass correction during the analysis in positive mode, whereas 112.9859 and 1033.9881 were used in negative modeFull scan data were recorded with Agilent Mass Hunter Data Acquisition software (version B.06.00) and processed with Agilent Mass Hunter Qualitative Analysis software . To identify the compounds, retention times of the analytes were compared with a standard compound (± 0.5 min), with the difference of the theoretical exact mass and the measured accurate masses of the analyte being ≤ 5 ppm. This information is provided in 4, 0.33 g NaCl, 0.17 g Na-citrate dibasic sesquihydrate and 0.33 g Na-citrate tribasic dehydrate) to separate the liquid phase and stabilize the compounds. For this, tubes were vigorously shaken again during 1 min with vortex. The tubes were centrifuged at 5000× g for 5 min (Sanyo MSE MISTRAL 2000 R) and kept at −20 °C for 1 h. Total supernatant was then purified by being transferred to another tube with 300 mg MgSO4, 50 mg PSA and 50 mg DSC-18. The tube was shaken similarly to the first step and then centrifuged again at 5000× g for 5 min. Total supernatant was transferred to a clean tube and evaporated until 1 mL under nitrogen stream to be transferred to a vial for HPLC analysis.In the methods A, B and C, 2 mL of sample (whole blood or plasma) was homogenized with 2 mL of extraction solvent (acetonitrile or methanol). Method A used a mixture of whole blood and methanol, and method B used a mixture of whole blood and acetonitrile. Method C used a mixture of plasma and methanol. In each case, the mix was shaken vigorously with a vortex for 1 min followed by the addition of extraction salts . These replicates were used to obtain a calibration curve by linear regression using the method of least squares, applied subsequently to calculate levels of pharmaceuticals in field samples. The acceptance criterion for linearity was a correlation coefficient r ≥ 0.9.For the technique selection, accuracy and repeatability parameters were calculated using five replicates of spiked samples at 100 ng mLeviously  was baseeviously , using fRSD (%) = (SD/Xm) × 100, where SD is the standard deviation of the whole series of measurements and Xm is the mean. RSD for both parameters should be ≤20% to accept the method. To calculate RSD of repeatability, four replicates of hen plasma samples spiked with the mix of pharmaceuticals at five levels  were analyzed. Reproducibility was calculated by analyzing four replicates of spiked hen plasma at 200 ng mL−1 with different analysts and on different days.To determine the precision of a method, repeatability and reproducibility tests should be applied, calculating relative standard deviation (RSD) as follows: −1), comparing peak areas of the extracted spiked samples with peak areas obtained in the working standard solutions using the following formula: Recovery (%) = (Cm/Cp) × 100, where Cm is the average area of each compound in the sample and Cp is the area of each compound in the standard solution. Blank plasma samples were analyzed to ensure that they were free from the compounds analyzed and to test for a matrix effect.To assess accuracy, recoveries were calculated by analyzing four replicates of spiked hen plasma at five levels  procedure extraction developed previously by Gómez-Ramírez et al.   was achieved by trimethoprim when we applied the QuEChERS method to plasma and used methanol as solvent. Moreover, the lowest recoveries were found in the case of method B, when acetonitrile was used as solvent.As shown in −1 and for all the concentrations tested to validate the technique . However, both compounds showed good linearity and precision values, which are needed to accept recoveries below 60% (but in any case >30%) in multiresidue methods [−1 for all the compounds except carprofen and meloxicam (50 ng mL−1). Chromatograms of each antibiotic and NSAID detected by the validated method are shown in On the contrary, method D (simple extraction of plasma with methanol) provided very good recoveries for all the compounds at 100 ng mLechnique . The hig methods . The limPlasma of 29 griffon vulture nestlings was tested for traces of the pharmaceuticals listed above. As shown in According to the “Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed from the Directorate-General for Health and Food Safety of the European Commission” , we achi−1) than in our method. Similarly, Hermo et al. [−1. We anticipate that further improvements to our method will enable to quantification of other veterinary drugs  to be achieved.Although the use of multiresidue methods to quantify veterinary drugs in small sample amounts (<1 g or mL) is becoming more popular , the numo et al.  also valAlthough other mass spectrometry detectors coupled to HPLC have been commonly used to detect pharmaceuticals , MS-TOF n = 25) [n = 61) and Navarra , Northern Spain, [−1) than that used in other studies  [Among all the antibiotics analyzed, enrofloxacin is the most commonly used for livestock, ranking as second most common product registered for veterinary use in Spain, after amoxicillin . This qun = 25)  and in fn Spain, . Howevern Spain,  and in on Spain, ,15. The o et al. ). These o et al. ). Studieo et al. , ∼50% ofo et al. ,17, althonachus) . TherefoTolfenamic acid was the only NSAID detected in the samples, but the levels were below the LOQ. Its presence is not surprising, as it is used for medication of livestock, including pigs, in Spain . To our To our knowledge, risk assessment studies concerning veterinary drugs in vultures have focused on a single group of compounds ,13,16,17The purpose of the validation of the analytical method has been fulfilled, since it allows the simultaneous quantification of low levels of seven antibiotics of different chemical classes and five NSAIDs in a single analysis, using a small volume of plasma.To our knowledge, this is the first study to detail the exposure assessment of different classes of veterinary pharmaceuticals in scavenger birds with the simultaneous detection of antibiotics and NSAIDs in the same individuals.