Extremely Simple and Rapid HPLC Analysis of Tocilizumab in Human Serum with Selective Precipitation Using Alkylamine.

An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40-1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5-114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.

A Turn-On Quinazolinone-Based Fluorescence Probe for Selective Detection of Carbon Monoxide.

Carbon monoxide (CO) is a toxic, hazardous gas that has a colorless and odorless nature. On the other hand, CO possesses some physiological roles as a signaling molecule that regulates neurotransmitters in addition to its hazardous effects. Because of the dual nature of CO, there is a need to develop a sensitive, selective, and rapid method for its detection. Herein, we designed and synthesized a turn-on fluorescence probe, 2-(2'-nitrophenyl)-4(3H)-quinazolinone (NPQ), for the detection of CO. NPQ provided a turn-on fluorescence response to CO and the fluorescence intensity at 500 nm was increased with increasing the concentration of CO. This fluorescence enhancement could be attributed to the conversion of the nitro group of NPQ to an amino group by the reducing ability of CO. The fluorescence assay for CO using NPQ as a reagent was confirmed to have a good linear relationship in the range of 1.0 to 50 µM with an excellent correlation coefficient (r) of 0.997 and good sensitivity down to a limit of detection at 0.73 µM (20 ppb) defined as mean blank+3SD. Finally, we successfully applied NPQ to the preparation of a test paper that can detect CO generated from charcoal combustion.

HPLC Fluorescence Method for Eugenols in Basil Products Derivatized with DIBI.

Eugenols (Eugs) such as eugenol (Eug), methyleugenol (MeEug), and linalool (Lin) in basil product are the main bioactive components of basil products and have a terminal double-bond. A sensitive HPLC-fluorescence method for Eugs derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) was developed. Good separation of DIB-Eugs was achieved within 20 min on an Atlantis T3 column (50 × 2.1 mm i.d., 3 µm) with a mobile phase of methanol-water. The calibration curves obtained with Eug standards showed good linearities in the range of 0.1-50 µM (r ≥ 0.999). The limits of detection at a signal-to-noise ratio (S/N) = 3 for Eug, MeEug, and Lin were 1.0, 6.0, and 4.8 nM, respectively. The limits of quantitation (S/N = 10) of the Eugs were lower than 19.9 nM. The accuracies for the Eugs were within 96.8-104.6%. The intra- and inter-day precisions as relative standard deviations for the Eugs were less than 1.2 and 9.6% (n = 3). The recoveries of Eug, MeEug, and Lin were 99.0 ± 0.1, 98.0 ± 0.2, and 96.0 ± 0.4% (n = 3), respectively. The DIB-Eugs were confirmed to be stable for 2 h (>90%) at room temperature and 24 h (>95%) at 4 °C. These parameters of the proposed method were useful for the simultaneous determination of Eugs in basil products. Therefore, the developed method may be a powerful tool for the quality evaluation of dried commercially available basil products.

Safety evaluation of recycling process used to produce recycled pulp for adult paper diapers.

Eight contaminants were used to perform a surrogate contamination test to evaluate safety of the process for producing recycled pulp for adult paper diapers. We performed a safety evaluation of the recycling process for producing recycled pulp following the European Food Safety Authority safety assessment for polyethylene terephthalate (PET) bottles using recycled PET. We also compared the first tier safety limit established on the basis of TTC (Climit) in the pulp that contained recycled pulp with residual contaminant concentrations (Cres). If Climit > Cres, there is no concern regarding safety of the recycling process. Climit was determined to be 0.46 mg/kg-pulp based on the human exposure threshold of 0.15 µg/person/day using the scenario in which a bedridden senior citizen uses four adult paper diapers manufactured with pulp containing 10% recycled pulp per day. Cres was derived from the initial concentration in used pulp (0.17 mg/kg-pulp), and decontamination rate was obtained from the surrogate contamination test. The Cres of the eight contaminants were between 0.0017 and 0.10 mg/kg-pulp, which were all below the Climit of 0.46 mg/kg-pulp. These results indicated there was no safety problem regarding this process for producing recycled pulp for adult paper diapers.

Stereodivergent Synthesis of Bispyrrolidinoindoline Alkaloidal Scaffolds and Generation of a Lead Candidate with Stereospecific Antiproliferative Activity.

