Capillary electrophoresis-based assessment of nanobody affinity and purity

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced “nanobody” EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50 mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0–12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (K_d) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the K_d of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE–MS using a BGE of 100 mM acetic acid (pH 2.8) in combination with a polybrene–dextran sulfate–polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE–MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.     

Determination of ethylenediaminetetraacetic acid, ethylenediaminedisuccinic acid and iminodisuccinic acid in cosmetic products by capillary electrophoresis and high performance liquid chromatography

A capillary electrophoresis (CE) and a high performance liquid chromatography (HPLC) method are described for the simultaneous determination of ethylenediaminetetraacetic acid (EDTA), S,S′-ethylenediaminedisuccinic acid (EDDS) and R,S-iminodisuccinic acid (IDS) complexing agents as their Fe(III) complexes in cosmetics like shower cream and foam bath. The non-biodegradable EDTA is used in combination with biodegradable analogues like EDDS and IDS in many commercial products. The HPLC method involves separation by reversed-phase ion pair chromatography on a C₁₈ column using methanol-formate buffer (20 mM tetrabutylammonium hydrogen sulfate, 15 mM sodium formate adjusted to pH 4.0 with formic acid) (10:90, v/v) as mobile solvent at a flow rate of 0.8 mL min⁻¹ at 24 °C using UV detection at 240 nm. The CE separation was performed in a fused silica capillary of 50 μm i.d. with the total length of 50 cm with a 10 mM MES and MOPSO (pH 5.5) at an applied voltage of −25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s. Absorbances at 215 and 225 nm were monitored for the detection of the complexes. The methodology performance of the two methods was evaluated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The LOD values obtained from HPLC are low when compared with CE. The applicability of both the methods was demonstrated for the analysis of cosmetic products such as shower cream and foam bath. The results obtained by both CE and HPLC were found to be comparable and in good agreement.     

Tungstate as complex-forming reagent facilitating separation of selected polyphenols by capillary electrophoresis and its comparison with borate

A novel electrophoretic BGE containing tungstate as complex-forming reagent is suitable for the separation of polyphenols. Similar to molybdate-containing BGE reported earlier (cf. M. Polásek, et al.., Talanta 2006, 69, 192) addition of tungstate to BGE affects significantly migration of compounds/ligands with vicinal -OH groups due to the formation of negatively charged complexes involving W(VI) as central ion. Baseline separation of mixtures of flavonoids (apigenin, luteolin, hyperoside, quercetin, and rutin) and phenolic acids (chlorogenic and p-coumaric acid) was achieved within 15 min with optimized BGE of pH 7.4 containing 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES), 2.2 mM tungstate, and 25% v/v of methanol. The separation was performed in a 75 cm (effective length 42 cm)x 75 microm id uncoated fused-silica capillary at 30 kV with spectrophotometric detection at 275 nm. The calibration curves were rectilinear for 25-175 microg/mL of all analytes (cinnamic acid as the internal standard). The LODs ranged from 1.8 to 6 microg/mL for all analytes except for chlorogenic acid. Intraday precision (n = 6) of migration times (RSD < or = 1.2%) and peak areas (RSD < or = 5.6%) was evaluated. The tungstate-based BGEs can be alternatively utilized for the analysis of polyphenols at considerably lower pH than with conventional alkaline borate-based BGEs.     

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability (σ_rel ≤ 4.4%), precision (inter-day σ_rel ≤ 8.13%, intra-day σ_rel ≤ 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) ≤ 4.5 μg mL⁻¹) and linearity (R² ≥ 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3-O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.     

Separation and determination of alpinetin and cardamonin in Alpinia katsumadai Hayata by flow injection-micellar electrokinetic chromatography

A simple, rapid, and accurate method for the separation and determination of alpinetin and cardamonin in Alpinia katsumadai Hayata was developed by combination of flow injection (FI)-micellar electrokinetic chromatography (MEKC) for the first time. The analysis was carried out using an unmodified fused-silica capillary (50 μm i.d.; total length 13.6 cm; effective length 10.3 cm) and direct ultraviolet (UV) detection at 214 nm. The sample throughput was 11-24 samples per hour using the background electrolyte (BGE) containing 4 mM sodium borate-8 mM NaH₂PO₄ (pH 8.1)-8 mM sodium dodecyl sulfate (SDS)-19% (v/v) ethanol. The repeatabilities (n = 4) reached relative standard deviation values (R.S.D.) of 3.0% and 2.5% for the peak areas and 2.5% and 3.1% for peak heights of alpinetin and cardamonin, respectively. Regression equations revealed linear relationships (r²: 0.9993-0.9994) between the peak area of each analyte and the concentration. Recoveries were in the range 90-92% and 99-105% for alpinetin and cardamonin, respectively.     

