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OBJECTIVE Ambrisentan, an endothelin receptor antagonist FDA-approved for the treatment of pulmonary arterial hypertension in adult patients, lacks an acceptable pediatric dosage form. The objective of this investigation was to determine the stability of an extemporaneously compounded ambrisentan suspension. METHODS Ambrisentan suspension was compounded to a concentration of 1 mg/mL using commercially available suspending agents. The suspension was then evenly split into 2 plastic amber prescription bottles. One bottle was stored at room temperature and under continuous fluorescent light while the other bottle was stored under refrigeration and protection from light. A fast and selective reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of ambrisentan. HPLC analysis was performed on samples withdrawn from the stock bottles at predetermined time intervals, up to 90 days. RESULTS The developed HPLC method enabled the elution and detection of ambrisentan peak at 4.4 minutes. HPLC analysis revealed that all samples from both storage conditions retained >90% potency throughout the study timeframe. There were no signs of any ambrisentan breakdown products on HPLC analysis. Color and odor of the final product was also consistent throughout the 90-day storage period. CONCLUSION Ambrisentan suspension, compounded to 1 mg/mL, is stable at room temperature or under refrigeration for up to 90 days.

The fruit of the Maclura pomifera tree is a sustainable source for the pharmacologically interesting isoflavones, osajin and pomiferin. A reversed-phase HPLC method was developed to identify osage orange samples with high isoflavone content and to determine the optimum conditions for sample preparation. Analytical run time was 8 min at a flow rate of 1 mL/min using a gradient of acetonitrile in H2O (0.1% formic acid) and UV peak detection at 274 nm. The method was validated for specificity, accuracy, precision, and limits of detection and quantitation (LOD/LOQ). The method was applied to determine the levels of osajin and pomiferin in extracts prepared from different samples of osage orange growing in the Midwest and southern United States. Results demonstrated the effect of different variables, such as sample preparation, geographical location, and growth stage, on the levels of osajin and pomiferin in analyzed samples.

Borututu (Cochlospermum angolensis) is an African tree whose bark has recently emerged as a herbal dietary supplement with claims for antioxidant activity. In order to substantiate the claimed activity of borututu supplements, we performed an activity-guided fractionation of the total extract utilizing a 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. Subsequent flash and centrifugal chromatography resulted in the isolation of gallic acid (1) and protocatechuic acid (2) as the main antioxidant constituents. Two apocarotenoids and one flavonoid were also isolated from the chloroform fraction and were identified as cochloxanthin (3), dihydrocochloxanthin (4), and 7,4′-dimethyltaxifolin (5), respectively. A High-performance liquid chromatography (HPLC) method was also developed for fingerprinting borututu samples, with Compounds 1–4 suggested as chemical markers for quality control purposes.

Nutmeg [Myristica fragrans Houtt. (Myristicaceae)] has a long-standing reputation of psychoactivity. Anecdotal reports of nutmeg use as a cheap marijuana substitute, coupled to previous studies reporting a cannabimimetic-like action, suggest that nutmeg may interact with the endocannabinoid system.The study evaluates nutmeg fractions for binding capacity with various CNS receptors and their potential interaction with the endocannabinoid system.Dichloromethane (DF) and ethyl acetate (EF) fractions were prepared from the methanol extract of powdered whole nutmeg. The HPLC-profiled fractions were assayed by the NIMH Psychoactive Drug Screening Program (PDSP) in a panel of CNS targets at a 10 μg/mL concentration. The fractions were also screened for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition, initially at a concentration of 500 μg/mL, then by concentration-dependent inhibition studies.None of the tested fractions showed significant binding to CNS receptors included in the PDSP panel. However, both fractions exerted significant inhibition of the FAAH and MAGL enzymes. The DF fraction inhibited FAAH and MAGL enzymes at ICThe study provides the first piece of evidence that nutmeg interacts with the endocannabinoid system via inhibition of the endocannabinoid catabolizing enzymes. This mechanism provides insight into reported cannabis-like action as well as expands the potential therapeutic utility of nutmeg.

