CN106093230B - N in a kind of measurement blood plasma1The LC-MS-MS methods of methyl niacinamide concentration - Google Patents

N in a kind of measurement blood plasma1The LC-MS-MS methods of methyl niacinamide concentration Download PDF

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CN106093230B
CN106093230B CN201610382961.2A CN201610382961A CN106093230B CN 106093230 B CN106093230 B CN 106093230B CN 201610382961 A CN201610382961 A CN 201610382961A CN 106093230 B CN106093230 B CN 106093230B
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mna
ms
μ
plasma
methyl
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CN201610382961.2A
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CN106093230A (en
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储继红
居文政
戴国梁
刘鸣
张军
李长印
吴婷
许美娟
刘史佳
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江苏省中医院
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Abstract

The invention discloses N in a kind of measurement blood plasma1Methyl niacinamide (N1MNA 50 μ L of plasma sample are added in) the LC MS MS methods of concentration in 1.5mL plastic centrifuge tubes, and 30ngmL is added‑120 μ L of internal standard N ' methyl niacinamide (N ' MNA) solution add 160 μ L acetonitriles, and vortex vibrates 2min, and Aspirate supernatant carries out LC MS MS analyses after centrifugation.The present invention establishes N1Assay methods of the MNA in human body, compared with the method for document report, this method pre-treatment is easier, and lower limit of quantitation is lower, and analysis time is shorter, less using plasma sample amount, to study N1MNA and fat, diabetes and coronary heart disease correlation, find new biomarker, improve clinical prevention and treatment level provides technical support.

Description

N in a kind of measurement blood plasma1The LC-MS-MS methods of methyl niacinamide concentration

Technical field

The invention belongs to analysis technical fields, and in particular to N in a kind of measurement blood plasma1Methyl niacinamide concentration LC-MS-MS methods.

Background technology

With the development of the social economy, raising and the living-pattern preservation of living standards of the people, fat, diabetes and hat The incidence of worry is in worldwide rapid increase trend, it has also become influences the Major health problems of people's quality of life. New biomarker is found, finds and quantifies to be of great significance to preventing and early diagnosing these diseases.

Niacinamide (Nicotinamide, NA, also known as niacinamide) system has important biological action in human body.It should System is by niacinamide and its metabolite, the compositions such as metabolic enzyme.Niacinamide is in Nicotinamide N-Methyltransferase Under the action of (Nicotinamide N-methyltransferase, NNMT), S-adenosylmethionine (S- is utilized Adenosylmethionine, SAM) it is used as methyl donor, methylation reaction occurs, generates N1Methyl niacinamide (N1- methylnicotinamide,N1-MNA).Recently, studies have found that generated N in NA metabolic processes1- MNA may influence Energy i (in vivo) is metabolized, and to influence blood glucose and blood fat, reduces fat and diabetes incidences.Therefore, we establish one kind Quickly, N in sensitive LC-MS-MS methods detection human plasma1The concentration of-MNA, to study this new biomarker and fertilizer The degree of correlation of fat, diabetes and coronary heart disease has established technical foundation.

Invention content

Technical problem to be solved by the invention is to provide N in a kind of quick, sensitive measurement blood plasma1Methyl Buddhist nun gram acyl The LC-MS-MS methods of amine concentration.

In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

N in a kind of measurement blood plasma1Methyl niacinamide (N1- MNA) LC-MS-MS methods, it includes the following steps:

(1) plasma sample pre-processes:50 μ L of plasma sample are added in 1.5mL plastic centrifuge tubes, 30ngmL is added-1It is interior 20 μ L of N '-methyl niacinamide (N '-MNA) solution are marked, 160 μ L acetonitriles are added, vortex vibrates 2min, supernatant is drawn after centrifugation Liquid carries out LC-MS-MS analyses;

(2) preparation of retinue standard curve sample:1.5mL plastic centrifuge tube number branch is taken, 50 μ L of blank plasma are added, respectively The N of various concentration is added in precision110 μ L of-MNA standard solution, vortex 10s mixings are made into containing N1The concentration of-MNA is respectively 80, 40,20,10,5,2.5ngmL-1Plasma containing drug;20 μ L of internal standard N '-MNA solution are added, add 150 μ L acetonitriles, vortex shakes 2min is swung, Aspirate supernatant carries out LC-MS-MS analyses after centrifugation;

