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FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography–tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002–10 μg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at −20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5–5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies. [ABSTRACT FROM AUTHOR]

Lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix) is an ethnopharmacological plant commonly known as English lavender. Linalool and linalyl acetate are putative phytoactives in lavender essential oil (LEO) derived from the flower heads. LEO has been used in aroma or massage therapy to reduce sleep disturbance and to mitigate anxiety. Recently, an oral LEO formulation was administered in human clinical trials designed to ascertain its anxiolytic effect. However, human pharmacokinetics and an LC–MS/MS method for the measurement of linalool are lacking. To address this deficiency, a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the analysis of linalool in human serum. Prior to the analysis, a simple sample preparation protocol including protein precipitation and liquid–liquid extraction of serum samples was created. The prepared samples were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to quantitatively determine linalool and IS according to transitions of m/z 137.1→95.1 (tR 0.79 min) and 205.2→149.1 (tR 1.56 min), respectively. The method was validated for precision, accuracy, selectivity, linearity, sensitivity, matrix effects, and stability, and it was successfully applied to characterize the oral pharmacokinetics of linalool in humans. The newly developed LC–MS/MS-based method and its application in clinical trial serum samples are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions. [ABSTRACT FROM AUTHOR]

Cissus quadrangularis is a nutrient-rich plant with a history of use in traditional medicine. It boasts a diverse range of polyphenols, including quercetin, resveratrol, β-sitosterol, myricetin, and other compounds. We developed and validated a sensitive LC-MS/MS method to quantify quercetin and t-res biomarkers in rat serum and applied this method to pharmacokinetic and stability studies. The mass spectrometer was set to negative ionization mode for the quantification of quercetin and t-res. Phenomenex Luna (C18(2), 100 A, 75 × 4.6 mm, 3 µ) column was utilized to separate the analytes using an isocratic mobile phase consisting of methanol and 0.1% formic acid in water (82:18). Validation of the method was performed using various parameters, including linearity, specificity, accuracy, stability, intra-day, inter-day precision, and the matrix effect. There was no observed significant endogenous interference from the blank serum. The analysis was completed within 5.0 min for each run, and the lower limit of quantification was 5 ng/mL. The calibration curves showed a linear range with a high correlation coefficient (r2 > 0.99). The precision for intra- and inter-day assays showed relative standard deviations from 3.32% to 8.86% and 4.35% to 9.61%, respectively. The analytes in rat serum were stable during bench-top, freeze-thaw, and autosampler (−4 °C) stability studies. After oral administration, the analytes showed rapid absorption but underwent metabolism in rat liver microsomes despite being stable in simulated gastric and intestinal fluids. Intragastric administration resulted in higher absorption of quercetin and t-res, with greater Cmax, shorter half-life, and improved elimination. No prior research has been conducted on the oral pharmacokinetics and stability of anti-diabetic compounds in the Ethanolic extract of Cissus quadrangularis EECQ, making this the first report. Our findings can provide the knowledge of EECQ's bioanalysis and pharmacokinetic properties which is useful for future clinical trials. [ABSTRACT FROM AUTHOR]

XYY-CP1106, a candidate compound synthesized from a hybrid of hydroxypyridinone and coumarin, has been shown to be remarkably effective in treating Alzheimer's disease. A simple, rapid and accurate high-performance liquid chromatography coupled with the triple quadrupole mass spectrometer (LC-MS/MS) method was established in this study to elucidate the pharmacokinetics of XYY-CP1106 after oral and intravenous administration in rats. XYY-CP1106 was shown to be rapidly absorbed into the blood (Tmax, 0.57–0.93 h) and then eliminated slowly (T1/2, 8.26–10.06 h). Oral bioavailability of XYY-CP1106 was (10.70 ± 1.72)%. XYY-CP1106 could pass through the blood–brain barrier with a high content of (500.52 ± 260.12) ng/g at 2 h in brain tissue. The excretion results showed that XYY-CP1106 was mainly excreted through feces, with an average total excretion rate of (31.14 ± 0.05)% in 72 h. In conclusion, the absorption, distribution and excretion of XYY-CP1106 in rats provided a theoretical basis for subsequent preclinical studies. [ABSTRACT FROM AUTHOR]

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats. [ABSTRACT FROM AUTHOR]

