JPH09138226A - Analytical method for drugs in sample containing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an analytical method in which a protein removal treatment (a pretreatment) by a solid-phase extraction method is performed quickly and in which drugs in a sample can be extracted, separated and analyzed by a method wherein an aqueous medium which contains an organic acid and an organic solvent is used as the elution liquid of a target component. SOLUTION: By using a column, for solid-phase extraction (pretreatment), for a protein removal treatment, a liquid which holds a target component and which elutes a protein is set as a mobile phase, and the protein removal treatment is executed. Then, drugs are eluted from the column for solid-phase extraction. While an aqueous medium which contains an organic acid and an organic solvent is set as a mobile phase for extraction. As samples which contains the protein, biological samples such as serum, blood plasma, urine and the like are enumerated as samples. As the mobile phase for the protein removal treatment, a buffer solution which contains organic salts is used generally. As the organic solvent which is used as a mobile phase for drug extraction, hexane, tetrahydrofuran, dichlormethane, acetone or the like can be used, and a polar solvent is especially preferable. In addition, as the acid which is contained in the aqueous medium, acetic acid, formic acid and the like are enumerated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to rapid deproteinization pretreatment (extraction) of a drug containing a protein, for example, a drug present in serum, plasma, etc., by a solid phase extraction method for deproteinization. The present invention relates to a method for performing, extracting, separating, and analyzing drugs in a sample.

[0002]

2. Description of the Related Art As a method for analyzing drugs present in serum and plasma without pretreatment by deproteinization operation such as precipitation method, a sample is preliminarily adsorbed with protein. It was introduced into a cartridge column for solid-phase extraction (pretreatment) that adsorbs drugs, and the mobile phase consisting of a buffer solution containing an inorganic salt was passed through this to deproteinize, and then adsorbed to the cartridge column. A solid phase extraction method is used in which a drug is eluted by flowing a mobile phase consisting of a mixed solution of a buffer solution containing an organic solvent and an inorganic salt, and the eluate is led to an analytical column to analyze drugs. A method for automatically performing a solid phase extraction method is known as column switching high performance liquid chromatography (hereinafter referred to as column switching method HPLC).

In the above-mentioned column switching method HPLC, in eluting the drug adsorbed on the column for pretreatment (solid phase extraction) after deproteinization, generally, from a mixed solution of a buffer solution containing an inorganic salt and an organic solvent. The drug is eluted from the pretreatment column by sending the mobile phase to the column at a flow rate of 1 ml / min to 2 ml / min.

However, when the drug is eluted as described above, a large amount of mobile phase needs to flow with respect to the amount of the adsorbed drug, which is not economical. Further, the analysis sensitivity is also lowered, which is not preferable, and there is a concern of environmental pollution. Furthermore, when the column switching method HPLC is connected to a mass spectrometer for high-sensitivity analysis, under the above elution conditions, the flow rate of the mobile phase is too fast and inorganic salts are contained, so that the inorganic salts adhere to the detector. Since the detector is contaminated, it has a drawback that it cannot be directly introduced into the detector for measurement, and it has been desired to solve these problems.

The present inventors have found that the column switching method HP
In the course of intensive research aimed at solving the above problems using LC, it was found that the above problems can be solved by using an aqueous medium containing an organic acid and an organic solvent as the eluate of the target component. The present invention has been completed.

The mobile phase for extraction consisting of an aqueous medium containing an organic acid and an organic solvent for eluting a drug as a target component from the column for solid phase extraction (pretreatment) of the present invention includes manual extraction and automatic extraction in solid phase extraction. It can be applied to any of the extractions. Since this mobile phase does not contain salts, it can be directly introduced into a detector, particularly a mass spectrometer, and thus it can be said that it is particularly suitable for column switching HPLC and LC-MS analysis.

