MSAll strategy for comprehensive quantitative analysis of PEGylated-doxorubicin, PEG and doxorubicin by LC-high resolution q-q-TOF mass spectrometry coupled with all window acquisition of all fragment ion spectra.

The covalent attachment of polyethylene glycol (PEG) to therapeutic compounds (known as PEGylation) is one of the most promising techniques to improve the biological efficacy of small molecular weight drugs. After administration, PEGylated prodrugs can be metabolized into pharmacologically active compounds so that PEGylated drug, free drug and released PEG are present simultaneously in the body. Understanding the pharmacokinetic behavior of these three compounds is needed to guide the development of pegylated theranostic agents. However, PEGs are polydisperse molecules with a wide range of molecular weights, so that the simultaneous quantitation of PEGs and PEGylated molecules in biological matrices is very challenging. This article reports the application of a data-independent acquisition method (MSAll) based on liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-q-q-TOF-MS) in the positive ion mode to the simultaneous determination of methoxyPEG2000-doxorubicin (mPEG2K-Dox) and its breakdown products in rat blood. Using the MSAll technique, precursor ions of all molecules are generated in q1, fragmented to product ions in q2 (collision cell), and subjected to TOF separation before precursor and product ions are recorded using low and high collision energies (CE) respectively in different experiments for a single sample injection. In this study, dissociation in q2 generated a series of high resolution PEG-related product ions at m/z 89.0611, 133.0869, 177.1102, 221.1366, 265.1622, 309.1878, and 353.2108 corresponding to fragments containing various numbers of ethylene oxide subunits, Dox-related product ions at m/z 321.0838 and 361.0785, and an mPEG2K-Dox specific product ion at m/z 365.0735. Detection of mPEGs and mPEG2K-Dox was based on high resolution extracted ions of mPEG and the specific compound. The method was successfully applied to a pharmacokinetic study of doxorubicin, mPEG2K (methylated polyethylene glycol 2K), and mPEG2K-doxorubicin in rats after a single intravenous injection of mPEG2K-doxorubicin. To the best of our knowledge, this is the first assay that simultaneously determines mPEG, Dox, and mPEG2K-Dox in a biological matrix. We believe the MSAll technique as applied in this study can be potentially extended to the determination of other PEGylated small molecules or polymeric compounds.

Comparative pharmacokinetics and the bioavailability of escin Ib and isoescin Ib following the administration of escin, pure escin Ib and isoescin Ib in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Adequate pharmacokinetic data of escin, a natural mixture of triterpene saponins used for the treatment of chronic venous insufficiency, hemorrhoids, inflammation and edema, is of special interest in view of the growing use of escin agent in clinical medicine. However, pharmacokinetic data are inadequate to support their clinical indication. Escin Ib and isoescin Ib are the chief active ingredients in escin, pharmacokinetics study of them would be helpful for improving the practice of escin application. The goals of this study are to determine the plasma concentration of escin Ib and isoescin Ib using an established liquid chromatography tandem mass spectrometry (LC-MS/MS) method and to compare the pharmacokinetics and bioavailability of these compounds in rats when administered as pure isomers or as sodium escinate. MATERIALS AND METHODS: Five groups of Wistar rats (n=6 per group) were treated with either an intravenous (IV) dose (2.78mg/kg) of sodium escinate (corresponding to 0.5mg/kg of escin Ib and 0.5mg/kg of isoescin Ib), an IV dose (0.5mg/kg) and an oral dose (4mg/kg) of pure escin Ib or isoescin Ib. The concentrations of escin Ib and isoescin Ib in rat plasma were determined by LC-MS/MS at various times following the administration of the drugs. The pharmacokinetic parameters were estimated by a non-compartmental analysis and then subjected to statistical analysis. RESULTS: The administration of sodium escinate, which contains the two isomers, gave rise to higher terminal phase half-life (t1/2) and mean residence time (MRT) values for both escin Ib and isoescin Ib compared to the corresponding compounds administered alone. The absorption of escin Ib and isoescin Ib was very poor, with the oral bioavailability (F) values of <2% observed for both compounds. The two compounds were found to isomerize in vivo, wherein the conversion of escin Ib to isoescin Ib was much easier than that of isoescin Ib to escin Ib. CONCLUSIONS: A comparison of the pharmacokinetics of escin Ib and isoescin Ib administered alone and together in rats suggests that the administration of herbal preparations of escin in a clinical setting may result in a longer duration of action than the administration of each isomer alone. The interconversion of escin Ib and isoescin Ib when administered alone indicates that the administration of one isomer results in exposure to the other isomer.

