Current Analytical Chemistry - Volume 14, Issue 5, 2018

Background: Metformin (Mf) (N,N'-Dimethylbiguanide) is used as the first-line medication for treating diabetes mellitus type II. It is also considered as a “gold standard” treatment for various diseases like hyperglycemia, insulin resistance, infertility, cardiovascular disease, renal dysfunction, polycystic ovary syndrome, obesity, cancer and ageing. Majority of the methods available for quantitative analysis of Mf are expensive, time-consuming, require sample pre-treatment and skilled persons to operate. Electrochemical sensors overcome these drawbacks, as these are fast, simple, cost-effective, specific and highly sensitive. Methods: The updated literature and online papers related to Mf sensors as well as other methods were collected, analyzed and reviewed to provide the comprehensive information about the Mf sensing. The review was critically evaluated and concluded, emphasizing on future perspectives for Mf sensors. Results: The review presented the basic principles, merits and demerits of various conventional techniques for metformin determination in pharmaceuticals and biological samples with special emphasis on electrochemical sensors. Further, the review highlighted the updated summary of the recent advances in terms of the latest nanomaterials modified electrodes used for fabrication of Mf sensors. Conclusion: Metformin sensors worked optimally within 0.6 to 120s, between pH, 7 to 12 and temperature 30°C to 40°C and in the concentration range, 0.015 nmol/L to 6500 nmol/L, with detection limits between 0.33nmol/L to 0.45μM/L. These sensors measured metformin level in the pharmaceutical preparations, sera and urine samples and reused over a period of 21 to 120 days. The technical challenges and future perspective for further improvement in Mf sensors and their miniaturization are also discussed.

Background: A cationic polyelectrolyte, polyhexamethylene biguanide hydrochloride (PHMB), is widely used for disinfectants in personal-care products for cosmetics and toiletries. Several colloidal titration methods for the determination of PHMB are very tedious and time-consuming. A Flow Injection Analysis (FIA) is a very advantageous analytical method because of its ease of handling, high reproducibility and high sampling rate. Therefore, the development of a simple and sensitive FIA method for the determination of PHMB is required. In this paper, we report the FIA method for the determination of PHMB based on the electrostatic and hydrophobic interactions between an anionic dye, Bromocresol purple (BCP) and PHMB. Methods: The FIA manifold consists of a two-channel system which is composed of a double plunger pump equipped with a sample injector, a flow-through type spectrophotometer and a strip chart recorder. A sample solution (140 μL) of PHMB was injected into a distilled and deionized water stream. This stream was subsequently merged with a reagent solution stream of a solution of 20 μM BCP adjusted to pH 8.0. The absorbance of ion associate formed between BCP and PHMB was measured at 588 nm. Results: The absorption spectra of several anionic dye (10 μM) with PHMB (50 μM) were measured at 400-750 nm by changing pH of the solution. The wavelength at the largest change in the absorbance between the mixed PHMB solution and the reagent blank, and the change of the absorbance at the wavelength was examined. When BCP is used as the anionic dye, the largest change in the absorbance was obtained at 588 nm and the change of the absorbance at the wavelength was -0.56 at pH = 8.0. From the results, BCP was selected as the anionic dye for the FIA determination of PHMB. Under the optimal FIA conditions, a linear relationship between the peak height and concentration of PHMB was obtained in the concentration range from 2.0 to 10.0 μM. The present FIA method was applied to the determination of PHMB in contact-lens detergents. Conclusion: A new spectrophotometric FIA method was developed for the determination of PHMB, based on electrostatic and hydrophobic interactions between PHMB and the anionic dye, BCP. Under the optimal flow conditions of the FIA system, a good linear relationship between peak heights and concentrations of PHMB in a concentration range from 2.0 to 10.0 μM was obtained. The present FIA method uses more simpler instrumentation, and is more rapid (sample throughput: 13 samples h-1) than other reported methods, and has the advantages of high sensitivity and no toxic organic solvent than other analytical methods. The present method will be very useful for the determination of PHMB in commercially available contact-lens detergents and also be applied to the determination of PHMB in the sample solution, which has similar matrix to the contact-lens detergent.