[0007]

The present invention provides a method for extracting and separating drugs in a sample containing a protein by using a solid phase extraction (pretreatment) column for deproteinization. The solution that retains the protein but elutes the protein is used as a mobile phase for deproteinization to perform deproteinization, and then an aqueous medium containing an organic acid and an organic solvent is used as a mobile phase for extraction from the above solid-phase extraction (pretreatment) column. The present invention provides a method for extracting / separating a drug for eluting the drug, and a method for separating / analyzing the eluate directly to an analytical column connected to a detector to analyze the drug. .

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a method for introducing a sample into a pretreatment column for adsorbing a drug without adsorbing a protein is as follows:
Known column switching method HPLC can be used.

The sample containing the protein is not particularly limited as long as it contains the protein as a contaminant and the drug.
Examples of such a sample include biological samples such as serum, plasma and urine.

The drug in the sample containing the protein to be analyzed according to the present invention is not particularly limited, but phenylpropanolamine hydrochloride, atenolol, atropine, diclofenac sodium, buspirone, tetracycline,
Daunorubicin, propranolol, pindolol, atenolol, nifedipine, nicardipine, ketoprofen, flurbiprofen, ibuprofen, indomethacin, alprazolam, diazepam, triazolam,
Acetaminophen, theophylline, acyclovir, cimetidine, sulfamethoxazole, chlorpheniramine, testosterone, reserpine, and other low-polarity drugs, and ampicillin, cefotaxime sodium, and other polar amino- and carboxyl-containing polar groups High drugs are included.

The pretreatment column used in the present invention is as follows:
There is no particular limitation as long as it retains the drug without adsorbing the protein. The stationary phase of the pretreatment (solid phase extraction) column has a hydrophilic group mainly on the outer surface of the particle and a hydrophobic group inside the particle. It is preferable to use a filler having a functional group or a filler having an anion exchange group inside the particles. For example, a filler having a structure in which the inner surface of the filler is a hydrophobic or ion-exchange group and the outer surface of the filler has a hydrophilic group such as a diol group, or a structure in which a protein is supported on the outer surface of the adsorbent. It is possible to use the adsorbent of the above, or an adsorbent composed of a porous polymer [TCPinkerton, Journal of Chromatograp
hy, 544 , 13 (1991)]. A commercially available pretreatment column can be used as the column packed with such a packing material. For example, L-column L-1180 (trade name) (manufactured by Japan Chemicals Inspection Society), Develosil PT C8-5
(Trade name) (manufactured by Nomura Chemical Co., Ltd.), Shim-pack SPCAE1 (trade name) (manufactured by Shimadzu Corporation), TSK precolumn PW (trade name) (manufactured by Tosoh Corporation), and the like.

The selection of the column for pretreatment (solid phase extraction) for the drug is appropriately determined by preliminary experiments. Generally, for low polarity drugs, the inner surface of the adsorbent is filled with a filler having a hydrophobic group. For a highly polar drug, a pretreatment column in which a packing material having an ion exchange group is packed on the inner surface of the adsorbent is preferably used.

The mobile phase for deproteinization is not particularly limited as long as it is a mobile phase suitable for combination with the above stationary phase, but in the method known as the mobile phase for deproteinization, a buffer solution containing an inorganic salt is generally used. Used. In this case, since salts are mixed in the eluate when the drug is eluted in the next step, for example,
When a mass spectrometer is connected for high-sensitivity analysis, it is not preferable because the operation such as flowing the deproteinizing mobile phase and further washing with water becomes complicated.

In the present invention, passing of water containing no inorganic salt is preferably used as the mobile phase for deproteinization. The liquid passing rate and the liquid passing time of the deproteinized mobile phase differ depending on the type of sample and the type of pretreatment column, and the optimum conditions are appropriately determined by preliminary experiments. Generally, 0.5 ml / min to 1 ml / min
The protein can be removed from the pretreatment column by passing the solution for 10 minutes.

Next, as the organic solvent used in the mobile phase for extracting the drugs, hexane, tetrahydrofuran,
Dichloromethane, acetone, acetonitrile, methanol or the like can be used, and a polar solvent is particularly preferable,
Examples include acetonitrile and methanol.
The content of the organic solvent in the mobile phase for extraction is 50 to 90%
, Preferably 50 to 70% by weight, and most preferably 50 to 60% by weight.

