Development of ICP-MS assays for the study and prediction of the efficacy and side effects of Pt-based drugs in cancer chemotherapy

Pt-based drugs are important cytotoxic agents that are used in the chemotherapeutic regimes of ~50% of all cancer patients. However, the efficacy of these drugs is often limited by drug toxicity and tumour resistance. Determination of the cellular pharmacokinetics and pharmacodynamics of Pt-drugs is important for understanding their molecular mechanisms of action and toxicity, and may be used, therefore, to predict the outcome of the treatment. ICP-MS is the most sensitive technique for the determination of Pt in biological samples and can offer robust, fast and accurate quantitations for studying pharmacokinetics and pharmacodynamics of Pt-drugs in patients. This thesis describes the development of a set of ICP-MS based assays for the determination of Pt-DNA adducts and Pt sub-cellular distribution in leukocytes of cancer patients and human cancer cell lines following treatment with Pt-based chemotherapy. It is ultimately aimed to use these assays in the clinic to predict the effectiveness and toxicity of Pt-based chemotherapy in individual patients, and offer those who would respond to the treatment personalised drug doses. Alternatively, patients who would not benefit from these drugs would be offered other forms of treatment. Pt DNA adduct formation was determined in leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 106 nucleotides using 10 µg of patient DNA. It was shown that Pt/P ratio was robust against DNA matrix effects, and was considered more reliable approach, with Eu as internal standard, for estimating Pt adducts per nucleotide compared to using Pt data in combination with DNA concentration measured by UV. Comparison of in vivo Pt-DNA adduct formation with the patients clinical notes suggested possible correlation between the adduct formation in leukocytes and toxicity. Speciation methods employing HPLC with complementary ICP-MS and ESI-Ion Trap-MS detection were developed and used for characterisation of oxaliplatin bi-functional adducts with mono-nucleotides and di-nucleotides. Further, a fast and sensitive LC-ICP-MS assay was developed and used for the quantification of oxaliplatin GG intra-strand adducts in human cancer cell lines. The assay, which has a detection limit of 0.22 Pt adduct per 106 nucleotides based on a 10 μg DNA sample, is suitable for in vivo assessment of the adducts in patients undergoing oxaliplatin chemotherapy. Combining the ICP-MS quantitation with a cell fractionation procedure allowed, for the first time, the detailed quantitation of entire sub-cellular Pt-drug partitioning in patient leukocytes in vivo, and in human cancer cell lines in vitro, following exposure to variety of Pt-drugs. The studies showed that Pt broadly follows the total protein content of the individual sub-cellular compartments with the majority being scavenged in the cytosol compartment

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L–1)-methanol-acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min–1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL–1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL–1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 µL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics

Determination of Pt-DNA adducts and the sub-cellular distribution of Pt in human cancer cell lines and the leukocytes of cancer patients, following mono- or combination treatments, by inductively-coupled plasma mass spectrometry

Determination of Pt-DNA adducts and the sub-cellular distribution of Pt in human cancer cell lines and the leukocytes of cancer patients, following mono- or combination treatments, by inductively-coupled plasma mass spectrometr

Determination of Pt–DNA adducts and the sub-cellular distribution of Pt in human cancer cell lines and the leukocytes of cancer patients, following mono- or combination treatments, by inductively-coupled plasma mass spectrometry

This is the author’s version of a work that was accepted for publication in the International Journal of Mass Spectrometry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published at: http://dx.doi.org/10.1016/j.ijms.2010.11.01

Development and Validation of a Rapid High-Performance Liquid Chromatography–Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (HPLC–ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC–MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid

Development and Validation of a Rapid High-Performance Liquid Chromatography–Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (HPLC–ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC–MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L−1)/methanol/acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min−1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL−1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL−1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 μL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics

Low Molecular Weight Chitosan-Coated PLGA Nanoparticles for Pulmonary Delivery of Tobramycin for Cystic Fibrosis

(1) Background: Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with Tobramycin were prepared using a solvent-evaporation method. (2) Methods: The NPs were coated with low molecular weight chitosan (LMWC) to enhance the mucoadhesiveness of PLGA-NPs. The following w/w ratios of tobramycin to LMWC were prepared: control (0:0.50), F0 (1:0.25), F0.5 (1:0.5), and F1 (1:1). (3) Results: The results showed that the size of the particles increased from 220.7 nm to 575.77 nm as the concentration of LMWC used in the formulation increased. The surface charge was also affected by the amount of LMWC, where uncoated-PLGA nanoparticles had negative charges (−2.8 mV), while coated-PLGA NPs had positive charges (+33.47 to +50.13 mV). SEM confirmed the size and the spherical homogeneous morphology of the NPs. Coating the NPs with LMWC enhanced the mucoadhesive properties of the NPs and sustained the tobramycin release over two days. Finally, all NPs had antimicrobial activity that increased as the amount of LMWC increased. (4) Conclusion: In conclusion, the formulation of mucoadhesive, controlled-release, tobramycin-LMWC-PLGA nanoparticles for the treatment of P. aeruginosa in cystic fibrosis patients is possible, and their properties could be controlled by controlling the concentration of LMWC

HPLC with Fluorescence and Photodiode Array Detection for Quantifying Capmatinib in Biological Samples: Application to In Vivo and In Vitro Studies

Capmatinib, a recently approved tyrosine kinase inhibitor, is used for the treatment of non-small cell lung cancer. We describe two new HPLC methods for capmatinib quantification in vivo and in vitro. HPLC with a fluorescence detection method was used to quantify capmatinib in plasma for the first time. The method was successfully applied in a pharmacokinetic study following a 10 mg/kg oral dose of capmatinib given to rats. The chromatographic separation was performed using a Eurospher II 100-3 C18H (50 × 4 mm, 3 µm) column and a mobile phase containing 10 mM of ammonium acetate buffer (pH 5.5): acetonitrile (70:30, v/v), at a flow rate of 2.0 mL min−1. The study also describes the use of HPLC-PDA for the first time for the determination of capmatinib in human liver microsomes and describes its application to study its metabolic stability in vitro. Our results were in agreement with those reported using LC-MS/MS, demonstrating the reliability of the method. The study utilized a Gemini-NX C18 column and a mobile phase containing methanol: 20 mM ammonium formate buffer pH 3.5 (53:47, v/v), delivered at a flow rate of 1.1 mL min−1. These methods are suitable for supporting pharmacokinetic studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities