A composite chromatographic column and a two-dimensional liquid chromatography system

Abstract

The invention discloses a composite chromatographic column, comprising a column body, wherein the column body is divided into an upper section and a lower section, wherein the filler used in the upper section is a filler with a naphthalene group bonded to the surface of a microsphere, and the filler used in the lower section is a filler with a benzenesulfonic acid group bonded to the surface of a microsphere. The composite chromatographic column has good analytical effect. The invention also discloses a two-dimensional liquid chromatography system, which uses the composite chromatographic column as an analytical column and further comprises an extraction column; the filler of the extraction column is a filler with a carboxylic acid group bonded to the surface of a microsphere. The composite liquid chromatography system can realize the simultaneous separation of four psychotropic drugs, and has good separation effect and short analysis time.

Description

Technical Field

[0001] The invention belongs to the field of chromatographic columns, and in particular relates to a chromatographic column and a two-dimensional liquid chromatography system for the determination and analysis of psychotropic drugs. Background Art

[0002] There are many kinds of psychotropic drugs commonly used at present, such as venlafaxine, desvenlafaxine, amisulpride, sulpiride, etc. In 2011, the psychiatric therapeutic drug monitoring (TDM) mainly used liquid chromatography and liquid chromatography-mass spectrometry for the analysis of psychotropic drugs in human plasma. For example, application number 201810574905.8 disclosed a simple and efficient method for detecting the concentration of venlafaxine and its active metabolite O-desvenlafaxine in human plasma. Application number 201711326671.7 disclosed a method for detecting sulpiride tablets using liquid chromatography. However, the above methods are only for the determination of a single psychotropic drug.

[0003] If the existing liquid chromatography analysis method or liquid chromatography-mass spectrometry detection method is used to simultaneously detect multiple drugs, there are the following disadvantages and difficulties:

[0004] 1. The chromatographic column used in general liquid chromatography is a C18 column. If more than two drugs are mixed together for analysis, the polarity of the column is very different, and the separation time between the two substances is long, resulting in a long analysis time.

[0005] 2. The presence of other substances in plasma interferes with the separation, resulting in poor separation.

[0006] 3. Analysis of plasma samples generally requires a complex pre-treatment process with cumbersome procedures, which makes the entire analysis process time-consuming.

[0007] 4. Mass spectrometers are expensive, increasing enterprise costs and subsequent maintenance costs.

[0008] The present invention intends to start from a chromatographic column and a chromatographic system composed of multiple chromatographic columns, and intends to achieve the purpose of simultaneously analyzing multiple psychotropic drugs through a conventional liquid chromatograph by improving the chromatographic column filler and combining different chromatographic columns. Summary of the invention

[0009] In view of the deficiencies in the prior art, the present invention aims to provide a composite chromatographic column and a two-dimensional liquid chromatography system, which can realize the detection of four psychotropic drugs, namely, venlafaxine, desvenlafaxine, amisulpride and sulpiride.

[0010] In order to achieve the above invention object, the technical solution of the present invention is:

[0011] A composite chromatographic column comprises a column body, wherein the column body is divided into an upper section and a lower section, wherein the filler used in the upper section is a filler with naphthalene groups bonded to the surface of microspheres, and the filler used in the lower section is a filler with benzenesulfonic acid groups bonded to the surface of microspheres.

[0012] In a preferred embodiment, the upper section and the lower section are connected via a connecting piece, which is further preferably a ferrule or a sealing ring.

[0013] In a preferred embodiment, the microspheres are made of silica gel, titanium dioxide or high molecular polymer.

[0014] A two-dimensional liquid chromatography system comprises the composite chromatography column.

[0015] Preferably, the length of the upper section is 20-50 mm, and the length of the lower section is 80-150 mm.

[0016] In a further preferred embodiment, the length of the upper section is 30 mm, and the length of the lower section is 100 mm.

[0017] A two-dimensional liquid chromatography system uses the composite chromatography column as an analysis column and also comprises an extraction column; the filler of the extraction column is a filler with carboxylic acid groups bonded to the surface of microspheres.

[0018] The liquid chromatograph can be an existing conventional two-dimensional liquid chromatograph, and we mainly replace the analysis column and extraction column therein.

