The use of plasmatic acceptors as specific ligands in affinity chromatography cleanup of veterinary drugs from biological matrices

Bovine α₁-acid glycoprotein (bAAG) and bovine serum albumin (BSA) are plasmatic acceptors working as carriers by the specific and reversible binding of several drugs in vivo. We synthesized affinity columns by coupling bAAG and BSA to an activated chromatographic support through their carbohydrate moieties, to preserve protein tertiary structure and, consequently, to improve the biological activity in vitro. The bAAG and BSA affinity columns were used to study the binding of acidic and basic drugs. Moreover, a purification strategy was developed for the cleanup of drug residues from biological matrices and foods, prior to screening and/or confirmatory analysis, on the basis of the specific molecular recognition between the protein and the drug. The aim of this work was to test the potency of bAAG- and BSA-based affinity chromatography to bind some veterinary drugs and purify them in the context of the official control of animal products. The efficiency of these homemade affinity columns in minimising matrix interference and in selective cleanup of different classes of substances was reported and discussed. Figure “Coupling strategies in synthesizing plasmatic acceptor affinity columns: the covalent coupling of bAAG and BSA through their carbohydrate moiety allows one to preserve the gross tertiary structure of the protein and thus its biological activity, whereas coupling through the ε-amine group of lysine residues can reduce the interactions with the binding sites of the plasmatic acceptor.”     

Separation of the enantiomers of basic drugs by affinity capillary electrophoresis using a partial filling technique and α1-acid glycoprotein as chiral selectorglycoprotein as chiral selector

Summary Separation of the enantiomers of a variety of basic drugs by affinity capillary electrophoresis has been investigated using α₁-acid glycoprotein (α₁-AGP) as chiral selector. In order to use a high concentration of α₁-AGP without causing low detection sensitivity, the partial filling technique was employed. Enantiomer separations were performed under conditions (a running buffer at pH 5.0 or 6.0) causing the protein to migrate toward the injection end. Twenty nine basic racemates were successfully separated by optimizing the protein concentration, buffer pH and organic modifier. α₁-AGP obtained from three different suppliers was used to investigate differences among the proteins from different sources. Although most of the racemates were similarly separated with any of the three types of α₁-AGP, some racemates, e.g. acebutolol behaved differently with the three types. The reasons for the different enantioselectivities of the three types of α₁-AGP has not yet been clarified. The method was used to test the optical purity of commercial sulpiride enantiomers and it was found that the method was suitable and applicable for the purpose.     

Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography

Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG₁ monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG₁ mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG₁ mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG₁ molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture.     

In situ aqueous derivatization and determination of non-steroidal anti-inflammatory drugs by salting-out-assisted liquid-liquid extraction and gas chromatography-mass spectrometry

A new analytical method for the determination of trace levels of five non-steroidal anti-inflammatory drugs (NSAIDs: clofibric acid, ibuprofen, naproxen, diclofenac and ketoprofen) in water samples is described. The analytical procedure involves in situ aqueous derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 2,2,2-trifluoroethylamine hydrochloride (TFEA) and salting-out liquid-liquid extraction (SALLE), followed by gas chromatography-programmed temperature vaporizer-mass spectrometry (GC-PTV-MS). The influence of several parameters on the efficiency of the derivatization (stirring time, reaction time, reagent concentration and pH), and the extraction (solvent, volume, salts and stirring time) and injection steps (liner, injection volume, liner temperature, injection time, venting time and venting flow) was investigated. The detection limits of the method in water varied from 0.042 μg/L for ibuprofen to 1.2 μg/L for ketoprofen. The relative standard deviations (RSD) values were found to be relatively low (<10% for all compounds). The methodology developed was applied to the determination of NSAIDs in several environmental matrices including tap, river, sea and influent and effluent waste water samples. The results obtained show the presence of ibuprofen and naproxen in the influent waste water sample.     

