Drug-drug cocrystalllization is a novel mechanism for effective pharmacological combination therapy. In this work, we have demonstrated the preparation of a drug-drug cocrystal of a hypertension drug (Telmisartan; TEL) with a hyperuricemia drug (Febuxostat; FEB) in 1:1 molar ratio using a solvent evaporation method for the first time. Generally, a multi-component system may yield either a eutectic, salt, and/or a cocrystal. This study adopted a methodical orthogonal framework to analyze the final solid form. A single crystal X-ray structural investigation revealed the formation of a heterosynthon with carboxylic and benzimidazole groups of FEB and TEL, respectively, in the triclinic P-1 space group. ΔpKa of the heterosynthon is ∼1.5, hence, based on the empirical rules, a salt-cocrystal continuum is hypothesized. Further, attenuated total reflectance Fourier transform infrared (ATR-FTIR), and Raman spectroscopy were employed to corroborate the hydrogen bond formation in the heterosynthon (-N---H-O-), which confirmed the propensity for cocrystal formation. An accelerated stability study and an in vitro biorelevant dissolution study of the cocrystal were performed, which demonstrated that it is physiochemically stable, but it resulted in a slower dissolution rate when compared with plain drugs.
Conventional sampling of biological fluids often involves a bulk quantity of samples that are tedious to collect, deliver and process. Miniaturized sampling approaches have emerged as promising tools for sample collection due to numerous advantages such as minute sample size, patient friendliness and ease of shipment. This article reviews the applications and advances of microsampling techniques in therapeutic drug monitoring (TDM), covering the period January 2015 - August 2020. As whole blood is the gold standard sampling matrix for TDM, this article comprehensively highlights the most historical microsampling technique, the dried blood spot (DBS), and its development. Advanced developments of DBS, ranging from various automation DBS, paper spray mass spectrometry (PS-MS), 3D dried blood spheroids and volumetric absorptive paper disc (VAPD) and mini-disc (VAPDmini) are discussed. The volumetric absorptive microsampling (VAMS) approach, which overcomes the hematocrit effect associated with the DBS sample, has been employed in recent TDM. The sample collection and sample preparation details in DBS and VAMS are outlined and summarized. This review also delineates the involvement of other biological fluids (plasma, urine, breast milk and saliva) and their miniaturized dried matrix forms in TDM. Specific features and challenges of each microsampling technique are identified and comparison studies are reviewed.
A simple adsorption/desorption procedure using a mixed matrix membrane (MMM) as extraction medium is demonstrated as a new miniaturized sample pretreatment and preconcentration technique. Reversed-phase particles namely polymeric bonded octadecyl (C18) was incorporated through dispersion in a cellulose triacetate (CTA) polymer matrix to form a C18-MMM. Non-steroidal anti-inflammatory drugs (NSAIDs) namely diclofenac, mefenamic acid and ibuprofen present in the environmental water samples were selected as targeted model analytes. The extraction setup is simple by dipping a small piece of C18-MMM (7 mm × 7 mm) in a stirred 10 mL sample solution for analyte adsorption process. The entrapped analyte within the membrane was then desorbed into 100 μL of methanol by ultrasonication prior to high performance liquid chromatography (HPLC) analysis. Each membrane was discarded after single use to avoid any analyte carry-over effect. Several important parameters, such as effect of sample pH, salting-out effect, sample volume, extraction time, desorption solvent and desorption time were comprehensively optimized. The C18-MMM demonstrated high affinity for NSAIDs spiked in tap and river water with relative recoveries ranging from 92 to 100% and good reproducibility with relative standard deviations between 1.1 and 5.5% (n=9). The overall results obtained were found comparable against conventional solid phase extraction (SPE) using cartridge packed with identical C18 adsorbent.
In this study, the potential for carbonaceous nanomaterials to be used as adsorbents for the mixed matrix membrane (MMM) microextraction and preconcentration of organic pollutants was demonstrated. For this method, multiwall carbon nanotubes (MWCNT) and single layer graphene (SLG) nanoparticles were individually incorporated through dispersion in a cellulose triacetate (CTA) polymer matrix to form a MWCNT-MMM and SLG-MMM, respectively. The prepared membranes were evaluated for the extraction of selected polycyclic aromatic hydrocarbons (PAHs) present in sewage pond water samples. The extraction was performed by dipping a small piece of membrane (7 mm × 7 mm) in a stirred 7.5 mL sample solution to initiate the analyte adsorption. This step was followed by an analyte desorption into 60 μL of methanol prior to high performance liquid chromatography (HPLC) analysis. When the optimum SLG-MMM microextraction technique was applied to spiked sewage pond water samples, the detection limit of the method for the PAHs were in the range of 0.02-0.09 ng/mL, with relative standard deviations of between 1.4% and 7.8%. Enrichment factors of 54-100 were achieved with relative recoveries of 99%-101%. A comparison was also made between the proposed approach and standard solid phase extraction using polymeric bonded octadecyl (C18) cartridges.
A new sample pre-treatment technique termed cone-shaped membrane liquid phase microextraction (CSM-LPME) was developed and combined with micro-liquid chromatography (micro-LC) for the determination of selected pesticides in water samples. Four pesticides (hexaconazole, procymidone, quinalphos and vinclozolin) were considered as target analytes. Several important extraction parameters such as types of extraction solvent, agitation rate, pH value, total exposure time and effect of salt and humic acids were optimized. Enrichment factors of > 50 folds were easily achieved within 20 min of extraction. The analytical data demonstrated relative standard deviations for the reproducibility of the optimized CSM-LPME method ranging from 6.3 to 7.5%. The correlation coefficients of the calibration curves were at least 0.9995 across a concentration range of 2-100 microg/L. The detection limits for all the analytes were found to be in the range of 1.1-1.9 microg/L.
A new approach for the quantification of tamoxifen and its metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in human plasma samples using NACE coupled with contactless conductivity detection (C(4) D) is presented. The buffer system employed consisted of 7.5 mM deoxycholic acid sodium salt, 15 mM acetic acid, and 1 mM 18-crown-6 in 100% methanol. The complete separation of all targeted compounds (including endoxifen racemate) could be achieved within 6 min under optimized conditions. The proposed method was validated and showed good linearity in the range from 100 to 5000 ng/mL with correlation coefficients between 0.9922 and 0.9973, LODs in the range of 25-40 ng/mL, and acceptable reproducibility of the peak area (intraday RSD 2.2-3.1%, n = 4; interday (3 days) RSD 6.0-8.8%, n = 4). The developed method was successfully demonstrated for the quantification of tamoxifen and its metabolites in human plasma samples collected from breast cancer patients undertaking tamoxifen treatment.
Micelle to solvent stacking was implemented for the recently established NACE-C(4) D method to determine tamoxifen and its metabolites in standard samples and human plasma of breast cancer patients. For stacking, the standard samples and extract after liquid-liquid extraction (LLE) were prepared in methanol and the resulting sample solution was pressure injected after a micellar plug of SDS. Factors that affected the stacking such as SDS concentration, micelle, and sample plug length were examined. The sensitivity enhancement factor (peak height from stacking/peak height from typical injection of sample in BGE) was 15-22. The method detection limits with LLE were in the range of 5-10 ng/mL, which was lower than the established method (where the LLE extract was also prepared in methanol) with reported method detection limits of 25-40 ng/mL. The intraday and interday repeatability were in the range of 1.0-3.4% and 3.8-6.5%, respectively.
A novel microextraction technique termed solid phase membrane tip extraction (SPMTE) was developed. Selected triazine herbicides were employed as model compounds to evaluate the extraction performance and multiwall carbon nanotubes (MWCNTs) were used as the adsorbent enclosed in SPMTE device. The SPMTE procedure was performed in semi-automated dynamic mode and several important extraction parameters were comprehensively optimized. Under the optimum extraction conditions, the method showed good linearity in the range of 1-100 microg/L, acceptable reproducibility (RSD 6-8%, n=5), low limits of detection (0.2-0.5 microg/L), and satisfactory relative recoveries (95-101%). The SPMTE device could be regenerated and reused up to 15 analyses with no analyte carry-over effects observed. Comparison was made with commercially available solid phase extraction-molecular imprinted polymer cartridge (SPE-MIP) for triazine herbicides as the reference method. The new developed method showed comparable or even better results against reference method and is a simple, feasible, and cost effective microextraction technique.
