A method for detecting trace amounts of L-proline in L-prolinol

Abstract

The invention belongs to the field of analysis, and discloses a method for detecting trace L-proline in L-prolinol. The invention adopts a derivatizing agent di-tert-butyl dicarbonate (i.e., Boc-anhydride) to derivatize L-prolinol and L-proline under alkaline conditions, thereby realizing the separation and determination of trace L-proline in L-prolinol using a common C18 chromatographic column. The method has the characteristics of short derivatization time, mild reaction conditions, simple and convenient operation, safety, accuracy, high efficiency, good reproducibility, and high sensitivity, and is suitable for the rapid detection of L-proline in L-prolinol.

Description

Technical Field

[0001] The invention belongs to the field of analysis, and particularly relates to a method for detecting trace amounts of L-proline in L-prolinol. Background Art

[0002] L-Prolinol is widely available, cheap, and environmentally friendly. It is an important chemical reagent. It is not only a raw material for synthesizing excellent optical materials N-(4-nitrophenyl)-(L)-prolinol (NPP) and chiral polymer materials, but also an important chiral source for synthesizing chiral drugs, such as the synthesis of the chiral drug avanafil. In the process of drug synthesis, people need to study and control the content of impurities, and the study and control of the impurity content of related materials further ensure the purity of the target drug. L-Prolinol is mainly generated by reducing cheap and easily available L-proline, and inevitably accompanied by trace amounts of L-proline. In order to ensure the purity of the target drug and effectively reduce the cost of purifying the target drug, the content of L-proline in L-prolinol needs to be controlled within 0.1% (w / w).

[0003] At present, the main methods for detecting and analyzing proline include electrophoresis, liquid chromatography-mass spectrometry, pre-column derivatization coupled with high performance liquid chromatography, etc. The reproducibility, accuracy and sensitivity of electrophoresis are worse than those of high performance liquid chromatography; the liquid chromatography-mass spectrometry instrument is expensive, relatively rare in conventional laboratories, and not widely popularized.

[0004] L-prolinol and L-proline have high polarity and are difficult to adsorb on the chromatographic column to form chromatographic peaks. They also have no characteristic ultraviolet absorption peaks, so liquid chromatography alone cannot meet the detection requirements. The combination of pre-column derivatization and high-performance liquid chromatography is the preferred method for analyzing and detecting L-proline. Commonly used derivatization reagents include phenyl isothiocyanate (PITC), 2,4-dinitrofluorobenzene (DNFB), 9-fluorenylmethoxyhydroxyl chloride (FMOC), and p-nitrobenzyl chloroformate. However, the operation of phenyl isothiocyanate (PITC) is complicated, and the derivatization time is too long; 9-fluorenylmethoxyhydroxyl chloride (FMOC) is easily hydrolyzed, unstable, and has poor results; 2,4-dinitrofluorobenzene (DNFB) melts at room temperature, affecting the operation, and the derived by-products affect the separation results; p-nitrobenzyl chloroformate is sensitive to moisture and may generate pressure during storage. It is incompatible with water, strong bases, alcohols, amines, and acids, and has the disadvantages of uneven solutions after derivatization and troublesome sampling. Therefore, there is currently a lack of a method that is simple to operate, has a short analysis time, is sensitive, has accurate and reliable results, and is widely used to detect the L-proline content in L-prolinol. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide a method for detecting trace L-proline in L-prolinol, which is achieved by pre-column derivatization combined with high performance liquid chromatography. The existing derivatization technology uses a reagent containing a benzene ring structure to derivatize the target compound, thereby introducing the differential changes in ultraviolet characteristic absorption and polarity and then detecting it, but such derivatization reagents are usually highly toxic and the derivatization conditions are harsh. The present invention uses di-tert-butyl dicarbonate (i.e., Boc-anhydride) to derivatize proline and prolinol, which not only has a short derivatization time, mild derivatization conditions, and a non-toxic green derivatizer, but also does not require the introduction of an aromatic group. The original ultraviolet terminal absorption of the compound itself is used as the detection wavelength, and the baseline separation of the two substances on the C18 chromatographic column can be achieved, which solves the problem that the derivatization operation of L-proline detection is complicated, time-consuming, and the derivatization process is harmful. The present invention provides an ideal method for detecting trace L-proline in L-prolinol.

