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Amino acid neurotransmitters, including glutamate, phenylalanine, tyrosine, alanine, and glycine, underlie the majority of the excitatory and inhibitory neurotransmission in the nervous system, and acute exercise has been shown to modulate their concentrations. We aimed to determine whether any correlation exists between the above-mentioned amino acid blood concentrations and the neuropsychological performance after an acute exercise intervention. Sixty basketball players were randomly assigned to one of two experimental conditions: exercise or inactive resting. All participants underwent a comprehensive neuropsychological assessment and blood samples were taken on a Guthrie card before and after the end of the experimental conditions. Amino acid blood concentrations were significantly elevated and cognitive performance significantly improved post-exercise on specific neuropsychological assessments. Significant intervention × group interaction effects were apparent for Trail Making Test part-B [F(1,58) = 20.46

Compounded medicinal products containing bupropion hydrochloride (BUP·HCl) and naltrexone hydrochloride (NTX·HCl) are available as adjunct therapy for the management of weight in obese/overweight adults. The present work describes the development and validation of a novel RP-HPLC method for a simultaneous quantitation during the dissolution of both drugs from compounded bilayer composition tablets. The method involves a Nucleosil 100-3 C-18 column (4.6 × 150 mm) and a mobile phase of a 70%/30% v/v ACN/KH2PO4·H2O aqueous solution of a 5 mM concentration. The flow rate was set at 1.35 mL/min and the detection was conducted using UV spectrophotometry (λmax 214 nm). The method was validated according to the ICH guidelines and fulfilled the specifications for the specificity, linearity, accuracy, precision and stability for both the sample and standard solutions. Furthermore, the robustness of the method was evaluated by applying a fractional factorial experimental design and by utilizing both graphical and statistical approaches to identify the HPLC factors that should be strictly controlled during the analysis. The method proved to be suitable for the analysis of the dissolution samples and, consequently, the release of BUP·HCl and NTX·HCl from the formulations.

Objectives Medium-chain (MCA) and long-chain acylcarnitine (LCA) blood concentrations play a significant role in the fatty acid (FA) oxidation process, especially during the first days of life. Identification of their abnormal concentrations, via expanded newborn screening, can lead to the diagnosis of FA oxidation disorders. This study aimed to demonstrate MCA and LCA concentrations in Dried Blood Spots (DBS) of full-term breastfed infants, in relation to their birth weight (BW) perinatally. Methods Breastfed full-term infants (n = 12,000, 6,000 males, 6,000 females) with BW 2,000–3,999 g were divided into four equal groups: Group A, 2,000–2,499 g, B 2,500–2,999 g, C 3,000–3,499 g, and D 3,500–3,999 g. Samples were collected as DBS and acylcarnitines were determined via a liquid chromatography tandem mass spectrometry method. Results MCA and LCA blood concentrations were determined significantly lower in group A (low birth weight infants) in both sexes. Infants with BW > 3,500 g (group D), were characterized by lower levels of C10, C10:1, C14, C14:1 acylcarnitines and higher levels of C16 and C18:1 acylcarnitines, as compared to the other groups of this study. Conclusions Concentration patterns in full-term breastfed newborns in relation to sex and mainly BW found in this study could be very helpful for neonatologists, especially for newborns of group A.

Background Free carnitine (C0) and short-chain acylcarnitine (SCA) blood concentrations play a significant role in fatty acid oxidation process during the first days of life. The aim of this study was to demonstrate C0 and SCA concentrations in Dried Blood Spots (DBS) of full term breastfed infants in relation to their birth weight (BW) perinatally. Methods Breastfed full term infants (n = 12000, 6000 males, 6000 females) with BW 2000–4000 g were divided into 4 equal groups: Group A, 2000–2500 g, B 2500–3000 g, C 3000–3500 g and D 3500–4000 g. Blood samples in the form of DBS were collected on the 3rd day of life and analyzed via a liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol. Results BW-related C0 and SCAs were found as follows: C0 was determined to be statistically significantly higher in group A (BW 2000–2500 g) in both males and females. Lower acetylcarnitine (C2) and hydroxybutyrylcarnitine (C4OH) blood concentrations were detected in group A of both sexes, whereas butyrylcarnitine (C4) concentrations were found to be lower in the same group of males only. Furthermore, high concentrations of C2 and C4OH were shown in group D (BW 3500-4000 g) in both sexes. SCA sum of means ± SD values in males and females of group A were statistically significantly lower as compared to other study groups. Conclusion Due to the number of the samples, data from this study could be applied as neonatal screening reference values for full term breastfed newborns in relation to their birth weight.