Coupled liquid-liquid extraction and column switching LC for the determination of a new antihistaminic H1 drug in human urine

Summary Mizolastine (SL 85.0324) is a new antihistaminic H₁ benzimidazole derivative which is excreted into urine almost completely metabolized; about 2% of the unchanged drug is excreted as conjugated compound which requires enzymatic deconjugation before analysis. Since the existing methods for plasma samples do not work on deconjugated human urine due to interferences, a new method was developed. The method is based on a diethyl-ether extraction of mizolastine and an internal standard from alkalinized urine. The ether extract is back-extracted with an aqueous buffer (pH=2.6), this extract is neutralized (pH=6.5) and an aliquot injected into a C₁₈ pre-column where clean up and preconcentration take place. The analytes are then desorbed from the pre-column and transferred to the analytical column. The analytical column is a C₁₈ type specially seactivated for basic compounds with an eluent of acetonitrile/phosphate solution (pH=4.5), 40/60, v/v, at a flow rate of 1 ml min^–1. Detection is at 285 nm. The method is linear in the range 10–500 ng ml^–1 with a lower limit of detection of 10 ng ml^–1. The precision and accuracy, evaluated during intra-day and inter-day assays, are satisfactory for pharmacokinetic investigations.     

Development of a methodology for the simultaneous determination of inorganic and organolead compounds using supercritical fluid extraction followed by gas chromatography-mass spectrometry and its application to environmental matrices

A method for the extraction of triethyl lead (TEL⁺), trimethyl lead (TML⁺), and Pb²⁺ from sand was developed using supercritical modified CO₂-CH₃OH extraction and in situ complexation with sodium diethyldithiocarbamate (NaDDTC) using a 2⁵ factorial exploratory design is described. The screened variables were (i) pressure (69-193 bar), (ii) temperature (40-150 °C), (iii) ligand amount (0-100 mg), (iv) methanol volume (0.0-0.5 mL) and (v) static time (0-45 min). The optimum extraction conditions found were as follow: pressure, 193 bar; temperature, 40 °C; amount of NaDDTC, 100 mg; methanol volume, 0.5 mL; static time 45 min; and CO₂ flow rate, 1 mL min⁻¹. Under these conditions the following recoveries were obtained (TML⁺ 97 ± 2%, TEL⁺ 70 ± 5%, and Pb²⁺ 100 ± 4%). The presence of NaDDTC is not necessary for the extraction of TML⁺ and TEL⁺, but it is a very significative parameter for Pb²⁺. A second experimental design 2² + star for temperature and pressure was realized, but the results were not better than those of the first model. SFE extract derivatization was achieved with pentylmagnesium bromide, and target analyte determination was carried out by gas chromatography-mass spectrometry. Detection limits in the full-scan mode were 4, 10, and 39 pg as lead for TMPeL, TEPeL and PbPe₄, respectively. The method was validated with urban dust containing TML⁺ (CRM 605. Pb 7.9 ±1.2 μg kg⁻¹) and river sediment containing inorganic lead (GBW08301. Pb 79.0 ± 12.0 mg kg⁻¹) as reference materials. The proposed method was applied to lead analysis in sand collected from an oil-polluted beach in Chile.     

Simultaneous determination of sex hormones in egg products by ZnCl2 depositing lipid, solid-phase extraction and ultra performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method was developed for simultaneous determination of residues of 17 sex hormones in egg products. Target compounds were extracted from samples with methanol in an ultrasonic bath, effectively separated from lipids in the extracts by ZnCl₂ depositing filtration and purified using a C₁₈ solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. The analytes were quantified by liquid chromatography using a BEH C₁₈ column coupled to an electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS) operating in negative mode for estrogens and in positive multiple reaction monitoring mode for androgens. The parameters of the mass spectrometer and the composition of mobile phase and additives were also optimized to enhance detection sensitivity. Average recoveries of the target compounds varied from 70.0% to 121.0% with relative standard deviations ranging from 2.3% to 11.2% at two fortification levels. The limits of detection (LOD) of the method were from 0.002 μg kg⁻¹ to 0.23 μg kg⁻¹ and the limits of quantification (LOQ) were in the range of 0.007-0.76 μg kg⁻¹.     

