Over the last ten years, helium direct analysis in real time time-of-flight mass spectrometry (He DART-TOFMS) has become an established technique in rapid screening of forensic drugs to decrease the time necessary to triage forensic drug cases, therefore contributing to backlog reduction and more timely criminal prosecution. Recently, we demonstrated that N2 DART was able to efficiently ionize all polar compounds except for a few extremely small ones such as methanol and acetonitrile. Therefore, N2 DART-TOFMS should be a suitable technique for rapid screening of forensic drugs.Nitrogen direct analysis in real time time-of-flight mass spectrometry (N2 DART-TOFMS) was performed using a JEOL AccuTOF mass spectrometer with an IonSense DART-100 ion source. A 3-min analytical protocol was used for the analysis of each sample. Sample introduction was accomplished by moving the closed end of a melting point capillary where approximately 1 μL sample solution was deposited or the exposed inside of a freshly cut tablet across the N2 gas stream between the DART-100 ion source and orifice 1 of the AccuTOF.Ten commonly abused drugs, eight synthetic cannabinoids and four controlled prescription drugs (CPDs) were analyzed. The limit of detection (LOD) was determined to be approximately 10 μg/mL or 10 pg in quantities. All drugs at the LOD level were positively identified using their [M + H]+ ions with mass errors less than 5 mDa. The identification were further supported by in-source fragment ions and characteristic N2 DART ions that are not commonly generated by He DART, e.g. [M + H + O]+ and [M + H + 2O]+ ions.It was concluded that the 3-min analytical protocol could be utilized in the analysis of seized drugs in the form of tablets and powders or prepared in solution. In consideration that N2 is readily available in the air and He is a non-renewable resource, N2 DART-TOFMS is a greener, cheaper and more convenient alternative to He DART-TOFMS in rapid screening of forensic drugs.
Nitrogen can be an inexpensive alternative to helium used by direct analysis in real time (DART), especially in consideration of the looming helium shortage. Therefore, the ionization mechanism of positive-ion N2 DART has been systematically investigated. Our experiments suggest that a range of metastable nitrogen species with a variety of internal energies existed and all of them were less energetic than metastable helium atoms. However, compounds with ionization energies (IE) equal to or lower than 10.2 eV (all organic compounds except the extremely small ones) can be efficiently ionized. Because N2 DART was unable to efficiently ionize ambient moisture and common organic solvents such as methanol and acetonitrile, the most important ionization mechanism was direct Penning ionization followed by self-protonation of polar compounds generating [M+H]+ ions. On the other hand, N2 DART was able to efficiently ionize ammonia, which was beneficial in the ionization of hydrogen-bonding compounds with proton affinities (PA) weaker than ammonia generating [M+NH4]+ ions and large PAHs generating [M+H]+ ions through proton transfer. N2 DART was also able to efficiently ionize NO, which led to the ionization of nonpolar compounds such as alkanes and small aromatics generating [M–(2m+1)H]+ (m=0,1…) ions. Lastly, metastable nitrogen species was also able to produce oxygen atoms, which resulted in increased oxygen adducts as the polarity of organic compounds decreased. In comparison with He DART, N2 DART was approximately one order of magnitude less sensitive in generating [M+H]+ ions, but could be more sensitive in generating [M+NH4]+ ions.
A new separation medium, poly(N-isopropylacrylamide)-g-poly(ethyleneoxide) (PNI-PAM-g-PEO) solution, used for double-stranded (ds) DNA separation by capillary electrophoresis (CE) is presented. This type of grafted copolymer has a good self-coating ability for quartz capillary tubing and a slightly temperature-dependent viscosity-adjustable property, making it easier to use. One bp resolution was achieved within 12.5 min by using 8% w/v PNIPAM-gPEO in 1 x TBE (Tris-borate-ethylenediaminetetraaceticacid) buffer with an effective column length of 10 cm and an applied electric field strength of 200 V/cm. The PNIPAM-g-PEO solutions had a high sieving ability for relatively small sized DNAs with the relative standard derivation for the first 10 runs being less than 0.9% by using the same polymer solution. With 8% w/v PNIPAM-g-PEO solution in a 1.5 cm column and 2400 V as the running voltage, phiX174/HaeIII digest could be clearly separated within 24 s.