There are twenty-one amino acids in the human body and some of them have neurotransmission action during exercise. The aim of this review is to present the increase of neurotransmission amino acids in the blood after training. Additionally, we highlight the neurotransmission action of certain amino acids and their beneficial effects on a wide range of neurological disorders. A Medline and Scopus search was performed to identify articles published on this topic. Only articles published in English were considered. Cognitive functioning of memory, attention, arousal, as well as motor control and emotion functioning, are related to modifications in the glutamatergic, monoaminergic and serotonergic systems in patients with some types of dementia. A direct effect of phenylalanine on certain neurotransmission enzymes is also described. Tyrosine is highly involved in dopamine production, resulting in amelioration of the symptoms of patients with moderate Parkinson’s disease via dopamine increase. Training could be beneficial, not only to healthy people but also to patients with moderate neurodegenerative disorders.

Background: Mesalazine (5-aminosalicylic acid, 5-ASA) is a drug substance with an antiinflammatory activity, which is mainly used in the symptomatic treatment of diseases, such as Ulcerative Colitis, the Crohn's disease and the idiopathic inflammatory bowel disease. Mesalazine exerts its effect locally in the inflamed area of the intestine and not through systematic absorption, therefore the investigation of its release characteristics from solid pharmaceutical formulations is of great importance. Objective: The development of novel mesalazine modified release formulations with improved properties, regarding drug release in the gastrointestinal tract, by utilisation of the Design of Experiments (DoE) approach. Methods: D-optimal experimental design was applied. A Simplex Lattice mixture design was used for the development of suitable capsules containing 4 mini tablets and a D-optimal mixture design was used for compression-coated tablets, with the following characteristics: ≤10% release in 2 h, to minimize its degradation in the upper gastrointestinal tract, 20-40% release in 5 h for mesalazine administration in the small intestine, and quantitative release in 12 h for colonic delivery. The dissolution experiments were conducted in gastrointestinal-like fluids and pectinases to simulate the pectinolytic enzymes present in the colon. Results: The optimal compositions were reached via the desirability function, as a compromise to the different responses. The optimal solutions for both formulations led to colon-specific delivery of the active substance with minimal 5-ASA release in the upper gastrointestinal tract and appeared to conform with the pre-determined characteristics. Hard gelatin capsules, when filled with mini-tablets led to the aimed modified release profile, having sigmoidal characteristics and compression coated tablets led to colonic delivery. Conclusion: Two novel mesalazine formulations were developed with the desirable colonic release, by conducting a minimal number of experiments, as suggested by DoE experimental design.

Background: The loop diuretic drug furosemide is widely used for the treatment of edema in various conditions, such as pulmonary, cardiac and hepatic edema, as well as cardiac infarction. Furosemide, due to its poor water solubility and low bioavailability after oral administration of conventional dosage form, is categorized as class IV in the biopharmaceutical classification system. Objective: In the case of furosemide, this release profile is responsible for various physiological problems, acute diuresis being the most serious. This adverse effect can be circumvented by the modified release of furosemide from tablet formulations compared to those forms designed for immediate release. Method: In this report, a D-optimal combined experimental design was applied for the development of furosemide containing bilayer and compression coated tablets, aiming at lowering the drug’s burst release in the acidic environment of the stomach. A D-optimal combined design was selected in order to include all requirements in one design with many levels for the factors examined. The following responses were selected as the ones reflecting better criteria for the desired drug release: dissolution at 120 min (30-40%), 300 min (60-70%) and 480 min >95%. The new formulations, suggested by the Doptimal combined design, incorporated different grades of Eudragit ® polymers (Eudragit® E100 and Eudragit® L100-55), lactose monohydrate and HPMC K15M. The dissolution profile of furosemide from these systems was probed via in vitro dissolution experiments in buffer solutions simulating the pH of the gastrointestinal tract. Results: The results indicate that the use of Eudragit® E100 in conjunction with lactose monohydrate led to 21.32-40.85 % drug release, in the gastric medium, in both compression-coated and bilayer tablets. This is lower than the release of the mainstream drug Lasix® (t=120 min, 44.5% drug release), implying longer gastric retention and drug waste minimization. Conclusion: Furosemide’s release in the intestinal environment, from compression coated tablets incorporating Eudragit® L100-55 and HPMC K15M in the inner core or one of the two layers of the bilayer tablets, was delayed, compared to Lasix®

