Impact of FLT3 Internal Tandem Duplication on the Outcome of Related and Unrelated Hematopoietic Transplantation for Adult Acute Myeloid Leukemia in First Remission: A Retrospective Analysis

Purpose Patients with acute myeloid leukemia (AML) and FLT3/internal tandem duplication (FLT3/ITD) have poor prognosis if treated with chemotherapy only. Whether this alteration also affects outcome after allogeneic hematopoietic stem-cell transplantation (HSCT) remains uncertain. Patients and Methods We analyzed 206 patients who underwent HLA-identical sibling and matched unrelated HSCTs reported to the European Group for Blood and Marrow Transplantation with a diagnosis of AML with normal cytogenetics and data on FLT3/ITD (present: n = 120, 58%; absent: n = 86, 42%). Transplantations were performed in first complete remission (CR) after myeloablative conditioning. Results Compared with FLT3/ITD-negative patients, FLT3/ITD-positive patients had higher median leukocyte count at diagnosis (59 v 21 x 10(9)/L; P < .001) and shorter interval from CR to transplantation (87 v 99 days; P = .04). Other characteristics were similar in the two groups. At 2 years, relapse incidence (RI; +/- standard deviation) was higher (30% +/- 5% v 16% +/- 5%; P = .006) and leukemia-free survival (LFS) lower (58% +/- 5% v 71% +/- 6%; P = .04) in FLT3/ITD-positive compared with FLT3/ITD-negative patients. In multivariate analyses, FLT3/ITD led to increased RI (hazard ratio [HR], 3.4; 95% CI, 1.46 to 7.94; P = .005), as did older age, female sex, shorter interval between CR and transplantation, and higher number of chemotherapy courses before achieving CR. FLT3/ITD positivity was associated with decreased LFS (HR, 0.37; 95% CI, 0.19 to 0.73; P = .002), along with older age and higher number of chemotherapy courses before achieving CR. Conclusion FLT3/ITD adversely affected the outcome of HSCT in the same direction it does after chemotherapy; despite this, more than half of the patients harboring this mutation who received transplants were alive and leukemia free at 2 years. To further improve the results, use of FLT3 inhibitors before or after HSCT deserves investigation.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min(-1) into a Phenomenex Gemini (R) C18, 5 mu m analytical column (150 x 4.6 mm id.). The calibration curve was linear over the range from 0.2 to 200 ng mL(-1) for dextromethorphan and doxylamine and 0.05 to 10 ng mL(-1) for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study. (C) 2012 Elsevier B.V. All rights reserved.

Relationship of short tandem repeats flanking leptin-melanocortin pathway genes with anthropometric profile and leptinemia in Brazilian individuals

Objective: To investigate the relationship of short tandem repeats (STR) near genes involved in the leptin-melanocortin pathway with body mass index (BMI) and leptinemia. Subjects and methods: Anthropometric variables and leptinemia were measured in 100 obese and 110 non-obese individuals. D1S200, D2S1788, DS11912, and D18S858 loci were analyzed by PCR and high-resolution electrophoresis. Results: Overall STR allele frequencies were similar between the obese and non-obese group (p > 0.05). Individual alleles D1S200 (17), D11S912 (43), D18S858 (11/12) were associated with obesity (p < 0.05). Individuals carrying these alleles showed higher BMI than non-carriers (p < 0.05). Moreover, a relationship between D18S858 11/12 alleles and increased waist circumference was found (p = 0.040). On the other hand, leptinemia was not influenced by the studied STRs (p > 0.05). Conclusions: D1S200, D11S912, and D18S858 loci are associated with increased BMI and risk for obesity in this sample. Arq Bras Endocrinol Metab. 2012;56(1):47-53

Development of Nanoinjector Devices for Electrospray Ionization - Tandem Mass Spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 mu L of sample.

Sensitive and fast determination of Sudan dyes in chilli powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 mu g/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective beta-blocker activity but both enantiomers present similar activity on a1-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T (R) (Teicoplanin) column. Protonated ions [M + H]+ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml-1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC08 205.52 vs. 82.61 (ng h) ml-1), with an enantiomer ratio of 2.48. Chirality, 2012. (C) 2012 Wiley Periodicals, Inc.

SENSITIVE METHOD FOR DETERMINATION OF PIPLARTINE, AN ALKALOID AMIDE FROM Piper SPECIES, IN RAT PLASMA SAMPLES BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

Corrole isomers: intrinsic gas-phase shapes via traveling wave ion mobility mass spectrometry and dissociation chemistries via tandem mass spectrometry

Corrole and four of its isomers with subtle structural changes promoted by exchange of nitrogen and carbon atoms in the corrole ring have been studied by traveling wave ion mobility mass spectrometry and collision induced dissociation experiments. Significant differences in shapes and charge distributions for their protonated molecules were found to lead to contrasting gas phase mobilities, most particularly for corrorin, the most "confused" isomer. Accordingly, corrorin was predicted by B3LYP/6-31g(d,p) and collisional cross section calculations to display the most compact tri-dimensional structure, whereas NCC4 and corrole were found to be the most planar isomers. Better resolution between the corrole isomers was achieved using the more polarizable and massive CO2 as the drift gas. Sequential losses of HF molecules were found to dominate the dissociation chemistry of the protonated molecules of these corrole isomers, but their unique structures caused contrasting labilities towards CID, whereas NCC4 showed a peculiar and structurally diagnostic loss of NH3, allowing its prompt differentiation from the other isomers.

