A method for detecting the quantitative fingerprint of free fatty acids in wool suet and application thereof

Abstract

The application discloses a kind of free fatty acid quantitative fingerprint detection methods of wool suet and application thereof, belong to analytical detection technical field, comprising the following steps: (1) take wool suet sample, after wool suet sample is melted, then dissolved with solvent, to prepare sample solution;Utilize reference substance to prepare reference substance solution;(2) the HPLC detection of sample solution and reference substance solution, record chromatogram;The chromatogram of reference substance solution is analyzed, and standard curve is drawn;(3) utilize the multiple chromatograms corresponding to different batches of wool suet obtained in steps (1) and (2), determine common peak, utilize standard curve to carry out quantification, realize the quantitative fingerprint detection of free fatty acid in wool suet.The method is simple, without derivatization and other operations, detection time is shorter, it is helpful to more objectively carry out quality detection, control and evaluation to wool suet.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a quantitative fingerprinting method for detecting free fatty acids in crude wool grease and its application. Background Technology

[0002] Lanolin is a multi-component mixture of lipid substances secreted by the sebaceous glands of sheep and adhering to the wool, recovered from wool washing wastewater. Crude lanolin contains a lot of water, is very dark in color, and is a blackish-brown or brownish viscous paste with an unpleasant odor, containing many impurities that affect the quality of lanolin.

[0003] Lanolin can be processed to obtain products including lanolin alcohol, lanolinic acid, and high-ester esters (as shown in Chinese patent documents CN115212142A and CN114426566A). High-ester esters are mixtures of lanolinic acids and can be used in industries such as leather processing. They differ from lanolin acid obtained through complete saponification and separation. High-ester esters are obtained by deacidifying lanolin with alkali, followed by pH adjustment of the resulting acid salts. The types and contents of fatty acids in lanolin significantly affect the composition and yield of high-ester esters; therefore, it is necessary to establish analytical methods for detecting fatty acids in different types of crude lanolin.

[0004] Lanolin's composition varies considerably depending on the wool's origin and season, and is quite complex. It consists of 95% esters formed from sterols, fatty alcohols, and triterpenoids with approximately equal amounts of fatty acids. The remainder comprises free fatty acids, fatty alcohols, and hydrocarbons. Currently, there are few methods for directly analyzing the ester components in lanolin. The primary method involves analyzing lanolin alcohols and lanonic acids obtained through hydrolysis (as described in Chinese patent document CN118130653A). However, there is currently no method for detecting free fatty acids in crude wool lint.

[0005] Traditional fatty acid detection methods require sample purification and derivatization, and detection using gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS). These methods have long pretreatment times and low specificity. Furthermore, the content of free fatty acids in crude wool lint is low, and most fatty acids do not have strong UV absorption, making detection with UV detectors insensitive.

[0006] Traditional Chinese medicine (TCM) fingerprinting can analyze the spectra or chromatograms of characteristic components in TCM preparations or raw materials using mathematical statistics, thus providing a better reflection of the overall quality of TCM. If TCM fingerprinting can be applied to the analysis of free fatty acids in crude wool lint, it is hoped that it can more comprehensively describe the types and contents of fatty acids in crude wool lint, enhance the understanding of the properties of different crude wool lint raw materials, and further promote the improvement of the control level in the processing of wool lint products. Summary of the Invention

[0007] This invention provides a quantitative fingerprinting method for detecting free fatty acids in crude wool grease. The method has a simple pretreatment process, does not require derivatization or other operations, and has a short detection time. It can fill the gap in existing methods for detecting free fatty acids in wool grease and helps to more objectively detect, control and evaluate the quality of crude wool grease.

[0008] The specific technical solution adopted is as follows:

[0009] A quantitative fingerprinting method for detecting free fatty acids in crude wool grease includes the following steps:

[0010] (1) Take the crude wool grease test sample, melt the crude wool grease test sample and then dissolve it in a solvent to prepare the test sample solution; prepare the reference solution using the reference standard;

[0011] (2) Perform HPLC detection on the test solution and reference solution prepared in step (1) and record the chromatograms; analyze the chromatogram of the reference solution and draw a standard curve;

[0012] The HPLC chromatographic conditions were as follows: Proshell 120EC C18 column; mobile phase A was an aqueous formic acid solution, and mobile phase B was a mixed solution of formic acid and acetonitrile, using gradient elution. The elution conditions were: 0-30 min, mobile phase A volume fraction 25%, mobile phase B volume fraction 75%; 30-60 min, mobile phase A volume fraction decreased from 25% to 21%, mobile phase B volume fraction increased from 75% to 79%; flow rate 0.75-0.80 ml / min; injection volume 8-10 μL.