The establishment of expanded newborn screening (NBS) not only results in the early diagnosis and treatment of neonates with inborn errors of intermediary metabolism disorders (IEMDs) but also helps the affected females to reach the reproductive age under medical and dietetic support, as well as to give birth to normal infants. In this review, we aimed to focus on laboratory investigation tests, dietetic management and medical support for most known IEMD pregnant and lactating women, such as those suffering from aminoacidopathies, carbohydrate metabolic diseases and fatty acid (FAO) oxidation disorders.

The core idea behind this study was the utilization of the principal components (PCs) as a substitute of the original dataset of molecular descriptors for the derivatization products of amino acid analogues with n-propyl chloroformate. The derivatives were described through principal component analysis (PCA) with a total of over 1200 different molecular descriptors, split into groups according to their prime chemical characteristic. The form of the chemical space was modeled by PCA and optimized through a supervised procedure; whose quality was tested employing an internal cross-validation leave-more-out methodology supplying the Q2X metric (> 0.90 on most sets). The independent “spaces” formed contributed a total set of 63 variables (their PCs), the potential of which was evaluated through their application in two independent tests and more specifically in the formation of quantitative structure–retention relationships for two different chromatography systems (gas and liquid), based on published experimental data on those systems. The first model was developed through projection to latent structures methodology, while the second on multilinear regression (MLR). In both cases, the new derivatives’ descriptor set formed models of good quality (Q2Y ≥ 0.9), validated through both internal and external test set validation procedures. Their ability to be used along with other variables in simpler modelling methods, like MLR, attests to their potential to be used in models of multivariate system calibration without blowing the dataset out of proportion. Regardless of the selected modelling method, data support their use for the composition of predictive models for analytical purposes.

In the present study we aimed at reaching a procedure for control of factors that have a potential to contribute to bioanalytical method bias and consistent dried blood spot (DBS) formation. The most influencing factors were identified via experimental design and image processing methodology. The effects of five qualitative factors - temperature of blood samples, type of pipettes, pipetting technique, age of blood samples and analysts were investigated by multilevel categorical D-optimal design. Due to well-known influence of hematocrit (Hct) level on rheological characteristic of blood samples, five responses were observed through the study (RSD22%Hct, RSD30%Hct, RSD39%Hct, RSD51%Hct, RSD62%Hct) for their ability to provide insights into influence of factor settings onto DBS areas consistency. The area of blood spots was determined by image processing after scanning DBS cards with four spot replicates, corresponding to particular combination of investigated factors, as defined by experimental plan. The qualitative linear mathematical models with added appropriate two-factor interactions were derived by the principle of backward elimination. It was concluded that %RSD value of DBS, for all investigated Hct levels, is completely independent of type of pipettes and age of blood samples, but can significantly be affected by blood sample temperature, pipetting technique and training of analyst. Therefore, the procedure for precise and accurate formation of DBS of uniform area, regardless the Hct value, implies samples at body/room temperature, reversed pipetting technique for rigorous delivery of a sample volume onto the card and a properly trained analyst for handling samples. Finally, a verification experiment was performed and the adequacy of the suggested procedure was confirmed. Identification of the factors affecting the consistency of DBS formation provided the evidence that this contribution to total assay bias can be successfully controlled and reduced.

