A method for separating and detecting formic acid in droxidopa

Abstract

The present invention relates to a method for separating and detecting formic acid in droxidopa, and belongs to the field of analytical chemistry. The method comprises: using octylsilane as a chromatographic column as a filler, the mobile phase is divided into phase A and phase B, the phase A is a perchloric acid aqueous solution, and the phase B is an alcohol. The method of the present invention has high sensitivity, good specificity, and reliable accuracy.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and in particular to a method for separating and detecting formic acid in droxidopa. Background Art

[0002] Droxidopa, CAS: 23651-95-8, has a chemical structure as shown in the following formula I:

[0003]

[0004] According to ICH Q3C solvent classification, formic acid belongs to Class III solvents. Formic acid is produced during the synthesis of droxidopa, so the residual formic acid needs to be controlled and detected.

[0005] At present, the methods for testing formic acid reported in the literature mainly include the following methods: 1) Reverse phase HPLC method for direct formic acid testing. Since formic acid retention is relatively weak, the solvent peak and impurities in droxidopa are likely to interfere with formic acid detection; 2) Exclusion column for direct testing. Since droxidopa is retained on the exclusion column, it is likely to interfere with formic acid detection; 3) IC method for direct testing. Since droxidopa is strongly retained on IC, it is difficult to elute, which can easily lead to a short life of the chromatographic column; 4) Pre-column derivatization followed by GC method for indirect testing. Compared with the direct testing method, it has the problem of relatively complicated operation.

[0006] Therefore, in order to better and more accurately control the impurities in droxidopa products and ensure the quality of the drugs, it is necessary to study an analytical method suitable for formic acid in droxidopa. Summary of the invention

[0007] The object of the present invention is to provide a method for separating and detecting formic acid in droxidopa. The method of the present invention can effectively separate and determine the content of formic acid in droxidopa, the method has high sensitivity, good specificity, reliable accuracy, and can be used for quality control of formic acid in droxidopa.

[0008] A method for separating and detecting formic acid in droxidopa comprises: using a chromatographic column filled with octylsilane, wherein the mobile phase is divided into a phase A and a phase B, wherein the phase A is a perchloric acid aqueous solution and the phase B is an alcohol.

[0009] In some embodiments, the filler of the chromatography column is octylsilane.

[0010] The chromatographic column is selected from at least one of reverse phase RX-C8, SB-C8 and SB-C18 chromatographic columns. In some embodiments, the chromatographic column is selected from at least one of reverse phase RX-C8 and SB-C8 chromatographic columns, which is beneficial for the separation and detection of formic acid.

[0011] In some embodiments, the chromatographic column is selected from at least one of an Agilent ZORBAX Rx C8 column and an Agilent ZORBAXSB C8 column.

[0012] In some embodiments, the chromatographic column is connected in series with two chromatographic columns of the same specification. In some embodiments, the chromatographic column is connected in series with two Agilent ZORBAX Rx C8 columns. In some embodiments, the chromatographic column is connected in series with two Agilent ZORBAX SB C8 columns.

[0013] In some embodiments, the chromatographic column is an Agilent ZORBAX Rx C8 column purchased from Agilent.

[0014] In some embodiments, the volume ratio of perchloric acid to water in phase A is 2:1000 to 4:1000. In some embodiments, the volume ratio of perchloric acid to water is 3:1000.

[0015] In phase B, the alcohol is at least one of methanol, ethanol or isopropanol. In some embodiments, the alcohol in phase B is methanol.

[0016] In some embodiments, the volume ratio of phase A to phase B is 98:2 to 96:4. In some embodiments, the volume ratio of phase A to phase B is 97:3.

[0017] In some embodiments, the method for separating and detecting formic acid in droxidopa may include or be implemented by the following steps:

[0018] 1) Take an appropriate amount of the sample to be tested containing formic acid, add solvent to dissolve, and mix well;

[0019] 2) Set the instrument parameters: detector, chromatographic column, wavelength, flow rate of mobile phase, column temperature;

[0020] 3) Taking a certain amount of the solution in step 1), injecting it into a high performance liquid chromatograph to complete the separation and determination of formic acid.

[0021] The solvent in step 1) is aqueous HCl. In some embodiments, the solvent is 0.1N HCl.

