A method for accurate quantitative detection of cholesterol and its synthetic intermediates based on triple quadrupole mass spectrometry

Abstract

The present invention discloses a method for accurately and quantitatively detecting cholesterol and its synthetic intermediates based on a triple quadrupole mass spectrometer, and the present invention mainly includes the following three parts: 1) Establishment of an MRM method for accurately and quantitatively detecting cholesterol and its synthetic intermediates based on LC-MS; 2) Quantitative detection and analysis of cholesterol and its synthetic intermediates in various biological samples; 3) Detection of cholesterol and its synthetic intermediates in biological samples after stable isotope labeling. The present invention provides a new method with high sensitivity and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolomics, and in particular to a method for accurately and quantitatively detecting cholesterol and its synthetic intermediates based on a triple quadrupole mass spectrometer. Background Art

[0002] Cholesterol synthesis is essential for mammalian cells, and any abnormalities in the synthesis process often lead to the accumulation of sterol intermediates, which can cause severe malformations in humans. These diseases include the accumulation of lanosterol and 24,25-dihydrolanosterol, which leads to Antley-Bixler syndrome characterized by skeletal and genital abnormalities; the accumulation of 14-norlanosterol and dihydro-14-norlanosterol, which leads to microcephaly and ichthyosis; and the accumulation of 7-Dehydro cholesterol, which leads to Smith–Lemli–Opitz syndrome with facial and neurological abnormalities. Therefore, quantitative detection of intermediate accumulation caused by internal or external genetic alterations can provide key insights into the mechanisms of action of genes and the resulting human diseases.

[0003] The multiple reaction monitoring (MRM) mode based on triple quadrupole mass spectrometry (QQQ) combined with LC is a classic method of targeted analysis strategy. It has high sensitivity, stability and excellent quantitative analysis performance. It is currently the "gold standard" for absolute quantification of low-concentration sterols in biological samples. However, cholesterol and intermediates are highly hydrophobic and non-volatile compounds, and the ionization effect using the conventional ESI source of mass spectrometry is extremely poor. Traditionally, the analysis of cholesterol and intermediates based on LC-MS / MS is usually derivatized with chemical reagents to improve ionization efficiency and sensitivity. However, most derivatization methods have disadvantages such as reagent interference, time-consuming derivatization process and low reproducibility of derivatization yield. Therefore, establishing a simple analytical method to improve the ionization efficiency of cholesterol and intermediates in mass spectrometry is an urgent problem to be solved. The APCI source is considered to be an ionization technology complementary to the ESI source, which is mainly suitable for the ionization of compounds with lower polarity. Studies have shown that the introduction of the APCI source can avoid the need for derivatization of sterol samples while still providing sufficient sensitivity, and has great application potential in the detection of cholesterol and intermediates. In addition to the problem that sterols are extremely difficult to ionize, the isomers of sterols are also the main bottleneck of mass spectrometry detection. Statistical analysis shows that in the Lipid Map Structure Database (LMSD), up to 86% of sterols have isomers. These sterols with the same molecular weight and similar structure are challenging or even impossible to distinguish using mass spectrometry alone, especially for compounds that are prone to large-scale fragmentation. For example, cholesterol and 7-enecholine are isomers of each other, and the high abundance of cholesterol can easily cover the chromatographic peak of 7-enecholine, making it difficult to distinguish between the two when testing biological samples. Therefore, good liquid phase conditions for separating sterols with very similar retention capabilities are also a key factor in identification.

[0004] In addition, considering that the accumulation of metabolites may be due to increased production or decreased consumption, and does not necessarily reflect an increase in the activity of metabolic pathways, stable isotope relabeling can be used to track and quantitatively measure metabolic flux, thereby gaining a more comprehensive understanding of the metabolic activity of organisms. Metabolic flux refers to the flow of substances through specific metabolic reactions in the metabolic pathways of organisms. The measurement of metabolic flux is usually measured in terms of the amount of substances passing through the pathway per unit time.

