WO2023065521A1 - Gas chromatography-mass spectrometry combined method for determining genotoxic impurity 1,3-dichloro-2-propanol - Google Patents

Gas chromatography-mass spectrometry combined method for determining genotoxic impurity 1,3-dichloro-2-propanol Download PDF

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WO2023065521A1
WO2023065521A1 PCT/CN2021/139655 CN2021139655W WO2023065521A1 WO 2023065521 A1 WO2023065521 A1 WO 2023065521A1 CN 2021139655 W CN2021139655 W CN 2021139655W WO 2023065521 A1 WO2023065521 A1 WO 2023065521A1
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solution
gas chromatography
mass spectrometry
propanol
dichloro
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PCT/CN2021/139655
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French (fr)
Chinese (zh)
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麻新华
周贝贝
陈延安
金美春
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浙江海正药业股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7206—Mass spectrometers interfaced to gas chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Definitions

  • the invention belongs to the field of drug analysis and detection, and in particular relates to a gas chromatography-mass spectrometry method for measuring genotoxic impurity 1,3-dichloro-2-propanol.
  • Linezolid is a synthetic oxazolidinone antibiotic that acts on the bacterial 50S ribosomal subunit, inhibits the connection between mRNA and ribosomes at the initial stage of the translation system, and prevents the formation of the 70S initiation complex. Thereby inhibiting bacterial protein synthesis.
  • G + Gram-positive cocci
  • the intermediate linezolid epoxy compound (formula II) is used in the synthesis process of linezolid, and the starting material used for synthesizing the intermediate is epichlorohydrin, and the preparation process of epichlorohydrin in industry will produce 1,3-Dichloro-2-propanol.
  • the list of carcinogens released by the International Agency for Research on Cancer of the World Health Organization was initially compiled for reference.
  • 1,3-dichloro-2-propanol has a low limit (15ppm) and no ultraviolet absorption, it cannot be directly detected by liquid chromatography, and it is not easy to ionize, so it can be directly detected by LC-MS or GC-MS. Signals are generally poor.
  • CN201910616208.9 discloses a detection method for residual chloropropanol compounds in ornidazole, which uses headspace gas chromatography and uses a hydrogen flame ionization detector (FID) for measurement, and the sensitivity of the instrument is relatively poor (the detection limit concentration 150ng/mL: prepare the reference substance stock solution of 7.5 ⁇ g/ml, then take 0.2ml to 10ml volumetric flask to constant volume, draw the detection limit concentration to be 150ng/mL), the sample usage is relatively large and the concentration of the sample
  • the pretreatment is comparatively loaded down with trivial details (take about 1.0g of ornidazole, weigh accurately, put 10mL iodine measuring bottle, add 4.0mL mixed solvent namely acetone: n-hexane (1:9) and seal, until the speed-regulating oscillator 50 times per minute Shake for 10 minutes, ice-water bath for 30 minutes, filter with a 0.22 ⁇ m nylon membrane, take the filtrate and return it to room
  • the invention provides a detection method of genotoxic impurity 1,3-dichloro-2-propanol, which has good linearity, strong specificity, good precision, high accuracy and high sensitivity, and can quickly and accurately measure 1, 3-dichloro-2-propanol content.
  • a gas chromatography-mass spectrometry detection method for genotoxic impurity 1,3-dichloro-2-propanol which is realized by the following scheme: (1) sample pretreatment: mix the sample to be tested with perfluoropropionic acid (formula IV ) are mixed, and the 1,3-dichloro-2-propanol (formula III) in the sample is derivatized with perfluoropropionic acid (formula IV), the catalyst of the derivatization reaction is a sulfuric acid solution, and the sulfuric acid The concentration of the solution is 15%-50%, preferably 25%-40%, most preferably 40%, the unit is v/v
  • GC-MS detection GC-MS instrument is used for detection to determine the content of 1,3-dichloro-2-propanol impurity in the sample.
  • the time for the derivatization reaction in the step (1) is 10 min-30 min, preferably 15 min-25 min, most preferably 20 min.
  • the temperature of the derivatization reaction in step (1) is 50°C-70°C, preferably 55°C-65°C, most preferably 60°C.
  • a diluent is also used in the derivatization reaction in the step (1), and the diluent is isopentane, cyclohexane, n-heptane, preferably n-heptane.
  • the method comprises:
  • Sample pretreatment mix the sample to be tested with perfluoropropionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction, react at 50°C-70°C for 10min-30min, add water and dilute Reagent extraction, the supernatant was taken to obtain the sample solution.
  • Detection by gas chromatography adopt gas chromatography to detect the sample solution described in step (1), record the spectrogram of the sample solution, and according to the standard of 1,3-dichloro-2-propanol obtained in advance Curve, calculate the content of 1,3-dichloro-2-propanol impurity in the sample to be tested according to the external standard method.
  • the standard curve of 1,3-dichloro-2-propanol in the step (2) is obtained by the following method: Get the reference substance of 1,3-dichloro-2-propanol, Dissolved in the diluent and configured as a linear solution stock solution of 1,3-dichloro-2-propanol in a linear concentration range; respectively take the linear solution stock solution of 1,3-dichloro-2-propanol and perfluorinated Mix propionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction.
  • the chromatographic column detected by gas chromatography in the step (2) is DB-5MS, HP-5MS, SH-Rxi-5Sil MS, preferably SH-Rxi-5Sil MS, and the specification is 30m* 250 ⁇ m, 0.25 ⁇ m.
  • the conditions of the GC-MS detection in the step (2) are: gas phase conditions: the carrier gas is high-purity helium; the carrier gas flow rate is 0.6-1.5mL/min, preferably 1.2mL/min ;The split ratio is 3:1 ⁇ 5:1, preferably 3:1; the temperature of the injection port: 230°C ⁇ 280°C, preferably 250°C; 25°C/min to 100°C-150°C for 0-2min, then 40°C/min to 240°C for 2min, preferably 60°C for 2min, then 20°C/min to 100°C for 2min, then Raise temperature at 40°C/min to 240°C and keep for 2min.
  • the conditions for the gas chromatography-mass spectrometry detection in the step (2) are: mass spectrometry conditions: the ionization source is an EI source, and the ion source temperature is 200°C to 250°C, preferably 230°C; analyzer: single four The stage rod mass analyzer, the monitoring mode is the selected ion monitoring mode SIM, and the extracted ions are 110,225.
  • the detection conditions of gas chromatography in the step (2) are as follows
  • Chromatographic column DB-5MS, HP-5MS, SH-Rxi-5Sil MS, preferably SH-Rxi-5Sil MS, with specifications of 30m*250 ⁇ m, 0.25 ⁇ m;
  • Carrier gas high purity helium
  • Carrier gas flow rate 0.6mL/min-1.5mL/min, preferably 1.2mL/min;
  • the split ratio is 3:1 ⁇ 5:1, preferably 3:1;
  • Injection port temperature 230°C ⁇ 280°C, preferably 250°C;
  • Injection volume 1.0-3.0 ⁇ L, preferably 1.0 ⁇ L;
  • Heating program keep at 50°C-80°C for 2 minutes, then raise the temperature at 15°C/min-25°C/min to 100°C-150°C and keep for 2 minutes, then raise the temperature at 40°C/min to 240°C and keep for 2 minutes; preferably keep at 60°C After 2 minutes, raise the temperature at 20°C/min to 100°C and keep it for 2 minutes, then raise the temperature at 40°C/min to 240°C and keep it for 2 minutes;
  • the injection method is direct injection
  • the ionization source is an EI source
  • the ion source temperature is 200°C to 250°C, preferably 230°C
  • the analyzer a single quadrupole mass analyzer
  • the monitoring mode is the selected ion monitoring mode SIM
  • the extracted ions are 110, 225 , solvent delay: 3min.
  • the GCMS detection instrument of the present invention is SHIMADZU GCMS QP2020 or Agilent7890B-5977B.
  • the sample to be tested is linezolid or a linezolid intermediate, preferably, the linezolid intermediate is a compound represented by formula II
  • the reagents and solvents used in the present invention are not particularly limited, and commercially available conventional reagents and solvents can be used.
  • the present invention has the following beneficial effects, but this should not be interpreted as that the detection method of the present invention only has the following effects:
  • the detection method of the present invention has high sensitivity, small amount of sample, few pretreatment steps, and its detection limit (1.5ppm) and quantification limit (4.5ppm) are far lower than the genotoxic impurity 1,3-dichloro-2-propanol The limit (15ppm).
  • the detection method of the present invention has a good linear relationship within the range of 60ng/mL-600ng/mL, and the linear correlation coefficient R2 is 0.9992.
  • the detection method of the present invention is accurate and feasible, the sample recovery rate is 106.09%, the RSD is 7.08%, and the instrument precision RSD is 2.05%.
  • the present invention has developed a new detection method for the genotoxic impurity 1,3-dichloro-2-propanol, and for the first time realized the detection of the genotoxic impurity 1,3-dichloro-2-propanol in linezolid or its intermediates.
  • the effective control of -2-propanol ensures the safety of medication for patients.
  • Fig. 1 is the chromatogram of the 1,3-dichloro-2-propanol reference substance of embodiment 1
  • Fig. 2 is the linear equation standard curve of 1,3-dichloro-2-propanol of embodiment 2
  • Fig. 3 is the chromatogram of the quantitative limit solution of embodiment 2
  • Fig. 4 is the chromatogram of the detection limit solution of embodiment 2
  • Fig. 5 is the chromatogram of the sample solution of embodiment 3.
