CN105136957B - Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G - Google Patents

Abstract

The invention relates to a detection method for simultaneously measuring OXC and metabolites MHD and MHD-G in human plasma, belonging to the technical field of biological detection. In this experiment, nitroazepam was used as the internal standard, and after pretreatment by protein precipitation, the plasma concentrations of the three compounds could be detected simultaneously by high performance liquid chromatography-tandem mass spectrometry. The method of the invention has the advantages of short time, strong specificity, high sensitivity and simple operation, and has been successfully applied to the analysis and research of oxcarbazepine and its metabolites in human plasma.

Description

An assay for the simultaneous determination of OXC and its metabolites MHD and MHD-G in human plasma method

technical field

The invention relates to a detection method for simultaneously measuring OXC and metabolites MHD and MHD-G in human plasma, belonging to the technical field of biological detection.

Background technique

Epilepsy (Epilepsy) is a common nervous system disease worldwide. Its clinical feature is brain dysfunction caused by repeated abnormal discharge of brain nerve cells. The most common and most serious disease. If it is not well controlled, it will cause irreversible damage to the cranial nerves, seriously affect the growth and development of children, and bring heavy psychological burden and economic pressure to children, families and society.

Oxcarbazepine (OXC) is a new drug for the treatment of partial seizures and generalized tonic-clonic seizures. After oxcarbazepine (OXC) enters the body, hepatic cytosolic enzymes (AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3) can rapidly convert it into the active metabolite 10,11-dihydro,10-hydroxycarbamazepine ( MHD), and play a pharmacological role, MHD is further combined with glucuronic acid to be excreted under the catalysis of UGT, a small part of MHD is oxidized into pharmacologically inactive 10,11-dihydroxycarbamazepine (DHD), more than 90% Drug excreted in urine as metabolites: glucuronide (49%), MHD (27%), DHD (3%), other metabolites (13%), OXC less than 1%; less than 4% The original drug is excreted in feces. It can be seen that metabolism is the main form of OXC elimination in vivo, and the combined reaction of MHD and UGT II is the most important way for OXC to undergo biotransformation in vivo, affecting UGT activity will interfere with the metabolism of MHD in vivo, thereby changing the blood concentration of MHD , lead to treatment failure or even serious adverse reactions, which will cause great harm to the safety of patients.

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is the most commonly used method for detecting drug concentration. It has the characteristics of high sensitivity and strong specificity, and can detect a variety of trace compounds at the same time. Therefore, by simultaneously detecting the blood drug concentrations of OXC, MHD, and MHD-G in the human body, it is possible to more accurately monitor the dynamic metabolic changes of OXC in the body, and determine whether the concentration of MHD therapeutic drugs is due to reduced OXC metabolism or UGT gene mutations. reduce.

At present, the HPLC-MS/MS method verified by the system can only detect OXC and MHD at the same time, but it also has defects: such as a long time for aliasing (10min) (High-performance liquid chromatography-tandem massspectrometry method for simultaneous quantification ofcarbamazepine, oxcarbazepine, and their main metabolites in human serum. Ther Drug Monit.2012, 34 (1): 53-58), the processing method is complex (extraction method) (Simultaneous quantitative analysis of

oxcarbazepine and 10,11-dihydro-10-hydroxycarbamazepine in human plasma by liquid chromatography-electrospray tandem mass spectrometry. J PharmBiomed Anal. 2007, 45(2):304-311). For the detection method of MHD-G, no relevant literature was found, and there was no literature report on the simultaneous detection of OXC, MHD, and MHD-G. The present invention firstly established HPLC-MS/MS in human plasma of OXC, MHD, and MHD-G The determination method has laid a solid methodological foundation for the detection of clinical blood drug concentration of OXC.

Contents of the invention

The purpose of the present invention is to overcome the above disadvantages and provide a detection method for simultaneously measuring OXC and metabolites MHD and MHD-G in human plasma.