[0005] Therefore, it is necessary to establish a sterol quantification method with high sensitivity, good stability and simple operation to fill the gap in current sterol detection technology, and also provide very practical significance for studying diseases related to the cholesterol synthesis pathway. Summary of the invention

[0006] The purpose of the present invention is to provide a method for accurately quantitatively detecting cholesterol and its synthetic intermediates based on a triple quadrupole mass spectrometer. Not only an MRM quantitative method for detecting cholesterol and its synthetic intermediates is established, but also various biological samples and stable isotope-labeled cholesterol and its synthetic intermediates are accurately quantitatively analyzed, providing a research method and idea for the treatment of cholesterol metabolism-related diseases, with high sensitivity, good stability and simple operation.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The object of the present invention is to provide a method for accurately and quantitatively detecting cholesterol and its synthetic intermediates based on a triple quadrupole mass spectrometer, the method comprising:

[0009] Extracting cholesterol or its synthetic intermediates from the biological sample to be tested or labeling the biological sample to be tested with an isotope before the extraction;

[0010] The extracted cholesterol and its synthetic intermediates are subjected to LC-MS / MS detection to obtain a spectrum; wherein the liquid chromatography conditions include: gradient elution, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is deionized water, and mobile phase B is pure methanol;

[0011] Obtain standard curves for cholesterol and intermediates;

[0012] The concentration of cholesterol or its synthetic intermediate in the biological sample to be tested is obtained by calculation based on the spectrum and the standard curve.

[0013] In the above technical solution, the cholesterol or its synthetic intermediates in the biological sample to be tested can be extracted and then subjected to LC-MS / MS detection, or the cholesterol or its synthetic intermediates in the biological sample to be tested can be extracted and then subjected to LC-MS / MS detection by first labeling the biological sample to be tested with an isotope. When the change range is small, labeling with an isotope first will make it clearer.

[0014] The present invention includes but is not limited to using [2- 13 After labeling biological samples with stable isotopes such as C]mevalonolactone, cholesterol and its synthetic intermediates are efficiently extracted, and LC-MS / MS detection is performed to accurately quantify cholesterol and its synthetic intermediates. The liquid chromatography settings, including information such as chromatographic columns, mobile phases, and liquid phase gradients, are used to accurately quantify cholesterol and its synthetic intermediates. The MRM mass spectrometry method settings, including mass spectrometer operating parameters, Q1, Q3, DP, CE, CXP, etc., are used to accurately quantify cholesterol and its synthetic intermediates. Thus, a sterol quantification method with high sensitivity, good stability, and simple operation is established.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] 1. The present invention focuses on the precise quantitative analysis of cholesterol and its synthetic intermediates based on LC-MS. Not only has the MRM quantitative method for the detection of cholesterol and its synthetic intermediates been established, but also precise quantitative analysis of various biological samples and stable isotope-labeled cholesterol and its synthetic intermediates has been performed, providing research methods and ideas for the treatment of cholesterol metabolism-related diseases.

[0017] 2. One aspect of the present invention is to develop a method for detecting cholesterol and its synthetic intermediates with good linear range and sensitive detection limit by independently establishing and optimizing the detection conditions of various cholesterol and its intermediates, such as chromatographic columns, liquid phase gradients and mass spectrometry parameters. Among the 10 cholesterol and intermediates, the detection limit and quantification limit of dihydro-14-norlanosterol are the lowest, which are 0.29ng / mL and 0.95ng / mL, respectively, while the detection limit and quantification limit of 7-enecholanol are the highest, which are 5.14ng / mL and 17.12ng / mL, respectively, indicating that the method has high sensitivity, and the stability range of the 10 cholesterol and intermediate standard solutions is 1.27% to 4.39%, which is less than 5%, indicating that the analyte has good stability in the solution. And the operation is simple.

[0018] 3. Another aspect of the present invention is to establish a method for extracting cholesterol and its intermediates from various biological samples, and to perform LC-MS analysis to detect cholesterol and its intermediates in biological samples, and to study the contents of various types of cholesterol and their synthetic intermediates in the liver of wild-type Hela cells and wild-type C57BL / 6J mice, providing data and method references for analyzing diseases with abnormal cholesterol synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 Standard curve and correlation coefficient of cholesterol and intermediates. The X-axis is concentration (ng / mL), the Y-axis is peak area ratio (cholesterol intermediate / IS), and IS is cholesterol-d7. The black dots represent the standard sample points, and the red line represents the fitting line. The equation and R2 value were obtained by linear regression fitting.

