Reproducibility in capillary supercritical fluid chromatography: Comparison of injection techniques

An analysis of the precision obtained using commercially available microvalve injectors is reported for three modes of injection: conventional split; timed-split; and direct. Results from this study show that good precision (< 3% RSD for external standard and < 1% RSD for internal standard methods) can be obtained with capillary supercritical fluid chromatography (SFC). However, particular attention must be paid to the type of valve used, the orientation of the column relative to the valve, the mode of interfacing or connecting the column to the valve, and the type of pressure or density programming used for the analysis as all of these factors will affect the reproducibility.     

Development and validation of an LC–MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside,and its application in a pharmacokinetic study of normal and diabetic nephropathy mice

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5′-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC–MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an Acquity HSS T₃ column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5–1,000 ng/ml for RDV and 5–10,000 ng/ml for both RM-442 and RN. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442 and RSD < 7.2% for RN) and accuracy (91.0–106.3% for RDV, 92.5–98.6% for RM-442 and 87.5–98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in the blood of normal and diabetic nephropathy DBA/2 J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration–time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).     

Preparation and characterization of nanotube Li-Ti-O by molten salt method

Nanotube Li-Ti-O compound with high surface (198.6 m²·g⁻¹) was prepared by a method involving the treatment of nanotube Na₂Ti₂O₅·H₂O in molten LiNO₃ and characterization by means of transmission electron microscopy (TEM), energy-dispersive spectra (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and thermogravimetry-differential thermal analysis (TG/DTG). Results show that the nanotube Li-Ti-O compound prepared by this method involves two crystal phases: spinel Li₂Ti₂O₄ and anatase Li_xTiO₂ (x < 0.1). Li⁺ exhibits different Li1s binding energy in the two crystal phases. In ambient air, the Li-Ti-O compound adsorbs water easily, and the chemically adsorbed water is difficult to remove below 400°C. Translated from Chinese Journal of Inorganic Chemistry, 2006, 22(12): 2135–2139 [译自: 无机化学学报]     

Chemiluminescent lipase determination based on the enhanced luminol/H2O2/horseradish peroxidase/fluorescein diacetate energy transfer system

A methodology for the determination of lipase, based on the coupled processes of energy transfer and enhancement of the chemiluminescence of the luminol-H₂O₂-horseradish peroxidase (HRP) system has been developed. Fluorescein diacetate (FDA) was hydrolyzed to fluorescein by the action of the enzyme lipase, and this compound acted as an enhancer of the chemiluminescent process and acceptor of the chemiluminescent emission from the luminol-H₂O₂-HRP system. By measuring the transferred emission to fluorescein at 525 nm, lipase (range 0.2–1.5 U/mL, RSD 2.3%) was determined. This methodology permited the determination of every compound of the system, thus, H₂O₂ (range 0.5–2 mM, RSD 6.9%) and HRP (range 5.5–49.5 U/mL, RSD 3.6%) could also be determined. Lipase was determined in rabbit serum with 96.7 ± 3.3% and 102.9 ± 5.4% recoveries for two different lipase concentrations. Besides, H₂O₂ was determined in the disinfectant solution for contact lenses. Received: 1 March 1999 / Revised: 6 May 1999 / Accepted: 12 May 1999     

Renewable sol-gel carbon ceramic electrodes modified with a Ru-complex for the amperometric detection of l-cysteine and glutathione

A renewable three-dimensional chemically modified carbon ceramic electrode containing Ru [(tpy)(bpy)Cl] PF₆ was constructed by sol-gel technique. It exhibits an excellent electro-catalytic activity for oxidation of l-cysteine and glutathione at pH range 2-8. Cyclic voltammetry was employed to characterize the electrochemical behavior of the chemically modified electrode. The electrocatalytic behavior is further exploited as a sensitive detection scheme for l-cysteine and glutathione by hydrodynamic amperometry. Optimum pH value for detection is 2 for both l-cysteine and glutathione. The catalytic rate constants for l-cysteine and glutathione were determined, which were about 2.1×10³ and 2.5×10³ M⁻¹ s⁻¹, respectively. Under the optimized condition the calibration curves are linear in the concentration range 5-685 and 5-700 μM for l-cysteine and glutathione determination, respectively. The detection limit (S/N=3) and sensitivity is 1 μM, 5 nA/μM for l-cysteine and 1 μM, 7.8 nA/μM for glutathione. The relative standard deviation (RSD) for the amperogram's currents with five injections of l-cysteine or glutathione at concentration range of linear calibration is <1.5%. The advantages of this amperometric detector are: high sensitivity, good catalytic effect, short response time (t<3 s), remarkable long-term stability, simplicity of preparation and reproducibility of surface fouling (RSD for six successive polishing is 3.31%). This sensor can be used as a chromatographic detector for analysis of l-cysteine and glutathione.     