Summary Ethyl 8-oxo-5,6,7,8-tetrahydrothiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124) is one of a new generation of ultra-short-acting hypnotics. A high-performance thin-layer chromatographic (HPTLC) method has been established as the first reported method for quantitative analysis of HIE-124 in plasma. Baseline resolution between HIE-124 and diazepam (internal standard) was achieved on HPTLC plates with chloroform–ethyl acetate 80:20 (v/v) as mobile phase. Densitometric detection was performed at 265 nm. The method was linear in the range 40–400 μg mL–1 (R = 0.997) and recoveries of the drug were in the range 93–102% at 80– 350 μg mL –1 . Within-run and between-run precision calculated as RSD [%], and accuracy calculated as percentage error were in the ranges 2.1–4.6% and 0.8–6.9%, respectively. The limits of detection (LOD) and quantification (LOQ) in spiked human plasma were 20 and 40 μg mL–1 (40 and 80 ng per band), respectively.

An HPTLC system commonly used for chromatographic separations and analytical quality control has been utilized non-chromatographically as a screening tool for antioxidant activity in the 2,2- diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay. The accurate and precise capabilities for sample application and densitometric analysis of dry spots were applied to determine the 50% inhibition of DPPH (IC50) by a number of natural antioxidants at 530 nm. Concentration-response curves were generated from 3rd degree polynomial fitting of data acquired for each antioxidant at 4 concentration levels. The IC50 values ranged from 9.0 - 332.8 μM with quercetin being the most active and silybin the least active of the pure compounds. Three antioxidant extracts were also evaluated and grapeseed extract was the most active (IC50 8.3 μg/ml). The coefficients of variation for all IC50 values were around 5% which indicated method reproducibility and the suitability of the used system for the intended purpose. Based on its densitometric evaluation of dry spots, the described technique provides an alternative to the spectrophotometric evaluation of samples in solution.

A high-performance thin-layer chromatographic (HPTLC) method has been established for determination of sildenafil citrate in commercial products. The compounds were separated on silica gel, with CHCl 3 -MeOH-diethylamine, 90 + 10 + 1 ( v/v ), as mobile phase, and the analyte spots were quantified densitometrically at λ = 305 nm. Recovery of sildenafil citrate was 100.6 and 98.2%, for pure and spiked samples, respectively. Method precision and specificity were also validated. The method was used for determination of sildenafil in four pharmaceutical products and three aphrodisiac herbal preparations which were claimed to be totally natural. Levels of sildenafil in the pharmaceutical products ranged from 49.7 to 50.5 mg per tablet. Sildenafil was also detected in one herbal preparation at a concentration of 85.0 mg per capsule.

The caffeine content of selected herbal products and energy drinks available in the Saudi market was determined by HPTLC-UV densitometric analysis. Pre-coated HPTLC silica gel plates (20cm x 10cm) were used for the analysis. The solvent system consisted of ethyl acetate-methanol (85:15, v/v), and caffeine was detected at 275nm. The developed method was validated for specificity, repeatability (C.V. < 5%), recovery (98.90 +/- 3.46), and accuracy (99.84 +/- 2.87). The levels of caffeine were 4.76-13.29% (w/w) and 0.011-0.032% (w/v), for the herbal products and the energy drinks, respectively.

A high-performance thin-layer chromatographic method for determination of parthenolide has been validated for recovery, accuracy, specificity, and precision and applied to four feverfew products. Chromatography was performed on silica gel plates with ethyl acetate- n -hexane, 3 + 2 ( v/v ) as mobile phase. Parthenolide was visualized by dipping plates into p -anisaldehyde reagent and heating at 105°C for 5 min. Densitometric quantification of analyte spots was achieved by scanning the plates at λ = 543 nm after derivatization. The method is precise with CV [%] < 5.00; calibration recovery = 101.12 ± 4.11% and overall accuracy = 101.14 ± 4.47%. The levels of parthenolide in the products analyzed ranged from 0.03 to 0.24%.

