KR101027433B1 - Method for quantifying campesterol of soybean using NIR - Google Patents

Abstract

The present invention relates to a method for quantifying vegetable sterols of soybeans by a rapid (dry) method using near infrared light. The first derivative of the values measured by using a near infrared spectroscopy for powdered soybeans The present invention relates to a method for quantifying vegetable sterols, in particular campesterol, by introducing a statistical multicrystal coefficient (r ² ) into a calibration curve with high accuracy of 0.84 or higher. The method of the present invention can quickly and in large quantities explore the content of vegetable sterols per unit weight of soybean without undergoing chemical and wet pretreatment of the soybean, which is useful for developing high quality maintenance materials or breeding or industrial materials. Can be.

Dry quantitative method, soybean, vegetable sterol, campestrol, NIR, mass search

Description

Method for quantifying campesterol of soybean using near infrared light {Method for quantifying campesterol of soybean using NIR}

The present invention relates to a method of quantifying soybean camphorsterol by a rapid (dry) method using near infrared light.

Soybeans contain vegetable sterols that are structurally similar to the common cholesterol found only in animal foods. Plant sterols contained in soybeans are known to reduce blood sterols and prevent heart disease, and they are also known to inhibit cancer by protecting the intestines from bile acids and harmful substances, which are cholesterol metabolites in the large intestine. In general, countries with low incidence of colon cancer have high phytosterol intake and have been reported to inhibit skin cancer. To this end, countries around the world are now focusing on developing vegetable oils, soybeans, and salad oils or margarine that have the effect of lowering blood cholesterol levels. If the recent diet is rapidly westernized and excessive intake of animal fat is a problem, soy foods containing vegetable sterols that do not accumulate body fat may be an alternative.

In addition, phytosterol contained in soybean is widely used as an active ingredient in cosmetics such as creams and lipsticks, and occupies an important area as a functional cosmetic raw material. Soybeans ingested as food constitute lipids of the skin. Soybean phytosterol was found to be effective in the recovery of the stratum corneum. Of formula kamto sterol (Campesterol) is C ₂₈ H _48O, and a molecular weight of 400.68 is found in many plant-based, also known as plant sterols, cholesterol and is chemically similar. As the second most abundant substance in phytosterol, camphorsterol differs from cholesterol in that methyl group is present at the 24th carbon site of the side chain, and is cholesterol-based 4-desmethylsterols. Are classified as). Present in soybeans, plant seeds, and sunflower seeds, used in salad dressings and margarine-type functional foods. Campesterol lowers your total cholesterol and LDL-cholesterol levels, reducing your risk of coronary heart disease. Campestrol inhibits dietary cholesterol absorption and endogenous cholesterol resorption of the gastrointestinal tract, and excretes cholesterol into feces to reduce serum cholesterol levels. It has also been shown to lower cholesterol levels by replacing cholesterol with bile salts or inhibiting cholesterol esterification in the intestinal mucosa. Conventionally, in order to analyze such camphorol, powdered soybeans are extracted, all fats and oils are extracted, and only camphorsterol is derivatized by methyl ester or silylation, and then quantified by gas chromatography. As a result of analysis through wet pretreatment of the reagents and complex procedures, it was not appropriate to test many samples at once. However, as the usefulness of the soybeans increases, there is a need for a method for quickly and mass-exploring the vegetable sterol content per unit weight.

Therefore, the inventors repeatedly obtained the near-infrared spectrum of the soybean that was not subjected to wet pretreatment, and designed a calibration curve having a coefficient of polycrystallization coefficient (r ² ) of 0.84 or more through statistical processing, and the calibration curve of the fast (dry) soybean. The present invention has been completed by confirming that it can be usefully used to quantify plant sterol properties.

It is an object of the present invention to provide a rapid (dry) quantitative method for the content of the camphorsterol component of soybeans.

In order to achieve the above object, the present invention 1) to classify the beans seeds according to varieties, put them in a test tube in a powder state into a near-infrared analyzer, and then irradiate near-infrared light to obtain an absorption spectrum of 1700 to 2100 nm region Making;

2) applying a first order differential method to the near infrared spectrum and obtaining a first derivative value with a gap of 10 nm for each wavelength measured;

3) It provides a dry quantitative method for the content of the camphorsterol component of the soybean, comprising the step of determining the camphorsterol content in the soybean sample by substituting the first derivative value for each wavelength into a function of the following formula (1).

