Ross F. Lawrence

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography-mass spectrometry and gas chromatography-nitrogen-phosphorus detection.

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC-NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile-methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration-time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.

Preparation and diels-alder reaction of N-carbomethoxy-5-ethyl-1.2-dihydropyridine: An approach for the synthesis of catharanthine

N-carbomethoxy-5-ethyl, 1,2-dihydropyridine (1) may be prepared by the bromination of N-carbomethoxy-3-ethyl-1,2,5,6-tetrahydropyridine (10), and subsequent double dehydrobromination with EtAlCl2/HMPA; this diene undergoes Diels-Alder reaction with dimethyl methylenemalonate (2) to give the isoquinuclidine (3), an intermediate in our planned approach to catharanthine.

Intramolecular hydride transfer and cyclization of 5-oxo-Na-tosyl-14,21 -dehydrosecodine

Abstract 5-Oxo-Na-tosyl-14,21-dehydrosecodine (8) was prepared by bromination of 6 followed by double dehydrobromination with ethyl aluminum dichloride in HMPA. When a dilute solution of 8 was heated, 10 was formed via intramolecular hydride transfer and cyclization.

Claisen ortho ester rearrangement with trimethyl β-(methoxy)orthopropionate: a thermally stable synthon for the preparation of methyl α-substituted acrylates☆

Abstract Trimethyl β-(methoxy)orthopropionate, a compound which is thermally stable at 190 °C for prolonged times, may be utilized as a synthon for the preparation of methyl α-substituted acrylates via Claisen ortho ester rearrangement with allylic alcohols followed by β-elimination of methanol with base.

Quantitation of Zoledronic Acid in Murine Bone by Liquid Chromatography Coupled with Tandem Mass Spectrometry

An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0μg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06±0.52 vs. 0.76±0.09ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment.

Application of negative-ion chemical ionization isotope dilution gas chromatography—mass spectrometry to single-dose bioavailability studies of mefloquine

An electron-capture negative-ion chemical ionization gas chromatographic-mass spectrometric assay for mefloquine, an antimalarial drug used in the treatment of drug-resistant Plasmodium falciparum malaria, is described. The method, developed in support of bioavailability studies involving the co-administration of different tableted formulations of the drug and an aqueous solution of its 13C3-labeled analog, enables quantification of both dosage forms. Quantitative analysis of extracted plasma samples was performed on the O-tert.-butyldimethylsilyl (t-BDMS) derivative of the drug by selected-ion monitoring, using a VG Trio 2000 quadrupole mass spectrometer and monitoring the [M-t-BDMSOH]-. ions of the analytes. The method, incorporating [2H6]mefloquine as an internal standard, demonstrated good accuracy and precision over the 1-200 ng ml-1 range, with correlation coefficients greater than 0.990 for all standard curves and a detection level of 50 fg on-column. Replicate analysis of plasma samples over a 90-day period exhibited a mean intra-day and inter-day variation of less than 4.5% and 5.5%, respectively. The high stability and sensitivity of the assay, combined with the inherent selectivity of mass spectrometric detection, make the method well-suited for such studies.

Analysis of hydroxylated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography–mass spectrometry

This report describes an assay for the H(1)-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization-liquid chromatography-mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H]+ ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5-250 and 12.5-1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes.