Determination of (4E,6E,12E)-Tetradecatriene-8,10-diyne-1,3-diyl Diacetate in Rat Plasma and Tissues by HPLC-UV Method and Their Application to a Pharmacokinetic and Tissue Distribution Study

In China Atractylodis Rhizoma is widely used for the treatment of rheumatic diseases and digestive disorders. Stir-frying with wheat bran is the most common processing method. In order to clarify the influence of processing on pharmacological properties of Atractylodis Rhizoma, an investigation was carried out to compare the pharmacokinetics and tissue distribution of typical constituent after oral administration of raw Atractylodis Rhizoma and processed ones. A simple, rapid, and sensitive high performance liquid chromatography with UV detection was developed and validated for the determination of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma. A chromatography was carried out on Diamonsil C18 (250 × 4.6 mm; 5 μm) analytical column, using a mobile phase which consisted of acetonitrile and 0.1% phosphoric acid water (60 : 40, v/v) at a flow rate of 1.0 mL·min−1. The wavelength was set at 336 nm. The LLOQ of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate was 0.00143 μg·mL−1. Both accuracy and precision were satisfactory. The pharmacokinetic results showed that the T max⁡ was 1 hour in advance and the C max⁡ was increased after processing. Tissue distribution showed that the highest level was in spleen. And the concentrations in the spleen were increased after stir-frying with bran.


Introduction
In traditional Chinese medicine, Atractylodis Rhizoma is the dried root and stems from Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. The medicinal herb is widely known as Cangzhu in China. And it is widely used for the treatment of rheumatic diseases, digestive disorders, mild diarrhea, and influenza [1]. In clinic, Atractylodis Rhizoma is often processed by stir-frying with wheat bran with the aim of reducing its dryness and increasing the function of tonifying spleen [2,3].
However, there have been few methods available for its quantification in biosamples and few reports on its pharmacokinetic study and tissues distribution until now. The in vivo study of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate, an active component of Atractylodis Rhizoma, would be necessary and helpful for further clinical application and explanation of the processing mechanism. The present paper developed a new and simple RP-HP LC method for quantification of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma and tissues after oral administration of raw and processed Atractylodis Rhizoma, respectively. This fully validated method was successfully applied to a pharmacokinetic and tissue distribution study of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rats for the first time.
HPLC grade acetonitrile was purchased from Fisher Scientific Company (New Jersey, USA) and Pure water was supplied by Wahaha Company (Hangzhou, China). Analytical grade ethanol and chloroform were from Baierdi Company (Beijing, China). Atractylodis Rhizoma was identified by Professor Li Feng (Liaoning University of TCM) according to the standards of Chinese Pharmacopoeia 2010. The processed Atractylodis Rhizoma comes from the same batch Atractylodis Rhizoma. The herb was stored in a cool and dry place.

Preparation of Atractylodis Rhizoma Solution.
Atractylodis Rhizoma (50 g) was crushed into powder and soaked into 600 mL of 80% ethanol for 24 h and then percolated at 2 mL⋅min −1 , and Ethanol was evaporated to near dryness under reduced pressure to get the residue. Distilled water was added into the residue and then vortexed for 10 min. The final concentration of Atractylodis Rhizoma solution was 2 g⋅mL −1 [9]. The sample was stored in dry and dark place before use.

Apparatus and HPLC Conditions.
The liquid chromatographic system consisted of an LC-10 AD pump (Shimadzu, Kyoto, Japan) with a 20 L loop (Cotata, CA, USA) and an SPD-10A ultraviolet-visile detector (Shimadzu, Kyoto, Japan). A LC-10 AD workstation was used for data acquisition. A Diamonsil C 18 analytical column (250 × 4.6 mm; 5 m) from Dikma Technologies (Dalian, China) was used. The mobile phase consisted of acetonitrile and 0.1% phosphoric acid water (60 : 40, v/v) at a flow rate of 1 mL⋅min −1 . The detection wavelength was set at 336 nm. All the measurements were performed at room temperature, and the injection volume was 20 L.

Sample Preparations.
For plasma samples, the 200 L of rat plasma was mixed with 20 L IS (0.00504 mg⋅mL −1 ). After protein was precipitated with 1,000 L of acetonitrile in 1.5 mL polypropylene tube by vortexing for 2 min, the sample was centrifuged at 10,000 rpm⋅min −1 for 5 min. The supernatant was transferred into a 5.0 mL tube and added with 1,000 L of chloroform, extract and the under organic phase was transferred to another tube and evaporated to dryness at 40 ∘ C with nitrogen. The resulting extract was dissolved in 50 L of methanol, and vortex mixed for 2 min. After centrifugation at 10,000 rpm⋅min −1 for 5 min, 20 L supernatant was injected for analysis [10][11][12]. For tissue homogenate, each weighed tissue sample was thawed and the homogenized in ice-cold physiological saline (2 mL). Then a 200 L of tissue homogenate was taken and processed further like the plasma samples. after an oral dose. All blank plasma samples and tissue homogenates were prepared and analyzed to ensure the absence of interfering peaks.

