5 / 63
K
line
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
3
E. Preparation of Extraction Solutions
(a)
Saturated sodium chloride solution.—
Add 50 g sodium
chloride to 100 mL freshly boiled purified water.
(b)
Ethyl acetate–methanol (90 + 10) solution.—
Combine
900 mL ethyl acetate with 100 mL methanol. Mix well.
(c)
Methanol–water (60 + 40) solution.—
Combine 600 mL
methanol with 400 mL purified water. Mix well.
F. Preparation of Standards
(a)
Stock standard solutions
.—(
1
)
Aloin A and B stock
standard solution.—
Accurately weigh about 5 mg each of aloin
A and B reference standards into the same 50 mL volumetric
flask. Dissolve and dilute to volume with methanol. Store the
stock standard solution in a refrigerator [2–8°C, concentration
of ~100 parts per million (ppm)].
(
2
)
Aloe-emodin stock standard solution
.—Accurately
weigh about 5 mg aloe-emodin reference standard into a 50 mL
volumetric flask. Dissolve and dilute to volume with methanol.
Store the stock standard solution in a refrigerator (2–8°C,
concentration of ~100 ppm).
(
3
)
Aloin A and B mid-standard solution
.—Pipet 100 µL
aloin A and B stock standard solution into a 10 mL volumetric
flask and dilute to volume with methanol–water (60 + 40). Mix
well (concentration of ~1 ppm).
(
4
)
Aloe-emodin mid-standard solution
.—Pipet 100 µL aloe-
emodin stock standard solution into a 10 mL volumetric flask
and dilute to volume with methanol–water (60 + 40). Mix well
(concentration of ~1 ppm).
(b)
Working standard solution
.—(
1
)
Standard [300 parts
per billion (ppb)]
.—Pipet 3 mL mid-standard solution into a
10 mL volumetric flask and dilute to volume with methanol–
water (60 + 40). Mix well.
(
2
)
Standard (80 ppb)
.—Pipet 2 mL mid-standard solution
into a 25 mL volumetric flask and dilute to volume with
methanol–water (60 + 40). Mix well.
(
3
)
Standard (20 ppb)
.—Pipet 200 µL mid-standard solution
into a 10 mL volumetric flask and dilute to volume with
methanol–water (60 + 40). Mix well.
G. Preparation of Samples
(a)
Rawmaterial (powder and liquid form)
.—(
1
) Accurately
weigh about 0.1 g powder raw material sample into a 15 mL
screw-cap test tube; pipet 1 mL purified water and mix the
sample well on a vortex mixer to dissolve the powder. For the
liquid raw material, weigh ~1 g raw material sample into a
15 mL screw-cap test tube.
(
2
) Pipet 1 mL reagent alcohol and 2 mL saturated sodium
chloride solution into the test tube. Mix the sample well on the
vortex mixer.
(
3
) Pipet 4 mL ethyl acetate–methanol (90 + 10) solution
into the test tube.
(
4
) Cap the test tube and mix the sample on the vortex mixer
for about 60 s at maximum speed.
(
5
) If necessary, centrifuge the sample at 2000 rpm for 5 min
to aid separation of layers.
(
6
) Transfer the top-most organic layer into a second
15 mL screw-cap test tube. Extract the sample from the
original test tube one more time using a 4 mL portion of
the ethyl acetate–methanol (90 + 10) solution. Mix the
sample on the vortex mixer for about 30 s. If necessary,
centrifuge the sample at 2000 rpm for 5 min to separate
layers. Transfer the top-most organic layer into the second
test tube.
(
7
) Evaporate the combined sample extract of the second test
tube to dryness using an N
2
purge in a 50°C water bath.
(
8
) Pipet 0.5 mL methanol–water (60 + 40) into the second
test tube containing the dried sample residue. Mix the sample on
a vortex mixer for 15 s.
(
9
) Filter the sample through a 0.2 µm PVDF filter into a
glass HPLC vial with an insert or a microvial.
(b)
Finished product (powder and liquid form)
.—
(
1
)
Powder product.
—Accurately weigh about 0.5 g powder
product into a 15 mL screw-cap test tube, pipet 1 mL purified
water, and mix the sample well on the vortex mixer to dissolve
the powder.
(
2
)
Liquid product
.—Accurately weigh about 1 g liquid
product into a 15 mL screw-cap test tube.
(
3
) Pipet 1 mL reagent alcohol and 2 mL saturated sodium
chloride solution into the test tube. Mix the sample well on the
vortex mixer.
(
4
) Pipet 4 mL ethyl acetate–methanol (90 + 10) solution
into the test tube.
(
5
) Cap the test tube and mix the sample on the vortex mixer
for 60 s at maximum speed.
(
6
) If necessary, centrifuge the sample at 2000 rpm for 5 min
to separate layers.
(
7
) Transfer the top-most organic layer into a second 15 mL
screw-cap test tube.
(
8
) Extract the sample from the original test tube one
more time using a 4 mL portion of the ethyl acetate–methanol
(90 + 10) solution. Mix the sample on a vortex mixer for 30 s.
If necessary, centrifuge the sample at 2000 rpm for 5 min to
separate layers. Transfer the top-most organic layer into the
second test tube.
(
9
) Evaporate the combined sample extract of the second test
tube to dryness using an N
2
purge in a 50°C water bath.
(
10
) Pipet 0.5 mL methanol–water (60 + 40) into the second
test tube containing the dried sample residue.
(
11
) Mix the sample on a vortex mixer for 15 s.
(
12
) Filter the sample through a 0.2 µm PVDF filter into a
glass HPLC vial with the insert or a microvial.
H. System Suitability
(a)
In chromatographing the standards, the correlation
coefficient (R) for the aloin A and B (and aloe-emodin, if
necessary) curves should not be less than 0.998.
(b)
Run a standard check (80 ppb standard) after every six
sample injections and at the end of the run. The peak area of
each check standard should be within 90–110% of the peak area
of the working standard from the calibration curve.
(c)
The theoretical plate count should not be less than 10000
for aloins A and B and aloe-emodin (if required).
(d)
The tailing factor should not be more than 2.0 for aloins
A and B and aloe-emodin (if required).
5