CN113030343A - Liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in blood plasma - Google Patents
Liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in blood plasma Download PDFInfo
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
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Abstract
The invention belongs to the technical field of biological analysis, and discloses a liquid chromatography tandem mass spectrometry detection method of pyrroloquinoline quinone in blood plasma, which comprises the following steps: acidifying plasma with strong acid to make the medicine in free state, and adding rhein as internal standard substance while adding strong acid; and performing liquid-liquid extraction by using a water-saturated n-butyl alcohol-ethyl acetate solution or sequentially using water-saturated n-butyl alcohol and ethyl acetate, taking supernate, volatilizing the solvent by using nitrogen, redissolving by using methanol, injecting a sample, and detecting the concentration of pyrroloquinoline quinone by using a liquid chromatography-tandem mass spectrometry. The method can effectively measure the concentration in the body of a rat to which the PQQ supplement is administered, and has the characteristics of simple extraction method, short detection time, good linearity, high accuracy, good precision, good reproducibility and the like.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and relates to a liquid chromatography tandem mass spectrometry detection method of pyrroloquinoline quinone (PQQ) in blood plasma.
Background
Pyrroloquinoline quinone is a cofactor for the redox of bacterial dehydrogenases, is classified as a member of the vitamin B family, and is widely present in various microorganisms and plants. Humans and mammals are unable to synthesize them themselves and are widely distributed in various tissues of the body by dietary supplementation. PQQ plays important roles in mammals, including prevention of liver, heart and brain damage, enhancement of DNA synthesis in human fibroblasts, promotion of nerve growth factor production, and the like. In addition, it has a defense against embryonic cataracts induced by hydrocortisone and acts as a free radical scavenger; it also inhibits the formation of amyloid fibrils and protects the toxic properties of the C-truncated alpha-synuclein variant, making it an effective agent for the prevention of parkinson's disease. Considering that PQQ has various physiological actions, it is necessary to develop a simple and efficient method for measuring the concentration of PQQ in vivo, because it is determined whether toxicity has occurred or not from biochemical indicators after administration as a supplement dosage setting and a plurality of pharmacological and toxicological tests have not been carried out.
Mizuho Fukuda the concentration of PQQ in human plasma was determined using the following method: 200 μ L plasma sample plus 200 μ L1 MNaH2PO4Acidifying plasma with HCl (pH 2), adding 300. mu.L of acidified plasma to an activated Varian Bond Elut C18 solid phase extraction column (activated with 4mL of methanol and 4mL of water in sequence), washing with 2mL of ultrapure water and 600. mu.L of 5% aqueous pyridine in sequence, eluting with 600. mu.L of 30% aqueous acetonitrile and collecting the eluate, introducing 20. mu.L of eluate into a liquid chromatograph, eluting from the chromatographic column with 0.15mM DTT and 1.5mM luminolMixing the mixture in a polytetrafluoroethylene tube for oxidation-reduction reaction, and measuring the concentration by adopting a chemiluminescence detector. The water phase is Tris-HNO3Buffer (50mM, pH8.8) was supplemented with 4mM tetrabutylammonium bromide ion-pairing reagent and each sample was analyzed for 15 min.
Takeshi Kumazawa determines PQQ concentrations in plasma and tissues in humans and rats using the following method: adding 1M HCl 4mL, 2-mercaptoethanol 50 μ L, potassium ferricyanide 10% 100 μ L and n-butanol 10mL into 1g or 1mL of biological sample (blood plasma, tissue), mixing, centrifuging, and collecting supernatant; adding 20mL of n-heptane, 1mL of pyridine, 0.1g of NaCl and 1mL of distilled water, fully mixing uniformly, centrifuging, taking supernatant, volatilizing, redissolving by 10mL of 0.1M HCl, adding into a Sep-Pak C18 cartridge solid phase extraction column, eluting impurities by 20mL of 1mM HCl, eluting PQQ by 3mL of 5% pyridine aqueous solution, volatilizing, redissolving by 100 mu LPTMA hydroxide, heating at 100 ℃ for 15min to fully methylate the PQQ, and feeding 1 mu L of sample into GC-MS; use of13C-labeled PQQ as an internal standard, measured in a linear range of 10-250pg, and a detection line of 0.1-1.0ng/g, is suitable for determining low concentrations of PQQ absorbed in vivo by dietary supplementation.
The PQQ plasma sample extraction method comprises solid-phase extraction, derivatization and multiple liquid-liquid extractions, and has the defects of troublesome operation, long consumed time, more organic solvent consumption, high cost, infrequent use of a detector, long analysis time and the like.
Disclosure of Invention
The invention aims to provide a method for detecting pyrroloquinoline quinone in plasma by liquid chromatography tandem mass spectrometry, which can simply and effectively extract PQQ in plasma through pretreatment and accurately detect PQQ in a plasma sample in a short measuring time.
The technical scheme of the invention is as follows:
a liquid chromatography tandem mass spectrometry detection method of pyrroloquinoline quinone in blood plasma comprises the following steps: acidifying plasma with strong acid to make the medicine in free state, and adding rhein as internal standard substance while adding strong acid; and performing liquid-liquid extraction with water-saturated n-butanol-ethyl acetate solution or sequentially with water-saturated n-butanol and ethyl acetate, collecting supernatant, volatilizing solvent with nitrogen, redissolving with methanol, introducing sample, and detecting pyrroloquinoline quinone concentration by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The strong acid is phosphoric acid, nitric acid, hydrochloric acid or sulfuric acid, the concentration of the phosphoric acid is 4% (mass fraction), the concentration of the nitric acid is 0.8mol/L, the concentration of the hydrochloric acid is 0.5mol/L, and the concentration of the sulfuric acid is 0.25 mol/L; preferably 4% phosphoric acid.
Preferably, the acidification treatment is as follows: dissolving rhein in 4% phosphoric acid as an internal standard substance, acidifying the plasma by using 4% phosphoric acid with the same volume as the plasma, so that the final concentration range of the rhein in the plasma is 250-500ng/mL, but all samples in each batch keep the concentration of the rhein consistent. The polarity and the molecular weight of the rhein are similar to those of PQQ, and meet the internal standard substance selection standard; rhein and PQQ adopt the same acidification form, thus not influencing rhein detection; the rhein is added into the acidifying agent in advance, and the plasma is added with the acidifying agent at the same time, so that the operation steps are simplified, the error is reduced, and the unused rhein is realized14And C isotope labeled PQQ is used as an internal standard substance for accurate quantification.
