Chemometrics-assisted spectroscopic methods for rapid analysis of combined anti-malarial tablets.

Combination of piperaquine (PQ) (320mg) and dihydroartemisinin (DHA) (40 mg) is an anti-malarial formulation, which is recommended by World Health Organization (WHO). Simultaneous analysis of PQ and DHA can be problematic due to the lack of chromophores or fluorophores in DHA molecule. Whereas PQ possesses strong UV absorption and it presents in 8 times of DHA contents in the formulation. In this study, two spectroscopic methods, Fourier transform infrared (FTIR) and Raman spectroscopy, were developed for the determination of both drugs in combined tablets. The FTIR and Raman spectra were recorded in the attenuate total reflectance (ATR) and scattering modes, respectively. The original and pretreated spectra from FTIR and handheld-Raman were subjected to Unscrambler® program to construct partial least squares regression (PLSR) model comparing with references values obtained from high performance liquid chromatography (HPLC)-UV method. The optimal PLSR models of PQ and DHA from FTIR spectroscopy were obtained from orthogonal signal correction (OSC) pretreatment at the wavenumbers 400-1,800 cm-1 and 1,400-4,000 cm-1, respectively. For Raman spectroscopy of PQ and DHA, the optimal PLSR models were obtained from standard normal variate (SNV) pretreatment at the wavenumbers 1,200-2,300 cm-1 and OSC pretreatment at the wavenumber 400-2,300 cm-1, respectively. Determination of PQ and DHA in tablets from the optimum model was compared with HPLC-UV method. Results were not significantly different at 95% confidence limit (p-value >0.05). The chemometrics-assisted spectroscopic methods were fast (1-3 min), economical and less labor intensive. Moreover, the handheld Raman spectrometer is portable and can be utilized for onsite analysis to facilitate the detection of counterfeit or substandard drugs at ports of entry.

Development of Miconazole-Loaded Microemulsions for Enhanced Topical Delivery and Non-Destructive Analysis by Near-Infrared Spectroscopy.

The antifungal drug miconazole nitrate has a low solubility in water, leading to reduced therapeutic efficacy. To address this limitation, miconazole-loaded microemulsions were developed and assessed for topical skin delivery, prepared through spontaneous emulsification with oleic acid and water. The surfactant phase included a mixture of polyoxyethylene sorbitan monooleate (PSM) and various cosurfactants (ethanol, 2-(2-ethoxyethoxy) ethanol, or 2-propanol). The optimal miconazole-loaded microemulsion containing PSM and ethanol at a ratio of 1:1 showed a mean cumulative drug permeation of 87.6 ± 5.8 µg/cm2 across pig skin. The formulation exhibited higher cumulative permeation, permeation flux, and drug deposition than conventional cream and significantly increased the in vitro inhibition of Candida albicans compared with cream (p < 0.05). Over the course of a 3-month study conducted at a temperature of 30 ± 2 °C, the microemulsion exhibited favorable physicochemical stability. This outcome signifies its potential suitability as a carrier for effectively administering miconazole through topical administration. Additionally, a non-destructive technique employing near-infrared spectroscopy coupled with a partial least-squares regression (PLSR) model was developed to quantitatively analyze microemulsions containing miconazole nitrate. This approach eliminates the need for sample preparation. The optimal PLSR model was derived by utilizing orthogonal signal correction pretreated data with one latent factor. This model exhibited a remarkable R2 value of 0.9919 and a root mean square error of calibration of 0.0488. Consequently, this methodology holds potential for effectively monitoring the quantity of miconazole nitrate in various formulations, including both conventional and innovative ones.

Optimization of Magnetic and Paper-Based Molecularly Imprinted Polymers for Selective Extraction of Charantin in Momordica charantia.

