Pomalidomide is an immunomodulatory agent (IMiD) that has been approved by the US Food and Drug Administration (FDA) for clinical treatment of patients with multiple myeloma. In this work, we developed a sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide over the range of 1.006-100.6 ng/mL (R-2 = 0.9991) in human plasma and pharmacokinetic studies. A liquid-liquid extraction method using ethyl acetate was applied to extract pomalidomide and afatinib (as an internal standard, IS) from human plasma. Chromatographic separation was performed on a Hedera ODS column (150 mm x2.1 mm, 5 mu m) with security guard C18 column (4 mm x2.0 mm) at 40 degrees C. Methanol and 10 mmol/L aqueous solution of ammonium acetate containing 0.1% formic acid were used as a gradient elution mobile phase, and the flow rate was 0.4 mL/min. A triple quadruple tandem mass spectrometer using multiplex reaction monitoring mode (MRM) with electrospray ionization (ESI) positive ionization was employed. The precursor to product ion transitions for the quantitative analysis of pomalidomide and the IS were m/z 274.2 -> 163.1 and m/z 486.1 -> 371.1, respectively. This established method has been validated according to regulatory guideline, and the results were all within the acceptance criteria. The validated LC-MS/MS method was successfully applied to analyze samples obtained from clinical pharmacokinetics study after oral administration of pomalidomide (4 mg) capsules in human. (C) 2021 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

A simple LC-tandem mass spectrometry (MS/MS) method to determine ebastine and carebastine (active metabolite) in human plasma was developed and validated. Analytes and internal standards were precipitated by protein precipitation and separated on Synergi Hydro-RP 80A column (4 μm, 50 mm × 2.0 mm; Phenomenex) by gradient elution with mobile phase A comprising 0.1% formic acid in 5 mm ammonium acetate (NH4 Ac) and B comprising 100% methanol at a flow rate 0.4 mL/min. Ions were detected in positive multiple reaction monitoring mode, and they exhibited linearity over concentration range 0.01-8.0 and 1.00-300 ng/mL for ebastine and carebastine, respectively. A clinical pharmacokinetic study was conducted in healthy Chinese volunteers under fasting and fed conditions after a single oral administration of 10 mg ebastine. The maximum plasma concentration (Cmax ), time to Cmax (Tmax ) and elimination half-life for ebastine were 0.679 ± 0.762 ng/mL, 1.67 ± 1.43 h and 7.86 ± 6.18 h, respectively, whereas these for carebastine were 143 ± 68.4 ng/mL, 5.00 ± 2.00 h and 17.4 ± 4.97 h, respectively under fasting conditions; the corresponding values under fed conditions were 4.13 ± 2.53 ng/mL, 3.18 ± 1.09 h and 21.6 ± 7.77 h for ebastine and 176 ± 68.4 ng/mL, 6.14 ± 2.0 h and 20.0 ± 4.97 h for carebastine.

Simple LC-MS/MS method to determine Ebastine and Carebastine (active metabolite) in human plasma was developed and validated. Analytes and internal standards precipitated by protein precipitation and separated on SynergiTM Hydro-RP 80A column (4 μm, 50 mm × 2.0 mm, Phenomenex) by gradient elution with mobile phase A: 0.1 % Formic Acid (FA) in 5 mM Ammonium Acetate (NH4 Ac) and B: 100% methanol, flow rate 0.4 mL/min. Ions were detected in positive multiple reaction monitoring mode. Exhibited linearity over concentration range 0.01-8.0 ng/mL and 1.00-300 ng/mL for Ebastine and Carebastine, respectively. A clinical pharmacokinetic study was conducted in healthy Chinese subjects under fasting and fed conditions after 10 mg Ebastine single oral administration. The maximum plasma concentration (Cmax ), time to Cmax (Tmax ) and elimination half-life for Ebastine were 0.679 ± 0.762 ng/mL, 1.67 ± 1.43 h and 7.86 ± 6.18 h respectively, and for Carebastine 143 ± 68.4 ng/mL, 5.00 ± 2.00 h and 17.4 ± 4.97 h respectively under fasting conditions, fed conditions 4.13 ± 2.53 ng/mL, 3.18 ± 1.09 h and 21.6 ± 7.77 h for Ebastine and 176 ± 68.4 ng/mL, 6.14 ± 2.0 h and 20.0 ± 4.97 h for Carebastine respectively.

