CN107167540A - The method for determining the DTPA Zn in human urine biological sample - Google Patents
The method for determining 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
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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 method for determining the DTPA Zn in human urine biological sample.Specifically, the present invention relates to the method for determining the DTPA Zn in human urine biological sample, this method is determined the content of the DTPA Zn in the human urine biological sample through processing using liquid chromatography tandem mass spectrometry, comprised the following steps：(1) processing of biological sample, (2) Liquid Chromatography-Tandem Mass Spectrometry is determined, (3) data process＆analysis.The inventive method 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, be related to a kind of reliable method to determine organism such as people, it is big
Mouse give biological sample after above-mentioned DTPA- metal-chelators such as blood, urine in above-mentioned metal-chelator content, to assess
The therapeutic effect of these metal-chelators or its behavior in vivo.
Especially, the present invention relates to a kind of method for determining the DTPA-Zn in human urine biological sample.
Background technology
The harm of lead contamination and lead poisoning to health has been received significant attention.Lead is human body non-essential element,
Absorption of human body is mainly entered by respiratory tract, alimentary canal, can be accumulated in vivo, it is toxic to each system of whole body and organ to make
With, main 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 used 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 complexing discharge internal zinc, calcium, manganese, iron, copper etc., can cause internal essential trace element
, there is toxic side effect in dysequilibrium, and most important toxic side effect is renal damage caused by zinc missing.
Ca-DTPA (DTPA-Ca can be denoted as not only) also known as Ca DTPA salt and Zn-DTPA (but also can
It is denoted as DTPA-Zn) also known as diethylene-triamine pentaacetic acid trisodium zinc salt, the two belongs to complexones together, in August, 2004 by U.S.
State FDA ratifies to list simultaneously, is stained with for entering the work of radionuclide severe contamination place 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 optionally with radionuclide lanthanum (140La), cerium (144Ce), promethium (147Pm), americium (124Am),
The soluble complexes of the cation formation stability of plutonium (239Pu) etc., are excreted through kidney, so as to reduce in vivo quickly
The deposition of radioactive substance.Because Ca2+ with DTPA complexation constant is less than Zn2+ [Xue HL.The chelator
Treatment of common metal intoxication [J] .Chem Ind Occup Saf Health (work by chemical industry
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 missing, so that the work of the enzyme relevant with zinc
Property influenceed by serious, including DNA and RNA polymerase, carboxypeptidase, carbonic anhydrase etc., liver kidney, intestinal mucosa can be caused
Adverse reaction [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 Medicines point
Volume)], 1980,4:211-215].Zn-DTPA security is higher, though the missing of the trace element such as manganese and magnesium can be also caused,
Zinc-deficiency will not be caused and serious toxic side effect is produced, 1/10~1/30 [the Zhang ZY, Xu that its toxicity is about Ca-DTPA
XW, Zhu Z.Pentetate zinc trisodium [J] .Chin Pharm J (Chinese Pharmaceutical Journal), 2007,42:557-
558]。
Existing document determines lead content using sampling Graphite Furnace Atomic Absorption spectrophotometer method, has investigated Zn-DTPA to chronic
Decorporation effect [Yang S, Chen L, Yin YY, the et al.Study of the effect of of mice with lead poisoning
pentetate zinc trisodium on excretion of lead in lead poisoned mice[J].Pharm
J Chin PLA (PLA's Acta Pharmaceutica Sinica), 2011,27:147-149], but sampling Graphite Furnace Atomic Absorption spectrophotometer method is surveyed
Determine that method is complicated, cost is high, sensitivity is low, be difficult to meet to determine for the amount in the biological sample such as blood, urine and require.
In addition, also document [Chen Li, etc. decorporation effects of the ICP-MS methods analysis Zn-DTPA to mice with lead poisoning, pharmacy
Journal (Acta Pharmaceutica Sinica) 2014,49 (11):1588-1592] report, using inductance coupled plasma
Constitution spectrum (ICP-MS) determines the concentration of lead in biological specimen, investigates decorporations of the nucleic decorporation medicine Zn-DTPA to mice with lead poisoning
Effect.Mouse disposable celiac injects acetic acid lead solution, and every dye lead 1mg sets up 4h abdominal cavities after acute lead poisoning model, dye lead
Inject Zn-DTPA, successive administration 5 days.Normal group, dye lead model group, Zn-DTPA and Ca-DTPA combination groups 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, clears up after processing,
Lead content is determined with ICP-MS.It is believed that as a result showing that Zn-DTPA can dramatically increase the discharge of lead in urine, reduction blood, femur and brain
Lead content in tissue.
However, because DTPA-Ca and DTPA-Zn can be used not only for excluding internal heavy metal lead, can also exclude in vivo
The heavy metal such as lanthanum, cerium, promethium, americium, plutonium even can also be used to make a definite diagnosis as clinical practice medicine 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.It is widely applied under environment, only investigates so
Lead distribution in vivo and content are obviously not enough to evaluate the internal behavior of this metal-chelators of DTPA.
Therefore, this area still expects have reliable method to give above-mentioned metal chelating to determine organism such as people, rat
The content of above-mentioned metal-chelator during biological sample is such as blood, urine after mixture, to assess the treatment of these metal-chelators
Effect or its behavior in vivo.
The content of the invention
It is an object of the invention to provide a kind of reliable method above-mentioned metal is given to determine organism such as people, rat
The content of above-mentioned metal-chelator during biological sample is such as blood, urine after chelating agent, 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.Particularly, the present invention is surveyed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method
Determine the DTPA-Zn in human urine biological sample, show excellent Methodological characteristics.The present invention is consequently found that and be accomplished.