[0022] In step 1), each 1 ml of the solvent may contain 2 mg to 8 mg of the sample to be tested. In some embodiments, in step 1), each 1 ml of the solvent may contain 5 mg of the sample to be tested.

[0023] In step 2), the detector is a VWD detector.

[0024] In step 2), the wavelength may be 210 nm to 215 nm. In some embodiments, the wavelength is 215 nm.

[0025] In step 2), the flow rate of the mobile phase may be 0.5 ml / min to 1.5 ml / min. In some embodiments, in step 2), the flow rate of the mobile phase is 0.5 ml / min. In some embodiments, in step 2), the flow rate of the mobile phase is 1.0 ml / min. In some embodiments, in step 2), the flow rate of the mobile phase is 1.5 ml / min.

[0026] In step 2), the temperature of the chromatographic column box is 25° C. to 35° C. In some embodiments, the temperature of the chromatographic column box is 25° C.; in some embodiments, the temperature of the chromatographic column box is 30° C.; in some embodiments, the temperature of the chromatographic column box is 35° C.

[0027] In step 3), the sample solution injection volume may be 2 μl to 10 μl. In some embodiments, the sample solution injection volume is 2 μl. In some embodiments, the sample solution injection volume is 5 μl. In some embodiments, the sample solution injection volume is 10 μl.

[0028] In some embodiments, the method for separating and detecting formic acid comprises or can be implemented according to the following steps:

[0029] 1) Take an appropriate amount of the sample containing formic acid, add 0.1N HCl to dissolve, and mix well;

[0030] 2) Set the flow rate of the mobile phase to 0.5 ml / min to 1.5 ml / min, the detector to a VWD detector, the chromatographic column to use two Agilent ZORBAX Rx C8 columns in series or two Agilent ZORBAX SB C8 columns in series, the flow rate of the mobile phase to 0.5 ml / min to 1.5 ml / min, and the column box temperature to 25°C to 35°C;

[0031] 3) Take 2 μl to 10 μl of the sample solution in step 1) and inject it into a high performance liquid chromatograph to complete the separation and determination of formic acid.

[0032] The high performance liquid chromatograph can be an American Agilent 1260 high performance liquid chromatography system and workstation or other suitable and feasible systems.

[0033] In some embodiments, the method for separating and detecting formic acid in droxidopa comprises: using a chromatographic column filled with octylsilane bonded phase, the mobile phase is divided into phase A and phase B, phase A is an aqueous solution of perchloric acid, and phase B is alcohol.

[0034] In some embodiments, the method for separating and detecting formic acid in droxidopa comprises: using a chromatographic column filled with octadecylsilane bonded phase, the mobile phase is divided into phase A and phase B, phase A is an aqueous solution of perchloric acid, and phase B is methanol.

[0035] In some embodiments, the method for separating and detecting formic acid in droxidopa comprises: using two Agilent ZORBAX Rx C8 columns in series, the mobile phase is divided into phase A and phase B, phase A is an aqueous solution of perchloric acid, and phase B is methanol.

[0036] In some embodiments, the method for separating and detecting formic acid in droxidopa comprises: using two Agilent ZORBAX SB C8 columns in series, the mobile phase is divided into phase A and phase B, phase A is an aqueous solution of perchloric acid, and phase B is methanol.

[0037] By adopting the separation method of the present invention, the time for separating and detecting formic acid can be within 15 minutes.

[0038] In the above or below, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. The value of each number may differ by 1%, 2%, 5%, 7%, 8% or 10%.

[0039] The method provided by the present invention can effectively separate formic acid impurities in the drug droxidopa, with a separation degree of more than 1.5 and complete baseline separation, and can solve the problems of weak retention of droxidopa in liquid chromatography, easy interference of formic acid detection, and inaccurate detection results of low-concentration formic acid. The method of the present invention is simple, fast, accurate and highly sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Blank solution chromatogram in Example 1;

[0041] Figure 2 Chromatogram of formic acid control solution in Example 1;

[0042] Figure 3 Chromatogram of droxidopa spiked solution in Example 1;

[0043] Figure 4 Blank solution chromatogram in Example 2;

[0044] Figure 5 Chromatogram of formic acid control solution in Example 2;

[0045] Figure 6 Chromatogram of droxidopa spiked solution in Example 2;

[0046] Figure 7 Blank solution chromatogram in Example 3;

[0047] Figure 8 Chromatogram of formic acid control solution in Example 3;