Essential, non-essential and conditionally essential amino acid blood concentrations play a critical role in newborns. We aimed to quantitate most of these amino acids in the blood of full-term breastfed infants, perinatally and correlate the obtained values with their birth weight. Breastfed full-term infants (

Objective Cystic fibrosis (CF) is a multisystemic inherited disease. The aim of this study was to determine free carnitine (FC) and acylcarnitine concentrations in CF newborns with various mutations of the CFTR gene perinatally.Study Design FC/acylcarnitines were determined in dried blood spots via liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the third day of life of full-term normal (n = 50) and CF (n = 28) newborns. For infants with elevated immunoreactive trypsinogen values, FC/acylcarnitines were quantified again 48 hours later, followed by mutational analysis of CFTR gene via Sanger sequencing.Results Initial FC and sums of acylcarnitine concentrations were statistically significantly lower in CF patients than in controls and even lower 48 hours later. The mutations F508del and 621 + 1G > T were predominantly identified among CF patients.Conclusion Low FC and acylcarnitine concentrations were measured perinatally in CF patients, for all CFTR mutations detected. Carnitine supplementation of breastfeeding mothers could be beneficial.

Purpose Fentanyl analogues are popular in recent years among drug addicts and have been related to many overdoses and deaths worldwide. Furanylfentanyl, ocfentanil, acetylfentanyl and butyrfentanyl are among the most common of these drugs. Methods for the determination of furanylfentanyl and ocfentanil by gas chromatography–mass spectrometry (GC–MS) in biological samples do not exist, and therefore, their development would be extremely useful for routine toxicological analysis. Methods A GC–MS method was developed and fully validated for the determination of furanylfentanyl and ocfentanil in whole blood. This method was also suitable for the determination of acetylfentanyl and butyrfentanyl. The method included solid-phase extraction after protein precipitation using acetonitrile, and it was applied during the toxicological investigation of forensic cases. Methadone-d3 was used as internal standard for the quantification of the analytes. Results The limit of detection and limit of quantification values were 0.30 and 1.0 ng/mL for furanylfentanyl and ocfentanil and 0.15 and 0.50 ng/mL for acetylfentanyl and butyrfentanyl, respectively. The calibration curves were linear (R2 ≥ 0.993) from 1.00 to 100 ng/mL for furanylfentanyl and ocfentanil and from 0.50 to 50.0 ng/mL for acetylfentanyl and butyrfentanyl. The recoveries were not lower than 85%, while accuracies and precisions were not greater than 6.0% (% error) and 8.0% (% relative standard deviation), respectively, for all four fentanyl analogues. Conclusions The developed method is the first one in the literature for the detection of furanylfentanyl and ocfentanil in biological fluids by GC–MS, and it provides very high sensitivity comparable to that by liquid chromatography–tandem mass spectrometry.

Currently, the growing demand on quick, easy and ecological sample pretreatment methods is unquestionable. Such challenge involves also approaches focusing on the analysis of pharmaceuticals and other endogenous compounds in biological matrices, termed as Bioanalysis. Solvent extraction such as liquid-liquid extraction (LLE), derived liquid phase microextraction (LPME) and related approaches such as solid liquid extraction (SLE), proved to be applicable in bioanalysis, as numerous papers have been published in this field. However, their manual performances may suffer from a long-term and laborious preparation, due to the inherent complexity of the biological samples. A high sample-throughput (enabling measurement of tens or hundreds of samples on a daily basis) can be achieved when automation of sample pretreatment is performed, resulting in decreased imprecision and low waste production of hazardous solvents and risky biological materials. Here, robotic systems have a key role, especially when multiple processing (e.g., 96-well plate format) and coupling to modern analytical instrumentation (e.g. LC-MS) are combined. A thorough overview on the up-to-date automations of LLE, LPME, SLE and solid LLE via robotics, is therefore presented. Pharmaceuticals and related compounds determined in classical liquid biological samples (i.e. plasma/serum, whole blood, urine, saliva etc.) and modern dried matrix spots (DMS) were considered as analytes of interest. The methodologies were critically compared to manual setups and among themselves.