Therefore, first aspect present invention is related to a kind of method for determining the DTPA-Zn in human urine biological sample, this method
The content of the DTPA-Zn in the human urine biological sample through processing is determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method,
Comprise the following steps：
(1) processing of biological sample：
(1a) takes the μ L of human urine 50 for containing or not contain DTPA-Zn, is placed in 1.5mL centrifuge tubes, plus inner mark solution is
The 25 μ g/mL μ L of the EDTA-Zn aqueous solution 20 are not added with inner mark solution, add the μ L of 0.01% ammoniacal liquor 900 and (are not added with interior timestamp to add
The μ L of 0.01% ammoniacal liquor 920, it is 970 μ L to make urine, ammoniacal liquor, optional inner mark solution cumulative volume), vortex mixing 3min, in whole
Sample is on solid phase extraction column；
Cleaned, finally eluted with methanol solutions of the 0.5mL containing 1% ammoniacal liquor with 2mL water after (1b) loading；
(1c) collects the eluent, and drying, residue is redissolved with 150 μ L water, draws 10 μ L and carries out LC-MS/MS analyses；
The preparation of (1d) standard curve：The μ L of DTPA-Zn standard serial solutions 100 of various concentrations are taken, 1.5mL centrifugations are placed in
Guan Zhong, adds blank human urine, vortex 3min, preparation is 10,20,50,100,200,500,1000 and 2000 μ equivalent to concentration
G/mL urine sample；The urine sample 50 μ L are taken, and sequentially add the μ L of inner mark solution 20, the μ L of 0.01% ammoniacal liquor 900, vortex is mixed
3min is closed, is all splined on solid phase extraction column, then (1b) and (1c) is operated according to more than, according to LC-MS/MS analysis results
Standard curve is drawn, the standard curve 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 is equipped with electric spray ion source and data handling system；
(2b) chromatographic condition：(its specification is for example for the chromatographic columns of SHISEIDO Proteonavi brands for analytical column used
For 5 μm, 250 × 4.6mm I.D.), C is connected before post18(its specification is, for example, 4 × 3.0mm I.D., such as U.S. to guard column
The guard column of Phenomenex companies), mobile phase is methanol -2mM ammonium formates (for example, wherein containing 0.03% ammoniacal liquor) (50:50,
V/v), flow velocity 0.45mL/min；
(2c) Mass Spectrometry Conditions：Electro-spray ionization source (such as using Turbo Ionspray sources), 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；How anti-scan mode be
(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 are 45V) and m/z353.0 → m/z 263.0 (ethylenediamine tetraacetic acid disodium zinc salt salt, CE is 15V), sweep
The time is retouched for 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
(such as Winnolin (Phoenix 6.3, the U.S.) is divided surveyed data (3b) application pharmacokinetics software
Analysis, obtain it is related to medicine 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 DTPA people obtains gives the urine that different time after Zn-DTPA is obtained as blank diaper sample, or from people
It is used as pastille urine sample.
Method described in any embodiment according to a first aspect of the present invention, human urine obtained in it being set to optionally
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, wherein being designated as EDTA-Zn in used
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, first activated with 1mL methanol, then use 1mL water balances；Or, first activated with 2mL methanol, then use 2mL water balances.
Method described in any embodiment according to a first aspect of the present invention, wherein the high performance liquid chromatograph is, for example,
The type liquid chromatographs of Agilent company of the U.S. Agilent 1200.
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.
The type tandem mass spectrometers of AB Sciex company API 4000；For example it is furnished with Turbo Ionspray ionization sources and Analyst
1.5 data handling system.
In the present invention, Proteonavi chromatographic columns are a kind of using in porous spherical silica filler surface bond butyl
(C4) high-performance protein-polypeptide analysis chromatographic column specially, the filler is both with the high separation capacity of silica type filler and resistance to
Pressure property, has the specialities such as acid resistance, the absorption for suppressing protein again.The chromatographic column is indicated with Proteonavi s5 sometimes.Although
The DTPA metal chelating agents that the present invention is analyzed are a kind of typical small-molecule substances, and molecular weight is much smaller than common protein-many
Peptide, however, having had now surprisingly been found that, the present invention is only under conditions of the chromatographic column using above-mentioned Proteonavi chromatographic columns
The inventive method could obtain excellent methodology performance.For example, using YMC-C18, Agilent Extard-C18, money life
During the C18 posts such as hall PAK CR-18 (it is same manufacturer with Proteonavi posts used herein), DTPA metal complexs are extremely
It is difficult to elute, such as using chromatographic condition of the present invention but DTPA-Za retention time is more than when using these C18 posts instead
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 posts).In another example, using ZORBAX EDIPSR-C8, Dikma diamonds-C8, taking
During the C8 posts such as sieve door Gemini-C8, DTPA metal complexs do not retain in the chromatography column, i.e., retention time is extremely short, for example
DTPA-Za retention time using chromatographic condition of the present invention but when using these C8 posts instead be less than 1.5min and with 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 modelWAX solid phase extraction column, it is for example purchased from Waters companies.Waters Oasis WAX solid phase extraction columns are in business
Be typically in industry be configured as mixed type weak anionic exchange reverse phase absorption agent, have to highly acid compound 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 columns
In the case of, the inventive method could obtain excellent methodology performance.And when the solid phase extraction column example for using other brands instead
Such as U.S. Ai Jieer Cleanert PWAX solid phase extraction columns, StrataTM-X solid phase extraction columns and even same
During the Oasis MAX type solid phase extraction columns of manufacturer, it can not much obtain above-mentioned Oasis WAX type solid phase extraction columns and be obtained
Good results.For example when being tested according to the method involved by table 4 below result, with above-mentioned three kinds other brand/models
Solid phase extraction column when, the μ g/mL of the rate of recovery 20,180 μ g/mL, 1800 μ g/mL, tri- kinds of concentration rate of recovery respectively 67~
73%th, in the range of 76~81%, 79~94% and various pillars under three kinds of various concentrations gained RSD in the range of 9~22%
Greatest differences and the result of RSD wide fluctuations are presented in the rate of recovery under fluctuation, this various concentrations is in biological sample analysis
It is unacceptable.In addition, when carrying out biological sample processing, after sample is loaded into solid phase extraction column, with containing a small amount of ammonia
The eluent of water carry out elution be it is necessary, otherwise the rate of recovery under each concentration than it in table 4 below rate of recovery result of the present invention
It is low more than 20 percentage points.
