CN107167540B - The method for measuring the DTPA-Zn in human urine biological sample - Google Patents
The method for measuring the DTPA-Zn in human urine biological sample Download PDFInfo
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The present invention relates to the methods of the DTPA-Zn in measurement human urine biological sample.Specifically, the present invention relates to the methods of the DTPA-Zn in measurement human urine biological sample, this method uses the content of the DTPA-Zn in the processed human urine biological sample of liquid chromatography-tandem mass spectrometry, include the following steps: the processing of (1) biological sample, (2) liquid chromatography tandom mass spectrometry determination, (3) data process&analysis.The method of the present invention shows the excellent effect as described in description of the invention, such as with excellent chromatographic isolation effect.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of reliable method to measure organism such as people, big
Mouse gives the content of the above-mentioned metal-chelator after above-mentioned DTPA- metal-chelator in biological sample such as blood, urine etc., with assessment
The therapeutic effect of these metal-chelators or its behavior in vivo.
Particularly, the present invention relates to the methods of the DTPA-Zn in measurement human urine biological sample a kind of.
Background technique
Lead contamination and lead poisoning have received significant attention the harm of human health.Lead is human body non-essential element,
Absorption of human body is mainly entered by respiratory tract, alimentary canal, can be accumulated in vivo, to each system of whole body and the toxic work of organ
With main to influence nerve, digestion, hematological system, lead contamination and lead poisoning are the public health problem [Hao for needing emphasis to solve
FT,Du XQ,Niu YM,et al.Progress in research of the lead intoxication[J].Chin J
Ind Med (Chinese industrial medical journal), 2008,21:200-202.1；Zhou QQ,Hu FF,Xia CY,et al.Study
progress on health hazards in occupational lead-exposed workers[J].Chin J Ind
Med (Chinese industrial medical journal), 2013,26:353-356].In terms for the treatment of, calcium disodium edetate is by as choice drug
For clinic [Zhou JY, Duan Z, Deng JX, et al.Clinical observation on chronic lead
poisoning treated with different dosages of CaNa-EDTA[J].Occup Health Emerg
Resue (occupational health and emergency management and rescue), 2002,20:159-160].Calcium disodium edetate treatment lead poisoning has good treatment
Effect, but while lead is complexed, also intracorporal zinc, calcium, manganese, iron, copper etc. is discharged in complexing, will cause internal essential trace element
, there is toxic side effect in dysequilibrium, and most important toxic side effect is renal damage caused by zinc lacks.
Ca-DTPA (DTPA-Ca can be denoted as not only) also known as Ca DTPA salt and Zn-DTPA (but also can
Be denoted as DTPA-Zn) also known as diethylene-triamine pentaacetic acid trisodium zinc salt, the two belong to complexones, in August, 2004 by beauty
State FDA ratifies while listing, and is stained with for entering the serious polluted place work of radionuclide or stopping preceding and radionuclide
Treatment [Cada DJ, Levien T, Baker DE.Pentetate calcium trisodium (Ca-DTPA) and after dye
pentetate zinc trisodium(Zn-DTPA)[J].Hospital pharmacy,2005,40:65-71]。Zn-DTPA
With Ca-DTPA in vivo can selectively with radionuclide lanthanum (140La), cerium (144Ce), promethium (147Pm), americium (124Am),
The cation of plutonium (239Pu) etc. forms the soluble complexes of stability, excretes quickly through kidney, to reduce in vivo
The deposition of radioactive substance.Since Ca2+ is less than Zn2+ [Xue HL.The chelator with the complexation constant of DTPA
Treatment of common metal intoxication [J] .Chem Ind Occup Saf Health (chemical industry labour
Protection), 1989,10:22-26], DTPA is easier to the cation complex with nucleic in Ca-DTPA, therefore the effect of its decorporation is better than
Zn-DTPA.But toxicologic study is shown, Ca-DTPA easily causes endogenic zinc to lack, so that the work of enzyme related with zinc
Property is by serious influence, including DNA and RNA polymerase, carboxypeptidase, carbonic anhydrase etc., can cause liver kidney, intestinal mucosa
Adverse reactions [Shen BY, Ruan TM, the Wu DC.Effect of Zn-DTPA on the such as damage and hematopoietic repair
decorporation of ultra-Uranium,ultra-Plutonium and Lanthanide and its
Application [J] .Foreign Med Sci (Radit Med Nucl Med) [foreign medical science (Radiation Medicine nuclear medicine point
Volume)], 1988,12:70-74；Zhao XC,Wu DC.Toxicity of DTPA and its application on the
Decorporation of nuclide [J] .Foreign Med Sci (Radit Med) [foreign medical science (Radiation Medicine point
Volume)], 1980,4:211-215].The safety of Zn-DTPA is higher, though the missing of the microelements such as manganese and magnesium can be also caused,
It will not cause zinc-deficiency and generate serious toxic side effect, 1/10~1/30 [the Zhang ZY, Xu that toxicity is about Ca-DTPA
XW, Zhu Z.Pentetate zinc trisodium [J] .Chin Pharm J (Chinese Pharmaceutical Journal), 2007,42:557-
558]。
Existing document measures lead content using sampling Graphite Furnace Atomic Absorption spectrophotometer method, has investigated Zn-DTPA to chronic
The decorporation of mice with lead poisoning acts on [Yang S, Chen L, Yin YY, et al.Study of the effect of
pentetate zinc trisodium on excretion of lead in lead poisoned mice[J].Pharm
J Chin PLA (liberation army Acta Pharmaceutica Sinica), 2011,27:147-149], however sampling Graphite Furnace Atomic Absorption spectrophotometer method is surveyed
Determine that method is complicated, at high cost, sensitivity is low, such as the amount in the biological samples such as blood, urine is difficult to meet measurement requires.
In addition, there are also document, [Chen Li analyzes Zn-DTPA etc., ICP-MS method and acts on the decorporation of mice with lead poisoning, pharmacy
Journal (Acta Pharmaceutica Sinica) 2014,49 (11): 1588-1592] report, using inductance coupled plasma
Constitution composes the concentration of lead in (ICP-MS) measurement biological sample, investigates nucleic decorporation medicine Zn-DTPA to the decorporation of mice with lead poisoning
Effect.Mouse disposable celiac injects acetic acid lead solution, and every dye lead 1mg establishes acute lead poisoning model, contaminates the abdominal cavity 4h after lead
Inject Zn-DTPA, successive administration 5 days.Normal group, dye lead model group, Zn-DTPA and Ca-DTPA combination group are set simultaneously.Often
It collects urine, in the dead some animals in the natural gift other places of the 2nd, 4 and 6 of experiment, takes whole blood, bilateral femur, brain, after resolution processing,
Lead content is measured with ICP-MS.It is believed that the result shows that Zn-DTPA can dramatically increase the discharge of lead in urine, reduction blood, femur and brain
Lead content in tissue.
However, can also be excluded in vivo since DTPA-Ca and DTPA-Zn can be used not only for excluding internal heavy metal lead
The heavy metals such as lanthanum, cerium, promethium, americium, plutonium be even used as clinical application drug can also be used to making a definite diagnosis or it is doubtful by plutonium, americium, hard iron in vivo
The treatment of subject is polluted, and improves the clearance rate of these metallic elements.In the case where being so widely applied environment, only investigate
Lead distribution in vivo and content are obviously not enough to evaluate the internal behavior of this metal-chelator of DTPA.
Therefore, this field still expects have reliable method to give above-mentioned metal chelating to measure organism such as people, rat
The content of above-mentioned metal-chelator after mixture in biological sample such as blood, urine etc., to assess the treatment of these metal-chelators
Effect or its behavior in vivo.
Summary of the invention
The purpose of the present invention is to provide a kind of reliable methods to give above-mentioned metal to measure organism such as people, rat
The content of above-mentioned metal-chelator after chelating agent in biological sample such as blood, urine etc., to assess controlling for these metal-chelators
Therapeutic effect or its behavior in vivo.It has been had now surprisingly been found that, use liquid chromatography-tandem mass spectrometry (LC-MS/ of the present invention
MS) method can effectively realize above-mentioned purpose.In particular, the present invention is surveyed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method
Determine the DTPA-Zn in human urine biological sample, shows excellent Methodological characteristics.The present invention is consequently found that and be accomplished.
