Development and Validation of a Stable Isotope Dilution Headspace-SPME-GC/MS Method for the Determination of Vanillin in Fragrant Vegetable Oils.

It has been reported that vanillin has been intentionally added to enhance the taste and flavor of low-quality vegetable oils. Therefore, it is crucial to investigate the accurate concentrations of vanillin in three types of fragrant vegetable oils commonly consumed in China. In this study, a method has been developed for the quantification of vanillin in commercial fragrant vegetable oils using the stable isotope dilution assay (SIDA) and headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography/mass spectrometry (GC/MS). The limit of detection (LOD) and limit of quantification (LOQ) of the analyte were determined to be 20 µg kg-1 and 50 µg kg-1, respectively. The validation study demonstrated that the recoveries ranged from 89% to 101%, with intra-day and inter-day precision being less than 7.46%. A survey of 80 commercially available fragrant vegetable oils was performed using the present method. Vanillin was found to be widely present in fragrant vegetable oils, with sesame oils showing the highest average content (842.6 µg kg-1), followed by rapeseed oils (262.1 µg kg-1) and peanut oils (115.0 µg kg-1). The results indicate that the proposed method is a simple, accurate, and eco-friendly approach for determining the presences of vanillin in fragrant vegetable oils.

Simple and Rapid Method for the Quantitation of Trace-Level 3-Alkyl-2-methoxypyrazines in Fragrant Vegetable Oils by Gas Chromatography-Tandem Mass Spectrometry.

3-Alkyl-2-methoxypyrazines (MPs) are characteristic aroma compounds found in fragrant vegetable oils, a type of specially processed oils with enhanced flavor. MP contents in these oils are usually at trace level, which makes their quantification a big challenge. In this work, we describe an optimized approach with a double-step acid/alkali extraction for the analysis of such compounds, namely, 3-isopropyl-2-methoxypyrazine, 3-isobutyl-2-methoxypyrazine, and 3-sec-butyl-2-methoxypyrazine, in those fragrant oils using gas chromatography-tandem mass spectrometry (GC-MS/MS). The sample preparation conditions including selections and percentages of acids, alkalis, and extraction solvents, as well as the stability of MPs, were optimized and examined. Method validation was conducted with a good linearity (r2 > 0.999), and average recoveries between 93.9 and 109.3% were achieved. The limit of detection ranged from 0.2 to 0.4 µg/kg, and the relative standard deviations varied from 0.4 to 12.2% for samples spiked with the MPs at different concentrations. Overall, the method satisfactorily meets the requirements for the measurement of trace-level MPs in the fragrant vegetable oils via odor activity value calculation, and the results indicate that the improved acid/alkali extraction method is suitable for the routine analysis of MPs in those vegetable oils.

Efficacy and safety of Shexiang Baoxin pill (MUSKARDIA) in patients with stable coronary artery disease: a multicenter, double-blind, placebo-controlled phase IV randomized clinical trial.

BACKGROUND: The Shexiang Baoxin Pill (MUSKARDIA) has been used for treating coronary artery disease (CAD) and angina for more than 30 years in China. Nevertheless, methodologically sound trials on the use of MUSKARDIA in CAD patients are scarce. The aim of the study is to determine the effects of MUSKARDIA as an add-on to optimal medical therapy (OMT) in patients with stable CAD. METHODS: A total of 2674 participants with stable CAD from 97 hospitals in China were randomized 1:1 to a MUSKARDIA or placebo group for 24 months. Both groups received OMT according to local tertiary hospital protocols. The primary outcome was the occurrence of a major adverse cardiovascular event (MACE), defined as a composite of cardiovascular death, non-fatal myocardial infarction (MI), or non-fatal stroke. Secondary outcomes included all-cause mortality, non-fatal MI, non-fatal stroke, hospitalization for unstable angina or heart failure, peripheral revascularization, angina stability and angina frequency. RESULTS: In all, 99.7% of the patients were treated with aspirin and 93.0% with statin. After 2 years of treatment, the occurrence of MACEs was reduced by 26.9% in the MUSKARDIA group (MUSKARDIA: 1.9% vs. placebo: 2.6%; odds ratio = 0.80; 95% confidence interval: 0.45-1.07; P  = 0.2869). Angina frequency was significantly reduced in the MUSKARDIA group at 18 months (P = 0.0362). Other secondary endpoints were similar between the two groups. The rates of adverse events were also similar between the two groups (MUSKARDIA: 17.7% vs. placebo: 17.4%, P = 0.8785). CONCLUSIONS: As an add-on to OMT, MUSKARDIA is safe and significantly reduces angina frequency in patients with stable CAD. Moreover, the use of MUSKARDIA is associated with a trend toward reduced MACEs in patients with stable CAD. The results suggest that MUSKARDIA can be used to manage patients with CAD. TRIAL REGISTRATION: chictr.org.cn, No. ChiCTR-TRC-12003513.

