JP2006112835A - Quantification method of dexmedetomidine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply measuring a dexmedetomidine in a specimen such as blood or the like with high sensitivity using a small amount of the specimen. <P>SOLUTION: The specimen containing dexmedetomidine is subjected to a solid phase extraction method to separate dexmedetomidine as an eluted component and separated dexmedetomidine is detected and quantified by a liquid chromatography/tandem mass analyzer using 4-[1-(2-methylphenyl)ethyl]-1H-imidazole as an internal standard substance. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for measuring dexmedetomidine in a specimen such as blood.

で表記される化合物であり、中枢性α２アドレナリン受容体を介して、大脳皮質等の上位中枢の興奮、覚醒レベル上昇を抑制することにより鎮静作用を発現する新しいクラスの鎮静剤である。特にデクスメデトミジンは、集中治療室において、静脈ライン（ｉｖ−ｌｉｎｅ）やカテーテル等を設置された患者が興奮したり、不安や苦痛を感じるのを軽減させることができる。（特許文献１参照。）。 Dexmedetomidine ((+)-(S) -4- [1- (2,3-dimethylphenyl) ethyl] -1H-imidazole) has the following structural formula:

And is a new class of sedatives that exert a sedative effect by suppressing the excitement of the upper central nervous system such as the cerebral cortex and the increase in the arousal level through the central α2 adrenergic receptor. In particular, dexmedetomidine can reduce the feeling of excitement and anxiety and distress in patients who have vein lines (iv-line) or catheters installed in the intensive care unit. (See Patent Document 1).

As a dosage for dexmedetomidine, adults are usually continuously infused intravenously for 10 minutes at 6 μg / kg / hr (initial load administration), and the optimal sedation level is obtained according to the patient's condition. The dexmedetomidine is continuously infused intravenously in a range of 0.2 to 0.7 μg / kg / hour as a maintenance amount so that the target plasma concentration of dexmedetomidine is 0.1 to 1.25 ng / mL. However, there are patients who cannot obtain the target plasma concentration even if the maintenance dose of dexmedetomidine is continuously infused intravenously, and conversely, there are patients who exceed the target plasma concentration, and the blood concentration of dexmedetomidine is controlled. Is important.
As a method of measuring the blood concentration of dexmedetomidine, gas chromatography / mass spectrometry (hereinafter, GC / MS method) is known. According to the GC / MS method, when hexane and pentafluorobenzoyl chloride are first added to plasma containing dexmedetomidine, dexmedetomidine is converted to pentafluorobenzoyl, and then the organic layer containing dexmedetomidine converted to pentafluorobenzoyl is separated. After collecting and concentrating, the concentrated solution is used as a sample and subjected to quantitative analysis by GC / MS (see Non-Patent Document 1). However, this method requires 2 mL or more of blood (1 mL or more as plasma), and dexmedetomidine must be pentafluorobenzoylated. Therefore, this method is complicated and unsatisfactory as a method for quickly managing and maintaining the blood dexmedetomidine concentration of a patient to whom dexmedetomidine is administered in the intensive care unit.
Special table 2002-509880 gazette Journal of Chromatography, 1989, Vol. 497, p. 282-287

  An object of the present invention is to provide a method for measuring dexmedetomidine in a sample such as blood easily and with high sensitivity using a small amount of sample.