Comparison of alpha1-adrenergic receptor cell-membrane stationary phases prepared from expressed cell line and from rabbit hepatocytes

A G-protein-coupled receptor-cell-membrane stationary phase (CMSP) has been prepared by immobilizing cell membranes on the surface of silica, as carrier. The resulting HEK293 α _1A adrenoceptor cell-membrane stationary phase can be used for rapid on-line chromatographic determination of potential subtype-selective α ₁ -adrenoceptor ligand-binding affinities for α ₁ -adrenoceptor subtypes. The objective of the research was to study whether cell lines stably overexpressing subtype receptors could improve the sensitivity and specificity of cell-membrane chromatography (CMC) compared with use of homogenized tissue and cells in primary culture. Effects of mobile-phase ionic strength, sample concentration, and the presence of competitive agents on ligand-receptor interaction in CMSP were also evaluated. We found that cell lines stably overexpressing subtype receptors led to improved sensitivity and specificity in CMC. The technique leads to useful procedures-cell-membrane stationary phases may, for example, facilitate exploration of ligand-receptor interaction and determination of ligand-receptor binding affinity in initial screening and separation of lead compounds or active components in Chinese traditional natural medicine and herbs. This might eventually be an important contribution and an addition to our collection of techniques.     

Determination of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair by solid-phase extraction and gas chromatography-mass spectrometry

Summary A method for the determination of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair has been developed and validated. Hair samples (200 mg) were dissolved in NaOH (1 M) and PhIP was isolated by successive solid-phase extraction on a polystyrene-divinylbenzene column and on a silica-based mixed-mode column with C₈ and-SO₃ ⁻ functional groups. Quantification was performed by gas chromatography-electron-impact ionization high-resolution mass spectrometry in selected-ion-monitoring mode. The method was validated for determination of PhIP in the concentration range 0.5–25 ng g⁻¹ hair with [²H₃]PhIP as internal standard. The limit of quantification was 0.26 ng g⁻¹ hair. Within-day and between-day precision were in the ranges 1–27% and 2–15% relative standard deviation, respectively. The hair sample used for method validation was found to contain 0.26 ng PhIP g⁻¹ hair.     

离子色谱法测定合成气制烯烃产物中的C_1~C_6有机酸

采用离子色谱建立了合成气制烯烃(SGTO)水相产物和油相产物中C1~C6有机酸的测定方法。对分离条件进行了优化,使用标准样品测定了线性范围和工作曲线,考察了方法的精密度和准确度,确定了SGTO油相产物样品的碱洗条件,并对SGTO水相产物和油相产物样品进行了测定。结果表明:C1~C6有机酸的质量浓度在各自配制的质量浓度范围内呈现良好的线性关系,相关系数均大于0.99。标准溶液的回收率测定表明回收率在95.6%~104.3%之间,5次重复测定的相对标准偏差(RSD)在0.4%~3.6%之间,表明该方法具有良好的准确性和精密度。SGTO油相产物中的加标回收率在91.1%~96.8%之间,5次重复测定的RSD为0.7%~2.3%,准确性可以满足实际分析的需要。实际SGTO水相产物和油相产物中C1~C6有机酸的分析结果表明,SGTO水相产物中C2~C4有机酸含量较高,而SGTO油相产物中C4~C6有机酸含量较高。本研究对SGTO反应研究、催化剂制备、工艺优化以及设备材料的选择具有重要意义。     

Comparison of the cold vapor generation using NaBH4 and SnCl2 as reducing agents and atomic emission spectrometry for the determination of Hg with a microstrip microwave induced argon plasma exiting from the wafer

