JP2002311010A - Method for determining dihydroavermectine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining 22,23-dihydroavermectine B1a in a sample at high sensitivity. SOLUTION: In this method for determining 22,23-dehydroavermectine B1a in a sample, a sample from which a specific impurity component is substantially eliminated is prepared, and 22,23-dihydroavermectine B1a in the sample is detected in a high speed liquid chromatography/mass spectrum/mass spectrum method (LC/MS/MS) using avermectine B1a as an inner reference substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for high performance liquid chromatography / mass spectrum / mass spectrometry (LC
/ MS / MS) for measuring the concentration of 22,23-dihydroavermectin B _{1a in} a sample with extremely high sensitivity.

[0002]

2. Description of the Related Art 22,23-Dihydroavermectin B _1a is a major component of ivermectin, which is a parasitic drug for animals, accounting for 80% or more.
As a preventive / control agent for dog hookworm, cat roundworm, cattle,
It is widely used as an insecticide for endoparasites and ectoparasites in livestock such as pigs, sheep, and horses. The measurement of the concentration of ivermectin in a biological sample is frequently performed in studies on the pharmacokinetics of ivermectin, but is not limited thereto, and the concentration of residual ivermectin in livestock foods, for example, meat, milk, etc., exceeds a regulated value. Measurement of ivermectin concentration is frequently performed to confirm whether or not the ivermectin is present.

However, since the concentration of residual ivermectin and the effective concentration of ivermectin in blood that are acceptable in foods are very low, it is required to use a very sensitive measuring method in their measurement.

As a method for measuring ivermectin in a biological sample, for example, a high-performance liquid chromatography method using a fluorescent derivative (J. Chromatogr., Vol. 190 (1980) 367-376; J. C)
hromatogr., vol.413 (1987) 326-331) and high-performance liquid chromatography using UV (J. Pharm. Sci., vol.72).
(1983) by 1447-1450), a method for measuring the concentration of 22,23-dihydroavermectin B _1a in plasma are generally known. However, these high-performance liquid chromatograph methods require about 1 to 5 mL of a sample as a specimen, and the lower limit of quantification is only about 1 ng.
The measurement time also requires about 30 minutes per measurement.

[0005] Some studies have been made on the method of measuring ivermectin using a device combining liquid chromatography and mass spectrometry. For example, liquid chromatography and chemical ionization mass spectrometry have been studied.
A method for measuring ivermectin concentration in tissue by combining mass spectra has been proposed (Biomed. Mass Spect.
rom., vol.11 (1984) 172-176). However, the detection limit of this method is only about 5 to 10 ppb (ng / g). In addition, since no internal standard is used, there is a high possibility that the measurement results will vary. A method of measuring ivermectin concentration in milk, liver, etc. using particle beam liquid chromatography / mass spectrometry has also been proposed (Biol. Mass Spectrom., Vol. 22 (19).
93) 184-193), also in this method, the detection limit is only about 2 ppb because the ion is selected only once by the mass spectrum, and it is not yet satisfactory.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for quantifying ivermectin contained in a trace amount in blood or food with high sensitivity, and as a result of pretreatment, specific contaminant components were reduced. Using the removed sample, it was found that 22,23-dihydroavermectin B _1a in the sample can be detected with high sensitivity by LC / MS / MS using inexpensive avermectin B _1a which is available as an internal standard and inexpensive. Thus, the present invention has been completed.

Thus, according to the present invention, contaminants having a retention time of 10 minutes or less in liquid chromatography using an acetonitrile / water mixture (70:30) as a mobile phase on an octadecyl silica-filled column are substantially removed. 22, 23-dihydroavermectin B _1a in the sample is detected by LC / MS / MS using avermectin B _1a as an internal standard substance. -A method for the quantification of dihydroavermectin B _1a is provided.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS When quantifying a trace component in a sample, it is recommended to use an internal standard substance having a physicochemical behavior similar to the substance to be measured in order to minimize the dispersion of analysis results. ing. When a trace component in a specimen is analyzed by LC / MS / MS, a compound in which the substance to be measured is labeled with a stable isotope such as deuterium is generally used as an internal standard substance, but a trace amount is always contained in the specimen. In order to eliminate the influence of the natural isotope, it is usually necessary to use an internal standard substance having a molecular weight that is 3 or more away from the compound to be measured. However, 22,23-dihydroavermectin B, which is measured by the present invention,
_1a is Streptomyces avermitilis (Streptomyc
es avermitilis) obtained by selectively hydrogenating avermectin B _1a contained in avermectins separated and purified from a fermentation broth of an avermectin-producing strain of avermectins.
5.11 is a compound derived from a natural product having a macrocyclic structure containing a complex 16-membered lactone,
Obtaining compounds labeled with stable isotopes is by no means easy and, if available, would be quite expensive.

