CN105675784B - UPLC-MS/MS methods measure the concentration of human plasma and urine Content of Chlorogenic Acid - Google Patents

Abstract

Present document relates to the concentration that UPLC MS/MS methods measure human plasma and urine Content of Chlorogenic Acid.The present invention provides the method for the concentration for measuring human body fluid Content of Chlorogenic Acid by ultra performance liquid chromatography tandem mass spectrometry, wherein the method uses 0.5% formic acid water of mobile phase: acetonitrile carries out.The analysis time of the single sample of the method for the present invention be 2min, chlorogenic acid linear relationship within the scope of blood plasma 5~2,000ng/mL and urine 50~20,000ng/mL is good, in a few days, day to day precision (RSD) be respectively less than 15%.The specificity, matrix effect, extraction recovery and stability of this research method are verified.The method of the present invention has been successfully applied to give the blood plasma of chlorogenic acid injection for treating patient and the Concentration Testing of urine Content of Chlorogenic Acid.

Description

UPLC-MS/MS methods measure the concentration of human plasma and urine Content of Chlorogenic Acid

Technical field

The present invention relates to the methods of the concentration of detection human body fluid Content of Chlorogenic Acid.Specifically, the present invention relates to pass through superelevation The method that effect liquid phase chromatogram tandem mass spectrometry (UPLC-MS/MS) measures the concentration of human body fluid Content of Chlorogenic Acid.

Background technology

Introduction

Chlorogenic acid is the depside formed by caffeic acid and chinic acid, it is edible with medicinal plant for example honeysuckle, Cortex Eucommiae, to There is wide research in day certain herbaceous plants with big flowers.Verified chlorogenic acid has anti-inflammatory, antibacterial and antiviral activity etc. extensive for a large amount of research Biological function [1-4].It is nearest that researches show that chlorogenic acids can induce human hepatocarcinoma cells, neuroglial cytoma and white The apoptosis [5-8] of blood disease cell.It is well known that cancer is a worldwide major public health problem, in recent years The quantity of cancer patient continues growing, and chlorogenic acid with active anticancer and hypotoxicity because constantly becoming focus of people's attention.It is green Ortho acid is low (i.e. adverse reaction is few) as biological response modifiers immunogenicity, by activating the immune system of body itself, makes Body itself targetedly generates specific immunity effect, realizes immunity of organism balance, can be lacked according to the immune of diseased region It loses, stablizes and moderately supplement and express endogenous immune effector molecule, correct the immune imbalance of tumor microenvironment, to more preferable Ground plays the effect to oncotherapy, has important clinical value.As the compound with potential applicability in clinical practice, treatment The I phase CLINICAL PHARMACOKINETIS STUDY ONs of late malignant tumour are in progress.

Some use Liquid Chromatography-Tandem Mass Spectrometry (liquid chromatography-tandem mass Spectrometry, LC-MS/MS) technology measure biological sample Content of Chlorogenic Acid method have been reported that [9-11].To being at present Only, the only concentration for determining human plasma Content of Chlorogenic Acid, and the detection time of single sample is 5min [11], and do not have The methodology measured in human urine Content of Chlorogenic Acid concentration is reported.

Ultra performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) chromatography is used Little particle (being less than 2 μm) increases efficiency, to increase the resolution of instrument.UPLC has high separation, high speed, high sensitivity The advantages of, analysis throughput is improved, sample and solvent analysis time are saved.However, UPLC is to mobile phase, chromatographic column, sample Pre-treatment, mass spectrometer interface, data collecting system are proposed tightened up requirement.

Although having carried out some researchs to conversion method, the condition of traditional high-efficient liquid phase chromatogram HPLC cannot be applied directly In UPLC, need to including that the terms and conditions such as sample pre-treatments, internal standard, mobile phase, chromatographic column selection carry out a large number of experiments, ability Match with this little particle filler, realizes that UPLC is quickly tested and analyzed.For example, research (HPLC and the UPLC colors of Zhou Xin et al. Spectral condition conversion method research, assay office, the 4th phase of volume 27, in April, 2008) show directly to be applied to HPLC conditions UPLC cannot achieve the purpose that detach test substance and analyze；It needs to include such as mobile phase selection and proportioning to experiment condition Equal progress, which are fully tested, could obtain the good UPLC methods of separate condition.

Invention content

Inventor has been set up quick, sensitive ultra performance liquid chromatography tandem mass spectrum (ultra high Performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS) technology inspection Survey the concentration of human plasma and urine Content of Chlorogenic Acid.The detection time of single sample can reach 2min.

In some embodiments, method of the invention may be used Puerarin and be carried out for internal standard.When using internal standard method, The selection of internal standard compound is a highly important job.Ideal internal standard compound should can be added to accurate, known amount in sample It goes, has essentially identical or as consistent as possible physicochemical properties (such as chemical constitution, polarity, volatility with analyzed sample And solubility etc. in a solvent), chromatographic behavior and response characteristic；Under chromatographiccondition, internal standard compound must be able to and sample Middle each component is sufficiently separated.In some embodiments, inventor has shown that the matrix effect of Puerarin is consistent with chlorogenic acid, It is capable of providing good linear relationship, precision, matrix effect, extraction recovery and the satisfactory detection method of stability.

In some embodiments, ACQUITY UPLC Shield RP18column may be used in method of the invention (100mm × 2.1mm, 1.7 μm) is carried out as analytical column.In chromatography, the selection of chromatographic column is particularly significant, to chromatographic column It is required that being column effect is high, selectivity is good, and analyze speed is fast etc..In the present invention, inventor has found to be embedded with hydrophilic amino formyl The Acquity BEH Shield RP 18colum (100mm × 2.1mm, 1.7 μm, Waters) of group can realize that chlorogenic acid is good Good separation and peak type, and other chromatographic columns such as Acquity UPLC C18 columns (50mm × 2.1mm, 1.7 μm, Waters) carry out in fact Chlorogenic acid is presented bimodal when testing, therefore is not suitable for using.

In some embodiments, mobile phase formic acid water may be used in method of the invention: acetonitrile carries out.In some implementations In scheme, the volume ratio of mobile phase is 0.5% formic acid water: -0.5% formic acid water of acetonitrile (85/15, v/v): acetonitrile (50/50, v/ Including 0.5% formic acid water v): -0.5% formic acid water of acetonitrile (80/20, v/v): acetonitrile (60/40, v/v), preferably for example, by using 0.5% formic acid water: acetonitrile (77/23, v/v) carries out.In order to improve chromatographic isolation selectivity, it may be considered that adjust the pole of mobile phase Property, modifying agent etc. is added into mobile phase.

In some embodiments, method of the invention is used to detect the concentration of human plasma Content of Chlorogenic Acid.The present invention's In method for detecting human plasma Content of Chlorogenic Acid concentration, it has been found that pass through precipitation of protein such as methanol and acetonitrile albumen precipitation Plasma sample is handled, there are apparent substrate inhibition effects, although this can be reduced by adding formic acid, matrix effect is still Cause same sample signal extremely unstable.Matrix refers to the component other than analyte in sample, usually to analyte Analytic process has significant interference, and the accuracy of impact analysis result, for example, the ionic strength of solution can be to analyte activity Coefficient has an impact, these are influenced and interference is referred to as matrix effect.In some embodiments, method of the invention uses liquid liquid Extraction carries out sample treatment.In some embodiments, method of the invention use ethyl acetate liquid-liquid extraction, preferably into One step is added hydrochloric acid and is analyzed.It has been found that hydrochloric acid is added in the extracting process of the present invention can improve the rate of recovery.One In a little embodiments, method of the invention is further redissolved using formic acid methanol-water, preferably uses 0.5% formic acid water: - 0.5% formic acid water of methanol (30/70, v/v): methanol (70/30, v/v), including 0.5% formic acid water: methanol (40/60, v/v)- 0.5% formic acid water: methanol (60/40, v/v), preferably for example, by using 0.5% formic acid water: methanol (50/50, v/v) carries out. It is found that when being diluted only with methanol-water, substrate inhibition effect is larger, and using instead when formic acid methanol-water dilutes can significantly improve Matrix effect.