  • Fig. 6 is the chromatogram of the sample solution of embodiment 3.
  • the linezolid epoxy compound (compound of formula II) used in the examples of the present invention was purchased from Jiangsu Alpha Pharmaceutical Co., Ltd.
  • the 1,3-dichloro-2-propanol reference substance used in the examples of the present invention was purchased from aladdin with a purity of 97%.
  • the instrument and model used in this embodiment are: SHIMADZU GCMS QP2020, and the sampling method adopted is direct sampling.
  • Embodiment 1 positioning experiment
  • Chromatographic column SH-Rxi-5Sil MS, 30m*250 ⁇ m, 0.25 ⁇ m; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 5:1, injection volume: 1.0 ⁇ L , Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, then raise the temperature to 120°C at 20°C/min, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
  • Mass spectrometry conditions ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, full scan range is 20-400, solvent delay: 3min.
  • Reference substance stock solution a Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 10mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the reference substance stock solution a with a concentration of 30 ⁇ g/mL.
  • Reference substance solution Pipette concentrated sulfuric acid solution (100 ⁇ L) + reference substance stock solution a (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) into 10 mL graduated glass tubes with stoppers, add water (5 mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a reference substance solution with a concentration of 3.0 ⁇ g/mL, inject 1.0ul, and record the spectrum ( Figure 1). It can be seen from the figure that there is no interference peak around the reference substance, and the method can be used for the detection of this impurity.
  • Embodiment 2 methodological investigation
  • Chromatographic column SH-Rxi-5Sil MS, 30m*250 ⁇ m, 0.25 ⁇ m; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume: 1.0 ⁇ L , Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, then raise the temperature to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
  • Mass spectrometry conditions ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
  • ionization source is EI source
  • ion source temperature 230°C
  • analyzer single quadrupole mass analyzer
  • monitoring mode is selected ion monitoring mode (SIM)
  • extracted ions 110, 225, solvent delay: 3min.
  • Reference substance stock solution b Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 50mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the reference substance stock solution b with a concentration of 6.0 ⁇ g/mL.
  • Linear solution stock solution 1 ⁇ 5 Pipette 1.0mL, 3.0mL, 5.0mL, 7.0mL, 10.0mL of reference substance stock solution b into 5 different 10mL volumetric flasks, dilute to the mark with diluent, and mix well to obtain linear solution stock solution 1 to linear solution stock solution 5, the concentrations of which are: 0.6 ⁇ g/mL, 1.8 ⁇ g/mL, 3.0 ⁇ g/mL, 4.2 ⁇ g/mL, 6.0 ⁇ g/mL, respectively.
  • Linear solution pipette 40% sulfuric acid solution (100 ⁇ L) + each linear solution stock solution (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) into a 10 mL graduated glass tube with stopper, add water (5 mL ), n-heptane (0.9mL), shake well, let stand to separate layers, and take the supernatant to obtain a solution with a concentration of 60ng/mL, 180ng/mL, 300ng/mL, 420ng/mL, 600ng/mL linear 1 ⁇ linear 5 , each injection 1.0ul, record the spectrum, take the concentration as the abscissa, the peak area as the ordinate, draw the standard curve (Fig. The linearity is good in the range of mL ⁇ 600ng/mL.
  • Quantitation limit solution stock solution pipette 1.5mL of control solution stock solution b into a 10mL volumetric flask, dilute to the mark with diluent, and mix well.
  • Detection limit solution stock solution pipette 0.5mL of control solution stock solution b into a 10mL volumetric flask, dilute to the mark with diluent, and mix well.
  • Limit of quantitation solution pipette 40% sulfuric acid solution (100ul) + limit of quantitation solution stock solution (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) into a 10mL graduated glass tube with stopper, add water (5mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a solution with a concentration of 90ng/mL at the limit of quantification, inject 1.0ul each, and record the spectrum (Fig. 3).
  • Detection limit solution pipette 40% sulfuric acid solution (100ul) + detection limit solution stock solution (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) into a 10mL graduated glass tube with stopper, add water (5mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a solution with a detection limit of 30ng/mL, inject 1.0ul each, and record the spectrum ( Figure 4).
  • the signal-to-noise ratio of the detection limit solution was greater than 3, and the signal-to-noise ratio of the quantification limit solution was greater than 10, indicating that the detection limit and quantification limit met the test requirements.
  • Embodiment 3 sample detection
  • Chromatographic column SH-Rxi-5Sil MS, 30m*250 ⁇ m, 0.25 ⁇ m; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume 1.0 ⁇ L, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
  • Mass spectrometry conditions ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
  • ionization source is EI source
  • ion source temperature 230°C
  • analyzer single quadrupole mass analyzer
  • monitoring mode is selected ion monitoring mode (SIM)
  • extracted ions 110, 225, solvent delay: 3min.
  • sample solution Preparation of sample solution: Weigh 20 mg of linezolid epoxy compound, put them in 10 mL graduated glass tubes with stoppers, respectively add 40% sulfuric acid solution (100 ⁇ L) + n-heptane (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L ), add water (5mL) and n-heptane (0.9mL) after water bath (20min) at 60°C, shake well, let stand to separate layers, prepare a 20mg/mL sample solution, inject 1.0uL respectively, and record the spectrum (Fig. 5 ⁇ 6), the result sample was not detected, and the report was less than the detection limit (1.5ppm).
  • Embodiment 4 catalyst concentrated sulfuric acid selection
  • Chromatographic column SH-Rxi-5Sil MS, 30m*250 ⁇ m, 0.25 ⁇ m; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 5:1, injection volume 1.0 ⁇ L, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
  • Mass spectrometry conditions ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
  • ionization source is EI source
  • ion source temperature 230°C
  • analyzer single quadrupole mass analyzer
  • monitoring mode is selected ion monitoring mode (SIM)
  • extracted ions 110, 225, solvent delay: 3min.
  • Reference substance stock solution c Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 100mL volumetric flask, dilute to the mark with diluent, mix evenly, precisely pipette 5.0mL of the above solution into a 10mL volumetric flask, dilute to the mark with diluent, mix well, and obtain the reference substance stock solution c with a concentration of 1.5 ⁇ g/mL.
  • Control solution In a 10mL stoppered graduated glass tube, add reference substance stock solution c (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) + concentrated sulfuric acid (100 ⁇ L) / no concentrated sulfuric acid, add water after 60°C water bath (20min) (5mL), n-heptane (0.9mL), shake well, let stand to separate layers, prepare 2 parts each of the control solution with the concentration of 150ng/mL adding concentrated sulfuric acid and without adding concentrated sulfuric acid, inject 1.0ul respectively, and record the spectrum , see Table 1 for data processing.
  • Sample spiking solution Weigh 10 mg of linezolid epoxy and place 4 parts in 10 mL graduated glass tubes with stoppers respectively, add reference substance stock solution c (100 ⁇ L) + perfluoropropionic acid (100 ⁇ L) + concentrated sulfuric acid ( 100 ⁇ L)/without adding concentrated sulfuric acid, add water (5mL) after 60°C water bath (20min), shake well with n-heptane (0.9mL), let stand to separate layers, and prepare 2 samples each with a concentration of 150ng/mL. Inject 1.0ul samples respectively, record the chromatograms, and see Table 1 for data processing. The results show that no concentrated sulfuric acid is added, the response is low, and there may be detection interference in the investigation of later quantification limits, detection limits and other items.
  • Embodiment 5 the screening test of sulfuric acid solution concentration
  • Chromatographic column SH-Rxi-5Sil MS, 30m*250 ⁇ m, 0.25 ⁇ m; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume 1.0 ⁇ L, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
  • Mass spectrometry conditions ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
  • ionization source is EI source
  • ion source temperature 230°C
  • analyzer single quadrupole mass analyzer
  • monitoring mode is selected ion monitoring mode (SIM)
  • extracted ions 110, 225, solvent delay: 3min.
  • Control solution stock solutions 1 to 3 Pipette 1.5mL, 2.5mL, and 5.0mL of control stock solution b into three different 10mL volumetric flasks, dilute to the mark with diluent, and mix well to obtain control solution stock solution 1 ⁇ Contrast solution stock solution 3, its concentration is respectively: 900ng/mL, 1500ng/mL, 3000ng/mL.
  • 25% sulfuric acid concentration sample spike solution Weigh 20 mg of linezolid epoxy, a total of 6 parts were placed in 10 mL graduated glass tubes with stoppers, respectively added 25% sulfuric acid solution (100 ⁇ L) + control solution stock solution 1 / control solution Stock solution 2/control solution stock solution 3 (both 100 ⁇ L) + perfluoropropionic acid (100 ⁇ L), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.
  • 40% sulfuric acid concentration sample spiked solution Weigh 20 mg of linezolid epoxy compound, a total of 6 parts, put them in 10 mL graduated glass tubes with stoppers, add 40% sulfuric acid solution (100 ⁇ L) + control solution stock solution 1/control solution Stock solution 2/control solution stock solution 3 (both 100 ⁇ L) + perfluoropropionic acid (100 ⁇ L), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.