As a result of intensive studies on a method for quantifying dexmedetomidine contained in a sample such as blood with high sensitivity, the present inventors have found that a sample containing dexmedetomidine is pretreated with a solid-phase extraction method and liquid chromatography. It is found that dexmedetomidine in a sample can be easily detected with high sensitivity by using a combination of detection with a tandem mass spectrometer (hereinafter abbreviated as LC / MS / MS system) and a quantitative method. It came to complete.
That is, the present invention
(1) A specimen containing dexmedetomidine is subjected to solid phase extraction to separate dexmedetomidine in the specimen as an elution component, and the separated dexmedetomidine is converted into 4- [1- (2-methylphenyl) ethyl] -1H. A method for quantifying dexmedetomidine in a specimen, characterized by detecting and quantifying with an LC / MS / MS system using imidazole as an internal standard substance,
(2) The quantitative method according to (1), wherein the solid phase extraction resin in the solid phase extraction method is a combination polymer or enhanced polymer having both hydrophilicity and hydrophobicity,
(3) The description in (1) or (2) above, wherein the eluent in the solid phase extraction method is a solution containing 60 to 100 V / V% of a polar organic solvent having water miscibility and volatility. Quantification method,
(4) The determination method according to (3), wherein the polar organic solvent is methanol or acetonitrile,
(5) The method according to (1) above, wherein the column in the high performance liquid chromatography of the LC / MS / MS system is a reverse phase chromatography column,
(6) The quantification method according to (5), wherein the mobile phase in the high performance liquid chromatography of the LC / MS / MS system is an acetate buffer / acetonitrile mixture,
(7) In the LC / MS / MS system, an ion near m / z 201 is selected as the precursor ion of dexmedetomidine, and an ion near m / z 95 is selected as the product ion, and 4- [1- (2-methylphenyl) is selected. ) The determination method according to (1) above, wherein ions near m / z 187 are selected as precursor ions of ethyl] -1H-imidazole and ions near m / z 95 are selected as product ions, and these ions are monitored.
(8) The method according to (1), wherein the specimen is animal plasma, serum, blood or urine.
About.

  According to the present invention, dexmedetomidine to be measured can be quantified easily and with high sensitivity by performing analytical quantification with an LC / MS / MS system using a sample pretreated by a solid phase extraction method. it can.

  In the present invention, dexmedetomidine may be in the form of not only a free base but also a salt such as hydrochloride. The specimen that can be used in the present invention is not particularly limited, and any specimen containing dexmedetomidine can be used, for example, blood, plasma, serum, saliva, urine, etc. of animals including humans. And various organs and tissues in animals.

  In the solid phase extraction method, for example, a solid phase extraction cartridge is preferably used. As the solid-phase extraction cartridge, any solid-phase extraction cartridge capable of extracting dexmedetomidine in a biological sample with high sensitivity can be preferably used. For example, Isolute C8 using octyl silica gel (made by International sorbent technology), Bond Elut C8 (manufactured by Varian), Sep-pak C8 (manufactured by Waters), etc .; Isolute C18 using octadecyl silica gel (manufactured by International sorbent technology), Bond Elut C18 (manufactured by Varian), Sep-pak C18 (manufactured by Varian) Solid phase extraction cartridge NEXUS (manufactured by Varian) using a combination polymer having both hydrophilicity and hydrophobicity , Oasis HLB (Waters); Enhanced Polymer solid phase extraction cartridge Focus Using (manufactured by Varian Co., Ltd.). Among these, a solid phase extraction cartridge (NEXUS, Oasis HLB) using a combination polymer having both hydrophilicity and hydrophobicity or a solid phase extraction cartridge (Focus) using an enhanced polymer is particularly preferable. The solid phase extraction column is not limited to the cartridge type, and a disk type or plate type can also be used as appropriate.

The eluent in the solid-phase extraction is preferably a solution containing 60 to 100 V / V% of a polar organic solvent that is miscible with water and easily volatilizes. The solution may contain water or a nonpolar solvent in addition to the polar solvent. Examples of such a polar organic solvent include methanol, ethanol, acetonitrile, and the like. One or more polar solvents may be mixed as appropriate. The polar solvent may be further acidified with a volatile acid such as acetic acid or hydrochloric acid (preferably about 0.01 to 1N hydrochloric acid). The acid should be added at about 0.01 to 10 V / V%, preferably about 0.05 to 5 V / V% with respect to the polar solvent. By making the elution solution acidic, dexmedetomidine adsorbed on the solid phase cartridge can be eluted even with a small amount of eluent (for example, about 1/2 volume or less of the volume of the eluate to which no acid is added). In consideration of the operation after elution, the eluate contains no water or water, but the water content is 40 V / V% or less, preferably 20 V / V% or less, more preferably 10 V / V% or less. .
The solution is usually injected into the cartridge using a pipette, a syringe or the like.

