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.

Claims

1. 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.

2. The process of claim 1 further comprising measuring the absorbance of the aqueous solution before separating the solution into the plurality of fractions.

3. The process of claim 1 wherein the separating step includes a column for reversed-phase separation.

4. The process of claim 1 wherein the aqueous solution of gardenia yellow comprises a plurality of crocetin derivatives.

5. The process of claim 4 wherein each of the separate fractions comprises a crocetin derivative characterized by a molecular weight and a peak area.

6. The process of claim 5 further comprising determining an absorption coefficient of total crocetin derivative, wherein the absorption coefficient of total crocetin derivative is calculated by a weighted sum of a corrected absorbance coefficient of crocetin for each crocetin derivative.

7. The process of claim 6 wherein the corrected absorbance coefficient of crocetin for each crocetin derivative is determined by the ratio of the molecular weight of crocetin to the molecular weight of the crocetin derivative, multiplied by the absorbance of a 1% (W/V) crocetin solution in a cell of 1 cm path-length monitored at 440 nm.

8. The process of claim 7 wherein the absorption coefficient of total crocetin derivative is calculated by a sum of each crocetin derivative peak area multiplied by the corrected absorbance coefficient of each crocetin derivative, divided by the sum of all crocetin derivative peak areas.

9. A method for analysis of a mixture of crocetin compounds, wherein the mixture of crocetin compounds is derived from an extract of fruit of a Gardenia jasminoides Ellis, comprising the steps: (i) preparing an aqueous solution of the mixture of crocetin compounds; (ii) separating the mixture of crocetin compounds into a plurality of individual fractions comprising a crocetin species using reversed phase high-performance liquid chromatography; (iii) characterizing each of the crocetin species by determining an absorption peak area at 440 nm (P.sub.i) and a molecular mass (M.sub.i); (iv) calculating a crocetin species absorption coefficient (A.sub.i) by multiplying a standard absorption coefficient for crocetin (A.sub.crocetin) by a ratio of a molecular mass of crocetin to the crocetin species molecular mass (M.sub.crocetin/M.sub.i) (v) determining a total crocetin absorption coefficient (A.sub.total) by multiplying the crocetin species absorption coefficient from step (iv) by the crocetin species absorption peak area from step (iii) divided by a sum of all the crocetin species absorption peak areas from step (iii) and summing this value for all of the crocetin species. $A_{\mathrm{total}}=\underset{i}{.Math.}.Math.\frac{A_{\mathrm{crocetin}}\left(\frac{M_{\mathrm{crocetin}}}{M_i}\right).Math.P_i}{\Sigma .Math..Math.P_i}$

10. The method of claim 9 further comprising determining the absorbance of the mixture of crocetin compounds before separation (A).

11. The method of claim 9 wherein the molecular mass of each crocetin derivative is substituted by its measured parent ion [M-1].sup.− mass reading [m/z] from MS.

12. The method of claim 9 wherein the absorption peak area at 440 nm is measured using a photodiode array detector.

13. The method of claim 9 wherein the molecular mass is measured using an atmospheric pressure chemical ionization mass spectrometer.

14. The method of claim 9, wherein the characterizing step is performed simultaneously with the separating step.

15. The method of claim 10 further comprising determining the amount of total crocetin derivative in the mixture of crocetin compounds.

16. A method of determining total crocetin content from a mixture of crocetin derivatives, comprising: (i) preparing an aqueous solution of the mixture of crocetin derivatives; (ii) determining the absorbance of the mixture of crocetin derivatives (A); (iii) separating the mixture of crocetin derivatives into a plurality of individual fractions comprising a crocetin species using reversed phase high-performance liquid chromatography; (iv) characterizing each of the crocetin species by determining an absorption peak area at 440 nm (P.sub.i) and a molecular mass (M.sub.i); (v) calculating a crocetin species absorption coefficient (A.sub.i) by multiplying a standard absorption coefficient for crocetin (A.sub.crocetin) by a ratio of a molecular mass of crocetin to the crocetin species molecular mass (M.sub.crocetin/M.sub.i) (vi) determining a total crocetin absorption coefficient (A.sub.total) by multiplying the crocetin species absorption coefficient from step (iv) by the crocetin species absorption peak area from step (iii) divided by a sum of all the crocetin species absorption peak areas from step (iii) and summing this value for all of the crocetin species; $A_{1.Math.{\mathrm{cm}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivstive}}}^{1.Math.\%}=\underset{i}{.Math.}.Math.\frac{A_{\mathrm{crocetin}}\left(\frac{M_{\mathrm{crocetin}}}{M_i}\right).Math.P_i}{\Sigma .Math..Math.P_i}$ (vii) calculating the total crocetin content according to the Beer-Lambert relationship, wherein the total crocetin absorption coefficient is used as a proxy for molar absorptivity.

17. The method of claim 16 wherein the molecular mass of each crocetin derivative is substituted by its measured parent ion [M-1].sup.− mass reading [m/z] from MS.