The liquid-liquid extraction with water saturated n-butanol-ethyl acetate solution is: mixing the acidified blood plasma with water saturated n-butanol-ethyl acetate solution, shaking, centrifuging, collecting supernatant, volatilizing solvent, and dissolving in methanol. Wherein the volume ratio of the water saturated n-butyl alcohol to the ethyl acetate in the water saturated n-butyl alcohol-ethyl acetate solution is 1:0.5-1:1.5v/v, preferably 1: 1; the volume ratio of the water saturated n-butanol-ethyl acetate solution to the blood plasma is 20: 1.
Sequentially adopting water saturated n-butanol and ethyl acetate to carry out liquid-liquid extraction: mixing the acidified blood plasma with water saturated n-butanol-ethyl acetate solution, shaking, centrifuging, collecting supernatant, volatilizing solvent, redissolving with methanol, adding ethyl acetate for extraction, shaking, centrifuging, collecting supernatant, volatilizing solvent, and redissolving with methanol. Wherein the volume ratio of the water saturated n-butanol to the plasma is 20: 1; the volume ratio of the ethyl acetate to the plasma is 20: 1.
In the invention, the rotation speed of the centrifugation is 18000rpm, and the time of the centrifugation is 5 min.
Liquid phaseThe chromatographic conditions were: waters
HSS T3column (50X 3.0mm,2.5 μm) chromatography column; column temperature: 40 ℃; mobile phase A: 2-10mM aqueous dibutylammonium acetate solution; mobile phase B: acetonitrile; flow rate: 0.4 mL/min; gradient elution procedure: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B; sample introduction volume: 5 μ L.
Preferably, the ratio of mobile phase a: 3mM aqueous dibutylammonium acetate, mobile phase B: and (3) acetonitrile.
The parameters of the mass spectrum are: an ion source: electrospray ion source, ion mode: negative ion mode, monitoring mode: and (5) monitoring multiple reactions. The mass spectrum detector has the advantages of high sensitivity, small sample amount, high analysis speed, simultaneous separation and identification of characteristic ions of the PQQ and an internal standard substance and the like, and realizes the quantification of the concentration of trace PQQ medicines in vivo.
Specifically, the liquid chromatography tandem mass spectrometry detection method of pyrroloquinoline quinone in blood plasma comprises the following steps: taking 50 mu L of plasma, acidifying the plasma by using 4% phosphoric acid containing internal standard substance rhein and having the same volume with the plasma, performing liquid-liquid extraction by using water saturated n-butyl alcohol-ethyl acetate (1:1v/v) with the volume of 20 times of the plasma volume, taking 800 mu L of supernatant, volatilizing nitrogen, redissolving 300 mu L of methanol, performing 5 mu L of sample injection analysis, obtaining the ratio of the peak area of PQQ to the peak area of the internal standard substance by adopting liquid chromatography tandem mass spectrometry, and calculating the concentration of PQQ in the plasma according to a standard curve.
The preparation method of the standard curve comprises the following steps: adding 5 muL of PQQ working solution with the concentration of 50, 100, 200, 500, 1000, 2000, 5000ng/mL into 45 muL of blank plasma to prepare a plasma sample with the final concentration of 5, 10, 20, 50, 100, 200, 500ng/mL of PQQ, acidifying the plasma by using 4% phosphoric acid containing internal standard rhein with the same volume as the plasma sample, extracting by using 20 times of plasma volume of water saturated n-butyl alcohol-ethyl acetate (1:1v/v) liquid, taking 800 muL of supernatant, volatilizing nitrogen, redissolving 300 muL of methanol, analyzing 5 muL of sample injection, detecting pyrroloquinoline quinone by using liquid chromatography tandem mass spectrometry, performing least square method (the weight coefficient is 1/x) regression operation by using the peak area ratio (y) of PQQ and the internal standard rhein the plasma sample as a dependent variable and the final concentration of PQQ in the plasma sample as an independent variable (x), a standard curve for PQQ was obtained.
The invention has the beneficial effects that:
(1) the invention establishes a quantitative detection method of a trace target compound PQQ in complex biological matrix plasma by using a chromatography-mass spectrometry combined technology, and can effectively determine the concentration of PQQ in a rat body after a PQQ supplement is given. The pretreatment process of the PQQ sample in the plasma sample is simple, and the used reagent is conventional and has low cost and convenient operation. The determination and analysis time is short, only 6min is needed for single sample injection detection, and the detection efficiency is improved. Has the advantages of simple extraction method and short detection time.
(2) The method has less sampling amount of the biological fluid sample, and can complete detection only by 50 mu L of sample volume.
(3) The liquid-liquid extraction method of the water saturated n-butyl alcohol-ethyl acetate solution can reduce the interference of matrix effect in blood plasma. Addition of dibutylammonium acetate to the mobile aqueous phase increases the retention of PQQ in the chromatography column.
(4) The invention uses the internal standard method to make the standard curve quantitative PQQ, the internal standard substance can correct the sample processing and eliminate the errors generated by the manual operation and the detection of the sample injection process instrument, so that the medicine to be detected is more accurate in quantification. The biological sample analysis method has the determination result that the linear regression equation is as follows: y is 0.0122x +0.01(r is 0.9987), PQQ is linear in the concentration range of 5-500 ng/mL; quantitative lower line 5ng (S/N >10), precision and accuracy within batch are 1.27-4.66% and 87.29-110.15% respectively, and precision and accuracy between batches are 1.68-9.46% and 90.95-108.04% respectively. The daily precision and accuracy, the daytime precision and accuracy, the matrix effect and the extraction recovery rate all accord with the verification guiding principle of the quantitative analysis method of the biological samples in the pharmacopoeia. Has the characteristics of good linearity, high accuracy, good precision, good reproducibility and the like.
Drawings
FIG. 1 shows PQQ and rhein (IS) in SynergiTMChromatograms at different aqueous phase conditions in a 4 μm Polar-RP 80A (50X 4.6mm) chromatography column; wherein (A) an aqueous phase:ultrapure water; (B) water phase: ultrapure water (0.1% formic acid +2mM ammonium formate); (C) water phase: ultrapure water (0.1% formic acid +2mM ammonium formate + 10% methanol); (D) water phase: ultrapure water (0.1% formic acid +2mM ammonium formate + 10% methanol + 2% isopropanol).
