Pharmacokinetic/Pharmacodynamic Assessment of the Structural Refinement of Clopidogrel Focusing on the Balance between Bioactivation and Deactivation.

The delicate balance between ischemic and bleeding risks is a critical factor in antiplatelet therapy administration. Clopidogrel and prasugrel, belonging to the thienopyridine class of antiplatelet drugs, are known for their variability in individual responsiveness and high incidence of bleeding events, respectively. The present study is centered on the development and assessment of a range of deuterated thienopyridine derivatives, leveraging insights from structure-pharmacokinetic relationships of clopidogrel and prasugrel. Our approaches were grounded in the molecular framework of clopidogrel and incorporated the C2-pharmacophore design from prasugrel. The selection of ester or carbamate substituents at the C2-position facilitated the generation of the 2-oxointermediate through hydrolysis, akin to prasugrel, thereby bypassing the issue of CYP2C19 dependency. The bulky C2-pharmacophore in our approach distinguishes itself from prasugrel's acetyloxy substituent by exhibiting a moderated hydrolysis rate, resulting in a more gradual formation of the active metabolite. Excessive and rapid release of the active metabolite, believed to be linked with an elevated risk of bleeding, is thus mitigated. Our proposed structural modification retains the hydrolysis-sensitive methyl ester of clopidogrel but substitutes it with a deuterated methyl group, shown to effectively reduce metabolic deactivation. Three promising compounds demonstrated a pharmacokinetic profile similar to that of clopidogrel at four times the dose, while also augmenting its antiplatelet activity. SIGNIFICANCE STATEMENT: Inspired by the structure-pharmacokinetic relationship of clopidogrel and prasugrel, a range of clopidogrel derivatives were designed, synthesized, and assessed. Among them, three promising compounds have been identified, striking a delicate balance between efficacy and safety for antiplatelet therapy. Additionally, the ozagrel prodrug conjugate was discovered to exert a synergistic therapeutic effect alongside clopidogrel.

Bioanalytical Assays for Pharmacokinetic and Biodistribution Study of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are produced by the chemical linkage of cytotoxic agents and monoclonal antibodies. The complexity and heterogeneity of ADCs and the low concentration of cytotoxic agent released in vivo poses big challenges to their bioanalysis. Understanding the pharmacokinetic behavior, exposure-safety, and exposure-efficacy relationships of ADCs is needed for their successful development. Accurate analytical methods are required to evaluate intact ADCs, total antibody, released small molecule cytotoxins, and related metabolites. The selection of appropriate bioanalysis methods for comprehensive analysis of ADCs is mainly dependent on the properties of cytotoxic agents, the chemical linker, and the attachment sites. The quality of the information about the whole pharmacokinetic profile of ADCs has been improved due to the development and improvement of analytical strategies for detection of ADCs, such as ligand-binding assays and mass spectrometry-related techniques. In this article, we will focus on the bioanalytical assays that have been used in the pharmacokinetic study of ADCs and discuss their advantages, current limitations, and potential challenges. SIGNIFICANCE STATEMENT: This article describes bioanalysis methods which have been used in pharmacokinetic study of ADCs and discusses the advantages, disadvantages and potential challenges of these assays. This review is useful and helpful and will provide insights and reference for bioanalysis and development of ADCs.

Differential Mobility Spectrometry-Tandem Mass Spectrometry with Multiple Ion Monitoring Coupled with in Source-Collision Induced Dissociation: A New Strategy for the Quantitative Analysis of Pharmaceutical Polymer Excipients in Rat Plasma.

