• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Long-term stability of gentamicin sulfate-ethylenediaminetetraacetic acid disodium salt(EDTA-Na2)solution for catheter locks

    2018-12-10 12:02:38AnneSophieFioletEliseJndotPulineDouceyCorlieCrtetliBrunelChristinePivotJenMrcGhigoChristopheBeloinDvidLeeuxFricePirot
    Journal of Pharmaceutical Analysis 2018年6期

    Anne-Sophie Fiolet,Elise Jndot,Puline Doucey,Corlie Crétet,Céli Brunel,Christine Pivot,Jen-Mrc Ghigo,Christophe Beloin,Dvid Leeux,c,d,Frice Pirot,e,*,2

    aService Pharmaceutique,Plateforme FRIPHARM,Groupe Hospitalier Centre Edouard Herriot,Hospices Civils de Lyon,5,Place d′Arsonval,F-69437 Lyon Cedex 03,France

    bUnité de Génétique des Bio films,Département de Microbiologie,Institut Pasteur,28 rue du docteur Roux,F-75724 Paris Cedex 15,France

    cService de Microbiologie,Unité Mobile de Microbiologie Clinique,Assistance Publique-H?pitaux de Paris,H?pital Européen Georges Pompidou,20 rue Leblanc,75015 Paris,France

    dUniversité Paris Descartes,12 rue de l′Ecole de Médecine,75270 Paris Cedex 06,France

    eLaboratoire de Recherche et Développement de Pharmacie Galénique Industrielle,UMR 5305,Plateforme FRIPHARM,F-69373,Faculté de Pharmacie,Université Claude Bernard Lyon 1,8,avenue Rockefeller,F-69373 Lyon Cedex 08,France

    Keywords:Gentamicin-EDTA-Na2loaded antimicrobial lock solution Pharmaceutical compounding Stability indicating HPLC assay method Totally implantable venous access ports

    A B S T R A C T A lock solution composed of gentamicin sulfate(5 mg/mL)and ethylenediaminetetraacetic acid disodium salt(EDTA-Na2,30 mg/mL)could fully eradicate in vivo bacterial biofilms in totally implantable venous access ports(TIVAP).In this study,fabrication,conditioning and sterilization processes of antimicrobial lock solution(ALS)were detailed and completed by a stability study.Stability of ALS was conducted for 12 months in vial(25°C ± 2°C,60% ± 5%relative humidity(RH),and at 40°C ± 2°C,RH 75% ± 5%)and for 24 h and 72 h in TIVAP(40°C ± 2°C,RH 75% ± 5%).A stability indicating HPLC assay with UV detection for simultaneous quantification of gentamicin sulfate and EDTA-Na2was developed.ALS was assayed by ion-pairing high performance liquid chromatography(HPLC)needing gentamicin derivatization,EDTA-Na2metallocomplexation of samples and gradient mobile phase.HPLC methods to separate four gentamicin components and EDTA-Na2were validated.Efficiency of sterility procedure and conditioning of ALS was confirmed by bacterial endotoxins and sterility tests.Physicochemical stability of ALS was determined by visual inspection,osmolality,pH,and sub-visible particle counting.Results confirmed that the stability of ALS in vials was maintained for 12 months and 24 h and 72 h in TIVAP.

    1.Introduction

    Central venous catheters(CVC)are frequently used in oncology,nephrology and intensive care units to administer medication or fluids to patients[1].The use of CVC is complicated by the risk of colonization by pathogenic microorganisms on their surface,causing infectious complications such as catheter-related bloodstream infections(CRBSI)[2].Optimal management of CRBSI involves systemic antimicrobial therapy and CVC removal,which might be questionable in specific clinical situations(e.g.,limited vascular access or bleeding disorders)[3].Therefore,direct instillation of a small volume of highly-concentrated antimicrobial solution into the lumen of the catheter,known as antimicrobial lock therapy(ALT),might be favored as an alternative strategy for both prevention and treatment of CRBSI[4,5].Several studies reported the use of antibiotics such as aminoglycosides,e.g.,gentamicin,[3],beta-lactams, fluoroquinolones,folate antagonists(sulfamethoxazole/trimethoprim), glycopeptides,glycylcyclines,lipopeptides,oxazolidinones,polymyxins,and tetracyclines in ALT[6].However,surface of medical device might be colonized by a reservoir of pathogenic microorganisms forming a biofilm,a complex microbial consortium surrounded by an heterogeneous matrix composed of water,polysaccharides,proteins and deoxyribonucleic acid[6],decreasing the antibiotic efficacy[7].Potential additives(e.g.,ethylenediaminetetraacetic acid disodium salt,EDTA-Na2,and sodium citrate)have shown their capacity to disrupt biofilm and to enhance bactericidal effect of current antibiotics[6,8–11].Recent in vitro and in vivo study demonstrated the beneficial effect of EDTA-Na2against Pseudomonas aeruginosa biofilm and the concomitant decrease of minimal inhibitory concentration of ciprofloxacin and ampicillin(30-fold)and gentamicin(two-fold)[12].An earlier in vivo study reported that an antimicrobial lock solution(ALS)composed of gentamicin(5 mg/mL)and EDTA-Na2(30 mg/mL)in association with systemic antibiotics completely eradicated biofilms formed by Escherichia coli,Pseudomonas aeruginosa,Staphylococcus epidermidis or Staphylococcus aureus on the surface of totally implantable venous access ports(TIVAP)[13].

    Gentamicin produced by Micromonospora purpurea is used as gentamicin sulfate which is a complex mixture of five structurally related components:C1,C1a,C2 and C2a and a minor component C2b[14].Previous basic stability study showed that gentamicin sulfate-EDTA-Na2antibiotic lock solution stored at 25 °C or 37 °C was visually stable for at least 72 h[3].

    In the present study,a process of pharmaceutical compounding and conditioning of gentamicin sulfate-EDTA-Na2lock solution is detailed and then completed by a study of stability according to the technical requirements of International Conference on Harmonization(ICH)for registration of pharmaceuticals for human use.The physicochemical stability of ALS conditioned in glass vial was analyzed(i)over 12 months of storage at 25°C ± 2 °C(relative humidity(RH)60%± 5%)and 40°C± 2°C(RH 75%± 5%),and(ii)after 24 h and 72 h of contact time in TIVAP at 40°C ±2°C(RH 75% ± 5%),by high performance liquid chromatography(HPLC)evidencing the impurities and degradation products of gentamicin sulfate and EDTA-Na2.

