Characterization of the chemical constituents of Croton crassifolius Geisel extract and plasma pharmacokinetics of 6 terpenoids after oral administration by UPLC-Q/TOF-MS.

INTRODUCTION: Croton crassifolius Geisel. (CCG) is a traditional Chinese medicine widely used in South China. It has various pharmacological effects and is often used in treating rheumatoid arthritis and gastric and duodenal ulcers. However, the chemical characteristics and its effective constituents are still scarcely studied. OBJECTIVE: To determine the phytochemical profile of the CCG extract and to investigate the chemical characteristics of terpenoids extracted from rat plasma following oral administration of CCG extract based on UPLC-Q/TOF-MS. Moreover, six terpenoids in CCG were quantified, and in vivo pharmacokinetic behavior after oral CCG extract was further explored. RESULTS: In total, 56 terpenoids were tentatively identified in the CCG extract and 16 terpenoids were detected in rat plasma after oral CCG extract. In addition, the contents of six terpenoids in CCG were clarified. The plasma quantification method of six terpenoids was further established, validated, and confirmed to have good sensitivity and specificity. The six analytes exhibited excellent linearity in respective concentration ranges (r ≥ 0.998). The intra-day and inter-day precisions relative standard deviation (RSD, %) were less than 11.27%, and the accuracies ranged from -7.06% to 9.91%. Stability, extraction recovery, and matrix effect in plasma were within the required limits (RSD < 15%). CONCLUSION: A total of 56 terpenoids were identified in CCG and 16 prototype components in plasma after oral CCG. The validated quantitative method was successfully applied to the simultaneous determination of six major terpenoids in plasma. The pharmacokinetic parameters are clarified, which can guide the clinical application of CCG.

Simultaneous LC-MS/MS bioanalysis of alkaloids, terpenoids, and flavonoids in rat plasma through salting-out-assisted liquid-liquid extraction after oral administration of extract from Tetradium ruticarpum and Glycyrrhiza uralensis: a sample preparation strategy to broaden analyte coverage of herbal medicines.

Herbal medicines have historically been practiced in combinatorial way, which achieves therapeutic efficacy by integrative effects of multi-components. Thus, the accurate and precise measurement of multi bioactive components in matrices is inalienable to understanding the metabolism and disposition of herbal medicines. In this study, aiming to provide a strategy that improves analyte coverage, evaluation of six protocols employing sample pretreatment methods- protein precipitation (PPT), liquid-liquid extraction (LLE), sugaring-out-assisted liquid-liquid extraction (SULLE), and salting-out-assisted liquid-liquid extraction (SALLE)- was performed by LC-MS/MS using rat plasma and a mixture of alkaloid (evodiamine, rutaecarpine, dehydroevodiamine), terpenoid (limonin, rutaevin, obacunone), and flavonoid (liquiritin, isoliquiritin, liquiritigenin) standards isolated from Tetradium ruticarpum and Glycyrrhiza uralensis. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) LLE with methyl tertiary-butyl ether-dichloromethane, (4) LLE with ethyl acetate-n-butanol, (5) SALLE with ammonium acetate, (6) SULLE with glucose. The results suggested that SALLE produced broader analyte coverage with satisfactory reproducibility, acceptable recovery, and low matrix interference. Then, sample preparation procedure of SALLE, chromatographic conditions, and mass spectrometric parameters were optimized, followed by method validation, showing that good sensitivity (LLOQ ≤ 1 ng mL-1), linearity (r ≥ 0.9933), precision (RSD ≤ 14.45%), accuracy (89.54~110.87%), and stability could be achieved. Next, the developed method was applied successfully to determine the pharmacokinetic behavior of the nine compounds in rat plasma after intragastric administration with an extract from Tetradium ruticarpum and Glycyrrhiza uralensis (Wuzhuyu-Gancao pair). Based on an extensive review and experiments, a sample preparation procedure that matches with LC-MS/MS technique and can get wider analyte coverage was outlined. The developed SALLE method is rapid, reliable, and suitable for bioanalysis of analytes with diverse polarity, which was expected to be a promising strategy for the pharmacokinetic studies of herbal medicines. Graphical abstract.