  • 60% sulfuric acid concentration control solution pipette 60% sulfuric acid solution (100 ⁇ L) + control solution stock solution 1/control solution stock solution 2/control solution stock solution 3 (100 ⁇ L each) + perfluoropropionic acid (100 ⁇ L) in 10 mL
  • adding water (5mL) and n-heptane (0.9mL) after a 60°C water bath (20min) shake well, let it stand and separate layers, and take the supernatant to obtain a concentration of 90ng/mL, 150ng/mL As well as 300ng/mL control 1 solution to control 3 solution, prepare 2 copies in parallel.
  • 60% sulfuric acid concentration sample spike solution Weigh 20 mg of linezolid epoxy compound, a total of 6 parts, put them in 10 mL graduated glass tubes with stoppers, add 60% sulfuric acid solution (100 ⁇ L) + control solution stock solution 1/control solution Stock solution 2/control solution stock solution 3 (both 100 ⁇ L) + perfluoropropionic acid (100 ⁇ L), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.

Abstract

A gas chromatography-mass spectrometry combined analysis method for genotoxic impurity 1,3-dichloro-2-propanol, the method comprising the following steps: (1) carrying out pretreatment on a sample to prepare a sample solution, wherein perfluoropropionic acid is used as a derivatization reagent; and (2) measuring the sample solution using a gas chromatography-mass spectrometry combined instrument so as to determine the content of the impurity 1,3-dichloro-2-propanol in the sample. A gas chromatography-mass spectrometry analysis method for genotoxic impurity 1,3-dichloro-2-propanol. The method comprises: (1) carrying out pretreatment on a sample to prepare a sample solution, wherein perfluoropropionic acid is used as a derivatization reagent; and (2) detecting the sample solution using a gas chromatography-mass spectrometer to determine the content of the impurity 1,3-dichloro-2-propanol in the sample.

Description

一种测定基因毒杂质1,3-二氯-2-丙醇的气相色谱质谱联用方法A gas chromatography-mass spectrometry method for the determination of genotoxic impurity 1,3-dichloro-2-propanol 技术领域technical field
本发明属于药物分析检测领域，具体涉及一种测定基因毒杂质1,3-二氯-2-丙醇的气相色谱质谱联用方法。The invention belongs to the field of drug analysis and detection, and in particular relates to a gas chromatography-mass spectrometry method for measuring genotoxic impurity 1,3-dichloro-2-propanol.
背景技术Background technique
利奈唑胺(Linezolid)是人工合成的恶唑烷酮类抗生素，作用于细菌50S核糖体亚单位，在翻译系统的起始阶段，抑制mRNA与核糖体连接，阻止70S起始复合物的形成，从而抑制了细菌蛋白质的合成。2000年获得美国FDA批准，用于治疗革兰阳性(G +)球菌引起的感染。其结构式如下式I： Linezolid is a synthetic oxazolidinone antibiotic that acts on the bacterial 50S ribosomal subunit, inhibits the connection between mRNA and ribosomes at the initial stage of the translation system, and prevents the formation of the 70S initiation complex. Thereby inhibiting bacterial protein synthesis. In 2000, it was approved by the US FDA for the treatment of infections caused by Gram-positive (G + ) cocci. Its structural formula is as follows formula I:
Figure PCTCN2021139655-appb-000001
Figure PCTCN2021139655-appb-000001
利奈唑胺的合成工艺中用到了中间体利奈唑胺环氧物(式II)，而合成该中间体所用到的起始物料为环氧氯丙烷，工业上环氧氯丙烷的制备过程会产生1,3-二氯-2-丙醇。2017年10月27日，世界卫生组织国际癌症研究机构公布的致癌物清单初步整理参考，1,3-二氯-2-丙醇在2B类致癌物清单中。The intermediate linezolid epoxy compound (formula II) is used in the synthesis process of linezolid, and the starting material used for synthesizing the intermediate is epichlorohydrin, and the preparation process of epichlorohydrin in industry will produce 1,3-Dichloro-2-propanol. On October 27, 2017, the list of carcinogens released by the International Agency for Research on Cancer of the World Health Organization was initially compiled for reference.
Figure PCTCN2021139655-appb-000002
Figure PCTCN2021139655-appb-000002
由于1,3-二氯-2-丙醇限度低(15ppm)，且无紫外吸收，无法用液相色谱法直接检测，而且其不容易电离，直接用液质连用或者是气质连用进行检测，信号一般都很差。Since 1,3-dichloro-2-propanol has a low limit (15ppm) and no ultraviolet absorption, it cannot be directly detected by liquid chromatography, and it is not easy to ionize, so it can be directly detected by LC-MS or GC-MS. Signals are generally poor.
国内以及国际上检测1,3-二氯-2-丙醇多是在食品领域，食品的处理方法需要加入硅藻土等吸附剂，再利用柱层析进行富集处理，样品前处理步骤繁多，且样品使用量大，食品的样品处理方法不适用于药品溶液的前处理(周相娟,谢精精,赵玉琪,等.气相色谱-质谱法测定酱油中氯丙醇类化合物[J].中国调味品,2011(05):88-90.)。Domestic and international detection of 1,3-dichloro-2-propanol is mostly in the food field. The food processing method needs to add adsorbents such as diatomaceous earth, and then use column chromatography for enrichment treatment. There are many steps in sample pretreatment. , and the amount of sample used is large, and the food sample processing method is not suitable for the pretreatment of drug solutions (Zhou Xiangjuan, Xie Jingjing, Zhao Yuqi, et al. Determination of chloropropanols in soy sauce by gas chromatography-mass spectrometry[J]. Chinese seasoning Product, 2011(05):88-90.).
CN201910616208.9公开了一种奥硝唑中残留氯丙醇化合物的检测方法，是用顶空气相色谱法，采用氢火焰离子化检测器(FID)进行测定，仪器灵敏度较差(检出限浓度为150ng/mL：配制得到7.5μg/ml的对照品储备液，再取0.2ml到10ml的容量瓶中定容，得出检出限浓度为150ng/mL)，样品使用量较大而且样品的前处理较为繁琐(取奥硝唑约1.0g，精密称定， 置10mL碘量瓶，加入4.0mL混合溶剂即丙酮:正己烷(1:9)并封口，至调速振荡器每分钟50次振荡10min，冰水浴30min，0.22μm尼龙膜过滤，取续滤液恢复至室温后作为供试品溶液)。CN201910616208.9 discloses a detection method for residual chloropropanol compounds in ornidazole, which uses headspace gas chromatography and uses a hydrogen flame ionization detector (FID) for measurement, and the sensitivity of the instrument is relatively poor (the detection limit concentration 150ng/mL: prepare the reference substance stock solution of 7.5μg/ml, then take 0.2ml to 10ml volumetric flask to constant volume, draw the detection limit concentration to be 150ng/mL), the sample usage is relatively large and the concentration of the sample The pretreatment is comparatively loaded down with trivial details (take about 1.0g of ornidazole, weigh accurately, put 10mL iodine measuring bottle, add 4.0mL mixed solvent namely acetone: n-hexane (1:9) and seal, until the speed-regulating oscillator 50 times per minute Shake for 10 minutes, ice-water bath for 30 minutes, filter with a 0.22 μm nylon membrane, take the filtrate and return it to room temperature as the test solution).
因此，开发一种灵敏度高，样品用量小并且能够快速准确检测基因毒杂质1,3-二氯-2-丙醇的方法是本领域技术人员亟需解决的问题。Therefore, it is an urgent problem to be solved by those skilled in the art to develop a method with high sensitivity, small amount of sample, and rapid and accurate detection of the genotoxic impurity 1,3-dichloro-2-propanol.
发明内容Contents of the invention
本发明提供了一种基因毒杂质1,3-二氯-2-丙醇的检测方法，其线性好，专属性强，精密度好，准确度高，灵敏度高，可快速准确的测定1,3-二氯-2-丙醇的含量。The invention provides a detection method of genotoxic impurity 1,3-dichloro-2-propanol, which has good linearity, strong specificity, good precision, high accuracy and high sensitivity, and can quickly and accurately measure 1, 3-dichloro-2-propanol content.
一种基因毒杂质1,3-二氯-2-丙醇的气相色谱质谱联用检测方法，其通过以下方案实现：(1)样品前处理：将待测样品与全氟丙酸(式IV)混合，使样品中的1,3-二氯-2-丙醇(式III)与全氟丙酸(式IV)进行衍生化反应，所述衍生化反应的催化剂为硫酸溶液，所述硫酸溶液的浓度为15％-50％，优选25％-40％，最优选40％，单位为v/vA gas chromatography-mass spectrometry detection method for genotoxic impurity 1,3-dichloro-2-propanol, which is realized by the following scheme: (1) sample pretreatment: mix the sample to be tested with perfluoropropionic acid (formula IV ) are mixed, and the 1,3-dichloro-2-propanol (formula III) in the sample is derivatized with perfluoropropionic acid (formula IV), the catalyst of the derivatization reaction is a sulfuric acid solution, and the sulfuric acid The concentration of the solution is 15%-50%, preferably 25%-40%, most preferably 40%, the unit is v/v
Figure PCTCN2021139655-appb-000003
Figure PCTCN2021139655-appb-000003
(2)气质联用检测：采用气质联用仪器进行检测，以确定样品中1,3-二氯-2-丙醇杂质的含量。(2) GC-MS detection: GC-MS instrument is used for detection to determine the content of 1,3-dichloro-2-propanol impurity in the sample.
