A new strategy for the computer-assisted development of reversed-phase liquid chromatography separation methods of unknown sample mixtures.

A new strategy for the computer-assisted methods development in the reversed-phase liquid chromatographic separations of unknown sample mixtures has been developed using the latent spectral information in chromatogram raw data files of appropriately designed experiments, rather than resorting to elemental information functions (e.g., the number of peaks in chromatograms or similar criteria). The strategy developed allows unification of the approach for samples of both known and unknown composition and, thus, provide a general strategy for computer-aided tools in the chromatography laboratory. The operation principle of this strategy departs from extracting the spectra of components in the mixture chromatograms by resorting to multivariate curve resolution-alternating least squares (MCR-ALS). This technique allows the estimation of the true spectra for the individual components except when they have identical spectra or are fully overlapped. Thus, a convenient experimental design will try to perform separations of the sample mixture having at least partial resolution of components in some runs. This will allow estimating the spectra of components and, then, assign these components to the peaks in each run chromatogram. In this way, a retention model can be built for each component so computerized optimization process can be developed to provide the chromatographer with the best possible separation programs. Following this approach, strategies for sample mixtures of known and unknown composition are only different in the need of an initial spectrum discovery process for unknown mixtures and therefore a real general approach for the computer-assisted LC methods development is now available for the first time.

Automatic assignment of 1H-NMR spectra of small molecules.

A novel data-evaluation procedure for the automatic atom to peak or multiplet assignment of 1H-NMR spectra of small molecules has been developed using a fast and robust expert system. The applicability and reliability of the method are demonstrated by comparison of a manually assigned database of 1H-NMR spectra with the assignments produced by the automatic procedure. The results of this analysis show an excellent success ratio, indicating that this new algorithm can have a major impact as a time saving tool for the organic chemist. A new graphical feature used to illustrate both the stability and quality of the elementary assignments is also introduced.

Robustness assessment in computer-assisted liquid chromatography procedures based on desirability functions.

A novel approach based on the use of desirability functions is presented for the robustness assessment of liquid chromatographic separations as derived from computer-assisted methods development processes. The approach is based on generally accepted hypothesis that a robust separation procedure will be inert to small random variations of the operational variables, typically encountered in the day-to-day routine analytical practice. This means that peak positions along the chromatograms must keep standstill or move insignificantly when operational variables are not intentionally changed. Thus, the degree of peak positions variation as evaluated from mathematical retention models can be used to assess the robustness of the developed procedures before testing the actual performance experimentally. In the approach proposed, this assessment is obtained by fixing a bilateral partial desirability window around each peak in the simulated chromatogram. The whole chromatogram robustness is characterized by an overall desirability value calculated as the geometric mean of the partial desirability windows evaluation. An added advantage of this approach is that the robustness value calculated is normalized between zero and one and thus, easy to interpret. Thus, when chromatograms are simulated and small random variations are introduced into the operational factors of the model, values for the overall desirability close to one means that the procedure performs robustly. On the contrary, low values for the overall desirability clearly indicated a serious lack of robustness. When used in conjunction with the Pareto optimality approach, as shown here, this robustness assessment strategy allows testing several Pareto front solutions before the final experimental testing which is always needed. In this way, a dramatical reduction of the experimental effort is obtained. Although the approach is theoretically applicable to any chromatographic separation, examples of reversed phase liquid chromatographic procedures are used to show the performance of the proposed methodology.

Validation of PROFUND prognostic index over a four-year follow-up period.

BACKGROUND/OBJECTIVES: The PROFUND index stratifies accurately the 12-month mortality risk of polypathological patients (PPs), but its fitness over a longer follow-up period remains unknown. We aimed to explore the calibration and discrimination power of PROFUND index over 4-years, in order to assess its follow-up interval generalizability. DESIGN: Multicenter prospective cohort-study. SETTING: 33 Spanish hospitals. PARTICIPANTS: PPs included after hospital discharge, outpatient clinics, or home hospitalization. MEASUREMENTS: Mortality over a 4-year follow-up period. METHODS: PROFUND index calibration was assessed by risk-quartiles predicted/observed mortality (Hosmer-Lemeshow goodness-of-fit test), and its discrimination power by ROC curves. RESULTS: A total of 768 patients were included (630 [82%] of them completed the 4-year follow-up). Global mortality rate was 63.5%. When assessing individual patient scores, mortality was 52% in the lowest risk group (0-2 points in PROFUND score); 73.5% in the low-intermediate risk group (3-6 points), 85% in the intermediate-high group (7-10 points); and 92% in the highest risk group (≥11 points). Accuracy testing of the PROFUND index showed good calibration (P=.8 in the Hosmer-Lemeshow goodness-of-fit test), and also a good discrimination power (AUC=0.71 [0.67-0.77] in ROC curve). CONCLUSIONS: The PROFUND index maintained its accuracy in predicting mortality of polypathological patients over a 4-year follow-up period. This index may be of potential usefulness in deciding the most appropriate health-care interventions in populations with multimorbidity.

