Simultaneous separation and detection of monochloramine, nitrite, and nitrate by step-gradient mixed-mode ion chromatography: Translation from benchtop to portable ion chromatograph.

BACKGROUND: Nitrite (NO2-) and nitrate (NO3-) can be produced in the distribution systems of chloraminated drinking water due to the nitrification of ammonia. The most applied inorganic chloramine for this purpose, namely monochloramine (NH2Cl), is also released into aquatic environments from water treatment plants' effluent and within industrial waste streams. Within the treatment process, the continuous monitoring of disinfectant levels is necessary to limit the harmful disinfectant by-product (DBP) formation. Currently, NH2Cl can interfere with nutrient analysis in water samples, and there are no analytical techniques available for the simultaneous analysis of NH2Cl, NO2-, and NO3-. RESULTS: A green analytical method based on mixed-mode ion chromatography, specifically ion exchange and ion exclusion modes, was developed for the simultaneous separation and detection of NH2Cl, NO2-, and NO3-. The separation was achieved using a Dionex IonPac AG15 column guard column and a step gradient elution involving deionized water and 120.0 mM NaCl. The method was developed using a benchtop HPLC with a custom-made multi-wavelength UV absorbance detector with a 50-mm flow cell to enable the sensitive detection of NH2Cl, NO2-, and NO3- at 240 nm, 220 nm, and 215 nm, respectively. The developed method was then transferred to a portable ion chromatography (IC) system, the Aquamonitrix analyser. The total run time was less than 10 min for both systems. The benchtop HPLC method had a limit of detection (LOD) of 0.07 µg mL-1 as Cl2 for NH2Cl, 0.01 µg mL-1 for NO2-, and 0.03 µg mL-1 for NO3-. The LODs obtained using the portable Aquamonitrix analyser were found to be 0.36 µg mL-1 as Cl2, 0.02 µg mL-1, and 0.11 µg mL-1 for NH2Cl, NO2-, and NO3-, respectively. Excellent linearity (r ≥ 0.9999) was achieved using the portable analyser over the studied concentration ranges. The developed system was applied to the analysis of spiked municipal drinking water samples and showed excellent repeatability for the three analytes at three different concentration levels (RSD of triplicate recovery experiments ≤ 1.9 %). Moreover, the variation in retention time was negligible for the three target analytes with RSD ≤ 0.8 % over 12 runs. SIGNIFICANCE: We are reporting the first ion chromatographic method for the simultaneous separation and detection of NH2Cl, NO2-, and NO3- in water samples. The monitoring of NH2Cl, NO2-, and NO3- is critical for the determination of disinfectant dosing, water quality, and nitrification status. The developed method can be applied using a benchtop HPLC or via the portable automated IC system to monitor for the three target compounds analysis in water treatment plants.

Application of a portable ion chromatograph for real-time field analysis of nitrite and nitrate in soils and soil pore waters.

Real-time monitoring of nitrite and nitrate is crucial for maintaining soil health and promoting plant growth. In this study, a portable ion-chromatograph (IC, Aquamonitrix) analyser, coupled with a field-applicable ultrasonic-assisted extraction method, was utilised for in-field determination of nitrate and nitrite in soils. This is the first application of this type of analyser to soil nutrients. On-site analysis of soil from a local sports field showed 94.8 ± 4.3 µg g-1 nitrate, with LODs of 32.0 µg g-1 for nitrate and 5.4 µg g-1 for nitrite. The results were in close agreement with those obtained using a conventional lab-based IC. Relative standard deviations (%RSDs) for soil analysis using Aquamonitrix were consistently below 10%. The obtained average recoveries of samples spiked with nitrite were 100% and 104% for the portable IC and conventional IC, respectively. Furthermore, to assess the suitability of portable IC for samples with high organic matter content, various natural organic fertilisers were extracted and analysed. The results showed 16.2 ± 0.7 µg g-1 nitrite and 28.5 ± 5.6 µg g-1 nitrate in sheep manure samples with LODs of 2.0 µg g-1 for nitrite and 12.0 µg g-1 for nitrate. The portable IC system was further demonstrated via real-time on-site analysis of soil pore-water acquired using a portable battery-based ceramic pore-water sampler. A continuous increase in nitrate concentration over time was observed (from 80 to 148 µg mL-1) in the soil pore-water in a vegetable garden four days after heavy rain. Unlike conventionally sampled natural waters, 7-day storage of the studied pore water samples revealed no changes in nitrate concentrations. An average of 558 ± 51 µg mL-1 nitrate was detected in the soil pore-water samples analysed on a spinach farm, immediately after irrigation.

