Method for determining content of penicillamine enantiomer by precolumn derivatization liquid chromatography

Abstract

The invention provides a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography, namely a method for detecting L-penicillamine in D-penicillamine or a method for detecting D-penicillamine in L-penicillamine, derivatization is carried out under conventional alkaline conditions, a termination reaction is carried out by conventional acid, the derivatization steps are simplified, the detection efficiency is improved, and the detection cost is reduced. According to the method, various interferences of other amino acids and corresponding isomers in the raw materials can be effectively avoided due to the gradient of the mobile phase, the damage to a chromatographic column is well reduced due to the fact that conventional reagents such as phosphoric acid and acetonitrile serve as the mobile phase, and the method is easy to operate, high in sample preparation speed and high in accuracy. The method has the advantages of strong anti-interference capability, good separation effect, high sensitivity, accurate result and good stability, realizes effective separation of penicillamine isomers, has a separation degree of greater than 2, can perform content determination on another main drug while performing impurity detection on one isomer, and has good practicability.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography. Background Art

[0002] Penicillamine (PA) is chemically named 2-amino-3-mercapto-3-methylbutanoic acid, and its molecular formula is C 5 H 11 NO 2 S, which contains a chiral carbon atom in its molecule, is an optically active substance with DL-type (CAS no.52-66-4), D-type (CAS no.52-67-5) and L-type (CAS no. 1113-41-3). Its structure is shown below:

[0003]

[0004] DL-PenicillamineD-PenicillamineL-Penicillamine

[0005] The penicillamine tablets currently used in clinical practice are D-penicillamine, i.e. D-3-mercaptovaline, which is classified as a heavy metal detoxifier and a national essential medicine. It is suitable for heavy metal poisoning, Wilson's disease, cystinuria and its stones, and also for the treatment of severe active rheumatoid arthritis and chronic active hepatitis that are ineffective with other drugs. L-penicillamine is a metabolite of penicillin antibiotics with unique toxicological properties. Its strong inhibitory effect on pyridoxal-5'-phosphate (PLP)-dependent enzymes leads to neurotoxicity in animal experiments, neuritis, bone marrow damage, visual impairment, kidney damage, bone tissue damage, olfactory impairment and cancer, so it has not been used clinically.

[0006] With further research on penicillamine, in addition to the discovery of new therapeutic indications such as D-penicillamine's ability to treat chronic aggressive hepatitis and scleral ulcers, and its preventive effect on premature infant retinopathy, L-penicillamine's medicinal value has been gradually explored, and its status has become increasingly important. It can be used as an inducer of spasms and convulsions, and can significantly reduce the hemodynamic response caused by peroxynitrite, and has potential clinical significance for the treatment of inflammatory diseases related to peroxynitrite production.

[0007] The quality standards of D-penicillamine are included in the Pharmacopoeia of the People's Republic of China (2020 edition), the United States Pharmacopoeia (2024 edition) and the European Pharmacopoeia (11.0 edition), and their quality standards do not include the determination of enantiomers. Currently, the detection of penicillamine isomers mainly includes electrochemistry, fluorescence molecular imprinting, capillary electrochromatography, capillary electrophoresis, circular dichroism, electrospray ionization mass spectrometry, ion mobility mass spectrometry, nanocluster aggregation-induced emission, high performance liquid chromatography, and micellar liquid chromatography. Among them, high performance liquid chromatography uses expensive chiral stationary phase (CSP) or chiral additives (CMPA) for direct detection. Li Yitian's team (Li Yitian, Liu Ying, Li Yang, HPLC pre-column derivatization method for L-penicillamine in penicillamine tablets, West China Journal of Pharmaceutical Sciences, 2022, 37 (1): 75-77) reported the use of ninhydrin as a derivatization reagent for pre-column derivatization determination of L-penicillamine, but there are problems such as insufficient separation, and copper chloride and L-proline are also needed as mobile phases, which increases the burden on the chromatographic column.

