Pharmacokinetic study of nobiletin and tangeretin in rat serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry

Nobiletin (NOB) and tangeretin (TAN), two of the main polymethoxylated flavones (PMFs) in citrus, influence a number of key biological pathways in mammalian cells. Although the impacts of NOB and TAN on glucose homeostasis and cholesterol regulation have been investigated in human clinical trials, much information is still lacking about the metabolism and oral bioavailability of these compounds in animals. In this study, NOB and TAN were administered to rats by gavage and intraperitoneal (ip) injection, and the blood serum concentrations of these compounds and their main metabolites were monitored by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). In addition to the administered compounds, two metabolites of TAN and eight metabolites of NOB were detected and measured over 24 h. With identical oral doses, nearly 10-fold higher absorption of NOB occurred compared to TAN. For both compounds, maximum levels of glucuronidated metabolites occurred in the blood serum at later time points (～5-8 h) compared to the earlier T(max) values for NOB and TAN. In most cases the glucuronides occurred at substantially higher concentrations than the aglycone metabolites. Low levels of NOB and TAN and their metabolites were detectable in rat blood serum even at 24 h after treatment.     

Polymethoxylated flavones and other phenolic derivates from citrus in their inhibitory effects on P-glycoprotein-mediated transport of talinolol in Caco-2 cells

Many studies investigating drug interactions with citrus compounds focus on the major grapefruit furanocoumarins bergamottin, dihydroxybergamottin, and the flavonoid naringenin. This study evaluated the influence of polymethoxylated flavones (PMFs), tangeretin, nobiletin, 3,5,6,7,8,3,4'-heptamethoxyflavone, and sinensetin, as well as other minor occurring citrus phenols, hesperetin, limettin, 7-OH-coumarin, 7-geranyloxycoumarin, and eriodictyol, on P-glycoprotein-mediated transport of the beta-blocker talinolol using the Caco-2 cell monolayer model and was used to determine the structure-function aspects of the interaction. The transport of talinolol across Caco-2 cells monolayers was determined in the absence and presence of distinct concentrations of the calcium-channel blocker verapamil (a known inhibitor of P-glycoprotein) and citrus compounds. A sigmoid dose-response model was used to fit the data and to estimate the IC50 values of the potential inhibitors. Results from this study show that PMFs significantly decreased talinolol transport from the basolateral to apical side, where tangeretin had the lowest IC50 of 3.2 micromol/L, followed by nobiletin, heptamethoxyflavone, and sinensetin with IC50 values of 3.5, 3.8, and 3.9 micromol/L, respectively. However, the efficacy of the compounds did not appear to be dependent on the number of methoxy groups. Other citrus compounds did not have any significant effect on the transport of talinolol. This study suggests that PMFs have a high potential in the interaction with P-gp-mediated talinolol transport in Caco-2 cells. Based on their relatively low concentrations (< or =3 microg/mL) in citrus, the clinical relevance of these interactions needs to be further elucidated in in vivo studies.     

The Composition of Volatile Aroma Components, Flavanones, and Polymethoxylated Flavones in Shiikuwasha (Citrus depressa Hayata) Peels of Different Cultivation Lines

Citrus peels are important sources of various pleasant aroma compounds and valuable bioactive substances. To investigate differences in the composition and content of Shiikuwasha (Citrus depressa Hayata) peels from different cultivation lines, the composition of volatile aroma components, flavanones, and polymethoxylated flavones (PMFs) in four Shiikuwasha cultivation lines was examined. The composition of volatile aroma components in cold-pressed extracts of Shiikuwasha peels was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectrophotometry. The extracts contained mainly monoterpene hydrocarbons (93.40-97.25%), including limonene (46.52-68.26%) and γ-terpinene (21.48-30.52%). Differences in the composition of volatile aroma compounds in the Shiikuwasha cultivation lines were revealed using principal component analysis. Additionally, the composition of flavanones and PMFs was determined using high-performance liquid chromatography methods. Neohesperidin (96.58%) was the predominant flavanone in 'Izumi kugani' peel, while the other peels had high hesperidin contents (89.26-98.66%). Moreover, the PMFs of Shiikuwasha peels were composed of nobiletin (56.74-64.77%) and tangeretin (23.17-34.70%).     

Molecular structures of citrus flavonoids determine their effects on lipid metabolism in HepG2 cells by primarily suppressing apoB secretion

This study investigated the underlying mechanisms of action for blood lipid lowering effects of citrus flavonoids and their methoxylated analogues (n = 19; dose range: 0-100 μM) in HepG2 cells. Cholesterol (CH) and triglyceride (TG) syntheses were assessed by measuring the incorporation of (14)C-acetate and (14)C-glycerol, respectively, whereas apoB secretion was determined by ELISA. Results show that two polymethoxylated citrus flavonoids (PMFs), tangeretin and nobiletin, potently inhibited apoB secretion (IC(50) = 13 and 29 μM, respectively) and modestly inhibited CH synthesis (IC(50) = 49 and 68 μM) and TG synthesis (IC(50) = 14 and 73 μM), without effecting LDL-receptor activity. Other PMFs (e.g., sinensetin) and non-PMFs (e.g., hesperetin and naringenin) had only weak effects on CH and TG syntheses and apoB secretion (IC(50) > 100 μM). The structure-activity analysis indicated that a fully methoxylated A-ring of the flavonoid structure was associated with a potent inhibitory activity on hepatic apoB secretion. In conclusion, this study using HepG2 cells indicates that citrus flavonoids with a fully methoxylated A-ring may lower blood CH and TG concentrations primarily by suppressing hepatic apoB secretion as a main underlying mode of action.     

Fourier transform infrared spectroscopic analysis of the polymethoxylated flavone content of orange oil residues

Preliminary studies have shown that orange peel polymethoxylated flavones (PMFs) exhibit beneficial biological properties in animals. These properties have increased the demands for these compounds as candidate nutraceuticals and specialty food ingredients. Orange oil residues are a likely commercial source of the PMFs, and a rapid, solvent-free method for the analysis of the PMFs in orange oil residues has been developed based on Fourier transform infrared (FTIR) spectroscopy. The intensities of the FTIR vibrations of the phenyl ring nu(C=C) stretch at 1515 cm(-1) of the PMFs can be used, relative to the intensity of the carbonyl stretch at 1733 cm(-1) of the non-PMF orange oil residue components, to measure PMF content. Excellent correlations for the ratios of the intensities of these vibrations and the total PMF content were observed irrespective of the source, viscosity, and presence of particulate material. The detection limit by this method is approximately 0.1% PMF.     

Variation in major antioxidants and total antioxidant activity of Yuzu (Citrus junos Sieb ex Tanaka) during maturation and between cultivars

Epidemiological studies suggest that a high consumption of fruits can reduce the risk of some cancers and cardiovascular disease, and this may be attributable to the antioxidant activity of vitamins and phenolic compounds. The present study investigated the variations in vitamin C, total phenolic, hesperidin, and naringin contents, and total antioxidant activity of yuzu (Citrus junos Sieb ex Tanaka)-which is a popular citrus fruit in Korea and Japan-between cultivars and during maturity. The amounts of phenolics and vitamin C and the antioxidant activity in all tested yuzu cultivars were higher in peel than in flesh. Ripening increased the total antioxidant activity and vitamin C content in both peel and flesh of yuzu. However, the amounts of all total phenolics, hesperidin, and naringin in peel increased with ripening, whereas they decreased slightly in flesh. There was a highly linear relationship between the vitamin C content and the total antioxidant activity in both peel (r(2) = 1.000) and flesh (r(2) =0.998), suggesting that vitamin C plays a key role in the antioxidant activity of yuzu. In addition, the contribution of each antioxidant to the total antioxidant activity of yuzu was determined using a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay and is expressed here in terms of the vitamin C equivalent antioxidant capacity (VCEAC). The means of vitamin C, naringin, and hesperidin in yuzu were 90.4, 63.8, and 65.7 mg/100 g fresh yuzu, respectively. The relative VCEAC values of these compounds were in the following order: vitamin C (1.00) > naringin (0.195) > hesperidin (0.162). Therefore, the estimated contribution of each antioxidant to the total antioxidant capacity of 100 g of fresh yuzus is as follows (in mg of VCEAC): vitamin C (90.36 mg) > naringin (12.44 mg) > hesperidin (10.64 mg). Our results indicate that mature yuzu contains higher amounts of vitamin C and phenolics than other citrus fruits and could therefore be used as a significant dietary source of antioxidants.     

