A liquid chromatography-tandem mass spectrometry platform for the routine therapeutic drug monitoring of 14 antibiotics: Application to critically ill pediatric patients.

BACKGROUND: The accurate measurement of plasma levels of antibiotics is crucial for the individualization of antimicrobial therapies based on PK/PD strategies. In this paper we describe a new rapid and simple LC-MS/MS platform for quantifying 14 antibiotics (amikacin, amoxicillin, ceftazidime, ciprofloxacin, colistin, daptomycin, gentamicin, linezolid, meropenem, piperacillin, teicoplanin, tigecycline, tobramycin and vancomycin) and a beta-lactamase inhibitor (tazobactam) starting from 50 µL plasma samples. METHODS: Analyses were performed on a Thermo Scientific™ Ultimate™ 3000 LC system (Thermo Fisher Scientific, Milan, Italy) coupled to a Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole mass spectrometer. After fast protein precipitation protocols and addition of deuterated internal standards, samples were subjected to a fast HPLC gradient separation and the 15 drugs were quantified using multiple reaction monitoring of specific transitions over a wide range of concentrations. The suitability of the assay for TDM was tested on plasma samples derived from pediatric patients under treatment with one or more antibiotics. RESULTS: The overall turnaround time of the assay was 20 min. The assay was validated following EMA guidelines for bioanalytical method validation and showed excellent accuracy (ranging from 85.3 and 112.7) and reproducibility (ranging from 1.3 to 9.7) as well as the absence of matrix effects (<15 %) for all the drugs tested. The lower limits of quantifications were between 0.1 and 2 mg/L. the recovery rate exceeded 85 % for all the drug tested. Stability was evaluated in different conditions thus allowing the setting up of reliable operative procedures. CONCLUSIONS: This work provides a LC-MS/MS platform validated for clinical use for a rapid quantification of a broad spectrum of drugs having different chemical characteristics in a small volume of plasma and is suitable for real-time TDM-guided personalization of antimicrobial treatment in critically ill patients.

A rapid and robust UHPLC-DAD method for the quantification of amphotericin B in human plasma.

Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC-DAD) method for the quantification of Amphotericin B in 200µL human plasma over a wide range of concentrations (0.125-10mg/L). The new method has been validated following international guidelines on bioanalytical method validation and showed high selectivity, high accuracy and precision and high process efficiency. The new UHPLC-DAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

Ultra high performance liquid chromatography-tandem mass spectrometry vs. commercial immunoassay for determination of vancomycin plasma concentration in children. Possible implications for everyday clinical practice.

BACKGROUND: Vancomycin therapeutic drug monitoring (TDM) is necessary for effective and safetherapy. The aim of the this paper was to develop a specific and robust ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for vancomycin quantification starting from low plasma volumes to be applied for the routine TDM in children. METHODS: Samples from children receiving intravenous vancomycin were analysed using a TSQ Quantum Access MAX Triple Quadrupole system coupled with an Accela 1250 UHPLC system after a rapid protein precipitation. Gradient separation chromatography was carried out using a Hypersil GOLD aQ C18 column (50 × 2.1 mm, particle size 1.9 µm). Method performance was validated following international guidelines. RESULTS: UHPLC-MS/MS allowed a rapid and specific quantification of vancomycin over the range 0.1-128 µg/mL from 50 µL of plasma with high reproducibility and accuracy in the absence of matrix effect. The comparison with the commercial immunoassay performed on 138 samples demonstrated the presence of a proportional bias. The concentrations of vancomycin measured with immunoassay were found to be 4.5% (95% CI: 1.3-7.7) higher than those determined with UHPLC-MS/MS. Importantly, a clinical discordance was found in about 10% of samples analysed. CONCLUSIONS: This new UHPLC-MS/MS method is accurate and specific for the measurement of vancomycin starting from small (50 µL) plasma volumes. The use of UHPLC-MS/MS is recommended to prevent a misclassification of therapeutic or toxic vancomycin levels in paediatrics.

Urinary homovanillic and vanillylmandelic acid in the diagnosis of neuroblastoma: report from the Italian Cooperative Group for Neuroblastoma.