[0008] Therefore, a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography is provided, in which the derivatization reagent is cheap and easily available, the derivatization step is simple, the anti-interference ability is strong, the separation effect is good, the sensitivity is high, the results are accurate, and the stability is good. This is a technical problem to be solved in the field of this technology. Summary of the invention

[0009] The purpose of the present invention is to provide a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography. The derivatization reagent is cheap and easy to obtain, the derivatization step is simple, the anti-interference ability is strong, the separation effect is good, the sensitivity is high, the result is accurate, the stability is good, and the system applicability, repeatability, specificity and accuracy are fully in line with the guiding principles of method validation of the Chinese Pharmacopoeia, and the method can be used for quality control of penicillamine isomers.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] In view of some problems of the existing analytical methods, the first aspect of the present invention provides penicillamine isomers, the structure of which is as follows:

[0012] , where R 1 , R 2 is selected from nitro or hydrogen; R 3 is selected from fluorine or chlorine; R 4 Each of them is independently selected from any one of hydrogen, methyl, ethyl, isopropyl, phenyl and benzyl.

[0013] As a preferred technical solution of the present invention, the R 1 is nitro; R 2 is nitro; R 3Selected from fluorine; R 4 It is any of methyl, isopropyl and benzyl.

[0014] The pre-column derivatization reaction of penicillamine provided by the present invention is an alkylation reaction between a derivatization reagent and racemic penicillamine DL-penicillamine, which increases the spatial structural difference of enantiomers, thereby achieving effective separation. The reaction equation of the separation method is as follows (derivation reaction of penicillamine):

[0015]

[0016]

[0017] D-configuration derivatives L-configuration derivatives

[0018] The present invention discloses a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography, namely, a method for detecting L-penicillamine in D-penicillamine or a method for detecting D-penicillamine in L-penicillamine. The detection method comprises the following steps:

[0019] (1) The sample stock solution and the derivatization solution are subjected to a derivatization reaction in an alkaline solution, and then an acid solution is added to terminate the reaction to obtain a derivatization reaction solution;

[0020] (2) The derivatization reaction liquid was detected by liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was a C18 column, the mobile phases were mobile phase A and mobile phase B for gradient elution, mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was acetonitrile, the column temperature was 20-40°C, and the detection wavelength was 335 nm.

[0021] The derivatization liquid described in step (1) is an organic solution of Marfey reagent and its derivatives. Specifically, the Marfey reagent and its derivatives are selected from NA-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDNP-Ala-NH2), NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide (FDNP-Val-NH2), NA-(2,4-dinitro-5-fluorophenyl)-L-phenylalanamide (FDNP-L-Phe-NH2), and NA-(2,4-dinitro-5-fluorophenyl)-L-prolinamide (FDNP-L-Pro-NH2).

[0022] The derivatization liquid is an organic solution of Marfey reagent and its derivatives, and the organic solution is selected from one or more of dimethyl sulfoxide, acetonitrile, acetone, methanol and ethanol.

[0023] More preferably, the organic solution is dimethyl sulfoxide.

[0024] In some embodiments of the present invention, the concentration of the derivative solution is 1-5g / L;

[0025] Preferably the concentration of the derivative solution is 1-3g / L;

[0026] More preferably, the concentration of the derivatization solution is 2 g / L.

[0027] In some embodiments of the present invention, the derivatization solution is prepared by weighing 400 mg of the derivatization reagent into a 200 mL volumetric flask, adding dimethyl sulfoxide to dissolve and dilute to the scale, and shaking well.

[0028] The alkaline solution described in step (1) is an aqueous solution of an alkali, and the alkali is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, dipotassium hydrogen phosphate, triethylamine, ethylenediamine, and aniline.

[0029] More preferably, the base is triethylamine.

[0030] In some embodiments of the present invention, the concentration of the alkaline solution is 0.01-0.1 mol / L;

[0031] Preferably, the concentration of the alkaline solution is 0.03-0.06mol / L;

[0032] More preferably, the concentration of the alkaline solution is 0.05 mol / L.

[0033] The acid solution described in step (1) is an aqueous solution of an acid, and the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, perchloric acid, trifluoroacetic acid, acetic acid, and formic acid.

[0034] More preferably, the acid is acetic acid.