FIG. 2 shows PQQ and rhein in Waters
HSS T3column (50 × 3.0mm,2.5um) chromatographic column, by adjusting pH of water phase and adding dibutylamine acetate ion with different concentrations to chromatogram under test strip; wherein (A) an aqueous phase: ultrapure water; (B) water phase: ultrapure water (pH 8 adjusted with ammonia); (C) water phase: ultrapure water (containing 2mM dibutylamine acetate); (D) water phase: ultrapure water (containing 3mM dibutylamine acetate); (E) water phase: ultrapure water (containing 5mM dibutylamine acetate); (F) water phase: ultrapure water (containing 10mM dibutylamine acetate).
FIG. 3 is a diagram of PQQ and rhein in SynergiTMChromatogram obtained by adding dibutyl acetate ion pair reagents with different concentrations into an aqueous phase of a 4-micron Polar-RP 80A (50X 4.6mm) chromatographic column; wherein (A) an aqueous phase: ultrapure water (containing 2mM dibutylamine acetate); (B) water phase: ultrapure water (containing 3mM dibutylamine acetate); (C) water phase: ultrapure water (containing 5mM dibutylamine acetate); (D) water phase: ultrapure water (containing 10mM dibutylamine acetate).
FIG. 4 is a fragment ion diagram of PQQ.
FIG. 5 is a chromatogram of 6 blank plasma samples with methanol blank injection and without PQQ working solution; wherein, A represents PQQ; b represents an internal standard rhein.
FIG. 6 quantitative bottom line 5ng/mL chromatogram.
FIG. 7 standard curve 100ng/mL chromatogram.
FIG. 8 is a PQQ internal standard curve.
Detailed Description
Rat plasma source: SPF SD rat (purchased from Beijing Wittingle laboratory animal technology Co., Ltd., qualification: 20201225Aazz0619000379) weighing 180-200g, after raising for one week, femoral artery blood was taken into 10mL EP tube previously added with heparin anticoagulation, centrifuged at 8000rpm for 5min, and the supernatant was taken to obtain rat blank plasma.
Preparing PQQ working solution: accurately weighing a reference PQQ, dissolving the reference PQQ with a proper amount of dimethyl sulfoxide, adding methanol to prepare a PQQ stock solution with the final concentration of 1mg/mL, and diluting the PQQ stock solution with methanol in a gradient manner to obtain a series of working solutions for standard koji with the concentrations of 50, 100, 200, 500, 1000, 2000 and 5000 ng/mL; diluting PQQ stock solution with final concentration of 1mg/mL with methanol to obtain working solution with final concentration of 50, 80, 600, 4500ng/mL for precision and accuracy determination; diluting PQQ stock solution with final concentration of 1mg/mL with methanol to obtain working solution with final concentration of 80 and 4500ng/mL for inspecting extraction recovery rate and matrix effect; PQQ stock solution with a final concentration of 1mg/mL was diluted with methanol to a final concentration of 8000ng/mL working solution for examination of dilution stability.
Preparing rhein stock solution: the rhein powder is accurately weighed and dissolved by dimethyl sulfoxide to prepare rhein stock solution with the final concentration of 1 mg/mL.
Preparing a 4% phosphoric acid solution: a volume of 85% phosphoric acid (analytical grade) was diluted with ultrapure water to a final concentration of 4% phosphoric acid solution.
Preparing 4% phosphoric acid (containing 500ng/mL internal standard rhein): adding a certain amount of 1mg/mL rhein stock solution to a certain volume of 4% phosphoric acid solution to dilute to a 4% phosphoric acid solution with a final concentration of 500ng/mL rhein.
0.8mol/L nitric acid (containing 500ng/mL internal standard rhein) preparation: diluting 16mol/L concentrated nitric acid (analytically pure) with ultrapure water to obtain a nitric acid solution with a final concentration of 0.8mol/L, and adding a certain amount of 1mg/mL rhein stock solution into the nitric acid solution with a certain volume of 0.8mol/L to obtain a nitric acid solution with a final concentration of 0.8mol/L containing 500ng/mL rhein.
Preparing 0.25mol/L sulfuric acid (containing 500ng/mL internal standard rhein): diluting a certain volume of 2.5mol/L sulfuric acid (analytically pure) with ultrapure water to obtain a sulfuric acid solution with a final concentration of 0.25mol/L, and adding a certain amount of 1mg/mL rhein stock solution into a certain volume of 0.25mol/L sulfuric acid to dilute the solution to obtain 0.25mol/L sulfuric acid with a final concentration of 500ng/mL rhein.
Preparing 0.5mol/L hydrochloric acid (containing 500ng/mL internal standard rhein): diluting a certain volume of 11.5mol/L hydrochloric acid (analytically pure) with ultrapure water to obtain a hydrochloric acid solution with a final concentration of 0.5mol/L, and adding a certain amount of 1mg/mL rhein stock solution into a certain volume of 0.5mol/L hydrochloric acid to dilute the hydrochloric acid solution to obtain a hydrochloric acid solution with a final concentration of 0.5mol/L containing 500ng/mL rhein.
Preparing a water phase: an amount of 0.5M dibutylammonium acetate solution was added to a volume of ultrapure water to prepare a final concentration of ultrapure water containing 3mM dibutylammonium acetate as an aqueous phase. Aqueous phases containing 2mM, 5mM, 10mM dibutylammonium acetate were prepared in the same manner.
The liquid chromatograph model is as follows: shimadzu high performance liquid chromatography system (LC-20A).
The mass spectrometer model: AB SCIEX 4000. The mass spectrometer parameters were set as: an ion source: electrospray ion source, ion mode: negative ion mode, monitoring mode: and (5) monitoring multiple reactions. The set source parameters are respectively: spray voltage (IS) -4500V, auxiliary gas nitrogen, auxiliary gas 1(GS 1)65Arb, auxiliary gas 2(GS 2)60Arb, auxiliary gas heating Temperature (TEM)550 ℃, air curtain gas (CUR)35Arb, and collision gas (CAD)10 Pa. The fragment ions of PQQ are shown in FIG. 4, PQQ (Q1:329.1, Q3: 241.0; DP: -30V, CE: -20eV), rhein (Q1:283.0, Q3: 239.0; DP: -60V, CE: -19 eV).
