High-performance Liquid Chromatographic Determination Research Articles

Pressurized liquid extraction followed by high-performance liquid chromatography for determination of beta-ecdysone extracted from Pfaffia glomerata (Spreng.) Pedersen

Pfaffia glomerata (Spreng.) Pedersen has among its main bioactive compounds saponins, with the phytoestroid β-ecdysone as its chemical marker. In this study, pressurized liquid extraction (PLE), a green extraction technique used to obtain bioactive compounds from plants, was employed to extract beta-ecdysone from P. glomerata leaves, stems, and roots. The 22 factorial design was used with the variables temperature (333 K and 353 K) and flow rate (1.5 and 2 mL min−1), pressure (300 Bar), time (60 min), and solvent [ethanol and distilled water (70:30 (v/v)] were kept constant for all parts of the plant. The results of experimental responses demonstrated that the factors temperature and flow rate significantly interfere with the yields of leaf (0.499%), root (0.65%) and stem (0.764%) extracts. The latter presented presents the highest yield compared to the other parts of the plant. HPLC results showed the presence of beta-ecdysone in all parts of the plant with concentrations of β-ecdysone 86.82, 76.53 and 195.86 mg L−1 to leaf, root and stem, respectively. FT Raman results exhibited typical peaks of beta-ecdysone, such as 3310 cm−1, 1654 cm−1, and 1073 cm−1 for all plant parts. Another interesting result was the presence of the peak at 1460 cm−1 in the PLE root extract can be associated with selenium. This foundational knowledge confirms that the PLE extraction process was efficient in obtaining the chemical marker of Pfaffia glomerata in all plant parts.

Co2+ coordination-assisted molecularly imprinted covalent organic framework for selective extraction of ochratoxin A

Covalent organic frameworks (COFs) are attractive materials for sample pretreatment due to their tunable structures and functions. However, the precise recognition of contaminant in complex environmental matrices by COFs remains challenging owing to their insufficient specific active sites. Herein, we report Co2+ coordination-assisted molecularly imprinted flexible COF (MI-COF@Co2+) for selective recognition of ochratoxin A (OTA). The MI-COF@Co2+ was prepared via one-step polymerization of 3,3-dihydroxybenzidine, 2,4,6-tris(4-formylphenoxy)− 1,3,5-triazine, Co2+ and template. The flexible units endowed COFs with the self-adaptable ability to regulate the molecular conformation and coordinate with Co2+ to locate the imprinted cavities. The coordination interaction greatly improved the adsorption capacity and selectivity of MI-COF@Co2+ for OTA. The prepared MI-COF@Co2+ was used as solid phase extraction adsorbent for high-performance liquid chromatography determination of OTA with the detection limit of 0.03 ng mL−1 and the relative standard deviation of < 2.5 %. In addition, this method permitted interference-free determination of OTA in real samples with recovery from 89.5 % to 102.8 %. This work provides a simple way to improve the selectivity of COFs for the determination of hazardous compounds in complex environments.

High-performance liquid chromatography determination of polyamine contents in poultry based on precolumn benzoyl chloride derivatization

Polyamines are widely present in animal cells and perform important physiological and biochemical functions. Moderate intake of polyamines contributes to good health. Poultry is one of the common sources of polyamines in daily life. However, different tissues from poultry contain different amounts of polyamine. In this study, an efficient and accurate method for the determination of polyamines was developed by high-performance liquid chromatography (HPLC) with precolumn benzoyl chloride derivatization. The linearity of the polyamines was good between 10–400 nmol/mL, with the limit of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 0.37 and 1.23 nmol/mL. The recoveries of the method ranged from 94.73 % to 106.58 % with high accuracy (RSD < 7 %) and no significant matrix effects or interferences. The RSD of intra-day, inter-day and inter-batch repeatability in the range of 0.07–1.57 %, 0.08–1.03 % and 1.46–4.57 %. The method was successfully used to detect polyamines in poultry tissue, with putrescine, spermidine and spermine levels ranging from 3.24 to 29.85 mg/kg, 0.46 to 164.11 mg/kg and 63.39 to 489.57 mg/kg, respectively. Polyamine levels were higher in the spleen, thymus and bursa of fabricius and lower in the heart and muscle. Furthermore, the method was employed to study the effect of exogenous spermine on polyamine deposition in different tissues of geese. In summary, the method is suitable for the detection of polyamine in animal tissues.

