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Note
: Nonspecific binding can occur with these analytes when using glassware, so plasticware should be
used at all times for standard/sample preparation. All laboratory plasticware should be single-use
whenever possible. Positive displacement pipets are also mandatory for pipeting to avoid
contamination and for accuracy with organic solvents.
B.
Chemicals and Reagents
(a)
Water
. – Optima MS grade (Thermo Fisher Scientific, Waltham, MA) or equivalent.
(b)
Acetonitrile
. – Optima MS grade (Thermo Fisher Scientific) or equivalent.
(c)
Ammonium formate
. – Optima MS grade (Thermo Fisher Scientific) or equivalent.
(d)
Formic acid
. – Optima MS grade (Thermo Fisher Scientific) or equivalent.
(e)
Nitric acid
. – 70% w/w, ACS grade (Avantor, Center Valley, PA) or equivalent.
(f)
Isopropanol
. – Optima MS grade (Thermo Fisher Scientific) or equivalent.
(g)
Desiccant
. – (VWR) or equivalent.
(h)
Reference Standard
. – L-Carnitine (USP, Rockville, MD) or equivalent.
(i)
Reference Standard
. – Choline Bitartrate (TCI, Tokyo, Japan) or equivalent.
(j)
Reference Internal Standard
. – L-Carnitine-d3 HCl (CDN Isotopes, Pointe Claire, Québec, Canada) or
equivalent.
(k)
Reference Internal Standard
. – Choline-1,1,2,2-d4 Chloride (CDN Isotopes) or equivalent.
Note
: All use of water in this method must be high purity MS grade water. It is recommended that all
preparation steps with nitric acid be performed within a fume hood, and the necessary personal
protective equipment used when handling.
C.
Mobile Phase Preparation
Mobile phase A (5mMammonium formate in 50:50 (v/v) water:acetonitrile with 0.2% formic acid) was
prepared by weighing 0.63 g of ammonium formate into a 1 L graduated cylinder. Water was added
along with a stir bar and mixed to dissolve before diluting to volume with water. The solution was
transferred to a 2 L mobile phase container along with 1 L of acetonitrile, 4mL of formic acid, stir bar,
and then thoroughly mixed. Mobile phase B (30mMammonium formate in 50:50 (v/v)
water:acetonitrile with 0.2% formic acid) was prepared by weighing 3.78 g of ammonium formate into a
1 L graduated cylinder. Water was added alongwith a stir bar and mixed to dissolve before diluting to
volume with water. The solutionwas transferred to a 2 L mobile phase container along with 1 L of
acetonitrile, 4mL of formic acid, stir bar, and then thoroughly mixed. Mobile phase B was also used for
the rinse solutions in the autosampler.
D.
Preparation of Standard Solutions
The carnitine stock standard was prepared at a concentration of 25mg/mL by weighing 0.25 g of L-
carnitine into a 20mL polypropylene tube followed by 10mL of water to dissolve. The purity of L-
carnitine from the Certificate of Analysis (CoA) and moisture determined by Karl Fisher immediately at
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