Analysis of Impurities in Curcumin - Shimadzu

SHIMADZU APPLICATION NEWS
● LIQUID CHROMATOGRAPHY MASS SPECTROMETRY
Ultra Fast Analysis (Part 2)
Analysis of Impurities in Curcumin
An example of ultra fast analysis using the
Prominence UFLC with the LCMS-2010EV was
presented in Application News No. C49. As for
improvement in throughput, this combination is, of
course, effective even in impurities analysis,
shortening analysis time which might ordinarily take
■ Analysis of Curcumin Standard Solutions
Curcumin is a type of polyphenol present in turmeric,
and is used as a food dye. Fig.1 shows the structrures
of curcumin in addition to two similar compounds
(curcuminoids). The measurement samples consisted
of commercial curcumin standards of different purity,
referred to as grade A and grade B. The standard
solutions were prepared by dissolving the standards in
methanol and adjusting the respective concentrations
to 1 mg/mL. PDA detection was conducted at a
wavelength of 425 nm. MS analysis was conducted
using electrospray ionization, and the deprotonated
molecules were detected. Grade A showed a relatively
low purity of curcumin, and more than 20 minutes was
Fig.1 Structures of Curcuminoids (1: Bisdemethoxycurcumin (MW 308),
2: Demethoxycurcumin (MW 338), 3: Curcumin (MW 368))
Column
(1)
(2)
(3)
Mobile phase
Flow rate
Column temperature
Injection volume
Detection
PDA
SPD-M20A
MS
Probe voltage
Nebulizing gas flow
Drying gas pressure
CDL temperature
HO OH
HO OH
HO OH
OCH3 OCH3
Table 1 Analytical conditions
: Shim-pack XR-ODS (75 mmL. × 2.0 mmI.D., 2.2 µm, UFLC)
Shim-pack VP-ODS (150 mmL. × 2.0 mmI.D., 5 µm, HPLC)
: 0.1 % Formic acid/Acetonitrile = 60/40
: 0.4 mL/min (UFLC), 0.2 mL/min (HPLC)
: 40 ˚C
: 1 µL
: 425 nm
H
O O
H
O O
H
O O
OCH3
: -3.5 kV (ESI-Negative mode)
: 1.5 L/min
: 0.15 MPa (UFLC), 0.10 MPa (HPLC)
: 250 ˚C
Block heater temperature : 200 ˚C
CDL, Q-array voltages : using Default values
Scan range
: m/z 150 - 500
LAAN-A-LM-E030
No.C59
twenty or thirty minutes to several hours, greatly
contributing to higher operations efficiency. Here we
present an example of analysis of a curcumin
standard solution using photodiode array (PDA)
detection and mass spectrometry (MS).
required to complete the analysis using a 5 µm
particle diameter column (fig.2 (a), (b)), while this
analysis time was shortened by 7 minutes using the
Prominence UFLC (fig.2 (c), (d)). The area
percentages in the PDA chromatogram were 1.11 %
for bisdemethoxycurcumin, 11.46 % for
demethoxycurcumin, and 87.19 % for curcumin. Other
than these compounds, minute peaks were also
noticeable at t R=1.34 min (λ max 338 nm, m/z 337),
t R=1.48 min (λ max 349 nm, m/z 367), and
t R=2.77 min (λ max 429 nm, m/z 383). As for the highpurity
grade B sample, neither bisdemethoxycurcumin
nor demethoxycurcumin were detected (fig.2 (e), (f)).
