Blank Plasma Research Articles

Application of Modified Polyether Sulfone Microspheres in Hyperbilirubinemia

To design and prepare a high efficiency bilirubin adsorbent with good mechanical properties and biocompatibility. In this study, quaternary ammonium pyridine was designed and synthesized, and then modified polyether sulfone microspheres, or PES/p(4-VP-co-N-VP)@6 microspheres, were prepared by phase conversion and electrostatic spraying. The morphology of the polymer components and the microspheres were studied by means of nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscopy. The basic properties of the microspheres and their bilirubin adsorption efficiency were tested, and the adsorption mechanism was further explored. Blood cell counts and the clotting time of the microspheres were also measured. The diameter of the modified polyether sulfone microspheres prepared in the study was approximately 700-800 μm. Compared with the original PES microspheres, the surface and internal structure of PES/p(4-VP-co-N-VP)@6 microspheres did not change significantly, and they also had a loose porous structure, with some micropores scattered around in addition to irregular large pores. Compared with the control group, the bilirubin removal effect of the modified microspheres was (94.91±0.73)% after static adsorption in bilirubin PBS buffer solution for 180 min, with the difference being statistically significant (P<0.0001). According to the findings for the clotting time, the activated partial thromboplastin time (APTT) of the blank plasma group, the control PES group, and the modified PES microsphere group were (27.57±1.25) s, (28.47±0.45) s, and (30.4±0.872) s, respectively, and the difference between the experimental group and the other two groups was statistically significant (P<0.01, P<0.05). There was no significant change in red blood cell and white blood cell counts. The microspheres prepared in the study have high efficiency in bilirubin adsorption, excellent mechanical properties and thermal stability, and good blood biocompatibility, and are expected to be used in the clinical treatment of patients with liver failure.

Development, validation and application of a UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its metabolites tolvaptan, DM-4103 and DM-4107 in human plasma

OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00–1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.

Chemiluminescence data modeling using parallel factor analysis for the simple and sensitive determination of diphenoxylate in human plasma and tablets

AbstractIn this study, a chemiluminescence (CL) method was developed to determine diphenoxylate in tablets and human plasma. This is the first CL method proposed to determine diphenoxylate. Creating three‐dimensional data caused the parallel factor analysis algorithm (PARAFAC) to be used for the first time in CL methods. The method is based on the fact that diphenoxylate enhances the weak CL produced in the reaction of Ru(phen)32+ and acidic Ce(IV), and the concentration of Ce(IV) solution has a different effect on the CL response of diphenoxylate and the blank plasma. The calibration curve was linear from 4.0 × 10−8 to 1.6 × 10−6 mol L−1 (R2 = 0.9954), and the detection limit was 1.3 × 10−8 mol L−1 (S/N = 3). The sampling rate was about 30 samples per hour, and the % RSD for 10 repeated measurements of 4 × 10−7 mol L−1 diphenoxylate was 5.4%. The interference effects of some ions, amino acids, and common additives were also investigated. The CL method was successfully used to determine diphenoxylate in tablets, and the results were statistically confirmed by the reference method. The proposed CL method and the PARAFAC algorithm were successfully used to determine the concentration of diphenoxylate in human blood plasma samples.

Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples

IntroductionMitotane (o,p’–DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. ObjectiveTo develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. MethodDrug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p’-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. ResultsThe present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4’-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00–50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). ConclusionDDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Integrated ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry-based components analysis and network pharmacology strategy of Gancao Xiexin Decoction in treating gastric ulcer.

