Ultrasound-enhanced enzymatic hydrolysis of conjugated female steroids as pretreatment for their analysis by LC-MS/MS in urine.

A fast, selective and sensitive method is here proposed for the analysis of female steroid hormones as conjugated forms (mainly, glucuronides and sulfates). The method has been applied to female urine samples to assess the metabolism of these compounds. The method implements an enzymatic hydrolysis (beta-glucuronidase with sulfatase activity) kinetically enhanced by ultrasonic energy in order to generate the free steroid forms. This enables a drastic shortening of the time required for this step as compared with conventional protocols (from 12-18 hours to 30 min). The reaction kinetics of the ultrasound-enhanced hydrolysis was characterized by comparison to that of the conventional protocol. After hydrolysis, the free steroid hormones were isolated and preconcentrated by automated solid-phase extraction and the eluate was subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The target analytes were confirmed and quantified by multiple reaction monitoring (MRM). The detection and quantification limits were within 0.06-0.8 ng mL(-1) and 0.19-2.69 ng mL(-1), respectively. The precision of the method, expressed as intra-day and inter-day variability, ranged between 2.1 and 5.2% and between 4.9 and 8.0%, respectively. A complementary study was carried out to assess the storage conditions of urine samples. This study is crucial in those applications involving metabolic processes as the integrity of the sample has to be preserved.

Ultrasonic enhancement of leaching and in situ derivatization of haloacetic acids in vegetable foods prior to gas chromatography-electron capture detection.

A continuous ultrasound-assisted approach to enhance the extraction of nine haloacetic acids (HAAs) from vegetables with in situ derivatization to methyl esters for their gas chromatography (GC) analysis is presented. The optimization of simultaneous extraction (using acidic methanol as extractant) and derivatization enabled the completion of both steps in 15 min. Ultrasound assistance has proved to enhance both linked steps, which results in a considerable shortening of the overall analysis time (i.e. 552.1 and 552.2 EPA methods for analysis of these compounds in drinking water require 1 and 2 h, respectively, only for derivatization). After sample preparation, the esterified HAAs were isolated by liquid-liquid extraction with n-hexane and analysed by GC-electron capture detection. The proposed method is an interesting alternative to present methods for the determination of HAAs in vegetable foods. This is an area unjustifiably forgotten by reference laboratory organisms as proved by the absence of official methods for analysis of the target compounds in these samples. The proposed method can be applied to the analysis of HAAs in any solid sample after optimization of the main variables involved in the extraction-derivatization step.

Automated solid-phase extraction for concentration and clean-up of female steroid hormones prior to liquid chromatography-electrospray ionization-tandem mass spectrometry: an approach to lipidomics.

A method for determination of free and glucuronide-conjugated female steroid hormones in urine at the pgmL(-1) level is here presented. For this purpose, a dual approach with or without beta-glucuronidase hydrolysis has been developed to succeed in this analysis. The target analytes were two progestogens - progesterone and pregnenolone - and three endogenous estrogens - estradiol, estriol and estrone. Separation and detection were carried out by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS-MS) with a triple quadrupole (qQq) mass detector. The determination step was optimized by multiple reaction monitoring for highly selective identification and sensitive quantification of female hormones in a complex sample such as human urine. As these compounds are present in urine at very low concentration (ngmL(-1) level), a preconcentration and clean-up step by solid-phase extraction was automatically carried out prior to the chromatographic step in order to improve the sensitivity of the method. This sample pretreatment was performed using a lab-on-valve (LOV) manifold which provided preconcentration factors ranging from 59.1 to 72.3 for 10mL urine. The detection and quantification limits were in the ranges 1.8-18pg and 6-61pg on-column, respectively, with precision values from 1.93 to 10.99%, expressed as relative standard deviation. These results enable to conclude the suitability of the LOV-LC-qQq approach for determination of the lipidomic profiling of the main female steroid hormones in a difficult matrix as human urine. The method can be potentially applied to the clinical and other metabolomic areas.

[Obtain best usage of meropenem dose in severe infections. Results of an observational multicenter study]. / Optimización de la dosis de meropenem en infecciones graves. Resultados de un estudio multicéntrico observacional.