A 2.45 GHz low power microwave microstrip plasma (MSP) exiting the wafer and operated with Ar at atmospheric pressure was used for the optical emission spectrometric determination of Hg with the aid of a miniaturized optical fiber spectrometer with a CCD detector and the cold vapor (CV) generation technique using NaBH₄ and SnCl₂ as reductants. The experimental conditions were optimized with respect to the relative intensity of the Hg I 253.6 nm line and its signal-to-background intensity ratio (SBR). So as to understand the results of the optimization experiments, the excitation temperatures as measured from Ar I lines (T _exc) and the electron number densities (n _e) for the Ar MSP loaded with Hg vapors were determined and found to be in the range from 5500 to 6300 K and from 1.4 to 2.0 × 10¹⁴ cm⁻³, respectively. Under the optimized conditions, the detection limit for Hg of the CV-MSP-OES using SnCl₂ as the reducing agent was found to be much lower (0.11 ng mL⁻¹) than in the case where NaBH₄ was used (9 ng mL⁻¹). The linearity range was found to be up to 1 μg mL⁻¹ while the precision was of the order of 0.7–5%. The procedure with SnCl₂ as reductant was used for the determination of Hg at a concentration of 0.2 μg mL⁻¹ in synthetic water samples containing 1 to 4% (m/v) of NaCl with an accuracy of 3% as well as in a solution of the domestic sludge standard reference material (NIST SRM 2781) with a certified concentration for Hg of 3.64 ± 0.25 μg g⁻¹ for which 3.55 ± 0.41 μg g⁻¹ was found. Correspondence: J. A. C. Broekaert, Institut für Anorganische und Angewandte Chemie, Universit?t Hamburg, 20146 Hamburg, Germany     

Analysis of ibuprofen,pantoprazole, and itopride combination therapeutic drugs in human plasma by solid phase membrane microtip extraction and high-performance liquid chromatography methods using new generation core shell C18 column

Iron nanocomposite adsorbent was synthesized by green technology with 90% yield. The surface was amorphous and irregular in nature. The iron nanocomposite adsorbent was applied in solid phase membrane microtip extraction (SPMMTE) procedure for the extraction of ibuprofen, pantoprazole, and itopride drugs. SPMMTE was used to extract these drugs from plasma. SunShell C₁₈ column was used with phosphate buffer (10 mM, pH 7.0):acetonitrile (70:30, v/v) as mobile phase at 1.0 mL min^?1 flow rate with a detection at 220 nm. The retention factor values were 2.23, 3.25, and 5.38. The values of separation and resolution factors were 1.41 and 1.65, and 5.00 and 12.14, respectively. The percentage recoveries were ibuprofen (90%), pantoprazole (80%), and itopride (75%) in standard solution. The reported SPMMTE and HPLC methods were fast, inexpensive, specific, precise, accurate, and robust for the analysis of the reported drugs. These results indicated that the reported nanocomposite adsorbent-based SPMMTE and HPLC methods may be used to monitor the reported drugs in any unknown matrices.     

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [¹²C]- and [¹³C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α₁-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.     

Determination of vitamin B<Subscript>1</Subscript> in seawater and microalgal fermentation media by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of vitamin B₁. Vitamin B₁ was converted into a fluorescent compound by treatment with hydrogen peroxide–horseradish peroxidase and the derivative was subsequently analyzed by HPLC on a Waters Spherisorb ODS2 column (250 mm×4.6 mm ID, 5 μm) with 40:60 methanol–pH 8.5 acetate buffer solution as mobile phase and fluorescence detection at 440 nm (with excitation at 375 nm). The calibration graph was linear from 5.00×10⁻¹⁰ mol L⁻¹ to 5.00×10⁻⁷ mol L⁻¹ for vitamin B₁ with a correlation coefficient of 0.9991 (n=9). The detection limit was 1.0×10⁻¹⁰ mol L⁻¹. The method was successfully used for determination of vitamin B₁ at pg mL⁻¹ levels in microalgal fermentation media and seawater after solid-phase extraction. Recovery was from 89 to 110% and the relative standard deviation was in the range 1.1 to 4.3%.     

Stir bar sorptive extraction of parabens, triclosan and methyl triclosan from soil, sediment and sludge with in situ derivatization and determination by gas chromatography-mass spectrometry

The aim of this research work was the evaluation of stir-bar sorptive extraction (SBSE) in combination with an in situ derivatization to determine parabens (methylparaben, isopropylparaben, n-propylparaben, butylparaben and benzylparaben), triclosan and methyltriclosan in soil samples. This is the first time that this approach has been applied to the determination of these compounds in soil samples, providing important advantages over conventional extraction techniques, such as minimization of sampling handling, complete elimination of the use of organic solvents and simplification of the analytical procedure with reduced time consumption. The enriched target analytes were desorbed thermally using a thermodesorption system coupled to a gas chromatograph and a mass spectrometer. The optimized derivatization and SBSE extraction conditions, as well as the analytical characteristics of the method were obtained using spiked soil samples. The proposed methodology proved to be easy to use and sensitive, with limits of detection between 80 ng/kg and 1.06 μg/kg, and reproducibility values below 13%. The accuracy of the method was evaluated at two concentration levels, obtaining apparent recoveries between 91% and 110%. The matrix composition significantly influenced the extraction procedure, and a need to adopt a standard additions protocol is apparent. The analytes assayed were determined successfully in different environmental soil samples.     

Development and validation of a new analytical method for the determination of 1,4-dichlorobenzene in honey by gas chromatography-isotope dilution mass spectrometry after steam-distillation

A simple, fast, sensitive and robust analytical method using gas chromatography (GC)-isotope dilution mass spectrometry (MS) was developed and validated for the identification and quantification of 1,4-dichlorobenzene (p-DCB) residues in honey samples. The proposed methodology is based on steam-distillation using a Clevenger-type apparatus followed by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode employing the isotopically labeled analogue d₄-1,4-dichlorobenzene (d₄-p-DCB) as internal standard (IS). Validation of the method was performed in two different GC-MS systems, using quadrupole MS (QMS) and ion-trap MS (ITMS) detectors, with no statistically significant differences between two. Recoveries were better than 91% with percent relative standard deviations lower than 12%. The instrumental limits of detection were 1 μg kg⁻¹ in the GC-ITMS system and 0.6 μg kg⁻¹ in the GC-QMS system. The expanded uncertainty was estimated as 17% at the currently accepted “action level” of 10 μg kg⁻¹. The method was applied to the analysis of 310 honey samples in an extensive national monitoring study. A quality control (QC) system applied during the assays has demonstrated a good performance and long-term stability over a period of more than 8 months of continuous operation.     

A new solvent system for the separation of Th4+, UO 2 2+ and Zr4+ in the presence of common anions by thin-layer chromatography

Summary A TLC method has been developed for separating Th⁴⁺, UO₂ ²⁺ and Zr⁴⁺ in the presence of some common anions using a dimethylamine/acetone/formic acid mobile phase. Capacity factors, separation factors and resolution for the separation of Th⁴⁺ from UO₂ ²⁺ have been evaluated. The effect of the pH of the sample on R_F values of Th⁴⁺, UO₂ ²⁺, Ni²⁺ and Cu²⁺ has also been examined.     

Valorization of Different Fractions from Butiá Pomace by Pyrolysis: H2 Generation and Use of the Biochars for CO2 Capture

This work valorizes butiá pomace (Butia capitata) using pyrolysis to prepare CO₂ adsorbents. Different fractions of the pomace, like fibers, endocarps, almonds, and deoiled almonds, were characterized and later pyrolyzed at 700 °C. Gas, bio-oil, and biochar fractions were collected and characterized. The results revealed that biochar, bio-oil, and gas yields depended on the type of pomace fraction (fibers, endocarps, almonds, and deoiled almonds). The higher biochar yield was obtained by endocarps (31.9%wt.). Furthermore, the gas fraction generated at 700 °C presented an H₂ content higher than 80%vol regardless of the butiá fraction used as raw material. The biochars presented specific surface areas reaching 220.4 m² g⁻¹. Additionally, the endocarp-derived biochar presented a CO₂ adsorption capacity of 66.43 mg g⁻¹ at 25 °C and 1 bar, showing that this material could be an effective adsorbent to capture this greenhouse gas. Moreover, this capacity was maintained for 5 cycles. Biochars produced from butiá precursors without activation resulted in a higher surface area and better performance than some activated carbons reported in the literature. The results highlighted that pyrolysis could provide a green solution for butiá agro-industrial wastes, generating H₂ and an adsorbent for CO₂.     