In some embodiments, the present invention is used to detect the concentration of human urine Content of Chlorogenic Acid.In some embodiments, The method direct dilution method of the present invention handles urine sample, without carrying out other pretreatments.It, can when using methanol-water dilution The substrate inhibition effect for observing 40-50%, when using the dilution of formic acid methanol-water instead, matrix effect is obviously improved.In some implementations In scheme, method of the invention is diluted using formic acid methanol-water, preferably uses 0.5% formic acid water: methanol (30/70, v/ V) -0.5% formic acid water: methanol (70/30, v/v), including 0.5% formic acid water: -0.5% formic acid of methanol (40/60, v/v): water beetle Alcohol (60/40, v/v), preferably for example, by using 0.5% formic acid water: methanol (50/50, v/v) carries out.It has been found that only with first When alcohol water dilutes, substrate inhibition effect is larger, and matrix effect can be significantly improved by using instead when formic acid methanol-water dilutes.

By means of the invention it is also possible to realize that the analysis time of single sample is the quick detection of 2min, chlorogenic acid exists Blood plasma 5~2,000ng/mL and urine 50~20, linear relationship is good within the scope of 000ng/mL, in a few days, day to day precision (RSD) it is respectively less than 15%.

Specificity, matrix effect, extraction recovery and the stability of the method for the present invention are verified, the side Method has been successfully applied to give the blood plasma of chlorogenic acid injection for treating tumor patient and the Concentration Testing of urine Content of Chlorogenic Acid.

Description of the drawings

Fig. 1：The molecular structure and second order ms figure of chlorogenic acid (left side) and internal standard (right side).

Fig. 2：Chlorogenic acid (left side) and internal standard Puerarin (right side) characteristic chromatogram：Double blank (Double-blank), blank (Blank sample), 1h plasma samples (a concentration of 568ng/ after the administration in first day of light basis weight lower limit (LLOQ) and subject mL)。

Fig. 3：Chlorogenic acid (left side) and internal standard Puerarin (right side) characteristic chromatogram：Double blank (Double-blank), blank (Blank sample), 6-9h urine samples (a concentration of 1310ng/ after the administration in first day of light basis weight lower limit (LLOQ) and subject mL)。

Fig. 4：Chlorogenic acid (blood plasma) standard curve.

Fig. 5：Chlorogenic acid (urine) standard curve.

Fig. 6：First day single-dose (0.5mg/kg.d of subject's chlorogenic acid^-1) blood concentration-time curve.

Fig. 7：Subject adds up m- curve (0.5mg/kg.d when excretion rate^-1)。

Specific implementation mode

1. method and result

1.1. drug and reagent

Chlorogenic acid and Puerarin standard items are purchased from National Institute for Food and Drugs Control, and (purity is respectively 96.6% He 95.5%).Chromatographic Pure Methanol, ethyl acetate, acetonitrile are purchased from Fisher companies, and formic acid is purchased from Sigma companies, and 1M hydrochloric acid is purchased from state Family's chemical reagent quality inspection center, experimental water is ultra-pure water.