[0006] The technology involved in the present invention is mainly realized by the following scheme:

[0007] A method for detecting trace amounts of L-proline in L-prolinol comprises the following steps:

[0008] 1) Weigh L-proline reference substance and dissolve it in ethanol to obtain L-proline reference substance stock solution;

[0009] 2) taking different volumes of the L-proline reference substance stock solution obtained in step 1), respectively adding Boc-anhydride solution and sodium hydroxide solution, shaking, to obtain derivatized L-proline reference substance solutions of different concentrations, i.e., Boc-L-proline reference substance solutions of different concentrations;

[0010] 3) subjecting the Boc-L-proline solution prepared in step 2) to high performance liquid chromatography analysis, plotting a standard curve with the concentration of Boc-L-proline as the abscissa and the corresponding peak area integral value as the ordinate, and calculating a regression equation;

[0011] 4) Weigh the L-prolinol sample, add the Boc-anhydride solution and the sodium hydroxide solution, and shake to obtain the derivatized test solution;

[0012] 5) The derivatized test solution prepared in step 4) is subjected to high performance liquid chromatography analysis to obtain the peak area integral value of Boc-L-proline, and the peak area integral value of Boc-L-proline is substituted into the regression equation obtained in step 3) to calculate the injection concentration of Boc-L-proline, and the injection concentration of L-proline is obtained by conversion.

[0013] The HPLC analysis conditions are as follows:

[0014] Octadecylsilane bonded silica gel is used as a filler, and the filler particle size is 1.8-6 μm, preferably 5 μm; the column length is 150-350 mm, preferably 250 mm; phosphoric acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; the volume ratio of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.4%, preferably 0.05-0.2%; the detection wavelength is 190-240 nm, preferably 200-220 nm; the column temperature is 15-40° C., preferably 20-30° C.; the flow rate is 0.5-1.5 mL / min, preferably 1.0 mL / min.

[0015] The Boc-anhydride solution in step 2) and step 4) is obtained by dissolving Boc-anhydride in ethanol, and the concentration is 10-50 mg / mL, preferably 20-40 mg / mL.

[0016] The concentration of the sodium hydroxide solution in step 2) and step 4) is 0.01-0.5M, preferably 0.05-0.2M.

[0017] Step 1) The concentration of the L-proline reference substance stock solution is 0.01-0.1 mg / mL, preferably 0.02-0.08 mg / mL.

[0018] Step 2) The concentration of Boc-L-proline in the Boc-L-proline reference solution is 0.9 to 46 μg / mL, preferably 2.3 to 27.6 μg / mL.

[0019] In step 2), the volume ratio of the L-proline reference substance stock solution to the Boc-anhydride solution is (0.1-5):(1-5); preferably (0.25-3):(3-5).

[0020] In step 4), the amount of the L-prolinol sample is such that the concentration of the L-prolinol sample in the test solution after derivatization is 2 to 8 mg / mL, preferably 3 to 6 mg / mL.

[0021] The volume ratio of the Boc-anhydride solution to the sodium hydroxide solution in step 2) and step 4) is (1-5):1, preferably (3-5):1.

[0022] The oscillation time in step 2) and step 4) is 5 to 30 minutes, preferably 10 minutes; after oscillation, the volume is fixed with an ethanol aqueous solution having a volume ratio of ethanol to water of 1:1.

[0023] The amount of the ethanol aqueous solution in step 2) and step 4) is such that the concentration of Boc-anhydride is diluted to 3-15 mg / mL, preferably 8-14 mg / mL.