在具体的实施方案中，所述步骤(1)中的衍生化反应的时间为10min-30min，优选为15min-25min，最优选20min。In a specific embodiment, the time for the derivatization reaction in the step (1) is 10 min-30 min, preferably 15 min-25 min, most preferably 20 min.
在具体的实施方案中，所述步骤(1)中的衍生化反应的温度为50℃-70℃，优选为55℃-65℃，最优选60℃。In a specific embodiment, the temperature of the derivatization reaction in step (1) is 50°C-70°C, preferably 55°C-65°C, most preferably 60°C.
在具体的实施方案中，所述步骤(1)中的衍生化反应中还使用了稀释剂，所述稀释剂为异戊烷，环己烷，正庚烷，优选为正庚烷。In a specific embodiment, a diluent is also used in the derivatization reaction in the step (1), and the diluent is isopentane, cyclohexane, n-heptane, preferably n-heptane.
在具体的实施方案中，所述方法包括：In specific embodiments, the method comprises:
(1)样品前处理：将待测样品与全氟丙酸、稀释剂、25％-40％的硫酸溶液混合进行衍生化反应，50℃-70℃下反应10min-30min后，加入水和稀释剂萃取，取上清液即得样品溶液。(1) Sample pretreatment: mix the sample to be tested with perfluoropropionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction, react at 50°C-70°C for 10min-30min, add water and dilute Reagent extraction, the supernatant was taken to obtain the sample solution.
(2)气质联用检测：采用气质联用仪器对步骤(1)所述样品溶液进行检测，记录样品溶液的谱图，并根据预先获得的1,3-二氯-2-丙醇的标准曲线，按外标法计算待测样品中1,3-二氯-2-丙醇杂质的含量。(2) Detection by gas chromatography: adopt gas chromatography to detect the sample solution described in step (1), record the spectrogram of the sample solution, and according to the standard of 1,3-dichloro-2-propanol obtained in advance Curve, calculate the content of 1,3-dichloro-2-propanol impurity in the sample to be tested according to the external standard method.
在具体的实施方案中，所述步骤(2)中1,3-二氯-2-丙醇的标准曲线通过下述方法获得： 取1,3-二氯-2-丙醇的对照品，溶解在稀释剂中，配置成线性浓度范围的1,3-二氯-2-丙醇的线性溶液储备液；分别取1,3-二氯-2-丙醇的线性溶液储备液与全氟丙酸、稀释剂、25％-40％的硫酸溶液混合进行衍生化反应，50℃-70℃下反应10min-30min后，加入水和稀释剂萃取，取上清液为线性溶液；将线性溶液进行气相色谱质谱联用检测，以线性溶液浓度为横坐标，峰面积为纵坐标，绘制标准曲线。In a specific embodiment, the standard curve of 1,3-dichloro-2-propanol in the step (2) is obtained by the following method: Get the reference substance of 1,3-dichloro-2-propanol, Dissolved in the diluent and configured as a linear solution stock solution of 1,3-dichloro-2-propanol in a linear concentration range; respectively take the linear solution stock solution of 1,3-dichloro-2-propanol and perfluorinated Mix propionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction. After reacting at 50°C-70°C for 10min-30min, add water and diluent for extraction, and take the supernatant as a linear solution; Carry out gas chromatography-mass spectrometry detection, with linear solution concentration as abscissa and peak area as ordinate, draw a standard curve.
在具体的实施方案中，本发明预先获得的1,3-二氯-2-丙醇的标准曲线为：y＝91.1711x+638.7254，R 2＝0.9992。 In a specific embodiment, the standard curve of 1,3-dichloro-2-propanol obtained in advance in the present invention is: y=91.1711x+638.7254, R 2 =0.9992.
在具体的实施方案中，所述步骤(2)中的气质联用检测的色谱柱为DB-5MS，HP-5MS，SH-Rxi-5Sil MS，优选SH-Rxi-5Sil MS，规格为30m*250μm，0.25μm。In a specific embodiment, the chromatographic column detected by gas chromatography in the step (2) is DB-5MS, HP-5MS, SH-Rxi-5Sil MS, preferably SH-Rxi-5Sil MS, and the specification is 30m* 250μm, 0.25μm.
在具体的实施方案中，所述步骤(2)中的气质联用检测的条件为：气相条件：载气为高纯氦气；载气流速为0.6-1.5mL/min，优选1.2mL/min；分流比为3：1～5：1，优选3：1；进样口温度：230℃～280℃，优选250℃；升温程序为50℃～80℃保持2min后，以15℃/min～25℃/min升温到100℃～150℃保持0～2min，再以40℃/min升温到240℃保持2min，优选为60℃保持2min后，以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。In a specific embodiment, the conditions of the GC-MS detection in the step (2) are: gas phase conditions: the carrier gas is high-purity helium; the carrier gas flow rate is 0.6-1.5mL/min, preferably 1.2mL/min ;The split ratio is 3:1～5:1, preferably 3:1; the temperature of the injection port: 230℃～280℃, preferably 250℃; 25°C/min to 100°C-150°C for 0-2min, then 40°C/min to 240°C for 2min, preferably 60°C for 2min, then 20°C/min to 100°C for 2min, then Raise temperature at 40°C/min to 240°C and keep for 2min.
在具体的实施方案中，所述步骤(2)中的气质联用检测的条件为:质谱条件：电离源为EI源，离子源温度200℃～250℃，优选230℃；分析器：单四级杆质量分析器，监测模式为选择离子监测模式SIM，提取离子为110，225。In a specific embodiment, the conditions for the gas chromatography-mass spectrometry detection in the step (2) are: mass spectrometry conditions: the ionization source is an EI source, and the ion source temperature is 200°C to 250°C, preferably 230°C; analyzer: single four The stage rod mass analyzer, the monitoring mode is the selected ion monitoring mode SIM, and the extracted ions are 110,225.
在具体的实施方案中，所述步骤(2)中气质联用的检测条件如下In a specific embodiment, the detection conditions of gas chromatography in the step (2) are as follows
a)气相条件：a) Gas phase conditions:
色谱柱：DB-5MS，HP-5MS，SH-Rxi-5Sil MS，优选SH-Rxi-5Sil MS，规格为30m*250μm，0.25μm；Chromatographic column: DB-5MS, HP-5MS, SH-Rxi-5Sil MS, preferably SH-Rxi-5Sil MS, with specifications of 30m*250μm, 0.25μm;
载气：高纯氦气；Carrier gas: high purity helium;
载气流速：0.6mL/min-1.5mL/min，优选1.2mL/min；Carrier gas flow rate: 0.6mL/min-1.5mL/min, preferably 1.2mL/min;
分流比为3：1～5：1，优选3：1；The split ratio is 3:1~5:1, preferably 3:1;
进样口温度：230℃～280℃，优选250℃；Injection port temperature: 230°C~280°C, preferably 250°C;
进样量：1.0-3.0μL，优选1.0μL；Injection volume: 1.0-3.0μL, preferably 1.0μL;
升温程序：50℃～80℃保持2min后，以15℃/min～25℃/min升温到100℃～150℃保持2min，再以40℃/min升温到240℃保持2min；优选为60℃保持2min后，以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min；Heating program: keep at 50°C-80°C for 2 minutes, then raise the temperature at 15°C/min-25°C/min to 100°C-150°C and keep for 2 minutes, then raise the temperature at 40°C/min to 240°C and keep for 2 minutes; preferably keep at 60°C After 2 minutes, raise the temperature at 20°C/min to 100°C and keep it for 2 minutes, then raise the temperature at 40°C/min to 240°C and keep it for 2 minutes;
进样方式为直接进样；The injection method is direct injection;
b)质谱条件：电离源为EI源，离子源温度200℃～250℃，优选230℃；分析器：单四级杆质量分析器，监测模式为选择离子监测模式SIM，提取离子为110，225，溶剂延迟：3min。b) Mass spectrometry conditions: the ionization source is an EI source, the ion source temperature is 200°C to 250°C, preferably 230°C; the analyzer: a single quadrupole mass analyzer, the monitoring mode is the selected ion monitoring mode SIM, and the extracted ions are 110, 225 , solvent delay: 3min.
在具体的实施方案中，本发明气质联用检测仪器为SHIMADZU GCMS QP2020或Agilent7890B-5977B。In a specific embodiment, the GCMS detection instrument of the present invention is SHIMADZU GCMS QP2020 or Agilent7890B-5977B.
在具体的实施方案中，所述待测样品为利奈唑胺或利奈唑胺中间体，优选的，所述利奈唑胺中间体为式II所示的化合物In a specific embodiment, the sample to be tested is linezolid or a linezolid intermediate, preferably, the linezolid intermediate is a compound represented by formula II
Figure PCTCN2021139655-appb-000004
Figure PCTCN2021139655-appb-000004
在具体的实施方案中，本发明的样品溶液的浓度是按照下述方法计算的：样品溶液的浓度＝线性3的浓度/1,3-二氯-2-丙醇的限度，进一步具体地，1,3-二氯-2-丙醇的限度为15ppm，线性3浓度为300ng/mL，计算得样品溶液的浓度为300ng·mL -1/15ppm＝20mg/mL。 In a specific embodiment, the concentration of the sample solution of the present invention is calculated according to the following method: the concentration of the sample solution=the limit of the concentration of linear 3/1,3-dichloro-2-propanol, further specifically, The limit of 1,3-dichloro-2-propanol is 15ppm, the linear concentration is 300ng/mL, and the calculated concentration of the sample solution is 300ng·mL -1 /15ppm=20mg/mL.
