不同来源高山红景天材料中有效成分的HPLC分析

本文用HPLC测定、比较了5种不同来源的高山红景天材料中有效成分—红景天甙及其甙无酪醇的含量,并建立了高山红景天有效成分分析的样品制备方法,结果表明:固体培养、液体培养及反应器培养的高山红景天组织中均含有上述两种有效成分.其中固体培养的材料中有效成分的含量最高.三种提取方法比较表明,用索氏提取器连续回流提取明显优于冷浸提取和批式回流提取.     

Interaction of the chemopreventive agent resveratrol and its metabolite,piceatannol, with model membranes

Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer.     

Effect of long-term piceatannol treatment on eNOS levels in cultured endothelial cells

Piceatannol (3, 3′, 4, 5′-tetrahydroxy-trans-stilbene) is a naturally occurring phytochemical found in passion fruit (Passiflora edulis) seeds. Previously, we demonstrated that piceatannol has acute vasorelaxant effects in rat thoracic aorta. It was suggested that endothelial NO synthase (eNOS) might be involved in piceatannol-induced acute vasorelaxation. Here, we investigated the expression of eNOS in EA.hy926 human umbilical vein cells after long-term treatment with piceatannol, and compared this effect with that of resveratrol, an analog of piceatannol. Long-term treatment with piceatannol up-regulated eNOS mRNA expression and increased eNOS protein expression in a dose-dependent manner. Moreover, piceatannol increased the levels of phosphorylated eNOS. Treatment with resveratrol also increased eNOS expression, but to a lesser degree than piceatannol. These findings indicate that piceatannol may improve vascular function by up-regulating eNOS expression.     

Inhibition of ATPase activity of Escherichia coli ATP synthase by polyphenols

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (～0 residual activity), inhibition by other compounds was partial with ～20% residual activity by quercetin, ～50% residual activity by quercetin-3-β-d glucoside, and ～60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC₅₀ ～14 μM) followed by quercetin (IC₅₀ ～33 μM), quercetin-3-β-d glucoside (IC₅₀ ～71 μM), resveratrol (IC₅₀ ～94 μM), quercitrin (IC₅₀ ～120 μM). Inhibition was identical in both F₁F_o membrane preparations as well as in isolated purified F₁. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.     

Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism.     

A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC‐MS/MS

Introduction – Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC‐MS/MS after sample extraction and preparation from ion‐exchange resins. Objective – To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid‐phase extraction equipment or procedures which significantly reduce sample preparation time. Methodology – A previously reported and validated sample preparation method using ion‐exchange resin was compared with methods using a commercially available solid‐phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC‐MS/MS. The measured concentration of swainsonine was then statistically compared between methods. Results – There were no statistically significant differences found between four of the five different sample preparation methods tested. Conclusion – A commercially available SPE cartridge can be used to replace the previously used ion‐exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation. Published in 2010 by John Wiley & Sons, Ltd.     

Piceatannol, natural polyphenolic stilbene, inhibits adipogenesis via modulation of mitotic clonal expansion and insulin receptor-dependent insulin signaling in early phase of differentiation

Piceatannol, a natural stilbene, is an analog and a metabolite of resveratrol. Despite a well documented health benefit of resveratrol in intervention of the development of obesity, the role of piceatannol in the development of adipose tissue and related diseases is unknown. Here, we sought to determine the function of piceatannol in adipogenesis and elucidate the underlying mechanism. We show that piceatannol inhibits adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner at noncytotoxic concentrations. This anti-adipogenic property of piceatannol was largely limited to the early event of adipogenesis. In the early phase of adipogenesis, piceatannol-treated preadipocytes displayed a delayed cell cycle entry into G(2)/M phase at 24 h after initiation of adipogenesis. Furthermore, the piceatannol-suppressed mitotic clonal expansion was accompanied by reduced activation of the insulin-signaling pathway. Piceatannol dose-dependently inhibited differentiation mixture-induced phosphorylation of insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/Akt pathway in the early phase of adipogenesis. Moreover, we showed that piceatannol is an inhibitor of IR kinase activity and phosphatidylinositol 3-kinase (PI3K). Our kinetics study of IR further identified a K(m) value for ATP of 57.8 μm and a K(i) value for piceatannol of 28.9 μm. We also showed that piceatannol directly binds to IR and inhibits IR kinase activity in a mixed noncompetitive manner to ATP, through which piceatannol appears to inhibit adipogenesis. Taken together, our study reveals an anti-adipogenic function of piceatannol and highlights IR and its downstream insulin signaling as novel targets for piceatannol in the early phase of adipogenesis.     