Integrated UPLC-MS/MS and UHPLC-Q-orbitrap HRMS Analysis to Reveal Pharmacokinetics and Metabolism of Five Terpenoids from Alpiniae oxyphyllae Fructus in Rats.

BACKGROUND: Alpiniae oxyphyllae Fructus (AOF), a traditional Chinese medicine (TCM), is widely used in the treatment of urinary, gastrointestinal and neurologic diseases in China. Although terpenoids are the main active ingredients of AOF, there are few researches on their pharmacokinetics and metabolism. METHODS: In this study, a sensitive, rapid, accurate and novel ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to evaluate the pharmacokinetic behavior of five terpenoids (oxyphyllenodiol B, (4S*,5E,10R*)-7-oxo-tri-nor-eudesm-5-en-4ß-ol, 7-epi-teucrenone, (+)- (4R,5S,7R)-13-hydroxynootkatone, (E)-labda-12,14-dien-15(16)-olide-17-oic acid) in rats after oral administration of AOF extracts. 27 metabolic metabolites of the five terpenoids were identified by ultra high performance liquid chromatography -Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) based on precise mass and fragment ions. RESULTS: The established pharmacokinetic analysis method showed good linearity over a wide concentration range, and the lower quantitative limit (LLOQ) ranged from 0.97 to 4.25 ng/mL. Other validation parameters were within the acceptable range. In addition, 27 metabolites were identified in plasma, urine and feces samples, and the metabolic pathways of five terpenoids were mainly focused on glucoside conjugation, dehydration, desaturation and glycine conjugation. CONCLUSION: This is the first study on the pharmacokinetics and metabolism of five terpenoids in AOF, illuminating the disposal process of terpenoids in vivo. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and the further pharmacological study of five terpenoids in AOF.

Nine absorbed components pharmacokinetic of raw and processed Moutan Cortex in normal and blood-heat and hemorrhage syndrome model rats.

Raw Moutan Cortex (RMC) and Processed Moutan Cortex (PMC) have a long history of use in China and other Asian countries. In this study, a rapid and accurate ultra-high-pressure liquid chromatography coupled with diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of nine absorbed compounds of RMC/PMC. After extraction by protein precipitation with methanol from plasma, the analytes were separated on an Acquity UPLC® BEH Shield RP18 column (2.1 × 100 mm, 1.7 µm, Waters, USA). Acetonitrile (A) and 0.1% (v/v) formic acid in water (B) were selected as the mobile phase to perform gradient elution. The linearity of nine analytes was >0.9915. The intra- and inter-assay precision (RSD) values were within 11.18%, and accuracy ranged from 91.32 to 101.29%. Suitable stability, matrix effect and extraction recoveries were also obtained. The validated method was applied to compare the pharmacokinetics of RMC and PMC in Blood-Heat and Hemorrhage Syndrome Model and normal rats. The results revealed that processing and the pathological state could influence the pharmacokinetic characteristics of compounds in RMC/PMC. The study willbe useful for further studies on pharmacokinetics and clinical application of raw and processed Moutan Cortex.

Structure-Activity Relationship Study of Acyclic Terpenes in Blood Glucose Levels: Potential α-Glucosidase and Sodium Glucose Cotransporter (SGLT-1) Inhibitors.

Twelve terpenoids were evaluated in the treatment of type 2 diabetes mellitus: seven monoterpenes (geranyl acetate (1), geranic acid (2), citral (3), geraniol (4), methyl geranate (5), nerol (6), and citronellic acid (7)), three sesquiterpenes (farnesal (8), farnesol (9), and farnesyl acetate (10)), one diterpene (geranylgeraniol (11)), and one triterpene (squalene (12)) were selected to carry out a study on normoglycemic and streptozotocin-induced diabetic mice. Among these, 2, 3, 7, 8, 9, and 10 showed antihyperglycemic activity in streptozotocin-induced diabetic mice. They were then selected for evaluation in oral sucrose and lactose tolerance tests (OSTT and OLTT) as well as in an oral glucose tolerance test (OGTT). In the OSTT and OLTT, compounds 3, 7, 8, 9, and 10 showed a reduction in postprandial glucose peaks 2 h after a sucrose or lactose load (comparable to acarbose). In the case of the OGTT, 2, 7, 8, 9, and 10 showed a reduction in postprandial glucose peaks 2 h after a glucose load (comparable to canagliflozin). Our results suggest that the control of postprandial hyperglycemia may be mediated by the inhibition of disaccharide digestion, such as sucrose and lactose, and the regulation of the absorption of glucose. The first case could be associated with an ∝ -glucosidase inhibitory effect and the second with an inhibition of the sodium-glucose type 1 (SGLT-1) cotransporter. Finally, five acyclic terpenes may be candidates for the development and search for new α-glucosidase and SGLT-1 cotransporter inhibitors.

