Journal Article Cloning of the E. coli O 6 -methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay Get access Geoffrey P. Margison, Geoffrey P. Margison 1Department of CarcinogenesisManchester, M20 9BX, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Donald P. Cooper, Donald P. Cooper 1Department of CarcinogenesisManchester, M20 9BX, UK 3 Present address: Cancer Research Unit, University of York, York YO 5DU. UK Search for other works by this author on: Oxford Academic PubMed Google Scholar John Brennand John Brennand 2Department of Biochemical Genetics, Paterson Laboratories, Christie HospitalManchester, M20 9BX, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 13, Issue 6, 25 March 1985, Pages 1939–1952, https://doi.org/10.1093/nar/13.6.1939 Published: 25 March 1985 Article history Received: 13 December 1984 Revision received: 25 February 1985 Accepted: 25 February 1985 Published: 25 March 1985
In this study, a combined immunoaffinity purification/32P-postlabelling procedure has been used to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) in human DNA. DNA digests are subjected to a two-stage immunopurification in which the acetone-eluted fraction from the first stage is reapplied to a second immuno-column, and the O6-MedGp specifically eluted using O6-methylguanosine (O6-MerG). O6-MedGp is then 32P-post-labelled in the presence of deoxyinosine-3'-monophosphate (dIp) as internal standard, separated by two-dimensional TLC and levels of the adduct quantified using storage phosphor technology. The recovery of O6-MedGp at levels between 0.4 and 500 fmol was 61%. Analysis of human DNA samples indicated that <1 fmol O6-methyldeoxy-guanosine-5'-monophosphate (O6-MepdG) could be detected with a high degree of precision (coefficient of variation <12%) during a 2 h exposure to a storage phosphor screen. The assay was then applied to 25 human samples from three separate populations, one of which was exposed to methylating agent chemotherapy, for which O6-methyl-deoxyguanosine (O6-MedG) levels had already been quantified by HPLC/radioimmunoassay. The results indicated a high degree of correlation between the two assays (r = 0.99). O6-MedGp was detected in all the samples analysed with levels ranging from 0.026 to 23.2 μmol O6-MedGp/ mol dG. The minimum amount of O6-MepdG detected was 0.2 fmol. As there was no detectable signal in the area to which O6-MepdG maps in negative control samples, a detection limit based upon the signal/noise ratio was impossible to quantify. However the limit of detection of the storage phosphor technology itself was estimated by quantifying a visually identifiable compound, which mapped to the same region. The amount of this compound was determined to be 32 ± 27 amol (n = 5). If a similar amount of O6-MepdG was detected from 50 (μg of DNA, and assuming that the labelling efficiency and recovery was similar to that found in this study, then this would correspond to an adduct level of -3 nmol O6-MedGp/mol dG.
Clinical LC-MS/MS assays traditionally require that samples be run in batches with calibration curves in each batch. This approach is inefficient and presents a barrier to random access analysis. We developed an alternative approach called multipoint internal calibration (MPIC) that eliminated the need for batch-mode analysis.The new approach used 4 variants of 13C-labeled methotrexate (0.026-10.3 µM) as an internal calibration curve within each sample. One site carried out a comprehensive validation, which included an evaluation of interferences and matrix effects, lower limit of quantification (LLOQ), and 20-day precision. Three sites evaluated assay precision and linearity. MPIC was also compared with traditional LC-MS/MS and an immunoassay.Recovery of spiked analyte was 93%-102%. The LLOQ was validated to be 0.017 µM. Total variability, determined in a 20-day experiment, was 11.5%CV. In a 5-day variability study performed at each site, total imprecision was 3.4 to 16.8%CV. Linearity was validated throughout the calibrator range (r2 > 0.995, slopes = 0.996-1.01). In comparing 40 samples run in each laboratory, the median interlaboratory imprecision was 6.55%CV. MPIC quantification was comparable to both traditional LC-MS/MS and immunoassay (r2 = 0.96-0.98, slopes = 1.04-1.06). Bland-Altman analysis of all comparisons showed biases rarely exceeding 20% when MTX concentrations were >0.4 µM.The MPIC method for serum methotrexate quantification was validated in a multisite proof-of-concept study and represents a big step toward random-access LC-MS/MS analysis, which could change the paradigm of mass spectrometry in the clinical laboratory.