Protective Effects of Salvianolate on Myocardial Injury or Myocardial Infarction after Elective Percutaneous Coronary Intervention in NSTE-ACS Patients: A Randomized Placebo-Controlled Trial.

OBJECTIVE: To evaluate the protective effects of salvianolate on percutaneous coronary intervention (PCI) related myocardial injury or myocardial infarction after elective PCI in non-ST-segment elevation acute coronary syndrome (NSTE-ACS) patients. METHODS: A total of 149 patients with NSTE-ACS who underwent elective PCI were enrolled. The patients were randomly allocated in a 1:1 ratio to the salvianolate group (74 cases) or the control group (75 cases). After exclusion criteria of coronary angiography, 60 patients with PCI therapy remained in the salvianolate group and 68 in the control group. The incidence and the severity of PCI related myocardial injury or myocardial infarction, in addition to major adverse cardiac events (MACEs) during 1 year follow-up after PCI were studied between the two groups. Multivariate logistic regression analysis was used to determine the independent factors for PCI related myocardial injury or myocardial infarction after elective PCI. RESULTS: Compared with the control group, salvianolate treatment reduced the incidence of PCI related severe myocardial injury or myocardial infarction (11.7% vs. 26.5%, P=0.035). The rate of MACEs or all-cause death within 1 month or 1 year after the procedure was not significantly different between the two groups. CONCLUSIONS: Periprocedural treatment with salvianolate reduces the incidence of PCI related severe myocardial injury or myocardial infarction, although it does not influence clinical prognosis. [Chinese clinical trial registry: ChiCTR1800016992].

Fast simultaneous determination of capsaicin, dihydrocapsaicin and nonivamide for detecting adulteration in edible and crude vegetable oils by UPLC-MS/MS.

Capsaicinoids are pungent components in hot peppers, which have been detected in waste cooking oil. However, trace analysis of capsaicinoids in edible and crude vegetable oils is a challenging task due to the complex matrix. In this study, a simple liquid-liquid extraction and solid phase extraction (SPE) coupled with RP-UPLC-ESI-MS/MS method was developed for the quantification of capsaicinoids in edible and crude vegetable oils to screen the adulteration with waste cooking oil. This method was used to simultaneously determine 3 capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) with capsaicin-d3, and dihydrocapsaicin-d3 as internal standards. This method allows the complete analysis of a sample in only an hour, even including sample preparation and chromatographic separation. The linear range of 3 capsaicinoids ranged between 0.5 and 40 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) for capsaicinoids were calculated as 0.15 and 0.5 µg/kg, respectively. Quantitative recoveries ranging from 92.9% to 105% were obtained by the analysis of spiked oil. The relative standard deviations were less than 5% (n = 6). The established method can potentially overcome the interference of triacylglycerols and fatty acids in edible and crude vegetable oils, and have been successfully applied to analyse real oil samples. This method provided a rapid and reliable method for the detection of adulteration of vegetable oils with waste cooking oils.

Profiling hydroxycinnamic acid glycosides, iridoid glycosides, and phenylethanoid glycosides in baobab fruit pulp (Adansonia digitata).