Method described in any embodiment according to a first aspect of the present invention, wherein also including one kind that following solution is prepared
Or a variety of operations：
The preparation of (10a) DTPA-Zn storing solutions：The Zn-DTPA injections for taking concentration to be 100mg/mL are stored up as DTPA-Zn
Standby liquid (it can be designated as A1), is transferred in tool plug test tube, and -20 DEG C of refrigerator freezings are preserved, standby；
The preparation of (10b) DTPA-Zn standard serial solutions：By DTPA-Zn storing solutions be A1 be 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 solutions：It is that A1 is diluted with water and is made down by DTPA-Zn storing solutions
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 substances EDTA-Zn in 2mL measuring bottles, uses water
Dissolve and constant volume, be made into the internal standard storing solution (it can be designated as IS1) that concentration is 10.0mg/mL, -20 DEG C of refrigerator freezings are preserved, standby
With；
Precision draws internal standard storing solution (i.e. IS1) 1250 μ L and, into 25mL measuring bottles, scale is settled to 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% ammoniacal liquor methanol solution：Ammoniacal liquor 10.0mL, methanol 990.0mL are measured, is mixed in conical flask
Produce；
Method described in any embodiment according to a first aspect of the present invention, wherein also including what following plasma sample was handled
One or more operations：
The preparation of (20a) blank diaper sample：
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, 0.01% ammoniacal liquor 920 μ L, vortex mixing 3min is added,
All it is splined on solid phase extraction column；
Cleaned, finally eluted with the methanol solution 0.5mL containing 1% ammoniacal liquor with 2mL water after loading；
Eluent is collected, 10 μ L is drawn and is fully mixed with 190 μ L water, is obtained for carrying out the molten of LC-MS/MS analysis measure
Liquid (desirable 10 μ L carry out LC-MS/MS analyses)；
(20b)The preparation of zero point urine sample：
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, inner mark solution 20 μ L, the μ L of 0.01% ammoniacal liquor 900 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：
The μ L of DTPA-Zn standard serial solutions 100 of various concentrations are taken (for example, taking " (10b) DTPA-Zn standard serial solutions
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 solutions), it is placed in 1.5mL centrifuge tubes, adds blank people
Urine, vortex 3min is prepared equivalent to the urine sample that concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL；
The μ L of urine sample 50 are taken, and sequentially add the μ L of inner mark solution 20,0.01% ammoniacal liquor 900 μ L, vortex mixing 3min, are 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
Take DTPA-Zn quality-control sample working solutions to be placed in 1.5mL centrifuge tubes, add blank human urine, vortex 3min, system
For equivalent to the urine sample that concentration is 20,180 and 1800 μ g/mL, the μ L of urine sample 50 are taken, and sequentially add inner mark solution 20
μ L, 0.01% ammoniacal liquor 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)
Take DTPA-Zn quality-control sample working solutions to add in blank human urine, be prepared into 10.0 μ g/mL LLOQ samples；
Remaining is pressed and operated under " preparation of (20d) quality-control sample " item, and carries out LC-MS/MS analyses；
The preparation of (20f) cross jamming test specimen
The μ L of DTPA-Zn working solutions 40 are taken, are placed in 1.5mL centrifuge tubes, blank human urine is added, be prepared by vortex 3min
Into the ULOQ samples of 2000 μ g/mL not containing the internal standards；In addition to internal standard is not added with, remaining is pressed and grasped under " preparation of (20d) quality-control sample " item
Make, and carry out LC-MS/MS analyses.
In the step of aforesaid operations method of the present invention, although specific steps that it is described are in some details or language
The step of described in example in description with following detailed description part, is otherwise varied, however, those skilled in the art
Approach described above step can be summarized according to the detailed disclosure of full text of the present invention completely.
Any embodiment of the either side of the present invention, can be combined with other embodiments, as long as they are not
Contradiction occurs.In addition, in any embodiment of either side of the present invention, any technical characteristic goes for other realities
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 is incorporated herein by reference, and if these are literary
Offer expressed implication with it is of the invention inconsistent when, be defined by the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explained that the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
The implication stated is defined.
The calcium metal chelating agent of DTPA metal chelating agents, such as DTPA or DTPA zinc metal chelating agent, their body
Outer detection, such as detection in some chemicals, preparation, are still that run into one of current those skilled in the art is huge
Problem is detected, and the detection difficulty in their vivo biodistribution samples is even more far more difficult than vitro detection.Particularly, current state
The inside and outside report all without analysis in these compound bodies.
DTPA calcium metal chelating agent, can be abbreviated as Ca-DTPA or DTPA-Ca, and it is generally also known as Ca-DTPA
(DTPA-CaNa3)；DTPA zinc metal chelating agent, can be abbreviated as Zn-DTPA or DTPA-Zn, and it is generally also known as Zn-
DTPA(DTPA-ZnNa3)。
The internal standard substance EDTA-Zn used in Ca-DTPA, Zn-DTPA and the present invention (is secondly sodium-salt hydrate
EDTA-Zn Na2xH2O) molecular formula be respectively with 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 indications of Ca-DTPA clinically with application be：Ca-DTPA
As it is a kind of alleviate radiate chelating agent be used to make a definite diagnosis or it is doubtful by plutonium, americium, huge internal pollution subject treatment, and improve this
The clearance rate of a little metallic elements.
Ca-DTPA dosage gives the Ca-DTPA of first dosage with administration aspect, FDA code requirement in contaminated 24h
Treated, after 24h, it is proposed that maintain chelating therapy using Zn-DTPA.Used in 24h after chelating therapy is contaminated in vivo
Effect is best；Chelating therapy should be given in time when making a definite diagnosis or suspecting and be contaminated；If can not 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, Ca chelate effect is optimal.Radioactivity after curative effect is chelated with internal pollution
Pollutant is isolated in liver and bone and reduced.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 exist multipath pollution
DTPA；5ml 3-4 minutes Ca-DTPA solution (1g/5ml) used time slow intravenous injection is administered, or 5ml Ca-DTPA is molten
Liquid is diluted to the administration of 100-250ml venous transfusions in 5% D/W, woods grignard lactic acid solution, physiological saline；If by
Examination person is contaminated due to suction, and the Ca-DTPA of atomization is given in 24h and can be used as therapy approach.
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 indications of Zn-DTPA clinically with application be：Zn-DTPA
Suitable for the internal pollution made a definite diagnosis or doubtful plutonium, americium, hard iron are caused, and accelerate its clearance rate.
Zn-DTPA dosage gives the Ca- of first dosage with administration aspect, FDA code requirement in contaminated 24h
DTPA is treated；Ca-DTPA is than Zn-DTPA better efficacy during this period；If Ca-DTPA is invalid, Zn-DTPA progress is given
Treat first.Given if second day extra chelation therapy of administration is proposed, conventional therapy is carried out 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 green grass or young crops
Teenager, is injected intravenously the Zn-DTPA of 1g dosage；For the children of less than 12 years old, 14mg/Kg Zn-DTPA, consumption are injected intravenously
It is sure not more than 1g.Chelating continued treatment after 24h uses Zn-DTPA, to adult and teenager, intravenous injection 1g dosage
Zn-DTPA, once a day；For the children of less than 12 years old, 14mg/Kg Zn-DTPA is injected intravenously, once a day, consumption is not
More than 1g.Using effect is best in 24h after DTPA chelating therapy is contaminated in vivo.It is contaminated making a definite diagnosis or suspecting
When should give chelating therapy in time.If can not 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-
DTPA chelate effect is optimal.Radioactive pollutant is isolated in liver and bone and dropped 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 exist multipath pollution；By 5ml Zn-DTPA solution (1g/
5ml) slow intravenous injection administration in used time 3-4 minutes, or by 5ml Zn-DTPA solution in 5% D/W, woods grignard
The administration of 100-250ml venous transfusions is diluted in lactic acid solution, physiological saline；If subject merely due to inhalation route and get dirty
The Zn-DTPA of atomization is given in dye, 24h to be used as therapy approach.
