Validation of a bioanalytical HPLC-UV method to quantify Α-Bisabolol in rat plasma applied to pharmacokinetic pilot study with the drug nanoemulsion.

α-Bisabolol (α-BIS) is a sesquiterpene alcohol present in chamomile essential oil [Chamomilla recutita (L.) Rauschert]. Despite its numerous pharmacological effects, its pharmacokinetics remain understudied. An analytical method capable of quantifying α-BIS in plasma is crucial to enable pharmacokinetic analysis. Presently, only one study has quantified it using mass spectrometry. Administering α-BIS requires a nanoemulsion for intravenous injection. This study aimed to develop and validate a bioanalytical method using high-performance liquid chromatography with an ultraviolet detector to quantify α-BIS in rat plasma. The method employed acetonitrile and ultrapure water (80:20, v/v) as the mobile phase, with a flow rate of 1 ml/min and concentrations ranging from 465 to 29.625 µg/ml. All US Food and Drug Administration-designated assays were successful, indicating the method's precision, accuracy, sensitivity and linearity in determining α-BIS in rat plasma. The developed nanoemulsion, assessed through dynamic light scattering analysis, the ensemble collection of particles and polydispersity index evaluation, proved safe and effective for intravenous administration. The pharmacokinetic parameters such as volume of distribution, clearance and half-life indicated that α-BIS tends to persist in the body. This study provides a foundation for further research to explore α-BIS's potential pharmaceutical applications in the future.

Validation of a HPLC method for quantification of midazolam in rat plasma: Application during a Maytenus ilicifolia-drug interaction study.

Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC-UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol-isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC-UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb-drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol-water (1:1). The analyte was eluted throughout a C18 column using sodium acetate buffer (10 mm, pH 7.4)-methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from -19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

Determination of l-(+)-bornesitol, the hypotensive constituent of Hancornia speciosa, in rat plasma by LC-MS/MS and its application on a pharmacokinetic study.

Hancornia speciosa is a medicinal plant with proven antihypertensive activity. The cyclitol l-(+)-bornesitol is the main constituent of its leaves and is a potent inhibitor of the angiotensin-converting enzyme. We herein investigated the pharmacokinetic properties of bornesitol administered orally to Wistar rats, as well as bornesitol permeation in Caco-2 cells. Bornesitol was isolated and purified from an ethanol extract of H. speciosa leaves. An ultra-high performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated to quantify bornesitol in rat plasma based on Multiple Reaction Monitoring, using pentaerythritol as an internal standard. Pharmacokinetics was evaluated by the administration of single doses via intravenous in bolus (3 mg/kg) and gavage (3, 15 and 25 mg/kg). Bornesitol permeation was assayed in a transwell Caco-2 cells model, tested alone, or combined with rutin, or as a constituent of H. speciosa extract, using a developed and validated UPLC-ESI-MS/MS method. All assayed validation parameters (selectivity, residual effect, matrix effect, linearity, precision, accuracy and stability of analyte in plasma and solution) for the bioanalytical method met the acceptance criteria established by regulatory guidelines. Bornestiol reached peak plasma concentration within approximately 60 min after oral administration with a half-life ranging from 72.15 min to 123.69 min. The peak concentration and area under the concentration-time curve of bornesitol did not rise proportionally with the increasing doses, suggesting a non-linear pharmacokinetics in rats and the oral bioavailability ranged from 28.5%-59.3%. Bornesitol showed low permeability in Caco-2 cells, but the permeability apparently increased when it was administered either combined with rutin or as a constituent of H. speciosa extract. In conclusion, bornesitol was rapidly absorbed after a single oral administration to rats and followed a non-linear pharmacokinetics. The obtained data will be useful to guide further pre-clinical development of bornesitol-containing herbal preparations of H. speciosa as an antihypertensive agent.

