Quantitative fingerprint spectrum detection method for free fatty acid in wool crude fat and application of quantitative fingerprint spectrum detection method

Abstract

The invention discloses a quantitative fingerprint spectrum detection method for free fatty acid in wool crude fat and application thereof, and belongs to the technical field of analysis and detection.The quantitative fingerprint spectrum detection method comprises the following steps that 1, a wool crude fat test sample is taken, the wool crude fat test sample is melted and then dissolved with a solvent, and a test sample solution is prepared; preparing a reference substance solution by using a reference substance; (2) performing HPLC (High Performance Liquid Chromatography) detection on the test solution and the reference solution, and recording chromatograms; analyzing the chromatogram of the reference substance solution, and drawing to obtain a standard curve; and (3) determining a common peak by utilizing a plurality of chromatograms corresponding to different batches of wool crude fat obtained in the steps (1) and (2), and quantifying by utilizing a standard curve to realize quantitative fingerprint detection of the free fatty acid in the wool crude fat. According to the method, the pretreatment method is simple, operation such as derivatization is not needed, the detection time is short, and quality detection, control and evaluation can be more objectively carried out on the wool crude fat.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a quantitative fingerprint spectrum detection method for free fatty acids in wool crude fat and an application thereof. Background Art

[0002] Lanolin is a multi-component mixture of lipid substances secreted by the sebaceous glands of sheep and adhering to the wool. It is recovered from the waste liquid of wool washing. Crude lanolin contains more water and is very dark in color. It is a dark brown or brown viscous paste with a bad smell and contains more impurities that affect the quality of wool grease.

[0003] Lanolin can be processed to obtain products including lanolin alcohol, lanolin acid and high acid esters (such as Chinese patent documents with publication numbers CN115212142A and CN114426566A). High acid esters are a mixture of lanolin acids and can be used in industries such as leather processing. Different from the lanolin acid obtained by completely saponifying and separating lanolin, lanolin is deacidified by alkali refining, and the resulting acid salt is treated by adjusting the pH value to obtain high acid esters. The type and content of fatty acids in lanolin have a great influence on the composition and yield of high acid esters, so it is necessary to establish an analytical method to detect fatty acids in different types of crude wool fat.

[0004] Lanolin has different components due to the different wool production areas and seasons, and its composition is relatively complex. 95% is esters formed by sterols, fatty alcohols and triterpenoid alcohols with approximately equal amounts of fatty acids, and the rest is free fatty acids, fatty alcohols and hydrocarbon substances. Currently, there are few methods for directly analyzing the ester components in lanolin. The main method is to analyze the lanolin alcohol and lanolin acid obtained by hydrolyzing lanolin (such as the Chinese patent document with publication number CN118130653A), so as to achieve the purpose of analyzing lanolin. However, there is currently no detection method for free fatty acids in crude wool fat.

[0005] Traditional fatty acid detection methods require sample purification and derivatization, and detection using gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), etc., which requires long pre-treatment time and low specificity. In addition, the content of free fatty acids in wool crude fat is low, and most fatty acids do not have strong ultraviolet absorption, so detection using an ultraviolet detector is not sensitive.

[0006] Traditional Chinese medicine fingerprints can analyze and process the spectra or chromatograms of characteristic components in traditional Chinese medicine preparations or traditional Chinese medicines in combination with mathematical statistics knowledge, thus better reflecting the overall quality of traditional Chinese medicine. If traditional Chinese medicine fingerprints can be introduced into the analysis of free fatty acids in wool crude fat, it is hoped that the types and contents of fatty acids in wool crude fat can be more comprehensively described, the understanding of the properties of different wool crude fat raw materials can be strengthened, and the control level of wool fat product processing can be further promoted. Summary of the invention

[0007] The present invention provides a quantitative fingerprint detection method for free fatty acids in wool crude fat. The method has a simple pretreatment process, does not require operations such as derivatization, and has a short detection time. The method can fill the gap in the existing methods for detecting free fatty acids in wool fat, and is helpful for more objective quality detection, control and evaluation of wool crude fat.

[0008] The specific technical solutions adopted are as follows:

[0009] A quantitative fingerprint detection method for free fatty acids in wool crude fat comprises the following steps:

[0010] (1) taking a wool crude grease test sample, melting the wool crude grease test sample and then dissolving it with a solvent to prepare a test sample solution; and using a reference sample to prepare a reference sample solution;

[0011] (2) performing HPLC detection on the test solution and the reference solution prepared in step (1), and recording the chromatogram; analyzing the chromatogram of the reference solution, and drawing a standard curve;

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

[0013] (3) Using steps (1) and (2) to obtain multiple chromatograms corresponding to different batches of wool crude fat, determine the common peaks, and use the standard curve for quantification to achieve quantitative fingerprint detection of free fatty acids in wool crude fat.

[0014] In step (1), the wool crude fat is heated and melted to form a liquid and then dissolved with a solvent. Preferably, the heating temperature is 40-50° C. and the heating time is 20-40 min.

[0015] The reference substance includes palmitic acid, oleic acid or stearic acid; the solvent of 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 membrane before HPLC detection.

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

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

[0019] Furthermore, the conditions of HPLC detection also include: column temperature 35°C; volume flow rate 0.8ml / min; injection volume 10μL; under the corresponding 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 with an atomization temperature of 35°C.

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

[0022] Specifically, using palmitic acid, oleic acid, and stearic acid as reference substances, in step (3), seven common peaks were determined.

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

[0024] The wool crude fat to be tested is made into a test solution, which is subjected to HPLC detection, and the chromatogram of the test product is recorded. The chromatogram of the test product is compared with the fingerprint spectrum composed of the multiple chromatograms obtained in the above steps for similarity, and quantification is performed using a standard curve. The quality of the wool crude fat raw material is detected and / or controlled based on the similarity result and the quantitative result.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

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

[0027] (2) The present invention draws on the concept of common peaks in the field of traditional Chinese medicine to introduce fingerprint spectrum into the detection of wool crude fat, which is convenient for determining common peaks, and combines chemical content determination and fingerprint spectrum to more comprehensively describe the types and contents of fatty acids in wool crude fat.

[0028] (3) The method of the present invention is convenient for raw material pretreatment, has a simple elution solvent, a short detection time, and each peak of the fingerprint spectrum is baseline separated. It has high sensitivity and can detect many compounds with weak ultraviolet absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the high-resolution mass spectrometry result of wool crude fat sample in negative ion mode;

[0030] Figure 2 To establish the fingerprint of free fatty acids in different batches of wool crude fat;

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

[0032] The present invention will be further described below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods of the following examples that do not specify specific conditions are usually performed under normal conditions or under conditions recommended by the manufacturer. The content that is not described in detail in this specification sheet belongs to the prior art known to professionals in the field. The experimental materials used in the following examples, if not otherwise specified, can be purchased by conventional biochemical reagent companies.

[0033] Example 1

[0034] 1 Experimental instruments and materials

[0035] 1.1 Instruments

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

[0037] 1.2 Reagents

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

[0039] Palmitic acid reference substance (99%), oleic acid reference substance (≥99%, HPLC grade), stearic acid reference substance (>99%, GC grade). Different batches of wool crude grease all come from enterprises.

[0040] 2 Experimental conditions

[0041] 2.1 Chromatographic conditions

[0042] Proshell 120EC C18 chromatographic column, column length 100mm×inner diameter 4.6mm, particle size 2.7μm; mobile phase A is 0.1% formic acid-water, mobile phase B is 0.1% formic acid-acetonitrile; gradient elution is adopted, and the elution conditions are: 0-30min, the volume fraction of mobile phase A is 25%, and the volume fraction of mobile phase B is 75%; 30-60min, the volume fraction of mobile phase A decreases from 25% to 21%, and the volume fraction of mobile phase B increases from 75 to 79%; after each needle, run with 0.1% formic acid-water: 0.1% formic acid-acetonitrile = 25:75 (v:v) for 10 minutes to balance the chromatographic column; volume flow rate: 0.80ml / min; column temperature: 35℃; nebulization temperature: 35℃; injection volume: 10μL.

[0043] 2.2 Preparation of reference substance mixed solution

[0044] Preparation of reference solution: Weigh accurately palmitic acid, oleic acid and stearic acid as reference substances, place in a volumetric flask, dilute to scale with chromatography grade heptane, shake well, and a reference solution containing 49.97 μg / mL palmitic acid, 33.94 μg / mL oleic acid and 45.43 μg / mL stearic acid can be prepared. Pipette the reference solution, inject into the HPLC, and record the chromatogram.

[0045] 2.3 Preparation of test solution

[0046] After melting different types of wool crude grease (40-50℃, 30min) to form a liquid, weigh an appropriate amount into a 10ml volumetric flask, use chromatography grade heptane as solvent to make up to the mark, shake well, filter through a 0.22μm filter membrane to take the filtrate and sample. The concentration of the prepared test solution is about 50-55mg / mL. Pipette the test solution, inject it into the high performance liquid chromatograph, and record the chromatogram.