Gancao Xiexin Decoction (GCXXD) is a traditional Chinese decoction that is often used in treating gastric ulcers. However, the substance basis and mechanism of action remain unclear. In this study, in vivo and in vitro components of GCXXD were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry. The compound Discover platform was used to ultimately enable rapid identification of compounds. Acquire X intelligent data acquisition technology software was innovatively adopted. In the process of collecting drug-containing plasma, all components detected in blank plasma samples were excluded to eliminate the interference and influence of endogenous components in plasma, making the analysis results more accurate and reliable. At the same time, the possibility of selecting precursor parent ions with low concentration levels within the chromatographic peak can be increased, improving the coverage and integrality of the detection of components in vivo. Also, the targeted network pharmacology strategy combined with molecular docking was established to explore the mechanism of GCXXD in treating gastric ulcers. As a result, 113 components were identified, 41 of which could enter the bloodstream and exert therapeutic effects in vivo. The main effective components are glycyrrhizic acid, 6-gingerol, jatrorrhizine, wogonin, palmatine, and liquiritigenin, main targets in vivo were related to ALB, IL6, and VEGF, which play an important role in anti-inflammatory and promoting angiogenesis. In summary, this study adopted a comprehensive analysis strategy to reveal the pharmacodynamic material basis and mechanism of GCXXD against gastric ulcers, providing a scientific basis for its clinical application.

Systematic characterization of the chemical constituents in vitro and in vivo of Qianghuo by UPLC-Q-TOF-MS/MS

The Chinese herb Qianghuo is an antiphlogistic herb with many effects and complex components. In this study, the chemical composition of Qianghuo and its components in rat plasma after oral administration were investigated using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). The extracts, blank plasma, and plasma containing the drug were analyzed by mass spectrometry, and data collected in both positive and negative ion modes were analyzed using Masslynx software, and the structures were confirmed by combining the compound fragment ions and mass spectrometry cleavage pathways. A total of 62 in vitro chemical components were identified, including 27 coumarins, 18 organic acids, 5 amino acids, 5 glycosides, 2 flavonoids, 4 nucleotides, and 1 ester, which were summarized from the obtained compounds in terms of their possible cleavage patterns. Among the identified 31 compounds in rat plasma, 21 were prototypes, mostly coumarins, organic acids, and flavonoids, and 10 were metabolites, which were mainly generated via hydroxylation and methylation pathways. Based on these, this study provides a theoretical foundation for quality control and basic research on Qianghuo medicinal substances.

Separation of bile acid isomer plays a pivotal role in bioequivalence evaluation of ursodeoxycholic acid

Based on our experiences in bile acid profiling, this work developed and validated a liquid chromatography electrospray ionization tandem mass spectrometry method to separate endogenous bile acid isomers and quantitatively determine ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. The separation was performed on a CORTECS C18 column with the mobile phase consisting of 1.0 mM ammonium acetate and acetonitrile-methanol (80:20, v/v). UDCA, GUDCA and TUDCA were detected in the negative mode on a triple-quadrupole mass spectrometer at the ion transitions of m/z 391 > 391, m/z 448 > 74, m/z 498 > 80, respectively. Phosphate buffer was employed as the surrogate matrix to establish the isotope internal standard corrected calibration curves of analytes. The background-method with a linearity range of 10–200 ng/mL was partially validated to determine the endogenous levels of analytes in blank human plasma, which was incorporated into the validation of bioequivalence-method with a linearity range of 50–10000 ng/mL. The bioequivalence (BE)-method was fully validated with special focus on matrix effects, which have been critically evaluated using the precision and accuracy of quality control samples prepared from the blank human plasma of 12 individuals. It is disclosed for the first time that the BE results of UDCA formulation may yield false results when the method is insufficient to separate UDCA from isoursodeoxycholic acid, a microbial metabolite of both endogenous and exogenous UDCA. The present method has established a milestone for the evaluation of UDCA formulations and is expected to provide a valuable reference for the bioanalytical development of endogenous medicinal products.