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: 1) sorting the bean seeds according to varieties and inserting them into a test tube in powder form into a near-infrared spectrometer, and irradiating near-infrared light to obtain an absorption spectrum in the region of 1700 to 2100 nm;

2) applying a first order differential method to the near infrared spectrum and obtaining a first derivative value with a gap of 10 nm for each wavelength measured;

3) by providing a first derivative of each wavelength in the function of Equation 1 below, to provide a dry quantification method for the content of the camphorsterol component of soybean, comprising determining the camphorsterol content in the soybean sample:

[Equation 1]

Campesterol Content (μg / g) = -159.7143 - 539.5369x_'1700+ 208.2029x_'1710+ 862.7268x_'1720+ 304.1911x_'1730- 65.8495x_'1740- 255.1128x_'1750- 212.5636x_'1760- 52.1982x_'1770+ 135.6333x_'1780+ 203.0269x_'1790+ 233.239x_'1800+ 104.6265x_'1810+ 75.9039x_'1820- 5.9972x_'1830+ 56.3546x_'1840+ 43.7998x_'1850+ 140.0899x_'1860+ 689.9338x_'1870+ 791.2035x_'1880+ 801.7698x_'1890- 284.5929x_'1900- 1425.0724x_'1910- 1286.3221x_'1920- 732.9667x_'1930+ 61.4209 x_'1940+ 424.8888x_'1950+ 568.9919x_'1960+ 601.3539x_'1970+ 406.6427x_'1980+ 344.6469x_'1990+ 278.9063x_'2000+ 148.3x_'2010+ 40.2344x_'2020+ 22.6729x_'2030- 28.6875x_'2040- 142.7464x_'2050- 305.8374x_'2060- 237.7981x_'2070- 112.3536x_'2080- 50.1014x_'2090+ 15.0298x_'2100.

_X'1700 to x _'2100 of Equation 1 refers to a first derivative value measured at intervals of 10 nm in a spectrum between 1700 and 2100 nm.

In a specific embodiment of the present invention, 152 soybean transgenic plants were subjected to wet plant sterol analysis (see Table 1) and dry plant sterol analysis (see Table 2) using a near-infrared spectrometer. Then, the results of the above analysis of the dry plant sterols, which were subjected to water treatment with a first or second differential method and a measurement wavelength gap of 10 nm, were analyzed by wet vegetable sterol analysis and modified partial least squares (MPLS). By investigating the significant correlation, a calibration curve for each plant sterol (see FIG. 2) and a squared coefficient of multiple determination in calibration (R ² ) of each calibration curve (see Table 2) were obtained. As a result, the multiple crystal coefficient of the calibration curve of campestrol (Equation 1) was found to be high accuracy of 0.84 or more.

Thus, by using the calibration curve of Equation 1 it may be possible to quickly and accurately measure the vegetable sterol content of the soybeans by the dry method.

The method of the present invention makes it easy to measure a sample as it is without chemical wet pretreatment, and does not require a pretreatment process such as quantitating, mixing, heating, and extracting a sample without using a reagent, and thus analyzing multiple components quickly and simultaneously. In addition to being able to do so, there is an advantage in that the sample can be recovered and used for use and storage or other analysis.

In the present invention, the near-infrared spectrum may be obtained by scanning in a soybean powder in any manner commonly used in the art, and may be preferably obtained by diffuse reflection or transmission.

The near-infrared spectrum obtained in step 2) is not well suited to Beer's law because of the complex and unidentified change factors that change the overlap and physical properties of the absorbing peaks. Specifically, the near-infrared spectrum causes baseline variations and absorbance disturbances. Changes in baseline include particle size, density of sample, country of origin, height, and short-term changes, and blockages of absorbance include broad, overlapping, complex, and uncertain conditions. To solve this problem, various statistical processes are performed to derive the optimal calibration curve. Specifically, in the present invention, a differential method is applied, and preferably, the spectrum is subjected to the mathematical pretreatment of the first derivative or the second derivative. The mathematical pretreatment primarily corrects the scattering effect of the light irradiated onto the sample, pretreatment of the sample as an essential tool for stabilizing the calibration curve, and serves as a separation column of the chromatography. In addition, in order to prevent misinterpretation of the spectrum such as over-fitting, the gap for each measurement wavelength is adjusted. In the present invention, 4 to 10 nm is preferable. In the determination of the camphorsterol content contained in the soybean of the present invention, it is most preferable to use a first-order differential method, and most preferably, a gap for each wavelength of measurement is 10 nm.