According to the technical scheme provided by the present invention, a method for simultaneously measuring OXC and metabolites MHD and MHD-G in human plasma, the steps are:

(1) Plasma sample pretreatment: take the plasma sample, add internal standard nitroazepam, then add acetonitrile to precipitate protein, speed centrifuge to take the supernatant, and obtain the pretreated plasma sample;

(2) Chromatography: the plasma sample after step (1) pretreatment is subjected to liquid chromatography separation: chromatographic column in chromatographic conditions: Phenomenex kinetex XB-C18 column; Mobile phase: acetonitrile: water volume ratio is 50:50, and water contains Formic acid with a mass concentration of 0.1%; flow rate: 250-300 μL/min; column temperature: 30-40 °C; injection volume: 10 μL;

(3) Mass Spectrometry: Ion Source: Electrospray Ionization ESI Ion Source, Mode Scanning: Selected Reaction Detection Scanning SRM, Spray Voltage: 3500~4000V, Sheath Gas: 20~50psi, Auxiliary Gas: 10~20arb, Capillary Temperature: 350 ℃, ion source temperature: 300 ℃;

(4) Calculation: Using the internal standard method, the peak area ratios of OXC, MHD, MHD-G and the internal standard nitrozepam were substituted into the standard curve equation to calculate the plasma concentrations of OXC, MHD, and MHD-G.

Step (1) is as follows: take 150 μL of plasma sample, add 15 μL of 1000 ng/mL internal standard nitrozepam solution, then add 285 μL of acetonitrile, vortex and mix for 5-10 min, centrifuge at 13000 rpm for 10-15 min, and take the supernatant after centrifugation.

The plasma sample in step (1) is plasma containing OXC, MHD, and MHD-G.

In step (2), the length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the filler particle size is 2.6 μm.

The detection ion in step (3) is specifically as follows: OXC selective reaction detection ion [M+H] ⁺ m/z253.2→m/z180.2CE:28eV; MHD selective reaction detection ion [M+H] ⁺ m/ z 255.2→m/z194.2CE:21eV; MHD-G selective reaction detection ion [M+H] ⁺ m/z 431.1→m/z194.2CE:28eV; internal standard nitroazepam selective reaction detection ion [M +H] ⁺ m/z 282.3 → m/z 236.2CE: 22eV.

Beneficial effects of the present invention:

(1) The pretreatment method is simple: a one-step protein precipitation method is adopted, which is suitable for routine detection.

(2) Strong specificity, adopt Phenomenex kinetex XB-C18 chromatographic column to separate, acetonitrile: water (water contains formic acid with a mass concentration of 0.1%) mixed solution as mobile phase, isocratic elution, the retention time of OXC is about 1.24min, the retention time of MHD is about 1.07min, the retention time of MHD-G is about 1.00min, and the retention time of internal standard Nitrazepam is about 1.55min. The four have good separation and can be separated within 3min. Complete the assay. In addition, endogenous substances do not interfere with the determination of both.

(3) High sensitivity: the lowest quantification limits of OXC, MHD, and MHD-G in plasma are 10, 50, and 125 ng/mL.

(4) Rapid detection: one measurement of a sample is completed in 3 minutes, and the time is short, so it is suitable for the detection of large quantities of biological samples.

(5) Small amount of sample: Only 150 μL of plasma sample is needed to determine the accurate concentration.

(6) The method of the present invention is fast, accurate, highly sensitive, and easy to operate, and provides a basis for the determination of blood drug concentrations of OXC, MHD, and MHD-G. The linear range of OXC, MHD, MHD-G plasma standard curves of this method is 10-750ng/mL, 50-10000ng/mL, 125-5000ng/mL, low QC intraday, interday precision and accuracy are less than 20%; Both medium and high QC intraday and interday precision and accuracy are less than 15%.

Description of drawings

Figure 1 is the mass spectrum of blank human plasma.

Figure 2 is the mass spectrogram of adding OXC, MHD, MHD-G and internal standard Nitrazepam to blank human plasma.

Figure 3 is a standard curve diagram of human plasma OXC.

Figure 4 is a standard curve diagram of human plasma MHD.

Fig. 5 is a standard curve diagram of human plasma MHD-G.

detailed description

According to the following examples, the present invention can be better understood, but those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention, and should not and will not limit the detailed description in the claims of the invention.

Example: Determination of Human Plasma OXC, MHD and MHD-G Concentrations

Experimental Materials and Instruments

Oxcarbazepine, purity >99.8%, batch number: 100657-201102, purchased from China National Institutes for Food and Drug Control; 10,11-dihydro-10-hydroxycarbamazepine, purity >98%, batch number: D449135, purchased from Bailingwei Company; 10,11-dihydro-10-hydroxycarbamazepine glucuronide, purity>95.5%, lot number: MD114541501, purchased from Carbosynth Company, UK; nitroazepam, lot number: 9201, purchased from National Narcotics Experiment Methanol, batch number: HX080973, acetonitrile, batch number: HX086245, all purchased from CNW Company; formic acid, batch number: T20090811, purchased from Sinopharm Chemical Reagent Co., Ltd.; water was made by ultrapure water (Milli-Q water machine).

Liquid chromatography system: ACCELA autosampler, ACCELA 1250pump, American Thermo Fisher Company; MS/MS system: TSQ QuantumAccess triple quadrupole tandem mass spectrometer, equipped with electrospray ionization ESI ion source, American Thermo Fisher Company; data acquisition: LC QUAN ^TM 2.6 software, Thermo Fisher Company, USA; XW-80A miniature vortex mixer, Shanghai Medical University Instrument Factory; Z233MK-2 high-speed centrifuge, HERMLE Company, Germany; BT25S analytical balance, Sartorius Company, Germany. HGC-12A Nitrogen Blowing Apparatus, Tianjin Hengao Company.

liquid condition

1. Liquid chromatography conditions:

Chromatographic column: Phenomenex kinetex XB-C18 (2.6μm, 2.1×100mm); mobile phase: acetonitrile:water (the water contains formic acid with a mass concentration of 0.1%)=50:50 (V/V); flow rate: 200～300μL/ min; column temperature: 30-35°C; injection volume: 10 μL.

2. Mass spectrometry conditions:

Ion source: Electrospray ionization ESI ion source, scanning mode: selective reaction detection scanning (SRM), spray voltage: 3500~4000V, sheath gas: 20~50psi, auxiliary gas: 10~20arb, capillary temperature: 350℃, ion Source temperature: 300°C. Detection ion: OXC selective reaction detection ion [M+H] ⁺ m/z 253.2→m/z180.2CE: 28eV; MHD selective reaction detection ion [M+H] ⁺ m/z 255.2→m/z194.2CE :21eV; MHD-G selective reaction detection ion [M+H] ⁺ m/z431.1→m/z194.2CE: 28eV; internal standard nitroazepam selective reaction detection ion [M+H] ⁺ m/z 282.3→m/z236.2CE:22eV;

experiment procedure

1. Preparation of OXC, MHD, MHD-G and internal standard nitroazepam standard solution

Accurately weigh a small amount of OXC standard substance, place it in a volumetric flask, and prepare 1 mg/mL OXC stock solution with methanol. Accurately measure an appropriate amount of OXC stock solution and dilute it sequentially with methanol to prepare OXC standard solutions with concentrations of 100, 200, 500, 1000, 2500, 5000, and 7500 ng/mL.

Accurately weigh a small amount of MHD standard substance, place it in a volumetric flask, and prepare a 1mg/mL MHD stock solution with methanol. Accurately measure an appropriate amount of MHD stock solution and dilute it sequentially with methanol to prepare MHD standard solutions with concentrations of 500, 1250, 5000, 20000, 50000, 80000 and 100000 ng/mL.

Accurately weigh a small amount of MHD-G standard substance, place it in a volumetric flask, and prepare a 1mg/mL MHD-G stock solution with methanol. Accurately measure an appropriate amount of MHD-G stock solution and dilute it sequentially with methanol to prepare MHD-G standard solutions with concentrations of 1250, 2500, 5000, 10000, 20000, 40000 and 50000 ng/mL.

Accurately weigh a small amount of the internal standard nitrozepam standard product, place it in a volumetric flask, and prepare a 1 mg/mL nitrozepam stock solution with methanol. Precisely measure an appropriate amount of stock solution and dilute it with methanol to prepare a standard solution of nitroazepam with a concentration of 1000ng/mL.

2. Specificity

Take 150 μL blank human plasma, add 300 μL acetonitrile to precipitate protein, vortex mix for 5-10 min, centrifuge at 13000 rpm for 10-15 min, take 10 μL supernatant for LC-MS/MS analysis. The mass spectrum of the blank human plasma sample is shown in Figure 1.

