Hyphenation of enzyme/graphene oxide-ionic liquid/glassy carbon biosensors with anodic differential pulse stripping voltammetry for reliable determination of choline and acetylcholine in human serum.

Acetylcholine (ACh) and its precursor choline (Ch) play important roles in many biological processes. It is expected that Alzheimer's disease is occurred due to the reduction in synthesis of ACh. On the other hand, the increase in the level of ACh results in a depression of heart rate and over production of saliva. Therefore, the quantitative determination of Ch and ACh is very important in biological media. In the current work, sensitive and selective biosensors composed of choline oxidase (ChO) and/or acetylcholine esterase (AChE) on graphene oxide-ionic liquid (GO-IL)/ glassy carbon electrode (GCE) hyphenated with anodic differential pulse stripping voltammetry (ADPSV) were firstly established for the determination of ACh and Ch in human serum samples. The molecular bond of ionic liquid 1-allyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (AMIM TFSI) with GO was investigated by FT-IR and UV-Vis techniques. Furthermore, the surface topography of ChO/GO-IL and AChE-ChO/GO-IL composites was investigated by SEM and XRD. Then, the electron transfer features of biosensors ChO/GO-IL/GCE and AChE-ChO/GO-IL/GCE were characterized by the electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. The ADPSV was further used for the determination of Ch and ACh. The experimental parameters such as differential pulse working potential, differential pulse scan rate, equilibrium time and long-term stability were further optimized. Detection limits of 0.885 and 1.352 nmol L-1 with excellent linearity (R2 = 0.9996) over the range of 5-1000 nmol L-1 were obtained for Ch and ACh, respectively. The developed analytical methods showed excellent accuracy and precision for the determination of Ch and ACh in human serum samples avoiding their pretreatment or purification.

Temperature controlled ionic liquid aqueous two phase system combined with affinity capillary electrophoresis for rapid and precise pharmaceutical-protein binding measurements.

Temperature controlled ionic liquid aqueous two phase system (ILATPS) was used to improve the precision of pharmaceutical-AGP (human alpha (α1)-acid glycoprotein) binding measurements by affinity capillary electrophoresis (ACE). The effect of different types of short-chain alkyl imidazolium ILs within the concentration range of 10.0-1000.0 µmol L-1 on a propranolol (PRO)-AGP model was firstly investigated by ILATPS/ACE system. Use of 100.0 µmol L-1 1-butyl-3-methylimidazolium chloride (BMImCl) in 67.0 mmol L-1 potassium phosphate buffer (pH 7.4) containing low concentrations of AGP (5.0-100.0 µmol L-1) gave the highest precision value (2.98 ±â€¯0.14 × 105 L/mol) which is in concord with the reported one (3.00 × 105) under similar experimental conditions. The proposed BMImCl/phosphate solution was a unique temperature controlled system to preserve AGP activity during the pre-analysis and within ACE measurements under lab conditions for about 30 days. This period could be prolonged by converting the one-phase solution into biphasic solution at 4 °C storage temperature and again it could get rapidly back into one-phase by raising the temperature with gentle shaking. This behavior could be attributed to the electrostatic interaction and π-π interaction between BMIm cations and negatively charged AGP ions (pI = 2.7). Moreover, the compatibility of ILATPS with ACE has been the critical factor to avoid precipitation of salts formed by anion exchange in the running buffer. The current ILATPS/ACE system was further used to rapidly and precisely estimate the binding constants of anticancer drugs methotrexate (MTX) and vinblastine (VBL) with human AGP. The obtained binding values have been in good agreements with their findings by high performance affinity chromatography (HPAC). This ILATP/ACE system could similarly be applied to improve the precision of other proteins binding measurements with consuming a small amount of protein and with shortening ACE analysis time.

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013-2015).

This review updates and follows-up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug-related substances, chiral CE, and chiral CE-MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor-ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.

