JPH09119916A - Method for quantitative analysis of very small amount of compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method for quantitatively analyzing a target compound of which content in an organism sample is extremely small, by a high-speed liquid chromatography/mass spectrometer system. SOLUTION: The inner diameter of the column for a high-speed liquid chromatography is set to 1.0-4.6mm, the length is set to 1-25cm, and the particle diameter of an inverse-phase system filler in the column is 3-10μm. A fixed amount of target compound which is equal to or more than 0.1pg/ml and less than 10pg/ml is injected into a mass spectrograph and an optimized product ion is set to a take-in width which is 1 mass unit to 2 mass units for measurement, thus achieving, with an improved reproducibility, a relative standard deviation of 20% or less for a quantitative lower limit which is 0.1pg to less than 10pg when the content of the quantitative target compound in a sample is less than 1μg/ml.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a high performance liquid chromatography / mass spectrometer system (LC-MS / MS).
The present invention relates to a method for quantitatively analyzing a target compound whose content in a sample is extremely small. This quantitative analysis method can be used for measuring a trace amount of drug concentration in a sample involved in a pharmacokinetic test associated with a safety test in drug development.

[0002]

2. Description of the Related Art As a technique for measuring the concentration of a trace amount of drug in a biological sample using chromatography, LC-UV method, LC-fluorescence method, GC has already been used.
-NPD method and the like are well known. However, these analytical methods have a lower limit of quantification at the nano or sub-nanogram level, and are insufficient for the drug analysis of a very small amount (picogram / ml level). Recently, there is a report of GC-MS / MS method as an analysis method for picogram / ml level drug (ANALYTIC
AL BIOCHEMISTRY 164 , 156-163 (1987)), but this method requires a two-step derivative modification of pentafluorobenzyl esterification and trimethylsiletherification, which limits the target compounds and complicated pretreatment. Since it requires a lot of labor, it has a drawback that it is not suitable for the analysis of a large number of samples. There is also a report that the lower limit of quantification of 10 pg / ml was achieved without derivatizing the drug by LC-MS / MS (APCI) method (Biol. Mass Spectrometry 21 , 585).
-589 (1992)) cannot quantify the drug at very low picogram / ml levels below this. The present invention provides "an analysis means by a combination of a liquid chromatography technique and a mass spectrometer use technique" capable of stably quantifying a picogram / ml level drug concentration for a long time without the above-mentioned drawbacks. Is.

[0003]

A quantitative analysis method using a high performance liquid chromatography / mass spectrometer system (LC-MS / MS) comprising a high performance liquid chromatography and a mass spectrometer having two ion separation chambers. (1) The inner diameter of the high performance liquid chromatography column is 1.0 to 4.6 mm, the length is 1 to 25 cm, and the particle size of the reversed-phase packing material in the column is 3 to 10 μm. ) The mass spectrometer injects 0.1 pg or more and less than 10 pg of the quantitative target compound, and optimizes the product ion to 1
Quantitative when the S / N ratio is 3 or more, and (3) the content of the target compound in the sample is less than 1 μg / ml, when the measurement is performed by setting the intake width from the mass unit to 2 mass units. Lower limit 0.1 pg / ml or more 10 pg / ml
It is a quantitative analysis method characterized in that a relative standard deviation of 20% or less is realized with good reproducibility.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION In the above packed column, the inner diameter of the column is 1.0 to 4.6 mm, preferably 1.
It is 0 to 2.5 mm, and more preferably 2.1 mm or less.

The length of the column is 1 to 25 cm, 5 to
15 cm is more preferable. The particle size of the filler is 3 to 1
It may be 0 μm.

As the packing material, a packing material suitable for reverse phase chromatography is used as a stationary phase in relation to the mobile phase. Usually, a bonded filler obtained by chemically modifying a hydroxyl group on a silica surface with a methyl (C ₁ )-, aminopropyl-, cyanopropyl-, phenyl-, octyl (C ₈ )-or octadecyl (C ₁₈ ) -silyl group is used. Although used, a filler in which cellulose, protein, pyrene or the like is immobilized, carbon black or the like can also be used. Preferred are phenyl-, octyl-, octadecyl-silyl, and most preferred are those modified with octadecylsilyl groups. As such a filler, a commercially available one can be used.

The mobile phase is an aqueous medium, and as the organic solvent, acetonitrile, methanol, acetone, dioxane, tetrahydrofuran, ethanol, isopropanol and the like are used. Use properly according to the separation situation.

