HPLC Method
1.0 Scope
This method is used for determination of total astaxanthin in Aquasta™ powder using
DMSO as extraction solvent, followed by partitioning into methylene chloride. A larger
sample size is used to decrease variability in the assay.
2.0 Equipment
2.1 High Performance Liquid Chromatograph (HPLC), with UV/VIS detector
2.2 Shimadzu CR-1 Integrator or equivalent or Integration software
2.3 1.0 ml, 2.0 ml, 5.0 ml and 25 ml volumetric pipets
2.4 15 ml and 50 ml plastic, screw-cap centrifuge tubes
2.5 Vortex
2.6 5 ml plastic syringes
2.7 13 mm 0.45 micron PTFE syringe filters
2.8 Spectrophotometer (visible range)
2.9 25 ml volumetric flasks with plastic stoppers
2.10 Analytical balance
2.11 Table top centrifuge
2.12 P1000 and P200 automatic pipettes
2.13 40 degree C water bath and/or 40 degree C incubator
3.0 Solvents/Reagents
3.1 Tank of Nitrogen Gas
3.2 HPLC grade Hexane
3.3 HPLC grade Acetone
3.4 HPLC grade Methylene Chloride
3.5 Crystalline astaxanthin >98% (Sigma-A9335)
3.6 Dimethylsulfoxide (DMSO) > 99.9% (Sigma-D8779)
3.7 Sodium sulfate anhydrous (20% w/v in deionized water ) ( Sigma-S6264)
4.0 Sample Preparation
4.1 Weigh out approximately 0.6 g of powder sample into a tared 50 ml centrifuge
tube. Record sample weight to 5 decimal places.
4.2 Add 25 ml of water to the powder using a volumetric pipet.
4.3 Vortex vigorously and sonicate until there are no clumps of powder in the
suspension.
4.4 Using a pipetman place 1.00 ml of the well mixed suspension from above into a
tared 25 ml volumetric flask. Cap the flask. Record the sample weight to 5
decimal places.
4.5 Add warm DMSO (40C) to the volumetric flask until the volume of the liquid
reaches the bottom of the neck of the flask.
4.6 Cap with stopper and vortex vigorously for 2 minutes.
4.7 Cool to 20 degrees C +/- 1 degree C.
4.5 Add additional DMSO to qs to the 25 ml mark. Mix well by inverting the flask
several times.
Note: The efficiency of extraction can be verified at this step by
centrifuging 1.5 ml of the extract in a microfuge followed by two
washings of 1.5 ml each with deionized water. The pellet should be tan
colored without any hint of yellow or orange. If not, vortex for three
additional minutes and test again. Do not proceed unless extraction is
complete.
4.6 Using a 1 ml Tc volumetric pipet, transfer 1.0 ml of the DMSO extract (cooled to
20 degrees C +/- 1 degree C) to a 15 ml centrifuge tube. Rinse pipet with 4 ml of
deionized water into the top of the pipet to flush the entire 1.0 ml sample. This
step is critical for accuracy and reproducibility. The remaining DMSO extract
can be stored frozen in the event that the assay needs to be repeated.
4.7 Fill to the 5.0 ml mark with deionized water. Vortex to mix well.
4.8 Add 1 ml of Sodium Sulfate solution. Vortex to mix well.
4.9 Using a 5 ml Td volumetric pipet, add 5.0 ml methylene chloride.
4.10 Add deionized water to the 13 ml mark.
4.11 Vortex vigorously for 2 minutes until pigment partitions completely into the
lower methylene chloride layer.
4.12 Centrifuge for a minute to separate layers.
4.13 Remove water to 7.0 ml mark. Be careful not to disturb the pigment containing
bottom methylene chloride layer.
4.14 Add deionized water to 13 ml mark.
4.15 Repeat steps 4.11 to 4.14 4 more times vortexing for 15 seconds between washes.
(Do not count the 1st spindown).
4.16 Remove the entire water layer.
4.17 Using a 2.0 ml Td volumetric pipet transfer 2.0 ml of the methylene chloride layer
to a 15ml plastic centrifuge tube.