Enantioselective three-dimensional high-performance liquid chromatographic determination of amino acids in the Hayabusa2 returned samples from the asteroid Ryugu

The chirality of amino acids in extraterrestrial materials may provide an insight into the origin of the essential l-enantiopure amino acids in the terrestrial biosphere. In 2020, the Hayabusa2 mission succeeded in bringing back surface materials from the C-type asteroid (162173) Ryugu to the Earth. Amino acids were one of the targeted organic molecules to be studied in the Ryugu samples. To analyze the various structural isomers of amino acids, which were expected to be present, from the limited amount of the returned samples, the development of a highly-sensitive and selective analytical method was necessary. In the present study, a three-dimensional high-performance liquid chromatography (3D-HPLC) system has been developed for the enantioselective determination of five proteinogenic and three non-proteinogenic amino acids in the Ryugu samples, in which amino acids in the sample were separated by reversed-phase, anion-exchange and enantioselective columns after the fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiozole. The applicability of the analytical system to the extraterrestrial samples was evaluated by analyzing several types of carbonaceous meteorites before applying the system to the Ryugu samples. In the analysis of the Ryugu samples, all of the target amino acids were successfully determined quantitatively. Non-proteinogenic amino acids including 2-amino-n-butyric acid, isovaline and norvaline, rarely present in the terrestrial environment, were found as almost racemic mixtures with 47.1 to 55.2%l.

Facile construction of a stable core-shell spherically magnetic polyimide covalent organic framework for efficient extraction of phenylurea herbicides

Efficient enrichment is crucial for the highly sensitive monitoring of phenylurea herbicides (PUHs) in various environmental waters. In this work, a stable core-shell spherically magnetic polyimide covalent organic framework (COF) was synthesized via a simple template-mediated precipitation polymerization method under mild conditions using tri(4-aminophenyl)amine (TAPA) and 3,3′,4,4′-biphenyltetracarboxylic dianhydride (BPDA) as the building units (denoted as Fe3O4@TAPA-BPDA). The Fe3O4@TAPA-BPDA exhibits remarkable adsorption performance for PUHs with an optimized adsorption time of only 10 min. The adsorption of PUHs by Fe3O4@TAPA-BPDA followed the pseudo-second-order kinetic model and the Langmuir model. Furthermore, hydrogen bonding, halogen bonding, hydrophobic interaction, electro donor-acceptor interaction and π-π interactions are identified as the dominant mechanisms contributing to excellent adsorption performance. It was demonstrated that halogen bonds play an important role in the adsorption of substances containing chlorine atoms. The Fe3O4@TAPA-BPDA is easy to operate and highly regenerable. A simple magnetic solid-phase extraction (MSPE) method based on the Fe3O4@TAPA-BPDA was then developed for the rapid extraction of five PUHs in real samples, coupled with high-performance liquid chromatography (HPLC) determination. The analytical method developed has a linear range of 0.5–50 ng/mL, and the limit of detection (LOD) ranges from 0.06 to 0.10 ng/mL. The method exhibits good accuracy with recoveries ranged from 74.5 % to 111.4 %. The analytical method was successfully applied to the highly sensitive detection of PUHs in environmental water samples, which highlighting the potential application of the Fe3O4@TAPA-BPDA in the sample pretreatment.

Fabrication of amino- and hydroxyl dual-functionalized magnetic microporous organic network for extraction of zearalenone from traditional Chinese medicine prior to the HPLC determination

Efficient enrichment of trace zearalenone (ZEN) from the complex traditional Chinese medicine (TCM) samples is quite difficult, but of great significance for TCM quality control. Herein, we reported a novel magnetic solid phase extraction (MSPE) strategy for ZEN enrichment using the amino- and hydroxyl dual-functionalized magnetic microporous organic network (Fe3O4@MON-NH2-OH) as an advanced adsorbent combined with the high-performance liquid chromatography (HPLC) determination. Efficient extraction of ZEN was achieved via the possible hydrogen bonding, hydrophobic, and π-π interactions between Fe3O4@MON-NH2-OH and ZEN. The adsorption capacity of Fe3O4@MON-NH2-OH for ZEN was 215.0 mg g−1 at the room temperature, which was much higher than most of the reported adsorbents. Under the optimal condition, the developed Fe3O4@MON-NH2-OH-MSPE-HPLC method exhibited wide linear range (5–2500 μg L−1), low limits of detection (1.4–35 μg L−1), less adsorbent consumption (5 mg), and large enhancement factor (95) for ZEN. The proposed method was successfully applied to detect trace ZEN from 10 kinds of real TCM samples. Conclusively, this work demonstrates the Fe3O4@MON-NH2-OH can effectively extract trace ZEN from the complex TCM matrices, which may open up a new way for the application of MONs in the enrichment and extraction of trace contaminants or active constituents from the complex TCM samples.