The method of the present invention can quickly and in large quantities explore the content of vegetable sterols per unit weight of soybean without undergoing chemical and wet pretreatment of the soybean, which is useful for developing high quality maintenance materials or breeding or industrial materials. Can be.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Target of Analysis

A total of 152 soybeans were sampled, some of which were distributed at the National Center for Agricultural Genetic Resources, and some of the soybeans used in the present study.

< Example  2> Investigation of vegetable sterol content per wet unit weight

After each 0.2 g of the soybean sample of Example 1 in a tube for centrifugation, 5 ml of extraction solvent (chloroform: methanol = 2: 1) and an internal standard [1000 ppm in MeOH dissolved in pentacanoic acid (pentadecanoic acid) ): Sigma-Aldrich, USA] 1 ml was added and sonicated for 1 hour. Then 5 ml of 0.58% NaCl solution (dissolved in water) was added and sonicated for 10 minutes. The extract was centrifuged at 2000 rpm for 15 minutes to take a chloroform fraction (lower layer) and concentrated by blowing the solvent with nitrogen gas. Then 0.5 ml toluene and 2 ml of 0.5N NaOH (dissolved in methanol) were added, followed by bathing in hot water (80 ° C.) for 5 minutes. After the hot solution was allowed to cool, 2.5 ml of 14% BF ₃ (Boron trifloride methanol solution 14%: Sigma-Aldrich, USA) was added thereto, and the mixture was allowed to stand for 5 minutes in hot water (80 ° C) and then allowed to cool. 10 mL of petroleum ether and 15 mL of distilled water were added to the sample, which was transferred to another test tube, vortexed, and centrifuged at 4 ° C. for 10 minutes at 2000 rpm. Take only the supernatant, filter it with a new test tube (sodium sulfate, Whatman filter, USA), and then concentrate the filtrate with nitrogen gas and dissolve in 1 ml of petroleum ether to GC / FID (Hewlett Packard, 5890 series II, USA). Analyzed.

GC-FID conditions used in this experiment were analyzed isothermally at 300 ° C. using an HP-5 column (60 m × 0.25 mm ID, 0.25 μm, Agilent Co., USA) as an analytical column. The detector temperature was maintained at 280 ° C and 290 ° C, respectively. The mobile phase (nitrogen gas) rate was 1 ml / min, the split ratio was 1: 8, and the injection amount was 1 µl. Quantitative phytosterols were calculated by comparing each pit area shown in the chromatogram with the internal standard 5α-cholestane. β-sitosterol was found at 17.9, 18.7 and 20.5 minutes, respectively.

As a result, as shown in Table 1, the contents of camphorsterol, stigmasterol, β-sitosterol and total vegetable sterols of 152 soybeans were measured.