For this purpose, first aspect present invention is related to a kind of method for measuring the DTPA-Zn in human urine biological sample, this method
The content of the DTPA-Zn in processed human urine biological sample is measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method,
Include the following steps:
(1) processing of biological sample:
(1a) takes the 50 μ L of human urine for containing or not contain DTPA-Zn, is placed in 1.5mL centrifuge tube, adds inner mark solution i.e.
Inner mark solution is not added in the 20 μ L of EDTA-Zn aqueous solution of 25 μ g/mL, and 0.01% ammonium hydroxide, 900 μ L (addition when internal standard is not added is added
0.01% ammonium hydroxide, 920 μ L makes urine, ammonium hydroxide, optional 970 μ L of inner mark solution total volume), vortex mixing 3min, in whole
Sample is on solid phase extraction column；
It is cleaned after (1b) loading with 2mL water, finally the methanol solution with 0.5mL containing 1% ammonium hydroxide elutes；
(1c) collects the eluent, and drying, residue is redissolved with 150 μ L water, draws 10 μ L and carries out LC-MS/MS analysis；
The preparation of (1d) standard curve: taking the 100 μ L of DTPA-Zn standard serial solution of various concentration, is placed in 1.5mL centrifugation
Blank human urine, vortex 3min is added in Guan Zhong, and it is 10,20,50,100,200,500,1000 and 2000 μ that preparation, which is equivalent to concentration,
The urine sample of g/mL；50 μ L of the urine sample is taken, and sequentially adds 20 μ L of inner mark solution, 0.01% ammonium hydroxide, 900 μ L, vortex is mixed
3min is closed, is all splined on solid phase extraction column, then according to above (1b) and (1c) operation, result is analyzed according to LC-MS/MS
Standard curve is drawn, which is used to calculate the target substance content in various samples；
(2) liquid chromatography tandom mass spectrometry determination:
(2a) provides high performance liquid chromatograph and tandem mass spectrometer, and the high performance liquid chromatograph is equipped with gradient pump and sample introduction
Device, the tandem mass spectrometer are equipped with electric spray ion source and data processing system；
(2b) chromatographic condition: analytical column used be SHISEIDO Proteonavi brand chromatographic column (its specification is for example
Be 5 μm, 250 × 4.6mm I.D.), C is connected before column18(its specification is, for example, 4 × 3.0mm I.D., such as the U.S. to guard column
The guard column of Phenomenex company), mobile phase be methanol -2mM ammonium formate (for example, wherein contain 0.03% ammonium hydroxide) (50:50,
V/v), flow velocity 0.45mL/min；
(2c) Mass Spectrometry Conditions: electro-spray ionization source (such as using the source Turbo Ionspray), negative-ion mode detection；
Injection electric is -4000V；Source temperature is 500 DEG C；Roller shutter gas (CUR) is 20；Collision gas (CAD) is 4；Scanning mode is mostly anti-
(MRM) should be monitored, the ionic reaction for quantitative analysis is respectively 226.6 → m/z of m/z 454.2 → m/z 364.3+m/z
204.5 (DTPA-Zn, CE 45V) and m/z353.0 → m/z 263.0 (ethylenediamine tetraacetic acid disodium zinc salt salt, CE 15V), sweep
Retouching the time is 150msec；
(3) data process&analysis
(3a) obtains the target substance content in biological sample by above-mentioned liquid chromatography tandom mass spectrometry determination；With optionally
's
(3b) (such as Winnolin (Phoenix 6.3, the U.S.) divides institute's measured data using pharmacokinetics software
Analysis, obtain it is relevant with drug exposure be selected from it is following once or multiple kinetic parameter Cmax、Tmax、AUC0-tAnd MRT0-t。
Method described in any embodiment according to a first aspect of the present invention, wherein the human urine is never to give Zn-
The urine that the people of DTPA obtains as blank diaper sample, or from people give Zn-DTPA after the urine that obtains of different time
As drug containing urine sample.
Method described in any embodiment according to a first aspect of the present invention is set to wherein human urine obtained is optional
Frozen in -20 DEG C of refrigerators, with etc. it is to be determined.
Method described in any embodiment according to a first aspect of the present invention, used in be designated as EDTA-Zn
Na2·xH2O。
Method described in any embodiment according to a first aspect of the present invention, wherein the solid phase extraction column is in loading
Before, it is first activated with 1mL methanol, then with 1mL water balance；Alternatively, first being activated with 2mL methanol, then with 2mL water balance.
Method described in any embodiment according to a first aspect of the present invention, wherein the high performance liquid chromatograph is, for example,
1200 type liquid chromatograph of Agilent company of the U.S. Agilent.
Method described in any embodiment according to a first aspect of the present invention, wherein the tandem mass spectrometer is, for example, the U.S.
4000 type tandem mass spectrometer of AB Sciex company API；Such as it is furnished with Turbo Ionspray ionization source and Analyst
1.5 data processing system.
In the present invention, Proteonavi chromatographic column is a kind of using in porous spherical silica filler surface bond butyl
(C4) high-performance protein-polypeptide analysis chromatographic column specially, the filler had both had the high separation capacity of silica type filler and resistance to
Pressure property, and with specialities such as acid resistance, the absorption for inhibiting protein.The chromatographic column is sometimes with Proteonavi s5 mark.Although
The DTPA metal chelating agent that the present invention analyzes is a kind of typical small-molecule substance, and molecular weight is more much smaller than common protein-
Peptide, however, having had now surprisingly been found that, the present invention is only stated in use under conditions of the chromatographic column of Proteonavi chromatographic column
The method of the present invention could obtain excellent methodology performance.For example, using YMC-C18, Agilent Extard-C18, money life
When the C18 columns such as hall PAK CR-18 (it is same manufacturer with Proteonavi column used herein), DTPA metal complex is extremely
It is difficult to elute, such as using chromatographic condition of the present invention but when using these C18 columns instead, the retention time of DTPA-Za is greater than
43min and hangover is serious, however it is unaccountable be internal standard substance but not so serious extension retention time (different
Retention time is about between 5~6min on C18 column).In another example using ZORBAX EDIPSR-C8, Dikma diamond-C8, taking
When the C8 columns such as sieve door Gemini-C8, DTPA metal complex does not retain in the chromatography column, i.e., retention time is extremely short, such as
The retention time of DTPA-Za using chromatographic condition of the present invention but when using these C8 columns instead be less than 1.5min and be easy and solvent
It can not be efficiently separated etc. mixed in together.
Method described in any embodiment according to a first aspect of the present invention, wherein the solid phase extraction column is modelThe solid phase extraction column of WAX, such as purchased from Waters company.Waters Oasis WAX solid phase extraction column is in quotient
Be usually to be configured as the exchange reverse phase absorption agent of mixed type weak anionic in industry, to highly acid compound have high selectivity and
The solid phase extraction column of sensitivity.It has been had now surprisingly been found that, only ought use above-mentioned Oasis WAX type solid phase extraction column
In the case where, the method for the present invention could obtain excellent methodology performance.And when the solid phase extraction column example for using other brands instead
Such as the Cleanert PWAX solid phase extraction column of U.S. Ai Jieer, StrataTM-X solid phase extraction column and even same
When the Oasis MAX type solid phase extraction column of manufacturer, it can not much obtain above-mentioned Oasis WAX type solid phase extraction column and be obtained
Good results.Such as when being tested according to method involved in following table 4 result, with above-mentioned three kinds other brand/models
Solid phase extraction column when, 20 μ g/mL of the rate of recovery, 180 μ g/mL, 1800 μ g/mL, tri- kinds of concentration rate of recovery respectively 67~
73%, in 76~81%, 79~94% ranges and various pillars under three kinds of various concentrations gained RSD in 9~22% ranges
It fluctuates, greatest differences are presented in the rate of recovery under this various concentration and the result of RSD wide fluctuations is in biological sample analysis
It is unacceptable.In addition, when carrying out biological sample processing, after sample is loaded to solid phase extraction column, with containing a small amount of ammonia
The eluent of water carry out elution be necessary, otherwise the rate of recovery ratio under each concentration in following table 4 rate of recovery result of the present invention
Low 20 percentage points or more.