[0024] The parameters of the gradient elution are shown in Table 1:

[0025] Table 1

[0026] Time, min Mobile phase A, % (V / V) Mobile phase B, % (V / V) 0 65-75 35-25 10 35-25 65-75 12 35-25 65-75 12.1 65-75 35-25 20 65-75 35-25

[0027] Preferably, the parameters of the gradient elution are as shown in Table 2:

[0028] Table 2

[0029] Time, min Mobile phase A, % (V / V) Mobile phase B, % (V / V) 0 70 30 10 30 70 12 30 70 12.1 70 30 20 70 30

[0030] The mechanism of the present invention is:

[0031]

[0032] The molecular weight of L-proline before derivatization is 115, and the molecular weight after derivatization is 215, which is 1.87 times that before derivatization.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] Provided is a novel method for detecting trace amounts of L-proline in L-prolinol. The method has the characteristics of short derivatization time, mild reaction conditions, simple operation, safety and greenness, accuracy, high efficiency, good reproducibility and high sensitivity, and can be used for the rapid detection of L-prolinol. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the blank derivatization agent chromatogram.

[0036] Figure 2 This is the chromatogram of the derivatized solution of 100% spiked test sample in Example 6.

[0037] Figure 3 This is the chromatogram of the test solution after derivatization in Example 1.

[0038] Figure 4 This is the linear relationship diagram of Boc-L-proline in Example 3. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0040] Unless otherwise specified, the reagents used in the examples can be purchased from the market.

[0041] Instruments: Thermo Fisher (U3000) high performance liquid chromatograph, diode array detector (DAD), Chromeleon data workstation; XP26 analytical balance (1 part per million, METTLER);

[0042] Reagents: L-prolinol sample (batch number: GLS210501-37109) was from Gill Biochemical (Shanghai) Co., Ltd., L-proline reference (batch number: 0407-RB-0001) was from Cato Research Chemicals Inc (CATO), with a purity of 99.3%, di-tert-butyl dicarbonate (i.e., Boc-anhydride; batch number: C2111027-25g, purity 99.0%) was from Aladdin, analytical grade; acetonitrile was chromatographic grade; other reagents were all analytical grade; water was purified water.

[0043] Example 1 Solution Preparation

[0044] Derivatization agent: Weigh 3 g of Boc-anhydride and dissolve it in 100 mL of ethanol.

[0045] L-proline reference substance stock solution: weigh 4.92 mg of L-proline reference substance into a 100 mL volumetric flask, dilute to volume with ethanol, and shake well.

[0046] Derivatized L-proline reference solution: Take 0.25-3 mL of L-proline reference stock solution and place it in a 10 mL volumetric flask, then add 4 mL of derivatizing agent and 1 mL of 0.1 M sodium hydroxide solution, shake vigorously for 10 min, then add ethanol: water (1:1, V / V) to the scale and shake well.

[0047] Derivatized test solution: weigh 50 mg of L-prolinol sample and place it in a 10 mL volumetric flask, add 4 mL of derivatizing agent and 1 mL of 0.1 M sodium hydroxide solution, shake vigorously for 10 min, then make up to volume with ethanol: water (1:1, V / V), and shake well.

[0048] Example 2 High performance liquid chromatography determination

[0049] Accurately pipette 10 μL of the derivatized spiked sample solution and the sample solution, respectively, and inject them into the liquid chromatograph for analysis according to the following chromatographic conditions:

[0050] Column: Syncronis C18, 4.6×250 mm, 5 μm

[0051] Mobile phase A: 0.1% (v / v) phosphoric acid aqueous solution

[0052] Mobile phase B: acetonitrile

[0053] Column temperature: 30°C

[0054] Flow rate: 1.0mL / min

[0055] Detection wavelength: 205nm

[0056] Gradient elution:

[0057] Table 3 Chromatographic gradient

[0058] Time, min Mobile phase A, % (V / V) Mobile phase B, % (V / V) 0 70 30 10 30 70 12 30 70 12.1 70 30 20 70 30

