CN115932123A - Method for detecting trace L-proline in L-prolinol - Google Patents

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 derivatization agent di-tert-butyl dicarbonate (namely Boc-anhydride) to derivatize L-prolinol and L-proline under the alkaline condition, thereby realizing the separation and determination of trace L-proline in L-prolinol by adopting 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 quickly detecting L-proline in L-prolinol.

Description

Method for detecting trace L-proline in L-prolinol

Technical Field

The invention belongs to the field of analysis, and particularly relates to a method for detecting trace L-proline in L-prolinol.

Background

The L-prolinol has wide sources, low price and environmental protection, is an important chemical reagent, is a raw material for synthesizing excellent optical materials N- (4-nitrophenyl) - (L) -prolinol (NPP) and chiral polymer materials, and is an important chiral source for synthesizing chiral drugs, such as the synthesis of chiral drug avanafil. In the process of synthesizing the medicine, people need to research and control the content of impurities, and the research and control on the content of the impurities of the related materials further ensure the purity of the target medicine. The L-prolinol is mainly generated by reducing cheap and easily available L-proline, and is inevitably accompanied by trace amount 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).

The existing proline detection and analysis methods mainly comprise an electrophoresis method, a liquid chromatography-mass spectrometry method, a pre-column derivatization-high performance liquid chromatography method and the like. The reproducibility, accuracy and sensitivity of the electrophoresis method are poorer than those of high performance liquid chromatography; the liquid chromatography-mass spectrometry instrument is expensive, rare in a conventional laboratory and not wide in popularity.

The L-prolinol and the L-proline have larger polarity, are difficult to be adsorbed on a chromatographic column to form a chromatographic peak, have no ultraviolet characteristic absorption peak, and can not meet the detection requirement only by liquid chromatography detection. The combination of pre-column derivatization and high performance liquid chromatography is the first choice for analyzing and detecting L-proline, and commonly used derivatization reagents include Phenyl Isothiocyanate (PITC), 2, 4-Dinitrofluorobenzene (DNFB), 9-Fluorenylmethoxylchloride (FMOC) and p-nitrobenzyl chloroformate. But the operation of Phenyl Isothiocyanate (PITC) is complex, and the derivatization time is too long; 9-Fluorenylmethoxylchloride (FMOC) is easy to hydrolyze, unstable and has poor effect; 2, 4-Dinitrofluorobenzene (DNFB) melts at room temperature, so that operation is influenced, and derived by-products influence a separation result; p-nitrobenzyl chloroformate is sensitive to moisture, may generate pressure during storage, is incompatible with water, strong alkali, alcohol, amine and acid, and has the defects of inhomogeneous solution after derivatization, troublesome sampling and the like. Therefore, the method for detecting the content of the L-proline in the L-prolinol is lack of a method which is simple to operate, short in analysis time, sensitive, accurate and reliable in result and wide in popularization.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a method for detecting trace L-proline in L-prolinol, which is realized by pre-column derivatization and high performance liquid chromatography. In the prior art, a target compound is derivatized by a reagent containing a benzene ring structure, so that the difference change of ultraviolet characteristic absorption and polarity is introduced for detection, but the derivatization reagent is usually high in toxicity and harsh in derivatization conditions. According to the method, the proline and the prolinol are derivatized by using di-tert-butyl dicarbonate (namely Boc-anhydride), so that the derivatization time is short, the derivatization condition is mild, the derivatization agent is non-toxic and green, no aromatizing group is required to be introduced, the original ultraviolet terminal absorption of the compound is used as the detection wavelength, the baseline separation of the two substances on a C18 chromatographic column can be realized, and the problems of complex operation, time consumption and great harm in the derivatization process in L-proline detection are solved. The invention provides an ideal method for detecting trace L-proline in L-prolinol.