Method described in any embodiment according to a first aspect of the present invention, wherein further including one kind that following solution is prepared
Or a variety of operation:
The preparation of (10a) DTPA-Zn stock solution: the Zn-DTPA injection that concentration is 100mg/mL is taken to store up as DTPA-Zn
Standby liquid (it can be denoted as A1), is transferred in tool plug test tube, and -20 DEG C of refrigerator freezings save, spare；
The preparation of (10b) DTPA-Zn standard serial solution: DTPA-Zn stock solution, that is, A1 is diluted with water be made it is following
The standard serial solution of DTPA-Zn concentration, and saved backup in 4 DEG C of refrigerators: 1.0 × 105μg/ml、1.0×104μg/ml、
5.0×103μg/ml、2.5×103μg/ml、103μg/ml,500μg/ml,250μg/ml,100μg/ml,50μg/ml；
The preparation of (10c) DTPA-Zn quality-control sample working solution: DTPA-Zn stock solution, that is, A1 is diluted with water and is made down
The quality-control sample working solution of DTPA-Zn concentration is arranged, and is saved backup in 4 DEG C of refrigerators: 1.0 × 105μg/ml、1.0×104μ
g/ml、9.0×103μg/ml、1.0×103μg/ml,900μg/ml,100μg/ml,50μg/ml；
The preparation of (10d) internal standard working solution: precision weighs 20.0mg internal standard substance EDTA-Zn in 2mL measuring bottle, uses water
Simultaneously constant volume is dissolved, is made into the internal standard stock solution (it can be denoted as IS1) that concentration is 10.0mg/mL, -20 DEG C of refrigerator freezings save, standby
With；
Precision draws internal standard stock solution (i.e. IS1) 1250 μ L and, into 25mL measuring bottle, is settled to scale with water, obtains 500 μ g/mL
Internal standard working solution (IS2), and save backup in 4 DEG C of refrigerators (as inner mark solution, concentration is 500 μ g/ml)；
The preparation of (10e) 1% ammonium hydroxide methanol solution: measuring ammonium hydroxide 10.0mL, and methanol 990.0mL is mixed in conical flask
To obtain the final product；
Method described in any embodiment according to a first aspect of the present invention, wherein further including what following plasma sample was handled
One or more operations:
The preparation of (20a) blank diaper sample:
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 0.01% ammonium hydroxide 920 μ L, vortex mixing 3min is added,
All it is splined on solid phase extraction column；
It is cleaned after loading with 2mL water, is finally eluted with the methanol solution 0.5mL containing 1% ammonium hydroxide；
Eluent is collected, 10 μ L is drawn and is mixed well with 190 μ L water, is obtained for carrying out the molten of LC-MS/MS analysis measurement
Liquid (desirable 10 μ L carry out LC-MS/MS analysis)；
(20b)The preparation of zero point urine sample:
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 20 μ L of inner mark solution, 0.01% ammonium hydroxide, 900 μ L is added,
Vortex mixing 3min, is all splined on solid phase extraction column；Then such as " preparation of (20a) blank diaper sample " described progress
Subsequent operation；
(20c)The preparation of standard curve:
Take the 100 μ L of DTPA-Zn standard serial solution of various concentration (for example, taking " (10b) DTPA-Zn standard serial solution
Preparation " in 1.0 × 104μg/ml、5.0×103μg/ml、2.5×103μg/ml、103μg/ml、500μg/ml、250μg/
Ml, 100 μ g/ml, 50 each 100 μ L of μ g/ml " DTPA-Zn standard serial solution), it is placed in 1.5mL centrifuge tube, blank people is added
Urine, vortex 3min, preparation are equivalent to the urine sample that concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL；
50 μ L of urine sample is taken, and sequentially adds 20 μ L of inner mark solution, 0.01% ammonium hydroxide 900 μ L, vortex mixing 3min is all splined on
On solid phase extraction column；Then such as " preparation of (20a) blank diaper sample " described carry out subsequent operation；
The preparation of (20d) quality-control sample
It takes DTPA-Zn quality-control sample working solution to be placed in 1.5mL centrifuge tube, blank human urine, vortex 3min, system is added
It is standby to be equivalent to the urine sample that concentration is 20,180 and 1800 μ g/mL, 50 μ L of urine sample is taken, and sequentially add inner mark solution 20
μ L, 0.01% ammonium hydroxide 900 μ L, vortex mixing 3min are all splined on solid phase extraction column；Then such as " (20a) blank diaper
The preparation of sample " the carry out subsequent operation；
The preparation of (20e) minimum lower limit of quantitation sample (LLOQ)
It takes DTPA-Zn quality-control sample working solution to add in blank human urine, is prepared into the LLOQ sample of 10.0 μ g/mL；
Remaining is pressed operates under " preparation of (20d) quality-control sample " item, and carries out LC-MS/MS analysis；
The preparation of (20f) cross jamming test specimen
40 μ L of DTPA-Zn working solution is taken, is placed in 1.5mL centrifuge tube, blank human urine, vortex 3min, preparation is added
At the ULOQ sample of 2000 μ g/mL not containing the internal standard；In addition to internal standard is not added, remaining presses " preparation of (20d) quality-control sample " Xiang Xiacao
Make, and carries out LC-MS/MS analysis.
In the aforesaid operations method of the present invention the step of, although the specific steps of its description are in certain details or language
In description with the example of following detailed description part described in step different from, however, those skilled in the art
The detailed disclosure of full text can summarize approach described above step completely according to the present invention.
Any embodiment in either present invention face can be combined with other embodiments, as long as they are not
It will appear contradiction.In addition, any technical characteristic can be adapted for other realities in any embodiment of either side of the present invention
The technical characteristic in scheme is applied, as long as they are not in contradiction.The invention will be further described below.
All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and
Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
Subject to the meaning stated.
DTPA metal chelating agent, for example, DTPA calcium metal chelating agent or DTPA zinc metal chelating agent, their body
Outer detection, such as the detection in some chemicals, preparation are still the Xiang Ju great that current those skilled in the art encounter
Problem is detected, and the detection difficulty in their vivo biodistribution samples is even more from far away than vitro detection difficulty.In particular, current state
The inside and outside report all without being analyzed in these compound bodies.
The calcium metal chelating agent of DTPA, can be abbreviated as Ca-DTPA or DTPA-Ca, usually be also known as Ca-DTPA
(DTPA-CaNa3)；The zinc metal chelating agent of DTPA, can be abbreviated as Zn-DTPA or DTPA-Zn, usually be also known as Zn-
DTPA(DTPA-ZnNa3)。
The internal standard substance EDTA-Zn used in Ca-DTPA, Zn-DTPA and the present invention is (for secondly sodium-salt hydrate
EDTA-Zn Na2xH2O) molecular formula be respectively following formula A, formula B and formula C:
Wherein, Ca-DTPA can be injected intravenously and inhalation, be provided respectively with injection and powders for inhalation dosage form
In clinic, first batch of sample got the Green Light in 2004 in the U.S..The indication and application of Ca-DTPA clinically are as follows: Ca-DTPA
It is used to make a definite diagnosis or the doubtful treatment by plutonium, americium, huge internal pollution subject as a kind of radiation chelating agent of alleviating, and improve this
The clearance rate of a little metallic elements.