Charantin is a mixture of ß-sitosterol and stigmastadienol glucosides, which effectively lowers high blood glucose. Novel molecularly imprinted polymers coated magnetic nanoparticles (Fe3O4@MIPs) and filter paper (paper@MIPs) were synthesized by sol-gel polymerization to selectively extract charantin. ß-sitosterol glucoside was selected as a template for imprinting a specific recognition owing to its larger molecular surface area than that of 5,25-stigmastadienol glucoside. Factorial designs were used to examine the effects of the types of porogenic solvents and cross-linkers on the extraction efficiency and imprinting factor before investigating other factors (for example, amounts of template and coated MIPs, and types of substrates for MIP immobilization). Compared to traditional liquid-liquid extraction, the optimal Fe3O4@MIP-based dispersive micro-solid phase extraction and paper@MIP extraction provided excellent extraction efficiency (87.5 ± 2.1% and 85.0 ± 2.9%, respectively) and selectivity. Charantin was well separated, and a new unidentified sterol glucoside was observed using the developed high-performance liquid chromatography with diode-array detection (Rs ≥ 2.0, n > 16,400). The developed methods were successfully utilized to extract and quantify charantin from M. charantia fruit powder and herbal products. Moreover, these methods are rapid (<10 min), inexpensive, simple, reproducible, and environmentally friendly.

Portable near-infrared and Raman spectroscopic devices as complementary tools for instantaneous quality control of turmeric powder.

INTRODUCTION: Process analytical technology (PAT) guidance is implemented in the quality assurance of phytocompounds to achieve the Industry 4.0 concept. Near-infrared (NIR) and Raman spectroscopies are feasible for rapid, reliable quantitative analysis through transparent packaging without removing the samples from their original containers. These instruments can serve PAT guidance. OBJECTIVE: This study aimed to develop online portable NIR and Raman spectroscopic methods for quantifying total curcuminoids in turmeric samples through a plastic bag. The method mimicked an in-line measurement mode in PAT compared with placing samples into a glass vessel (at-line mode). MATERIALS AND METHODS: Sixty-three curcuminoid standard-spiked samples were prepared. Then, 15 samples were randomly selected as fixed validation samples, and 40 of the 48 remaining samples were chosen as calibration set. The results obtained from the partial least square regression (PLSR) models constructed by using the spectra acquired from NIR and Raman were compared with the reference values from high-performance liquid chromatography (HPLC). RESULTS: The optimum PLSR model of at-line Raman was achieved with three latent variables and a root mean square error of prediction (RMSEP) of 0.46. Meanwhile, the PLSR model of at-line NIR with one latent variable offered an RMSEP of 0.43. For the in-line mode, PLSR models created from Raman and NIR spectra had one latent variable with RMSEP of 0.49 and 0.42, respectively. The R2 values for prediction were 0.88-0.92. CONCLUSION: The models established from the spectra from portable NIR and Raman spectroscopic devices with the appropriate spectral pretreatments allowed the determination of total curcuminoid contents through plastic bag.

Response Surface Methodology for Optimization of Hydrogel-Forming Microneedles as Rapid and Efficient Transdermal Microsampling Tools.

Microneedles (MNs) have shown a great potential for the microsampling of dermal interstitial fluid (ISF) in a minimally invasive manner for point-of-care testing (POCT). The swelling properties of hydrogel-forming microneedles (MNs) allow for passive extraction of ISF. Surface response approaches, including Box-Behnken design (BBD), central composite design (CCD), and optimal discrete design, were employed for the optimization of hydrogel film by studying the effects of independent variables (i.e., the amount of hyaluronic acid, GantrezTM S-97, and pectin) on the swelling property. The optimal discrete model was selected to predict the appropriate variables, due to the good fit of the experimental data and the model validity. The analysis of variance (ANOVA) of the model demonstrated p-value < 0.0001, R2 = 0.9923, adjusted R2 = 0.9894, and predicted R2 = 0.9831. Finally, the predicted film formulation containing 2.75% w/w hyaluronic acid, 1.321% w/w GantrezTM S-97, and 1.246% w/w pectin was used for further fabrication of MNs (525.4 ± 3.8 µm height and 157.4 ± 2.0 µm base width), which possessed 1508.2 ± 66.2% swelling, with 124.6 ± 7.4 µL of collection volume, and could withstand thumb pressure. Moreover, almost 50% of MNs achieved a skin insertion depth of approx. 400 µm, with 71.8 ± 3.2% to 78.3 ± 2.6% recoveries. The developed MNs show a promising prospect in microsample collection, which would be beneficial for POCT.