LE300 is a novel dopamine receptor antagonist used to treat cocaine addiction. In the current study, a sensitive and fast liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been established and validated for the simultaneous analysis of LE300 and its N-methyl metabolite, MLE300, in rat plasma with an application in a pharmacokinetic study. The chromatographic elution of LE300, MLE300, and Ponatinib (IS, internal standard), was carried out on a 50 mm C18 analytical column (ID: 2.1 mm and particle size: 1.8 μm) maintained at 22 ± 2 °C. The run time was 5 min at a flow rate of 0.3 mL/min. The mobile phase consisted of 42% aqueous solvent (10 mM ammonium formate, pH: 4.2 with formic acid) and 58% organic solvent (acetonitrile). Plasma samples were pretreated using protein precipitation with acetonitrile. The electrospray ionization (ESI) source was used to generate an ion-utilizing positive mode. A multiple reaction monitoring mass analyzer mode was utilized for the quantification of analytes. The linearity of the calibration curves in rat plasma ranged from 1 to 200 ng/mL (r2 = 0.9997) and from 2 to 200 ng/mL (r2 = 0.9984) for LE300 and MLE300, respectively. The lower limits of detection (LLOD) were 0.3 ng/mL and 0.7 ng/mL in rat plasma for LE300 and MLE300, respectively. Accuracy (RE%) ranged from −1.71% to −0.07% and −4.18% to −1.48% (inter-day), and from −3.3% to −1.47% and −4.89% to −2.15% (intra-day) for LE300 and MLE300, respectively. The precision (RSD%) was less than 2.43% and 1.77% for the inter-day, and 2.77% and 1.73% for intra-day of LE300 and MLE300, respectively. These results are in agreement with FDA guidelines. The developed LC-MS/MS method was applied in a pharmacokinetic study in Wistar rats. Tmax and Cmax were 2 h and 151.12 ± 12.5 ng/mL for LE300, and 3 h and 170.4 ± 23.3 ng/mL for MLE300. [ABSTRACT FROM AUTHOR]

Sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-4(methylthio)butanoate (GMDTC) is the first compound to use cadmium repellent as an indication. In this paper, we established and validated a bioanalytical method for the determination of GMDTC in rat plasma, and used it to determine the drug concentrations in the plasma of rats after intravenous dosing in different genders and dosages. After pretreating the plasma samples with an acetonitrile–water–ammonia solution (70:30:1.25, v/v/v), liquid chromatographic separations were efficiently achieved with a XBridge C18 column using a 5 min gradient system of aqueous ammonium bicarbonate and 95% acetonitrile–water solution (95:5, v/v) as the eluent. The GMDTC and metolazone (internal standard, IS) detection were carried out using high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (LC–MS/MS), monitored at m/z 390.06–324.1 (for the GMDTC, tR: 2.03 min) and m/z 366.0–259.2 (for IS, tR: 3.88 min). The GMDTC was stable under various testing conditions, and this analytical method conforms to the verification standard of biological analysis methods. The half-life (t1/2) was determined to be 0.54–0.65 h for the intravenous, mean distribution volume and clearances were 1.08–2.08 L/kg and 1–3 L/h/kg, respectively. The AUC0-t and AUC0-∞ found after increasing the dosage exhibited a linear relationship with the administered dose. There were no statistically significant differences in the values obtained for the different genders at dosages of 50, 100 and 250 mg/kg, respectively (p > 0.05). This is the first report of a bioanalytical method to quantify GMDTC in rat plasma using LC–MS/MS, which provides useful information for the study of its pharmacological effects and clinical applications. [ABSTRACT FROM AUTHOR]

Benzisothiazolinone (BIT), a biocide widely used as a preservative in household cleaning and personal care products, is cytotoxic to lung cells and a known skin allergen in humans, which highlights the importance of assessing its toxicity and pharmacokinetics. In this study, a simple, sensitive, and accurate LC–MS/MS method for the quantification of BIT in rat plasma, urine, or tissue homogenates (50 μL) using phenacetin as an internal standard was developed and validated. Samples were extracted with ethyl acetate and separated using a Kinetex phenyl–hexyl column (100 × 2.1 mm, 2.6 μm) with isocratic 0.1% formic acid in methanol and distilled water over a run time of 6 min. Positive electrospray ionization with multiple reaction monitoring transitions of m/z 152.2 > 134.1 for BIT and 180.2 > 110.1 for phenacetin was used for quantification. This assay achieved good linearity in the calibration ranges of 2–2000 ng/mL (plasma and urine) and 10–1000 ng/mL (tissue homogenates), with r ≥ 0.9929. All validation parameters met the acceptance criteria. BIT pharmacokinetics was evaluated via an intravenous and dermal application. This is the first study that evaluated BIT pharmacokinetics in rats, providing insights into the relationship between BIT exposure and toxicity and a basis for future risk assessment studies in humans. [ABSTRACT FROM AUTHOR]