Table of Vegetable Sterol Content Per Weight of Soybean sample Concentration (㎍ / g) all Campestrol Stigmasterol β-sitosterol Μg / g IT154763 10.8 8.7 2.9 22.4 IT141616 1.1 9.0 18.9 29.0 bean119-15 8.2 8.9 14.5 31.5 bean148-6 8.3 7.6 15.8 31.7 IT161404 8.1 1.0 23.0 32.1 IT142885 8.8 7.2 16.3 32.3 bean139-11 9.8 7.9 15.1 32.8 IT153428 9.2 7.7 16.3 33.2 bean13-5 9.2 8.6 15.5 33.3 bean58-12 8.4 8.0 17.3 33.7 bean111-5 8.3 8.0 17.6 33.9 IT154761 6.9 8.5 18.6 34.0 IT115489 9.3 9.4 15.8 34.4 IT121449 9.5 8.3 17.0 34.8 bean18-4 7.8 8.6 18.5 35.0 IT195204 9.3 8.1 18.1 35.5 IT115933 10.8 8.6 16.2 35.5 IT212847 10.5 7.9 17.5 35.9 IT143148 9.3 8.4 18.2 35.9 IT115729 11.1 9.8 15.2 36.0 IT153719 7.8 8.9 19.6 36.2 bean100-3 9.0 9.6 17.6 36.3 bean35-7 8.3 9.2 18.9 36.5 IT108998 9.5 9.5 17.6 36.6 bean1-6 12.0 8.7 15.9 36.6 IT154405 8.3 8.8 19.6 36.7 IT154741 7.7 8.9 20.1 36.7 IT115763 10.2 8.8 17.8 36.8 bean131-16 7.7 8.4 20.9 37.1 bean146-3 8.7 8.5 20.0 37.2 bean160-6 10.8 8.4 18.0 37.2 IT162450 7.5 8.6 21.2 37.3 IT154760 8.6 8.0 20.8 37.4 bean105-3 7.8 9.0 20.6 37.5 IT119898 9.5 8.3 20.0 37.8 bean136-3 9.9 9.5 18.6 38.0 bean108-5 9.9 8.7 19.3 38.0 IT154588 8.2 9.0 21.3 38.6 IT115431 10.0 8.5 20.2 38.7 IT142924 9.4 9.2 20.2 38.8 IT212822 10.8 8.1 20.2 39.1 IT162955 10.1 9.1 20.0 39.2 bean54-9 9.6 8.5 21.1 39.2 bean74-11 10.6 9.0 19.6 39.2 IT115882 9.4 10.1 20.0 39.6 bean135-5 11.0 9.4 19.4 39.7 bean62-5 10.6 8.9 20.4 39.9 IT212824 10.4 9.3 20.3 40.0 bean156-8 9.6 10.3 20.2 40.1 IT141595 11.2 10.0 19.0 40.2 bean69-6 10.7 9.3 20.3 40.3 IT211818 9.7 10.0 20.8 40.4 IT181939 9.6 9.6 21.4 40.6 IT208933 9.5 9.7 21.4 40.6 IT162495 9.7 8.1 22.8 40.7 bean157-19 11.6 8.3 20.9 40.7 IT177345 10.9 8.9 20.9 40.8 bean137-5 10.0 9.9 21.1 41.0 IT154573 7.7 10.3 23.1 41.1 IT211829 10.2 8.9 22.1 41.2 IT195600 9.1 8.4 23.8 41.3 bean80-5 10.8 9.0 21.8 41.6 bean33-6 9.5 9.5 22.8 41.8 IT189273 10.5 9.0 22.2 41.8 IT186278 9.1 10.2 22.6 41.8 IT115605 13.5 9.1 19.2 41.8 IT115451 11.9 10.3 19.7 41.9 bean72-6 11.7 9.2 21.1 42.0 IT171212 10.3 10.4 21.5 42.1 bean138-8 12.2 9.5 20.4 42.2 IT153739 9.5 10.2 23.2 42.8 IT165447 8.8 9.9 24.2 42.9 bean91-7 10.3 9.8 22.8 42.9 IT160299 11.6 8.3 23.0 43.0 IT161546 10.5 9.6 22.8 43.0 IT167245 11.4 10.5 21.1 43.0 IT142744 11.1 9.1 22.8 43.1 bean45-7 11.0 10.0 22.2 43.2 IT115486 11.1 9.2 23.0 43.3 bean82-7 10.5 9.5 23.4 43.4 IT162405 9.5 9.2 24.7 43.4 IT180039 11.2 10.0 22.4 43.6 IT024888 12.9 11.3 19.4 43.6 bean89-5 10.9 8.7 24.1 43.7 IT186142 8.8 10.6 24.4 43.8 IT160152 10.3 7.8 25.8 43.9 bean159-4 12.1 8.1 23.7 43.9 IT162133 8.6 9.0 26.4 44.0 IT141988 10.6 10.7 22.6 44.0 IT165073 9.3 10.3 24.5 44.1 IT177734 10.0 9.4 24.7 44.1 IT162055 11.4 9.3 23.5 44.2 IT161510 10.4 10.6 23.3 44.3 IT154195 10.5 11.1 22.8 44.3 IT189206 10.0 10.6 23.9 44.4 bean50-11 12.0 9.9 22.6 44.6 bean94-8 11.5 10.1 23.2 44.7 IT154615 8.3 9.0 27.4 44.7 bean65-12 11.2 10.4 23.1 44.7 bean25-1 11.6 9.7 23.5 44.8 IT115400 11.4 9.7 23.7 44.8 IT108931 12.6 9.6 22.7 44.9 IT180021 12.2 10.2 22.6 44.9 bean81-13 12.4 9.7 22.9 45.0 bean115-10 12.4 9.8 23.1 45.4 IT154517 10.2 10.1 25.3 45.5 IT180019 12.3 10.9 22.4 45.5 IT154291 10.3 10.1 25.1 45.6 bean123-8 10.9 9.4 25.4 45.7 IT162267 10.7 10.6 24.5 45.8 IT180028 12.2 10.0 23.6 45.8 IT165506 9.5 9.3 27.3 46.1 IT161617 10.3 9.6 26.6 46.5 IT180589 11.8 10.2 24.6 46.6 IT189269 11.5 9.9 25.3 46.7 IT180851 11.3 9.9 25.5 46.7 IT161737 9.2 9.5 28.2 46.8 bean40-5 12.2 10.4 24.4 47.0 bean151-5 9.8 12.5 24.7 47.0 IT186206 10.7 10.4 25.9 47.1 bean158-3 12.2 8.5 26.4 47.2 bean163-3 11.2 9.3 26.7 47.2 bean24-5 11.8 9.8 25.8 47.3 bean42-6 11.5 11.0 24.9 47.4 IT180190 12.9 9.2 25.3 47.4 IT153365 12.0 11.4 24.2 47.6 IT162546 11.0 10.3 26.4 47.7 bean78-4 12.9 10.4 24.4 47.7 IT181551 10.9 12.3 24.7 47.8 IT161673 12.7 9.5 25.6 47.8 IT161826 10.3 11.0 27.0 48.3 IT154596 9.9 11.3 27.4 48.6 IT162651 13.2 10.3 25.1 48.6 bean10-12 12.2 13.0 23.7 48.9 bean29-6 13.3 10.3 25.4 49.0 bean3-3 14.0 10.3 25.0 49.3 bean125-3 12.2 9.8 27.5 49.4 IT180321 10.6 11.4 27.9 49.9 IT212829 11.6 10.2 28.6 50.3 IT154444 11.5 11.3 27.7 50.5 IT209385 11.7 9.8 29.0 50.5 bean5-10 14.0 11.6 25.6 51.2 IT181569 11.3 11.9 28.8 52.0 IT189224 11.4 10.4 30.3 52.1 IT162163 12.8 11.4 28.7 52.9 IT180023 13.3 11.8 27.8 52.9 IT1622277 13.7 10.4 30.6 54.8 IT181509 13.6 13.3 31.9 58.8 IT186179 12.6 13.7 35.1 61.4 IT175884 13.4 12.6 35.7 61.7 IT194542 17.1 14.3 30.9 62.4 IT025061 25.1 30.4 25.4 80.9 Average 10.6 9.8 22.5 42.9 SD 2.1 2.2 4.2 6.9 Minimum value 1.1 1.0 2.9 22.4 Maximum value 25.1 30.4 35.7 80.9

