Current status of in vivo bioanalysis of nano drug delivery systems

The development of nano drug delivery systems(NDDSs)provides new approaches to fighting against diseases.The NDDSs are specially designed to serve as carriers for the delivery of active pharmaceutical ingredients(APIs)to their target sites,which would certainly extend the benefit of their unique physicochemical characteristics,such as prolonged circulation time,improved targeting and avoiding of drugresistance.Despite the remarkable progress achieved over the last three decades,the understanding of the relationships between the in vivo pharmacokinetics of NDDSs and their safety profiles is insufficient.Analysis of NDDSs is far more complicated than the monitoring of small molecular drugs in terms of structure,composition and aggregation state,whereby almost all of the conventional techniques are inadequate for accurate profiling their pharmacokinetic behavior in vivo.Herein,the advanced bioanalysis for tracing the in vivo fate of NDDSs is summarized,including liquid chromatography tandemmass spectrometry(LC-MS/MS),Forster resonance energy transfer(FRET),aggregation-caused quenching(ACQ)fluorophore,aggregation-induced emission(AIE)fluorophores,enzyme-linked immunosorbent assay(ELISA),magnetic resonance imaging(MRI),radiolabeling,fluorescence spectroscopy,laser ablation inductively coupled plasma MS(LA-ICP-MS),and size-exclusion chromatography(SEC).Based on these technologies,a comprehensive survey of monitoring the dynamic changes of NDDSs in structure,composition and existing form in system(i.e.carrier polymers,released and encapsulated drug)with recent progress is provided.We hope that this review will be helpful in appropriate application methodology for investigating the pharmacokinetics and evaluating the efficacy and safety profiles of NDDSs.

Quantitation of bivalirudin,a novel anticoagulant peptide,in human plasma by LC-MS/MS:Method development,validation and application to pharmacokinetics

A rapid and sensitive method based on liquid chromatographtandem mass spectrometry （LC-MS/MS） for the determination of a novel anticoagulant peptide bivalirudin in human plasma has been developed and validated. Plasma samples were precipitated protein with acetonitrile and reextracted with dichloromethane, after which the analyte and triptorelin as an internal standard （IS） were separated on a 300SB-Cl8 column （150 mm x 4.6 mm i.d., 5 gm particle size） using 0.1% formic acid：methanol （45：55, v/v） as mobile phase. The triple-quadrupole mass spectrometer, equipped with electrospray ionization （ESI） interface, was operated in the positive ion mode, and the multiplereaction monitoring （MRM） transitions of bivalirudin and IS were at m/z 1091.0-650.4 and m/z656.5 - 249.3, respectively. The lower limit of quantification （LLOQ） was 1 ng/mL for 100 ng/mL plasma sample and the assay was linear over the concentration range 1 1000 ng/mL. The accuracy was within a range from -0.4% to 0.5% in terms of relative error （RE） and the intra- and inter-day precisions in terms of relative standard deviation （RSD） were 〈2.92 and 〈 3.36, respectively. The method was successfully applied to a pharmacokinetic study involving intravenous administration of bivalirudin （0.5 mg/kg） to Chinese volunteers.

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of valproic acid and its ene-metabolites in epilepsy patient plasma

A simple and high throughput method was developed and validated for simultaneous determination of valproic acid and its two toxicant ene-metabolites, 2-enevalproic acid and 4-enevalproic acid in epilepsy patient plasma using liquid chromatography-tandem mass spectrometry. Probenecid was used as in- ternal standard and solid-phase extraction was selected for sample preparation. A chromatographic separation was performed on an Agilent Poroshell SB-C18 column （50 mm × 4.6 mm i.d., 2.7μm） by an optimized gradient elution at a flow rate of 0.9 mL/min. The total run time was 7 rain. Electrospray ionization was used in negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 143.0→143.0 for valproic acid, m/z 140.9 →140.9 for 2-enevalproic acid and 4-enevalproic acid for their poor fragments, and m/z 283.9→239,9 for probenecid. The results showed good linearity ofvalproic acid, 2-enevalproic acid and 4-enevalproic acid in their respective linear ranges. The correlation coefficients were more than 0.998, The intra- and inter-day precision of the assay was less than 11.0% and the accuracy ranged from 2% to 12%. This analytical method was successfully applied to assay plasma concentrations of valproic acid and its two ene-metabolites in epilepsy patient plasma and used for therapeutic drug monitoring.

Impact of molecular weight on the mechanism of cellular uptake of polyethylene glycols(PEGs) with particular reference to P-glycoprotein

Polyethylene glycols(PEGs)in general use are polydisperse molecules with molecular weight(MW)distributed around an average value applied in their designation e.g.,PEG 4000.Previous research has shown that PEGs can act as P-glycoprotein(P-gp)inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs.However,questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed.In this study,we examined the mechanism of uptake of PEGs by MDCK-mock cells,in particular,the effect of MW and interaction with P-gp by MDCK-hMDRl and A549 cells.The results show that:(a)the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors;(b)PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil;and(c)uptake of PEGs of low MW(<2000 Da)occurs by passive diffusion whereas uptake of PEGs of hish MW(>5000 Da)occurs by a combination of passive diffusion and caveolae-mediated endocytosis.These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems.

LC-MS/MS Method for the Quantitation of Cefotetan in Human Plasma and Its Application to Pharmacokinetic Study

A rapid and sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS） method for the de- termination of cefotetan in human plasma was developed and validated. After the protein precipitation of sample with acetonitrile, the analyte and internal standard（IS）, tramadol, were separated on a Zorbax XDB C8 column using ace- tonitrile/1%（volume fraction） formic acid（volume ratio 35：65, pH=2.5） as mobile phase at a flow rate of 1.0 mL/min with a 1 ： 1 split. The detection was performed by electrospray ionization with positive ion mode, followed by multiple reaction monitoring of the transitions for cefotetan at m/z 576.3→460.2（quantifier） and m/z 576.3→432.2（qualifier） and for IS at m/z 264.1→58.1. Cefotetan and IS were eluted at 1.86 and 1.87 rain, respectively. The assay was linear over the concentration range of 0.1-100 gg/mL for 20 μL of human plasma only with intra- and inter-day preci- sions（expressed as the relative standard deviation） of less than 6.62% and accuracies（as relative error） of ＋1.31%. The method was applied to the pharmacokinetic study of a l-h intravenous infusion of 1.0 g of cefotetan disodium for human volunteers（n=6）.

Rapid Quantification of Astilbin in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry and Its Application to Pharmacokinetic Study

Astilbin is a potential immunosuppressive agent with minor cytotoxicity. Its oral bioavailability is supposed to be rather low and therefore a sensitive analytical method is required for its pharmacokinetic study after oral administration. A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed and validated for the determination of astilbin in rat plasma. Plasma samples were subjected to liquid-liquid extraction with ethyl acetate and separated by reversed phase high performance liquid chromatography（HPLC） with methanol-0.01%（volume fraction） formic acid（50：50, volume ratio） as mobile phase. Quantitive determination was achieved on negative LC-MS/MS by a multiple reaction moitoring method with transitions m/z 449.1→150.9（quantifier） and m/z 449.1→284.9（qualifier） for astilbin and m/z 128.9→42.0 for internal standard（IS）. A lower limit of quantification（LLOQ） of ng/mL was achieved within a short cycle time of 3.4 min. The method was successfully applied to a pharmacokinetic study involving oral and intravenous administrations of 6 mg/kg astilbin to six rats.