The combination regimen targeting BRAF and MEK inhibition, for instance, encorafenib (Braftovi™, ENF) plus binimetinib (Mektovi®, BNB), are now recommended as first-line treatment in patients with unresectable or metastatic melanoma with a BRAF V600-activating mutation. Patients treated with combination therapy of ENF and BNB demonstrated a delay in resistance development, increases in antitumor activity, and attenuation of toxicities compared with the activity of either agent alone. However, the pharmacokinetic profile of the FDA-approved ENF and BNB is still unclear. In this study, a rapid and sensitive LC-MS/MS bioanalytical method for simultaneous quantification of ENF and BNB in rat plasma was developed and validated. Chromatography was performed on an Agilent Eclipse plus C18 column (50 mm × 2.1 mm, 1.8 µm), with an isocratic mobile phase composed of 0.1% formic acid in water/acetonitrile (67:33, v/v, pH 3.2) at a flow rate of 0.35 mL/min. A positive multiple reaction monitoring (MRM) mode was chosen for detection and the process of analysis was run for 2 min. Plasma samples were pre-treated using protein precipitation with acetonitrile containing spebrutinib as the internal standard (IS). Method validation was assessed as per the FDA guidelines for the determination of ENF and BNB over concentration ranges of 0.5–3000 ng/mL (r2 ≥ 0.997) for each drug (plasma). The lower limits of detection (LLOD) for both drugs were 0.2 ng/mL. The mean relative standard deviation (RSD) of the results for accuracy and precision was ≤ 7.52%, and the overall recoveries of ENF and BNB from rat plasma were in the range of 92.88–102.28%. The newly developed approach is the first LC–MS/MS bioanalytical method that can perform simultaneous quantification of ENF and BNB in rat plasma and its application to a pharmacokinetic study. The mean result for Cmax for BNB and ENF was found to be 3.43 ± 0.46 and 16.42 ± 1.47 µg/mL achieved at 1.0 h for both drugs, respectively. The AUC0-∞ for BNB and ENF was found to be 18.16 ± 1.31 and 36.52 ± 3.92 µg/mL.h, respectively. On the other hand, the elimination half-life (t1/2kel) parameters for BNB and ENF in the rat plasma were found to be 3.39 ± 0.43 h and 2.48 ± 0.24 h, and these results are consistent with previously reported values. [ABSTRACT FROM AUTHOR]

Physalin A is a promising natural product with excellent anti-inflammatory and anti-tumor activities. However, the pharmacokinetic profile of physalin A is still unclear. In this study, a rapid and sensitive analytical method based on LC–MS/MS for the quantitation of physalin A in rat plasma with special consideration to its chemical stability was developed and validated. To avoid the degradation of physalin A, the separation of plasma was conducted at 4 °C directly after the blood samples were collected. Meanwhile, plasma samples were immediately precipitated with acetonitrile containing tolbutamide (internal standard, IS) and the pH of the supernatant was adjusted to 1.5 with formic acid. Chromatographic separation of physalin A and IS was achieved on an ACQUITY UPLC BEH-C18 column (2.1 × 50 mm, 1.7 μm) using 0.1% formic acid and acetonitrile as mobile phase delivered at 0.3 mL/min in a gradient elution mode. Physalin A and IS were detected through negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The MS/MS ion transitions for physalin A and IS were m/z 525.1–148.9 and m/z 269.8–169.9, respectively. The developed method showed good linearity over the range of 2.00–400 ng/mL. This method was successfully applied to the pharmacokinetic study of physalin A in rats following its intragastric administration and the findings were beneficial for future studies of physalin A. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the pharmacokinetics of acipimox in rats under simulated high altitude hypoxia conditions. A sensitive and reliable LC–MS/MS method has been established for the quantitation of acipimox in rat plasma and tissue homogenate and validated according to the guidelines of the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA). Western blotting and enzyme linked immunosorbent assay (ELISA) were used to investigate the expression of lipid metabolism-related proteins and free fatty acid (FFA) levels, respectively. Cell viability was detected using a Cell Counting kit-8 assay (CCK-8). The method was then successfully applied in a pharmacokinetic comparison between normoxic and hypoxic rats. The results indicated that there were significant differences in the main pharmacokinetics parameters of acipimox between normoxic and hypoxic rats. HCAR2 expression in the hypoxia group was upregulated compared to that in the normoxia group and the levels of FFA decreased more in the hypoxia group. Under the hypoxia condition, the proliferation of HK2 cells was inhibited with increasing concentrations of acipimox. The results provide important and valuable information for the safety and efficacy of acipimox, which indicated that the dosage of acipimox might be adjusted appropriately during clinical medication in hypoxia. [ABSTRACT FROM AUTHOR]