Calibration
Procedure. The linearity of the method was assessed by plotting calibration curves in plasma at seven concentration levels in triplicate on three consecutive days. The lower limit of quantification (LLOQ) was defined as the lowest concentration of the calibration curve that was measured with accuracy and precision by analyzing samples in six replicates at the concentration of 0.00143 g⋅mL −1 for (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate.

Accuracy and Precision.
Intraday precision and accuracy were evaluated by analysis of the three QC samples with six determinations per concentration at the same day, whilst the interday precision and accuracy were measured over three consecutive days. The precision was defined as the relative standard deviation (RSD%), while accuracy was determined by calculating the percentage deviation observed in the analysis of QC samples and expressed by relative

Extraction Recovery and Stability.
The extraction recoveries of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate were determined at low, medium, and high level of QC samples. Recoveries were calculated by comparing the observed peak area ratios in biosamples to those nonprocessed standard solutions at the same concentrations. The recovery of IS was determined in the same way at the concentration of 0.00504 mg⋅mL −1 .
The stability of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in plasma and tissue was determined under different storage or handling conditions. Short-term stability was assessed by analyzing QC samples kept at room temperature for 8 h. Freeze-thaw stability was evaluated at three consecutive freeze-thaw cycles. Long-term stability was studied by assaying samples following a period of 10 days of storage at −20 ∘ C.

Applications in Pharmacokinetic Studies.
All the studies on animals were in accordance with the Guidelines for the Care and Use of Laboratory Animals. Healthy Sprague-Dawley rats (250 ± 20 g) were purchased from The Medical University of Dalian (Dalian, China) and acclimated in the laboratory for one week before to the experiments. Rats for oral ingestion were fasted for 12 h with free access to water. Rats were oral administration raw and processed Atractylodis Rhizoma at a single dose of 40 g⋅kg −1 , respectively.

Tissue Distribution Study.
For tissue distribution study, forty-eight rats were divided into eight groups ( = 6 per group) randomly.
After oral administration four groups rats raw Atractylodis Rhizoma at a single dose of 40 g⋅kg −1 and the other four groups rats processed Atractylodis Rhizoma at a single dose of 40 g⋅kg −1 . Heart, liver, spleen, lung, kidney, stomach, small intestine, and large intestine were collected at 0.5, 2, 4, and 8 h. Tissue samples were weighed 0.1 g rapidly, rinsed with physiological saline to remove the blood or content, blotted on filter paper, and then stored at −20 ∘ C and dark place until use.

Recovery and Stability.
The extraction recoveries of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate ranged from 86.6% to 91.6% in plasma and tissue samples and the method recovery ranged from 85.6% to 94.9%, while the recovery of IS was above 80%. These data indicated that the biosample preparation procedure was satisfied and can achieve the acceptable extraction recovery. Stability of analytes showed no significant sample loss over 12 h at room temperature, three freeze-thaw cycles, and 10 days storage condition. The RE of three conditions were within ±15%. (4E,6E,12E)-Tetradecatriene-8,10diyne-1,3-diyl Diacetate in Rats. The assay was applied to a preliminary pharmacokinetic experiment after oral administration of 40 g⋅kg −1 raw and processed Atractylodis Rhizoma to rats, respectively. Mean concentration-time curves were shown in Figures 3 and 4. The pharmacokinetic parameters were shown in Table 2.

Pharmacokinetics of
A significant result of this study is finding that (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate showed double peaks after oral administration, which demonstrated that a hepatoenteral circulation may exist. For raw Atractylodis Rhizoma the absorption peaks in rat plasma was at 2 h and 4 h, respectively and the max was 38 ± 24 g⋅L −1 . And for processed Atractylodis Rhizoma the absorption peaks was at 1 h and 3 h, respectively and the max is 42 ± 17 ⋅gL −1 . So the time of absorption peak was 1 hour in advance and the concentration of rat plasma was increased after processing. The value of max and 1/2 indicated that the (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate was rapidly distributed but slowly eliminated. The reason for this result also requires further study.

Conclusion
A simple, specific, and rapid RP-HPLC method with UV detection for quantification of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma has been developed for the first time. It has been successfully applied   to a preliminary pharmacokinetic and tissue distribution study of (4E,6E,12E)-tetradedecatriene-8,10-diyne-1,3diyl diacetate after oral administration of 40 g⋅kg −1 raw and processed Atractylodis Rhizoma, respectively. We found that the max have significant difference ( < 0.05), and other pharmacokinetics have no significant difference after using Student's t-test. The result indicated that processing can promote and accelerate the absorption and the concentration of (4E,6E,12E)-tetradecatrinen-8,10-diyne-1,3-diyl diacetate is the highest in the spleen Which proved that the traditional theory of processing Atractylodis Rhizoma can increase its function of tonifying the spleen.