A Comparative Study of Benchtop and Portable NIR and Raman Spectroscopic Methods for the Quantitative Determination of Curcuminoids in Turmeric Powder.

Turmeric consumption is continually increasing worldwide. Curcuminoids are major active constituents in turmeric and are associated with numerous health benefits. A combination of spectroscopic methods and chemometrics shows the suitability of turmeric for food quality control due to advantages such as speed, versatility, portability, and no need for sample preparation. Five calibration models to quantify curcuminoids in turmeric were proposed using benchtop and portable devices. The most remarkable results showed that Raman and NIR calibration models present an excellent performance reporting RMSEP of 0.44% w/w and 0.41% w/w, respectively. In addition, the five proposed methods (FT-IR, Raman, and NIR) were compared in terms of precision and accuracy. The results showed that benchtop and portable methods were in good agreement and that there are no significant differences between them. This study aims to foster the use of portable devices for food quality control in situ by demonstrating their suitability for the purpose.

Detection of pesticides in food products using paper-based devices by UV-induced fluorescence spectroscopy combined with molecularly imprinted polymers.

In this proof-of-concept study, we explore the detection of pesticides in food using a combined power of sensitive UV-induced fingerprint spectroscopy with selective capture by molecularly imprinted polymers (MIPs) and portable cost-effective paper-based analytical devices (PADs). The specific pesticides used herein as model compounds (both pure substances and their application products for spraying), were: strobilurins (i.e. trifloxystrobin), urea pesticides (rimsulfuron), pyrethroids (cypermethrine) and aryloxyphenoxyproponic acid herbicides (Haloxyfop-methyl). Commercially available spraying formulations containing the selected pesticides were positively identified by MIP-PADs swabs of sprayed apple and tomato. The key properties of MIP layer - imprinting factor (IF) and selectivity factor (α) were characterized using trifloxystrobin (IF-3.5, α-4.4) was demonstrated as a potential option for in-field application. The presented method may provide effective help with in-field testing of food and reveal problems such as false product labelling.

Antiviral Dosing Modification for Coronavirus Disease 2019-Infected Patients Receiving Extracorporeal Therapy.

Previous literature regarding coronavirus disease 2019 outlined a presence of organ dysfunction including acute respiratory distress syndrome and acute kidney injury that are linked to mortality. Several patients require extracorporeal therapy. This review aims to gather available published resources including physicochemical and pharmacokinetic properties and suggests antiviral drug dosing adaptation for coronavirus disease 2019-infected critically ill patients receiving extracorporeal therapy. A literature search was performed using PubMed, clinical trial registries, and bibliographic review of textbooks and review articles. Unfortunately, no standard of pharmacologic management and recommendations of drug dosing for coronavirus disease 2019 infection for critically ill patients receiving extracorporeal therapy exist due to the limited data on pharmacokinetic and clinical studies. All available extracted data were analyzed to suggest the appropriate drug dosing adjustment. Antiviral drug dosing adjustments for critically ill patients receiving extracorporeal membrane oxygenation and continuous renal replacement therapy are presented in this review. Considering pathophysiologic changes, drug properties, and extracorporeal modalities, applying our suggestions is recommended.

Distance-based paper device using polydiacetylene liposome as a chromogenic substance for rapid and in-field analysis of quaternary ammonium compounds.