In Situ Imaging of Subcutaneous Drug Delivery Systems Using Microspatially Offset Low-Frequency Raman Spectroscopy.

The noninvasive in situ monitoring of the status of drug retention and implant integrity of subcutaneous implants would allow optimization of therapy and avoid periods of subtherapeutic delivery kinetics. A proof-of principle study was conducted to determine the use of microspatially offset low-frequency Raman spectroscopy (micro-SOLFRS) for nonintrusive in situ analysis of subcutaneous drug delivery systems. Caffeine was used as the model drug, and it was embedded in a circular-shape Soluplus matrix via vacuum compression molding. For the exploratory analysis, prototype implants were positioned underneath skin tissue samples, and various caffeine concentrations (1-50% w/w) and micro-SOLFRS displacement settings (Δz = 0-8 mm) were tested from the pseudo three-dimensional (3D)-imaging perspective. This format allowed the optimization of real-time micro-SOLFRS analysis of implants through skin tissue that was embedded in an agarose hydrogel. Notably, this analytical approach allowed the temporal and spatial erosion of the implant and solid-state transformations of caffeine to be distinguished. The spectrometric results correlated with complementary high-performance liquid chromatography (HPLC) determination of changes in drug concentration, illustrating drug dissipation/diffusion characteristics. The discovered capability of micro-SOLFRS for in situ measurements of drugs and implants makes it attractive for biomedical diagnostics that, ultimately, could result in development of a new point-of-care technology.

2,4-Dinitrophenylhydrazine Pre-column Derivatization: High Performance Liquid Chromatography Determination of Residual Formaldehyde in Pharmaceutical Industry and Key Parameters Analysis

2,4-Dinitrophenylhydrazine Pre-column Derivatization: High Performance Liquid Chromatography Determination of Residual Formaldehyde in Pharmaceutical Industry and Key Parameters Analysis

Optimizing Ultrasonic Extraction and Purification of Nuciferine With Response Surface Method

Background Lotus leaves are obtained from plants belonging to the genus Nelumbo in the Nymphaeaceae family. They serve both as agricultural produce and traditional Chinese medicinal herbs. Nuciferine, an amorphine alkaloid found in lotus leaves, holds significance due to its anti-inflammatory, lipid-lowering, and hypoglycemic properties. Objective The factors influencing nuciferine extraction from lotus leaves using the dipping-acid-assisted ultrasonic extraction method (DAUEM) have received limited attention. This study aims to optimize DAUEM conditions (ethanol concentration, solvent-to-material ratio, and ultrasonic extraction time) through a Box-Behnken response surface design. Methods Nuciferine purification from lotus leaves is achieved using D101 macroporous adsorptive resin. A single-factor test, with ethanol concentration ( V/V), solvent-to-material ratio ( V/M), and ultrasonic extraction time as variables, serves as a basis for optimizing the nuciferine extraction process via the response surface methodology. Results The highest nuciferine yield (0.1035 ± 0.005)% was obtained using 74% ethanol ( V/V), a 26:1 solvent-to-material ratio, and an 82min extraction time. Nuciferine purity reached 67.14% using 70% ethanol as the eluent and high-performance liquid chromatography for determination. DAUEM effectively extracts nuciferine from lotus leaves, with optimization achieved using Box-Behnken response surface methodology. D101 macroporous adsorption resin efficiently separates and purifies nuciferine in lotus leaves. Conclusion This experiment demonstrates good precision and accuracy, making it suitable for the extraction, separation, and purification of nuciferine in lotus leaves. The method lays the foundation for the development and utilization of nuciferine.