[0021] Figure 2 [2- 13 Schematic diagram of the synthesis pathway of C]mevalonolactone-labeled cholesterol. Carbon No. 2 is 13 C-labeled mevalonolactone is converted to mevalonate and incorporated into the cholesterol biosynthesis pathway to ultimately obtain labeled [1,7,5,22,26- 13 C] cholesterol. Cholesterol and its intermediate molecules are 13 C-labeled carbons are shown in red.

[0022] Figure 3 M represents all carbon atoms. 12 C cholesterol molecule, M+1 means only one carbon atom is 13 C-substituted cholesterol molecules. MRM method for cholesterol M+0 to M+5 isotopes was established: (A) 10 mM [2- 13 C] mevalonolactone was treated in HeLa cells for 5 hours to produce [1,7,5,22,26- 13 C] Cholesterol. (B) MS spectrum of the labeled sample obtained by the first quadrupole scan, the inset shows the isotopic peak distribution of cholesterol. (C) Parent ion and product ion information of cholesterol M+0 to M+5 isotopes.

[0023] Figure 4 [2- 13C] mevalonolactone labeled cholesterol M+0 to M+5 isotope peaks and percentages. 12 UHPLC-LC-MS / MS ion chromatogram peaks (A) and distribution percentages (B) of cholesterol M+0 to M+5 isotopes in Hela cells treated with [2- 13 UHPLC-LC-MS / MS ion chromatogram peaks (C) and distribution percentages (D) of cholesterol M+0 to M+5 isotopes after 5 h of mevalonolactone treatment in Hela cells. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0027] The present application will be described in detail below with reference to embodiments and experimental data.

[0028] Example 1 Establishment of LC-MS method for accurate quantification of cholesterol and its intermediates

[0029] In order to establish an LC-MS method that can accurately quantify cholesterol and its synthetic intermediates, we tested and optimized the liquid chromatography column, mobile phase, liquid phase gradient, mass spectrometry parameters and MRM information of cholesterol and its intermediates, and finally obtained the LC-MS method that can identify and accurately quantify cholesterol and its synthetic intermediates as follows.

[0030] 1. Chromatographic column

[0031] Waters Acquity UPLC BEH C18 Column(2.1mm×100mm,1.7μm,Waters)

[0032] 2. Mobile phase

[0033] Mobile phase A: deionized water

[0034] Mobile phase B: pure methanol

[0035] 3. Liquid phase gradient

[0036] Table 1

[0037]

[0038]

[0039] 4. Mass spectrometry parameters

[0040] The APCI source related parameters were set as follows: Curtain Gas: 35 psi;

[0041] Nebulizer Current: 3.0mA;

[0042] Temperature: 400℃;

[0043] Ion Source Gas 1: 50 psi.

[0044] 5. Cholesterol intermediate MRM information

[0045] Table 2

[0046]

[0047] Construct a standard curve:

[0048] A standard curve was drawn with a series of dilution concentrations of 10 kinds of cholesterol and intermediates (10000 ng / mL, 5000 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, 0 ng / mL) as the horizontal axis and the ratio of their peak area to the internal standard peak area (cholesterol-d7) as the vertical axis.

[0049] The standard curve of cholesterol is y=0.03646x+0.1279.

[0050] The intermediates specifically include: lanosterol, 24,25-dihydrolanosterol, 14-demethyl-14-dehydrolanosterol, 14-demethyllanosterol, dihydro-14-demethyllanosterol, yeast sterol, 7-ene cholesterol, 7-dehydrocholesterol, dehydrocholesterol, cholesterol; the standard curves are: y = 0.03646x + 0.1279 (R 2 =0.9980);y=0.05864x+0.7222(R 2 =0.9985);y=0.06278x+1.073(R 2=0.9952);y=0.04521x+0.3868(R 2 =0.9993);y=0.1306x+0.1475(R 2 =0.9981);y=0.02797x+0.1645(R 2 =0.9996);y=0.3562x+0.1625(R 2 =0.9968);y=0.03136x+0.05287(R 2 =0.9980);y=0.04284x+0.2729(R 2 =0.9992); y=0.04212x+0.4705 (R2=0.9976).