In this work, a new variation of the electromembrane extraction (EME) approach employing a hollow polymer inclusion membrane (HPIM) was developed. In this method, a HPIM was prepared by casting a solution of the desired proportions of cellulose acetate (CTA), tris(2-ethylhexyl)phosphate (TEHP) and di-(2-ethylhexyl)phosphoric acid (D2EHPA) in dichloromethane on glass capillary tubing. Three basic drugs namely amphetamine, methamphetamine, and 3,4-methylenedioxy-N-methylamphetamine (MDMA) were selected as model analytes to evaluate the extraction performance of this new approach. The drugs were extracted from human plasma samples, through a 20μm thickness HPIM, to an aqueous acceptor solution inside the lumen of the hollow membrane. Parameters affecting the extraction efficiency were investigated in detail. Under the optimized conditions, enrichment factors in the range of 97-103-fold were obtained from 3mL of sample solution with a 10min extraction time and an applied voltage of 300V across the HPIM. The detection limits of the method for the three drugs were in the range of 1.0-2.5ng/mL (at a signal/noise ratio of three), with relative standard deviations of between 6.4% and 7.9%. When the method was applied to spiked plasma samples, the relative recoveries ranged from 99.2% to 100.8%. Enrichment factors of 103, 99 and 97 were obtained for amphetamine, methamphetamine, and MDMA, respectively. A comparison was also made between the newly developed approach and EME using supported liquid membranes (SLM) as well as standard sample preparation methods (liquid-liquid extraction) used by the Toxicology Unit, Department of Chemistry, Malaysia.
A new dispersive inclusion complex microextraction (DICM) approach coupled with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the determination of n-nitrosamine impurities in different medicinal products is demonstrated for the first time. The proposed DICM procedures consist of a dispersive liquid phase microextraction steps employing cyclodextrin as an inclusion complex agent to extract n-nitrosamines namely N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiisopropylamine (NDIPA), N-ethyl-N-nitrosoisopropylamine (NEIPA) and N-nitroso-di-n-butylamine (NDBA) present in the medicinal products. The sample solutions were prepared by mixing 5% (m/v) NaCl solution with 1.5 mM β-cyclodextrin and 20 mM sodium dodecyl sulphate to form a stable inclusion complex and subsequently extracted into dichloromethane as an extraction solvent. The enriched solution was reconstituted into aqueous solution prior to UPLC-MS/MS analysis. The method showed good linearity in the range of 0.036-1 ng/mL with a correlation coefficient of at least 0.995, acceptable reproducibility (RSD 0.5-5.8%, n=5), low limits of detection (0.011-0.018 ng/mL), and satisfactory relative recoveries (96-105%). The results obtained were found to be at least 10-fold more sensitive comparable to those obtained using validated direct sample dissolutions coupled with UPLC-MS/MS approach.
A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 microM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01-200 microg/L (GLYP) and 0.1-400 microg/L (AMPA), acceptable reproducibility (RSD 5-7%, n=5), low limits of detection of 0.005 microg/L for GLYP and 0.06 microg/L for AMPA, and satisfactory relative recoveries (90-94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success.
A polymer inclusion membrane (PIM) based sampling probe was developed for electrokinetic extraction of drugs from biological fluids. The probe was fabricated by dip-coating a nonconductive glass capillary tube in a homogeneous PIM solution for three cycles. The PIM solution comprised cellulose triacetate (CTA), 2-nitrophenyl octyl ether (NPOE), and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [EMIM][NTf2] in a ratio of 5:4:2. The developed probe electrokinetically extracted doxorubicin from human plasma, human serum, and dried blood spot (DBS). The practicability and reliability of the electrokinetic extraction were evaluated by LC-MS/MS to quantify the desorption of extracted doxorubicin. Under the optimized conditions, a quantification limit of 0.2-2 ng/mL was achieved for the three biological samples. The probe was further integrated into a portable battery-powered device for safe low-voltage (36 V) electrokinetic extraction. The developed technique is envisioned to provide a more efficient analytical workflow in the laboratory.
