Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Development and validation of a rapid LC-MS/MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study.

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2 → 53.2, m/z 142.2 → 44.2 and m/z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5-100 ng/mL for arecoline, 5-5000 ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20 mg/kg arecoline to rats.

Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application.

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300µL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1-1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.

Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with ß-pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6×75mm, 3.5µm) using methanol: 5mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2-500ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC-MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration.

Quantification of blood betel quid alkaloids and urinary 8-hydroxydeoxyguanosine in humans and their association with betel chewing habits.

Chewing betel quid is a common habit in Taiwan and associated with the risk of oral cancer. Betel quid contains arecoline and arecaidine, which may serve as the exposure biomarkers of a chewing habit. We developed a liquid chromatography-tandem mass spectrometry method for the quantitative analysis of blood arecoline and arecaidine. Because 8-hydroxydeoxyguanosine (8-OH-dG) level in urine is only one early health marker of carcinogenesis, we also examined its association with chewing habit. We found a significant positive correlation between the quantities of betel quid used before the day of drawing blood and arecoline [(Spearman correlation coefficient (r) = 0.81; p value < 0.01) or arecaidine levels (r = 0.86; p value < 0.01)]. Habitual use quantity (quids/day) showed moderate correlation with both arecoline (r = 0.52; p value < 0.05) and arecaidine concentrations (r = 0.51; p value < 0.05). However, there were no significant correlations between total chewing years and concentrations of arecoline and arecaidine in serum or 8-OH-dG in urine. In conclusion, serum arecoline and arecaidine levels are measurable and good indicators for recent betel quid use.

A new matrix for analyzing low molecular mass compounds and its application for determination of carcinogenic areca alkaloids by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Arecoline is the main alkaloid present in the areca nut (or betel nut) and it has central nervous system effects. Its pharmacological activities induce the constriction of the bronchial smooth muscles, and stimulation of the lacrimal and intestinal glands. Chewing areca nut is harmful to health because this habit may increase the risk of the development of oral cancer. In this study, a fast method was provided for the determination of areca alkaloids by matrix-assisted laser desorption ionization (MALDI) mass spectrometer with a time-of-flight (TOF) analyzer. Traditionally the MALDI-TOF method was not suitable for the analysis of small molecular weight (m/z<600) compounds because of the high background of the matrix. In this study, a new matrix was utilized to decrease the background interference effectively. After simple sample preparation, 1 microL sample supernatant was mixed with 1 microL matrix and then deposited on the target plate. This new matrix was also used to test the MALDI imaging experiment. Application of this MALDI-TOF method for trace analysis of arecoline by this new matrix in human plasma at sub microM level proved workable.

Areca nut, energy metabolism and hunger in Asian men.

BACKGROUND: The nut of the Areca catechu palm has long been attributed effects on hunger and the digestive process. OBJECTIVES: The objectives were to assess experimentally effects of areca nut on fasting and postprandial energy metabolism, substrate utilization and hunger. SUBJECTS AND METHODS: Two randomized, placebo-controlled, double-blind studies were undertaken. In study 1, eight Indian men received bioadhesive gels delivering 0, 5, 10 or 20 mg arecoline to the buccal sulcus after an overnight fast. Resting energy expenditure and substrate utilization were determined by ventilated hood calorimetry over 6 h during which hunger was rated on five occasions. In study 2, 15 Indian men received gels delivering 0 or 10 mg arecoline after consuming a 2.5 MJ meal, and the same protocol was then applied as in study 1. RESULTS: Fasting resting energy expenditures exceeded basal metabolic rate (BMR) by 5.4+/-0.8% (Mean+/-SE) after placebo, and 5.1+/-0.7% after 20 mg arecoline, but by 0.9+/-0.8% and 0.7+/-0.5% following 5 mg and 10 mg arecoline, respectively. Carbohydrate (CHO) utilization rates rose after areca nut compared to placebo (F(3,252)= 7.3, p< 0.001). Hunger varied across doses (chi(2) = 10.5, p < 0.02), being lowest after 10 mg and highest after 20 mg, and was influenced by interaction of dose with delta resting energy expenditure. In study 2, areca dose interacted with fat-free mass (FFM) to lower by 5.4+/-11.2% the thermic effect of a meal (F(1,28) = 4.9, p = 0.05), and retarded peak 'digestive-phase' thermogenesis by 60 min (F(1,58) = 5.7, p = 0.02). Postprandial delta CHO utilization was greater (F(1,28) = 4.5, p = 0.05), and hunger was lower (chi:(2) = 3.8, p = 0.05), after areca nut. The areca nut altered relationships of hunger to thermic effects of the meal, and to delta substrate utilization, in ways consistent with appetite suppression. CONCLUSION: Areca nut constituents modulate metabolic signals regulating appetite in man. This concurs with customary belief.

Clinical pharmacokinetics of arecoline in subjects with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease. METHODS: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS. RESULTS: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects. CONCLUSIONS: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Quantitative determination of arecoline in plasma by gas chromatography chemical ionization mass spectrometry.

A capillary gas chromatography/mass spectrometry (GC/MS) method for the quantitative analysis of arecoline in plasma has been developed for concentrations in the range 1-50 ng ml-1. Hexadeuterated arecoline was utilized as the internal standard. The removal of drug from plasma was accomplished by a two-step liquid/liquid extraction procedure involving a wash step followed by extraction with 5% triethyl amine in ethyl acetate. The GC/MS determinations were carried out with temperature-programmed capillary GC and ammonia chemical ionization mass spectrometry. The [M + H]+ ions of both analyte and internal standard were monitored at m/z 156 and 162, respectively. The method is linear and has sufficient sensitivity, precision, accuracy and selectivity for analysis of drug levels in human plasma.