[0051] The results showed that the linear range of cholesterol and intermediates was approximately 0.5-1500 ng / mL. The correlation coefficients R2 of the standard curves of 10 cholesterol and intermediates within the linear range were all greater than 0.9900, indicating that the detection method was quantitatively accurate ( Figure 1 ).

[0052] We then tested the detection limits of cholesterol and its synthetic intermediates, as shown in Tables 3 and 4.

[0053] Table 3 Linear range, detection limit and quantification limit of cholesterol and intermediates

[0054]

[0055] Table 4 Intra-day and inter-day precision and stability of cholesterol and intermediates

[0056]

[0057]

[0058] It can be seen from Tables 3 and 4 that the method of the present invention has high sensitivity (Table 3) and good intra-day and inter-day stability (Table 4). Implementation Example 2 Accurate quantitative detection of cholesterol and its synthetic intermediates in biological samples

[0059] 1. Accurate quantitative detection of cholesterol and its synthetic intermediates in cell samples

[0060] (1) Extraction of cholesterol and its synthetic intermediates from cell samples

[0061] 1) Add 500 μL of pre-cooled 80% ethanol (containing 50 ng / mL cholesterol-d7) to the cells;

[0062] 2) Vortex for 30 seconds and ultrasonicate in an ice bath for 10 minutes;

[0063] 3) Freeze in liquid nitrogen for 1 min, thaw at 30°C, and ultrasonicate in an ice bath for 5 min, repeat 3 times;

[0064] 4) Add 1 mL of n-hexane, vortex for 30 seconds, and extract at 23°C, 1200 rpm for 20 minutes;

[0065] 5) Centrifuge at 4°C, 12000 rpm for 5 min, transfer the upper phase to a new 1.5 mL centrifuge tube, and vacuum dry;

[0066] 6) Add 600 μL of n-hexane to the lower phase, vortex for 30 seconds, and extract at 23°C and 1200 rpm for 15 minutes.

[0067] 7) Centrifuge at 4°C, 12000 rpm for 5 min, combine the upper phases, and vacuum dry;

[0068] 8) Redissolve in 100 μL isopropanol, vortex for 30 seconds, and sonicate on ice for 10 minutes;

[0069] 9) Centrifuge at 4°C, 12,000 rpm for 5 min, transfer the supernatant to the inner liner of the injection bottle for LC-MS / MS analysis.

[0070] (2) We performed precise quantitative analysis of cholesterol and its intermediates in Hela cells according to the above-mentioned cholesterol and its intermediates extraction process and the LC-MS method in Example 1. The results are shown in Table 5.

[0071] Table 5 Cholesterol and intermediate contents in Hela cells. Cholesterol and intermediates were extracted from wild-type Hela cells using 80% ethanol for LC-MS / MS analysis. The contents of each cholesterol and intermediate were absolute contents (ng / mg protein) normalized to protein amount and displayed as mean±SEM (n=3).

[0072]

[0073]

[0074] As shown in Table 5, accurate quantification of cholesterol and its synthetic intermediates can be achieved in wild-type Hela cells (Table 3).

[0075] 2. Accurate quantitative detection of cholesterol and its synthetic intermediates in tissue samples

[0076] (1) Extraction of cholesterol and its synthetic intermediates from tissue samples

[0077] 1) Add 500 μL of pre-cooled 80% ethanol (containing 50 ng / mL cholesterol-d7) to the tissue;

[0078] 2) Use a high-throughput tissue grinder to break up the tissue, vortex for 30 seconds, and sonicate in an ice bath for 10 minutes;

[0079] 3) Freeze in liquid nitrogen for 1 min, thaw at 30°C, and ultrasonicate in an ice bath for 5 min, repeat 3 times;

[0080] 4) Add 1 mL of n-hexane, vortex for 30 seconds, and extract at 23°C, 1200 rpm for 20 minutes;

[0081] 5) Centrifuge at 4°C, 12000 rpm for 5 min, transfer the upper phase to a new 1.5 mL centrifuge tube, and vacuum dry;

[0082] 6) Add 600 μL of n-hexane to the lower phase, vortex for 30 seconds, and extract at 23°C and 1200 rpm for 15 minutes.

