Carn-06_Book_Final

Carn-06 (Choline) FOR ERP USE ONLY DO NOT DISTRIBUTE

( 1 ) For high concentration samples a dilution step with water should be performed before addition of the internal standard.. ( 2 ) Accurately weigh the digestion vessels. ( 3 ) Transfer 1 – 5 g accurately weighed, of the clear extraction liquid to the digestion vessels. ( 4 ) Add 10 g accurately weighed ISTD standard solution to the digestion vessels. ( 5 ) Add 3 mL of 2M Potassium hydroxide solution, and 47 mL of water, close with stopper. ( 6 ) Hydrolyse in oven at 102°C for 4 h. ( 7 ) Cool to ambient temperature. ( 8 ) Add 4 mL 1.6M Hydrochloric acid ( 9 ) Dilute to 250 mL volume with water and accurately weight the solution. Cap and mix well. Transfer to an autosampler vial and cap. G. Instrument operation conditions ( a ) LC. – Isocratic run 0-2 min. 98% A; 2% B. Flow 0,5 mL/min; injection volume, 2 μL; column temperature, 25°C; autosampler temperature, ambient. ( b ) MS/MS.—Ionization mode, positive-ion electrospray ionization (ESI+);Curtain gas, 30 psi;Collision gas, Med; Ionspray voltage , 5.5 kV; source temperature, 700°C; Ion Source Gas 1, 65 psi;Ion source gas 2, 65 psi. ( c ) Parameters for MS/MS measurement see table 1. Table 1. Analyte Q1 a Q3 b DP c EP d CE e CXP f Choline ion 104.2 60.1 41.0 5.0 23.0 4.0 Choline ion 104.2 58.2 41.0 5.0 33.0 4.0 Choline-1,1,2,2-d 4 108.2 60.1 41.0 5.0 23.0 4.0 Choline-1,1,2,2-d 4 108.2 58.2 41.0 5.0 33.0 4.0 d EP = Entrance potential. e CE = Collision energy. f CXP = Collision cell exit potential. ( d ) Data acquisition was done in the multiple-reaction monitoring (MRM) mode. ( e ) Quantitation of choline.—The MRM chromatogram for choline is a sum of signals for the transitions m/z 104 > 60 and 104 > 58. Likewise, the MRM chromatogram for the ISTD is a sum of signals for the transitions m/z 108 > 160 and 108 > 58. Analyst automatically calculates a response ratio, which is defined as the choline peak area divided by the ISTD peak area. ( f ) UPLC analysis.—After verifying equilibration of the UPLC system, inject the working standards (L1 to L6) followed by a reagent blank, control sample, and sample extracts. ( g ) Inject working standards after approximately every 20 sample extraction, injected after the analysis of the last sample extract. Notes: Calibration curves must have a correlation coefficient r 2 of >0.990. ( 1 ) Plot each calibration standard response ratio versus its corresponding concentration to obtain calibration curves for choline. Apply a nonweighted linear regression to the data, and obtain an equation for the best-fit line. = + H. Calculation of results ( a ) Manual calculations.— a Q1 = Quadrupole mass filter 1. b Q3 = Quadrupole mass filter 3. c DP = Declustering potential.

vial and cap. ( 4 ) Level 4 calibration standard. - Accurately weigh 20 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 5 ) Level 5 calibration standard. - Accurately weigh 26 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 6 ) Level 6 calibration standard. - Accurately weigh 40 g standard solution and 2 g ISTD standard solution into a 50 mL flask. Dilute to volume with water and accurately weight the solution. Cap and mix well. Transfer to an autosampler vial and cap. F. Sample preparation and Extraction ( a ) Sample weighing. – ( b ) Accurately weighed the extraction vessels. ( 1 ) For dry blended/nonhomogeneous powder samples, transfer 25 g, accurately weighed to a 250 mL volumetric flask. Dissolve using warm distilled water ~40°C, cool, and make up to 250 g accurately weighed with distilled water. Transfer 10 - 15 g accurately weighed of reconstituted sample to the extraction vessels. ( 2 ) For wet blended homogeneous powder samples, transfer 2 - 3 g, accurately weighed directly to the extraction vessels. ( 3 ) For ready-to-feed samples or concentrated liquid products, transfer 5 to 10 g, accurately weighed of thoroughly agitated sample directly to the extraction vessel. ( 4 ) For dry food or animal feed samples, transfer 5 - 10 g, accurately weighed of sample directly to the extraction vessel. ( 5 ) For wet food or animal feed samples, transfer 10 - 15 g, accurately weighed of sample directly to the extraction vessel. ( 6 ) For nutritional supplements, transfer 1 – 1.5 g, accurately weighed of sample directly to the extraction vessel. ( c ) Ekstraktion. – ( 1 ) For liquid samples no added water is necessary. For wet samples add 5 mL of water. For dry samples add 10 ml of water. ( 2 ) Add 100 ml of Methanol (HPLC-grade), and cap. ( 3 ) Mix by shaking until well dispersed. Place flask on shaking table at 37°C for 12 h. adjust shaking to obtain good mixing. ( 4 ) Cool to ambient temperature. ( 5 ) Accurately weigh the extraction vessel. ( 6 ) Transfer part of the extraction liquid to a 50 mL centrifuge tube, and cap. ( 7 ) Centrifuge the extraction liquid to obtain a clear liquid, ( 1 ) For high concentration samples a dilution step with water should be performed before addition of the internal standard. ( 2 ) Accurately weigh the digestion vessels. ( 3 ) Transfer 1 – 5 g accurately weighed, of the clear extraction liquid to the digestion vessels. ( 4 ) Add 10 g accurately weighed ISTD standard solution to the digestion vessels. ( 5 ) Dilute to 250 mL volume with water and accurately weight the solution. Cap and mix well. Transfer to an auto- sampler vial and cap. ( e ) Determination of Total choline. – (approximately 200 rpm for 10 min). ( d ) Determination of free choline. –