C18 Reversed-phase HPLC Research Articles

B-197 Separation of Bile Acid Muricholic Acid Enantiomers by C18 Reversed-phase HPLC as a Function of Component Polarity Values of the Mobile Phase

Abstract Objective Optimize the reversed phase HPLC technique for the separation of alpha-muricholic acid and beta-muricholic bile acid enantiomers, by investigating the effects of the mobile phase’s three polarity components [hydrogen bond donor acidity (α), hydrogen bond donor basicity (β) and dipolarity/polarizability (π)] on the separation of the enantiomers. Background The importance of drug enantiomers in clinical pharmacology is well recognized, where often only one of the enantiomer pair effectively binds to the target protein site to exert its therapeutic effect. Same for some endogenous compounds, only one of the enantiomer pair molecules binds to an enzyme or receptor to exert its physiological action, while the other molecule is inactive. It is essential for the analytical method to separately determine the two compounds of the pair. Usual HPLC techniques for separating enantiomers employ specialized chiral columns. In this work, steroid isomers are separated on a C18 column, with the separation controlled by mobile phase (MP) component polarity adjusted with different proportions of binary organic modifiers added to the MP. Methods The bile acid enantiomers alpha-muricholic acid and beta-muricholic acid were separated on a C18 column (150 mm × 2.1 mm, 3.5 μm, 40 °C) using two different organic modifiers in the MP at a flow rate of 0.2 mL/min, with ESI-LC/MS detection. The isocratic MP consisted of 0.1% formic acid in 100% water for MP A and 0.1% formic acid and five or more different proportions (separate runs) of the two organic modifiers for MP B to adjust MP polarity. MP component polarities was determined from: 1) the literature values for the total polarity (P) for each of the pure solvents in the MP; 2) published values of the fraction of the total polarity for the polarity components (α + β + π = 1) for each solvent; and 3) the fraction proportion of each solvent in the MP. The experimentally determined selectivity factor is then plotted vs the polarity component value of the MP. Results When the selectivity factor vs the values of the MP polarity components was plotted for six different organic modifier pair combinations on the same plot, the summed dipolarity/polarizability (and basicity polarity (β) components of the MP showed reasonable correlation (R2 = 0.72), displaying mostly a linear superimposed trend for all the organic modifier pairs, while the acidity component (α, R2 = 0.13) and total polarity (P) (R2 = 0.002) of the MP did not. Thus, the π + β summed MP polarities is a significant determiner of selectivity, irrespective of the organic modifier pair used. The bile acid enantiomers were baseline separated, with a selectivity factor of 1.3 being the highest value obtained. Conclusion For the laboratory determination of steroid enantiomers, our results suggest that calculated component polarity values of the MP can be used to direct laboratorians for achieving desired separation of steroid enantiomers (as opposed to a trial-and-error approach for achieving the separation). This study shows that the separation of these bile acid enantiomers significantly depends on the summed π and β polarities of the MP, with poor correlation for total polarity (P) and α.

Photoselective Shade Films Mitigate Heat Wave Damage by Reducing Anthocyanin and Flavonol Degradation in Grapevine (Vitis vinifera L.) Berries

Wine grape production is challenged by forecasted increases in air temperature and droughts due to climate change and photoselective overhead shade films are promising tools in hot viticulture areas to overcome climate change related factors. The aim of this study was to evaluate the vulnerability of ‘Cabernet Sauvignon’ grape berries to solar radiation overexposure, optimize shade film use for preserving berry composition. An experiment was conducted for two years with four shade films (D1, D3, D4, D5) with differing solar radiation spectra transmittance and compared to an uncovered control (C0). Integrals for leaf gas exchange and mid-day stem water potential were unaffected by the shade films in both years. At harvest, berry primary metabolites were not affected by treatments applied in either year. Despite precipitation exclusion during the dormant seasons in shaded treatments, and cluster zone temperatures reaching 58°C in C0, yield was not affected. Berry skin anthocyanin and flavonol composition and content were measured by C18 reversed-phase HPLC. In 2020, total skin anthocyanins (mg·berry-1) in the shaded treatments were 27% greater than C0 during berry ripening and at harvest. Conversely, flavonol content in 2020 decreased in partially shaded grapevines compared to C0. Berry flavonoid content in 2021 increased until harvest while flavonol degradation was apparent from veraison to harvest in 2020 across partially shaded and control grapevines. Untreated control showed lower di- to tri-hydroxylated flavonol ratios closer to harvest. Our results provided evidence that overhead partial shading of vineyards mitigate anthocyanin degradation by reducing cluster zone temperatures and is a useful tool in combatting climate change in hot climate regions.