This work presents an affordable distance-based microfluidic paper-based device (µPAD), using polydiacetylene (PDA) liposome as a chromogenic substance with a smartphone-based photo editor, for rapid and in-field analysis of quaternary ammonium compounds (QACs) (e.g., didecyldimethylammonium chloride (DDAC), benzyldimethyltetradecyl ammonium chloride (BAC), and cetylpyridinium chloride (CPC)). In-field analysis of these compounds is important to ensure their antimicrobial activity and user safety since they are widely utilized as disinfectants in households and hospitals. The µPAD featured a thermometer-like shape consisting of a sample reservoir and a microchannel as the detection zone, which was pre-deposited with PDA liposome. The color change from blue to red appeared in the presence of QACs and the color bar lengths were proportional to the QAC concentrations. Reactions of QACs with the PDA required a specific pH range (from pH 4.0 to 10.0) and a readout time of 7 min. Analytical performance characteristics of the device were tested with DDAC, BAC, and CPC showing acceptable specificity, accuracy (96.1-109.4%), and precision (%RSDs ≤ 9.3%). Limits of detection and quantitation were in the ranges of 20 to 80 and 70 to 250 µM, respectively. Feasibility of the newly developed device was demonstrated for in-field analysis of QACs in fumigation solution providing comparable results with those obtained from a colorimetric assay (P > 0.05). The proposed device shows potentials for further applications of other analytes since it offers speed, simplicity, and affordability for in-field analysis, especially in remote areas where expertise, resources, and infrastructures are limited. Graphical abstract.

Paper-based molecularly imprinted-interpenetrating polymer network for on-spot collection and microextraction of dried blood spots for capillary electrophoresis determination of carbamazepine.

Carbamazepine is an antiepileptic drug with a narrow therapeutic index, which requires an efficient method for blood level monitoring. Finger-prick dried blood spot (DBS) collection is an alternative microsampling technique, which is less invasive than conventional venipuncture. Paper-based molecularly imprinted-interpenetrating polymer networks (MI-IPN) were developed as blood collection devices, which allowed for selective on-spot microextraction of carbamazepine from DBS. A hybrid of homogeneous polystyrene and silica gel polymer was synthesized and coated on a Whatman® Grade 1 filter paper. Proteins and other interferences in the blood samples were eliminated by using the MI-IPN collection devices, and the resulting DBS extracts were suitable for direct injection into the capillary electrophoretic instrument. The lower limit of quantitation of 4 µg/mL in capillary blood was achieved by the sweeping-micellar electrokinetic chromatography method using a KCl-containing matrix, which was sufficient for the therapeutic drug monitoring purposes. Method accuracies were in the range of 88.4 ± 4.5% to 94.5 ± 2.7% with RSD values ≤ 5.1%. The developed paper-based MI-IPN provided superior extraction efficiencies (92.2 ± 2.5%) in comparison with commercially available DBS collection cards, i.e., Whatman® 903 protein saver card (59.8 ± 2.8%) and GenCollect™ 2.0 card (47.2 ± 1.4%). The paper-based MI-IPN devices for DBS collection and on-spot extraction were characterized by simple fabrication, low costs, disposability, and reduction in sample preparation steps, and their further developments might open new perspectives in clinical applications, such as in therapeutic drug monitoring. Graphical abstract.

Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement.

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 µM with good precision (RSDs < 4.1%). Furthermore, the assay of CYP3A4 activity on the developed paper-based device provided comparable results with those obtained from conventional well-plates (p > 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 ±â€¯0.35 µM and 0.43 ±â€¯0.05 µM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 °C can increase storage stability, offering the activity of 86.3 ±â€¯0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.

Exosome aggregation mediated stop-flow paper-based portable device for rapid exosome quantification.

Exosome quantification is important for estimation of informative messengers (e.g., proteins, lipids, RNA, etc.) involving physiological and pathological effects. This work aimed to develop a simple and rapid distance-based paper portable device using exosome-capture vesicles (polydiacetylene conjugated with antiCD81) for exosome quantification in cell cultures. This novel concept relied on distinct aggregation of exosomes and exosome-capture vesicles leading to different solvent migration. Distances of the migration were used as signal readouts, which could be detected by naked eye. PDA-antiCD81 as exosome-capture vesicles were optimized (e.g., size, reaction ratio, and concentration) and the paper designs were investigated (e.g., diameter of sample reservoir and lamination layer) to enhance the solvent stop-flow effects. Finally, exosome screening on three cell culture samples (COLO1, MDA-MB-231, and HuR-KO1 subclone) was demonstrated. The method could linearly measure exosome concentrations in correlation with solvent migration distances in the range of 106 -1010 particles/mL (R2  > 0.98) from the cell culture samples. The exosome concentration measurements by the developed device were independently assessed by nanoparticle tracking analysis. Results demonstrated no statistically significant difference (p > 0.05) by t-test. This low-cost and rapid device allows a portable platform for exosome quantification without the requirement of expensive equipment and expertise of operation. The developed device could potentially be useful for quantification of other biomarker-related extracellular vesicles.