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.     

Biocatalytic synthesis of 3,4,5,3′,5′-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3′,5′-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100?mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9?mM (1.8?g L^?1) in 48?h.     

Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells

Neuronal cell death caused by oxidative stress is common in a variety of neural diseases and can be investigated in detail in cultured HT22 neuronal cells, where the amino acid glutamate at high concentrations causes glutathione depletion by inhibition of the glutamate/cystine antiporter system, intracellular accumulation of reactive oxygen species (ROS) and eventually oxidative stress-induced neuronal cell death. Using this paradigm, we have previously reported that resveratrol (3,5,4′-trans-trihydroxystilbene) protects HT22 neuronal cells from glutamate-induced oxidative stress by inducing heme oxygenase (HO)-1 expression. Piceatannol (3,5,4′,3′-trans-trihydroxystilbene), which is a hydroxylated resveratrol analog and one of the resveratrol metabolites, is estimated to exert neuroprotective effect similar to that of resveratrol. The aim of this study, thus, is to determine whether piceatannol, similarly to resveratrol, would protect HT22 neuronal cells from glutamate-induced oxidative stress. Glutamate at high concentrations induced neuronal cell death and ROS formation. Piceatannol reduced glutamate-induced cell death and ROS formation. The observed cytoprotective effect was much higher when HT22 neuronal cells were pretreated with piceatannol for 6 or 12 h prior to glutamate treatment than when pretreated for 0.5 h. Piceatannol also increased HO-1 expression and HO activity via its activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, neuroprotective effect of piceatannol was partly (but not completely) abolished by either down-regulation of HO-1 expression or blockage of HO-1 activity. Taken together, our results suggest that piceatannol, similar to resveratrol, is capable of protecting HT22 neuronal cells against glutamate-induced cell death, at least in part, by inducing Nrf2-dependent HO-1 expression.     

Synthesis and evaluation of water-soluble resveratrol and piceatannol via BGLation

Synthesis of four water-soluble resveratrol and piceatannol derivatives bearing symmetrically branched glyceryl trimer (BGL003) with a non-biocleavable linkage, and their biological evaluation as a mitochondrial fusion-inducing agent with cellular fat-reducing effect from cells, is described. The effect of Piceatannol-BGL003 conjugate was as high as that of original stilbenoids.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

Solid-phase extraction–high-perfomance liquid chromatography determination of verapamil and norverapamil enantiomers in urine

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C₈ or C₁₈ bonded phase (C₈ in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5–300 ng/ml. Intra-day and inter-day reproducibilities were 10.5–14.2% at 3 ng/ml, 4.8–9.3% at 138.5 ng/ml and 7.8–10.1% at 280 ng/ml level, respectively, for all the enantiomers.     

α-Glucosidase inhibitory effect of resveratrol and piceatannol

Dietary polyphenols have been shown to inhibit α-glucosidase, an enzyme target of some antidiabetic drugs. Resveratrol, a polyphenol found in grapes and wine, has been reported to inhibit the activity of yeast α-glucosidase. This triggered our interest to synthesize analogs and determine their effect on mammalian α-glucosidase activity. Using either sucrose or maltose as substrate resveratrol, piceatannol and 3′-hydroxypterostilbene showed strong inhibition of mammalian α-glucosidase activity; pinostilbene, cis-desoxyrhapontigenin and trans-desoxyrhapontigenin had moderate inhibition. Compared to acarbose (IC₅₀ 3–13 μg/ml), piceatannol and resveratrol inhibited mammalian α-glucosidase to a lesser extent (IC₅₀ 14–84 and 111–120 μg/ml, respectively). 3′-Hydroxypterostilbene (IC₅₀ 105–302 μg/ml) was 23–35-fold less potent than acarbose. We investigated the effect of piceatannol and resveratrol on postprandial blood glucose response in high-fat-fed C57Bl/6 mice. Animals administered resveratrol (30 mg/kg body weight [BW]) or piceatannol (14 mg/kg BW) 60 min prior to sucrose or starch loading had a delayed absorption of carbohydrates, resulting in significant lowering of postprandial blood glucose concentrations, similar to the antidiabetic drug acarbose, while no significant effect was observed with the glucose-loaded animals. Our studies demonstrate that the dietary polyphenols resveratrol and piceatannol lower postprandial hyperglycemia and indicate that inhibition of intestinal α-glucosidase activity may be a potential mechanism contributing to their antidiabetic property.     