Simultaneous determination of seven effective components of Tripterygium glycosides in human biological matrices by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

This paper developed a novel, sensitive, and selective ultra-performance liquid chromatography-triple quad mass spectrometry method to simultaneously determine seven effective constituents (triptolide, triptophenolide, celastrol, wilforgine, wilforine, wilfordine and wilfortrine) of Tripterygium glycosides (GTW) in human serum and urine. The chromatographic separation was performed on the C18 column using an ammonium acetate buffer solution-acetonitrile (both containing 0.1% formic acid) in a gradient program with a flow rate of 0.3 mL/min. Monitoring reaction mode was applied to target compounds quantitative analysis in the positive electrospray ionization (ESI) mode. The analysis process took 11 min in total. This method was fully validated with a linear range of 1-200 ng/mL for triptolide, 0.4-80 ng/mL for celastrol, 0.1-20 ng/mL for triptophenolide, wilforgine, wilforine, wilfordine, and wilfortrine. The intra-day and inter-day accuracy and precision of the target compounds all met the 15% criterion in both serum and urine. Extraction recovery, matrix effect, and dilution integrity were also validated. The short-term and long-term stability results indicated that all the constituents were stable in human serum and urine under the investigated storage conditions. 10 patients' specimens were collected and analyzed. Most of the compounds exhibited the tendency of higher concentration in urine than that in serum. The concentration that was detected in the serum and in the urine of alkaloids showed a positive-correlation property. This is the first time that triptophenolide was quantified in human bio-matrices. The method is feasible for multi-components therapeutic monitoring or pharmacokinetics study in clinical pharmaceutical research of Tripterygium glycosides.

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC-Q-TOF/MS/MS.

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid-liquid extraction and separated on a Shim-pack XR-ODS C18 column (75 × 3.0 mm, 2.2 µm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone-related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid-related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid-related metabolites. It is concluded the developed UHPLC-Q-TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.

Bioavailability of Terpenes and Postprandial Effect on Human Antioxidant Potential. An Open-Label Study in Healthy Subjects.

SCOPE: To assess bioavailability of terpenes in human plasma and their effect on oxidative stress biomarkers. METHODS AND RESULTS: In this open-label and single arm postprandial trial, seventeen healthy male volunteers (20-40 years old) follow a low-phytochemical diet for 5 days. Next, after overnight fasting, volunteers consume Mastiha powder (a natural resin rich in terpenes) dispersed in water. Blood samples are collected on time points 0 h (before ingestion) and 0.5, 1, 2, 4, 6, and 24 h (post-ingestion). Ultra-high-pressure liquid chromatography high-resolution MS (UHPLC-HRMS/MS) is applied for high throughput analysis of plasma. Serum resistance to oxidation and oxidized LDL (oxLDL) levels are measured. UHPLC-HRMS/MS analysis shows that major terpenes are bioavailable since 0.5 h after administration, reaching a peak between 2 h and 4 h. Serum resistance to oxidation, expressed as difference of tLAG (time point-0 h), starts to increase from 0.5 h. This increase reaches statistical significance at 4 h (402.3 ± 65.0 s), peaks at 6 h (524.6 ± 62.9 s), and remains statistically significant until 24 h (424.2 ± 48.0 s). oxLDL levels, expressed as %change from 0 h, are reduced significantly from time point-1 h until time point-6 h. CONCLUSION: Results demonstrate the terpene bioavailability pattern after oral administration of Mastiha. Terpenes are potential mediators of antioxidant defense in vivo.

Simultaneous quantification of multiple components in rat plasma by UPLC-MS/MS and pharmacokinetic study after oral administration of Huangqi decoction.