Multi-Pesticide Analysis in Sediment by GC-EI-MS/MS Using Programmed Temperature Vaporization–Large Volume Injection Technique

A sensitive, selective and high throughput gas chromatography–tandem mass spectrometry method using programmable temperature vaporization–large volume injection mode (PTV-LVI-GC-MS/MS) for the analysis of 30 organochlorine pesticides (OCPs) including toxaphenes in sediments was developed. The PTV-LV injection settings, viz. inlet temperature, split flow, injection phase time, and injection speed were optimized for 50 µL injection. A significant increase in sensitivity was accomplished as compared with that obtained by the conventional 1 µL cold splitless injection. Average LVI recoveries for OCPs were in the range 58–133 % with low % RSD in instrument precision (<12 %). The method detection limits achieved were 0.04–0.92 µg kg^?1. The method recovery ranged from 80 to 120 % with <10 % RSD for more than 83 % of targeted analytes fortified at 10 µg kg^?1 in sediments. The PTV-LVI-GC-MS/MS allows simultaneous determination and unambiguous confirmation of trace OCPs and toxaphene congeners, which significantly streamlines and improves the trace organic analysis in the environmental surveillance and monitoring.     

Universal approach for mesofluidic handling of bead suspensions in lab-on-valve format

In the present report, new protocols are introduced for automatic mesofluidic handling of irregularly shaped and non-uniformly sized bead materials for renewable micro-solid phase extraction (μSPE) under the lab-on-valve (LOV) format. To this end, two alternative strategies were studied comprising either (i) the direct aspiration of bead suspension placed at a container attached to LOV device or (ii) the aspiration of beads after a resuspension step, allowing the formation of a fluidized bed inside the beads’ container. Suspensions with homogeneously dispersed beads were also tested in the first strategy above, as prepared by increasing the viscosity of the suspension milieu with 75:15:10 glycerol/MeOH/H₂O (in wt). The bead injection protocols were applied to four commercial reversed-phase sorbent materials with different sorptive surfaces: Oasis HLB, SupelMIP β-receptors, Lichrolut EN and Discovery DSC-MCAX, and the mass of sorbent packed in each microcolumn was assessed. Direct aspiration of methanolic suspensions gave rise to bead stacking and clogging of the LOV microconduits, resulting in a source of results with poor precision (RSD: 3.8-67.6%). The use of glycerolic suspensions was merely effective for repeatable sampling and packing of Oasis HLB and SupelMIP β-receptor beads without sorbent settlement along time. The resuspension strategy was able to handle all the materials tested with acceptable precision (RSD: 1.6-13.8%). Enhanced precision was attained (RSD <4.1%) when the sorbent bed was physically restricted to the volume of the LOV microchannel cavity. Different volumes of suspension aiming at a target mass of sorbent of 10 mg were successfully handled (RSD: 3.1-13.8%), showing the reliability of the bead resuspension approach for varied nominal bead sampling volumes. The proposed bead handling protocols were applied to μSPE of propranolol taken as a model of β-blocker from aqueous solutions by SupelMIP β-receptors and Discovery DSC-MCAX beads with high repeatability (RSD <6%) and absolute recoveries between 69 and 74% in a bead-injection mode.     

Extraction of trace amounts of silver on various amino-functionalized nanoporous silicas in real samples

Aminopropyl-functionalized mesoporous silicas, NH₂-MCM-41 and NH₂-SBA-15, as absorbents were utilized for rapid extraction, preconcentration and determination of trace amounts of silver. Flow rates of sample and eluent, pH, eluent solution, type, concentration and the least amount of eluent for desorption of silver ions were optimized; moreover, break through volume and the effect of various cationic interferences on the sorption of silver were evaluated. The extraction efficiency of silver ions was greater than 95% for MCM-41-NH₂ and 85% for SBA-15-NH₂ and the limit of detection (LOD) was less than 4 ng mL^?1 for both functionalized mesoporous silicas. The preconcentration factor was greater than 210 and the relative standard deviation (RSD) was <2%. The adsorption capacity of the mesoporous silicas is higher than 143 mg g^?1 for NH₂-MCM-41 and 137 mg g^?1 for NH₂-SBA-15. Under similar experimental conditions the results for these solid phases were compared with each other. NH₂-SBA-15, in spite of larger pore size diameter and adsorption of silver ions in higher flow rates has lower recovery and a higher RSD compared to MCM-41. This method has been applied to determine silver in photographic emulsions and real samples.     