The technology related by the invention is mainly realized by the following scheme:

a method for detecting trace L-proline in L-prolinol comprises the following steps:

1) Weighing an L-proline reference substance, and dissolving with ethanol to obtain an L-proline reference substance stock solution;

2) Taking the L-proline reference substance stock solution obtained in the step 1) with different volumes, respectively adding Boc-anhydride solution and sodium hydroxide solution, and oscillating to obtain derivative L-proline reference substance solutions with different concentrations, namely Boc-L-proline reference substance solutions with different concentrations;

3) Performing high performance liquid chromatography analysis on the Boc-L-proline solution prepared in the step 2), drawing a standard curve by taking the concentration of the Boc-L-proline as an abscissa and the corresponding peak area integral value as an ordinate, and calculating to obtain a regression equation;

4) Weighing an L-prolinol sample, adding a Boc-anhydride solution and a sodium hydroxide solution, and oscillating to obtain a derivatized test sample solution;

5) And (3) carrying out high performance liquid chromatography analysis on the derived sample solution prepared in the step 4) to obtain a peak area integral value of Boc-L-proline, substituting the peak area integral value of Boc-L-proline into the regression equation obtained in the step 3), calculating the sample injection concentration of Boc-L-proline, and converting to obtain the sample injection concentration of L-proline.

The analysis conditions of the high performance liquid chromatography are as follows:

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

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

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

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

The concentration of Boc-L-proline in the Boc-L-proline reference solution in the step 2) is 0.9-46 mu g/mL, preferably 2.3-27.6 mu g/mL.

Step 2), the volume ratio of the L-proline reference substance stock solution to the Boc-anhydride solution is (0.1-5) to (1-5); preferably (0.25-3) to (3-5).

The dosage of the L-prolinol sample in the step 4) meets the following requirements: the concentration of the L-prolinol sample in the test solution after derivatization is 2 to 8mg/mL, preferably 3 to 6mg/mL.

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

The oscillation time of the step 2) and the step 4) is 5-30 min, preferably 10min; after shaking with ethanol: and (4) adding ethanol water with the water volume ratio of 1.

The dosage of the ethanol water solution in the step 2) and the step 4) meets the following requirements: the Boc-anhydride concentration is diluted to 3-15 mg/mL, preferably 8-14 mg/mL.

The parameters of the gradient elution are as in table 1:

TABLE 1

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

Preferably, the parameters of the gradient elution are as in table 2:

TABLE 2

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

The mechanism of the invention is as follows:

the molecular weight of L-proline before derivatization is 115, the molecular weight after derivatization is 215, and the molecular weight after derivatization is 1.87 times that before derivatization.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the method has the characteristics of short derivation time, mild reaction condition, simplicity and convenience in operation, safety, greenness, accuracy, high efficiency, good reproducibility and high sensitivity, and can be used for quickly detecting the L-prolinol.

Drawings

FIG. 1 is a blank derivatizing agent chromatogram.

FIG. 2 is a chromatogram of a 100% spiked test sample derived solution in example 6.

FIG. 3 is a chromatogram of the test solution after the derivatization in example 1.

FIG. 4 is a linear relationship chart of Boc-L-proline in example 3.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The reagents used in the examples are commercially available without specific reference.

The instrument comprises the following steps: a Sammerfet (U3000) high performance liquid chromatograph, a Diode Array Detector (DAD), a Chromeleon data workstation; analytical balance model XP26 (METTLER);

reagent: l-prolinol samples (batch No.: GLS 210501-37109) were from Gill Biochemical (Shanghai) Co., ltd, L-proline reference (batch No.: 0407-RB-0001) was derived from Cato Research Chemicals Inc (CATO) with a purity of 99.3%, di-tert-butyl dicarbonate (i.e., boc-anhydride; batch No.: C2111027-25g, purity 99.0%) was derived from alatin, analytically pure; the acetonitrile is chromatographically pure; other reagents are analytically pure; the water is purified water.

EXAMPLE 1 solution preparation

A derivatizing agent: weighing 3g of Boc-anhydride, and adding 100mL of ethanol for dissolving to obtain the Boc-anhydride.

L-proline control stock: weighing 4.92mg of L-proline reference substance, placing in a 100mL measuring flask, adding ethanol to a constant volume, and shaking uniformly to obtain the final product.

