Assay of Ractopamine Hcl 2

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  DETERMINATION OF RACTOPAMINE HYDROCHLORIDE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Preparation of Standard Solution(s) a. Prepare the following standard solutions for the analysis of liver at 25 - 300 ppb: i. Ractopamine HCl liver stock solution - 1.00 mg/mL Note: The stock solution must be adjusted for purity during preparation. Do not correct for the HCl since ractopamine HCl is the analyte of interest. Prepare a ractopamine HCl standard stock solution by adding 100 ± 1 mg of ractopamine HCl reference standard to a 100 mL volumetric flask and diluting to volume with the 1:1 sample diluent plus methanol mixture. This solution is stable for three months at 2 - 8 °C. CAUTION:  Wear gloves when handling reference standard. Do not inhale the dust of the primary reference standard. ii. Ractopamine HCl intermediate liver standard (10 µg/mL): Pipet 1.0 mL of 1.00 mg/mL ractopamine HCl standard stock solution into a 100 mL volumetric flask and dilute to volume with the 1:1 sample diluent plus methanol mixture. Mix well. This solution is stable for one month at 2 - 8 °C. iii. Ractopamine HCl fortification liver standard (1.5 µg/mL): Pipet 15 mL of 10 µg/mL ractopamine HCl intermediate standard solution into a 100 mL volumetric flask and dilute to volume with the 1:1 sample diluent plus methanol mixture. Mix well. This solution is stable for one month at 2 - 8 °C. b. Prepare the following standard solutions for the analysis of muscle at 0.5 - 2.0 ppb: i. Ractopamine HCl muscle stock solution - 1.00 mg/mL Note: The stock solution must be adjusted for purity during preparation. Prepare a ractopamine hydrochloride standard stock solution by adding 100 ± 1 mg of ractopamine hydrochloride reference standard to a 100 mL volumetric flask and diluting to volume with methanol. This solution is stable for three months at 2 - 8 °C. CAUTION:  Wear gloves when handling reference standard. Do not inhale the dust of the primary reference standard. ii. Ractopamine HCl Intermediate muscle standard (10 µg/mL):   Pipet 1.0 mL of standard stock solution into a 100 mL volumetric flask and dilute to volume with sample diluent. Mix well. This solution is stable for one month at 2 - 8 °C. iii. Ractopamine HCl intermediate muscle standard (100 ng/mL): Pipet 1.0 mL of 10 µg/mL ractopamine HCl intermediate standard solution into a 100 mL volumetric flask and dilute to volume with sample diluent. Mix well. This solution is stable for one month at 2 - 8 °C. iv. Ractopamine HCl fortification muscle standard (10 ng/mL): Pipet 10 mL of 100 ng/mL ractopamine HCl intermediate standard solution into a 100 mL volumetric flask and dilute to volume with sample diluent. Mix well. This solution is stable for one month at 2 - 8 °C. Resolution Standard Preparation: Note: Prepare these solutions as necessary to check column resolution. a. For the analysis of liver at 25 - 300 ppb. i. Ritrodrine HCl liver stock solution (1 mg/mL): Weigh 50 ± 0.1 mg of ritodrine HCl reference standard into a 50 mL volumetric flask and dilute to volume with the 1:1 sample diluent plus methanol mixture. This solution is stable for three months at 2 - 8 °C. ii. Ritrodrine HCl intermediate liver standard (10 µg/mL): Pipet 1.0 mL of 1 mg/mL ritrodrine HCl standard stock solution into a 100 mL flask and dilute to volume with the 1:1 sample diluent plus methanol mixture. Mix well. This solution is stable for one month at 2 - 8 °C. iii. Liver resolution solution, mixed external standard (25 ng/mL): Pipet 250 µL of the 10 µg/mL ritodrine HCl intermediate solution and 250 µL of the 10 µg/mL ractopamine HCl intermediate solution into a 100 mL flask and dilute to volume with the 1:1 sample diluent plus methanol mixture. Mix well. This solution is stable for one month at 2 - 8 °C. b. For the analysis of muscle at 0.5 - 2.0 ppb: i. Ritrodrine HCl muscle stock solution (1 mg/mL): Weigh 50 ± 0.1 mg of ritodrine hydrochloride reference standard into a 50 mL volumetric flask and dilute to volume with methanol. ii. Ritrodrine HCl intermediate muscle standard (10  g/mL): Pipet 1.0 mL of standard stock solution into a 100 mL flask and dilute to volume with sample diluent. Mix well.   iii. Ritodrine HCl intermediate muscle standard (100 ng/mL): Pipet 1 mL of 10 µg/mL ritodrine HCl intermediate standard solution into a 100 mL volumetric flask and dilute to volume with sample diluent. Mix