Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13164
標題: 歐索林酸在中華絨螯蟹之藥物動力學與殘留檢測研究
Pharmacokinetic and residues determination of oxolinic acid in the Chinese mitten crab, Eriocheir sinensis
作者: 涂青宇
Tu, Ching-Yu
關鍵字: oxolinic acid
歐索林酸
Chinese mitten crab
HPLC
pharmacokinetic
haemolymph
muscle
hepatopancreas
testis
hair
中華絨螯蟹
高效液相層析法
藥物動力學
血淋巴液
肌肉
肝胰腺
精巢
絨毛
出版社: 獸醫學系暨研究所
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摘要: 本研究之目的在於開發以高效液相層析法搭配螢光檢測器檢測中華絨螯蟹血清與各組織中之歐索林酸(oxolinic acid),並分析經口服投予歐索林酸後中華絨螯蟹體內之藥物動力學。藥物標準品以0.05 M 磷酸鹽緩衝食鹽水(PBS, pH7.4)稀釋後分別加入中華絨螯蟹血清和均質之肌肉、肝胰腺與精巢,與在5N NaOH溶解後以等量5N HCl中和之絨毛組織液,所有組織分別經超音波水浴震盪後離心。將血清、絨毛上清液以及經n-hexane去脂之肌肉、肝胰腺與精巢水層液分別通過C18固相萃取管柱,管柱中結合藥物以methanol/30% NH4OH(75: 25, v/v)沖提,並經氮氣吹乾後進行高效液相層析。所開發出檢測技術條件為:Cosmosil 5C18-MS(5 µm, 4.6 × 150 mm i.d.)管柱以25 : 75之acetonitrile/0.02 M PBS(pH 3)為移動相,流速1 mL/min,螢光檢測器激發波長335 nm,散射波長378 nm。結果顯示8分鐘左右有一明顯之歐索林酸吸收波峰,波峰面積與濃度回歸曲線相關係數高於0.999,歐索林酸在所有組織中的回收率則介於72~146%之間,不同組織中變異係數不超過2.32%,標準品之檢測極限(limit of detection)可達到0.5 ng/mL(s/n≧3),定量極限(limit of quantitation)則為1 ng/mL(s/n≧10)。為了解歐索林酸在中華绒螯蟹體內之藥物動力學分布情形,口服投予50 mg/kg b.w.之歐索林酸至平均體重127 ± 34 g 之雄蟹,維持水溫為23 ℃,灌食藥物後第0.5、1、2、3、4、8、12、16、24、36 hr以及第2、3、4、7、14、21、28、35與42天後均採樣3隻進行分析。結果顯示血清中最高藥物濃度(Cmax)為9.863 ± 0.94 µg/mL,出現時間(Tmax)為2 hr,經WinNonlin軟體分析,歐索林酸在中華絨螯蟹血清與組織中之分布均為二室開放模式與一級速率排除方程式,血清中藥物分佈半衰期(t1/2α)為16.14 hr,排除半衰期(t1/2β)為52.14 hr。歐索林酸之曲線下面積(Area under curve)在血淋巴液、肌肉、肝胰腺、精巢與絨毛則分別為195.23 µg*hr/mL、147.80 µg*hr/g、151.61 µg*hr/g、121.58 µg*hr/g 與 96.19 µg*hr/mg。根據我國歐索林酸在水產品中最高殘留容許量(maximum residue limit, MRL)50 ppb之標準,所有可食組織以肝胰腺藥物排除較慢,其藥物濃度實測值與預測值低於50 ppb之時間點分別為35天與27天,因此建議在臨床上對中華絨螯蟹單一劑量口服投予歐索林酸後之停藥期至少應在35天以上,當體內組織歐索林酸濃度排除至低於MRL時,體外絨毛中藥物之實測值與預測值分別為44.25 ng/mg與48.7 ng/mg,由於絨毛具有所有組織中最長的藥物排除半衰期(705.53 hr),若以絨毛建立體外檢測之標準,則可能不用犧牲蟹體便能評估體內組織中藥物殘留。本研究開發之檢測方法確能有效應用於中華絨螯蟹或相關水產品組織內歐索林酸之殘留檢測與藥物動力學之研究。
The aim of this study is to develop a technique to determinate the residual oxolinic acid in the sera and tissues of the Chinese mitten crab (Eiocheir sinensis) by high-performance liquid chromatography (HPLC) with fluorescence detector. A standard solution of oxolinic acid (OA) was prepared by dilution with 0.05 M phosphate buffered saline (PBS) (pH 7.4). Male crabs were sampled in this study by withdrawing haemolymph Muscle, hepatopancreas and testis were completely homogenized prior to addition of spiked OA. Before mixing with OA, the hair was dissolved with 5N NaOH then neutralized by equal volume 5N HCl. All tissues diluted with PBS (pH 7.4) were transferred into ultrasonic water bath and then centrifuged. The supernatants of serum, hair and the water layer of n-hexane defatted muscle, hepatopancreas and testis were passed through C18 solid phase extraction columns. Finally, OA in the column was eluted with methanol/30% NH4OH (75: 25, v/v), and then was evaporated to dry by nitrogen gas. Extracted OA was determined by HPLC with a fluorescence detector (excitation/emission = 335 nm/ 378 nm) and a Cosmosil 5C18-MS reverse phase