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(
4
) Store LC vial at 4°C protected from light until analysis.
F. LC Analysis
(
a
)
Setup.-
(
1
) Set detector to 425 nm; column temperature to 55°C; injection volume to 0.8 μL;
and flow rate to 1.4 mL/min.
(
2
) Program the gradient shown in Table
2016.16D
.
(
3
) Equilibrate the column with mobile phase A.
(
b
)
Procedure.-
(
1
) Make single injections of one set of calibration standards (Std 1-Std 7).
(
2
) Make single injections of each LC sample.
(
3
) After approximately every 10 sample injections, and at the end of the run, re-inject one of
the calibration standards for quality control purposes.
G. Calculations
(
a
)
Calibration standards.-
(
1
) Measure peak areas for CUR, DMC, and BDMC in the set of
calibration standards and the re-injected calibration standards.
(
2
) Ensure that the re-injected calibration standard peak areas are within 5% of the initial
calibration standard peak areas.
(
3
) Construct a plot of analyte concentration (x-axis) versus individual peak area (y-axis) for CUR,
DMC, and BDMC. Use least squares analysis to determine the slope, intercept, and correlation
coefficient (r
2
) of the best-fit line for each analyte.
(
b
)
Unknown samples.-
(
1
) From the standard curves and the peak areas of each analyte in the
samples, calculate the concentration of CUR, DMC, and BDMC in each sample solution. If the
peak area of any analyte is above the standard curve for that analyte, dilute the extract 1/10 or
1/20 in methanol, filter, and repeat the analysis for that extract.
Curcuminoid Concentration (
mg
L
) =
curcuminoid peak area − intercept of linear regression
slope of linear regression
(
2
) Calculate the amount of curcuminoid in the original sample as:
( ) =
( ) ×
( )
( )
×
where C is the concentration of analyte from the standard curve (mg/L); V is the extract volume
(0.025 L); W is the weight of the test portion (g); and D is the dilution factor.
(
3
) Report the curcuminoid concentrations in the original samples as the mean concentrations
with 95% confidence intervals as follows:
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