[0059] Table 4 Retention time of substances

[0060] Substance name Retention time RT (min) Boc-L-prolinol 8.607 Boc-L-Proline 7.397

[0061] Example 3 Linear Relationship Investigation

[0062] Take 0.25mL, 0.5mL, 1mL, 1.5mL, and 3mL of the L-proline reference stock solution prepared in Example 1 and place them in 5 10mL volumetric flasks, respectively, and then add 4mL of the derivatizing agent and 1mL of 0.1M sodium hydroxide solution, respectively, and add ethanol: water (1:1, V / V) to the scale after strong shaking for 10min, shake well, and then stand for determination according to the chromatographic conditions shown in Example 2. Determine the peak area integral value, with the injection concentration of the reference substance after derivatization (Boc-L-proline) as the abscissa and the corresponding peak area integral value as the ordinate, draw a standard curve, and calculate the regression equation. The regression equation of Boc-L-proline is: y=0.0644x+0.0008, r=1.0000; the results show that Boc-L-proline has a good linear relationship in the range of 2.30μg / mL to 27.49μg / mL.

[0063] Table 5 Boc-L-proline linear relationship investigation results

[0064] Boc-L-proline (μg / mL) Peak area 2.30 0.146 4.58 0.298 9.16 0.591 13.74 0.886 27.49 1.770

[0065] Example 4 Investigation of detection limit of Boc-L-proline

[0066] Take 1mL of the L-proline reference substance stock solution prepared in Example 1 and place it in a 25mL volumetric flask, add ethanol to dilute to the scale to obtain a dilution, take 1mL of the dilution, place it in a 10mL volumetric flask, add 4mL of a derivatizing agent, 1mL of a 0.1M sodium hydroxide solution, vigorously shake for 10min, then add ethanol: water (1:1, V / V) to make up the volume, shake well to obtain a Boc-L-proline detection limit solution, and after standing, determine according to the chromatographic conditions shown in Example 2. The Boc-L-proline signal-to-noise ratio is 6, and the corresponding injection concentration is 0.368μg / mL (calculated by the preparation method, that is, the L-proline reference substance sample weight divided by the dilution multiple, and then converted to the mass concentration of Boc-L proline) is the detection limit of this method. The present invention provides a highly sensitive detection means for the detection of the L-proline content in L-prolinol.

[0067] Example 5 Repeatability Study

[0068] 1 mL of the L-proline reference stock solution prepared in Example 1 was placed in a 10 mL volumetric flask, 4 mL of a derivatizing agent and 1 mL of a 0.1 M sodium hydroxide solution were added, and after vigorous shaking for 10 min, ethanol: water (1:1, V / V) was added to make up the volume, and after standing, the measurement was repeated 6 times according to the chromatographic conditions shown in Example 2. The results showed that the RSD of the Boc-L-proline peak area was less than 5.0%, and the method had good repeatability.

[0069] Table 6 Boc-L-proline repeatability test (n=6)

[0070]

[0071] Example 6: Sample recovery rate study

[0072] Test solution: Prepare the test solution after derivatization as in Example 1, prepare two portions in parallel. Inject and analyze the prepared solution according to the chromatographic conditions shown in Example 2 to obtain the peak area integral value of Boc-L-proline, substitute it into the regression equation obtained in Example 3, and the average concentration of Boc-L-proline is 0.374 μg / mL, that is, the average concentration of L-proline is 0.2 μg / mL. It can be calculated that the average content of L-proline in the L-prolinol sample is 0.004% (w / w).

[0073] Add sample recovery solution: Take 9 portions of L-proline alcohol of the same batch number, 50 mg each, accurately weigh, and place in 9 10 mL volumetric bottles, numbered 1 to 9. Take 0.5 mL of the L-proline reference stock solution prepared in step 1) of Example 1, 4 mL of the derivatizing agent, and 1 mL of 0.1 M sodium hydroxide solution and add them to volumetric bottles 1 to 3, shake vigorously for 10 minutes, and then add ethanol: water (1:1, V / V) to make up the volume, as a 50% spiked test solution; take 1 mL of the L-proline reference stock solution prepared in step 1) of Example 1, 4 mL of the derivatizing agent, and 1 mL of 0.1 M sodium hydroxide solution and add them to volumetric bottles 4 to 6, shake vigorously for 10 minutes, and then add ethanol: water (1:1, V / V) to make up the volume, as a 100% spiked test solution; take 1.5 mL of the L-proline reference stock solution prepared in step 1) of Example 1, 4 mL of the derivatizing agent, and 1 mL of 0.1 M sodium hydroxide solution and add them to volumetric bottles 4 to 6, shake vigorously for 10 minutes, and then add ethanol: water (1:1, V / V) to make up the volume, as a 100% spiked test solution. 0.1 M sodium hydroxide solution was added to No. 7-9 volumetric flasks, vigorously shaken for 10 min, and then ethanol: water (1:1, V / V) was added to make up to volume to prepare the 150% spiked test solution; after standing, each solution was measured according to the chromatographic conditions shown in Example 2 to obtain the Boc-L-proline peak area integral value, which was substituted into the regression equation obtained in Example 3 to obtain the corresponding Boc-L-proline concentration.

[0074] In Table 7, the theoretical amount added is the corresponding content of Boc-L-proline after derivatization of L-proline reference substance in the prepared solution; the measured amount is the corresponding content of Boc-L-proline in the prepared solution measured by this method; the background amount is the sample amount of L-prolinol * the average value of Boc-L-proline content in the test solution (0.004%, w / w); recovery rate = (measured amount - background amount) / theoretical amount added * 100%. The results show that the recovery rate of Boc-L-proline is between 80% and 120%, and the recovery rate of this method is good.

[0075] Table 7 Boc-L-proline recovery determination results

[0076]

[0077] Example 7 Solution Stability Study

[0078] The solution prepared by the repeatability test of Example 5 was sampled and analyzed after 0 h, 2 h, 6 h, 10 h, and 24 h according to the chromatographic conditions shown in Example 2. The results showed that the RSD of the peak area of ​​Boc-L-proline was less than 5.0%, and the solution prepared by this method was basically stable within 24 h, and could provide accurate analysis results.

[0079] Table 8 Boc-L-proline stability test results

[0080]

[0081] Example 8 Sample Determination

[0082] Take 50 mg of L-prolinol sample of the same batch number, put it into a 10 mL volumetric flask, prepare the test solution after derivatization as in Example 1, prepare 6 copies in parallel, inject and analyze the prepared solution according to the chromatographic conditions shown in Example 2, and obtain the Boc-L-proline peak area integral value. Substitute it into the regression equation obtained in Example 3, and the average concentration of Boc-L-proline is 0.374 μg / mL, that is, the average concentration of L-proline is 0.2 μg / mL. It can be calculated that the average content of L-proline in the L-prolinol sample is 0.004% (w / w), which is less than 0.10%, meeting the requirements.