1.2. equipment and analysis condition

2.2.1 chromatographic condition：

Mobile phase：0.5% formic acid water: acetonitrile (77/23, v/v)

Flow velocity：0.3mL/min

Sample size：2μL

Column temperature：40℃

Temperature in injector：4℃

2.2.2 Mass Spectrometry Conditions：

Electro-spray ionization source (ESI), MRM multiion reaction monitorings, positive ion mode, spray voltage 5500v, temperature 500 DEG C, GAS1 flow velocitys are 50mL/min, and GAS2 flow velocitys are 45mL/min, and CUR 15mL/min, EP and CXP values are respectively 110V And 13V.Chlorogenic acid and interior target DP are respectively 110V and 130V.Under ESI Ionization modes, chlorogenic acid mainly generate [M+H]+ Quasi-molecular ion peak is m/z 355.1, and main fragment ion is m/z 163.1, and CE is 20 (Fig. 1).Internal standard Puerarin is mainly given birth to At [M+H]+quasi-molecular ion peak, be m/z 417.4, main fragment ion be m/z 297.1, CE be 35 (Fig. 1).

1.3. prepared by standard solution and internal standard working solution

The preparation of chlorogenic acid standard solution：Take chlorogenic acid reference substance appropriate, it is accurately weighed, it is dissolved by solvent of methanol, it is fixed Amount is configured to chlorogenic acid concentration and is the standard solution storing solution of 1mg/mL, then is quantitatively diluted to 50,100,200,500 with methanol, 1000,2000,5000,10000,20000ng/mL (blood plasma use) and 500,1000,2000,5000,20000,50000, The standard curve working solution and 150 of 100000,200000ng/mL (urine use), 1500,15000ng/mL (blood plasma) and The Quality Control solution of 1500,15000,150000ng/mL (urines).

The preparation of Puerarin (internal standard) solution：Take Puerarin reference substance appropriate, it is accurately weighed, it is dissolved by solvent of methanol, It is quantitatively configured to the standard solution storing solution of a concentration of 1mg/mL of Puerarin, then 250ng/mL is quantitatively diluted to methanol respectively The standard working solution of (blood plasma) and 500ng/mL (urine).

The above solution is placed in -20 DEG C and saves backup.

1.4. plasma sample is handled

Precision measures 100 μ L of plasma sample, sets in the Ep pipes of 1.5mL, and 20 μ L of internal standard standard working solution are added in precision (250ng/mL) and 30 μ L 1M hydrochloric acid, vortex mixing 10s, again be added 1mL ethyl acetate, vortex mixing, in 12000rpm from Heart 5min draws whole supernatants in new Ep pipes, after nitrogen drying, with 0.5% formic acid water: methanol (50/50, v/v) is multiple Molten, 12000rpm centrifuges 5min, and 2 μ L supernatants is taken to carry out concentration of the UPLC-MS/MS analyses for measuring blood plasma Content of Chlorogenic Acid.

1.5. urine sample is handled

Precision measures 100 μ L of urine sample, sets in the Ep pipes of 1.5mL, and 50 μ L of internal standard standard working solution are added in precision (500ng/mL) and 900 μ L, 0.5% formic acid waters: methanol (50/50, v/v), vortex mixing 10s, 12000rpm centrifugation 5min take 2 μ L supernatants carry out concentration of the UPLC-MS/MS analyses for measuring urine Content of Chlorogenic Acid.

1.6. method validation

With reference to FDA biology sample detection guidelines, methodology validation includes that the sensitivity of method, specificity, standard are bent Line and the range of linearity, precision and accuracy, sample stability, extraction recovery, matrix effect, methodology Quality Control etc..

2.6.1. specificity and sensitivity

The 100 μ L of blank plasma for taking 6 different subjects respectively press " processing of 2.4 plasma samples " item in addition to being not added with internal standard Under operate in accordance with the law, carry out UPLC-MS/MS analyses, obtain blank plasma samples chromatogram (Fig. 2).

The 90 μ L of blank plasma for taking 6 different subjects respectively, are added the 10 μ L of chlorogenic acid standard solution of 50ng/mL, press It is operated in accordance with the law under " processing of 2.4 plasma samples " item, carries out UPLC-MS/MS analyses, obtain blank plasma samples+chlorogenic acid+internal standard Reference substance chromatogram (Fig. 2).