The baobab (Adansonia digitata L.) is a magnificent tree revered throughout Africa and is becoming recognized for its high nutritional and medicinal values. Despite numerous reports on the pharmacological potential, little is known about its chemical compositions. In this study, four hydroxycinnamic acid glycosides (1-4), six iridoid glycosides (5-10), and three phenylethanoid glycosides (11-13) were isolated from the dried baobab fruit pulp. Their structures were determined by means of spectroscopic analyses, including HRMS, 1H and 13C NMR and 2D experiments (COSY, HSQC, HMBC, and ROESY). All 13 compounds isolated were reported for the first time in the genus of Adansonia. An ultra high-performance liquid chromatography high-resolution accurate-mass mass spectrometry (UHPLC HRAM MS) method was used to conduct further investigation of the chemical compositions of the hydro-alcohol baobab fruit pulp extract. Hydroxycinnamic acid glycosides, iridoid glycosides and phenylethanoid glycosides were found to be the main components in baobab fruit pulp.

Separating four diastereomeric pairs of dihydroflavonol glycosides from Engelhardia roxburghiana using high performance counter-current chromatography.

Four pairs of diastereomers were successfully isolated and separated from the water extract of Engelhardia roxburghiana by high performance counter-current chromatography (HPCCC) using a two-step procedure. The diastereomers were initially separated by a two-phase solvent system composed of n-hexane-n-butanol-0.1% trifluoroacetic acid (1:2:3, v/v/v) and followed by the same solvent system using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as an additive. The chromatographic conditions, elution mode, and concentrations of the additive were refined. The two-step HPCCC isolation yielded 43.7mg (2S, 3S)-astilbin, 27.6mg (2R, 3R)-astilbin, 5.9mg (2S, 3R)-astilbin, 4.8mg (2R, 3S)-astilbin, 6.9mg (2S, 3S)-engelitin, 3.1mg (2R, 3R)-engelitin, 8.2mg (2S, 3R)-engelitin, and 6.0mg (2R, 3S)-engelitin from 384mg crude extract in four runs with purities of 99.3%, 96.2%, 99.8%, 99.9%, 97.0%, 96.5%, 96.1%, and 96.8%, respectively. The present study revealed that HP-ß-CD can be used as an additive in HPCCC to effectively improve the resolution of the diastereomers. The established HPCCC method may serve as an approach to obtain high purity diastereomers on a large scale.

Simultaneous HPLC quantification of five major triterpene alcohol and sterol ferulates in rice bran oil using a single reference standard.

A high performance liquid chromatography (HPLC) method was developed for simultaneous quantification of five major triterpene alcohol and sterol ferulates in rice bran oils (RBO) with a single internal standard, cycloartenyl ferulate. The five compounds are cycloartenyl ferulate (1), 24-methylene cycloartanyl ferulate (2), campesteryl ferulate (3), sitosteryl ferulate (4) and stigmastanyl ferulate (5). All five compounds had good linear concentration-measurement relationships (r(2) ≥ 0.9995) and possessed similar relative response factors. The relative deviation of this method was less than 2.5% for intra- and inter-day assays, and the average recovery varied from 95.1% to 99.4%. The new method was validated by comparing the amount of 24-methylene cycloartanyl ferulate (2) in 17 RBO samples obtained with this method and that with an external standard method. This method was also successfully applied to determine five major triterpene alcohol and sterol ferulates in 17 batches of RBO samples. The results demonstrated that the present method could be utilised for quality control of RBO since some of the reference standards are not commercially available.

Two novel anti-inflammatory 21-nordammarane saponins from tetraploid Jiaogulan ( Gynostemma pentaphyllum ).

Two novel 20-oxo-21-nordammar-22, 24-diene saponins, 21-norgypenosides A (1) and B (2), were characterized from the aerial parts of tetraploid Jiaogulan ( Gynostemma pentaphyllum ), a popular tea ingredient. Their structures, including the absolute configurations, were comprehensively elucidated by HRESIMS, 1D and 2D NMR data, chemical degradation, and through comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The two compounds suppressed the expression of interleukin (IL)-1ß, cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α mRNAs in the lipopolysaccharide-induced RAW 264.7 mouse macrophage cells at 10 and 100 µg/mL, suggesting their potential anti-inflammatory effects.

Two new saponins from tetraploid jiaogulan (Gynostemma pentaphyllum), and their anti-inflammatory and α-glucosidase inhibitory activities.