Mass spectrometric identification and high-performance liquid chromatographic determination of a flavonoid glycoside naringin in human urine

The aim of this study was to evaluate the absorption of a citrus flavonoid, naringin, as its glycosylated form. Six healthy volunteers (three males and three females) were studied. After a single oral administeration of 500 mg of naringin, intact naringin was isolated from 2-4 h urine. Isolated naringin was identified by the LC/electrospray ionization mass spectrometry (ESI-MS), MS/MS, and MS/MS/MS techniques. The cumulative urinary excretion of naringin and its metabolites (naringenin and naringenin glucuronides) was determined by HPLC for 0-24 h. Approximately 0.02% of the administered dose was recovered in urine as unchanged naringin, whereas urinary recoveries of naringenin and naringenin glucuronides were approximately 0.4 and 3.6% of the administered dose, respectively. It was concluded that trace amounts of orally administered naringin can be absorbed as the glycoside. However, it is not clear whether the glycoside is cleaved before or after absorption to generate naringenin.     

Structure-activity relationship of citrus polymethoxylated flavones and their inhibitory effects on Aspergillus niger

Citrus peels are rich in polymethoxylated flavones (PMFs) and are potential sources of natural preservatives. Six PMFs extracts, isolated and purified from the peels of three mandarins (Citrus reticulata) and three sweet oranges (Citrus sinensis), were identified and quantitated. Their inhibitory effects on Aspergillus niger were evaluated using a microbroth dilution assay. The Red tangerine variety exhibited the greatest antifungal activity (MIC = 0.2 mg/mL), while Jincheng showed the lowest activity (MIC = 1.8 mg/mL). An analysis of principal components was applied to the results in order to elucidate the structure-activity relationships of the citrus PMFs. The structure-activity relationship analysis revealed that, for good inhibitory effect, the 5-OH, 3-OCH?, and 8-OCH? functionalities were essential, while the presence of 3-OH and 3'-OCH? greatly reduced inhibition. The findings of this study provide important information for the exploitation and utilization of citrus PMFs as natural biopreservatives.     

Phenols in citrus peel byproducts. Concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel molasses.

In addition to the main flavanone glycosides (i.e., hesperidin and naringin) in citrus peel, polymethoxylated flavones and numerous hydroxycinnamates also occur and are major phenolic constituents of the molasses byproduct generated from fruit processing. Although a small number of the hydroxycinnamates in citrus occur as amides, most occur as esters and are susceptible to alkaline hydrolysis. This susceptibility to alkaline hydrolysis was used in measuring the concentrations of hydroxycinnamates in citrus peel molasses. The highest concentrations of hydroxycinnamates occurred in molasses of orange [C. sinensis (L.) Osbeck] and tangerine (C. reticulata Blanco.) compared to grapefruit (C. paradisi Macf.) and lemon [C. limon (L.) Burm.]. Concentrations of two phenolic glucosides, phlorin (phloroglucinol-beta-O-glucoside) and coniferin (coniferyl alcohol-4-beta-O-glucoside), were also measured. Measurements of the polymethoxylated flavones in molasses from several tangerine and orange varieties showed that these compounds occurred in the highest amounts in Dancy tangerine, whereas samples from two other tangerine molasses contained significantly lower levels, similar to those in the molasses samples from late- and early/mid-season oranges.     

Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach

In recent years much attention has been focused on the pharmaceutical relevance of bioflavonoids, especially hesperidin and its aglycon hesperetin in terms of their antioxidant and anti-inflammatory actions. However, the bioactivity of their metabolites, the real molecules in vivo hesperetin glucuronides/sulfates produced after ingestion, has been poorly understood. Thus, the study using an ex vivo approach is aimed to compare the antioxidant and anti-inflammatory activities of hesperidin/hesperetin or hesperetin metabolites derived from hesperetin-administered rat serum. We found that hesperetin metabolites (2.5-20 μM) showed higher antioxidant activity against various oxidative systems, including superoxide anion scavenging, reducing power, and metal chelating effects, than that of hesperidin or hesperetin. The data also showed that pretreatment of hesperetin metabolites (1-10 μM) within the range of physiological concentrations, compared to hesperetin, significantly inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as evidenced by the inhibition of their precursors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels without appreciable cytotoxicity on LPS-activated RAW264.7 macrophages or A7r5 smooth muscle cells. Concomitantly, hesperetin metabolites dose-dependently inhibited LPS-induced intracellular reactive oxygen species (ROS). Furthermore, hesperetin metabolites significantly downregulate LPS-induced nuclear factor-κB (NF-κB) activation followed by the suppression of inhibitor-κB (I-κB) degradation and phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and p38 MAPKs after challenge with LPS. Hesperetin metabolites ex vivo showed potent antioxidant and anti-inflammatory activity in comparison with hesperidin/hesperetin.     

Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells

The citrus flavanones hesperidin, hesperetin, and neohesperidin are known to exhibit antioxidant activities and could traverse the blood-brain barrier. H2O2 formation induces cellular oxidative stress associated with neurodegenerative diseases. In this study, protective effects of pretreatments (6 h) with hesperidin, hesperetin, and neohesperidin (0.8, 4, 20, and 50 microM) on H2O2-induced (400 microM, 16 h) neurotoxicity in PC12 cells were evaluated. The results showed that hesperetin, hesperidin, and neohesperidin, at all test concentrations, significantly ( p < 0.05) inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, increased catalase activity, and attenuated the elevation of intracellular free Ca2+, the decrease of mitochondrial membrane potential (except those of 0.8 microM neohesperidin-treated cells) and the increase of caspase-3 activity in H2O2-induced PC12 cells. Meanwhile, hesperidin and hesperetin attenuated decreases of glutathione peroxidase and glutathione reductase activities and decreased DNA damage in H2O2-induced PC12 cells. These results first demonstrate that the citrus flavanones hesperidin, hesperetin, and neohesperidin, even at physiological concentrations, have neuroprotective effects against H2O2-induced cytotoxicity in PC12 cells. These dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.     

HL-60 differentiating activity and flavonoid content of the readily extractable fraction prepared from citrus juices

Citrus plants are rich sources of various bioactive flavonoids. To eliminate masking effects caused by hesperidin, naringin, and neoeriocitrin, the abundant flavonoid glycosides which make up 90% of the conventionally prepared sample, the readily extractable fraction from Citrus juice was prepared by adsorbing on HP-20 resin and eluting with EtOH and acetone from the resin and was subjected to HL-60 differentiation assay and quantitative analysis of major flavonoids. Screening of 34 Citrus juices indicated that King (C. nobilis) had a potent activity for inducing differentiation of HL-60, and the active principles were isolated and identified as four polymethoxylated flavonoids, namely, nobiletin, 3,3',4',5,6,7, 8-heptamethoxyflavone, natsudaidain, and tangeretin. HPLC analysis of the readily extractable fraction also indicated that King contained high amounts of these polymethoxylated flavonoids among the Citrus juices examined. Principal component and cluster analyses of the readily extractable flavonoids indicated peculiarities of King and Bergamot.     

Bioevaluation of human serum albumin-hesperidin bioconjugate: insight into protein vector function and conformation

Hesperidin is a flavanone glycoside widely available for dietary intake in citrus fruits or citrus fruit derived products; however, exhaustive and reliable data are scarcely available for biological activity when it exerts protective health effects in humans. The principal intent of this work is to check binding domain and structural changes of human serum albumin (HSA), the primary carrier of flavonoids, in blood plasma association with hesperidin by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) methods. From molecular modeling simulations, subdomains IIA and IIIA, which correspond to Sudlow's sites I and II, respectively, were earmarked to possess affinity for hesperidin, but the affinity of site I with flavanone is greater than that of site II. This corroborates the site-specific probe and hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results placing the hesperidin at warfarin-azapropazone and indole-benzodiazepine sites. Steady state and time-resolved fluorescence manifested that static type, due to HSA-hesperidin complex formation (1.941 × 10(4) M(-1)), is the operative mechanism for the diminution in the tryptophan (Trp)-214 fluorescence. Moreover, via alterations in three-dimensional fluorescence and CD spectral properties, we can securely draw the conclusion that the polypeptide chain of HSA is partially destabilized after conjugation with hesperidin. We anticipate that this study can provide better knowledge of bioavailability such as absorption, biodistribution, and elimination, of hesperidin in vivo, to facilitate the comprehension of the biological responses to physiologically relevant flavanones.     

Separation of flavanone-7-O-glycoside diastereomers and analysis in citrus juices by multidimensional liquid chromatography coupled with mass spectrometry

The major flavanone-7-O-glycoside constituents in citrus fruit juices (naringin, hesperidin, neohesperidin, narirutin, and eriocitrin) were separated as diastereomers by multidimensional liquid chromatography. The method consisted of coupling two HPLC columns: a reversed-phase (RP(18)) column was used for the separation of flavanone glycosides, which were, then, individually switched into a carboxymethylated beta-cyclodextrin (beta-CD)-based column and resolved as the corresponding stereoisomers. The method was used for the full analysis of flavanone glycosides in fresh hand-squeezed and commercial fruit juices by combining the quantitative estimation with the diastereomeric analysis. Quantitative data were in general consistent with previously reported data in this field. CC-LC isomer analysis was carried out by coupling the beta-CD column with a mass spectrometer operated with negative ion electrospray ionization (ESI-MS). The results showed that hesperidin was present in orange juices almost exclusively as the 2S isomer, whereas narirutin had mainly the 2R configuration. In grapefruit juices (2S)-naringin prevailed with the respect to the 2R isomer, whereas the opposite was true for narirutin. Lemon juices contained eriocitrin stereoisomers in equal amount (50% each), but hesperidin was almost exclusively found as the 2S isomer. Significant differences of the diastereomeric ratios were observed between freshly squeezed juices and juices from commercial sources.     

Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (Citrus sinensis) peel

Polymethoxyflavones (PMFs) from citrus genus have been of particular interest because of their broad spectrum of biological activities, including antiinflammatory, anticarcinogenic, and antiatherogenic properties. There have been increasing interests in the exploration of health beneficial properties of PMFs in citrus fruits. Therefore, the isolation and characterization of PMFs from sweet orange (Citrus sinensis) peel will lead to new applications of the byproducts from orange juice processes and other orange consumption in nutraceutical and pharmaceutical products. In our study, eight hydroxylated PMFs, six PMFs, one polymethoxyflavanone, one hydroxylated polymethoxyflavanone, and two hydroxylated polymethoxychalcones were isolated from sweet orange peel and their structures were elucidated by various MS, UV, and different NMR techniques. Some of the hydroxylated PMFs and chalcones are newly isolated from sweet orange peel.     

Flavonoids in pigmented orange juice and second-pressure extracts

Flavanone glycosides (FGs) and polymethoxylated flavones (PMFs) have been studied in pigmented orange (Citrus sinensis) juices and second-pressure extracts (SPEs) by high-performance liquid chromatography and diode array detector. Detection was performed simultaneously at two different wavelengths: 278 nm (for determination of FGs) and 325 nm (for determination of PMFs). Qualitative distribution patterns of FGs and PMFs in juices and SPEs were similar, although the quantitative results are quite different. An increased narirutin/hesperidin ratio after centrifugation and the presence of high amounts of PMFs in SPEs, which remain unchanged after centrifugation, were observed. Therefore, a simple and affordable procedure to distinguish an orange juice from SPEs was proposed.     