Isolation of the hemeoxygenase-1 inducer from rice-derived peptide.

Bioactive peptides with various health benefits have been reported from rice protein hydrolysates. We previously showed that rice-derived peptides (RP) increased intracellular glutathione levels and induced the expression of γ-glutamylcysteine synthetase, which is regulated by nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is an important Nrf2 downstream antioxidant enzyme that protects against oxidative stress. This study aimed to investigate the protective effects of RP on hydrogen peroxide (H2O2)-induced oxidative stress in human hepatoblastoma cell line HepG2 and identified HO-1 induced peptides from RP. Pretreatment of cells with RP reduced the cytotoxicity caused by H2O2 in a dose-dependent manner. Moreover, RP induced HO-1 expression in a concentration- and time-dependent manner. Next, we attempted to isolate the HO-1 inducer from RP by bioactivity-guided fractionation. Purification of the active peptides using a Sep-Pak C18 cartridge and reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of the three peptides. These peptides effectively reduced H2O2-induced oxidative stress. Among them, only P3 (peptide sequence: RSAVLLSH) increased HO-1 protein expression. Additionally, the knockdown of Nrf2 suppressed the induction of HO-1 expression by P3. Our results indicated that P3 identified from RP induced HO-1 by activating the Nrf2 signaling pathway.

Pharmacokinetics of genistein distribution in blood and retinas of diabetic and non-diabetic rats

Genistein, a natural tyrosine kinase inhibitor, may act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by its nonlinear pharmacokinetics. We aimed to determine genistein's kinetics and retinal tissue distributions in normal and diabetic rats. We developed an isocratic, reverse-phase C18 HPLC system to measure genistein concentration in blood and retinas of streptozotocin (65 mg/kg IV)-diabetic and non-diabetic rats receiving two types of genistein-rich diet (150 and 300 mg/kg) for ten days. Genistein's decay exhibited a two-compartmental open model. Half-lives of distribution and elimination were 2.09 and 71.79 min, with no difference between groups. Genistein steady-state concentration in blood for 150 and 300 mg/kg diet did not differ between diabetic (0.259 ± 0.07 and 0.26 ± 0.06 μg/ml) and non-diabetic rats (0.192 ± 0.05 and 0.183 ± 0.09 μg/ml). In retina, genistein concentration was significantly higher in diabetic rats (1.05 ± 0.47 and 0.997 ± 0.47 μg/gm wt. vs. 0.087 ± 0.11 and 0.314 ± 0.18 μg/gm wt., p < 0.05). The study determined that increasing genistein dose did not change its bioavailability, perhaps due to the poor aqueous solubility. The retina's increased genistein could be due to increased permeability of blood-retinal barrier that occurs early in diabetes.

Neuroprotective Effect of β-secretase Inhibitory Peptide from Pacific Hake (Merluccius productus) Fish Protein Hydrolysate.