18. The method of claim 16 wherein the characterizing step is performed simultaneously with the separation step.

19. 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). $\begin{array}{cc}\mathrm{Relative}.Math..Math.\mathrm{peak}.Math..Math.{\mathrm{area}}_i=\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{{\Sigma }_{i=1}^n.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}& \left(1\right)\end{array}$ Where Relative peak area.sub.i=Relative peak area of crocetin derivative i in total crocetin derivative Peak area.sub.i=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, A.sub.1 cm.sup.1%=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].sup.− mass reading [m/z] from MS. $\begin{array}{cc}{A}_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}=A_{1.Math.{\mathrm{cm}}_{\mathrm{crocetin}}}^{1.Math.\%}\times \frac{\mathrm{Molecular}.Math..Math.{\mathrm{mass}}_{\mathrm{crocetin}}}{\mathrm{Molecular}.Math..Math.{\mathrm{mass}}_i}& \left(2\right)\end{array}$ Where A.sub.1 cm.sub.i.sup.1%=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. A.sub.1 cm.sub.Crocetin.sup.1%=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 mass.sub.crocetin=Molecular mass of crocetin. Published value of 328 is applied hereon. Molecular mass.sub.i=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).
A.sub.1 cm.sub.Total crocetin derivative.sup.1%=Σ.sub.i=1.sup.n Relative peak area.sub.i×A.sub.1 cm.sub.i.sup.1%  (3) Where Relative peak area.sub.i=Relative peak area of crocetin derivative i A.sub.1 cm.sub.Total crocetin derivative.sup.1%=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. A.sub.1 cm.sub.i.sup.1%=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). $\begin{array}{cc}\mathrm{Relative}.Math..Math.{\mathrm{content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}=\frac{{\Sigma }_{i=1}^n.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{{\Sigma }_{j=1}^m.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_j}.Math..Math.C_{\mathrm{Total}.Math..Math.\mathrm{ester}}\left[\%\right]=\frac{{\Sigma }_{i=1}^m.Math.\mathrm{Fi}}{{\Sigma }_{j=1}^n.Math.\mathrm{Fj}}& \left(4\right)\end{array}$ Where Relative content.sub.Total crocetin derivative=Relative content of total crocetin derivative in total pigment Peak area.sub.i=Peak area of crocetin derivative i Peak area.sub.j=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). $\begin{array}{cc}{\mathrm{Amount}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\left[g\right]=\frac{A\times \mathrm{Relative}.Math..Math.{\mathrm{content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\times V\times N}{A_{1.Math.{\mathrm{cm}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}}^{1.Math.\%}\times 100}& \left(5\right)\end{array}$ Where Amount.sub.Total 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 content.sub.Total crocetin derivative=Relative content of total crocetin derivative in total pigment A.sub.1 cm.sub.Total crocetin derivative.sup.1%=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). $\begin{array}{cc}{\mathrm{Content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\left[\%\right]=\frac{{\mathrm{Amount}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}}{M}\times 100.Math.\%& \left(6\right)\end{array}$ Where Content.sub.Total crocetin derivative[%]=Content of total crocetin derivative in gardenia yellow sample Amount.sub.Total 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). $\begin{array}{cc}\mathrm{Relative}.Math..Math.{\mathrm{content}}_i\left[\%\right]=\frac{\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{A_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}}}{{\Sigma }_{i=1}^n.Math.\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{A_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}}}\times 100.Math.\%& \left(7\right)\end{array}$ Where Relative content.sub.i=Relative content of each crocetin derivative in total crocetin derivative Peak area.sub.i=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).
Amount.sub.i[g]=Relative content.sub.i×Amount.sub.Total crocetin derivative  (8) Where Amount.sub.i=Amount of each crocetin derivative from gardenia yellow sample Relative content.sub.i=Relative content of each crocetin derivative in total crocetin derivative Amount.sub.Total crocetin derivative=Amount of total crocetin derivative in gardenia yellow sample i=Fraction number of crocetin derivative.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 illustrates a flow chart for determining the total amount of crocetin derivative amount, in accordance with an exemplary embodiment of the disclosure.

[0084] FIG. 2 illustrates a flow chart for determining each crocetin derivative amount in accordance with an exemplary embodiment of the disclosure.

[0085] FIG. 3 illustrates a flow chart for determining the amount of crocetin in accordance with an exemplary embodiment of the disclosure.

DETAILED DESCRIPTION

[0086] Although preferred embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the preferred embodiments, specific terminology will be resorted to for the sake of clarity.

[0087] It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0088] Also, in describing the preferred embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

[0089] Ranges can be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.

[0090] By “comprising” or “comprising” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0091] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0092] For the gardenia yellow product quality control scheme, UV-VIS spectroscopic method, given in China National Food Safety Standards, Food additive, Gardenia yellow, for the specific absorption coefficient assessment of total crocetin derivative and other pigments was applied. In some cases, C18-HPLC method for crocin amount determination may be employed.

[0093] (1) UV-VIS Spectroscopic Method for the Specific Absorption Coefficient

[0094] The accurately weighed amount of gardenia yellow is dissolved in an aliquot of water and methanol mixture (1:1, V/V) to prepare a sample solution at a fixed concentration. The absorbance of final diluted sample solution is measured at 440±5 nm in a 1 cm cuvette on UV-VIS spectrophotometer, with water as control. The specific absorption coefficient of the sample is calculated as follow:

[00009]$E_{1.Math.\mathrm{cm}}^{1.Math.\%}\left(440\pm 5\right).Math.\mathrm{nm}=\frac{A}{C}\times \frac{1}{100}$

Where

[0095] E.sub.1 cm.sup.1%(440±5 nm): The specific absorption coefficient, is defined as the absorbance of the product solution of 1% concentration, i. e. 1 g in 100 mL, in a 1 cm path-length cuvette.
A: Actual absorbance of product solution
C: Concentration [g/mL] of product solution
The final result is calculated from the arithmetic mean of parallel measurements. The absolute difference of two independent observations is not more than the 5% of their arithmetic mean under the same conditions.

[0096] It is an object of this method to provide the E.sub.1 cm.sup.1%(440±5 nm) of the product but not total and individual component amounts.

