US 2017/0248561 A1 · METHOD TO DETERMINE THE CROCETIN AND ITS DERIVATIVE AMOUNTS AND THE DERIVATIVE COMPOSITION FROM GARDENIA YELLOW

Abstract

The invention relates to a method for the determination of crocetin and its derivative amounts and the derivative composition from gardenia yellow. The method mainly comprises of following steps: the absorbance measurement of a gardenia yellow aqueous solution at known concentration by UV-VIS, the absorption coefficient measurement of total crocetin derivative, the total crocetin derivative amount calculation according to Lambert-Beer law, the relative amount calculation of each crocetin derivative from its absorption coefficient and peak area on HPLC, the amount calculation of each crocetin derivative from the total amount of crocetin derivative and the relative amount of each crocetin derivative, and finally total crocetin amount calculation. In practice, the absorption coefficient of each crocetin derivative is calculated from that of its root structure, crocetin, based on the negative correlation-ship of absorbance with molecular mass of the molecule while the molecular mass of each crocetin derivative is substituted by the m/z value of its parent ion.

Claims19

Dependent claims

A process comprising the ordered series of steps as follows:

(i) producing an aqueous solution of gardenia yellow;
(ii) separating the solution into a plurality of fractions using high performance liquid chromatography;
(iii) measuring absorbance and molecular weight of each of the fractions using a photo-diode array coupled to an atmospheric pressure chemical ionization MS-MS.

A method to determine derivative amounts and composition of crocetin in Gardenia yellow, the method includes the following steps:

1) dissolving M grams gardenia yellow powder dissolved in V mL in water, which is then diluted by water to N-fold, and the absorbance (A) of diluted sample solution recorded at the wavelength of 440 nm.
2) acquiring a chromatographic profile of gardenia yellow aqueous solution of the sample aqueous solution, by filtering an aliquot of gardenia yellow aqueous solution before dilution on 0.45μ filter, injecting onto an HPLC with C18-HPLC column, the fractions of crocetin, its derivatives and other pigments separated under conditions comprising
As a stationary phase, a reversed phase C18 column (250×4.6 mm, 5 μm);
As a mobile phase, mobile phase A:
Water-acetonitrile-acetic acid at 74.95:25:0.05 (V/V/V), mobile phase B:
Acetonitrile;
and a linear gradient elution of mobile phase B from 0 to 13.1% (V/V) in 20 minutes, and isocratic elution from 20 to 35 minutes;
As a Flow rate:
0.8 mL/min;
An Injection volume:
20 μL;
and
a Column temperature at room temperature;
wherein the monitoring wavelength:
440 nm, and the electronic absorption spectrum of each fraction is acquired from 250 to 700 nm for fraction identification if a PDA equipped, and the peak are of each fraction is integrated on the chromatogram.
3) identifying, on a C18-HPLC-PDA-APCI-MS-MS, each fraction containing crocetin structure as crocetin derivative, usually as crocetin ester, on the HPLC-MS under the chromatographic conditions as outlined above and MS conditions comprising:
Spray voltage:
−4 Kv;
Sheath gas:
30 arb;
Auxiliary gas:
10 arb;
Purge gas:
0 arb;
Capillary temperature:
350° C.;
Negative ion detection mode:
Data dependency scan;
CID collision energy:
35%;
Fragment mass scanning range:
150-2000 (m/z).
4) identifying the parent ion and m/z value of each crocetin derivative on MS
5) calculating the relative peak area (%) of each crocetin derivative according to formula (1).
Relative


peak


area
i
=
Peak


area
i
Σ
i
=
1
n


Peak


area
i
(
1
)
Where
Relative peak areai=Relative peak area of crocetin derivative i in total crocetin derivative
Peak areai=Peak area of crocetin derivative i
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative
6) calculating the absorption coefficient of each crocetin derivative from published absorption coefficient, A1 cm1%=3820 (in EtOH), of crocetin as root structure, in accordance with the negative dependence of crocetin derivative UV-VIS absorbance on its side-chain quantity and mass indicated by molecular mass, as shown in formula (2), where the molecular mass of each crocetin derivative is substituted by its measured parent ion [M-1]− mass reading [m/z] from MS.
A
1