Various methodologies have been employed for the therapeutic interpolation of the progressive brain disorder Alzheimer's disease. Thus, β-secretase inhibition is significant to prevent disease progression in the early stages. This study seeks to purify and characterize a novel β-secretase inhibitory peptide from Pacific hake enzymatic hydrolysate. A potent β-secretase inhibitory peptide was isolated by sequential purifications using Sephadex G-25 column chromatography and octadecylsilane (ODS) C18 reversed-phase HPLC. A total of seven peptides were synthesized using the isolated peptide sequences. SH-SY5Y cells stably transfected with the human ''Swedish'' amyloid precursor protein (APP) mutation APP695 (SH-SY5YAPP695swe) were used as an in-vitro model system to investigate the effect of Leu-Asn peptide on APP processing. The β-secretase inhibitory activity (IC50) of the purified peptide (Ser-Leu-Ala-Phe-Val-Asp- Asp-Val-Leu-Asn) from fish protein hydrolysate was 18.65 μM and dipeptide Leu-Asn was the most potent β-secretase inhibitor (IC50 value = 8.82 µM). When comparing all the seven peptides, the inhibition pattern of Leu-Asn dipeptide was found to be competitive by Lineweaver-Burk plot and Dixon plot (Ki value = 4.24 µM). The 24 h treatment with Leu-Asn peptide in SH-SY5Y cells resulted in reducing the β-amyloid (Aβ) production in a dose-dependent manner. Therefore, the results of this study suggest that β-secretase inhibitory peptides derived from marine organisms could be potential candidates to develop nutraceuticals or pharmaceuticals as antidementia agents.

ACP-TX-I and ACP-TX-II, Two Novel Phospholipases A2 Isolated from Trans-Pecos Copperhead Agkistrodon contortrix pictigaster Venom: Biochemical and Functional Characterization.

This work reports the purification and biochemical and functional characterization of ACP-TX-I and ACP-TX-II, two phospholipases A2 (PLA2) from Agkistrodon contortrix pictigaster venom. Both PLA2s were highly purified by a single chromatographic step on a C18 reverse phase HPLC column. Various peptide sequences from these two toxins showed similarity to those of other PLA2 toxins from viperid snake venoms. ACP-TX-I belongs to the catalytically inactive K49 PLA2 class, while ACP-TX-II is a D49 PLA2, and is enzymatically active. ACP-TX-I PLA2 is monomeric, which results in markedly diminished myotoxic and inflammatory activities when compared with dimeric K49 PLA2s, confirming the hypothesis that dimeric structure contributes heavily to the profound myotoxicity of the most active viperid K49 PLA2s. ACP-TX-II exhibits the main pharmacological actions reported for this protein family, including in vivo local myotoxicity, edema-forming activity, and in vitro cytotoxicity. ACP-TX-I PLA2 is cytotoxic to A549 lung carcinoma cells, indicating that cytotoxicity to these tumor cells does not require enzymatic activity.

Anthranilic Acid as a Versatile Fluorescent Tag and Linker for Functional Glycomics.

The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.

Bioassay-Guided Fractionation of Wound Healing Active Compounds From Piper nigrum L. Berries Extract in Malaysia

The study aimed to further evaluate the wound healing property of P. nigrum L. using bioassay-guided fractionation method. The ethanolic extract of Piper nigrum L. was fractionated in two stages using column chromatography and preparative reversed phase C18 HPLC, respectively. Significant wound healing properties screened using the scratch wound assay was observed through the cell migration assay in fraction number three (PNE3) out of the 14 fractions, exhibiting 36.7% and 43.8% closure of wound gap within 20 hours at concentration of 0.3 and 1.0 µg/mL, respectively. Sub-fractions which were further fractionated from PNE3 showed comparatively reduced wound healing activity using the same bioassay. The sub-fractions were also compared to Piperine, a major component of P. nigrum L. and the results were comparable. This experimental study revealed that Piperine works together with other compounds in P. nigrum L. to improve wound healing as claimed by those home remedy.

Liquid chromatography–tandem MS/MS method for simultaneous quantification of paracetamol, chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study

A facile, fast and specific method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid-liquid extraction. The analysis was performed on a reversed-phase C18 HPLC column (5 μm, 4.6 × 50 mm) using acetonitrile-10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 μL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple-reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03-30.0, 0.015-15.00 and 0.15-15.00 μg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers.

A Novel Peptide from Vespa ducalis Induces Apoptosis in Osteosarcoma Cells by Activating the p38 MAPK and JNK Signaling Pathways.