[0097] (2) Crocin Amount Assay by HPLC

[0098] The accurately weighed amount of gardenia yellow is dissolved in an aliquot of water and methanol mixture (1:1, V/V) to prepare a sample solution at a fixed concentration. The external reference sample (commercialized Crocin I) is dissolved in the same solvent to make its mother and work solutions. The sample solution is introduced into C18-HPLC for fraction separation and quantification by external reference under following conditions: Stationary phase: C18 column (4.6×150 mm, 5 μm); Column temperature: 40° C.; Mobile phase A: 4 L H2O (with 0.04% TFA); Mobile phase B: Acetonitrile (with 0.0.2% TFA); Linear gradient: Mobile phase B increased from 0 to 60% in 10 minutes then to 100% in 5 minutes followed by an isocratic elution for 5 minutes: Flow rate: 1 mL/min; Monitoring wavelength: 440 nm; Sample injection volume: 5 □L.

[0099] A linear equation (R.sup.2=0.9994) was obtained by correspondence of peak areas with concentrations of external reference sample. Sample with crocin concentration from 7.44 μg/mL to 119 μg/mL conforms to the linear relation. The crocin amount of gardenia yellow sample is then calculated from the peak area of crocin fraction and the linear equation.

[0100] While the existing analytical method to determine the absorption coefficient of gardenia yellow is applied to the quality control scheme of the product, it would be appreciated that the present invention provides a method to determine the crocetin content and the total amount and composition of its derivative from gardenia yellow. Under such circumstance, the amount of functional factors and coloring substances are determined. This effort is going to form a base to demonstrate the dose-effect relationship between the amount of functional factors and their biological functions.

[0101] It should be appreciated that the present method may be applied although the structure elucidation work of some crocetin derivatives has not finished yet from gardenia yellow.

[0102] The present method is easy to be performed and with a satisfied reproducibility.

[0103] To facilitate the understanding of the invention, a number of terms are herewith defined. Terms defined herein have meanings as commonly understood by a professional individual with ordinary skill in the fields that is relevant to the invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but to multiple entities which a specific example may be applied. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0104] To achieve the objectives as indicated above, the present invention employs the following technical approaches:

[0105] A. Determination of Total Crocetin Derivative Amount

[0106] 1. To determine the absorbance of gardenia yellow aqueous solution at known concentration, M grams gardenia yellow powder is accurately weighed. The sample powders are dissolved and make volume to V mL in water. The solution is then diluted by water to N-fold. The absorbance (A) of diluted sample solution is recorded at the wavelength of 440 nm.

[0107] 2. To acquire the HPLC profile of sample aqueous solution, an aliquot of gardenia yellow aqueous solution before dilution is filtered by 0.45μ filter and immediately for HPLC injection. With C18-HPLC, the fractions of crocetin, its derivatives and other pigments are readily separated under following conditions: Stationary phase: reversed phase C18 column (250×4.6 mm, 5 μm); Mobile phase A: Water-acetonitrile-acetic acid at 74.95:25:0.05 (V/V/V), mobile phase B: Acetonitrile; Linear gradient elution: Mobile phase B from 0 to 13.1% (V/V) in 20 minutes consequently followed by an isocratic elution from 20 to 35 minutes; Flow rate: 0.8 mL/min; Injection volume: 20 μL; Column temperature: At room temperature; Monitoring wavelength: 440 nm. The electronic absorption spectrum of each fraction is acquired from 250 to 700 nm for fraction identification if a PDA equipped. The chromatographic profile of gardenia yellow aqueous solution is acquired on HPLC. The peak are of each fraction is integrated on the chromatogram.

[0108] 3. With C18-HPLC-PDA-APCI-MS-MS, each fraction containing crocetin structure is identified as crocetin derivative, usually as crocetin ester, on the HPLC-MS under the chromatographic conditions as outlined above and MS conditions as follow: 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).

[0109] 4. The parent ion is identified of each crocetin derivative on MS. The m/z value of each parent ion is recorded.

[0110] 5. The relative peak area (%) of each crocetin derivative is calculated according to formula (1).

[00010]$\begin{array}{cc}\mathrm{Relative}.Math..Math.\mathrm{peak}.Math..Math.{\mathrm{area}}_i=\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{{\Sigma }_{i=1}^n.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}& \left(1\right)\end{array}$

Where

[0111] Relative peak area Relative peak area of crocetin derivative i in total crocetin derivative
Peak area.sub.i=Peak area of crocetin derivative i
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative

[0112] 6. The absorption coefficient of each crocetin derivative is calculated from published absorption coefficient, A.sub.1 cm.sup.1%=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). The molecular mass of each crocetin derivative is hereon substituted by its measured parent ion [M-1].sup.− mass reading [m/z] from MS.

[00011]$\begin{array}{cc}{A}_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}=A_{1.Math.{\mathrm{cm}}_{\mathrm{crocetin}}}^{1.Math.\%}\times \frac{\mathrm{Molecular}.Math..Math.{\mathrm{mass}}_{\mathrm{crocetin}}}{\mathrm{Molecular}.Math..Math.{\mathrm{mass}}_i}& \left(2\right)\end{array}$

Where

[0113] A.sub.1 cm.sub.i.sup.1%=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.
A.sub.1 cm.sub.Crocetin.sup.1%=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 mass.sub.crocetin=Molecular mass of crocetin. Published value of 328 is applied hereon.
Molecular mass.sub.i=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

[0114] 7. The absorption coefficient of total crocetin derivative is calculated 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).