[0019] In a preferred embodiment, the two-dimensional liquid chromatography system comprises: a flow channel switching valve, a waste liquid flow channel, a first flow channel for conveying a one-dimensional mobile phase, a second flow channel for conveying a one-dimensional mobile phase, an analysis flow channel connected to an analysis column, and an extraction column, wherein the waste liquid flow channel, the first flow channel, the second flow channel, and the analysis flow channel are respectively connected to any one port of the flow channel switching valve, and the extraction column is connected between any two remaining ports of the flow channel switching valve.

[0020] In a further preferred embodiment, the flow channel switching valve is a six-way valve.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] 1. The fillers of the composite chromatographic column (analytical column) and the extraction column of the present invention are carefully designed and have good separation effect. The filler used in the upper section of the analytical column is a filler with a naphthyl group bonded to the surface of the microsphere, and the filler used in the lower section is a filler with a benzenesulfonic acid group bonded to the surface of the microsphere; the extraction column is a filler with a carboxylic acid group bonded to the surface of the microsphere; the filler with a benzenesulfonic acid group bonded to the surface has the strongest retention effect on amisulpride and sulpiride, and has a stronger retention effect on desvenlafaxine, the filler with a naphthyl group bonded to the surface of the microsphere has a stronger retention effect on venlafaxine, and the filler with a carboxylic acid group bonded to the surface of the microsphere has a very strong capture effect on these four drugs. Through this combination, these four drugs can be well separated.

[0023] 2. The composite liquid chromatograph of the present invention is provided with an improved analytical column and, through the cooperation with an extraction column, a special chromatographic column combination is constructed, which can be used to analyze a variety of psychotropic drugs simultaneously.

[0024] 3. The composite liquid chromatograph of the present invention has good effect on the simultaneous separation of four psychotropic drugs and short analysis time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the structure of the composite two-dimensional liquid chromatography of the present invention;

[0026] Figure 2 Schematic diagram of the analytical column structure;

[0027] Figure 3 is the chromatogram of blank human plasma;

[0028] Figure 4 The chromatograms are of venlafaxine, desvenlafaxine, amisulpride and sulpiride standard solutions;

[0029] Figure 5 Chromatogram of blank human plasma spiked with venlafaxine, desvenlafaxine, amisulpride, and sulpiride (493.86 ng / mL, 497.81 ng / mL, 977.64 ng / mL, and 985.99 ng / mL);

[0030] Figure 6 This is the standard curve of venlafaxine;

[0031] Figure 7 This is the standard curve of desvenlafaxine;

[0032] Figure 8 This is the standard curve of amisulpride;

[0033] Fig. 9 This is the standard curve of sulpiride.

[0034] Wherein: SI: one-dimensional mobile phase; C1: extraction column (one-dimensional chromatographic column) S2: two-dimensional mobile phase; C2: analytical column (two-dimensional chromatographic column) DE: UV detector P1: extraction pump P2: analytical pump V1: flow channel switching valve AS: automatic sampler L1: first flow channel L2: second flow channel L3: analytical flow channel L4: extraction column connecting flow channel L5: waste liquid flow channel;

[0035] 1 is the upper section of the analytical column, 2 is the lower section of the analytical column, and 3 is the connecting piece. DETAILED DESCRIPTION

[0036] The present invention will be further explained and illustrated below in conjunction with the accompanying drawings and embodiments.

[0037] like Figure 1 As shown: a composite two-dimensional liquid chromatograph, a flow channel switching valve V1, a waste liquid flow channel L5, a first flow channel L1 for conveying a one-dimensional mobile phase S1, a second flow channel L2 for conveying a one-dimensional mobile phase S2, an analysis flow channel L3 connected to an analysis column C2, and an extraction column C1, the waste liquid flow channel L5, the first flow channel L1, the second flow channel L2, and the analysis flow channel L3 are respectively connected to any one port of the flow channel switching valve V1, and the extraction column C1 is connected between any two remaining ports of the flow channel switching valve V1.

[0038] like Figure 2 As shown, the analytical column is divided into an upper section 1 and a lower section 2. The filler used in the upper section 1 is a silica gel filler with naphthalene groups bonded to the microsphere surface, and the filler used in the lower section 2 is a silica gel filler with benzenesulfonic acid groups bonded to the microsphere surface. The upper section and the lower section are connected by a connector 3. The length of the upper section is 30 mm, and the length of the lower section is 100 mm.