    2.Materials and methods

    2.1.Reagents

    Gentamicin sulfate and EDTA-Na2dihydrate physico chemical properties are reported in Table 1.Ready-to-use gentamicin sulfate sterile solutions(40 mg-2 mL and 80 mg–2 mL),and EDTA-Na2dihydrate (pharmaceutical grade) were purchased from Panpharma?(Beignon,France)and Inresa(Bartenheim,France)laboratories,respectively.Nitrilotriacetic acid and trifluoroacetic acid(HPLC grade,TFA)were purchased from Carl Roth(Lauterbourg,France)and Rh?ne Poulenc(Lyon,France),respectively.Hydrochloric acid(HCl)and sodium hydroxide(NaOH)were provided by Carlo Erba Reagents(Peypin,France).Hydrogen peroxide solution(3%)was purchased from Gifrer(Décines,France).Gentamicin for peaks identification(CRS,European Pharmacopoeia reference standard),gentamicin USP standard,sisomicin sulfate,EDTA disodium salt standard,ortho-phtalaldehyde(OPA),thioglycolic acid,boric acid and potassium hydroxide(KOH)were obtained from Sigma-Aldrich(Saint Quentin Fallavier,France).Acetonitrile and methanol(HPLC grade)were purchased from Merck Millipore(Molsheim,France).Copper sulfate was obtained from Cooper(Melun,France).Peptone water(0.1%)was purchased from Oxoid(Basingstoke,United Kingdom).Culture media for anaerobic bacteria( fluid thioglycollate medium),aerobic bacteria and fungi(soya bean casein digest medium),and neutralizing pharmacopoeia diluent used for the gentamicin neutralization were obtained from Biomérieux(Lyon,France).Water for injection was delivered by Lavoisier(Paris,France).

    2.2.Pharmaceutical compounding and conditioning of ALS

    EDTA-Na2solution(40 mg/mL)was prepared by dissolution of 22.5 g of EDTA-Na2in 562.5 mL of water for injection.Then,EDTANa2solution(40 mg/mL)was mixed to 187.5 mL of gentamicin sterile solution for injection(40 mg–2 mL).Finally,the pH value of the ALS was adjusted to 8.5 by adding few drops of 5 M NaOH solution.ALS was filtered through 0.22 μm sterile filter(Millex?GS,Millipore?,Molsheim,France),and then sampled(5 mL)in 150 individual amber type 1 glass injection vials(15 mL)hermetically sealed by bromobutyl stoppers in cleanroom.Finally,ALS vials were autoclaved at 121°C for 20 min[15,16].Final concentrations of gentamicin(5 mg/mL)and EDTA-Na2(30 mg/mL)in ALS were assayed by HPLC(see Section 2.6).

    2.3.Storage stability testing

    The environmental factors(temperature,RH)and time of storage upon degradation of gentamicin sulfate and EDTA-Na2in ALS conditioned in vials and then stored at 25°C± 2°C,RH 60% ± 5%and 40°C ± 2°C,RH 75% ± 5%in climatic test chambers(Froilabo,Meyzieu,France)for 12 months were investigated using HPLC(see Section 2.6)[17].Complementary physicochemical andmicrobiological controls of ALS were conducted over the period of storage(see Section 2.4).TIVAP,supplied by Perouse Medical(polysite 4000,4008 IPS,Ivry le Temple,France),was used to test compatibility between ALS and implantable port.This medical device is made up of a chamber in titanium/polyoxymethylene and a silicone catheter.Total dead space of implantable port was 1.38 mL and internal surface of silicone catheter was 6.6 cm2.One mL of ALS was injected into the device and left for 24 h and 72 h at 40 °C ± 2°C,RH 75% ± 5%.To detect a potential water loss through polymeric wall,implantable port was weighted before and after filling throughout storage.Gentamicin and EDTA-Na2contents after 24 h and 72 h of contact time in chambers(n=3)were assessed by HPLC(see Section 2.6).

    Table 1 Physicochemical properties of gentamicin sulfate and EDTA-Na2dihydrate(from Pubmed open chemistry database,National Institute of Health).

    2.4.Physicochemical and microbiological controls of ALS

    Physicochemical controls of ALS included pH measurement(EcopHtest2,Thermo Fisher Scientific,Massachusetts,USA)and determination of osmolality(Fiske?,Norwood,MA,USA).Controls of visual aspect,particulate contamination of ALS by visible particles(2.9.20 European Pharmacopoeia Monograph,2016)and subvisible particles(2.9.19 European Pharmacopoeia Monograph,2016,method 1:light obscuration particle count test,Hiac/Royco 9703+,Pacific Scientific Instruments,IL,USA)were carried out after autoclaving.Microbiological assays of ALS were carried out by membrane filtration using Steritest?closed filtration device[18]with special low-binding Durapore?membranes for products with inhibitory properties,e.g.antibiotics(Millipore,Merck KGaA,Darmstadt,Germany),peptone water(0.1%)as rinse fluid,neutralizing pharmacopoeia diluent and both soya-bean casein digest medium and fluid thioglycollate medium(2.6.1 European Pharmacopoeia Monograph,2016).

    2.5.Preparation of gentamicin sulfate and EDTA-Na2standard and ALS assay solutions

    A derivatization reaction of gentamicin sulfate with OPA allowing a detection gentamicin OPA derivative was adapted from previous reports[19–21].A stock solution of derivatizing agent was prepared by dissolving 250 mg of OPA in 1.25 mL of methanol,then addition of 23.75 mL of boric acid solution(2.47%,m/v;pH adjusted to 10.4 by 8 M KOH solution)and 0.5 mL of thioglycolic acid.Finally,the pH value of derivatizing agent solution was adjusted to 10.4 using 8 M KOH solution,for pre-column derivatization.The stock solution of derivatizing agent,stored at 4°C and protected from light,was stable for three days.Therefore,2.5 mL of gentamicin sulfate standard solution or ALS was then mixed with 0.5 mL of stock solution of derivatizing agent and 2.75 mL of methanol( final volume:5.75 mL).Although usually derivatization was carried out at 60°C constant temperature[20,22],no heating was applied to gentamicin–OPA based solution,taking into account the lack of stability of such chemical reaction and the short half-life of reaction products,as reported elsewhere[23].