Polylactic acids (PLAs) are synthetic polymers composed of repeating lactic acid subunits. For their good biocompatibility, PLAs have been approved and widely applied as pharmaceutical excipients and scaffold materials. Liquid chromatography-tandem mass spectrometry is a powerful analytical tool not only for pharmaceutical ingredients but also for pharmaceutical excipients. However, the characterization of PLAs presents particular problems for mass spectrometry techniques. In addition to their high molecular weights and wide polydispersity, multiple charging and various adductions are intrinsic features of electrospray ionization. In the present study, a strategy combining of differential mobility spectrometry (DMS), multiple ion monitoring (MIM) and in-source collision-induced dissociation (in source-CID) has been developed and applied to the characterization and quantitation of PLAs in rat plasma. First, PLAs will be fragmented into characteristic fragment ions under high declustering potential in the ionization source. The specific fragment ions are then screened twice by quadrupoles to ensure a high signal intensity and low interference for mass spectrometry detection. Subsequently, DMS technique has been applied to further reduce the background noise. The appropriately chosen surrogate specific precursor ions could be utilized for the qualitative and quantitative analysis of PLAs, which provided results with the advantages of low endogenous interference, sufficient sensitivity and selectivity for bioassay. The linearity of the method was evaluated over the concentration range 3-100 µg/mL (r2 = 0.996) for PLA 20,000. The LC-DMS-MIM coupled with in source-CID strategy may contribute to the pharmaceutical studies of PLAs and the possible prospects of other pharmaceutical excipients.

The In Vivo Pharmacokinetics of Block Copolymers Containing Polyethylene Glycol Used in Nanocarrier Drug Delivery Systems.

Polyethylene glycol (PEG) is one of the most commonly used synthetic macromolecular polymers for modifying small molecule drugs, peptides, proteins, or nanodrug delivery systems to improve their water solubility, biocompatibility, and stability. Block copolymers containing PEG have been widely used in nanodrug delivery systems such as solid lipid nanoparticles, polymeric nanoparticles, polymeric micelles, and liposomes. To date, although numerous PEGylated nanodrug delivery systems have been developed, only a few have been approved for clinical application. Poor safety and effectivity are important reasons for the high failure rate of nanodrug delivery system clinical trials. These factors are not only related to the loaded drugs and released drugs but are also related to the nanocarriers. Therefore, investigating the in vivo spatiotemporal fate of block copolymers containing PEG used in nanodrug delivery systems is necessary and important for evaluating their safety, efficacy, and toxicity. In this article, we will review the information that has been reported about the absorption, distribution, metabolism, and excretion of block copolymers containing PEG. We believe this review is helpful to understand the biologic fate of block copolymers containing PEG.This review describes pharmacokinetic study of block copolymers containing polyethylene glycol. The main focus of this paper is the in vivo fate of these polyethylene glycol-related copolymers after their release from nanocarriers. This review is helpful for understanding of the in vivo fate of block copolymers containing polyethylene glycol used in nanocarrier drug delivery systems.

Antibiotics-induced disruption of gut microbiota increases systemic exposure of clopidogrel active metabolite in type 2 diabetic rats.

Gut microbiota play an important role in the pathophysiology of type 2 diabetic mellitus (T2DM) and biodisposition of drugs. Our previous study demonstrated that T2DM rats had the decreased plasma exposure of clopidogrel active metabolite (Clop-AM) due to upregulation of P-glycoprotein (P-gp). However, whether the change to clopidogrel (Clop) disposition under T2DM condition is associated with gut microbiota needs to be elucidated. In the study, we used an antibiotic cocktail consisting of ampicillin, vancomycin, metronidazole, and neomycin to disrupt gut microbiota and observed their influence on pharmacokinetic profiles of Clop-AM. Antibiotic administration markedly alleviated T2DM rats' phenotype including hyperglycemia, insulin resistance, oxidative stress, inflammation, hyperlipidemia, and liver dysfunction. Meanwhile, treatment with antibiotics significantly reversed the reduced systemic exposure of Clop-AM in T2DM rats relative to control rats, which was associated with the decreased intestinal P-gp level that might promote Clop absorption, resulting in more Clop transformation to Clop-AM. Fecal microbiome analysis exhibited a serious disruption of gut microbiota after antibiotic treatment with the sharply reduced microbial load and the altered microbial composition. Interestingly, an in vitro study showed that antibiotics had no influence on P-gp mRNA leve in SW480 cells, suggesting the microbiome disruption, not the direct role of antibiotics on P-gp expression, contributes to the altered P-gp level and Clop disposition in T2DM rats. The findings add new insights into the potential impact of gut microbiota on Clop biodisposition. Significance Statement 1.Antibiotics increase systemic exposure of Clop-AM in T2DM rats, which is associated with the downregulation of P-gp level.2.Antibiotics-induced disruption of gut microbiota, not direct effect of antibiotics on P-gp and CYPs expression, contributes to the altered Clop disposition.3.Antibiotics also alleviate T2DM phenotype including hyperglycemia, hyperlipidemia, insulin resistance, liver dysfunction and inflammation.