A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of cyclosporin A (CsA) and creatinine using capillary blood has been developed. Venous and capillary blood samples were taken predose and at C2 from 65 heart and lung transplant recipients (65 x 4 samples). For comparisons, serum creatinine and blood CsA concentrations were measured by the Jaffe and EMIT methods, respectively, using an Olympus AU600 analyzer. For the LC-MS/MS assay, samples were prepared in a 96 x 700-microL well block by adding 10 microL of blood (or serum) to 40 microL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 microL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 microL of the supernatant was injected into the LC-MS/MS system. A Waters 2795 high-performance liquid chromatography (HPLC) system was used to elute a C18 cartridge (3 mm x 4 mm) at 0.6 mL/min with a step gradient of 50-100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. The column was maintained at 55 degrees C, and the retention times were creatinine, 0.4 minutes; ascomycin, 0.98 minutes; and CsA, 1.2 minutes. Cycle time was 2.5 minutes, injection to injection. The analytes were monitored using a Quattro microtandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: creatinine, m/z 114>44; d3-creatinine (IS), m/z 117>47; ascomycin (IS), m/z 809>756; and CsA, m/z 1,220>1,203. Assay characteristics were CsA intraassay CV, 3.6-3.0% (33-1,500 microg/L); CsA interassay CV, 6.7-2.5% (10-5,000 microg/L); LC-MS/MS capillary [CsA] = 0.99 x LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 x EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 micromol/L); interassay CV, 5.7-3.3% (80-590 micromol/L); LC-MS/MS capillary [creatinine] = 0.99 Jaffe plasma [creatinine] -42.6, R = 0.87. Total time for the preparation and analysis of 30 samples was approximately 2 hours. This assay will provide a flexible, robust, and cost-effective solution for the monitoring of CsA and creatinine in transplant recipients with potential applications in pediatric medicine and pharmacokinetic studies, in which frequent sampling is required.
INTRODUCTION: It is recognised that the desired therapeutic effect of most Anti-epileptic Drugs (AEDs) is achieved within a specific concentration range, with lower levels giving an unsatisfactory response and higher levels possibly giving undesirable side-effects. Large inter-individual differences in drug disposition occur, and many refractory patients require treatment with several AEDs. Thus, monitoring serum/plasma concentrations of AEDs has proved to be an extremely useful means of assisting in the individualisation of treatment. A large number of AEDs are available and it is necessary to quantitatively determine each compound that a patient is prescribed. Assay methods include a range of gas- and liquid- chromatographic procedures, and specific immunoassays, although the latter are not available for all AEDs. This work describes an Ultra Performance Liquid Chromatography – tandem mass spectrometry (UPLC/MS/MS) method capable of analysing a panel of AEDs, and, where appropriate, the active metabolite, simultaneously in a single procedure. METHODOLOGY: Sample preparation consisted of a simple protein precipitation using aqueous zinc sulphate and methanol. The resulting supernatant was diluted with mobile phase to give a response in the linear range of the analyte. A reversed phase UPLC method, using a Waters ACQUITY UPLC™ BEH C18 2.1x50mm 1.7µm column was developed to meet the demands of the clinical laboratory for speed of analysis, chromatographic resolution and sensitivity. The mobile phase consisted of :- Solvent A: aqueous ammonium acetate and solvent B: methanolic ammonium acetate; A five-minute gradient elution from 2-75% organic phase, produced an effective separation. Detection was undertaken using a Waters Quattro Premier XE™ tandem quadrupole mass spectrometer operating in Multiple Reaction Monitoring (MRM) mode with fast (20ms) positive and negative ion mode switching and short (20ms) dwell times, commensurate with the UPLC peak widths. RESULTS: The assay for Lamotrigine was linear over the analytical range 2.9 -15.3 mg/L, with a correlation coefficient (r 2 ) of 0.995, and a precision %RSD = 4.4% at the lower concentration (n=6). The assay for Primidone was linear over the analytical range 4.3-30.0mg/L with a r 2
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