A.sub.1 cm.sub.Total crocetin derivative.sup.1%=Σ.sub.i=1.sup.n Relative peak area.sub.i×A.sub.1 cm.sub.i.sup.1%  (3)

Where

[0115] Relative peak area.sub.i=Relative peak area of crocetin derivative i
A.sub.1 cm.sub.Total crocetin derivative.sup.1%=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.
A.sub.1 cm.sub.i.sup.1%=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

[0116] 8. The relative content of total crocetin derivative in total pigment is calculated from the peak area of each fraction monitored at 440 nm according to formula (4).

[00012]$\begin{array}{cc}{C}_{\mathrm{Total}.Math..Math.\mathrm{ester}}\left[\%\right]=\frac{{\Sigma }_{i=1}^m.Math.\mathrm{Fi}}{{\Sigma }_{j=1}^n.Math.\mathrm{Fj}}.Math..Math.\mathrm{Relative}.Math..Math.{\mathrm{content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}=\frac{{\Sigma }_{i=1}^n.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{{\Sigma }_{j=1}^m.Math..Math.\mathrm{Peak}.Math..Math.{\mathrm{area}}_j}& \left(4\right)\end{array}$

Where

[0117] Relative content.sub.Total crocetin derivative=Relative content of total crocetin derivative in total pigment
Peak area.sub.i=Peak area of crocetin derivative i
Peak area.sub.i=Peak area of fraction j
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative
j=Fraction number
m=Total fraction number

[0118] According to Lambert-Beer law, the amount of total crocetin derivative in gardenia yellow sample is calculated from the absorbance of gardenia yellow aqueous solution (Section A.1), the relative content of total crocetin derivative in total pigment (Section A.8) and the absorption coefficient of total crocetin derivative (Section A.7), as shown by formula (5).

[00013]$\begin{array}{cc}{\mathrm{Amount}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\left[g\right]=\frac{A\times \mathrm{Relative}.Math..Math.{\mathrm{content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\times V\times N}{A_{1.Math.{\mathrm{cm}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}}^{1.Math.\%}\times 100}& \left(5\right)\end{array}$

Where

[0119] Amount.sub.Total 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 content.sub.Total crocetin derivative=Relative content of total crocetin derivative in total pigment
A.sub.1 cm.sub.Total crocetin derivative.sup.1%=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.

[0120] 10. The content of total crocetin derivative in gardenia yellow sample is calculated from the amount of total crocetin derivative (Section A.9) and sample weight (M) (Section A.1) according to formula (6).

[00014]$\begin{array}{cc}{\mathrm{Content}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}\left[\%\right]=\frac{{\mathrm{Amount}}_{\mathrm{Total}.Math..Math.\mathrm{crocetin}.Math..Math.\mathrm{derivative}}}{M}\times 100.Math.\%& \left(6\right)\end{array}$

Where

[0121] Content.sub.Total crocetin derivative[%]=Content of total crocetin derivative in gardenia yellow sample
Amount.sub.Total crocetin derivative[g]=Amount of total crocetin derivative in gardenia yellow sample
M=Mass of gardenia yellow sample

[0122] B. Determination of Crocetin Derivative Composition

[0123] 1. According to Lambert-Beer law, the relative content of each crocetin derivative in total crocetin derivative is calculated from the peak area (Section A.2) and absorption coefficients of each crocetin derivative (Section A.6), as shown by formula (7).

[00015]$\begin{array}{cc}\mathrm{Relative}.Math..Math.{\mathrm{content}}_i\left[\%\right]=\frac{\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{A_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}}}{{\Sigma }_{i=1}^n.Math.\frac{\mathrm{Peak}.Math..Math.{\mathrm{area}}_i}{A_{1.Math.{\mathrm{cm}}_i}^{1.Math.\%}}}\times 100.Math.\%& \left(7\right)\end{array}$

Where

[0124] Relative content.sub.i=Relative content of each crocetin derivative in total crocetin derivative
Peak area.sub.i=Peak area of crocetin derivative i
i=Fraction number of crocetin derivative
n=Total number of crocetin derivative

[0125] 2. The amount of each crocetin derivative from gardenia yellow sample is calculated from the relative content of each crocetin derivative (Section B.1) and the amount of total crocetin derivative (Section A.9) according to formula (8).

Amount.sub.i[g]=Relative content.sub.i×Amount.sub.Total crocetin derivative  (8)

Where

[0126] Amount.sub.i=Amount of each crocetin derivative from gardenia yellow sample
Relative content.sub.i=Relative content of each crocetin derivative in total crocetin derivative
Amount.sub.Total crocetin derivative=Amount of total crocetin derivative in gardenia yellow sample
i=Fraction number of crocetin derivative

[0127] C. Determination of Crocetin Amount

[0128] 1. According to formula (9), the relative crocetin content of each crocetin derivative is calculated from the molecular masses of crocetin and its derivative, 328 Da and the parent ion mass of the derivative, respectively.

[00016]$\begin{array}{cc}\mathrm{Relative}.Math..Math.\mathrm{crocetin}.Math..Math.{\mathrm{content}}_i=\frac{\mathrm{Crocetin}.Math..Math.\mathrm{molecular}.Math..Math.\mathrm{mass}}{\mathrm{Crocetin}.Math..Math.\mathrm{derivative}.Math..Math.\mathrm{molecular}.Math..Math.{\mathrm{mass}}_i}& \left(9\right)\end{array}$

Where

[0129] Relative crocetin content.sub.i=Relative crocetin content of each crocetin derivative
Crocetin molecular mass=328 Da (Published value)
Crocetin derivative molecular mass.sub.i: The molecular mass of crocetin derivative I is hereon substituted by its parent ion [M-1].sup.− mass value [m/z] from MS (Section A.4).
i=Fraction number of crocetin derivative

[0130] 2. The relative crocetin content of total crocetin derivative is calculated from the relative crocetin content of each crocetin derivative (Section C.1) and its relative peak area (Section A.2) by weighted sum.