Although the presence of harman alkaloids in the genus Passiflora has been established in a few species, their presence in the medicinally significant P. incarnata (passion flower) is still debatable. Powdered samples from 91 Passiflora species were subjected to accelerated speed extraction and analyzed for their content of five harman alkaloids by reversed-phase HPLC with photodiode array detection. The utilized analytical method was capable of detecting harman alkaloids in more than 50% of the analyzed samples at ppm levels. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied Passiflora species.

Clutia richardiana L. is a traditional medicinal herb with demonstrated hypoglycemic effects. An HPLC method (Luna((R)) C(18) column, gradient elution, UV detection at 220 nm) has been utilized to determine the levels of the major diterpenes of C. richardiana in extracts obtained by using three different solvents. Extraction with the organic solvents methanol and acetonitrile was more efficient than aqueous extraction with hot water, but nine markers could be detected equally in all extracts. The aqueous extract was non-toxic against a number of human cell lines, which is a promising indication for its use as a safe and effective antidiabetic herbal preparation.

An approach comprising accelerated solvent extraction followed by quantitative HPLC analysis has been adopted in fingerprinting 115 samples of different species of the genus Passiflora. The C-flavonoid glycosides schaftoside (1), isoschaftoside (2), isoorientin (3), orientin (4), isovitexin (5) and vitexin (6) were chosen as analytical standards and their overall prevalence in all samples was determined. The developed HPLC method utilizes gradient elution on an analytical Phenomenex Hypersil ODS column and UV detection at 280 nm. The availability of unique fingerprints, as well as quantitative data, for each species can provide a number of benefits including, but not limited to (a) authentication of samples, (b) determination of chemotaxonomic markers, (c) identification of constituent patterns related to specific geographical locations, (d) supportive data in genetic studies, (e) identifying possible substitutes for Passiflora incarnata, (f) differentiating between closely related species, and (g) relati...

The migraine prophylactic herb feverfew (Tanacetum parthenium L.) is marketed in the United States in a variety of forms and compositions. Although its therapeutic efficacy is still uncertain, the sesquiterpene lactone parthenolide is the constituent recommended to be measured for quality control of feverfew preparations. A validated liquid chromatographic method was developed and used to estimate parthenolide in a number of U.S. feverfew market products formulated as capsules, tablets, or crude powder. The method uses a Lichrosphere 5 C18 column, a mobile phase consisting of 50mM NaH2PO4 in H2O (solvent A), and CH3CN–MeOH (90 + 10, v/v; solvent B). Elution was run at a flow rate of 1.0 mL/min with a linear gradient of 50–15% A in B over 20 min and UV detection at 210 nm. The correlation coefficient for the calibration curve was 0.9999 over the range of 0.00–0.400 mg/mL. Overall recovery of parthenolide was 103.1%.

Lotaustralin was isolated as a mixture of two diastereoisomeric forms from the methanol extract of Rhodiola rosea roots, together with the known compounds rosavin, rosarin, rosin, rosiridin, salidroside, and beta-sitosterol. The structure of lotaustralin was established by 1D and 2D-NMR spectroscopy, including 1H-1H COSY, NOESY, HMQC, and HMBC, and FAB and HR MS.

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kernel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5′-methoxylicarin B (12), licarin B (13), and 2-(3′-allyl-2′,6′-dimethoxy-phenyloxy)-1-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Microbial transformation of silybin ( 1 ), the major flavonolignan of milk thistle ( Silybum marianum , Asteraceae), resulted in the isolation of four metabolites. The structures of the isolated metabolites were determined by spectroscopic methods. One phase I metabolite was produced by Beauveria bassiana and was characterized as 8-hydroxysilybin ( 2 ). Three phase II metabolites were produced by two Cunninghamella species and were identified as 2,3-dehydrosilybin-3- O -β- d -glucoside ( 3 ), obtained from Cunninghamella species; and silybin-7-sulfate ( 4 ) and 2,3-dehydrosilybin-7-sulfate ( 5 ), obtained from Cunninghamella blakesleana . Compared to 1 (IC 50 284 μg/mL), the generated metabolites displayed varying levels of antioxidant activities in the DPPH free-radical scavenging assay.