In-capillary derivatization with fluorescamine for the rapid determination of adamantane drugs by capillary electrophoresis with UV detection.

In-capillary derivatization using fluorescamine as the labeling reagent was proposed to enhance the detectability of adamantine drugs (memantine, amantadine and rimantadine) by spectrophotometric detection. Fluorescamine and the drugs were delivered to the capillary electrophoresis instrument at a ratio of 10:1 by zone injection. The derivatization reaction occurred following the application of voltage (20 kV). The derivatized products, hydrolyzed- fluorescamine and excess fluorescamine were separated in 7 min using 100 mM borate buffer (pH 10.0) containing 0.1% w/v of Brij®-35 and 20% v/v of acetonitrile. Validation data showed good linearity (r2  > 0.98), precision (%RSDs < 3.4), and accuracy (recoveries ranging from 98.0 to 102.0%). The detection and quantitation limits are in the range of 6.0-8.5 and 18-25 µM, respectively. The validation data is comparable to reported methods, however, the current method offers better precision with enhanced sensitivity (up to six times). Applications of the method show percent labeled amounts found in the studied samples within 100.6-109.3%, which complied with the United States Pharmacopeia limit (90.0-110.0%). The method was simple, rapid and, automated, which required no extra instrumentation or skillful operators.

Brompheniramine as a novel probe for indirect UV detection and its application for the capillary electrophoresis of adamantane drugs.

Brompheniramine, an antihistamine drug, was employed as a novel UV probe for capillary electrophoresis with indirect UV detection of adamantane drugs (memantine, amantadine, and rimantadine). The probe possesses high molar absorptivity of 24 × 103 L/mol cm at 6 mM, which enables the measurement of these nonchromophore analytes without derivatization. The simple background electrolyte (10 mM sodium dihydrogen phosphate (pH 5.0) containing 5 mM brompheniramine and 6 mM ß-cyclodextrin) provided the separation of the analytes in a short time (7.5 min). Under these conditions, brompheniramine had similar mobility to that of the analyte ions resulting in symmetric peaks with minimal electrodispersion. The analytes displace the probe at a one-to-one ratio with transfer values close to unity. ß-Cyclodextrin played a role in the resolution of the structurally similar adamantane derivatives. Method validation showed good linearity (r2  > 0.98), precision (%RSD ≤ 3.30), and accuracy (recoveries ranging from 98 to 109%). The proposed method was successfully applied to determine the adamantane content in pharmaceutical products.

A novel highly flexible, simple, rapid and low-cost fabrication tool for paper-based microfluidic devices (µPADs) using technical drawing pens and in-house formulated aqueous inks.

Paper-based microfluidic devices (µPADs) are capable of achieving rapid quantitative measurements of a variety of analytes inexpensively. µPADs rely on patterning hydrophilic-hydrophobic regions on a sheet of paper in order to create capillary channels within impermeable fluidic brakes on the paper. Here, we present a novel, highly flexible and low-cost fabrication method using a desktop digital craft plotter/cutter and technical drawing pens with tip size of 0.5 mm. The pens were used with either commercial black permanent ink for drawing fluidic brakes, or with specialty in-house formulated aqueous inks. With the permanent marker ink it was possible to create barriers on paper rapidly and in a variety of designs in a highly flexible manner. For instance, a design featuring eight reservoirs can be produced within 10 s for each µPAD with a consistent line width of brakes (%RSD < 1.5). Further, we investigated the optimal viscosity range of in-house formulated inks controlled with additions of poly(ethylene glycol). The viscosity was measured by capillary electrophoresis and the optimal viscosity was in the range of ∼3-6 mPa s. A functional test of these µPADs was conducted by the screening of antioxidant activity. Colorimetric measurements of flavonoid, phenolic compounds and DPPH free radical scavenging activity were carried out on µPADs. The results can be detected by the naked eye and simply quantified by using a camera phone and image analysis software. The fabrication method using technical drawing pens provides flexibility in the use of in-house formulated inks, short fabrication time, simplicity and low cost.