Changes in the levels of polymethoxyflavones and flavanones as part of the defense mechanism of Citrus sinensis (cv. Valencia Late) fruits against Phytophthora citrophthora

Phytophthora citrophthora causes serious losses in Citrus fruits through brown rot lesion. The effect of infection with P. citrophthora on Citrus sinensis (cv. Valencia Late) fruits was studied, with particular reference to the levels of the flavanones hesperidin and isonaringin and the polymethoxyflavones sinensetin, nobiletin, tangeretin, and heptamethoxyflavone, because flavonoids are most probably involved as natural defense or resistance mechanisms in this genus. Changes in the levels of these flavonoids were detected after infection. The hesperidin and isonaringin contents fell by 13 and 67%, respectively, whereas the contents of their corresponding aglycons, hesperetin and naringenin, increased, suggesting the hydrolyzing effect of this fungus on the glycosylated flavanones. The heptamethoxyflavone, nobiletin, sinensetin, and tangeretin levels increased by 48, 28, 26, and 24%, respectively. The in vitro study revealed that these compounds acted as antifungal agents, the most active being the aglycons (naringenin and hesperetin), followed by the polymethoxyflavones and flavanone glycosides. The participation of these flavonoids in the defense mechanism of this Citrus species is discussed.     

Recovery of hesperidin from orange peel by concentration of extracts on styrene-divinylbenzene resin.

This paper describes a new procedure for obtaining hesperidin from the waste orange peel of the citrus industry. It is based on the adsorption of dilute extracts of hesperidin on a styrene-divinylbenzene (SDVB) resin and the desorption in much more reduced volumes by means of alkaline eluents. Hesperidin immediately precipitates with good yield and high purity after acidification of the concentrated solutions, thus overcoming disadvantages due to the high dilution. Different experiments were carried out to examine operating conditions in each phase of the process. Hesperidin was extracted from peel with an aqueous saturated Ca(OH)(2) solution, allowing precipitation of calcium pectates from colloidal pectins that can interfere in the subsequent phases of adsorption and separation of hesperidin. The clear extracts were neutralized to optimize adsorption on resin. The most effective eluent was 0.5 N NaOH solution containing 10% ethanol. Recycling of the crystallization liquor improved the yield and purity of the product and reduced the acid amount required for neutralizing fresh alkaline extracts. Resin must be washed after each adsorption-desorption cycle and regenerated after five cycles. Results can constitute a useful starting point for an industrial application. A flow scheme of the process is also reported.     

Characterization of Polymethoxylated Flavonoids (PMFs) in the Peels of 'Shatangju' Mandarin ( Citrus reticulata Blanco) by Online High-Performance Liquid Chromatography Coupled to Photodiode Array Detection and Electrospray Tandem Mass Spectrometry

A sensitive HPLC-DAD-ESI-MS/MS method was established to screen and identify the polymethoxylated flavonoids (PMFs) in the peels of 'Shatangju' mandarin ( Citrus reticulata Blanco). Eight PMF standards, including four polymethoxylated flavones, two polymethoxylated flavanones, and two polymethoxylated chalcones, were first to be analyzed in positive mode by CID-MS/MS. On the basis of the ESI-MS(n) characteristics of PMFs and the results of EIC-MS/MS experiment, 32 PMFs including 24 flavones and 8 flavanones or chalcones were screened from the complex extract of the peels of 'Shatangju' mandarin. Among them, 10 PMFs were hydroxylated polymethoxyflavonoids (OH-PMFs), and the rest were all permethoxylated PMFs. This was the first systematic report of the presence of PMFs in the peels of 'Shatangju' mandarin, especially for polymethoxylated flavanones and chalcones. Meanwhile, the contents of the three main PMFs and total flavonoids in the peels of 'Shatangju' were determined by HPLC and UV spectrophotometry, respectively. The results indicated that the developed analytical method could be employed as an effective technique for the characterization of PMFs.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.