The 100 μ L of blank diaper for taking 6 different subjects respectively press " processing of 2.5 urine samples " item in addition to being not added with internal standard Under operate in accordance with the law, carry out UPLC-MS/MS analyses, obtain blank diaper sample chromatogram figure (Fig. 3).

The 90 μ L of blank plasma for taking 6 different subjects respectively, are added the 10 μ L of chlorogenic acid standard solution of 500ng/mL, press " processing of urine sample " operates in accordance with the law, carries out UPLC-MS/MS analyses, obtains blank diaper sample+chlorogenic acid+internal standard control Product chromatogram (Fig. 3).

2.6.2. preci-sion and accuracy

Preparation chlorogenic acid plasma concentration is 15,150,1500ng/mL and urine concentration is 150,1500,15000ng/mL Basic, normal, high 3 concentration sample, each concentration prepares 6 parts, as a collection of preci-sion and accuracy sample, presses " 2.4 respectively It is operated under the processing of plasma sample " and " processing of 2.5 urine samples " item, prepares within one day a collection of preci-sion and accuracy sample, It is 3 days different, three batches of preci-sion and accuracy samples are measured, chromatogram is recorded, calculate drug peak area As and internal standard peak area Ai Ratio f, the standard curve for substituting into the same day acquires measured concentration, calculate batch in and betweenrun precision and accuracy.Precision and Accuracy indicates that absolute value is less than 15% for qualification, and low-quality control point is less than 20% for qualification with RSD% and RE% respectively.Knot Fruit is shown in Table 1.

The preci-sion and accuracy of table 1. chlorogenic acid blood plasma and urinary assay

2.6.3. standard curve and light basis weight lower limit

90 μ L of blank plasma are taken, 10 μ L of chlorogenic acid standard solution are separately added into, are configured to be equivalent to blood plasma Content of Chlorogenic Acid dense Degree is 5,10,20,50,100,200,500,1000,2000ng/mL plasma sample, is pressed under " processing of 2.4 plasma samples " item Same operation, records chromatogram from " 20 μ L of inner mark solution are added in precision "；It is with chlorogenic acid concentration and internal standard concentration proportion (X) The peak area ratio f of abscissa, chlorogenic acid and internal standard compound is ordinate, is returned with weighting (W=1/X2) least square method Operation, the linear regression equation acquired are standard curve.As a result (Fig. 4) shows chlorogenic acid in 5~2000ng/mL concentration ranges Interior linear relationship is good.Six groups of lower limit of quantitation samples are prepared with method, measured concentration is acquired according to same day standard curve, are prepared daily One batch, is detected for three days on end.

90 μ L of blank diaper are taken, 10 μ L of chlorogenic acid standard solution are separately added into, are configured to be equivalent to urine Content of Chlorogenic Acid dense Degree is 50,100,200,500,1000,2000,5000,10000,20000ng/mL urine sample, presses " 2.5 urine samples The same operation from " 20 μ L of inner mark solution are added in precision " under processing " item, records chromatogram；With chlorogenic acid concentration and internal standard concentration Ratio (X) is abscissa, and the peak area ratio f of chlorogenic acid and internal standard compound is ordinate, with weighting (W=1/X2) least square method Regressing calculation is carried out, the linear regression equation acquired is standard curve.As a result (Fig. 5) shows chlorogenic acid in 50~20000ng/ Linear relationship is good in mL concentration ranges.Six groups of lower limit of quantitation samples are prepared with method, it is dense to acquire actual measurement according to same day standard curve Degree prepares a batch, detects for three days on end daily.

