nanomaterials
Article
Determination of Non-Transferrin Bound Iron, Transferrin Bound Iron,Drug Bound Iron and Total Iron in Serum in a Rats after IV Administration of Sodium Ferric Gluconate Complex by Simple
Ultrafiltration Inductively Coupled Plasma Mass Spectrometric Detection
Murali K.Matta Christopher R.Beekman1,Adarsh Gandhi1,Suresh Narayanasamy1, Christopher D.Thomas1,Adil Mohammad2,Sharron Stewart1,Lin Xu1,
Ashok Chockalingam1,Katherine Shea1,Vikram Patel1and Rodney Rou1,*
1U.S.Food and Drug Administration,Center for Drug Evaluation and Rearch,Office of Translational Science,Office of Clinical Pharmacology,Division of Applied Regulatory Science,Silver Spring,MD20993, USA;murali.matta@v(M.K.M.);christopher.beekman@v(C.R.B.);
adarsh.gandhi@v(A.G.);Suresh.narayanasamy@v(S.N.);thomdc15@wfu.edu(C.D.T.);
sharron.stewart@v(S.S.);lin.xu@v(L.X.);ashok.chockalingam@v(A.C.);
katherine.shea@v(K.S.);vikram.patel@v(V.P.)
融资性担保公司管理暂行办法2U.S.Food and Drug Administration,Center for Drug Evaluation and Rearch,Office of Pharmaceutical Quality,Office of Testing and Rearch,Division of Product Quality Rearch,Silver Spring,MD20993,USA;
*u@v;Tel.:+1-301-796-3914
Received:13December2017;Accepted:8February2018;Published:11February2018
喜耕田的故事
Abstract:A rapid,nsitive and specific ultrafiltration inductively-coupled plasma mass spectrometry method was developed and validated for the quantification of non-transferrin bound iron(NTBI), transferrin bound iron(TBI),drug bound iron(DI)and total iron(TI)in the same rat rum sample after intravenous(IV)administration of iron gluconate nanoparticles in sucro solution(Ferrlecit®). Ultrafiltration with a30kDa molecular cut-offfilter was ud for sample cleanup.Different elution solvents were ud to parate each form of iron from sample rum.Isolated fractions were subjected to inductively-coupled mass spectrometric analysis after microwave digestion in4%nitric acid. The reproducibility of the method was evaluated by precision and accuracy.The calibration curve demonstrated linearity from5–500ng/mL with a regression(r2)of more than0.998.This method
was effectively implemented to quantify rat pharmacokinetic study samples after intravenous administration of Ferrlecit®.The method was successfully applied to a pharmacokinetic(PK)study of Ferrlecit in rats.The colloidal iron followedfirst order kinetics with half-life of2.2h and reached background or pre-do levels after12h post-dosing.The drug shown a clearance of0.31mL/min/kg and volume of distribution of0.05L/kg.19.4±2.4mL/h/kg.
Keywords:inductively coupled plasma-mass spectrometry;ultrafiltration;pharmacokinetics;iron; iron gluconate;nanoparticles
1.Introduction
Iron is an esntial component of every cell in the body.Iron acts as a carrier for electrons,a catalyst for oxygenation and hydroxylation,and is necessary for cellular growth and proliferation.However,its most critical role is as a component of hemoglobin in the transport and storage of oxygen[1].Without Nanomaterials2018,8,101;doi:10.3390//journal/nanomaterials
a sufficient supply of iron,hemoglobin cannot be synthesized and the number of erythrocytes in the blood cannot be maintained at an adequate level[2].Iron supplements are widely administered to treat iron deficiency anemia,particularly in chronic dias such as kidney dia[1],heart failure[3]
or inflammatory bowel dia[4].In the dia conditions,intravenous(IV)iron colloidal products are being ud to treat rious iron deficiency anemias particularly,tho requiring dialysis.
Iron colloidal products are iron carbohydrate complexes consisting of a mineral core,compod of polynuclear iron(III)-hydroxide surrounded by a carbohydrate ligand[5].The main function of the ligand is to stabilize the complex and to protect it against further polynuclearization.In the first step of drug uptake,the stable iron-carbohydrate complexes are taken up by macrophages of the reticuloendothelial system(RES)[5]followed by fusion of the endosome with a lysosome releasing iron from the complex.The Fe2+generated is transported by the divalent metal transporter 1(DMT1)across the endolysosomal membrane to enter the labile iron pool within the macrophage cytoplasm.From there,it can be incorporated into ferritin and remain transiently stored within the macrophage or can be transported out of the macrophage by the transmembrane protein ferroportin (as Fe2+).The exported Fe2+is immediately oxidized by ceruloplasmin to Fe3+which is questered by transferrin for transport in the rum to sites of utilization,for example,in the bone marrow for hemoglobin synthesis or in the liver for storage in ferritin.