well. This solution is stable for one month at 2 - 8 °C. iv. Muscle Resolution solution, mixed external standard (1 ng/mL): Pipet 1 mL of the 100 ng/mL ritodrine HCl intermediate solution and 1 mL of the 100 ng/mL ractopamine HCl intermediate solution into a 100 mL flask and dilute to volume with sample diluent. Mix well. This solution is stable for one month at 2 - 8 °C. Preparation of External Calibration Curve a. Prepare the following standard solutions for the analysis of liver at 25 - 300 ppb: i. Ractopamine HCl liver external standard curve solutions (25, 50, 75, 150 and 300 ng/mL): Prepare volumetric dilutions of the 10 µg/mL ractopamine HCl intermediate standard using the 1:1 sample diluent plus methanol mixture. For 25, 50, 75, 150 and 300 ng/mL solutions make 250 µL to 100 mL, 500 µL to 100 mL, 750 µL to 100 mL, 1.5 mL to 100 mL and 3.0 mL to 100 mL volumetric dilutions, respectively. These solutions are stable for one month at 2 - 8 °C. b. Prepare the following standard solutions for the analysis of muscle at 0.5 - 2.0 ppb: i. Ractopamine HCl muscle external standard curve solutions (0.5, 0.75, 1.0, 1.5, 2.0 ng/mL): Prepare volumetric dilutions of the 100 ng/mL ractopamine HCl intermediate standard using sample diluent. For 0.5, 0.75, 1.0, 1.5, and 2.0 ng/mL solutions make 500 µL to 100 mL, 750 µL to 100 mL, 1000 µL to 100 mL, 1500 µL to 100 mL, and 2000 µL to 100 mL volumetric dilutions, respectively. These solutions are stable for one month when maintained at 2 - 8 °C. SAMPLE PREPARATION 1. Preparation and Storage of Tissues a. Initial processing includes grinding or blending of the tissues using a food grinder (or cryogenic grinding) to produce homogenous samples. Grind a minimum 500 g sample of tissue when possible. b. Store all tissues at < -10 °C when not processing or sub-sampling. Ractopamine has been shown to be stable in frozen tissue for one year. Note: Extreme care should be taken to make sure all tissue residue containing ractopamine is cleaned from glassware and other laboratory items in contact with   samples and standards. It is recommended that disposable items be used whenever possible and that labware used with standards and other sources containing high levels of ractopamine be kept separate from that used to prepare samples in the low ppb range. ANALYTICAL PROCEDURE 1. Preparation of Controls a. For liver i. Prepare blank and recovery samples by weighing two 10 ± 0.2 g blank tissues as part of the sample set. ii. For the analysis of liver at 25 - 300 ppb, prepare a 150 ppb recovery by adding 1 mL of 1.5 µg/mL fortification standard (D.2.a.iii) to one of the tissue blanks. Continue at step F.2.b. b. For muscle i. Prepare blank and recovery samples by weighing two 10 ± 0.2 g blank tissues as part of the sample set. ii. For the analysis of muscle at 0.5 - 2.0 ppb, prepare a 1 ppb recovery by adding 1 mL of the 10 ng/mL fortification standard (D.2.b.iii) to one of the tissue blanks. Continue at step F.2.b. 2. Extraction Procedure a. Tissue Extraction Weigh 10.0 ± 0.2 g of frozen or partially thawed ground sample tissue into a suitable container such as a 50 mL polypropylene centrifuge tube. b. Add 20 ± 1 mL of methanol to the sample. c. Homogenize the tissue slurry for approximately one minute using an ultrasonic cell disrupter equipped with a 1/4 inch micro tip. Alternatively, the tissue may be blended for approximately one minute using a suitable blender to produce a uniform slurry. Let the sample stand at room temperature for 10 - 15 minutes to enhance solvent contact with tissue. d. Add 20 ± 1 mL of methanol to a clean 50 mL polypropylene centrifuge tube. Rinse the probe by immersing it in this solution and blending for at least 30 seconds. Use this solution as the second 20 mL rinse in Step F.2.g. The probe must be cleaned between samples with methanol and water rinses, and given a final methanol rinse. A detergent may also be used to assist in cleaning mechanical homogenizers (e.g. UltraTurrix/Polytron). e. Centrifuge the tissue slurry at approximately 1500 RCF for 10 minutes. Exact speed and centrifugal force is not critical provided a good sediment pack is obtained. Refrigeration may be used, but is not necessary. f. Decant the supernatant into a 100 mL graduated mixing cylinder or other appropriate graduated glassware. g. Add the 20 mL of methanol from Step F.2.d to the tissue, vigorously suspend the
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