Jiaogulan tea has been commercialised globally. This study investigated the chemical components and health properties of a new jiaogulan genotype, tetraploid Gynostemma pentaphyllum. Two new saponins, (23S)-21ß-O-methyl-3ß,20ξ-dihydroxy-12-oxo-21,23-epoxydammar-24-ene-3-O-[α-L-rhamnopyranosyl(1→2)][ß-D-glucopyranosyl(1→3)]-α-L-arabinopyranoside (4) and 23ß-H-3ß,20ξ-dihydroxy-19-oxo-21,23-epoxydammar-24-ene-3-O-[α-L-rhamnopyranosyl(1→2)][ß-D-xylopyranosyl(1→3)]-α-L-arabinopyranoside (5), together with one lactone, 3,5-dihydroxyfuran-2(5H)-one (1), and two flavonoids, rutin (2) and kaempferol 3-O-rutinoside (3), were characterised in the aerial parts of tetraploid jiaogulan. The chemical structures of the five isolated compounds were elucidated by NMR, HR-MS spectra and chemical degradation. The five compounds were also examined and compared with the methanol extract and n-butanol soluble fraction of the jiaogulan for their inhibitory activities on lipopolysaccharide (LPS)-induced IL-1ß, IL-6 and COX-2 mRNA expression in RAW 264.7 mouse macrophages, and their in vitro α-glucosidase suppressing capacities. The results from this study may be used to promote the potential application of jiaogulan in functional foods.

Identification and quantification of phytochemical composition and anti-inflammatory, cellular antioxidant, and radical scavenging activities of 12 Plantago species.

Twenty-eight seed samples of 12 Plantago species were investigated for their chemical compositions and anti-inflammatory, cellular antioxidant, and radical scavenging properties. A new UPLC-UV procedure was developed and applied to quantify acteoside and geniposidic acid, the characteristic constituents of the genus Plantago. The amounts of acteoside and geniposidic acid ranged from 0.07 to 15.96 mg/g and from 0.05 to 10.04 mg/g in the tested samples, respectively. Furthermore, 26 compounds were tentatively identified by UPLC/Q-TOF-MS analysis. The Plantago samples significantly differed in their phytochemical compositions. The extracts of Plantago seeds also showed inhibitory effects on LPS-induced IL-1ß, IL-6, and COX-2 mRNA expression in RAW 264.7 mouse macrophage cells. Additionally, significant variations were observed among different samples on cellular antioxidant activities in HepG2 cells, as well as DPPH and hydroxyl radical scavenging capacities. The results from this study may be used to promote the use of the genus Plantago in improving human health.

Preparative separation of triterpene alcohol ferulates from rice bran oil using a high performance counter-current chromatography.

A novel method for the separation of two major triterpene alcohol ferulates from rice bran oil (RBO) was developed using a high performance counter-current chromatography (HPCCC). A two-phase solvent system of n-hexane-acetonitrile (1:1, v/v) was applied to purify cycloartenyl ferulate (CAF) and 24-methylene cycloartanyl ferulate (24-mCAF) from RBO. The yields were 20.50±2.60 mg CAF and 12.62±1.15 mg 24-mCAF from 390 mg RBO through a two-step separation procedure. The purities of the two compounds were 97.97±0.90% and 95.50±0.75%, respectively, as determined by high performance liquid chromatography (HPLC). Their chemical structures were confirmed by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and (1)H, (13)C and 2D nuclear magnetic resonance (NMR). This represents the first report on direct separation of CAF and 24-mCAF from RBO by HPCCC.

Free radical mediated formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters.

The present study was conducted to test the hypothesis that a free radical was formed and mediated the formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters, a group of food contaminants, from diacylglycerols at high temperature under a low-moisture condition for the first time. The presence of free radicals in a vegetable oil kept at 120 °C for 20 min was demonstrated using an electron spin resonance (ESR) spectroscopy examination with 5,5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. ESR investigation also showed an association between thermal treatment degree and the concentration of free radicals. A Fourier transform infrared spectroscopy (FT-IR) analysis of sn-1,2-stearoylglycerol (DSG) at 25 and 120 °C suggested the possible involvement of an ester carbonyl group in forming 3-MCPD diesters. On the basis of these results, a novel free radical mediated chemical mechanism was proposed for 3-MCPD diester formation. Furthermore, a quadrupole-time of flight (Q-TOF) MS/MS investigation was performed and detected the DMPO adducts with the cyclic acyloxonium free radical (CAFR) and its product MS ions, proving the presence of CAFR. Furthermore, the free radical mechanism was validated by the formation of 3-MCPD diesters through reacting DSG with a number of organic and inorganic chlorine sources including chlorine gas at 120 and 240 °C. The findings of this study might lead to the improvement of oil and food processing conditions to reduce the level of 3-MCPD diesters in foods and enhance food safety.