[0048] Fig. 9 Chromatogram of droxidopa spiked solution in Example 3;

[0049] Fig.10 Blank solution chromatogram in Example 4;

[0050] Fig.11 Chromatogram of formic acid control solution in Example 4;

[0051] Fig.12 Chromatogram of droxidopa spiked solution in Example 4;

[0052] Fig.13 Blank solution chromatogram in Example 5;

[0053] Fig.14 Chromatogram of formic acid control solution in Example 5;

[0054] Fig.15 Chromatogram of droxidopa spiked solution in Example 5;

[0055] Fig.16 Blank solution chromatogram in Example 6;

[0056] Fig.17 Chromatogram of formic acid control solution in Example 6;

[0057] Fig.18 Chromatogram of droxidopa spiked solution in Example 6;

[0058] Fig.19 Blank solution chromatogram in Example 7;

[0059] Fig. 20 Chromatogram of formic acid control solution in Example 7;

[0060] Fig.21 Chromatogram of droxidopa spiked solution in Example 7;

[0061] Fig. 22 Blank solution chromatogram in Example 8;

[0062] Fig.23 Chromatogram of formic acid control solution in Example 8;

[0063] Fig.24 Chromatogram of droxidopa spiked solution in Example 8;

[0064] Fig.25 Blank solution chromatogram in Example 9;

[0065] Fig.26 Chromatogram of formic acid control solution in Example 9;

[0066] Fig. 27 Chromatogram of droxidopa spiked solution in Example 9;

[0067] Fig.28 Blank solution chromatogram in Example 10;

[0068] Fig.29 Chromatogram of formic acid control solution in Example 10;

[0069] Fig.30 Chromatogram of droxidopa spiked solution in Example 10;

[0070] Fig.31 Blank solution chromatogram in Example 11;

[0071] Fig.32 Chromatogram of formic acid control solution in Example 11;

[0072] Fig.33 Chromatogram of droxidopa spiked solution in Example 11;

[0073] Fig.34 Blank solution chromatogram in Example 12;

[0074] Fig.35 Chromatogram of formic acid control solution in Example 12;

[0075] Fig.36 Chromatogram of the droxidopa spiked solution in Example 12. DETAILED DESCRIPTION

[0076] The embodiment of the present invention discloses a method for separating and detecting formic acid. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0077] In the embodiment of the present invention,

[0078] The residual formic acid content in the test sample was calculated according to the following formula:

[0079]

[0080] Where: A T is the peak area of ​​the impurity to be detected in the test solution;

[0081] AS The average peak area of ​​the impurity to be tested after three consecutive injections of the reference solution;

[0082] W T is the sample weight of the test solution, mg;

[0083] W S The mass of the impurity to be tested weighed to prepare the reference solution, mg;

[0084] D T is the dilution factor of the test solution;

[0085] D S is the dilution multiple of the reference solution;

[0086] P is the content of the reference substance, %.

[0087] The recovery rate is calculated as follows:

[0088]

[0089] Where: A T+S is the peak area of ​​formic acid in the test sample spike solution;

[0090] A T is the formic acid peak area in the test solution;

[0091] A S is the average value of the formic acid peak area in 3 reference solutions;

[0092] W T is the weight of the test sample in the test solution, mg;

[0093] W T+S is the weight of the test sample in the test sample spike solution, mg.

[0094] In the present invention, N / A means none.

[0095] In the present invention, 0.1N HCl means a 0.1 mol / L hydrochloric acid aqueous solution.

[0096] In the present invention, VWD represents a ultraviolet detector.

[0097] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0098] Instrument: American Agilent 1260 high performance liquid chromatography system and workstation; automatic injection.

[0099] Example 1

[0100] Instruments and conditions

[0101] Chromatographic conditions:

[0102] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm;

[0103] Detector: VWD;

[0104] Detection wavelength: 215nm;

[0105] Flow rate: 1.0ml / min;

[0106] Column temperature: 25°C;

[0107] Injection volume: 10 μl;

[0108] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0109] Mobile phase B: methanol;

[0110] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0111] Running time: 15min;

[0112] Experimental procedures

[0113] Solution preparation:

[0114] Blank solution / diluent: 0.1N HCl.

[0115] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0116] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0117] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0118] operate:

[0119] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Figure 1 , Figure 2 , Figure 3 .