[0047] 2.4 Liquid chromatography-high resolution mass spectrometry conditions

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

[0049] The mass spectrometry conditions are as follows: positive and negative ion scanning modes; scanning range: m / z 50-1000; nebulizer gas (GS1): 55psi; nebulizer gas (GS2): 55psi; curtain gas (CUR): 35psi; ion source temperature (TEM): 600℃ (positive) 550℃ (negative); ion source voltage (IS): -4500V (negative) 5500V (positive); primary scan: declustering voltage (DP): ±80V; focusing voltage (CE): ±10V; secondary scan: use TOF MS-TOF MSMS-IDA mode to collect mass spectrometry data, declustering voltage (DP): ±80V; focusing voltage (CE): ±35±15V. Before injection, use CDS pump to perform mass axis correction to make the mass axis error less than 2ppm.

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

[0051] Combined with the comparison of the chromatograms of the reference substances, peaks 2, 3 and 6 were identified as palmitic acid, oleic acid and stearic acid, respectively, and these three components were determined as the components for content determination.

[0052] Table 1 High resolution mass spectrometry results of some compounds in wool crude fat of batch 1

[0053]

[0054] *Indicates ingredients confirmed by comparison with reference substances

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

[0056] 3. Investigation of fingerprint research methodology

[0057] The methodological validation of fingerprint analysis mainly includes the investigation of injection precision, repeatability and sample stability.

[0058] 3.1 Injection precision experiment

[0059] Take the same sample solution and inject it 6 times in succession. Take palmitic acid as the reference peak, calculate the ratio of retention time and peak area of ​​each common peak to the reference peak, and obtain the relative retention time of each common peak in Table 2, and the relative peak area in Table 3. The relative retention time RSD (relative standard deviation) of each common peak is between 0.06% and 0.18%, and the relative peak area RSD is between 1.49% and 3.24%, indicating that the injection precision is good.

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

[0061]

[0062] Table 3 Precision test results of relative peak area

[0063]

[0064] 3.2 Method repeatability experiment

[0065] Six parallel prepared test solutions were taken and analyzed separately. With palmitic acid as the reference peak, the ratio of retention time to peak area of ​​each common peak to the reference peak was calculated, and the relative retention time of each common peak was shown in Table 4, and the relative peak area was 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 that the method had good repeatability.

[0066] Table 4 Relative retention time repeatability experimental results

[0067]

[0068] Table 5 Reproducibility test results of relative peak area

[0069]

[0070] 3.3 Sample stability test

[0071] Take the same sample solution and inject and analyze at 0, 3, 6, 9, 12, 18, and 24 hours. Take palmitic acid as the reference peak, calculate the ratio of the retention time and peak area of ​​each common peak to the reference peak, and obtain the relative retention time of each common peak in Table 6 and the relative peak area in Table 7. The relative retention time RSD (relative standard deviation) of each common peak is between 0.07% and 1.19%, and the relative peak area RSD is between 1.39% and 3.25%, indicating good stability.

[0072] Table 6 Stability test results relative to retention time

[0073]

[0074] Table 7 Precision test results of relative peak area

[0075]

[0076] 4 Free fatty acid fingerprint of wool crude fat

[0077] The original data of wool crude fat chromatograms from different batches were imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)”. The chromatogram of sample S1 was used as the reference spectrum. The median method was used with a time window width of 0.1 min. After multi-point correction, full spectrum peak matching was performed to establish superimposed spectra and reference spectra. Figure 2 .

[0078] 4.1 Common peak calibration

[0079] There are 7 common peaks in the wool crude fat sample. After high-resolution mass spectrometry and comparison with reference substances, peaks 2, 3 and 6 were identified as palmitic acid, oleic acid and stearic acid, respectively. Peak 2 (palmitic acid) with moderate retention time, relatively stable peak area, good separation from adjacent chromatographic peaks and stable baseline was selected as the reference peak.

[0080] 4.2 Evaluation of wool crude fat similarity

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

[0082] Table 8 Fingerprint similarity

[0083]

[0084] According to the production and processing experience of the enterprise, batch S0 is a raw material batch suitable for processing high acid esters, so it is used as the control fingerprint. According to the similarity results, the similarity between S7 and S0 is greater than 0.9, which is recorded as similar to this type of lanolin and suitable for processing this type of lanolin into high acid ester products.

[0085] 5. Content Assay Methodology

[0086] 5.1 Linearity and range investigation

[0087] Take mixed reference solutions of different concentrations and inject 10 μL for analysis. Use the peak area of ​​each component as the ordinate and the concentration as the abscissa to make a standard curve, and obtain the linear regression equation (standard curve) and the analysis range. Since CAD is a mass detector, it is linear within a certain range, but due to the large difference in sample concentration, the overall relationship is nonlinear. The power function PFV is used to optimize the detection signal and mathematically compensate for the nonlinear relationship between the concentration of the analytical substance and the CAD response signal. The regression equations and determination coefficients of each component after different power function processing are shown in Table 9, and the linear results are shown in Table 10.