Example 1 acidulant selection
By Waters
HSST 3column (50X 3.0mM,2.5 μm), mobile phase A (aqueous phase, 3mM dibutylammonium acetate ion-pair reagent added), and mobile phase B (acetonitrile), were examined for the effect of 0.7mol/L phosphoric acid (i.e., 4% phosphoric acid solution), 0.8mol/L nitric acid, 0.5mol/L hydrochloric acid, and 0.25mol/L sulfuric acid on the extraction recovery of PQQ from plasma and the matrix effect according to the matrix effect and extraction recovery examination method. When 4% phosphoric acid elution gradients of 0.8mol/L nitric acid, 0.5mol/L hydrochloric acid, 0.25mol/L sulfuric acid, and 0.7mol/L phosphoric acid (i.e., 4% phosphoric acid solution) were used, various splitting and too broad peaks, tailing, etc. occurred in the peak shape, and thus the optimal elution gradient after acidifying the plasma samples with 3 other acidifying agents was investigated.
Matrix effect and extraction recovery investigation method: blank plasma samples were taken from six different rats. A first group: 5 μ L of 80ng/mL and 450 respectivelyAdding 0ng/mLPQQ working solution into 45 mu L of blank plasma to prepare rat plasma samples with final PQQ concentrations of 8 and 450ng/mL respectively, preparing six parts of the rat plasma samples with each concentration in parallel, adding 50 mu L of acidifying agent (containing 500ng/mL rhein) to acidify the plasma, shaking for 5min, adding 1mL of water-saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing the supernatant by using a room-temperature nitrogen blower, redissolving at 300 mu L of methanol, shaking for 5min, centrifuging at 18000rpm for 5min, taking the supernatant, and carrying out sample injection analysis at 5 mu L. Second group: adding 50 mul of acidifier (containing 500ng/mL rhein) into 45 mul of blank plasma to acidify the plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, respectively adding 5 mul of 80ng/mL PQQ working solution and 4500ng/mL PQQ working solution, shaking for 5min, preparing six parts for each concentration simultaneously in parallel, centrifuging for 5min at 18000rpm, taking 800 mul of supernatant, volatilizing by a room temperature nitrogen blower, redissolving by 300 mul of methanol, shaking for 5min, centrifuging for 5min at 18000rpm, taking the supernatant, and sampling for analysis by 5 mul. And in the third group, 45 mu L of ultrapure water is used for replacing 45 mu L of blank plasma, 50 mu L of acidifying agent (containing 500ng/mL rhein) is added into the ultrapure water, the mixture is shaken for 5min, 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v) is added, the mixture is shaken for 5min, 5 mu L of 80ng/mL PQQ working solution and 4500ng/mL PQQ working solution are respectively added, the shaking is carried out for 5min, six parts are prepared in parallel at each concentration, the mixture is centrifuged for 5min at 18000rpm, 800 mu L of supernatant is taken, a room temperature nitrogen blower is used for volatilizing, 300 mu L of methanol is used for redissolving, the shaking is carried out for 5min at 18000rpm, the supernatant is. Comparing the average peak area of PQQ measured after the first group of treatments with the average peak area measured after the second group of treatments, and calculating the extraction recovery rate of PQQ in different biological matrixes; meanwhile, the average peak area of PQQ measured after the second group of treatments is compared with the average peak area measured after the third group of treatments, and the matrix effect of PQQ in different biological matrixes is calculated. Wherein, the chromatographic conditions are as follows: a chromatographic column: waters
HSST 3column (50X 3.0mm,2.5 μm); column temperature: 40 ℃; mobile phase A: ultrapure water (containing 3mM dibutylammonium acetate); mobile phase B: acetonitrile; flow rate: 0.4 mL/min. The mass spectrum parameters were set as: an ion source: electrospray ion source, ion mode: negative ion mode, monitoring mode: multiple reaction monitoring。
When 0.8mol/L nitric acid is used as an acidifying agent, gradient elution is carried out: 0-1.5 min, 5% B; 1.5-2 min, 5-50% B; 2-4.5 min, 50% B; 4.5-5 min, 50-5% B; 5-7 min, 5% B, PQQ retention time 3.29min, matrix effect and extraction recovery of 76% and 83% respectively.
Gradient elution with 0.25mol/L sulfuric acid as acidifying agent: 0-1.5 min, 10% B; 1.5-2 min, 10-50% B; 2-3.5 min, 50% B; 3.5-4 min, 50-10% B; 4-6 min, 10% B, PQQ retention time 3.24 min. The matrix effect and extraction recovery were measured to be 77% and 93%, respectively.
When 0.5mol/L hydrochloric acid is used as an acidifying agent, gradient elution is carried out: 0-1.5 min, 5% B; 1.5-2 min, 5-50% B; 2-3.5 min, 50% B; 3.5-4 min, 50-5% B; 4-6 min, 5% B, PQQ retention time 3.28 min. The matrix effect and extraction recovery were measured to be 100% and 102%, respectively, but the PQQ response was low and PQQ remained in the chromatogram.
When 0.7mol/L phosphoric acid is used as an acidifying agent, gradient elution is carried out: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B. The matrix effect and extraction recovery were measured to be 88% and 107%, respectively.
Considering both the matrix effect and the extraction recovery, it is preferable to acidify the plasma with equal volume of 4% phosphoric acid (containing 500. mu.g/mL rhein as internal standard) in a plasma sample (the volume of the plasma sample is the sum of the volume of the blank plasma and the volume of the PQQ working solution).
Example 2 extraction reagent selection
Plasma samples are acidified by equal volume of 4% phosphoric acid (containing 500 mug/mL rhein as an internal standard substance) in plasma samples, and after the plasma is acidified, the influence of different sample pretreatment modes on the matrix effect and the extraction recovery rate of PQQ in the plasma is examined (the matrix effect and extraction recovery rate examination method is the same as that in example 1). By Waters
HSS T3column (50X 3.0mM,2.5 μm), mobile phase A (aqueous phase, 3mM dibutylammonium acetate ion pairing reagent added), mobile phase B(acetonitrile), gradient elution: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B.
Pretreatment method 1: after adding 300. mu.L of methanol to the plasma sample, centrifuging twice at 18000rpm 5min, taking 150. mu.L of supernatant, analyzing by 5. mu.L of sample injection, the matrix effect and the extraction recovery rate were determined to be about 35.01. + -. 5.21% and 101.00. + -. 14.63%.
Pretreatment method 2: adding 1mL of ethyl acetate (volume ratio of the plasma sample to the ethyl acetate is 1:20) into the plasma sample to extract PQQ, centrifuging at 18000rpm for 5min, taking 800. mu.L of supernatant, volatilizing nitrogen, redissolving at 300. mu.L of methanol, centrifuging at 18000rpm for 5min, taking supernatant, carrying out 5. mu.L injection analysis, and determining that the matrix effect and the extraction recovery rate are 77.32 +/-5.42% and 92.60 +/-18.04%, respectively.
Pretreatment method 3: adding 1mL of water-saturated n-butanol (volume ratio of the plasma sample to the water-saturated n-butanol is 1:20) into the plasma sample to extract PQQ, centrifuging at 18000rpm for 5min, taking 800 μ L of supernatant, volatilizing in a vacuum volatilizing instrument, redissolving with 300 μ L of methanol, analyzing with 5 μ L of sample injection, and determining that the matrix effect and the extraction recovery rate are 77.43 +/-5.42% and 75.20 +/-3.70% respectively.
Pretreatment method 4: adding 1mL of water saturated n-butanol into the plasma sample (namely the volume ratio of the plasma sample to the water saturated n-butanol is 1:20) for extraction, centrifuging for 5min at 18000rpm, taking 800 mu L of supernatant, volatilizing the supernatant in a vacuum volatilizing instrument, redissolving by 100 mu L of methanol, adding ethyl acetate (namely the volume ratio of the plasma sample to the ethyl acetate is 1:20) for extraction, centrifuging for 5min at 18000rpm, taking 800 mu L of supernatant, volatilizing nitrogen, redissolving by 300 mu L of methanol, centrifuging for 5min at 18000rpm, analyzing by 5 mu L of sample injection, and measuring the matrix effect and the extraction recovery rate to be 98.26 +/-4.34% and 93.65 +/-15.82% respectively.
Pretreatment method 5: adding 1mL of water saturated n-butyl alcohol-ethyl acetate (volume ratio of water saturated n-butyl alcohol to ethyl acetate is 1:1) into the plasma sample for liquid-liquid extraction, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, drying by a room temperature nitrogen blower, centrifuging at 18000rpm for 5min, redissolving at 300 mu L of methanol, centrifuging at 18000rpm for 5min, and analyzing at 5 mu L of sample injection, wherein the matrix effect and the extraction recovery rate are respectively 84.40 +/-4.58% and 105.70 +/-6.40%.
According to the extraction recovery rate and the matrix effect result, better effect can be obtained by sequentially adopting water saturated n-butyl alcohol and ethyl acetate for extraction or adopting water saturated n-butyl alcohol-ethyl acetate for one-time extraction. Preferably, the method adopts water saturated n-butyl alcohol-ethyl acetate (1:1v/v) with the volume 20 times that of the plasma sample for liquid-liquid extraction, has relatively simple operation steps, can extract PQQ from the acidified plasma sample at one time, and has high extraction recovery rate and small matrix effect interference. The redissolution volume is 300 mu L, which not only ensures high signal response intensity of PQQ sample injection detection, but also can sample for multiple times for determination.
Example 3 different chromatography columns and mobile phase conditioning
Adding 5 μ L of 500ng/mL QQ working solution into 45 μ L of blank plasma, adding 50 μ L of 4% phosphoric acid (containing 500ng/mL rhein internal standard substance) to acidify the plasma, shaking for 5min, adding 1mL of water saturated n-butanol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 μ L of supernatant, volatilizing the supernatant in a room temperature nitrogen blower, redissolving at 300 μ L of methanol, centrifuging at 18000rpm for 5min, taking the supernatant, and carrying out sample injection analysis at 5 μ L. And (3) investigating the influence of different chromatographic columns and flowing phase changes on the detection result.
SynergiTM4μm Polar-RP
50x 4.6mm chromatography column: column temperature: 40 ℃, mobile phase B: acetonitrile, flow rate 0.4mL/min, 90% B isocratic elution, and PQQ retention and peak shape in the column were examined when the aqueous phase was added to one or more of formic acid, ammonium formate, isopropanol, methanol. When the aqueous phase was ultrapure (see FIG. 1A), the PQQ response was low with almost no peak; when 0.1% (volume fraction) formic acid and 2mM ammonium formate (see FIG. 1B) were added to the aqueous phase, PQQ showed a front peak and a broad peak profile; when 0.1% (volume fraction) formic acid, 2mM ammonium formate and 10% (volume fraction) methanol were added simultaneously to the aqueous phase (see FIG. 1C), the peak shape was not improved as compared to FIG. 1B; when 0.1% (volume fraction) formic acid, 2mM ammonium formate, 10% (volume fraction) methanol and 2% (volume fraction) isopropanol were added simultaneously to the aqueous phase (see FIG. 1D), the PQQ peak shape narrowed but still had a leading peak. The results showed that the aqueous phase was simultaneously charged with 0.1% formic acid, 2mM ammonium formate,2% isopropanol and 10% methanol, the best peak shape optimized (see fig. 1D) was obtained, under which conditions PQQ was retained in the column for about 1.32min, but a leading peak was present and the response of the internal standard was about e4Internal standards are not applicable under these conditions. Waters
HSS T3column (50X 3.0mm,2.5 μm): column temperature: 40 ℃, mobile phase B: acetonitrile; the effect of mobile phase a (aqueous phase) on column retention and peak shape was examined. The liquid phase gradient elution conditions are consistent: flow rate of 0.4mL/min, gradient elution procedure: 0-1.5 min, 5% B; 1.5-2 min, 5-60% B; 2-3.5 min, 60% B; 3.5-4 min, 60-5% B; 4-6 min, 5% B. When the aqueous phase was ultrapure, PQQ had little response and the internal standard response was too low (see fig. 2A); when the pH value of the aqueous phase is adjusted to be 8 by ammonia water, the peak shape is improved and good (see figure 2B), the retention time of PQQ in the chromatographic column is 0.98min, the peak shapes of PQQ and an internal standard are symmetrical and the response is good, but the retention time of PQQ in the chromatographic column is short, which can cause the interference of plasma endogenous matrixes and influence the accurate determination of PQQ.
The fixed gradient elution procedure was: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B, acetonitrile in the organic phase, and observing that when the water phase is added with the dibutylammonium acetate ion pair reagent with different concentrations, PQQ is in Waters
HSS T3column (50X 3.0mm,2.5 μm) and SynergiTM4μm Polar-RP
Retention time and peak shape, response change in 50x 4.6 mm. By Waters
HSST 3column (50X 3.0mM,2.5 μm) column, PQQ retention time about 1.38min when dibutylammonium acetate ion pair reagent concentration is 2mM (see FIG. 2C); PQQ retention at a dibutylammonium acetate concentration of 3mMAbout 1.45min (see fig. 2D); PQQ retention time was about 1.72min when dibutylammonium acetate ion was present at a concentration of 5mM to reagent (see FIG. 2E); the PQQ retention time was about 1.96min when the dibutylammonium acetate ion pair reagent concentration was 10mM (see FIG. 2F). Using SynergiTM4μm Polar-RP
50X 4.6mM column, no peak of PQQ was observed at a concentration of dibutylammonium acetate ion pair reagent of 2mM (see FIG. 3A); when the dibutylammonium acetate concentration was 3mM, the PQQ retention time was about 2.12min, the PQQ response was low, and the peak was asymmetric (see FIG. 3B); when the dibutylammonium acetate concentration was 5mM, the PQQ retention time was about 2.16min, the PQQ response was low, and the peak was asymmetric (see fig. 3C); when the dibutylammonium acetate ion concentration was 10mM, the PQQ retention time was about 2.27min, the PQQ response was improved (see FIG. 3D), and the internal standard response was low over all concentration ranges and not suitable for internal standard detection under this column and elution gradient conditions.
In conclusion, Waters is adopted
HSS T3column (50X 3.0mM,2.5 μm) chromatography column, when aqueous phase contains 2, 3, 5, 10mM dibutylammonium acetate ion pairing reagent, peak shape of PQQ and internal standard substance are good; as the concentration of dibutylammonium acetate ion on the reagent increases, the retention of PQQ on the column increases, but the PQQ response signal decreases (see fig. 2C, D, E, F); after addition of dibutylammonium acetate ion pairing reagent, the retention increased compared to when the pH of the aqueous phase was adjusted to 8 (see fig. 2B). Using SynergiTM4μm Polar-RP
50x 4.6mm column, by adding one or more of formic acid, ammonium formate, isopropanol, methanol to the aqueous phase, neither peak shape nor response of PQQ and internal standard (see figure 1) is ideal. Comparison of the same concentration of dibutylamine acetate ion-pairing reagent with the same elution gradient revealed the use of Waters
HSST 3column (50X 3.0mm,2.5 μm), PQQ and internal standard peak shape, response change (see FIG. 2) are all compared in SynergiTM4μm Polar-RP
Better in 50x 4.6mm (see fig. 3). Thus, the optimal chromatographic conditions were determined to be: waters
HSS T3column (50X 3.0mm,2.5 μm) chromatography column; column temperature: 40 ℃; mobile phase A: ultrapure water (containing 3mM dibutylammonium acetate); mobile phase B: acetonitrile; flow rate: 0.4mL/min, assay time: 6 min; gradient elution: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B; retention time: t is tR,PQQ≈1.45min；tR, rhein≈3.84min。
Example 4
The method for extracting PQQ from the rat plasma comprises the steps of mixing 4% of phosphoric acid acidified plasma with the same volume as the rat plasma and 20 times of water saturated n-butyl alcohol-ethyl acetate (1:1v/v) for extraction of PQQ, drying by a room-temperature nitrogen blowing instrument, redissolving by 300 mu L of methanol, centrifuging at 18000rpm for 5min, carrying out 5 mu L of sample injection volume, and carrying out methodology verification on the established sample extraction and detection method by adopting the chromatographic conditions and mass spectrum parameters determined in example 3.
(1) And detecting ion pairs: the PQQ standard is precisely weighed and dissolved by a proper amount of dimethyl sulfoxide, and methanol is added to prepare a stock solution with the final concentration of 1 mg/mL. Diluting the stock solution of 1mg/mL with methanol to a solution with PQQ concentration of 1 mug/mL, and respectively selecting ions by mass spectrum full scanning and meeting the actual sample selectivity requirement: PQQ (Q1:329.1, Q3:241.0), a declustering voltage (DP) value of-30V, a collision voltage (CE) value of-20 eV; rhein (Q1:283.0, Q3:239.0), a declustering voltage (DP) value of-60V and a collision voltage (CE) value of-19 eV.
(2) And special investigation: replacing PQQ working solution with 5 μ L of methanol, taking 45 μ L of blank plasma from 6 different rats, adding 5 μ L of methanol, shaking and mixing uniformly, adding 50 μ L of 4% phosphoric acid acidified plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 μ L of supernatant, volatilizing at room temperature nitrogen blower, redissolving with 300 μ L of methanol, shaking for 5min, centrifuging at 18000rpm for 5min, taking supernatant, and sampling 5 μ L for analysis to obtain chromatogram (see figure 5). The result shows that the PQQ and internal standard detection response values in the blank plasma are lower, and the established chromatographic tandem mass spectrometry detection method can eliminate the interference of other endogenous components in the plasma matrix on the detection of the target analyte PQQ and the internal standard rhein. The invention has good specificity for determining PQQ and internal standard rhein.
(3) Detection linear range and detection line measurements: accurately weighing a certain amount of PQQ as a reference substance, dissolving the PQQ with a proper amount of dimethyl sulfoxide, adding methanol to prepare a PQQ stock solution with the final concentration of 1mg/mL, diluting the PQQ stock solution with methanol to obtain 50, 100, 200, 500, 1000, 2000 and 5000ng/mL series working solutions, respectively taking 5 mu L of the working solutions, adding the working solutions into 45 mu L blank plasma, acidifying the plasma with 50 mu L of 4% phosphoric acid (containing 500ng/mL rhein internal standard substance), shaking for 5min, adding 1mL of water-saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing the supernatant by using a room temperature nitrogen blower, redissolving 300 mu L of methanol, shaking for 5min, centrifuging at 18000rpm for 5min, taking the supernatant, and injecting 5 mu L of sample for analysis. The linear equation y is measured to be 0.0122x +0.018(r is 0.9987) and is linear in the concentration range of 5-500ng/mL, and the standard curve is shown in figure 8; the quantitative bottom line 5ng/mL concentration signal response (S/N >10) is shown in FIG. 6, and the standard curve 100ng/mL chromatogram is shown in FIG. 7.