[0120] Experimental results:

[0121] Figure 3The chromatographic peak with a retention time of about 3.4 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 3.6 minutes is the chromatographic peak of droxidopa.

[0122] Figure 3 It was demonstrated that droxidopa and formic acid can be separated using an Agilent ZORBAX Rx C8 column (4.6*250 mm, 5 μm), and this condition can be used for the detection of residual formic acid in droxidopa.

[0123] Example 2

[0124] Instruments and conditions

[0125] Chromatographic conditions:

[0126] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0127] Detector: VWD;

[0128] Detection wavelength: 215nm;

[0129] Flow rate: 1.0ml / min;

[0130] Column temperature: 25°C;

[0131] Injection volume: 10 μl;

[0132] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0133] Mobile phase B: methanol;

[0134] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0135] Running time: 15min;

[0136] Experimental procedures

[0137] Solution preparation:

[0138] Blank solution / diluent: 0.1N HCl.

[0139] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0140] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0141] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0142] operate:

[0143] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Figure 4 , Figure 5 , Figure 6 .

[0144] Experimental results:

[0145] Figure 4 is the chromatogram of the blank solution. Figure 5 is the chromatogram of formic acid reference solution, Figure 6 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 7.2 minutes is the chromatographic peak of droxidopa.

[0146] Figure 3 and Figure 6 The comparison proves that the separation degree between droxidopa and formic acid will be better after the chromatographic columns are connected in series, and the durability of the method will be better. This method can be used to detect the residual formic acid in droxidopa.

[0147] Example 3

[0148] Instruments and conditions

[0149] Chromatographic conditions:

[0150] Chromatographic column: Agilent ZORBAX SB C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0151] Detector: VWD;

[0152] Detection wavelength: 215nm;

[0153] Flow rate: 1.0ml / min;

[0154] Column temperature: 25°C;

[0155] Injection volume: 10 μl;

[0156] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0157] Mobile phase B: methanol;

[0158] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0159] Running time: 15min;

[0160] Experimental procedures

[0161] Solution preparation:

[0162] Blank solution / diluent: 0.1N HCl.

[0163] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0164] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0165] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0166] operate:

[0167] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Figure 7 , Figure 8 , Fig. 9 .

[0168] Experimental results:

[0169] Fig. 9 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 7.2 minutes is the chromatographic peak of droxidopa.

[0170] Fig. 9 and Figure 6 The comparison shows that droxidopa and formic acid can be separated in the method using two Agilent ZORBAX SB C8 columns (4.6*250 mm, 5 μm), and this condition can be used for the detection of residual formic acid in droxidopa.

[0171] Example 4

[0172] Instruments and conditions

[0173] Chromatographic conditions:

[0174] Chromatographic column: Agilent ZORBAX SB C18 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0175] Detector: VWD;

[0176] Detection wavelength: 215nm;

[0177] Flow rate: 1.0ml / min;

[0178] Column temperature: 25°C;

[0179] Injection volume: 10 μl;

[0180] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0181] Mobile phase B: methanol;

[0182] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0183] Running time: 15min;

[0184] Experimental procedures

[0185] Solution preparation:

[0186] Blank solution / diluent: 0.1N HCl.

[0187] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0188] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0189] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0190] operate:

[0191] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.10 , Fig.11 and Fig.12 .

[0192] Experimental results:

[0193] Fig.11 The chromatographic peak with a retention time of about 6.2 minutes is the chromatographic peak of formic acid.

[0194] Fig.11 and Figure 8 Compared with the C18 column, the retention is weaker and Fig.12It can be seen that the impurity peaks of formic acid and droxidopa overlap on the C18 chromatographic column, and the formic acid content cannot be accurately quantified. Therefore, this condition is not conducive to the detection of formic acid in droxidopa.

[0195] Example 5

[0196] Instruments and conditions

[0197] Chromatographic conditions:

[0198] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0199] Detector: VWD;

[0200] Detection wavelength: 215nm;

[0201] Flow rate: 0.5ml / min;

[0202] Column temperature: 25°C;

[0203] Injection volume: 10 μl;

[0204] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0205] Mobile phase B: methanol;

[0206] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0207] Running time: 20 minutes;

[0208] Experimental procedures

[0209] Solution preparation:

[0210] Blank solution / diluent: 0.1N HCl.