Multi-wavelength deep-ultraviolet absorbance detector based upon program-controlled pulsing light-emitting diodes.

A novel approach for multi-wavelength ultraviolet (UV) absorbance detection has been introduced employing a single board computer (SBC) with a field programmable gate array (FPGA), Red Pitaya SBC, to generate separated micro pulses for three deep-ultraviolet light-emitting diodes (DUV-LEDs), λmax = 235, 250, and 280 nm, along with data acquisition and processing via a custom-made program. The pulse set generation and data acquisition were synchronized using the SBC. The outputs of the three pulsing DUV-LEDs were combined and transmitted to the flow cell via a solarisation resistant trifurcated optical fiber (OF). An ultra-fast responding photodiode was connected to the optical-fiber-compatible flow cell to record the intensity of the DUV pulses. Upper limit of detector linearity (A95 %) was found to be 1917 mAU, 2189 mAU, and 1768 mAU at 235 nm, 250 nm, and 280 nm, respectively, with stray light ≤0.9 %. In addition, the effective path length (Leff) was estimated to be ≥98.0 % of the length of the used flow cell (50 mm). The new pulsed multi-LEDs absorbance detector (PMLAD) has been successfully coupled with a standard liquid chromatograph and utilized for the analysis of pharmaceuticals. Paracetamol, caffeine, and aspirin were simultaneously determined at 250, 280, and 235 nm, respectively, using the PMLAD. The absorbance ratios between the different wavelengths were applied to further confirm the identity of the studied compounds. Excellent linearity was achieved over a range of 0.1-3.2 µg/mL for paracetamol, 0.4-6.4 µg/mL for caffeine, and 0.8-12.8 µg/mL for aspirin with a regression correlation coefficient (r2) ≥ 0.99996. The quantitation limits (LOQs) were 0.10 µg/mL, 0.38 µg/mL, and 0.66 µg/mL for paracetamol, caffeine, and aspirin, respectively.

Green and sensitive spectrofluorimetric determination of Remdesivir, an FDA approved SARS-CoV-2 candidate antiviral; application in pharmaceutical dosage forms and spiked human plasma.

Fluorescence spectroscopy has gained much attention over the last few years. Its advantages cover both analytical performance as well as ecological greenness. The quality control of pharmaceuticals requires sensitive, fast and economic methodologies to provide a high throughput at desirable costs. The low energy and decreased solvent consumption make this technique green as well as economic, and hence it is much preferred by pharmaceutical industries. Remdesivir is a recent antiviral, previously used for the treatment of Ebola virus infections, which was approved by the US-FDA in 2020 for treatment of infections caused by SARS-CoV-2 virus. Formulation and wide-scale production of this drug started recently and hence methodologies related to its quality control are highly required. A smart spectrofluorimetric method was validated as per the US-FDA guidelines for the facile estimation of Remdesivir. The assay of Remdesivir was based on its native fluorescence measurements at pH 4 and wavelengths of 244/405 nm. Calibration was achieved over the range of 1.0-65.0 ng mL-1. Different variables affecting the proposed methodology were studied to achieve maximum sensitivity, at limits of detection and quantification of 0.287 and 0.871 ng mL-1, respectively. The developed method is regarded as the first spectrofluorimetric one for the estimation of Remdesivir. The developed method was also utilized for the determination of the drug in its formulated IV infusion and in spiked human plasma. Statistical analysis verified that this method is accurate and reproducible. Moreover, the ecological impact of the developed procedures was evaluated on two recently reported assessment metrics, the Green Analytical Procedure Index (GAPI) and AGREE-analytical greenness metric, to emphasize the greenness of the procedure.

Hyphenated sample preparation-electrospray and nano-electrospray ionization mass spectrometry for biofluid analysis.

Stand-alone electrospray ionization mass spectrometry (ESI-MS) has been advancing through enhancements in throughput, selectivity and sensitivity of mass spectrometers. Unlike traditional MS techniques which usually require extensive offline sample preparation and chromatographic separation, many sample preparation techniques are now directly coupled with stand-alone MS to enable outstanding throughput for bioanalysis. In this review, we summarize the different sample clean-up and/or analyte enrichment strategies that can be directly coupled with ESI-MS and nano-ESI-MS for the analysis of biological fluids. The overview covers the hyphenation of different sample preparation techniques including solid phase extraction (SPE), solid phase micro-extraction (SPME), slug flow micro-extraction/nano-extraction (SFME/SFNE), liquid extraction surface analysis (LESA), extraction electrospray, extraction using digital microfluidics (DMF), and electrokinetic extraction (EkE) with ESI-MS and nano-ESI-MS.