Background: Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and infant health outcome. Normal fetal growth and development depend on a continuous supply of nutrients via the placenta. The placenta transports, utilizes, produces, and interconverts amino acids (AAs).Findings: Concentrations of both nonessential and essential AAs in maternal plasma decrease in early pregnancy and persist at low concentrations throughout. The decline is greatest for the glucogenic AAs and AAs of the urea cycle. Additionally, there is a large placental utilization of the branched-chain AAs, some of which are transaminated to alpha ketoacids and contribute to placental ammonia production. Both nonessential and essential AAs regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of organisms. Some of the nonessential AAs (e.g. glutamine, glutamate, and arginine) play also important roles in regulating gene expression, cell signaling, antioxidant responses, immunity, and neurological function.Conclusions: Nutritional support during pregnancy is of great interest focusing not only to common pregnancies but also to those with low socioeconomic status, vegan-vegetarian groups, and pregnant women with metabolic disorders, the most known maternal phenylketonuria. The latter is of great interest because phenylalanine must be within the recommended range throughout pregnancy in addition to other nutrients such as vitamin B12, folate, etc. Loss of the adherence to this specific diet results in congenital malformations of the fetus. In addition to the routine laboratory test, quantitation of plasma AAs may be necessary throughout pregnancy.

To detect an analyte, typically at the sub-nanomolar scale, extremely sensitive analytical tools are required. Fluorescence is the spectroscopy of choice to achieve such a level due to its non-invasive nature and efficiency in accurately probing the sub-nanomolar concentration range. Here, we report the design, synthesis and photophysical characterization of a fluorogenic derivatization reagent with exclusive selectivity for primary amines. This xanthene-based dye owns an exacerbated fluorogenic character making the derivatized amine absorbing in yellow and emitting in the vermilion edge. In addition to being fluorogenic, this derivatization method also has the crucial advantage of being chromogenic (colorless => fuchsia). Chemical quantum calculations give an insight into the dye's molecular properties, while the development of an LC analytical method provides proof of concept regarding its application for the analysis of primary amines in a complex matrix.

A reversed-phase high-pressure liquid chromatography (RP-HPLC) method was developed and subsequently validated for the simultaneous determination of butamirate citrate (BC) and benzoic acid (BA) in cough syrup. The separation was performed employing a cyanopropyl column with a mobile phase consisting of 50%/50% v/v MeOH/NaH2PO4 * H2O 50 mM aqueous solution pH = 3.0. The quantitation was achieved with a diode array detector (DAD) at 210 nm. The method demonstrated a congenitally satisfactory separation, yet the acquired peaks were asymmetrical. This effect was eliminated by using 1% triethylamine in the buffer solution as a silanol blocker. In addition, the method was found to unequivocally assess the target analytes in the sample matrix and fulfilled the required specifications in relevance to specificity, linearity, accuracy, precision and stability of both the standard solutions and of the sample solutions. Lastly, an experimental design was designed in order to assess the robustness of the proposed assay. To this purpose, a graphical and a statistical approach were utilized and compared to identify the factors that should be strictly controlled during each execution of the method.

A fully automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of omeprazole in human plasma. Utilization of 96-well plates and robotic liquid handling workstations, rendered the whole procedure very fast, compared to the manual respective procedure of Liquid-Liquid Extraction (LLE). Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring (MRM). The method required low plasma volumes and analysis of samples was completed in short run times. It was fully validated and applied to a pharmacokinetic study after per os administration of 20mg tablet formulations of omeprazole. The obtained concentrations were used for the calculation of the basic omeprazole pharmacokinetic parameters. Some variations observed in pharmacokinetic parameters among subjects were attributed to differences of CYP2C19 genotype. Therefore, a novel molecular method was developed in which DNA analysis was conducted by using Real Time-Polymerase Chain Reaction (Real Time-PCR). As source of biological material, Dried Blood Spots (DBS) were utilized, offering an alternative and advantageous strategy for such kind of studies.