Determination of polyphenolic compounds by liquid chromatography–mass spectrometry in Thymus species

Polyphenolic compounds represent a wide group of phytochemicals, including well-known subgroups of phenolic acids, flavonoids, natural dyes, lignans etc., which are produced by plants. These natural bioactive compounds possess a variety of beneficial effects including antioxidant and anticarcinogenic activities, protection against coronary diseases as well as antimicrobial properties. Thymus species have already been reported as sources of different phenolic acids and flavonoids. Moreover, the composition and content of flavonoids in Thymus species play important role as taxonomic markers providing distinction of species. High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and on-line mass spectrometry (ESI-MS) method was used for analysis. The method was evaluated for a number of validation characteristics (repeatability and intermediate precision, LOD, LOQ, calibration range, and recovery). The polyphenolic pattern of five native Hungarian Thymus species (T. glabrescens Willd., T. pannonicus All., T. praecox Opiz, T. pulegioides L., and T. serpyllum L.) was characterized. The dominant compound was rosmarinic acid, which ranged between 83.49 μg g⁻¹ and 1.436 mg g⁻¹. Other phenolic acids (ferulic acid, caffeic acid and its other derivatives, chlorogenic acid and p-coumaric acids) were present in every examined Thymus species, as well as flavanones: naringenin, eriodictyol and dihydroquercetin; flavones: apigenin and apigenin-7-glucoside, flavonols: quercetin and rutin. The polyphenolic pattern was found to be a useful additional chemotaxonomic tool for classification purposes and determination of the locality of origin.     

Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-beta-cyclodextrin as modifier

A fast capillary zone electrophoresis (CZE) method, using dimethyl-β-cyclodextrin (DM-β-CD) as modifier, has been developed for the determination of three flavonoids (quercetin (QU), kaempferol (KA) and isorhamnetin (IS)) in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon tablet). Optimum separation was achieved with 20 mM borate buffer at pH 10.0 containing 5 mg ml⁻¹ of DM-β-CD. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992 and 0.9996) between the peak area of each compound (QU, KA and IS) and its concentration. The relative standard deviations of migration times and peak areas were <1.53 and 4.14%, respectively. The effects of several CE parameters on the resolution were studied systematically. The contents of three flavonoids in H. rhamnoides were successfully determined with 4.5 min, with satisfactory repeatability and recovery. It was also tested that the possibilities of using this method for the determination of flavonoids in Chinese medicinal preparation.     

Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

Absorption and emission behaviour of trans-p-coumaric acid in aqueous solutions and some organic solvents

The absorption and fluorescence behaviour of trans-p-coumaric acid (trans-4-hydroxycinnamic acid) is investigated in buffered aqueous solution over a wide range from pH 1 to pH 12, in un-buffered water, and in some organic solvents. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and degrees of fluorescence polarisation are measured. p-Coumaric acid exists in different ionic forms in aqueous solution depending on the pH. There is an equilibrium between the neutral form (p-CAH₂) and the single anionic form (p-CAH⁻) at low pH (pK_na ≈ 4.9), and between the single anionic and the double anionic form (p-CA²⁻) at high pH (pK_aa ≈ 9.35). In the organic solvents studied trans-p-coumaric acid is dissolved in its neutral form. The fluorescence quantum yield of trans-p-coumaric acid in aqueous solution is ?_F ≈ 1.4 × 10⁻⁴ for the neutral and the single anionic form, while it is ?_F ≈ 1.3 × 10⁻³ for the double anionic form. For trans-p-coumaric acid in organic solvents fluorescence quantum yields in the range from 4.8 × 10⁻⁵ (acetonitrile) to 1.5 × 10⁻⁴ (glycerol) were measured. The fluorescence spectra are 7700–10,000 cm⁻¹ Stokes shifted in aqueous solution, and 5400–8200 cm⁻¹ Stokes shifted in the studied organic solvents. Decay paths responsible for the low fluorescence quantum yields are discussed (photo-isomerisation and internal conversion for p-CA²⁻, solvent-assisted intra-molecular charge-transfer or ππ^∗ to nπ^∗ transfer and internal conversion for p-CAH₂ and p-CAH⁻). The solvent dependence of the first ππ^∗ electronic transition frequency and of the fluorescence Stokes shift of p-CAH₂ is discussed in terms of polar solute–solvent interaction effects. Thereby the ground-state and excite-state molecular dipole moments are extracted.     