Post-derivatization L-proline control solution: placing 0.25-3 mL of L-proline reference substance stock solution into a 10mL volumetric flask, adding 4mL of derivative agent and 1mL of 0.1M sodium hydroxide solution, strongly oscillating for 10min, and adding ethanol: and (3) adding water (1, V/V) to a constant volume to a scale, and shaking up to obtain the product.

Test solution after derivatization: weighing 50mg of L-prolinol sample, placing the L-prolinol sample in a 10mL measuring flask, adding 4mL of derivatization agent and 1mL of 0.1M sodium hydroxide solution, strongly shaking for 10min, and adding ethanol: and (3) adding water (1, V/V) to a constant volume, and shaking up to obtain the product.

Example 2 high Performance liquid chromatography assay

Precisely sucking 10 μ L of each of the derivatized standard sample solution and the derivatized sample solution, injecting into a liquid chromatograph, and analyzing by sample injection according to the following chromatographic conditions:

a chromatographic column: syncronis C18, 4.6X 250mm,5 μm

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

And (3) mobile phase B: acetonitrile

Column temperature: 30 deg.C

Flow rate: 1.0mL/min

Detection wavelength: 205nm

Gradient elution:

TABLE 3 chromatographic gradient

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

TABLE 4 retention time of substances

Name of substance	Retention time RT (min)
		Boc-L-prolinol	8.607
Boc-L-proline	7.397

Example 3 examination of the Linear relationship

Taking 0.25mL,0.5mL,1mL,1.5mL and 3mL of the L-proline reference substance stock solution prepared in example 1, respectively placing the stock solution in 5 10mL volumetric flasks, respectively adding 4mL of derivative and 1mL of 0.1M sodium hydroxide solution, strongly shaking for 10min, then adding ethanol: water (1, V/V) was added to the volume to the scale, shaken, allowed to stand, and then measured under the chromatographic conditions shown in example 2. Measuring the peak area integral value, taking the sample injection concentration of a reference substance after derivation (Boc-L-proline) as an abscissa and the corresponding peak area integral value as an ordinate, drawing a standard curve, and calculating a regression equation, wherein the Boc-L-proline regression equation is as follows: y =0.0644x +0.0008, r =1.0000; the results showed that Boc-L-proline had a good linear relationship in the range of 2.30. Mu.g/mL to 27.49. Mu.g/mL.

TABLE 5 Boc-L-proline Linear relationship investigation results Table

Boc-L-proline (μ g/mL)	Peak area
		2.30	0.146
4.58	0.298
		9.16	0.591
13.74	0.886
		27.49	1.770

Example 4 Boc-L-proline detection Limit study

Taking 1mL of the stock solution of the L-proline reference substance prepared in the example 1, placing the stock solution into a 25mL measuring flask, adding ethanol to dilute the stock solution to a scale to obtain a diluent, taking 1mL of the diluent, placing the diluent into a 10mL measuring flask, adding 4mL of a derivative and 1mL of 0.1M sodium hydroxide solution, strongly oscillating for 10min, and then adding ethanol: water (1, V/V) was added to a constant volume, and shaken to obtain a Boc-L-proline detection limiting solution, which was then allowed to stand and measured under the chromatographic conditions shown in example 2. The signal-to-noise ratio of the Boc-L-proline is 6, and the corresponding sample injection concentration is 0.368 mu g/mL (which can be calculated by a preparation method, namely the mass concentration of the Boc-L proline is converted by dividing the sample weighing of the L-proline reference substance by the dilution factor). The invention provides a high-sensitivity detection means for detecting the content of L-proline in L-prolinol.

Example 5 repeatability test

Taking 1mL of the L-proline reference stock solution prepared in example 1, placing the L-proline reference stock solution in a 10mL measuring flask, adding 4mL of a derivatization agent and 1mL of a 0.1M sodium hydroxide solution, strongly shaking for 10min, and adding ethanol: water (1, V/V) was added to the constant volume, and the measurement was repeated 6 times under the chromatographic conditions shown in example 2 after leaving it. The results show that: the RSD of the peak area of the Boc-L-proline is less than 5.0 percent, and the method has good repeatability.

