Detection method of N-methyl-D-prolinol isomer
Through derivatization reaction of N-methyl-D-proline and high-performance liquid chromatography detection, the problem that the prior art cannot detect its isomers is solved, and the detection effect of high accuracy and sensitivity is achieved.
Patent Information
- Application Number
- CN202411125000.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-08-18
- Filing Date
- 2024-08-16
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art cannot effectively detect isomers of N-methyl-D-proline, which leads to huge difficulties in the development of its detection methods.
Derivative reaction of N-methyl-D-proline with acid chloride derivatization reagents was performed to generate derivatized products, and the detection was performed by high-performance liquid chromatography. The content of isomers was calculated by area normalization method. .
The detection of the isomer of N-methyl-D-proline isomers with high accuracy, sensitivity and repetition were achieved, and its isomers were effectively isolated and quantitatively analyzed.
Smart Images
Figure BDA0004996138310000011 
Figure BDA0004996138310000012 
Figure BDA0004996138310000041
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of drug detection, and in particular to a method for detecting N-methyl-D-prolinol isomers. Background Art
[0002] N-methyl-D-prolinol is an important intermediate material with the chemical structure:
[0003]
[0004] Its isomer is N-methyl-L-prolinol, and its chemical structure is:
[0005]
[0006] Since the compound has a simple structure and no chromophore, conventional chiral separation methods cannot detect the content of isomers, and there is currently no literature or other information reporting on methods for detecting isomers, which brings great difficulties to its method development.
[0007] The present disclosure provides a method for detecting N-methyl-D-prolinol isomers, by derivatizing N-methyl-D-prolinol with the aid of some compounds, thereby improving its chromatographic retention, increasing optical groups, and achieving separation, qualitative and quantitative analysis of its isomers. Summary of the invention
[0008] The present invention relates to a method for detecting N-methyl-D-prolinol isomers with high accuracy, high sensitivity and good repeatability, and the method can effectively separate N-methyl-D-prolinol and isomers.
[0009] The present disclosure provides a method for detecting N-methyl-D-prolinol isomers, the method comprising the following steps:
[0010] Mixing N-methyl-D-prolinol with a derivatization reagent, performing a derivatization reaction, adding a diluent, and obtaining a derivatization product;
[0011] The derivatized product solution was detected by high performance liquid chromatography, and the content of isomers was calculated by area normalization method.
[0012] In some embodiments, the conditions used in the high performance liquid chromatography method include: mobile phase A is aqueous ammonium bicarbonate solution, and mobile phase B is acetonitrile.
[0013] In some embodiments, the concentration of the aqueous ammonium bicarbonate solution is 10-30 mmol. In some embodiments, the concentration of the aqueous ammonium bicarbonate solution is 20 mmol.
[0014] In some embodiments, the derivatization agent is selected from acyl chloride reagents, including but not limited to one or more of benzoyl chloride, p-methoxybenzoyl chloride, p-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride, and 9-fluorenylmethyl chloroformate. In some embodiments, the derivatization agent is selected from benzoyl chloride.
[0015] In some embodiments, the solvent environment of the derivatization reaction is an acetonitrile solution.
[0016] In some embodiments, the elution gradient is: the volume ratio of mobile phase A to mobile phase B is selected from 10:90 to 90:10. In some embodiments, the volume ratio of mobile phase A to mobile phase B is 50:50.
[0017] In some embodiments, the chromatographic column in the high performance liquid chromatography method is a chiral chromatographic column, the filler particle size is 1-10 μm, the chromatographic column length is 50-250 mm, and the inner diameter of the chromatographic column is 2-10 mm.
[0018] In some embodiments, the chiral chromatographic column is of model CHIRALPAK AY-3R, CHIRALPAK AZ-3R or CHIRALPAK AD-3R, the filler particle size is 3 μm, the chromatographic column length is 150 mm, and the inner diameter of the chromatographic column is 4.6 mm.
[0019] In some embodiments, the column temperature is 5-25° C. Preferably, the column temperature is 20° C.
[0020] In some embodiments, the injection volume is 5-20 μL. Preferably, the injection volume is 10 μL.
[0021] In some embodiments, the flow rate is 0.1-1.5 mL / min. Preferably, the flow rate is 0.5 mL / min.
[0022] In some embodiments, the detection wavelength ranges from 200-254 nm. Preferably, the detection wavelength is 231 nm.
[0023] In some embodiments, the diluent is a mixture of aqueous ammonium bicarbonate solution and acetonitrile. In some embodiments, the volume ratio of aqueous ammonium bicarbonate solution to acetonitrile is 1:9-9:1, preferably 1:1. In some embodiments, the concentration of aqueous ammonium bicarbonate solution is 10-30 mmol, preferably 20 mmol.