The dosage and administration aspect of Ca-DTPA, the code requirement of FDA is in the contaminated Ca-DTPA for giving first dosage interior for 24 hours
Treated, for 24 hours after, it is proposed that using Zn-DTPA maintain chelating therapy.After chelating therapy is contaminated in vivo for 24 hours in use
Effect is best；Chelating therapy should be given when being contaminated in time making a definite diagnosis or suspecting；If cannot treat at once, permit in condition
Xu Shiying gives chelating therapy at once.A period of time chelating therapy after polluting in vivo is still effective.Radioactive pollutant exists
When during body-internal-circulation or in interstitial fluid, the chelate effect of Ca is best.Radioactivity after curative effect is chelated with internal pollution
Pollutant is isolated in liver and bone and reduces.If making a definite diagnosis or doubtful internal pollution not being caused by plutonium, americium, hard iron, need to carry out
Other treatment.Specific medication is that pollution channel is injected intravenously Ca- when not determining or there may be multipath pollution
DTPA；3-4 minutes Ca-DTPA solution (1g/5ml) used time of 5ml slow intravenous injection is administered, or the Ca-DTPA of 5ml is molten
Liquid is diluted to the administration of 100-250ml venous transfusion in 5% glucose solution, woods grignard lactic acid solution, physiological saline；If by
Examination person is contaminated due to sucking, and the interior Ca-DTPA for giving atomization can be used as therapy approach use for 24 hours.
In addition, Zn-DTPA can be injected intravenously and inhalation, provided respectively with injection and powders for inhalation dosage form
In clinic, first batch of sample got the Green Light in 2004 in the U.S..The indication and application of Zn-DTPA clinically are as follows: Zn-DTPA
Suitable for make a definite diagnosis or doubtful plutonium, americium, hard iron caused by internal pollution, and accelerate its clearance rate.
The dosage and administration aspect of Zn-DTPA, the code requirement of FDA, in the contaminated Ca- for giving first dosage interior for 24 hours
DTPA is treated；Curative effect is better by Ca-DTPA ratio Zn-DTPA during this period；If Ca-DTPA is invalid, Zn-DTPA progress is given
It treats for the first time.It is given if second day additional chelation therapy of administration is proposed, carries out conventional therapy using Zn-DTPA.If Zn-
DTPA is invalid, and chelating therapy is maintained using Ca-DTPA.Adjoint mineral supplements containing Zn should be given.To adult and blueness
Teenager is injected intravenously the Zn-DTPA of 1g dosage；For 12 years old children below, it is injected intravenously the Zn-DTPA of 14mg/Kg, dosage
It is sure not more than 1g.Chelating continued treatment after for 24 hours uses Zn-DTPA, to adult and teenager, is injected intravenously 1g dosage
Zn-DTPA, once a day；For 12 years old children below, it is injected intravenously the Zn-DTPA of 14mg/Kg, once a day, dosage is not
More than 1g.After the chelating therapy of DTPA is contaminated in vivo for 24 hours in using effect it is best.It is contaminated making a definite diagnosis or suspecting
When should give chelating therapy in time.If cannot treat at once, chelating therapy should be given at once in conditions permit.It is dirty in vivo
A period of time chelating therapy after dye is still effective.Radioactive pollutant in vivo in cyclic process or in interstitial fluid when, Zn-
The chelate effect of DTPA is best.Radioactive pollutant is isolated in liver and bone and drops after curative effect is chelated with internal pollution
It is low.If making a definite diagnosis or doubtful internal pollution not being caused by plutonium, americium, hard iron, other treatment need to be carried out.Specific medication is,
Pollution channel is injected intravenously Zn-DTPA when not determining or there may be multipath pollution；By the Zn-DTPA solution (1g/ of 5ml
5ml) slow intravenous injection administration in used time 3-4 minutes, or by the Zn-DTPA solution of 5ml in 5% glucose solution, woods grignard
The administration of 100-250ml venous transfusion is diluted in lactic acid solution, physiological saline；If subject merely due to inhalation route and get dirty
Dye, the interior Zn-DTPA for giving atomization can be used as therapy approach use for 24 hours.
By establishing the method for the present invention, animal (such as rat, people etc.) is being given afterwards in vivo for the metallo-chelate of DTPA
The assay of these substances provides possibility in biological sample (such as blood, urine, saliva etc.).The analysis that the present invention establishes
Method has excellent methodology performance.
Detailed description of the invention
The second level full scan mass spectrogram of Fig. 1: DTPA-Zn and internal standard EDTA-Zn；In figure, (A) DTPA-Zn, (B) EDTA-
Zn。
The MRM chromatogram of Fig. 2: DTPA-Zn and EDTA-Zn；Wherein, A: blank diaper, B: mark-on blank diaper, C: administration
Urine sample；In figure, I, II: DTPA-Zn, III: internal standard are represented.
Fig. 3: the chromatogram of DTPA-Zn and internal standard EDTA-Zn cross jamming in urine are investigated；In figure, (A) blank diaper mark
Quasi- addition DTPA-Zn is 2000 μ g/mL to concentration, and (B) blank diaper standard addition internal standard EDTA-Zn to concentration is 200 μ g/
mL；Wherein, DTPA-Zn, III: internal standard I, II: are represented.
Fig. 4: LC-MS/MS method measures DTPA-Zn concentration standard curve citing in urine.
Specific embodiment
Following embodiment provided by the present invention is only used for task of explanation rather than is used for, and is also not necessarily to be construed as with any
Mode limits the present invention.Those skilled in the art will recognize that not past the spirit or scope of the present invention
Conventional change and modification can be made to following embodiment.
It is an object of the present invention to more fully understand the disposition of DTPA-Zn rule, the present invention, which tests, to be established simultaneously
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method of the content of DTPA-Zn in quantitative detection biological sample is demonstrated, is investigated simultaneously
DTPA-Zn is in the intracorporal pharmacokinetics behavior of people.
1 test is summarized
Zn-DTPA (diethyl pentetic acid trisodium zinc salt, DTPA-Zn) is clinically as a kind of alleviation radiation chelating
Agent is used to make a definite diagnosis or the doubtful treatment by plutonium, americium, hard iron pollution subject, and improves the clearance rate of these metallic elements.This examination
Testing purpose is to evaluate Zn-DTPA injection in the intracorporal pharmacokinetics of health volunteer by DTPA-Zn concentration in measurement human urine
Behavior.This report describes analysis side liquid chromatography-tandem mass spectrometry (LC-MS/MS) of DTPA-Zn in measurement human urine sample
Method.
2 experimental materials
2.1 drugs, reagent and material
2.2 solution
2.2.1 the preparation of DTPA-Zn stock solution and standard serial solution
Zn-DTPA injection 1 (500mg/5mL) is taken as DTPA-Zn stock solution (A1), be transferred in tool plug test tube-
20 DEG C of refrigerator freezings save.
DTPA-Zn standard serial solution is prepared by the following method: DTPA-Zn stock solution (A1) is diluted to water as shown in table 1
The standard serial solution of following concentration, and saved backup in 4 DEG C of refrigerators.
The preparation (water dissolution and constant volume) of table 1:DTPA-Zn standard serial solution
| Solution numbers | Preparation method | Zn-DTPA concentration (μ g/mL) |
| A1 | 1.0×105 | |
| A2 | A1 solution, 100 μ L → 1mL | 1.0×104 |
| A3 | A1 solution, 50 μ L → 1mL | 5.0×103 |
| A4 | A1 solution, 25 μ L → 1mL | 2.5×103 |
| A5 | A2 solution, 100 μ L → 1mL | 103 |
| A6 | A3 solution, 100 μ L → 1mL | 500 |
| A7 | A4 solution, 100 μ L → 1mL | 250 |
| A8 | A5 solution, 100 μ L → 1mL | 100 |
| A9 | A6 solution, 100 μ L → 1mL | 50 |
2.2.2 the preparation of DTPA-Zn quality-control sample working solution
DTPA-Zn stock solution is separately prepared by the following method: taking 1 Zn-DTPA injection (500mg/5mL) as stock solution
(B1), -20 DEG C of refrigerator freezings in tool plug test tube are transferred to save.
DTPA-Zn quality-control sample working solution is prepared by the following method: by DTPA-Zn stock solution (B1) with water by dilute shown in table 2
It is interpreted into the quality-control sample working solution of following concentration, and is saved backup in 4 DEG C of refrigerators.