Upon IV administration of iron colloidal products,depending on the do,there could be a saturation of the blood transporter protein,transferrin,resulting in circulating non-transferrin protein bound(Fe3+)
(NTBI)iron in rum.This NTBI is potentially harmful due to its ability to form free radicals,thereby inducing oxidative stress and cellular toxicity[6,7].Hence,it is very uful to monitor the real NTBI,or“free iron”,in circulating blood to evaluate the possibility of toxicity upon IV administration of iron colloidal products.Accurate measurement of NTBI is difficult in the prence of other iron sources like transferrin bound iron(TBI)and drug bound iron(DI).DI is the iron that forms the nanoparticle core encapsulated inside the carbohydrate shell.DI is stable in rum with particle uptake and iron extraction facilitated by macrophages,as described above[5,8].While many analytical methods are available in the literature for the determination of iron[8–20],no suitable analytical method was available for accurately measuring all iron forms including total iron(TI),TBI,NTBI,and DI in a pharmacokinetic rat study.A few discrete methods were reported for determination of free iron and TBI.Kolb et al.[17]compared veral available methods for measurement of free iron,of which five were bad on detection with iron chelators and one assay measured redox-active iron using bleomycin.However,it was evident from reports in the literature that the nature and concentration of the chelating agent influenced the measurement of free iron[17].Internal unpublished studies suggested that in the prence of DI,bleomycin incubation requirements resulted in relea of iron from the drug and caud the over estimation of NTBI.
The very small sample volumes obtained in rial sampling of rats preclude the u of other analytical methods for determination of TBI or and total iron[18,19]that either require larger sample volumes and/or do not allow concurrent quantification of NTBI and DI.To support a rat pharmacokinetic study,an analytical method capable of quantifying NTBI,TBI,DI and TI in small sample volumes was required.To date no suitable analytical method had been described in the literature to accurately measure the all the fractions in small sample volumes.Hence,the authors developed a simple and robustfiltration technique capable of parating NTBI,TBI,and DI species (fractions)with a parate simple measure of TI.Iron concentration of all fractions and TI were then determined by inductively coupled plasma mass spectrometry(ICP-MS).During method development, the authors investigated the impact of a commonly employed chelator and bleomycin incubation conditions on estimate of NTBI to inform a method less biad toward NTBI over estimation.This less biad and validated method was then effectively applied for quantification of all forms of iron species in a rat pharmacokinetic study.
2.Materials and Methods
2.1.Chemicals
All the chemicals ud were of analytical reagent grade.Nitrilotriacetic acid(NTA),sodium thioglycollate(TGA),BPP3-morpholinopropane-1-sulfonic acid,3-(N-morpholino)propane sulfonic acid(MOPS),ascorbic acid,sodium dithionite,holo-transferrin,DNA from calf thymus,bleomycin sulfate,magnesium chloride,thiobarbituric acid,butanol,hydrochloric acid,bovine rum albumin and magnesium chloride were purchad from Sigma Aldrich(St.Louis,MI,USA).Formic acid was procured from Fluka(St.Louis,MI,USA).Amicon®Ultra0.5mLfilters(30kDa)for protein purification and concentration were purchad from EMD Millipore(Billerica,MA,USA).Ultra-pure nitric acid was obtained from Fisher Scientific(Waltham,MA,USA).Ferrlecit®.Two different standard solutions of iron(Fe)(1000ppm)were purchad from two parate sources Perkin Elmer(Shelton, CT,USA)and High Purity Standards(Charleston,SC,USA).A solution of1000ppm Indium(In) was purchad from Perkin Elmer(Shelton,CT,USA).Commercial drug product was obtained from
邮政日a retail pharmacy.
2.2.Instrumentation
A Perkin Elmer(Shelton,CT,USA)Nexlon300D inductively coupled plasma mass spectrometry (ICP-MS)was ud for analysis.A Synergy MX,BioTek,spectrophotometer(Winooski,VT,USA),was ud for
colorimetric experiments.A Heraeus Multifuge X1R(Thermo Scientific,Waltham,MA,USA) centrifuge was employed in the preparation of the rum ultrafiltrates.
2.3.Propod Bioanalytical Method
The propod bioanalytical method has three different steps:ultrafiltration,sample digestion of filtered samples and ICP-MS analysis.