[0083] 7) Centrifuge at 4°C, 12000 rpm for 5 min, combine the upper phases, and vacuum dry;

[0084] 8) Redissolve in 100 μL isopropanol, vortex for 30 seconds, and sonicate on ice for 10 minutes;

[0085] 9) Centrifuge at 4°C, 12,000 rpm for 5 min, transfer the supernatant to the inner liner of the injection bottle for LC-MS / MS analysis.

[0086] (2) We carried out precise quantitative analysis of cholesterol and its intermediates in mouse liver tissue according to the above process and the LC-MS method in Example 1. In the liver, cholesterol is a precursor for the synthesis of bile acids and steroid hormones. The results are shown in Table 6.

[0087] Table 6 Cholesterol and intermediate contents in mouse liver tissue. Wild-type C57BL / 6J mice were sacrificed and the tissues were weighed immediately. Cholesterol and intermediates were extracted with 80% ethanol for LC-MS / MS analysis. The content of each cholesterol and intermediate is the absolute content normalized by tissue weight (ng / mg tissue) and is shown as mean±SEM (n=4). - indicates that the content is below the detection limit and not detected.

[0088]

[0089]

[0090] As shown in Table 6, cholesterol and its synthetic intermediates can be accurately quantified in mouse liver tissue. The cholesterol content in liver tissue is relatively abundant, which is closely related to the function of liver tissue (Table 6).

[0091] Example 3 Based on [2- 13 Detection of C]mevalonolactone-labeled cholesterol and its intermediates

[0092] We used 10 mM [2- 13 Wild-type Hela cells were labeled with C]mevalonolactone, and cholesterol and its intermediates were extracted according to Example 2. Then, a metabolic flux detection method for cholesterol and its intermediates was established according to Example 1. The specific MRM information is as follows, and the other parameters are consistent with those in Example 1.

[0093] Table 7

[0094]

[0095] The results show that we can well detect the above stable isotope labeled cholesterol and its synthetic intermediate molecules. 13 Taking C-labeled cholesterol as an example, the analysis process is as follows: According to the cholesterol synthesis pathway, we analyzed 10mM [2- 13 Cholesterol process after C]mevalonolactone labeling ( Figure 2 ), and then create a 13 C-labeled cholesterol MRM method ( Figure 3 ), and finally we used 10mM [2- 12 C]mevalonolactone and 10 mM [2- 13 C]mevalonolactone was used to treat wild-type Hela cells. It was found that only M+0 to M+3 were detected in the unlabeled sample, while M+0 to M+5 could be detected in the labeled sample, indicating that the isotope peaks of M+4 and M+5 are due to [2- 13 C]mevalonolactone labeling. Hela cells were incubated with 10 mM [2- 13 After 5 hours of exposure to C]mevalonolactone to replace M+0 cholesterol, more than 8.5% of the cholesterol molecules were labeled with four or five 13 C( Figure 4 ).

