Three-dimensional graphing representing six variables for speed and separation performance in liquid chromatography.

The two variables, flow rate and column length, enable naive determination of the number of theoretical plates (N) in isocratic elution; this, in turn, enables the formation of a three-dimensional graph with N as the z-axis. An alternate three-dimensional graph with N as the z-axis can be drawn, then, with the alternate basal plane illustrating the pressure drop and hold-up time. In this article, the pressure drop and hold-up time are formulated so as to be represented unitarily in the former graph, because the flow rate and column length interact simultaneously as operational variables. This formulation manipulates both the pressure drop and the hold-up time as logarithmic axes, to evaluate the landscape. Also of use is the representation, in the same graph, of the height equivalent to a theoretical plate, as the fundamental property of the packing supports. For this purpose, the number of theoretical plates per unit length are here introduced as the sixth variable, instead of the height equivalent to a theoretical plate. Representing the six variables in three-dimensional graphs enables a clear understanding both of the separation condition optimization methods and the relation among variables for the speed and separation performance. The linear velocity, column length, N, velocity-length product, hold-up time, and number of theoretical plates per unit length, are here selected as the six elementary variables for the three-dimensional graphs; and, based on the packing supports of 2, 3, and 5-µm particle and monolithic columns. Finally, the usage of logarithmic three-dimensional graph is illustrated for understanding the speed and separation performance.

Three approaches to improving performance of liquid chromatography using contour maps with pressure, time, and number of theoretical plates.

Attempts to improve HPLC performance often focus on increasing the speed or separation performance. In this article, both the flow rate and column length are optimized as separation conditions, while observing the number of theoretical plates and hold-up time with isocratic elutions. In addition, the upper pressure limit must be simultaneously considered as the boundary condition. Approaches based on the optimal velocity (Opt.) are often adopted; but the kinetic performance limit (KPL) in Desmet's method can also be utilized for three-dimensional graphing with axes of pressure, time, and number of theoretical plates. Here, two approaches involving pressure increase are introduced, beginning with the condition of optimal linear velocity: one aimed at greater speed and the other at higher resolution. Coefficients of pressure-application are derived to measure the effectiveness of the intermediate conditions between the Opt. and KPL methods. In the third approach, the hold-up time is extended while maintaining a fixed pressure. Coefficients of time-extension are also derived, to determine the effectiveness to improve the separation performance.

Three-dimensional Representation Method Using Pressure, Time, and Number of Theoretical Plates to Analyze Separation Conditions in HPLC Columns.

There has been considerable discussion of the speed performance of HPLC separation, especially regarding the relationship between theoretical plates and hold-up time. The fundamental discussion focuses on the optimal velocity, u0,opt, which gives a minimal height equivalent to a theoretical plate of the van Deemter plot. On the other hand, Desmet's method, using the kinetic performance limit (KPL), calculates the highest performance with a constant pressure drop, without focusing solely on the optimal velocity. In this paper, a precise method based on the KPL is proposed, to understand how increasing pressure enhances both theoretical plates and hold-up time. A three-dimensional representation method that combines the pressure drop with two axes of time and theoretical plates will be useful for discussing the effect of pressure in pressure-driven chromatography. Using three dimensions, the methods based on u0,opt and the KPL can be combined, because u0,opt can be visualized three-dimensionally, including the neighbor of u0,opt; and the question of whether the KPL is an asymptotic or effective limit can be investigated. Three performances of high resolution, high speed, and low pressure can be understood on different packing supports at a glance.

Localized surface plasmon resonance-mediated fluorescence signals in plasmonic nanoparticle-quantum dot hybrids for ultrasensitive Zika virus RNA detection via hairpin hybridization assays.

The current epidemic caused by the Zika virus (ZIKV) and the devastating effects of this virus on fetal development, which result in an increased incidence of congenital microcephaly symptoms, have prompted the World Health Organization (WHO) to declare the ZIKV a public health issue of global concern. Efficient probes that offer high detection sensitivity and specificity are urgently required to aid in the point-of-care treatment of the virus. In this study, we show that localized surface plasmon resonance (LSPR) signals from plasmonic nanoparticles (NPs) can be used to mediate the fluorescence signal from semiconductor quantum dot (Qdot) nanocrystals in a molecular beacon (MB) biosensor probe for ZIKV RNA detection. Four different plasmonic NPs functionalized with 3-mercaptopropionic acid (MPA), namely MPA-AgNPs, MPA-AuNPs, core/shell (CS) Au/AgNPs, and alloyed AuAgNPs, were synthesized and conjugated to L-glutathione-capped CdSeS alloyed Qdots to form the respective LSPR-mediated fluorescence nanohybrid. The concept of the plasmonic NP-Qdot-MB biosensor involves using LSPR from the plasmonic NPs to mediate a fluorescence signal to the Qdots, triggered by the hybridization of the target ZIKV RNA with the DNA loop sequence of the MB. The extent of the fluorescence enhancement based on ZIKV RNA detection was proportional to the LSPR-mediated fluorescence signal. The limits of detection (LODs) of the nanohybrids were as follows: alloyed AuAgNP-Qdot646-MB (1.7 copies/mL)) > CS Au/AgNP-Qdot646-MB (LOD =2.4 copies/mL) > AuNP-Qdot646-MB (LOD =2.9 copies/mL) > AgNP-Qdot646-MB (LOD =7.6 copies/mL). The LSPR-mediated fluorescence signal was stronger for the bimetallic plasmonic NP-Qdots than the single metallic plasmonic NP-Qdots. The plasmonic NP-Qdot-MB biosensor probes exhibited excellent selectivity toward ZIKV RNA and could serve as potential diagnostic probes for the point-of care detection of the virus.

Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays.

Chemical reactivity of acyl glucuronides (AGs) is believed to be involved in the toxicity of carboxylic acid-containing drugs. Both direct and immune-mediated toxicity have been suggested as possible mechanisms of toxicity; however, it remains unclear. In the present study, we performed assays of half-lives, peptide adducts, and immunostimulation to evaluate the potential risk of AGs of 21 drugs and analyzed the relationship to the toxic category. AGs of all withdrawn drugs tested in this study showed short half-lives and peptide adducts formation, but so did those of several safe drugs. In contrast, only AGs of withdrawn and warning drugs induced interleukin-8 (IL-8) in human peripheral blood mononuclear cells (hPBMCs). Using a DNA microarray assay, we found that zomepirac AG induced the mRNAs of 5 genes, including IL-8 in hPBMCs. In addition, withdrawn and warning drugs were distinguished from safe drugs by an integrated score of relative mRNA expression levels of 5 genes. The immunostimulation assay showed higher sensitivity, specificity, and accuracy compared with other methods. In preclinical drug development, the evaluation of the reactivity of AGs using half-lives and peptide adducts assays followed by the evaluation of immunostimulation by highly reactive AGs using hPBMCs can contribute to improved drug safety.

Rapid method for the simultaneous determination of flavonol aglycones in food using u-HPLC coupled with heating block acidic hydrolysis.

A rapid method for the simultaneous determination of flavonol aglycones in food using ultra-high-performance LC (u-HPLC) coupled with a heating-block acidic hydrolysis method was validated in terms of precision, accuracy, and linearity. The u-HPLC separation was performed on an RP C18 column (particle size 2 micro m id, 2 mm, length 100 mm) with a photodiode array detector. The LOD and LOQ of the u-HPLC analyses were 0.15 and 0.47 mg/kg for myricetin, 0.09 and 0.28 mg/kg for quercetin, 0.16 and 0.49 mg/kg for kaempferol, and 0.08 and 0.25 mg/kg for isorhamnetin. The intraday and interday precisions of the individual flavonol aglycones were less than 9.31%. All calibration curves exhibited good linearity (r2 = 0.99) within the tested ranges. Total run time of u-HPLC was 13 min. The rapid u-HPLC method presented herein significantly improved the speed, sensitivity, and resolution of the analyses of myricetin, quercetin, kaempferol, and isorhamnetin in food.

Simultaneous determination of free capsorubin and capsanthin in red pepper powder using u-HPLC.

A simultaneous ultra-HPLC (u-HPLC) method for the determination of free capsorubin and capsanthin in red pepper powder was validated in terms of its precision, accuracy, and linearity. The u-HPLC separation was performed on an RP C18 column (particle size 2 pm, id 2 mm length 100 mm) and with a photodiode-array detector. The recoveries of capsorubin were greater than 83.8 +/- 1.7%; the LOD and LOQ of the u-HPLC analyses were 0.043 and 0.129 mg/kg, respectively. The intraday and interday precisions for capsorubin were less than 9.01%. The recoveries of capsanthin were greater than 87.7 +/- 1.5%, and the LOD and LOQ were 0.101 and 0.306 mglkg, respectively. The intraday and interday precisions for capsanthin were less than 12.66%. All calibration curves for capsorubin and capsanthin exhibited good linearity (r2 = 0.99) within the tested ranges.

Determination of 22 ginsenosides in ginseng products using ultra-high-performance liquid chromatography.

A reverse-phase ultra-high-performance liquid chromatography (u-HPLC) method was developed for the rapid quantification of 22 ginsenosides in ginseng products. The proposed method for the analysis of ginsenosides is based on a heating-block method without further treatment. The u-HPLC separation was performed on a reversed C18 column (100 × 2 mm id, particle size 2 µm) followed by ultraviolet detection at 203 nm. Aqueous 50% methanol was used as the extraction solvent. The optimum amount of extraction solvent and the optimum extraction time were 20 mL and 20 min (extracted twice with 10 mL), respectively. The method validation parameters yielded good results for linearity, precision, accuracy and recovery. The recovery of ginsenosides from ginseng powder was greater than 98.1% and the limits of detection and quantification of the u-HPLC analysis were >0.6 and >1.8 mg/kg for ginsenosides. The calibration graphs for ginsenosides were linear from approximately 2.6 to 40.4 mg/kg for u-HPLC. The inter-day and intra-day precisions (relative standard deviation values) were <14.6 and 14.7%, except for Rg2(R) + Rh1(R).