[0024] The present disclosure provides a method for detecting isomers in N-methyl-D-prolinol, which specifically comprises the following steps:
[0025] Mixing N-methyl-D-prolinol with an acetonitrile solution of benzoyl chloride to carry out a derivatization reaction, and adding a diluent to obtain a derivatized product, wherein the diluent is a mixture of 20 mmol of an aqueous solution of ammonium bicarbonate and acetonitrile in a volume ratio of 1:1;
[0026] The derivatized product solution was detected by high performance liquid chromatography, and the content of the isomers was calculated by area normalization method. The chromatographic conditions were as follows:
[0027] Mobile phase A: 20 mmol ammonium bicarbonate aqueous solution
[0028] Mobile phase B: acetonitrile
[0029] Chromatographic column: CHIRALPAK AY-3R (4.6*150mm, 3μm)
[0030] Gradient: Mobile phase A: Mobile phase B = 50:50 (V / V), isocratic elution for 30 min
[0031] Column temperature: 20℃
[0032] Flow rate: 0.5mL / min
[0033] Detection wavelength: 231nm
[0034] Injection volume: 10 μL BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 :Chromatograms of N-methyl-D-prolinol and its isomers after derivatization
[0036] Figure 2 : RP-CAD model direct measurement diagram
[0037] Figure 3 :Direct determination of gas phase chiral column DETAILED DESCRIPTION
[0038] The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or commodity manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.
[0039] Experimental Materials
[0040] name factory batch number Benzoyl chloride MacLean's reagent C14362481 Ammonium bicarbonate Sinopharm Group 20210219 Acetonitrile Merck SHBN9562
[0041] Example 1: Liquid Chromatographic Analysis of N-Methyl-D-Prolinol and Its Isomer Derivatization Products
[0042] Preparation of derivatization reagent: Accurately pipette 0.5 mL of benzoyl chloride into a 100 mL volumetric flask, dilute to volume with acetonitrile, and shake well to obtain a 0.5% v / v benzoyl chloride acetonitrile solution.
[0043] Preparation of test solution: Pipette 10 μL of N-methyl-D-prolinol into a 100 mL volumetric flask, dissolve it with 2.5 mL of acetonitrile, then dropwise add 2.5 mL of the derivatization reagent, shake gently for 3-5 seconds, and then dilute to the mark with a quencher (a mixture of 20 mmol of aqueous ammonium bicarbonate solution and acetonitrile, in a volume ratio of 1:1).
[0044] Chromatographic conditions:
[0045] Mobile phase A: 20 mmol ammonium bicarbonate aqueous solution
[0046] Mobile phase B: acetonitrile
[0047] Chromatographic column: CHIRALPAK AY-3R (4.6*150mm, 3μm)
[0048] Gradient: Mobile phase A: Mobile phase B = 50:50 (V / V), isocratic elution for 30 min
[0049] Column temperature: 20℃
[0050] Flow rate: 0.5mL / min
[0051] Detection wavelength: 231nm
[0052] Injection volume: 10 μL
[0053] Sample plate temperature: 5°C
[0054] Test results: See attached Figure 1 , wherein RT=7.694min is the peak of the N-methyl-D-prolinol isomer derivatization product, and RT=10.345min is the peak of the N-methyl-D-prolinol derivatization product.
[0055] Calculation formula:
[0056] The isomer content was calculated using the area normalization method.
[0057] Isomer content (%) = L / (D+L), L is the peak area of N-methyl-D-prolinol isomer, and D is the peak area of N-methyl-D-prolinol.
[0058] Example 2: Solution stability experiment
[0059] Referring to the chromatographic conditions and sample preparation method in Example 1, a sample solution was injected at 0h and 24h respectively, and the results are as follows:
[0060] Table 1 Stability results of test solutions
[0061] time Isomer peak area Main peak area Isomer content% Starting 84.0 10477.1 0.80％ 24h 83.4 10358.5 0.80％
[0062] Conclusion: It can be seen from the above table that the test solution is stable within 24 hours under this condition.
[0063] Example 3: Repeatability and recovery test
[0064] Background: Measure 10 μL of N-methyl-D-prolinol into a 100 mL volumetric flask, add 2.5 mL of acetonitrile to dissolve, then slowly add 2.5 mL of the derivatization reagent, shaking while adding, dilute to volume with the mobile phase after the addition is complete, shake well, and use this as the test solution. Prepare 6 copies in parallel;
[0065] Isomer stock solution: accurately weigh about 100 mg of N-methyl-L-prolinol into a 100 mL volumetric flask, dissolve it with acetonitrile and dilute to the mark, then accurately transfer 5 mL to a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well;
[0066] Low concentration point accuracy: Accurately weigh about 100 mg of N-methyl-D-prolinol into a 25 mL volumetric flask, and accurately transfer 1 mL of the isomer stock solution into it, dilute to volume with acetonitrile, then transfer 2.5 mL to a 100 mL volumetric flask, add 2.5 mL of the derivatization solution, and then dilute to volume with the quencher, and prepare three parallel copies.
[0067] Accuracy at mid-concentration point: Accurately weigh about 100 mg of N-methyl-D-prolinol into a 25 mL volumetric flask, and accurately pipette 3 mL of the isomer stock solution into it, dilute to volume with acetonitrile, then pipette 2.5 mL into a 100 mL volumetric flask, add 2.5 mL of the derivatization reagent, and then dilute to volume with the quencher, and prepare three parallel copies.