Supercritical fluid extraction of selected pharmaceuticals from water and serum

Selected drugs from benzodiazepine, anabolic agent and non-steroidal anti-inflammatory drug (NSAID) therapeutic classes were extracted from water and serum using a supercritical CO₂ mobile phase. The samples were extracted at a pump pressure of 329 MPa, an extraction chamber temperature of 45°C, and a restrictor temperature of 60°C. The static extraction time for all samples was 2.5 min and the dynamic extraction time ranged from 5 to 20 min. The analytes were collected in appropriate solvent traps and assayed by modified literature HPLC procedures. Analyte recoveries were calculated based on peak height measurements of extracted vs. unextracted analyte. The recovery of the benzodiazepines ranged from 80 to 98% in water and from 75 to 94% in serum. Anabolic drug recoveries from water and serum ranged from 67 to 100% and 70 to 100%, respectively. The NSAIDs were recovered from water in the 76 to 97% range and in the 76 to 100% range from serum. Accuracy, precision and endogenous peak interference, if any, were determined for blank and spiked serum extractions and compared with classical sample preparation techniques of liquid-liquid and solid-phase extraction reported in the literature. For the benzodiazepines, accuracy and precision for supercritical fluid extraction (SFE) ranged from 1.95 to 3.31 and 0.57 to 1.25%, respectively (n=3). The SFE accuracy and precision data for the anabolic agents ranged from 4.03 to 7.84 and 0.66 to 2.78%, respectively (n=3). The accuracy and precision data reported for the SFE of the NSAIDs ranged from 2.79 to 3.79 and 0.33 to 1.27%, respectively (n=3). The precision of the SFE method from serum was shown to be comparable to the precision obtained with other classical preparation techniques.     

Piceatannol is more effective than resveratrol in restoring endothelial cell dimethylarginine dimethylaminohydrolase expression and activity after high-glucose oxidative stress

Glucose-induced oxidative stress is involved in endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) and arginase are regulators of the endothelial NO synthase (eNOS). This study aimed to compare the effect of two polyphenolic antioxidants, resveratrol and piceatannol, on DDAH and arginase pathways in bovine aortic endothelial cells under 25 mM glucose for 24 h. DDAH activity and expression were decreased in these cells as compared to control cells, whereas arginase activity was unchanged. DDAH inhibition led to intracellular accumulation of asymmetric dimethylarginine (ADMA), a natural inhibitor of eNOS. Under these conditions, cell pre-treatment with resveratrol (0.1-10 μM) restored basal DDAH activity and ADMA level with a dose-dependent effect. Piceatannol acted as resveratrol on DDAH pathway but at 10-fold lower concentrations. Resveratrol and piceatannol restored DDAH activity even in the presence of splitomicin, a specific inhibitor of Sirtuin 1. These results suggest potential therapeutic intervention targeting resveratrol or piceatannol administration to improve endothelial dysfunction.     

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.     

Determination of paclitaxel in mouse plasma and brain tissue by liquid chromatography-mass spectrometry

Paclitaxel is an anticancer agent extracted from the bark of the yew tree and is widely used in chemotherapy for solid tumors, including non-small cell lung cancer and ovarian carcinoma. Most assays to measure paclitaxel in plasma require a large amount of sample (0.4-1 ml) to achieve the necessary sensitivity, and are not suitable when only small sample sizes are available. To circumvent this latter limitation, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) method for the determination of paclitaxel in plasma based on the use of small sample volumes (50 microl plasma). A solid phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of paclitaxel were 98 and 83% from plasma and brain tissues, respectively. The mobile phase consisted of 50% acetonitrile in 0.1% formic acid that was pumped at 0.2 ml/min to yield a retention time for paclitaxel of 6.2 and 5.4 min for cephalomannine, the internal standard. The method has been validated at paclitaxel plasma concentrations from 0.036 to 9.9 microg/ml, and from 0.054 to 1.96 microg/ml in brain homogenates. A sensitive and specific assay for paclitaxel has been developed that has the advantages of using small sample sizes, and a single extraction step without solvent evaporation.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.