2.3.1.Ultrafiltration Method
A simple ultrafiltration technique was ud with a modification of the method propod by Kolb et al.[17].Serum samples were frozen at−20◦C until time of analysis and thawed before u. An aliquot of25µL of rum was mixed with225µL of magnesium chloride solution(0.2M)and allowed to stand at room temperature for20min.The solution was then ultra-filtered using Amicon ultrafilters(MW30,000Da cutoff),with an applied centrifugal force of14,000g for10min to parate the resulting free iron as ferric chloride complex from TBI and DI.As a cond step,the samefilter was treated with200µL of magnesium chloride solution(0.5%formic acid)and thefilter was again centrifuged at14,000g for10min to parate TBI from thefilter while retaining DI.As afinal step, the samefilter was treated with50µL sodium dithionite(1M)and150µL of magnesium chloride solution.The 杨戬的师傅
solution was ultra-filtered at14,000g for10min to elute the DI into thefiltrate.A parate sample was prepared for the estimation of total iron by microwave digestion.The detailedflow chart of sample preparation is reprented in Figure1.
2.3.2.Sample Digestion
Each fraction offiltrate was pre-digested in4%nitric acid in microwave before ICP-MS analysis. The parameters of the microwaving cycle were as follows:power1200W;temperature,200◦C; pressure,200psi;control style,ramp to temperature;hold time,20min;and stirring,off.A t of quality control(QC)samples were prepared to evaluate the efficiency,accuracy and reproducibility of the propod ultrafiltration steps.Iron is an endogenous compound obrved even in blank rum samples.Hence,5%bovine rum albumin(BSA)solution in water was ud as a surrogate matrix for the preparation of quality control(QC)samples.The QC samples at four levels were prepared by castte spiking of elemental iron,holo-transferrin and drug formulation reflecting the anticipated葫芦条
pharmacokinetic samples after IV administration of iron colloidal formulation.The QC samples were subjected to filtration as mentioned earlier and analyd by the ICP-MS method after microwave digestion of each
sample.
Figure 1. Detailed methodology of ultra-filtration procedure. 2.3.2. Sample Digestion
Each fraction of filtrate was pre-digested in 4% nitric acid in microwave before ICP-MS analysis. The parameters of the microwaving cycle were as follows: power 1200 W; temperature, 200 °C;
pressure, 200 psi; control style, ramp to temperature; hold time, 20 min; and stirring, off. A t of
quality control (QC) samples were prepared to evaluate the efficiency, accuracy and reproducibility of the propod ultrafiltration steps. Iron is an endogenous compound obrved even in blank rum samples. Hence, 5% bovine rum albumin (BSA) solution in water was ud as a surrogate matrix for the preparation of quality control (QC) samples. The QC samples at four levels were prepared by castte spiking of elemental iron, holo-transferrin and drug formulation reflecting the anticipated pharmacokinetic samples after IV administration of iron colloidal formulation. The QC samples
were subjected to filtration as mentioned earlier and analyd by the ICP-MS method after microwave digestion of each sample. 2.3.3. ICP-MS Method The optimized operating conditions ud for ICP-MS are summarized in Table 1. A peristaltic pump was ud to deliver the samples (0.1 mL/min) to the nebulizer, which in turn converted the sample into a spray mist using argon (Ar) gas. Instrumental ttings were optimized daily prior to
analysis using a tuning solution consisting of Boron (B), Barium (Be), Iron (Fe), Indium (In), Lithium
(Li), Magnesium (Mg), Lead (Pb) and Uranium (U) to establish system suitability for daily operation.
Automated adjustments were made for torch alignment, detector voltage and ion lens voltages for
optimized resolution, nsitivity, and stability across a broad range of atomic mass. The doubly
charged ion/charge ratio (Ce 2+ 69.95 to Ce 139.90) and oxide ratio (CeO + 155.90 to Ce 139.90) were also monitored and were maintained below 3% and 2.5% (intensity) respectively. The PrepFast system was interfaced to the ICP-MS and ud to auto-dilute the stock solutions to generate the calibration curves and QCs samples. The 30 ppb indium (In) solution was also mixed in-line by the Prep-Fast during the analysis as internal standard to all the analyzed samples and standards. The method was evaluated for its lectivity, nsitivity, linearity, precision and accuracy. Figure 1.Detailed methodology of ultra-filtration procedure.
