KR101092371B1 - Simultaneous quantitaive analysis method for tobacco elements and metabolites thereof in human urine - Google Patents

Abstract

Disclosed is a method for the simultaneous analysis of tobacco components and their metabolites in urine using a liquid chromatography-mass spectrometer. Simultaneous analysis method can save time and money by analyzing the substances that require individual analysis through a single measurement process, it is possible to easily detect the risk for diseases associated with smoking.

Nicotine, NNK, Liquid Chromatography, Mass Spectrometry, Solid Phase Extraction, MRM

Description

Simultaneous analysis of tobacco components and their metabolites in urine {Simultaneous quantitaive analysis method for tobacco elements and metabolites of in urine}

The present invention relates to a method for the simultaneous analysis of tobacco components and their metabolites present in urine.

Nicotine and NNK (NNK; 4- (Methylnitrosamino) -1- (3-pyridyl) -1-butanone) are major components of tobacco and carcinogens.They are absorbed into the body and undergo various metabolic pathways. Excreted

In order to detect nicotine, NNK and their metabolites, gas chromatography-mass spectrometers, liquid chromatography-ultraviolet detectors, liquid chromatography-mass in the urine, plasma, saliva or tissues of smokers, non-smokers or cancer patients, etc. Methods using an analyzer or the like were used. However, these assays were limited in that they should be individually analyzed for nicotine and nicotine major metabolites or NNK and NNK major metabolites. Therefore, in order to analyze the overall metabolites excreted through urine, it is necessary to perform a separate experiment by varying the process for each substance, there was a problem that takes a lot of time and money.

Therefore, there is a need to develop an economical extraction method for simultaneously analyzing various substances including nicotine and its metabolites and NNK and its metabolites, and to develop stable detection methods that can obtain excellent analytical sensitivity even in a small amount of urine samples.

It is an object of one embodiment of the present invention to provide a high sensitivity simultaneous analysis of tobacco components and their metabolites.

The analytical method according to the present invention is characterized by simultaneously analyzing the tobacco components and their metabolites in urine using a liquid chromatography-mass spectrometer.

Simultaneous analysis of tobacco components and metabolites in urine according to the present invention can significantly reduce time and cost by simultaneously quantitatively analyzing nicotine and NNK and their overall metabolites, including highly polar substances. Through this, it is possible to provide effective and appropriate judgment indicators suitable for the treatment of diseases related to smoking.

Simultaneous analysis method according to an embodiment of the present invention, characterized in that the analysis of tobacco components and their metabolites in the urine simultaneously using liquid chromatography and mass spectrometry. Simultaneous analysis method according to the present invention has the advantage that it can analyze various nicotine, NNK and their metabolites at the same time. In addition, existing methods can improve the sensitivity of using a large amount of samples (about 1 ml or more) in order to analyze nicotine, NNK and their metabolites. .

In one embodiment, look at the simultaneous analysis method according to the present invention in more detail as follows. The simultaneous analysis method,

An extraction step of extracting the analyte from the sample urine;

A separation step of separating the extracted material by using liquid chromatography; And

An analytical step of analyzing the concentration of the tobacco component and its metabolites using mass spectrometry for the analyte that passed the liquid chromatography in the separation process.

The tobacco component and its metabolites are meant to include tobacco components and various physiological metabolites of the components. In one embodiment, the metabolites of tobacco components include nicotine and its metabolites, NNK (4- (Methylnitro samino) -1- (3-pyridyl) -1-butanone) and its metabolites.

In one embodiment, the extraction process may extract the analyte through a solid phase extraction process. In general, nicotine, NNK and their metabolites are characterized by a generally strong polarity. Therefore, for the simultaneous analysis of strong nicotine and ENK metabolites from the urine sample, the sample was extracted by solid phase extraction using the mixed mode Cation Exchange mode, concentrated by evaporation to dryness and high performance. Concentration is analyzed by applying to High Performance Liquid Chromatography-Mass Spectrometry.