[0083] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting trace amounts of L-proline in L-prolinol, characterized in that The steps include: 1) Weigh L-proline reference substance and dissolve it in ethanol to obtain L-proline reference substance stock solution; 2) taking different volumes of the L-proline reference substance stock solution obtained in step 1), respectively adding Boc-anhydride solution and sodium hydroxide solution, shaking, to obtain L-proline reference substance solutions after derivatization with different concentrations, i.e., Boc-L-proline reference substance solutions with different concentrations; 3) subjecting the solution prepared in step 2) to high performance liquid chromatography analysis to obtain the peak area integral value of Boc-L-proline; plotting a standard curve with the concentration of Boc-L-proline as the abscissa and the corresponding peak area integral value as the ordinate, and calculating a regression equation; 4) Weigh the L-prolinol sample, add Boc-anhydride solution and sodium hydroxide solution, and shake to obtain the derivatized test solution; 5) subjecting the solution prepared in step 4) to high performance liquid chromatography analysis to obtain the peak area integral value of Boc-L-proline; substituting the peak area integral value of Boc-L-proline into the regression equation obtained in step 3) to obtain the injection concentration of Boc-L-proline, and converting it to obtain the injection concentration of L-proline; The HPLC analysis conditions are as follows: Octadecylsilane bonded silica gel was used as the filler, and the filler particle size was 1.8~6μm; the column length was 150~350mm; phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B for gradient elution; the volume content of phosphoric acid in the phosphoric acid aqueous solution was 0.05~0.4%; the detection wavelength was 190~240nm; the column temperature was 25~40℃; the flow rate was 0.5~1.5mL / min; Step 2) the concentration of Boc-L-proline in the Boc-L-proline reference solution is 0.9-46 μg / mL; Step 2) the volume ratio of the L-proline reference substance stock solution to the Boc-anhydride solution is 0.1~5: (1~5); The oscillation time in step 2) and step 4) is 5 to 30 minutes; after oscillation, the volume is fixed with ethanol aqueous solution; The amount of the ethanol aqueous solution in step 2) and step 4) is such that the concentration of Boc-anhydride is diluted to 3-15 mg / mL; The parameters of the gradient elution are shown in Table 1: Table 1 。 2. The method for detecting trace amounts of L-proline in L-prolinol according to claim 1, wherein: Step 2) the concentration of Boc-L-proline in the Boc-L-proline reference solution is 2.3-27.6 μg / mL; Step 2) The volume ratio of the L-proline reference substance stock solution and the Boc-anhydride solution is (0.25-3):(3-5); The amount of the ethanol aqueous solution in step 2) and step 4) is such that the concentration of Boc-anhydride is diluted to 8-14 mg / mL.

3. A method for detecting trace amounts of L-proline in L-prolinol according to claim 1, characterized in that: The concentration of the Boc-anhydride solution in step 2) and step 4) is 10-50 mg / mL.

4. A method for detecting trace amounts of L-proline in L-prolinol according to claim 3, characterized in that: The concentration of the Boc-anhydride solution in step 2) and step 4) is 20-40 mg / mL.

5. The method for detecting trace amounts of L-proline in L-prolinol according to claim 1, wherein: The concentration of the sodium hydroxide solution in step 2) and step 4) is 0.01-0.5M.

6. A method for detecting trace amounts of L-proline in L-prolinol according to claim 5, characterized in that: The concentration of the sodium hydroxide solution in step 2) and step 4) is 0.05-0.2 M.

7. The method for detecting trace amounts of L-proline in L-prolinol according to claim 1, characterized in that: Step 1) The concentration of the L-proline reference substance stock solution is 0.01-0.1 mg / mL.

8. The method for detecting trace amounts of L-proline in L-prolinol according to claim 7, characterized in that: Step 1) The concentration of the L-proline reference substance stock solution is 0.02-0.08 mg / mL.

9. The method for detecting trace amounts of L-proline in L-prolinol according to claim 1, characterized in that: Step 4) The amount of the L-prolinol sample is such that the concentration of the L-prolinol sample in the test solution after derivatization is 2-8 mg / mL.

10. The method for detecting trace amounts of L-proline in L-prolinol according to claim 9, characterized in that: Step 4) The amount of the L-prolinol sample is such that the concentration of the L-prolinol sample in the test solution after derivatization is 3-6 mg / mL.

11. The method for detecting trace amounts of L-proline in L-prolinol according to claim 1, characterized in that: The volume ratio of the Boc-anhydride solution to the sodium hydroxide solution in step 2) and step 4) is (1-5):

1.

12. The method for detecting trace amounts of L-proline in L-prolinol according to claim 11, characterized in that: The volume ratio of the Boc-anhydride solution to the sodium hydroxide solution in step 2) and step 4) is (3-5):1.

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