An online preconcentration method, namely electrokinetic supercharging (EKS), was evaluated for the determination of tamoxifen and its metabolites in human plasma in nonaqueous capillary electrophoresis with ultraviolet detection (NACE-UV). This method was comprehensively optimized in terms of the leading electrolyte (LE) and terminating electrolyte (TE) injection lengths, as well as electrokinetic sample injection time. The optimized EKS conditions employed were as follows: hydrodynamic injection (HI) of 10mM potassium chloride as LE at 150mbar for 36s (4% of total capillary volume). The sample was injected at 10kV for 300s, followed by HI of 10mM pimozide as TE at 150mbar for 36s (4% of total capillary volume). Separation was performed in 7.5mM deoxycholic acid sodium salt, 15mM acetic acid and 1mM 18-crown-6 in 100% methanol at +25kV with UV detection at 205nm. Under optimized conditions, the sensitivity was enhanced between 160- and 600-fold when compared with our previously developed method based on HI at 150mbar for 12s. The detection limit of the method for tamoxifen and its metabolites were 0.05-0.25ng/mL, with RSDs between 2.1% and 3.5%. Recoveries in spiked human plasma were 95.6%-99.7%. A comparison was also made between the proposed EKS approach and the standard field-amplified sample injection (FASI) technique. EKS proved to be 3-5 times more sensitive than the FASI. The new EKS method was applied to the analysis of tamoxifen and its metabolites in plasma samples from breast cancer patients after liquid-liquid extraction.
Point-of-collection (POC) devices aim for a fast, on-site detection for medical and environmental purposes. In this area, microfluidic Paper-based Analytical Devices (μPADs) have recently gained popularity because these are potentially cheap and environmentally friendly to produce, and easy to use. From an analytical perspective, paper is well known for its use as a substrate for chromatography, but less known for its use in electrophoretic separations. With the recent interest in μPADs, most applications are based on rather simple assays with relatively few applications incorporating an analytical separation. The focus of this review is on paper-based electrophoresis, originating with the key developments in the 1940s and 1950s as well as the recent developments of electrophoretic μPADs, and concluding with a critical discussion of the opportunities and challenges for electrophoretic μPADS in the future.
The low conductivity of separation electrolytes employed in nonaqueous capillary electrophoresis (NACE) limits the use of on-line sample concentration or stacking by field enhancement. Herein, micelle-to-solvent stacking (MSS) was performed by the simple injection of a micellar solution plug prior to electrokinetic injection of sample prepared under field-enhanced stacking conditions (known as field-enhanced sample injection, FESI). The proposed approach allowed a 214-625-fold improvement in peak signals for targeted anticancer drugs (e.g., tamoxifen) and its major metabolites in NACE using 100% methanol-based separation electrolyte that comprised of 7.5mM deoxycholic acid sodium salt, 15mM acetic acid and 1mM 18-crown-6. These improvements yielded tamoxifen and its metabolites with 2-5 times better stacking efficiency as compared to those obtained without micellar solution injection or FESI only. This is comparable to the results typically achieved when FESI is combined with isotachophoresis (electrokinetic supercharging). The FESI-MSS-NACE was tested for the measuring levels of target drugs in plasma. The analytical figures of merit are also reported.
A method for on-line matrix elimination to enable selective quantification of ultraviolet absorbing analytes by a flow-injection analysis procedure is described. Selectivity is achieved by electric field driven extraction across a polymer inclusion membrane. The method was demonstrated on the example of the determination of naproxen from spiked human urine. Membranes of 10 μm thickness were employed which consisted of 7.5 mg cellulose triacetate as base polymer, 5 mg of o-nitrophenyl octyl ether as plasticizer and 7.5 mg of Aliquat 336 as cationic carrier. Ten μL of sample was introduced into a continuous stream of background solution consisting of 100 µM aqueous NaClO₄ with a flow rate of 2 μL/min while applying a voltage of 150 V to the extraction cell. The target ion was electrokinetically transported across the membrane and enriched in 1.5 μL of a stagnant acceptor solution. This was subsequently pumped past a flow-through UV detector for quantification. The method showed a linear range from 5 to 200 µM with a correlation coefficient of 0.9978 and a reproducibility of typically 7% (n = 8). The detection limit of the method for naproxen was 2 µM.