Characterization and determination of genipin-1-β-gentiobioside in gardenia fruit by high-performance liquid chromatography with ultraviolet detection following electrospray ionization mass spectrometry

A practical method for the analysis of genipin-1-β-gentiobioside in gardenia fruit was developed by high-performance liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization (HPLC/UV/ESI-MS). Efficiency of ultrasonic extraction (UE), accelerated solvent extraction (ASE) and high-speed homogenizer extraction (HHE) on genipin-1-β-gentiobioside in gardenia fruit was compared. UE gave higher extraction efficiency and lower matrix interference, and it was employed. Analysis of genipin-1-β-gentiobioside was performed using a Supelco C₁₈ column with methanol–20 mM NH₄Ac (30:70, v/v) as the eluent. Flow rate was at 0.8 ml/min, and the detection was at 240 nm. The limit of detection (LOD), using HPLC/UV, was about 0.25 μg/ml, whereas the overall recovery was better than 96.0%. Both full scan MS and MS² of genipin-1-β-gentiobioside with positive polarity were obtained and elucidated. The specific ions were chosen to characterize genipin-1-β-gentiobioside in gardenia fruit sample. The proposed method has been successfully applied to measure the real samples and the content profiles of genipin-1-β-gentiobioside in gardenia fruit samples were obtained and evaluated.     

Investigating the binding measurements of human α-acid glycoprotein with chlorambucil and dacarbazine in the presence of imidazolium based -ionic liquid by affinity capillary electrophoresis

Human alpha (α1)-acid glycoprotein (AGP) is an acute phase protein whose plasma concentration increases several-folds in the presence of various diseases. The variability in AGP plasma concentration is expected to have a huge impact on the drug binding equilibrium. Therefore, a precise measurement of AGP-drug binding is of great demand for drug development. In the current study, an ionic liquid-based aqueous two-phase system combined with affinity capillary electrophoresis (ILATPS/ACE) was utilised in order to improve the accuracy of AGP-drug binding analysis through the measurements of electrophoretic mobilities. The utilisation of ILATPS has shown to have a positive impact on the stability of AGP activity solution during the storage for an extended period of time. In addition, the effect of various alkyl chains (C2-C10) of imidazolium-based ILs with concentrations ranging between 10.00 and 1000.0 μmol L⁻¹ on the AGP binding with the anti-cancer drugs chlorambucil (CHL) and dacarbazine (DAC) was examined by the system developed (ILATPS/ACE). A 100.00 μmol L⁻¹ 1-ethyl-3-methylimidazolium chloride (EMImCl) prepared in the physiological buffer conditions containing AGP (5.00–100.00 µmol L⁻¹) has provided an accurate apparent binding constant of 1.99 ± 0.11 and 6.95 ± 0.14 L mmol⁻¹ with CHL and DAC respectively. Apart from the ACE analysis, EMImCl/phosphate buffer solution was found to be a distinguished system that could lengthen the stability of AGP activity for a period of time reaching 90 days during the solution storage at 4.00 °C. This effect is thought to be due to the easy conversion of one-phase EMImCl/phosphate buffer/AGP at the ambient lab temperature into the two-phase solution at refrigerator temperature, 4.00 °C, and vice versa. Therefore, the ILATP/ACE system could be used to enhance the accuracy for other AGP-drug bindings with a fast, easy to use, and cost-effective analysis.