High-throughput bioanalysis of sitagliptin in plasma using direct analysis in real time mass spectrometry and its application in the pharmacokinetic study thereof.

Sitagliptin is a dipeptidyl peptidase-IV inhibitor for the treatment of type 2 diabetes mellitus. In the present study, a sensitive and high-throughput quantitative method based on the direct analysis in real time tandem mass spectrometry has been developed and validated for the bioanalysis of sitagliptin in rat plasma without chromatographic separation. Sitagliptin and its internal standard retagliptin were detected in positive ion mode by multiple reaction monitoring transitions at m/z 408.2→235.0 and 465.2→260.1, respectively. The method includes a simple solid-phase extraction sample preparation procedure, through which appropriate and reproducible analytical results within the linear concentration range of 20-2000 ng/mL have been achieved. The intra- and interday precisions were <10.6% and the accuracies were ranging from -8.17 to 2.60%. This method has been successfully applied to the pharmacokinetic study of sitagliptin after single intravenous administration in rats. This approach shows considerable promise of direct analysis in real time tandem mass spectrometry method in the high-throughput bioanalysis.

High-throughput and trace analysis of diazepam in plasma using DART-MS/MS and its pharmacokinetic application.

A high-throughput quantitative analytical method based on Direct Analysis in Real Time tandem mass spectrometry (DART-MS/MS) has been developed and validated for the determination of diazepam in rat plasma, whereby analyzing of each sample needs merely 25 µL plasma, simple solid phase extraction sample preparation and 15 s acquisition time. The multiple reaction monitoring (MRM) transitions at m/z 285.2 â†’ 193.1 and 316.0 â†’ 270.0 were selected for the monitoring of diazepam and its internal standard clonazepam respectively. A good linearity within the range of 10-2000 ng/mL, an intra- and inter-day precisions within <7.78% as to an accuracy ranging from 1.04% to 7.92% have been achieved. The method has been successfully applied to the pharmacokinetic study of diazepam in rats' plasma after a single intragastric administration at a dose of 10 mg/kg. The results indicate that this method fulfills the requirements of the bioanalysis in sensitivity and accuracy. It shows considerable promise for application of DART-MS to the quantitative investigation of other drugs.

Comprehensive Bioanalysis of Ultrahigh Molecular Weight, Highly Disperse Poly(ethylene oxide) in Rat via Microsolid Phase Extraction and RPLC-Q-Q-TOF Coupled with the MSALL Technique.

Ultrahigh molecular weight (UHMW) poly(ethylene oxide) (PEO) is a synthetic hydrophilic polymer with wide dispersity which shows considerable promise as a hemostatic agent in the treatment of gastrointestinal bleeding. Currently there is no analytical method for the determination of highly disperse UHMW PEO in biological samples that would allow its characterization in vivo and support its clinical development. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful bioanalytical tool, it faces major challenges when applied to UHMW PEO. In this work, we report a novel bioanalytical method for the determination of UHMW PEO involving microsolid phase extraction (µ-SPE), chromatography on a PLRP-S 1000 Å reversed phase column and detection by positive ion Q-Q-TOF MS using the MSALL technique. In this mode, dissociation of all precursor ions in Q2 generated a series of product ions at m/z 89.0715, 133.0854, 177.1047, and 221.1475 of which the product ion at m/z 133.0854 was common to all precursor ions and enabled quantitation of all polymers in UHMW PEO. The method was successfully applied to the determination of UHMW PEO polymers in rat plasma, urine, and feces after oral administration of 1700 kDa PEO. The results show that UHMW PEO is not absorbed into the blood and is largely eliminated unchanged in feces over 48 h. We maintain the method is sufficiently robust to be used in routine bioanalysis of polymers with UHMW and wide dispersity.

DDR2, a discoidin domain receptor, is a marker of periosteal osteoblast and osteoblast progenitors.