本发明所使用的试剂和溶剂没有特别的限制，可采用商购的常规试剂和溶剂。The reagents and solvents used in the present invention are not particularly limited, and commercially available conventional reagents and solvents can be used.
应当强调的是，本发明技术方案中所涉及的数值或数值端点，其含义或意欲的保护范围并不局限于该数字本身，本领域技术人员能够理解，它们包含了那些已被本领域广为接受的可允许误差范围，例如实验误差、测量误差、统计误差和随机误差等等，而这些误差范围均包含在本发明的范围之内。It should be emphasized that the numerical values or numerical endpoints involved in the technical solutions of the present invention, their meanings or intended protection scope are not limited to the numbers themselves, and those skilled in the art can understand that they include those that have been widely recognized in the art. Acceptable permissible error ranges, such as experimental errors, measurement errors, statistical errors, random errors, etc., are included within the scope of the present invention.
相较于现有技术，本发明具有下述有益效果，但不应将此理解为本发明所述检测方法仅具有下列效果：Compared with the prior art, the present invention has the following beneficial effects, but this should not be interpreted as that the detection method of the present invention only has the following effects:
(1)本发明检测方法灵敏度高，样品用量小，前处理步骤少，其检测限(1.5ppm)和定量限(4.5ppm)远低于基因毒杂质1,3-二氯-2-丙醇的限度(15ppm)。(1) The detection method of the present invention has high sensitivity, small amount of sample, few pretreatment steps, and its detection limit (1.5ppm) and quantification limit (4.5ppm) are far lower than the genotoxic impurity 1,3-dichloro-2-propanol The limit (15ppm).
(2)本发明检测方法在60ng/mL～600ng/mL范围内线性关系良好，线性相关系数R 2为0.9992。 (2) The detection method of the present invention has a good linear relationship within the range of 60ng/mL-600ng/mL, and the linear correlation coefficient R2 is 0.9992.
(3)本发明检测方法准确可行，加样回收率为106.09％，RSD为7.08％，仪器精密度RSD为2.05％。(3) The detection method of the present invention is accurate and feasible, the sample recovery rate is 106.09%, the RSD is 7.08%, and the instrument precision RSD is 2.05%.
(4)本发明开发了一种新的基因毒杂质1,3-二氯-2-丙醇的检测方法，首次实现了对利奈唑胺或其中间体中基因毒杂质1,3-二氯-2-丙醇的有效控制，保证了患者的用药安全。(4) The present invention has developed a new detection method for the genotoxic impurity 1,3-dichloro-2-propanol, and for the first time realized the detection of the genotoxic impurity 1,3-dichloro-2-propanol in linezolid or its intermediates. The effective control of -2-propanol ensures the safety of medication for patients.
附图说明：Description of drawings:
图1为实施例1的1,3-二氯-2-丙醇对照品的色谱图Fig. 1 is the chromatogram of the 1,3-dichloro-2-propanol reference substance of embodiment 1
图2为实施例2的1,3-二氯-2-丙醇的线性方程标准曲线Fig. 2 is the linear equation standard curve of 1,3-dichloro-2-propanol of embodiment 2
图3为实施例2的定量限溶液的色谱图Fig. 3 is the chromatogram of the quantitative limit solution of embodiment 2
图4为实施例2的检测限溶液的色谱图Fig. 4 is the chromatogram of the detection limit solution of embodiment 2
图5为实施例3的样品溶液的色谱图Fig. 5 is the chromatogram of the sample solution of embodiment 3
图6为实施例3的样品溶液的色谱图Fig. 6 is the chromatogram of the sample solution of embodiment 3
具体实施方式Detailed ways
以下实施例是对本发明的内容作进一步的详细说明，但不应理解为本发明上述主题的范围仅限于以下实施例。The following examples are to further describe the content of the present invention in detail, but it should not be understood that the scope of the above subject of the present invention is limited to the following examples.
本发明实施例中使用的利奈唑胺环氧物(式II化合物)购买自江苏阿尔法药业有限公司。The linezolid epoxy compound (compound of formula II) used in the examples of the present invention was purchased from Jiangsu Alpha Pharmaceutical Co., Ltd.
本发明实施例中使用的1,3-二氯-2-丙醇对照品，购买自aladdin，纯度为97％。The 1,3-dichloro-2-propanol reference substance used in the examples of the present invention was purchased from aladdin with a purity of 97%.
本实施例所采用的仪器及型号为：SHIMADZU GCMS QP2020，采用的进样方式为直接进样。The instrument and model used in this embodiment are: SHIMADZU GCMS QP2020, and the sampling method adopted is direct sampling.
本发明实施例中使用到的下述溶液的配制方法具体为：The preparation method of the following solutions used in the embodiments of the present invention is specifically:
25％硫酸溶液：移取7.5mL的水于20mL的烧杯，缓慢加入2.5mL的浓硫酸，混匀。25% sulfuric acid solution: pipette 7.5mL of water into a 20mL beaker, slowly add 2.5mL of concentrated sulfuric acid, and mix well.
40％硫酸溶液：移取6.0mL的水于20mL的烧杯，缓慢加入4.0mL的浓硫酸，混匀。40% sulfuric acid solution: pipette 6.0mL of water into a 20mL beaker, slowly add 4.0mL of concentrated sulfuric acid, and mix well.
60％硫酸溶液：移取4.0mL的水于20mL的烧杯，缓慢加入6.0mL的浓硫酸，混匀。60% sulfuric acid solution: pipette 4.0mL of water into a 20mL beaker, slowly add 6.0mL of concentrated sulfuric acid, and mix well.
实施例1：定位实验Embodiment 1: positioning experiment
气相条件：色谱柱：SH-Rxi-5Sil MS,30m*250μm,0.25μm；载气：高纯氦气；载气流速：1.2mL/min，分流比为5：1，进样量：1.0μL，进样口温度：250℃，采用的是程序升温：60℃保持2min后，以20℃/min升温到120℃，再以40℃/min升温到240℃保持2min。Gas phase conditions: Chromatographic column: SH-Rxi-5Sil MS, 30m*250μm, 0.25μm; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 5:1, injection volume: 1.0μL , Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, then raise the temperature to 120°C at 20°C/min, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
质谱条件：电离源为EI源，离子源温度：230℃，分析器：单四级杆质量分析器，全扫范围为20～400，溶剂延迟：3min。Mass spectrometry conditions: ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, full scan range is 20-400, solvent delay: 3min.
稀释剂：正庚烷Diluent: n-heptane
对照品储备液a：精密称取1,3-二氯-2-丙醇对照品30mg至100mL容量瓶中，加稀释剂溶解，并稀释至刻度，混匀；精密移取上述溶液1.0mL至10mL容量瓶，用稀释剂稀释至刻度，混匀，得到浓度为30μg/mL的对照品储备液a。Reference substance stock solution a: Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 10mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the reference substance stock solution a with a concentration of 30μg/mL.
对照品溶液：分别移取浓硫酸溶液(100μL)+对照品储备液a(100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为3.0μg/mL的对照品溶液，进样1.0ul，记录图谱(图1)。由图可见对照品周边无干扰峰，方法可以用于该杂质的检测。Reference substance solution: Pipette concentrated sulfuric acid solution (100 μL) + reference substance stock solution a (100 μL) + perfluoropropionic acid (100 μL) into 10 mL graduated glass tubes with stoppers, add water (5 mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a reference substance solution with a concentration of 3.0μg/mL, inject 1.0ul, and record the spectrum (Figure 1). It can be seen from the figure that there is no interference peak around the reference substance, and the method can be used for the detection of this impurity.
实施例2：方法学考察Embodiment 2: methodological investigation
气相条件：色谱柱：SH-Rxi-5Sil MS,30m*250μm,0.25μm；载气：高纯氦气；载气流速：1.2mL/min，分流比为3：1，进样量：1.0μL，进样口温度：250℃，采用的是程序升温：60℃ 保持2min后，以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。Gas phase conditions: Chromatographic column: SH-Rxi-5Sil MS, 30m*250μm, 0.25μm; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume: 1.0μL , Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, then raise the temperature to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
质谱条件：电离源为EI源，离子源温度：230℃，分析器：单四级杆质量分析器，监测模式为选择离子监测模式(SIM)，提取离子为110，225，溶剂延迟：3min。Mass spectrometry conditions: ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
稀释剂：正庚烷。Diluent: n-heptane.
对照品储备液b：精密称取1,3-二氯-2-丙醇对照品30mg至100mL容量瓶中，加稀释剂溶解，并稀释至刻度，混匀；精密移取上述溶液1.0mL至50mL容量瓶，用稀释剂稀释至刻度，混匀，得到浓度为6.0μg/mL的对照品储备液b。Reference substance stock solution b: Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 50mL volumetric flask, dilute to the mark with diluent, and mix well to obtain the reference substance stock solution b with a concentration of 6.0μg/mL.
1.线性与范围1. Linearity and range
线性溶液储备液1～5：分别精密移取对照品储备液b 1.0mL、3.0mL、5.0mL、7.0mL、10.0mL至5个不同的10mL容量瓶中，用稀释剂稀释至刻度，混匀，得到线性溶液储备液1～线性溶液储备液5，其浓度分别为：0.6μg/mL，1.8μg/mL，3.0μg/mL，4.2μg/mL，6.0μg/mL。Linear solution stock solution 1~5: Pipette 1.0mL, 3.0mL, 5.0mL, 7.0mL, 10.0mL of reference substance stock solution b into 5 different 10mL volumetric flasks, dilute to the mark with diluent, and mix well to obtain linear solution stock solution 1 to linear solution stock solution 5, the concentrations of which are: 0.6 μg/mL, 1.8 μg/mL, 3.0 μg/mL, 4.2 μg/mL, 6.0 μg/mL, respectively.
线性溶液：分别移取40％硫酸溶液(100μL)+各线性溶液储备液(100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为60ng/mL，180ng/mL，300ng/mL，420ng/mL，600ng/mL线性1～线性5溶液，各进样1.0ul，记录图谱，以浓度为横坐标，峰面积为纵坐标，绘制标准曲线(图2)，得到标准曲线方程y＝91.1711x+638.7254，R 2＝0.9992，表明在60ng/mL～600ng/mL范围内线性良好。 Linear solution: pipette 40% sulfuric acid solution (100 μL) + each linear solution stock solution (100 μL) + perfluoropropionic acid (100 μL) into a 10 mL graduated glass tube with stopper, add water (5 mL ), n-heptane (0.9mL), shake well, let stand to separate layers, and take the supernatant to obtain a solution with a concentration of 60ng/mL, 180ng/mL, 300ng/mL, 420ng/mL, 600ng/mL linear 1 ~ linear 5 , each injection 1.0ul, record the spectrum, take the concentration as the abscissa, the peak area as the ordinate, draw the standard curve (Fig. The linearity is good in the range of mL～600ng/mL.
2.检测限和定量限2. Limit of detection and limit of quantitation
定量限溶液储备液：移取对照溶液储备液b 1.5mL到10mL容量瓶中，用稀释剂稀释至刻度，混匀。Quantitation limit solution stock solution: pipette 1.5mL of control solution stock solution b into a 10mL volumetric flask, dilute to the mark with diluent, and mix well.
检测限溶液储备液：移取对照溶液储备液b 0.5mL到10mL容量瓶中，用稀释剂稀释至刻度，混匀。Detection limit solution stock solution: pipette 0.5mL of control solution stock solution b into a 10mL volumetric flask, dilute to the mark with diluent, and mix well.
定量限溶液：移取40％硫酸溶液(100ul)+定量限溶液储备液(100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL的定量限溶液，各进样1.0ul，记录图谱(图3)。Limit of quantitation solution: pipette 40% sulfuric acid solution (100ul) + limit of quantitation solution stock solution (100μL) + perfluoropropionic acid (100μL) into a 10mL graduated glass tube with stopper, add water (5mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a solution with a concentration of 90ng/mL at the limit of quantification, inject 1.0ul each, and record the spectrum (Fig. 3).
检测限溶液：移取40％硫酸溶液(100ul)+检测限溶液储备液(100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为30ng/mL的检测限溶液，各进样1.0ul，记录图谱(图4)。Detection limit solution: pipette 40% sulfuric acid solution (100ul) + detection limit solution stock solution (100μL) + perfluoropropionic acid (100μL) into a 10mL graduated glass tube with stopper, add water (5mL ), n-heptane (0.9mL), shake well, let stand to separate layers, take the supernatant to obtain a solution with a detection limit of 30ng/mL, inject 1.0ul each, and record the spectrum (Figure 4).
检测限溶液的信噪比大于3，定量限溶液的信噪比大于10，表明检测限和定量限满足测试需求。The signal-to-noise ratio of the detection limit solution was greater than 3, and the signal-to-noise ratio of the quantification limit solution was greater than 10, indicating that the detection limit and quantification limit met the test requirements.
3.加样回收率3. Sample recovery rate
精密称取利奈唑胺环氧物20mg共9份分别置于10mL具塞刻度玻璃管中，分成三组(每组3份)，每组分别加入40％硫酸溶液(100μL)+定量限溶液储备液/线性溶液储备液2/线性溶液储备液3(均各为100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液，配制成浓度为90ng/mL的定量限溶液加标溶液、180ng/mL的线性2溶液加标溶液以及300ng/mL的线性3溶液加标溶液各3份，分别进样1.0ul，记录图谱，计算回收率(n＝9)为106.09％，RSD为7.08％。Precisely weigh 9 parts of 20 mg of linezolid epoxy and place them in 10 mL graduated glass tubes with stoppers, divide them into three groups (3 parts in each group), and add 40% sulfuric acid solution (100 μL) + limit of quantitation solution to each group for reserve solution/linear solution stock solution 2/linear solution stock solution 3 (100 μL each) + perfluoropropionic acid (100 μL), add water (5 mL) after 60 ° C water bath (20 min), and shake well with n-heptane (0.9 mL) , stand for stratification, take the supernatant, and prepare 3 parts each of 90ng/mL limit of quantitation solution spiked solution, 180ng/mL linear 2 solution spiked solution and 300ng/mL linear 3 solution spiked solution , injecting 1.0 ul respectively, and recording the spectrum, the calculated recovery (n=9) was 106.09%, and the RSD was 7.08%.
4.精密度4. Precision
称取利奈唑胺环氧物20mg共6份分别置于10mL具塞刻度玻璃管中，分别加入40％硫酸溶液(100μL)+线性溶液储备液3(100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液，配制成浓度为300ng/mL的线性3溶液加标溶液，分别进样1.0ul，记录图谱，计算回收率(n＝6)为110.03％，RSD为2.05％。Weigh 20 mg of linezolid epoxy compound and place 6 parts in total into 10 mL graduated glass tubes with stoppers, respectively add 40% sulfuric acid solution (100 μL) + linear solution stock solution 3 (100 μL) + perfluoropropionic acid (100 μL), Add water (5mL) and n-heptane (0.9mL) after 60°C water bath (20min), shake well, let stand to separate layers, take the supernatant, and prepare a linear 3 solution spiked solution with a concentration of 300ng/mL. Sample 1.0ul, record the spectrum, the calculated recovery rate (n=6) is 110.03%, RSD is 2.05%.
实施例3：样品检测Embodiment 3: sample detection
气相条件：色谱柱：SH-Rxi-5Sil MS,30m*250μm,0.25μm；载气：高纯氦气；载气流速：1.2mL/min，分流比为3：1，进样量1.0μL，进样口温度：250℃，采用的是程序升温：60℃保持2min后，开始以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。Gas phase conditions: Chromatographic column: SH-Rxi-5Sil MS, 30m*250μm, 0.25μm; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume 1.0μL, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
质谱条件：电离源为EI源，离子源温度：230℃，分析器：单四级杆质量分析器，监测模式为选择离子监测模式(SIM)，提取离子为110，225，溶剂延迟：3min。Mass spectrometry conditions: ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
样品溶液配制：称取利奈唑胺环氧物20mg共2份分别置于10mL具塞刻度玻璃管中，分别加入40％硫酸溶液(100μL)+正庚烷(100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，配制成20mg/mL的样品溶液，分别进样1.0uL，记录图谱(图5～6)，结果样品未检出，报告小于检测限(1.5ppm)。Preparation of sample solution: Weigh 20 mg of linezolid epoxy compound, put them in 10 mL graduated glass tubes with stoppers, respectively add 40% sulfuric acid solution (100 μL) + n-heptane (100 μL) + perfluoropropionic acid (100 μL ), add water (5mL) and n-heptane (0.9mL) after water bath (20min) at 60°C, shake well, let stand to separate layers, prepare a 20mg/mL sample solution, inject 1.0uL respectively, and record the spectrum (Fig. 5 ~6), the result sample was not detected, and the report was less than the detection limit (1.5ppm).
实施例4：催化剂浓硫酸选择Embodiment 4: catalyst concentrated sulfuric acid selection
气相条件：色谱柱：SH-Rxi-5Sil MS,30m*250μm,0.25μm；载气：高纯氦气；载气流速：1.2mL/min，分流比为5：1，进样量1.0μL，进样口温度：250℃，采用的是程序升温：60℃保持2min后，开始以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。Gas phase conditions: Chromatographic column: SH-Rxi-5Sil MS, 30m*250μm, 0.25μm; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 5:1, injection volume 1.0μL, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
质谱条件：电离源为EI源，离子源温度：230℃，分析器：单四级杆质量分析器，监测模式为选择离子监测模式(SIM)，提取离子为110，225，溶剂延迟：3min。Mass spectrometry conditions: ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
稀释剂：正庚烷Diluent: n-heptane
对照品储备液c：精密称取1,3-二氯-2-丙醇对照品30mg至100mL容量瓶中，加稀释剂溶解，并稀释至刻度，混匀；精密移取上述溶液1.0mL至100mL容量瓶，用稀释剂稀释至刻 度，混匀，精密移取上述溶液5.0mL至10mL容量瓶，用稀释剂稀释至刻度，混匀，得到浓度为1.5μg/mL的对照品储备液c。Reference substance stock solution c: Accurately weigh 30mg of 1,3-dichloro-2-propanol reference substance into a 100mL volumetric flask, add diluent to dissolve, dilute to the mark, and mix well; precisely pipette 1.0mL of the above solution to 100mL volumetric flask, dilute to the mark with diluent, mix evenly, precisely pipette 5.0mL of the above solution into a 10mL volumetric flask, dilute to the mark with diluent, mix well, and obtain the reference substance stock solution c with a concentration of 1.5μg/mL.