[0035] In some embodiments of the present invention, the concentration of the acid solution is 0.01-0.1 mol / L;

[0036] Preferably, the concentration of the acid solution is 0.03-0.06mol / L;

[0037] More preferably, the concentration of the acid solution is 0.05 mol / L.

[0038] Specifically, the derivatization reaction is: firstly react the sample stock solution with the derivatization solution in the presence of an alkaline solution, and then add an acidic solution.

[0039] Preferably, the concentration of the sample stock solution is 0.5-3 mg / mL;

[0040] More preferably, the concentration of the sample stock solution is 2 mg / mL.

[0041] Specifically, the volume ratio of the sample stock solution to the derivatization solution is 1:(1-10);

[0042] Preferably, the volume ratio of the sample stock solution to the derivatization solution is 1: (4-6);

[0043] More preferably, the volume ratio of the sample stock solution to the derivatization solution is 1:5.

[0044] Specifically, the volume ratio of the sample stock solution to the alkaline solution is 1:(0.1-5);

[0045] Preferably, the volume ratio of the sample stock solution to the alkaline solution is 1: (0.5-2);

[0046] More preferably, the volume ratio of the sample stock solution to the alkaline solution is 1:1.

[0047] Specifically, the reaction time of the sample stock solution and the derivatization solution is 1-90 min;

[0048] Preferably, the reaction time of the sample stock solution and the derivatization solution is 50-70min;

[0049] More preferably, the reaction time between the sample stock solution and the derivatization solution is 60 minutes.

[0050] Specifically, the volume ratio of the sample stock solution to the acid solution is 1:(0.1-5);

[0051] Preferably, the volume ratio of the sample stock solution to the acid solution is 1: (1-3);

[0052] More preferably, the volume ratio of the sample stock solution to the acid solution is 1:2.

[0053] Specifically, the C18 column described in step (2) includes any one of Agilent Z0RBAX SB-C18, Agilent Poroshell 120 EC-C18, Welch Ultimate XB-C18, Welch Ultimate LP-C18, Welch Ultimate AQ-C18, Welch Ultimate PLUS C18, Welch Ultimate ALK-C18, Welch Ultimate XS-C18, YMC Triart C18, Thermo BDS Hypersil C18, Waters SunFire C18, and Waters XBridge C18;

[0054] Preferably, the C18 column in step (2) is Agilent Poroshell 120 EC-C18.

[0055] Specifically, the specifications of the C18 column described in step (2) are 4.6 mm×150 mm×2.7 μm.

[0056] Specifically, the gradient elution procedure described in step (2) is:

[0057] 0min, 40-60%A, 40-60%B;

[0058] 15min, 40-60%A, 40-60%B;

[0059] 25min, 50-70%A, 30-50%B;

[0060] 25.5min, 30-40%A, 60-70%B;

[0061] 27min, 30-40%A, 60-70%B;

[0062] 27.1min, 40-60%A, 40-60%B;

[0063] 33min, 40-60%A, 40-60%B.

[0064] In some embodiments of the present invention, the detection wavelength is 300 nm-340 nm, preferably 330-340 nm, and more preferably 335 nm.

[0065] In some embodiments of the present invention, the temperature of the chromatography column is 30-40°C, preferably 35°C.

[0066] In some embodiments of the present invention, the flow rate of the mobile phase is 0.9-1.1 mL / min, preferably 1.0 mL / min.

[0067] In some embodiments of the present invention, preferably, the injection volume is 5 µL.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The invention adopts derivatization under conventional alkaline conditions and termination reaction with conventional acid, thereby simplifying the derivatization steps and improving the detection efficiency. The invention provides a method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography, which enhances the response value of penicillamine and uses common liquid phase ultraviolet coupling technology to achieve a quantitative limit of 0.3µL / mL. The mobile phase gradient of the method of the invention can effectively avoid various interferences of other amino acids and corresponding isomers in the raw materials. The invention uses conventional reagents such as phosphoric acid and acetonitrile as mobile phases, which greatly reduces the damage to the chromatographic column. The method of the invention is simple to operate, fast in sample preparation speed, and the derivatization reagent is cheap and readily available, has strong anti-interference ability, good separation effect, high sensitivity, accurate results and good stability. The invention realizes the effective separation of penicillamine isomers, with a separation degree greater than 2, and can determine the content of another main drug while detecting an isomer impurity, thereby having good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the result diagram of the specificity investigation;