[0068] High concentration point accuracy: Accurately weigh about 100 mg of N-methyl-D-prolinol into a 25 mL volumetric flask, and accurately transfer 5 mL of the isomer stock solution into it, dilute to the mark with acetonitrile, then transfer 2.5 mL to a 100 mL volumetric flask, add 2.5 mL of the derivatization reagent, and then dilute to the mark with the quencher, and prepare three parallel copies.
[0069] 3.1 Repeatability
[0070]
[0071]
[0072] Conclusion: The repeatability is good.
[0073] 3.2 Sample recovery rate
[0074]
[0075] Conclusion: The recovery rate calculated by area normalization method is between 90% and 110%, which is a good recovery rate.
[0076] Since the compound has no ultraviolet absorption, a reversed-phase HPLC-CAD method was attempted for direct determination. The chromatographic conditions are as follows:
[0077] Mobile phase A: 10 mmol ammonium acetate aqueous solution;
[0078] Mobile phase B: acetonitrile
[0079] Chromatographic column: Chiralcel OX-3R, 4.6*150mm, 3μm
[0080] Gradient: Mobile phase A: Mobile phase B = 90:10, isocratic 15 min
[0081] Flow rate: 0.5ml / min
[0082] Injection volume: 20 μL
[0083] Column temperature: 25°C
[0084] Detector: CAD
[0085] Test solution: Accurately measure 10 μL of N-methyl-D-prolinol and place it in 1 ml of water. Shake to dissolve.
[0086] Conclusion: As attached Figure 2 As shown, due to the high polarity of this compound, the reverse phase mode could not be retained when directly measured.
[0087] Comparative Example 2: GC direct determination
[0088] The boiling point of the compound was found to be 68-71°C, and gas chromatography was attempted using a chiral column for separation. The chromatographic conditions were as follows:
[0089] Injection method: direct injection
[0090] Chromatographic column: CP-Chiralsil-L-VAL, 25m*0.25mm, 0.12μm
[0091] Inlet temperature: 200°C
[0092] Flow rate: 1mL / min
[0093] Program temperature: starting temperature 40℃, heating to 100℃ at 5℃ / min Detector temperature: 260℃
[0094] Split ratio: 1:1
[0095] Carrier gas: Nitrogen
[0096] Detector: FID
[0097] Test solution: Accurately measure 10 μL of N-methyl-D-prolinol in 1 ml of methanol and shake to dissolve. Conclusion: Figure 3 As shown, the main peak cannot be separated from the isomers and the peak shape is poor.
Claims
1. A method for detecting N-methyl-D-prolinol isomers, comprising the following steps: Mixing N-methyl-D-prolinol with a derivatization reagent to perform a derivatization reaction, adding a diluent to obtain a derivatization product solution; Using high performance liquid chromatography to detect the derivatized product solution; in, The conditions adopted by the high performance liquid chromatography method include: mobile phase A is ammonium bicarbonate aqueous solution, and mobile phase B is acetonitrile.
2. The detection method according to claim 1, wherein the derivatization reagent is selected from acyl chloride reagents, preferably one or more of benzoyl chloride, p-methoxybenzoyl chloride, p-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride, and 9-fluorenylmethyl chloroformate, and most preferably benzoyl chloride.
3. According to the detection method of claim 1 or 2, the solvent environment of the derivatization reaction is acetonitrile solution.
4. The detection method according to any one of claims 1 to 3, wherein the volume ratio of mobile phase A to mobile phase B is 10:90-90:10, preferably 50:
50.
5. The detection method according to any one of claims 1 to 4, wherein the column temperature of the HPLC is 5-25°C, preferably 20°C.
6. According to the detection method according to any one of claims 1 to 5, the injection volume of the high performance liquid chromatography is 5-20 μL, preferably 10 μL.
7. According to the detection method according to any one of claims 1 to 6, the flow rate of the high performance liquid chromatography is 0.1-1.5 mL / min, preferably 0.5 mL / min.
8. According to the detection method according to any one of claims 1 to 7, the detection wavelength of the high performance liquid chromatography is 200-254 nm, preferably 231 nm.
9. The detection method according to any one of claims 1 to 8, wherein the diluent is a mixture of aqueous ammonium bicarbonate solution and acetonitrile.
10. A method for detecting N-methyl-D-prolinol isomers, comprising the following steps: Mixing N-methyl-D-prolinol with an acetonitrile solution of benzoyl chloride to carry out a derivatization reaction, and adding a diluent to obtain a derivatized product, wherein the diluent is a mixture of 20 mmol of an aqueous solution of ammonium bicarbonate and acetonitrile in a volume ratio of 1:1; The derivatized product solution was detected by high performance liquid chromatography to obtain the content of isomers in N-methyl-D-prolinol. The chromatographic conditions were as follows: Mobile phase A: 20 mmol ammonium bicarbonate aqueous solution Mobile phase B: acetonitrile Chromatographic column: CHIRALPAK AY-3R (4.6*150mm, 3μm) Gradient: Mobile phase A: Mobile phase B = 50:50 Column temperature: 20℃ Flow rate: 0.5mL / min Detection wavelength: 231nm Injection volume: 10 μL.