Miniaturised medium pressure capillary liquid chromatography system with flexible open platform design using off-the-shelf microfluidic components.

Trends towards portable analytical instrumentation of the last decades have not been equally reflected in developments of portable liquid chromatography (LC) instrumentation for rapid on-site measurements. A miniaturised medium pressure capillary LC (MPLC) system with gradient elution capability has been designed based on a flexible modular microfluidic system using primarily off-the-shelf low cost components to ensure wide accessibility to other analysts. The microfluidic platform was assembled on a breadboard and contained microsyringe pumps and switch valves, complemented with an injection valve and on-capillary detectors, all controlled by a PC. Four miniaturised microsyringe pumps, with 5, 20 and 100 µL syringe volume options, formed the basis of the pumping system. Two pairs of pumps were used for each mobile phase to create gradient elution capability. The two microsyringe pumps in each pairs were linked by two electrically operated microfluidic switching valves and both pairs of pumps were connected through a zero void volume cross-connector, thus providing a low hold-up volume for gradient formation. Sample was injected by a 20 nL nano-LC sampling valve, directly connected to a 18 cm long 100 µm i.d. Chromolith CapRod RP-18 monolithic capillary column. On-capillary LED-based UV-vis photometric detection was conducted through a piece of equal diameter fused silica capillary connected after the column. The performance of the portable LC system was evaluated theoretically and experimentally, including the maximum operating pressure, gradient mixing performance, and the performance of the detectors. The 5 µL microsyringe pump offered the best performance, with typical maximum operating pressures up to 11.4 ± 0.4 MPa (water) and gradient pumping repeatability of between 4 and 9% for gradients between 0.10% s(-1) and 0.33% s(-1). Test analytes of charged and uncharged dyes and pharmaceuticals of varying hydrophobicity showed typical RSD values of 0.7-1.4% and 3.3-4.8% in isocratic mode and 1.2-4.6% and 3.2-6.4% in gradient mode, respectively for retention time and peak area repeatability.

Recent applications of microchip electrophoresis to biomedical analysis.

Many separation methods have been developed for biomedical analysis, including chromatographic (e.g. high performance liquid chromatography (HPLC) and gas chromatography (GC)) and electrophoretic methods (e.g. gel electrophoresis and capillary electrophoresis (CE)). Among these techniques, CE provides advantages in terms of high separation efficiency, simplicity, low sample and solvent volume consumption, short analysis time and applicability to a wide range of biomedically important substances. Microchip electrophoresis (ME) is a miniaturized platform of CE and is now considered as a simpler and more convenient alternative, which has demonstrated potential in analytical chemistry. High-throughput, cost-effective and portable analysis systems can be developed using ME. The current review describes different separation modes and detectors that have been employed in ME to analyze various classes of biomedical analytes (e.g. pharmaceuticals and related substances, nucleic acids, amino acids, peptides, proteins, antibodies and antigens, carbohydrates, cells, cell components and lysates). Recent applications (during 2010-2014) in these areas are presented in tables and some significant findings are highlighted.

Potential of capillary electrophoresis (CE) and chip-CE with dual detection (capacitively-coupled contactless conductivity detection (C4D) and fluorescence detection) for monitoring of nicotine and cotinine derivatization.

Capillary electrophoresis (CE) coupled with capacitively-coupled contactless conductivity (C(4)D) and fluorescence (FD) detectors and chip-CE for monitoring of nicotine and cotinine derivatization was demonstrated. Separation of the substrates and intermediates could be achieved by CE-C(4)D in 7 min (R(s) > 2.7) using 45 mM acetic acid (pH 3.0) and this system was applied to detect the intermediate formation. Final fluorescent products could be analyzed by micellar electrokinetic chromatography (MEKC-FD) in 5 mM borate buffer (pH 9.0) containing 10 mM sodium dodecylsulfate (SDS) (%RSD < 3.00%). Transferring of MEKC-FD to chip-CE allowed for shorter analysis time (2.5 min) and decreased sample consumption. The chip-CE-FD shows good detection and quantitation limits (< 7.5 µM) and precision (%RSD < 2.81%) and was employed to determine nicotine and cotinine in artificial urine. This work reveals the potential of CE and chip-CE with dual detectors as simultaneous, convenient and rapid methods for monitoring pyridine derivatization.