    The determination of EDTA-Na2in ALS was allowed by conversion to Cu(II)EDTA complex[24]using copper sulfate containing solution and subsequent separation from gentamicin,formulation excipients,impurities,and potential degradation products by HPLC(see Section 2.6).Therefore,2.5 mL of EDTA-Na2standard solution or ALS were then mixed with 2.5 mL of copper sulfate solution(10 mg/mL)and 0.75 mL of water for injection( final volume:5.75 mL).

    Gentamicin sulfate-standard EDTA-Na2solutions were prepared daily by dissolving the appropriate amount of EDTA-Na2in water for injection,and then mixing with gentamicin sulfate sterile solution for injection(40 mg–2 mL or 80 mg–2 mL)to obtain,after gentamicin sulfate derivatization orEDTA-Na2complexation, final concentrations of gentamicin sulfate and EDTA-Na2in the range of 87-260 μg/mL and 0.52-1.56 mg/mL,respectively.

    2.6.Stability indicating HPLC assay

    An HPLC Agilent?(Agilent?1290 In finity Quaternary LC System,Les Ulis,France),equipped with a binary pump with integrated vacuum degasser,a thermostated column compartment(40°C),an autosampler and a diode array detector,was used for gentamicin and EDTA dosages.The separation of gentamicin and EDTA-Na2was accomplished using Security Guard?cartridge and column Kinetex? (C18100 ?,100 mm × 4.6 mm,2.6 μm)Core-Shell Technology(Phenomenex?,Le Pecq,France).The detection wavelength was set at 330 nm and injection volume was 10 μL.The mobile phase was a binary mixture of 100 mM TFA,used as pairing reagent in water(phase A,the pH value was adjusted to 3 by adding 5 M NaOH solution)and acetonitrile(phase B)in a gradient elution mode at a flow-rate of 1 mL/min[25,26].The mobile phase A was filtered and degassed through nylon membranes(0.20-μm pore size)under vacuum before use.A linear gradient elution was programed as 95%A-5%B(v/v;0 min),95%A-5%B(v/v;2 min),50%A-50%B(v/v;10 min),and 40%A-60%B(v/v;15 min).Therefore,the resolved peaks of gentamicin sulfate OPA derivatives,C1,C1a,C2a and C2,and EDTA-Na2were identified from gentamicin for peaks identification(CRS,European Pharmacopoeia reference standard),gentamicin(216 μg/mL)USP standard solutions,and EDTA(1.3 mg/mL)standard solution.Sisomicin and nitrilotriacetic acid,as gentamicin sulfate and EDTA-Na2respective impurities,were identified from sisomicin(4.65 mg/mL)and nitrilotriacetic acid(43.5 μg/mL)standard solutions.

    2.7.HPLC assay validation criteria

    The HPLC-UV analytical methods for gentamicin sulfate and EDTA-Na2were validated for specificity,precision(repeatability,intermediate precision),linearity,limits of detection(LOD)and quantification(LOQ),accuracy/recovery and robustness to include the essential requirements of ICH guidelines[27,28].The validation protocol was conducted on three consecutive days by the same operator and fresh solutions were prepared daily.Five standard gentamicin sulfate and EDTA-Na2solutions were prepared to enable the determination of drugs concentrations in ALS.Furthermore,forced degradation of ALS was investigated(i)by acidic(0.1–1 M HCl)and alkaline(0.1–1 M NaOH),(ii)heating(80°C for 1 h)treatments,(iii)under oxidative conditions using 3%H2O2then heating at 80 °C for 3 h,and(iv)from UVA(320–400 nm)irradiation for 6 h[29].

    3.Results and discussion

    3.1.Physicochemical and microbiological controls of ALS

    Results of visual inspections for color,clarity,and particles completed by sub-visible particles counting,pH and osmolality assessments of ALS throughout the 12 months storage at 25°C ±2°C,RH 60% ± 5%and 40°C ± 2°C,RH 75% ± 5%are summarized in Table 2.No significant macroscopic and/or microscopic alterations of ALS were evidenced.Efficiency of sterility procedure and conditioning of ALS was confirmed by the absence of microbial growth and endotoxin.No impact of autoclaving either upon particle quality or colligative property of ALS was observed.Interestingly,ALS was isosmotic to serum compared to hypoosmotic commercial gentamicin sulfate solution(Panpharma?)in favor of further easy handling in TIVAP filling and blood compatibility.

    Table 2 Characterization of ALS stability over 12 months of storage at 25°C± 2°C,RH 60% ± 5%(at 40°C ± 2°C,RH 75% ± 5%).Each data is the mean± standard deviation of three experimental determinations.nd:Not determined.

    Fig.1.(A)A typical chromatogram of gentamicin standard solution(USP)(216 μg/mL)after OPA derivatization.(B)A typical chromatogram of sisomicin after OPA derivatization(4.65 mg/mL).(C)A typical chromatogram of ALS after OPA derivatization(gentamicin 217 μg/mL).(D)Analysis of ALS(gentamicin 217 μg/mL)under oxidative conditions(3%H2O2,80°C for 3 h)after OPA derivatization.

    Table 3 Identification of the gentamicin sulfate components in gentamicin USP standard solution(217 μg/mL)and in ALS according to the requirements of US Pharmacopeia USP37-NF32 monograph.Relative content(%)of each component was calculated as the area of each individual peak divided by the sum of all peak areas.The sum of all peak areas(C1a,C2 C2a,C2b and C1)corresponds to 100%.The elution order is gentamicin C1,C1a,C2a and C2.

