TW515888B - Method for quantitatively analyzing misoprostol acid in plasma - Google Patents

Abstract

The present invention discloses a method of using a liquid chromatography connected in series with a mass spectrometer (LC/MS/MS) to analyze the quantity of misoprostol acid in plasma, which is characterized in that two separation columns packed with different packing elements are connected in series, together with a specific plasma sample extraction method and a specific proportion of mobile phase, to achieve the objective of quantitatively analyzing tiny amount of misoprostol acid in plasma.

Description

515888

V. Description of the Invention (1) Field of the Invention The present invention relates to a method for quantitatively analyzing the concentration of misoprostol acid in blood, and more particularly to an application of liquid chromatography-tandem mass spectrometry (LC / MS). / MS) method to quantify the concentration of misoprost acid in the blood to 50 pg / mL. BACKGROUND OF THE INVENTION Misoprostol is a synthetic prostaglandin derivative with a structure similar to that of prostaglandin Ei (PGEi). It is an effective gastric acid secretion inhibitor and can be mixed with other drugs to form it. Misoprostol will be replaced soon after taking it into the gastrointestinal tract. Placing the misoprost bank in the plasma '3 7 C in vitro test shows that a lot of misoprostol metabolism will be formed in the plasma However, the misoprostol itself was not found after 6.4 minutes (Schoenhard, G. Tatar, Digestive Diseases and Sciences, 1 98 5 Zan J, 30 (11) · 126S-128S). In plasma, a biologically effective metabolite, misoprostol acid, can be measured; this is a lipidated derivative of misoprostol. Within 15 minutes after taking misoprostol tablets, humans can measure misoprost acid in plasma 'and the metabolic life half-life (ha 1 f-1 ife) of misoprost acid in plasma is very high Short, less than 30 minutes. Because the dose of misoprostol tablets (100 micrograms / tablet, 200 micrograms / tablet) is much lower than the general suspect dose (more than milligrams) and after oral administration,

Page 4 V. Explanation of the invention (2) As for the metabolite misoprost acid, the oral coating period of misoprostol is less than 30 minutes due to its very low content, and its heat resistance is not equal to miso in vivo. Formic acid red, therefore, the general method of analysis ^ two, ~ 弋 (heat 1 i abl e), chromatographic mass spectrometer, etc. quantification of Fang Sheng, 'Achieve liquid chromatography, gas phase

The requirements of Pg / mL, plus the current 1 / # method field reached / quantity limit (LOQ) 50 column, can not be misoprost acid: two-performance liquid chromatography tubes PGEl, PGA] and- Some eggs] The other factors of blood sacrifice (for example, Gulu * ti MW) are effectively separated, that is, the interference of other components in plasma is eliminated by the current technology and ... The report of the known literature of the 'Guanguan misoprost acid blood concentration report (Schoenhard "Chu / ^ rd, G. et al., Ibid.") Shows that only: species, radioimmunoassay kit ⑴A The method of ki " can reach the setting limit of 50 pg / mL. However, this kit is the method used by the manufacturer of misoglitacol in lutein to analyze misoprostic acid in blood. The entire set has not been disclosed to the public, and it uses radioactive species 氚 OH ^ tritium), which is also dangerous and inconvenient in operation. Misoprostol is a generic drug whose patent has expired. Regulation 'Before the domestic market, a BE study of misoprostol tablets is required, that is, the analysis method used must be able to analyze the content of misoprost acid in plasma to The limit of quantification is 50 pg / mL. Therefore, the purpose of the present invention is to solve the bottleneck encountered in the above analysis, and to develop a simpler and safer method than R 丨 A to effectively and accurately quantify the forefront of miso Acid in plasma To the limit of quantification of 50 pg / mL.

Page 5 515888

5. Description of the invention (3) Summary of the invention In view of this, the present invention provides a method for analyzing the content of misoprost acid in plasma, including the following steps: (a) preparing a mobile phase; (b) Extract the misoprost acid sample from the plasma ^; (0) connect two reverse phase high performance liquid chromatography columns in series; (d) install the serial columns in a liquid chromatography series Mass spectrometer (LC / MS / MS); and (e) injecting the sample into the liquid chromatography tandem mass spectrometer and analyzing the misoprost acid sample. Another aspect of the present invention is to provide an extracted plasma The method of the sample 'includes adding phosphoric acid to the plasma sample and mixing uniformly; adding an internal standard to the above mixture and mixing uniformly; adding an acid to maintain the above mixture in an acidic environment; extracting the mixture with an organic solvent; removing the organic Layer 'and drying at room temperature; and dissolving the dried sample in a mobile phase. ^ The method of extracting a plasma sample according to the present invention described above can effectively ^ heat-labile misoprost acid in plasma, and Analysis by the method of the invention the knife may be accurately quantified in blood misoprostol acid content to quantify the degree of 50 pg / mL. ·