Background: The development of nanomaterials modified electrode can provide more opportunities for the enhancement of the analytical performance in constructing electrochemical sensor and biosensor. LiFePO4 (LFP) is a promising candidate to develop new modified electrodes owing to its advantages such as cheap cost, environmental compatibility, high safety, non-toxicity, large theoretical capacity, long cycle life and abundant earth materials. Objective: The aim of this paper was to construct nanosized LFP modified electrodes, which could be applied as working electrode for rutin analysis and as an electrochemical biosensor for direct electrochemistry of Hemoglobin (Hb). Method: Nanosized LFP was characterized by scanning electron microscopy. The LFP modified electrodes with Carbon Ionic Liquid Electrode (CILE) as substrate electrode were fabricated by dropping method with chitosan (CTS) as film. Cyclic voltammetry and electrochemical impedance spectroscopy were carried out to examine the electrochemical performance of the modified electrodes. Differential pulse voltammetry was used to establish determination methods. Results: Two new LFP-based modified electrodes (CTS/LFP/CILE and CTS/Hb-LFP/CILE) were fabricated. The electrochemical behaviors of rutin on CTS/LFP/CILE were achieved with a pair of enhanced redox peaks. The linear range for rutin analysis was from 4.00-8 to 1.00-4 mol L-1 with a detection limit of 8.00-9 mol L-1. The direct electrochemistry of Hb in CTS/Hb-LFP/CILE was realized with a pair of obvious redox peaks. The electrocatalytic activity of CTS/Hb-LFP/CILE toward trichloroacetic acid and H2O2 reduction were investigated, and the detection limits of 0.068 mmol L-1 for TCA and 0.07 μmol L-1 for H2O2 were obtained. Conclusion: Two kinds of LFP modified electrodes were successfully constructed with improved electrochemical performances. Electrochemistry of rutin and Hb were realized on LFP based electrodes with enhanced responses, and the application of LFP nanoparticle in electrochemical sensor and biosensors was extended.

Background: Microscale in vitro assays are fast, simple, and inexpensive, with reduced reagent quantities, waste, and experimental animal use. However, they have low reproducibility and low correlation with the results of in vivo models, possibly due to differences in precision and accuracy in methodologies between laboratories. Objective: The objective was the optimization and validation of an in vitro assay, carried out on microscale, to assess the inhibition of α-glucosidase activity, which is indicative of antihyperglycemic activity. Methods: The optimization was carried out using a fractional factorial design taking into account the best inhibition percentage and the absorbance of the controls. With the optimized experimental conditions in hand, we carried out method validation. Results: The optimized conditions were as follows: enzyme concentration, 0.55 U/mL; substrate concentration, 111.5 μM; and 17.5 min incubation at 37°C. A linear range between 100 and 310.2 μg/mL of acarbose (r2 0.994) was established. The RSD was <2% and the % error was <3%. The Z factor was >0.96. This method was applied to four plant extracts, one of which was found to be very active. Conclusion: The method was found to be accurate, precise, selective, linear, and reliable in evaluating the antihyperglycemic activity of natural extracts in vitro.

Background: Honeysuckle, as one of the most important traditional Chinese medicines (TCMs), has attracted more and more attention due to the increasing number of people using it as selfmedication or alternative therapy. In order to determine whether honeysuckle is a special matrix that may be less susceptible to be infected by toxigenic molds and consequently to less mycotoxins contamination due to its biological and pharmacological effects such as antibacterial, antiviral and antioxidant activities, a total of 60 dried samples randomly collected from main producing areas were analyzed. Methods: Conventional mycological investigations and molecular studies were performed for isolation and identification of the fungi, while the produced mycotoxins were determined by ultra-highperformance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After validation by determining linearity, recovery, sensitivity and precision, the analytical method has been successfully applied in real samples. Results: Alternaria, Aspergillus, Chaetomium, Penicillium, Phoma, Mucor, Arthrinium and Trichoderma were recovered, whereas occurrence of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), zearalanone (ZAN) and zearalenone (ZEA) was positively identified in 14 samples with the concentration levels up to 1.2, 9.6, 10.7, 34.9, 5.6 and 27.4 μg kg-1, respectively. Conclusion: To the best of our knowledge, this is the first report for the assessment of toxigenic fungal and mycotoxins contamination in honeysuckle. A total of 30 strains were isolated and identified, while mycotoxins were positively detected in honeysuckle by the established sensitive and reliable UHPLCMS/ MS method, indicating the potential health risks to consumers.