Osteosarcoma (OS) is a typical bone cancer, and most frequently used cancer treatments for OS are limited due to severe drug-related toxicities. Wasp venoms contain functional components that may offer pharmaceutical components for the treatment of cancers. This study aimed to isolate and characterize a novel peptide (venom anti-cancer peptide 1, VACP1) derived from the wasp venom of Vespa ducalis SMITH. Toxins from Vespa ducalis crude venom were separated by gel filtration and purified by C18 reverse-phase HPLC. As examined by Edman degradation, the amino acid sequence of VACP1 is AQKWLKYWKADKVKGFGRKIKKIWFG. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that VACP1 inhibited the cell proliferation of MG-63, U-2 OS and Saos-2 cells. Furthermore, annexin V and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining revealed that VACP1 could induce the apoptosis of OS cell lines. In addition, VACP1 increased the protein levels of cleaved poly ADP-ribose polymerase (PARP), caspase 3, but decreased B-cell lymphoma 2 (Bcl-2). Apoptotic signaling pathway screening in MG-63 cells via an antibody array revealed that VACP1 activated the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. The present study demonstrates that VACP1 potently suppressed cell proliferation and induced the cell apoptosis of OS cells by inducing the activation of the p38 MAPK and JNK signaling pathways, suggesting that VACP1 is a promising agent for OS therapy.

Isolation of the New Antiplasmodial Butanolide, Malleastrumolide A, from Malleastrum sp. (Meliaceae) from Madagascar.

An extract of Malleastrum sp. (Meliaceae) collected in Madagascar by the Madagascar International Cooperative Biodiversity Group was found to have antimalarial activity, with an IC50 value between 2.5 and 5μgml-1 . After purification by liquid-liquid partition, chromatography on a Diaion open column, C18 SPE and C18 reversed phase HPLC, the new butanolide, malleastrumolide A, was isolated. The structure of malleastrumolide A was determined by mass spectrometry, NMR, and ECD. The double bond position was determined by cross-metathesis and mass spectrometry. The compound has antiproliferative activity against the A2780 ovarian cancer cell line with an IC50 value of 17.4μm and antiplasmodial activity against the drug-resistant Dd2 strain of Plasmodium falciparum with an IC50 value of 2.74μm.

Analysis of iodinated quorum sensing peptides by LC–UV/ESI ion trap mass spectrometry

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%).

Antiplasmodial Sesquiterpenoid Lactones from Trichospira verticillata: Structure Elucidation by Spectroscopic Methods and Comparison of Experimental and Calculated ECD Data.

A dichloromethane extract of Trichospira verticillata from the Natural Products Discovery Institute was discovered to have good antiplasmodial activity (IC50 ∼5 μg/mL). After purification by liquid-liquid partition and C18 reversed-phase HPLC, four new germacranolide-type sesquiterpenoid lactones named trichospirolides A-D (1-4) were isolated. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and MS data. The relative and absolute configurations were assigned based on a comparison of calculated and experimental ECD and UV spectra, specific rotations, internuclear distances, and coupling constants for all possible diastereomers for each compound. Among these four compounds, the conjugated dienone 1 displayed the most potent antiplasmodial activity, with an IC50 value of 1.5 μM.

Microcystin-LRDetectedinaLowMolecularWeight FractionfromaCrudeExtractofZoanthussociatus.

Cnidarian constitutes a great source of bioactive compounds. However, research involving peptides from organisms belonging to the order Zoanthidea has received very little attention, contrasting to the numerous studies of the order Actiniaria, from which hundreds of toxic peptides and proteins have been reported. In this work, we performed a mass spectrometry analysis of a low molecular weight (LMW) fraction previously reported as lethal to mice. The low molecular weight (LMW) fraction was obtained by gel filtration of a Zoanthus sociatus (order Zoanthidea) crude extract with a Sephadex G-50, and then analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) in positive ion reflector mode from m/z 700 to m/z 4000. Afterwards, some of the most intense and representative MS ions were fragmented by MS/MS with no significant results obtained by Protein Pilot protein identification software and the Mascot algorithm search. However, microcystin masses were detected by mass-matching against libraries of non-ribosomal peptide database (NORINE). Subsequent reversed-phase C18 HPLC (in isocratic elution mode) and mass spectrometry analyses corroborated the presence of the cyanotoxin Microcystin-LR (MC-LR). To the best of our knowledge, this finding constitutes the first report of MC-LR in Z. sociatus, and one of the few evidences of such cyanotoxin in cnidarians.

