Bonnie A. Avery

Rapid determination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative light scattering detector

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.

Off-line and on-line measurements of drug-loaded hot-melt extruded films using Raman spectroscopy.

The objective of this study was to investigate the use of Raman spectroscopy for the quantitative and qualitative analysis of an active ingredient in hot-melt extruded film formulations. Clotrimazole and ketoprofen were used as the active pharmaceutical ingredients (APIs) in the subject formulations. Films were prepared with contents varying from 1 to 20% of the respective API. Raman spectroscopy was used to quantify these APIs, both off-line and on-line. The spectral data were also used to ascertain the physical status of these APIs in the formulations. For off-line analysis, the films were cut into small rectangles, and the amount of the API was measured using a fiber optic probe equipped with a non-contact optic (NCO). For on-line analysis, real-time measurements were accomplished by fixing the probe over the extruded film for continuous data collection. Raman spectroscopy can be a convenient alternative to HPLC and other techniques currently employed for the quantification of the API in these formulations. Because Raman is also sensitive to changes in crystallinity, employment of the technique provided additional information to deduce the crystalline status of the API. The results reported in this paper suggest the suitability of Raman for PAT applications because of the on-line capability.

Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra performance liquid chromatography with mass spectrometry

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2-5.0ng/microL, and limits of detection (LOD) were lower than 0.01ng/microL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4+/-21.3% to 92.4+/-3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC-MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.

Carrier-Mediated Partitioning of Artemisinin into Plasmodium falciparum-Infected Erythrocytes

ABSTRACT The purpose of the present study was to characterize the partitioning of artemisinin into both uninfected and Plasmodium falciparum-infected red blood cells (RBCs). The partitioning of [14C](+)-artemisinin into RBCs was studied at four different hematocrit levels and eight time periods. At the optimum time of 2 h, the partitioning process was investigated with eight different drug concentrations ranging from 0.88 to 3.52 μM at 37 and 4°C. The effect of the presence of unlabeled artemisinin on the partitioning of the same concentration of [14C]artemisinin was studied. About 35 to 40% of the drug was seen to partition into uninfected RBCs at a hematocrit of 33%, irrespective of the incubation period or the drug concentration used. In contrast, infected RBCs showed an increase in partitioning of the drug with time until saturation was achieved at 1 h. While the partitioning of artemisinin into parasitized RBCs at 37°C was found to be significantly higher than that in nonparasitized RBCs, at 4°C both parasitized and nonparasitized RBCs showed identical partitioning of the drug. The partitioning of [14C]artemisinin into parasitized RBCs was completely inhibited in the presence of the same concentration of unlabeled artemisinin. However, no such effect was observed in nonparasitized cells, and no evidence suggesting that binding of the drug in parasitized RBCs is reversible was found. The partitioning of artemisinin into parasitized RBCs was found to be rapid, saturable, temperature dependent, irreversible, and subject to competitive inhibition with unlabeled artemisinin. The results obtained suggest the involvement of carrier mediation in the partitioning of artemisinin across the parasitized RBC membrane. In contrast, simple passive diffusion of artemisinin was seen in nonparasitized RBCs.

New positron emission tomography (PET) radioligand for imaging σ-1 receptors in living subjects.

σ-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([(18)F]FTC-146, [(18)F]13). [(18)F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/μmol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [(18)F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [(18)F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

The Use of Near‐Infrared Spectroscopy for the Quantitation of a Drug in Hot‐Melt Extruded Films

The objective of the study was to demonstrate the utility of near‐infrared spectroscopy (NIRS) for quantitative analysis of a model drug in hot‐melt extruded film formulations. Polyethylene oxide (PEO) films with clotrimazole (CT) as a model drug were prepared by hot‐melt extrusion (HME) incorporating drug concentrations ranging from 0–20% and analyzed using a Fourier transform near‐infrared (FT‐NIR) spectrophotometer in the reflectance mode. High performance liquid chromatography (HPLC) was the reference method used for this study. The NIR calibration model derived for CT was composed of 21 frequency ranges that were correlated to the values quantified using the HPLC reference method. The NIR method developed resulted in an assayed CT amount in the film matrix to be within 3.5% of the quantity determined by the reference method. These studies clearly demonstrate that NIRS is a powerful method for the quantitation of active drug substances contained in films produced by HME and warrants further investigation.