Effects of central epinephrine synthesis inhibition on stress-induced prolactin secretion in male rats.

1. The present study was designed to examine the role of central epinephrine pathways in the control of stress-induced prolactin secretion in male adult Wistar rats. 2. Intracerebroventricular administration of two epinephrine synthesis inhibitors, SKF 64139 (5 and 10 micrograms/rat) and LY 134046 (10 and 20 micrograms/rat), 6 h before the onset of immobilization stress blocked prolactin secretion in a dose-dependent manner. Prolactin values before stress were about 4.0 ng/ml and increased to almost 50 ng/ml in the control group. SKF 64139 injection in the higher dose (10 micrograms/rat) induced a complete blockade of the stress-induced prolactin release, whereas partial blockade was observed after the higher dose (20 micrograms/rat) of LY 134046. 3. Salbutamol pretreatment (10 micrograms/rat) completely restored stress-induced prolactin secretion in animals receiving a central injection of both epinephrine synthesis inhibitors under the same conditions as described above. 4. It is suggested that epinephrine pathways in the brain play an important role in the control of prolactin release occurring during immobilization stress.

Desipramine blocks stress-induced prolactin release in rats: role of central beta-2 adrenoceptors.

1. The present study was designed to examine the relationship between beta-adrenoceptors and the enhanced, sustained prolactin secretion induced by immobilization stress in rats. 2. Chronic administration of desipramine (15 mg kg-1 day-1, intraperitoneally) for 7 days, a procedure that desensitizes central beta-adrenoceptors, partially inhibits stress-induced prolactin release. 3. Intracerebroventricular administration of the beta-2 adrenoceptor agonist salbutamol (1 microgram/rat) to rats pretreated with desipramine for 7 days, 15 min before immobilization, significantly relieved the inhibition by desipramine 5 and 10 min after the initiation of stress but the effect was not demonstrable after 20 and 40 min. 4. We conclude that beta-2 adrenoceptors play a role in the control of prolactin release in response to stress.

Desipramine blocks stress-induced prolactin release in rats: role of central beta-2 adrenoceptors

The present study was designed to examine the relationship between beta-adrenoceptors and the enhanced, sustained prolactin secretion induced by immobilization stress in rats. Chronic administration of desipramine (15 mg kg**-1 day**-1, intraperitoneally) for 7 days, a procedure that desensitizes central beta-adrenoceptors, partially inhibits stress-induced prolactin release. Intracerebroventricular adminsitration of the beta-2 adrenoceptor agonist salbutamol (1 microng/rat) to rats pretreated with desipramine for 7 days, 15 min before immobilization, significantly relieved the inhibition by desipramine 5 and 10 min after the initiation of stress but the effect was not demonstrable after 20 and 40 min. We conclude that beta-2 adrenoceptors play a role in the control prolactin release in response to stress

Effects of central epinephrine synthesis inhibition on stress-induced prolactin secretion in male rats

1. The present study was designed to examine the role of central epinephrine pathways in the control of stress-induced prolactin secretion in male adulto Wistar rats. 2. Intracerebroventricular adminsitration of two epinephrine synthesis inhibitors, SKF64139 (5 and 10 µg/rat) and LY 134046 (10 and 20 µg/rat), 6 h before the onset of immobilization stress blocked prolactin secretion in a dose-dependent manner. Prolactin values before stress were about 4.0 ng/ml and increased to almost 50 ng/ml in the control group. SKF 64139 injection in the higher dose (10 µg/rat) induced a complete blockade of the stress-induced prolactin release, whereas partial blockade was observed after the higher dose (20 µg/rat) of LY 134046. 3.Salbutamol pretreatment (10 µg/rat) completely restored stress-induced prolactin secretion in animals receiving a central injection of both epinephrine synthesis inhibitors under the same conditions as described above. 4. It is suggested that epinephrine pathways in the brain play an important role in the control of prolactin release occuring during immobilization stress