High-throughput and sensitive screening by ultra-performance liquid chromatography tandem mass spectrometry of diuretics and other doping agents.

The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC- MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL(-1) to 200 ng mL(-1), and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.

Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation: comparison of recipients of positive CD34+ selected grafts with recipients of unmanipulated grafts.

We compared the kinetic recovery of lymphocytes and their subsets in two groups of patients submitted to allogeneic peripheral blood progenitor cell transplantation (allo-PBT): those receiving lymphocyte-depleted leukaphereses by positive selection of CD34+ cells (group 1, n = 18) and those receiving unmanipulated leukaphereses (group 2, n = 15). Patients were conditioned with cyclophosphamide (120 mg/kg) and fractioned total body irradiation (13 Gy, group 1; 12 Gy, group 2). The mean number (x 10(6)/kg) of CD34+ and CD3+ cells infused was 4.0 and 0.67, respectively, in group 1 patients, and 4.7 and 274, respectively, for group 2 patients. Graft-versus-host disease prophylaxis consisted of cyclosporin A + methylprednisolone for group 1 and cyclosporin A + methotrexate for group 2. Median follow-up was 7 months (range 2-8 months) for both groups. During the first 6 months post-transplant, CD4+ cell counts were lower in group 1 as compared with group 2 (p = 0.014, 0.010, 0.011, 0.0003, and 0.052 at 0.5, 1, 2, 3, and 6 months, respectively), whereas there was no difference at 8 months. The number of CD4+CD45RA+ cells was very low throughout the study in both groups, being lower in group 1 than in group 2, especially during the first 3 months post-transplant (p = 0.007 and 0.0006 at 1 and 3 months). Normal levels of CD8+ cells were reached by 1 month post-transplant in both groups. TCR gamma delta + cell counts were lower in group 1 than in group 2 during the first 4 months post-transplant (p = 0.001, 0.004, and 0.04 at 1, 3, and 4 months). A normal number of natural killer cells (CD3-CD56+) was achieved 1 month post-transplant in both groups. B lymphocytes (CD19+) showed low or undetectable counts throughout the first 4 months in both groups, achieving the normal range at 8 months. These results show that, during the first 6 months following allo-PBT with CD34+ selected grafts, the number of CD4+, CD4+CD45RA+, and TCR gamma delta + cells is significantly lower than after unmanipulated allo-PBT; these differences disappeared at 8 months. In contrast, there are no differences between transplant groups in the recovery of CD8+, CD19+, and natural killer cells.

Effects of short-term administration of G-CSF (filgrastim) on bone marrow progenitor cells: analysis of serial marrow samples from normal donors.

To determine the effect of G-CSF administration on both the total number of CD34+ cells and the primitive CD34+ subsets in bone marrow (BM), we have analyzed BM samples serially obtained from 10 normal donors in steady-state and during G-CSF treatment. Filgrastim was administered subcutaneously at a dosage of 10 microg/kg/day (n = 7) or 10 microg/kg/12 h (n = 3) for 4 consecutive days. Peripheral blood sampling and BM aspirates were performed on day 1 (just before G-CSF administration), day 3 (after 2 days of G-CSF), and day 5 (after 4 days of G-CSF). During G-CSF administration, a significant increase in the total number of BM nucleated cells was observed. The percentage (range) of CD34+ cells decreased in BM from a median of 0.88 (0.47-1.44) on day 1 to 0.57 (0.32-1.87), and to 0.42 (0.16-0.87) on days 3 and 5, respectively. We observed a slight increase in the total number of BM CD34+ cells on day 3 (0.66 x 10(9)/l (0.13-0.77)), and a decrease on day 5 (0.23 x 10(9)/l (0.06-1.23)) as compared with steady-state (0.40 x 10(9)/l (0.06-1.68)). The proportion of primitive BM hematopoietic progenitor cells (CD34+CD38-, CD34+HLA-DR-, CD34+CD117-) decreased during G-CSF administration. In parallel, a significant increase in the total number of CD34+ cells in peripheral blood was observed, achieving the maximum value on day 5. These results suggest that in normal subjects the administration of G-CSF for 5 days may reduce the number of progenitor cells in BM, particularly the most primitive ones.