  The column in the high performance liquid chromatography (LC) of the LC / MS / MS system is preferably a reverse phase chromatography column. As the reverse phase chromatography column, an octadecyl silica gel (ODS) column is preferable. For example, Inertsil ODS (manufactured by GL Science Co., Ltd.), L-column ODS (manufactured by Chemicals Evaluation and Research Institute), Develosil ODS UG-5 ( Nomura Chemical Co., Ltd.), CAPCELL PAK C18 MGII (Shiseido Co., Ltd.), ZORBAX XDB-C18 (Yokogawa Analytical Systems Co., Ltd.), Symmetry C18 (Waters Co., Ltd.), Nucleosil C18 (M. Nagel Co., Ltd.) However, the ODS column is not limited to these.

  In the present invention, an acetate buffer solution mobile phase is preferably used as the mobile phase in the high performance liquid chromatography. Examples of the acetate buffer include an acetic acid solution, an ammonium acetate solution, and an ammonium acetate buffer. The acetic acid concentration in the acetic acid solution may be in the range of about 0.1 to 1 V / V%, more preferably about 0.4 to 0.6 V / V%. The concentration of ammonium acetate in the ammonium acetate solution can generally be in the range of about 0.01 to 100 mM, preferably about 1 to 50 mM, more preferably about 1 to 10 mM. The ammonium acetate buffer is preferably prepared by adding acetic acid to the ammonium acetate solution to a pH in the range of about 3-5. Examples of the acetate buffer system mobile phase include acetic acid solution / methanol mixture, acetic acid solution / methanol acetate mixture, acetic acid solution / acetonitrile mixture, acetic acid solution / acetonitrile mixture, acetic acid solution / methanol / acetonitrile mixture, ammonium acetate solution or Examples thereof include ammonium acetate buffer / methanol mixture, ammonium acetate solution or ammonium acetate buffer / acetonitrile mixture, ammonium acetate solution or ammonium acetate buffer / methanol / acetonitrile mixture, and the like. The acetic acid solution and the ammonium acetate solution may be an acetic acid aqueous solution and an ammonium acetate aqueous solution, respectively. The mixing ratio of methanol or acetonitrile to acetic acid solution or ammonium acetate solution or ammonium acetate buffer is generally about 1:99 to about 99: 1, preferably about 20:80 to about 80:20, more preferably, by volume. It is in the range of about 30:70 to about 70:30, most preferably about 35:65 to about 60:40.

  Examples of the ionization method in the LC / MS / MS system include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), thermospray ionization (TSP), and fast atom bombardment (FAB). Among these, ESI is preferable, and the ESI turbo ion spray method is more suitably used. In the present invention, MS / MS is a precursor ion obtained in MS 1 (here, a hydrogen-added ion), MS 2 is used to decompose the precursor ion with nitrogen gas or argon gas, and the resulting product ions are detected. This method has a high sensitivity and an improved S / N ratio due to high selectivity.

  As a method for detecting the generated product ions, a full scan method, a selective reaction monitoring (SRM) method, a multi-reaction monitoring (MRM) method, or the like is generally used. In the present invention, the MRM method is preferably used. In this MRM method, for example, only ions near m / z 201 are selected as precursor ions for dexmedetomidine, and then when these ions collide with an inert gas such as nitrogen gas or argon gas to be decomposed. The object of the present invention is to select only ions having a maximum peak in the range of m / z 10 to 201, preferably ions in the vicinity of m / z 95, and monitor the amount of the generated product ions. Dexmedetomidine in a sample can be quantified with high sensitivity. At this time, an ion near m / z 187 is selected as a precursor ion of 4- [1- (2-methylphenyl) ethyl] -1H-imidazole as an internal standard substance, and m / z of 10 to 187 is selected as a product ion. An ion showing the maximum peak within the range, preferably an ion in the vicinity of m / z 95, should be selected.