Mixtures of two polymers with totally different chemical structures, polyacrylamide and polyvinylpyrrolidone (PVP) have been successfully used for double-stranded DNA separation. By polymerization of acrylamide in a matrix of PVP solution, the incompatibility of these two polymers was suppressed. Laser light scattering (LLS) studies showed that highly entangled interpenetrating networks were formed in the solution. Further systematic investigation showed that double-stranded DNA separation was very good in these interpenetrating networks. With a concentration combination of as low as 2% w/v PVP (weight-average molecular mass Mr = 1×106 g/mol) + 1% w/v polyacrylamide (Mr = 4×105 g/mol), the 22 fragments in pBR322/HaeIII DNA, including the doublet of 123/124 bp, have been successfully separated within 6.5 min. Under the same separation conditions, similar resolution could only be achieved by using polyacrylamide (Mr = 4×105 g/mol) with concentrations higher than 6% w/v and could not be achieved by using only PVP (Mr = 1×106 g/mol) with a concentration as high as 15% w/v. It is noted that the interpenetrating network formed by 2% PVP and 1% polyacrylamide has a very low viscosity and can dynamically coat the inner wall of a fused-silica capillary. The separation reached an efficiency of more than 107 theoretical plate numbers/m and a reproducibility of less than 1% relative standard deviation of migration time in a total of seven runs. The interpenetrating network could stabilize polymer chain entanglements. Consequently, the separation speed was increased while retaining resolution.
Two high molecular weight copolymers of poly(N-isopropylacrylamide) (PNIPAM) densely grafted with short poly(ethylene oxide) (PEO) chains (PNIPAM-g-PEO) were studied by NMR and laser light scattering. The long PNIPAM chains with densely grafted PEO branches had a random coil conformation at very dilute concentrations and low temperatures (i.e., T ≤ 30 °C). When the temperature was increased above 31 °C, the copolymers could undergo a broad "coil-to-globule" transition. The collapsed copolymer chains had a 〈Rg〉/〈Rh〉 value of about 1.0 with PNIPAM chains inside the core and the hydrophilic PEO chains on the surface. This kind of PNIPAM-g-PEO copolymers was studied as a DNA separation medium in capillary electrophoresis. Several advantages of the copolymers as a separation medium for DNA fragments were achieved, such as an automatic coating ability for the capillary inner wall, an easier injection into the capillary channel due to the slightly adjustable viscosity with temperature (up to 31 °C), a high resolution (i.e., one base pair resolution), and fast separation time. In contrast, the homo-PNIPAM or PEO showed worse DNA separation efficiency under similar conditions. The high DNA separation efficiency of the PNIPAM-g-PEO copolymers is related to the polymer chain conformation. The long copolymer chains in a random coil conformation with densely grafted PEO branches could form a physical network with a relatively stable and uniform pore size at high concentrations (i.e., ≥10 wt %). The separation medium has a high sieving ability for DNA separation in terms of DNA migration mechanisms. The collapsed copolymer chains in the globule state could destroy the chain network and thus lose the DNA separation ability.