[0071] Figure 2 This is the result diagram of system suitability investigation;

[0072] Figure 3 It is the linear relationship diagram of the pre-column derivatization working curve of penicillamine L-isomer;

[0073] Figure 4 This is the linear relationship diagram of the pre-column derivatization working curve of penicillamine D-isomer. DETAILED DESCRIPTION

[0074] In order to make the technical solutions, creative features and technical advantages of the present invention easy to understand and appreciate, the technical solutions of the present invention are further clearly and completely explained in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0075] Examples 1 to 7 of the present invention disclose the methodological verification experiments of the present invention.

[0076] Example 1 Specificity Investigation

[0077] 1. Experimental Methods

[0078] Derivatization reagent 1: The structure is as follows:

[0079] , where R 1 , R 2 is nitro; R 3 is fluorine; R 4 It is methyl.

[0080] 1.1 Solution preparation

[0081] Derivatization solution: weigh 400 mg of NA-(2,4-dinitro-5-fluorophenyl)-L-alaninamide into a 200 mL volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, and shake well.

[0082] 0.05 mol / L triethylamine solution: Take 500 mg of triethylamine in a 100 mL volumetric flask, add water to dilute to the scale line, and shake well.

[0083] 0.05 mol / L acetic acid solution: Take 300 mg of acetic acid and put it into a 100 mL volumetric flask, add water to dilute to the mark and shake well.

[0084] Sample stock solution: weigh about 20 mg of the test sample into a 10 mL volumetric flask, add water to dilute to the scale line, and shake well.

[0085] D isomer stock solution: weigh about 20 mg of D isomer into a 20 mL volumetric flask, add water to dilute to the mark, and shake well.

[0086] L isomer stock solution: weigh about 20 mg of L isomer into a 20 mL volumetric flask, add water to dilute to the mark, and shake well.

[0087] Blank solution: Take 1 mL of water in a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, and 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ constant temperature bath and heat for 60 minutes, take out, cool to room temperature, make up to volume with 0.05 mol / L acetic acid solution, and shake well.

[0088] DL sample stock solution: Pipette 1 mL of sample stock solution into a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, 1 mL of 0.05 mol / L triethylamine solution, shake well, heat in a 60 ℃ constant temperature bath for 60 minutes, take out, cool to room temperature, dilute with 0.05 mol / L acetic acid solution, shake well, and prepare 6 copies in parallel. (200 μg / L)

[0089] D isomer localization solution: Pipette 1 mL of D isomer stock solution into a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ constant temperature bath and heat for 60 minutes, take out, cool to room temperature, dilute with 0.05 mol / L acetic acid solution, and shake well. (100 μg / L)

[0090] L isomer localization solution: Pipette 1 mL of L isomer stock solution into a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ constant temperature bath and heat for 60 minutes, take out, cool to room temperature, dilute to volume with 0.05 mol / L acetic acid solution, and shake well. (100 μg / L)

[0091] 1.2 Chromatographic conditions

[0092] This example discloses a specificity investigation experiment of the method of the present invention. The instrument and chromatographic conditions are as follows:

[0093] Agilent 1260 or equivalent instrument; chromatographic column: poroshell 120 EC-C18 (4.6mm×150mm×2.7μm); 0.1% phosphoric acid aqueous solution (take 1g of phosphoric acid, add water to 1000ml) as mobile phase A, acetonitrile as mobile phase B for gradient elution; column temperature 35℃; flow rate 1.0mL / min; detection wavelength UV-335nm; injection volume 5µL.

[0094] The gradient elution program is as follows:

[0095] Table 1 Gradient elution program

[0096]

[0097] 1.3 Specificity experiment

[0098] Take blank solution, DL sample stock solution, D isomer positioning solution, and L isomer positioning solution and inject them into liquid chromatograph respectively. Measure according to the above chromatographic conditions and record the chromatogram. The results are shown in the attached figure. Figure 1 shown.