A rapid, sensitive and accurate UPLC-MS/MS method was developed for the simultaneous quantification of components of Huangqi decoction (HQD), such as calycosin-7-O-ß-d-glucoside, calycosin-glucuronide, liquiritin, formononetin-glucuronide, isoliquiritin, liquiritigenin, ononin, calycosin, isoliquiritigenin, formononetin, glycyrrhizic acid, astragaloside IV, cycloastragenol, and glycyrrhetinic acid, in rat plasma. After plasma samples were extracted by protein precipitation, chromatographic separation was performed with a C18 column, using a gradient of methanol and 0.05% acetic acid containing 4mm ammonium acetate as the mobile phase. Multiple reaction monitoring scanning was performed to quantify the analytes, and the electrospray ion source polarity was switched between positive and negative modes in a single run of 10 min. Method validation showed that specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability for 14 components met the requirements for their quantitation in biological samples. The established method was successfully applied to the pharmacokinetic study of multiple components in rats after intragastric administration of HQD. The results clarified the pharmacokinetic characteristics of multiple components found in HQD. This research provides useful information for understanding the relation between the chemical components of HQD and their therapeutic effects.

Metabolism and antioxidant effect of malaxinic acid and its corresponding aglycone in rat blood plasma.

Malaxinic acid (MA) is a phenolic acid compound, found mainly in pear fruits (Pyrus pyrifolia N.), that is isoprenylated on the C-3 position of benzoic acid. Recently, the effects of prenylated phenolics on health have received much interest owing to their reported potent beneficial biological effects. We conducted a comparative study in rats to determine the metabolism, pharmacokinetics, and antioxidative activities of MA and its corresponding aglycone (MAA). MA and MAA were orally administered to rats (Sprague-Dawley, male, 6 weeks old) and their metabolites in plasma were analyzed. In addition, the MA metabolites in plasma were separated and the structures were confirmed via NMR and HR-MS analyses. The antioxidative activities of MA and MAA were evaluated by measuring their inhibitory effects on the 2,2'-azobis(2-amidinopropane)dihydrochloride- or copper ion-induced lipid peroxidation of rat plasma. MA was not absorbed in the intact form (the glucoside); both MA and MAA were absorbed as MAA and its metabolite form (glucuronide or sulfate). Moreover, the observed metabolite was the glucuronate of MAA rather than the glucuronide or sulfate. Concentrations of the free form of aglycone (MA administration, 4.6 ± 2.2µM; MAA administration, 7.2 ± 2.3µM) and total MAA (MA administration, 19.6 ± 4.4µM; MAA administration, 21.7 ± 3.3µM) in plasma reached a maximum at 15min after the oral administration of MA and MAA, respectively. The relative inhibitory effects on the formation of cholesteryl ester hydroperoxides in plasma collected at 15min after the oral administration of MA, MAA, and p-hydroxybenzoic acid (p-HBA) were as follows: MAA > MA ≥ p-HBA > control. Although the majority of MA and MAA is metabolized to conjugates, the compounds may contribute to the antioxidant defenses in the blood circulation owing to the presence of a phenolic hydroxyl group in the free form.

Pharmacokinetic properties, in vitro metabolism and plasma protein binding of govaniadine an alkaloid isolated from Corydalis govaniana Wall.

Govaniadine (GOV) is an alkaloid isolated from Corydalis govaniana Wall. It has been reported to show a different number of biological activities including anti-urease, leishmanicidal and antinociceptive. The present study aims to characterize the GOV in vitro metabolism after incubation with rat and human liver microsomes (RLM and HLM, respectively) and to evaluate its pharmacokinetic properties. The identification of GOV metabolites was conducted by different mass analyzers: a micrOTOF II-ESI-ToF Bruker Daltonics® and an amaZon-SL ion trap (IT) Bruker Daltonics®. For the pharmacokinetic study of GOV in rats after intravenous administration, a LC-MS/MS method was developed and applied to. The analyses were performed using an Acquity UPLC® coupled to an Acquity TQD detector equipped with an ESI interface. The liver microsomal incubation resulted in new O-demethylated, di-hydroxylated and mono-hydroxylated compounds. Regarding the method validation, the calibration curve was linear over the concentration range of 2.5-3150.0ngmL-1, with a lower limit of quantitation (LLOQ) of 2.5ngmL-1. This method was successfully applied to a pharmacokinetic study. The profile was best fitted to a two-compartment model, the first phase with a high distribution rate constant (α) 0.139±0.086min-1, reflected by the short distribution half-life (t1/2α) 9.2±8.9min and the later one, with an elimination half-life (t1/2ß) 55.1±37.9min. The main plasma protein binding was 96.1%. This is a first report in this field and it will be useful for further development of govaniadine as a drug candidate.