OBJECTIVE: To describe the effectiveness and tolerability of the dose adjustment of meropenem in empirical treatment of nosocomial infections in critically-ill patients admitted to intensive care medicine services. METHODS: Prospective, observational and multicenter study in patients admitted to 17 intensive care medicine services with nosocomial infection, who were initially treated with meropenem, 1 g every 8 h, were eligible. The initial dose was adjusted to 0.5 g every 8 h if there were: a) a favorable clinical course, and b) microbiological isolation of meropenem-susceptible pathogens or absence of pathogens in cultures. RESULTS: Ninety-two patients in whom meropenem doses were adjusted to 0.5 g every 8 h were included. Ventilator-associated pneumonia followed by bacteremia was the most frequently treated infections. Microbiological studies were positive in 53 patients, with a predominance of gram-positive bacteria (53.7%), especially methicillin-susceptible Staphylococcus aureus, followed by gram-negative bacteria (42.7 %). A total of 18 patients were not evaluable at the end of treatment. Sixty-seven (90.5 %) of the 74 evaluable patients had a favorable clinical course (54 patients cured and 13 improved). In 50 out of 53 microbiologically evaluable cases, eradication or apparent eradication of initial microorganisms was achieved. In 3 cases, the initial pathogen persisted: Acinetobacter baumannii (2 cases) and Pseudomonas aeruginosa (1 case). On three occasions, new pathogens developed during treatment: A. baumannii (2 cases) and methicillin-resistant S. aureus (1 case). Adverse events occurred in 3 patients (4%), none of which was considered severe, and withdrawal of meropenem was not necessary. A total of 25 (27.2 %) patients died, three of them in relation to the infectious process. CONCLUSIONS: Dose adjustment of meropenem to 0.5 g every 8 h is a useful tool in the treatment of severe nosocomial infections in patients admitted to services of intensive care medicine except in cases in which causative pathogens are non-fermenting Gram-negative bacteria.

[Prognostic factors and etiology in patients with severe community-acquired pneumonia admitted at the ICU. Spanish multicenter study. Study Group on Severe Community-Acquired Pneumonia in Spain]. / Factores pronósticos y etiología de los pacientes con neumonía comunitaria grave ingresados en UCI. Estudio multicéntrico español. Grupo de Estudio de Neumonía Comunitaria Grave en Espanña.

OBJECTIVE: To determine the techniques used for the etiological diagnosis of community-acquired pneumonia in patients admitted to the intensive care unit (ICU) and to describe the predominant causative organisms as well as prognostic factors of ICU mortality. PATIENTS AND METHODS: A total of 262 patients with community-acquired pneumonia admitted to 26 ICUs between 1 November of 1991 and 31 October of 1992 were included in a prospective, open, multicenter study. RESULTS: The diagnostic techniques most frequently used were blood culture (243 cases) and simple tracheal aspirate (166 cases). Simple tracheal aspirate (58.4%), bronchoalveolar lavage (47.7%), and protected-specimen brush (44.2%) were the techniques that showed the highest diagnostic reliability. In 220 cases, techniques considered of high diagnostic probability were employed. With the use of these procedures, the most frequent causative pathogens were Streptococcus pneumoniae (13.6%) and Legionella pneumophila (9.5%). In 100 cases (45.5%), no pathogen was isolated. A total of 88 patients (33.6%) died during the ICU stay. Predictive variables of poor outcome selected by means of a multivariate analysis were as follows: multisystemic failure (OR = 28.6; 95% CI: 12.8-65.1; p = 0.0001), APACHE II at the time of ICU admission (OR = 5.3; 95% CI: 2.5-11.3; p = 0.0001), progression and/or spread of lung infection (OR = 4.5; 95% CI: 2.4-8.4; p = 0.0001), and shock on admission (OR = 8.48; 95% CI: 4.5-15.9; p = 0.0001). CONCLUSIONS: In 45.5% of patients with community-acquired pneumonia admitted to ICU, no causative pathogen was identified. The prognosis of these patients was influenced by the severity of disease assessed by APACHE II score and presence of multisystemic failure and shock at the time of ICU admission.