IWR-1-endo, a small molecule that potently inhibits the Wnt/β-catenin signaling pathway by stabilizing the AXIN2 destruction complex, can inhibit drug efflux at the blood–brain barrier. To conduct murine cerebral microdialysis research, validated, sensitive, and reliable liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods were used to determine IWR-1-endo concentration in the murine plasma and brain microdialysate. IWR-1-endo and the internal standard (ISTD) dabrafenib were extracted from murine plasma and microdialysate samples by a simple solid-phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation was executed on a Kinetex C18 (100A, 50 × 2.1 mm, 4 µm particle size) column with a binary gradient of water and acetonitrile, each having 0.1% formic acid, pumped at a flow rate of 0.6 mL/min. Detection by mass spectrometry was conducted in the positive selected reaction monitoring ion mode by monitoring mass transitions 410.40 > 344.10 (IWR-1-endo) and 520.40 > 307.20 (ISTD). The validated curve range of IWR-1-endo was 5–1000 ng/mL for the murine plasma method (r2 ≥ 0.99) and 0.5–500 ng/mL for the microdialysate method (r2 ≥ 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for the murine plasma and microdialysate sample analysis method, respectively. Negligible matrix effects were observed in murine plasma and microdialysate samples. IWR-1-endo was extremely unstable in murine plasma. To improve the stability of IWR-1-endo, pH adjustments of 1.5 were introduced to murine plasma and microdialysate samples before sample storage and processing. With pH adjustment of 1.5 to the murine plasma and microdialysate samples, IWR-1-endo was stable across several tested conditions such as benchtop, autosampler, freeze–thaw, and long term at −80 °C. The LC-MS/MS methods were successfully applied to a murine pharmacokinetic and cerebral microdialysis study to characterize the unbound IWR-1-endo exposure in brain extracellular fluid and plasma. [ABSTRACT FROM AUTHOR]

Urea transporter (UT) inhibitors are a class of promising novel diuretics that do not cause the imbalance of Na+, K+, Cl−, and other electrolytes. In our previous studies, 25a, a promising diuretic candidate inhibiting UT, was discovered and showed potent diuretic activities in rodents. Here, a sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of 25a in rat plasma, urine, feces, bile, and tissue homogenates was developed and validated to support the preclinical pharmacokinetic studies. The tissue distribution, excretion, and plasma protein binding were investigated in rats. After a single oral dose of 25a at 25, 50, and 100 mg/kg, the drug exposure increased linearly with the dose. The drug accumulation was observed after multiple oral doses compared to a single dose. In the distribution study, 25a exhibited a wide distribution to tissues with high blood perfusion, such as kidney, heart, lung, and spleen, and the lowest distribution in the brain and testis. The accumulative excretion rate of 25a was 0.14%, 3.16%, and 0.018% in urine, feces, and bile, respectively. The plasma protein binding of 25a was approximately 60% in rats and 40% in humans. This is the first study on the preclinical pharmacokinetic profiles of 25a. [ABSTRACT FROM AUTHOR]

ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical trials. However, there is a lack of bioanalytical assays for ARV-110 determination in biological samples. In this study, we developed and validated an LC-MS/MS method for the quantitation of ARV-110 in rat and mouse plasma and applied it to pharmacokinetic studies. ARV-110 and pomalidomide (internal standard) were extracted from the plasma samples using the protein precipitation method. Sample separation was performed using a C18 column and a mobile phase of 0.1% formic acid in distilled water–0.1% formic acid in acetonitrile (30:70, v/v). Multiple reaction monitoring was used to quantify ARV-110 and pomalidomide with ion transitions at m/z 813.4 → 452.2 and 273.8 → 201.0, respectively. The developed method showed good linearity in the concentration range of 2–3000 ng/mL with acceptable accuracy, precision, matrix effect, process efficiency, and recovery. ARV-110 was stable in rat and mouse plasma under long-term storage, three freeze-thaw cycles, and in an autosampler, but unstable at room temperature and 37 °C. Furthermore, the elimination of ARV-110 via phase 1 metabolism in rat, mouse, and human hepatic microsomes was shown to be unlikely. Application of the developed method to pharmacokinetic studies revealed that the oral bioavailability of ARV-110 in rats and mice was moderate (23.83% and 37.89%, respectively). These pharmacokinetic findings are beneficial for future preclinical and clinical studies of ARV-110 and/or other PROTACs. [ABSTRACT FROM AUTHOR]