[0096] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0097] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for accurately and quantitatively detecting cholesterol and its synthetic intermediates based on a triple quadrupole mass spectrometer, characterized in that: The method comprises: Extracting cholesterol or its synthetic intermediates in the biological sample to be tested or labeling the biological sample to be tested with an isotope before the extraction; the biological sample to be tested includes at least one of human cells and mouse liver tissue; The extracted cholesterol and its synthetic intermediates are subjected to LC-MS / MS detection to obtain a spectrum; wherein the liquid chromatography conditions include: gradient elution, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is deionized water, and mobile phase B is pure methanol; In the LC-MS / MS detection, the chromatographic column was a Waters Acquity UPLC BEH C18 column, and the mobile phase flow rate was 0.4 mL / min; The gradient elution specifically comprises: The mass spectrometry conditions include: ion source: atmospheric pressure chemical ionization source, scanning mode: multiple reaction monitoring, ionization mode: positive ion; the parameters of the ion source are set as follows: curtain gas: 35±5 psi; needle current: 3.0±1 mA; temperature: 400±5°C; spray gas: 50±1psi; For the intermediates lanosterol, 24,25-dihydrolanosterol, 14-demethyl-14-dehydrolanosterol, 14-demethyllanosterol, dihydro-14-demethyllanosterol, yeast sterol, 7-ene cholesterol, 7-dehydrocholesterol, dehydrocholesterol, and cholesterol, the parameters in the mass spectrometer are set as follows: Lanosterol: Q1=409.4, Q3=191.2, DP=44.19, CE=14.22, EXP=11.50; 24,25-Dihydrolanosterol: Q1=411.4, Q3=203.2, DP=41.03, CE=25.99, EXP=6.88; 14-Demethyl-14-dehydrolanosterol: Q1=393.4, Q3=185.2, DP=84.13, CE=39.96, EXP=21.24; 14-Demethyllanosterol: Q1=395.4, Q3=243.3, DP=81.05, CE=27.18, EXP=18.98; Dihydro-14-desmethyllanosterol: Q1=397.4, Q3=243.1, DP=88.96, CE=29.76, EXP=19.52; Yeastosterol: Q1=367.4, Q3=215.1, DP=87.23, CE=28.9, EXP=22.05; 7-enecholanol: Q1=369.4, Q3=215.2, DP=71.06, CE=25.03, EXP=16.98; 7-dehydrocholesterol: Q1=409.4, Q3=191.2, DP=71.06, CE=25.03, EXP=16.98; Dehydrocholesterol: Q1=367.4, Q3=161.2, DP=91.04, CE=23.00, EXP=5.80; Cholesterol: Q1=369.4, Q3=147.2, DP=55.28, CE=29.15, EXP=10.07; Obtain standard curves for cholesterol and intermediates; Calculating according to the graph and the standard curve to obtain the concentration of cholesterol or its synthetic intermediate in the biological sample to be tested; In providing 13 After C-labeled mevalonic acid, according to theory, M+5 cholesterol, that is, cholesterol with 5 carbon atoms labeled, and other labeled intermediates, hereinafter represented by M+n, n represents the labeled 13 C number; For the intermediate lanosterol (M+6), 14-demethyl-14-deaminolanosterol (M+6), 14-demethyllanosterol (M+6), dihydro-14-demethyllanosterol (M+6), yeast sterol (M+5), dehydrocholesterol (M+5), cholesterol (M+1), cholesterol (M+2), cholesterol (M+3), cholesterol (M+4), cholesterol (M+5), the parameters in the mass spectrum are set as follows: Lanosterol (M+6): Q1=415.4, Q3=415.4, DP=44.19, CE=14.22, EXP=11.50; 14-Demethyl-14-deaminolanosterol (M+6): Q1=399.4, Q3=399.4, DP=54.14, CE=16.70, EXP=11.81; 14-Demethyllanosterol (M+6): Q1=401.4, Q3=401.4, DP=73.95, CE=14.73, EXP=10.96; Dihydro-14-demethyllanosterol (M+6): Q1=397.4, Q3=397.4, DP=81.85, CE=18.20, EXP=11.93; Yeastosterol (M+5): Q1=372.3, Q3=372.3, DP=51.9, CE=17.8, EXP=13.88; Dehydrocholesterol (M+5): Q1=372.3, Q3=372.4, DP=94.12, CE=12.60, EXP=11.94; Cholesterol (M+1): Q1=370.4, Q3=370.4, DP=86.10, CE=18.02, EXP=10.87; Cholesterol (M+2): Q1=371.4, Q3=371.4, DP=86.10, CE=18.02, EXP=10.87; Cholesterol (M+3): Q1=372.4, Q3=372.4, DP=86.10, CE=18.02, EXP=10.87; Cholesterol (M+4): Q1=373.4, Q3=373.4, DP=86.10, CE=18.02, EXP=10.87; Cholesterol (M+5): Q1=374.4, Q3=374.4, DP=86.10, CE=18.02, EXP=10.87.

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