The acid contained in the aqueous medium constituting the mobile phase for elution is preferably an organic acid, for example, butyric acid, propionic acid, acetic acid, formic acid, etc., preferably acetic acid or formic acid. Is. The amount of organic acid contained in the mobile phase for extraction is 0.05 to 2% by weight, preferably 0.1 to 1% by weight, most preferably 0.1 to 1% by weight.
0.2% by weight.

The flow rate of the mobile phase for elution can be appropriately determined by preliminary experiments, but it is generally 0.1 ml / min to 0.4 ml.
/ Min, preferably 0.1 to 0.3 ml / min, most preferably 0.2 to 0.25 ml / min for 5 minutes to 15 minutes.

The eluate containing the drug eluted by the drug-eluting mobile phase is then introduced into an analytical column to separate the individual drugs. As the packing material for the analytical column and the mobile phase, those used in the commonly used method can be adopted.

As the mobile phase for the analytical column, the mobile phase for drug elution used for eluting the drug from the pretreatment column of the present invention can be continuously flowed as it is.

The analysis column of the present invention is not particularly limited as long as it can analyze drugs, but a column packed with octadecylsilylated silica gel having a particle size of 1 to 15 μm is generally employed. For example commercially available
SUMIPAX ODS-A series, SUMIPAX ODS-C series, SUM
IPAX ODS-J series, SUMIPAX ODS-K series, SUMIPAX
ODS-L series (above manufactured by Sumika Chemical Analysis Service Co., Ltd.), Wako
The sil series (Wako Pure Chemical Industries, Ltd.) and the like are exemplified, and in particular, an inner diameter of 4.6 mm and a length of 3 cm filled with octadecyl silylated silica gel having a particle size of 3 μm are preferably used.

Drugs eluting from the analytical column are sequentially introduced to an HPLC detector such as an ultraviolet spectrophotometric detector, a fluorescence detector and a mass spectrometer provided at the outlet of the column to be detected.

When a mass spectrometer is used as a detector, the ionization method is not particularly limited, but in general, the electrospray method and the atmospheric pressure ionization method are excellent in sensitivity and versatility and can be preferably used. Although a single mass spectrometer can be used, if a tandem type (LC-MS / MS) in which two mass spectrometers are connected in series is used, there is no interference of components in a biological sample. More selective analysis is possible.

[0023]

EFFECTS OF THE INVENTION In the present invention, a drug elution mobile phase consisting of water, a polar organic solvent and an organic acid having a large elution effect is used for elution of a drug without using inorganic salts. The drug can be efficiently eluted from the pretreatment column. Further, since the amount used is about 1/5 to 1/10 less than when using a known mobile phase, (2) it can be quickly eluted from the pretreatment column. Due to the effects of (1) and (2) above, the solute retained in the pretreatment column can be introduced into the separation column in a non-diffused state. Furthermore, since a solution consisting of a mixed solution of water, an organic solvent and an organic acid is used at a minimum, it is possible to directly analyze a high-sensitivity analyzer such as a mass spectrometer without performing complicated operations, and analyze as it is. It is also preferable in terms of analytical sensitivity. The amount of the mobile phase used in the analytical column of the present invention is extremely small when a known mobile phase is used, so that it is economical and helps prevent environmental pollution.

[0024]

EXAMPLES Examples will be described below to explain the present invention in detail, but these examples do not limit the present invention.

Example 1 A pretreatment column having a hydrophilic group on the outer surface of the particle and a packing having a hydrophobic group inside the particle (L-column L- 1180, inner diameter 4.6mm, length 5c
m) and an octadecyl-filled packing material having a particle size of 3 μm (SUMIPA manufactured by Sumika Chemical Analysis Service Co., Ltd.)
X ODS J-03-4603, inner diameter 4.6 mm, length 3 cm) were connected.