Development and Validation of a Bioanalytical method for the Determination of Levamisole Residue in Backyard Poultry Egg

Development of a bioanalytical method is often tricky and labour intensive due to its nature or the criteria set by regulatory bodies. In the present study, a simple, reliable and sensitive analytical method was developed for the determination of levamisole residue in chicken eggs. Levamisole and ronidazole (internal standard, IS) were separated on a C18 reversed-phase HPLC column. Following liquid-liquid extraction, chromatographic separation was accomplished with a mobile phase consisting of acetonitrile: 0.05M KH2PO4 at ratio of 20: 80, and the drug was detected at 235nm using a UV detector at a flow rate of 1.0ml/min at the ambient temperature. Linearity was obtained over the range 0.3125–10.0μg/ml for levamisole hydrochloride with lower limit of quantitation of 0.3125μg/ml. For each level of quality control samples, inter-and intra-day precision (%CV) was within ±15%. Absolutes extraction recovery of drug from plasma was ≥;60%. The residue limit of levamisole gradually increased from day 1 to day 3 and then declined from day4 to day 7. The highest level of residue after oral administration of 80mg/kg levamisole to chickens was on day 3 (0.599μg/g). This level was lower than the recommended maximum residue limit (MRL), thus, no withholding period was required for egg consumption following the administration of the levamisole solution.

Evaluation of sulfur isotopic enrichment of urine metabolites for the differentiation of healthy and prostate cancer mice after the administration of 34S labelled yeast

Sulfur isotopic enrichment of urine metabolites in healthy and prostate cancer mice using 34S enriched yeast and High Performance Liquid Chromatography coupled to Multicollector Inductively Coupled Plasma Mass Spectrometry (HPLC-MC-ICP-MS) has been evaluated. A 30 weeks experiment (since the eleventh to the fortieth week of life) was carried out collecting the urine of three healthy mice and three transgenic mice with prostate cancer during 24h after a single oral administration of a 34S enriched yeast slurry. The isotopic enrichment of different sulphur metabolites was monitored by coupling a C18 reverse phase HPLC column with a multicollector ICP-MS using a membrane desolvating system. Quantification of sulfur in the chromatographic peaks was carried out by post-column isotope dilution using a 33S enriched spike. Differences between the 34S enrichment in the urine metabolites of healthy and prostate cancer mice were found from the beginning of the disease. Both populations could be differentiated using a principal component analysis (PCA). Finally, 7 unknown mice were correctly classified in each population using a linear discriminant analysis.

메기의 껍질로부터 항균성 펩타이드의 정제

An antibacterial peptide from skin extract of the catfish Silurus asotus was purified and characterized. The acidified skin extract was put through a Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient and divided into flow-through (F.T.), 10% methanol-elute (RM10), 60% methanol- elute (RM60), and 100% methanol-elute (RM100) fractions. RM10, RM60, and RM 100 showed anti-microbial activity against Escherichia coli D31. On the other hand, the F.T. fraction did not show anti-microbial activity. Among the various fractions, RM 60 had the highest activity. RM 60 was partially purified on a cation exchange column (CM52) by a stepwise gradient. The ammonium acetate (pH 5.15) 0.02 M – 0.8 M fraction showed antimicrobial activity. Then an antimicrobial peptide was puri-fied using a 0.6M fraction with strong antibacterial activity through a series of five C18 reversed-phase HPLC columns. For the characterization of the purified peptide, the molecular weight and amino acid sequence were analyzed by MALDI-TOF MS and Edman degradation. The molecular weight of this peptide was about 4182.1 [M+H]