π i Ύ makes the above and other purposes, features, and advantages of this & Ming obvious and easy to understand, and ordered 4 main L_ nn, Bu also cited the preferred embodiment and cooperated with the attached diagram, the state of the year is as follows : Simple description of the icon

Page 6 515888 5. Description of the invention (4) Figure 1 shows LC / MS / MS chromatograms of pure standards of misoprost acid; Figures 2 (a) and (b) show LC of blank plasma / MS / MS chromatogram 'where' (a) is for monitoring misoprost acid, and (b) is for monitoring the internal standard para-fluorocinnamic acid (p-fluorocinnamic acid); sections 3 (a) and ( b) The LC / MS / MS chromatogram showing the misoprost acid added to plasma with 50 pg / mL, where (a) is the monitoring of misoprost acid and (b) is the monitoring of internal standards P-fluorocinnamic acid (P-fluorocinnamic acid); Figures 4 (a) and (b) show LC / MS / MS chromatograms of misoprost acid added to plasma at 4 50 pg / mL , Where (0 is the monitoring of misoprost acid, and (b) is the monitoring of the internal standard para-fluorocinnamic acid (^-f 1 uor oc i nnam ic acid); and Figure 5 shows the misoprost Standard curve diagram of acid quantification. Detailed description of the invention The invention provides a novel LC / MS / MS quantification method that can be used to analyze the content of misoprost acid in plasma, including: (a) preparation-mobile, phase; (B) extracting a misoprost acid sample from plasma; (c) connecting two reverse-phase high performance liquid chromatography columns in series; (d) installing the series columns in a liquid chromatography tandem mass spectrometer Instrument (LC / MS / MS); and inject the sample into the liquid chromatography tandem mass spectrometer and analyze a sample of misoprost acid.

515888 V. Description of the invention (5) The specific mobile phase used in the present invention is composed of 1.5 mM ammonium acetate (95% acetate) dissolved in 95% ethyl acetate (CH3CN) and 2% isopropyl acetate. Composed of iso-propan〇i, by using this specific mobile phase, LC / MS / MS can achieve the highest sensitivity. Because of the traditional LC / MS / MS method, and The influence from interfering substances in plasma cannot be overcome. Therefore, the present invention uses two serially connected separation columns to achieve the purpose of separation. The separation column is selected from a reverse phase chromatography column, preferably, a Q column and a phenyl j phenyl) column, and more preferably, a tandem sequence of the reverse phase chromatography column The Q-column string is behind the rigid tube and the stupid column. There are no special restrictions on the size, length, and pore size of the chromatography column. It should depend on the parameters of the LC / MS / MS instrument and the volume of the analysis sample. The selection of these conditions is also understood and achieved by those skilled in the art. . " Another feature of the present invention is a method for extracting a misoprost acid sample. Due to the thermal instability of misoprost acid and the short half-life of metabolic life in the blood, the appropriate extraction step plays a key role in the success of the overall quantitative analysis. The present invention therefore provides a method for extracting a plasma sample so that the misoprost acid in the sample can maintain stability for subsequent quantitative analysis. 2 = The method includes the following steps: adding phosphoric acid to the plasma sample and mixing and adding an internal standard In the above mixture, and mix well, take the mixture with two = dissolve; add human acid 'to keep the above mixture in an acidic environment, which is conducive to the extraction of misoprost acid; take out the organic layer, ^ in

515888 V. Description of the invention (6) Dry to temperature; and dissolve the dried sample in mobile phase. According to the extraction method provided by the present invention, the step of adding phosphoric acid to the plasma sample is to maintain the stability of misoprost acid in plasma, and it is preferable to select 85% phosphoric acid in a volume of 1/1000. Add to plasma sample. In addition, an internal standard can be added to the plasma sample prior to extraction as a reference point for subsequent quantitative analysis. The selection of the standard should be suitable for the operation characteristics and parameters of misoprost acid 2LC / MS / MS ’and consider the stability during extraction (丨). (2)