Green micellar solvent-free HPLC and spectrofluorimetric determination of favipiravir as one of COVID-19 antiviral regimens.

Quality control (QC) of pharmaceutical products requires fast, sensitive as well as economic methodologies in order to provide high through output at low cost which are the main aspects considered by such economic facilities. Meanwhile, the ecological impacts must be considered by researchers to minimize the hazardous effects of research laboratories. Favipiravir (FAV) is an antiviral agent recently approved for treatment of COVID-19 infections during 2020 pandemic crisis, so the size of its production by international pharmaceutical corporations evolved dramatically within the past few months. Two novel simple, sensitive, and green methods were developed and validated for FAV determination based on solvent-free micellar LC and spectrofluorimetry techniques. To improve FAV native fluorescence, several factors were studied including solvent type, buffering, pH and added surfactants. The best sensitivity for FAV fluorescence was obtained in Britton-Robinson buffer (pH 4) at 436 nm after excitation at 323 nm within concentration range of 20-350 ng mL-1. Another HPLC method was validated using C18-RP (5 µm, 250 × 4.6 mm) stationary phase and solvent-free mobile phase consisting of (0.02 M Brij-35, 0.15 M SDS, and 0.02 M disodium hydrogen phosphate, pH 5.0) isocratically eluted at a flow rate of 1 mL min-1 and detection wavelength of 323 nm. LC method was validated across concentration range of 10-100 µg mL-1 and FAV eluted in 3.8 min. The methods were validated according to the FDA guidelines and were applied successfully for determination of FAV in its marketed tablet dosage forms and in spiked human plasma samples. The proposed methods are eco-friendly since they are typically based on biodegradable reagents in aqueous solvent-free phases, which was proven by their assessment on two recent greenness metrics (GAPI and AGREE) to prove their eco-friendly properties.

In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 µg mL-1 and 2.9 µg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

In-Syringe Electrokinetic Ampholytes Focusing Coupled with Electrospray Ionization Mass Spectrometry.

A 25 µL analytical glass syringe has been used for isoelectric focusing (IEF) utilizing the stainless-steel needle and plunger as electrodes. The generation of protons and hydroxyl ions at the electrodes facilitated a neutralization reaction boundary (NRB) mechanism to focus different amphoteric compounds, such as hemoglobin, bovine serum albumin, R-phycoerythrin, and histidine, within minutes. After optimization of different experimental parameters affecting the IEF process and the coupling of the IEF syringe with electrospray ionization mass spectrometry (ESI-MS), a BGE composed of NH4Ac, 1.0 mM, pH 4.0, in 70.0% (v/v) acetonitrile was used for the IEF of histidine. A voltage of -200 V was applied for 5.0 min to accomplish the IEF and increased to -400 V during the infusion to ESI-MS at a flow rate of 4.0 µL/min. The coaxial sheath liquid consisting of 0.2% (v/v) formic acid was added at 4.0 µL/min. The detection limit was found to be 2.2 µg/mL and a nonlinear quadratic fit calibration curve was constructed for histidine over the range of 4.0-64.0 µg/mL with a correlation coefficient ( r) = 0.9998. The determination of histidine in spiked urine samples as relevant for the diagnosis of histidinemia was demonstrated by the IEF syringe-ESI-MS system with accuracy from 88.25% to 102.16% and a relative standard deviation less than 11%.

Development and Validation of a New HPLC Method for the Simultaneous Determination of Paracetamol, Ascorbic Acid, and Pseudoephedrine HCl in their Co-formulated Tablets. Application to in vitro Dissolution Testing.

The first HPLC method was developed for the simultaneous determination of paracetamol (PC), ascorbic acid (AA), and pseudoephedrine HCl (PE) in their co-formulated tablets. Separation was achieved on a C18 column in 5 min using a mobile phase composed of methanol-0.05 M phosphate buffer (35:65, v/v) at pH 2.5 with UV detection at 220 nm. Linear calibration curves were constructed over concentration ranges of 1.0 - 50.0, 3.0 - 60.0 and 3.0 - 80.0 µg mL(-1) for PC, AA, and PE, respectively. The method was validated and applied for the simultaneous determination of these drugs in their tablets with average % recoveries of 101.17 ± 0.67, 98.34 ± 0.77, and 98.95 ± 1.11%, for PC, AA, and PE, respectively. The proposed method was also used to construct in vitro dissolution profiles of the co-formulated tablets containing the three drugs.