< Example  3> soybean NIR  Create spectrum profile

1) Near-infrared spectrophotometer 6500 (FOSS NIRsystems, INc., Korea) performed a full diagnostic test (Diagnodtics) of its own to pass all the equipment check. After creating a new file, the spectra were set to produce spectra at wavelengths of 1100-2350 nm.

2) 0.7 g of the soybean powder (120 mesh or less) of Example 1 dried at room temperature for 3 days or more were loaded into a sample cell (18 mm diameter, 0.4 mm depth: IH-0348, FOSS NIRsystems, Inc., USA). . Spectral profiles were prepared at wavelength 1100-2350 nm for 1 minute per total 152 soybean samples. At this time, as an internal standard material (Ceramic; FOSS NIRsystems, INc., USA)

As a result, a spectral profile for 152 beans as shown in FIG. 1 was obtained.

< Example  4> Calibration curve for quantifying soybean vegetable sterol

The near-infrared spectrum of FIG. 1 was subjected to water treatment with a first or second differential method and a measurement wavelength gap of 10 nm. After performing statistical analysis using Modified Partial Least Squares (MPLS) again, an appropriate calibration equation showing a highly significant correlation between the wet quantitative value of Example 2 and the dry quantitative value of Example 3 was obtained. Derived. When the validation calibration equation is obtained, it is necessary to verify that the application of the calibration equation is reasonable. Therefore, the cross validation (CV) was used internally in the calibration set when creating the calibration equation.