对照溶液：在10mL具塞刻度玻璃管中，加入对照品储备液c(100μL)+全氟丙酸(100μL)+浓硫酸(100μL)/不加浓硫酸，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，配制成浓度为150ng/mL的加入浓硫酸和不加浓硫酸的对照溶液各2份，分别进样1.0ul，记录图谱，数据处理见表1。Control solution: In a 10mL stoppered graduated glass tube, add reference substance stock solution c (100μL) + perfluoropropionic acid (100μL) + concentrated sulfuric acid (100μL) / no concentrated sulfuric acid, add water after 60°C water bath (20min) (5mL), n-heptane (0.9mL), shake well, let stand to separate layers, prepare 2 parts each of the control solution with the concentration of 150ng/mL adding concentrated sulfuric acid and without adding concentrated sulfuric acid, inject 1.0ul respectively, and record the spectrum , see Table 1 for data processing.
样品加标溶液：称取利奈唑胺环氧物10mg共4份分别置于10mL具塞刻度玻璃管中，分别加入对照品储备液c(100μL)+全氟丙酸(100μL)+浓硫酸(100μL)/不加浓硫酸，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，配制成浓度为150ng/mL的样品加标溶液各2份，分别进样1.0ul，记录图谱，数据处理见表1，结果显示不加入浓硫酸，响应较低，后期定量限，检测限等项目考察可能存在检测干扰，为了达到限度要求，需要加大样品量，但是样品量加大又会对对照(1，3-二氯-2-丙醇)检测造成干扰；直接加入浓硫酸响应较高，但其回收率超出实验室要求(气质联用要求回收率50％～150％)。Sample spiking solution: Weigh 10 mg of linezolid epoxy and place 4 parts in 10 mL graduated glass tubes with stoppers respectively, add reference substance stock solution c (100 μL) + perfluoropropionic acid (100 μL) + concentrated sulfuric acid ( 100μL)/without adding concentrated sulfuric acid, add water (5mL) after 60℃ water bath (20min), shake well with n-heptane (0.9mL), let stand to separate layers, and prepare 2 samples each with a concentration of 150ng/mL. Inject 1.0ul samples respectively, record the chromatograms, and see Table 1 for data processing. The results show that no concentrated sulfuric acid is added, the response is low, and there may be detection interference in the investigation of later quantification limits, detection limits and other items. In order to meet the limit requirements, it is necessary to increase Sample size, but the increase of sample size will cause interference to the detection of the control (1,3-dichloro-2-propanol); directly adding concentrated sulfuric acid has a higher response, but its recovery rate exceeds the laboratory requirements (requirements for gas chromatography-mass spectrometry Recovery rate 50%～150%).
表1浓硫酸的加样实验结果Table 1 The results of the sample addition experiment of concentrated sulfuric acid
Figure PCTCN2021139655-appb-000005
Figure PCTCN2021139655-appb-000005
实施例5：硫酸溶液浓度的筛选试验Embodiment 5: the screening test of sulfuric acid solution concentration
气相条件：色谱柱：SH-Rxi-5Sil MS,30m*250μm,0.25μm；载气：高纯氦气；载气流速：1.2mL/min，分流比为3：1，进样量1.0μL，进样口温度：250℃，采用的是程序升温：60℃保持2min后，开始以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。Gas phase conditions: Chromatographic column: SH-Rxi-5Sil MS, 30m*250μm, 0.25μm; carrier gas: high-purity helium; carrier gas flow rate: 1.2mL/min, split ratio 3:1, injection volume 1.0μL, Injection port temperature: 250°C, using a temperature program: keep at 60°C for 2 minutes, start to heat up to 100°C at 20°C/min and hold for 2 minutes, then raise the temperature to 240°C at 40°C/min and hold for 2 minutes.
质谱条件：电离源为EI源，离子源温度：230℃，分析器：单四级杆质量分析器，监测模式为选择离子监测模式(SIM)，提取离子为110，225，溶剂延迟：3min。Mass spectrometry conditions: ionization source is EI source, ion source temperature: 230°C, analyzer: single quadrupole mass analyzer, monitoring mode is selected ion monitoring mode (SIM), extracted ions are 110, 225, solvent delay: 3min.
对照品储备液b和稀释剂：同实施例2。Reference substance stock solution b and diluent: the same as in Example 2.
对照溶液储备液1～3：分别精密移取对照储备液b 1.5mL、2.5mL、5.0mL至3个不同的10mL容量瓶中，用稀释剂稀释至刻度，混匀，得到对照溶液储备液1～对照溶液储备液3，其浓度分别为：900ng/mL，1500ng/mL，3000ng/mL。Control solution stock solutions 1 to 3: Pipette 1.5mL, 2.5mL, and 5.0mL of control stock solution b into three different 10mL volumetric flasks, dilute to the mark with diluent, and mix well to obtain control solution stock solution 1 ～Contrast solution stock solution 3, its concentration is respectively: 900ng/mL, 1500ng/mL, 3000ng/mL.
25％硫酸浓度对照溶液：移取25％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度 为90ng/mL，150ng/mL以及300ng/mL的对照1溶液～对照3溶液，平行配制2份。25% sulfuric acid concentration control solution: pipette 25% sulfuric acid solution (100 μL) + control solution stock solution 1/control solution stock solution 2/control solution stock solution 3 (100 μL each) + perfluoropropionic acid (100 μL) in 10 mL In a stoppered graduated glass tube, add water (5mL) and n-heptane (0.9mL) after a 60°C water bath (20min), shake well, let it stand and separate layers, and take the supernatant to obtain a concentration of 90ng/mL, 150ng/mL As well as 300ng/mL control 1 solution to control 3 solution, prepare 2 copies in parallel.
25％硫酸浓度样品加标溶液：称取利奈唑胺环氧物20mg共6份分别置于10mL具塞刻度玻璃管中，分别加入25％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL对照1溶液加标溶液，150ng/mL对照2溶液加标溶液以及300ng/mL的对照3溶液加标溶液各2份。25% sulfuric acid concentration sample spike solution: Weigh 20 mg of linezolid epoxy, a total of 6 parts were placed in 10 mL graduated glass tubes with stoppers, respectively added 25% sulfuric acid solution (100 μL) + control solution stock solution 1 / control solution Stock solution 2/control solution stock solution 3 (both 100 μL) + perfluoropropionic acid (100 μL), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.
40％硫酸浓度对照溶液：移取40％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL，150ng/mL以及300ng/mL的对照1溶液～对照3溶液，平行配制2份。40% sulfuric acid concentration control solution: pipette 40% sulfuric acid solution (100 μL) + control solution stock solution 1/control solution stock solution 2/control solution stock solution 3 (100 μL each) + perfluoropropionic acid (100 μL) in 10 mL In a stoppered graduated glass tube, add water (5mL) and n-heptane (0.9mL) after a 60°C water bath (20min), shake well, let it stand and separate layers, and take the supernatant to obtain a concentration of 90ng/mL, 150ng/mL As well as 300ng/mL control 1 solution to control 3 solution, prepare 2 copies in parallel.
40％硫酸浓度样品加标溶液：称取利奈唑胺环氧物20mg共6份分别置于10mL具塞刻度玻璃管中，分别加入40％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL对照1溶液加标溶液，150ng/mL对照2溶液加标溶液以及300ng/mL的对照3溶液加标溶液各2份。40% sulfuric acid concentration sample spiked solution: Weigh 20 mg of linezolid epoxy compound, a total of 6 parts, put them in 10 mL graduated glass tubes with stoppers, add 40% sulfuric acid solution (100 μL) + control solution stock solution 1/control solution Stock solution 2/control solution stock solution 3 (both 100 μL) + perfluoropropionic acid (100 μL), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.
60％硫酸浓度对照溶液：移取60％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)于10mL具塞刻度玻璃管中，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL，150ng/mL以及300ng/mL的对照1溶液～对照3溶液，平行配制2份。60% sulfuric acid concentration control solution: pipette 60% sulfuric acid solution (100 μL) + control solution stock solution 1/control solution stock solution 2/control solution stock solution 3 (100 μL each) + perfluoropropionic acid (100 μL) in 10 mL In a stoppered graduated glass tube, add water (5mL) and n-heptane (0.9mL) after a 60°C water bath (20min), shake well, let it stand and separate layers, and take the supernatant to obtain a concentration of 90ng/mL, 150ng/mL As well as 300ng/mL control 1 solution to control 3 solution, prepare 2 copies in parallel.
60％硫酸浓度样品加标溶液：称取利奈唑胺环氧物20mg共6份分别置于10mL具塞刻度玻璃管中，分别加入60％硫酸溶液(100μL)+对照溶液储备液1/对照溶液储备液2/对照溶液储备液3(均各为100μL)+全氟丙酸(100μL)，60℃水浴(20min)后加入水(5mL)，正庚烷(0.9mL)摇匀，静置分层，取上清液即得浓度为90ng/mL对照1溶液加标溶液，150ng/mL对照2溶液加标溶液以及300ng/mL的对照3溶液加标溶液各2份。60% sulfuric acid concentration sample spike solution: Weigh 20 mg of linezolid epoxy compound, a total of 6 parts, put them in 10 mL graduated glass tubes with stoppers, add 60% sulfuric acid solution (100 μL) + control solution stock solution 1/control solution Stock solution 2/control solution stock solution 3 (both 100 μL) + perfluoropropionic acid (100 μL), add water (5 mL) after 60 ° C water bath (20 min), shake well with n-heptane (0.9 mL), let stand and divide layer, and the supernatant was taken to obtain 90 ng/mL control 1 solution spiked solution, 150 ng/mL control 2 solution spiked solution and 300 ng/mL control 3 solution spiked solution.