The preparation (water dissolution and constant volume) of table 2:DTPA-Zn Quality control samples working solution
| Solution numbers | Preparation method | DTPA-Zn concentration (μ g/mL) |
| B1 | 1.0×105 | |
| B2 | B1 solution, 100 μ L → 1mL | 1.0×104 |
| B3 | B1 solution, 90 μ L → 1mL | 9.0×103 |
| B4 | B2 solution, 100 μ L → 1mL | 1.0×103 |
| B5 | B3 solution, 100 μ L → 1mL | 900 |
| B6 | B4 solution, 100 μ L → 1mL | 100 |
| B7 | B4 solution, 50 μ L → 1mL | 50 |
2.2.3 the preparation of internal standard working solution
Precision weighs EDTA-Zn (internal standard) 20.0mg in 2mL measuring bottle, and with water dissolution and constant volume, being made into concentration is
The stock solution (IS1) of 10.0mg/mL, -20 DEG C of refrigerator freezings save.
Precision draws stock solution (IS1) 1250 μ L and, into 25mL measuring bottle, is settled to scale with water, obtains the internal standard of 500 μ g/mL
Working solution (IS2), and saved backup in 4 DEG C of refrigerators.
2.2.4 the preparation of 1% ammonium hydroxide methanol solution
Measure ammonium hydroxide 10.0mL, methanol 990.0mL is mixed in conical flask to obtain the final product.
3 instruments and experimental condition
3.1 LC-MS/MS conditions
Chromatographic condition: analytical column be SHISEIDO Proteonavi column (5 μm, 250 × 4.6mm I.D., Japan
SHISEIDO company), C18Guard column (4 × 3.0mm I.D., Phenomenex company, the U.S.), mobile phase are methanol -2mM formic acid
Ammonium (containing 0.03% ammonium hydroxide) (50:50, v/v), flow velocity 0.45mL/min.
Mass Spectrometry Conditions: electro-spray ionization source, negative-ion mode detection；Injection electric is -4000V；Source temperature is 500
℃；Roller shutter gas (CUR) is 20；Collision gas (CAD) is 4；Scanning mode is multiple-reaction monitoring (MRM), for quantitative analysis from
Son reaction is respectively 226.6 → m/z of m/z454.2 → m/z 364.3+m/z 204.5 (DTPA-Zn, CE 45V) and m/z
353.0 → m/z 263.0 (ethylenediamine tetraacetic acid disodium zinc salt salt, CE 15V), sweep time 150msec.
3.2 key instrument
Liquid chromatograph-mass spectrometer (LC-MS/MS): 4000 triple quadrupole bar tandem mass spectrometer (American AB of API
Sciex company), it is furnished with 1.5 data processing system of Turbo Ionspray ionization source and Analyst, liquid chromatographic system
1200 binary gradient of Agilent pump and CTC autosampler.BT224S type precise electronic assay balance (German Sartorius
Company)；DMT-2500 type multitube eddy mixer (Hangzhou meter Ou Instrument Ltd.)；BF-2000 type is dried with nitrogen instrument (Beijing
All directions Centrix Technology Ltd.).
3.3 urine sample processing methods
3.3.1 the preparation of blank diaper sample
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 0.01% ammonium hydroxide 920 μ L, vortex mixing 3min is added,
All it is splined on solid phase extraction column.It is first activated with 2mL methanol before solid phase extraction column loading, then with 2mL water balance.Loading
It is cleaned with 2mL water, is finally eluted with 1% ammonium hydroxide methanol solution of 0.5mL afterwards.Eluent is collected, 10 μ L is drawn and is filled with 190 μ L water
Divide and mix, 10 μ L is taken to carry out LC-MS/MS analysis.
3.3.2 the preparation of zero point urine sample
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 20 μ L of inner mark solution, 0.01% ammonium hydroxide, 900 μ L is added,
Vortex mixing 3min, is all splined on solid phase extraction column.Remaining is operated with " 3.3.1 ".
3.3.3 the preparation of standard curve
100 μ L of DTPA-Zn standard serial solution (A2~A9 solution) is taken, is placed in 1.5mL centrifuge tube, blank human urine is added
Liquid, vortex 3min, preparation are equivalent to the urine sample that concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL.It takes
50 μ L of urine sample, and 20 μ L of inner mark solution, 0.01% ammonium hydroxide 900 μ L, vortex mixing 3min are sequentially added, it is all splined on solid
Mutually on extraction pillar.Remaining is operated with " 3.3.1 ".
3.3.4 the preparation of quality-control sample
250 μ L of DTPA-Zn quality-control sample working solution (B3, B5, B6) is taken, is placed in 1.5mL centrifuge tube, blank people is added
Urine, vortex 3min, preparation are equivalent to the urine sample that concentration is 20,180 and 1800 μ g/mL.Take 50 μ L of urine sample, and according to
Secondary 20 μ L of addition inner mark solution, 0.01% ammonium hydroxide 900 μ L, vortex mixing 3min are all splined on solid phase extraction column.Remaining
It operates with " 3.3.1 ".
3.3.5 the preparation of minimum lower limit of quantitation sample (LLOQ)
250 μ L of DTPA-Zn quality-control sample working solution (B7) is taken, is added in blank human urine, 10.0 μ g/mL's of preparation
LLOQ sample.Remaining step is pressed to be operated under " preparation of 3.3.4 quality-control sample " item, and carries out LC-MS/MS analysis.
3.3.6 the preparation of cross jamming test specimen
40 μ L of DTPA-Zn working solution (A2 solution) is taken, is placed in 1.5mL centrifuge tube, blank human urine is added, is vortexed
3min is prepared into the ULOQ sample of 2000 μ g/mL not containing the internal standard.Remaining step presses " preparation of 3.3.4 quality-control sample " Xiang Xiacao
Make, and carries out LC-MS/MS analysis.
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, zero point urine sample is prepared, presses " 3.3.2 zero point urine
It is operated under the preparation of sample " item.
3.3.7 the preparation of extraction recovery sample
The 50 μ L of blank human urine for taking 6 kinds of separate sources, is placed in 1.5mL centrifuge tube, and 0.01% ammonium hydroxide, 920 μ L is added,
Vortex mixing 3min, is all splined on solid phase extraction column.It is first activated, then used with 2mL methanol before solid phase extraction column loading
2mL water balance.It is cleaned after loading with 2mL water, is finally eluted with 1% ammonium hydroxide methanol solution of 0.5mL.Eluent is collected, draws 10
μ L and the mixed standard solution of 190 μ L respective concentrations mix well, and 10 μ l is taken to carry out LC-MS/MS analysis.
3.3.8 the preparation of matrix effect test specimen
20 μ L of 10 μ L of DTPA-Zn quality-control sample working solution (B3, B5, B6) and inner mark solution (IS2) is taken, 1.5mL is placed in
In centrifuge tube, 40 DEG C of nitrogen stream dryings are configured to the mixed standard solution of respective concentration with water, draw 190 μ L and 10 μ L 1%
After ammonium hydroxide methanol solution mixes, 10 μ L is taken to carry out LC-MS/MS analysis.Each concentration carries out 6 sample analyses, obtains corresponding peak
Area.
3.3.9 the preparation of urine sample stability test sample
100 μ L of quality-control sample working solution (B3, B6) is taken, is placed in 1.5mL centrifuge tube, blank human urine is added, is vortexed
3min is prepared into the urine sample of high and low two concentration of DTPA-Zn, takes 50 μ L urine samples sample after being placed at room temperature for 4h, processing
Product be placed at room temperature for for 24 hours, -80 DEG C, -20 DEG C place 26 days, after experience 3 freeze-thaws circulation, each concentration carries out 4 samples point
Analysis.
3.3.10 the preparation of unknown sample
50 μ L of urine sample is taken, is placed in 1.5mL centrifuge tube, 20 μ L of internal standard working solution (IS2) is sequentially added, is added
0.01% ammonium hydroxide 900 μ L, vortex mixing 3min are all splined on solid phase extraction column.Remaining is operated with " 3.3.1 ".