Relative crocetin content.sub.Total crocetin derivative=Σ.sub.i=1.sup.n Relative crocetin content.sub.i×Relative peak areai  (10)

Where

[0131] Relative crocetin content.sub.Total crocetin derivative=Relative crocetin content of total crocetin derivative
Relative crocetin content.sub.i=Relative crocetin content of each crocetin derivative
Peak area.sub.1=Peak area of crocetin derivative i
i=Fraction number of crocetin derivative

[0132] 3. The crocetin amount of gardenia yellow sample is calculated from the relative crocetin content of total crocetin derivative (Section C.2) and the amount of total crocetin derivative (Section A.9).

Crocetin amount[g]=Relative crocetin content.sub.Total crocetin derivative×Amount.sub.Total crocetin derivative  (11)

Relative crocetin content.sub.Total crocetin derivative=Relative crocetin content of total crocetin derivative
Amount.sub.Total crocetin derivative=Amount of total crocetin derivative in gardenia yellow sample

[0133] D. Recovery, Reproducibility and Minimum Detection Limit

[0134] To facilitate the evaluation and acceptance of the method described in the present invention, certain amount (usually equal to the amount of crocin in sample) of external reference, crocin (Sigma, C/N:17304), is added to sample before the analysis for the recovery, reproducibility and RSD assessment of the method. Additionally, minimum detection limit is also detected with added crocin.

[0135] Data from those assessments suggested: (1) The recovery of added reference sample, crocin, was more than 97±1.61%. (2) The RSD of repeated (more than six times) experiments is less than 3.17%. This observation indicates that the method described in the invention exhibits a good reproducibility. (3) The minimum detection limit is more than 40 ng/mL on UV-VIS.

[0136] E. Advantages, Importance and Significance of the Invention

[0137] 1. The invention provides a method to quantify the total amount and composition of crocetin derivative and crocetin amount from gardenia yellow. This effort results in the quantitative color ability and dose-effect safety evaluations of gardenia yellow.

[0138] 2. The invention provides a method to quantify the total amount and composition of crocetin derivative and crocetin amount from gardenia yellow while the molecular structure of certain derivatives have not yet been elucidated and clarified.

[0139] 3. The results from those three assays are fundamentally from the absorbance of the sample solution on UV-VIS. It is therefore for most individuals who work in this filed to be easy to perform while the recovery and reproducibility are also good.

[0140] The above description is intended to enable the person skilled in the science to practice the invention. It is not intended to detail all of the possible modifications and variations which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such modifications and variations are included within the scope of the invention which is seen in the above description.

EXAMPLES

[0141] Following examples are presented to further explain of the analytical method described in the invention and are not to be taken as limiting in any regard.

Example 1

[0142] This example provides a preferred, but not limited, method to determine the crocetin and its derivative amounts and the derivative composition from one of commercialized gardenia yellow products most commonly encountered in current food additive market with relative higher absorption coefficient than others.

[0143] A. Determination of Total Crocetin Derivative Amount

[0144] In practice, gardenia yellow product with the label of E500 is selected as tested sample. According to procedure described as above, following steps are undertaken:

[0145] 10.0502 g sample powder is accurately (0.0001 g) weighed. The weighed sample is then completely dissolved and makes volume to 50 mL in water. After the solution was diluted 100 times, the absorbance (A) of the solution is measured on UV-VIS spectrophotometer at 440 nm. A=0.5064 is recorded.

[0146] 2. To acquire the HPLC profile of sample aqueous solution, an aliquot of gardenia yellow aqueous solution before dilution is filtered by 0.45□ filter and immediately for HPLC injection. With C18-HPLC, the fractions of crocetin, its derivatives and other pigments are readily separated under following conditions: Stationary phase: reversed phase C18 column (250×4.6 mm, 5 μm); Mobile phase A: Water-acetonitrile-acetic acid at 74.95:25:0.05 (V/V/V), mobile phase B: Acetonitrile; Linear gradient elution: Mobile phase B from 0 to 13.1% (V/V) in 20 minutes consequently followed by an isocratic elution from 20 to 35 minutes; Flow rate: 0.8 mL/min; Injection volume: 20 □L; Column temperature: At room temperature; Monitoring wavelength: 440 nm. The electronic absorption spectrum of each fraction is acquired from 250 to 700 nm for fraction identification if a PDA equipped. The chromatographic profile of gardenia yellow aqueous solution is acquired on HPLC. It is observed that each fraction is readily separated under HPLC conditions as mentioned above. The peak are of each fraction is then integrated on the chromatogram and shown in Table 1.

[0147] 3. With HPLC-ACPI-MS-MS, each fraction containing crocetin structure is identified as crocetin derivative, usually as crocetin ester, on the HPLC-MS under the chromatographic conditions as outlined above and MS conditions as follow: 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). The fractions containing crocetin structure are given in Table 1.

[0148] 4. The parent ion is identified of each crocetin derivative on MS. The m/z value of each parent ion is recorded in Table 1.

[0149] 5. According to formula (1), the relative peak area (%) of each crocetin derivative is calculated and illustrated in Table 1.

