A rapid and sensitive LC-MS/MS method for the determination of vanillic acid in rat plasma with application to pharmacokinetic study.

Vanillic acid, a phenolic compound isolated from Angelica sinensis and green tea, exhibits excellent antioxidant and anti-inflammatory activities. In this study, a rapid and sensitive ultra-high-performance liquid chromatography tandem mass spectrometry method was established and validated for the determination of vanillic acid in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Vanillic acid and caffeic acid (internal standard, IS) were quantified by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with a correlation coefficient of >0.99. The carryover, matrix effect, extraction recovery, dilution effect, intra- and interday precision and accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of vanillic acid in rats. After oral administration at doses of 2, 5 and 10 mg/kg, the plasma concentration reached peaks of 0.42 ± 0.09, 0.73 ± 0.21 and 0.92 ± 0.28 µg/ml at the time of 0.55-0.64 h, respectively. The oral bioavailability was calculated as 25.3-36.2% in rat plasma. The result provided pre-clinical information for further application of vanillic acid.

Offline derivatization LC-MS/MS method for simultaneous estimation of vanillin and vanillic acid in guinea pig plasma.

AIM: Vanillin used as a positive control substrate of aldehyde oxidase activity gets metabolized to vanillic acid. Low MW and low sensitivity in negative ion mode are challenges with these analytes. Our objective was to develop a simple offline derivatization LC-MS/MS method to address these challenges. METHODOLOGY/RESULTS: A simple dansyl chloride derivatization of the phenolic groups on vanillin and vanillic acid was adopted to enable easy ionization in commonly used acidic mobile phases. Calibration curves were linear over the concentrations of 4.88-1250 nM with an LLOQ of 0.64 fmoles on column for both analytes. CONCLUSION: The qualified method was successfully applied to simultaneously measure vanillin and vanillic acid in plasma and urine from a guinea pig pharmacokinetic study.

Influence of Intestinal Microbiota on the Catabolism of Flavonoids in Mice.

Although in vitro studies have shown that flavonoids are metabolized into phenolic acids by the gut microbiota, the biotransformation of flavonoids by intestinal microbiota is seldom studied in vivo. In this study, we investigated the impact of the gut microbiota on the biotransformation of 3 subclasses of flavonoids (flavonols, flavones, and flavanones). The ability of intestinal microbiota to convert flavonoids was confirmed with an in vitro fermentation model using mouse gut microflora. Simultaneously, purified flavonoids were administered to control and antibiotic-treated mice by gavage, and the metabolism of these flavonoids was evaluated. p-Hydroxyphenylacetic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, hydrocaffeic acid, coumaric acid, and 3-(4-hydroxyphenyl)propionic acid were detected in the serum samples from the control mice after flavonoid consumption. The serum flavonoid concentrations were similar in both groups, whereas the phenolic metabolite concentrations were lower in the antibiotic-treated mice than in the control mice. We detected markedly higher flavonoids excretion in the feces and urine of the antibiotic-treated mice compared to the controls. Moreover, phenolic metabolites were upregulated in the control mice. These results suggest that the intestinal microbiota are not necessary for the absorption of flavonoids, but are required for their transformation.

Online restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry for the simultaneous determination of vanillin and its vanillic acid metabolite in human plasma.

An automated online solid-phase extraction with restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry was developed and validated for the simultaneous quantification of vanillin and its vanillic acid metabolite in human plasma. After protein precipitation by methanol, which contained the internal standards, the supernatant of plasma samples was injected to the system, the endogenous large molecules were flushed out, and target analytes were trapped and enriched on the adsorbent, resulting in a minimization of sample complexity and ion suppression effects. Calibration curves were linear over the concentrations of 5-1000 ng/mL for vanillin and 10-5000 ng/mL for vanillic acid with a coefficient of determination >0.999 for the determined compounds. The lower limits of quantification of vanillin and vanillic acid were 5.0 and 10.0 ng/mL, respectively. The intra- and inter-run precisions expressed as the relative standard deviation were 2.6-8.6 and 3.2-10.2%, respectively, and the accuracies expressed as the relative error were in the range of -6.1 to 7.3%. Extraction recoveries of analytes were between 89.5 and 97.4%. There was no notable matrix effect for any analyte concentration. The developed method was proved to be sensitive, repeatable, and accurate for the quantification of vanillin and its vanillic acid metabolite in human plasma.

Comparative Pharmacokinetics of three major bioactive components in rats after oral administration of Typhae Pollen-Trogopterus Feces drug pair before and after compatibility.

BACKGROUND: Typhae Pollen (TP) and Trogopterus Feces (TF) are well-known traditional medicine in china which widely used for thousands of years as drug pair called Shixiao San for treatment of blood stasis syndrome, specially shown great efficacy in gynecological disease. Typhaneoside, vanillic acid and p-coumaric acid are the main bioactive components of Typhae Pollen. This study was carried out for comparing the pharmacokinetic profile of these three major bioactive components in rats after oral administration of Typhae Pollen-Trogopterus Feces (TP-TF) drug pair before and after compatibility. METHODS: A sensitive and rapid UPLC-TQ/MS method has been developed for simultaneous quantification of the three main bioactive compounds in blood at different time points after oral administration of Typhae Pollen (TP) and the combination with Trogopterus Feces (TF). RESULTS: There were significant differences of C(max), T(max), T(1/2) and AUC(0~t) for three bioactive compounds among the groups, for typhaneoside with the most highest plasma concentration of 370.86 ± 315.71 ng/mL and more longer T(max) in TP-TF co-decoction group (C(M)); for vanillic acid, TP-TF co-decoction group (C(M)) had a good absorption with C(max) (3870.99 ± 2527.99 ng/mL) and T(max) (1.47 ± 3.20 h); for p-coumaric acid, it had similar pharmacokinetic characteristics with vanillic acid. CONCLUSIONS: The three bioactive components in Typhae Pollen (TP) were simultaneously determined by UPLC-TQ/MS and had a good absorption in rat plasma after the combination with Trogopterus Feces (TF).

Phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus cerasus) and influence of phenolic acids on vascular smooth muscle cells in vitro.

PURPOSE: To investigate the phytochemical uptake following human consumption of Montmorency tart cherry (L. Prunus cerasus) and influence of selected phenolic acids on vascular smooth muscle cells in vitro. METHODS: In a randomised, double-blinded, crossover design, 12 healthy males consumed either 30 or 60 mL of Montmorency tart cherry concentrate. Following analysis of the juice composition, venous blood samples were taken before and 1, 2, 3, 5 and 8 h post-consumption of the beverage. In addition to examining some aspects of the concentrate contents, plasma concentrations of protocatechuic acid (PCA), vanillic acid (VA) and chlorogenic (CHL) acid were analysed by reversed-phase high-performance liquid chromatography (HPLC) with diode array for quantitation and mass spectrometry detection (LCMS) for qualitative purposes. Vascular smooth muscle cell migration and proliferation were also assessed in vitro. RESULTS: Both the 30 and 60 mL doses of Montmorency cherry concentrate contained high amounts of total phenolics (71.37 ± 0.11; 142.73 ± 0.22 mg/L) and total anthocyanins (62.47 ± 0.31; 31.24 ± 0.16 mg/L), as well as large quantities of CHL (0.205 ± 0.24; 0.410 ± 0.48 mg/L) and VA (0.253 ± 0.84; 0.506 ± 1.68 mg/L). HPLC/LCMS identified two dihydroxybenzoic acids (PCA and VA) in plasma following MC concentrate consumption. Both compounds were most abundant 1-2 h post-initial ingestion with traces detectable at 8 h post-ingestion. Cell migration was significantly influenced by the combination of PCA and VA, but not in isolation. There was no effect of the compounds on cell proliferation. CONCLUSIONS: These data show new information that phenolic compounds thought to exert vasoactive properties are bioavailable in vivo following MC consumption and subsequently can influence cell behaviour. These data may be useful for the design and interpretation of intervention studies investigating the health effects of Montmorency cherries.

UHPLC-MS simultaneous determination and pharmacokinetic study of three aromatic acids and one monoterpene in rat plasma after oral administration of Shaofu Zhuyu decoction.

We developed a sensitive and rapid method for determination of ferulic acid, caffeic acid, vanillic acid, and paeoniflorin in rat plasma based on ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The separation of the four compounds was carried out on an AcQuity UHPLC™ BEH C18 column using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). Electrospray ionization in positive and negative ion mode and multiple reaction monitoring was used to identify and quantify active components. All calibration curves gave good linearity (r > 0.991) over the concentration range from 4.24-2875 ngmL(-1) for all components. The precision of the in vivo study was evaluated by intraday and interday assays and the percentages of RSD were all within 10.6%. The recovery ranged from 60.2 to 77.9%. The method was successfully applied to pharmacokinetic study of all three aromatic acids and one monoterpene in rat plasma. Furthermore, we compared the pharmacokinetics profile of the four compounds in normal and primary dysmenorrhea rats' plasma following oral administration of Shaofu Zhuyu decoction (SFZYD) and its ethanol supernatant extract (SFE).

Reverse-phase HPLC determination and pharmacokinetic study of vanillic acid in the plasma of rats treated with the traditional Chinese medicinal preparation Di-Gu-Pi decoction.

A sensitive, simple, and accurate method for the determination and pharmacokinetic study of vanillic acid in rat plasma was developed using reverse-phase HPLC with UV detection after oral administration of the traditional Chinese medicine preparation of the Di-Gu-Pi decoction. Plasma samples taken from rats were extracted with methanol. The constituent vanillic acid was separated on a C(18) stationary phase and a mobile phase of acetonitrile-water (15:85, v/v) (adjusted to pH 3.0 using phosphoric acid), with a UV detector setting at 260 nm. The validated HPLC method developed was used to determine the pharmacokinetic profile of vanillic acid in rat plasma after administration of the Di-Gu-Pi decoction.

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine.

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)-acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 microg/ml and the urine method was linear from 2 to 40 microg/ml. The method had a detection limit of 1 microg/ml for vanillin and vanillic acid using 5 microl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.

[Follow up control of stage IV neuroblastoma: 123I-MIBG scintigraphy, bone scintigraphy and catecholamine metabolites]. / Verlaufskontrolle des Neuroblastoms Stadium IV: 123I-mIBG-Szintigraphie, Knochenszintigraphie und Katecholaminmetabolite.

AIM: The diagnostic value of 123I-mIBG-scintigraphy, bone scintigraphy and catecholamine metabolites in the follow up of neuroblastoma stage IV will be evaluated. METHODS: Nineteen children suffering from neuroblastoma were analysed retrospectively by 123I-mIBG-scintigraphy, bone scintigraphy and measurement of homovanillic acid, vanillic acid, neuronspecific enclose, lactate dehydrogenase, and ferritine. Follow up was 7-132 (median 36) months. RESULTS AND CONCLUSION: The significance of the methods was dependent on the time of diagnostic use. In principal, 123I-mIBG-scintigraphy has the highest diagnostic impact. For initial staging and diagnosis of recurrence a combination of all three methods can be used. On the contrary, follow up during chemotherapy is best documented by 123I-mIBG-scintigraphy, whereas bone scintigraphy is of limited and measurement of catecholamine metabolites of less diagnostic value.