In another embodiment, in the separation step, liquid chromatography is dissolved by dissolving the material extracted in the extraction step in a solution or methanol having a ratio of water and methanol in a volume ratio of 3: 7 to 9: 1. The column can be passed through. More specifically, the material extracted in the extraction process may be dissolved in a solution having a ratio of water and methanol of 1: 1 to pass through an analytical column of liquid chromatography. This is because, in preparing the solution for dissolving the extracted material, the resolution of the specific material may vary depending on the mixing ratio of water and methanol. Therefore, this range is to select the optimum range for the simultaneous separation of various metabolites.

In one embodiment, the analysis process may include a step of quantifying with MRM (Multiple Reaction Monitoring) in the MS-MS mode. Selected Ion Monitoring (SIM) is a method of using ions generated by collisions once in the source portion of a mass spectrometer. On the other hand, MRM is a method of using specific ions from the broken ions one more time to pass through another MS source connected in succession and collide one more time to use the ions obtained therefrom. More specifically, in SIM, there is a problem that the selected ions may interfere with quantification when the selected ions are ions that are also detected in plasma. On the other hand, in case of using MRM, even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. You get a line. Thus, by using the MRM mode in mass spectrometry, it is possible to simultaneously analyze the desired materials with better analytical sensitivity.

In one embodiment, the nicotine and its metabolites may include various metabolites generated during physiological metabolism along with nicotine. Nicotine and its metabolites include, for example, nicotine, nicotine-N-oxide, nornicotine, nicotine-N-glucuronide, cotinine ( Cotinnie, Cotinine-N-oxide, Norcotinine, Hydroxycotinine and the like, and may include one or more selected from the group consisting of. Simultaneous analysis method according to the present invention has the advantage that the simultaneous analysis of various metabolites that can be generated in the physiological metabolic process of nicotine is possible.

In another embodiment, the NNK (4- (Methylnitrosamino) -1- (3-pyridyl) -1-butanone) and its metabolites include various metabolites that occur during physiological metabolism along with ENNK. As NNK and NNK metabolites, for example, NNK, NNK-N-oxide; 4- (Methyl nitrosamino) -1- (3-pyridyl-N-oxide) -1- butanone), NNAL; 4- (Methyl nitrosamino) -1- (3-pyridyl) -1-butanol) and NNAL-N-oxide; 4- (Methylnitrosamino) -1- (3-pyridyl-N-oxide) -1-butanol) may include one or more selected from the group consisting of.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and the like, but this is only to illustrate the present invention, but the scope of the present invention is not limited thereto.

<Examples>

1. Solid-Phase Extraction Process

Nicotine-N-glucuronide (NIC-N-GLU), Cotinine-N-glucuronide (COT-N-GLU), Hydroxyen-glucoronide (Hydroxycotinin- Analytes, such as N-glucuronide, THOC-N-GLU, and NNAL-N-glucuronide, NNAL-N-GLU, are hydrophilic and lipophilic polymer forms HLB (Hydrophlic-liophilic balance) When extracted and analyzed using a filler, it was found that the extraction efficiency was very low, making it difficult to analyze.

Therefore, in order to analyze these highly polar analytes simultaneously, the extraction is performed using a mixed mode Cation exchange mode, which uses a filler that shows high extraction efficiency for the basic material and has a selective retention between the filler and the analyte. Extracted by.

2 mL of human urine is taken and placed in a 15 mL polypropylene tube, followed by the internal standard Nicotine-methyl-d ₃ and Cotinine-methyl-d. _3), hydroxy cotinine methyl-D _{3 (Hydroxycotinine-methyl-d 3} ) 10 ㎍ / ml and NK yen methyl-D _{3 (NNK-methyl-d 3} ), ¥ ¥ AL methyl -3 (NNAL-methyl-d ₃ ) 20 µl of 1 µg / ml mixed solution was added. Then, the prepared solution was vortexed for 1 minute and then subjected to solid-phase extraction.

