A method for determining clozapine and norclozapine plasma concentrations by a two-dimensional liquid phase method

Abstract

The present application relates to clozapine and norclozapine blood concentration determination technical field, specifically a kind of two-dimensional liquid phase method for determining the method for determining clozapine and norclozapine blood concentration, specifically comprising the following steps: selecting two-dimensional liquid phase instrument: Hunan Demet instrument Co., Ltd. FLC2701SPD-M30A full-automatic two-dimensional liquid phase system;Select calibration and quality control product source: Hunan Demet instrument Co., Ltd. Calibration batch number: 20221219, quality control product batch number: 20230221;Chromatographic conditions are as follows: one-dimensional column: SXI3.5*25mm*5um;Intermediate column: SCB3.0*10mm*2.8um;Two-dimensional column: MSCB3.0*50mm*3.3um.The present application has the advantages that: step design is reasonable, simple operation, low cost, while it can guarantee the accuracy and reliability of the determination of clozapine norclozapine blood concentration analysis result, for blood concentration detection work, promote two-dimensional liquid phase method for clinical detection, from saving cost, accelerate inspection TAT have higher value, have good popularization prospect and economic value.

Description

Technical Field

[0001] This invention relates to the field of clozapine and norclozapine blood concentration determination technology, specifically to a two-dimensional liquid chromatography method for determining the blood concentration of clozapine and norclozapine. Background Technology

[0002] Clozapine, also known as 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazapine The molecular formula is C18H19ClN4. It is a pale yellow crystalline powder at room temperature, odorless and tasteless, readily soluble in chloroform, soluble in ethanol, and practically insoluble in water. It has a good therapeutic effect on both positive and negative symptoms of schizophrenia. It belongs to the dibenzodiazepine class. It has a strong blocking effect on 5-HT2A and dopamine (DA1) receptors in the brain, and also has a blocking effect on dopamine (DA4) receptors, but a weaker blocking effect on dopamine (DA2) receptors. In addition, it has anticholinergic (M1), antihistamine (H1), and anti-α-adrenergic receptor effects. Extrapyramidal reactions and tardive dyskinesia are mild, and it generally does not cause an increase in serum prolactin levels. It can directly inhibit the ascending activating system of the brainstem reticular formation, and has a strong sedative-hypnotic effect, used to treat various types of schizophrenia.

[0003] Two-dimensional liquid chromatography (2D liquid chromatography) is less expensive than mass spectrometry (MS) with self-calibrated internal standards. However, due to the difference in detection sensitivity compared to mass spectrometry, it has difficulty detecting low concentrations of drugs. The sample injection volume is relatively large, the retention time is generally 5-10 minutes, the detection speed is slow, and the efficiency is not high. Currently, the range of drugs that can be detected is limited. However, for newly established laboratories with small sample volumes and limited detection needs, since the detection instruments and detection costs are lower than those of mass spectrometry isotope methods and self-calibrated internal standards, 2D liquid chromatography can be promoted first, and then upgraded to a new mass spectrometry detection system after three to five years, depending on the needs. Summary of the Invention

[0004] The purpose of this invention is to provide a two-dimensional liquid chromatography method for determining the blood concentrations of clozapine and norclozapine, in order to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a method for determining the blood concentrations of clozapine and norclozapine using a two-dimensional liquid chromatography method, specifically including the following steps:

[0006] 1) Selected two-dimensional liquid chromatography instrument: Hunan Demeter Instrument Co., Ltd. FLC2701 SPD-M30A fully automatic two-dimensional liquid chromatography system;

[0007] 2) Source of calibrators and quality control products: Hunan Demeter Instrument Co., Ltd., calibrator batch number: 20221219, quality control product batch number: 20230221;

[0008] 3) The chromatographic conditions are as follows:

[0009] One-dimensional chromatographic column: SXI 3.5*25mm*5um;

[0010] Intermediate column: SCB 3.0*10mm*2.8um;

[0011] Two-dimensional column: MSCB 3.0*50mm*3.3um;

[0012] One-dimensional reagent: CAA-1D mobile phase model CAA-1B;

[0013] Two-dimensional reagent: BPI3-2A: API3-2A: MPI-2A: OPI-2A = 0:74:0:26;

[0014] Auxiliary reagent: pure water;

[0015] Processing method ORG-1 processing temperature: 50℃;

[0016] Detection wavelength CH1: 290nm; Injection volume: 50uL;

[0017] CH2: 262nm Method Name: Clozapine - Secondary Simplex Method - 50 μL / 1 cm;

[0018] Flow rates: Pump A: 0.8 ml / min; Pump B: 0.01 ml / min; Pump C: 0.7 ml / min;

[0019] Time program: The secondary simplex constant pressure pump stops automatically.

[0020] The advantages of this invention are: reasonable step design, simple operation, and low cost. At the same time, it can ensure the accuracy and reliability of the blood drug concentration analysis results of clozapine and norclozapine. For blood drug concentration detection, promoting the use of two-dimensional liquid chromatography for clinical testing has high value in terms of cost saving and speeding up the testing of TAT, and has good prospects for promotion and economic value. Attached Figure Description

[0021] Figure 1 This is a time-sharing flowchart for measuring the blood concentration of clozapine and norclozapine.

[0022] Figure 2 This is a confirmation LC2 chromatogram of clozapine and norclozapine blood drug concentration measurement. The peak area of ​​norclozapine is 235696, and the peak area of ​​clozapine is 226217.