Sensitive method for determination of piplartine, an alkaloid amide from Piper species, in rat plasma samples by liquid chromatography-tandem mass spectrometry

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.

Hydroxocobalamin quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a pharmacokinetic study

A rapid, sensitive and specific method for quantifying hydroxocobalamin in human plasma using paracetamol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethanol 100%; -20°C). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on Prevail C8 3 μm, analytical column (2.1×100 mm i.d.). The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 5-400 ng.mL-1 (r>0.9983). The limit of quantification was 5 ng.mL-1. The method was also validated without the use of the internal standard. The precision in the intra-batch validation with IS was 9.6%, 8.9%, 1.0% and 2.8% whereas without IS was 9.2%, 8.2%, 1.8% and 1.5% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in intra-batch validation with IS was 108.9%, 99.9%, 98.9% and 99.0% whereas without IS was 101.1%, 99.3%, 97.5% and 92.5% for 5, 15, 80 and 320 ng/mL, respectively. The precision in the inter-batch validation with IS was 9.4%, 6.9%, 4.6% and 5.5% whereas without IS was 10.9%, 6.4%, 5.0% and 6.2% for 5, 15, 80 and 320 ng/mL, respectively. The accuracy in inter-batch validation with IS was 101.9%, 104.1%, 103.2% and 99.7% whereas without IS was 94.4%, 101.2%, 101.6% and 96.0% for 5, 15, 80 and 320 ng/mL, respectively. This HPLC-MS-MS procedure was used to assess the pharmacokinetics of Hydroxo cobalamin following intramuscular injection 5000 μg in healthy volunteers of both sexes (10 males and 10 females). The volunteers had the following clinical characteristics (according to gender and expressed as mean ± SD [range]): males: age: 32.40 ± 8.00 y [23.00-46.00], height: 1.73 ± 0.07 m [1.62-1.85], body weight: 72.48 ± 10.22 Kg [60.20- 88.00]; females: age: 28.60 ± 9.54 y [18.00-44.00], height: 1.60 ± 0.05 m [1.54-1.70], body weight: 58.64 ± 6.09 Kg [51.70- 66.70]. The following pharmacokinetic parameters were obtained from the hydroxocobalamin plasma concentration vs. time curves: AUClast, T1/2, Tmax, Vd, Cl, Cmax and Clast. The pharmacokinetic parameters were 120 (± 25) ng/mL for Cmax, 2044 (± 641) ng.h/mL for AUClast, 8 (± 3.2) ng.mL-1 for Clast, 38 (± 15.8) hr for T1/2 and 2.5 (range 1-6) hr for Tmax. Female volunteers presented significant (p=0.0136) lower AUC (1706 ± 704) ng.h/mL) and larger (p=0.0205) clearance (2.91 ± 1.41 L/hr), as compared to male 2383 ± 343 ng.h/mL and 1.76 ± 0.23 L/hr, respectively. These pharmacokinetic differences could explain the higher prevalence of vitamin B12 deficiency in female patients. The method described validated well without the use of the internal standard and this approach should be investigated in other HPLC-MS-MS methods.

The role of wake stiffness on the wake-induced vibration of the downstream cylinder of a tandem pair

When a pair of tandem cylinders is immersed in a flow the downstream cylinder can be excited into wake-induced vibrations (WIV) due to the interaction with vortices coming from the upstream cylinder. Assi, Bearman & Meneghini ( J. Fluid Mech. , vol. 661, 2010, pp. 365–401) concluded that the WIV excitation mechanism has its origin in the unsteady vortex–structure interaction encountered by the cylinder as it oscillates across the wake. In the present paper we investigate how the cylinder responds to that excitation, characterising the amplitude and frequency of response and its dependency on other parameters of the system. We introduce the concept of wake stiffness , a fluid dynamic effect that can be associated, to a first approximation, with a linear spring with stiffness proportional to Re and to the steady lift force occurring for staggered cylinders. By a series of experiments with a cylinder mounted on a base without springs we verify that such wake stiffness is not only strong enough to sustain oscillatory motion, but can also dominate over the structural stiffness of the system. We conclude that while unsteady vortex–structure interactions provide the energy input to sustain the vibrations, it is the wake stiffness phenomenon that defines the character of the WIV response