Background: Molecularly Imprinted Polymers (MIPs) are used as artificial receptors in biosensors for the detection of a wide range of analytes from small drug molecules to large molecular weight biomolecules. Quantification of Digoxin (DIG) in human serum and pharmaceutical samples has very high importance due to its low margin of safety. In this report, a highly sensitive molecularly imprinted polymer for DIG is prepared and the detection of analyte was conducted using electrochemical impedance spectroscopy. Methods: Bulk polymerization method was chosen for the preparation of MIP because of its better preservation of the pockets formed during the polymerization. DIG imprinted polymer was prepared by thermal polymerization of Methacrylic Acid (MAA) cross-linked with Ethyleneglycol Dimethacrylate (EGDMA) in the presence of DIG. The molecular interactions via hydrogen bonding between template and monomer in MIP and Non-Imprinted Polymer (NIP) were confirmed using Fourier Transform Infrared (FTIR) spectroscopy. Morphological studies were performed before and after extraction of the template using Scanning Electron Microscopy (SEM). MIP modified carbon paste electrode (MIPCPE) was fabricated by mixing optimized quantities of graphite, the polymer, and eicosane. The change in charge transfer Resistance (Rct) at the electrode-electrolyte (MIPCPE-electrolyte) interface before and after addition of DIG solution was studied using electrochemical impedimetric sensing of DIG. Results: The surface morphology of the MIPCPE shows better porosity in comparison to non-imprinted polymer CPE (NIPCPE) and MIPCPE before extraction of the template. In contrast to NIPCE, the fabricated MIPCPE shows a significant increase in Rct after addition of DIG. The MIPCPE based impedimetric sensor is able to detect DIG over a wide range of concentration from 1.0 10-9 M to 0.5 10-7 M with a detection limit 6.95 10-11 M. The selectivity coefficients obtained for the MIPCPE and NIPCPE reveal specific recognition MIPCPE sensor towards DIG. The recovery rates of DIG from the spiked blood serum and pharmaceutical samples are in the range of sensor 89-101%. Conclusion: In the present work, the capability of MIPs as molecular recognition elements is proved by reporting a selective and sensitive impedimetric sensor for DIG. The selectivity coefficients (K) of MIPCPE and NIPCPE obtained for the DIG and interferents show better selectivity of the sensor towards DIG in the presence of interferents. The proposed sensor also shows satisfactory stability, reproducibility, and repeatability for DIG. The proposed sensor was successfully applied for the quantitative estimation of DIG in serum and pharmaceutical samples with appreciable recoveries.

Background: Considering the economic loss by columnaris disease in fish, a rapid, simpleto- use and visual detection technology for the detection of Flavobacterium columnare (F. columnare) is very important. Methods: This assay utilizes LAMP for F. columnare-specific target gene amplification, and the resulting LAMP amplicons were further analyzed by Immunochromatographic Strip (ICS) for the rapid visual detection of F. columnare. The performance of such method includeing specificity, sensitivity, repeatability and practical use were validated. Results: This assay demonstrated high specificity, repeatability and has a limit of detection of 2.5 102 F. columnare CFU. The reliability of this assay was further confirmed by the equencing and alignment of LAMP amplicon with the DNA sequence of F. columnare. The whole detection process was less than 50 min. Conclusion: The LAMP-ICS assay presented here is simple, rapid, and reliable. Moreover, it could be easily adapted to detect other fish pathogens, and it may be more suitable for future use in the resourcelimit region for the detection of fish pathogen on site.

Background: Multivariate transfer techniques have become a widely accepted concept over the past few years, since they avoid full recalibration procedures when instruments are changed to analyze a specific sample. Objective: This paper reports a multivariate control chart transfer approach between two near infrared (NIR) spectrometers for simultaneous determination of rifampicin and isoniazid in pharmaceutical formulation using Direct Standardization (DS). Method: The control charts are based on the calculation of Net Analyte Signal (NAS) models and the transfer samples are selected by the Kennard-Stone (KS) algorithm. Three control charts (NAS, interference and residual) transferred on both the master and slave instruments were measured. Results: As a result, a classification model for rifampicin and isoniazid developed on a primary instrument has been successfully transferred to a secondary instrument. The spectral differences after the standardization procedure were considerably reduced and errors values found in the charts for both analytes were comparable with the errors obtained for the original chart models. Conclusion: The proposed approach appears to be a valid alternative to the commonly used transfer of multivariate calibration models in simultaneous determination of isoniazid and rifampicin in pharmaceutical formulation.