[0023] Figure 3 This is a confirmation graph of the online blood concentration measurement of clozapine and norclozapine. The peak area of ​​norclozapine is 211936; the peak area of ​​clozapine is 219311.

[0024] Figure 4 This is a confirmation graph of the standard curve for measuring clozapine blood concentration.

[0025] Figure 5 This is a confirmation graph of the standard curve for measuring the blood concentration of norclozapine.

[0026] Figure 6 The QCL chart confirms the quality control results of clozapine blood concentration: Deviation: 3.42%.

[0027] Figure 7 The QCM chart confirms the quality control results of clozapine blood concentration: Deviation: 3.47%.

[0028] Figure 8 The QCH graph confirms the quality control results of clozapine blood concentration: Deviation: 4.97%. Detailed Implementation

[0029] For the purposes of the detailed description below, it should be understood that the invention may take various alternative variations and sequences of steps unless expressly stated otherwise. Furthermore, except in any operational instance, or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term “about.” Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention. It is not at all an attempt to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0030] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0031] Furthermore, it should be understood that any numerical range described herein is intended to include all subranges falling within it. For example, the range “1 to 10” is intended to include all subranges between the minimum value 1 and the maximum value 10, i.e., a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0032] Example

[0033] A two-dimensional liquid chromatography method for determining the blood concentrations of clozapine and norclozapine specifically includes the following steps:

[0034] 1) Selected two-dimensional liquid chromatography instrument: Hunan Demeter Instrument Co., Ltd. FLC2701 SPD-M30A fully automatic two-dimensional liquid chromatography system;

[0035] 2) Source of calibrators and quality control products: Hunan Demeter Instrument Co., Ltd., calibrator batch number: 20221219, quality control product batch number: 20230221;

[0036] 3) The chromatographic conditions are as follows:

[0037] One-dimensional chromatographic column: SXI 3.5*25mm*5um;

[0038] Intermediate column: SCB 3.0*10mm*2.8um;

[0039] Two-dimensional column: MSCB 3.0*50mm*3.3um;

[0040] One-dimensional reagent: CAA-1D mobile phase model CAA-1B;

[0041] Two-dimensional reagent: BPI3-2A: API3-2A: MPI-2A: OPI-2A = 0:74:0:26;

[0042] Auxiliary reagent: pure water;

[0043] Processing method ORG-1 processing temperature: 50℃;

[0044] Detection wavelength CH1: 290nm; Injection volume: 50uL;

[0045] CH2: 262nm Method Name: Clozapine - Secondary Simplex Method - 50 μL / 1 cm;

[0046] Flow rates: Pump A: 0.8 ml / min; Pump B: 0.01 ml / min; Pump C: 0.7 ml / min;

[0047] Time program: The secondary simplex constant pressure pump stops automatically.

[0048] in conclusion

[0049] ● The transfer recovery rate of clozapine was 96.9%, and the transfer recovery rate of norclozapine was 90.0%, which meets the transfer recovery rate requirement of <90%-110%>;

[0050] ●Clozapine standard curve R 2 =0.9999244, R value of demeclozapine standard curve 2=0.9999624 meets the methodological validation requirements;

[0051] ● The quality control results meet the method validation requirements, and the chromatograms are pure and free of interference.

[0052] The comparison between two-dimensional liquid chromatography (2D-LC) and mass spectrometry (MS / ISO) for determining clozapine and norclozapine blood concentrations concluded that MS / ISO is the reference method for blood drug concentration detection. Comparison of the research data showed a high degree of consistency between the two-dimensional liquid chromatography and IOSO results. With a CV of 10%, only 5 samples with lower concentrations showed 2D-LC results outside the acceptable range compared to the IOSO results out of range. This indicates that 2D-LC can be used for routine clinical detection of clozapine and norclozapine blood concentrations.

[0053] In summary, for blood drug concentration detection, mass spectrometry isotope method is used for research, while promoting mass spectrometry self-calibration internal standard method and two-dimensional liquid chromatography method for clinical testing is of great value in terms of cost saving and speeding up TAT testing.

[0054] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for determining the blood concentrations of clozapine and norclozapine using two-dimensional liquid chromatography, characterized in that, Specifically, the following steps are included: 1) Selected two-dimensional liquid chromatography instrument: Hunan Demeter Instrument Co., Ltd. FLC2701 SPD-M30A fully automatic two-dimensional liquid chromatography system; 2) Source of calibrators and quality control products: Hunan Demeter Instrument Co., Ltd., calibrator batch number: 20221219, quality control product batch number: 20230221; 3) The chromatographic conditions are as follows: One-dimensional chromatographic column: SXI 3.5*25mm*5um; Intermediate column: SCB 3.0*10mm*2.8um; Two-dimensional column: MSCB 3.0*50mm*3.3um; One-dimensional reagent: CAA-1D mobile phase model CAA-1B; Two-dimensional reagent: BPI3-2A: API3-2A: MPI-2A: OPI-2A = 0:74:0:26; Auxiliary reagent: pure water; Processing method ORG-1 processing temperature: 50℃; Detection wavelength CH1: 290nm; Sample volume: 50uL; CH2:262nm Method Name: Clozapine - Secondary Simplex Method - 50uL.lcm; Flow rates: Pump A: 0.8 ml / min; Pump B: 0.01 ml / min; Pump C: 0.7 ml / min; Time program: The secondary simplex constant pressure pump stops automatically.

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