By setting up the inventive method, the metallo-chelate for being DTPA is to give animal (such as rat, people) internal afterwards
The assay of these materials provides possible in biological sample (such as blood, urine, saliva).The analysis that the present invention is set up
Method has excellent methodology performance.
Brief description of the drawings
Fig. 1：DTPA-Zn and internal standard EDTA-Zn two grades of full scan mass spectrograms；In figure, (A) DTPA-Zn, (B) EDTA-
Zn。
Fig. 2：DTPA-Zn and EDTA-Zn MRM chromatograms；Wherein, A:Blank diaper, B:Mark-on blank diaper, C:Administration
Urine sample；In figure, I, II:Represent DTPA-Zn, III:Internal standard.
Fig. 3：Investigate the chromatogram of DTPA-Zn and internal standard EDTA-Zn cross jammings in urine；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, I, II:Represent DTPA-Zn, III:Internal standard.
Fig. 4：LC-MS/MS methods determine DTPA-Zn concentration standard curves in urine and illustrated.
Embodiment
Following examples provided by the present invention are only used for task of explanation rather than are used for, and are also not necessarily to be construed as with any
Mode limits the present invention.Those skilled in the art will recognize that in the case of not past the spirit or scope of the present invention
Following examples can be made with conventional change and modifications.
It is an object of the present invention to more fully understand DTPA-Zn disposition rule, present invention experiment is set up simultaneously
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method of DTPA-Zn content in quantitative detection biological sample is demonstrated, is investigated simultaneously
Pharmacokinetics behaviors of the DTPA-Zn in human body.
1 experiment general introduction
Zn-DTPA (diethyl pentetic acid trisodium zinc salt, DTPA-Zn) is clinically to alleviate radiation chelating as a kind of
Agent is used to make a definite diagnosis or the doubtful treatment for being polluted subject by plutonium, americium, hard iron, and improves the clearance rate of these metallic elements.This examination
It is by determining DTPA-Zn concentration in human urine, evaluating pharmacokinetics of the Zn-DTPA parenteral solutions in health volunteer's body to test purpose
Behavior.This report describes liquid chromatography-tandem mass spectrometry (LC-MS/MS) the analysis side for determining DTPA-Zn in human urine sample
Method.
2 experiment materials
2.1 medicines, reagent and material
2.2 solution
2.2.1 the preparation of DTPA-Zn storing solutions and standard serial solution
Zn-DTPA injections 1 (500mg/5mL) are taken as DTPA-Zn storing solutions (A1), be transferred in tool plug test tube-
20 DEG C of refrigerator freezings are preserved.
DTPA-Zn standard serial solutions are prepared by the following method：DTPA-Zn storing solutions (A1) are diluted to water as shown in table 1
The standard serial solution of following concentration, and saved backup in 4 DEG C of refrigerators.
Table 1：The preparation (water dissolving and constant volume) of DTPA-Zn standard serial solutions
| 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 samples working solution
Separately DTPA-Zn stock solutions are prepared by the following method：1 Zn-DTPA injection (500mg/5mL) is taken as storing solution
(B1) -20 DEG C of refrigerator freezings in tool plug test tube, are transferred to preserve.
DTPA-Zn quality-control sample working solutions are prepared by the following method：By DTPA-Zn storing solutions (B1) with water as shown in table 2 it is dilute
The quality-control sample working solution of following concentration is interpreted into, and is saved backup in 4 DEG C of refrigerators.
Table 2：The preparation (water dissolving and constant volume) of DTPA-Zn Quality control samples working solutions
| 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 bottles, and with water dissolving and constant volume, being made into concentration is
10.0mg/mL storing solution (IS1), -20 DEG C of refrigerator freezings are preserved.
Precision draws storing solution (IS1) 1250 μ L and, into 25mL measuring bottles, scale is settled to water, obtains 500 μ g/mL internal standard
Working solution (IS2), and saved backup in 4 DEG C of refrigerators.
2.2.4 the preparation of 1% ammoniacal liquor methanol solution
Ammoniacal liquor 10.0mL, methanol 990.0mL are measured, mixes and produces in conical flask.
3 instruments and experimental condition
3.1 LC-MS/MS conditions
Chromatographic condition：Analytical column is SHISEIDO Proteonavi posts (5 μm, 250 × 4.6mm I.D., Japan
SHISEIDO companies), C18Guard column (4 × 3.0mm I.D., Phenomenex companies of the U.S.), mobile phase is methanol -2mM formic acid
Ammonium (containing 0.03% ammoniacal liquor) (50:50, v/v), flow velocity is 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；Scan mode be 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 are 45V) and m/z
353.0 → m/z 263.0 (ethylenediamine tetraacetic acid disodium zinc salt salt, CE is 15V), sweep time is 150msec.
3.2 key instrument
Liquid chromatograph-mass spectrometer (LC-MS/MS)：The triple quadrupole bar tandem mass spectrometer (American ABs of API 4000
Sciex companies), equipped with Turbo Ionspray ionization sources and the data handling systems of Analyst 1.5, liquid chromatographic system
The binary gradient pumps of Agilent 1200 and CTC automatic samplers.BT224S type precise electronic assay balances (German Sartorius
Company)；DMT-2500 type multitubes eddy mixer (Hangzhou meter Ou Instrument Ltd.)；BF-2000 types nitrogen drying instrument (Beijing
All directions Centrix Technology Ltd.).
3.3 urine sample processing methods
3.3.1 the preparation of blank diaper sample
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, 0.01% ammoniacal liquor 920 μ L, vortex mixing 3min is added,
All it is splined on solid phase extraction column.First activated before solid phase extraction column loading with 2mL methanol, then use 2mL water balances.Loading
Cleaned, finally eluted with the ammoniacal liquor methanol solutions of 0.5mL 1% with 2mL water afterwards.Eluent is collected, 10 μ L is drawn and is filled with 190 μ L water
Divide and mix, take 10 μ L to carry out LC-MS/MS analyses.