High-Performance Liquid Chromatographic Determination of Folic Acid Content in Pharmaceutical Formulations marketed in Southwest Nigeria

High-Performance Liquid Chromatographic Determination of Folic Acid Content in Pharmaceutical Formulations marketed in Southwest Nigeria

Simultaneous high-performance liquid chromatography determination of three active compounds in snake fruit peel extracts

Simultaneous high-performance liquid chromatography determination of three active compounds in snake fruit peel extracts

Vortex-assisted microextraction of melamine from milk samples using green short chain ionic liquid solvents coupled with high performance liquid chromatography determination

Melamine is added illegally to milk and dairy products to increase the amount of apparent protein. This organic nitrogen rich chemical compound has been of great challenge in food safety based on its adverse effect on health. Therefore, the extraction and determination of melamine from milk is necessary. Recently, ionic liquid (ILs) as solvent usage has been noticeable for low melting point, low toxicity, high thermal stability, and high extraction capabilities in a wide range of separation processes. ILs are introduced as organic–inorganic salts and green solvents in microextraction preparation. Therefore, in this study, three ionic liquids ([C6mim][NTF2], [C4mim][NTF2] and [C2mim][NTF2] ILs) were prepared and employed as an extraction solvent in dispersive liquid–liquid microextraction (DLLME) of melamine from milk samples followed by HPLC-UV. The selected ILs were designed using three types of alkyl-imidazolium (as the short organic cations) and bis (tri fluoro methyl sulfonyl) imide as anion and characterized by ATR-FTIR spectra, carbon, and hydrogen Nuclear Magnetic Resonance spectroscopy (H&C-NMR) and energy-dispersive X-ray spectroscopy (EDX). These techniques confirmed the formation of functional groups, the structure of hydrogen and carbon atoms, and various elements of ionic bond between imidazolium and bis (tri fluoro methyl sulfonyl) imide. In the next step, the effect of significant parameters, including type and volume of ILs, adsorption time, pH of the sample solution, and sample volume, were optimized. Under the optimal conditions, the limits of detection (LOD), limits of quantification (LOQ), and linearity range were obtained 63.64 µg kg−1, 210.03 µg kg−1, and 210.03–1000 µg kg−1, respectively, for as prepared [C6mim][NTF2] as the best ILs. Notably, the achieved LOQ was lower than the maximum residue level (MRL) for the melamine residue in dairy products. Eventually, the proposed method was applied to detect melamine in milk samples, and the relative recoveries were examined as 79.6–105.0 %.

Application of instantaneous nebulization dispersive liquid-phase microextraction combined with HPLC for the determination of chalcone and isoflavone in traditional Chinese medicines.

A simple and rapid instantaneous nebulization dispersive liquid-phase microextraction method was developed, and combined with high-performance liquid chromatography for determination of the contents of seven analytes in traditional Chinese medicines. In this study, using the sprinkler device to achieve instantaneous synchronous dispersion and extraction, only one spray can rapidly achieve the concentration and enrichment of seven kinds of chalcone and isoflavones. The key factors affecting the extraction efficiency were optimized including the type and volume of extractant, the pH and salt concentration of the sample phase, and the number of dispersion. Under the optimal conditions, the enrichment factor of the target analytes ranged from 103.1 to 180.9, with good linearity and correlation coefficients above 0.9970. The limits of detection ranged from 0.02 to 0.15ng/mL, with good accuracy (recoveries 91.1 to 108.9%) and precision (relative standard deviations 1.5-7.1%). This method has short extraction time (2 s), low organic solvent consumption and high enrichment effect, so it has a wide application prospects.

Polyamidoamine with a hyper-branched structure grafted on modified magnetic graphene oxide for the trace separation of diclofenac and acetaminophen followed by high-performance liquid chromatography determination

Different generations of polyamidoamine dendrimers were synthesized on a focal core of magnetic graphene oxide modified with 3-aminopropyltriethoxysilane. After the characterization of synthesized dendrimers, its second generation was employed as a suitable sorbent for simultaneous separation/preconcentration of diclofenac and acetaminophen by a dispersive magnetic solid phase microextraction. The extracted analytes were then quantified by high-performance liquid chromatography with ultraviolet detection. Under optimized conditions, the limits of detection were 0.3 µg/L for diclofenac and 0.1 µg/L for acetaminophen. The intra-day relative standard deviations at 50 μg L−1 levels were 1.8% for diclofenac and 2.1% for acetaminophen, while the inter-day relative standard deviations were 3.6% and 4.5% for diclofenac and acetaminophen, respectively. The calibration graphs were linear in ranges of 1.0–500.0 µg/L and 0.5–600.0 µg/L for diclofenac and acetaminophen, respectively, with good coefficients of determination (r2 > 0.998). The method was successfully applied to the determination of diclofenac and acetaminophen in water, milk, and biological samples.

Hexafluorobutanol primary alcohol ethoxylate-based supramolecular solvent formation and their application in direct microextraction of malachite green and crystal violet from lake sediments.