Study of sample preparation for metabolomic profiling of human saliva by liquid chromatography-time of flight/mass spectrometry.

Saliva has recently gained popularity as a potential tool for diagnosis and biomarkers monitoring, as saliva composition may potentially reflect plasma metabolite levels and, therefore, may be used as an indicator of the physiological state. The aim of the present study was to optimize a sample preparation protocol to obtain a metabolite profiling analysis of human saliva by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS) in high-accuracy mode. Under the optimum sample preparation conditions, identification of potential molecular features was carried out. This resulted in 75 compounds tentatively identified from an acidic extract and 33 from an alkaline extract, with a mass tolerance window below 10 ppm. Amino acids, lipids antioxidants and other potentially interesting biomarkers such as polyamines, vitamin B3, and ethylphosphate have been identified. This study covers the gap of knowledge about this biofluid and opens new possibilities for the selection of saliva as source of metabolite biomarkers representative of specific disorders.

Impact of liposomal amphotericin B on renal function in critically ill patients with renal function impairment.

OBJECTIVE: To assess the tolerability of liposomal amphotericin B (L-AmB) in critically ill patients with elevated serum creatinine concentrations (Cr) (> 1.5 mg/dL) at starting L-AmB therapy. METHODS: Retrospective, multicenter, comparative study of two cohorts of critically ill patients treated with L-AmB during 3 or more days, the difference between them was the level of Cr at the beginning of treatment. A cutoff value of Cr of 1.5 mg/dL was established. Patients undergoing extrarenal depuration procedures before or 48 hours after starting L-AmB were excluded. The primary endpoint was the difference between Cr values at the end of treatment as compared with Cr at starting L-AmB. Secondary endpoints were treatment-related withdrawals, need of extrarenal depuration techniques, and treatment-related severe adverse events. Demographic data, underlying illness, indication of L-AmB therapy, concomitant risk factors of nephrotoxicity, and vital status at ICU and hospital discharge were recorded. RESULTS: A total of 122 patients admitted to 26 ICUs (16 with Cr > 1.5 g/dL; 106 with normal Cr levels) were recruited. Main reasons for the use of L-AmB in both groups were the broad spectrum of the drug and the presence of hemodynamic instability. L-AmB was administered as first-line treatment in 68.8% of patients with elevated Cr and in 52.8% with normal Cr. The APACHE II score on ICU admission was 25 in patients with elevated Cr and 17 in those with normal Cr values (p < 0.001). Duration of treatment with L-AmB was 16 and 12 days in patients with elevate and normal Cr values, respectively, with a mean dose of 3.5 vs 3.9 mg/kg/day. The use of concomitant nephrotoxic drugs, mortality rate, and ICU and hospital length of stay were similar in both cohorts. In patients with renal function impairment at the initiation of L-AmB treatment, an absolute decrease of Cf-Ci of 1.08 mg/dL was observed (P < 0.001). A decrease of Cr levels to normal limits was observed in 50% of the patients; in 37.5% of patients there was a decrease but normal levels were not achieved, whereas a Cr increased occurred in only one (6.25%) patient. None of the patients required withdrawal of L-AmB or use of extrarenal depuration procedures. Treatment-related severe adverse events were not reported. CONCLUSIONS: In critically ill patients with impaired renal function, the impact of L-AmB on renal function was minimal. L-AmB can be used for the treatment of fungal infections in critically ill patients independently of renal function at the initiation of treatment.

Automated determination of folate catabolites in human biofluids (urine, breast milk and serum) by on-line SPE­HILIC­MS/MS.

A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography­tandem mass spectrometry (HILIC­MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.

Use of liposomal amphotericin B in critically ill patients: a retrospective, multicenter, clinical study.