BACKGROUND: Urinary homovanillic and vanillylmandelic acid (HVA and VMA) are well known biomarkers for the management of neuroblastoma (NB). Very few and contradictory publications on their diagnostic performance are present in the literature. The aim of this study is to review the results of HVA/Cr and VMA/Cr obtained by the reference laboratory of the Italian Cooperative Group for NB within a 7-year period using HPLC-EC. PROCEDURE: Updated reference intervals based on age as a continuous variable were calculated by using a multivariate statistical analysis. The diagnostic performance of the two biomarkers has been established by calculating their specificity and sensitivity and by receiver operating characteristics (ROC) curves for different ages and stages of disease. RESULTS: Accurate age-related reference intervals were obtained from 648 HVA/Cr and 671 VMA/Cr results derived from patients in which the diagnosis of neuroendocrine tumors was excluded. Sensitivity, specificity and ROC curves were obtained from 169 HVA/Cr and 179 VMA/Cr results from confirmed NB patients. The best diagnostic performance was obtained in stage 4S tumors and in children <18months. CONCLUSIONS: This is the first report, to our knowledge, that analyzes in depth the diagnostic performance of HVA/Cr and VMA/Cr for NB in different stages and age subgroups. In addition, the present work provides cut-off points able to discriminate between NB patients and negative subjects suspected to have NB and could be of help in taking medical decisions.

Prognostic value of ferritin, neuron-specific enolase, lactate dehydrogenase, and urinary and plasmatic catecholamine metabolites in children with neuroblastoma.

Different plasma and urinary parameters have been tested as valuable prognostic markers for children with neuroblastoma (NB), but conclusive results from multivariate analyses are still lacking. Samples collected at diagnosis from 505 patients diagnosed in Italy between June 1994 and November 2010 were analyzed at the Italian reference laboratory according to standard methodologies. Patient clinical data were retrieved from the Italian NB Registry. For statistical analysis, patients were grouped according to stage, age, MYCN status, and outcome. Cumulative survival was calculated by the Kaplan-Meier procedure using the first quartile of the marker distribution as a cut-off value to stratify the patients. Multivariate analysis was performed by the Cox regression model by considering only the significant variables. When the entire cohort of patients was considered, none of the different parameters had an independent prognostic value. However, in patients with localized disease without MYCN amplification the significant positive associations between urinary and plasmatic vanillylmandelic acid (VMA)/homovanillic acid (HVA) ratio and a better prognosis remained significant (P < 0.05 and P < 0.01, respectively), as well as, the positive association between high lactate dehydrogenase (LDH) values and a worse prognosis (P < 0.001). Moreover, in stage 4 patients without MYCN amplification, neuron-specific enolase levels above 200 ng/mL and LDH levels above 2500 IU/mL maintained their significant association with a worse outcome (P = 0.01 and P = 0.0001, respectively). In conclusion, LDH had an independent prognostic value in patients of all stages without MYCN amplification. Moreover, the urinary and plasmatic VMA/HVA ratio was an independent predictor of prognosis in patients with localized disease without MYCN amplification. Since LDH and catecholamine metabolites are measured in all patients at diagnosis, these findings may be helpful for an easy, cost-effective, patient risk stratification.

Identification and structural characterization by LC-ESI-IONTRAP and LC-ESI-TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients.

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow-up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC-EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC-EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) and liquid chromatography-ion trap tandem mass spectrometry (LC-IT-MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with ß-glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol- and acyl-glucuronide of HVA. The latter was the unknown peak observed in LC-EC separations of urine samples from NB patients.

Erythrocyte Galactose-1-phosphate measurement by GC-MS in the monitoring of classical galactosemia.

BACKGROUND: Classical galactosemia is a rare but very severe disease characterized by a deficiency of the galactose-1-phosphate uridyltransferase enzyme. The confirmed galactosemic patients are treated with a galactose-restricted diet. Nevertheless, metabolites such as galactose-1-phosphate can accumulate in red blood cells of treated patients and its measurement is a standard practice for their monitoring. At present, no commercial methods for measuring galactose-1-phosphate in erythrocytes are available. METHODS: In this study, we will describe the optimization and laboratory validation of a previously published quantitative gas chromatographic-mass spectrometric method and its clinical validation on normal donors and galactosemic patients both at the diagnosis and during the follow-up. RESULTS: The method was technically optimized and validated for its clinical use on normal donors and galactosemic newborns, children and adults. The method was suitable for the monitoring of dietary compliance. Galactose-1-phosphate levels were found to be well correlated with the clinical signs in the galactosemic patients at the follow-up. CONCLUSIONS: This paper provides information on the measurement of Galactose-1-phosphate levels that can be very useful for the management of classical galactosemia.

Physical properties of root cementum: Part 10. Comparison of the effects of invisible removable thermoplastic appliances with light and heavy orthodontic forces on premolar cementum. A microcomputed-tomography study.

INTRODUCTION: Orthodontic treatment with clear sequential removable thermoplastic appliances (TAs) is gaining popularity as an alternative to treatment with fixed appliances. The amount of orthodontically induced inflammatory root resorption generated by such appliances has not been investigated. In this prospective randomized clinical trial, we used x-ray microtomography to quantify resorption generated by treatment with ClearSmile appliances (ClearSmile, Woollongong, Australia) and compared the effects with those of heavy and light conventional orthodontic forces and no force. METHODS: The sample consisted of 54 maxillary first premolars in 27 patients who required bilateral extractions as part of their planned orthodontic treatment. The subjects were randomly assigned to 3 groups, each with 9 subjects. A split-mouth design was used, and forces were applied to the first premolars. In group 1, TAs were used to move teeth on 1 side in a buccal direction at a rate of 0.5 mm every 2 weeks (TA movement); the contralateral teeth were not moved and served at controls. In group 2, TA movement was used on 1 side. A buccal force of 225 g from a beta-titanium alloy cantilever spring (heavy force) was used on the contralateral side. In group 3, TA movement was used on 1 side. A buccal force of 25 g from a cantilever spring (light force) was used on the contralateral side. The treatment duration was 8 weeks (56 days +/- 1 day). The TAs were changed every 14 days, and each patient used 4 appliances. The springs were not reactivated. At the end of the study period, the teeth were extracted according to a strict protocol to prevent root damage. Resorption was measured with an x-ray microtomograph (1072, SkyScan, Aartselaar, Belgium). Software analysis determined quantity, location, and distribution of root resorption craters. RESULTS: The control teeth had the least amount of resorption. The light-force teeth had approximately 5 times more resorption than the control teeth (P <.001). The TA teeth had similar but slightly greater resorption than the light-force teeth, or approximately 6 times greater than the control teeth (P <.001). The heavy-force teeth had the most resporption, about 9 times greater than the controls (P <.001). CONCLUSIONS: Clear removable TAs have similar effects on root cementum as light (25 g) orthodontic forces with fixed appliances.

A novel pressure film approach for determining the force imparted by clear removable thermoplastic appliances.

The force imparted by removable thermoplastic appliances (RTA) onto teeth has not been investigated in the past. This investigation was designed to explore a novel methodology to measure the magnitude and identify the pattern of this force. Eight patients with moderate malocclusion were selected. In each patient, the palatally mal-positioned upper first premolar was corrected by wearing a series of four ClearSmile RTA over a duration of 8 weeks. When constructing RTA, the ClearSmile Company was advised that the amount of movement to be programmed into each appliance was 0.5 mm. The Pressurex film was used to measure the pressure generated by ClearSmile RTA against the palatal surface of the upper first premolar for buccal tipping movement. Three measurements were conducted respectively upon the issue and retrieval of each appliance (after 2 weeks of wear), resulting in 24 pressure measurements for each patient. Digital imaging and spectrophotometry analysis were employed to quantify the stain intensity mounted by the pressure on the films. The irrelevant forces were subtracted out to allow an assessment of the force purely acting to buccally repositioning the tooth. The results revealed that (1) the mean force magnitude over 2 weeks of RTA wear was 1.12 N (SE = 0.72 N); (2) the higher force magnitude of 5.12 N (SE = 0.80 N) seen at the issue of the appliance declined drastically to -2.67 N at the time of retrieval. These findings suggest that ClearSmile RTA exerts a high level of force against the tooth to be moved at the initial stage followed by a rapid force diminish.