Synthesis,Characterization and Oxide Ionic Conductivity of Binary β‐(Bi2O3)1‐x(Lu2O3)x System

In this research, the effects of doping Lu₂O₃ to α‐Bi₂O₃ in the range of 0.01 < x < 0.10 in a series of different mole fractions (1% < n < 10% mole ratios) was studied. Beside, heat treatment was performed by applying a cascade temperature rise in the range of 700‐800 °C for 72 hours and new phases were obtained in the (Bi₂O₃)_1‐x(Lu₂O₃)_x system. After heat treatment for 72 hours at 800 °C; mixtures, containing 2‐8% mole Lu₂O₃, formed a tetragonal phase. As a result of subjecting mixtures, containing 9% and 10% mole Lu₂O₃, to a quenching process at 825 °C, tetragonal phases were obtained. With the help of XRD, the crystal systems and lattice parameters of the solid solutions were obtained and their characterization was carried out. Thermal measurements were made by using a simultaneous DTA/TG system. The total conductivity (σ_T) in the β‐Bi₂O₃ doped with Lu₂O₃ was measured using the four‐probe DC method.     

Coulometric Determination of Chromium(VI) and Copper(II) Present Simultaneously

It was shown that chromium(VI) can be determined by coulometry at a Pt electrode in HNO₃and H₂SO₄solutions. A procedure for the determination of 1–4 mg of chromium in 0.5–1 M HNO₃solutions by controlled-potential coulometry with RSD = 0.2% was developed. It was demonstrated that the degree of the reduction of Cr(VI) to Cr(III) reached 99% (RSD = 0.5%) in the determination of chromium by coulometry at a slow potential sweep (v= 1–2 mV/s) in HNO₃solutions. A procedure was proposed for the determination of Cr(VI) and Cu(II) simultaneously present in 0.25–0.5 M H₂SO₄solutions by controlled-potential coulometry with RSD = 0.3%.     

Electrochemistry in ionic liquids: Case study of a manganese corrole

Voltammetry of [5,10,15-tris(pentafluorophenylcorrole)]Mn(III) was investigated in four different ionic liquids (ILs): 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMIm-TFSI); 1-ethyl-3-methylimidazolium ethylsulfate (EMIm-EtOSO3); 1-ethyl-3-methylimidazolium triflate (EMIm-OTf); and 1-ethyl-3-methylimidazolium tetracyanoborate (EMIm-TCB). We found that MnIV/III E _1/2 values depend on IL counter anion: OTf–< EtOSO₃ ^? < TFSI^? < TCB^?. In EMIm-TCB and BMIm- TFSI, reversible, diffusion-controlled Mn^IV/III reactions occurred, as evidenced in each case by the ratio of anodic to cathodic diffusion coefficients over a range of scan rates. Axial coordination was evidenced by a cathodic to anodic diffusion coefficient ratio greater than one, an increasing cathodic to anodic peak current ratio with increasing scan rate, and a split Soret band in the UV-vis spectrum of the complex.     

Influence of a Second Cation (M = Ca2+, Mg2+) on the Phase Evolution of (BaxM1–x)F2 Starting from Amorphous Deposits

A second type of cation (Mg²⁺, Ca²⁺) was introduced into BaF₂ by low‐temperature atomic beam deposition. The structure evolution from low‐temperature (–150 °C) amorphous deposits to high‐temperature (< 1000 °C) annealed crystalline phases was studied by in‐situ transmission electron microscopy and X‐ray diffraction. Amorphous (Ba_0.5, Ca_0.5)F₂ crystallizes in a first step to metastable solid solution phase (fluorite‐type), which then decomposes into the pure phases of CaF₂ and BaF₂ at higher temperature. The crystallization behavior of amorphous (Ba_xMg_1–x)F₂ is completely different. When the Mg/Ba atomic ratio is around 1:1, the mixture transforms to the ternary compound BaMgF₄ at annealing, and no decomposition occurs by further heating up to 1000 °C. When the Ba concentration is below 15 % in atomic ratio (x < 0.15), the mixture forms a solid solution phase (rutile type) with the lattice expanded by +1 % compared to rutile type MgF₂. The difference between the phase evolutions of the two mixture systems is discussed.     

Development and Validation of a Stability-Indicating LC Method to Quantify Hydrochlorothiazide in Oral Suspension for Pediatric Use

A stability-indicating reversed-phase high-performance liquid chromatography (LC) method was developed and validated for the determination of hydrochlorothiazide in an oral suspension. Isocratic chromatography was performed on a C₁₈ column with 0.1 M sodium phosphate buffer pH 3.0/acetonitrile (70:30 v/v) as mobile phase, at a flow rate of 1.3 mL min⁻¹, and UV detection at 254 nm. The method was linear (r ² = 0.9998), accurate (mean recovery = 100.3%), and precise (RSD <2%). It was also validated for specificity and robustness. The method was successfully applied for the quality control analysis of a new pharmaceutical formulation of HCTZ for pediatric use.     