Solubility of organic solvents and mobile phases; and (3) Factors such as whether the standard can be distinguished from factors of plasma components in the analysis chart 碏. Can be used in the internal standard of the present invention, for example, para-fluorocinamic acid (P-f1uorocinnamic acid). In addition, in the extraction step, an acid needs to be added so that the above mixture is maintained in an acidic environment to avoid carboxyl groups. Dissociate into carboxylate (c0__); wherein, preferably, the acidic environment is in the range of pH 1 to 4. The usable acid is not particularly limited, and it is preferable to use an inorganic acid including hydrochloric acid, phosphoric acid, sulfuric acid, or acetic acid. The extraction step of the present invention is to use an organic solvent to extract misoprost acid. Among them, the organic solvents that can be used are not particularly limited and are applicable to those skilled in the art, including ethyl acetate, ether (Ether), acetone (acetone), chloroform (chl0f0rm) and benzene (benzene). After extraction, the separation of the organic layer and the water layer can be accelerated by centrifugation or freezing. The step of centrifugation includes a temperature of 4 ° C at 3,000 revolutions per minute (rpm). 10 minutes

515888 V. Description of the invention (7) Minutes, and the freezing step chooses to freeze at the temperature for 15 minutes. After delamination, it is ready to be used. The drying method is not special to avoid the misoprost acid being dried by heat (for example, nitrogen). It should be noted that 'After performing the above to avoid the temperature to accurately measure the sample after subsequent extraction of the quantitative work', it will be entered into LC / MS / MS. The following examples will only be used as examples. purpose. The gas phase is below -7 (TC (for example, _86), it is an organic phase, and the temperature is limited at room temperature, but the operation must be unstable below room temperature. The best way is that the entire inert extraction step should produce any changes at low temperature or rissolenic acid. The sample is dissolved back to the transfer prepared by the present invention for analysis. The invention is further explained, However, as an example, it is not intended to limit the cream of the present invention. Example 1: Preparation of the sample (1) Preparation of stock solution. Weigh 3 milligrams (mg) of misoprost acid (provided by Taiwan Yongguang Chemical Co., Ltd.). In 1 ml of methanol, the misoprost acid at a concentration of 3 mg / mL was obtained. Then the sample was serially diluted to a mobile phase at a multiple of 100 times (1.5 mM ammonium acetate in 95% acetonitrile). And 2% isopropanol) 3 times to obtain a misoprost acid at a concentration of 3 ng / mL. Then add 1 ml of a 3 ng / mL sample to 5 ml of a mobile phase to obtain a concentration of 0.5 ng / mL Misoprost acid. Freeze at -86 ° C after dispensing.

Page 10 515888 V. Description of the invention (8) (2) Preparation of standard samples Prepare standard curve sample concentrations according to the following table, which are 50, 7, 5, 100, 200, 300, 450, 750 and 1200 respectively. pg / mL. Among them, the number represents the volume of the mother liquid sample (microliter; # L) that needs to be added to each milliliter of blank plasma (purchased from Taipei Blood Donation Center), which means that it is not added. Mother liquor sample standard Quxiang L sample concentration (pg / mL) 50 75 100 200 300 450 750 1200 0-5 ng / mL 100 150 200 400 _ — _ — 3 ng / mL one — one one 100 150 250 400 (3) The ua tube sample (qc samp 1 e) was prepared according to the following table to prepare tube sample concentrations, which were 50, 150, 60 0, 90 0, and 1 200 pg / mL, respectively. Among them, the number represents the volume of mother liquid sample (#L) to be added in each milliliter of blank plasma, and '-π means not to add. -^ Quality control sample concentration (丨 pg / mL) Mother liquor sample 50 -------- one-150 600 900 1200 0.5 ng / mL 100 300---3 ng / mL-one 200 300 400

Page 11 515888 V. Description of the invention (9) Example 2: Extraction of the sample Take 1 ml of the above-mentioned plasma sample and place it in a test tube, and add 85% phosphoric acid (Merck, Cat · No. 1 · 〇〇573 · 100〇) 10 microliters and vortex for 1 minute to mix well. Add 100 microliters of the internal standard mouthpiece's p-fluorocinnamic acid (Al d r i c h, Cat · N 0 · 16384-8) at a concentration of j OO ng / mL, and shake for 1 minute. Next, 5 ml of ethyl acetate (Merck, Cat · No · 1.0 0868 · 25 00) and 50 µl of 1 N HC1 (Merck, Cat · No · 1.0 03 1 7) were added, and the mixture was shaken for 10 minutes for extraction. The mixture was centrifuged at 3,000 rpm at 4 ° C for 10 minutes, and then frozen at -86 ° C for 15 minutes. After the upper layer was decanted, it was blow-dried with nitrogen at room temperature and dissolved in 70 microliters of mobile phase. Example 3 · Analysis of the sample (1) LC part: using C18 column (Phenomenex, 75 X 4 · 6 mm, 3 //, C18 (2)) and phenyl column (Cosmosil, 250 χ 4 · 6 mm, 5-ph) in series; LC pump is Shimadzu LC-10AT and LC / MS / MS is PE sc i ex API 300 system. The flow rate of the LC was set to 0.8 ml / min; before entering the MS / MS, the flow was split at a ratio of 1: 9 (splitter), and 1/10 of the amount entered the MS / MS. The mobile phase was 1 · 5 mM ammonium acetate (Merck, Cat. No. 1.00 0 30 · 2500) in 95% acetonitrile (Merck,