[0211] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0212] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0213] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0214] operate:

[0215] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.13 , Fig.14 , Fig.15 .

[0216] Experimental results:

[0217] Fig.15 The chromatographic peak with a retention time of about 13.3 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 14.4 minutes is the chromatographic peak of droxidopa.

[0218] Fig.15 It was demonstrated that droxidopa and formic acid could be separated at a flow rate of 0.5 ml / min, and this condition could be used for the detection of residual formic acid in droxidopa.

[0219] Example 6

[0220] Instruments and conditions

[0221] Chromatographic conditions:

[0222] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0223] Detector: VWD;

[0224] Detection wavelength: 215nm;

[0225] Flow rate: 1.5ml / min;

[0226] Column temperature: 25°C;

[0227] Injection volume: 10 μl;

[0228] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0229] Mobile phase B: methanol;

[0230] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0231] Running time: 15min;

[0232] Experimental procedures

[0233] Solution preparation:

[0234] Blank solution / diluent: 0.1N HCl.

[0235] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0236] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0237] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0238] operate:

[0239] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.16 , Fig.17 , Fig.18 .

[0240] Experimental results:

[0241] Fig.18 The chromatographic peak with a retention time of about 4.4 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 4.8 minutes is the chromatographic peak of droxidopa.

[0242] Fig.18 It was demonstrated that droxidopa and formic acid could be separated at a flow rate of 1.5 ml / min, and this condition could be used for the detection of residual formic acid in droxidopa.

[0243] Example 7

[0244] Instruments and conditions

[0245] Chromatographic conditions:

[0246] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0247] Detector: VWD;

[0248] Detection wavelength: 215nm;

[0249] Flow rate: 1.0ml / min;

[0250] Column temperature: 35 °C;

[0251] Injection volume: 10 μl;

[0252] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0253] Mobile phase B: methanol;

[0254] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0255] Running time: 15min;

[0256] Experimental procedures

[0257] Solution preparation:

[0258] Blank solution / diluent: 0.1N HCl.

[0259] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0260] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0261] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0262] operate:

[0263] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.19 , Fig. 20 , Fig.21 .

[0264] Experimental results:

[0265] Fig.21 The chromatographic peak with a retention time of about 6.5 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 6.8 minutes is the chromatographic peak of droxidopa.

[0266] Fig.21 It was demonstrated that droxidopa and formic acid could be separated when the column temperature was 35°C, and this condition could be used for the detection of residual formic acid in droxidopa.

[0267] Example 8

[0268] Instruments and conditions

[0269] Chromatographic conditions:

[0270] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0271] Detector: VWD;

[0272] Detection wavelength: 215nm;

[0273] Flow rate: 1.0ml / min;

[0274] Column temperature: 25°C;

[0275] Injection volume: 10 μl;

[0276] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0277] Mobile phase B: methanol;

[0278] Elution ratio: mobile phase A: mobile phase B = 98:2 (V:V);

[0279] Running time: 15min;

[0280] Experimental procedures

[0281] Solution preparation:

[0282] Blank solution / diluent: 0.1N HCl.

[0283] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0284] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0285] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0286] operate:

[0287] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig. 22 , Fig.23 , Fig.24 .

[0288] Experimental results:

[0289] Fig.24 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 7.3 minutes is the chromatographic peak of droxidopa.

[0290] Fig.24 It was demonstrated that droxidopa and formic acid could be separated in a method with a mobile phase ratio of 98:2, and this condition could be used for the detection of residual formic acid in droxidopa.

[0291] Example 9

[0292] Instruments and conditions

[0293] Chromatographic conditions:

[0294] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0295] Detector: VWD;

[0296] Detection wavelength: 215nm;

[0297] Flow rate: 1.0ml / min;

[0298] Column temperature: 25°C;

[0299] Injection volume: 10 μl;

[0300] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0301] Mobile phase B: methanol;

[0302] Elution ratio: mobile phase A: mobile phase B = 96:4 (V:V);

[0303] Running time: 15min;

[0304] Experimental procedures

[0305] Solution preparation:

[0306] Blank solution / diluent: 0.1N HCl.

[0307] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0308] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0309] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0310] operate:

[0311] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.25 , Fig.26 , Fig. 27 .