Take several 1.5mL EP tubes, add 15μL each of OXC, MHD, and MHD-G standard solutions, blow dry with nitrogen, add 150μL blank human plasma, 15μL 1000ng/mL internal standard nitroazepam solution and 285μL acetonitrile to precipitate protein, vortex Mix for 5-10 minutes, centrifuge at 13,000 rpm for 10-15 minutes, and take 10 μL of the supernatant for LC-MS/MS analysis.

The mass spectrum of adding OXC, MHD, MHD-G and internal standard Nitrazepam to blank human plasma is shown in Fig. 2 .

Note: channel a: OXC selective reaction detection ion [M+H] ⁺ m/z 253.2→m/z180.2CE: 28eV; channel b: MHD selective reaction detection ion [M+H] ⁺ m/z 255.2→ m/z194.2CE: 21eV; channel c: internal standard nitroazepam selective reaction detection ion [M+H] ⁺ m/z 282.3→m/z 236.2CE: 22eV; channel d: MHD-G selective reaction detection Ion [M+H] ⁺ m/z 431.1 → m/z 194.2 CE: 28eV.

The results showed that under the LC-MS/MS conditions used in this experiment, there were no impurities in the plasma that interfered with the detected substances, the retention time of OXC was about 1.24min, the retention time of MHD was about 1.07min, and the retention time of MHD-G The time is about 1.00min, the retention time of the internal standard nitroazepam is about 1.55min, and the determination is completed within 3min. OXC, MHD, MHD-G and internal standard nitroazepam do not interfere with each other, the peak shape is good, and the baseline is stable.

3. Standard curve

Take several 1.5mL EP tubes, add OXC, MHD, MHD-G standard series solutions respectively, after blowing dry with nitrogen, add 150 μL blank human plasma, and prepare the concentration as (OXC: 10, 20, 50, 100, 250, 500 , 750ng/mL; MHD: 50, 125, 500, 2000, 500, 8000, 10000ng/mL; MHD-G: 125, 250, 500, 1000, 2000, 4000, 5000ng/mL) standard drug-containing plasma. Then add 15 μL of 1000 ng/mL internal standard nitrozepam solution, 285 μL of acetonitrile to precipitate protein, vortex and mix for 5-10 min, centrifuge at 13000 rpm for 10-15 min, and take 10 μL of supernatant for LC-MS/MS analysis. Calculate the ratio f (f=As/Ai) of the peak area As and the internal standard peak area Ai of OXC, MHD, MHD-G respectively, take the analyte concentration c as the abscissa, take f as the ordinate, and use the weighted ( Weight coefficient: 1/x ² ) The least squares method is used to perform linear regression operation to obtain the regression equation.

OXC: f=0.0055663+0.00615469*c, ^r2 =0.9985;

MHD: f=0.0245203+0.000508853*c, ^r2 =0.9953;

MHD-G: f=-0.00106133+8.3264e-005*c, r ² =0.9967.

OXC had a good linear relationship in the range of 10-750ng/mL, and the lowest limit of quantification was 10ng/mL.

MHD has a good linear relationship in the range of 50-10000ng/mL, and the lowest limit of quantification is 50ng/mL.

MHD-G had a good linear relationship in the range of 125-5000ng/mL, and the lowest limit of quantification was 125ng/mL.

The specific standard curve is shown in Figure 3-5.

4. Lower limit of quantitation (LLOQ)

Take several 1.5mL EP tubes, add OXC, MHD, MHD-G standard solution respectively, after blowing dry with nitrogen, add 150μL blank human plasma, and prepare the concentration as (OXC: 10ng/mL; MHD: 50ng/mL; MHD- G: 125ng/mL) standard drug-containing plasma. Add 15 μL of 1000 ng/mL internal standard nitrozepam solution and 285 μL of acetonitrile to precipitate protein, vortex and mix for 5-10 minutes, centrifuge at 13000 rpm for 10-15 minutes, and take 10 μL of supernatant for LC-MS/MS analysis. Set up 5 parallel samples at the same time. Calculate the ratio f of the peak area As of OXC, MHD, MHD-G and the peak area Ai of the internal standard, and substitute it into the plasma standard curve of the day to obtain the measured concentration of OXC, MHD, MHD-G. The precision (RSD%) and accuracy (RE%) of the lower limit of quantitation (LLOQ) were calculated from the measured concentrations.

As can be seen from the results in Table 1, the RSD% and RE% of OXC, MHD, MHD-G are all within 15%, and the lower limit of quantitation (LLOQ) of OXC, MHD, MHD-G is respectively 10, 50, 125ng/mL (S/ N>10).

The lower limit of quantification (LLOQ) of OXC, MHD, MHD-G in table 1 LC-MS/MS method determination in human plasma

OXC (10ng/mL) RE% MHD(50ng/mL) RE% MHD-G (125ng/mL) RE% 1 10.60 106.04 42.18 84.36 130.23 104.18 2 10.06 100.61 47.39 94.78 122.59 98.07 3 9.31 93.09 48.12 96.23 100.02 80.02 4 9.84 98.42 45.54 91.07 110.87 88.70 5 10.45 104.46 48.54 97.07 131.07 104.85 average value 10.05 46.35 118.96 RSD% 5.11 5.61 11.21

5. Precision and accuracy

Prepare plasma samples containing OXC (20, 100, 500ng/mL), MHD (125, 2000, 8000ng/mL), MHD-G (250, 1000, 4000ng/mL) according to the human plasma standard curve method as low, medium and high Quality control (QC) samples. Add 15 μL of 1000 ng/mL internal standard nitrozepam solution and 285 μL of acetonitrile to precipitate protein, vortex and mix for 5-10 min, centrifuge at 13000 rpm for 10-15 min, and take 10 μL of the supernatant for LC-MS/MS analysis. For 3 consecutive days, 5 parallel samples of each concentration every day, calculate the ratio f of the peak area As of OXC, MHD, MHD-G and the peak area Ai of the internal standard, and substitute it into the plasma standard curve of the day to obtain the concentration of OXC, MHD, MHD-G For the measured concentration of -G, calculate the intra-day and inter-day precision from the measured concentration, which is the relative standard deviation (RSD%), and the ratio of the measured concentration to the theoretical concentration is the accuracy (RE%).

It can be seen from Table 2-4 that the low QC intraday and interday precision and accuracy of OXC, MHD and MHD-G are all less than 20%; the medium and high QC intraday and interday precision and accuracy are all less than 15%.

Table 2 The intraday and interday precision and accuracy of the LC-MS/MS method for the determination of OXC in human plasma

Table 3 The intraday and interday precision and accuracy of the LC-MS/MS method for the determination of MHD in human plasma

Table 4 The intraday and interday precision and accuracy of the LC-MS/MS method for the determination of MHD-G in human plasma

6. Recovery rate

Take several 1.5mL blank centrifuge tubes, add 15 μL each of OXC, MHD and MHD-G standard solutions of different concentrations, and blow dry with _N2 , then add 150 μL human blank plasma to prepare three concentrations of standard solutions with low, medium and high concentrations. Drug plasma (OXC plasma concentrations were 20, 100, 500ng/mL, MHD plasma concentrations were 125, 2000, 8000ng/mL, MHD-G plasma concentrations were 250, 1000, 4000ng/mL). Add 15 μL of 1000 ng/mL internal standard nitrozepam solution and 285 μL of acetonitrile to precipitate protein, vortex and mix for 5-10 minutes, centrifuge at 13000 rpm for 10-15 minutes, and take 10 μL of supernatant for LC-MS/MS analysis. The corresponding drug peak area and internal standard peak area were obtained, which were As(H) and Ai(H) respectively; 5 samples were analyzed for each concentration.

Take several 1.5mL blank centrifuge tubes, add 150μL human blank plasma, then add 300μL acetonitrile to precipitate protein, vortex and mix for 5-10min, centrifuge at 13000rpm for 10-15min, and set aside. Take several 1.5mL blank centrifuge tubes, add OXC, MHD and MHD-G quality control (QC) solutions of different concentrations and 15 μL of 1000ng/mL internal standard nitroazepam solution respectively, blow dry under _N2 , add Above-mentioned 450 μ L of acetonitrile centrifuges the blank plasma supernatant, vortex dissolves, is mixed with the reference substance solution of three concentrations of low, middle and high, injects and analyzes, obtains corresponding drug peak area and internal standard peak area, respectively As(D ), Ai(D), and 5 samples were analyzed for each concentration. The extraction recovery rate is calculated according to the following formula = [As(H)/As(D) average]×100%.