Cat. No. 1.0116) and 2% isopropanol (Merck, Cat. No. 1. 1 0 1 40. 2500). (2) MS part:

Page 12 515888 V. Description of the Invention (1) Use the Ion spray inlet 'to select the negative ion and adjust it to the best state; 2. Enter the sample control software of LC / MS / MS, this software is divided into experiment editing and method editing And sample editing three parts; 3. Set the q1, q3 and residence time of misoprost acid to 366, 248 and 500 msecs respectively in the routine editing part, and set the Ql, Q3 and residence time of the internal standard respectively It is 164, 95, and 50 0 msecs. In addition, the scan type is MRM, the pause time is 50 ms, and the scan time is 1 s. 4 • Set the experiment file, status file, and calibration in the method editing section. In addition, the duration is 1 5 Minutes; 5 · In the sample editing section, choose the method to use, create a new folder to store the file, and enter the basic data; 6. Select "quote start run" in the option "sample ·" to start execution. Results · In order to verify that the analytical method disclosed in the present invention can meet the requirements of L0Q 50 0 pg / mL and can effectively analyze samples for biological equality test, the formulation concentration range of the present invention is from 50 to 1 2 0 0 pg / mL mL of misoprost There are a total of 8 concentrations in human plasma. In addition, 5 plasma control samples of different concentrations are prepared. All sample analysis data are substituted into the standard calibration curve to calculate the nasal analysis. The precision of the analytical method is not estimated by β (precisi ο η) and accuracy. The validation methodology used in the present invention can be referred to

Page 13 515888 V. Description of the invention (11) See "Test Standards and Related Materials for Drugs' Bioavailability and Bioequivalence Test" Published by the Department of Health of the Executive Yuan. 1. Specificity ··

Refer to Figures 1-4, where Figure i is a chromatogram of misoprost acid dissolved in mobile phase. Figure 2 is a chromatogram of blank plasma after extraction treatment. Figures 3 and 4 The figure shows the chromatograms of misoprost acid concentration of 50 and 450 pg / mL (in plasma) after extraction. The retention time of lysoxolide I and the internal standard (P-fluorocinnamic acid). (Retention time) were 5.38 minutes and 7.06 minutes, respectively. It can be clearly observed that after the treatment of the extraction and analysis steps of the present invention, the 'misoprost acid is not disturbed by the plasma and the extraction reagent, but can be effectively separated. 2 · Linear range test (η nearrange): The standard calibration curve range is a linear function (Y = a + bX), with the peak-to-surface ratio (Y) versus> degree (X) for weighted linear regression. regression). Measured by LC / MS / MS

The ratio of the peak area of the analysis of soprostanoic acid to the internal standard was brought into the linear function Y = a + bX, and the concentration of misoprost acid in plasma was calculated. Figure 5 shows the calibration curve of misoprost acid for the intraday test. The peak area ratio is plotted against the concentration. The data is shown in Table 1.

Page 14 515888 V. Description of the invention (12) Table 1: Peak area-wave degree relationship number on the same day of the misoprost acid standard curve Roll degree (pg / mL) Wave area of MPA1_area IS2 Wave area ratio Touch calculation value (pg / rriL) Deviation value ⑽ 1 50 941 50046 0.019 49.938 -0. 124 2 7 5 1104 48825 0.023 77.788 3.717 3 100 157 6 5955 1 0.026 102.245 2.245 4 200 2587 63057 0.041 201.879 0.94 5 300 2817 56376 0.05 260.85 -13.05 6 450 3646 4337 5 0.084 496.265 10.281 7 7 50 4888 40027 0.122 7 56.658 0.888 8 1200 8175 44153 0.185 1187.871 -1.01 1 1: MP A stands for misoprost acid 9