(3) liquid phase chromatogram condition is:

Chromatographic column:Waters Spherisorb CNRP, 4.6 × 150mm, 5 μm;

Mobile phase:A:Aqueous solution containing 0.1% (v/v) formic acid and 5mM/L ammonium formates;B:Acetonitrile;

Gradient elution is shown in Table 1;

Flow velocity:200mL·min-1

Column temperature:35℃;

Sampling volume:3μL;

Table 1

Time A:B volume ratio percentages % 0min 55:45 3.3min 90:10 3.6min 90:10 3.7min 55:45 5.5min 55:45

(4) mass spectroscopy condition is:

Ion source:Electro-spray ionization ionization source ESI;

Ion detection mode:Multiple-reaction monitoring;

Ion polarity:Cation;

Ion source capillary voltage:4000V;

Ion source temperature:350℃;

Dry gas:10L/min;

Atomization gas:20psi;

Detect the ion selector channel of object:N1- MNA, [M+H]+, m/z 137.1 → 94.1, fragmentor:100 V, collision energy:20V;Internal standard N '-MNA, [M+H]+, m/z 137.1 → 80.1, fragmentor:100 V, collision energy:25V;

(5) it calculates:Using internal standard method, with sample N1It is bent that the peak area ratio of-MNA and internal standard N '-MNA substitute into retinue standard Line equation calculates N1The plasma concentration of-MNA.

In step (1), the blood plasma is human plasma or animal blood plasma.

In step (1) and (2), internal standard the N '-MNA solution is prepared as follows to obtain:Weigh N '-MNA pairs It is placed in 10mL volumetric flasks according to product 10.33mg, methanol dissolving is added, is settled to scale, shakes up to get 1.033mgmL-1N’- MNA storing solutions, by N '-MNA storing solutions with dilution in acetonitrile to 30ngmL-1Inner mark solution.

In step (1) and (2), the centrifugation, condition is preferably 12000rmin-1Centrifuge 10min.

In step (2), the N1- MNA standard solution is prepared as follows to obtain:Weigh N1- MNA reference substances 10.40mg is placed in 10mL volumetric flasks, and methanol dissolving is added, is settled to scale, shakes up to get 1.040mgmL-1 N1-MNA Storing solution.By N1- MNA storing solutions are diluted successively with acetonitrile, be made into concentration be respectively 400,200,100,50,25,12.5ng mL-1Standard solution.

In step (2), the blank plasma is inactivation of virus refrigerated plasma, is the blood of human body product of Clinical practice, Anti-coagulants is sodium citrate, is purchased from Jiangsu Blood Center.Because background interference is extremely low, it is selected as the blank plasma of this experiment.

In step (5), the retinue calibration curve equation is the N by calculating retinue standard curve sample1-MNA The ratio f (f=As/Ai) of peak area As and internal standard peak area Ai, with f to N-1MNA plasma concentrations C, which makees to return, to be calculated, and is obtained Regression equation.

Advantageous effect:The present invention establishes N1Assay methods of-the MNA in human body, with document report [Journal of Chromatography B, 878 (2010) 895-902] method compare, this method pre-treatment it is easier (acetonitrile precipitation, on Clear liquid direct injection analysis eliminates concentration and volatilizes the processes such as redissolution), lower limit of quantitation is lower (by 10ngmL-1It is down to 2.5ng·mL-1), analysis time is shorter (being down to 5.5min by 15min), and less (50 are down to by 100 μ L using plasma sample amount μ L), to study N1- MNA and fat, diabetes and coronary heart disease correlation, find new biomarker, improve clinical prevention Technical support is provided with treatment level.The concentration of quantitative unknown sample generally requires and prepares retinue standard curve with blank medium, For endogenous material, when establishing detection method, how obtaining blank medium or how removing background interference is technology hardly possible Point.This experiment prepare used when standard curve inactivation of virus refrigerated plasma background interference it is extremely low, be the reason developed for method Think blank medium.

Description of the drawings

Fig. 1 is the LC-MS-MS figures of blank plasma.