Biotransformation of 10-deoxoartemisinin to its 7β-hydroxy derivative by Mucor ramannianus

Abstract10-Deoxoartemisinin at 0.2 mg ml−1 medium was transformed to 7β-hydroxy deoxoartemisinin by Mucor ramannianus growing on sucrose, 20 g l−1, and peptone, 10 g l−1, at pH 4 and 26 °C. The yield of product was increased from 16% to 45% by selecting optimal culture conditions using a 25−2 factorial design.

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of Artemisinin in rat serum and its application in pharmacokinetics

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify artemisinin in rat serum. The lower limit of quantification (LLOQ) was 4 ng/mL. The calibration curve was linear from 4 ng/mL to 10,000 ng/mL (R=0.998). The assay was based on the selected reaction monitoring (SRM) transitions at m/z 305.4-151.10 for artemisinin and m/z 335.2-163.10 for arteether (internal standard). The artemisinin and internal standard can be separated from endogenous interferences in rat serum. Inter- and intra-day assay variation was less than 15%. The extraction recoveries ranged from 80.0 to 107.3% at the three concentrations (5000, 2000, and 200 ng/mL). This method was successfully applied to pharmacokinetic studies of artemisinin after intravenous and oral administration to rats.

Evaluation of σ-1 Receptor Radioligand 18F-FTC-146 in Rats and Squirrel Monkeys Using PET

The noninvasive imaging of σ-1 receptors (S1Rs) could provide insight into their role in different diseases and lead to novel diagnostic/treatment strategies. The main objective of this study was to assess the S1R radiotracer 18F-FTC-146 in rats. Preliminary squirrel monkey imaging and human serum/liver microsome studies were performed to gain information about the potential of 18F-FTC-146 for eventual clinical translation. Methods: The distribution and stability of 18F-FTC-146 in rats were assessed via PET/CT, autoradiography, γ counting, and high-performance liquid chromatography (HPLC). Preliminary PET/MRI of squirrel monkey brain was conducted along with HPLC assessment of 18F-FTC-146 stability in monkey plasma and human serum. Results: Biodistribution studies showed that 18F-FTC-146 accumulated in S1R-rich rat organs, including the lungs, pancreas, spleen, and brain. Pretreatment with known S1R compounds, haloperidol, or BD1047, before radioligand administration, significantly attenuated 18F-FTC-146 accumulation in all rat brain regions by approximately 85% (P < 0.001), suggesting radiotracer specificity for S1Rs. Similarly, PET/CT and autoradiography results demonstrated accumulation of 18F-FTC-146 in rat brain regions known to contain S1Rs and that this uptake could be blocked by BD1047 pretreatment. Ex vivo analysis of 18F-FTC-146 in the brain showed that only intact radiotracer was present at 15, 30, and 60 min, whereas rapid metabolism of residual 18F-FTC-146 was observed in rat plasma. Preliminary monkey PET/MRI studies demonstrated specific accumulation of 18F-FTC-146 in the brain (mainly in cortical structures, cerebellum, and vermis) that could be attenuated by pretreatment with haloperidol. HPLC of monkey plasma suggested radioligand metabolism, whereas 18F-FTC-146 appeared to be stable in human serum. Finally, liver microsome studies revealed that 18F-FTC-146 has a longer half-life in human microsomes, compared with rodents. Conclusion: Together, these results indicate that 18F-FTC-146 is a promising tool for visualizing S1Rs in preclinical studies and that it has potential for mapping these sites in the human brain.

Content Analysis and Stability Evaluation of Selected Commercial Preparations of St. John's Wort

Content analysis and stability studies were performed for the commercial products of St. Johns wort. Six marketed formulations were analyzed for their hypericin and pseudohypericin content. These products were standardized to contain 0.3% hypericin. Results revealed total hypericin as 7.72–38.57% of the labeled claim with varying concentrations of pseudohypericin. Stability studies were carried out under three different storage conditions: 1) 25 ± 2°C, 60 ± 5%RH for six months, 2) 40 ± 2°C, 75 ± 5%RH for six months, and 3) 50°C for one month. Tablet formulations were also analyzed for their hardness and friability. Stability studies revealed significant decrease in the content of the marker compounds with time.