Method for measuring concentration of phenylephrine in plasma by LC-MS/MS, and pretreatment method of sample

Abstract

The invention relates to the technical field of medical detection, and specifically relates to a method for measuring the concentration of phenylephrine in plasma by LC-MS / MS. The method comprises thefollowing steps: primary extraction: adding an internal standard working solution and an extracting solution into a sample, centrifugally layering the solution after mixing the solutions uniformly, and collecting the supernatant to obtain first supernatant; derivatization reaction: concentrating and drying the first supernatant, adding a NaHCO3 buffer salt for dissolution, and then adding an acetonitrile solution of dansyl chloride to perform a derivatization reaction; and secondary extraction: adding the extracting solution into the reaction solution after the derivatization reaction for secondary extraction, centrifuging to obtain second supernatant, concentrating and drying the second supernatant, and redissolving the product to obtain a sample solution. By adoption of the pretreatment method of the sample provided by the invention, the extraction rate of low-level phenylephrine can be maximally improved, the interference of a plasma matrix is effectively removed, the specificityis improved, the minimum detection limit is reduced to 0.02ng / ml, and the sensitivity is increased to 0.1nM to adapt to the detection requirements of the concentration of the low-concentration phenylephrine in drug clinical studies.

Description

technical field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for measuring the concentration of phenylephrine in blood plasma by LC-MS / MS, and an applicable pretreatment method for analyzing biological samples. Background technique

[0002] Phenylephrine is a catecholamine selective α1 receptor agonist, mainly used to prevent and treat hypotension caused by spinal anesthesia, general anesthesia, application of chlorpromazine, etc. It is also used for supraventricular tachycardia and mydriasis Check, phenylephrine is an over-the-counter drug that can be substituted for pseudoephedrine.

[0003] Currently, the phenylephrine-like nasal decongestants used clinically usually need to be administered every four hours to relieve nasal congestion due to the short plasma half-life of the active ingredient phenylephrine in the body. Therefore, in order to delay the release time, improve the therapeutic effect and reduc...

Examples

Embodiment 1

[0067] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170401, each containing hydrochloric acid to Oxynephrine 5mg), each subject was given one tablet, and the subject’s forearm vein was embedded with an indwelling needle. 1 mL of blood was collected at different time points at 40, 60, 90, 120, 240, 360, and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0068] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0069] (a) Sample pretreatment

[0070] (1) Preparation of standard curve samples: Accurately weigh pheny...

Embodiment 2

[0085] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170301, each containing hydrochloric acid to Oxynephrine 10mg), each subject took one tablet, and the subjects were respectively before administration (0min) and after administration 10, 15, 20, 25, 30, 40, 60, 90, 120, 240, 1 mL of blood was collected at different time points at 360 and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0086] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0087] (a) Sample pretreatment

[0088] (1) Preparation of standard curv...

Embodiment 3

[0101] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170402, each containing hydrochloric acid to Oxynephrine 20mg), each subject took one tablet, and the subjects were respectively before administration (0min) and after administration 10, 15, 20, 25, 30, 40, 60, 90, 120, 240, 1 mL of blood was collected at different time points at 360 and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0102] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0103] (a) Sample pretreatment

[0104] (1) Preparation of standard curv...

technical field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for measuring the concentration of phenylephrine in blood plasma by LC-MS / MS, and an applicable pretreatment method for analyzing biological samples. Background technique

[0002] Phenylephrine is a catecholamine selective α1 receptor agonist, mainly used to prevent and treat hypotension caused by spinal anesthesia, general anesthesia, application of chlorpromazine, etc. It is also used for supraventricular tachycardia and mydriasis Check, phenylephrine is an over-the-counter drug that can be substituted for pseudoephedrine.

[0003] Currently, the phenylephrine-like nasal decongestants used clinically usually need to be administered every four hours to relieve nasal congestion due to the short plasma half-life of the active ingredient phenylephrine in the body. Therefore, in order to delay the release time, improve the therapeutic effect and reduc...

Examples

Embodiment 1

[0067] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170401, each containing hydrochloric acid to Oxynephrine 5mg), each subject was given one tablet, and the subject’s forearm vein was embedded with an indwelling needle. 1 mL of blood was collected at different time points at 40, 60, 90, 120, 240, 360, and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0068] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0069] (a) Sample pretreatment

[0070] (1) Preparation of standard curve samples: Accurately weigh pheny...

Embodiment 2

[0085] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170301, each containing hydrochloric acid to Oxynephrine 10mg), each subject took one tablet, and the subjects were respectively before administration (0min) and after administration 10, 15, 20, 25, 30, 40, 60, 90, 120, 240, 1 mL of blood was collected at different time points at 360 and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0086] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0087] (a) Sample pretreatment

[0088] (1) Preparation of standard curv...

Embodiment 3

[0101] Collection of plasma samples to be tested: 12 subjects were fasted overnight for at least 10 hours the day before the administration, and at 0 min, the subjects took renin Namin tablets (batch number: 20170402, each containing hydrochloric acid to Oxynephrine 20mg), each subject took one tablet, and the subjects were respectively before administration (0min) and after administration 10, 15, 20, 25, 30, 40, 60, 90, 120, 240, 1 mL of blood was collected at different time points at 360 and 480 min, and placed in a 2 Put in vacuum blood collection tubes with EDTA anticoagulant, mix well, let stand, centrifuge, take 200 μL of the upper layer of plasma, cover the tube tightly, and store in an ultra-low temperature freezer at -60°C to -90°C.

[0102] The method for measuring the concentration of phenylephrine in plasma by LC-MS / MS described in this embodiment specifically includes the following steps:

[0103] (a) Sample pretreatment

[0104] (1) Preparation of standard curv...

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