[0039] The extraction column is a silica gel filler with carboxylic acid groups bonded to the surface of microspheres.

[0040] Specific applications:

[0041] Simultaneous determination of venlafaxine, desvenlafaxine, amisulpride and sulpiride concentrations in human plasma

[0042] 1. Drugs and reagents

[0043] Venlafaxine (content: 99.9%, China Food and Drug Administration);

[0044] Desvenlafaxine (content: 98.0%, San Chemical Technology Co., Ltd.);

[0045] Amisulpride (content: 99.9%, China Food and Drug Administration);

[0046] Sulpiride (content: 100.00%, trc);

[0047] Methanol (chromatographic grade, ACS);

[0048] Acetonitrile (chromatographic grade, ACS);

[0049] Phosphoric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0050] Diammonium hydrogen phosphate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0051] Ammonia (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0052] Perchloric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0053] Ammonium acetate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0054] Isopropanol (chromatographic grade, ACS);

[0055] Ethylene glycol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

[0056] Purified water is produced by a water purifier (not lower than the implementation standard of GB17323).

[0057] 2. Instruments and analytical conditions

[0058] The instruments and equipment are as described above.

[0059] The method for simultaneously measuring multiple psychotropic drugs comprises the following steps:

[0060] (1) Starting the first-dimension pump, introducing the sample to be tested into the extraction column through the one-dimensional mobile phase, and the target components in the sample are enriched and captured on the extraction column;

[0061] (2) starting the second dimension pump to deliver the target components in the sample into the analytical column through the two-dimensional mobile phase. The target components in the sample are separated on the analytical column and enter the detector for detection;

[0062] Table 1 Process diagram corresponding to the time program

[0063]

[0064] Analysis conditions:

[0065] Column temperature: 40℃.

[0066] First-dimension mobile phase: methanol: acetonitrile: 10mmoL / L ammonium phosphate aqueous solution = 1:3:3 (v / v / v), ammonia water adjusted pH to 7.00; first-dimension pump flow rate: 0.7mL·min -1 ;

[0067] Second dimension mobile phase: methanol: acetonitrile: 25mmoL / L ammonium phosphate aqueous solution = 1:2:3 (v / v / v), ammonia water adjusted pH to 7.00; second dimension pump flow rate: 1.2mL·min -1 ;

[0068] Detection wavelength of venlafaxine and desvenlafaxine: 280nm.

[0069] Detection wavelength of amisulpride and sulpiride: 292nm.

[0070] 3. Sample processing

[0071] Protein precipitation method: Accurately pipette 1000 μL of acetonitrile into a 1.5 mL EP tube, then accurately add 400 μL of plasma sample, vortex for 1 min, and centrifuge at high speed (14500 r / min). -1 ) for 8 min, and transfer 1000 μL of the supernatant to a sample injection bottle for testing.

[0072] 4. Technical verification results

[0073] 4.1 Specificity

[0074] Figure 3-5 The chromatograms are blank human plasma, venlafaxine and desvenlafaxine, amisulpride, sulpiride standard solutions, and blank human plasma with standard solutions added. The retention time of venlafaxine is 5.882 min, the retention time of desvenlafaxine is 3.415 min, the retention time of amisulpride is 4.605 min, and the retention time of sulpiride is 3.718 min. Endogenous substances in plasma and other impurities do not interfere with the separation and determination of samples.

[0075] 4.2 Linear range and limit of quantification

[0076] The prepared standard curve samples were measured, and the peak areas of venlafaxine, desvenlafaxine, amisulpride and sulpiride were used for linear regression of concentrations to obtain the regression equation:

[0077] Venlafaxine: y = 230.8x-1030.1, r = 0.9999, linear range 24.37 ~ 1218.43ng ml -1

[0078] Desvenlafaxine: Y = 262.3x-1293.6, r = 0.9999, linear range 24.11 ~ 1205.4ng ml -1

[0079] Amisulpride Y = 2246.0x + 18034.6, r = 0.9999, linear range 48.15 ~ 2407.59 ng ml -1

[0080] Sulpiride: y = 459.4x + 2092.4, r = 0.9999, linear range 48.4 ~ 2420ng ml -1 .