The fungal secondary metabolites (+)-WIN 64821 and (-)-ditryptophenaline are biosynthesized through condensation of l-tryptophan and l-phenylalanine, followed by reductive dimerization with generation of stereochemical variations. Inspired by the stereodivergent biogenetic process, we designed and synthesized a collection of bispyrrolidinoindoline diketopiperazine alkaloids and their analogues with systematic diversification of the stereochemistry of the privileged structural motif of the fungal alkaloids. Not only the stereochemical modifications of (+)-WIN 64821 at the 3-/3'-, 11-/11'-, and 15-/15'-positions, but also ring cleavage of the diketopiperazine moieties, allowed the generation of a lead compound exhibiting potent growth inhibitory activity (IC50 =3.03 µm) toward human colon cancer cells. Structure-activity relationship studies revealed that all six stereogenic centers were essential for the pharmacophore. High cell densities dramatically intensified the cytotoxic activities of the lead compound.

Quantification of three triterpenic acids in dried rosemary using HPLC-fluorescence detection and 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride derivatization.

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) and detected using HPLC-fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB-Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB-triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 µm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λex ) and 490 nm (λem ) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5-100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.

Proposal of a flow scheme for the chemical-form-based quantitative analysis of chlorine compounds in pulp for sanitary products and verification of safety.

To determine the amounts and chemical forms of chlorine compounds in elemental chlorine-free (ECF) bleached pulp for sanitary products, a chemical-form-based quantitative analysis flow scheme was created. The scheme involves quantitative determination of compounds eluted in elution tests assuming dermal exposure. The results indicated that most of the chlorine compounds in pulp used for sanitary products were insoluble organically bound chlorine and water-soluble chlorides consisting of chloride ions. The total amount of chlorine obtained by analysis of the total chlorine in the pulp products was close to the sum of the amounts of water-soluble chlorides, residual chlorine, chloroacetic acids, and insoluble organically bound chlorine. Therefore, the balance of chlorine measured by the flow scheme was acceptable. Little residual chlorine was detected in the pulp products, suggesting that the chlorine dioxide used for bleaching was almost completely converted into inactive inorganic chlorides and organic chlorine compounds and that these substances do not affect the health risk posed by pulp products. A risk assessment of the detected chloroacetic acid and dichloroacetic acid revealed that there is no concern about the health risk posed by use of these products.

Quantitative and antioxidative behavior of Trolox in rats' blood and brain by HPLC-UV and SMFIA-CL methods.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10(3) antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0-360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood-brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified.

Validation of an LC-MS/MS Method for the Determination of Propofol, Midazolam, and Carbamazepine in Rat Plasma: Application to Monitor Their Concentrations Following Co-administration.

Propofol (PRO) is a hypnotic used to induce and maintain general anesthesia. A risk of drug-drug interactions exists in cases of clinical co-administration of PRO and midazolam (MDZ) or carbamazepine (CBZ). Therefore a sensitive and rapid assay is needed to monitor these drugs. In this study, a sensitive and selective liquid chromatography-tandem mass spectrometry technique was developed for simultaneous determination of PRO, MDZ, and CBZ in plasma. Simultaneous selected reaction monitoring in the positive and negative ionization modes was used for mass detection. Analytes were isolated from plasma samples by a simple, economic, and rapid solid-phase extraction method. Chromatographic separations were achieved using a Chromolith Performance RP-18e analytical column (100×4.6 mm i.d.) with a mixture of acetonitrile-ammonium acetate buffer (10 mM, pH 3.5) (90 : 10, v/v) as the mobile phase. The method was fully validated for PRO, MDZ, and CBZ over concentrations ranging at 1-100, 2-100, and 7-1000 ng/mL, respectively, with acceptable validation parameters. Furthermore, the method was applied to monitor PRO and MDZ or CBZ following co-administration in rats.

Investigation of the amount of transdermal exposure of newborn babies to phthalates in paper diapers and certification of the safety of paper diapers.

A risk assessment study of seven phthalates in paper diapers for newborn babies produced in Japan was performed. The diapers were purchased and the contents of the seven phthalates were determined and estimated amounts of exposure were calculated based on the eluted rate into artificial medium of urine or sweat, average weight of infants, and frequency of use. Di-2-ethylhexyl phthalate and di-n-butyl phthalate were detected in the topsheets and determined to be 0.6 µg/g and 0.2 µg/g, respectively. The daily estimated exposure volume was calculated to be in the range of 1.86 × 10(-10)-2.98 × 10(-6) mg/kg/day as follows: content of seven phthalates in the topsheet (0.1-1 µg/g) × eluted rate of phthalates into artificial sweat (0.0006-2.4%) × weight of the topsheet of a diaper (1.5 g) × the number of diapers used per day (12 sheets) × skin absorption rate (0.005-0.1)/average body weight (2.9 kg). For hazard assessment, we used 0.2-300 mg/kg/day for the seven phthalates based on the data available at international agencies. The margin of exposure to the seven phthalates was 6.71 × 10(4)-1.99 × 10(11), indicating that the risk of exposure to phthalates from the diapers produced in Japan was negligible.