4.18 Dry the samples under nitrogen or vacuum dryer.
4.19 Resuspend the dried down sample with 2.0 ml of mobile phase.
4.20 Filter this resuspended sample through a 0.45u syringe filter into an HPLC vial.
5.0 Standard Preparation
5.1 To a 15 ml test tube, add 1 ml of methylene chloride.
5.2 With a micro spatula, obtain approximately 1mg of crystalline astaxanthin. The
weight of the crystalline astaxanthin is not critical because the standard curve will
rely on the absorbance of the solution at 475 nm.
5.3 Dissolve the crystalline astaxanthin in 1.0 ml of methylene chloride and vortex.
This is your stock standard solution.
5.4 To a 15ml tube, pipet 25ul of the stock standard solution. Add 5mls of mobile
phase to the tube, shake to dissolve and read the absorbance at 475 nm. The
absorbance of this solution should be between 0.140 and 0.160. If the absorbance
does not fall within this range, adjust the stock accordingly until it does.
5.5 To four centrifuge tubes, pipet 100 Pl, 75 Pl, 50 Pl, and 25Pl of this adjusted
stock standard solution and dry under nitrogen.
5.6 Add 5 mls of mobile phase (hexane/acetone) to each of the dried
standards, and cap immediately. Dissolve in solution using an ultrasonic water
bath by sonicating for 5 minutes.
5.7 Using the 3 ml plastic syringes, filter approximately 2mls of each solution
through a 0.45 micron filter and into four HPLC vials. With the remaining
solutions, read the absorbance of each at 475 nm in the spectrophotometer against
a blank containing mobile phase. Record absorbance for calculation of the
standard curve.
6.0 HPLC Conditions
6.1 Mobile Phase: 85.5 : 14.5 Hexane/Acetone
6.2 Run Time: 15 minutes, isocratic
6.3 Column: Phenomenex Luna - Silica, 3 micron, 46 mm x 150
mm. Modified by pumping a solution of orthophosphoric
acid in methanol (1 g/100 ml) through the column for 1
hours at 1.0 ml/min. followed by equilibration in mobile
phase. This needs to be done only once. Use 100% acetone
as a transition between aqueous and non-aqueous phases.
6.4 Temperature: 25 degrees C
6.5 Flow Rate: 1.2 ml/min.
6.6 Injection volume: 50 ul
6.7 Detector wavelength: 475 nm
7.0 Calculations
7.1 Determine the peak areas for the standard* and the sample. For both standard and
sample:
Total area = trans area + (1.11 x di-cis area) + (1.2 x 9-cis area) +
(1.56 x 13-cis area) + (1.56 x 15-cis area)
This calculation takes into account that the extinction coefficients for di-cis, 9-cis,
13-cis and 15-cis astaxanthin are 1900, 1750, 1350 and 1350, respectively, at 475
nm. (Bjerkeng, 1997)
*Standards will usually not contain any 15-cis isomers, so these should be
excluded from the “total area” calculation for the standard.
7.2 Determine the concentration (ppm) of the four standard points using the
following formula:
ppm (standard) = absorbance at 475nm x 10,000
*E
*E = (di-cis area / total area x 1900) + (trans area / total area x 2100) + (9-cis area
/ total area x 1750) + (13-cis area / total area x 1350)
7.3 Plot concentration (ppm) versus area counts for each of the four standards
to establish a standard curve. Use linear regression to determine the line of best
fit for the four points.
7.4 With the slope and Y-intercept from the standard regression, use the
following formula to determine the concentration of the sample:
mgs/L astaxanthin = [(total area x slope) + Y-intercept] x dilution factor
The dilution factors for this spreadsheet = 25*Contraction Factor*2/2
25 = the 25 ml of DMSO used for extraction
Contraction Factor = 4.375 ml is the final reduced volume of the
Methylene Chloride layer after the washings.
2/2 = 2 ml methylene chloride dried down and resuspended in 2 ml mobile
phase
ppm astaxanthin = mgs/ L astaxanthin
dry sample weight
Dry Sample Weight = weight of asta sample _ x wgt. of sample supension
25.0+ weight of asta sample
8.0 Representative Chromatograph-Aquasta™S powder sample (attached)
9.0 References
9.1 Bjerkeng, B., Folling, M., Lagocki, S., Storebakken, T., Olli, J., Alsted, N.
Bioavailability of all-E-astaxanthin and Z-isomers of astaxanthin in rainbow trout
(Oncorhyncus mykiss). Aquaculture 157 (1997), 63-82.