    3.2.HPLC assay validation

    Gentamicin sulfate and EDTA-Na2contents in ALS conditioned in amber type I glass vials were analyzed during the storage using HPLC method adapted,for a part,from US Pharmacopoeia Monograph[22]and earlier studies[20,21,24].The chemical structure of gentamicin reveals the lack of chromophore in the molecule,making the direct detection of the antibiotic difficult.Furthermore,besides methods based on microbiological assay[30],enzymeimmunoassay,polarization fluoroimmunoassay[31,32],direct detection methods,e.g.,electrochemical detection[14,33,34],evaporative light scattering detection[35,36],charged aerosol detection[26,37],direct capillary electrophoresis[38]require specific and costly instrumentation(e.g.,tandem mass spectrometer,[39])are not commonly found in quality control laboratories.In the present study,gentamicin sulfate components were assayed by UV-detection after pre-column derivatization with OPA based reagent which reacts only with three primary amines of gentamicin to form UV-absorbing fluorophores,ionpairing chromatographic gradient and separation by a combination of electrical(charge-charge between two underivatized secondary amines of gentamicin positively charged at acidic pH of mobile phase)and hydrophobic interactions with the stationary phase and ions of the mobile phase[25].No pre-heating of gentamicin-OPA based mixture was carried out,as described by previous authors[19],eliminating tedious manual procedures,reducing error,and thereby increasing method reproducibility.The column was maintained at 40°C,leading likely to more reproducible in situ derivatization of gentamicin components passing through the C18stationary phase.

    Fig.2.(A)A typical chromatogram of EDTA-Na2(USP)(1.3 mg/mL)after CuSO4complexation.(B)A typical chromatogram of nitrilotriacetic acid(43 μg/mL)after CuSO4 complexation.(C)A typical chromatogram of ALS(EDTA-Na2:1.3 mg/mL)after CuSO4complexation.(D)Analysis of ALS(EDTA-Na2:1.3 mg/mL)under stress oxidative conditions(3%H2O2,80°C for 3 h)after CuSO4complexation.

    Fig.3.(A)A typical calibration curve of the cumulative peak area(C1,C1a,C2a,C2)of gentamicin sulfate(Panpharma?)(a)and four components(b,c,d and e)ranged from 87 to 260 μg/mL.(B)A typical EDTA-Na2calibration curve ranged from 0.52 to 1.56 mg/mL.

    Fig.4.Linear relationship between water loss through TIVAP and time of storage at 40°C ± 2°C,RH 75% ± 5%.

    Fig.1A shows typical chromatogram of gentamicin standard solution.The four components of gentamicin(C1,C1a,C2a and C2)were eluted as distinct peaks with retention time of 9.7,12.6,13.3 and 13.5 min,respectively in the order reported by US Pharmacopoeia Monograph[22].Gentamicin C2b component was below LOD.Furthermore,the relative content of each gentamicin component in gentamicin standard was found in agreement with US Pharmacopoeia Monograph requirements,as reported in Table 3.These findings were similar to those reported in previous study[20],in which the chromatogram of gentamicin sulfate solution(2 mg/mL)prepared from the gentamicin sulfate powder met the USP test specifications.The USP method describes the elution order as C1,C1a,C2a and C2 gentamicin with a 5 mm×10 cm column that contains 5μm packing L1(octadecyl silane chemically bonded to porous or nonporous silica or ceramic microparticles,1.5–10 μm in diameter,or a monolithic silica rod).In the present method,Kinetex? (C18100 ?,100 mm × 4.6 mm,2.6 μm,Core-Shell Technology)column was preferred to Luna?C18(2)column used by earlier authors[20].Therefore,the elution of last gentamicin component(C2)was nearly 2.5 times shorter(13.5 min versus 31 min)in the present method than that reported previously[20],con firming that the combination of the small particle size and narrow particle size distribution coupled with the significantly shorter diffusion path resulted in a material that yielded significantly increased column efficiency and chromatographic resolution.Chromatographic separation was shortened mandatory,considering that isoindoles formed during derivatization are generally highly reactive compounds.As a result,it was considered that any derivative breakdown during chromatography would be time-dependent and that component derivatives with long retention time(C2a and C2)would be particularly affected by degradation prior to detection[40].Previous analysis of gentamicin in raw material used column(150 mm×4.6 mm)packed with Ul trasphere?ODS(C18)and elution gradient(A:5% acetic acid-25%water-70%methanol and B:100%methanol)for faster elution of C1(5 min)and similar elution time for C2(13.7 min)[41].In the present study,longer elution time for C1(~10 min,Fig.1C)was preferred to elute first the most hydrophilic components of ALS(e.g.,excipients from gentamicin sulfate sterile solution for injection,Panpharma?).Interestingly,it was noticed that the relative content of gentamicin components was proportional to molecular weight of C1,C1a,C2a and C2 as equimolar amounts,con firming that the sum of the areas of the four gentamicin components was a relevant measure of the gentamicin concentration.Sisomicin chromatogram shows one main peak surrounded by three minor peaks(Fig.1B).Possible partial derivatization of one,two or three primary amines of sisomicin might explain the presence of numerous peaks.

    The determination of EDTA-Na2in ALS by direct UV detection was challenging since EDTA-Na2does not contain a significant chromophore.In the present study,EDTA-Na2in ALS was assayed by ion-pairing HPLC and metallocomplex formation by using TFA in mobile phase and copper sulfate in sample preparation.Typical chromatogram of EDTA-Na2eluted as EDTA-Cu2+is shown in Fig.2A.As reported in previous study assaying EDTA-Na2in pharmaceutical formulation,EDTA-Cu2+,as ionic analyte,showed minimal retention(Fig.2C,<1 min)through reversed-phase by using liquid chromatography with ion-pairing and elution gradient[42],and co-elution of nitrilotriacetic acid-Cu2+complex(Fig.2B).

    3.3.HPLC validation criteria

    3.3.1.Specificity

    The specificity of the assay was conducted to evaluate the assay for potential sources of interfering peaks from the matrices used in sample preparation.No interfering peaks were seen from waterused to prepare mobile phase,the mobile phase or a blank filter media.No significant macroscopic and/or microscopic alterations of ALS were evidenced in acidic(0.1–1 M HCl,at 80 °C for 1 h)and basic(0.1–1 M NaOH,at 80 °C for 1 h)conditions.ALS was found stable under heat conditions(at 80 °C)and under UVA(320–400 nm)irradiation for 6 h.Under oxidative stress conditions with 3%H2O2at 80°C for 3 h,EDTA-Na2was degraded to about 60%(Fig.2D)while all of gentamicin sulfate was degraded(Fig.1D).Four main degradation products were generated with retention time of 6.4,7.2,8.8 and 9.9 min that could correspond to the impurity A(e.g.,sisomicin,Fig.1B).

