Methods to be Reviewed by OMB in 2016

1388

Bidlack et al.:

ournal of

AOAC I

nternational

ol.

98, N

5, 2015

will be. Degas the mobile phase and postcolumn electrolyte

solutions by bubbling helium through them at a flow rate just

fast enough to cause small ripples on the surface of the mobile

phase and postcolumn solutions. To maximize the life of the

zinc reactor column, degas the mobile phase and postcolumn

electrolyte solution for at least 30 min before connecting the zinc

reactor column or do not pump mobile phase and postcolumn

electrolyte solutions until at least 30 min after degassing begins.

Once the mobile phase and postcolumn electrolyte solutions

have been degassed, allow the column and postcolumn reactor

to equilibrate with mobile phase flowing at 0.4 mL/min and

postcolumn electrolyte solution flowing at 0.4 mL/min for at

least 30 min prior to the first injection if the zinc reactor has

been used for previous analyses or several hours if the zinc

postcolumn reactor has been freshly packed. Once the mobile

phase and postcolumn solutions have been degassed, reduce the

helium flow rate so that only a small stream of helium bubbles

are visible in the mobile phase and postcolumn solutions and

there is minimal disturbance to the surface of these solutions.

Bubble helium very slowly through the mobile phase and

postcolumn electrolyte solutions continuously throughout the

entire run. Once the run has started, do not adjust the helium

flow rate. Allow the fluorescence detector lamp to warm up

30 min prior to the first injection. (

Note

: When the mobile phase

and postcolumn electrolyte solution are continuously sparged

with helium throughout a run, it is not necessary to pack the

postcolumn reactor with zinc at the beginning of every run. It

should be possible to analyze hundreds of samples before the

zinc reactor column must be repacked.)

(

)

HPLC of standards and samples

.—Inject the most

concentrated standard (approximately 80 µg/L) onto the

column and observe the response on the fluorescence detector.

If necessary, adjust the detector gain and sensitivity settings so

that the standard response is within the range of the detector.

Once the detector settings have been determined, inject the

most concentrated standard 3–4 times and note the peak areas.

If the system is equilibrated, the RSD of the standard peak areas

should be ≤2%, and the peak areas should not steadily increase

or decrease by more than 4% from the first injection to the third

or fourth injection. If the RSD is >2%, locate the source of the

imprecision and correct it before beginning the sample analysis.

If peak areas steadily increase or decrease by more than 4%, the

system is not equilibrated and must be allowed to equilibrate

longer. Once the system has reached equilibrium and the

RSD is ≤2%, inject a set of standards, unknown samples, and

another set of standards. Every set of unknown samples must be

bracketed by standards.

(

)

Instrument shut down

.—After analyzing a set of

samples, simultaneously turn off the flow on the mobile phase

and postcolumn electrolyte solution pumps. Remove the helium

sparge lines from the mobile phase and postcolumn electrolyte

solution and turn off the helium. Turn off the fluorescence

detector lamp.

F. Calculations

The vitamin K

concentrations of samples analyzed on the

HPLC system are determined by comparison of peak areas from

samples of known weight with the peak areas of standards of

known concentration. Because the

cis

and

trans

vitamin K

retention times may shift slightly during a run, peak areas must

be used to quantitate

trans

vitamin K

(a)

Calculation of the standard concentrations:

(

) 1,000,000,000

(

)

1 2 3

1 2

3 4

W V V V

D D D D

= × × × ×

× × ×

where C

is the working standard concentration in µg/L; W is

the weight of standard in g; V

, V

, and V

are the aliquots of

stock, intermediate I, and intermediate standard II solutions,

respectively, in mL; 1,000,000,000 is the conversion factor

from g/mL to mcg/L; and D

, D

, and D

are the dilution

volumes of the stock, intermediate I, intermediate II, and

working standard solutions, respectively, in mL.

(b)

Peak areas are measured with a data system. Before

calculating concentrations, review all chromatograms to make

sure that

cis

and

trans

vitamin K

are baseline separated and that

there are no interfering peaks.

Trans

vitamin K

concentrations

cannot be calculated for any samples with interfering peaks

or poor separation between the

cis

and

trans

isomers (

see

Figures

2015.09B–D

). Check the integration of the

cis

and

trans

vitamin K

peaks.

Cis

vitamin K

elutes 1 to 3 min before

trans

vitamin K

depending on the analytical column used. If the peak

Figure 2015.09D. Chromatogram of an adult nutritional.

Candidates for 2016 Method of the Year

115