According to the present invention, the molecular weight of 22,23-dihydroavermectin B _1a to be measured is determined by performing LC / MS / MS using a sample from which a specific contaminant component has actually been removed. 2 only an easy and inexpensive avermectin B _1a to obtain because it is commercially available but does not differ it is possible to use as an internal standard, moreover, highly sensitive surprisingly 22,23-dihydroavermectin B _1a in the specimen Can be quantified.

[0010] As a specimen that can be used in the present invention is not particularly limited, it may be used either as long as it contains a 22,23-dihydro-avermectin B _1a, for example, animal blood, plasma, Biological samples such as serum, saliva, urine, etc .; various organs and tissues in animals;
Examples include edible liver and food derived from livestock such as dairy cows.

In the present invention, it is necessary to prepare a specimen from which specific contaminants have been substantially removed. Here, the specific contaminant component is, specifically, an acetonitrile / water mixed solution (70:
A component having a retention time of 10 minutes or less in high performance liquid chromatography using 30). For example, when using plasma as a specimen, such contaminants include proteins,
Water-soluble substances such as protein conjugates, carbohydrates and various low molecular polar substances; non-polar substances such as lipids are included. In the present invention, a sample from which such contaminant components have been substantially removed is used. Perform quantification.

Examples of the column packed with octadecyl silica used in the high performance liquid chromatography include ZORBAX XDB-C18 (manufactured by Hewlett-Packard), Inertsil ODS (manufactured by GL Science), Capcell pak
C18 (manufactured by Shiseido Co., Ltd.), L-column ODS (manufactured by the Chemical Inspection Association), Develosil ODS (manufactured by Nomura Chemical Co., Ltd.), Sym
measurement C18 (manufactured by Waters), Nucleosil C18 (manufactured by M. Nagel) and the like.

The removal of specific contaminant components from a sample can be performed by a purification method known per se, such as deproteinization, filtration, ultrafiltration, dialysis, liquid-liquid extraction, column chromatography, or the like, depending on the type of the sample. High-performance liquid chromatogram, liquid-solid extraction and the like can be used in appropriate combination.

For example, when plasma is used as a sample, the sample is firstly treated with Isolute C8 (International sorbent technotechnique).
Logic), Bond Elut C8 (Varian), Sep-pak C8
Octyl silica gel such as (Waters); Isolute C18
(Manufactured by International sorbent technology), Bond Elu
t C18 (manufactured by Varian), Sep-pak C18 (manufactured by Waters) and other octadecyl silica gel; applied to a reversed-phase solid-phase extraction column such as a polymer resin such as OASIS HLB; Elut CN (Varian), IsoluteCN (Inter
cyanopropyl silica gel such as national sorbent technology; Bond Elut SI (Varian), Isolute
By applying to a normal-phase solid-phase extraction column such as silica gel such as SI (manufactured by International sorbent technology),
A sample from which specific contaminant components usable in the present invention have been substantially removed can be obtained. The solid phase extraction column is not limited to the cartridge type,
Disc-type ones can also be used in appropriate combination. As the elution solvent, in the case of reversed phase solid phase extraction, for example, a mixed solution of methanol / water, a mixed solution of acetonitrile / water and the like can be mentioned, and among them, a methanol / water based solvent is suitably used. As an elution solvent for normal phase solid phase extraction, for example,
Hexane / ethyl acetate mixed liquid, hexane / toluene mixed liquid and the like can be mentioned, and among them, a hexane / ethyl acetate solvent is preferably used.

When the sample contains a large amount of contaminant components, for example, blood, tissues, organs, etc., as a pretreatment, a solvent is extracted for deproteinization or defatting, and then a reverse phase system is used. It is effective to apply to a solid phase extraction column and a normal phase solid phase extraction column. Examples of the extraction solvent in this pretreatment include hexane, isooctane, diethyl ether, toluene, ethyl acetate and the like.

These series of sample preparations were carried out on 22, 23
-4 to avoid degradation of dihydroavermectin B _1a
It is desirable to carry out at 0 ° C. or lower.

The LC / MS / MS can be carried out by appropriately selecting ordinary operating conditions used in this type of measurement, and there is no particular limitation. In the present invention, an ammonium-based mobile phase is preferably used. Examples of the ammonium-based mobile phase include an acetonitrile / ammonium formate aqueous solution mixture, an acetonitrile / ammonium acetate aqueous solution mixture, a methanol / ammonium formate aqueous solution mixture, and a methanol / ammonium acetate aqueous solution mixture. A mixed mobile phase is preferred. Here, the concentration of the aqueous solution of ammonium formate is generally 0.01 to 300 mM, preferably 30 to 7 mM.
0 mM, and the mixing ratio of acetonitrile and ammonium formate is generally 50:50.
１００100: 1, especially 70:30 to 95: 5.

Examples of the method of ionizing a sample in LC / MS / MS include soft ionization methods such as electrospray ionization, atmospheric pressure chemical ionization, and thermospray ionization. Among them, the electrospray ionization method is preferably used. Can be

The method for detecting the generated ions generally includes a full scan method, a selective ion monitoring (SIM) method, a selective reaction monitoring (SRM) method, and the like. In the present invention, the SRM method is preferably used.
In this SRM method, for example, as a precursor ion of 22,23-dihydroavermectin B _1a , m /
Only the ion exhibiting the largest peak in the range of z 892 to 893, preferably only the ion near m / z 892.5, is selected, and then the product ion generated when the ion is bombarded with argon gas and decomposed M / z 569-5
The ion showing the largest peak within the range of 70, preferably
By selecting only ions near m / z 569.5 and monitoring the amount of the ions, 22,23-dihydroavermectin B _1a in the target sample of the present invention can be quantified with high sensitivity. . In the above SRM method, by using the ammonium-based mobile phase, the peak intensity of the precursor ion to which ammonia is added can be increased.

Avermectin B _1a as the internal standard used in the present invention is, as described above, 22,23-
Since the molecular weight differs from dihydroavermectin B _{1a by} only 2, the m / z 89 of the natural isotope of avermectin B _1a is determined by ordinary LC / MS / MS measurement.
M / z 56 from precursor ions around 2.5
Due to the effect of product ions around 9.5,
The quantitative value of the product ion near m / z 569.5 to be monitored as the measured value of 3-dihydroavermectin B _1a may be high. However, in the present invention, as described above, the mobile phase and the analytical column The present invention solves this problem by separating both compounds in a short period of time by high performance liquid chromatography by combining the above.

According to the present invention, ammonia is added by using a sample from which specific contaminant components have been substantially removed as described above, and further by using an ammonium-based moving layer by LC / MS / MS. By efficiently generating a precursor ion, for example, when using plasma as a specimen, detection and measurement of 22,23-dihydroavermectin B _1a can be performed with a high sensitivity of 100 pg with a specimen volume of only 0.5 mL.

As described above, the use of the quantification method of the present invention makes it possible to quantify 22,23-dihydroavermectin B _1a in a sample extremely sensitively, in a short time, and at low cost.

[0023]

EXAMPLES The present invention will be described more specifically with reference to the following examples. Example 1 (1) Removal and Analysis of Contaminant Components Plasma was applied to Isolute C8 pre-washed with methanol and water. After washing with water and 50% methanol, the fraction eluted with 2 mL of methanol was collected. After evaporating the solvent, 3 mL of diethyl ether and 0.5 mL of water were added to the residue to give 10
Shake for minutes. After centrifugation, the organic layer was dried. The residue was dissolved in 0.5 mL of a hexane / ethyl acetate mixed solution (7/3), and washed with a hexane / ethyl acetate mixed solution (7/3).
Applied to nd ElutCN. After washing with a hexane / ethyl acetate mixed solution (7/3), further washing with 1 mL of a hexane / ethyl acetate mixed solution (1/1), and then collecting a fraction eluted with 3 mL of a hexane / ethyl acetate mixed solution (1/1). Water 0.
5 mL was added and shaken for 10 minutes. After centrifugation, the organic layer was separated, and the solvent was distilled off under a nitrogen stream on a water bath to obtain an analytical sample of contaminant components.