    Table 4 Determination of gentamicin and EDTA-Na2concentrations in ALS after 24 h and 72 h of contact time in TIVAP.Each data is the mean±standard deviation of three experimental determinations of concentrations in three TIVAP.Relative standard deviation(RSD)(%)of concentrations was calculated as follows:RSD(%)=(initial concentration–experimental concentration)/initial concentration.

    3.3.2.Precision

    Repeatability and intermediate precision of gentamicin sulfate-EDTA-Na2assays were found to be 1.82%-1.23%,and 2.16%-1.47%,respectively,con firming the overall precision of HPLC methods involving pre-column derivatization and metallocomplex procedure.

    3.3.3.Linearity

    The linearity of assay was determined to have a correlation coefficient(r)for gentamicin sulfate of 0.999(in the range of 87-260 μg/mL)and for EDTA-Na2of 0.993(in the range of 0.52-1.56 mg/mL)as shown in Figs.3A and B,respectively.

    3.3.4.Accuracy

    The recovery of gentamicin sulfate and EDTA-Na2 was found to be in the range of 97.96%–99.81%,and 100.72%–102.72%over all concentration range,respectively.

    3.3.5.LOD and LOQ

    LOD and LOQ for gentamicin were 8 μg/mL and 24 μg/mL,respectively.The LOD and LOQ for EDTA were found to be 45 μg/mL and 136 μg/mL,respectively.

    3.3.6.Robustness

    The robustness of the assay in terms of varied injection volume(5–15 μL)and flow rate(0.5 mL/min)did not significantly change the time retention,asymmetry and the percentage of target amounts in the samples of gentamicin sulfate and EDTA-Na2.

    3.3.7.Stability study of ALS in glass injection vials and in TIVAP

    Stability of gentamicin sulfate and EDTA-Na2in vials stored for 12 months was confirmed by minimal variation of drug concentration in ALS,as reported in Table 2.A loss of ALS was highlighted by weighing the device throughout contact time in TIVAP.Loss solution per cm2of catheter is represented in Fig.4.Solution loss was perfectly linear in function of time.After 24 h and 72 h,124 μL and 363 μL of ALS were evaporated through polymeric wall,respectively.At the same time,HPLC assay of ALS after contact in TIVAP showed that gentamicin and EDTA concentrations increased compared to initial concentrations in ALS(Table 4).After recalculation considering the extent of water loss(124 μL for 24 h and 363 μL for 72 h),gentamicin and EDTA concentrations were in accordance with initial concentrations in ALS(Table 4).

    4.Conclusion

    In this study,a process of pharmaceutical compounding and conditioning of ALS was performed.Numerous physicochemical analysis,including the development of validated HPLC methods for dual gentamicin and EDTA-Na2assays,showed a stability of ALS conditioned in amber type I glass vials for 12 months at 25°C ±2°C(RH 60% ± 5%)and 40°C ± 2°C(RH 75% ± 5%).Furthermore,complementary findings reported satisfactory stability of ALS in TIVAP for 24 h and 72 h.At the outset,the present study confirmed the pharmaceutical relevance of gentamicin-EDTA-Na2combined solution as a new antimicrobial lock therapy.

    Conflicts of interest

    The authors declare that there are no conflicts of interest.

    Acknowledgments

    This study was supported by Centre de Recherche Translationnelle de I′Institut Pasteur,grant Number S-PI15007-02A.JMG,CB and DL are supported by the French Government's Investissement d′Avenir program:Laboratoire d′Excellence ‘Integrative Biology of Emerging Infectious Diseases’(grant no.ANR-10-LABX-62-IBEID.),the Fondation pour la Recherche Médicale(grant no.DEQ.20140329508)and the Center for Translational Science of the Institut Pasteur(S-PI15007-02A).