Simultaneous determination of acrylamide, asparagine and glucose in food using short chain methyl imidazolium ionic liquid based ultrasonic assisted extraction coupled with analyte focusing by ionic liquid micelle collapse capillary electrophoresis.

Acrylamide (AA) is a known lethal neurotoxin and carcinogen. AA is formed in foods during the browning process by the Maillard reaction of glucose (GL) with asparagine (AS). For the first time, the simultaneous online preconcentration and separation of AA, AS and GL using analyte focusing by ionic liquid micelle collapse capillary electrophoresis (AFILMC) was presented. Samples were prepared in a 1-butyl-3-methylimidazolium bromide (BMIMBr) micellar matrix with a conductivity 4 times greater than that of the running buffer (12.5 mmol L(-1) phosphate buffer at pH 8.5). Samples were hydrodynamically injected into a fused silica capillary at 25.0 mbar for 25.0 s. Separations were performed by applying a voltage of 25.0 kV and a detection at 200.0 nm. To sufficiently reduce BMIMBr adsorption on the interior surface of capillary, an appropriate rinsing procedure by hydrochloric acid and water was optimized. AFILMC measurements of analytes within the concentration range of 0.05-10.0 µmol L(-1) achieved adequate reproducibility and accuracy with RSD 1.14-3.42% (n=15) and recovery 98.0-110.0%, respectively. Limits of detections were 0.71 ng g(-1) AA, 1.06 ng g(-1) AS and 27.02 ng g(-1) GL with linearity ranged between 2.2 and 1800 ng g(-1). The coupling of AFILMC with IL based ultrasonic assisted extraction (ILUAE) was successfully applied to the efficient extraction and determination of AA, AS and GL in bread samples. The structure of ILs has significant effects on the extraction efficiency of analytes. The optimal extraction efficiency (97.8%) was achieved by an aqueous extraction with 4:14 ratio of sample: 3.0 mol L(-1) BMIMBr followed by sonication at 35 °C. The proposed combination of ILUAE and AFILMC was simple, ecofriendly, reliable and inexpensive to analyze a toxic compound and its precursors in bread which is applicable to food safety.

A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, ß-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

Eco-friendly ionic liquid assisted capillary electrophoresis and α-acid glycoprotein-assisted liquid chromatography for simultaneous determination of anticancer drugs in human fluids.

In the current work, two eco-friendly analytical methods based on capillary electrophoresis (CE) and reversed phase liquid chromatography (RPLC) were developed for simultaneous determination of the most commonly used anticancer drugs for Hodgkin's disease: methotrexate (MTX), vinblastine, chlorambucil and dacarbazine. A background electrolyte (BGE) of 12.5 mmol/L phosphate buffer at pH 7.4 and 0.1 µmol/L 1-butyl-3-methyl imidazolium bromide (BMImBr) ionic liquid (IL) was used for CE measurements at 250 nm detection wavelength, 20 kV applied voltage and 25 °C. The rinsing protocol was significantly improved to reduce the adsorption of IL on the interior surface of capillary. Moreover, RPLC method was developed on α-1-acid glycoprotein (AGP) column. Mobile phase was 10 mmol/L phosphate buffer at pH 6.0 (100% v/v) and flow rate at 0.1 mL/min. As AGP is a chiral column, it was successfully separated l-MTX from its enantiomer impurity d-MTX. Good linearity of quantitative analysis was achieved with coefficients of determinations (r(2) ) >0.995. The stability of drugs measurements was investigated with adequate recoveries up to 24 h storage time under ambient temperature. The limits of detection were <50 and 90 ng/mL by CE and RPLC, respectively. The using of short-chain IL as an additive in BGE achieved 600-fold sensitivity enhancement compared with conventional Capillary Zone Electrophoresis (CZE). Therefore, for the first time, the proposed methods were successfully applied to determine simultaneously the analytes in human plasma and urine samples at clinically relevant concentrations with fast and simple pretreatments. Developed IL-assisted CE and RPLC methods were also applied to measure MTX levels in patients' samples over time.