2. Dissolution and Separation Process of Mixed Solution of Water and Methanol

The extracted solid phase extract is separated from the analyte by using a selective retention with the filler, and is subjected to a process of obtaining the remaining residue after evaporation using nitrogen gas. However, in the obtained residues, substances having opposite polarities are mixed. Therefore, in order to dissolve the residue containing these opposite polar substances, a solution of water and methanol in an appropriate ratio is required. In order to analyze both polar and nonpolar materials simultaneously, the appropriate mixing ratio for water and methanol must be determined. Therefore, in order to obtain a higher recovery rate, an experiment was conducted to select an appropriate mixing ratio of water and methanol.

The experimental process is as follows. First, the mixing ratio of water and methanol (water: methanol) was obtained from a solution in which the mixing ratio was adjusted to 3: 7, 5: 5, 7: 3, 9: 1, 0: 1 (pure methanol) based on the volume ratio. The residue was dissolved. The recovered residues were then compared when analyzed by high performance liquid chromatography-mass spectrometry.

Experimental results show that NNK is used when 30% methanol is used, Cotinine is used when 70% methanol is used, Cotinine and Cotinine-N- is used when 90% methanol is used. oxide, 100% methanol, nicotine-N-oxide, nornicotine, cotinine and cotinine-N-oxide are not detected. appear. Therefore, the experiment was conducted using a 50% methanol solution showing a relatively high recovery rate.

First, the extract extracted through the solid phase extraction method was dried with a vacuum evaporator (vacuum evaporator). After the drying, 100 µl of a solution of 5: 5 mixed with water and methanol was added to the residue, and vortexed for 1 minute to dissolve, followed by 10 µl injection into a high performance liquid chromatography-mass spectrometer for analysis.

3. Concentration Analysis of Nicotine and NNK by Liquid Chromatography-Mass Spectrometry

Instruments used to determine the concentrations of nicotine and its metabolites, NNK and its metabolites include high performance liquid chromatography (Agilent Co., Palo Alto, USA) and 6410 quadrupole mass spectrometers equipped with Agilent's binary pump. (triple quardruple mass spectrometer, Agilent Co., Palo Alto, USA). The column used is a Discovery column from Summer Spellco (Discovery HS F5 C18, 50 mm × 2.1 mm, Supleco, Bellefornte, PA, USA USA), 50 mm long, 2.1 mm inner diameter and 3 μm particle size. As a developing solvent, an aqueous solution containing 10 mM ammonium acetate and 0.1% acetic acid (solvent A) and 100% methanol (solvent B) were used. The flow rate was flowed at 200 μl / min, and the developing solvent conditions in the analysis were as follows.

In other words, the start was flowed into solvent A for 3 minutes, and then the solvent B was increased to 10% by 3.5 minutes, and then the ratio of solvent B was maintained at 10% by 6 minutes thereafter. Thereafter, solvent B was increased to 70% by 9 minutes, and solvent B was increased to 100% by 10 minutes again. Stayed here for 4 minutes thereafter.

Nicotine and its metabolites, NNK and its metabolites that passed through the analysis column were analyzed by applying to a mass spectrometer (MS, Mass Spectrophotometer). The mass spectrometer used for the analysis was equipped with an electrospary ionizer, an ion spray voltage of 4 KV, a nebulizer gas pressure of 40 arb (arbitrary unit), and a capillary temperature of 350 ° C. Mass spectrometry used a positive ionization method. In order to improve the signal-to-noise ratio, the analysis was performed in MRM (Multiple Reaction Monitoring) mode, which detects product ions generated by selectively catching ions corresponding to the molecular weight of the parent drug and then colliding again. .

The HPLC chromatogram of the analyzed nicotine and its metabolites, NNK and its metabolites is shown in Figure 1, the quantitative ions are shown in Table 1 below.