A mass spectrometer as a detector uses an integrated mass spectrometer having two ion separation chambers.
The analyzer used for carrying out the quantitative analysis method of the present invention is required to have an S / N ratio of 3 or more when injecting 0.1 pg or more and less than 10 pg of the quantitative target compound.

The S / N ratio is the ratio of the intensity of the target ion peak of the actual test chromatogram to the intensity of the noise peak in the blank test chromatogram. S / N of 3 or more
In order to obtain the ratio, the amount of the compound to be analyzed when introducing the compound to be analyzed into the ionization chamber after the start of operation of the mass spectrometer is 500 μg or less per time,
It is desirable to adjust the operation history by using it so that it is less than 00 μg and less than 50 mg per year.

The content of the quantitative target compound in the sample is 1 μg
When the product ion (daughter ion) optimized using the above-mentioned mass spectrometer is detected with a capture width of 1 mass unit or more and 2 mass units or less when the amount is less than 1 ml / ml, the relative standard deviation is 20% or less. Lower limit 0.1 pg / m
It can be measured with good reproducibility for 1 or more and less than 10 pg / ml. The lower limit of quantification refers to the minimum measurable quantity whose accuracy and precision in the test method have been confirmed. Relative standard deviation (RS
D.) refers to the percentage of the standard deviation of n measured values with respect to the average of the measured values.

In using the quantitative analysis method of the present invention, usually, pretreatment (cleanup) for removing biological components other than the target compound as much as possible is dissolved in, for example, a mixed solvent of water and an organic solvent. A sample solution), 1 to 100 μl of which is accurately weighed and introduced into the injection part of the high performance liquid chromatograph.

[0012]

【Example】

Example 1 Preparation of standard solution After dissolving 10 mg of hyoscyamine with a small amount of methanol,
To exactly 50 ml with methanol, this was used as a stock solution (200 μg / ml). Each stock solution was prepared by diluting this stock solution as follows. 10 μg / ml: 5 ml of the stock solution was taken and made exactly 100 ml with methanol. 0.5 μg / ml: Take 5 ml of the above and make exactly 100 ml with methanol. 0.05 μg / ml: Take 5 ml of the above and make exactly 50 ml with methanol. 0.02 μg / ml: 2 ml of the above was taken and made exactly 50 ml with methanol. 0.01 μg / ml: The above 4 ml was taken and made exactly 20 ml with methanol. 0.005 μg / ml: The above 5 ml was taken and made exactly 50 ml with methanol. 0.002 μg / ml: The above 5 ml was taken and made exactly 50 ml with methanol. 0.001 μg / ml: The above 2 ml was taken and made exactly 20 ml with methanol. 0.0005 μg / ml: 1 ml of the above was taken and made exactly 20 ml with methanol.

2. Pretreatment methods (see FIG. 1) internal standard serum 1 ml (IS): DL-methanol solution (25 ng / ml) of hyoscyamine _{-d 3 (N-methyl-d} 3 98%) added to, and mixed with 10 [mu] l, 14%
(V / v) Add 20 μl of ammonia water to add
H 9) and 1-butanol / 1-chlorobutane (1/9 v / v) under salting out by adding 250 mg of sodium chloride.
Add 5 ml, shake for 5 minutes, and centrifuge for 5 minutes (3000
rpm) and then the organic layer was separated. 1-butanol / 1-chlorobutane (1/9 v / v) was added again to the remaining aqueous layer.
Add 5 ml, shake for 5 minutes, and centrifuge for 5 minutes (3000
rpm), the organic layer was separated and combined with the previous organic layer. To this, add 300 μl of 1% (v / v) acetic acid and add 5
After shaking for 5 minutes and centrifugation (3000 rpm) for 5 minutes,
The organic layer was discarded, and the remaining aqueous layer was subjected to LC-MS under the following conditions 1
00 μl was injected.