で表記される化合物（以下、内標化合物と略記する。）である。この内標化合物は、遊離の塩基の状態のみならず、例えば塩酸塩などの塩の状態であってもよい。前記内標化合物は、測定の対象であるデクスメデトミジンと比して分子量が約１４小さいので、ＬＣ／ＭＳ／ＭＳシステムでは、デクスメデトシミジン化合物の検出ピークと異なる位置にピークを得ることができ、かつそのピークは血漿等生体成分の検出ピークの位置とも異なる。また、上記内標化合物は、血漿等の生体成分中において安定で経時的変化が少なく、固相抽出法による処理を行っても回収率が良好で、取り扱い易い化合物である。このため上記内標化合物は、内部標準物質として使用することが可能となり、しかも、前記内標化合物を使用することにより検体中のデクスメデトミジンを驚くべき高感度で定量することができる。上記内標化合物は、例えば特公平３−７２６２１号公報に記載の方法等に準じて製造することができる。 4- [1- (2-methylphenyl) ethyl] -1H-imidazole used as an internal standard used in the present invention has the following structure;

(Hereinafter abbreviated as internal standard compound). This internal standard compound may be in the form of not only a free base but also a salt such as hydrochloride. Since the internal standard compound has a molecular weight of about 14 smaller than that of dexmedetomidine to be measured, the LC / MS / MS system can obtain a peak at a position different from the detection peak of the dexmedethymidine compound. And the peak is different from the position of the detection peak of biological components such as plasma. The internal standard compound is a compound that is stable in biological components such as plasma and has little change with time, has a good recovery rate even when treated by a solid phase extraction method, and is easy to handle. Therefore, the internal standard compound can be used as an internal standard substance, and dexmedetomidine in a sample can be quantified with a surprisingly high sensitivity by using the internal standard compound. The internal standard compound can be produced, for example, according to the method described in JP-B-3-72621.

Below, the preferable process of this invention is illustrated. For example,
(A) a step of diluting and pretreating the specimen;
(B) a step of separating dexmedetomidine as an elution component from the pretreated product obtained in the step (a) by a solid phase extraction method;
(C) a step of measuring dexmedetomidine in the eluted component obtained in the step (b) using an LC / MS / MS system, and (e) a sample from the measured value obtained in the step (d) Calculating the concentration of dexmedetomidine in the
Is mentioned.
Hereinafter, each of the above preferred steps will be described more specifically.

  In the step (a), for example, plasma is separated from blood collected from a patient administered with dexmedetomidine, and an internal standard compound is added to about 100 to 250 μL of plasma containing the separated dexmedetomidine, and water, or The plasma is finally diluted about 1.5 to 10 times in volume with acid (1N hydrochloric acid) or / and alkali (1N sodium hydroxide). The diluted plasma containing the internal standard compound is preferably centrifuged at about 0 ° C. to room temperature, preferably about 4 to 10 ° C. at about 10,000 to 20000 rpm for about 5 to 10 minutes, and the supernatant is used. . In addition, you may dilute to the above final volume times after centrifugation.

  In the step (b), the diluted or optionally centrifuged plasma obtained in the step (a) is applied to the solid phase extraction cartridge previously activated with a polar organic solvent and water by a conventional method. Injected. As said polar organic solvent, alcohol (for example, methanol, ethanol, etc.) or acetonitrile etc. are preferable, for example. The solid phase extraction cartridge into which the plasma has been injected is preferably washed with about 0.5 to 2 mL of water, then about 50 V / V% or less (preferably about 2 to 40 V / V%) of methanol or acetonitrile aqueous solution. . Thereby, dexmedetomidine and the internal standard compound can be adsorbed on the solid phase of the solid phase extraction cartridge. Next, about 0.5 to 2 mL of the elution solution is injected into the solid phase extraction cartridge, and dexmedetomidine and the internal standard compound are eluted from the solid phase extraction cartridge as elution components. The eluate containing the eluted dexmedetomidine and the internal standard compound is concentrated to dryness at about 20 to 50 ° C. in a nitrogen stream.