3.3.2 the preparation of zero point urine sample
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, inner mark solution 20 μ L, the μ L of 0.01% ammoniacal liquor 900 is added,
Vortex mixing 3min, is all splined on solid phase extraction column.Remaining operation is with " 3.3.1 ".
3.3.3 the preparation of standard curve
The μ L of DTPA-Zn standard serial solutions (A2~A9 solution) 100 are taken, are placed in 1.5mL centrifuge tubes, blank human urine is added
Liquid, vortex 3min is prepared equivalent to the urine sample that concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL.Take
The μ L of urine sample 50, and inner mark solution 20 μ L, 0.01% ammoniacal liquor 900 μ L, vortex mixing 3min are sequentially added, all it is splined on solid
Mutually on extraction pillar.Remaining operation is with " 3.3.1 ".
3.3.4 the preparation of quality-control sample
The μ L of DTPA-Zn quality-control samples working solution (B3, B5, B6) 250 are taken, are placed in 1.5mL centrifuge tubes, blank people is added
Urine, vortex 3min is prepared equivalent to the urine sample that concentration is 20,180 and 1800 μ g/mL.Take the μ L of urine sample 50, and according to
It is secondary to add inner mark solution 20 μ L, 0.01% ammoniacal liquor 900 μ L, vortex mixing 3min, all it is splined on solid phase extraction column.Remaining
Operation is with " 3.3.1 ".
3.3.5 the preparation of minimum lower limit of quantitation sample (LLOQ)
The μ L of DTPA-Zn quality-control samples working solution (B7) 250 are taken, are added in blank human urine, prepare 10.0 μ g/mL's
LLOQ samples.Remaining step is pressed and operated under " preparation of 3.3.4 quality-control samples " item, and carries out LC-MS/MS analyses.
3.3.6 the preparation of cross jamming test specimen
The μ L of DTPA-Zn working solutions (A2 solution) 40 are taken, are placed in 1.5mL centrifuge tubes, blank human urine is added, are vortexed
3min, is prepared into the ULOQ samples of 2000 μ g/mL not containing the internal standards.Remaining step is pressed and grasped under " preparation of 3.3.4 quality-control samples " item
Make, and carry out LC-MS/MS analyses.
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, zero point urine sample are prepared, by " 3.3.2 zero point urines
Operated under the preparation of sample " item.
3.3.7 the preparation of extraction recovery sample
The μ L of blank human urine 50 of 6 kinds of separate sources are taken, are placed in 1.5mL centrifuge tubes, the μ L of 0.01% ammoniacal liquor 920 are added,
Vortex mixing 3min, is all splined on solid phase extraction column.First activated, then used with 2mL methanol before solid phase extraction column loading
2mL water balances.Cleaned, finally eluted with the ammoniacal liquor methanol solutions of 0.5mL 1% with 2mL water after loading.Eluent is collected, 10 are drawn
μ L and the mixed standard solution of 190 μ L respective concentrations are fully mixed, and take 10 μ l to carry out LC-MS/MS analyses.
3.3.8 the preparation of matrix effect test specimen
The μ L of DTPA-Zn quality-control samples working solution (B3, B5, B6) 10 and the μ L of inner mark solution (IS2) 20 are 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 ammoniacal liquor methanol solution is mixed, 10 μ L are taken to carry out LC-MS/MS analyses.Each concentration carries out 6 sample analyses, obtains corresponding peak
Area.
3.3.9 the preparation of urine sample stability test sample
The μ L of quality-control sample working solution (B3, B6) 100 are taken, are placed in 1.5mL centrifuge tubes, blank human urine is added, are vortexed
3min, is prepared into the urine sample of high and low two concentration of DTPA-Zn, takes 50 μ L urine samples to place sample after 4h, processing in room temperature
Product room temperature place 24h, -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
The μ L of urine sample 50 are taken, are placed in 1.5mL centrifuge tubes, the μ L of internal standard working solution (IS2) 20 are sequentially added, added
0.01% ammoniacal liquor 900 μ L, vortex mixing 3min, are all splined on solid phase extraction column.Remaining operation is with " 3.3.1 ".
The preparation of 3.4 storing solutions and working solution stability sample
3.4.1 the preparation of DTPA-Zn storing solutions stability sample
The same day is determined by under " preparation of 2.2.1DTPA-Zn storing solutions and standard serial solution " item, takes 1 Zn-DTPA to note
Agent is penetrated to be transferred in tool plug test tube as storing solution A1.Precision pipettes the fresh μ L of DTPA-Zn storing solutions (A1) 50 to 1.0mL amounts
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 bottles, uses water
Dilution constant volume obtains A6 solution (500 μ g/mL).
Take the freshly prepared μ L of A6 solution 10, be placed in 1.5mL centrifuge tubes, add 490 μ L water mix after, draw 10 μ L with
190 μ L water are fully mixed, and take 10 μ L to carry out LC-MS/MS analyses.
The preparation of DTPA-Zn storing solutions 56 days stability samples of -20 DEG C of placements：
Precision pipettes DTPA-Zn storing solutions (A1, -20 DEG C are placed 56 days) 50 μ L and, into 1.0mL measuring bottles, constant volume is diluted with water
Obtain A3 solution (5.0 × 103μg/mL)；Precision pipettes (A3) 100 μ L into 1.0mL measuring bottles, and constant volume is diluted with water and obtains A6 solution
(500μg/mL)。
The μ L of A6 solution 10 are placed in 1.5mL centrifuge tubes, added after the mixing of 490 μ L water, drawn 10 μ L and filled with 190 μ L water
Divide and mix, take 10 μ L to carry out LC-MS/MS analyses.
DTPA-Zn storing solutions room temperature places the preparation of 4h stability samples：
Precision pipettes the same day fresh μ L of DTPA-Zn storing solutions (A1) 50 into 1.0mL measuring bottles, is placed in room temperature after 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 bottles, and it is fixed to be diluted with water
Hold to obtain A6-1 solution (500 μ g/mL).
The μ L of A6-1 solution 10 are taken, are placed in 1.5mL centrifuge tubes, adds after the mixing of 490 μ L water, draws 10 μ L and 190 μ L water
Fully mix, take 10 μ L to carry out LC-MS/MS analyses.