A new type of hexafluorobutanol (HFB) primary alcohol ethoxylate (AEO)-based supramolecular solvent (SUPRAS) with density higher than water was prepared for the first time. HFB acted as AEO micelle-forming agent and density-regulating agent for SUPRAS formation. The prepared SUPARS was applied as extraction solvent for vortex-assisted direct microextraction of malachite green (MG) and crystal violet (CV) from lake sediment followed by high-performance liquid chromatographic determination. In the present work, SUPRASs prepared from AEO with different carbon chains as the amphiphiles and various coacervation agents were investigated. SUPARS formed from MOA-3 and HFB provided better extraction efficiency in comparison with other SUPRASs. Parameters influencing the extraction recovery of target analytes including the type and volume of AEO, volume of HFB, and vortex time were investigated and optimized. Under optimized conditions, linearity in the range of 2.0-400μgg-1 for MG and 2.0-500μgg-1 for CV with acorrelation coefficient higher than 0.9947 was obtained. Limits of detection of 0.5μgg-1 and relative standard deviations in the range of 0.9-5.8% were obtained. Compared to conventional extraction techniques for analysis of analytes in solid samples, the proposed method reduced sample usage and eliminated a primary extraction process by using a toxic organic solvent. The proposed method is simple, fast, and green and can be used for the analysis of target analytes in solid samples.

Microextraction by packed sorbent and high-performance liquid chromatography for determination of benznidazole in human plasma

Benznidazole is the main drug used in Chagas disease and its determination in plasma samples is useful in several situations. Hence, robust and accurate bioanalytical methods are needed. In this context, sample preparation deserves special attention, as it is the most error-prone, labor-intensive and time-consuming step. Microextraction by packed sorbent (MEPS) is a miniaturized technique, developed to minimize the use of hazardous solvents and sample amount. In this context, this study aimed to develop and validate a MEPS coupled to high performance liquid chromatography method for the analysis of benznidazole in human plasma. MEPS optimization was performed by a 24 full factorial experimental design, which resulted in about 25 % of recovery. The best condition was achieved when 500 µL of plasma,10 draw-eject cycles, sample volume drawn of 100 µL, and desorption with three times of 50 µL of acetonitrile were used. The chromatographic separation was performed with a C18 (150 × 4.5 mm, 5 µm) column. The mobile phase was composed of water:acetonitrile (60:40) at a flow rate of 1.0 mL min−1. The developed method was validated and proved to be selective, precise, accurate, robust and linear in the range from 0.5 to 6.0 µg mL−1. The method was applied to three healthy volunteers that made use of benznidazole tablets and showed to be adequate to assess this drug in plasma samples.

Quantification of particle number concentration in liposomal suspensions by Laser Transmission Spectroscopy (LTS)

Laser Transmission Spectroscopy (LTS) is an experimental technique able to determine the particle number concentration and the size of colloidal suspensions by a single measurement of the transmittance of a laser beam through the suspension of particles as a function of the wavelength. In this protocol, we show that LTS represents a unique and powerful tool to investigate suspensions of liposomes, where the precise quantification of the number concentration is particularly relevant for the complete definition of the colloidal properties of the suspension. We study a model formulation of Soy-PC:Chol liposomes and we validate LTS results by comparison with High-Performance Liquid Chromatography determination of lipid mass. Then LTS protocols is applied to state-of-art liposomal nanocarrier suspensions. We explain details of data analysis to obtain the particle number concentration by using the Lambert-Beer law and by calculating the extinction cross section, within the framework of Mie theory for spherical vesicles. We also determine the liposome radius and compare it with the hydrodynamic radius measured by Dynamic Light Scattering. As future perspective, we aim to extend LTS analysis to other nanostructures with different geometries and to contribute to the development of new quantitative strategies for the accurate characterization of nanocarriers and other nanoparticles.

