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1388 Bidlack et al.: J ournal of AOAC I nternational V ol. 98, N o. 5, 2015

Figure 2015.09D. Chromatogram of an adult nutritional.

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. ( 4 )  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. 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 1 retention times may shift slightly during a run, peak areas must be used to quantitate trans vitamin K 1. (a)  Calculation of the standard concentrations: F. Calculations The vitamin K 1 is the working standard concentration in µg/L; W is 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 1 , D 2 , D 3 , and D 4 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 1 are baseline separated and that there are no interfering peaks. Trans vitamin K 1 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 1 peaks. Cis vitamin K 1 elutes 1 to 3 min before trans vitamin K 1 depending on the analytical column used. If the peak the weight of standard in g; V 1 , V 2 , and V 3 ( W V V V s = × × × × ) 1,000,000,000 ( 1 2 3 1 2 D D D D × × × 3 4 ) C where C s

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.) ( 3 )  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