2.3.3.ICP-MS Method The optimized operating conditions ud for ICP-MS are summarized in Table 1.A peristaltic pump was ud to deliver the samples (0.1mL/min)to the nebulizer,which in turn converted the sample into a spray mist using argon (Ar)gas.Instrumental ttings were optimized daily prior to analysis using a tuning solution consisting of Boron (B),Barium (Be),Iron (Fe),Indium (In),Lithium (Li),Magnesium (Mg),Lead (Pb)and Uranium (U)to establish system suitability for daily o方展发
peration.Automated adjustments were made for torch alignment,detector voltage and ion lens voltages for optimized resolution,nsitivity,and stability across a broad range of atomic mass.The doubly charged ion/charge ratio (Ce 2+69.95to Ce 139.90)and oxide ratio (CeO +155.90to Ce 139.90)were also monitored and were maintained below 3%and 2.5%(intensity)respectively.The PrepFast system was interfaced to the ICP-MS and ud to auto-dilute the stock solutions to generate the calibration curves and QCs samples.The 30ppb indium (In)solution was also mixed in-line by the Prep-Fast during the analysis as internal standard to all the analyzed samples and standards.The method was evaluated for its lectivity,nsitivity,linearity,precision and accuracy.Table 1.Inductively coupled plasma mass spectrometry (ICP-MS)operating conditions and acquisition parameters.ICP Parameters
RF Power 1600W Plasma gas flow rate 18.0L/min Auxiliary gas flow rate 1.2L/min Nebulizer gas flow rate 1.0L/min Spray chamber Peltier-cooled (2◦C)baffled quartz cyclonic spray chamber Torch
Quartz Sampler and skimmer cones
Platinum Hyper skimmer cones Nickel
Table1.Cont.
一二年级消防绘画Mass Spectrometer Parameters
小草湖天气预报Resolution0.7amu at10%peak maximum
Dwell time50ms
Sweeps20
Readings1
Replicates3
Autolens ON
Internal Standard In(115amu)
Mode of detection Kinetic Energy Differentiation(KED)
2.4.In Vitro Incuation Studies to Evaluate Advantages of Propod Method
In the literature,two different methods were described for the measurement of NTBI in rum sample
s,one method involves the u of chelating agent(NTA)in the ultrafiltration step[17]and the other one is incubating with non-chelating agent(bleomycin)[20]followed by colorimetric detection. However,the methods were not thoroughly evaluated for accuracy in prence of DI,hence a t of incubation studies were designed to evaluate the effect of assay conditions/reagents on the artifacts of the assay measurements and the efficiency of the propod ultrafiltration method using MgCl2 solution over the reported methods for NTBI measurements.
Incubation studies with chelating agent(NTA):Studies were conducted to asss the impact of incubation with chelating agent(NTA)and non-chelating agent like bleomycin on measured free iron concentrations.The potential iron leaching effect of NTA on iron colloidal formulation was evaluated by incubating the iron colloidal drug formulation(100µg/mL in5%BSA)with0.8M of NTA and collecting samples after15,30,60and120min.The results were then compared to aliquots of the same samples ud with magnesium chloride solution as per the propod ultrafiltration method.The concentration of all the samples were measured by the reported spectrophotometric method[17] and validation results were prented in Table S1.
Incubation studies with nonchelating agent(Bleomycin):The bleomycin assay requires incubation of the samples with bleomycin at37◦C for two hours.Experiments were designed to evaluate the effect
of high temperature and bleomycin on measured free iron concentrations.To evaluate the impact of bleomycin incubation on measured free iron concentrations in the bleomycin assay,rum samples containing all iron species(harvested15min after IV drug bolus)were obtained and incubated at 37◦C for two hours with and without bleomycin.To asss the impact of temperature aliquots of the same samples were kept at room temperature with and without bleomycin.All samples were pasd through the30kDa ultrafiltration step using MgCl2as described in propod method.The ultra filtrates were ud for the bleomycin assay[20],which was validated at in-hou and results were reported in Table S2.The effects of bleomycin incubation on free iron measures in the prence of holo-transferrin iron and drug iron were investigated in vitro.
2.5.Pharmacokinetic Study in Sprague Drawley Rats
Animals:All animal experiments were conducted under an approved protocol and oversight of the Food and Drug Administration’s White Oak Federal Rearch Center Institutional Animal Care and U Committee.Animal rearch was conducted in accordance with the Guide for the Care and U of Laboratory Animals,8th Edition(NRC2011).Dual cannulated male Sprague Dawley rats approximately300–350g were purchad from Taconic Farms(Derwood,MD,USA).Each rat had a cannula placed in the right jugular vein and in the left femoral vein.Animals were kept on a12-h light
cycle and received food and water ad libitum.All rats were acclimated for a minimum of7days prior to initiation of experiments.
Pharmacokinetic study design:The developed method was ud to analyze rum samples from a rat pharmacokinetic study.Pharmacokinetic studies were conducted in16male Sprague Drawly