[0312] Experimental results:

[0313] Fig. 27 The chromatographic peak with a retention time of about 6.5 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 6.9 minutes is the chromatographic peak of droxidopa.

[0314] Fig. 27 It was demonstrated that droxidopa and formic acid could be separated in a method with a mobile phase ratio of 96:4, and this condition could be used for the detection of residual formic acid in droxidopa.

[0315] Example 10

[0316] Instruments and conditions

[0317] Chromatographic conditions:

[0318] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0319] Detector: VWD;

[0320] Detection wavelength: 210nm;

[0321] Flow rate: 1.5ml / min;

[0322] Column temperature: 25°C;

[0323] Injection volume: 10 μl;

[0324] Mobile phase A: 0.3% perchloric acid aqueous solution;

[0325] Mobile phase B: methanol;

[0326] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0327] Running time: 15min;

[0328] Experimental procedures

[0329] Solution preparation:

[0330] Blank solution / diluent: 0.1N HCl.

[0331] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0332] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0333] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0334] operate:

[0335] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.28 , Fig.29 , Fig.30 .

[0336] Experimental results:

[0337] Fig.30 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 7.2 minutes is the chromatographic peak of droxidopa.

[0338] Fig.30 It was demonstrated that droxidopa and formic acid can be separated in a method with a detection wavelength of 210 nm, and this condition can be used for the detection of residual formic acid in droxidopa.

[0339] Embodiment 11

[0340] Instruments and conditions

[0341] Chromatographic conditions:

[0342] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0343] Detector: VWD;

[0344] Detection wavelength: 215nm;

[0345] Flow rate: 1.0ml / min;

[0346] Column temperature: 25°C;

[0347] Injection volume: 10 μl;

[0348] Mobile phase A: 0.2% perchloric acid aqueous solution;

[0349] Mobile phase B: methanol;

[0350] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0351] Running time: 15min;

[0352] Experimental procedures

[0353] Solution preparation:

[0354] Blank solution / diluent: 0.1N HCl.

[0355] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0356] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0357] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0358] operate:

[0359] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.31 , Fig.32 , Fig.33 .

[0360] Experimental results:

[0361] Fig.33 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 6.9 minutes is the chromatographic peak of droxidopa.

[0362] Fig.33 It was demonstrated that droxidopa and formic acid can be separated in the 0.2% HClO4 method, and this condition can be used for the detection of residual formic acid in droxidopa.

[0363] Example 12

[0364] Instruments and conditions

[0365] Chromatographic conditions:

[0366] Chromatographic column: Agilent ZORBAX Rx C8 column, 4.6*250mm, 5μm (two chromatographic columns of the same specification connected in series);

[0367] Detector: VWD;

[0368] Detection wavelength: 215nm;

[0369] Flow rate: 1.0ml / min;

[0370] Column temperature: 25°C;

[0371] Injection volume: 10 μl;

[0372] Mobile phase A: 0.4% perchloric acid aqueous solution;

[0373] Mobile phase B: methanol;

[0374] Elution ratio: mobile phase A: mobile phase B = 97:3 (V:V);

[0375] Running time: 15min;

[0376] Experimental procedures

[0377] Solution preparation:

[0378] Blank solution / diluent: 0.1N HCl.

[0379] Formic acid control stock solution: Accurately weigh 62.5 mg of formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake well.

[0380] Formic acid control solution: Accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume.

[0381] Droxidopa spiked solution: Take 250 mg of drooxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 minutes to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0382] operate:

[0383] Take blank, formic acid control solution, and droxidopa spiked solution, and perform HPLC analysis under the above conditions. Record the chromatogram. The results are shown in Fig.34 , Fig.35 , Fig.36 .

[0384] Experimental results:

[0385] Fig.36 The chromatographic peak with a retention time of about 6.6 minutes is the chromatographic peak of formic acid, and the chromatographic peak with a retention time of about 7.2 minutes is the chromatographic peak of droxidopa.

[0386] Fig.36 It was demonstrated that droxidopa and formic acid could be separated in the presence of 0.4% HClO4 and that this condition could be used for the detection of residual formic acid in droxidopa.