  In the step (c), the concentrated dried product obtained in the step (b) is dissolved in a mobile phase used for high performance liquid chromatography in an LC / MS / MS system and injected into the LC / MS / MS system. And dexmedetomidine is measured. Examples of the LC / MS / MS system include API 4000 LC / MS / MS system (manufactured by AB / MDS SCIEX), 4000 Q TRAP LC / MS / MS system (manufactured by AB / MDS SCIEX), and Q TRAP LC / MS / MS system. (Manufactured by AB / MDS SCIEX), API2000 LC / MS / MS system (manufactured by AB / MDS SCIEX), TSQ QUANTUM LC / MS / MS system (manufactured by Thermo Electron) or TSQ 7000 LC / MS / MS system (manufactured by Thermo Electron) ) And the like.

High-performance liquid chromatography (LC) uses the above-described ODS column and mobile phase, and the column temperature is preferably room temperature to about 50 ° C., preferably about 35 to 45 ° C.
Next, components separated and eluted by high performance liquid chromatography are introduced into an ionization chamber of a tandem mass spectrometer (MS / MS) and ionized. Precursor ions are selected by the first-stage mass spectrometer (MS), and then collide with nitrogen gas or the like in a collision chamber called a collision cell to dissociate the precursor ions and generate new ions. An ion having the maximum peak in the new ion group is selected as a product ion and analyzed by a second-stage mass spectrometer (MS).
A mass chromatogram at the time of injection of 10 μL of dexmedetomidine 0.2 ng / mL and internal standard compound 10 ng / mL is shown in FIG. In this figure, dexmedetomidine is eluted at about 3.8 minutes, and the internal standard compound is eluted at about 2.7 minutes.

The step (e) is not particularly limited as long as the concentrations of dexmedetomidine and the internal standard compound in the sample can be calculated from the measurement values obtained in the step (d). Examples of the concentration calculating means used in this step include means for performing using a calibration curve prepared using a standard solution of dexmedetomidine. Specifically, the calibration curve is created from measured values obtained by performing the above steps (a) to (d) using a sample obtained by adding an internal standard compound to a standard solution of dexmedetomidine.
The calibration curve used in this step is a calibration curve whose accuracy is within ± 20% at the lower limit of quantification, within ± 15% at other concentrations, and whose correlation coefficient is 0.99 or more. Is preferred. When using an internal standard compound, for example, obtain a ratio between the peak area value of dexmedetomidine and the peak area value of the internal standard compound in the sample, and plot this ratio on the graph to achieve a highly reliable calibration. A line can be created. In addition, by using a standard solution in which dexmedetomidine is dissolved in a sample (eg, plasma) of a healthy person, the influence of endogenous factors contained in the plasma can be eliminated, for example. That is, by using such a calibration curve, the concentration of dexmedetomidine in the patient can be directly calculated, which is convenient. The lower limit of quantification means the lowest concentration of dexmedetomidine in the sample among samples added with dexmedetomidine used for preparing a calibration curve. The accuracy is calculated by dividing the value obtained by subtracting the addition concentration from the average of the quantitative values by the addition concentration. The correlation coefficient can be calculated using, for example, the least square method.

  If the quantification method of the present invention is used, for example, when plasma is used as a sample, dexmedetomidine in the sample can be quantified with a sample amount of only about 200 μL.

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
As the internal standard compound, 4- [1- (2-methylphenyl) ethyl] -1H-imidazole hydrochloride produced according to Japanese Patent Publication No. 3-72621, Example 7 was used.