The clinical use of liposomal amphotericin B in 179 patients admitted to 30 medical-surgical intensive Care Units (ICUs) treated with this agent in 2006 was analyzed. Invasive fungal infections were proven, probable and possible in 44%, 16%, and 25% of cases, respectively. Fungi isolated were Candida albicans (38%), non-albicans Candida spp. (15%) and Aspergillus spp. (7%). The mean duration of treatment was 15 days (mean dose 3.7 mg/kg/day). The drug was used as rescue treatment after fluconazole or caspofungin in 47% of patients and as first line in 52% with a satisfactory clinical response in 54% of cases (72.6% with proven infection). Microbiological eradication was achieved in 68% of cases. Adverse events occurred in 51 patients but were severe in only 4. The use of liposomal amphotericin B both as first line and rescue treatment and mainly for proven invasive fungal infection was associated with a high rate of satisfactory clinical response.

[Use of antibiotics for the treatment of multiresistant gram positive cocci infections in critical patients]. / Utilización de antibióticos en el tratamiento de infecciones por cocos grampositivos multirresistentes en pacientes críticos.

INTRODUCTION: This study has been designed to know the use of these antibiotics (ATB) in Intensive Care Units (ICUs). DESIGN AND PATIENTS: A multicentric, prospective, observational study was conducted. In cluded as cases were the indications of vancomycin (VAN), teicoplanin (TPN), quinupristin/dalfopristin (Q/D) and linezolid (LZD). RESULTS: A total of 826 indications (VAN 52.1%, TPN 36.6%, LZD 11.6% and Q/D 0%) were analyzed, 793 (96%) as treatment and 33 (4%) as prophylaxis in 818 patients. Serious sepsis or septic should occurred in 55.9% of the patients treated. The most common infections were pneumonia and catheter-related bacteremia, 48.3% of all the infections being acquired in ICUs. LZD was used mostly in mechanical-ventilation related pneumonia (p = 0.001), VAN in community Central Nervous System infections (p = 0.01) and in catheter-related bacteremia (p = 0.001), TPN in community pneumonia (p = 0.01) and in catheter-related bacteremia (p = 0.001). Treatments were empirical in 65.8% and diagnosis of gram positive cocci was confirmed in 48.3% of them. Staphylococcus aureus was isolated more in the LZD-treated group. Initial treatment was modified 224 times, this occurring more frequently in the VAN-treated group. The reason for this change was clinical failure 59 times, this being more frequent in the TPN group and less frequent in the patients treated with LZD, which was the drug used the most in rescue therapies (43/69, 62.3%). Adverse events, probably related to the ATB, were identified in 36/826 (4.4%) cases. CONCLUSIONS: The most common use of this ATB was to treat ICU-acquired infections. VAN was the most frequently used drug. Treatments with LZD were modified less frequently for clinical failure and this drug was the one most used in rescue therapies. This information indicates an appropriate use of these ATB in an important percentage of critically patients.

[Abnormalities in coagulation and fibrinolysis in septic shock with purpura]. / Alteraciones del sistema de la coagulación y la fibrinólisis en el shock séptico asociado a púrpura.

OBJECTIVE: To describe abnormalities in coagulation and fibrinolysis in septic shock with purpura and to assess the relationship between plasma plasminogen activator inhibitor-1 (PAI-1) concentrations and multiple organ system failure (MOSF). METHODS: Observational study in the pediatric intensive care unit of a tertiary care hospital. The presence of early MOSF was assessed at admission in 15 children with septic shock and purpura consecutively admitted to the pediatric intensive care unit. Blood samples were taken to determine coagulation and fibrinolysis parameters. RESULTS: At admission, MOSF was diagnosed in 7 patients (46.7 %), acute respiratory distress syndrome (ARDS) in 6 (40 %), consumption coagulopathy in 7 (46.7 %) and acute renal failure in 1 (6.7 %). The overall mortality rate was 40 %. Coagulation parameters were generally affected but statistically significant differences were found only in concentrations of fibrinogen and antithrombin III, which were lower in patients with MOSF than in those without organ dysfunction. Fibrinolysis parameters were increased in all patients but plasma PAI-1 concentrations were significantly elevated only in patients with MOSF and in those with ARDS. CONCLUSION: These data indicate that impaired fibrinolysis could play a major role in the development of MOSF in children with septic shock and purpura.