The analysis of the contaminants was performed for 30 minutes using high performance liquid chromatography (Waters 2690 separation module) under the following conditions. Column: Capcell pak C18 (3 μm, 4.6φ X100
mm), mobile phase: acetonitrile / water (70:30),
Flow rate: 1.0 mL, column temperature: 40 ° C., injection volume: 50
Dissolve in 100 μL of 100% methanol and inject 70 μL.

The analysis results are shown in FIG. 1 and FIG.

The chart of FIG. 1 is a chromatogram of a mixture of 22,23-dihydroavermectin B _1a and avermectin B _1a . The chart at the top of FIG.
The chromatogram of the blank plasma and the lower chart are the chromatograms after removing contaminants from the blank plasma.

In the charts of FIGS. 1 and 2, "AU" on the vertical axis is an abbreviation of "absorbance unit" and represents the magnitude of an electric signal of absorbance (unit is mV), and the vertical axis is chromatographic time (unit). Represents minutes). In addition, "IS."
Is the internal standard avermectin B _1a , and “I
"VM" means 22,23-dihydroavermectin B _1a , which is the main component of ivermectin. (2) Measurement of 22,23-dihydroavermectin B _1a concentration in dog plasma Reagents and Materials Ivermectin standard is European Pharmacopoeia Commi
Obtained from ssion. 22, 23-
The content of dihydroavermectin B _1a was 83.9%. Avermectin B _1a, an internal standard, is 20 ng / m
L was used as a methanol solution.

As the water, distilled water is converted to ultrapure water production equipment (trade name "MI
LLI-Q-Labo ", manufactured by Nippon Millipore). All other reagents and solvents are commercial grade products and HP
LC standard products were used. Canine drug-free plasma was stored frozen until use. The solid phase extraction column is Isolute C8 (200mg
/ 3 mL, manufactured by International Sorbent Technology) and Bond Elut CN (500 mg / 10 cc, manufactured by Varian)
It was used. 2. Preparation of standard curve sample and spiked recovery sample 22,23-dihydroavermectin B _1a 100,20
0, 500, 1000, 2000, 5000, 1000
To 0 pg, 2 ng of avermectin B _1a (100 μL as a methanol solution), 1 mL of water and 0.5 mL of drug-free dog plasma were added to prepare a calibration curve sample.

The spiked and recovered samples were as described above, with 22,23-dihydroavermectin B _1a content of 100, 2000, 10
000 pg was prepared. 3. Extraction method The above sample was washed with Isolute C8 with methanol and water.
Applied. After washing with water and 50% methanol, the fraction eluted with 2 mL of methanol was collected. After the solvent was distilled off, 3 mL of diethyl ether and 0.5 mL of water were added to the residue, and the mixture was shaken for 10 minutes. After centrifugation, the organic layer was dried. The residue was mixed with a hexane / ethyl acetate mixture (7/3) 0.5
The mixture was dissolved in mL, and applied to BondElut CN that had been pre-washed with a hexane / ethyl acetate mixture (7/3). After washing with a hexane / ethyl acetate mixture (7/3), further washing with 1 mL of a hexane / ethyl acetate mixture (1/1), and then washing with hexane / ethyl acetate
The fraction eluted with 3 mL of an ethyl acetate mixture (1/1) was collected. 0.5 mL of water was added and shaken for 10 minutes. After centrifugation, the organic layer was separated, and the solvent was distilled off under a nitrogen stream on a water bath to obtain an actually measured sample. The measured sample was dissolved in 100 μL of acetonitrile, and 10 μL of the solution was injected into LC. 4. Measurement equipment and measurement conditions The LC / MS / MS equipment used was an HP1100 (manufactured by Hewlett-Packard) in the LC section and a Quattro II in the MS section.
(Manufactured by Micromass). The measurement conditions are as follows. Column: ZORBAX XDB-C18 (3.5 μm, 2.1φ × 30
mm, Hewlett-Packard), column temperature: 4
0 ° C., mobile phase: acetonitrile / 50 mM / L ammonium formate mixed solution (90:10), flow rate: 0.2 mL / mi
n, ionization method: electrospray positive ion, measurement ion 22,23-dihydroavermectin B _1a precursor ion m / z 892.5 (M + NH ₄ ) ⁺ , product ion m / z 569.5, avermectin B _1a precursor ion m / z 890.5 (M + NH ₄ ) ⁺ , product ion m / z 567.5.