column (5 µm, 4.6 X 150 mm i.d.). A mixture of acetonitrile and 0.02 M PBS pH 3.0 (25: 75) was used as the mobile phase at a flow rate of 1.0 mL /min. A peak with retention time around 8 min was observed. The standard curves spiked OA at different levels showed a good linear correlation coefficient (R2 > 0.999). Our results indicated that the recoveries of OA from all tissues were about 72% ~146%. Meanwhile, the mean of coefficient of variation (CV) value in different tissue was less than 2.32%. The limit of detection in standard was 0.5 ng/mL (s/n≧3). The limit of quantitation in standard was 1 ng/mL (s/n≧10). In order to realize the pharmacokinetic of OA in the Chinese mitten crab, the usage of oral administration at a dose of 50 mg/kg-b.w. in male crabs, body weight as 127 ± 34 g, at water temperature of 23 °C. Three crabs were sacrificed to extract and analyze the residual OA in each time point, 0.5, 1, 2, 3, 4, 8, 12, 16, 24, 36 hr, 2nd, 3rd, 4th, 7th, 14th, 21st, 28th, 35th, and 42nd day after feeding OA. The results revealed that the time to peak serum concentration, Tmax, was 2 hr and peak concentration, Cmax, was 9.863 ± 0.94 µg/mL. After analyzed by the WinNonlin software, the pharmacokinetics of OA in sera and tissues of Chinese mitten crab were fitted to a two-compartment open model, 1st order elimination. The distribution half-life (t1/2α) was16.14 hr while the elimination half-life (t1/2β) was 52.14 hr in crab haemolymph. The estimated area under the curve (AUC) in haemolymph, muscle, haptopancreas, testis and hair were 195.23 µg*hr/mL, 147.80 µg*hr/g, 151.61 µg*hr/g, 121.58 µg*hr/g and 96.19 µg*hr/mg, respectively. According to the rules of aquatic animal drug administration, the maximum residue limit (MRL) of OA should be 50 ppb. As the elimination rate of hepatopancreas was slower than other edible tissue of Chinese mitten crab. Once the concentration of residual OA was lower than 50 ppb, the observed value and predicted value appeared around 35th day and 27th day, respectively. Therefore, we suggested that the withdrawal period in Chinese mitten crab should be longer than 35 days with single oral administration of OA in clinic application. When the concentration of residual OA inside crab decreasing under MRL, the observed value and predicted value of outside hair were 44.25 ng/mL and 48.7 ng/mL, respectively. Since the crab hair showed the longest half-life (705.53 hr) to eliminate OA. It's possible to establish a standard for outside determination with the hair instead of taking life from crabs to estimate possible residue inside. In conclusion, the developed technique may be employed as a practical method for detection of residual OA and pharmacokinetic studies in Chinese mitten crab or other aquaculture products.
URI: http://hdl.handle.net/11455/13164
其他識別: U0005-0702200716110000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0702200716110000
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