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 chromatography-tandem 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 re-extracted with dichloromethane, after which the analyte and triptorelin as an internal standard (IS) were separated on a 300SB-C18 column (150 mm×4.6 mm i.d., 5 µm 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 multiple-reaction monitoring (MRM) transitions of bivalirudin and IS were at m/z 1091.0→650.4 and m/z 656.5→249.3, respectively. The lower limit of quantification (LLOQ) was 1 ng/mL for 100 µL 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.

Pharmacokinetics and bioavailability of cimicifugosides after oral administration of Cimicifuga foetida L. extract to rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cimicifuga foetida L., a traditional Chinese medicine, has been used as an anti-inflammatory, antipyretic and analgesic remedy. The primary active constituents are believed to be present in the triterpene glycoside fraction. MATERIALS AND METHODS: To develop an LC-MS/MS assay for four major cimicifugosides [cimicifugoside H-1 (Cim A), 23-epi-26-deoxyactein (Cim B), cimigenolxyloside (Cim C) and 25-O-acetylcimigenoside (Cim D)] obtained from C. foetida L. and apply it to investigate their pharmacokinetic (PK) properties and bioavailabilities through oral administration of C. foetida L. extract (12.5, 25 and 50mg/kg) and single intravenous (i.v.) doses (5mg/kg) of the individual cimicifugosides in rat. PK parameters were estimated by non-compartmental analysis. RESULTS: All calibration curves showed excellent linear regressions (all r>0.995) within the range of tested concentrations. The intra- and inter-day variations were <15% in terms of RSD. The molar ratio of Cims A, B, C, and D in the extract was 20.7:1.4:2.9:1. PK parameters for Cims A, B, C, and D following oral administration of the extract were respectively: C(max) 4.05-17.69, 90.93-395.7, 407.1-1180 and 21.56-45.09pmol/mL; T(max) 0.46-1.28, 2.00-4.67, 14.67-19.67 and 8.08-14.27h; absolute oral bioavailability (F) 1.86-6.97%, 26.8-48.5%, 238-319% and 32.9-48%. PK parameters after i.v. administration of individual cimicifugosides were respectively: elimination half-life 1.1, 2.5, 5.7 and 4.2h; clearance 15.7, 0.48, 0.24 and 1.13mL/hkg. CONCLUSIONS: Systemic exposure to Cims B, C and D following oral administration of the extract was significantly greater than to Cim A despite the predominance of Cim A in the extract. Significantly different clearance and interconversion from Cim A to Cim C probably accounts for the different exposure to the four cimicifugosides.

Sensitive liquid chromatographic assay for the simultaneous determination of 5-fluorouracil and its prodrug, tegafur, in beagle dog plasma.

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of 5-fluorouracil (5-FU) and its prodrug, tegafur (TF), in dog plasma. 5-FU, the internal standard, 5-bromouracil, and TF were separated on a C18 Spherisorb ODS2 column using isocratic elution with retention times of 4.4, 8.0 and 21.2 min, respectively. Detection by UV absorption at 260 nm gave a limit of quantitation of 4 microg/l for 5-FU in plasma. Calibration curves for 5-FU and TF were linear over the ranges of 4-160 microg/l and 0.48-19.2 mg/l, respectively. Intra- and inter-day precision over these concentration ranges were <10.9 and <13.6% for 5-FU and TF, respectively, with good accuracy for both compounds. The method was successfully applied to define plasma concentration-time curves of TF and 5-FU in dogs administered a single oral dose containing TF (100 mg) and uracil (224 mg).

Isolation and identification of the glucuronide of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid from rabbit urine.

The metabolic profile of 3H-1,2-dihydro-2-(4-methylphenylamino)methyl-1-pyrrolizinone (SFZ-47), a putative non-steroidal anti-inflammatory pro-drug, has been studied in rabbit urine. Semi-preparative reversed-phase HPLC of 24 h urine from two rabbits given single oral doses of SFZ-47 (200 mg) allowed the separation of SFZ-47 together with the oxidative metabolite 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid (SFZ-47-COOH) and its glucuronide conjugate. The glucuronide was characterized by ESI-MS(n) and (1)H NMR and shown to be the 1-O-acyl beta-D-glucuronide conjugate of SFZ-47-COOH. The method gave excellent resolution of the glucuronide from endogenous constituents in urine and may be suitable for the preparation of glucuronide metabolites of other drugs.