INTRODUCTION: The periosteum has a bilayered structure that surrounds cortical bone. The outer layer is rich in connective tissue and fibroblasts, while the inner layer in contact with the cortical surface of the bone predominantly consists of osteoblasts and osteoblast progenitors. The identification of cell-specific surface markers of the bilayered structure of the periosteum is important for the purpose of tissue regeneration. MATERIALS AND METHODS: We investigated the expression of the discoidin domain tyrosine kinase receptor DDR2, fibroblast specific protein-1 (FSP-1) and alkaline phosphatase (ALP) in the periosteum of cortical bone by immunohistochemistry. Osteogenic differentiation was compared between DDR2- and FSP-1-expressing cells flow-sorted from the periosteum. RESULTS: We showed that DDR2 predominantly labeled osteogenic cells residing in the inner layer of the periosteum and that Pearson's coefficient of colocalization indicated a significant correlation with the expression of ALP. The mineralization of DDR2-expressing osteogenic cells isolated from the periosteum was significantly induced. In contrast, FSP-1 predominantly labeled the outer layer of periosteal fibroblasts, and Pearson's coefficient of colocalization indicated that FSP-1 was poorly correlated with the expression of DDR2 and ALP. FSP-1-expressing periosteal fibroblasts did not exhibit osteogenic differentiation for the induction of bone mineralization. CONCLUSION: DDR2 is a novel potential cell surface marker for identifying and isolating osteoblasts and osteoblast progenitors within the periosteum that can be used for musculoskeletal regenerative therapies.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals.

Polyethylene glycols are synthetic polymers composed of repeating oxyethylene subunits, which have been known for non-toxic, non-immunogenic, non-antigenic, good solubility in water and therefore approved for pharmaceutical applications. Recently, attachment or amalgamation of polyethylene glycols to therapeutic small molecules, peptides, proteins, or nanoparticles has become a mature technology for the sake of improving their pharmacokinetic and pharmacological profiles, also referred to as PEGylation. By comparison, there are only a few PEGylated pharmaceuticals have been registered for further clinical trials and even less was approved for marketing. High failure rate of PEGylated pharmaceuticals in pre-clinical and clinical trials could be majorly attributed to their unclear pharmacokinetic behaviors. Therefore, the in vivo fate of the PEGylated pharmaceuticals for the various routes of administration needs to be thoroughly investigated An accurate in vivo pharmacological study thereof highly depends on the precise detection of polyethylene glycols as well as their fragments in biological matrixes. The goal of this review is to highlight the analytical methods that were developed and applied to evaluate the polyethylene glycols in pharmaceutical ingredients and excipients, which bring us closer to bridging the gap between the development of polyethylene glycol-based drug delivery systems and their clinical application.

Simultaneous determination of ferulic acid, paeoniflorin, and albiflorin in rat plasma by ultra-high performance liquid chromatography with tandem mass spectrometry: Application to a pharmacokinetic study of Danggui-Shaoyao-San.

A rapid, selective, and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of ferulic acid, paeoniflorin, and albiflorin, the major active constituents of Danggui-Shaoyao-San, in rat plasma using geniposide as the internal standard. The plasma samples were processed by protein precipitation with acetonitrile, and then separated on a Shim-Pack XR-ODS C18 column (75 mm × 3.0 mm, 2.2 µm) using gradient elution program with a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.4 mL/min. The detection was achieved on a 3200 QTRAP mass spectrometer equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reaction monitoring mode by monitoring the fragmentation of m/z 192.9→134.0 for ferulic acid, m/z 525.0→120.9 for paeoniflorin, m/z 525.2→121.0 for albiflorin, and m/z 433.1→225.1 for the internal standard, respectively. The calibration curve was linear in the range of 5-2500 ng/mL for all the three analytes (r ≥ 0.9972) with the lower limit of quantitation of 5 ng/mL. The intraday and interday precisions were below 12.1% for all the analytes in terms of relative standard deviation, and the accuracy was within ±11.5% in terms of relative error. The extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of ferulic acid, paeoniflorin, and albiflorin after oral administration of Danggui-Shaoyao-San to rats.