Background The amino acids glutamine plus glutamate, phenylalanine and tyrosine are implicated in neurotransmission. We aimed to evaluate these amino acid blood concentrations in full-term breastfed infants with different birth weight (BW) perinatally. Methods Breastfed full-term infants (n = 6000, males 3000, females 3000) BW 2000–4000 g were divided into four equal groups. Both males and females Groups A, 2000–2500 g, B 2500–3000 g, C 3000–3500 g, D 3500–4000 g. Blood samples on Guthrie cards, were taken on the 3rd day of life and quantified via a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results Glutamine plus glutamate mean values were found to be statistically significantly different between males vs. females in all the studied groups. The highest values were determined in both males and females in group D. Statistically significantly higher values of phenylalanine appeared in group D vs. other groups. Tyrosine mean values were calculated to be statistically significantly different in both sexes in group A compared to other groups. Conclusions Differences of glutamine plus glutamate, phenylalanine and tyrosine levels among full-term newborns with different BW are presented for the first time in the literature. Newborns with BW 3000–4000 g are benefited by having higher concentrations of the mentioned neurotransmission related amino acids. Neonatal screening reference values for these amino acids in relation to BW could be established, not only for preterm and low BW infants but also for full-term newborns with BW >3000 g.

Background Arginine family amino acids (AFAAs) include glutamine (Gln) plus glutamate (Glu), ornithine (Orn), proline (Pro), citrulline (Cit) and arginine (Arg). We aimed to quantitate these amino acids in the blood of full-term infants in relation to their birth weight (BW) perinatally. Methods Breastfeeding full-term infants (n = 2000, 1000 males, 1000 females) with a BW of 2000–4000 g were divided into four equal groups: group A, 2000–2500 g; B, 2500–3000 g; C, 3000–3500 g and D, 3500–4000 g. Blood samples as dried blood spots (DBS) were collected on the third day of life and analyzed via a liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol. Results Gln plus Glu mean values were found to be statistically significantly different between males and females in all studied groups. The highest values of these amino acids were detected in both males and females in group D. Orn mean values were found to be statistically significantly different between males and females of the same BW in all groups except the last one. The lower mean value was determined in group A, whereas the highest was determined in group D. Cit and Arg mean values were determined to be almost similar in all studied groups. Conclusions Gln plus Glu and Orn blood concentrations were directly related to infants’ BW. Conversely, Cit and Arg did not vary significantly in all groups.

Dried blood spots (DBS) are formed by deposition of a small amount of blood on specific adsorbent paper and its physical drying. DBS are employed as a sampling method in several fields of life sciences and drug research. A concern about DBS is the so-called 'Hematocrit (Ht) effect', as a different Ht leads, due to different viscosity, to different spot size, affecting assay bias. Solutions have been proposed, including the correction of quantified concentrations with a suitable correction factor. In order to quantitatively assess Ht impact on the DBS measurements, a computational approach was developed and implemented in R® language. First, the % relative error was modeled with respect to Ht. Then, Monte Carlo simulations were performed in virtual men/women populations with different Ht levels and the % relative error in relation to the Ht used for calibrators was quantified. An upper level for % relative error being a 'tolerable contribution' of Ht effect to % total analytical error was finally suggested, defining, for the first time, a potential Ht range for analysis of adults' samples, where correction of concentrations of unknown samples may be omitted. Such tolerable level for % relative error may be defined in each laboratory, also based on experimental parameters (type of paper and blood volume). Using a Ht calibration value representing the study population is fully rationalized, leading to reduced probability for concentration corrections. Regulatory criteria for bioanalysis can thus be targeted, moving towards wider utilization of DBS in human pharmacokinetic and clinical trials.

In the current study, a rapid and sensitive LC-QTOF-MS/MS method for the determination of brinzolamide in dried blood spots (DBS) was developed and validated. This novel sample collection, storage and transfer technique was suitable for analyzing a drug with high distribution into red blood cells and negligible plasma levels. The method included an isocratic mobile phase consisting of methanol and 10 mM ammonium formate (90:10, v/v) and detection in positive electrospray mode (ESI+). The flow rate was adjusted to 0.350 mL/min yielding retention times of 1.7 min for both brinzolamide and internal standard (IS) rabeprazole on a Cyano analytical column, respectively. The validation of the proposed method over the concentration range 0.500-20.0 mu g/mL was performed in compliance with EMEA and FDA guidelines, assessing all major performance characteristics. Inter- and intra- assay precisions were less than 14%, while inter- and intra- assay accuracies varied from 922 to 111%. No matrix effect was observed and the mean brinzolamide extraction recovery was 93.5%. The method was successfully applied to real DBS samples from patients in steady state condition, receiving brinzolamide ophthalmic suspension 1% (w/v) for several months. Initial concentrations were corrected due to hematocrit effect, using image processing algorithm written in Matlab.