Tetracycline 20 μg, 50 μg, 1
50 μl of a sample solution prepared by dissolving 00 μg and 200 μg in human serum 1 ml was applied to a pretreatment column at a flow rate of 1 ml / min, a deproteinizing mobile phase (water) was sent for 5 minutes, and then column switching was performed, A mobile phase for drug elution (water / acetonitrile / methanol / formic acid = 100/100/30 /
1) was transferred at a flow rate of 0.2 ml / min for 20 minutes. The tetracycline eluted from the analytical column was measured at a wavelength of 360 nm using an ultraviolet spectrophotometric detector.

As a result, an elution pattern as shown in FIG. 1 was obtained, the elution of the drug was about 9.5, and the required analysis time was 1
5 minutes. From the result of performing the same operation individually on human serum to which tetracycline was not added and the tetracycline solution, it was possible to identify that the peak in FIG. 1 was derived from tetracycline. Further, linear regression analysis was carried out using tetracycline added weight as X and the peak area value obtained from each as y, and good linearity with a correlation coefficient of 0.9996 or more was obtained.

Comparative Example 1 A method similar to that of Example 1 except that the mobile phase for drug elution of Example 1 was changed to methanol and phosphate buffer commonly used in HPLC and the flow rate was changed to 1 ml / min. The tetracycline was eluted at. As a result, the elution time of the drug was about 10 minutes and the time required for analysis was 15 minutes. Therefore, about 5 times the amount of solvent was required as compared with Example 1.

Example 2 50 μl of a sample solution prepared by dissolving 100 μg of daunorubicin in 1 ml of human serum was applied to the same pretreatment column as in Example 1 for deproteinization, and then column switching was performed to perform the pretreatment column and Example 1. The mobile column for analysis (water: acetonitrile: acetic acid = 100 /
100 / 0.2) was sent for 20 minutes at a flow rate of 0.2 ml / min. The same column as in Example 1 was used for analysis in the same mobile phase, and daunorubicin eluted from the analytical column was measured at a wavelength of 380 nm using an ultraviolet spectrophotometric detector. Table 1 shows the results.

Example 3 100 μg of buspirone was dissolved in 1 ml of human serum, 50 μl of a sample solution was subjected to analysis in the same mobile phase as in Example 2, and buspirone eluted from the analytical column was analyzed by an ultraviolet spectrophotometric detector. , Measured at a wavelength of 300 nm. Table 1 shows the results.

Example 4 50 μl of a sample solution prepared by dissolving 100 μg of atropine in 1 ml of human serum was subjected to analysis in the same column and mobile phase as in Example 2, and atropine eluted from the analytical column was analyzed using an ultraviolet spectrophotometric detector. Was measured at a wavelength of 210 nm. Table 1 shows the results.

Example 5 50 μl of a sample solution prepared by dissolving 100 μg of atenolol in 1 ml of human serum was subjected to analysis in the same mobile phase as the same column as in Example 2, and atenolol eluted from the analytical column was analyzed using an ultraviolet spectrophotometric detector. Was measured at a wavelength of 225 nm. Table 1 shows the results.

Example 6 100 μg of phenylpropanolamine hydrochloride was added to human serum 1
50 μl of the sample solution dissolved in ml was subjected to analysis in the same mobile phase as the same column as in Example 2, and phenylpropanolamine hydrochloride eluted from the analytical column was measured at a wavelength of 210 nm using an ultraviolet spectrophotometric detector. Table 1 shows the results. The chromatogram is shown in FIG.

Comparative Example 2 A test was conducted in the same manner as in Example 6 except that the analytical column was changed to a column packed with octadecylsilylated silica gel having a particle size of 5 μm and having an inner diameter of 2.1 mm and a length of 15 cm. The chromatogram is shown in FIG. As a result, as can be understood by comparing FIG. 2 and FIG. 3, when the column of Example 6 having a particle size of 3 μm, an inner diameter of 4.6 mm, and a length of 3 cm was used, the baseline fluctuation and the system peak were smaller, It has been found that the analysis can be suitably performed.