The preparation of 3.4 stock solutions and working solution stability sample
3.4.1 the preparation of DTPA-Zn stock solution stability sample
The same day is measured by under " preparation of 2.2.1DTPA-Zn stock solution and standard serial solution " item, 1 Zn-DTPA is taken to infuse
Agent is penetrated to be transferred in tool plug test tube as stock solution A1.Precision pipettes fresh 50 μ L of DTPA-Zn stock solution (A1) to 1.0mL amount
In bottle, constant volume is diluted with water and obtains A3 solution (5.0 × 103μg/mL)；Precision pipettes (A3) 100 μ L and, into 1.0mL measuring bottle, uses water
Dilution constant volume obtains A6 solution (500 μ g/mL).
Take freshly prepared 10 μ L of A6 solution, be placed in 1.5mL centrifuge tube, be added 490 μ L water mix after, draw 10 μ L with
190 μ L water mix well, and 10 μ L is taken to carry out LC-MS/MS analysis.
The preparation of 56 days stability samples of -20 DEG C of DTPA-Zn stock solution placements:
Precision pipettes DTPA-Zn stock solution (A1, -20 DEG C are placed 56 days) 50 μ L and, into 1.0mL measuring bottle, constant volume is diluted with water
Obtain A3 solution (5.0 × 103μg/mL)；Precision pipettes (A3) 100 μ L into 1.0mL measuring bottle, and constant volume is diluted with water and obtains A6 solution
(500μg/mL)。
It is placed in 10 μ L of A6 solution in 1.5mL centrifuge tube, after the mixing of 490 μ L water is added, draws 10 μ L and filled with 190 μ L water
Divide and mix, 10 μ L is taken to carry out LC-MS/MS analysis.
DTPA-Zn stock solution is placed at room temperature for the preparation of 4h stability sample:
Precision pipettes the same day fresh 50 μ L of DTPA-Zn stock solution (A1) into 1.0mL measuring bottle, after being placed at room temperature for 4h,
Constant volume is diluted with water and obtains A3 solution (5.0 × 103μg/mL)；Precision pipettes (A3) 100 μ L into 1.0mL measuring bottle, and it is fixed to be diluted with water
Hold to obtain A6-1 solution (500 μ g/mL).
10 μ L of A6-1 solution is taken, is placed in 1.5mL centrifuge tube, after the mixing of 490 μ L water is added, draws 10 μ L and 190 μ L water
It mixes well, 10 μ L is taken to carry out LC-MS/MS analysis.
3.4.2 the preparation of DTPA-Zn standard working solution stability sample
The same day is measured by under " preparation of 2.2.2 quality-control sample working solution " item, fresh preparation DTPA-Zn Quality Control work is molten
Liquid (B3, B6).
Freshly prepared 10 μ L of B3, B6 solution is taken, is placed in 1.5mL centrifuge tube, after the mixing of 490 μ L water is added, draws 10 μ
L is mixed well with 190 μ L water, and 10 μ L is taken to carry out LC-MS/MS analysis.
The preparation of 56 days stability samples of -20 DEG C of DTPA-Zn standard working solution placements:
The 10 μ L of B3, B6 solution for taking -20 DEG C of preservations, is placed in 1.5mL centrifuge tube, after the mixing of 490 μ L water is added, draws 10
μ L is mixed well with 190 μ L water, and 10 μ L is taken to carry out LC-MS/MS analysis.
DTPA-Zn standard working solution is placed at room temperature for the preparation of 4h stability sample:
Precision pipettes the same day freshly prepared 10 μ L of B3, B6 solution, is placed in 1.5mL centrifuge tube, after being placed at room temperature for 4h,
After the mixing of 490 μ L water is added, draws 10 μ L and mixed well with 190 μ L water, 10 μ L is taken to carry out LC-MS/MS analysis.
3.4.3 the preparation of internal standard stock solution and internal standard working solution stability sample
The same day is measured by under " preparation of 2.2.3 internal standard working solution " item, precision pipettes freshly prepared internal standard stock solution
(IS1) 50 μ L are into 1mL measuring bottle, are diluted with water and are settled to scale and obtain IS2 solution (500 μ g/mL).
Freshly prepared 20 μ L of IS2 solution is taken, is placed in 1.5mL centrifuge tube, after the mixing of 480 μ L water is added, draws 10 μ L
It is mixed well with 190 μ L water, 10 μ L is taken to carry out LC-MS/MS analysis.
The preparation of 57 days stability samples of -20 DEG C of internal standard stock solution placements:
Precision is pipetted, into 1mL measuring bottle, to be used in -20 DEG C of internal standard stock solutions saved (IS1, -20 DEG C are placed 57 days) 50 μ L
Water dilution is settled to scale and obtains IS2 solution (500 μ g/mL).
20 μ L of IS2 solution is taken, is placed in 1.5mL centrifuge tube, after the mixing of 480 μ L water is added, 10 μ L is drawn and is filled with 190 μ L water
Divide and mix, 10 μ L is taken to carry out LC-MS/MS analysis.
Internal standard stock solution is placed at room temperature for the preparation of 4h stability sample:
Precision pipettes 50 μ L of the same day freshly prepared (IS1) into 1mL measuring bottle, and after being placed at room temperature for 4h, it is fixed to be diluted with water
Hold to scale and obtains IS2-1 solution (500 μ g/mL).
20 μ L of IS2-1 solution is taken, is placed in 1.5mL centrifuge tube, after the mixing of 480 μ L water is added, draws 10 μ L and 190 μ L water
It mixes well, 10 μ L is taken to carry out LC-MS/MS analysis.
The preparation of 57 days stability samples of -20 DEG C of internal standard working solution placements:
IS2 solution (- 20 DEG C place 57 days) the 20 μ L for taking -20 DEG C of preservations are added after 480 μ L water mix, draw 10 μ L with
190 μ L water mix well, and 10 μ L is taken to carry out LC-MS/MS analysis.
Internal standard working solution is placed at room temperature for the preparation of 4h stability sample:
Freshly prepared 20 μ L of IS2 solution is taken, is placed in 1.5mL centrifuge tube, after being placed at room temperature for 4h, 480 μ L water are added
After mixing, draws 10 μ L and mixed well with 190 μ L water, 10 μ L is taken to carry out LC-MS/MS analysis.
3.5 data process&analysis
Institute's measured data is analyzed using Winnolin (Phoenix 6.3, the U.S.) pharmacokinetics software, obtain with
The relevant kinetic parameter C of drug exposuremax、Tmax、AUC0-tAnd MRT0-t。
4 test results
The verifying of 4.1 LC-MS/MS quantitation methodologies
4.1.1 method choice
The second level full scan mass spectrogram of determinand is shown in Fig. 1.
The 50 μ L of blank human urine for taking 6 kinds of separate sources, is placed in 1.5mL centrifuge tube, presses " 3.3.1 blank diaper sample
Preparation " operate under item, carry out LC-MS/MS analysis, the chromatogram of blank diaper sample obtained, such as attached drawing 2A；By a certain concentration
Standard solution be added in blank diaper, take 50 μ L of urine sample, by operating under " preparation of 3.3.3 standard curve " item, obtain
Chromatogram, such as attached drawing 2B, wherein the retention time of DTPA-Zn is about 4.67min, and the retention time of internal standard EDTA-Zn is about
5.02min；The 50 μ L of urine sample acquired after medicine is drawn, by operating under " preparation of 3.3.10 unknown sample " item, such as attached drawing 2C.
The result shows that endogenous material does not interfere determinand and interior target to measure in blank diaper.
4.1.2 the cross jamming test of method
By operating under " preparation of 3.3.6 cross jamming test specimen " item, zero is carried out after the ULOQ sample of not containing the internal standard
Point urine sample analysis, obtains attached drawing 3.The result shows that without cross jamming between DTPA-Zn and internal standard.