Polymeric microspheres containing a magnetic core have been used in cancer therapy for biophysical targeting of antitumor agents and in magnetic resonance imaging as contrasting agents. For the Human Genome Project, deoxyribose nucleic acid (DNA) capillary electrophoresis has become the most widely used analytical technique where a key component is the design of an effective separation medium. The synthesis and optical characterization of polymeric coated superparamagnetic nanoparticles and of (self-assembled) polymer networks by means of a range of physical techniques, including laser light scattering and laser-induced fluorescence detection, are presented. (1) Polymeric microspheres with a superparamagnetic core. A water-in-oil microemulsion approach has been used successfully to synthesize the superparamagnetic core and the polymeric microsphere in one continuous step. The synthesis permits us to control the magnetic nanoparticle size and the thickness of the hydrogel, ranging from 80 to 320 nm. Magnetite concentration in the microspheres, calculated by vibrating-sample magnetometry, was found to be up to 3.3 wt %. The internal structure of the microspheres, as observed by atomic force microscopy, confirmed a core-shell model. (2) Development of new separation media for DNA capillary electrophoresis. Block copolymers in selective solvents can self-assemble to form supramolecular structures in solution. The nanostructures can be characterized in the dilute concentration regime by means of laser light scattering. At semidilute concentrations, the mesh size, the supramolecular structure, and the surface morphology can be investigated by means of small angle x-ray scattering and atomic force microscopy. The structural knowledge and the information on chain dynamics can then be correlated with electrophoresis using laser-induced fluorescence detection to provide a deeper understanding for the development of new separation media.
To quantify phytocannabinoids in hemp, liquid chromatography diode array detector (LC-DAD) methods are favored, but their selectivity depends on baseline separation of all phytocannabinoids and unknown compounds in an extract. Therefore, development of a LC-DAD method with a different selectivity has become highly desirable. Currently, most LC-DAD methods use the water/acetonitrile eluting system, while this study aimed to use the water/methanol eluting system. A systematic investigation of various chromatographic parameters on LC separation of eighteen phytocannabinoids, the maximum number that has been quantified in hemp so far, plus two potential internal standards, led to a four-step isocratic mobile phase that was able to baseline separate the twenty compounds with a significantly different eluting order from published methods. Although changes in the mobile phase composition caused baseline drifts, consequent difficulty in quantification was avoided through detection at wavelengths longer than 230 nm. Subsequently, the method was validated according to the ISO 17025 guidelines, calibrated between 0.04 to 50 µg/mL, and used to analyze phytocannabinoids in nine strains of hemp flowers that were extracted using methanol between 0.04 to 50% (w/w). Extraction recovery was tracked in real-time by spiking one of the two potential internal standards, i.e., abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp. Method selectivity was further assessed by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS), indicating minimum interferences. In addition, five untargeted/unknown phytocannabinoids were identified by ESI/TOFMS, including two structural isomers of Δ9-tetrahydrocannabinol (Δ9-THC), two structural isomers of Δ9-tetrahydrocannabinolic acid (Δ9-THCA), and one structural isomer of Δ9-THC acetate.
The ionization mechanism of negative ion-direct analysis in real time (NI-DART) has been investigated using over 42 compounds, including fullerenes, perfluorocarbons (PFC), organic explosives, phenols, pentafluorobenzyl (PFB) derivatized phenols, anilines, and carboxylic acids, which were previously studied by negative ion-atmospheric pressure photoionization (NI-APPI). NI-DART generated ionization products similar to NI-APPI, which led to four ionization mechanisms, including electron capture (EC), dissociative EC, proton transfer, and anion attachment. These four ionization mechanisms make both NI-DART and NI-APPI capable of ionizing a wider range of compounds than negative ion-atmospheric pressure chemical ionization (APCI) or negative ion-electrospray ionization (ESI). As the operation of NI-DART is much easier than that of NI-APPI and the gas-phase ion chemistry of NI-DART is more easily manipulated than that of NI-APPI, NI-DART can be therefore used to study in detail the ionization mechanism of LC/NI-APPI-MS, which would be a powerful methodology for the quantification of low-polarity compounds. Herein, one such application has been further demonstrated in the detection and identification of background ions from LC solvents and APPI dopants, including water, acetonitrile, chloroform, methylene chloride, methanol, 2-propanol, hexanes, heptane, cyclohexane, acetone, tetrahydrofuran (THF), 1,4-dioxane, toluene, and anisole. Possible reaction pathways leading to the formation of these background ions were further inferred. One of the conclusions from these experiments is that THF and 1,4-dioxane are inappropriate to be used as solvents and/or dopants for LC/NI-APPI-MS due to their high reactivity with source basic ions, leading to many reactant ions in the background.
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