Development of two step liquid-liquid extraction tandem UHPLC-MS/MS method for the simultaneous determination of Ginkgo flavonoids, terpene lactones and nimodipine in rat plasma: Application to the pharmacokinetic study of the combination of Ginkgo biloba dispersible tablets and Nimodipine tablets.

A sensitive, reliable and accurate UHPLC-MS/MS method has been firstly established and validated for the simultaneous quantification of ginkgo flavonoids, terpene lactones and nimodipine in rat plasma after oral administration of Ginkgo biloba dispersible tablets, Nimodipine tablets and the combination of the both, respectively. The plasma samples were extracted by two step liquid-liquid extraction, nimodipine was extracted by hexane-ether (3:1, v/v) at the first step, after that ginkgo flavonoids and terpene lactones were extracted by ethyl acetate. Then the analytes were successfully separated by running gradient elution with the mobile phase consisting of 0.1% formic acid in water and methanol at a flow rate of 0.6mL/min. The detection of the analytes was performed on a UHPLC-MS/MS system with turbo ion spray source in the negative ion and multiple reaction monitoring (MRM) mode. The calibration curves for the determination of all the analytes showed good linearity (R(2)>0.99), and the lower limits of quantification were 0.50-4.00ng/mL. Intra-day and inter-day precisions were in the range of 3.6%-9.2% and 3.2%-13.1% for all the analytes. The mean extraction recoveries of the analytes were within 69.82%-103.5% and the matrix were within 82.8%-110.0%. The validated method had been successfully applied to compare the pharmacokinetic parameters of ginkgo flavonoids, terpene lactones and nimodipine in rat plasma after oral administration of Ginkgo biloba dispersible tablets, Nimodipine tablets with the combination of the both. There were no statistically significant differences on the pharmacokinetic behaviors of all the analytes between the combined and single administration groups. Results showed that the combination of the two agents may avoid dosage adjustments in clinic and the combination is more convenient as well as efficient on different pathogenesis of cerebral ischemia.

Developing an Absorption-Based Quality Control Method for Hu-Gan-Kang-Yuan Capsules by UFLC-QTOF-MS/MS Screening and HPLC-DAD Quantitative Determination.

Traditional Chinese Medicine Preparations (TCMPs) contain massive numbers of ingredients responsible for their multiple efficacies. An absorption-based quality control method for complicated TCMPs using Hu-gan-kang-yuan Capsule (HGKYC) as an example was developed. To select proper chemical markers for quality control of HGKYC, an ultra-fast liquid chromatography (UFLC) coupled with electrospray ionization quadrupole time-off light mass spectrometry (UFLC-QTOF-MS/MS) method was used for the rapid separation and structural identification of the constituents in the HGKYC extract and the rat serum after oral administration of HGKYC. As a result, one hundred and seven prototype constituents including flavonoids, organic acid, phenylpropanoids, anthraquinones, saponins, alkaloids, terpenes, phenols and amino acids in HGKYC extract, and 43 compounds found in rat serum after oral administration of HGKYC were unambiguously identified or tentatively characterized by comparing retention times and MS information with those of authentic standards or available literature references. Finally, a simple, low-cost and effective method of simultaneous determination for baicalein, wogonin, paeonol and emodin in HGKYC was developed using high performance liquid chromatography coupled with a diode array detector. In conclusion, an absorption-based quality control pattern was developed and successfully used for evaluating HGKYC.

Development and Validation of an UPLC-Q-TOF-MS Method for Quantification of Fuziline in Beagle Dog After Intragastric and Intravenous Administration.