Assessment of dispersive liquid–liquid microextraction for the simultaneous extraction,preconcentration, and derivatization of Hg2+ and CH3Hg+ for further determination by GC–MS

This work reports the development of a dispersive liquid – liquid microextraction method for the simultaneous extraction, preconcentration, and derivatization of Hg²⁺ and CH₃Hg⁺ species from water samples for further determination by GC – MS. Some parameters of the proposed method, such as volume and type of disperser and extraction solvent, and Na[B(C₆H₅)₄] concentration were investigated using response surface methodology. Suitable recoveries were obtained using 80 μL C₂Cl₄ (as extraction solvent), 1000 μL ethanol (as disperser solvent), and 300 μL 2.1 mmol/L Na[B(C₆H₅)₄] (as derivatizing agent). Accuracy was evaluated in terms of recovery and ranged from 87 to 99% with RSD values <7%. In addition, a certified reference material of water (NIST 1641d) was analyzed and agreed with the certified value about 107% (for Hg²⁺), with RSD values <8.5%. LODs were 0.3 and 0.2 μg/L, with enrichment factors of 112 and 115 for Hg²⁺ and CH₃Hg⁺, respectively. The optimized method was applied for the determination of Hg²⁺ and CH₃Hg⁺ in tap, well, and lake water samples.     

Determination of the pesticide naptalam and its degradation products by positive and negative ion mass spectrometry

N-1-Naphthylphtalamic acid (naptalam) and its degradation products, 1-naphthylamine and N-(1-naphthyl) phthalimide were simultaneously determined in river water by two independent mass spectrometric (MS) methods. These were negative ion MS (NIMS) and programmable temperature vaporizer gas chromatography mass spectrometry (PTV-GC MS) with electron impact ionization (positive ions). Prior to the NIMS analysis, the samples were preconcentrated by solid phase extraction (SPE) of C₁₈ membrane discs. The PTV-GC MS studies were performed without any preconcentration procedure. Selected ion monitoring (SIM) and internal standardization with naphthalene were applied in both methods. The limits of determination (LOD) of NIMS studies were 230, 270 and 260 ng L^–1 for naptalam, 1-naphthylamine and N-(1-naphthyl) phthalimide, respectively, with relative standard deviation (RSD) < 1% (n = 5) and of PTV-GC MS 17, 11 and 15?ng L^–1 (RSD < 0.7%, n = 5). The LOD, linearity, RSD and time required for these methods are far better than for HPLC analyses.     

Application of a hexapole collision and reaction cell in ICP-MS Part II: Analytical figures of merit and first applications

The application of an ion-guiding gas-filled hexapole collision/reaction cell in ICP-MS has been studied to characterize the analytical figures of merit that can be achieved with this approach. For the elements investigated, application of a buffer and a reaction gas resulted in improved sensitivities which are lowest for Be with about 7 · 10⁷ cps per μg mL^–1 and highest for Ba with about ?6 · 10⁸ cps per μg mL^–1. Relative standard deviations (RSD) < 0.1% were obtained. Application of the reaction gas H₂ was used to suppress polyatomic ions caused by argon. The reduction amounted up to four orders of magnitude so that elements such as Ca, K, Cr, Fe, As and Se could be analyzed in nitric and hydrochloric acid or in methanol. Detection limits of 6 pg mL^–1 for Cr in 2% methanol, 23 pg mL^–1 for As and 9 pg mL^–1 for Se in 0.28 M HCl were achieved. For other elements detection limits ?< 1 pg mL^–1 were realized in the medium and high mass range. Accuracy was proved using the NIST 1643d standard reference material. Received: 10 May 1999 / Revised: 4 June 1999 / Accepted: 12 June 1999     

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C₁₈ column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC₅₀ values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of the rate of production and dissolution of biosilica in marine waters by thermal ionisation mass spectrometry

A new method is described for a precise and simultaneous determination of the rate of production and dissolution of biosilica in marine waters, using isotopic dilution technique. No HF or F₂ is required for chemical preparations as the change in isotopic composition is measured on silica producing SiO₂⁻ ions. The seawater sample flask is spiked with ³⁰Si(OH)₄ (<10% of increase in situ concentration) and incubated in in situ conditions. At the end of incubation, changes of the ³⁰Si:²⁸Si ratios in particulate and liquid phases are measured by using a thermal ionisation mass spectrometer Finnigan THQ. The relative analytical precision of the isotopic ratio measurements is <0.5%. The limit of detection of the change in isotopic ratio during incubation is 0.02 atom%. The overall repeatability determined on eight subsamples (average production: 0.23 μM day⁻¹; average dissolution: 0.07 μM day⁻¹) is ±0.02 and ±0.01 μM day⁻¹ for production and dissolution, respectively. Using mass and isotopic balances of the particulate and dissolved phases in the incubation flask, the best estimates for production and dissolution rates are calculated iteratively. This method was applied to 112 samples of marine waters (production, range: 0.00-2.38 μM day⁻¹; dissolution, range: 0.00-1.18 μM day⁻¹).