Fig. 2 is that (blood plasma contains N to lower limit of quantitation figure1- MNA is:2.5ng·mL-1, containing the internal standard is:12ng·mL-1)。

Fig. 3 is that (blood plasma contains N to upper limit of quantification figure1- MNA is:80ng·mL-1, containing the internal standard is:12ng·mL-1)。

Fig. 4 is the LC-MS-MS figures of examinee's plasma sample.

Specific implementation mode

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited Invention.

Embodiment 1:

(1) experiment material and instrument

N1- MNA reference substances:It is provided by Sigma Co., USA, lot number:SML0704, content:>=99.5% (HPLC); N '-MNA reference substances:It is provided by Sigma Co., USA, lot number:M5402, content:>=97.0% (HPLC);Test water:It is ultrapure Water;Acetonitrile:Chromatographically pure (Merck Company).

Agilent 6430Triple Quad LC/MS combined instruments (Agilent companies of the U.S., MassHunter Workstation Sofeware,Version B.05.00);Plum Teller MT-5 electronic balances (Mei Tele companies of Switzerland); LEGEND MICRO17R high-speed refrigerated centrifuges (Thermo companies of the U.S.);Millipore Drict-Q5 ultrapure water machines (France Millipore companies).

(2) liquid matter condition

1. liquid phase chromatogram condition

Chromatographic column:Waters Spherisorb CNRP(4.6×150mm,5μm);

Mobile phase:A:Aqueous solution containing 0.1% (v/v) formic acid and 5mM/L ammonium formates;B:Acetonitrile

Gradient elution is shown in Table 1;

Flow:200μL·min-1

Column temperature:35℃;

Sampling volume:3μL;

Analysis time:5.5min.

2. Mass Spectrometry Conditions

Ion source:Electro-spray ionization ionization source ESI;

Ion detection mode:Multiple-reaction monitoring (MRM);

Ion polarity:Cation;

Ionization mode:Electro-spray ionization;

Detect object:N1- MNA, [M-H]+, m/z 137.1 → 94.1 (fragmentor 100V, collision energy 20V);

N '-MNA, [M-H]+, m/z 137.1 → 80.1 (fragmentor 100V, collision energy 25V);

Ion source capillary voltage:4000V;

Ion source temperature:350℃;

Dry gas:10L/min;

Atomization gas:20psi.

(3) experimentation:

1.N1The preparation of-MNA and N '-MNA standard solution:

Precision weighs N1- MNA reference substances 10.40mg is placed in 10mL volumetric flasks, and methanol dissolving is added, is settled to scale, It shakes up to get 1.040mgmL-1N1- MNA storing solutions.By N1- MNA storing solutions are diluted successively with acetonitrile, are made into concentration and are respectively 400、200、100、50、25、12.5ng·mL-1Working solution.

Precision weighs N '-MNA reference substances 10.33mg and is placed in 10mL volumetric flasks, and methanol dissolving is added, is settled to scale, It shakes up to get 1.033mgmL-1N '-MNA storing solutions, by N '-MNA storing solutions with dilution in acetonitrile to 30ngmL-1Internal standard Solution.

2. the processing of human plasma sample:

It is accurate in 1.5mL plastic centrifuge tubes that 50 μ L of examinee's plasma sample are added, internal standard N '-MNA solution is added (30ng·mL-1) 20 μ L, 160 μ L acetonitriles are added, vortex vibrates 2min, 12000rmin-110min is centrifuged, supernatant is drawn Liquid carries out LC-MS-MS analyses.

3. specificity:

N in the blood plasma that this experiment is surveyed1- MNA is endogenous material, is more or less all existed in vivo.How reality is obtained The blank medium for testing needs is our problems to be solved.The inactivation of virus frozen blood that we buy from Jiangsu Blood Center N in slurry1The content of-MNA is generally very low, generally within the 20% of lower limit of quantitation or even 10%.Therefore, we select N1-MNA The very low inactivation of virus refrigerated plasma of content is (referred to as:Blank plasma) it is used as replacement blank medium.