Selective determination of formaldehyde by high-performance liquid chromatography with porous graphitic carbon column using N,N′-bis(9-anthrylmethyl)propane-1,3-diamine as derivatizing reagent

Aromatic compounds containing two secondary amino groups were designed and prepared as new derivatizing reagents for aldehydes. One of them, N,N'-bis(9-anthrylmethyl)propane-1,3-diamine (APD), could achieve selective determination of formaldehyde (FA) on a porous graphitic carbon (PGC) column using xylenes, chlorobenzene, and 1-chloronaphthalene as mobile phases by high-performance liquid chromatography (HPLC). The APD-FA derivative was eluted from the PGC column, while the other APD-aldehyde derivatives remained on the column during the HPLC measurements. This specific elution was not observed using mobile phases such as acetonitrile, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, chloroform, benzene, toluene, benzyl alcohol, 2-ethyl-1-hexanol, and pyridine. The APD-FA derivative had a six-membered ring of two tertiary amines identified using 1H NMR spectroscopy. When the π-π interaction of the solvent molecule of the mobile phase with PGC overcame that between the APD-FA derivative and PGC, the APD-FA derivative could be eluted from the column. The best resolution between the peak of the APD-FA derivative and that of free APD was observed when using o-xylene. The optimum derivatization and the HPLC conditions for selective HPLC determination of FA were to conduct the derivatization of FA by heating in an aqueous phase with APD in o-xylene at 100°C. In this method, FA could be derivatized with APD at a mildly neutral pH of 6.7, unlike the low pH required for the derivatization of aldehydes with 2,4-dinitrophenylhydrazine (DNPH), which is commonly used for the derivatization of aldehydes. The detection and quantification limits of FA were 0.8 and 3.5ngmL-1 in this HPLC method with fluorescent detection, respectively. This selective HPLC method could be applied to the determination of FA in various water samples. It was found that only APD among the derivatizing reagents containing two secondary diamines was useful for the selective determination of FA.

Capsule Phase Microextraction Combined with Chemometrics for the HPLC Determination of Amphotericin B in Human Serum

This article discusses the use of a sorbent-based microextraction technique employing a capsule device to isolate amphotericin B (AMB) from human serum before analysis by high performance liquid chromatography (HPLC). AMB is a macrocyclic compound used for the treatment of invasive fungal infections. Before determining AMB in human serum by HPLC, a sample preparation step is required. Capsule phase microextraction (CPME) integrates the stirring and filtration mechanisms in a single unit, simplifying the sample preparation procedure. Moreover, it results in fast extraction kinetics and high extraction efficiency, while it has proved to be a powerful tool for bioanalysis. Different sol–gel sorbent encapsulated microextraction capsules were investigated, and sol–gel Carbowax 20 M was finally chosen as the basis for the microextraction device. Accordingly, the sample preparation protocol was investigated using a face-centered central composite design to achieve good extraction performance. The optimum protocol was validated in terms of linearity, selectivity, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. The linear range of the developed approach was 0.10–10.0 μg mL−1. The LOD value was 0.03 μg mL−1, and the LOQ value was 0.10 μg mL−1. Method accuracy (expressed as relative recovery) was 87–113%, while the relative standard deviation of the repeatability (sr) and within-laboratory reproducibility (sR) were &lt;12.4%. The sol–gel sorbent encapsulated microextraction capsules were reusable for at least 10 extraction cycles. All things considered, the proposed method exhibited good overall performance, and it could be used in bioanalysis for quality control, therapeutic drug monitoring and research purposes.

Evolution of Hydroxytyrosol, Hydroxytyrosol 4-β-d-Glucoside, 3,4-Dihydroxyphenylglycol and Tyrosol in Olive Oil Solid Waste or “Alperujo”

The main by-product generated from the olive oil two-phase extraction system, or alperujo, is undoubtedly a rich source of bioactive components, among which phenolics are one of the most important. The evolution of four of its main phenolics: hydroxytyrosol (HT), hydroxytyrosol 4-β-d-glucoside (Glu-HT), 3,4-dihydroxyphenylglycol (DHPG) and tyrosol (Ty) was studied over two seasons and in ten oil mills under similar climatological and agronomic conditions, for the first time using organic extraction and high-performance liquid chromatography (HPLC-DAD) determination. The results show that HT (200-1600 mg/kg of fresh alperujo) and Ty (10-570 mg/kg) increase, while DHPG (10-370 mg/kg) decreases only in the last month of the season and Glu-HT (1400-0 mg/kg) decreases drastically from the beginning. This evolution is similar between different seasons, with a high correlation between Glu-HT, HT, and Ty. On the other hand, it has been verified that a mixture of alperujos from all the oil mills, which is what the pomace extractor receives, is a viable source of a liquid fraction which is rich in the phenolics studied through organic extractions and especially after the application of a thermal treatment, obtaining values of 4.2 g/L of HT, 0.36 g/L of DHPG, and 0.49 g/L of Ty in the final concentrated liquid fraction.