Comparative study of bioactive constituents in crude and processed Glycyrrhizae radix and their respective metabolic profiles in gastrointestinal tract in vitro by HPLC-DAD and HPLC-ESI/MS analyses.

Two HPLC methods with diode array detection (HPLC-DAD) and electrospray ionization-mass spectrometry (HPLC-ESI/MS), respectively, were developed to investigate the differences of chemical constituents and their metabolism in gastrointestinal tract in vitro between two decoctions of crude and processed Glycyrrhizae radix. Total of eleven constituents (liquiritin apioside, liquiritin, licuraside, isoliquiritin, ononin, glycyrrhizin, liquiritigenin-7,4'-diglucoside, licorice saponin A3, 22ß-acetoxylglycyrrhizic acid, licorice saponin G2, and yunganoside E2) were identified in the two decoctions, whereas lower contents of these constituents were usually found in the decoction of processed Glycyrrhizae Radix. [corrected] Furthermore, these constituents were metabolized into their respective aglycons in human intestinal bacteria juice, and the metabolism ratios were all higher in processed Glycyrrhizae Radix [corrected] decoction. No change was found in artificial gastric or intestinal juice. This study revealed that the processing can alter the contents of main constituents in crude G. radix and their metabolism in gastrointestinal tract, in which intestinal bacteria play an important role in the metabolism of licorice constituents.

Isolation and characterization of five glycerol esters from Wuhan propolis and their potential anti-inflammatory properties.

Five new glycerol esters including 2-acetyl-1-coumaroyl-3-cinnamoylglycerol (13), (+)-2-acetyl-1-feruloyl-3-cinnamoylglycerol (14), (-)-2-acetyl-1-feruloyl-3-cinnamoylglycerol (15), 2-acetyl-1,3-dicinnamoylglycerol (16), and (-)-2-acetyl-1-(E)-feruloyl-3-(3″(ζ),16″)-dihydroxy-palmitoylglycerol (17) were isolated from methanolic extract of Wuhan propolis. The chemical structures of the five new compounds were confirmed by (1)H and (13)C NMR and HR-MS spectra. Twelve minor constituents in the Wuhan propolis extract were tentatively identified by UPLC-Q-TOF-MS, according to their characteristic UV spectrum, retention times, and accurate MS data. The anti-inflammatory activities of the five new compounds were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophage cells. All five compounds exhibited remarkable inhibitory effects on interleukin (IL)-1ß, IL-6, and cyclooxygenase (COX)-2 mRNA expressions at 10 and 100 µM, suggesting that Wuhan propolis may serve as a potential anti-inflammatory functional food ingredient or nutraceutical.

Chemical composition and anti-proliferative and anti-inflammatory effects of the leaf and whole-plant samples of diploid and tetraploid Gynostemma pentaphyllum (Thunb.) Makino.

Leaf and whole-plant samples of the diploid and tetraploid Gynostemma pentaphyllum (GP) were investigated and compared for their chemical compositions, and their potential anti-proliferative and anti-inflammatory effects. The highest levels of total flavonoids and phenolics were observed in the diploid leaf botanical (2L3) at 36.84mg rutin equiv/g and 41.15mg gallic acid equiv/g, respectively. The diploid leaf sample (2L2) had the highest amount of rutin and quercetin contents of 77.7µmol quercetin equiv/g. The tetraploid whole-plant botanical (4L3) had the highest total saponin content of 227.1mg gypenoside equiv/g. Extracts from all tested GP samples showed time- and dose-dependent antiproliferative effects in HT-29 cells, and the diploid leaf samples had the overall highest inhibitory activity. These extracts had different order of antiproliferative properties in the LNCaP cells, suggesting the potential selective inhibition of GP extracts against different types of cancer cells and the effect of the cell model in screening and evaluation of antiproliferative components. In addition, the diploid leaf extracts showed the strongest inhibitory effects on the expression of TNF-α, IL-6 and COX-2 mRNA at final concentrations of 0.2 and 1mg botanical equiv/ml media. The results from this study will be used to develop new nutraceutical products from G. pentaphyllum.