3.4.2 the preparation of DTPA-Zn standard working solutions stability sample
The same day is determined by under " preparation of 2.2.2 quality-control sample working solutions " item, fresh preparation DTPA-Zn Quality Controls work is molten
Liquid (B3, B6).
The freshly prepared μ L of B3, B6 solution 10 are taken, are placed in 1.5mL centrifuge tubes, adds after the mixing of 490 μ L water, draws 10 μ
L is fully mixed with 190 μ L water, takes 10 μ L to carry out LC-MS/MS analyses.
The preparation of DTPA-Zn standard working solutions 56 days stability samples of -20 DEG C of placements：
The μ L of B3, B6 solution 10 of -20 DEG C of preservations are taken, are placed in 1.5mL centrifuge tubes, adds after the mixing of 490 μ L water, draws 10
μ L are fully mixed with 190 μ L water, take 10 μ L to carry out LC-MS/MS analyses.
DTPA-Zn standard working solutions room temperature places the preparation of 4h stability samples：
Precision pipettes the same day freshly prepared μ L of B3, B6 solution 10, is placed in 1.5mL centrifuge tubes, is placed in room temperature after 4h,
Add after the mixing of 490 μ L water, draw 10 μ L and fully mixed with 190 μ L water, take 10 μ L to carry out LC-MS/MS analyses.
3.4.3 the preparation of internal standard storing solution and internal standard working solution stability sample
The same day is determined by under " preparation of 2.2.3 internal standard working solutions " item, precision pipettes freshly prepared internal standard storing solution
(IS1) 50 μ L are into 1mL measuring bottles, are diluted with water and are settled to scale and obtain IS2 solution (500 μ g/mL).
The freshly prepared μ L of IS2 solution 20 are taken, are placed in 1.5mL centrifuge tubes, adds after the mixing of 480 μ L water, draws 10 μ L
Fully mixed with 190 μ L water, take 10 μ L to carry out LC-MS/MS analyses.
The preparation of internal standard storing solution 57 days stability samples of -20 DEG C of placements：
Precision is pipetted, into 1mL measuring bottles, to be used in internal standard storing solution (IS1, -20 DEG C are placed 57 days) 50 μ L of -20 DEG C of preservations
Water dilution is settled to scale and obtains IS2 solution (500 μ g/mL).
The μ L of IS2 solution 20 are taken, are placed in 1.5mL centrifuge tubes, are added after the mixing of 480 μ L water, 10 μ L is drawn and is filled with 190 μ L water
Divide and mix, take 10 μ L to carry out LC-MS/MS analyses.
Internal standard storing solution room temperature places the preparation of 4h stability samples：
Precision pipettes the μ L of the same day freshly prepared (IS1) 50 into 1mL measuring bottles, is placed in room temperature after 4h, and it is fixed to be diluted with water
Hold to scale and obtain IS2-1 solution (500 μ g/mL).
The μ L of IS2-1 solution 20 are taken, are placed in 1.5mL centrifuge tubes, adds after the mixing of 480 μ L water, draws 10 μ L and 190 μ L water
Fully mix, take 10 μ L to carry out LC-MS/MS analyses.
The preparation of internal standard working solution 57 days stability samples of -20 DEG C of placements：
Take IS2 solution (- 20 DEG C place 57 days) 20 μ L of -20 DEG C of preservations, add after 480 μ L water mix, draw 10 μ L with
190 μ L water are fully mixed, and take 10 μ L to carry out LC-MS/MS analyses.
Internal standard working solution room temperature places the preparation of 4h stability samples：
The freshly prepared μ L of IS2 solution 20 are taken, are placed in 1.5mL centrifuge tubes, are placed in room temperature after 4h, 480 μ L water are added
After mixing, draw 10 μ L and fully mixed with 190 μ L water, take 10 μ L to carry out LC-MS/MS analyses.
3.5 data process＆analysis
Surveyed data are analyzed using Winnolin (Phoenix 6.3, the U.S.) pharmacokinetics software, obtain with
The related kinetic parameter C of medicine exposuremax、Tmax、AUC0-tAnd MRT0-t。
4 result of the tests
4.1 LC-MS/MS quantitation methodologies are verified
4.1.1 method choice
Two grades of full scan mass spectrograms of determinand are shown in Fig. 1.
The μ L of blank human urine 50 of 6 kinds of separate sources are taken, are placed in 1.5mL centrifuge tubes, by " 3.3.1 blank diaper samples
Preparation " operate under item, carry out LC-MS/MS analyses, obtain the chromatogram of blank diaper sample, such as accompanying drawing 2A；By finite concentration
Standard liquid be added in blank diaper, take the μ L of urine sample 50, by under " preparation of 3.3.3 standard curves " item operate, obtain
Chromatogram, such as accompanying drawing 2B, wherein DTPA-Zn retention time are about 4.67min, and internal standard EDTA-Zn retention time is about
5.02min；The μ L of urine sample 50 gathered after medicine are drawn, are operated by under " preparation of 3.3.10 unknown samples " item, such as accompanying drawing 2C.
As a result show, endogenous material does not disturb determinand and interior target to determine in blank diaper.
4.1.2 the cross jamming experiment of method
Operated by under " preparation of 3.3.6 cross jamming test specimens " item, zero is carried out after the ULOQ samples of not containing the internal standard
Point urine sample analysis, obtains accompanying drawing 3.As a result show, 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, DTPA-Zn linear regression equation is tried to achieve, 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 of method and the degree of accuracy
Operated by under " preparation of the minimum lower limit of quantitation samples (LLOQ) of 3.3.5 " and " preparation of 3.3.4 quality-control samples " item,
It (is respectively 20,180 to prepare the minimum lower limit of quantitation of DTPA-Zn (LLOQ, concentration is 10 μ g/mL) sample and basic, normal, high 3 concentration
With 1800 μ g/mL) QC samples, each concentration carries out 6 sample analyses, and METHOD FOR CONTINUOUS DETERMINATION three days calculates the survey of LLOQ and QC samples
Concentration is obtained, with preparing concentrations control, the veracity and precision of this law is tried to achieve, the result of 3 analyses batch is subjected to variance analysis
Obtain preci-sion and accuracy (being shown in Table 3).As a result show, the preci-sion and accuracy of method meets biological sample quantitative analysis
It is required that.
Table 3：DTPA-Zn veracity and precision (n=18)
4.1.5 extraction recovery and matrix effect
Operated by under " preparation of 3.3.4 quality-control samples " and " preparation of 3.3.7 extraction recovery samples " item.Prepare 3
The human urine QC samples 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 obtains extraction recovery (being shown in Table 4) compared with the peak area of extraction recovery sample.