LC-MS / MS device and measurement conditions (1) Device Triple stage quadrupole mass spectrometer TSQ7000 (manufactured by Finnigan MAT) LC-MS interface TSQ / SSQ700 API system (manufactured by Finnigan MAT) Liquid chromatograph 626 (Waters) Auto-injector 717plus (Waters) (2) Measurement conditions Analytical column SUMIPAX ODS C-210 2.1mm id x 150mmL Mobile phase 20mM Ammonium acetate / methanol = 4/6 (v / v) Column temperature Room temperature Flow rate 0.2ml / min Ionization method Electrospray ionization method Ion detection Positive ion detection Spray voltage 4.5kV Sheath gas pressure 70psi (N ₂ ) Auxiliary gas flow 10unit (N ₂ ) Capillary temperature 200 ℃ Collision gas pressure 1.5mTorr (Ar) Collision energy 30eV Electro Multipla 2.0kV Ear voltage monitor Ion Hyoscyamine m / z 290 → m / z 124 IS m / z 293 → m / z 127

3. Preparation of addition calibration curve To 1.0 ml of serum, the following standard solution was added and mixed well, and then treated according to the pretreatment method of 2. S-0 0 pg / ml serum: no additive S-1 5 pg / ml serum: standard solution 10 μl S-2 10 pg / ml serum: standard solution 10 μl S-3 20 pg / ml serum: standard solution 10 μl S-4 50 pg / ml serum: standard solution 10 μl S-5 100 pg / ml serum: standard solution 10 μl S-6 200 pg / ml serum: standard solution 10 μl S-7 500 pg / ml serum: standard solution The above sample was measured every 10 μl of measurement, and the concentration of hyoscyamine added x
And the relationship of the ratio y of the peak area of hyoscyamine to the peak area of the internal standard was determined by the weighted least squares method, and a calibration curve was prepared. As the weight in the least square method, the square of the reciprocal of the measured value, 1 / y ^2, was used.

4. Results (1) Lower limit of quantification in 5 measurements (S-of addition calibration curve
As a result of calculating the S / N ratio from the chromatogram of 1), the values were 8.0, 7.3, 6.5, 10.2 and 6.8, respectively. An example of the lower limit of quantification chromatogram is shown in FIG. (2) Confirmation of measurement range Table 1 shows the results. Addition calibration curve (5-500 pg / ml
The linearity of serum) was good with a correlation coefficient of 0.99 or more. In addition, in all concentration ranges, 2.2% to
It showed an accuracy of 16.9%.

[0017]

[Table 1] Accuracy of measurement range Concentration peak area ratio (pg / ml) 1st 2nd 3rd 4th 5th Mean ± SDRSD S0 (0.0) 0.0000 0.0000 0.0000 0.0000 0.0000 S1 (5.0) 0.1210 0.1281 0.1303 0.1137 0.1365 0.1259 ± 0.0088 7.0 S2 (10.1) 0.2519 0.1879 0.2346 0.1639 0.2058 0.2088 ± 0.0353 16.9 S3 (20.2) 0.4073 0.3699 0.4278 0.3281 0.4987 0.4064 ± 0.0641 15.8 S4 (50.4) 0.9121 0.8998 1.0686 0.7618 0.8529 0.8990 ± 0.1117 12.4 S5 (100.8) 1.7857 1.76064 1.4373 1.6836 1.6615 ± 0.1474 8.9 S6 (201.6) 3.7621 3.4589 3.1689 3.5479 3.8852 3.5646 ± 0.2783 7.8 S7 (504.0) 8.3288 8.2082 8.1567 8.0341 8.5182 8.2492 ± 0.1838 2.2 inclination 0.01722 0.01678 0.01675 0.01518 0.01738 0.01666 ± 0.0009 intercept 0.03836 0.03467 0.05512 0.02867 0.04821 0.04101 ± 0.0106 Relation number 0.99599 0.99801 0.99288 0.99392 0.99105 0.99437

(3) Intraday variation and daily variation Table 2 shows the results. The measurement accuracy in daily fluctuation (simultaneous reproducibility test) was 5.6 to 12.8%, and the average accuracy was 98.5.
-10 to 108.4%, the measurement accuracy in daily fluctuation (daily reproducibility test) is 8.6 to 10.2%, the average accuracy is 10
2.1 to 104.4 are shown.