以上各溶液分别进样1.0uL，记录图谱，数据处理见表2，结果显示加入25％-40％的浓硫酸后，响应均较高，且回收率均符合要求，其中加入40％硫酸的响应值以及加样回收率都是最优的。Inject 1.0uL of each of the above solutions, record the chromatograms, and see Table 2 for data processing. The results show that after adding 25%-40% concentrated sulfuric acid, the response is high, and the recovery rate meets the requirements. Among them, the response of adding 40% sulfuric acid The value and the recovery rate of the sample addition are optimal.
表2不同浓度硫酸的回收率试验结果The recovery test result of table 2 different concentration sulfuric acid
Figure PCTCN2021139655-appb-000006
Figure PCTCN2021139655-appb-000006
Figure PCTCN2021139655-appb-000007
Figure PCTCN2021139655-appb-000007

Claims (10)

  1. 一种1,3-二氯-2-丙醇的气相色谱质谱联用检测方法，其特征在于，A gas chromatography-mass spectrometry detection method for 1,3-dichloro-2-propanol, characterized in that,
    (1)样品前处理：将待测样品与全氟丙酸(式IV)混合，使样品中的1,3-二氯-2-丙醇(式III)与全氟丙酸(式IV)进行衍生化反应；(1) Sample pretreatment: mix the sample to be tested with perfluoropropionic acid (formula IV), so that 1,3-dichloro-2-propanol (formula III) and perfluoropropionic acid (formula IV) in the sample carry out a derivatization reaction;
    Figure PCTCN2021139655-appb-100001
    Figure PCTCN2021139655-appb-100001
    (2)气质联用检测：采用气质联用仪器进行检测，以确定样品中1,3-二氯-2-丙醇杂质的含量；(2) Detection by GC-MS: detection by GC-MS to determine the content of 1,3-dichloro-2-propanol impurities in the sample;
    其中，步骤(1)中所述衍生化反应的催化剂为硫酸溶液，所述硫酸溶液的浓度为15％-50％，优选25％-40％，最优选40％，单位为v/v。Wherein, the catalyst for the derivatization reaction in step (1) is sulfuric acid solution, the concentration of the sulfuric acid solution is 15%-50%, preferably 25%-40%, most preferably 40%, and the unit is v/v.
  2. 根据权利要求1所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(1)中的衍生化反应的时间为10min-30min。The gas chromatography-mass spectrometry detection method according to claim 1, characterized in that the time of the derivatization reaction in the step (1) is 10min-30min.
  3. 根据权利要求1或2所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(1)中的衍生化反应的温度为50℃-70℃。The gas chromatography-mass spectrometry detection method according to claim 1 or 2, characterized in that the temperature of the derivatization reaction in the step (1) is 50°C-70°C.
  4. 根据权利要求1-3任一项所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(1)中的衍生化反应中还使用了稀释剂，所述稀释剂为异戊烷，环己烷，正庚烷。The gas chromatography-mass spectrometry detection method according to any one of claims 1-3, wherein a diluent is also used in the derivatization reaction in the step (1), and the diluent is isopentane , cyclohexane, n-heptane.
  5. 根据权利要求1-4任一项所述的气相色谱质谱联用检测方法，其特征在于，所述方法包括：The gas chromatography-mass spectrometry detection method according to any one of claims 1-4, wherein the method comprises:
    (1)样品前处理：将待测样品与全氟丙酸、稀释剂、25％-40％的硫酸溶液混合进行衍生化反应，50℃-70℃下反应10min-30min后，加入水和稀释剂萃取，取上清液即得样品溶液。(1) Sample pretreatment: mix the sample to be tested with perfluoropropionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction, react at 50°C-70°C for 10min-30min, add water and dilute Reagent extraction, the supernatant was taken to obtain the sample solution.
    (2)气质联用检测：采用气质联用仪器对步骤(1)所述样品溶液进行检测，记录样品溶液的谱图，并根据预先获得的1,3-二氯-2-丙醇的标准曲线，按外标法计算待测样品中1,3-二氯-2-丙醇杂质的含量。(2) Detection by gas chromatography: adopt gas chromatography to detect the sample solution described in step (1), record the spectrogram of the sample solution, and according to the standard of 1,3-dichloro-2-propanol obtained in advance Curve, calculate the content of 1,3-dichloro-2-propanol impurity in the sample to be tested according to the external standard method.
  6. 根据权利要求1-5任一项所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(2)中1,3-二氯-2-丙醇的标准曲线通过下述方法获得：取1,3-二氯-2-丙醇的对照品，溶解在稀释剂中，配制成线性浓度范围的1,3-二氯-2-丙醇的线性溶液储备液；分别取1,3-二氯-2-丙醇的线性溶液储备液与全氟丙酸、稀释剂、25％-40％的硫酸溶液混合进行衍生化反应，50℃-70℃下反应10min-30min后，加入水和稀释剂萃取，取上清液即得线性溶液；将线性溶液进行气相色谱质谱联用检测，以线性溶液浓度为横坐标，峰面积为纵坐标，绘制标准曲线。According to the gas chromatography-mass spectrometry detection method described in any one of claims 1-5, it is characterized in that, in the described step (2), the standard curve of 1,3-dichloro-2-propanol is obtained by the following method : Take the reference substance of 1,3-dichloro-2-propanol, dissolve it in the diluent, and prepare the linear solution stock solution of 1,3-dichloro-2-propanol in the linear concentration range; take 1,3-dichloro-2-propanol respectively The linear solution stock solution of 3-dichloro-2-propanol is mixed with perfluoropropionic acid, diluent, and 25%-40% sulfuric acid solution for derivatization reaction. After reacting at 50°C-70°C for 10min-30min, add Extract with water and diluent, take the supernatant to obtain a linear solution; perform gas chromatography-mass spectrometry detection on the linear solution, draw the standard curve with the concentration of the linear solution as the abscissa and the peak area as the ordinate.
  7. 根据权利要求1-6任一项所述的气相色谱质谱联用检测方法，其特征在于，所述步骤 (2)中的气质联用检测的色谱柱为DB-5MS，HP-5MS，SH-Rxi-5Sil MS，优选SH-Rxi-5Sil MS，规格为30m*250μm，0.25μm。According to the gas chromatography-mass spectrometry detection method described in any one of claims 1-6, it is characterized in that the chromatographic column detected by gas chromatography-mass spectrometry in the step (2) is DB-5MS, HP-5MS, SH- Rxi-5Sil MS, preferably SH-Rxi-5Sil MS, the specification is 30m*250μm, 0.25μm.
  8. 根据权利要求1-7任一项所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(2)中的气质联用检测的条件为：气相条件：载气为高纯氦气；载气流速为0.6-1.5mL/min，优选1.2mL/min；分流比为3：1～5：1；进样口温度：230℃～280℃，优选250℃；升温程序为50℃～80℃保持2min后，以15℃/min～25℃/min升温到100℃～150℃保持0～2min，再以40℃/min升温到240℃保持2min，优选60℃保持2min后，以20℃/min升温到100℃保持2min，再以40℃/min升温到240℃保持2min。According to the gas chromatography-mass spectrometry detection method described in any one of claims 1-7, it is characterized in that, the gas chromatography-mass spectrometry detection condition in the described step (2) is: gas phase condition: carrier gas is high-purity helium The carrier gas flow rate is 0.6-1.5mL/min, preferably 1.2mL/min; the split ratio is 3:1~5:1; After keeping at 80°C for 2 minutes, raise the temperature at 15°C/min~25°C/min to 100°C~150°C and keep at 0~2min, then raise the temperature at 40°C/min to 240°C and keep at 2min, preferably at 60°C for 2min, then at 20°C ℃/min to 100°C for 2 minutes, then 40°C/min to 240°C for 2 minutes.
  9. 根据权利要求1-8任一项所述的气相色谱质谱联用检测方法，其特征在于，所述步骤(2)中的气质联用检测的条件为：质谱条件：电离源为EI源，离子源温度200℃～250℃，优选230℃；分析器：单四级杆质量分析器，监测模式为选择离子监测模式SIM，提取离子为110，225。According to the gas chromatography-mass spectrometry detection method described in any one of claims 1-8, it is characterized in that the conditions for the gas chromatography-mass spectrometry detection in the step (2) are: mass spectrometry conditions: the ionization source is an EI source, and the ionization The source temperature is 200°C-250°C, preferably 230°C; analyzer: single quadrupole mass analyzer, the monitoring mode is selected ion monitoring mode SIM, and the extracted ions are 110,225.
  10. 根据权利要求1-9中任一项所述的气相色谱质谱联用检测方法，其特征在于，所述待测样品为利奈唑胺或利奈唑胺中间体，优选利奈唑胺中间体，进一步优选的，所述利奈唑胺中间体为式II所示的化合物The gas chromatography-mass spectrometry detection method according to any one of claims 1-9, wherein the sample to be tested is linezolid or a linezolid intermediate, preferably a linezolid intermediate, more preferably Yes, the linezolid intermediate is a compound shown in formula II
    Figure PCTCN2021139655-appb-100002
    Figure PCTCN2021139655-appb-100002
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