Protein drugs showing strong pharmaceutical activity, high specificity, and low toxicity and side effects have drawn extensive attention in the field of life sciences and medicine. Precise evaluation of the function of these drugs requires accurate and sensitive detection methods. Here, we report a novel chromatography-tandem mass spectrometry (LC-MS/MS) method for sensitive and selective detection of protein drugs. Magnetic nanoparticles (Apt29@MNPs) were functionalized by thrombin aptamers, and quantum dots (Apt15@ss@QDs) were dual-functionalized with quantitative thrombin aptamers and small molecules with high ionization efficiency as the mass barcode. After Apt29@MNPs specifically purify and enrich thrombin from biological samples, they can form a nano "sandwich structure" when Apt15@ss@QDs are added, resulting in the release of the mass barcode for LC-MS/MS analysis via the cutting of the disulfide bond. Since there is a higher quantitative molecular ratio of mass barcode to thrombin in the nano-"sandwich structure", quantitative detection of thrombin with high sensitivity and selectivity can be achieved via the LC-MS/MS detection of the mass barcode with high ionization efficiency rather than thrombin, which effectively avoids the disadvantages of direct protein detection by mass spectrometry. The established method for thrombin detection shows a good linear relationship in a concentration range of 0.00115-1.15 nM with a limit of detection (LOD) of 0.0007 nM. The present work provides a new approach for the effective and sensitive quantitative analysis of protein drugs and would be of great significance in promoting the development of protein drugs and clinical applications.

A mass barcode mediated signal amplification strategy was developed and applied to the determination of protein. A new compound, N '-((2-aminopyridin-3-yl)methylene)-5-(1,2-dithiolan-3-yl)pentanehydrazide (TAPA), was synthesized from the linker and the signal barcode, and used as the bonding barcode. For the realization of signal transduction, TAPAs and the target catcher aptamers, were both modified on gold nanoparticles (AuNPs) to establish the relationship between TAPAs and the target. Owing to the fact that the amount of TAPAs was much greater than the target, the signal of the target was not only transduced to the signal of the mass barcodes, but also amplified greatly. Thrombin, an important biomarker for coagulation abnormality diseases, was selected as a model analyte. Two kinds of thrombin recognition aptamers, aptamer 29 (apt29) and aptamer 15 (apt15), were modified onto the magnetic beads (MBs) and AuNPs, respectively. The modified AuNPs were further functionalized with lots of TAPA and formed apt15-AuNPs-TAPA. MBs-apt29 and apt15-AuNPs-TAPA could both recognize the target thrombin and form the sandwich complex (MBs-apt29/thrombin/apt15-AuNPs-TAPA). After the complex was separated by an extra magnetic field, NaClO oxidant solution was added to release the signal barcodes, 2-Amino-3-pyridinecarboxaldehyde (APA), which were then collected after centrifuging and analyzed by LC-MS/MS. Under optimized conditions, the mass response intensity was proportional to thrombin concentration in the range of 0.05-10 nM, with a 0.007 nM detection limit. This method was applied to the determination of thrombin in spiked serum samples, and the average recoveries ranged from 89.6% to 110.4%, which confirmed the applicability of this method.

In this study, the hydrophobic drug Tanshinone IIA (Tan IIA) as a therapeutic drug was loaded into naphthalene acetic acid-phenylalanine-phenylalanine-glycine hydrogel (Nap-FFG) to construct the supramolecular hydrogels. Nap-FFG hydrogel fibers crosslinked into a network-like structure by self-assembly of polypeptides and then achieved drug loading. The rheological assay of Tan IIA-loaded Nap-FFG hydrogel (Tan@Nap-FFG) had excellent shear resistance, allowing it to maintain gel state. Anticoagulant and anti-oxidant assay showed Tan@Nap-FFG hydrogels synergistically resisted hydrogen peroxide damage and inhibited platelet aggregation caused by ADP, which was suitable for the therapy of cardiovascular diseases. In-vitro release assays and in-vivo pharmacokinetic had demonstrated that hydrogels released drugs slowly, reduced drug toxicity, and improved pharmacokinetic behavior. In summary, Tan@Nap-FFG hydrogels was expected to be developed as a novel injection for cardio-vascular therapy.