Operated by under " preparation of 3.3.7 extraction recovery samples " and " preparation of 3.3.8 matrix effect samples " item.Take 6
The blank human urine of separate sources is planted, extraction recovery sample is prepared；Take DTPA-Zn quality-control samples working solution (B3, B4, B7)
The 10 μ L and μ L of inner mark solution (IS2) 20, prepare 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), experiment knot compared with the peak area of matrix effect sample
Fruit shows that DTPA-Zn matrix effects are between 85%~115% in human urine, and urine matrix does not show to the measure of determinand
The influence of work.
Table 4：DTPA-Zn extraction recovery (n=6)
| Indicate 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 |
Table 5：DTPA-Zn matrix effect (n=6)
4.1.6 urine sample study on the stability
Stability of the DTPA-Zn urine samples under various experiment conditions is investigated.As a result show, DTPA-Zn urine samples
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：DTPA-Zn stability (n=4) in urine
4.1.7 storing solution and working solution study on the stability
Operated by under " preparation of 2.5.1DTPA-Zn storing solution stability samples " item, investigated DTPA-Zn storing solutions, work
Make solution, internal standard storing solution, working solution are in the stability that room temperature is placed, -20 DEG C are placed.Calculated and laid in using one point external standard method
The measured concentration of liquid and working solution, it is compared with theoretical value calculating RE (%)；Storing solution and working solution room are calculated simultaneously
Temperature places the RSD (%) of both front and back solution peak area.
As a result show：Determinand (DTPA-Zn) storing solution, working solution room temperature place 4h, 56 days stabilizations of -20 DEG C of placements.
Internal standard (EDTA-Zn) storing solution, working solution room temperature place 4h, 57 days stabilizations of -20 DEG C of placements.It the results are shown in Table 7.
Table 7：DTPA-Zn, internal standard storing solution and working solution stability (n=6)
4.1.8 the residual of method is investigated
Prepare DTPA-Zn highest quantitative limits ULOQ (2000 μ g/mL) urine sample and blank diaper sample.
In the laggard line blank urine sample analysis of ULOQ samples.Compare blank diaper sample and LLOQ chromatogram, blank
Determinand response is not to be exceeded the 20% of LLOQ in urine sample.DTPA-Zn and interior target signal ring in blank diaper sample
Answer that intensity is respectively LLOQ 6.2% and 0.3%.As a result show ULOQ samples to blank diaper sample without significantly interfering with.
4.2 unknown urine samples are determined and quality control
Urine sample is tested to determine by operation under " prepared by the unknown urine samples of 3.3.10 " item, and with the standard curve on the same day
The concentration of DTPA-Zn in each time point sample is calculated, the choice of same day data is determined by QC samples.It is required that the QC of each analysis batch
In the range of the 85%~115% of the degree of accuracy of sample, the concentration point that can have 1/3 is transfinited, but same concentration at least 50% is accorded with
Standardization.
5 DTPA-Zn pharmacokinetics
12 health volunteer's drip-feeds give Zn-DTPA parenteral solutions (single intravenous infusion 250mg, single intravenous infusion 500mg,
Single intravenous infusion 1000mg, multiple intravenous infusion 500mg), urine is taken in different time points, DTPA-Zn drug concentrations in urine are determined, and
Mean drug concentration-time graph is drawn, is as a result shown, pharmaceutical concentration-time curve coincide substantially during with dose, different agent
Pharmaceutical concentration-time curve variation tendency is identical during amount, and related pharmacokinetic parameters are identical.
6 conclusions
This experiment determines the concentration of DTPA-Zn in human urine sample using LC-MS/MS methods.Urine sample is through SPE
After method processing, using ESI sources, the endogenous material in quantitative analysis, urine is carried out with MRM scan modes and does not disturb DTPA-
Zn measure.This method is accurate, selectivity is strong, sensitivity is high, and meets " the chemicals medicine human-body biological of CFDA promulgations
Availability and bioequivalence investigative technique guideline " ([H] GCL2-1) is relevant to be required, is adapted to Zn-DTPA in human urine
Pharmacokinetic study.
The spirit of the present invention is elaborated above by present pre-ferred embodiments.Those skilled in the art manage
Solution, every any modification, equivalent variations and modification substantially made according to the technology of the present invention to above example, all falls within this hair
In bright protection domain.
Claims (10)
1. determining the method for the DTPA-Zn in human urine biological sample, this method is passed through using LC-MS/MS
The content of DTPA-Zn in the human urine biological sample of processing, comprises the following steps：
(1) processing of biological sample：
(1a) takes the μ L of human urine 50 for containing or not contain DTPA-Zn, is placed in 1.5mL centrifuge tubes, plus inner mark solution is 25 μ g/
The mL μ L of the EDTA-Zn aqueous solution 20 are not added with inner mark solution, add the μ L of 0.01% ammoniacal liquor 900 and (are not added with interior timestamp to add
The μ L of 0.01% ammoniacal liquor 920, it is 970 μ L to make urine, ammoniacal liquor, optional inner mark solution cumulative volume), vortex mixing 3min, in whole
Sample is on solid phase extraction column；
Cleaned, finally eluted with methanol solutions of the 0.5mL containing 1% ammoniacal liquor with 2mL water after (1b) loading；
(1c) collects the eluent, and drying, residue is redissolved with 150 μ L water, draws 10 μ L and carries out LC-MS/MS analyses；
The preparation of (1d) standard curve：The μ L of DTPA-Zn standard serial solutions 100 of various concentrations are taken, 1.5mL centrifuge tubes are placed in
In, blank human urine is added, vortex 3min, preparation is 10,20,50,100,200,500,1000 and 2000 μ g/ equivalent to concentration
ML urine sample；The urine sample 50 μ L are taken, and sequentially add the μ L of inner mark solution 20, the μ L of 0.01% ammoniacal liquor 900, vortex mixing
3min, is all splined on solid phase extraction column, and then (1b) and (1c) is operated according to more than, is painted according to LC-MS/MS analysis results
Standard curve processed, the standard curve 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 injector,
The tandem mass spectrometer is equipped with electric spray ion source and data handling system；
(2b) chromatographic condition：(its specification is, for example, 5 μ to analytical column used for the chromatographic columns of SHISEIDO Proteonavi brands
M, 250 × 4.6mm I.D.), C is connected before post18(its specification is, for example, 4 × 3.0mm I.D., such as U.S. to guard column
The guard column of Phenomenex companies), mobile phase is methanol -2mM ammonium formates (for example, wherein containing 0.03% ammoniacal liquor) (50:50,
V/v), flow velocity 0.45mL/min；
(2c) Mass Spectrometry Conditions：Electro-spray ionization source (such as using Turbo Ionspray sources), negative-ion mode detection；Injection
Voltage is -4000V；Source temperature is 500 DEG C；Roller shutter gas (CUR) is 20；Collision gas (CAD) is 4；Scan mode is many reaction prisons
Survey (MRM), 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 are 45V) and 353.0 → m/z of m/z 263.0 (ethylenediamine tetraacetic acid disodium zinc salt salt, CE is 15V), during scanning
Between be 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) application pharmacokinetics software (such as Winnolin (Phoenix 6.3, the U.S.) is analyzed surveyed data,
Obtain it is related to medicine exposure be selected from it is following once or multiple 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 urine work that Zn-DTPA people obtains
For blank diaper sample, or from people urine that different time after Zn-DTPA obtains is given as pastille urine sample.