Example 7 50 μl of a sample solution prepared by dissolving 100 μg of diclofenac sodium in 1 ml of human serum was applied to the same pretreatment column as in Example 2 for deproteinization, and then column switching was performed to perform the pretreatment column and the example. The same analytical column as in 2 was connected, and the mobile phase for analysis (water: acetonitrile: acetic acid = 20/180 / 0.2) was sent at a flow rate of 0.2 ml / min. Diclofenac sodium eluted from the analytical column was measured at a wavelength of 275 nm using an ultraviolet spectrophotometric detector. Table 1 shows the results.

Example 8 A pretreatment column having a hydrophilic group on the outer surface of a particle and a packing having an ion-exchange group inside the particle (Shimadzu Corporation, Shim-pack SPC AE1, inner diameter 4 mm, long length) 1 cm)
And an analytical column (Wakosil 3C18HG, manufactured by Wako Pure Chemical Industries, Ltd., inner diameter 4.6 mm, length 5 cm) packed with a packing composed of octadecylation having a particle size of 3 μm were connected.

50 μl of a sample solution prepared by dissolving 100 μg of cefotaxime sodium in 1 ml of human serum was added at a flow rate of 1 ml /
The pre-treatment column for 5 minutes and add 5 times the mobile phase (water) for deproteinization.
Liquid is sent for a minute, then column switching is performed, and the pretreatment column and the analytical column are connected to each other, and the analytical mobile phase (water / acetonitrile / methanol / formic acid = 100 /
100/30/1) was sent at a flow rate of 0.2 ml / min.
Cefotaxime sodium eluted from the analytical column was measured at a wavelength of 235 nm using an ultraviolet spectrophotometric detector.
Table 1 shows the results.

Example 9 50 μl of a sample solution prepared by dissolving 100 μg of ampicillin in 1 ml of human serum was applied to the same pretreatment column as in Example 8 for deproteinization, and then column switching was performed to perform the pretreatment column and Example 8. Mobile phase for analysis (water: acetonitrile: acetic acid = 100/1) with the same analytical column as
00 / 0.2) was sent at a flow rate of 0.2 ml / min. Ampicillin eluted from the analytical column was measured at a wavelength of 210 nm using an ultraviolet spectrophotometric detector. Table 1 shows the results.

[Table 1] Example Drug name Retention time Recovery rate 2 Daunorubicin 9.5 minutes 93.2% 3 Buspirone 9.6 minutes 107.0% 4 Atropine 9.4 minutes 96.5% 5 Atenolol 9.5 minutes 94 4% 6 Phenylpropanolamine hydrochloride 9.6 min 102.0% 7 Diclofenac sodium 10.0 min 103.6% 8 Cefotaxime sodium 11.5 min 98.8% 9 Ampicillin 8.3 min 93.9%

Example 10 As a column for the column switching method HPLC, a pretreatment column having a hydrophilic group on the outer surface of the particle and a packing having a hydrophobic group inside the particle (Chemicals inspection L-column L-1180 manufactured by the association, inner diameter 4.6 mm, length 5 cm) and an analytical column packed with a packing composed of octadecylation with a particle size of 3 μm (SUMICAX ODS manufactured by Sumika Chemical Analysis Service Co., Ltd.)
Using J-03-4603, an inner diameter of 4.6 mm, and a length of 3 cm, they were connected to LC-MS (TSQ-7000, manufactured by Finnigan Matt).

Tetracycline 20 ng, 50 ng, 1
50 ng of a sample solution prepared by dissolving 00 ng and 200 ng in 1 ml of human serum was applied to a pretreatment column at a flow rate of 1 ml / min, a deproteinizing mobile phase (water) was sent for 5 minutes, and then column switching was performed. A mobile phase for drug elution (water / acetonitrile / methanol / formic acid = 100/100/30 /
1) was transferred at a flow rate of 0.2 ml / min for 20 minutes. LC-MS / MS of tetracycline eluted from the analytical column
Was measured. As a result, a peak of tetracycline appeared at a retention time of 9.8 minutes, and the recovery rate was 97.5%. The sensitivity was improved about 1000 times as compared with the case of using the ultraviolet spectrophotometric detector.

Example 11 50 μl of a sample solution prepared by dissolving 100 ng of daunorubicin in 1 ml of human serum was applied to the same pretreatment column as in Example 1 to perform deproteinization, and then column switching was performed to carry out the pretreatment column and Example 1. The mobile column for analysis (water: acetonitrile: acetic acid = 100 /
100 / 0.2) was sent for 20 minutes at a flow rate of 0.2 ml / min. The same column as in Example 10 was used for analysis in the same mobile phase, and daunorubicin eluted from the analytical column was analyzed by LC-M.
S was measured. Table 2 shows the results.

Example 12 50 μl of a sample solution prepared by dissolving 100 ng of buspirone in 1 ml of human serum was subjected to analysis in the same column and the same mobile phase as in Example 11, and buspirone eluted from the analytical column was added to L.
It was measured by C-MS / MS. The results are shown in Table 2.

Example 13 50 μl of a sample solution in which 100 ng of atropine was dissolved in 1 ml of human serum was subjected to analysis in the same column and mobile phase as in Example 11, and atropine eluted from the analytical column was added to L.
It was measured by C-MS / MS. The results are shown in Table 2.

Example 14 50 μl of a sample solution prepared by dissolving 100 ng of atenolol in 1 ml of human serum was subjected to analysis in the same mobile phase as the same column as in Example 2, and atenolol eluted from the analytical column was measured by LC-MS / MS. did. The results are shown in Table 2.

Example 15 100 ng of phenylpropanolamine hydrochloride was added to human serum 1
50 μl of the sample solution dissolved in ml was subjected to analysis in the same mobile phase as the same column as in Example 2, and phenylpropanolamine hydrochloride eluted from the analytical column was subjected to LC-MS / M.
S was measured. The results are shown in Table 2.

Example 16 50 μl of a sample solution prepared by dissolving 100 ng of diclofenac sodium in 1 ml of human serum was applied to the same pretreatment column as in Example 11 to perform deproteinization, and then column switching was performed to carry out the pretreatment column and the example. An analytical mobile phase (water: acetonitrile:
Acetic acid = 20/180 / 0.2) at a flow rate of 0.2 ml / min.
The solution was sent for 0 minutes. Diclofenac sodium eluted from the analytical column was measured by LC-MS / MS. The results are shown in Table 2.

Example 17 Pretreatment column having a hydrophilic group on the outer surface of the particle and a packing having an ion-exchange group on the inner surface of the particle (Shimadzu Corporation, Shim-pack SPC AE1, inner diameter 4 mm, long length) 1 cm)
And an analytical column (Wakosil 3C18HG, manufactured by Wako Pure Chemical Industries, Ltd., inner diameter 4.6 mm, length 5 cm) packed with a packing composed of octadecylation having a particle size of 3 μm were connected.

50 μl of a sample solution in which 100 ng of cefotaxime sodium was dissolved in 1 ml of human serum, a flow rate of 1 ml /
The pre-treatment column for 5 minutes and add 5 times the mobile phase (water) for deproteinization.
Liquid is sent for a minute, then column switching is performed, and the pretreatment column and the analytical column are connected to each other, and the analytical mobile phase (water / acetonitrile / methanol / formic acid = 100 /
100/30/1) was sent for 20 minutes at a flow rate of 0.2 ml / min. Cefotaxime sodium eluted from the analytical column was measured by LC-MS / MS. The results are shown in Table 2.

Example 18 100 μg of ampicillin was dissolved in 1 ml of human serum, and 50 μl of the sample solution was applied to the same pretreatment column as in Example 17 to perform deproteinization, and then column switching was performed to perform the pretreatment column and Example 17. A mobile phase for analysis (water: acetonitrile: acetic acid = 100) with the same analytical column as the above is connected.
/100/0.2) was sent for 20 minutes at a flow rate of 0.2 ml / min. Ampicillin eluted from the analytical column was analyzed by LC-M
It was measured by S / MS. The results are shown in Table 2.