[0150] 6. The absorption coefficient (A.sub.1 cm.sub.i.sup.1%) of each crocetin derivative is calculated from published absorption coefficient, A.sub.1 cm.sup.1%=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). The molecular mass of each crocetin derivative is hereon substituted by its measured parent ion [M-1].sup.− mass reading [m/z] from MS. The absorption coefficient of each crocetin derivative is given in Table 1.

[0151] 7. According to formula (3), the absorption coefficient (A.sub.1 cm.sub.Total crocetin derivative.sup.1%) of total crocetin derivative is calculated by weighted sum from the relative peak area and absorption coefficients of each crocetin derivative as the results of formulae (1) and (2), respectively. A.sub.1 cm.sub.Total crocetin derivative.sup.1%=1125 is recorded.

[0152] 8. According to formula (4), the relative content of total crocetin derivative in total pigment is calculated from the peak area of each fraction monitored at 440 nm. Relative content.sub.Total crocetin derivative=97.20% is recorded.

[0153] 9. According to Lambert-Beer law, the amount of total crocetin derivative in gardenia yellow sample is calculated from the absorbance of gardenia yellow aqueous solution (Section A.1), the relative content of total crocetin derivative in total pigment (Section A.8) and the absorption coefficient of total crocetin derivative (Section A.7), as shown by formula (5). Amount.sub.Total crocetin derivative=0.0225 g is recorded.

[0154] 10. According to formula (6), the content of total crocetin derivative in gardenia yellow sample is calculated from the amount of total crocetin derivative and sample weight. Content.sub.Total crocetin derivative=44.82% is recorded.

[0155] B. Determination of Crocetin Derivative Composition

[0156] 1. According to Lambert-Beer law, the relative content of each crocetin derivative in total crocetin derivative is calculated from the peak area and absorption coefficients of each crocetin derivative, as shown by formula (7). Relative content of each crocetin derivative in total crocetin derivative is shown in Table 1.

[0157] 2. According to formula (8), the amount of each crocetin derivative from gardenia yellow sample is calculated from the relative content of each crocetin derivative and the amount of total crocetin derivative. Amount of each crocetin derivative from gardenia yellow sample is illustrated in Table 1.

TABLE-US-00001 TABLE 1 The chromatographic behaviors, MS characteristics and amounts of each fraction from gardenia yellow with marked absorption coefficient of 500 (E500) Relative Relative Fraction Retention Peak area peak Parent Crocetin content.sub.i Amount.sub.i No. time [min] [mAU * min] area [%] ion[m/z] piece[m/z] A.sub.1cm.sub.i.sup.1% [%] [g] 1 3.260 0.464 0.88 2 4.343 0.201 0.38 3 4.650 0.202 0.38 4 7.093 26.386 49.92 1089 327 1151 51.19 0.0115 5 8.037 1.617 3.06 1089 327 1151 3.14 0.0007 6 9.190 0.507 0.96 1089 327 1151 0.98 0.0002 7 11.203 4.000 7.57 1137 327, 651, 1102 8.10 0.0018 813 8 14.083 0.075 0.14 1241 327 1010 0.17 0.0000 9 14.807 0.568 1.07 1241 327 1010 1.26 0.0003 10 16.073 1.415 2.68 1241 327 1010 3.13 0.0007 11 16.593 0.449 0.85 12 18.150 0.165 0.31 13 21.517 0.638 1.21 819 327 1530 0.93 0.0002 14 22.350 7.005 13.25 1241 327 1010 15.49 0.0035 15 23.793 0.527 1.00 963 327 1301 0.90 0.0002 16 24.597 0.721 1.36 1241 327 1010 1.59 0.0004 17 25.883 6.743 12.76 951 327 1318 11.42 0.0026 18 28.273 0.477 0.90 813 327, 651 1541 0.69 0.0002 19 28.783 0.694 1.31 813 327, 651 1541 1.01 0.0002 Sum 52.854 100.0 100.0 0.0225

[0158] C. Determination of Crocetin Amount

[0159] 1. According to formula (9), the relative crocetin content of each crocetin derivative is calculated from the molecular masses of crocetin and its derivative, 328 Da and the parent ion mass of the derivative, respectively. Relative crocetin content of each crocetin derivative is given in Table 2.

[0160] 2. The relative crocetin content of total crocetin derivative is calculated from the relative crocetin content of each crocetin derivative and its relative peak area by weighted sum. The relative crocetin content of total crocetin derivative is 29.35%.

[0161] 3. The crocetin amount of gardenia yellow is calculated from the relative crocetin content of total crocetin derivative and the amount of total crocetin derivative. The crocetin amount of total crocetin derivative in the gardenia yellow sample (0.0502 g) is 0.0066 g. The crocetin amount of gardenia yellow is 13.15%.