Identification of the absorbed constituents and metabolites of Huangqi Guizhi Wuwu decoction by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Huangqi Guizhi Wuwu decoction (HGWWD) is a widely used traditional Chinese medicine (TCM) preparation for the treatment of ischemic stroke and diabetes peripheral neuropathy. However, the material basis for the efficacy of HGWWD remains unclear. In this study, a rapid, sensitive and selective ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed to separate and identify the absorbed components and metabolites of HGWWD in rat plasma after oral administration for the first time. By comparing the retention time, high-resolution mass spectrometry primary and secondary mass spectrometry data of blank plasma and drug-containing plasma, a total of 42 constituents, including 24 prototype compounds and 18 metabolites, were identified or tentatively characterized. The results indicated that monoterpenes, flavonoids, organic acids, amino acids, gingerols and alkaloids were main prototype compounds in rat plasma, and flavonoid-related metabolites, organic acid-related metabolites and gingerol-related metabolites were major metabolites. It is concluded the developed UHPLC-Q-TOF-MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HGWWD, and the results will provide important data for further study on the relationship between the chemical constituents and pharmacological activities of HGWWD.

A picogram BA-ELISA quantification assay for rLj-RGD3, a platelet fibrinogen receptor antagonist, in the rat plasma and its application to a pharmacokinetic study.

rLj-RGD3, a new member of the RGD (Arginine-Glycine-Aspartate)-motif toxin protein family obtained from Lampetra japonica by means of recombinant DNA techniques, has been demonstrated to be a platelet fibrinogen receptor antagonist and holds potential as a drug candidate for a specific indication. The present article reports an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) to provide a bio-analytical method for pharmacokinetic (PK) studies of rLj-RGD3. The concentration of picogram level rLj-RGD3 in rat plasma was measured using the developed double sandwich BA-ELISA assay, which used two mouse anti-rLj-RGD3 monoclonal antibodies that recognize different epitopes for capture and detection. This method was verified to be highly specific (blank plasma did not interfere with detection), precise (RSD <15%), and accurate (86%-113%). Absolute recovery was in the 94%-119% range. The calibration curve showed good linearity within the 50 to 1600 pg/mL range. The LOQ was as low as 50 pg/mL. The above validated assay was successfully employed to assess PK of rLj-RGD3 in rats. After i.v. and s.c. dosing with 30 μg/kg, the rLj-RGD3 plasma concentration declined bi-exponentially with time. This decay was best fitted to a two-compartment model. In conclusion, the BA-ELISA method described here meets all requirements for PK studies of rLj-RGD3 with an effective pharmacological dose in the μg/kg BW range.

An electrochemical sensor based on CS-MWCNT and AuNPs for the detection of mycophenolic acid in plasma

For patients receiving organ transplants, monitoring the blood concentration of MPA can provide timely information on whether MPA has reached the effective therapeutic window to better function while reducing the incidence of rejection or adverse reactions. In this study, an electrochemical sensor for the detection of MPA was built using a nanocomposite made of CS-MWCNT and AuNPs. At the same time, the high performance liquid phase (HPLC) method for MPA was established and compared with this sensor. The surface morphology, structure, and roughness of the material on the electrode were characterized by scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FTIR), and atomic force microscopy (AFM). In addition, the electrochemical behavior of the modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The standard curve was obtained in blank plasma, not pure buffer solution. The peak current was linearly related to the MPA concentration in the linear range of 0.001–0.1 mM with a detection limit of 0.05 μM and good anti-interference ability. Moreover, the sensor was employed with success for the determination of MPA in rat plasma with good recovery. The electrochemical sensor presented here is eco-friendly, and sensitive, and offers a great possibility for practical applicability.

Evaluation of Small Molecules in Blank EDTA Plasma Tubes and Optimization of Metabolomic Workflow for Biomarker Studies Using Plasma Samples.

As the first step of metabolomic analysis in biomarker identification studies, various types of blood collection tubes are used in clinical practice. However, little attention is paid to potential contamination caused by the blank tube itself. Here, we evaluated small molecules in blank EDTA plasma tubes through LC-MS-based untargeted metabolomic analysis and identified small molecules with markedly varied levels among different production batches or specifications. Our data demonstrate possible contamination and data interference caused by blank EDTA plasma tubes when employing large clinical cohorts for biomarker identification. Therefore, we propose a workflow of filtering metabolites in blank tubes prior to statistical analysis to improve the fidelity of biomarker identification.