(4) Accuracy and precision experiments: and 4 quality control samples with the concentration of 5ng/mL, 8ng/mL, 60ng/mL and 450ng/mL are selected for checking the precision and accuracy of the established analysis method. Respectively adding 5 mu L of 50ng/mL, 80ng/mL, 600ng/mL and 4500ng/mLPQQ working solution into 45 mu L of blank plasma to prepare PQQ plasma samples with final plasma concentrations of 5ng/mL, 80ng/mL, 60ng/mL and 4500ng/mL, parallelly measuring 6 samples in each concentration, adding 50 mu L of 4% phosphoric acid (containing 500ng/mL rhein internal standard substance) to acidify plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing by using a room temperature nitrogen blower, redissolving by 300 mu L of methanol, centrifuging at 18000rpm for 5min, taking supernatant, and carrying out sample injection analysis by 5 mu L. And continuously performing 3 analysis batches, calculating the actually measured concentration of the PQQ in each batch of quality control plasma samples according to the following standard curve of each batch, and calculating the precision and the accuracy according to the actually measured concentration of the PQQ compared with the added known standard concentration. The detection results are shown in table 1, the precision in the batch and between the batches is less than 10.0% (n is 6), and the accuracy is 87.29% -110.15%.
Table 1. precision accuracy of PQQ in rat plasma within and during day (n ═ 6)
(5) Matrix effect and extraction recovery: blank plasma samples were taken from six different rats. A first group: respectively adding 5 mu L of 80ng/mL and 4500ng/mLPQQ working solution into 45 mu L of blank plasma to prepare rat plasma samples with final concentrations of 8 and 450ng/mL, preparing six parts for each concentration simultaneously in parallel, adding 50 mu L of 4% phosphoric acid aqueous solution to acidify the plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing the supernatant by a room temperature nitrogen blower, redissolving at 300 mu L of methanol, centrifuging at 18000rpm for 5min, taking the supernatant, and carrying out sample injection analysis at 5 mu L. Second group: adding 50 mu L of 4% phosphoric acid aqueous solution into 45 mu L of blank plasma to acidify the plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, respectively adding 5 mu L of 80ng/mL and 4500ng/mL of PQQ working solution, shaking for 5min, preparing six parts for each concentration simultaneously in parallel, centrifuging for 5min at 18000rpm, taking 800 mu L of supernatant, volatilizing by a room temperature nitrogen blower, redissolving 300 mu L of methanol, centrifuging for 5min at 18000rpm, taking the supernatant, and carrying out sample injection analysis at 5 mu L. And in the third group, 45 mu L of ultrapure water is used for replacing 45 mu L of blank plasma, 50 mu L of 4% phosphoric acid aqueous solution is added into the ultrapure water for acidification, the mixture is shaken for 5min, 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v) is added, the mixture is shaken for 5min, and 5 mu L of 80ng/mL and 4500ng/mL of PQQ working solution are respectively added, and the mixture is shaken for 5 min. Six parts are prepared in parallel at each concentration, centrifuged at 18000rpm for 5min, 800 μ L of supernatant is taken, dried by a nitrogen blower at room temperature, redissolved in 300 μ L of methanol, shaken for 5min, centrifuged at 18000rpm × 5min, taken supernatant, and 5 μ L of sample is injected for analysis. Comparing the average peak area of PQQ measured after the first group of treatments with the average peak area measured after the second group of treatments, and calculating the extraction recovery rate of PQQ in different biological matrixes; meanwhile, the average peak area of PQQ measured after the second group of treatment is compared with the average peak area measured after the third group of treatment, the substrate effect of PQQ in different biological substrates is calculated, the result is shown in Table 2, the substrate effect value is 95-115%, the range of +/-15% specified by the verification guide principle of the quantitative analysis method of biological samples is met, and the endogenous substances in blood plasma can be considered not to interfere with the measurement of PQQ in the sample pretreatment process and the chromatographic separation process.
Table 2 extraction recovery and matrix effect (n ═ 6)
(6) Dilution stability: adding 5 mu L of 8000ng/mLPQQ working solution into 45 mu L of blank plasma to prepare rat plasma samples with final concentration of 800ng/mL respectively, preparing six parts in parallel, adding 50 mu L of 4% phosphoric acid (containing 500ng/mL rhein internal standard substance) to acidify the plasma, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing by a room temperature nitrogen blower, and redissolving by 300 mu L of methanol; preparing a blank diluent: adding 5 μ L methanol into 45 μ L blank plasma, adding 50 μ L4% phosphoric acid (containing 500ng/mL rhein internal standard substance) to acidify plasma, shaking for 5min, adding 1mL water saturated n-butanol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, collecting 800 μ L supernatant, volatilizing with room temperature nitrogen blower, and redissolving 300 μ L methanol. Taking 100 mu L of PQQ redissolved sample, adding 100 mu L of blank diluent, diluting by 1 time, shaking for 5min, centrifuging for 5min at 18000rpm, carrying out 5 mu L sample injection analysis, determining the ratio of the peak area of PQQ to the peak area of an internal standard as a y value, substituting the standard curve y as 0.0122x +0.01(r as 0.9987), and determining the concentration of PQQ 392.01 +/-20.46 ng/mL, wherein compared with the theoretical value of 400ng/mL after diluting by 1 time at 800ng/mL, the accuracy is 98.00 +/-5.11%, the result accords with the +/-15% range specified by the verification and verification principle of the biological sample quantitative analysis method, and the sample with the concentration of 500ng/mL on the linear range is considered to be quantitatively detected after being diluted by one time.
Example 5PQQ quantitative determination
Preparation of a standard curve: adding 5 mu L of a series of PQQ working solutions with the concentration of 50, 100, 200, 500, 1000, 2000 and 5000ng/mL into 45 mu L of blank plasma (the final concentration of PQQ in the plasma is respectively 5, 10, 20, 50, 100, 200 and 500ng/mL), adding 50 mu L of 4% phosphoric acid (containing 500ng/mL rhein) for acidification, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing at room temperature by a nitrogen blower, redissolving 300 mu L of methanol, centrifuging at 18000rpm for 5min, taking the supernatant, taking 5 mu L of sample; and (3) taking the peak area ratio (y) of the PQQ and the internal standard rhein in the plasma measured at each concentration as a dependent variable and the corresponding final concentration of the PQQ as an independent variable (x), and performing least square method (weight coefficient is 1/x) regression operation to obtain a standard curve of the PQQ in the plasma.