[0019]

[Table 2] Daily fluctuation Addition amount Days Quantitative value Average quantified value SDRSD Accuracy Average accuracy (pg / ml) (pg / ml) (pg / ml) (%) (%) 4.6 92.0 5.5 110.0 Day 1 5.6 5.0 0.6 11.1 112.0 99.2 4.4 88.0 4.7 94.0 5.0 100.0 5.2 104.0 5.0 Day 2 5.7 5.4 0.3 5.6 114.0 108.4 5.6 112.0 5.6 112.0 5.5 110.0 4.4 88.0 Day 3 4.3 5.0 0.6 12.8 86.0 100.8 5.3 106.0 5.7 114.0 50.6 100.4 54.2 107.5 Day 1 56.2 49.6 6.0 12.1 111.5 98.5 41.7 82.7 45.5 90.3 49.7 98.6 43.0 85.3 50.4 Day 2 52.8 51.5 5.5 10.7 104.8 102.2 57.3 113.7 54.8 108.7 58.5 116.1 54.5 108.1 Day 3 51.3 53.2 3.5 6.6 101.8 105.5 49.4 98.0 52.2 103.6 523.6 103.9 452.7 89.8 1 Day 578.3 502.9 49.5 9.8 114.7 99.8 472.0 93.7 488.0 96.8 581.3 115.3 533.9 105.9 504.0 Day 2 509.1 531.6 43.7 8.2 101.0 105.5 562.6 111.6 470.9 93.4 531.9 105.5 593.4 117.7 Day 3 580.4 543.4 41.7 7.7 115.2 107.8 498.9 99.0 512.4 101.7 S.D .: Standard deviation R.S.D .: Relative standard deviation Accuracy (%) = quantitative value / added amount × 100

Daily variation Addition amount Quantitative value Average quantified value SDRSD Average accuracy (pg / ml) (pg / ml) (pg / ml) (%) 4.6 5.5 Day 1 5.6 4.4 4.7 5.0 5.2 5.0 Day 2 5.7 5.1 0.52 10.2 102.8 5.6 5.6 5.5 4.4 Day 3 4.3 5.3 5.7 50.6 54.2 Day 1 56.2 41.7 45.5 49.7 43.0 50.4 Day 2 52.8 51.4 5.0 9.7 102.1 57.3 54.8 58.5 54.5 Day 3 51.3 52.2
523.6 452.7 Day 1 578.3 472.0 488.0 581.3 533.9 504.0 Day 2 509.1 526.0 45.3 8.6 104.4 562.6 470.9 531.9 593.4 Day 3 580.4 498.9 512.4

[0021]

EFFECTS OF THE INVENTION According to the quantitative analysis method of the present invention, the derivative modification operation of the compound to be measured is not required, and the picogram /
The compound concentration at the ml level can be stably measured over a long period of time. For example, this quantitative analysis method can be used for measuring a trace amount of drug concentration in a biological sample involved in a pharmacokinetic test accompanying a safety test in drug development. In recent years, pharmaceuticals tend to be administered more and more in order to reduce adverse effects such as side effects. In response to this, measurement of the drug concentration in a biological sample is required to be further reduced. Under such circumstances, the usefulness of the present invention is extremely great.

[Brief description of the drawings]

FIG. 1 is a flowchart of a pretreatment method.

FIG. 2 is a chromatogram showing an example of measurement of the lower limit of quantification (hyoscyamine).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuo Mizooku 3-1,135 Kasugadenaka, Konohana-ku, Osaka City, Osaka Prefecture Sumika Analytical Center Co., Ltd. (72) Inventor Kaji Sawa, Osaka City, Osaka Prefecture 3-1-135 Kasugaden-ku, Sumitomo Chemical Analysis Center Co., Ltd. (72) Inventor, Hajime Kitahara 3-1-135 Kasukadinaka, Konohana-ku, Osaka, Osaka

Claims (2)

[Claims] 1. A high performance liquid chromatography / mass spectrometer system (LC-MS) comprising high performance liquid chromatography and a mass spectrometer having two ion separation chambers.
/ MS), (1) The above-mentioned high performance liquid chromatography column has an inner diameter of 1.0 to 4.6 mm, a length of 1 to 25 cm, and particles of a reversed-phase packing material in the column. The diameter is 3 to 10 μm, and (2) the mass spectrometer injects a quantitative target compound of 0.1 pg or more and less than 10 pg, and optimizes the product ions to a capture width of 1 mass unit or more and 2 mass units or less. When set and measured, the S / N ratio is 3 or more, and (3) when the content of the target compound for quantitative determination in the sample is less than 1 μg / ml, the lower limit of quantitative determination is 0.1 pg / ml or more and 10 pg
A quantitative analysis method characterized in that a relative standard deviation of 20% or less per 1 ml / ml is realized with good reproducibility. 2. The quantitative analysis method according to claim 1, wherein the inner diameter of the column is 1.0 to 2.5 mm.