[0013] (3) Using steps (1) and (2), multiple chromatograms corresponding to different batches of crude wool grease are obtained, common peaks are determined, and quantitative analysis is performed using a standard curve to realize the quantitative fingerprint detection of free fatty acids in crude wool grease.

[0014] In step (1), the wool grease is heated and melted to form a liquid state and then dissolved in a solvent. Preferably, the heating temperature is 40-50℃ and the heating time is 20-40min.

[0015] The reference standard includes palmitic acid, oleic acid, or stearic acid; the solvent for the test solution and the reference solution is preferably heptane, and the concentration of the test solution is preferably 50-55 mg / mL.

[0016] Furthermore, the test solution was filtered through a 0.22 μm filter before HPLC detection.

[0017] Furthermore, the parameters of the Proshell 120EC C18 column are: column length 100mm × inner diameter 4.6mm, particle size 2.7μm.

[0018] Preferably, mobile phase A is a 0.1% (volume fraction) aqueous solution of formic acid, and mobile phase B is a 0.1% (volume fraction) mixture of formic acid and acetonitrile.

[0019] Furthermore, the HPLC detection conditions also include: column temperature 35℃; flow rate 0.8 ml / min; injection volume 10 μL; under these parameters, the running time can be shortened, the separation is good, the peak area is large, and the response is good.

[0020] Furthermore, HPLC detection was performed using an electrospray detector at an atomization temperature of 35°C.

[0021] Preferably, since the electrospray detector CAD is a mass detector, it exhibits a linear law within a certain range. However, due to the large differences in the concentration of the test sample solution, the power function PFV is used to analyze the nonlinear relationship between the substance concentration and the CAD response signal through mathematical compensation. The PFV value is 1.1 for palmitic acid and 1.2 for oleic acid and stearic acid.

[0022] Specifically, palmitic acid, oleic acid and stearic acid were used as reference standards. In step (3), seven common peaks were identified.

[0023] The present invention also provides the application of the quantitative fingerprinting method for detecting free fatty acids in wool crude grease in the quality detection and / or control of wool crude grease raw materials.

[0024] The crude wool grease to be tested is prepared into a test solution, and HPLC analysis is performed on it. The chromatogram of the test solution is recorded. The similarity of the chromatogram of the test solution with the fingerprint spectrum composed of multiple chromatograms obtained in the above steps is compared. Quantification is performed using a standard curve. The quality detection and / or control of the crude wool grease raw material is carried out based on the similarity results and the quantitative results.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention establishes a quantitative fingerprint spectrum detection method for free fatty acids in crude wool grease. This method can fill the current gap in the understanding of the properties of different crude wool grease raw materials, thereby improving the control of the properties of grease raw materials and distinguishing crude wool grease that is more conducive to the production of high ester products.

[0027] (2) This invention draws on the concept of common peaks in the field of traditional Chinese medicine to introduce fingerprint spectrum in the detection of crude wool fat, which makes it easier to determine common peaks. Combined with chemical content determination and fingerprint spectrum, it can more comprehensively describe the types and contents of fatty acids in crude wool fat.

[0028] (3) The method of the present invention is convenient for raw material pretreatment, simple for elution solvent, short for detection time, and all peaks of the fingerprint spectrum reach baseline separation. It has high sensitivity and can detect many compounds with weak ultraviolet absorption. Attached Figure Description

[0029] Figure 1 High-resolution mass spectrometry results of the crude lint sample for wool in negative ion mode;

[0030] Figure 2 To establish fingerprint profiles of free fatty acids in crude wool grease from different batches;

[0031] Figure 3 The chromatograms are representative of the test solution and the mixed reference solution, where A is the test solution and B is the mixed reference solution. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically described in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the embodiments below can be purchased from conventional biochemical reagent companies.

[0033] Example 1

[0034] 1. Experimental Instruments and Materials

[0035] 1.1 Instruments

[0036] Electronic balance (YP20002, Shanghai Hengji Scientific Instruments Co., Ltd.); high-speed centrifuge (Minispin, Eppendorf, Germany); digital display constant temperature water bath (HH4, Shanghai Lichen Instrument Technology Co., Ltd.); high-performance liquid chromatograph (Vanquish, Thermo Fisher Scientific, USA), equipped with an electrospray detector (CAD); high-resolution liquid chromatography-mass spectrometry (ABSciex, X500B), with Peak View software (version 1.2) for data processing. The fingerprint similarity evaluation software was the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (National Pharmacopoeia Commission 2012 edition).

[0037] 1.2 Reagents

[0038] Acetonitrile (chromatographic grade), isopropanol (chromatographic grade), formic acid (chromatographic grade, ≥98%), n-heptane (chromatographic grade, ≥99% (GC)), and ultrapure water.

[0039] Palmitic acid reference standard (99%), oleic acid reference standard (≥99%, HPLC grade), stearic acid reference standard (>99%, GC grade). Different batches of wool grease all came from the company.

[0040] 2 Experimental conditions

[0041] 2.1 Chromatographic conditions

[0042] The Proshell 120EC C18 column was used, with a length of 100 mm and an inner diameter of 4.6 mm, and a particle size of 2.7 μm. Mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile. Gradient elution was employed under the following conditions: 0-30 min, mobile phase A was 25% (v / v), and mobile phase B was 75% (v / v); 30-60 min, the volume fraction of mobile phase A decreased from 25% to 21%, and the volume fraction of mobile phase B increased from 75% to 79%; after each injection, the column was equilibrated for 10 min with 0.1% formic acid-water:0.1% formic acid-acetonitrile = 25:75 (v:v); flow rate: 0.80 ml / min; column temperature: 35 °C; nebulization temperature: 35 °C; injection volume: 10 μL.

[0043] 2.2 Preparation of the reference standard mixed solution

[0044] Preparation of the reference solution: Accurately weigh palmitic acid, oleic acid, and stearic acid as reference standards, place them in a volumetric flask, and dilute to the mark with chromatographic-grade heptane. Shake well to prepare a mixed reference solution containing 49.97 μg / mL palmitic acid, 33.94 μg / mL oleic acid, and 45.43 μg / mL stearic acid. Inject the reference solution into the high-performance liquid chromatograph (HPLC) and record the chromatogram.

[0045] 2.3 Preparation of the test solution

[0046] Different types of crude wool grease were melted (40-50℃, 30 min) to form a liquid state. An appropriate amount was weighed into a 10 ml volumetric flask, and the volume was adjusted to the mark using chromatographic grade heptane as the solvent. The solution was shaken well, filtered through a 0.22 μm filter membrane, and the filtrate was loaded onto the sample. The concentration of the prepared test solution was approximately 50-55 mg / mL. The test solution was injected into the high-performance liquid chromatograph, and the chromatogram was recorded.

[0047] 2.4 Liquid Chromatography-High Resolution Mass Spectrometry Conditions

[0048] The liquid chromatography analysis conditions described in section 2.1 above are used as the chromatographic conditions for liquid chromatography-high resolution mass spectrometry analysis.

[0049] The mass spectrometry conditions were as follows: positive and negative ion scanning modes were used; scanning range: m / z 50-1000; nebulizer gas (GS1): 55 psi; nebulizer gas (GS2): 55 psi; curtain gas (CUR): 35 psi; ion source temperature (TEM): 600℃ (positive) 550℃ (negative); ion source voltage (IS): -4500V (negative) 5500V (positive); first-stage scan: declustering voltage (DP): ±80V; focusing voltage (CE): ±10V; second-stage scan: mass spectrometry data were acquired using TOF MS-TOF MS-IDA mode, declustering voltage (DP): ±80V; focusing voltage (CE): ±35±15V. Before sample injection, mass axis calibration was performed using a CDS pump to ensure that the mass axis error was less than 2ppm.

[0050] After determining the analytical method, the test sample was detected using high-resolution mass spectrometry. The results are shown in [Figure number missing]. Figure 1 Based on the accurate relative molecular mass obtained from high-resolution mass spectrometry and the fragmentation information from secondary mass spectrometry, the chemical composition of the seven common peaks was preliminarily inferred, and the results are shown in Table 1.

[0051] By comparing the chromatograms of the reference standard, peaks 2, 3, and 6 were identified as palmitic acid, oleic acid, and stearic acid, respectively, and these three components were determined to be the components for content determination.

[0052] Table 1. High-resolution mass spectrometry results of partial compounds in crude wool lint from batch 1.

[0053]

[0054] * Indicates components determined by comparison with a reference standard.

[0055] The chromatograms of the representative test solution and the mixed reference solution obtained by the above method are shown below. Figure 3 As shown in A and B in the diagram.

[0056] 3. Methodological Examination of Fingerprint Spectrum Research

[0057] Methodological validation of fingerprinting mainly includes testing injection precision, repeatability, and sample stability.

[0058] 3.1 Sample injection precision experiment

[0059] The same sample solution was injected six times consecutively. Using palmitic acid as the control peak, the ratios of retention time to peak area for each common peak and the reference peak were calculated. The relative retention times of each common peak are shown in Table 2, and the relative peak areas are shown in Table 3. The relative retention time RSD (relative standard deviation) of each common peak was between 0.06% and 0.18%, and the relative peak area RSD was between 1.49% and 3.24%, indicating good injection precision.