[Table 2] Example Drug name Retention time Recovery rate 2 Daunorubicin 9.1 minutes 95.0% 3 Buspirone 9.3 minutes 99.0% 4 Atropine 9.1 minutes 106.9% 5 Atenolol 9.2 minutes 104 5% 6 Phenylpropanolamine hydrochloride 9.3 min 102.7% 7 Diclofenac sodium 9.9 min 96.9% 8 Cefotaxime sodium 10.8 min 97.8% 9 Ampicillin 7.9 min 94.9%

[Brief description of the drawings]

FIG. 1 shows a chromatogram obtained by an ultraviolet spectrophotometric detector in Example 1.

FIG. 2 shows a chromatogram obtained by an ultraviolet spectrophotometric detector using a column having an inner diameter of 4.6 mm and a length of 3 cm packed with octadecylsilylated silica gel having a particle diameter of 3 μm of Example 6.

FIG. 3 shows a chromatogram obtained by an ultraviolet spectrophotometric detector using a column having an inner diameter of 2.1 mm and a length of 15 cm packed with octadecylsilylated silica gel having a particle size of 5 μm of Comparative Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuo Mizooku 3-1,135 Kasugadenaka, Konohana-ku, Osaka-shi, Osaka Sumitomo Chemical Analysis Center Co., Ltd. (72) Inventor Takashi Suzuki, Konohana-ku, Osaka-shi, Osaka 3-1-135 Kasugade, Sumika Analysis Center Co., Ltd. (72) Inventor, Kimiaki Matsuda 3-1-1135 Kasugade, Konohana-ku, Osaka City, Osaka

Claims (13)

[Claims] 1. A method for extracting and separating a drug from a sample containing a protein, comprising using a solid phase extraction column for deproteinization to remove a liquid that retains the drug but elutes the protein. Deproteinization is performed as a mobile phase for protein treatment, and then drugs are eluted from the solid phase extraction column using an aqueous medium containing an organic acid and an organic solvent as a mobile phase for extraction to obtain drugs from the eluate. A method characterized by. 2. A method for separating and analyzing drugs in a sample containing protein, which comprises introducing the eluate obtained in claim 1 to an analytical column connected to a detector to analyze the drugs. A method characterized by. 3. The method according to claim 1, wherein the organic solvent is a polar solvent. 4. The method of claim 3, wherein the polar solvent is acetonitrile or methanol. 5. The method according to claim 1, wherein the organic acid is acetic acid or formic acid. 6. The flow rate of the mobile phase for extraction is 0.1 ml.
The method according to claim 1 or 2, wherein the flow rate is from 0.4 ml / min to 0.4 ml / min. 7. The packing material for the solid phase extraction column has a hydrophilic group mainly on the outer surface of the particle and a hydrophobic group inside the particle, or an anion exchange group inside the particle. The method according to claim 1 or 2, which is a filler having. 8. The method according to claim 1, wherein the sample containing the protein is serum or plasma. 9. The method according to claim 1 or 2, wherein the deproteinizing mobile phase is water. 10. The drug according to claim 1, wherein the drug is any one or more of phenylpropanolamine hydrochloride, atenolol, atropine, diclofenac sodium, ampicillin, buspirone, tetracycline, cefotaxime sodium and daunorubicin. the method of. 11. The method according to claim 2, wherein the analytical column is a column having a length of 1 cm to 3 cm packed with a packing material composed of octadecyl-silylated silica gel having a particle size of 3 μm. 12. The method according to claim 2, wherein the detector connected to the analytical column is a liquid chromatography detector or a mass spectrometer. 13. The ionization method in the mass spectrometer is an electrospray method or an atmospheric pressure ionization method, and the mass separation method is a single type or a tandem type.
The method according to claim 12.