Simultaneous determination of propofol and remifentanil in rat plasma by liquid chromatography-tandem mass spectrometry: application to preclinical pharmacokinetic drug-drug interaction analysis.

Propofol (Pro) is an ultra-short-acting hypnotic agent used for general anesthesia that has no analgesic properties. Remifentanil (Rem) is an ultra-short-acting opioid administered concomitantly as an analgesic with Pro. To evaluate the pharmacokinetic interactions between Pro and Rem, we developed and validated a method combining high-performance liquid chromatography with tandem mass spectrometry for simultaneous determination of Pro and Rem. The proposed method was successfully used to study the pharmacokinetic interactions of Pro and Rem coadministered to rats.

Nickel-catalyzed dimerization of pyrrolidinoindoline scaffolds: systematic access to chimonanthines, folicanthines and (+)-WIN 64821.

While metal-promoted activation of tertiary alkyl halides often causes elimination and hydrodehalogenation, we have developed a nickel-catalyzed reductive dimerization that allows the generation of a potently reactive tertiary radical equivalent to form a very congested C(sp(3))-C(sp(3)) bond even below room temperature. The catalytic protocol is applicable to the dimerization of several pyrrolidinoindoline scaffolds through an appropriate choice of catalyst to accommodate different substrate reactivities with functional group compatibilities. The efficiency of the nickel-catalyzed protocol was successfully demonstrated through a systematic total synthesis of chimonanthines, folicanthines and (+)-WIN 64821.

Risk assessment study of dioxins in sanitary napkins produced in Japan.

A risk assessment study of dioxins in sanitary napkins produced in Japan was performed. The daily estimated exposure volume to dioxins was compared with the tolerable daily intake (TDI). The concentrations of dioxins such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) in seven sanitary napkins were measured using gas chromatography and mass spectroscopy analytical methods. Among the seven napkins, a range of 0.0044-0.076pg TEQ/g dioxins was measured. Daily estimated exposure volume from sanitary napkins was calculated as follows: (dioxin volumes in a sanitary napkin (0.0044-0.076pg TEQ/g)×pulp weight in a sanitary napkin (11.2g)×used napkin numbers/d (7.5)×the number of days/month that women use sanitary napkins (7)×skin absorption rate (0.03)×used years (40))/(average body weight of women (50kg)×the number of days in the month (30)×life years (86)). Daily exposure volumes were estimated to be 0.000024-0.00042pg TEQ/kg/d. For hazard assessment, we used 0.7pg TEQ/kg/d which was the lowest level of TDI among TDI values reported by international agencies. When the daily exposure volume was compared with the TDI, the former was approximately 1666-29,166 times less than the latter. This fact indicated that the risk of exposure to dioxins from sanitary napkins produced in Japan was negligible.

Quantitation of sulfur-containing amino acids, homocysteine, methionine and cysteine in dried blood spot from newborn baby by HPLC-fluorescence detection.

Sulfur-containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC-fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies (n=200). Sample pretreatment involving reduction, derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, and liquid-liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04-0.14 µm as the limit of detection at a signal-to-noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5-106.2%) and intra- (≤ 9.0%) and inter-day precisions (≤ 8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies.

Determination of folates by HPLC-chemiluminescence using a ruthenium(II)-cerium(IV) system, and its application to pharmaceutical preparations and supplements.