[0081] 4.3 Accuracy and precision

[0082] The quality control samples of low, medium and high concentrations of venlafaxine and desvenlafaxine were processed and measured. Five parallel groups were set for each concentration. The measured results were substituted into the linear regression equation to calculate the concentration. The concentration was compared with the theoretical added concentration to obtain the method's added recovery rate and coefficient of variation, and the accuracy and intra-day precision were tested. Quality control samples were prepared and measured for 3 consecutive days to investigate the method's inter-day precision. From the results in Table 2-3, it can be seen that the recovery rate of the established venlafaxine analysis method is between 98.4% and 102.7%, and the intra-day and inter-day coefficients of variation are both less than 8.5%; the recovery rate of the desvenlafaxine analysis method is between 102.9% and 111.9%, and the intra-day and inter-day coefficients of variation are both less than 4.7%; the recovery rate of the amisulpride analysis method is between 98.7% and 101.3%, and the intra-day and inter-day coefficients of variation are both less than 3.3%; the recovery rate of the sulpiride analysis method is between 101.7% and 102.8%, and the intra-day and inter-day coefficients of variation are both less than 3.9%. This analysis method has high sensitivity and good stability, and meets the methodological requirements of bioequivalence studies.

[0083] Table 2 Method recovery and intra-day precision (n=5)

[0084]

[0085]

[0086] Table 3 Inter-day precision of the method (n=5)

[0087]

[0088]

Claims

1. A two-dimensional liquid chromatography system, comprising an extraction column and an analysis column, characterized in that: A composite chromatographic column is used as an analytical column, the composite chromatographic column comprises a column body, the column body is divided into an upper section and a lower section, the upper section and the lower section are connected by a connector, the length of the upper section is 20-50 mm, and the length of the lower section is 80-150 mm; the filler used in the upper section is a filler with naphthyl groups bonded to the surface of silica gel, and the filler used in the lower section is a filler with benzenesulfonic acid groups bonded to the surface of silica gel; the filler of the extraction column is a filler with carboxylic acid groups bonded to the surface of silica gel; the two-dimensional liquid chromatography system is a chromatographic system for detecting four psychotropic drugs, namely venlafaxine, desvenlafaxine, amisulpride and sulpiride; The method for detecting four psychotropic drugs, namely, venlafaxine, desvenlafaxine, amisulpride and sulpiride, comprises the following analytical conditions: a sample to be tested is introduced into an extraction column through a one-dimensional mobile phase, and a target component in the sample is enriched and captured on the extraction column, and the working time is 0-2.75 min; the one-dimensional mobile phase is: methanol: acetonitrile: 10 mmol / L ammonium phosphate aqueous solution = 1:3:3, the above ratio is a volume ratio, and ammonia water is adjusted to pH 7.00; flow rate: 0.7 mL×min -1 ; The target components in the sample are sent into the analytical column through the two-dimensional mobile phase, and the target components in the sample are separated on the analytical column. The working time is 2.75-6.9 min. The two-dimensional mobile phase is: methanol: acetonitrile: 25mmoL / L ammonium phosphate aqueous solution = 1:2:3, the above ratio is volume ratio, and the pH is adjusted to 7.00 with ammonia water; flow rate: 1.2 mL×min -1 ; Column temperature: 40℃.

2. The two-dimensional liquid chromatography system according to claim 1, characterized in that: The connecting piece is a ferrule or a sealing ring.

3. The two-dimensional liquid chromatography system according to claim 1, characterized in that: The length of the upper section is 30 mm, and the length of the lower section is 100 mm.

4. The two-dimensional liquid chromatography system according to claim 1 or 2, characterized in that it comprises: A flow channel switching valve (V1), a waste liquid flow channel (L5), a first flow channel (L1) for conveying a one-dimensional mobile phase (S1), a second flow channel (L2) for conveying a two-dimensional mobile phase (S2), an analysis flow channel (L3) connected to an analysis column (C2), and an extraction column (C1), wherein the waste liquid flow channel (L5), the first flow channel (L1), the second flow channel (L2), and the analysis flow channel (L3) are respectively connected to any one port of the flow channel switching valve (V1), and the extraction column (C1) is connected between any two remaining ports of the flow channel switching valve (V1).

5. The two-dimensional liquid chromatography system according to claim 4, characterized in that: The flow channel switching valve (V1) is a six-way valve.

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