2.6.4. extraction recovery and matrix effect

Blood plasma

Group 1：Precision draws 90 μ L of blank plasma, sets in the Ep pipes of 1.5ml, adds 30 μ L 1M hydrochloric acid and 1mL ethyl acetate, Vortex 1min centrifuges 10min in 12000rpm, draws whole supernatants in another Ep pipes, precision is added concentration and is respectively 150,1500,15000ng/mL 10 μ L of chlorogenic acid standard working solution, 20 μ L of inner mark solution are added in precision, after vortex mixing, in 12000rpm centrifuges 5min, draws whole supernatants in new Ep pipes, after nitrogen drying, with 100 μ L0.5% formic acid waters: first Alcohol (50/50, v/v) redissolves, and 12000rpm centrifuges 5min, and 2 μ L supernatants is taken to carry out LC-MS/MS analyses.Each concentration respectively prepares 6 Part, chromatogram is recorded, each concentration chlorogenic acid and interior target peak area are recorded.

Group 2：The 10 μ L of chlorogenic acid standard working solution of accurate addition a concentration of 150,1500,15000ng/mL in Ep pipes, 20 μ L (250ng/mL) of internal standard standard working solution and 30 μ L1M hydrochloric acid are added in precision, and 1mL second is added in vortex mixing 10s again Acetoacetic ester, vortex mixing centrifuge 5min in 12000rpm, draw whole supernatants in new Ep pipes, after nitrogen drying, with 0.5% formic acid water: methanol (50/50, v/v) redissolves, 12000rpm centrifuges 5min, and 2 μ L supernatants is taken to carry out LC-MS/MS analyses.Note Chromatogram is recorded, each concentration chlorogenic acid and interior target peak area are recorded.

Group 3：It is 15,150.1500ng/ to be configured to blood plasma chlorogenic acid concentration by " plasma sample standard curve " assay method Each 6 parts of the plasma sample of basic, normal, high three kinds of various concentrations of mL is equally operated by above-mentioned " 2.4 plasma sample processing method ", UPLC-MS/MS analyses are carried out, chromatogram is recorded, records each concentration chlorogenic acid and interior target peak area.

2 are the results are shown in Table, shows that the extraction recovery of blood plasma Content of Chlorogenic Acid is all higher than 65% in various concentration, there is certain base Mass effect, internal standard extraction recovery are 38.8%, and matrix effect is consistent with chlorogenic acid.

Urine

Group 1：Precision measures the 100 μ L of urine sample of 6 Different Individuals, sets in the Ep pipes of 1.5mL, and internal standard mark is added in precision 50 μ L (500ng/mL) of quasi- working solution and 900 μ L, 0.5% formic acid waters: methanol (50/50, v/v), vortex mixing 10s, 12000rpm centrifuges 5min, and 2 μ L supernatants is taken to carry out LC-MS/MS analyses.Each concentration respectively prepares 6 parts, records chromatogram, record Each concentration chlorogenic acid and interior target peak area.

Group 2：Precision measures 100 μ L of distilled water, sets in the Ep pipes of 1.5mL, and 50 μ L of internal standard standard working solution are added in precision (500ng/mL) and 900 μ L, 0.5% formic acid waters: methanol (50/50, v/v), vortex mixing 10s, 12000rpm centrifugation 5min take 2 μ L supernatants carry out UPLC-MS/MS analyses.Each concentration respectively prepares 6 parts, records chromatogram, records each concentration chlorogenic acid and internal standard Peak area.

2 are the results are shown in Table, shows that the matrix of urine Content of Chlorogenic Acid is imitated between various concentration unanimously, RSD, which is less than, is respectively less than 15% It does not influence to measure, interior target matrix effect is consistent with chlorogenic acid.

2. chlorogenic acid of table and interior target extraction recovery and matrix effect (n=6)

2.6.5 stability

Stability assessment covers the detection process of entire sample, and basic, normal, high three concentration is arranged, each concentration repeats Five samples, the results are shown in Table 3.