Simultaneous determination of eight bioactive constituents of Zhi-Zi-Hou-Po decoction in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Zhi-Zi-Hou-Po Decoction, consisting of Gardenia jasminoides Ellis, Magnolia officinalis Rehd. et Wils., and Citrus aurantium L, is a classical Traditional Chinese Medicine formula for the treatment of depression. In order to make good and rational use of this formula in the future, a sensitive, selective, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of two iridoid glycosides (geniposide and genipin gentiobioside), two lignans (honokiol and magnolol), four flavonoid glycosides (isonaringin, naringin, hesperidin, and neohesperidin), the major bioactive constituents of Zhi-Zi-Hou-Po Decoction, in rat plasma using paeoniflorin as internal standard. Plasma samples were pretreated by a simple protein precipitation with acetonitrile. Chromatographic separation was performed on a shim-pack XR-ODS C18 column (75 × 3.0 mm, 2.2 µm) using gradient elution with mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Mass spectrometric detection was conducted on a 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reactions monitoring mode. Calibration curves exhibited good linearity (r > 0.9947) over a wide concentration range for all analytes, and the lower limits of quantification were 10, 5, 1, 5, 1, 5, 1, and 5 ng/mL for geniposide, genipin gentiobioside, honokiol, magnolol, isonaringin, naringin, hesperidin, and neohesperidin, respectively. The intraday and interday precisions at three quality control levels were less than 12.3% and the accuracies ranged from -11.2 to 10.7%. Extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of the eight analytes after oral administration of Zhi-Zi-Hou-Po decoction to rats.

Enhanced Platelet Response to Clopidogrel in Zucker Diabetic Fatty Rats due to Impaired Clopidogrel Inactivation by Carboxylesterase 1 and Increased Exposure to Active Metabolite.

Clopidogrel (Clop), a thienopyridine antiplatelet prodrug, is metabolized by cytochrome P450s (CYPs) to an active metabolite, Clop-AM, and hydrolyzed by carboxylesterase (CES)1 to the inactive Clop-acid. Patients with type 2 diabetes (T2DM) tend to have a poor response to Clop due to reduced generation of Clop-AM. Whether a similar response occurs in the Zucker diabetic fatty (ZDF) rat, a commonly used animal model of T2DM, has not been explored. In this work, we compared ZDF and control rats for hepatic CES1- and CYP-mediated Clop metabolism; pharmacokinetics of Clop, Clop-AM, and Clop-acid; and the antiplatelet efficacy of Clop. In contrast to clinical findings, Clop-treated ZDF rats displayed significantly less (50%) maximum platelet aggregation at 4 hours than control rats; the enhanced efficacy was accompanied by higher formation of Clop-AM and lower formation of Clop-acid. In vitro studies showed that hepatic levels of CES1 protein and activity and Ces1e mRNA were significantly lower in ZDF than in control rats, as were the mRNA levels of CYP2B1/2, CYP2C11, and CYP3A2, and levels of CYP2B6-, CYP2C19-, and CYP3A4-related proteins and enzymatic activities in liver microsomes of ZDF rats. Interestingly, liver microsomes of ZDF rats produced higher levels of Clop-AM than that of control rats despite their lower CYP levels, although the addition of fluoride ion, an esterase inhibitor, enhanced Clop-AM formation in control rats more than in ZDF rats. These results suggest that the reduction in CES1-based Clop inactivation indirectly enhances Clop efficacy in ZDF rats by making more Clop available for CYP-mediated Clop-AM formation.

Recent advances in single-cell analysis by mass spectrometry.

Cells are the most basic structural units that play vital roles in the functioning of living organisms. Analysis of the chemical composition and content of a single cell plays a vital role in ensuring precise investigations of cellular metabolism, and is a crucial aspect of lipidomic and proteomic studies. In addition, structural knowledge provides a better understanding of cell behavior as well as the cellular and subcellular mechanisms. However, single-cell analysis can be very challenging due to the very small size of each cell as well as the large variety and extremely low concentrations of substances found in individual cells. On account of its high sensitivity and selectivity, mass spectrometry holds great promise as an effective technique for single-cell analysis. Numerous mass spectrometric techniques have been developed to elucidate the molecular profiles at the cellular level, including electrospray ionization mass spectrometry (ESI-MS), secondary ion mass spectrometry (SIMS), laser-based mass spectrometry and inductively coupled plasma mass spectrometry (ICP-MS). In this review, the recent advances in single-cell analysis by mass spectrometry are summarized. The strategies of different ionization modes to achieve single-cell analysis are classified and discussed in detail.

Ultrahigh-throughput absolute quantitative analysis of linezolid in human plasma by direct analysis in real time mass spectrometry without chromatographic separation and its application to a pharmacokinetic study.

Therapeutic drug monitoring (TDM) is necessary in the clinical management of linezolid to improve its efficacy and reduce the risk of time- and dose-dependent toxicity. A novel and ultrahigh-throughput analytical method for the determination of linezolid in human plasma was developed based on direct analysis in real-time tandem mass spectrometry (DART-MS/MS) without chromatographic separation. After solid-phase extraction with Waters Oasis HLB, the linezolid and internal standard linezolid-d3 were detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 338.1 → 296.2 and 341.2 → 297.3, respectively. The use of DART-MS obviates the need for chromatographic separation and allowed determination of linezolid in a total run time of only 24 s per sample. The method was linear in the concentration range 0.20-25 µg mL-1 with intraday and interday precision <14.5% and accuracy ranging from -3.85% to 12.7%. The method was successfully applied to a pharmacokinetic study of linezolid in healthy male volunteers after oral administration of a 600 mg tablet. DART-MS/MS provides a rapid and sensitive method for the determination of linezolid that does not require chromatographic separation. It is eminently suitable to meet the high-throughput challenge of clinical TDM. Graphical abstract.

Liquid chromatography tandem mass spectrometry with triple stage fragmentation for highly selective analysis and pharmacokinetics of alarelin in rat plasma.

Alarelin, a gonadotropin-releasing hormone analogue, is widely used in China for the treatment of endometriosis and uterine leiomyoma. In order to investigate its pharmacokinetic behavior and support the preclinical application of new formulations, we have developed a novel and highly selective bioanalytical method to determine alarelin in rat plasma based on liquid chromatography tandem mass spectrometry with triple stage fragmentation. After sample preparation by protein precipitation followed by reversed phase solid phase extraction, alarelin and triptorelin (internal standard) were chromatographed on an Ascentis® Express C18 column (50 mm × 4.6 mm, 2.7 µm) using gradient elution with 0.1% formic acid in water and acetonitrile at a flow rate of 1 mL/min. Detection was by positive mode electrospray ionization followed by triple stage fragmentation using the transitions at m/z 584.6→249.1→221.0 for alarelin and 656.5→249.1→176.0 for triptorelin, The assay was linear in the concentration range 0.3-10 ng/mL with excellent precision and accuracy. It was successfully applied to a pharmacokinetic study in rats administered a dose of 13.5 µg/kg alarelin by intramuscular injection. The results show that the triple stage fragmentation strategy allows highly selective analysis of alarelin and has the potential to be widely applied to the bioassay of other peptidic drugs.

The Effect of Molecular Structure on Cytotoxicity and Antitumor Activity of PEGylated Nanomedicines.

Fundamental studies on the cellular uptake and drug release of PEGylated nanomedicines are beneficial to understand their fate in vivo and construct ideal nanoparticle formulations. In this work, the detailed metabolic process of PEGylated doxorubicin (Dox) nanomedicines were investigated via confocal laser scanning microscopy (CLSM), flow cytometry (FCM), cytotoxicity test, fluorescence imaging in vivo (FLIV) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Among them, only LC-MS/MS could accurately determine the content of PEGylated Dox and Dox in vitro and in vivo. To the best of our knowledge, this was the first time the PEGylated Dox and released Dox were simultaneously quantified. The interplay of molecular structures, cellular uptake, drug release, and antitumor effect was well characterized. PEG with high molecular weight impeded the cellular uptake of nanoparticles, and the acid-labile hydrazone bond between Dox and PEG promoted Dox release significantly. Cellular uptake and drug release play decisive roles in cytotoxicity and antitumor effect, as evidenced by LC-MS/MS. We emphasized that LC-MS/MS would be a practicable method to quantify PEGylated drugs without complex tags, which could be more in-depth to understand the interaction between PEGylated nanomedicines and their antitumor efficacy.

[Pharmacokinetics of Phosphate Retagliptin Tabletin in Patients with Renal Dysfunction].