2: IS stands for Neuzou Standard. Y = 0.01 151 + 0.00015X τ R2 = 0.997. Table 2 is the data of the calibration curve of misoprost acid in the interday test. The minimum linear correlation coefficient (R2) is 0.999, and the maximum value of the coefficient of variation (CV%) of each concentration is 11. 5 98%, the deviation value (from -4 · 793 to 5 · 2 86, all meet the specification value

Page 15 515888 V. Description of the invention (13) Table 2: Inter-day variation of the back calculated concentration of the misoprost acid standard calibration curve itK zk back calculated concentration of the standard wearing distance R2 test additional concentration (pg / mL) slope number 50 75 100 200 300 450 750 1200 1 47360 72963 105294 1 Difficulty 9 329596 471.727 660311 1263.793 0.000330 0.0175 0.995 2 57.084 68.931 104.709 221.614 256.134 421.435 772.927 1237.198 0.000198 «0.0001 0.996 3 58.781 72753 107.776 178.193 306.306 306.130 306.130 306.130 105219 188557 338.016 461307 851.875 1097.019 0.000260 0.0213 0.993 5 50.768 69.739 109.846 199272 315.793 431.807 688.451 1272365 0.000352 -0.0076 0.999 6 51.00 80217 98.869 215222 260.408 429.872 850.793 1154269 O.OO8.1 501.501 5.203 25.03 0.03 0.003 0.03 0.030.0 3.702 16.455 34.911 19.626 80.464 68.665 0.0001 CV% 10364 7.624 1516 8261 11 * 598 4.431 10581 5.683 324202 Deviation < 1% 3234 A793 5286 -0.402 0338 -1575 13 95 0.678 3. Intraday and Interday:

The precision and accuracy of the standard calibration curve and quality control samples were used as the basis for evaluating the same-day and inter-day tests. Precision is evaluated by the coefficient of variation (CV), while accuracy is evaluated by the deviation; its normative value is at the quantitative limit L0Q

515888 V. Description of the invention (14) Within 20/6 'Other concentrations are within ± 5% (1) Standard calibration curve ^ The deviation values of 8 concentrations of the standard calibration curve of the test on the same day are calculated and recorded in Table 丨, 'The deviation is from -13.05% to 10.28%. There are 6 batches of standard calibration curves for the inter-day test. The deviation values and coefficients of variation of 8 concentrations in each batch are recorded in Table 2. The deviation values are from-4.793% to 5.286%. The coefficient of variation is from 3.516% to 1 1.598%. (2) Quality control samples were tested on the same day to analyze 5 concentrations of 50, 150, 600, 900, and 1200 Pg / mL. Each concentration was repeated for 6 samples. The analysis results are recorded in Table 4. The deviation value is from-4.793% to 5.286%, coefficient of variation from 3.516% to 11.598% 〇 Inter-day test to analyze 5 concentrations such as 50, 150, 600, 900 and 1200 Pg / mL 'mother concentrations were repeated 2 samples, a total of 6 different batches were performed Second, the analysis results are recorded in Table 3, where the deviation values are from-; 539% to 3563%, and the coefficients of variation are from 7.793% to 1 1.468%.

Page 17 515888 V. Description of the invention (15) Table 3: Precision and accuracy of inter-day validity plus concentrated soil sample calculated value (pg / mL) CV partial value is close to mean SD (pg ^ nL) 蹁Washing Da ^ rl Όδζτ2 Όδζτ3 Da5r4 Ώ ^ / 5 Ό ^ 6 (%) C%) 50 1 51.136 51.189 42.869 59.131 56.183 48.S76 49.231 5.646 11.46S -1.539 CLOQ) 2 46.571 51.646 40.187 51.365 41712 49.903 1 146.259 140.259 140.259 140. 162.162 1341261502 131.733 145.111 154 549 135.827 148.407 12.896 141.228 152.338 8.690 585.277 555.069 -1.062 521.948 652.183 1 616.940 675.616 594.133 621.899 525.562 6002 687.750 683.872 656.373 614.719 59.102 9.614 2.453 782.251 848.520 1 940.183 876.402 979.812 1001.42 9002 1033.09 855.232 897.476 969.283 962.370 832.048 914.840 77.301 8.450 1.649 1 1253.32 1261.55 1307.41 108873 10S1.01 1310.15 1200 2 1119.63 1342.88 1361.96 1241.48 1234.20 1310.82 1242.76 96.847 7.793 3.563 imi Page 18 515888