[0060] Table 2. Precision experimental results of relative retention time

[0061]

[0062] Table 3. Precision experimental results of relative peak area

[0063]

[0064] 3.2 Method repeatability experiment

[0065] Six parallel-prepared test solutions were injected and analyzed separately. Using palmitic acid as the control peak, the ratios of retention time to peak area for each common peak and the reference peak were calculated. The relative retention times of each common peak are shown in Table 4, and the relative peak areas are shown in Table 5. The relative retention time RSD (relative standard deviation) of each common peak was between 0.68% and 1.89%, and the relative peak area RSD was between 0.05% and 1.04%, indicating good method repeatability.

[0066] Table 4. Repeatability test results of relative retention time

[0067]

[0068] Table 5. Repeatability test results of relative peak area

[0069]

[0070] 3.3 Sample stability test

[0071] The same sample solution was injected and analyzed at 0, 3, 6, 9, 12, 18, and 24 hours. Using palmitic acid as the control peak, the ratios of retention time to peak area for each common peak and the reference peak were calculated. The relative retention times of each common peak are shown in Table 6, and the relative peak areas are shown in Table 7. The relative retention time RSD (relative standard deviation) of each common peak was between 0.07% and 1.19%, and the relative peak area RSD was between 1.39% and 3.25%, indicating good stability.

[0072] Table 6. Stability test results based on relative retention time

[0073]

[0074] Table 7. Precision experimental results of relative peak area

[0075]

[0076] 4. Fingerprint of free fatty acids in crude lanolin

[0077] Raw data from the chromatograms of crude lanolin from different batches were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using the chromatogram of sample S1 as the reference chromatogram, the median method was employed with a time window width of 0.1 min. After multi-point correction, full-spectrum peak matching was performed to establish overlay chromatograms and reference chromatograms. (See attached image.) Figure 2 .

[0078] 4.1 Calibration of common peaks

[0079] Seven common peaks were identified in the crude lint sample. After high-resolution mass spectrometry and comparison with reference standards, peaks 2, 3, and 6 were identified as palmitic acid, oleic acid, and stearic acid, respectively. Peak 2 (palmitic acid), which had a moderate retention time, relatively stable peak area, good separation from adjacent chromatographic peaks, and a stable baseline, was selected as the reference peak.

[0080] 4.2 Evaluation of the similarity between wool crude grease and wool

[0081] The chromatograms of 15 batches of crude wool grease samples were imported and their similarities were calculated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012)". The similarity results are shown in Table 8.

[0082] Table 8. Fingerprint Similarity

[0083]

[0084] Based on the company's production and processing experience, batch S0 is a raw material batch suitable for processing high esters, so it is used as a reference fingerprint spectrum. According to the similarity results, the similarity between S7 and S0 is >0.9, which is recorded as similar to this type of lanolin and suitable for processing this type of lanolin into high ester products.

[0085] 5. Content Determination Methodology

[0086] 5.1 Linearity and Range Examination

[0087] Mixed reference solutions of different concentrations were injected in 10 μL for analysis. A standard curve was constructed with the peak area of ​​each component as the ordinate and concentration as the abscissa, yielding the linear regression equation (standard curve) and analytical range. Since CAD is a mass-type detector, it exhibits a linear relationship within a certain range. However, due to significant differences in sample concentrations, the overall relationship is non-linear. A power function (PFV) was used to optimize the detection signal, mathematically compensating for the non-linear relationship between the analytical substance concentration and the CAD response signal. The regression equations and coefficients of determination for each component after different power function treatments are shown in Table 9, and the linearity results are shown in Table 10.

[0088] Table 9 shows the regression equations and coefficients of determination for each component after different power function treatments.

[0089]

[0090] Table 10 shows the regression equations, coefficients of determination, and ranges of linear analysis for each component.

[0091]

[0092] After R 2 For comparison, the PFV with the highest coefficient of determination was selected as the result of the power-law treatment. The PFV value of palmitic acid was 1.1, and the PFV value of oleic acid and stearic acid was 1.2.