For the first time, a fast, high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the new ultra-short hypnotic HIE-124 and its metabolite in mice serum. Each compound, together with carbamazepine (internal standard) was extracted from the serum matrix using liquid-liquid extraction (LLE). Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column (100 mm × 4.6 mm i.d.) under isocratic conditions using a mobile phase of 65:35 (v/v), 20 mM phosphate buffer (pH 7.0 adjusted with phosphoric acid)-acetonitrile. The elution of the analytes were monitored at 240 nm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 2.5 mL min(-1). The total run time of the assay was 2 min. The method was validated over the range of 60-2000 ng mL(-1) for HIE-124 and 200-1600 ng mL(-1) for the metabolite (r(2) = 0.99). The limit of detection (LOD) for HIE-124 and its metabolite were 20 ng mL(-1) and 65 ng mL(-1), respectively. The proposed method was validated in compliance with ICH guidelines, in terms of accuracy, precision, limits of detection and quantitation and other aspects of analytical validation. The developed method could be used for the trace analyses of HIE-124 and its metabolite in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.

Microemulsion electrokinetic capillary chromatography (MEEKC) with sample stacking induced by reverse migrating pseudostationary phase (SRMP) technique in a suppressed electro-osmotic flow (EOF) strategy was investigated for analysing the new ultra-short hypnotic HIE-124 in mice serum. The proposed method utilized fused-silica capillary with a total length of 50 cm (effective length 40 cm), applied voltages for stacking and separation were 5.0 kV for 4.30 min and subsequently 25 kV, respectively, with a sample injection of 0.5 psi for 90 s. All the runs were carried out at 25 °C and detected at 213 nm. The optimum microemulsion background electrolyte (BGE) solution consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium dodecyl sulfate (SDS), and 89.6 mL with 25 mM phosphate buffer pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. The proposed method was validated carefully with respect to high specificity of the method, good linearity ( r = 0.9994), fair wide linear concentration range (66–1500 ng mL −1 ), limit of detection and quantitation were 21.6 and 65.5 ng mL −1 , respectively. The mean relative standard deviation (RSD) of the results of intra- and inter-day precision and accuracy were less than 6.0%, and overall recovery higher than 95% of HIE-124 in mice serum. The developed method could be used for the trace analyses of HIE-124 in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.

This study examined the intestinal transport of five harman alkaloids using the Caco-2 cell monolayer as a model of the human intestinal mucosa. Transport parameters, permeability coefficients and percent transports, were calculated and compared under identical conditions with atenolol. Permeability coefficients were also compared with the reported values for model compounds like mannitol, propranolol and glucose. Sodium fluorescein was used as the marker for paracellular leakage. These alkaloids, in the concentration range of 250-500 microM, demonstrated substantial transport across the monolayer with moderate to high efflux rates and permeability coefficients. The transport was linear with time and was concentration dependent.

Goldenseal ( Hydrastis canadensis L., Ranunculaceae) is an ingredient of various dietary supplements intended for enhancing general body immunity. Many goldenseal products are currently available in the United States, either alone or in combination with echinacea. In most products, the content of the main active alkaloids of goldenseal, hydrastine and berberine, is not indicated on the label. A high‐performance liquid chromatography (HPLC) method has been developed for the detection and quantification of hydrastine and berberine in a number of products obtained from the United States market. The method uses a Phenomenex® Luna C 18 column, a mobile phase consisting of solvent A (100 mM sodium acetate/acetic acid, pH 4.0) and solvent B (acetonitrile/methanol; 90/10, v/v). Elution was run at a flow rate of 1.0 mL/min, with a linear gradient of 80– 40% A in B over 20 min and ultraviolet detection at 290 nm. A wide range of content variation was observed for both alkaloids in the tested samples. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:817–822, 2001

A simple HPLC method was developed to quantify the major daucane sesquiterpene esters present in herb and roots of Ferula hermonis Boiss. The method utilized a C(18) reversed phase analytical column with isocratic elution for 30 minutes and UV detection at 240 nm. Extracts from two crude plant samples and two commercial products were fingerprinted and quantitatively analyzed.