The results in Table 5-7 show that the recovery rate of OXC is 85.48%-96.24%, the recovery rate of MHD is 90.73%-97.95%, and the recovery rate of MHD is 72.30%-80.63%.

Table 5 LC-MS/MS method determines the recovery rate of OXC in plasma (n=5)

Table 6 LC-MS/MS method determines the recovery rate of MHD in plasma (n=5)

Table 7 LC-MS/MS method determines the recovery rate of MHD-G in plasma (n=5)

7. Test results

The trough concentrations of plasma OXC, MHD, and MHD-G were detected in 10 children after taking oxcarbazepine (OXC), see Table 8.

Table 8

sample OXC(ng/mL) MHD(ng/mL) MHD-G(ng/mL) Sample-1 54.20 3629.71 694.95 Sample-2 124.30 5731.20 1607.33 Sample-3 96.70 6055.96 1459.04 Sample-4 55.26 4047.15 1286.54 Sample-5 51.38 4339.84 507.61 Sample-6 77.90 4360.72 578.98 Sample-7 48.84 2826.03 856.97 Sample-8 120.55 5892.58 619.28 Sample-9 75.14 5244.91 614.33 Sample-10 77.00 6466.42 1667.10

It can be seen from Tables 1-8 that the method of the present invention is fast, accurate, highly sensitive, and easy to operate, and provides a basis for the determination of blood drug concentrations of OXC, MHD, and MHD-G. The linear range of OXC, MHD, MHD-G plasma standard curves of this method is 10-750ng/mL, 50-10000ng/mL, 125-5000ng/mL, low QC intraday, interday precision and accuracy are less than 20%; Both medium and high QC intraday and interday precision and accuracy are less than 15%.

Claims (1)

1. measure OXC and a detection method of metabolite MHD and MHD-G in human plasma simultaneously, it is characterized in that step is:

(1) plasma sample pretreatment: take plasma sample, adds internal standard nitrodiazepam, adds acetonitrile precipitation albumen, in centrifuging and taking Clear liquid, obtains plasma sample；

(2) chromatograph: step (1) pretreated plasma sample is carried out liquid chromatograph separation: chromatographic column in chromatographic condition: Phenomenex kinetex XB-C18 post；Flowing phase: acetonitrile: water volume ratio is 50:50, water containing mass concentration is The formic acid of 0.1%；Flow velocity: 250 ~ 300 μ L/min；Column temperature: 30 ~ 40 DEG C；Sample size: 10 μ L；

(3) mass spectrum: ion source: electro-spray ionization ESI ion source, scan mode: selective response detection scanning SRM, spraying electricity Pressure: 3500 ~ 4000V, sheath gas: 20 ~ 50psi, assists gas: 10 ~ 20arb, capillary temperature: 350 DEG C, ion source temperature: 300 ℃；

(4) calculate: use internal standard method, substitute into standard curve with the peak area ratio of OXC, MHD and MHD-G and internal standard nitrodiazepam Equation, calculates the blood drug level of OXC, MHD and MHD-G；

Step (1), particularly as follows: take plasma sample 150 μ L, adds 1000ng/mL internal standard nitrodiazepam solution 15 μ L, adds second Nitrile 285 μ L, vortex mixing 5 ~ 10min, 13000rpm are centrifuged 10 ~ 15min, take supernatant after being centrifuged；Step (1) described blood plasma sample Product are the blood plasma containing OXC, MHD and MHD-G；

In step (2), chromatogram column length is 100mm, and internal diameter is 2.1mm, and packing material size is 2.6 μm；

In step (3), detection ion is specific as follows: OXC selective reaction detection ion [M+H]⁺ m/z 253.2→ m/z180.2 CE: 28eV；MHD selective reaction detection ion [M+H]⁺m/z 255.2→ m/z194.2 CE: 21eV；MHD-G selects Property reaction detection ion [M+H]⁺m/z 431.1→ m/z194.2 CE: 28eV；Internal standard nitrodiazepam selective reaction detects Ion [M+H]⁺ m/z 282.3 → m/z 236.2 CE: 22eV。