[0093] 5.2 Sample injection precision, repeatability and stability experiments

[0094] Injection precision test: The same sample solution was injected six times consecutively, and the RSD value of the peak area of ​​each component was calculated. Repeatability test: Six parallel prepared sample solutions were injected and analyzed separately, and the RSD value of the content of each component was calculated. Sample stability test: The same sample solution was injected and analyzed at 0, 3, 6, 9, 12, 18, and 24 hours, and the RSD value of the peak area of ​​each component was calculated. The results of the injection precision, repeatability, and stability tests are shown in Table 11. The RSD values ​​of precision, repeatability, and stability were all less than 3%, which meets the requirements of the Chinese Pharmacopoeia, indicating that the method has good precision and repeatability, and the sample solution is stable within 24 hours.

[0095] Table 11 RSD values ​​of sample solution injection precision, method repeatability, and sample stability

[0096]

[0097] 5.3 Spiking and Recovery Experiment

[0098] Six aliquots of the test solution with known content were taken, and the ratio of the amount of reference standard added to the amount of the analyte in the test solution was controlled to be approximately 1:1 for analysis. The spiking recovery results are shown in Table 12. The spiking recoveries of the three components were all between 96% and 103%, and the RSDs were all less than 3%, indicating that the method is accurate and reliable and can be used for the detection of oleic acid, palmitic acid, and stearic acid content in different batches of wool crude grease.

[0099] Table 12 Results of the recovery experiment

[0100]

[0101] 5.4 Sample Content Determination

[0102] The results of quantitative component content determination of 15 different batches of test sample solutions are shown in Table 13. The fatty acid content of crude lanolin varied significantly among different batches, with palmitic acid content ranging from 0.352 to 14.61 mg / g, oleic acid content from 0.053 to 8.428 mg / g, and stearic acid content from 0.195 to 9.189 mg / g. Batches S0 and S7 had significantly higher contents of palmitic acid, oleic acid, and stearic acid, making them more suitable for processing high ester products.

[0103] Table 13 Results of Quantitative Component Content Determination in 15 Batches of Test Sample Solutions

[0104]

[0105] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for quantitative fingerprinting of free fatty acids in crude wool grease, characterized in that, Includes the following steps: (1) Take the crude wool grease test sample, melt the crude wool grease test sample and then dissolve it with a solvent to prepare the test sample solution; prepare the reference solution using the reference standard; (2) Perform HPLC detection on the test solution and reference solution prepared in step (1) and record the chromatograms; analyze the chromatogram of the reference solution and draw a standard curve; The HPLC chromatographic conditions were as follows: Proshell 120 EC C18 column; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-acetonitrile mixture, with gradient elution. The elution conditions were: 0-30 min, mobile phase A volume fraction 25%, mobile phase B volume fraction 75%; 30-60 min, mobile phase A volume fraction decreased from 25% to 21%, and mobile phase B volume fraction increased from 75% to 79%; flow rate 0.75-0.80 ml / min; injection volume 8-10 μL. (3) Using steps (1) and (2), multiple chromatograms corresponding to different batches of crude wool grease are obtained, common peaks are determined, and quantitative analysis is performed using a standard curve to realize the quantitative fingerprint detection of free fatty acids in crude wool grease. The reference standard includes palmitic acid, oleic acid, or stearic acid; the solvent for both the test solution and the reference solution is heptane; HPLC detection was performed using an electrospray detector at an atomization temperature of 35°C. The power function PFV was used to analyze the nonlinear relationship between the substance concentration and the response signal of the electrospray detector through mathematical compensation. The PFV value was 1.1 for palmitic acid and 1.2 for oleic acid and stearic acid.

2. The method for quantitative fingerprinting detection of free fatty acids in crude wool grease according to claim 1, characterized in that, The test solution was filtered through a membrane before HPLC analysis.

3. The method for quantitative fingerprinting of free fatty acids in crude wool grease according to claim 1, characterized in that, The parameters of the Proshell 120 EC C18 column are: column length 100 mm × inner diameter 4.6 mm, particle size 2.7 μm.

4. The method for quantitative fingerprinting of free fatty acids in crude wool grease according to claim 1, characterized in that, The HPLC detection conditions also included: column temperature 35℃; flow rate 0.8 ml / min; injection volume 10 μL.

5. The application of the quantitative fingerprinting method for detecting free fatty acids in crude wool grease according to any one of claims 1-4 in the quality detection and / or control of crude wool grease raw materials.

6. The application of the quantitative fingerprinting method for detecting free fatty acids in crude wool grease according to claim 5 in the quality detection and / or control of crude wool grease raw materials, characterized in that, The crude wool grease to be tested is prepared into a test solution, and HPLC is used to detect it. The chromatogram of the test solution is recorded. The similarity of the chromatogram of the test solution with a fingerprint spectrum composed of multiple chromatograms is compared. Quantification is performed using a standard curve. The quality detection and / or control of the crude wool grease raw material is carried out based on the similarity results and the quantitative results.

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