Rapid method for the determination of vitamins A and E in foods using ultra-high-performance liquid chromatography.

A rapid and novel ultra-HPLC (u-HPLC) method for the determination of vitamins A (retinol) and E (alpha-, gamma-, and delta-tocopherol) in foods was validated in terms of its precision, accuracy, and linearity. The u-HPLC separation was performed on an RP C18 column (particle size 2 microm, id 2 mm, and length 75 mm), followed by fluorescence detection. The recovery of retinol was more than 84.58%; the LOD and LOQ of the u-HPLC analysis were 0.015 and 0.045 mg/kg, respectively. The intraday and interday precision was less than 9.12%. The recoveries of alpha-, gamma-, and delta-tocopherol were more than 81.37%; the LOD and the LOQ were 0.014, 0.002, and 0.001 mg/kg and 0.042, 0.005, and 0.004 mg/kg, respectively. All calibration curves had good linearity (r2 = 0.99) within the test ranges. The novel, rapid method coupled to u-HPLC can provide significant improvements in the speed, sensitivity, and resolution compared with a conventional HPLC method.

Determination of parabens in cosmetic products by solid-phase microextraction of poly(ethylene glycol) diacrylate thin film on fibers and ultra high-speed liquid chromatography with diode array detector.

The fabrication of a solid-phase microextraction (SPME) fiber through UV-induced polymerization of poly(ethylene glycol) diacrylate (PEG-DA) for determination of parabens in cosmetic products is presented in this work. The PEG-DA polymer coating was covalently attached to the fiber by introducing a surface modification with 3-(trichlorosilyl)propyl methacrylate (TPM). The PEG-DA polymer thin film coated on the fiber was homogeneous and wrinkled, which led to an increase of the surface area and high extraction efficiency. The extraction performances of the prepared SPME fibers were assessed by preconcentration of parabens including methylparaben, ethylparaben, propylparaben and benzylparaben from cosmetic products. The analysis was performed on an ultra high-speed liquid chromatography with diode array detector. The prepared SPME fibers exhibited good repeatability (for one fiber) and reproducibility (fiber-to-fiber) with RSDs of 5.4 and 6.9%, respectively. The optimized SPME method supported a wide linear range of 0.50-160 µg/mL and the detection limits for parabens were in the range of 0.12-0.15 µg/mL (S/N=3). The developed method was successfully applied for determination of parabens in cosmetic products with different natures.

Rapid method for the determination of capsaicin and dihydrocapsaicin in Gochujang using ultra-high-performance liquid chromatography.

A sensitive and specific heating block method coupled with ultra-HPLC (u-HPLC) was developed for the analysis of capsaicin in Gochujang and validated by comparing with a conventional HPLC (AOAC Method 995.03). The method validation parameters yielded good results, including linearity, precision, accuracy, and recovery. The u-HPLC separation was performed on a reversed C18 column (50 x 2 mm id, particle size 2 microm), followed by fluorescence detection (excitation 280 nm, emission 325 nm). Methanol was used as the extracting solvent, and the amount of sample taken was approximately 0.2 g; the optimum amount of extraction solvent and extraction time were 15 mL and 1 h, respectively. The recovery of capsaicin in Gochujang was more than 93%, and the LOD and LOQ of the u-HPLC analysis were 0.05 and 0.16 microg/g for capsaicin and 0.05 and 0.16 microg/g for dihydrocapsaicin. The calibration graphs for capsaicin and dihydrocapsaicin were linear from 0.2 to 10.0 microg/mL for u-HPLC. The interday and intraday precisions (RSD values) were < 6.27%.

[A case of choriocarcinoma of suspected lung origin manifesting pulmonary embolism].

A 28-year-old woman visited a local hospital with a complaint of progressive dyspnea after a normal delivery. She was given a diagnosis of COP based on TBLB specimen and CT findings and received steroid pulse therapy at the hospital. Three months after her first visit she was referred to our hospital because her symptoms had not resolved despite receiving steroid therapy. The CT findings at our hospital suggested pulmonary thromboembolism. Although anticoagulation therapy was initiated, the CT findings and symptoms worsened. Thus thrombi were removed by open chest surgery and a diagnosis of choriocarcinoma was established. No primary lesion was found on gynecological examinations. Complete remission was achieved after systemic chemotherapy and there have been no event in the two years since diagnosis. Choriocarcinoma is a rare malignant tumor developing after pregnancy, with frequent metastases to the lung. However, when the tumors embolize into the pulmonary arteries, they resemble pulmonary thromboembolisms making correct diagnosis difficult. Since, owing to recent advances of cancer chemotherapy, choriocarcinoma is now curable tumor, physicians must suspect this disease in cases of pulmonary embolisms in women.

Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells.

Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F(6) progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator.

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice.

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6, 12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.