A Simplified Method for Determining Blood-to-Plasma Ratios in vitro and ex vivo by Matrix Matching with Blank Blood or Plasma.

This work describes a simplified, 96-well plate method for determining the blood-to-plasma concentration ratio (BP ratio) for small molecules. The need for calibration curves was eliminated using a matrix-matching approach in which blood samples were mixed with blank plasma and plasma samples were mixed with blank blood. As a result, both blood- and plasma-origin samples shared an equivalent matrix ahead of bioanalysis. In the in vitro assay, identical sample matrices were achieved by using the same source of blank plasma and blood. In humans, a good correlation (R2 = 0.84) was observed between the data obtained in this matrix-matching method and literature values for 11 commercial compounds possessing a wide range of logD values across multiple chemical classes. In addition, this method showed good agreement with in vitro BP ratios for 10 proprietary compounds determined radiometrically (R2 = 0.72) in human and preclinical species. Finally, the in vitro matrix matching method compared favorably to BP ratios determined ex vivo for 13 proprietary and literature compounds (R2 = 0.87) in rat. This method, suitable for in vitro and ex vivo BP ratio determinations, is operationally efficient, robust, and a useful improvement upon previously published methods.

Network pharmacology-based approach uncovers the pharmacodynamic components and mechanism of Fructus Tribuli for improving endothelial dysfunction in hypertension

Ethnopharmacological relevanceFructus Tribuli (FT), a traditional Chinese medicinal herbal, has been used for the clinical treatment of cardiovascular diseases for many years and affects vascular endothelial dysfunction (ED) in patients with hypertension. Aim of the studyThis study aimed to demonstrate the pharmacodynamic basis and mechanisms of FT for the treatment of ED. Materials and methodsThe present study used ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to analyze and identify the chemical components of FT. The active components in blood were determined after the oral administration of FT by comparative analysis to blank plasma. Then, based on the active components in vivo, network pharmacology was performed to predict the potential targets of FT in treating ED. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also performed, and component-target-pathway networks were constructed. Interactions between the major active components and main targets were verified by molecular docking. Moreover, spontaneously hypertensive rats (SHRs) were divided into the normal, model, valsartan, low-dose FT, medium-dose FT, and high-dose FT experimental groups. In pharmacodynamic verification studies, treatment effects on blood pressure, serum markers (nitric oxide [NO], endothelin-1 [ET-1,], and angiotensin Ⅱ [Ang Ⅱ)]) of ED, and endothelial morphology of the thoracic aorta were evaluated and compared between groups. Finally, the PI3K/AKT/eNOS pathway was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot of the thoracic aorta of rats in each group to detect the mRNA expression of PI3K, AKT, and eNOS and the protein expression of PI3K, AKT, p-AKT, eNOS, and p-eNOS. ResultsA total of 51 chemical components were identified in FT, and 49 active components were identified in rat plasma. Thirteen major active components, 22 main targets, and the PI3K/AKT signaling pathway were screened by network pharmacology. The animal experiment results showed that FT reduced systolic blood pressure and ET-1 and Ang Ⅱ levels and increased NO levels in SHRs to varying degrees. The therapeutic effects were positively correlated with the oral dose of FT. Hematoxylin-eosin (HE) staining confirmed that FT could alleviate the pathological damage of the vascular endothelium. qRT-PCR and Western blot analysis confirmed that up-regulated expression of the PI3K/AKT/eNOS signaling pathway could improve ED. ConclusionsIn this study, the material basis of FT was comprehensively identified, and the protective effect on ED was confirmed. FT had a treatment effect on ED through multi-component, multi-target, and multi-pathways. It also played a role by up-regulating the PI3K/AKT/eNOS signaling pathway.

Factors affecting untargeted detection of doping agents in biological samples.