[Advances in studies on metabolism and biotansformation of ginsenosides in vitro].

As a famous traditional Chinese medicine, ginseng's metabolism has been a hot spot in recent years. In this review, relevant literatures on the research progress of the metabolism of ginsenosides in vitro have been introduced. This review focuses on artifical juice metabolism, intestinal bacteria metabolism, microbial and enzymatic biotransformation of ginsenosides. Main metabolic pathways of ginsenosides in vitro are also analyzed, which will provide a reference for the systematic study on metabolism of ginsenosides.

Isolation and characterization of two flavonoids, engeletin and astilbin, from the leaves of Engelhardia roxburghiana and their potential anti-inflammatory properties.

Engeletin, a flavonoid compound, was isolated from the leaves of Engelhardia roxburghiana for the first time, along with astilbin, another flavonoid. The chemical structures of engeletin and astilbin were confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra, and their anti-inflammatory activities were studied in lipopolysaccharide (LPS)-stimulated mouse J774A.1 macrophage cells. LPS induced the inflammatory state in macrophage cells and increased mRNA expressions of pro-inflammatory cytokines. Engeletin and astilbin exhibited remarkable inhibitory effects on interleukin (IL)-1ß and IL-6 mRNA expression. Significant inhibition of LPS-mediated mRNA expressions were also seen in LPS binding toll-like receptor (TLR)-4, pro-inflammatory cytokine tumor necrosis factor (TNF)-α, IL-10, chemoattractant monocyte chemotactic protein (MCP)-1, and cyclooxygenase (COX)-2 genes. The reduced expression of these cytokines may alleviate immune response and reduce inflammatory activation, indicating that engeletin and astilbin may serve as potential anti-inflammatory agents.

Docking-based virtual screening of potential human P2Y12 receptor antagonists.

Platelet plays essential roles in hemostasis and its dysregulation can lead to arterial thrombosis. P2Y12 is an important platelet membrane adenosine diphosphate receptor, and its antagonists have been widely developed as anti-coagulation agents. The current P2Y12 inhibitors available in clinical practice have not fully achieved satisfactory anti-thrombotic effects, leaving room for further improvement. To identify new chemical compounds as potential anti-coagulation inhibitors, we constructed a three-dimensional structure model of human P2Y12 by homology modeling based on the recently reported G-protein coupled receptor Meleagris gallopavo ß1 adrenergic receptor. Virtual screening of the modeled P2Y12 against three subsets of small molecules from the ZINC database, namely lead-like, fragment-like, and drug-like, identified a number of compounds that might have high binding affinity to P2Y12. Detailed analyses of the top three compounds from each subset with the highest scores indicated that all of these compounds beard a hydrophobic bulk supplemented with a few polar atoms which bound at the ligand binding site via largely hydrophobic interactions with the receptor. This study not only provides a structure model of P2Y12 for rational design of anti-platelet inhibitors, but also identifies some potential chemicals for further development.

[Determination of thymine, hypoxanthine and uracil contents in medical pipefish by HPLC].

OBJECTIVE: To establish a RP-HPLC method for simultaneous determination of thymine, hypoxanthine and uracil contents in medical pipefish. METHOD: Samples were extracted with distilled water by ultrasonic wave and separated on Waters C18 column eluted with a mobile phase of 0.05 mol x L(-1) KFI2PO4-acetonitrile (97:3). The flow rate was 0.6 mL x min(-1). The determination wavelength was 260 nm and the column temperature was set at 40 degrees C. RESULT: The method had good linearity in the range of 0.033-0.660 (r = 0.9996), 0.620-12.400 (r = 0.9999), 0.048-0.960 microg (r = 0.9995), with average recoveries of 98.67% (RSD 1.6%), 99.03% (RSD 0.74%), 98.65% (RSD 1.8%), for thymine, hypoxanthine and uracil respectively. CONCLUSION: The simultaneous determination method of thymine, hypoxanthine and uracil in medical pipefish is established by RP-HPLC for the first time. The contents of the three constituents in different kinds of medical pipefish are significantly different. The method is simple, rapid and sensitive, and can be used for control the quality of medical pipefish.