TABLE 6 Boc-L-proline repeatability test (n = 6)

Example 6 sample recovery test

Test solution: two portions were prepared in parallel as the test solutions after the derivatization in example 1. And (3) carrying out sample injection analysis on the prepared solution according to the chromatographic condition shown in the example 2 to obtain the peak area integral value of the Boc-L-proline, substituting the peak area integral value into the regression equation obtained in the example 3 to obtain the average concentration of the Boc-L-proline of 0.374 mu g/mL, namely the average concentration of the L-proline of 0.2 mu g/mL, and calculating to obtain the L-prolinol sample with the average content of the L-proline of 0.004% (w/w).

Sample adding and solution recovering: taking 9 parts of L-prolinol of the same batch, weighing 50mg respectively, and placing the weighed materials into 9 10mL measuring bottles with the labels of 1-9. Respectively taking 0.5mL,4mL of derivative and 1mL of 0.1M sodium hydroxide solution of the L-proline reference substance stock solution prepared in the step 1) of the example 1, adding the derivative and the 1mL of 0.1M sodium hydroxide solution into a No. 1-3 measuring flask, strongly shaking for 10min, and adding ethanol: water (1, V/V) is added to the mixture to form a constant volume, and the constant volume is used as a 50% added standard test solution; taking 1mL of the L-proline reference substance stock solution prepared in the step 1) of the example 1, 4mL of the derivative and 1mL of the 0.1M sodium hydroxide solution, adding the solution into a No. 4-6 measuring flask, strongly shaking for 10min, and then adding ethanol: water (1, V/V) is added to the sample solution to form a constant volume, and the constant volume is taken as 100% added standard sample solution; 1.5mL,4mL of derivative and 1mL of 0.1M sodium hydroxide solution of the L-proline reference substance stock solution prepared in the step 1) of the example 1 are respectively added into a No. 7-9 measuring flask, strongly shaken for 10min, and then added with ethanol: water (1, V/V) is added to a constant volume to be used as a 150% standard sample solution; after standing, each solution was measured under the chromatographic conditions shown in example 2 to obtain an integrated Boc-L-proline peak area value, which was substituted into the regression equation obtained in example 3 to obtain the corresponding Boc-L-proline concentration.

In Table 7, the theoretical amount added is the Boc-L-proline content after derivatization of the L-proline reference in the prepared solution; the measured amount is the corresponding content of Boc-L-proline in the prepared solution measured by the method; the background amount is L-prolinol sample weighing amount, the average value (0.004%, w/w) of Boc-L-proline content in the test solution; recovery = (measured-background)/theoretical addition 100%. The result shows that the recovery rate of the Boc-L-proline is between 80 and 120 percent, and the method has better recovery rate.

TABLE 7 Boc-L-proline recovery assay results Table

Example 7 solution stability Studies

The solutions obtained in example 5 were repeatedly analyzed at 0h,2h,6h,10h and 24h, and then subjected to sample injection analysis under the chromatographic conditions shown in example 2. The results show that: the RSD of the peak area of the Boc-L-proline is less than 5.0%, the solution prepared by the method is basically stable within 24h, and an accurate analysis result can be provided.

TABLE 8 Boc-L-proline stability assay results Table

EXAMPLE 8 sample determination

Taking 50mg of the same batch of L-prolinol sample, placing the sample in a 10mL measuring flask, preparing 6 parts in parallel with the sample solution after deriving in the example 1, carrying out sample injection analysis on the prepared solution according to the chromatographic conditions shown in the example 2 to obtain the peak area integral value of Boc-L-proline, substituting the peak area integral value into the regression equation obtained in the example 3 to obtain the average concentration of Boc-L-proline of 0.374 mu g/mL, namely the average concentration of L-proline of 0.2 mu g/mL, and calculating to obtain the L-prolinol sample with the average content of L-proline of 0.004% (w/w) which is less than 0.10%, thus meeting the requirement.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A method for detecting trace L-proline in L-prolinol is characterized by comprising the following steps:

1) Weighing an L-proline reference substance, and dissolving with ethanol to obtain an L-proline reference substance stock solution;

2) Taking the L-proline reference substance stock solution obtained in the step 1) with different volumes, respectively adding Boc-anhydride solution and sodium hydroxide solution, and oscillating to obtain derivative L-proline reference substance solutions with different concentrations, namely Boc-L-proline reference substance solutions with different concentrations;

3) Carrying out high performance liquid chromatography analysis on the solution prepared in the step 2) to obtain a peak area integral value of Boc-L-proline; taking the concentration of Boc-L-proline as an abscissa and the integral value of the corresponding peak area as an ordinate, drawing a standard curve, and calculating to obtain a regression equation;

4) Weighing an L-prolinol sample, adding a Boc-anhydride solution and a sodium hydroxide solution, and oscillating to obtain a derivatized test solution;

5) Carrying out high performance liquid chromatography analysis on the solution prepared in the step 4) to obtain a peak area integral value of Boc-L-proline; substituting the integrated value of the peak area of the Boc-L-proline into the regression equation obtained in the step 3) to obtain the sample injection concentration of the Boc-L-proline, and converting to obtain the sample injection concentration of the L-proline.

2. The method for detecting trace L-proline in L-prolinol according to claim 1, which is characterized in that the conditions of high performance liquid chromatography are as follows:

octadecylsilane chemically bonded silica is used as a filler, and the particle size of the filler is 1.8-6 mu m; the length of the column is 150-350 mm; taking phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B for gradient elution; the volume content of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.4%; the detection wavelength is 190-240 nm; the column temperature is 25-40 ℃; the flow rate is 0.5-1.5 mL/min.

3. The method for detecting the trace amount of L-proline in L-prolinol according to claim 1, which is characterized in that: the concentration of the Boc-anhydride solution in the step 2) and the step 4) is 10-50 mg/mL, and preferably 20-40 mg/mL.

4. The method for detecting trace L-proline in L-prolinol according to claim 1, which comprises the following steps: the concentration of the sodium hydroxide solution in the step 2) and the step 4) is 0.01-0.5M, preferably 0.05-0.2M.

5. The method for detecting trace L-proline in L-prolinol according to claim 1, which comprises the following steps: the concentration of the L-proline reference substance stock solution in the step 1) is 0.01-0.1 mg/mL, preferably 0.02-0.08 mg/mL.

6. The method for detecting the trace amount of L-proline in L-prolinol according to claim 1, which is characterized in that:

the concentration of Boc-L-proline in the Boc-L-proline reference substance solution in the step 2) is 0.9-46 mu g/mL, preferably 2.3-27.6 mu g/mL;

the volume ratio of the L-proline reference substance stock solution to the Boc-anhydride solution in the step 2) is 0.1-5 (1-5), preferably (0.25-3) to (3-5).

7. The method for detecting trace L-proline in L-prolinol according to claim 1, which comprises the following steps: the dosage of the L-prolinol sample in the step 4) meets the following requirements: the concentration of the L-prolinol sample in the test solution after derivatization is 2 to 8mg/mL, preferably 3 to 6mg/mL.

8. The method for detecting trace L-proline in L-prolinol according to claim 1, which comprises the following steps: the volume ratio of the Boc-acid anhydride solution to the sodium hydroxide solution in the steps 2) and 4) is (1-5) to 1, preferably (3-5) to 1.

9. The method for detecting trace L-proline in L-prolinol according to claim 1, which comprises the following steps:

the oscillation time of the step 2) and the step 4) is 5-30 min; after oscillation, the volume is determined by ethanol water;

the dosage of the ethanol water solution in the step 2) and the step 4) meets the following requirements: the Boc-anhydride concentration is diluted to 3 to 15mg/mL, preferably 8 to 14mg/mL.

10. The method for detecting trace amount of L-proline in L-prolinol according to claim 2, which comprises the following steps: the parameters of the gradient elution are as in table 1:

TABLE 1

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

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