In this work, we developed and validated the specific, sensitive and simple LC–MS/MS method for quantification of eslicarbazepine in human plasma. The analyte samples were prepared through a simple one-step protein precipitation method by acetonitrile. The chromatographic separation was operated on an economical Hanbon ODS-2 C18 column (150 mm × 2.1 mm, 10 μm) with isocratic elution using 10 mM ammonium acetate containing 0.01% formic acid and acetonitrile (72:28, v/v) as the mobile phase at the flow rate of 0.5 mL/min. The mass quantification was carried on the multiple reaction monitoring (MRM) of the transitions of m/z 255.1 → 194.1 for eslicarbazepine and m/z 446.1 → 321.1 for glipizide (the internal standard), respectively. The established method was validated with acceptable specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability in accordance with FDA regulations. At last, the validated method was successfully applied to determination of eslicarbazepine in human plasma obtained from clinical study.

A sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of ramelteon and its active metabolite M-II in human plasma. After extraction from 200 μL of plasma by protein precipitation, the analytes and internal standard (IS) diazepam were separated on a Hedera ODS-2 (5 μm, 150 × 2.1 mm) column with a mobile phase consisted of methanol-0.1% formic acid in 10 mm ammonium acetate solution (85:15, v/v) delivered at a flow rate of 0.5 mL/min. Mass spectrometric detection was operated in positive multiple reaction monitoring mode. The calibration curves were linear over the concentration range of 0.0500-30.0 ng/mL for ramelteon and 1.00-250 ng/mL for M-II, respectively. This method was successfully applied to a clinical pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ramelteon. The maximum plasma concentration (Cmax ), the time to the Cmax and the elimination half-life for ramelteon were 4.50 ± 4.64ng/mL, 0.8 ± 0.4h and 1.0 ± 0.9 h, respectively, and for M-II were 136 ± 36 ng/mL, 1.1 ± 0.5 h, 2.1 ± 0.4 h, respectively.

The present work is focused on the design and development of novel amphotericin B (AmB)-conjugated biocompatible and biodegradable polypeptide hydrogels to improve the antifungal activity. Using three kinds of promoting self-assembly groups (2-naphthalene acetic acid (Nap), naproxen (Npx) and dexamethasone (Dex)) and polypeptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK(AmB)Y gels, Npx-FFDK(AmB)Y gels and Dex-FFDK(AmB)Y gels. The AmB-conjugated hydrogelators are highly soluble in different aqueous solutions. The cryo-transmission electron microscopy and scanning electron microscopy micrographs of hydrogels afford nanofibres with a width of 20–50 nm. Powder X-ray diffraction analyses demonstrate that the crystalline structures of the AmB and Dex are changed into amorphous structures after the formation of hydrogels. Circular dichroism spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the AmB-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drugs. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.

The authors describe magnetic nanoparticles comprising of a Fe3O4 core and a polyvinyl alcohol (PVA) coating for use in dispersive solid phase extraction (DSPE) of aminoglycoside antibiotics (AAs). The sorbent was investigated by Transmission electron microscope (TEM), Fourier transform infrared spectrometer (FT-IR), thermo-gravimetric analysis (TGA), nitrogen adsorption-desorption isotherms and so on. The extraction conditions consisting of the proportion of ACN, pH value, buffer concentration and sorbent dosage were optimized. The nanoparticles have a large surface area (73.28 m(2) g(-1)), a high binding capacity (11.33 mu mol g(-1)) and a fast binding time (30 s). The Fe3O4@PVA is shown to be an effective adsorbent for the enrichment of AAs (streptomycin, dihydrostreptomycin and kanamycin) in spiked honey. The limits of detection are as low as 0.993, 0.913 and 1.23 mu g kg(-1) for streptomycin, dihydrostreptomycin and kanamycin, respectively. The recoveries varied from 82.9% to 100.7% at the three spiking levels tested (40, 400, 4000 mu g.kg(-1)). Intra-day and inter-day assay precision were < 12.1% (n = 6) and <12.8% (n = 3) at three spiking levels. These data showed that the method could be applied to the extraction of AAs in honey samples. (C) 2018 Elsevier B.V. All rights reserved.