A specific and sensitive UPLC-Q-TOF-MS method operated in the positive ion mode was developed and validated for the quantification of Fuziline in Beagle dog plasma. Fuziline and Neoline internal standard were separated on an Acquity UPLC BEH C18 column with the total running time of 4 min using gradient elution at the flow rate of 0.25 mL/min. The calibration curves for Fuziline showed good linearity in the concentrations ranging from 2 to 400 ng/mL with correlation coefficients (r) greater than 0.9971. The lower limit of quantification was 0.8 ng/mL. Intra- and interbatch relative standard deviations ranged from 2.11 to 3.11% and 3.12 to 3.81%, respectively. Fuziline was stable under different sample storage and processing conditions. The developed method was successfully applied to the comparative pharmacokinetic study of Fuziline in Beagle dog after intravenous and oral administration. Low absolute bioavailability of Fuziline (1.45 ± 0.76%) suggested a significant metabolism transformation extent in Beagle dog.

Simultaneous quantification of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid in rat plasma by HPLC-MS/MS: application to a pharmacokinetic study of Longhu Rendan pills.

A sensitive, specific, accurate HPLC-MS/MS method was developed and validated for the simultaneous quantification of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid from Longhu Rendan pills in rat plasma. Chromatographic separation was performed with a Hypersil Gold C18 column using a gradient of methanol and 0.01% acetic acid containing 0.2 mm ammonium acetate as mobile phase. The analytes were quantified on a triple quadrupole mass spectrometer, operating in selected reaction monitoring mode and switching the electrospray ion source polarity between positive and negative modes in a single run. The calibration curves of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid were linear over the concentration ranges of 5-2000, 5-2000, 0.5-200, 0.5-200, 0.25-100, 0.25-100, 0.025-10 and 0.50-200 ng mL(-1) , respectively. The intra- and inter-assay precisions and accuracies were <11.6 and 91.9-108.2%, respectively, for all analytes. Matrix effects for all analytes were between 88.2 and 114.2%. Stability testing showed that all analytes were stable in plasma at 24 °C for 3 h, at 4 °C for 24 h, after three freeze-thaw cycles, and at -80 °C for 15 days. The method was successfully applied to an in vivo study evaluating the pharmacokinetics of multiple nonvolatile compounds following intragastric administration of Longhu Rendan pills to rats. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetics of reduced iso-α-acids in volunteers following clear bottled beer consumption.

Reduced iso-α-acids (reduced IAA) consisting of the rho-, tetrahydro- and hexahydro-IAA groups (RIAA, TIAA and HIAA, respectively) are ingredient congeners specific to beer and generally found in clear and also occasionally green bottled beer. Concentrations of reduced IAA were determined in the blood and urine of five volunteers over 6h following the consumption of small volumes of beer containing each of the reduced IAA. The reduced IAA were absorbed and bioavailable with peak concentrations at 0.5h followed by a drop of generally fivefold by 2h. Preliminary pharmacokinetics of these compounds in humans shows relatively small inter-individual differences and an estimated short half-life varying between ∼38 and 46min for the three groups. Comparison of RIAA analyte ratios within the group indicate that some analytes eliminate relatively faster than others and the formation of metabolite products was observed. Preliminary urine analysis showed only unmodified RIAA analytes were detectable throughout 6h and suggests extensive phase I metabolism of TIAA and HIAA analytes. In authentic forensic casework where clear or green bottled beers are consumed, the identification of reduced IAA groups may provide a novel method to target ingredient congeners consistent with beer ingestion and suggest the type of beer consumed.

Simultaneous determination by UPLC-MS/MS of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets: application to a pharmacokinetic study.

A rapid, reliable, and sensitive method was developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with an electrospray ionization (ESI) source for determination of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets (GBTs). The method simultaneously detects bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), quercetin (QCT), kaempferol (KMF), and isorhamnetin (ISR) for pharmacokinetic study. The analytes and internal standard (IS) were extracted from rat plasma by acetidin. An MS/MS detection was conducted using multiple reaction monitoring (MRM) and operating in the negative ionization mode. The calibration curve ranges were 5-500, 5-500, 2.5-250, 1-100, 1-100, 1-100, and 1-100 ng/ml for BB, GA, GB, GC, QCT, KMF, and ISR, respectively. The mean recovery of the analytes ranged from 68.11% to 84.42%. The intra- and inter-day precisions were in the range of 2.33%-9.86% and the accuracies were between 87.67% and 108.37%. The method was used successfully in a pharmacokinetic study of GBTs. The pharmacokinetic parameters of seven compounds were analyzed using a non-compartment model. Plasma concentrations of the seven compounds were determined up to 48 h after administration, and their pharmacokinetic parameters were in agreement with previous studies.