V. Description of the invention (16) Table 4: Intertemporal change of the returned concentration of the quality control sample Batch ____Recalculated concentration of the standard test Additional concentration (pg / mL) No. 50 150 600 900 1200 1 53.868 168.189 685.679 953.070 1287.029 2 59.967 167.009 612.370 993.618 1308.029 3 49.263 168.423 662.736 971.459 1284.396 4 59.682 168.798 596.484 965.759 1110.680 5 48.993 155.441 603.186. SD. 62.264. 4.852. 3.348 5.936 4.423 7.527 Deviation value (° / 〇) 9.038 9.810 4.583 6.569 4.058 4 · Limitation of quantitation (LQ) · The lowest concentration in the standard calibration curve is the quantitative limit. In Table 3, the deviation value And the coefficient of variation were -1.539% and 11.468%, respectively; in the reference, its deviation value and coefficient of variation were 9.058% and 8.82%, respectively. "From the above experimental results, it can be found that the analysis method of the present invention can meet the standard that human analysis method is effective, and can make the analysis of misoprostic acid reach $; the limit is 50 pg / mL. The above is disclosed in a preferred embodiment, but it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and retouches without departing from the spirit and scope of the present invention.

Page 19 515888 V. Description of the invention (17) The scope shall be defined by the scope of the attached patent application Ι ^ ί Page 20

Claims (1)

------ 1 515888 _Announcement I ----- 6. Scope of patent application L ......... 1. A type for analyzing plasma The method for misoprosto1 ac id content includes the following steps: (a) preparing a mobile phase (b) extracting a misoprost acid sample in a pulp; (c) mixing two Reverse phase high-performance liquid chromatography columns are connected in series; (d) the serial columns are installed in a liquid chromatography tandem mass spectrometer (LC / MS / MS); and (e) the A sample was injected into the liquid chromatography tandem mass spectrometer, and the misoprost acid sample was analyzed. 2. The method according to item 1 of the scope of patent application, wherein the mobile phase is composed of 1.5 mM ammonium acetate dissolved in 95% acetonitrile and 2% isopropanol. 3. The method as described in item 1 of the scope of patent application, wherein the reversed phase analytical column includes a C18 column and a phenyl column. 3 4 · The method as described in item 3 of the scope of patent application, wherein the tandem sequence of the reversed phase analytical column is that the C18 column is first and the stupid base column is second. 5. The method according to item 1 of the scope of patent application, wherein the extraction method of step (b) includes the following steps: (th) adding phosphoric acid to the plasma sample and mixing uniformly; (b2) adding an internal standard to the above mixture (B3) extract the mixture with an organic solvent; (ratio) add an acid so that the mixture is maintained in an acidic environment; 515888 6. Scope of patent application (h) Take out the organic layer and dry it at room temperature; and (ratio) dissolve the dried sample with the mobile phase. 6. The method as described in item 5 of the scope of patent application, wherein the step (dagger) is to add 85% phosphoric acid to the plasma sample in a volume of 1/1000. 17 · The method as described in item 5 of the scope of patent application, wherein the internal standard of step (b) includes para-fluoroluocinnamic acid (P -f luorocinnamic acid). 0 · As the item 5 of scope of patent application In the method, the acidic environment of step (%) is in the range of pH 1 to 4. 3 9. The method according to item 8 of the scope of patent application, wherein the acid is an inorganic acid. 10, The method according to item 9 of the scope of patent application, wherein the inorganic acid includes hydrochloric acid, phosphoric acid, sulfuric acid, or acetic acid. π. The method according to item 5 of the scope of the patent application, wherein the organic solvent in step (b4) is selected from the group consisting of ethyl acetate, ether, acetone, chloroform and benzene. 12. The method according to item 5 of the scope of the patent application, wherein the step of centrifuging and accelerating the layer is further included after extracting the mixture. 13. The method according to item 12 of the scope of the patent application, wherein the centrifugation is performed at 4 C for 3 minutes at 3,000 rpm. 1 4 · The method as described in item 12 of the scope of patent application, further comprising the step of freezing by low μ to separate the organic layer. !, ^ ^ ^ ^ ^ ^ 々 method, in which the cold heading is 15 · As described in item 4 of Shenyan's patent, it is frozen at -70 ° C for 15 minutes. Page 22 515888 6. Scope of patent application 1 6. The method as described in item 5 of the scope of patent application, wherein step (b) is dried by blowing nitrogen at room temperature.