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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
(
o
) Syringe filters.— 0.45 μm.
(
p
) Pipets.—5000 μL adjustable
(
q
) Pipet tips.
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).
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. –
(
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
concentration is calculated on mass to mass basis.
(
f
) L-Carnitine inner salt intermediate solution,
(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
Carn-06
FOR ERP USE ONLY
DO NOT DISTRIBUTE