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form. The microwave digestion volume is now 10 mL instead of 20 mL. Also, the data presented in
March only represented 3 independent analyses of the SPIFAN materials. The present submission CARN-
05 has a full set of SLV data with the method in its final form. It is submitted in response to a second Call
for Methods for carnitine, but full SLV data is also presented for choline in case the ERP also wants to
advance it for this use.
Method Outline
Below is an outline of the method. A more complete version is appended.
1.
Weigh RTF or recon powder (1-5g) into microwave vessel.
2.
Add 125 µL internal standards (d
3
-carnitine and d
9
-choline) and water to 15 mL total volume (if
only free determined; however, if Total is needed add only 10 mL water as the additional 5 mL
will come in steps 3 and 4). Proceed to Step 5 for Free analysis (no ammonia or acid added). DF
at this stage (using typical 3g sample size), is 15 mL/3.0g = 5.0
3.
Total: Add 2.2 mL 70% HNO
3
, seal, digest for 40 minutes at 120°C. Cool.
4.
Add 3.5 mL 30% ammonium hydroxide to each vessel. Mix and stand for 30 minutes.
5.
Add 1 mL of above solution to 25 mL water in disposable tube, mix, and filter with 0.45µ Nylon
syringe filter. (25x dilution)
6.
Makeup 0.1 mL of above filtrate with 0.9 mL ACN and inject. (10x dilution)
7.
Simultaneous choline/carnitine on BEH HILIC column
8.
Standards are nominally 30, 150, and 300 ppb choline (as hydroxide) and 8.0, 40, and 80 ppb
carnitine with about 30 ppb choline-d9 internal standard and about 15 ppb carnitine-d3 IS.
9.
Total dilution is approximately 5 x 25 x 10 = 1250, but depends on sample weight
Features
•
Carnitine is the subject of this SLV, but total choline is also obtained simultaneously with proven
accuracy
•
Time to Result: < 8 hours possible, but in most cases LC-MS/MS runs overnight
•
Advantages of internal standard, added up front for good precision
•
HILIC column compatible with many methods (e.g. melamine)
•
Validated on two substantially different Waters models of LC MS/MS. Note that other similar
publications had used single quad MS.
System Suitability
•
Calibration residuals <4%
•
Internal Standard recovery 75-125% required (exact range TBD)
•
Confirmatory transition required (ratio limits relative to standard TBD)
•
Control sample SRM 1849a must be within limits
Candidates for 2016 Method of the Year
321