Background: Imatinib (Gleevec) is an antineoplastic agent acting as tyrosine kinase inhibitor used for the treatment of cancer such as lymphoblastic leukemia. The dosage of the anticancer drugs plays a critical role in the survival of the patients. Therefore, each patient should be administered with a customized dosage of the antineoplastic agent. For this reason, patients' plasma and urine samples should be monitored to obtain the necessary information regarding the toxicity of the drug. Objective: To determine the trace levels of imatinib, the trend solvent bar microextraction and hollow fiber liquid phase microextraction technique coupled with High-Performance Liquid Chromatography- Ultraviolet (HPLC-UV) detection were employed and optimized using Minitab. Method: In this method, pH gradient plays a critical role in obtaining the optimum results. Imatinib is a weak base so that the donor phase containing the drug was adjusted to 10.5 and the acceptor phase (free of drug) was adjusted to pH 2.8. N-octanol was used as the organic solvent to impregnate the pores of the hollow fiber so that the drug could enter the solvent bar. To enhance the results and shorten the time required, the other parameters such as stirring rate (750 rpm), time (20 min), temperature (25°C) and salt addition (14.2%) were optimized. Results: Under the optimum condition, the Limit Of Detection (LOD), Limit Of Quantification (LOQ), and Preconcentration Factor (PF) were obtained as 7.0 ng mL-1, 20.0 ng mL-1, and 107, respectively. The relative standard deviations of analysis were 4.9% within a day (n=3) and 5.7% between days (n=9). The calibration curves represented good linearity for urine and plasma samples with coefficient estimations higher than 0.99 with a linearity range of 20.0-6000.0 ng mL-1. Conclusion: Considering the results, it can be concluded that the present technique can be a great method in clinical applications, customized treatment and for clean validation in pharmaceutical industries.

Background: Two enantiomers of a drug which might have various pharmacological, physiological, and toxicological properties are well-known. Enantioseparation of chiral drugs is becoming significant in various fields. Objective: The aim was to obtain better detection sensitivity and good enantioresolution of Norepinephrine (NE), Epinephrine (EP) and Isoproterenol (ISO)at the same time. Method: A new and sensitive method for NE, EP and ISO was developed by Capillary Electrophoresis- Indirect Electrochemilumine-Scence (CE-IECL) based on their quenching effects on the Ru(bpy)3 2+/tripropylamine (Ru(bpy)3 2+/TPA) system, using negatively charged carboxymethyl-betacyclodextrin( CM-β-CD) as chiral selectors. Results: The enantiomers of NE, EP and ISO were characterized with baseline enantioseparation and highly sensitive detection. Conclusion: A wide and excellent linear relationship of each enantiomer was observed in the range of 0.10 to 1000 μM with detection limit (S/N=3) ranging from 0.03 to 0.10 μM, with the RSD of peak area and peak height from 3.1-3.5% and 2.9-3.4%, respectively. This CE-IECL method was successfully applied for the enantioseparation of NE, EP and ISO in serum sample; the recoveries of NE, EP and ISO in serum ranged from 95.0% to 104.0% with relative standard deviations less than 3.4%.

Background: Glutathione peroxidase (GSH-Px) is one of the major enzyme in the antioxidative defense mechanism present in the cells. Methods: In the present study a novel and simple microplate-based method was developed, using the cupric neocuproine complex (Cu(Nc)2 2+) as a chromogenic oxidizing reagent, for the assessment of GSH-Px activity of biological samples for the first time. In this GSH-Px enzyme activity measurement, the GSH-Px catalyzed oxidation of Reduced Glutathione (GSH) gives rise to oxidized form of GSH (GSSG). The recommended method was based on the reduction of Cu(Nc)2 2+ to highly colored Cu(I)- neocuproine complex (Cu(Nc)2 +) by the unconsumed GSH, and measurement spectrophotometrically at 450 nm, the difference being correlated to GSH-Px activity of the analytes. Results: Under the optimum conditions, a linear calibration graph was obtained in the range of 18.5- 92.5 μM of GSH with Limit Of Detection (LOD) of 1.03 μM. Application of developed method to tissue homogenates (n = 9) provided GSH-Px activity values in agreement with those of the reference GSH-Px-5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) method. Conclusion: Cupric Ion Reducing Antioxidant Capacity (CUPRAC) antioxidant method was implemented in a microformat (96 well plates) and the reaction time of original CUPRAC method was significantly shortened from 30 to 4 min. Multi-sample can be simultaneously detected thanks to a novel microplate method.