[0387] Example 13 (Analytical Method Verification)

[0388] Instruments and conditions

[0389] Table 1: Chromatographic conditions

[0390]

[0391] Experimental procedures

[0392] Solution preparation:

[0393] Blank solution / diluent: 0.1N HCl aqueous solution (pipette 9 ml of concentrated hydrochloric acid into 1 L of ultrapure water and mix well);

[0394] Formic acid control stock solution: accurately weigh 62.5 mg formic acid into a 50 ml volumetric flask, add diluent to dissolve, and shake to volume;

[0395] Formic acid control solution (reference solution): accurately transfer 1 ml of formic acid control stock solution to a 50 ml volumetric flask, dilute with diluent, and shake to volume;

[0396] Detection limit solution: Accurately pipette 1 ml of formic acid control solution into a 100 ml volumetric flask, dilute with diluent, and shake to volume;

[0397] Test solution: Take 250 mg of droxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 min to dissolve, dissolve and make up to volume with diluent, and shake well;

[0398] Test sample spiked solution: Take 250 mg of droxidopa sample, accurately weigh it into a 50 ml volumetric flask, add about 30 ml of diluent, sonicate for 2 min to dissolve, accurately transfer 1 ml of formic acid control stock solution into the volumetric flask, dilute with diluent, and shake to volume.

[0399] operate:

[0400] Take blank solution, detection limit solution, reference solution, test solution, and test spiked solution and analyze them according to the following sequence table (Table 2) and the above conditions, record the chromatograms, and the results are shown in Tables 3-7.

[0401] Table 2: Injection sequence table

[0402] Solution name Number of injection needles blank solution 2-pin Detection limit solution 3-pin Reference solution 3-pin Test solution 1 needle Test sample spike solution 1 1 needle Test sample spike solution 2 1 needle Test sample spike solution 3 1 needle blank solution 1 needle Reference solution (stability solution) 1 needle Test sample spike solution (stability solution) 1 needle blank solution 1 needle Reference solution (stability solution) 1 needle Test sample spike solution (stability solution) 1 needle blank solution 1 needle Reference solution (stability solution) 1 needle Test sample spike solution (stability solution) 1 needle

[0403] Result analysis:

[0404] Table 3: System suitability results

[0405]

[0406]

[0407] Table 4: Specificity results

[0408]

[0409] Table 5: Detection limit results

[0410]

[0411] Table 6: Detectability results

[0412]

[0413]

[0414] Table 7: Solution stability results

[0415]

[0416] It can be seen from Example 13 that the method of the present invention has the advantages of being simple, rapid, highly sensitive and highly accurate in detecting formic acid in droxidopa.

Claims

1. A method for separating and detecting formic acid in droxidopa, characterized in that: include: A chromatographic column using octylsilane as filler is used, and the mobile phase is divided into phase A and phase B, phase A is a perchloric acid aqueous solution, and phase B is an alcohol; the alcohol is selected from methanol; the volume ratio of perchloric acid to water in the perchloric acid aqueous solution in phase A is 2:1000 to 4:1000; and the volume ratio of phase A to phase B is 98:2 to 96:

4.

2. The method according to claim 1, wherein the chromatographic column is selected from at least one of reverse phase RX-C8 and SB-C8 chromatographic columns.

3. The method according to claim 1 or 2, wherein the chromatographic column is selected from at least one of an Agilent ZORBAX Rx C8 column and an Agilent ZORBAX SB C8 column.

4. According to any one of the methods of claims 1-2, the chromatographic columns are two chromatographic columns of the same specification connected in series.

5. The method according to claim 3, wherein the chromatographic columns are connected in series using two chromatographic columns of the same specification.

6. The method according to any one of claims 1-2, characterized in that: The method comprises the following steps: 1) Take an appropriate amount of the sample to be tested containing formic acid, add solvent to dissolve, and mix well; 2) Set the instrument parameters: detector, chromatographic column, wavelength, flow rate of mobile phase, column temperature; 3) Taking a certain amount of the solution in step 1), injecting it into a high performance liquid chromatograph to complete the separation and determination of formic acid.

7. The method according to claim 6, characterized in that The solvent is an aqueous solution of HCl; and / or each 1 ml of the solvent contains 2 mg to 8 mg of the sample to be tested.

8. The method according to claim 6, wherein: The flow rate of the mobile phase is 0.5 ml / min to 1.5 ml / min; the detector is VWD; the wavelength is 210 nm to 215 nm; the temperature of the chromatographic column box is 25° C. to 35° C.; the sample solution injected in step 3) is 2 μl to 10 μl.

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