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Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 μg/mL for HS and from 1.56 to 100 μg/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spot (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed clear deficit in the enzymatic activity, and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patient and for monitoring the efficacy of therapeutic treatments. On the other hand, ARSA activity was measured in DBS for the first time without an antibody. Currently, this new ARSA DBS assay serves as a second-tier test following the sulfatide measurement in DBS in our large-scale de-identified pilot study for the screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay. We are also evaluating its potential utility as the first-tier test for the newborn screening of MLD.

To investigate the feasibility of a study based on treatment with topiramate (TPM) added to moderate hypothermia in newborns with hypoxic ischemic encephalopathy (HIE).Multicenter randomized controlled trial. Term newborns with precocious metabolic, clinical and electroencephalographic (EEG) signs of HIE were selected according to their amplified integrated EEG pattern and randomized to receive either TPM (10 mg/kg once a day for the first three days of life) plus moderate hypothermia or hypothermia alone. Safety was assessed by monitoring cardiorespiratory parameters and blood samples collected to check renal, liver, metabolic balance and TPM pharmacokinetics. Efficacy was evaluated by the combined frequency of mortality and severe neurological disability as primary outcome. Incidence of magnetic resonance injury, epilepsy, blindness, hearing loss, neurodevelopment at 18-24 months of life was assessed as secondary outcomes.Forty-four asphyxiated newborns were enrolled in the study. Twenty one newborns (10 with moderate and 11 with severe HIE) were allocated to hypothermia plus TPM and 23 (12 moderate and 11 severe HIE) to hypothermia. No statistically or clinically significant differences were observed for safety, primary or secondary outcomes. However, a reduction in the prevalence of epilepsy was observed in newborns co-treated with TPM.Results of this pilot trial suggest that administration of TPM in newborns with HIE is safe but does not reduce the combined frequency of mortality and severe neurological disability. The role of TPM co-treatment in preventing subsequent epilepsy deserves further studies.

This article provides supplementary data for the paper “LC–MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization” (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.

A 15-year-old girl was referred to our hospital in 2015 with a 3-month history of spontaneous mucocutaneous bleeding. Her clinical history showed she had suffered from a major stressful event (Abruzzo earthquake in 2006). Initially the bleeding was sporadic, clear and localised to the eyelids, but after a few weeks became darker, more frequent and widespread (figure 1). Before and after the episodes, no signs of wounds or scrapes were observed. The episodes were usually asymptomatic. …

A newborn with unresectable kaposiform hemangioendothelioma associated with Kasabach Merritt phenomenon, unresponsive to vincristine and prednisone, received second-line treatment with propranolol at a dose of 2 mg/kg/day, starting at 2 months of life and continued for 13 months. There was only slight reduction in tumor mass, but measurement of propranolol levels showed extremely low plasma concentrations. The propranolol dose was progressively increased to 3.5 mg/kg/day, leading to a substantial increase in plasma levels associated with clinically relevant tumor reduction. This case highlights the importance of relating propranolol dose to its plasma concentration before considering the treatment ineffective for this vascular tumor.

Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. This study evaluated safety and efficacy of propranolol eye micro-drops in preterm newborns with ROP. A multicenter open-label trial, planned according to the Simon optimal two-stage design, was performed to analyze safety and efficacy of propranolol micro-drops in newborns with stage 2 ROP. To this end, hemodynamic and respiratory parameters were monitored, and blood samples were collected weekly, for 3 wk. Propranolol plasma levels were also monitored. The progression of the disease was evaluated with serial ophthalmologic examinations. Twenty-three newborns were enrolled. Since the fourth of the first 19 newborns enrolled in the first stage of the study showed a progression to stage 2 or 3 with plus, the second stage was prematurely discontinued. Even though the objective to complete the second stage was not achieved, the percentage of ROP progression (26%) was similar to that obtained previously with oral propranolol administration. However, no adverse effects were observed and propranolol plasma levels were significantly lower than those measured after oral administration. Propranolol 0.1% eye micro-drops are well tolerated, but not sufficiently effective. Further studies are required to identify the optimal dose and administration schedule.

Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear.We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life.We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency.The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value,1.5 μmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value,0.07 μmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis.Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency.