Pharmacokinetics of iso-α-acids in volunteers following the consumption of beer.

Hop-derived iso-α-acid (IAA) ingredient congeners are specific to beer. Concentrations of IAAs were determined in blood of five volunteers over 6 h following the consumption of small volumes of beer containing relatively high (Pale Ale beer) or low (wheat beer) concentrations of IAAs. IAAs were quickly absorbed with peak trans-IAA concentrations at 0.5 h followed by a drop of generally 10-fold at 2 h and low or not detectable trans-IAA levels at 6 h. However, the qualitative monitoring showed that the cis-IAAs were detected at all time-points. Preliminary pharmacokinetics of these compounds in humans shows relatively small interindividual differences and an estimated short half-life of ∼30 min. Comparison of 0.5 and 2 h blood specimens demonstrated that the trans isomers were eliminated faster than the cis counterparts. Preliminary urine analysis showed only unmodified 'co' analytes detectable throughout the 6 h. In authentic forensic casework where typically large amounts of conventionally hopped beer are consumed, this approach may provide a novel method to target ingredient congeners consistent with beer ingestion.

The stability of iso-α-acids and reduced iso-α-acids in stored blood specimens.

The long-term stability of the iso-α-acids, and three structurally similar but chemically altered iso-α-acids (known as 'reduced iso-α-acids' and consisting of the rho-, tetrahydro- and hexahydro-iso-α-acid groups) were investigated in whole blood. Pools of blank blood spiked with the four beer-specific ingredient congener groups at two different concentration levels were stored at 20°C, 4°C and -20°C; and extracted in duplicate in weeks 1, 3, 5 and 8, using a previously published method. A loss of 15% of the initial concentration was considered to indicate possible instability and losses greater than 30% demonstrated significant losses. The individual analytes within the four iso-α-acid groups were also measured to determine which iso-α-acids were subject to greater degradation and were responsible for the overall group instability. All four iso-α-acid groups showed significant losses after 8 weeks of storage under room temperature conditions in particularly the natural iso-α-acid group where major losses were observed (96% and 85% losses for low and high concentrations, respectively). Some degradation in all iso-α-acid groups were seen at 4°C samples predominantly due to the 'n' analogs of the groups showing an increased instability in blood. The -20°C storage conditions resulted in minimal changes in concentrations of all analytes. Higher than frozen storage temperatures can result in substantial changes on the stability of the iso-α-acid type groups in blood. The aim of this study was to highlight the stabilities of the IAA analytes in order to assist in the interpretation of IAA in stored blood specimens.

Pharmacokinetic comparisons of different combinations of Shaoyao-Gancao-Decoction in rats: simultaneous determination of ten active constituents by HPLC-MS/MS.

A specific HPLC-MS/MS method was developed and validated for simultaneous determination of ten constituents including albiflorin, oxypaeoniflorin, paeoniflorin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, ononin, glycyrrhizin and glycyrrhetinic acid in rat plasma using genistein as an internal standard (IS). The rat plasma samples were prepared by a one-step direct protein precipitation procedure with methanol. HPLC separation was achieved on a Zorbax XDB-C18 column (2.1mm×50mm i.d., 3.5µm) with gradient elution (A: 0.1% aqueous formic acid; B: methanol with 0.1% formic acid) at a flow rate of 0.5mL/min in a run time of 7min. All analytes and IS were detected by multiple reaction monitoring scanning with electrospray ionization in the negative ion mode. Calibration curves showed good linearity (r>0.998) over a wide concentration range for all analytes. The intra- and inter-day precisions were all within 15% and the accuracies were in the range of -6.2% to 10.1%. The validated method was successfully applied to determination and comparative pharmacokinetics investigation of the ten constituents in rat plasma after oral administration of different combinations (Radix Paeoniae Alba:Glycyrrhiza uralensis=1:1 or 4:1) of Shaoyao-Gancao-Decoction (SGD) extracts. Pharmacokinetic parameters were evaluated by a compartment model. There were perceptible differences in pharmacokinetic parameters (Cmax, AUC0-t, CL) of the analytes except for liquiritin between the two groups of SGD.