TABLE-US-00002 TABLE 2 The chromatographic behaviors, MS characteristics and crocetin content of each fraction from gardenia yellow with marked absorption coefficient of 500 (E500) Relative Relative Relative Fraction Retention Peak peak Parent Crocetin crocetin content.sub.i No. time [min] area [mAU * min] area [%] ion [m/z] piece [m/z] centent i [%] 1 3.26 0.464 0.88 2 4.343 0.201 0.38 3 4.65 0.202 0.38 4 7.093 26.386 49.92 1089 327 0.3002 14.99 5 8.037 1.617 3.06 1089 327 0.3002 0.92 6 9.19 0.507 0.96 1089 327 0.3002 0.29 7 11.203 4 7.57 1137 327 0.2875 2.18 8 14.083 0.075 0.14 1241 327 0.2634 0.04 9 14.807 0.568 1.07 1241 327 0.2634 0.28 10 16.073 1.415 2.68 1241 327 0.2634 0.71 11 16.593 0.449 0.85 12 18.15 0.165 0.31 13 21.517 0.638 1.21 819 327 0.3992 0.48 14 22.35 7.005 13.25 1241 327 0.2634 3.49 15 23.793 0.527 1 963 327 0.3395 0.34 16 24.597 0.721 1.36 1241 327 0.2634 0.36 17 25.883 6.743 12.76 951 327 0.3438 4.39 18 28.273 0.477 0.9 813 327 0.4022 0.36 19 28.783 0.694 1.31 813 327 0.4022 0.53 Sum 29.35

[0162] D. Recovery and Reproducibility

[0163] To facilitate the evaluation and acceptance of the method described in the present invention, certain amount (usually equal to the amount of crocin in sample) of external reference, crocin (Sigma, C/N:17304), is added to sample before the analysis for the recovery, reproducibility and RSD assessment of the method. Additionally, minimum detection limit is also detected with added crocin.

[0164] Data from those assessments suggested: (1) The recovery of added reference sample, crocin, was more than 97±1.61%. (2) The RSD of repeated (more than six times) experiments is less than 3.17%. This observation indicates that the method described in the invention exhibits a good reproducibility.

[0165] This method is preferably employed to perform the determination of total amount and composition of crocetin derivative and crocetin amount of product with relative higher pigment concentration for its safety evaluation and quality control schemes.

Example 2

[0166] This example provides a preferred, but not limited, method to determine the crocetin and its derivative amounts and the derivative composition from one of commercialized gardenia yellow products most commonly encountered in current food additive market with relative lower absorption coefficient than others.

[0167] A. Determination of Total Crocetin Derivative Amount

[0168] In practice, gardenia yellow product with the label of E450 is selected as tested sample. According to procedure described as above, following steps are undertaken:

[0169] 10.0531 g sample powder is accurately (0.0001 g) weighed. The weighed sample is then completely dissolved and makes volume to 50 mL in water. After the solution was diluted 100 times, the absorbance (A) of the solution is measured on UV-VIS spectrophotometer at 440 nm. A=0.4971 is recorded.

[0170] 2. To acquire the HPLC profile of sample aqueous solution, an aliquot of gardenia yellow aqueous solution before dilution is filtered by 0.45□ filter and immediately for HPLC injection. With C18-HPLC, the fractions of crocetin, its derivatives and other pigments are readily separated under following conditions: Stationary phase: reversed phase C18 column (250×4.6 mm, 5 μm); Mobile phase A: Water-acetonitrile-acetic acid at 74.95:25:0.05 (V/V/V), mobile phase B: Acetonitrile; Linear gradient elution: Mobile phase B from 0 to 13.1% (V/V) in 20 minutes consequently followed by an isocratic elution from 20 to 35 minutes; Flow rate: 0.8 mL/min; Injection volume: 20 □L; Column temperature: At room temperature; Monitoring wavelength: 440 nm. The electronic absorption spectrum of each fraction is acquired from 250 to 700 nm for fraction identification if a PDA equipped. The chromatographic profile of gardenia yellow aqueous solution is acquired on HPLC. It is observed that each fraction is readily separated under HPLC conditions as mentioned above. The peak are of each fraction is then integrated on the chromatogram and shown in Table 3.

[0171] 3 With HPLC-ACPI-MS-MS, each fraction containing crocetin structure is identified as crocetin derivative, usually as crocetin ester, on the HPLC-MS under the chromatographic conditions as outlined above and MS conditions as follow: 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). The fractions containing crocetin structure are given in Table 3.

[0172] 4. The parent ion is identified of each crocetin derivative on MS. The m/z value of each parent ion is recorded in Table 3.

[0173] 5. According to formula (1), the relative peak area (%) of each crocetin derivative is calculated and illustrated in Table 3.

[0174] 6. The absorption coefficient (A.sub.1 cm.sub.i.sup.1%) of each crocetin derivative is calculated from published absorption coefficient, A.sub.1 cm.sup.1%=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). The molecular mass of each crocetin derivative is hereon substituted by its measured parent ion [M-1].sup.− mass reading [m/z] from MS. The absorption coefficient of each crocetin derivative is given in Table 3.

[0175] 7. According to formula (3), the absorption coefficient (A.sub.1 cm.sub.Total crocetin derivative.sup.1%) of total crocetin derivative is calculated by weighted sum from the relative peak area and absorption coefficients of each crocetin derivative as the results of formulae (1) and (2), respectively. A.sub.1 cm.sub.Total crocetin derivative.sup.1%1127 is recorded.

[0176] 8. According to formula (4), the relative content of total crocetin derivative in total pigment is calculated from the peak area of each fraction monitored at 440 nm. Relative content.sub.Total crocetin derivative=99.09% is recorded.

[0177] 9. According to Lambert-Beer law, the amount of total crocetin derivative in gardenia yellow sample is calculated from the absorbance of gardenia yellow aqueous solution (Section A.1), the relative content of total crocetin derivative in total pigment (Section A.8) and the absorption coefficient of total crocetin derivative (Section A.7), as shown by formula (5). Amount.sub.Total crocetin derivative=0.0221 g is recorded.

[0178] 10. According to formula (6), the content of total crocetin derivative in gardenia yellow sample is calculated from the amount of total crocetin derivative and sample weight. Content.sub.Total crocetin derivative=41.62% is recorded.