Determination of quinolizidine alkaloids in Sophora medicinal plants by capillary electrophoresis

A new capillary electrophoresis (CE) method for the determination of quinolizidine alkaloids in Sophora medicinal plants was developed. A total of seven alkaloid components (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) were separated within 15 min. The running buffer was a 50 mM phosphate buffer containing 1%HP-β-CD and 3.3% isopropanol. The linear calibration ranges were 5.50-88.0 μg ml⁻¹ for cytisine and lehmannine, 5.00-88.0 μg ml⁻¹ for sophocarpine and sophoranol, 5.60-89.6 μg ml⁻¹ for matrine and oxysophocarpine, and 24.0-384 μg ml⁻¹ for oxymatrine. The recoveries of the seven alkaloids were 96.0-102.9% with relative standard deviations from 1.50 to 3.00% (n = 5). The method was successfully applied to different Sophora medicinal plants including Sophora flavescens, Sophora tonkinensis and Sophora alopecuroides.     

Combined microwave-assisted isolation and solid-phase purification procedures prior to the chromatographic determination of phenolic compounds in plant materials

A fast, sensitive and selective procedure employing a combination of microwave-assisted extraction (MAE) and solid phase extraction (SPE) was applied prior to liquid chromatographic identification and quantification of phenolic compounds in plant materials. MAE has been tested and optimized for the isolation of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanilic, caffeic, syringic, p-coumaric, ferulic, sinapic, benzoic, m-coumaric, o-coumaric, rosmarinic, cinnamic acids) and 3,4-dihydroxybenzaldehyde, syringaldehyde, p-hydroxybenzaldehyde, and vanillin in various plants. The effects of experimental conditions on MAE efficiency, such as solvent composition, temperature, extraction time, have been studied. The extraction efficiencies were compared with those obtained by computer-controlled, two-step Soxhlet-like extractions. Plant extracts were purified and phenolic compounds were pre-concentrated using SPE on polymeric RP-105 SPE sorbent prior to HPLC analysis. Chromatographic separation was carried out on a Hypersil BDS C₁₈ column using a mobile phase consisted of 0.3% (v/v) acetic acid in water (solvent A) and methanol (solvent B) at flow rate 0.6 ml min⁻¹ and column temperature 30 °C with gradient elution.     

Determination of flavonoids and saponins in Gynostemma pentaphyllum (Thunb.) Makino by liquid chromatography-mass spectrometry

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chinese herb possessing antitumor and antioxidant activities, has been shown to contain several functional components like saponins and flavonoids. However, their identities remain uncertain. The objectives of this study were to develop an appropriate extraction, purification and HPLC-MS method to determine saponins and flavonoids in G. pentaphyllum. Both flavonoids and saponins were extracted with methanol, followed by purification with a C18 cartridge to elute the former with 50% methanol and the latter with 100% methanol. A total of 34 saponins were separated within 40 min by a Gemini C18 column and a gradient mobile phase of acetonitrile and 0.1% formic acid in water, in which 18 saponins were identified by LC-MS with ESI mode and Q-TOF (LC/MS/MS). Similarly, a total of eight flavonoids were separated within 45 min by the same column and a gradient solvent system of methanol and 0.1% formic acid in water, with identification being carried out by a post-column derivatization method and LC-MS with ESI mode. The amounts of flavonoids in G. pentaphyllum ranged from 170.7 to 2416.5 μg g⁻¹, whereas saponins were from 491.0 to 89,888.9 μg g⁻¹.     

Trace determination of short-chain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

In this study, a new capillary electrophoresis (CE) method is described originally for the sensitive and selective determination of short-chain aliphatic amines in biological samples. These amines were converted into their N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) derivatives and measured by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. The derivatization conditions and separation parameters for the aliphatic amines were optimized in detail. The SIFA-labeled amines were fully separated within 8.5 min using 25 mM pH 9.6 boric acid electrolyte containing 60 mM sodium dodecyl sulfate (SDS). The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.02 to 0.1 nM, which was the lowest value reported by CE methods. The developed method was successfully employed to monitor aliphatic amines in serum and cells samples. After comparison of other CE methods using different fluorescent probes, the present method represents a powerful tool for the trace determination of aliphatic amines in complex biological samples.     