mAU (× 100)
5.0
(a)
4.0
3.0
2.0
1.0
0.0
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
2
1
3
Inten. (× 1,000,000)
0 10 20 min 0 10 20 min
mAU (× 1,000)
Inten. (× 1,000,000)
(c)
3
3.0 (d)
1
2
0.0 2.5 5.0 min 0.0 2.5 5.0 min
mAU (× 1,000)
Inten. (× 1,000,000)
(e) 3 3.0 (f)
3
0.00
0.0
0.0 2.5 5.0 min 0.0 2.5 5.0 min
■ Peaks
1.Bisdemethoxycurcumin, 2. Demethoxycurcumin, 3. Curcumin
Fig.2 PDA Chromatograms, Total Ion Chromatograms (TIC) of
Curcumin Standard Solutions
(a): PDA chromatogram of curcumin (grade A) by HPLC,
(b): TIC of curcumin (grade A) by HPLC-MS
(c): PDA chromatogram of curcumin (grade A) by UFLC
(d): TIC of curcumin (grade A) by UFLC-MS
(e): PDA chromatogram of curcumin (grade B) by UFLC
(f): TIC of curcumin (grade B) by UFLC-MS
3.0
2.0
1.0
0.0
2.0
1.0
0.0
2.0
1.0
(b)
1
1
2
2
3
3

No.C59
■ PDA Data
Fig.3 shows the UV spectra of curcuminoids. All of
them clearly possess a maximum absorption
wavelength near 420 nm. When the peak purity is
mAU
17.5
(1)
15.0
12.5
10.0
7.5
5.0
2.5
200
246
Fig.3 UV Spectra of Curcuminoids (1: Bisdemethoxycurcumin, 2: Demethoxycurcumin, 3: Curcumin)
Fig.4 Purity Curves of Curcuminoids (1: Bisdemethoxycurcumin, 2: Demethoxycurcumin, 3: Curcumin)
■ Another Impurity
Focusing on the peak (compound X) at 5.35 minutes
in the mass chromatograms of fig.4 (2), the UV
spectrum and mass spectrum were obtained for this
peak, as shown in the fig.5. The maximum absorption
wavelength is in the vicinity of 370 nm, and the m/z
417
mAU mAU
150
125
(2) 421
1000
900
800
(3)
600
0.0 0 0
Fig.5 Mass chromatograms of curcumin standard solution (grade A, (a)) and UV spectrum (b), mass spectrum (c) of compound X
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100
75
50
25
240
calculated from the grade A data, an impurity was
detected at 5.35 minutes (fig.4 (2)).
300 400 500 nm 200 300 400 500 nm 200 300 400 500 nm
0.85
0.80
0.75
0.70
0.65
0.60
Purity Curves
Zero Line
mAU
peak 15.0
(1) 14.0
13.0
12.0
11.0
Purity Curves
Zero Line
0.6
0.5
0.4
mAU
peak
130
(2)
120
110
100
0.9
Purity Curves
Zero Line
0.8
0.7
mAU
peak 900
(3)
800
700
0.55
10.0
90 0.6
600
0.50
0.45
0.40
9.0
8.0
0.3
0.2
80
70
0.5
500
0.35
7.0
60
0.4
400
0.30
0.25
0.20
6.0
5.0
4.0
0.1
0.0
50
40
0.3
0.2
300
0.15
0.10
0.05
3.0
2.0
-0.1
30
20 0.1
200
100
0.00
1.0 -0.2
10
0.0
0.0 0
0
4.2 4.3 4.4 4.5 min 5.00 5.25
min
5.75 6.00 6.25 6.50 min
Inten. (× 1,000,000)
3.50
3.25 (a)
3.00
2.75
2.50
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0
■Peaks
1. Bisdemethoxycurcumin,
2. Demethoxycurcumin,
3. Curcumin
1
2
X
3
TIC
m/z307
m/z337
m/z369
m/z367
1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
NOTES:
✽This Application News has been produced and edited using information that was available when the
data was acquired for each article. This Application News is subject to revision without prior notice.
700
600
500
400
300
200
100
263
426
369 negative ion was detected. Therefore, the
molecular weight of compound X is presumed to 370.
Thus, by using both PDA and MS in UFLC detection,
greater efficiency is believed to be possible.
10.0
(× 1,000,000)
(b)
7.5
5.0
373
2.5
0.0
255
282
200 300 400 500 nm
Inten. (× 10,000)
6.0
5.0
4.0
3.0
2.0
(c)
369
1.0
169
397
0.0
200 300 400 m/z
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