3. chlorogenic acid blood plasma of table and urine stability (n=5, mean value (% deviations))

2. pharmacokinetics application

Screening previously received invalid or recurrence after one or more platiniferous standard chemotherapy regimen, or without standard regimens Locally Advanced and/or metastatic malignant tumor patient, give chlorogenic acid injection, intramuscular injection continuous 28 days, is given once a day Pharmaceutical quantities are 0.5mg/kg.d-1.Subject in d1 administration before (0.05h) and administration after 10min, 20min, 30min, 45min, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h and 6h respectively acquire ulnar vein blood 3ml.Blood specimen collection is placed on has posted mark in advance In the test tube of label, (3000rpm, 10min) separated plasma is centrifuged in 10 minutes, blood plasma, which is transferred in EP pipes, sets -80 DEG C of refrigerators It preserves.1-3h, 3-6h, 6-9h, 9-12h and 12-24h collect the urine of subject respectively before administration and after administration simultaneously, Urine volume is recorded, takes 1mL to set -80 DEG C of refrigerators after mixing and preserves.The pharmacokinetic parameter of single-dose is by software WinNolin 6.3 calculate acquisition with non-compartment model, and Fig. 6 shows that the subject takes orally the Drug-time curve after chlorogenic acid piece, and Fig. 7 is that urine is tired Meter excretion curve.

4. discussing

In the selection of sample pre-treatments, we first attempt to using methanol and acetonitrile albumen precipitation processing plasma sample, It was found that there are apparent substrate inhibition effect, inhibiting rate reaches 80-85%.By adding 0.1% formic acid, 35-40% can be reduced Substrate inhibition effect, however, it is observed that since matrix effect causes same sample signal extremely unstable.Therefore, we The liquid-liquid extraction method using ethyl acetate is attempted, as a result shows that matrix effect is substantially reduced, but extraction recovery is extremely low, less than 1/ 100.It is analyzed for this purpose, 30 μ L 1M hydrochloric acid are added in we in 100 μ L blood plasma, extraction recovery greatly increases (at least 60%).For urine sample, it has been found that direct dilution method processing urine sample may be used.When dilute using 50% methanol-water When releasing, the substrate inhibition effect of 40-50% can be observed, use 0.5% formic acid water instead: when methanol (50/50, v/v), matrix effect It is obviously improved.

In the optimization of chromatographic condition, consider that Acquity UPLC BEH chromatographic columns can be used for the pH value range of mobile phase Wider (1-12), and it is capable of providing a good separation and peak shape, we use Acquity UPLC C18 columns (50mm for the first time × 2.1mm, 1.7 μm, Waters), as a result chlorogenic acid is presented bimodal.We have found that the chromatography embedded with hydrophilic amino formic acid group Column Acquity BEH Shield RP 18colum (100mm × 2.1mm, 1.7 μm, Waters), are realized under Gradient elution Chlorogenic acid good separation and peak type.In order to advanced optimize separation and peak type, we by adjusting formic acid methanol ratio, most 0.5% formic acid water of mobile phase is selected eventually: acetonitrile (77/23, v/v), chlorogenic acid and interior target retention time are respectively 1.16 Hes 1.09min。

So far, the report that the concentration of human plasma Content of Chlorogenic Acid is measured using LC-MS/MS methods is less, and the inspection of single sample The survey time is 5min [11], and without measuring the methodology report in human urine Content of Chlorogenic Acid concentration.In our research, I Develop the concentration that a kind of quick, sensitive method measures human plasma and urine Content of Chlorogenic Acid, the detection time of single sample For 2min.The malignant tumor patient blood plasma and urine for receiving that single dose is 0.5mg/Kg.d-1 chlorogenic acids has been applied successfully in this method In determination of drug concentration, have important clinical value.

Bibliography：

[1] Shi H, Dong L, Dang X, et al.Effect of chlorogenic acid on LPS- induced proinflammatory signaling in hepatic stellate cells.INFLAMM RES 2013； 62：581-587.

[2] Karunanidhi A, Thomas R, van Belkum A, Neela V.In vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia including the trimethoprim/sulfamethoxazole resistant strain.BIOMED RES INT 2013；2013：392058.