Background: The ICH guidelines Q8 (Analytical Quality by Design (AQbD) technique) give the procedure for optimization of analytical parameter involved method development and validation of Sirolimus (SL) in spiked Plasma and in dosage form by using advanced analytical technique. Objective: This research paper is based on Quality by Design (QbD) finalized conditions for a method for the determination of concentration of Sirolimus by using Liquid Chromatography–Tandem Mass Spectrometry (LC-ESI/MS). Method: Sirolimus is a potent immunosuppresant drug. Critical Process Attributes (CPA) is considered to be an influential parameter in separation, identification and quantification processes by UHPLC-ESIMS/ MS which are pH modifier,organic content, buffer strength, flow rate, Ionization chamber temperature, sheath gas, spray voltage and auxiliary gas that alters Critical Analytical Attributes (CAA), like peak area and Retention Time (Rt). These factors were evaluated first in a factorial design (TAGUCHI) and then extensively in a Central Composite Design (CCD) to zero-in on the mobile phase for the quantification of Sirolimus (SL) standard drug and along with its internal standard ( SL IS) in spiked plasma samples and in the formulation. Pareto chart from initial factorial design (Taguchi) model suggested for which of the CPA factors be given the importance that is to be exhaustively analyzed in the CCD and response surface analysis. An UHPLC instrument with the octadecylsilica column (C18 ) of dimensions (5 μm, 2.1 50 mm) was used and selectivity of the column was modified using methanol: water (65:35) methanol as an organic modifier as the mobile phase at a flow rate of 0.6 mL min-1. While spray voltage and Ionization chamber temperature for the method is maintained at the level predicted by the response analysis. Detection was performed using Triple quadrupole MS/MS by multiple-reaction monitoring via a positive electrospray ionization source. The ICH guidelines had elaborated about the parameters to be studied in method validation, i.e., selectivity, linearity, accuracy, precision repeatability system-suitability tests, method robustness,ruggedness, sensitivity and stability were accomplished. Results: The results are very clearly indicated that linearity with r value = 0.9980 in the concentration range of 10–500.0 ng mL-1, an intra- and inter-assay precision of 2.4 and 4.1%, respectively, and recovery studies were found to be between 100.8 and 105.6%. The lower limit of quantification was 0.086 ng/mL in 50 μL of human plasma sample. Conclusion: The present method gives a robust QbD-compliant quantitative UHPLC –ESI-MS/MS method for Sirolimus drug containing plasma samples (spiked).

Background: This paper is focused on application of a recently developed disposable array of carbon composite electrodes and carbon paste electrode for the determination of indoxyl sulphate. Results: Measurement of basic electrochemical behaviour of indoxyl sulphate on working electrode resulted in the selection of BR buffer pH 2.0 and pH 3.0 as the optimum medium for carbon composite film electrode and carbon paste electrode, respectively. In this medium, concentration dependences of indoxyl sulphate were measured; calculated quantification limits were 0.72 μmol L-1 for the carbon composite film electrode and 1.7 μmol L-1 for the carbon paste electrode. Pasivation of the electrodes during repeated measurements and negligible indoxyl sulphate accumulation was observed. Applicability of the method for the indoxyl sulphate determination in spiked urine samples was tested in combination with the solid phase extraction, where methanol, BR buffer, deionised water and their combination were used as elution reagents. Conclusion: Selected graphite-based working electrodes were successfully applied for the development of suitable voltammetric method for the determination of indoxyl sulphate in BR buffer. Both electrodes showed similar behaviour and sufficient sensitivity and accuracy was reached; however, solid phase extraction was not successful due to ineffectual removal of interferents from human urine.