Analyte Light bulb
[Parent ion] Generated ions
Daughter ion Nicotine 163.1 84.1 Nicotine-N-oxide 179.0 132.0 Nornicotine 149.0 80.0 Nicotine-N-glucuronide 339.0 163.1 Cotinine 177.0 80.0 Cotinine-N-oxide 193.0 95.9 Norcotinine 163.1 80.0 Hydroxycotinine 193.0 79.9 NNK 208.1 122.0 NKK-N-oxide 224.1 122.0 NNAL 210.1 92.9 NNAL-N-oxide 226.1 93.0

4. Quantitative process

A calibration curve for quantifying nicotine and its metabolites, NNK and its metabolites was shown, through which the concentrations of nicotine and its metabolites and NNK and their metabolites in urine were quantified. The curve shown is shown in FIG. 2. As indicated by the calibration curve, all drugs showed good linearity of R = 0.993 or greater in the concentration range of 0.02 to 1000 ng / ml. Both the coefficient of variation (CV) and the accuracy showed good reproducibility and accuracy within 20%.

The results of quantifying the amount of nicotine and its metabolites, NNK and its metabolites actually detected in human urine using the curve are shown in Table 2 below.

Analyte Measure concentration
(ng / mL) Nicotine 427.96 ± 404.07 Nicotine-N-oxide 361.98 ± 221.68 Nornicotine 52.47 ± 32.27 Nicotine-N-glucuronide 434.44 ± 275.86 Cotinine 1056.35 ± 524.76 Cotininie-N-oxide 47.77 ± 19.01 Norcotinine 30122 ± 202.45 Hydroxycotinine 1151.49 ± 458.63 NKK 0.25 ± 0.15 NNK-N-oxide N.D. NNAL N.D. NNAL-N-oxide N.D.

N.D. Not Detected

1 is a chromatogram of nicotine and its metabolites, NNK and their metabolites extracted by a detection method according to an embodiment of the present invention using a high performance liquid chromatography / mass spectrometer;

Figure 2 shows a calibration curve for quantifying the concentration of nicotine and its metabolites, NNK and its metabolites.

Claims (7)

An extraction step of extracting the analyte from the sample urine into a solid phase by using a filler in a mixed mode cation exchange mode; A separation step of separating the extracted material by using liquid chromatography; And An analytical step of simultaneously analyzing the concentrations of tobacco components and their metabolites using mass spectrometry for the analyte that has passed liquid chromatography in the separation step; The mass spectrometry performs quantitative analysis in MRM (Multiple Reaction Monitoring), The tobacco component and its metabolites are nicotine, its metabolites and ENK (NNK; 4- (Methylnitro samino) -1- (3-pyridyl) -1-butanone) and their metabolites Method of simultaneous analysis of components and their metabolites. delete The method of claim 1, In the separation step, the extracted material is dissolved in a solution or methanol having a ratio of water and methanol in a ratio of 3: 7 to 9: 1, based on the volume ratio, and passed through an analytical column of liquid chromatography. Simultaneous analysis of tobacco components and their metabolites. The method of claim 3, wherein In the separation process, the extracted substance is dissolved in a solution having a ratio of water and methanol in a volume ratio of 1: 1 and passed through an analytical column of liquid chromatography, and its metabolites in urine. Simultaneous analysis method. delete The method of claim 1, Nicotine and its metabolites include Nicotine, Nicotine-N-oxide, Nornicotine, Nicotinine-N-glucuronide, Cotinine, Cotinine A method for the simultaneous analysis of tobacco components and metabolites in urine, characterized in that one or more selected from the group consisting of Cotinine-N-oxide, Norcotinine and Hydrooxycotinine. The method of claim 1, NK and its metabolites include NNK, NNK-N-oxide (4- (Methylnitrosamino) -1- (3-pyridyl-N-oxide) -1-butanone), JP NNAL; 4- (Methyl nitrosamino) -1- (3-pyridyl) -1-butanol) and NNAL-N-oxide; 4- (Methylnitrosamino) -1- (3-pyridyl -N-oxide) -1-butanol) at the same time selected from the group consisting of one or more tobacco components in the urine characterized in that the metabolic method.

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