A full-scale tracing study of "ghost peaks" encountered in impurity analysis of budesonide based on experimental operation inspection-liquid chromatography/mass spectrometry fingerprint-mechanism based stress studies integrated strategy.

Budesonide is an active pharmaceutical ingredient used in various dosage forms of finished products for the treatment of asthma. During the process of drug development, unbiased analysis of related substances is of utmost significance for both pharmaceutical research and quality control purposes. In this work, the official method documented in the United States Pharmacopoeia was selected to determine the related substances of budesonide considering the pros and cons of critical chromatographic parameters, compared to the European Pharmacopoeia. In doing so, several unpredictable interference peaks, namely "ghost peaks", were observed occasionally during analysis. A strategy that integrated information derived from experimental operation inspection, liquid chromatography/mass spectrometry fingerprint analysis, and mechanism-based stress studies was then proposed for comprehensively and quickly exploring those non-degradable and degradable peaks. Some ghost peaks were found to originate from nylon syringe filter, illumination, and alkali borosilicate glass high-performance liquid chromatography vials. Besides, degradation pathways under alkaline conditions were also unraveled through liquid chromatography-mass spectrometry qualitative analysis. Overall, an optimization of the analytical methodology based on the United States Pharmacopoeia for its application in impurity analysis of budesonide and corresponding formulations was carried out with the design of experiments, by which "ghost peaks" could be suppressed or prevented. The results obtained herein are not only crucial to studies on budesonide's stability or degradation kinetics but also contribute to clarifying the impurity research of other drugs.

Novel strategy using tryptic peptide immunoaffinity-based LC-MS/MS to quantify denosumab in monkey serum.

AIM: Denosumab is a recombinant fully human IgG2 that has a high affinity and specificity for human RANKL. Commercially available RANKL labeled with an Fc fragment cannot be used to establish an indirect ELISA. To characterize denosumab pharmacokinetic a robust and accuracy method should be developed urgently. RESULTS: In this study, an immunoaffinity enrichment method coupled with LC-MS/MS was established. The LC-MS/MS method acquired a linear range from 0.1 to 30 µg/ml. The intra- and inter-run precision (CV%) was within 11.5 and 10.5%, respectively. More importantly, the LC-MS/MS pharmacokinetic data were consistent with ELISA. CONCLUSION: This approach accelerated the quantification, reduced the costs and provided an alternative in case of lacking the special antigen to denosumab or a RANKL-biotinylated reagent.

Validated LC-MS/MS method for the simultaneous determination of rotigotine and its prodrug in rat plasma and an application to pharmacokinetics and biological conversion in vitro.

Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been synthesized for use in a sustained delivery system. The aim of the present report was to develop and validate a simple, sensitive and reliable LC-MS/MS method for the simultaneous determination of RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-CN chromatography column (2.1mm×100mm, 3.5µm) by isocratic elution using a 0.2% formic acid aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample preparation method employed 50µL of a plasma sample and liquid-liquid extraction with a mixture of diethyl ether-dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and stability. Good linearity was found within the range of 0.1-10.0ng/mL for both analytes (r>0.996). This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation in rats following a single intramuscular injection and biological conversion in vitro.

An LC-MS/MS method for the simultaneous determination of goserelin and testosterone in rat plasma for pharmacokinetic and pharmacodynamic studies.

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed, using testosterone-d3 as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-(13)C3 for testosterone or testosterone-d3. The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C18 column (Agilent, 2.1 × 50 mm, 1.8 µm, Stockport, UK) in a single chromatographic run at a flow rate of 400 µL/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis(®) HLB solid-phase extraction column. The method was validated in the concentration range of 0.01-30.0 ng/mL for goserelin and 0.05-30.0 ng/mL for testosterone-d3, respectively. The within- and between-run precisions were 1.7-9.2% and 2.1-6.9%, respectively. The within- and between-run accuracies were -1.8 to 5.3% and -4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats.

An analytical strategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC-MS/MS analysis of triptorelin and endogenous testosterone in rat plasma.

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1 × 50 mm, 1.8 µm) with a 0.05% propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-(13)C3, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D3 as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 µL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose.

Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry.

A highly sensitive ultra-high-performance liquid chromatographic-tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantification of the synthetic peptide drug of exenatide in monkey plasma. Sample preparation was carried out by solid-phase extraction (SPE), and bivalirudin was used as the internal standard (IS). An excellent chromatographic separation was obtained on a reversed-phase C18 column with a gradient elution. Detection utilized a Qtrap 5500 system operated in the positive ion mode with multiple reaction monitoring (MRM). The proposed method was validated by assessing the specificity, linearity, intra- and inter-day precision and accuracy, recovery, and stability. The method resulted in a linear calibration range of 0.10-30 ng/mL, extracting with only 50 µL monkey plasma aliquots. The intra- and inter-day precisions (as relative standard deviation) were less than 7.5% and 9.6%, respectively. The method could be successfully utilized for the pharmacokinetic study of exenatide in monkeys following a single subcutaneous injection of Byetta.

Quantitation of slow release triptorelin in beagle dog plasma by liquid chromatography-tandem mass spectrometry.

A sensitive method based on liquid chromatography-tandem mass spectrometry has been developed for the determination of triptorelin levels in beagle dog plasma. Plasma samples were applied to Oasis(®) HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 µl methanol:water:formic acid (60:40:0.08, v/v/v). The separation was achieved on a Venusil MP-C18 column (2.1 mm × 50 mm, 3 µm, Agela) with a gradient elution. Detection utilized a Qtrap5500 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 656.5→249.1 and of the I.S. at m/z 510.8→120.1. The proposed method was validated by assessing the specificity, linearity, precision and accuracy, recovery, matrix effects, and stability. Linear calibration curves were obtained in the concentration range of 0.01-10 ng/ml (the correlation coefficients were above 0.995). The lower limit of quantification (LLOQ) of the method was 0.01 ng/ml. The method was successfully applied to a pharmacokinetic study of a slow release triptorelin formulation in beagle dogs following a single intramuscular injection.

Quantitation of the tacrine analogue octahydroaminoacridine in human plasma by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of octahydroaminoacridine in human plasma using tramadol as internal standard (I.S.). Sample preparation involved pH adjustment with sodium carbonate followed by solvent extraction with dichloromethane:ethyl ether (40:60, v/v). Chromatographic separation was achieved on a Venusil MP-C18 column (5 microm, 100 mm x 4.6mm) using acetonitrile:10mM ammonium acetate:formic acid (30:70:1, v/v/v) as mobile phase. Detection utilized an API 4000 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 203.1-->175.1 and of the I.S. at m/z 264.1-->58.0. The method was linear in the range 0.01-10 ng/ml with a lower limit of quantitation of 0.0 1ng/ml. Intra- and inter-day precisions measured as relative standard deviation were <3.15% and <5.01%, respectively. The method was successfully applied to a pharmacokinetic study involving oral administration of a tablet containing 4 mg octahydroaminoacridine succinate to healthy volunteers. Pharmacokinetic parameters for octahydroaminoacridine include C(max) 1.19+/-0.53 ng/ml, T(max) 0.77+/-0.17 h, AUC(0-t) 3.42+/-1.01 ng h/ml and t(1/2) 2.89+/-0.56 h.

Rapid and sensitive LC-MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma.

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh(2), were extracted from plasma by liquid-liquid extraction and separated on a Zorbax extend C(18) analytical column using methanol-acetonitrile-10mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1-100.0ng/ml with a limit of detection of 0.05ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25mg capsule.