[0099] The results showed that the blank derivative solution and DL sample did not affect the detection of isomers, and the L isomer eluted earlier than the D isomer. In the DL sample stock solution, i.e. the specific solution, the minimum separation between the peaks was 2.51, which was greater than 1.5; the main peak tailing factor was 1.14, which was less than 2.0; and the theoretical plate number was 35268, which was greater than 3000, all meeting the specificity requirements.

[0100] Example 2 System suitability investigation

[0101] This example discloses a system suitability test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0102] System suitability solution, i.e., mixed reference solution (resolution): Pipette 1 mL of D isomer stock solution and 1 mL of L isomer stock solution into the same 10 mL volumetric flask, add 5 mL of derivatization solution and 1 mL of 0.05 mol / L triethylamine solution, shake well, heat in a 60 ℃ constant temperature bath for 60 minutes, take out, cool to room temperature, make up to volume with 0.05 mol / L acetic acid solution, and shake well.

[0103] Take the mixed reference solution and inject it into the liquid chromatograph. Measure it according to the above chromatographic conditions and record the chromatogram. The results are as shown in the attached Figure 2 As shown: the separation degree of D isomer and L isomer in the system suitability solution is 4.65, which is greater than 2.0; the main peak tailing factor is 1.12, which is less than 2.0; and the theoretical plate number is 36335, which is greater than 3000.

[0104] The method of the present invention meets the requirements for system suitability verification.

[0105] Example 3 Detection Limit and Quantification Limit

[0106] This example discloses the detection limit and quantification limit investigation test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0107] When the signal-to-noise ratio S / N ≥ 3, calculate the detection limit, and when the signal-to-noise ratio S / N ≥ 10, calculate the quantification limit. Inject the sample 6 times continuously.

[0108] LOQ solution (sensitivity solution): Pipette 0.3 mL of D isomer stock solution and 0.3 mL of L isomer stock solution into the same 100 mL volumetric flask, dilute to volume with water, and shake well. Pipette 1 mL of the above solution into a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, and 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ thermostatic bath and heat for 60 minutes, take out, cool to room temperature, dilute to volume with 0.05 mol / L acetic acid solution, and shake well.

[0109] LOD solution: Pipette 0.1mL D isomer stock solution and 0.1mL L isomer stock solution into the same 100mL volumetric flask, dilute to volume with water, and shake well. Pipette 1mL of the above solution into a 10mL volumetric flask, add 1mL water, 5mL derivatization solution, 1mL 0.05mol / L triethylamine solution, shake well, place in a 60℃ thermostatic bath and heat for 60 minutes, take out, cool to room temperature, dilute to volume with 0.05mol / L acetic acid solution, and shake well.

[0110] 1. Limit of quantitation

[0111] Table 2 Quantitation limit test results

[0112]

[0113] The results showed that the quantification limit of L-penicillamine was 0.3 μg / mL, the minimum S / N was 25, both greater than 10, and the peak area RSD was 1.36%, less than 15.0%, accounting for 0.3% of the sample detection concentration;

[0114] The quantitative limit of D-penicillamine was 0.3 μg / mL, the minimum S / N was 18, both were greater than 10, and the peak area RSD was 0.91%, less than 15.0%, accounting for 0.3% of the sample detection concentration.

[0115] The above results show that the detection sensitivity of the method of the present invention meets the requirements.

[0116] 2. Detection limit

[0117] Table 3 Detection limit test results

[0118]

[0119] The results showed that the detection limit of L-penicillamine was 0.1 μg / mL, and the S / N was 8, which was greater than 3, accounting for 0.1% of the sample detection concentration;

[0120] The detection limit of D-penicillamine was 0.1 μg / mL, and the S / N was 7, both greater than 3, accounting for 0.1% of the sample detection concentration.

[0121] The above results show that the method of the present invention meets the detection limit verification requirements.

[0122] Example 4 Linearity and range investigation

[0123] This example discloses the linearity and range investigation test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0124] Take 0.8, 0.9, 1.0, 1.1, 1.2 mL of L isomer stock solution and D isomer stock solution respectively, put them into a 10 mL volumetric flask, add 1 mL of water, 5 mL of derivatization solution, 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ constant temperature bath and heat for 60 minutes, take out, cool to room temperature, dilute with 0.05 mol / L acetic acid solution, shake well, and prepare reference solutions of different concentrations. Use the derivative peak area as the ordinate and the concentration (μg / mL) as the abscissa to make a standard curve. The results are shown in the attached Figure 3 , showing a good linear relationship in the concentration range of 80-120μg / mL, the L-isomer regression equation is y = 64.8953x -271.7206, the correlation coefficient R = 0.9993, greater than 0.999, the y-axis intercept deviation is -4.37, less than 25%; the results are shown in the attached Figure 4The regression equation of D-isomer is y = 60.2483 x - 473.7801, the correlation coefficient R = 0.9969, which is greater than 0.99, and the y-intercept deviation is -8.53, which is less than 25%.

[0125] Example 5 Precision Investigation

[0126] This example discloses a precision test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0127] 1. Repeatability

[0128] The sample stock solution was prepared 6 times, and the test results are shown in Table 4.

[0129] Table 4 Repeatability test results

[0130]

[0131] The results of the repeatability test showed that when the DL racemate content was determined, the average mass content of the L-isomer was 51.4%, and the RSD was 1.48%, which was less than 3.0%; the average mass content of the D-isomer was 52.93%, and the RSD was 1.64%, which was less than 3.0%.

[0132] 2. Precision

[0133] The mixed reference solution was tested 6 times. The test results are shown in Table 5.

[0134] Table 5 Precision test results

[0135]

[0136] The results of the repeatability test showed that when the mixed reference solution was measured, the mean mass content of the L-isomer was 50.9%, and the RSD was 0.23%, which was less than 6.0%; the mean mass content of the D-isomer was 49.1%, and the RSD was 0.19%, which was less than 6.0%.

[0137] Example 6 Accuracy Investigation

[0138] This example discloses an accuracy test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0139] Spiked recovery solution: weigh about 10 mg of the test sample in a 10 mL volumetric flask, dilute to the mark with water, and shake well. Pipette 1 mL of the above solution into a 10 mL volumetric flask, pipette 0.5 mL of the D isomer stock solution and 0.5 mL of the L isomer stock solution into the same 10 mL volumetric flask, add 5 mL of the derivatization solution and 1 mL of 0.05 mol / L triethylamine solution, shake well, place in a 60 ℃ thermostatic bath and heat for 60 minutes, take out, cool to room temperature, add 0.05 mol / L acetic acid solution to the volume, and shake well. Prepare 6 portions in parallel.

[0140] Table 6 Accuracy test results

[0141]

[0142] The results showed that the recovery rate of L-penicillamine was 99.92-101.99%, with an average recovery rate of 100.75%, which met the standard requirement of an average recovery rate between 92.0% and 105.0%, and the RSD was 0.72%, less than 3.0%; the recovery rate of D-penicillamine was 101.65-103.64%, with an average recovery rate of 100.75%, which met the standard requirement of an average recovery rate between 92.0% and 105.0%, and the RSD was 0.71%, less than 3.0%.

[0143] The above results show that the method of the present invention is accurate in detecting isomers.

[0144] Example 7 Study on the Durability of the Method

[0145] This example discloses a method durability test of the method of the present invention, which is carried out using the instrument and chromatographic conditions of Example 1.

[0146] Take LOQ solution, mixed reference solution, and DL sample stock solution, adjust the flow rate to ±1%, and the column temperature to ±2°C. The results are shown in Table 7.

[0147] Table 7 Method durability test results

[0148]

[0149] The results show that compared with the basic chromatographic conditions, the system suitability separation degree of this embodiment is greater than 1.5 at a flow rate of 0.9-1.1 ml / min and a column temperature of 33-37°C. Compared with normal conditions, the maximum absolute value of the content deviation of each solution measured is 1.64%, which is within 5%. The mobile phase flow rate and column temperature are adjusted to meet the durability verification requirements. The method is durable under the above conditions.

