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1660 Butler-Thompson et al.: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015

Figure 2011.10B. System setup and configuration: Configuration 2.

( 3 )  HPLC of standards and samples .—Make 3–4 injections of a working standard and verify the precision of those injections is ≤3%. If the system is working properly, inject a set of 3–6 working standards once, a set of 1–14 samples, and another set of 3–6 working standards. Every set of 1–14 samples should be bracketed by standards of appropriate concentration. F. Calculations (a)  Chromatography .—Visually inspect each standard and sample chromatogram and verify that vitamin B 12 is resolved from all other peaks in the chromatograms (Figures 2011.10D and E ). (b)  Measurement of peak area .—Peak areas are measured with a data system. Before calculating the vitamin B 12 concentrations of samples, compare the vitamin B 12 peak areas of the standards with the vitamin B 12 peak areas of the samples and verify that the vitamin B 12 peak areas of the samples are within the range of the vitamin B 12 peak areas of the standards. (c)  Calculation of standard concentration .— WS = S w × P × A/200 where WS = working standard concentration in μg/L; S w  = amount of vitamin B 12 standard weighed in mg; P = purity of USP reference standard in μg cyanocobalamin (vitamin  B 12 )/mg of the standard; A = aliquot of vitamin B 12 intermediate standard used (0.5, 1, 2, 3, 4, or 5) in mL; and 200 = dilution volume in mL. (d) Preparation of standard curves .—( 1 ) At each standard concentration, average the peak area of the standard injected at the beginning of a set of samples with the peak area of the standard injected at the end of the set of samples. Prepare a standard curve by performing linear least squares regression

Figure 2011.10A. System setup and configuration: Configuration 1.

laboratory water and discard eluent. Air-dry each cartridge by pulling a vacuum until no more effluent is observed. Close each stopcock. Place a 5 or 10 mL volumetric flask under each cartridge. Add 4.4 mL 30% acetonitrile to each cartridge. Open each stopcock and elute vitamin B 12 into the volumetric flasks. ( 4 )  Final dilution .—For samples collected in 10 mL volumetric flasks, dilute to volume with water. For samples collected in 5 mL volumetric flasks, in a hood add 0.1 mL freshly prepared 0.4% KCN to each volumetric flask. Place prepared samples in a 95°C oven for at least 1.5 h, but for no more than 4 h. After at least 1.5 h, remove samples from the oven and cool to room temperature. Dilute to volume with laboratory water. Filter an aliquot of each standard and prepared sample through a 0.45 µm syringe filter into an autosampler vial. (c) HPLC analysis .—( 1 )  System setup and configuration.— See Figures 2011.10A and B for configurations. ( 2 )  Instrument operation conditions .—( a )  Run time .—30–35 min. ( b )  Injection volume .—900 µL to 2.0 mL. ( c )  System configuration.—See Table 2011.10E . ( d )  Isocratic pump .—Mobile phase D: 2.5% acetonitrile. Flow rate: Adjust so that vitamin B 12 elutes from the size- exclusion column between 10.5 and 14.5 min. Typical flow rates, 1.1–1.2 mL/min. Note : To determine an appropriate flow rate, connect the size-exclusion column directly to the UV-Vis detector and inject the high standard. Adjust flow rate as necessary so that vitamin B 12 elutes between 10.5 and 14.5 min. ( e )  Gradient pump .—Mobile phase compositions: mobile phaseA, 0.4%TEAin laboratory water, pH 5–7; mobile phase B, 0.4% TEA and 25% acetonitrile in H 2 O, pH 5–7; mobile phase C, 0.4% TEA and 75% acetonitrile in H 2 O, pH 5–7. Determine an appropriate gradient to elute vitamin B 12 in 23–30 min and resolve vitamin B 12 from riboflavin using the information in Table 2011.10F . ( See Figure 2011.10C .) ( f ) Gradient pump flow rate .—1.0 mL/min. ( g )  Detector settings .—Detection wavelengths and bandwidth, 550 and 10 nm, respectively.

Table 2011.10E. System configuration Time, min

Valve configuration

0.00–10.5 10.5–14.5

Configuration 1 Configuration 2 Configuration 1

14.5–30.0 to 33