Aim: To evaluate the relationship between the pharmacokinetic (PK) parameters and therapeutic and adverse effects of rufinamide (RUF) in children with epileptic encephalopathies (EE) aged Methods: PK analysis was conducted at the steady state using a previously validated liquid chromatography tandem-mass spectrometric method in 15 children aged 6-42 months treated with RUF in add-on. Responders were defined as patients who achieved >50% decrease of seizures. Tolerability was evaluated by analysis of a parental report of adverse effects, a clinical examination and laboratory tests. Results: Maximum plasma concentration (47.40 ± 35.36 mg/l), average plasma concentration (39.94 ± 24.53 mg/l) and half-life (13.66 ± 4.43 h) were extremely variable and considerably higher than those reported in older children treated with the same dose regimen. At the last evaluation, 9 patients (60%) were responders. Conclusion: RUF is efficacious and is well tolerated in children with EE. Nonetheless, a correlation between dose, serum concentration and efficacy could not be demonstrated. The variability in measured concentrations may be related to polytherapy that is necessary for controlling seizures in this very severe form of epilepsy, in which the off-label use of RUF is justified.

A newborn with unresectable kaposiform hemangioendothelioma associated with Kasabach Merritt phenomenon, unresponsive to vincristine and prednisone, received second-line treatment with propranolol at a dose of 2 mg/kg/day, starting at 2 months of life and continued for 13 months. There was only slight reduction in tumor mass, but measurement of propranolol levels showed extremely low plasma concentrations. The propranolol dose was progressively increased to 3.5 mg/kg/day, leading to a substantial increase in plasma levels associated with clinically relevant tumor reduction. This case highlights the importance of relating propranolol dose to its plasma concentration before considering the treatment ineffective for this vascular tumor.

Background Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). Methods Dried blood spots (DBS) samples were extracted with 200 μL of methanol and 100 μL of water (by two steps). Internal standards were added at a final concentration of 10 μmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. Results The assay was linear over a concentration range of 0.05-50 μmol/L (R2>0.999) for dGuo and 0.5-50 μmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 μmol/L and 0.5 μmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. Conclusions The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Tyrosinemia type 1 is caused by deficiency of fumarylacetoacetate hydrolase. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, causing accumulation of succinylacetone which induces severe liver and kidney dysfunction along with mutagenic changes and hepatocellular carcinoma. Treatment is based on nitisinone (NTBC), an enzymatic inhibitor which suppresses succinylacetone production. NTBC, which has dramatically changed the disease course improving liver and kidney functions and reducing risk of liver cancer, causes a side effect of the increase of tyrosine levels. Treatment is therefore based on the combination of NTBC with a protein-restricted diet to prevent the potential toxicity of excessive tyrosine accumulation. Long-term therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include succinylacetone, and a selected panel of circulating aminoacids. We have developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, succinylacetone, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run. A single assay suitable for quantitative evaluation of all biochemical markers is of great advance over conventional methods, especially in pediatric patients, since it reduces laboratory costs and blood sampling, is less invasive and particularly suitable for pediatric patients, and allows easier storage and shipping.

Carbamazepine (CBZ) is a first-line drug for the treatment of different forms of epilepsy and the first choice drug for trigeminal neuralgia. CBZ is metabolized in the liver by oxidation into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent and known to contribute to the pharmacological activity of CBZ. The aim of the present study was to develop and validate a reliable, selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of CBZ and its active metabolite in dried blood spots (DBS). The extraction process was carried out from DBS using methanol-water-formic acid (80:20:0.1, v/v/v). Chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50mL/min. The method was linear over the range 1-40mg/L and 0.25-20mg/L for CBZ and CBZE, respectively. The limit of quantification was 0.75mg/L and 0.25mg/L for CBZ and CBZE. Intra-day and inter-day assay precisions were found to be lower than 5.13%, 6.46% and 11.76%, 4.72% with mean percentage accuracies of 102.1%, 97.5% and 99.2%, 97.8% for CBZ and CBZE. We successfully applied the method for determining DBS finger-prick samples in paediatric patients and confirmed the results with concentrations measured in matched plasma samples. This novel approach allows quantification of CBZ and its metabolite from only one 3.2mm DBS disc by LC-MS/MS thus combining advantages of DBS technique and LC-MS/MS in clinical practice.

Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders. The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage. The aim of this study was to develop a LC-MS/MS method for the quantification of PHT on DBS. This analytical method was validated and gave good linearity (r(2)=0.999) in the range of 0-100mg/l. LOQ and LOD were 1.0mg/l and 0.3mg/l, respectively. The drug extraction from paper was performed in a few minutes using a mixture composed of organic solvent for 80%. The recovery ranged from 85 to 90%; PHT in DBS showed to be stable at different storage temperatures for one month. A good correlation was also obtained between PHT plasma and DBS concentrations. This method is both precise and accurate and appears to be particularly suitable to monitor treatment with a simple and convenient sample collection procedure.