Doping control is essential for sports, and untargeted detection of doping agents (UDDA) is the holy grail for anti-doping strategies. The present study examined major factors impacting UDDA with metabolomic data processing, including the use of blank samples, signal-to-noise ratio thresholds, and the minimum chromatographic peak intensity. Contrary to data processing in metabolomics studies, both blank sample use (either blank solvent or plasma) and marking of background compounds were found to be unnecessary for UDDA in biological samples, the first such report to the authors' knowledge. The minimum peak intensity required to detect chromatographic peaks affected the limit of detection (LOD) and data processing time for untargeted detection of 57 drugs spiked into equine plasma. The ratio of the mean (ROM) of the extracted ion chromatographic peak area of a compound in the sample group (SG) to that in the control group (CG) impacted its LOD, and a small ROM value such as 2 is recommended for UDDA. Mathematical modeling of the required signal-to-noise ratio (S/N) for UDDA provided insights into the effect of the number of samples in the SG, the number of positive samples, and the ROM on the required S/N, highlighting the power of mathematics in addressing issues in analytical chemistry. The UDDA method was validated by its successful identification of untargeted doping agents in real-world post-competition equine plasma samples. This advancement in UDDA methodology will be a useful addition to the arsenal of approaches used to combat doping in sports.

The integrated analysis strategy of unstable hypoxanthine, a potential quality marker in Shuxuetong injection based on standard addition method and multi-level pharmacokinetics by LC-MS/MS

Objective: As an injection made from traditional Chinese medicine, Shuxuetong (SXT) injection is used for the treatment of ischemic stroke. Hypoxanthine is regarded as one of its potential quality markers. The purpose of this study is to lay the foundation for the quality control of SXT injection by the analysis of the quantitation and pharmacokinetic behavior for hypoxanthine. Methods: A quantitative method of hypoxanthine in SXT injection based on standard addition method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established for the first time. On the other hand, a determination method of hypoxanthine in rat plasma samples after administration of SXT was also successfully established based on LC-MS/MS. Results: It was found that the content of hypoxanthine was higher using conventional liquid-mass spectrometry technology compared to the application of LC-MS/MS combined standard addition method in the same batch of SXT injection. The ratio of low, medium and high dose of intravenous SXT were 1: 2: 4, and the AUC0-t was 848.34 ± 324.53, 1483.94 ± 497.74, and 3074.84 ± 910.29 μg·h/L, respectively. AUC0-t shows good linear dose-dependent relationship. Conclusions: The influences of endogenous substance tend to be eliminated through calibrating the concentration level of the target compound by the introduction of standard addition method. The added allopurinol could inhibit the conversion of the target compound, and ensure the accuracy of the detection during the pharmacokinetic studies. “Blank biological matrix” obtained from pretreatment of blank plasma successfully distinguished endogenous and drug-derived hypoxanthine. There is a good linear relationship between the blood concentration of intravenous hypoxanthine and the dosage of administration. Similarly, there was no drug accumulation in the multiple medium-dosage group, which is similar to the pharmacokinetic characteristics of the single medium-dosage group.

Development of polyanion-metal ion solution systems to overcome phospholipids-related matrix effects in LC-MS/MS-based bioanalysis