Preparation of calibration curve A calibration curve was created according to the following procedure.
(1) Preparation of dexmedetomidine standard solution: 11.8 mg of dexmedetomidine hydrochloride (10 mg as dexmedetomidine) was dissolved in purified water to 10 mL to prepare dexmedetomidine standard stock solution (1 mg / mL). The standard stock solution was diluted with purified water to prepare dexmedetomidine 0.08, 0.2, 0.8, 4.0, 20.0, 40.0, and 80.0 ng / mL standard solutions.
Preparation of internal standard solution: 10.0 mg of the internal standard compound was accurately weighed and dissolved in a small amount of purified water, and then the internal standard stock solution (1 mg / mL) was prepared to exactly 10 mL with purified water. This internal standard stock solution was accurately diluted with purified water to prepare an internal standard solution (40 ng / mL, 100 μg / mL or 5 μg / mL).
(2) To 200 μL of human plasma, 50 μL of dexmedetomidine standard solution (0.02, 0.05, 0.2, 1.0, 5.0, 10.0, 20.0 ng of dexmedetomidine per mL of human plasma) 50 μL of internal standard solution and 700 μL of purified water were added to obtain a solution.
(3) The solution of (2) was injected into a solid phase extraction cartridge Oasis HLB (30 mg / 1 mL; manufactured by Waters) that had been activated by injecting 1 mL of methanol and 1 mL of water in advance. Next, 1 mL of purified water was injected twice into Oasis HLB, and further 1 mL of 50 V / V% methanol aqueous solution was injected to wash Oasis HLB.
(4) 1 mL of methanol was injected into the washed Oasis HLB of (3), and the eluate eluted from the Oasis HLB was collected. The recovered eluate was concentrated to dryness under a nitrogen stream. 75 μL of 5 mmol / L acetate buffer (pH 5.0) / acetonitrile (70:30, V / V) was added to the concentrated dried product, and the concentrated dried product was dissolved to obtain a sample for high performance liquid chromatography.
(5) 10 μL of the sample for high performance liquid chromatography of (4) was injected into an LC / MS / MS system [API4000 LC / MS / MS system (manufactured by AB / MDS SCIEX)], and dexmedetomidine was measured. The LC / MS / MS system was performed under the following conditions.

LC (High Performance Liquid Chromatography):
Analytical column: CAPCELL PACK C18 MGII (2.0 × 75 mm, 3 μm; manufactured by Shiseido Co., Ltd.)
Guard column: GUARD CARTRIDGE CAPCELL C18 MGIIS-3 (2.0 × 10 mm, 3 μm; manufactured by Shiseido Co., Ltd.)
Mobile phase: 5 mmol / L acetate buffer (pH 5.0) / acetonitrile (70:30, V / V)
Needle cleaning solution: methanol / 0.05V / V% acetic acid solution (90:10, V / V)
Flow rate: 0.2 mL / min
Column temperature: 40 ° C
Autosampler set temperature: 4 ° C
Sample injection volume: 10 μL
MS / MS (tandem mass spectrometer)
Ionization method: Electrospray ionization (ESI)
(Turbo Ion Spray)
Ion spray voltage: 5500V
Heater gas temperature: 700 ° C
Multiplier voltage: 1800V
Nebulizer gas (GS1): 80 psi (about 552 kPa)
Turbo gas (GS2): 70 psi (about 483 kPa)
Curtain gas (Nitrogen): 20 psi (about 138 kPa)
Collision gas (Nitrogen): 3 psi (about 21 kPa)
Detection mode: Positive ion detection mode
Multiple reaction monitoring (MRM)
Monitor ion:

(6) The measurement data obtained was as shown in Table 2.
It was found that a calibration curve (r = 0.9997) having a high correlation coefficient was obtained in the plasma dexmedetomidine concentration range of 0.02 to 20 ng / mL. Moreover, when a calibration curve was similarly prepared for a dexmedetomidine concentration in the range of 0.075 to 40 ng / mL in plasma, it was confirmed that a calibration curve with high linearity (r = 0.9908) was obtained. The quantification range of dexmedetomidine is consistent with the target plasma concentration of dexmedetomidine to be administered of 0.1 to 1.25 ng / mL, and it has been found that this can be a method for quantifying the plasma concentration of dexmedetomidine.

Addition / recovery test of dexmedetomidine using human plasma 50 μL of dexmedetomidine standard solution (0.08, 4.0, 80 ng / mL) prepared in Example 1 (1) was added to 200 μL of human plasma (dexmede per 1 mL of human plasma). Medetomidine was added at 0.02, 1.0, and 20.0 ng.), 50 μL of internal standard solution (40 ng / mL) and 700 μL of purified water were added, and the mixture was stirred with a vortex mixer. Subsequent operations were carried out in the same manner as in the procedures (3) to (5) for preparing the calibration curve.
The results are shown in Table 3. This result shows that analysis and quantification are possible with an accuracy of 96% or higher in recovery rate at a target plasma concentration of dexmedetomidine of 0.1 to 1.25 ng / mL.