It is accurate in 1.5mL plastic centrifuge tubes that 50 μ L of people's blank plasma are added, 180 μ L acetonitriles, vortex oscillation is added 2min, 12000rmin-1Centrifuge 10min, Aspirate supernatant carries out LC-MS-MS analyses (see Fig. 1).

It is accurate in 1.5mL plastic centrifuge tubes that 50 μ L of people's blank plasma are added, N is added1- MNA standard solution (12.5ng·mL-1) 10 μ L, internal standard N '-MNA solution (30ngmL is added-1) 20 μ L, 150 μ L of acetonitrile, vortex oscillation is added 2min, 12000rmin-1Centrifuge 10min, Aspirate supernatant carries out LC-MS-MS analyses (lower limit of quantitation is shown in Fig. 2).

It is accurate in 1.5mL plastic centrifuge tubes that 50 μ L of people's blank plasma are added, N is added1- MNA standard solution (400ng mL-1) 10 μ L, internal standard N '-MNA solution (30ngmL is added-1) 20 μ L, 150 μ L of acetonitrile are added, vortex vibrates 2min, 12000r·min-1Centrifuge 10min, Aspirate supernatant carries out LC-MS-MS analyses (upper limit of quantification is shown in Fig. 3).

It is accurate in 1.5mL plastic centrifuge tubes that 50 μ L of examinee's blood plasma to be measured are added, internal standard N '-MNA solution is added (30ng·mL-1) 20 μ L, 160 μ L of acetonitrile are added, vortex vibrates 2min, 12000rmin-1Centrifuge 10min, Aspirate supernatant Carry out LC-MS-MS analyses (see Fig. 4).

The results show that under the conditions of LC-MS-MS used by this experiment, N1- MNA retention times in 3.03min or so, Internal standard N '-MNA retention times are in 1.85min or so.N1- MNA and internal standard are not interfere with each other, and peak shape is good, no miscellaneous peak interference, baseline Steadily.

4. linear test

1.5mL plastic centrifuge tube number branch is taken, 50 μ L of blank plasma are added, respectively the accurate N that various concentration is added1-MNA 10 μ L of standard solution, vortex 10s mixings are made into containing N1The concentration of-MNA is respectively 80,40,20,10,5,2.5 ngmL-1's Plasma containing drug.Internal standard N '-MNA solution (30ngmL is added-1) 20 μ L, 150 μ L acetonitriles are added, vortex vibrates 2min, 12000r·min-110min is centrifuged, Aspirate supernatant carries out LC-MS-MS analyses.Calculate N1- MNA peak areas As and internal standard peak The ratio f (f=As/Ai) of area Ai, with f to N-1MNA plasma concentrations C, which makees to return, to be calculated, and regression equation is obtained:

F=0.0631 × C+0.0119, r=0.9995, weight coefficient w=1/C

The N measured in this way1The minimum of the plasma concentration of-MNA is quantitatively limited to 2.5ngmL-1

5. accuracy and precision

1.5mL plastic centrifuge tube number branch is taken, is prepared by standard curve preparation method and contains N1- MNA concentration is respectively 3.75, 15,60ngmL-1Blood plasma quality-control sample and a retinue standard curve, operated by under " linear test " item.Do a batch daily Quality-control sample and a retinue standard curve, continuously do 3 days, three batches, each concentration of every batch of makees 5 parts of samples, calculates N totally1-MNA The ratio f of peak area As and internal standard peak area Ai are substituted into the standard curve on the same day and are acquired actual measurement N1- MNA concentration, it is dense by surveying Degree calculates batch interior and betweenrun precision, and measured concentration and the ratio that concentration is added are accuracy.The results show that in a few days and in the daytime Precision RSD is respectively less than 10%, and accuracy meets the requirements.

6. measurement result

We have detected N in 343 parts of examinee's plasma samples1The content of-MNA, the mean concentration measured are 9.33ng mL-1(SD=± 8.42).