[0179] B. Determination of Crocetin Derivative Composition

[0180] 1. According to Lambert-Beer law, the relative content of each crocetin derivative in total crocetin derivative is calculated from the peak area and absorption coefficients of each crocetin derivative, as shown by formula (7). Relative content of each crocetin derivative in total crocetin derivative is shown in Table 3.

[0181] 2. According to formula (8), the amount of each crocetin derivative from gardenia yellow sample is calculated from the relative content of each crocetin derivative and the amount of total crocetin derivative. Amount of each crocetin derivative from gardenia yellow sample is illustrated in Table 3.

TABLE-US-00003 TABLE 3 The chromatographic behaviors, MS characteristics and amounts of each fraction from gardenia yellow with marked absorption coefficient of 450 (E450) Relative Relative Fraction Retention Peak area peak Parent Crocetin amount.sub.i Amount.sub.i No. time[min] [mAU * min] area[%] ion[m/z] piece[m/z] A.sub.1cm.sub.i.sup.1% [%] [g] 1 4.343 0.269 0.40 2 4.607 0.265 0.40 3 4.743 0.018 0.03 4 5.107 0.395 0.59 1173 327, 651, 1068 0.63 0.0001 813, 5 5.773 0.055 0.08 6 7.083 49.324 73.85 1089 327 1151 73.43 0.0162 7 8.010 1.943 2.91 1089 327 1151 2.89 0.0006 8 9.173 0.889 1.33 1089 327 1151 1.32 0.0003 9 11.227 4.274 6.40 1137 327, 651, 1102 6.64 0.0015 813 10 14.873 0.785 1.18 1241 327 1010 1.33 0.0003 11 16.143 2.029 3.04 1241 327 1010 3.44 0.0008 12 21.587 0.263 0.39 819 327 1530 0.29 0.0001 13 22.407 4.765 7.13 1241 327 1010 8.08 0.0018 14 24.703 0.158 0.24 1241 327 1010 0.27 0.0001 15 25.975 0.754 1.13 951 327 1318 0.98 0.0002 16 28.890 0.604 0.90 813 327, 651 1541 0.67 0.0001 Sum 66.790 100.0 100.0 0.0221

[0182] C. Determination of Crocetin Amount

[0183] 1. According to formula (9), the relative crocetin content of each crocetin derivative is calculated from the molecular masses of crocetin and its derivative, 328 Da and the parent ion mass of the derivative, respectively. Relative crocetin content of each crocetin derivative is given in Table 4.

[0184] 2. The relative crocetin content of total crocetin derivative is calculated from the relative crocetin content of each crocetin derivative and its relative peak area by weighted sum. The relative crocetin content of total crocetin derivative is 29.41%.

[0185] 3. The crocetin amount of gardenia yellow is calculated from the relative crocetin content of total crocetin derivative and the amount of total crocetin derivative. The crocetin amount of total crocetin derivative in the gardenia yellow sample (0.0502 g) is 0.0065 g. The crocetin amount of gardenia yellow is 12.24%.

TABLE-US-00004 TABLE 4 The chromatographic behaviors, MS characteristics and crocetin content of each fraction from gardenia yellow with marked absorption coefficient of 450 (E450) Relative Relative Relative Fraction Retention Peak area peak Parent Crocetin crocetin content.sub.i No. time [min] [mAU * min ] area [%] ion [m/z] piece [m/z] centent i [%] 1 4.343 0.269 0.4 2 4.607 0.265 0.4 3 4.743 0.018 0.03 4 5.107 0.395 0.59 1173 327 0.2788 0.16 5 5.773 0.055 0.08 6 7.083 49.324 73.85 1089 327 0.3003 22.18 7 8.01 1.943 2.91 1089 327 0.3003 0.87 8 9.173 0.889 1.33 1089 327 0.3003 0.40 9 11.227 4.274 6.4 1137 327 0.2876 1.84 10 14.873 0.785 1.18 1241 327 0.2635 0.31 11 16.143 2.029 3.04 1241 327 0.2635 0.80 12 21.587 0.263 0.39 819 327 0.3993 0.16 13 22.407 4.765 7.13 1241 327 0.2635 1.88 14 24.703 0.158 0.24 1241 327 0.2635 0.06 15 25.975 0.754 1.13 951 327 0.3438 0.39 16 28.89 0.604 0.9 813 327 0.4022 0.36 Sum 29.41

[0186] D. Recovery and Reproducibility

[0187] To facilitate the evaluation and acceptance of the method described in the present invention, certain amount (usually equal to the amount of crocin in sample) of external reference, crocin (Sigma, C/N:17304), is added to sample before the analysis for the recovery, reproducibility and RSD assessment of the method. Additionally, minimum detection limit is also detected with added crocin.

[0188] Data from those assessments suggested: (1) The recovery of added reference sample, crocin, was more than 96±2.12%. (2) The RSD of repeated (more than six times) experiments is less than 4.03%. This observation indicates that the method described in the invention exhibits a good reproducibility.

[0189] This method is preferably employed to perform the determination of total amount and composition of crocetin derivative and crocetin amount of product with relative lower pigment concentration for its safety evaluation and quality control schemes.

[0190] Finally, it should be noted that: Obviously, the examples described above intend only to illustrate the invention clearly and not limitations to provide a way to implement the invention. For the individuals who are skilled in the related fields of science, various changes, modifications or variations in the invention based on description as above can also be made. There is no need to implement all the way or to be exhaustive to raise any other examples. Any change or variation in the invention is still in the protection scope of the invention.

[0191] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0192] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based can be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.