[0150] Example 8 Stability Study

[0151] This example discloses a stability test of the method of the present invention, which is performed using the instrument and chromatographic conditions of Example 1.

[0152] Take the DL sample stock solution, place it at room temperature for 24 hours, and take samples every 4 hours to examine the solution stability. The results are shown in Table 8.

[0153] Table 8 Stability test results

[0154]

[0155] The results showed that compared with the newly prepared solution (0h), the maximum absolute value of the content deviation of each solution measured after storage was 0.11%, all within 5%, indicating that the derivatized solution samples remained stable within 24 hours.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, let alone the patent scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the main design concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography, characterized in that: The method comprises the following steps: Step 1: The sample stock solution and the derivatization solution are subjected to a derivatization reaction in an alkaline solution, and then an acid solution is added to terminate the reaction to obtain a derivatization reaction solution; Step 2: The derivatization reaction solution was detected by liquid chromatography, and the chromatographic conditions were: the chromatographic column was a C18 column, the mobile phase was mobile phase A and mobile phase B for gradient elution, mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was acetonitrile, the column temperature was 20-40°C, and the detection wavelength was 335nm; The derivatization solution in step 1 is an organic solution of Marfey reagent and its derivatives, penicillamine isomers, and the structure is as follows: , wherein R1 and R2 are selected from nitro or hydrogen; R3 is selected from fluorine or chlorine; and R4 is independently selected from any one of hydrogen, methyl, ethyl, isopropyl, phenyl and benzyl.

2. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The reaction equation of the separation method is as follows: D-configuration derivatives and L-configuration derivatives.

3. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: R1 is nitro; R2 is nitro; R3 is selected from fluorine; R4 is any one of methyl, isopropyl and benzyl.

4. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The preparation method of the derivatization solution is as follows: weigh 400 mg of the derivatization reagent into a 200 mL volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the scale, and shake well; the concentration of the derivatization solution is 1-5 g / L.

5. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The organic solution is selected from one or more of dimethyl sulfoxide, acetonitrile, acetone, methanol, and ethanol; the alkaline solution is an aqueous solution of alkali, and the alkali is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, dipotassium hydrogen phosphate, triethylamine, ethylenediamine, and aniline; the concentration of the alkaline solution is 0.01-0.1 mol / L; the acid solution is an aqueous solution of acid, and the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, perchloric acid, trifluoroacetic acid, acetic acid, and formic acid; the concentration of the acid solution is 0.01-0.1 mol / L.

6. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The concentration of the sample stock solution is 0.5-3 mg / mL; the volume ratio of the sample stock solution to the derivatization solution is 1:5; the volume ratio of the sample stock solution to the alkaline solution is 1:(0.5-2); the reaction time of the sample stock solution and the derivatization solution is 1-90 min; the volume ratio of the sample stock solution to the acid solution is 1:(0.1-5).

7. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The C18 column includes any one of Agilent Z0RBAX SB-C18, Agilent Poroshell 120 EC-C18, Welch Ultimate XB-C18, Welch Ultimate LP-C18, Welch Ultimate AQ-C18, Welch Ultimate PLUS C18, Welch Ultimate ALK-C18, Welch Ultimate XS-C18, YMCTriart C18, Thermo BDS Hypersil C18, Waters SunFire C18, and Waters XBridge C18.

8. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The gradient elution procedure described in step (2) is: 0min, 40-60%A, 40-60%B; 15min, 40-60%A, 40-60%B; 25min, 50-70%A, 30-50%B; 25.5min, 30-40%A, 60-70%B; 27min, 30-40%A, 60-70%B; 27.1min, 40-60%A, 40-60%B; 33min, 40-60%A, 40-60%B.

9. The method for determining the content of penicillamine enantiomers by pre-column derivatization liquid chromatography according to claim 1, characterized in that: The detection wavelength is 300nm-340nm; the chromatographic column temperature is 30-40℃; the mobile phase flow rate is 0.9-1.1mL / min.

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