Claims (6)

1. N in a kind of measurement blood plasma1The LC-MS-MS methods of methyl niacinamide concentration, which is characterized in that it includes following step Suddenly:
(1) plasma sample pre-processes:50 μ L of plasma sample are added in 1.5mL plastic centrifuge tubes, 30ngmL is added-1Internal standard 20 μ L of N '-methyl niacinamides solution add 160 μ L acetonitriles, and vortex vibrates 2min, and Aspirate supernatant carries out LC- after centrifugation MS-MS is analyzed;
(2) preparation of retinue standard curve sample:1.5mL plastic centrifuge tube number branch is taken, 50 μ L of blank plasma are added, are separately added into The N of various concentration110 μ L of methyl niacinamide standard solution, vortex 10s mixings are made into containing N1The concentration of methyl niacinamide Respectively 80,40,20,10,5,2.5ng.mL-1Plasma containing drug;Internal standard N ' -20 μ L of methyl niacinamide solution are added, then add Enter 150 μ L acetonitriles, vortex vibrates 2min, and Aspirate supernatant carries out LC-MS-MS analyses after centrifugation;
(3) liquid phase chromatogram condition is:
Chromatographic column:Waters Spherisorb CNRP, 4.6 × 150mm, 5 μm;
Mobile phase:A:Aqueous solution containing 0.1% (v/v) formic acid and 5mM/L ammonium formates;B:Acetonitrile;
Gradient elution is shown in Table 1;
Flow velocity:200mL.min-1
Column temperature:35℃;
Sampling volume:3μL;
Table 1
Time A: B volume ratio percentage % 0min 55∶45 3.3min 90∶10 3.6min 90∶10 3.7min 55∶45 5.5min 55∶45
(4) mass spectroscopy condition is:
Ion source:Electro-spray ionization ionization source ESI;
Ion detection mode:Multiple-reaction monitoring;
Ion polarity:Cation;
Ion source capillary voltage:4000V;
Ion source temperature:350℃;
Dry gas:10L/min;
Atomization gas:20psi;
Detect the ion selector channel of object:N1- MNA, [M+H]+, m/z 137.1 → 94.1, fragmentor:100V, collision energy:20V;Internal standard N '-MNA, [M+H]+, m/z 137.1 → 80.1, fragmentor:100V, collision energy:25V;
(5) it calculates:Using internal standard method, with sample N1The peak area ratio of methyl niacinamide and internal standard N '-methyl niacinamide Retinue calibration curve equation is substituted into, N is calculated1The plasma concentration of methyl niacinamide;
In step (2), the blank plasma is inactivation of virus refrigerated plasma, and anti-coagulants is sodium citrate.
2. according to the method described in claim 1, it is characterized in that, in step (1), the blood plasma is human plasma or animal Blood plasma.
3. according to the method described in claim 1, it is characterized in that, in step (1) and (2), the internal standard N '-methyl Buddhist nun gram Amide solution is prepared as follows to obtain:N '-methyl niacinamide reference substances 10.33mg is weighed to be placed in 10mL volumetric flasks, Methanol dissolving is added, is settled to scale, shakes up to get 1.033mgmL-1N '-methyl niacinamide storing solutions, by N '-methyl Niacinamide storing solution is with dilution in acetonitrile to 30ngmL-1Inner mark solution.
4. according to the method described in claim 1, it is characterized in that, in step (1) and (2), the centrifugation, condition is 12000r·min-1Centrifuge 10min.
5. according to the method described in claim 1, it is characterized in that, in step (2), the N1Methyl niacinamide standard is molten Liquid is prepared as follows to obtain:Weigh N1Methyl niacinamide reference substance 10.40mg is placed in 10mL volumetric flasks, and first is added Alcohol dissolves, and is settled to scale, shakes up to get 1.040mgmL-1N1- MNA storing solutions;By N1- MNA storing solutions with acetonitrile successively Dilution, be made into concentration be respectively 400,200,100,50,25,12.5ng.mL-1Standard solution.
6. according to the method described in claim 1, it is characterized in that, in step (5), the retinue calibration curve equation is Pass through the N of calculating retinue standard curve sample1The ratio f of methyl niacinamide peak area As and internal standard peak area Ai, with f couples N1Methyl niacinamide plasma concentration, which is made to return, to be calculated, obtained regression equation.
CN201610382961.2A 2016-06-01 2016-06-01 N in a kind of measurement blood plasma1The LC-MS-MS methods of methyl niacinamide concentration CN106093230B (en)

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