    啪啪无遮挡十八禁网站| 午夜日韩欧美国产| 蜜桃久久精品国产亚洲av| 18禁黄网站禁片午夜丰满| 久久九九热精品免费| 一边摸一边抽搐一进一小说| 欧美成人a在线观看| 极品教师在线视频| 免费看美女性在线毛片视频| 亚洲av成人av| 久久欧美精品欧美久久欧美| 身体一侧抽搐| 小说图片视频综合网站| 日本成人三级电影网站| 欧美激情久久久久久爽电影| 欧美日韩国产亚洲二区| 免费av观看视频| 脱女人内裤的视频| 国产大屁股一区二区在线视频| 色av中文字幕| 可以在线观看的亚洲视频| 男插女下体视频免费在线播放| 91久久精品国产一区二区成人| 一个人免费在线观看的高清视频| 国产精品一区二区三区四区久久| 国内揄拍国产精品人妻在线| 五月伊人婷婷丁香| 大型黄色视频在线免费观看| 成年女人看的毛片在线观看| 激情在线观看视频在线高清| 18+在线观看网站| 成人一区二区视频在线观看| 国产av一区在线观看免费| 91麻豆av在线| 日本黄色片子视频| 国产麻豆成人av免费视频| 亚洲欧美日韩高清专用| 男插女下体视频免费在线播放| 最近最新免费中文字幕在线| 精品免费久久久久久久清纯| 亚洲狠狠婷婷综合久久图片| 国产三级黄色录像| 亚洲熟妇中文字幕五十中出| 久久精品国产清高在天天线| 搡老岳熟女国产| 午夜影院日韩av| 日韩人妻高清精品专区| 精品一区二区三区人妻视频| 亚洲 欧美 日韩 在线 免费| 亚洲成人免费电影在线观看| 国产亚洲精品久久久久久毛片| h日本视频在线播放| 老熟妇乱子伦视频在线观看| 欧美+日韩+精品| 免费av观看视频| 91在线观看av| 欧美日韩国产亚洲二区| 免费av观看视频| 亚洲成av人片在线播放无| 国产在线男女| 少妇的逼水好多| 国产亚洲精品av在线| 一个人看视频在线观看www免费| 欧美日韩国产亚洲二区| 国产毛片a区久久久久| 在线十欧美十亚洲十日本专区| 757午夜福利合集在线观看| 欧美日韩瑟瑟在线播放| 国产精品不卡视频一区二区 | 欧美成狂野欧美在线观看| 免费大片18禁| 久久精品夜夜夜夜夜久久蜜豆| 国产伦一二天堂av在线观看| 精品国内亚洲2022精品成人| 国产aⅴ精品一区二区三区波| 最近最新中文字幕大全电影3| 夜夜躁狠狠躁天天躁| 亚洲三级黄色毛片| 美女被艹到高潮喷水动态| 欧美潮喷喷水| 国产探花在线观看一区二区| 国内精品久久久久精免费| 看免费av毛片| 国产精品电影一区二区三区| 麻豆一二三区av精品| 亚洲国产日韩欧美精品在线观看| 欧美绝顶高潮抽搐喷水| 日韩精品中文字幕看吧| 黄色日韩在线| 波野结衣二区三区在线| 精品国产亚洲在线| 一进一出抽搐gif免费好疼| 午夜福利在线在线| 亚洲乱码一区二区免费版| 草草在线视频免费看| 欧美黄色片欧美黄色片| 国产av不卡久久| 18禁在线播放成人免费| 免费大片18禁| 18美女黄网站色大片免费观看| 男人的好看免费观看在线视频| av女优亚洲男人天堂| 国产成人aa在线观看| 久久精品国产99精品国产亚洲性色| 午夜福利成人在线免费观看| 我的老师免费观看完整版| 精品不卡国产一区二区三区| 久久久久免费精品人妻一区二区| 熟妇人妻久久中文字幕3abv| 网址你懂的国产日韩在线| 亚洲中文字幕日韩| 亚洲精品亚洲一区二区| 美女大奶头视频| 国产精品免费一区二区三区在线| 亚洲国产欧洲综合997久久,| a级毛片免费高清观看在线播放| 搡老妇女老女人老熟妇| 男人的好看免费观看在线视频| 在线免费观看不下载黄p国产 | 久久午夜福利片| 十八禁人妻一区二区| 国产成人av教育| 精品99又大又爽又粗少妇毛片 | 国产色爽女视频免费观看| a级一级毛片免费在线观看| 亚洲最大成人中文| 一级黄色大片毛片| 又粗又爽又猛毛片免费看| 97人妻精品一区二区三区麻豆| 搡女人真爽免费视频火全软件 | 非洲黑人性xxxx精品又粗又长| 极品教师在线视频| 国产大屁股一区二区在线视频| 麻豆一二三区av精品| 亚洲最大成人中文| 在线天堂最新版资源| 国产男靠女视频免费网站| 99久久精品热视频| 精品久久久久久久久av| 最近视频中文字幕2019在线8| 久久亚洲真实| 1024手机看黄色片| 欧美3d第一页| 国产综合懂色| 欧美日本视频| 欧美最新免费一区二区三区 | 我的女老师完整版在线观看| 亚洲七黄色美女视频| 51国产日韩欧美| 91麻豆av在线| 欧美高清性xxxxhd video| 午夜免费男女啪啪视频观看 | 嫁个100分男人电影在线观看| 老司机午夜十八禁免费视频| 内地一区二区视频在线| 99久久精品一区二区三区| a级一级毛片免费在线观看| 亚洲午夜理论影院| 亚洲国产高清在线一区二区三| 精品人妻视频免费看| 美女黄网站色视频| 久久久久久国产a免费观看| 免费搜索国产男女视频| 一本一本综合久久| 老司机午夜十八禁免费视频| 黄色丝袜av网址大全| 国产午夜精品论理片| 成人特级黄色片久久久久久久| 深夜a级毛片| 天堂√8在线中文| 免费搜索国产男女视频| 成年免费大片在线观看| 国产又黄又爽又无遮挡在线| 欧美激情久久久久久爽电影| 国产高潮美女av| 国产一区二区在线观看日韩| 亚洲18禁久久av| 两个人视频免费观看高清| 国产精品嫩草影院av在线观看 | 亚洲aⅴ乱码一区二区在线播放| 日韩亚洲欧美综合| 国产精品伦人一区二区| 99热精品在线国产| 757午夜福利合集在线观看| 国产精品一区二区性色av| 久久精品夜夜夜夜夜久久蜜豆| 成年女人永久免费观看视频| 亚洲欧美清纯卡通| 欧美极品一区二区三区四区| 欧美最黄视频在线播放免费| 欧美日韩瑟瑟在线播放| 悠悠久久av| 午夜影院日韩av| АⅤ资源中文在线天堂| 国产精品久久久久久人妻精品电影| 久99久视频精品免费| 中文字幕免费在线视频6| 看片在线看免费视频| h日本视频在线播放| 99国产极品粉嫩在线观看| 国产精品,欧美在线| 丝袜美腿在线中文| 成年免费大片在线观看| 成人美女网站在线观看视频| 网址你懂的国产日韩在线| 亚洲成人精品中文字幕电影| 国产成人福利小说| 午夜福利在线观看吧| 欧美成人免费av一区二区三区| 国产精品精品国产色婷婷| 禁无遮挡网站| 九色国产91popny在线| 亚洲精品在线美女| 长腿黑丝高跟| 久久久国产成人免费| 亚洲在线观看片| 人妻夜夜爽99麻豆av| 国产精品一区二区三区四区久久| 给我免费播放毛片高清在线观看| 国产又黄又爽又无遮挡在线| 国产免费一级a男人的天堂| 波多野结衣高清作品| 国产精品嫩草影院av在线观看 | 日韩有码中文字幕| 国产亚洲精品综合一区在线观看| av中文乱码字幕在线| 国产精品精品国产色婷婷| 久久久国产成人精品二区| 一个人看视频在线观看www免费| 美女大奶头视频| 国产精华一区二区三区| 在线观看舔阴道视频| 色噜噜av男人的天堂激情| 中国美女看黄片| 国产成人a区在线观看| 精品一区二区三区视频在线观看免费| 色综合站精品国产| 无人区码免费观看不卡| 变态另类成人亚洲欧美熟女| 一级毛片久久久久久久久女| 小蜜桃在线观看免费完整版高清| 国产精品久久久久久精品电影| 亚洲人成伊人成综合网2020| 国产爱豆传媒在线观看| 午夜精品在线福利| 九九在线视频观看精品| 亚洲av熟女| 久久久久久久久中文| 免费电影在线观看免费观看| 欧美3d第一页| 久久久久亚洲av毛片大全| 日韩高清综合在线| 国产精品1区2区在线观看.| 国产精品精品国产色婷婷| 夜夜躁狠狠躁天天躁| 国内揄拍国产精品人妻在线| 免费看日本二区| 成人毛片a级毛片在线播放| 国产精品久久视频播放| 欧美不卡视频在线免费观看| 搡老妇女老女人老熟妇| 色5月婷婷丁香| 国产欧美日韩精品亚洲av| 人妻久久中文字幕网| 国产乱人视频| 国产人妻一区二区三区在| 欧美最新免费一区二区三区 | 脱女人内裤的视频| 久久99热这里只有精品18| 色视频www国产| 成人毛片a级毛片在线播放| 亚洲精品久久国产高清桃花| 免费无遮挡裸体视频| 99国产综合亚洲精品| 免费人成在线观看视频色| 久久久成人免费电影| 99热这里只有是精品在线观看 | 日韩有码中文字幕| 悠悠久久av| 日韩欧美精品免费久久 | 久久午夜福利片| 9191精品国产免费久久| 90打野战视频偷拍视频| 精品久久久久久久人妻蜜臀av| 97超视频在线观看视频| 亚洲天堂国产精品一区在线| 亚洲美女视频黄频| 精品日产1卡2卡| 婷婷精品国产亚洲av在线| 男人和女人高潮做爰伦理| av福利片在线观看| 精品久久久久久久末码| 亚洲色图av天堂| 日韩欧美免费精品| 国产熟女xx| 国产精品三级大全| 日韩大尺度精品在线看网址| 丰满人妻一区二区三区视频av| 成熟少妇高潮喷水视频| www日本黄色视频网| 一级黄片播放器| 国内精品美女久久久久久| 欧美成人一区二区免费高清观看| av福利片在线观看| 最近最新免费中文字幕在线| 亚洲精品一卡2卡三卡4卡5卡| 狠狠狠狠99中文字幕| 免费av观看视频| 哪里可以看免费的av片| 亚洲在线观看片| 高清日韩中文字幕在线| 级片在线观看| 国产私拍福利视频在线观看| 最近最新中文字幕大全电影3| 亚洲美女视频黄频| 色尼玛亚洲综合影院| 精品久久久久久久久亚洲 | 久久久久免费精品人妻一区二区| 舔av片在线| 国产精品电影一区二区三区| 有码 亚洲区| 亚洲av成人精品一区久久| 久久久久久久久大av| 欧洲精品卡2卡3卡4卡5卡区| 久久这里只有精品中国| 内地一区二区视频在线| 久久婷婷人人爽人人干人人爱| 色综合欧美亚洲国产小说| 亚洲美女黄片视频| 久久精品国产99精品国产亚洲性色| 亚洲av成人不卡在线观看播放网| 一级作爱视频免费观看| 免费在线观看影片大全网站| 真人一进一出gif抽搐免费| 日韩中文字幕欧美一区二区| 给我免费播放毛片高清在线观看| 国产极品精品免费视频能看的| 一个人观看的视频www高清免费观看| 国产精品乱码一区二三区的特点| 久久6这里有精品| 一个人看视频在线观看www免费| 黄色视频,在线免费观看| 看黄色毛片网站| 国产成人福利小说| 少妇的逼水好多| 国产亚洲欧美在线一区二区| 人人妻人人看人人澡| 久久久久久国产a免费观看| 国产精品电影一区二区三区| 丰满乱子伦码专区| 日日摸夜夜添夜夜添av毛片 | 可以在线观看的亚洲视频| 三级国产精品欧美在线观看| 好男人在线观看高清免费视频| 国产一级毛片七仙女欲春2| 高清毛片免费观看视频网站| 欧美日韩黄片免| 给我免费播放毛片高清在线观看| 悠悠久久av| 国产淫片久久久久久久久 | 18禁裸乳无遮挡免费网站照片| 免费av不卡在线播放| 欧美在线一区亚洲| 欧美精品啪啪一区二区三区| 一个人观看的视频www高清免费观看| 日韩精品中文字幕看吧| 亚洲精华国产精华精| 白带黄色成豆腐渣| 免费人成视频x8x8入口观看| 白带黄色成豆腐渣| 日韩高清综合在线| 色哟哟哟哟哟哟| 男插女下体视频免费在线播放| 亚洲精品一卡2卡三卡4卡5卡| 中亚洲国语对白在线视频| 波野结衣二区三区在线| 一边摸一边抽搐一进一小说| 国产极品精品免费视频能看的| 欧美一级a爱片免费观看看| 深夜精品福利| 99精品在免费线老司机午夜| 综合色av麻豆| 成人永久免费在线观看视频| 观看美女的网站| 免费av观看视频| 国产一区二区三区在线臀色熟女| 欧美高清成人免费视频www| 欧美性猛交黑人性爽| 赤兔流量卡办理| a级毛片免费高清观看在线播放| 色综合婷婷激情| 欧美性猛交╳xxx乱大交人| 成人永久免费在线观看视频| 两个人视频免费观看高清| 老熟妇乱子伦视频在线观看| 俺也久久电影网| 久久精品91蜜桃| 久久久色成人| 日韩精品青青久久久久久| 老司机午夜十八禁免费视频| 亚洲成人中文字幕在线播放| 99久久精品国产亚洲精品| 婷婷精品国产亚洲av在线| aaaaa片日本免费| 国产三级在线视频| 亚洲男人的天堂狠狠| 久久这里只有精品中国| 日本一本二区三区精品| 欧美一区二区国产精品久久精品| 一本综合久久免费| 色播亚洲综合网| h日本视频在线播放| 日韩欧美精品免费久久 | 无人区码免费观看不卡| 深夜a级毛片| 国产v大片淫在线免费观看| 国内揄拍国产精品人妻在线| 十八禁国产超污无遮挡网站| 天堂动漫精品| 国产伦精品一区二区三区四那| 中文字幕熟女人妻在线| 亚洲一区二区三区不卡视频| 亚洲aⅴ乱码一区二区在线播放| 亚洲精品一卡2卡三卡4卡5卡| 一本久久中文字幕| 亚洲专区中文字幕在线| 成年免费大片在线观看| 色噜噜av男人的天堂激情| 尤物成人国产欧美一区二区三区| 一二三四社区在线视频社区8| 麻豆一二三区av精品| 深爱激情五月婷婷| 亚洲av二区三区四区| 美女cb高潮喷水在线观看| 国产精品综合久久久久久久免费| 国产亚洲av嫩草精品影院| 久久久久久久久久成人| 91在线精品国自产拍蜜月| 精品福利观看| 免费观看的影片在线观看| 国内精品美女久久久久久| 欧美xxxx性猛交bbbb| av天堂在线播放| 久久草成人影院| 国内揄拍国产精品人妻在线| 国产在线精品亚洲第一网站| 麻豆国产97在线/欧美| 亚洲午夜理论影院| 免费人成视频x8x8入口观看| 精品国产亚洲在线| 很黄的视频免费| 精品久久久久久久久亚洲 | 一级黄色大片毛片| 国产淫片久久久久久久久 | 久久久国产成人免费| 午夜福利在线在线| 精品99又大又爽又粗少妇毛片 | 久久久色成人| 日本五十路高清| 欧美日韩亚洲国产一区二区在线观看| 97超视频在线观看视频| 久久久久久久久中文| 日韩欧美免费精品| 在线a可以看的网站| 日韩亚洲欧美综合| 国产三级在线视频| 国内久久婷婷六月综合欲色啪| 亚州av有码| 人人妻人人看人人澡| 日日干狠狠操夜夜爽| 国产亚洲精品久久久久久毛片| 久9热在线精品视频| 国产男靠女视频免费网站| 亚洲avbb在线观看| 88av欧美| 亚洲无线观看免费| 在线观看午夜福利视频| 国内少妇人妻偷人精品xxx网站| 日本黄色片子视频| 欧美午夜高清在线| 真人做人爱边吃奶动态| 岛国在线免费视频观看| 不卡一级毛片| 国产精品亚洲av一区麻豆| 男女做爰动态图高潮gif福利片| 日韩大尺度精品在线看网址| 日本免费a在线| 欧美区成人在线视频| 国产成人aa在线观看| 精华霜和精华液先用哪个| 国产大屁股一区二区在线视频| 久久精品综合一区二区三区| 亚洲国产精品合色在线| 午夜精品在线福利| 日韩成人在线观看一区二区三区| 国产aⅴ精品一区二区三区波| 午夜福利免费观看在线| 免费无遮挡裸体视频| 国产欧美日韩一区二区精品| 久久国产乱子伦精品免费另类| 尤物成人国产欧美一区二区三区| 欧美性猛交黑人性爽| 激情在线观看视频在线高清| 99精品久久久久人妻精品| 91av网一区二区| 国产色爽女视频免费观看| 中文字幕熟女人妻在线| 久久国产精品影院| 老司机午夜十八禁免费视频| 国产蜜桃级精品一区二区三区| 丁香六月欧美| 免费人成视频x8x8入口观看| ponron亚洲| 午夜福利成人在线免费观看| 国产av不卡久久| 日本熟妇午夜| 精品一区二区三区视频在线| 一级a爱片免费观看的视频| 久久久久久久亚洲中文字幕 | 变态另类成人亚洲欧美熟女| 在线免费观看不下载黄p国产 | 午夜福利在线观看吧| 亚洲专区中文字幕在线| 欧洲精品卡2卡3卡4卡5卡区| 久久精品综合一区二区三区| 网址你懂的国产日韩在线| 可以在线观看的亚洲视频| 在线观看一区二区三区| 天堂av国产一区二区熟女人妻| 久久6这里有精品| 日韩欧美 国产精品| 色av中文字幕| 女生性感内裤真人,穿戴方法视频| 婷婷亚洲欧美| 欧美xxxx性猛交bbbb| 男女做爰动态图高潮gif福利片| 真人做人爱边吃奶动态| 久久天躁狠狠躁夜夜2o2o| 日韩欧美国产一区二区入口| 尤物成人国产欧美一区二区三区| 中文字幕熟女人妻在线| 亚洲人成网站在线播放欧美日韩| 老司机午夜十八禁免费视频| 久久中文看片网| 久久久久久久久久成人| 在现免费观看毛片| 精华霜和精华液先用哪个| 脱女人内裤的视频| 99久久无色码亚洲精品果冻| 日韩精品青青久久久久久| 久久久久精品国产欧美久久久| 舔av片在线| 成人特级黄色片久久久久久久| 最新在线观看一区二区三区| 在线免费观看的www视频| 欧美日韩国产亚洲二区| 给我免费播放毛片高清在线观看| av国产免费在线观看| 国内精品久久久久精免费| 日日干狠狠操夜夜爽| ponron亚洲| 直男gayav资源| 国产精品自产拍在线观看55亚洲| 国产精品久久久久久久电影| 国产成人欧美在线观看| 久久人人爽人人爽人人片va | 听说在线观看完整版免费高清| 99视频精品全部免费 在线| 国产69精品久久久久777片| 在线免费观看的www视频| 伊人久久精品亚洲午夜| 日韩有码中文字幕| 国产免费av片在线观看野外av| 午夜免费男女啪啪视频观看 | av视频在线观看入口| 在线观看av片永久免费下载| 日本三级黄在线观看| 极品教师在线免费播放| 亚洲 欧美 日韩 在线 免费| 欧美成人免费av一区二区三区| 久久久久久国产a免费观看| 小说图片视频综合网站| 久久精品国产99精品国产亚洲性色| 国产av不卡久久| 亚洲精品一卡2卡三卡4卡5卡| 99热这里只有是精品在线观看 | 国产精品99久久久久久久久| 久久九九热精品免费| eeuss影院久久| 在线国产一区二区在线| 亚洲人成网站在线播| 噜噜噜噜噜久久久久久91| 午夜两性在线视频| 欧美中文日本在线观看视频| 国产一区二区在线av高清观看| 日本成人三级电影网站| 又爽又黄无遮挡网站| 国产精品三级大全| 久久香蕉精品热| 国产精品伦人一区二区| 国产一区二区亚洲精品在线观看| 在线观看66精品国产| 热99re8久久精品国产| 久久久久久久久久黄片| 国产毛片a区久久久久| 午夜激情欧美在线| 亚洲av.av天堂| 日韩国内少妇激情av| 51国产日韩欧美| 亚洲av免费在线观看| 国产白丝娇喘喷水9色精品| 欧美色欧美亚洲另类二区| АⅤ资源中文在线天堂| 麻豆成人av在线观看| 91麻豆av在线|