As a result, as shown in FIG. 2, a calibration curve for each plant sterol was obtained, and as shown in Table 2, a multipled coefficient of multiple determination in calibration (R ² ) of each calibration curve was obtained. The stigmasterol and β-sitosterol were found to be difficult to use as a standard quantitative curve with 0.236 and 0.501 polycrystalline coefficients, and the multicrystal coefficient of the campestrol (Equation 1) calibration curve was 0.842 with high accuracy.

ingredient α-
Tocopherol β-
Tocopherol γ-
Tocopherol δ-
Tocopherol Campestrol Stigmasterol β-sitosterol all Coefficient of determination
(r ² ) 0.190 - 0.526 0.478 0.842 0.236 0.501 0.474

Campesterol Content (μg / g) = -159.7143 - 539.5369x _'1700 + 208.2029x _'1710 + 862.7268x _'1720 + 304.1911x _' 1730- 65.8495x _' 1740- 255.1 128x _'1750- 212.5636x _' 1760- 52.1982x _'1770 + 135.6333x _'1780 + 203.0269x _'1790 + 233.239x _'1800 + 104.6265x _'1810 + 75.9039x _' 1820- 5.9972x _'1830 + 56.3546x _'1840 + 43.7998x _'1850 + 140.0899x _'1860 + 689.9338x _'1870 + 791.2035x _'1880 + 801.7698x _'1890- 284.5929x _' 1900- 1425.0724x _'1910- 1286.3221x _' 1920- 732.9667x _'1930 + 61.4209x _'1940 + 424.8888x _'1950 + 568.9919x _'1960 + 601.3539x _'1970 + 406.6427x _'1980 + 344.6469x _'1990 + 278.9063x _'2000 + 148.3x _'2010 + 40.2344x _'2020 + 22.6729x _' 2030- 28.6875x _' 2040- 142.7464 x _' 2050- 305.8374x _' 2060- 237.7981x _' 2070- 112.3536x _' 2080- 50.1014x _'2090 + 15.0298x _'2100 .

1 is a diagram showing an NIR spectrum of a 152-point soybean sample.

2 is a diagram showing a calibration curve for soybean vegetable sterols:

Slope: slope, RSQ: multiple crystal coefficient, SEP: standard prediction error, BIAS: intercept, SEP (C): standard prediction error;

a: sigmasterol;

b: β-sitosterol; And,

c: camphorsterol.

Claims (3)

1) sorting the bean seeds according to the varieties into powder test tubes and inserting them into a near-infrared spectrometer, and irradiating near-infrared light to obtain an absorption spectrum in the region of 1700 to 2100 nm; 2) applying a first order differential method to the near infrared spectrum and obtaining a first derivative value with a gap of 10 nm for each wavelength measured; 3) dry quantification method for the content of the camphorsterol component of the soybean, comprising the step of determining the camphorsterol content in the soybean sample by substituting the first derivative value for each wavelength into a function of Equation 1 below: [Equation 1] Campesterol Content (μg / g) = -159.7143 - 539.5369x _'1700 + 208.2029x _'1710 + 862.7268x _'1720 + 304.1911x _' 1730- 65.8495x _' 1740- 255.1 128x _'1750- 212.5636x _' 1760- 52.1982x _'1770 + 135.6333x _'1780 + 203.0269x _'1790 + 233.239x _'1800 + 104.6265x _'1810 + 75.9039x _' 1820- 5.9972x _'1830 + 56.3546x _'1840 + 43.7998x _'1850 + 140.0899x _'1860 + 689.9338x _'1870 + 791.2035x _'1880 + 801.7698x _'1890- 284.5929x _' 1900- 1425.0724x _'1910- 1286.3221x _' 1920- 732.9667x _'1930 + 61.4209x _'1940 + 424.8888x _'1950 + 568.9919x _'1960 + 601.3539x _'1970 + 406.6427x _'1980 + 344.6469x _'1990 + 278.9063x _'2000 + 148.3x _'2010 + 40.2344x _'2020 + 22.6729x _' 2030- 28.6875x _' 2040- 142.7464 x _' 2050- 305.8374x _' 2060- 237.7981x _' 2070- 112.3536x _' 2080- 50.1014x _'2090 + 15.0298x _'2100 . The dry quantitative method of claim 1, wherein the multiple crystal coefficient R ² of the function of Equation 1 is 0.8423. The method of claim 1, wherein x _'1700 to x _{' 2100} is a first derivative value measured at intervals of 10 nm in a spectrum between 1700 and 2100 nm.

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