Quantitative Assay Validation for Oxandrolone in Human Plasma Using LC–MS-MS

A high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of oxandrolone concentration in human plasma (0.5 mL) was developed and validated according to the 2001 FDA Bioanalytical Guidelines. Oxandrolone is an anabolic steroid used to promote weight gain for cachectic patients with severe burn injuries, HIV/AIDS, hepatitis C and other wasting syndromes. The assay procedure involved a liquid-liquid extraction of oxandrolone and methyltestosterone (the internal standard, IS) from plasma with n-butyl chloride. The organic layer was clarified by centrifugation and evaporated to dryness under a stream of air. The residue was reconstituted in a solution containing 25% methanol and 75% Milli-Q water, and injected onto a Luna C18 reversed-phase HPLC column (30 mm × 2.0 mm, 2 μm). Separation of oxandrolone and methyltestosterone was achieved with a mobile phase starting composition of 55% methanol and 45% ammonium formate buffer at a flow rate of 0.1 mL/min. The total run time was 21 min per sample. Selected reaction monitoring mode was used for quantifying oxandrolone (m/z 307 → 271) and the IS, methyltestosterone (m/z 301 → 149). To the authors' knowledge, this is the first LC-MS-MS method validated for oxandrolone quantification in human plasma. This method can be used in future pharmacokinetic studies involving oxandrolone.

Characterization of β-secretase inhibitory peptide purified from skate skin protein hydrolysate

The objective of this study was to purify and characterize the β-secretase inhibitor from enzymatic hydrolysates of skate skin, for the development of a novel antidementia agent that may be utilized in the drug or functional food industries. β-secretase inhibitory peptide was purified from various enzymatic hydrolysates of skate skin. Among six enzymatic hydrolysates, the Neutrase hydrolysate showed the highest β-secretase inhibitory activity. Consecutive purification of the skate skin hydrolysate using Sephadex G-25 column chromatography and octadecylsilane C18 reversed phase HPLC techniques was used to isolate a potent β-secretase inhibitory peptide composed of 12 amino acids, Gln–Gly–Try–Arg–Pro–Leu–Arg–Gly–Pro–Glu–Phe–Leu (MW: 1,391 Da). The purified peptide had strong β-secretase inhibitory activity, with IC50 value of 24.26 μM, and displayed a non-competitive mode of inhibition. Among the synthesized β-secretase inhibitory peptides, the tetrapeptide Pro–Glu–Phe–Leu had the highest β-secretase inhibitory activity. The result of this study suggests that the β-secretase inhibitory peptide derived from skate skin could be potential candidates to develop nutraceuticals and pharmaceuticals.

A Simple HPLC Bioanalytical Method for the Determination of Doxorubicin Hydrochloride in Rat Plasma: Application to Pharmacokinetic Studies

Purpose: To develop a simple, accurate, and precise high performance chromatography (HPLC) method with spectrophotometric detection for the determination of doxorubicin hydrochloride in rat plasma. Methods: Doxorubicin hydrochloride and daunorubicin hydrochloride (internal standard, IS) were separated on a C18 reversed-phase HPLC column. Following protein precipitation extraction, chromatographic separation was accomplished with a mobile phase consisting of acetonitrile: water at ratio of 30:70 (pH 3.0), and the drug was detected at 233 nm using a UV detector at flow rate of 1.0 ml/min and ambient temperature. Results: Linearity was obtained over the range 1.0 – 50.0 μg/ml for doxorubicin hydrochloride with lower limit of quantitation of 1.0 μg/ml. For each level of quality control samples, inter- and intra-day precision (% CV) was < 9.6 and 5.1 %, respectively. Stability of doxorubicin hydrochloride in plasma was within the acceptance limit (± 15 %) with no evidence of degradation during sample processing and 30 days storage in a deep freezer at -70 ± 5 °C. Absolutes extraction recovery of drug from plasma was ≥ 86 %. Conclusion: The method is highly selective and rugged for the determination of doxorubicin hydrochloride in rat plasma and should be suitable for conducting pharmacokinetic studies and therapeutic drug monitoring.