4.1.3 standard curve
Using testing concentration as abscissa, the peak area ratio of determinand and internal standard compound is ordinate, with weighting (W=1/
X2) least square method progress regressing calculation, the linear regression equation of DTPA-Zn is acquired, standard curve is shown in Fig. 4.Determinand is 10
In the range of~2000 μ g/mL, there is good linear relationship, (r between concentration and peak area ratio2>0.99)。
4.1.4 the precision and accuracy of method
By being operated under " preparation of the minimum lower limit of quantitation sample (LLOQ) of 3.3.5 " and " preparation of 3.3.4 quality-control sample " item,
Prepare the minimum lower limit of quantitation of DTPA-Zn (LLOQ, concentration are 10 μ g/mL) sample and basic, normal, high 3 concentration (respectively 20,180
With the QC sample of 1800 μ g/mL), each concentration carries out 6 sample analyses, METHOD FOR CONTINUOUS DETERMINATION three days, calculates the survey of LLOQ and QC sample
Concentration, and prepare concentrations control, acquire the veracity and precision of this law, the result of 3 analyses batch is subjected to variance analysis
It obtains preci-sion and accuracy (being shown in Table 3).The results show that the preci-sion and accuracy of method meets biological sample quantitative analysis
It is required that.
The veracity and precision (n=18) of table 3:DTPA-Zn
4.1.5 extraction recovery and matrix effect
By being operated under " preparation of 3.3.4 quality-control sample " and " preparation of 3.3.7 extraction recovery sample " item.Prepare 3
The human urine QC sample of concentration, each concentration carries out 6 sample analyses, and takes the blank human urine of 6 kinds of separate sources, prepares and extracts
Rate of recovery sample, the peak area of quality-control sample obtain extraction recovery (being shown in Table 4) compared with the peak area of extraction recovery sample.
By being operated under " preparation of 3.3.7 extraction recovery sample " and " preparation of 3.3.8 matrix effect sample " item.Take 6
The blank human urine of kind separate sources, prepares extraction recovery sample；Take DTPA-Zn quality-control sample working solution (B3, B4, B7)
20 μ L of 10 μ L and inner mark solution (IS2), prepares matrix effect sample, and each concentration carries out 6 sample analyses, obtains corresponding peak face
Product.The peak area of extraction recovery sample obtains matrix effect (being shown in Table 5) compared with the peak area of matrix effect sample, experiment knot
Fruit shows that DTPA-Zn matrix effect is between 85%~115% in human urine, and urine matrix does not show the measurement of determinand
The influence of work.
The extraction recovery (n=6) of table 4:DTPA-Zn
| It indicates concentration (μ g/mL) | The rate of recovery (Mean ± SD) (%) | RSD (%) |
| 20 | 91.5±4.7 | 5.2 |
| 180 | 93.5±3.3 | 3.5 |
| 1800 | 90.1±3.1 | 3.4 |
The matrix effect (n=6) of table 5:DTPA-Zn
4.1.6 urine sample study on the stability
Stability of the DTPA-Zn urine sample under various experiment conditions is investigated.The result shows that DTPA-Zn urine sample
The RSD of product stability is respectively less than 6.1%, and relative deviation RE (is shown in Table 6) in the range of 3.1~6.4%, the stability symbol of the two
This test requirements document is closed, detailed data sees attached list 5.
Table 6: the stability (n=4) of DTPA-Zn in urine
4.1.7 stock solution and working solution study on the stability
By operating under " preparation of 2.5.1DTPA-Zn stock solution stability sample " item, DTPA-Zn stock solution, work have been investigated
Make solution, internal standard stock solution, working solution be placed at room temperature for, the stability of -20 DEG C of placements.It is calculated and is laid in using one point external standard method
The measured concentration of liquid and working solution calculates it RE (%) compared with theoretical value；Stock solution and working solution room are calculated simultaneously
Temperature places the RSD (%) of both front and back solution peak area.
The result shows that: determinand (DTPA-Zn) stock solution, working solution are placed at room temperature for 56 days 4h, -20 DEG C of placements stabilizations.
Internal standard (EDTA-Zn) stock solution, working solution are placed at room temperature for 57 days 4h, -20 DEG C of placements stabilizations.It the results are shown in Table 7.
Table 7:DTPA-Zn, internal standard stock solution and working solution stability (n=6)
4.1.8 the residual of method is investigated
Prepare the urine sample and blank diaper sample of DTPA-Zn highest quantitative limit ULOQ (2000 μ g/mL).
In the laggard line blank urine sample analysis of ULOQ sample.Compare blank diaper sample and the chromatogram of LLOQ, blank
The 20% of LLOQ is not to be exceeded in determinand response in urine sample.DTPA-Zn and interior target signal are rung in blank diaper sample
Answering intensity is respectively the 6.2% and 0.3% of LLOQ.The result shows that ULOQ sample is to blank diaper sample without significantly interfering with.
4.2 unknown urine sample measurements are controlled with quality
Test urine sample measurement is pressed to be operated under " the unknown urine sample preparation of 3.3.10 " item, and with the standard curve on the same day
The concentration for calculating DTPA-Zn in each time point sample, the choice of same day data is determined by QC sample.It is required that the QC of each analysis batch
In 85%~115% range of the accuracy of sample, there can be 1/3 concentration point to transfinite, but same concentration at least 50% accords with
Standardization.
The pharmacokinetics of 5 DTPA-Zn
12 health volunteer's intravenous drips give Zn-DTPA injection (single intravenous infusion 250mg, single intravenous infusion 500mg,
Single intravenous infusion 1000mg, multiple intravenous infusion 500mg), urine is taken in different time points, measures DTPA-Zn drug concentration in urine, and
Mean drug concentration-time graph is drawn, the results show that coincide substantially with pharmaceutical concentration-time curve when dose, different agent
Pharmaceutical concentration-time curve variation tendency is identical when amount, and related pharmacokinetic parameters are identical.
6 conclusions
This test measures the concentration of DTPA-Zn in human urine sample using LC-MS/MS method.Urine sample is through Solid Phase Extraction
After method processing, using the source ESI, quantitative analysis is carried out with MRM scanning mode, the endogenous material in urine does not interfere DTPA-
The measurement of Zn.This method is accurate, selectivity is strong, high sensitivity, and meets " the chemicals medicine human-body biological of CFDA promulgation
Availability and bioequivalence investigative technique guideline " ([H] GCL2-1) is related to be required, and is suitble to Zn-DTPA in human urine
Pharmacokinetic study.
Spirit of the invention is elaborated above by present pre-ferred embodiments.Those skilled in the art's reason
Solution, all any modification, equivalent variations and modification to the above embodiments according to the technical essence of the invention, all falls within this hair
In bright protection scope.