OBJECTIVE: To compared the differences in pharmacokinetics of phosphate retagliptin tablets in patients with varying degrees of renal dysfunction. METHODS: A total of 32 patients were categorized into five groups according to their renal function: normal,mild dysfunction, moderate dysfunction,severe dysfunction,and end stage renal dysfunction (ESRD). All of the patients took a single dose of 50 mg phosphate retagliptin tablet. Their plasma and urinary concentrations of phosphate retagliptin (SP2086) and phosphate retagliptin acid (SP2086 acid) were determined using LC-MS/MS methods. The plasma pharmacokinetic parameters were calculated using WinNolin 6.1 software. RESULTS: Peak concentrations (Cmax) of SP2086 reached at (1.07±0.35) h in the patients with mild renal dysfunction,(1.50±0.89) h in the patients with moderate renal dysfunction,(1.67±2.16) h in the patients with severe renal dysfunction,(2.42±2.15) h in the patients with ESRD,and (1.75±1.21) h in the normal participants,with a clearance (CL/F) of (23.50±6.01) ,(12.90±4.34) ,(6.70±1.55) ,(3.10±0.48) ,and (30.50±10.70) L/h,respectively. With the increasing damages in renal function presented an incease in Cmax,time to reach Cmax (Tmax),and area under curve (AUC), a decrease in CL/F, of SP2086 and SP2086 acid. The 0-96 hurine cumulative excretion percentage (Ae%) of SP2086 ranged from 0.441% to 4.530%. The Ae% of SP2086 acid reached (71.7±14.3) % in the patients with mild renal dysfunction, (59.5±22.7) % in the patients with moderate renal dysfunction, (63.3±13.9) % in the patients with severe renal dysfunction, (34.1±20.0) % in the patient with ESRD,and (74.2±14.6) % in the normal participants, with a renal clearance (CL/R) of (220.0±51.2),(105.0±64.5),(54.5±7.6),(13.5±7.8),and (289.0±73.7) mL/min,respectively. Compared with the participants with normal renal function,the AUCs of SP2086 and SP2086 acid were 1.44 times and 2.32 times higher in the patients with moderate renal dysfunction,2.20 times and 4.39 times higher in the patients with severe renal dysfunction, and 2.83 times and 9.28 times higher in the patients with ESRD. CONCLUSION: The dosage of phosphate retagliptin tablet is recommended at 100 mg/d for patients with normal renal function and those with mild renal dysfunction,at 50 mg/d for patients with moderate renal dysfunction,and at 25 mg/d for patients with severe renal dysfunction. No phosphate retagliptin tablet is recommended for patients with ESRD.

Development and Application of an MSALL-Based Approach for the Quantitative Analysis of Linear Polyethylene Glycols in Rat Plasma by Liquid Chromatography Triple-Quadrupole/Time-of-Flight Mass Spectrometry.

Polyethylene glycols (PEGs) are synthetic polymers composed of repeating ethylene oxide subunits. They display excellent biocompatibility and are widely used as pharmaceutical excipients. To fully understand the biological fate of PEGs requires accurate and sensitive analytical methods for their quantitation. Application of conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is difficult because PEGs have polydisperse molecular weights (MWs) and tend to produce multicharged ions in-source resulting in innumerable precursor ions. As a result, multiple reaction monitoring (MRM) fails to scan all ion pairs so that information on the fate of unselected ions is missed. This Article addresses this problem by application of liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry (LC-Q-TOF MS) based on the MSALL technique. This technique performs information-independent acquisition by allowing all PEG precursor ions to enter the collision cell (Q2). In-quadrupole collision-induced dissociation (CID) in Q2 then effectively generates several fragments from all PEGs due to the high collision energy (CE). A particular PEG product ion (m/z 133.08592) was found to be common to all linear PEGs and allowed their total quantitation in rat plasma with high sensitivity, excellent linearity and reproducibility. Assay validation showed the method was linear for all linear PEGs over the concentration range 0.05-5.0 µg/mL. The assay was successfully applied to the pharmacokinetic study in rat involving intravenous administration of linear PEG 600, PEG 4000, and PEG 20000. It is anticipated the method will have wide ranging applications and stimulate the development of assays for other pharmaceutical polymers in the future.