A chemiluminescence (CL) reaction of folic acid (FA) with ruthenium (II) and cerium (IV) was applied to quantify FA-related compounds such as FA, dihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid and methotrexate (MTX). Among the FAs, 5-methyltetrahydrofolic acid provided the highest CL intensity. HPLC-CL detection of FA was applied to quantify FA in pharmaceutical preparations and supplements. Analytical samples were separated on a semi-micro ODS column with a mixture of 20 mM phosphate buffer (pH 5.7) and acetonitrile (94 : 6, v/v %). The separated samples were mixed with a post-column CL reagent consisting of 1.5 mM Ru(bipy)3 (2+) and 1.0 mM Ce(SO4)2 , then the generated CL was monitored. The calibration range for FA was 10-100 µM and the limit of detection was 1.34 µM (signal-to-noise ratio of 3). Repeatabilities were 4.2, 4.6 and 5.0 RSD% (10, 25, 50 µM), and the recoveries for FA supplement, vitamin B complex supplement and FA-containing medication (tablet) were 102.4 ± 10.5, 103.3 ± 13.3 and 100.3 ± 8.5%, respectively. The described method is robust against changes in the chromatographic parameters of ± 3.3 or ± 1.5%. The measured FA content corresponded well to the labeled content of FA-containing products (100.6-104.9%), demonstrating the precision and accuracy of this method for the evaluation of FA pharmaceutical preparations.

In vitro screening of Fe2+-chelating effect by a Fenton's reaction-luminol chemiluminescence system.

In vitro screening of a Fe(2+) -chelating effect using a Fenton's reaction-luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe(2+) using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 µL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined. EC50 values for these compounds (except 1,10-phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 µM (n = 3). Rapid measurement of the Fe(2+)-chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed.

LC-MS/MS analysis of components in smoke from e-cigarettes that use guarana extract as the caffeine source.

Currently, e-cigarette products to inhale caffeine (Caf) are commercially available and widely used. Guarana extract (GE) is used as the caffeine source in some e-cigarette products. In this study, an LC-MS/MS analysis of components in the smoke from e-cigarettes with GE was performed. The concentration ranges of Caf and the minor components theophylline (TP), theobromine (TB), and paraxanthine (PX) in e-liquid and cigarette smoke extract (CSE) of five e-cigarette products were determined. The concentration ranges of e-liquid and CSE were 2.17-8.62 mg/mL and 0.17-1.17 µg/puff for Caf, 0.09-37.58 µg/mL and 0.03-11.88 ng/puff for TB, 50.28-185.26 ng/mL and 0.00-0.05 ng/puff for TP, and 0.44-4.09 µg/mL and 0.03-0.20 ng/puff for PX, respectively. By comparing the peak area ratios of e-liquid and CSE, we clarified that the heat degradation of Caf to its related components in GE products was accelerated. Epicatechin, which is another typical component in GE, was determined for CSE, but not for e-liquid.

4-Iodobenzonitrile as a fluorogenic derivatization reagent for chromatographic analysis of L-p-boronophenylalanine in whole blood samples using Suzuki coupling reaction.

BACKGROUND: L-p-Boronophehylalanine (BPA) is used in boron neutron capture therapy (BNCT), which is a novel selective cancer radiotherapy technique. It is important to measure BPA levels in human blood for effective radiotherapy; a prompt gamma-ray spectrometer, ICP-AES, and ICP-MS have been used for this purpose. However, these methods require sophisticated and expensive apparatuses as well as experienced analysts. Herein, we propose an HPLC-FL method for the determination of BPA after precolumn derivatization. A new fluorogenic reagent for aryl boronic acid derivatives, namely, 4-iodobenzonitrile, was employed for the fluorogenic derivatization of BPA based on the Suzuki coupling reaction. RESULTS: After the fluorogenic derivatization, a fluorescent cyanobiphenyl derivative is formed with maximum fluorescence at 335 nm after excitation at 290 nm. The developed method showed good linearity (r2=0.997) over the concentration range of 0.5-1000 nmol/L, and the detection limit (S/N = 3) was 0.26 nmol/L. The proposed method is more sensitive than previously reported methods for the determination of BPA, including the ICP-MS. Finally, the proposed method was successively applied to the measurement of BPA in human whole blood samples with a good recovery rate (≥95.7 %) using only 10 µL of blood sample. The proposed method offers a simple and efficient solution for monitoring BPA levels in BNCT-treated patients. SIGNIFICANCE: 4-Iodobenzonitrile was investigated as a new fluorogenic reagent for BPA based on Suzuki coupling. A new HPLC-FL method for BPA in whole blood samples with ultrasensitivity was developed. The developed method is superior in sensitivity to all previously reported methods for BPA. The method requires only a very small sample volume, making it suitable for micro-blood analysis of BPA via fingerstick sampling.

Development and validation of the first assay method coupling liquid chromatography with chemiluminescence for the simultaneous determination of menadione and its thioether conjugates in rat plasma.

Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.