Phospholipids-related matrix effects are a major source impacting the reproducibility of analyte quantification in LC-MS/MS-based bioanalysis. This study intended to evaluate different combinations of polyanion-metal ion based solution system for phospholipids removal and elimination of matrix effects in human plasma. Blank plasma samples or plasma samples spiked with model analytes were proceeded with different combinations of polyanions (dextran sulfate sodium (DSS) and alkalized colloidal silica (Ludox)) and metal ions (MnCl2, LaCl3, and ZrOCl2) followed with acetonitrile-based protein precipitation. The representative classes of phospholipids and model analytes (acid, neutral, and base) were detected using multiple reaction monitoring mode. The polyanion-metal ion systems were explored for providing balanced analyte recovery and phospholipids removal by optimizing reagent concentrations or adding formic acid and citric acid as the shielding modifiers. The optimized polyanion-metal ion systems were further evaluated for eliminating matrix effects of non-polar and polar compounds. Any combinations of polyanions (DSS and Ludox) and metal ions (LaCl3 and ZrOCl2) could completely remove phospholipids at best-case scenario, while the analyte recovery is low for compounds with special chelation groups. Addition of formic acid or citric acid can improve analyte recovery but significantly decrease the removal efficiency of phospholipids. Optimized ZrOCl2-Ludox/DSS systems provided efficient phospholipids removal (>85%) and adequate analyte recovery, and the systems also correctly eliminated ion suppression or enhancement of the non-polar and polar drugs. The developed ZrOCl2-Ludox/DSS systems are cost-effective and versatile for balanced phospholipids removal and analyte recovery and provide adequate elimination of matrix effects.

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes.

There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.

Comparison of the pharmacokinetics of timosaponin AIII, timosaponin BIII, and mangiferin extracted from crude and salt-processed Anemarrhenae Rhizoma by UPLC-MS/MS.

Processing is a traditional method for preparing decoctions of traditional Chinese medicine (TCM) that is imperative for reducing toxicity, increasing efficacy, and adjusting the properties of pharmacologically active components of the TCM. Salt processing of Anemarrhenae Rhizoma (AR), a traditional Chinese herb, has been employed since the Song dynasty and can enhance the ability of AR to enriching the Yin and downbearing fire according to the traditional theory recorded in the Enlightenment on Materia Medica. Previous research found that the hypoglycemic effect of AR was enhanced after salt processing, and the concentrations of three components, namely timosaponin AIII, timosaponin BIII, and mangiferin, all of which have hypoglycemic activities, have been found to be significantly increased after salt processing. In this study, we established an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to ultimately measure the concentrations of timosaponin AIII, timosaponin BIII, and mangiferin in rat plasma after administration of unprocessed AR and salt-processed AR (SAR) to the rats to further elucidate how salt processing affects the pharmacokinetic profiles of each of these compounds. Separation was achieved on an Acquity UPLC HSS T3 column. The 0.1% formic acid aqueous solution (v/v) and acetonitrile were used as the mobile phase system. Calibration curves of each compound in blank rat plasma, as well as the accuracy, precision, stability, and recovery of the total three analytes, were then measured to validate the method. The C max and AUC0-t values of timosaponin BIII and mangiferin in the SAR group were significantly higher than those of the AR group, while the T max values of timosaponin BIII and mangiferin in the SAR group were shorter than in the AR group. These results indicated that salt processing improved the absorption and bioavailability of timosaponin BIII and mangiferin in Anemarrhenae Rhizoma, and they provide a rationale for how the salt processing enhances the hypoglycemic effect of Anemarrhenae Rhizoma.

LC-MS/MS determination of HY072808, a novel candidate for treating atopic dermatitis, and its active metabolite: Application to a first-in-human pharmacokinetic study.

HY072808 is a novel phosphodiesterase 4 inhibitor currently under clinical development to treat atopic dermatitis. The first step is to address the pharmacokinetics and safety after topical administration of HY072808 ointments in healthy humans. In this study, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method to determine plasma HY072808 and its active metabolite, ZZ24, in tiny amounts. The plasma samples were prepared using a simple liquid-liquid extraction method. Liquid chromatographic separation was achieved by gradient elution. The MS/MS quantification was performed in positive ion mode via multiple reaction monitoring. The method showed satisfactory linearity from 10 to 4,000 pg/ml for HY072808 and ZZ24. There was no significant interference from blank plasma. The method was validated for accuracy and precision, matrix effect and extraction recovery, dilution integrity, injection carryover and stability according to the related guidelines of the regulatory authorities. The HY072808 and ZZ24 concentrations in human plasma from a clinical trial were determined using this method. In conclusion, the validated method was robust and could be utilized to support the clinical development of HY072808.