Carn-06_Book_Final

Carn-06 FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of Carnitine in Infant Formula, Adult/Pediatric Nutritional Formula, food and feed ESI LC-MS/MS (applicable to the determination of free and total carnitine in Food/feed, infant formula, adult nutritionals and nutritional supplement.) A. Principle Carnitine and esters of this are extracted by methanol and water. Free carnitine is determined on the extract. Total carnitine is measured after alkaline hydrolysis at elevated temperature of the extract, thereby converting the bound forms in to free carnitine. The resulting free Carnitine is quantified by isotope dilution LC-MSMS. B. Apparatus ( a ) Column. - Synergi 2,5μ polar-RP 100 x 3.00 mm (Phenomenex), or equivalent. ( b ) Liquid chromatograph. - Thermo Dionex Ultimate 3000 RS 600bar HPLC system, or equivalent. ( c ) Detector. - AB Sciex Q-Trap 3200 mass spectrometer, or equivalent. ( d ) Data system. – Analyst 1.5.1 and Dionex Chromatography MS link 2.10.0.2906 ( e ) Balance.—Readable to at least 0.0001 g (Satorius, or equivalent). ( f ) Balance.—Readable to at least 0.001 g (Kern, or equivalent). ( g ) Incubator. – Capable of 37 ± 5°C ( h ) Centrifuge. ( i ) Shaking table. (Inside Incubator) ( j ) Oven. - Capable of 102 ± 2°C ( k ) Centrifuge tubes.—plastic , 50 mL with screw cap. ( l ) Extraction vessels. – conical flasks, 250 ml with stoppers. ( m ) Digestion vessels. Conical flasks, 500 ml with stoppers. ( n ) Volumetric flasks. 50, 100 and 500 ml and 1 L C. Reagents ( a ) Purified Water, USP; ≥18 Mohm/cm. ( b ) Methanol. – HPLC-grade (for sample preparation) ( c ) Methanol. – LC-MS grade (for LC-MS/MS mobile phases preparation) ( d ) Ammonium formate. LC-MS grade, crystalline, ≥99.0% ( e ) Formic acid. LC-MS grade, ≥99.9% ( f ) Potasium hydroxide. – p.a., crystaline, ≥85% ( g ) Hydrochloric acid. –p.a., 37% D. Standards ( a ) L-Carnitine reference standard.—U.S. Pharmacopeia (Rockville, MD; Cat. No. 1359903). ( b ) L-Carnitine-d3 HCl (methyl-d3).—C/D/N Isotopes (Quebec, Canada; Cat. No. D-5069). ( o ) Syringe filters.— 0.45 μm. ( p ) Pipets.—5000 μL adjustable ( q ) Pipet tips.

( 1 ) Mobile phase A. – Measure 200 mL ammonium formate buffer into a 1 L volumetric flask and dilute to the mark with water. ( 2 ) Mobile phase B. – Methanol LC-MS grade ( c ) Potassium hydroxide solution. – 2 M Dissolve 56.1 g reagent grade KOH pellets in water in a 500 mL volumetric fl ask, and dilute to volume with water. Storage: plastic bottle, room temperature. Expiration: 1 year. ( d ) Hydrochloric acid.- 1.6 M. Measure 67.5 mL concentrated HCl into 500 mL volumetric flask and dilute to the mark with water. Standardization is not required. Expiration: 1 year. ( e ) L-Carnitine inner salt solution (approximately 4 mg/g). – Accurately weigh 400 mg L-carnitine reference standard as quickly as possible (because of hygroscopicity), and transfer to a 100 mL flask. Dissolve L-carnitine in water, and dilute to volume with water and accurately weigh the solution. The (approximately 400 µg/g). — Accurately weigh 10 g L- Carnitine inner salt solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. ( g ) L-Carnitine Stock solution, (approximately 12 µg/g). — Accurately weigh 15 g L-Carnitine inner salt intermediate solution into a 500 mL flask. Dilute to volume with water and accurately weigh the solution. Dispense the solution into plastic centrifuge tubes, each receiving ≥10 mL. Storage: freezer, –20°C. Expiration: 1 year. ( h ) L-Carnitine-d 3 Stock solution, (approximately 80 µg/g). – Accurately weigh 50 mg L-carnitine-d 3 HCl (methyl-d 3 ) as quickly as possible, and transfer to a 500 mL flask. Dissolve L-carnitine-d 3 HCl (methyl-d 3 ) in water, and dilute to volume with water and accurately weigh the solution. Dispense the solution into plastic centrifuge tubes, each receiving ≥15 mL. Storage: freezer, –20°C. Expiration: 1 year. ( i ) ISTD standard solution (approximately 2.3 µg/g L- carnitine-d 3 ). – Accurately weigh 14 g L-Carnitine Stock solution into a 200 mL flask. Add 4 mL of 1.6 M Hydrochloric acid and 3 ml of 2 M Potassium hydroxide solution. Dilute to volume with water and accurately weigh the solution. This amount of internal standard is sufficient to 6 standards and 18 samples. ( j ) Standard solution (approximately 1.2 µg/g of L-carnitine and choline). – Accurately weigh 10 g L-Carnitine Stock solution and 10 g Choline Stock solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. ( k ) Working standards. — Prepare fresh on day of analysis. ( 1 ) Level 1 calibration standard. - Accurately weigh 1 g standard solution and 4 g ISTD standard solution into a 100 mL flask. Dilute to volume with water and accurately weigh the solution. Cap and mix well. Transfer to an autosampler vial and cap. ( 2 ) Level 2 calibration standard. - Accurately weigh 3 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. ( 3 ) Level 3 calibration standard. - Accurately weigh 10 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. ( 4 ) Level 4 calibration standard. - Accurately weigh 20 g concentration is calculated on mass to mass basis. ( f ) L-Carnitine inner salt intermediate solution,

E. Solutions Preparation ( a ) Ammonium formate buffer. – 100mM, pH 3.0

Quantitatively transfer 1.12 ±0.01 g ammonium formate into a 1 L beaker add 800 ml water, dissolve, adjust pH to 3,0 with concentrated formic acid, transfer to volumetric flask and fill to volume with water. ( b ) Mobile phase. –