As an orally active iron chelator, deferasirox forms its ion complexes in the prepared plasma samples and LC-MS mobile phase where ferric ion exists, and then comparing with the nominal concentration level, a lower detected concentration level of deferasirox would be obtained after LC-MS analysis, if no proper treatment was adopted. Meanwhile, the phenomenon would be observed that multiple repeat injections of the same deferasirox plasma sample in the same tube would show the lower and lower detected concentration levels of deferasirox, which caused by more and more ferric ions from the injection needle dissolved in the sample solution as multiple repeated injections. The addition of a proper concentration of EDTA in the mobile phase and the sample will competitively inhibit deferasirox from complexing with ferric ion, and prevent the decrease of deferasirox concentration. In this paper, an LC-MS/MS method was developed and validated for the determination of deferasirox in human plasma. To achieve the protein precipitation, the analytes were extracted from aliquots of 200 mu L human plasma with acetonitrile. Chromatographic separation was performed on an ODS-C18 column with the mobile phase consisted of methanol and 0.1% formic acid containing 0.04 mM ethylenediamine tetraacetate dihydrate (EDTA) (80:20, v/v) at a flow rate of 0.5 mL/min. Deferasirox and the internal standard (IS, mifepristone) were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-product ion transitions m/z 374.2 -> 108.1 for deferasirox and m/z 430.1 -> 372.2 for the IS. The method exhibited good linearity over the concentration range of 0.04-40 mu g/mL for deferasirox. The method was successfully applied to a pharmacokinetic study in 10 Chinese healthy volunteers after oral administration of deferasirox. (C) 2018 Elsevier B.V. All rights reserved.

A hollow porous molecularly imprinted polymer (HPMIP) is described for use in dispersive solid phase extraction of macrolide antibiotics (MACs). The HPMIP was prepared by using spiramycin as the template, methacrylic acid as the functional monomer, and mesoporous MCM-41 (Mobile Composition of Matter No. 41; with a size of about 100 nm) as a sacrificial support. The sorbent was characterized by Fourier transform infrared spectrometry, transmission electron microscopy, nitrogen adsorption and thermo-gravimetric analysis. Several parameters affecting the extraction efficiency were optimized. The material has a large surface area (359 m2·g−1), and most recognition sites are located on the surface of the HPMIPs. This results in high binding capacity (120 μmol·g−1) and fast binding (20 min) in comparison to either MCM-41-core surface MIPs or solid MIPs. The method was applied to the extraction of the MACs azithromycin, spiramycin, tilmicosin, tylosin, clarithromycin, roxithromycin and josamycin from spiked honey. The recoveries, determined by HPLC-MS/MS, ranged from 88.0% to 117% at the three spiking levels tested (1, 5 and 20 μg·kg−1). Intra-day and inter-day assay precision at three spiking levels are <10.7% (for n = 6) and 12.6% (n = 3), respectively. The limits of detection are between 3 and 17 ng·kg−1. This indicates the superiority of the method in selective extraction of macrolides even from complex matrices.

Alkaline phosphatase (ALP) is an important biomarker for many diseases. Therefore, the sensitive and accurate detection of ALP activity is essential for fundamental biochemical processes and clinic diagnosis. Herein, we design a fluorescent on-off-on switch for sensitive and visual detection of ALP activity. Meanwhile, mass barcode-modified quantum dots (QDs) amplified the LC-MS/MS detection signal in complex biological samples. Firstly, the QDs were modified with phosphorylated Gly-Gly-Phe-Phe-Tyr (OPO3H2) peptide (GGFFYp) and the mass barcode. The fluorescence of QDs-SS-Yp was quenched by fluorescence resonance energy transfer (FRET) between QDs-SS-Yp and dansyl chloride (DNS). ALP can hydrolyze the phosphorylated peptide to form peptide self-assemblies on the QDs-SS-Yp surfaces. The effective separation distance between the QDs-SS-Yp donor and DNS acceptor becomes larger, restricting FRET between the QDs-SS-Yp and DNS. At this point, the obvious QDs-SS-Yp fluorescence signal can be restored. However, the absence of ALP results in no peptide self-assembly on the QDs-SS-Yp surface and no obvious QDs-SS-Yp fluorescence signal was detected. Therefore, the ALP activity can be analyzed according to the degree of fluorescence restoration by the fluorescence on-off-on switch. Finally, the small tag molecules obtained by cleaving the disulfide bond of the QDs-SS-Yp as a mass barcode were used to amplify the LC-MS/MS detection signal. The proposed approach shows a good linear relationship (from 0.01 to 2.4 U L-1) and has the significant advantage of a low detection limit of 0.001 U L-1.

In this work, we studied the synthesis, characterization, anticancer effect and pharmacokinetics of supramolecular hydrogels composed of 2-naphthalene acetic acid-Gly-Arg-Gly-Asp-Phe-Phe-Gly poly-peptide (Nap-peptide) and TanshinoneIIA (TsIIA) derivative (Ts-OH) which had excellent selectivity for inhibiting growth of cancer cells. The Nap-Ts hydrogels exhibited long, narrow and flexible nanofibers entangling each other to form three-dimensional networks, which further self-assembling into nanoparticles with diameters from 200 to 300 nm. The hydrogels drug delivery was suitable for the sustained release, which exhibited both diffusion and relaxation release mechanisms. Compared to TsIIA, the Ts-OH, Nap-Ts hydrogels exhibited higher anticancer effect to SCG7901 cells, MCF-7 cells, HepG2 cells and MDA-MB-231 cells in vitro. The pharmacokinetic behaviours of Nap-Ts hydrogels were evaluated in rats. The Nap-Ts hydrogels possessed better biocompatibility, sustain drug release and anticancer effect than TsIIA. The results demonstrated the potential drug delivery system of TsIIA improving the anticancer activity.

Rabeprazole is an effective proton pump inhibitor to treat acid-related diseases. To achieve the simultaneous determination of rabeprazole enantiomers in human plasma, a chiral LC-MS/MS method was developed and validated. Acetonitrile including 0.1% ammonium were used as protein precipitating agent. Analytes were separated within 8 minutes on a Chiralpak IC column (4.6 mm × 150 mm, 5 μm). The mobile phase was 10 mM ammonium acetate including 0.2% acetic acid-acetonitrile (35:65, v/v). An API 4000 mass spectrometer was used as detector for the analysis, and the multiple reactions monitoring transitions of m/z 360.1 → 242.2 and 346.1 → 198.1 were opted for quantifying rabeprazole enantiomers and internal standard. Matrix effects were not apparent for each enantiomer and internal standard (esomeprazole), the calibration curves were linear over the concentration of 0.500 to 400 ng·mL-1 , the intra-run precisions were below 5.4%, the inter-run precisions were below 9.9%, and the accuracy was between -9.2% and 9.3%. There was no chiral inversion observed during sample storage, preparation procedure, and analysis, demonstrating that analytes were stable in this study. This method was applied to the stereoselective pharmacokinetic study of (R)-(+)- and (S)-(-)-rabeprazole after oral administration of 10-mg rabeprazole sodium enteric-coated tablet in healthy Chinese subjects.

A method is described for the determination of proteins with LC-MS/MS enabled by a small molecule (adenosine) barcode and based on a double-recognition sandwich structure. The coagulation protein thrombin was chosen as the model analyte. Magnetic nanoparticles were functionalized with aptamer29 (MNP/apt29) and used to capture thrombin from the samples. MNP/apt29 forms a sandwich with functionalized gold nanoparticles modified with (a) aptamer15 acting as thrombin-recognizing element and (b) a large number of adenosine as mass barcodes. The sandwich formed (MNP/apt29-thrombin-apt15/AuNP/adenosine) can ben magnetically separated from the sample. Mass barcodes are subsequently released from the sandwiched structure for further analysis by adding 11-mercaptoundecanoic acid. Adenosine is then detected by LC-MS/MS as it reflects the level of thrombin with impressively amplified signal. Numerous adenosines introduced into the sandwich proportional to the target concentration further amplify the signal. Under optimized conditions, the response is linearly proportional to the thrombin concentration in the range of 0.02 nM to 10 nM, with a detection limit of 9 fM. The application of this method to the determination of thrombin in spiked plasma samples gave recoveries that ranged from 92.3% to 104.7%.

The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.