High temperature liquid chromatography using water-rich and superheated water eluent is evaluated as a new approach for the separation of selected triazole fungicides, hexaconazole, tebuconazole, propiconazole, and difenoconazole. Using a polybutadiene-coated zirconia column at temperatures of 100-150 degrees C, clear separations were achieved when 100% purified water was utilized as organic-free eluent. Excellent limits of detection down to pg level were obtained for the separation of the triazole fungicides under optimum conditions. Van't Hoff plots for the separations were linear suggesting that no changes occurred in the retention mechanism over the temperature range studied.
An electrokinetic platform was developed for extracting small-molecule pharmaceuticals from a dried blood spot. Through the exclusion of liquid reagents and use of low field strength (6 V cm-1 ), the electroextraction of a drug from a dried blood spot, deposited on a polymer inclusion membrane (PIM), could be realised while in transit in the mail. In transit sample preparation provides a potential solution to in situ sample degradation and may accelerate the workflow upon arrival of a patient sample at the analytical facility. The electroextraction method was enabled through our discovery of the use of 15-20 μm thin PIMs as electrophoretic separation medium in absence of liquid reagents. Here, a PIM consisting of cellulose triacetate as polymer base, 2-nitrophenyl octyl ether as plasticizer and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide as carrier was used. The PIM, was packaged with two 12 V batteries to supply the separation voltage. A blood spot containing berberine chloride was deposited and dried before the applying the separation potential, allowing for the electroextraction while the packaged device was shipped in internal mail. Upon arrival in the analytical laboratory, the PIM was analysed using a fluorescence microscope with photon multiplier tube, quantifying the berberine extracted away from the sample matrix. This platform represents a new opportunity for processing clinical samples during transport to the laboratory, saving time and manual handling to accelerate the time to result.
A new approach based on the integration of the free liquid membrane (FLM) into electrokinetic supercharging (EKS) was demonstrated to be a new powerful tool used in order to enhance online preconcentration efficiency in capillary electrophoresis (CE). A small plug of water immiscible organic solvent was used as a membrane interface during the electrokinetic sample injection step in EKS in order to significantly enhance the analyte stacking efficiency. The new online preconcentration strategy was evaluated for the determination of paraquat and diquat present in the environmental water samples. The optimised FLM-EKS conditions employed were as follows: hydrodynamic injection (HI) of 20mM potassium chloride as leading electrolyte at 50mbar for 75s (3% of the total capillary volume) followed by the HI of tris(2-ethylhexyl) phosphate (TEHP) as FLM at a 1mm length (0.1% of the capillary volume). The sample was injected at 10kV for 360s, followed by the HI of 20mM cetyl trimethylammonium bromide (CTAB) as terminating electrolyte at 50mbar for 50s (2% of the total capillary volume). The separation was performed in 12mM ammonium acetate and 30mM NaCl containing 20% MeOH at +25kV with UV detection at 205nm. Under optimised conditions, the sensitivity was enhanced between 1500- and 1866-fold when compared with the typical HI at 50mbar for 50s. The detection limit of the method for paraquat and diquat was 0.15-0.20ng/mL, with RSDs below 5.5%. Relative recoveries in spiked river water were in the range of 95.4-97.5%. A comparison was also made between the proposed approach with sole preconcentration of the field-enhanced sample injection (FASI) and EKS in the absence of the FLM.
A sample pre-treatment method based on a dynamic mixed matrix membrane tip extraction followed by capillary electrophoresis with contactless conductivity detection (CE-C4D) was evaluated for the determination of tobramycin in human plasma. The extraction tip device consisted of a cellulose triacetate membrane tip wall immobilised with 15% (w/w) of hydrophilic lipophilic balance (HLB) nanoparticles as adsorbent. The extraction was performed dynamically by withdrawing/dispensing the plasma sample through the tip device followed by desorption into 20 μL of acidified aqueous solution at pH 3 prior to the CE-C4D analysis. Under the optimum conditions, the detection limit of the method for tobramycin was 10 ng/mL, with intraday and interday repeatability RSDs of 3.5% and 4.5%, respectively. Relative recoveries in spiked human plasma were 99.6%-99.9%. The developed approach was successfully demonstrated for the quantification of tobramycin in human plasma samples.