[0088] Table 9 Regression equations and determination coefficients of each component after different power function processing

[0089]

[0090] Table 10 Regression equation, determination coefficient and linear analysis range of each component

[0091]

[0092] Through R 2 The PFV with the highest determination coefficient was selected as the power treatment result. 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 Injection precision, repeatability and stability experiments

[0094] Injection precision experiment: Take the same sample solution, inject it 6 times in a row, and calculate the RSD value of the peak area of ​​each component. Repeatability experiment: Take six parallel prepared sample solutions, inject and analyze them separately, and calculate the RSD value of the content of each component. Sample stability experiment: Take the same sample solution, inject and analyze it at 0, 3, 6, 9, 12, 18, and 24 hours, and calculate the RSD value of the peak area of ​​each component. The results of injection precision, repeatability, and stability tests are shown in Table 11, among which the precision, repeatability, and stability RSD are all less than 3%, which meets the requirements of the Chinese Pharmacopoeia, indicating that this method has good precision and repeatability, and the sample solution is stable within 24 hours.

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

[0096]

[0097] 5.3 Sample recovery experiment

[0098] Take 6 portions of the test solution with known content, control the ratio of the reference substance to the amount of the component to be tested in the test solution to be about 1:1, and analyze. The sample recovery results are shown in Table 12. The sample recovery rates of the three components are all between 96% and 103%, and the RSDs are all less than 3%, indicating that this method is accurate and reliable and can be used to detect the content of oleic acid, palmitic acid and stearic acid in different batches of wool crude fat.

[0099] Table 12 Sample recovery test results

[0100]

[0101] 5.4 Sample content determination

[0102] The quantitative component content determination results of 15 different batches of test solution are shown in Table 13. The fatty acid content of wool crude fat varies greatly from batch to batch, with palmitic acid content ranging from 0.352 to 14.61 mg / g, oleic acid content ranging from 0.053 to 8.428 mg / g, and stearic acid content ranging from 0.195 to 9.189 mg / g. The palmitic acid, oleic acid and stearic acid contents of batches S0 and S7 are significantly higher, and are more suitable for processing high acid ester products.

[0103] Table 13 Results of quantitative component content determination of 15 batches of test solution

[0104]

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

Claims

1. A quantitative fingerprint detection method for free fatty acids in wool crude fat, characterized in that: The following steps are involved: (1) taking a wool crude grease test sample, melting the wool crude grease test sample and then dissolving it with a solvent to prepare a test sample solution; and using a reference sample to prepare a reference sample solution; (2) performing HPLC detection on the test solution and the reference solution prepared in step (1), and recording the chromatogram; analyzing the chromatogram of the reference solution, and drawing a standard curve; The chromatographic conditions of the HPLC detection are: Proshell 120EC C18 chromatographic column; mobile phase A is a formic acid aqueous solution, mobile phase B is a mixed solution of formic acid and acetonitrile, gradient elution is adopted, and the elution conditions are: 0-30min, the volume fraction of mobile phase A is 25%, and the volume fraction of mobile phase B is 75%; 30-60min, the volume fraction of mobile phase A decreases from 25% to 21%, and the volume fraction of mobile phase B increases from 75 to 79%; Volume flow rate 0.75-0.80ml / min; injection volume 8-10μL; (3) Using steps (1) and (2) to obtain multiple chromatograms corresponding to different batches of wool crude fat, determine the common peaks, and use the standard curve for quantification to achieve quantitative fingerprint detection of free fatty acids in wool crude fat.

2. The quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 1, characterized in that: The reference substance includes palmitic acid, oleic acid or stearic acid; the solvent of the test solution and the reference solution is heptane.

3. The quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 1, characterized in that: The test solution was filtered through a membrane and then tested by HPLC.

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

5. The quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 1, characterized in that: Mobile phase A was 0.1% formic acid in water, and mobile phase B was a 0.1% formic acid-acetonitrile mixture.

6. The quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 1, characterized in that: The conditions of HPLC detection also include: column temperature 35°C; volume flow rate 0.8 ml / min; injection volume 10 μL.

7. The quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 1, characterized in that: HPLC detection was performed using an electrospray detector with an atomization temperature of 35°C.

8. Application of the quantitative fingerprint detection method for free fatty acids in wool crude fat according to any one of claims 1 to 7 in the quality detection and / or control of wool crude fat raw materials.

9. Application of the quantitative fingerprint detection method for free fatty acids in wool crude fat according to claim 8 in quality detection and / or control of wool crude fat raw materials, characterized in that: The wool crude fat to be tested is made into a test solution, which is subjected to HPLC detection, and the chromatogram of the test product is recorded. The chromatogram of the test product is compared with a fingerprint spectrum composed of multiple chromatograms for similarity, and quantification is performed using a standard curve. The quality of the wool crude fat raw material is detected and / or controlled based on the similarity results and the quantitative results.

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