Following gavage or intravenous administration of PQQ to rats, post-administration plasma samples were obtained by sampling the orbital blood at various times post-administration. Taking 50 mu L of plasma sample after administration, adding 50 mu L of 4% phosphoric acid (containing 500ng/mL rhein) for acidification, shaking for 5min, adding 1mL of water saturated n-butyl alcohol-ethyl acetate (1:1v/v), shaking for 5min, centrifuging at 18000rpm for 5min, taking 800 mu L of supernatant, volatilizing by a room temperature nitrogen blower, redissolving by 300 mu L of methanol, shaking for 5min, centrifuging at 18000rpm for 5min, taking supernatant, taking 5 mu L of sample injection, adopting liquid chromatography tandem mass spectrometry to obtain the ratio of the peak area of PQQ to the peak area of an internal standard substance, and calculating the concentration of PQQ in plasma according to a standard curve.
The liquid chromatograph model is as follows: shimadzu high performance liquid chromatography System (LC-20A), Waters
HSS T3column (50X 3.0mm,2.5 μm) chromatography column; column temperature: 40 ℃; mobile phase A: ultrapure water (containing 3mM dibutylammonium acetate); mobile phase B: acetonitrile; flow rate: 0.4mL/min, assay time: 6 min; gradient elution: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B.
The mass spectrometer model: AB SCIEX 4000. The mass spectrometer parameters were set as: an ion source: electrospray ion source, ion mode: negative ion mode, monitoring mode: and (5) monitoring multiple reactions. The set source parameters are respectively: spray voltage (IS) -4500V, auxiliary gas nitrogen, auxiliary gas 1(GS 1)65Arb, auxiliary gas 2(GS 2)60Arb, auxiliary gas heating Temperature (TEM)550 ℃, air curtain gas (CUR)35Arb, and collision gas (CAD)10 Pa.
The method can accurately quantify the concentration of 5-500ng/mL, the concentration of the high-concentration sample can still be accurately quantified within the concentration range of 5-500ng/mL after being diluted by one time according to the embodiment 4(6), and the final concentration of the sample is obtained by multiplying the actually-measured concentration after being diluted by 2.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for detecting pyrroloquinoline quinone medicine liquid chromatogram and mass spectrum in blood plasma is characterized in that: the method comprises the following steps: adopting strong acid to carry out acidification treatment on the plasma, and adding rhein as an internal standard substance while adding the strong acid; and performing liquid-liquid extraction by using a water-saturated n-butyl alcohol-ethyl acetate solution or sequentially using water-saturated n-butyl alcohol and ethyl acetate, taking supernate, volatilizing the solvent by using nitrogen, redissolving by using methanol, injecting a sample, and detecting the concentration of pyrroloquinoline quinone by using a liquid chromatography-tandem mass spectrometry.
2. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: the strong acid is phosphoric acid, nitric acid, hydrochloric acid or sulfuric acid.
3. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: the strong acid is 4% phosphoric acid.
4. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: the acidification treatment comprises the following steps: dissolving rhein in 4% phosphoric acid as an internal standard substance, acidifying the plasma by using 4% phosphoric acid with the same volume as the plasma, and enabling the final concentration of the rhein in the plasma to be 250-500 ng/mL.
5. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: the liquid-liquid extraction with water saturated n-butanol-ethyl acetate solution is: mixing the acidified blood plasma with water saturated n-butanol-ethyl acetate solution, shaking, centrifuging, collecting supernatant, volatilizing solvent, and re-dissolving with methanol; wherein the volume ratio of the water saturated n-butyl alcohol to the ethyl acetate in the water saturated n-butyl alcohol-ethyl acetate solution is 1:0.5-1:1.5 v/v; the volume ratio of the water saturated n-butanol-ethyl acetate solution to the blood plasma is 20: 1.
6. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 5, which is characterized in that: the volume ratio of the water saturated n-butyl alcohol to the ethyl acetate in the water saturated n-butyl alcohol-ethyl acetate solution is 1:1 v/v.
7. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: sequentially adopting water saturated n-butanol and ethyl acetate to carry out liquid-liquid extraction: mixing the acidified blood plasma with water saturated n-butanol-ethyl acetate solution, shaking, centrifuging, collecting supernatant, volatilizing solvent, redissolving with methanol, adding ethyl acetate for extraction, centrifuging, collecting supernatant, volatilizing solvent, and redissolving with methanol; wherein the volume ratio of the water saturated n-butanol to the plasma is 20: 1; the volume ratio of the ethyl acetate to the plasma is 20: 1.
8. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 1, which is characterized in that: the conditions of the liquid chromatography were: waters
HSS T3column, specification: 50mm × 3.0mm,2.5 μm; column temperature: 40 ℃; mobile phase A: 2-10mM aqueous dibutylammonium acetate solution; mobile phase B: acetonitrile; flow rate: 0.4 mL/min; gradient elution procedure: 0-2 min, 20% B; 2-2.5 min, 20-50% B; 2.5-4 min, 50% B; 4-4.5 min, 50-20% B; 4.5-6 min, 20% B; sample introduction volume: 5 μ L.
9. The method for detecting pyrroloquinoline quinone medicine in blood plasma by liquid chromatography-mass spectrometry according to claim 8, wherein: mobile phase A: 3mM aqueous dibutylammonium acetate solution; mobile phase B: and (3) acetonitrile.
10. A liquid chromatogram tandem mass spectrum detection method of pyrroloquinoline quinone in blood plasma is characterized in that: the method comprises the following steps: taking 50 mu L of plasma, acidifying the plasma by using 4% phosphoric acid containing internal standard rhein and having the same volume with the plasma, performing liquid-liquid extraction by using water saturated n-butyl alcohol-ethyl acetate (1:1v/v) with the volume of 20 times of the plasma, taking 800 mu L of supernatant, volatilizing nitrogen, redissolving 300 mu L of methanol, performing sample injection analysis by using 5 mu L of supernatant, and detecting the concentration of pyrroloquinoline quinone by using liquid chromatography tandem mass spectrometry.
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Application publication date: 20210625 Assignee: Nanjing Guangqi Pharmaceutical Technology Co.,Ltd. Assignor: CHINA PHARMACEUTICAL University Contract record no.: X2023980053893 Denomination of invention: A liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in plasma Granted publication date: 20211228 License type: Common License Record date: 20231225 |