Claims (10)
1. the method for measuring the DTPA-Zn in human urine biological sample, this method are passed through using liquid chromatography-tandem mass spectrometry
The content of DTPA-Zn in the human urine biological sample of processing, includes the following steps:
(1) processing of biological sample:
(1a) takes the 50 μ L of human urine for containing or not contain DTPA-Zn, is placed in 1.5mL centrifuge tube, adds inner mark solution i.e. 25 μ g/
Inner mark solution is not added in the 20 μ L of EDTA-Zn aqueous solution of mL, and 0.01% ammonium hydroxide, 900 μ L is added；The addition when internal standard is not added
0.01% ammonium hydroxide, 920 μ L makes urine, ammonium hydroxide, optional 970 μ L of inner mark solution total volume；Vortex mixing 3min, whole loadings
In on solid phase extraction column；
It is cleaned after (1b) loading with 2mL water, finally the methanol solution with 0.5mL containing 1% ammonium hydroxide elutes；
(1c) collects the eluent, and drying, residue is redissolved with 150 μ L water, draws 10 μ L and carries out LC-MS/MS analysis；
The preparation of (1d) standard curve: the 100 μ L of DTPA-Zn standard serial solution of various concentration is taken, 1.5mL centrifuge tube is placed in
In, blank human urine, vortex 3min is added, it is 10,20,50,100,200,500,1000 and 2000 μ g/ that preparation, which is equivalent to concentration,
The urine sample of mL；50 μ L of the urine sample is taken, and sequentially adds 20 μ L of inner mark solution, 0.01% ammonium hydroxide, 900 μ L, vortex mixing
3min is all splined on solid phase extraction column, then according to above (1b) and (1c) operation, is analyzed result according to LC-MS/MS and is drawn
Standard curve processed, the standard curve are used to calculate the target substance content in various samples；
(2) liquid chromatography tandom mass spectrometry determination:
(2a) provides high performance liquid chromatograph and tandem mass spectrometer, and the high performance liquid chromatograph is equipped with gradient pump and sample injector,
The tandem mass spectrometer is equipped with electric spray ion source and data processing system；
(2b) chromatographic condition: analytical column used is the chromatographic column of SHISEIDO Proteonavi brand, and specification is 5 μm,
250 × 4.6mm I.D. connects C before column18Guard column, specification are 4 × 3.0mm I.D., and mobile phase is volume ratio 50:50's
Methanol -2mM ammonium formate contains 0.03% ammonium hydroxide, flow velocity 0.45mL/min in mobile phase；
(2c) Mass Spectrometry Conditions: electro-spray ionization source uses the source Turbo Ionspray, negative-ion mode detection；Injection electric
For -4000V；Source temperature is 500 DEG C；Roller shutter gas is 20；Collision gas is 4；Scanning mode is multiple-reaction monitoring, is used for quantitative analysis
Ionic reaction be respectively as follows: the 226.6 → m/z of m/z 454.2 → m/z 364.3+m/z 204.5 and its CE of DTPA-Zn and be
353.0 → the m/z of m/z 263.0 and its CE of 45V and ethylenediamine tetraacetic acid disodium zinc salt salt are 15V, sweep time 150msec；
(3) data process&analysis
(3a) obtains the target substance content in biological sample by above-mentioned liquid chromatography tandom mass spectrometry determination；With, optional
(3b) analyzes institute's measured data using the 6.3 pharmacokinetics software of Phoenix of U.S. Winnolin, obtain with
Drug exposure is relevant to be selected from following one or more kinetic parameter Cmax、Tmax、AUC0-tAnd MRT0-t。
2. according to the method described in claim 1, wherein the human urine is never to give the people of the Zn-DTPA urine work obtained
For blank diaper sample, or from people give Zn-DTPA after the urine that obtains of different time as drug containing urine sample.
3. according to the method described in claim 1, wherein optional being placed in -20 DEG C of refrigerators of human urine obtained freezes,
With etc. it is to be determined.
4. according to the method described in claim 1, being designated as EDTA-Zn Na in used in it2·xH2O。
5. according to the method described in claim 1, wherein the solid phase extraction column before loading, first with 1mL methanol activate, then
With 1mL water balance；Alternatively, first being activated with 2mL methanol, then with 2mL water balance.
6. according to the method described in claim 1, wherein the high performance liquid chromatograph is Agilent company of the U.S. Agilent
1200 type liquid chromatographs.
7. according to the method described in claim 1, wherein the tandem mass spectrometer is 4000 type of American AB Sciex company API
Tandem mass spectrometer is furnished with 1.5 data processing system of Turbo Ionspray ionization source and Analyst.
8. according to the method described in claim 1, wherein the solid phase extraction column is modelThe Solid Phase Extraction of WAX
Pillar.
9. according to the method described in claim 1, wherein further including one or more operations that following solution is prepared:
The preparation of (10a) DTPA-Zn stock solution: the Zn-DTPA injection that concentration is 100mg/mL is taken to lay in as DTPA-Zn
Liquid is denoted as A1, is transferred in tool plug test tube, and -20 DEG C of refrigerator freezings save, spare；
The preparation of (10b) DTPA-Zn standard serial solution: DTPA-Zn stock solution, that is, A1 is diluted with water, following DTPA-Zn is made
The standard serial solution of concentration, and saved backup in 4 DEG C of refrigerators: 1.0 × 105μg/ml、1.0×104μg/ml、5.0×103μ
g/ml、2.5×103μg/ml、103μg/ml,500μg/ml,250μg/ml,100μg/ml,50μg/ml；
The preparation of (10c) DTPA-Zn quality-control sample working solution: DTPA-Zn stock solution, that is, A1 is diluted with water be made it is following
The quality-control sample working solution of DTPA-Zn concentration, and saved backup in 4 DEG C of refrigerators: 1.0 × 105μg/ml、1.0×104μg/
ml、9.0×103μg/ml、1.0×103μg/ml,900μg/ml,100μg/ml,50μg/ml；
The preparation of (10d) internal standard working solution: precision weighs 20.0mg internal standard substance EDTA-Zn in 2mL measuring bottle, is dissolved with water
And constant volume, it is made into the internal standard stock solution that concentration is 10.0mg/mL, is denoted as IS1, -20 DEG C of refrigerator freezings save, spare；
Precision draws 1250 μ L of internal standard stock solution into 25mL measuring bottle, is settled to scale with water, obtains the internal standard work of 500 μ g/mL
Solution is denoted as IS2, and saves backup in 4 DEG C of refrigerators, and as inner mark solution, concentration is 500 μ g/ml；
The preparation of (10e) 1% ammonium hydroxide methanol solution: measuring ammonium hydroxide 10.0mL, and methanol 990.0mL is mixed in conical flask to obtain the final product.
10. according to the method described in claim 1, wherein further including one or more operations of following plasma sample processing:
The preparation of (20a) blank diaper sample:
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 0.01% ammonium hydroxide 920 μ L, vortex mixing 3min is added, all
It is splined on solid phase extraction column；
It is cleaned after loading with 2mL water, is finally eluted with the methanol solution 0.5mL containing 1% ammonium hydroxide；
Eluent is collected, 10 μ L is drawn and is mixed well with 190 μ L water, obtains the solution for carrying out LC-MS/MS analysis measurement；
The preparation of (20b) zero point urine sample:
50 μ L of blank human urine is taken, is placed in 1.5mL centrifuge tube, 20 μ L of inner mark solution, 0.01% ammonium hydroxide, 900 μ L, vortex is added
3min is mixed, is all splined on solid phase extraction column；Then as " preparation of (20a) blank diaper sample " described progress is subsequent
Operation；
The preparation of (20c) standard curve:
The 100 μ L of DTPA-Zn standard serial solution for taking various concentration, is placed in 1.5mL centrifuge tube, and blank human urine, whirlpool is added
Revolve 3min, the urine sample that preparation concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL；Take 50 μ of urine sample
L, and sequentially add 20 μ L of inner mark solution, 0.01% ammonium hydroxide 900 μ L, vortex mixing 3min are all splined on solid phase extraction column
On；Then such as " preparation of (20a) blank diaper sample " described carry out subsequent operation；
The preparation of (20d) quality-control sample
It takes DTPA-Zn quality-control sample working solution to be placed in 1.5mL centrifuge tube, blank human urine is added, vortex 3min is prepared dense
Degree is the urine sample of 20,180 and 1800 μ g/mL, takes 50 μ L of urine sample, and sequentially add 20 μ L of inner mark solution, 0.01%
Ammonium hydroxide 900 μ L, vortex mixing 3min are all splined on solid phase extraction column；Then such as the " system of (20a) blank diaper sample
It is standby " the carry out subsequent operation；
The preparation of (20e) minimum lower limit of quantitation sample (LLOQ)
It takes DTPA-Zn quality-control sample working solution to add in blank human urine, is prepared into the LLOQ sample of 10.0 μ g/mL；Remaining
By operating under " preparation of (20d) quality-control sample " item, and carry out LC-MS/MS analysis；
The preparation of (20f) cross jamming test specimen
40 μ L of DTPA-Zn working solution is taken, is placed in 1.5mL centrifuge tube, blank human urine is added, vortex 3min is prepared into
The ULOQ sample of 2000 μ g/mL not containing the internal standard；In addition to internal standard is not added, remaining is pressed operates under " preparation of (20d) quality-control sample " item,
And carry out LC-MS/MS analysis.
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