LC-MS/MS determination of deoxyelephantopin, a novel anti-tumor candidate in rat plasma and its application to a pharmacokinetic study in rats

Abstract A specific, sensitive and robust LC-MS/MS method was developed and validated for the quantification of deoxyelephantopin in rat plasma using simvastatin as an internal standard as per regulatory guidelines of Bioanalytical Method Validation. Plasma sample was prepared through liquid-liquid extraction. Chromatographic separation was performed on an Agela-C18 analytical column (1.8 µm, 2.1 mm × 50 mm) with an isocratic mobile phase consisting of 0.05% formic acid (dissolved in acetonitrile) and water (55:45, v/v) at a flow rate of 0.5 ml/min. The column oven was maintained at 40 ºC and the injection volume was 4 µl. Elution of deoxyelephantopin and the internal standard occurred at 5.1 and 6.3 min, respectively. The total chromatographic run time was 7.5 min. A linear response function was constructed in the concentration range of 13.2-2640 ng/ml. The intra- and inter-day precision and accuracy were in the range of 1.4-14.8% and -11.7 to 14.1%, respectively. The validated LC-MS/MS was successfully applied to the pharmacokinetic study of deoxyelephantopin after intravenous injection of 1, 2 and 4 mg/kg and oral administration of 7.5, 15 and 30 mg/kg deoxyelephantopin in rats. After oral and intravenous administration, the C max and AUC values of deoxyelephantopin increased dose-dependently.

Pharmacokinetic profile and oral bioavailability of Kaurenoic acid from Copaifera spp. in rats.

Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological activities, such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are no pharmacokinetic data available about this molecule. The purpose of the study was to determine the pharmacokinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein cannulation received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed intravenous administration and serial blood collection along 10 h. Analytical quantification was performed by reversed phase HPLC-UV and mobile phase composed by acetonitrile:acidified water (70:30 v/v). The validated analytical method showed precision, accuracy, robustness, reliability and linearity between 0.75 and 100 µg/mL. KA administered intravenously showed a linear and two-compartment kinetic behavior at the tested dose. The following pharmacokinetic parameters were determined: Cmax = 22.17 ±â€¯1.65 mg/L; V = 14.53 ±â€¯1.47 L/kg; CL = 17.67 ±â€¯1.50 mL/min/kg; AUC0-∞ = 2859.65 ±â€¯278.42 mg·min/L, K = 0.073 ±â€¯0.005 h-1 and t1/2ß = 9.52 ±â€¯0.61 h. Oral treatment did not provide detectable plasma levels of KA, avoiding the determination of its bioavailability.

Simultaneous determination of six bioactive components of total flavonoids of Scorzonera austriaca in rat tissues by LC-MS/MS: application to a tissue distribution study

ABSTRACT A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of six bioactive constituents including vitexin, orientin, isoorientin, 2"-O-β-D-xylopyranosyl isoorientin, 2"-O-β-D-xylopyranosyl isovitexin, and 6-C-L-α-arabipyranosyl vitexin in rats' various tissues using isoquercitrin as the internal standard. Biological samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. All analytes and internal standard were quantitated through electrospray ionization in negative ion selected reaction monitoring mode. The mass transitions were as follows: m/z 431 → 311 for vitexin, m/z 447 → 327 for orientin or isoorientin, m/z 579 → 459 for 2"-O-β-D-xylopyranosyl isoorientin, m/z 563 → 293 for 2"-O-β-D-xylopyranosyl isovitexin, m/z 563 → 353 for 6-C-L-α-arabipyranosyl vitexin, and m/z 463 → 300 for the internal standard, respectively. The lower limits of quantification for the six analytes in different tissue homogenates were 0.8-2.2 ng/ml. The validated assay was successfully applied to a tissue distribution study of the six components in rats after intravenous administration of total flavonoids of Scorzonera austriaca Willd; Asteraceae. The results of the tissue distribution study showed that the high concentrations of six components were mainly in the kidney.

Pharmacokinetics of oleracimine in rats by ultra-high-performance liquid chromatography

The novel alkaloid, oleracimine, presented remarkable anti-inflammatory bioactivity, and therefore, its pharmacokinetics was investigated in rat plasma after intravenous and oral administration by using a rapid ultra-high-performance liquid chromatography (UHPLC) method with UV detection at 270 nm. The analysis was performed on a shim-pack ODS column (75 mm×2 mm, 1.6 µm particle size, Shimadzu, Japan) column using isocratic elution with a mobile phase consisting of methanol-water (62:38, v/v) within 3 min. The results indicated that oleracimine was rapidly distributed with Tmax for 11.7 min after oral administration, which presented the double-peak phenomenon in the pharmacokinetic profile with a higher oral absolute bioavailability of 55.1% ± 7.83%.