3. according to the method described in claim 1, being placed in -20 DEG C of refrigerators optionally of the human urine obtained in it freezes,
With etc. it is to be determined.
4. according to the method described in claim 1, wherein being designated as EDTA-Zn Na in used2·xH2O。
5. according to the method described in claim 1, wherein the solid phase extraction column is before loading, first activated with 1mL methanol, then
Use 1mL water balances；Or, first activated with 2mL methanol, then use 2mL water balances.
6. according to the method described in claim 1, wherein the high performance liquid chromatograph is, for example, Agilent company of the U.S.
The type liquid chromatographs of Agilent 1200.
7. according to the method described in claim 1, wherein the tandem mass spectrometer is, for example, American AB Sciex company API
4000 type tandem mass spectrometers；For example it is furnished with Turbo Ionspray ionization sources and the data handling systems of Analyst 1.5.
8. according to the method described in claim 1, wherein the solid phase extraction column is modelWAX SPE
Pillar.
9. according to the method described in claim 1, wherein also including one or more operations that following solution is prepared：
The preparation of (10a) DTPA-Zn storing solutions：The Zn-DTPA injections that concentration is 100mg/mL are taken as DTPA-Zn storing solutions
(it can be designated as A1), is transferred in tool plug test tube, and -20 DEG C of refrigerator freezings are preserved, standby；
The preparation of (10b) DTPA-Zn standard serial solutions：It is that A1 is diluted with water following DTPA-Zn is made by DTPA-Zn storing solutions
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 solutions：By DTPA-Zn storing solutions be A1 be 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 substances EDTA-Zn in 2mL measuring bottles, is dissolved with water
And constant volume, the internal standard storing solution (it can be designated as IS1) that concentration is 10.0mg/mL is made into, -20 DEG C of refrigerator freezings are preserved, standby；
Precision draws internal standard storing solution (i.e. IS1) 1250 μ L and, into 25mL measuring bottles, scale is settled to water, obtains the interior of 500 μ g/mL
Working solution (IS2) is marked, and (as inner mark solution, concentration is 500 μ g/ml) is saved backup in 4 DEG C of refrigerators；
The preparation of (10e) 1% ammoniacal liquor methanol solution：Ammoniacal liquor 10.0mL, methanol 990.0mL are measured, mixes and produces in conical flask.
10. according to the method described in claim 1, wherein also including one or more operations that following plasma sample is handled：
The preparation of (20a) blank diaper sample：
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, 0.01% ammoniacal liquor 920 μ L, vortex mixing 3min are added, all
It is splined on solid phase extraction column；
Cleaned, finally eluted with the methanol solution 0.5mL containing 1% ammoniacal liquor with 2mL water after loading；
Eluent is collected, 10 μ L is drawn and is fully mixed with 190 μ L water, is obtained for carrying out the solution that LC-MS/MS analyses are determined
(desirable 10 μ L carry out LC-MS/MS analyses)；
(20b)The preparation of zero point urine sample：
The μ L of blank human urine 50 are taken, are placed in 1.5mL centrifuge tubes, inner mark solution 20 μ L, the μ L of 0.01% ammoniacal liquor 900, vortex is added
3min is mixed, is all splined on solid phase extraction column；Then as " preparation of (20a) blank diaper sample " described carry out is follow-up
Operation；
(20c)The preparation of standard curve：
Taking the μ L of DTPA-Zn standard serial solutions 100 of various concentrations, (" (10b) DTPA-Zn standard serial solutions are matched somebody with somebody for example, take
1.0 × 10 in system "4μg/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 solutions), it is placed in 1.5mL centrifuge tubes, adds blank human urine
Liquid, vortex 3min is prepared equivalent to the urine sample that concentration is 10,20,50,100,200,500,1000 and 2000 μ g/mL；Take
The μ L of urine sample 50, and the μ L of inner mark solution 20,0.01% ammoniacal liquor 900 μ L, vortex mixing 3min are sequentially added, all it is splined on solid
Mutually on extraction pillar；Then such as " preparation of (20a) blank diaper sample " described carry out subsequent operation；
The preparation of (20d) quality-control sample
Take DTPA-Zn quality-control sample working solutions to be placed in 1.5mL centrifuge tubes, add blank human urine, vortex 3min prepares phase
When in the urine sample that concentration is 20,180 and 1800 μ g/mL, take the μ L of urine sample 50, and sequentially add the μ L of inner mark solution 20,
0.01% ammoniacal liquor 900 μ L, vortex mixing 3min, are all splined on solid phase extraction column；Then such as " (20a) blank diaper sample
The preparation of product " the carry out subsequent operation；
The preparation of (20e) minimum lower limit of quantitation sample (LLOQ)
Take DTPA-Zn quality-control sample working solutions to add in blank human urine, be prepared into 10.0 μ g/mL LLOQ samples；Remaining
Operated by under " preparation of (20d) quality-control sample " item, and carry out LC-MS/MS analyses；
The preparation of (20f) cross jamming test specimen
The μ L of DTPA-Zn working solutions 40 are taken, are placed in 1.5mL centrifuge tubes, blank human urine is added, vortex 3min is prepared into
The ULOQ samples of 2000 μ g/mL not containing the internal standards；In addition to internal standard is not added with, remaining is pressed and operated under " preparation of (20d) quality-control sample " item,
And carry out LC-MS/MS analyses.
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