[3] Du WY, Chang C, Zhang Y, et al.High-dose chlorogenic acid induces inflammation reactions and oxidative stress injury in rats without implication of mast cell degranulation.J ETHNOPHARMACOL 2013；147：74-83.

[4] Lou Z, Wang H, Zhu S, Ma C, Wang Z.Antibacterial activity and mechanism of action of chlorogenic acid.J FOOD SCI 2011；76：M398-M403.

[5] Yan Y, Li J, Han J, Hou N, Song Y, Dong L.Chlorogenic acid enhances the effects of 5-fluorouracil in human hepatocellular carcinoma cells through the inhibition of extracellular signal-regulated kinases.Anticancer Drugs 2015； 26：540-546.

[6] Belkaid A, Currie JC, Desgagnes J, Annabi B.The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression.CANCER CELL INT 2006；6：7.

[7] Bandyopadhyay G, Biswas T, Roy KC, et al.Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase- dependent apoptosis in chronic myelogenous leukemic cells.BLOOD2004；104：2514- 2522.

[8] Liu YJ, Zhou CY, Qiu CH, Lu XM, Wang YT.Chlorogenic acid induced apoptosis and inhibition of proliferation in human acute promyelocytic leukemia HL60 cells.MOL MED REP 2013；8：1106-1110.

[9] Yang J, Lv F, Chen XQ, et al.Pharmacokinetic study of major bioactive components in rats after oral administration of extract of Ilex hainanensis by high-performance liquid chromatography/electrospray ionization mass spectrometry.J Pharm Biomed Anal 2013；77：21-28.

[10] Wang XY, Ma XH, Li W, et al.Simultaneous determination of five phenolic components and paeoniflorin in rat plasma by liquid chromatography- tandem mass spectrometry and pharmacokinetic study after oral administration of Cerebralcare granule((R)).J Pharm Biomed Anal 2013；86：82-91.

[11] Zhang J, Chen M, Ju W, et al.Liquid chromatograph/tandem mass spectrometry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study.J Pharm Biomed Anal 2010；51：685-690.

[12]US Department of Health and Human Services Food and Drug Administration, 2001.Website：http：//www.fda.gov/cder/guidance/index.htm.

[13] Chinses Food and Drug Administration, 2005.Website：http：// www.sda.gov.cn/gsz05106/08.pdf.

[14] Zhou W, Liu S, Ju W, et al.Simultaneous determination of phenolic acids by UPLC-MS/MS in rat plasma and its application in pharmacokinetic study after oral administration of Flos Lonicerae preparations.J Pharm Biomed Anal 2013；86：189-197.

[15] Yang H, Lee DY, Jeon M, Suh Y, Sung SH.Determination of five active compounds in Artemisia princeps and A.capillaris based on UPLC-DAD and discrimination of two species with multivariate analysis.ARCH PHARM RES 2014； 37：617-625.

Claims (6)

1. the method that the concentration of human body fluid Content of Chlorogenic Acid is measured by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), Wherein the method uses ACQUITY UPLC Shield RP18 analytical columns, is carried out by mobile phase of 0.5% formic acid water-acetonitrile Gradient elution, wherein 0.5% formic acid water:The volume ratio of acetonitrile is 85:15-50:50；The wherein described body fluid is human urine, institute State human urine directly dilution after mixing, centrifuge, supernatant taken to be measured, wherein it is described dilution by 0.5% formic acid water-methanol into Row.

2. 0.5% formic acid water of mobile phase that method described in claim 1, wherein the method use:The volume ratio of acetonitrile is 77:23。

3. method described in claim 1, wherein the method using ACQUITY UPLC Shield RP18,100mm × 2.1mm, 1.7 μm carry out as analytical column.

4. method described in claim 1, wherein the method use Puerarin to be carried out for internal standard.

5. method described in claim 1, wherein 0.5% formic acid water used by dilution:The volume ratio of methanol is 30:70 to 70:30。

6. method described in claim 1, wherein 0.5% formic acid water used by dilution:The volume ratio of methanol is 50:50.