Cyclodextrin Micellar LC for Direct Selective Analysis of Combined Dosage Drugs in Urine.

Two cyclodextrin micellar liquid chromatographic methods were developed and applied to the simultaneous determination of bisoprolol/hydrochlorothiazide and atenolol/chlorthalidone combinations in urine matrices without sample pretreatment. These combined ß-blockers and diuretics chemotherapies are commonly used in the treatment of hypertension and cardiovascular diseases. Hybrid isocratic mobile phases containing hydroxypropyl-ß-cyclodextrin, sodium dodecyl sulfate, phosphate buffer and methanol on a Luna C18 column with 0.5 mL min(-1) flow rate and 25.0°C column temperature were used. The methods were sensitive enough for the determination of analytes at the therapeutic urine levels with limits of detections down to 1.0 µg mL(-1); relative standard deviations and recoveries were ranged between 1.5-4.4% and 98.00-109.52%, respectively. Urinary excretion studies showed that the detection of drugs is possible up to 24 h after their ingestion. The selective proposed separations with less consumption of organic solvents over the hitherto ones could be attributed to the four point competitive interactions among analysts, pseudostationary phases and a real stationary phase.

Use of short chain alkyl imidazolium ionic liquids for on-line stacking and sweeping of methotrexate, flinic acid and folic acid: their application to biological fluids.

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on-line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping-MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping-MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused-silica capillary pre-filled with phosphate buffer containing 3.0 mol/L of 1-butyl-3-methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping-MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping-MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π-π interactions. In sweeping-MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22- and 5-fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.

Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers.

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF6)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF6 which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2×10(5)±0.3 L mol(-1) and 0.8×10(4)±0.4 L mol(-1) for L- and D-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers-HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that L-fenoprofen replaced D-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.

Simultaneous separation and determination of Tarabine PFS and Adriblastine using micellar electrokinetic chromatography and high performance liquid chromatography. Application to some biological fluids.

The simultaneous determination of Tarabine PFS and Adriblastine by two independent techniques, viz. micellar electrokinetic chromatography (MEKC) and high performance liquid chromatography (HPLC), has been studied. For MEKC analysis, separations and identifications were accomplished using uncoated fused-silica capillaries and injections were performed in the hydrodynamic mode. The running buffer consisted of 0.05 M borate/phosphate pH 8.70, with 0.10 M SDS at an operating voltage of 15.0 kV and the temperature held at 25.0 degrees C. Under these conditions, the migration times of Tarabine PFS and Adriblastine were 2.70 and 6.40 min, respectively. Calibration curves were established for 0.010-0.300 microg/mL (r = 0.99) Tarabine PFS and 8.000-120.0 microg/mL (r = 0.99) Adriblastine. The limit of detection (LOD) was estimated and found to be 0.003 and 3.000 microg/mL of Tarabine PFS and Adriblastine, respectively. The limit of quantitation (LOQ) was found to be 0.009 and 8.000 microg/mL of Tarabine PFS and Adriblastine, respectively. For HPLC analysis, separations and determinations were performed on teicoplanin stationary phase with reversed mobile phase containing methanol:buffer pH 4.05 (20.0:80.0%, v/v) at 285 nm. Calibration curves were established for 3.000-90.00 microg/mL (r = 0.99) Tarabine PFS and for 10.00-120.0 microg/mL (r = 0.99) Adriblastine. LOD and LOQ were estimated and found to be 0.950 and 2.050 microg/mL of Tarabine PFS and 3.130 and 9.250 microg/mL of Adriblastine, respectively. Both MEKC and HPLC methods were applied for the simultaneous determination of analytes in urine samples. It was found that 8.00-10.0% (Tarabine PFS) and 13.0-15.0% (Adriblastine) of the injected dose was recovered in urine samples with 99.5-102% recovery.