In-vial liquid–liquid microextraction-capillary electrophoresis method for the determination of phenolic acids in vegetable oils

An in-vial liquid–liquid microextraction method was developed for the selective extraction of the phenolic acids (caffeic, gallic, cinnamic, ferulic, chlorogenic, syringic, vanillic, benzoic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, o-coumaric, m-coumaric and p-coumaric) in vegetable oil samples. The optimised extraction conditions for 20 g sample were: volume of diluent (n-hexane), 2 mL; extractant, methanol: 5 mM sodium hydroxide (60:40; v/v); volume of extractant, 300 μL (twice); vortex, 1 min; centrifugation, 5 min. Recoveries for the studied phenolic acids were 80.1–119.5%. The simultaneous determination of the phenolic acid extracts was investigated by capillary electrophoresis (CE). Separations were carried out on a bare fused-silica capillary (50 μm i.d. × 40 cm length) involving 25 mM sodium tetraborate (pH 9.15) and 5% methanol as CE background electrolyte in the normal polarity mode, voltage of 30 kV, temperature of 25 °C, injection time of 4 s (50 mbar) and electropherograms were recorded at 200 nm. The phenolic acids were successfully separated in less than 10 min. The validated in-vial LLME-CE method was applied to the determination of phenolic acids in vegetable oil samples (extra virgin olive oil, virgin olive oil, pure olive oil, walnut oil and grapeseed oil). The developed method shows significant advantages over the current methods as lengthy evaporation step is not required.     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Voltammetric determination of boron by using Alizarin Red S

Cordycepin is the main active metabolite of Cordyceps militaris extracts; according to recent studies it has interesting therapeutic activities. A new capillary electrophoresis (CE) procedure with UV detection at 254 nm for determination of cordycepin was developed and optimized. Optimal conditions found were 20 mM sodium borate buffer with 28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 s and temperature 25 °C. Linearity was found over the 20-100 μg/mL concentration ranges of cordycepin. The developed method has been applied for determination of cordycepin in various pharmaceutical products. A comparison was made between CE and a high performance liquid chromatography (HPLC) method. Both of these methods gave comparable results. The shorter analysis time and low running cost are the main advantages of CE method.     

Fast determination of flavonoids in Glycyrrhizae radix by capillary zone electrophoresis

A fast capillary zone electrophoresis (CZE) method has been developed for the determination of four flavonoids (liquiritin, licoisoflavone A, licochalconel A and calycosin) in Glycyrrhizae radix. After a series of optimization experiments, 100 mM borate buffer (pH 10.5), 30 kV applied voltage and 35 °C temperature were selected. The contents of four flavonoids in cultivated and wild crude drugs of Glycyrrhizae radix with different growth periods from one to four years, collected from different areas were successfully determined within 8 min, with satisfactory repeatability and recovery.     

Simultaneous determination of nine flavonoids in Anaphalis margaritacea by capillary zone electrophoresis

A capillary zone electrophoresis method was developed for simultaneous determination of nine flavonoids, including two rare flavonols, in Tibetan medicine Anaphalis margaritacea. Baseline separation was performed at pH 9.6 with 25 mM Na₂B₄O₇ and 10 mM NaH₂PO₄ buffer solution, 20 kV as driving voltage and 275 nm as detection wavelength. Repeatability tests showed that the R.S.D. of both intra- and inter-day migration times and peak areas were less than 5%. Recovery results ranged from 87.9% to 106.1%. Samples of A. margaritacea extracts were analyzed using the validated method, which is useful for its quality control.     

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: Application to labetalol hydrochloride

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C⁴D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol⁺) and its anionic counter-ion (Cl⁻) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software.This novel CE-C⁴D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time < 10 min), precise (repeatability of migration times < 0.7% and of corrected-peak areas < 3.3%; n = 6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.     

High dielectric constant PrYxOy sensing films electrolyte-insulator-semiconductor pH-sensor for the detection of urea

In this paper, we describe the structural and sensing properties of high-k PrY_xO_y sensing films deposited on Si substrates through reactive co-sputtering. Secondary ion mass spectrometry and atomic force microscopy were employed to analyze the compositional and morphological features of these films after annealing at various temperatures. The electrolyte-insulator-semiconductor (EIS) device incorporating a PrY_xO_y sensing membrane that had been annealed at 800 °C exhibited good sensing characteristics, including a high sensitivity (59.07 mV pH⁻¹ in solutions from pH 2 to 12), a low hysteresis voltage (2.4 mV in the pH loop 7 → 4 → 7 → 10 → 7), and a small drift rate (0.62 mV h⁻¹ in the buffer solution at pH 7). The PrY_xO_y EIS device also showed a high selective response towards H⁺. This improvement can be attributed to the small number of crystal defects and the large surface roughness. In addition, the enzymatic EIS-based urea biosensor incorporating a high-k PrY_xO_y sensing film annealed at 800 °C allowed the potentiometric analysis of urea, at concentrations ranging from 1 to 16 mM, with a sensitivity of 9.59 mV mM⁻¹.