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat. [ABSTRACT FROM AUTHOR]

This study was performed to evaluate and compare the pharmacokinetic parameters between two dosage formulations of hesperidin and naringenin: mixture and tablet. Our objective was to determine that the flavonoid tablet does not significantly modify the pharmacokinetic parameters compared with the mixture. For this study, we administered 161 mg/kg of either mixture (Mix-160) or tablet composed of hesperidin and by intragastric administration. Blood microsamples were collected from tail vein up to 24 h. Serum flavonoid extraction was performed by solid phase extraction and analyzed by LC-MS/MS of triple quadrupole (QqQ). Serum concentration vs. time plot showed that data fitted for a first-order model. The pharmacokinetic parameters were calculated by a noncompartmental model. The results showed that the absorption constant is higher than the elimination constant. The first concentration was found at five minutes, and minimal concentration at 24 h after administration, suggesting a enterohepatic recirculation phenomena and regulation of liver cytochromes' activity. We did not find meaningful differences between the pharmacokinetic parameters of both samples. We concluded that tablet form did not interfere with the bioavailability of hesperidin and naringenin, and it could be a suitable candidate for developing a drug product. [ABSTRACT FROM AUTHOR]

AWRK6 was a synthesized peptide developed based on the natural occurring peptide dybowskin-2CDYa, which was discovered in frog skin in our previous study. Here, a quantitative determination method for AWRK6 analysis in rat plasma by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and validated following U.S. FDA guidelines. A combination of plasma precipitation and liquid–liquid extraction was applied for the extraction. For pharmacokinetics study, the rats were administrated with AWRK6 via intraperitoneal and intravenous injection. The prepared plasma samples were separated on an ODS column and analyzed by tandem MS using precursor-to-product ion pairs of m/z: 533.4→84.2 for AWRK6 and m/z: 401.9→101.1 for internal standard Polymyxin B sulfate in multiple reaction monitoring mode. AWRK6 concentrations in rat plasma peaked at about 1.2 h after intraperitoneal injections at 2.35, 4.7 and 9.4 mg/kg bodyweight. The terminal half-life was around 2.8 h. The absolute bioavailability of AWRK6 was 50% after 3 doses via injection, and the apparent volume of distribution was 4.884 ± 1.736 L. The obtained determination method and pharmacokinetics profiles of AWRK6 provides a basis for further development, and forms a benchmark reference for peptide quantification. [ABSTRACT FROM AUTHOR]

Folate is a vitamin beneficial for humans that plays an important role in metabolism, but it cannot be well supplemented by food; it is necessary to supplement it in other ways. Based on this consideration, a novel crystal form C of 6S-5-methyltetrahydrofolate calcium salt (MTHF CAC) was obtained. To explore the difference between MTHF CAC and the crystal form Ⅰ of 6S-5-methyltetrahydrofolate calcium salt (MTHF CA) as well as an amorphous product of 6S-5-methyltetrahydrofolate glucosamine salt (MTHF GA), their stability and pharmacokinetic behaviours were compared. The results of high-performance liquid chromatography coupled with ultraviolet detection analysis indicated that MTHF CAC showed a better stability than MTHF CA and MTHF GA. After oral administration of MTHF CAC, MTHF CA, and MTHF GA to male rats, the MTHF concentrations were analysed using a validated liquid chromatography–tandem mass spectrometry, and the pharmacokinetic parameters were compared. The mean residence times (0–t) of MTHF CAC, MTHF CA, and MTHF GA were 3.7 ± 1.9 h, 1.0 ± 0.2 h (p < 0.01), and 1.5 ± 0.3 h (p < 0.05), respectively. The relative bioavailability of MTHF CAC was calculated to be 351% and 218% compared with MTHF CA and MTHF GA, respectively, which suggests that MTHF CAC can be better absorbed and utilized for a longer period of time. [ABSTRACT FROM AUTHOR]

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid–liquid extraction with only one step—protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r2 ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased. [ABSTRACT FROM AUTHOR]