In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.

Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine

Propranolol, a non-selective beta blocker drug, is used in young infants and newborns for treating several heart diseases; its pharmacokinetics has been extensively evaluated in adult patients using extrapolation to treat pediatric population. The purpose of the present study was to develop and validate a method to measure propranolol levels in dried blood spots. The analysis was performed by using liquid chromatography/tandem mass spectrometry operating in multiple reaction monitoring mode. The calibration curve in matrix was linear in the concentration range of 2.5–200 μg/L with correlation coefficient r = 0.9996. Intra-day and inter-day precisions and biases were less than 8.0% ( n = 10) and 11.5% ( n = 10) respectively. The recoveries ranged from 94 to 100% and the matrix effect did not result in a severe signal suppression. Propranolol on dried blood spot showed a good stability at three different temperatures for one month. This paper describes a micromethod for measuring propranolol levels on dried blood spot, which determines a great advantage in neonates or young infants during pharmacokinetic studies because of less invasive sampling and small blood volume required.

Background Despite progresses in neonatal care, the mortality and the incidence of neuro-motor disability after perinatal asphyxia have failed to show substantial improvements. In countries with a high level of perinatal care, the incidence of asphyxia responsible for moderate or severe encephalopathy is still 2–3 per 1000 term newborns. Recent trials have demonstrated that moderate hypothermia, started within 6 hours after birth and protracted for 72 hours, can significantly improve survival and reduce neurologic impairment in neonates with hypoxic-ischemic encephalopathy. It is not currently known whether neuroprotective drugs can further improve the beneficial effects of hypothermia. Topiramate has been proven to reduce brain injury in animal models of neonatal hypoxic ischemic encephalopathy. However, the association of mild hypothermia and topiramate treatment has never been studied in human newborns. The objective of this research project is to evaluate, through a multicenter randomized controlled trial, whether the efficacy of moderate hypothermia can be increased by concomitant topiramate treatment. Methods/Design Term newborns (gestational age ≥ 36 weeks and birth weight ≥ 1800 g) with precocious metabolic, clinical and electroencephalographic (EEG) signs of hypoxic-ischemic encephalopathy will be randomized, according to their EEG pattern, to receive topiramate added to standard treatment with moderate hypothermia or standard treatment alone. Topiramate will be administered at 10 mg/kg once a day for the first 3 days of life. Topiramate concentrations will be measured on serial dried blood spots. 64 participants will be recruited in the study. To evaluate the safety of topiramate administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of topiramate, the neurologic outcome of enrolled newborns will be evaluated by serial neurologic and neuroradiologic examinations. Visual function will be evaluated by means of behavioural standardized tests. Discussion This pilot study will explore the possible therapeutic role of topiramate in combination with moderate hypothermia. Any favourable results of this research might open new perspectives about the reduction of cerebral damage in asphyxiated newborns. Trial registration Current Controlled Trials ISRCTN62175998; ClinicalTrials.gov Identifier NCT01241019; EudraCT Number 2010-018627-25

Objective To evaluate safety and efficacy of oral propranolol administration in preterm newborns affected by an early phase of retinopathy of prematurity (ROP). Study design Fifty-two preterm newborns with Stage 2 ROP were randomized to receive oral propranolol (0.25 or 0.5 mg/kg/6 hours) added to standard treatment or standard treatment alone. To evaluate safety of the treatment, hemodynamic and respiratory variables were continuously monitored, and blood samples were collected weekly to check for renal, liver, and metabolic balance. To evaluate efficacy of the treatment, the progression of the disease (number of laser treatments, number of bevacizumab treatments, and incidence of retinal detachment) was evaluated by serial ophthalmologic examinations, and plasma soluble E-selectin levels were measured weekly. Results Newborns treated with propranolol showed less progression to Stage 3 (risk ratio 0.52; 95% CI 0.47-0.58, relative reduction of risk 48%) or Stage 3 plus (relative risk 0.42 95% CI 0.31-0.58, relative reduction of risk 58%). The infants required fewer laser treatments and less need for rescue treatment with intravitreal bevacizumab (relative risk 0.48; 95% CI 0.29-0.79, relative reduction of risk 52 %), a 100% relative reduction of risk for progression to Stage 4. They also had significantly lower plasma soluble E-selectin levels. However, 5 of the 26 newborns treated with propranolol had serious adverse effects (hypotension, bradycardia), in conjunction with episodes of sepsis, anesthesia induction, or tracheal stimulation. Conclusion This pilot study suggests that the administration of oral propranolol is effective in counteracting the progression of ROP but that safety is a concern.