[Examples 3 to 5]
Examination of solid-phase extraction cartridge in solid-phase extraction method-Addition and recovery test of dexmedetomidine using rat plasma-
Add 50 μL of dexmedetomidine standard solution (added 100 μg / mL of dexmedetomidine per 1 mL of rat plasma), 50 μL of internal standard solution (100 μg / mL) prepared in Example 1 (1) and 400 μL of 1N sodium hydroxide solution to 200 μL of rat plasma. The mixture was stirred with a vortex mixer and centrifuged at 15000 rpm, 4 ° C. for 5 minutes. A concentrated dried solid was prepared from the supernatant after centrifugation using the solid phase extraction cartridge and eluate shown in Table 4. As a control, plasma added with 50 μL of purified water instead of 50 μL of dexmedetomidine standard solution and 50 μL of internal standard solution was used.

200 μL of the mobile phase described below used for high performance liquid chromatography was added to each of the obtained dried products, the dried products were dissolved, and filtered through a Milex HV 0.45 μm filter to prepare a sample for high performance liquid chromatography. 50 μL of each of the obtained samples for high performance liquid chromatography was injected into the liquid chromatography, and dexmedetomidine was measured. In this example, dexmedetomidine was detected with a UV detector (wavelength 220 nm), and the peak area value (Sample peak area value) of dexmedetomidine was calculated. The peak area value is automatically calculated by a computer connected to high performance liquid chromatography. On the other hand, 50 μL of dexmedetomidine standard solution (100 μg / mL), 50 μL of internal standard solution (100 μg / mL) and 100 μL of the mobile phase described below were mixed to prepare a standard (STD) solution. 50 μL of the STD solution was injected into liquid chromatography, dexmedetomidine was detected in the same manner as described above, and the peak area value of the obtained dexmedetomidine was taken as the STD peak area value. The recovery rate of dexmedetomidine was calculated according to the following.
Recovery rate = (Sample peak area value / STD peak area value) × 100
It has been confirmed that the detection limit of dexmedetomidine by the UV detector is 0.125 μg / mL.

The high performance liquid chromatography was performed under the following conditions.
Analysis column: Inertsil ODS-3 (2.1 × 150 mm, 5 μm; manufactured by Tosoh Corporation)
Guard column: TSK GUARDGEL ODS-80Ts (1.5 × 20 mm, 5 μm; manufactured by Tosoh Corporation)
Mobile phase: 0.5 V / V% acetic acid / methanol (65:35, V / V)
Flow rate: 0.3 mL / min
Column temperature: 40 ° C
Sample injection volume: 50 μL

Table 5 shows the recovery rate of dexmedetomidine from rat plasma. It has been found that any solid phase extraction cartridge of combination polymer or enhanced polymer that has both hydrophilicity and hydrophobicity can be used for the quantification of the present invention.

[Examples 6 to 12]
Examination of eluate in solid-phase extraction method-Addition recovery test of dexmedetomidine using rat plasma-
50 μL of dexmedetomidine standard solution (added 10 μg / mL of dexmedetomidine per 1 mL of human plasma), 50 μL of the internal standard solution (5 μg / mL) prepared in Example 1 (1) and 200 μL of 1N hydrochloric acid solution were added to 200 μL of rat plasma. After stirring with a vortex mixer, the mixture was centrifuged at 15000 rpm, 4 ° C. for 5 minutes. To the supernatant after centrifugation, 400 μL of 1N sodium hydroxide solution was uniformly mixed. The mixed solution was poured into a solid phase extraction cartridge Oasis HLB (10 mg / 1 cc; manufactured by Waters) or Focus (manufactured by Varian) activated with 1 mL of methanol and 1 mL of purified water. The phase extraction cartridge was washed. Using the eluates in Tables 6 and 7, dexmedetomidine and the internal standard compound were eluted from the solid phase extraction cartridge, and the eluate was centrifuged to dryness (120 min, 50 ° C.).

[Comparative Examples 1-3]
Except for using the eluate in Comparative Examples 1 to 3 in Table 7, dexmedetomidine hydrochloride and the internal standard compound were eluted from the solid-phase extraction cartridge, and the eluate was centrifuged to dryness. (120 min, 50 ° C.).

  To each dried product obtained in Examples 6 to 7 and Comparative Examples 1 to 3, 200 μL of the mobile phase described below used for high performance liquid chromatography was added to dissolve the dried product, which was then filtered with a Milex HV 0.45 μm filter. A sample for high performance liquid chromatography was prepared by filtration. 50 μL of each obtained sample for high performance liquid chromatography was injected into high performance liquid chromatography, and dexmedetomidine was measured. In this example and comparative example, the amount of added dexmedetomidine was detected with a UV detector (wavelength 220 nm), and the recovery rate was calculated in the same manner as in Examples 3-5.

The high performance liquid chromatography was performed under the following conditions.
Analytical column: L-column ODS (2.1 × 150 mm, 3 μm; manufactured by Chemical Substance Evaluation Research Organization)
Guard column: TSK GUARDGEL ODS-80Ts (4.0 × 20 mm; manufactured by Tosoh Corporation)
Mobile phase: 5 mM ammonium acetate solution: methanol (40:60, V / V)
Flow rate: 0.25 mL / min
Column temperature: 40 ° C
Sample injection volume: 100 μL

  Tables 8 and 9 show the recovery rates of dexmedetomidine from rat plasma. In the solid-phase extraction method of dexmedetomidine in plasma, the recovery rate of dexmedetomidine from rat plasma is 75% by using 60% or more methanol aqueous solution (polar solvent solution) as eluent, which can be used for quantification. It has been found.

  The present invention is useful for quantifying the plasma concentration of dexmedetomidine within the range of a target plasma concentration of dexmedetomidine of 0.1 to 1.25 ng / mL. Moreover, since the plasma concentration of dexmedetomidine can be quantified easily and with high sensitivity, it can be used to determine the continuous dose of dexmedetomidine for maintaining the optimum sedation level from the plasma concentration of dexmedetomidine.

In the figure, A is a diagram showing a mass chromatogram when 10 μL of dexmedetomidine standard solution (dexmedetomidine 0.2 ng / mL) is injected into LC / MS / MS. B is a diagram showing a mass chromatogram when 10 μL of an internal standard solution (internal standard compound 10 ng / mL) is injected into LC / MS / MS.

Claims (8)

  A sample containing dexmedetomidine is subjected to solid phase extraction to separate dexmedetomidine in the sample as an elution component, and the separated dexmedetomidine is converted into 4- [1- (2-methylphenyl) ethyl] -1H-imidazole. A method for quantifying dexmedetomidine in a specimen, wherein the dexmedetomidine is detected and quantified by a liquid chromatography / tandem mass spectrometer using it as an internal standard substance.   2. The method according to claim 1, wherein the solid phase extraction resin in the solid phase extraction method is a combination polymer or enhanced polymer having both hydrophilicity and hydrophobicity.   The quantification method according to claim 1 or 2, wherein the eluate in the solid phase extraction method is a solution containing 60 to 100 V / V% of a polar organic solvent that is miscible with water and easily volatilizes.   The method according to claim 3, wherein the polar organic solvent is methanol, ethanol or acetonitrile.   The method according to claim 1, wherein the column in the high performance liquid chromatography of the liquid chromatography / tandem mass spectrometer is a reverse phase chromatography column.   6. The quantification method according to claim 5, wherein the mobile phase in the high performance liquid chromatography of the liquid chromatography / tandem mass spectrometer is an acetate buffer / acetonitrile mixture.   In a liquid chromatography / tandem mass spectrometer, an ion near m / z 201 is selected as a precursor ion of dexmedetomidine, and an ion near m / z 95 is selected as a product ion. 4- [1- (2-methylphenyl) The quantification method according to claim 1, wherein ions near m / z 187 are selected as precursor ions of ethyl] -1H-imidazole, ions near m / z 95 are selected as product ions, and these ions are monitored. 2. The method according to claim 1, wherein the specimen is animal plasma, serum, blood or urine.

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