Exploring chip-capillary electrophoresis-laser-induced fluorescence field-deployable platform flexibility: separations of fluorescent dyes by chip-based non-aqueous capillary electrophoresis.

Microfluidic chip electrophoresis (chip-CE) is a separation method that is compatible with portable and on-site analysis, however, only few commercial chip-CE systems with laser-induced fluorescence (LIF) and light emitting diode (LED) fluorescence detection are available. They are established for several application tailored methods limited to specific biopolymers (DNA, RNA and proteins), and correspondingly the range of their applications has been limited. In this work we address the lack of commercially available research-type flexible chip-CE platforms by exploring the limits of using an application-tailored system equipped with chips and methods designed for DNA separations as a generic chip-CE platform - this is a very significant issue that has not been widely studied. In the investigated Agilent Bioanalyzer chip-CE system, the fixed components are the Agilent chips and the detection (LIF at 635 nm and LEDIF at 470 nm), while the chemistry (electrolyte) and the programming of all the high voltages are flexible. Using standard DNA chips, we show that a generic CE function of the system is easily possible and we demonstrate an extension of the applicability to non-aqueous CE (NACE). We studied the chip compatibility with organic solvents (i.e. MeOH, ACN, DMF and DMSO) and demonstrated the chip compatibility with DMSO as a non-volatile and non-hazardous solvent with satisfactory stability of migration times over 50h. The generic CE capability is illustrated with separations of fluorescent basic blue dyes methylene blue (MB), toluidine blue (TB), nile blue (NB) and brilliant cresyl blue (BC). Further, the effects of the composition of the background electrolyte (BGE) on the separation were studied, including the contents of water (0-30%) and buffer composition. In background electrolytes containing typically 80 mmol/L ammonium acetate and 870 mmol/L acetic acid in 100% DMSO baseline separation of the dyes were achieved in 40s. Linearity was documented in the range of 5-28 µmol/L, 10-100 µmol/L, 1.56-50 nmol/L and 5-75 nmol/L (r(2) values in the range 0.974-0.999), and limit of detection (LOD) values were 90 nmol/L, 1 µmol/L 1.4 nmol/L, and 2 nmol/L for MB, TB, NB and BC, respectively.

Rapid separations of nile blue stained microorganisms as cationic charged species by chip-CE with LIF.

Rapid detection of microorganisms by alternative methods is desirable. Electromigration separation methods have the capability to separate microorganisms according to their charge and size and laser-induced fluorescence (LIF) detection have single-cell detection capability. In this work, a new combined separation and detection scheme was introduced using chip-based capillary electrophoresis (chip-CE) platform with LIF detection. Three microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans were selected as representatives of Gram-positive bacteria, Gram-negative bacteria, and fungi. While their cells carry an overall negative charge in neutral to alkaline pH, staining them with nile blue (NB) provided highly sensitive LIF detection with excitation and emission wavelengths at 635 nm and 685 nm, respectively, and at the same time, the overall charge was converted to positive. Electrolyte pH and concentration of polyethylene oxide (PEO) significantly affected the resolution of the microorganisms. Their optimal separation in the 14 mm separation channel was achieved in less than 30 s (R(s) > 5.3) in an electrolyte consisting of 3.94 mM Tris, 0.56 mM boric acid, 0.013 mM ethylenediaminetetraacetic acid disodium salt dihydrate (pH 10.5), and 0.025% PEO, with injection/separation voltages of +1000/+1000 V. The separation mechanism is likely employing contributions to the overall cationic charge from both the prevalently anionic membrane proteins and the cationic NB. Importantly, the resulting cationic NB-stained cells exhibited excellent separation selectivity and efficiency of ∼38000 theoretical plates for rapid separations within 30-40 s. The results indicate the potential of chip-CE for microbial analysis, which offers separations of a wide range of species with high efficiency, sensitivity, and throughput.