cm
i
1

%
=
A
1

cm
crocetin
1

%
×
Molecular


mass
crocetin
Molecular


mass
i
(
2
)
Where
A1 cmi1%=Absorbance coefficient of crocetin derivative i, defined as the theoretical absorbance of a 1% (W/V) crocetin derivative i solution in a cell of 1 cm path-length at monitored 440 nm.
A1 cmCrocetin1%=Absorbance coefficient of crocetin, defined as the theoretical absorbance of a 1% (W/V) crocetin solution in a cell of 1 cm path-length monitored at 440 nm. Published value of 3820 (in methanol) is applied hereon.
Molecular masscrocetin=Molecular mass of crocetin. Published value of 328 is applied hereon.
Molecular massi=Molecular mass of crocetin derivative i. Molecular mass of each crocetin derivative is hereon substituted by its parent ion mass [m/z] from MS.
i=Fraction number of crocetin derivative
7) calculating the absorption coefficient of total crocetin derivative by weighted sum from the relative peak area and absorption coefficients of each crocetin derivative as the results of formulae (1) and (2), respectively, according to formula (3).
A1 cmTotal crocetin derivative1%=Σi=1n Relative peak areai×A1 cmi1% (3)
Where
Relative peak areai=Relative peak area of crocetin derivative i
A1 cmTotal crocetin derivative1%=Absorption coefficient of total crocetin derivative, defined as the theoretical absorbance of a 1% (W/V) total crocetin derivative solution in a cell of 1 cm path-length monitored at 440 nm.
A1 cmi1%=Absorbance coefficient of crocetin derivative i, defined as the theoretical absorbance of a 1% (W/V) crocetin derivative i solution in a cell of 1 cm path-length monitored at 440 nm.
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative
8) calculating the relative content of total crocetin derivative in total pigment from the peak area of each fraction monitored at 440 nm according to formula (4).
Relative


content
Total


crocetin


derivative
=
Σ
i
=
1
n


Peak


area
i
Σ
j
=
1
m


Peak


area
j


C
Total


ester

[
%
]
=
Σ
i
=
1
m

Fi
Σ
j
=
1
n

Fj
(
4
)
Where
Relative contentTotal crocetin derivative=Relative content of total crocetin derivative in total pigment
Peak areai=Peak area of crocetin derivative i
Peak areaj=Peak area of fraction j
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative
j=Fraction number
m=Total fraction number
9) Calculating the amount of total crocetin derivative in gardenia yellow sample according to Lamber-Beer law from the absorbance of gardenia yellow aqueous solution, the relative content of total crocetin derivative in total pigment and the absorption coefficient of total crocetin derivative, as shown by formula (5).
Amount
Total


crocetin


derivative

[
g
]
=
A
×
Relative


content
Total


crocetin


derivative
×
V
×
N
A
1

cm
Total


crocetin


derivative
1

%
×
100
(
5
)
Where
AmountTotal crocetin derivative[g]=Amount of total crocetin derivative in gardenia yellow sample
A=Absorbance of sample solution
V=Solvent volume for sample originally dissolving
N=Dilution fold of sample solution
Relative contentTotal crocetin derivative=Relative content of total crocetin derivative in total pigment
A1 cmTotal crocetin derivative1%=Absorption coefficient of total crocetin derivative, defined as the theoretical absorbance of a 1% (W/V) total crocetin derivative solution in a cell of 1 cm path-length monitored at 440 nm.
10) Calculating the content of total crocetin derivative in gardenia yellow sample from the amount of total crocetin derivative and sample weight (M) according to formula (6).
Content
Total


crocetin


derivative

[
%
]
=
Amount
Total


crocetin


derivative
M
×
100

%
(
6
)
Where
ContentTotal crocetin derivative[%]=Content of total crocetin derivative in gardenia yellow sample
AmountTotal crocetin derivative[g]=Amount of total crocetin derivative in gardenia yellow sample
M=Mass of gardenia yellow sample
11) calculating according to Lamber-Beer law, the relative content of each crocetin derivative in total crocetin derivative from the peak area (Section A.2) and absorption coefficients of each crocetin derivative (Section A.6), as shown by formula (7).
Relative


content
i

[
%
]
=
Peak


area
i
A
1

cm
i
1

%
Σ
i
=
1
n

Peak


area
i
A
1

cm
i
1

%
×
100

%
(
7
)
Where
Relative contenti=Relative content of each crocetin derivative in total crocetin derivative
Peak areai=Peak area of crocetin derivative i
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative
12) calculating the amount of each crocetin derivative from gardenia yellow sample from the relative content of each crocetin derivative and the amount of total crocetin derivative according to formula (8).
Amounti[g]=Relative contenti×AmountTotal crocetin derivative (8)
Where
Amounti=Amount of each crocetin derivative from gardenia yellow sample
Relative contenti=Relative content of each crocetin derivative in total crocetin derivative
AmountTotal crocetin derivative=Amount of total crocetin derivative in gardenia yellow sample
i=Fraction number of crocetin derivative.

338 more elements

Dependencies are inferred from the claim text.

Method to determine the crocetin and its derivative amounts and the derivative composition from gardenia yellow

Life of this patent

Method to determine the crocetin and its derivative amounts and the derivative composition from gardenia yellow

Life of this patent

Abstract

Claims19