22,23-dihydroavermectin B _1a
The LC / MS / MS spectrum of is shown in FIG. In addition,
3, the upper part is a mass spectrum of product ions when the 22,23-mass spectrum of dihydro avermectin B _1a, the lower part of the m / z 893 and the precursor ion.

In FIG. 3, the vertical axis represents the intensity ratio of the other peaks when the ion showing the maximum peak is taken as 100%, and the horizontal axis represents the numerical value obtained by dividing the molecular weight by the electric charge. In addition, “ES +” means that the measurement was performed using positive ions in the electrospray ionization method, and the numerical values “2.19e4” and “537” described below are values on a 100% scale in each chart ( Numerical value obtained by integrating the area). 5. Calibration curve calibration curve peak area ratio obtained from samples (22,23-dihydroavermectin B _{1 a} / avermectin B _1a) and 2
The amount of 2,23-dihydroavermectin B _1a added was plotted, a regression linear equation (weighted 1 / x) was obtained by the least squares method, and the curve was used as a calibration curve.

FIG. 4 shows the obtained calibration curve.

In FIG. 4, the vertical axis represents the peak area ratio, and the horizontal axis represents the concentration (unit is ng). 6. Spike and Recovery Tests Tables 1 and 2 show the accuracy and accuracy of simultaneous reproducibility and daily reproducibility when 100, 2,000 and 10,000 pg of 22,23-dihydroavermectin B _1a were added to 0.5 mL of dog plasma.

[0034]

[Table 1]

[0035]

[Table 2]

From Tables 1 and 2, according to the method of the present invention,
Even samples in dog plasma 0.5 mL 22,23-dihydro-avermectin B _1a is not contained only trace amount of 100 pg, accuracy is within 7.4% and Accuracy is ±
It is 19.0%, which indicates that the quantification can be sufficiently performed by this measurement method.

[Brief description of the drawings]

FIG. 1 is a chromatogram of a mixture of 22,23-dihydroavermectin B _1a and avermectin B _1a .

FIG. 2 is a chromatogram of blank plasma, and the lower chart is a chromatogram after removing contaminants from the blank plasma.

FIG. 3. L of 22,23-dihydroavermectin B _1a
It is a spectrum of C / MS / MS.

FIG. 4 is a graph of a calibration curve.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/26 G01N 30/26 A 30/48 30/48 K 30/72 30/72 C (72) Invention Person Ryota Suzuki 5-14-6 Uedanaka, Nakahara-ku, Kawasaki-shi, Kanagawa

Claims (6)

[Claims] 1. A sample from a column packed with octadecyl silica, which is substantially free of contaminants having a retention time of 10 minutes or less in liquid chromatography using an acetonitrile / water mixture (70:30) as a mobile phase, 22, 23-dihydroavermectin B _1a in the sample is detected by high performance liquid chromatography / mass spectrum / mass spectrometry (LC / MS / MS) using avermectin B _1a as an internal standard. 22,23-Dihydroavermectin B _1a
Quantitation method. 2. The method according to claim 1, wherein the sample from which the contaminant components have been actually removed is prepared by purifying the sample by a combination of reversed-phase solid phase extraction and normal-phase solid phase extraction. 3. The method according to claim 1, wherein in LC / MS / MS, ions near m / z 892.5 are selected as precursor ions and ions near m / z 569.5 are selected and monitored as product ions. . 4. The method according to claim 1, wherein the mobile phase in the high performance liquid chromatography of LC / MS / MS is an ammonium-based mobile phase. 5. The method according to claim 4, wherein the ammonium-based mobile phase is a mixed solution of acetonitrile and an aqueous solution of ammonium formate. 6. The method according to claim 1, wherein the sample is animal plasma, serum, blood, urine, meat or milk.