Very recently, it was reported that a patient with classical galactosemia and a very high intelligence quotient (IQ) score obtained a university degree. In the present study, two siblings with classical galactosemia (homozygous for Q188R mutation) received upper normal IQ scores when tested with psychometric tools. Additionally, the same IQ scores were determined in their healthy brother when tested at the same age. It was concluded that patients could achieve upper normal IQ scores when on diet and followed up closely. Family and especially maternal care may ameliorate the psychomotor development.

Phenylalanine hydroxylase (PAH) deficiency, commonly named phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism inherited with an autosomal recessive trait. It is characterized by high blood and cerebral Phe levels, resulting in intellectual disabilities, seizures, etc. Early diagnosis and treatment of the patients prevent major neuro-cognitive deficits. Treatment consists of a lifelong restriction of Phe intake, combined with the supplementation of special medical foods, such as Amino Acid medical food (AA-mf), enriched in tyrosine (Tyr) and other amino acids and nutrients to avoid nutritional deficits. Developmental and neurocognitive outcomes for patients, however, remain suboptimal, especially when adherence to the demanding diet is poor. Additions to treatment include new, more palatable foods, based on Glycomacropeptide that contains limited amounts of Phe, the administration of large neutral amino acids to prevent phenylalanine entry into the brain and tetrahydrobiopterin cofactor capable of increasing residual PAH activity. Moreover, further efforts are underway to develop an oral therapy containing phenylalanine ammonia-lyase. Nutritional support of PKU future mothers (maternal PKU) is also discussed. This review aims to summarize the current literature on new PKU treatment strategies.

Summary Background & aims Pregnancy is characterized by a complexity of metabolic processes that may impact fetal health and development. Women's nutrition during pregnancy and lactation is considered important for both mother and infant. This review aims to investigate the significant role of fatty acids and carnitine during pregnancy and lactation in specific groups of pregnant and lactating women. Methods The literature was reviewed using relevant data bases (e.g. Pubmed, Scopus, Science Direct) and relevant articles were selected to provide information and data for the text and associated Tables. Results Dynamic features especially of plasma carnitine profile during pregnancy and lactation, indicate an extraordinarily active participation of carnitine in the intermediary metabolism both in pregnant woman and in neonate and may also have implications for health and disease later in life. Maternal diets rich in trans and saturated fatty acids can lead to impairments in the metabolism and development of the offspring, whereas the consumption of long chain-polyunsaturated fatty acids during pregnancy plays a beneficial physiologic and metabolic role in the health of offspring. Conclusions Pregnant women who are underweight, overweight or obese, with gestational diabetes mellitus or diabetes mellitus and those who choose vegan/vegetarian diets or are coming from socially disadvantaged areas, should be nutritionally supported to achieve a higher quality diet during pregnancy and/or lactation.

Luminol-peroxidase-H2O2 is among the most popular systems in chemiluminescence-based analytical applications. The addition of a forth compound into this system has been thoroughly explored over the years, namely an enhancer promoting potency in the signal attained. Improvements in this regard serve to increase sensitivity and applicability of various biochemical methods. Studies exploring the ability of compounds to act as enhancers have proposed several chemical groups as possible candidates, amongst which 4-substituted phenols have been widely employed. Although some studies have explored the effect of the substituent on enhancer potency, no quantitative structure – property relationships (QSPR) employing molecular descriptors for this group of compounds have been presented. Current study provides two such cross- and externally validated models, constructed by the projection to latent structures by means of partial least squares (PLS) multivariate linear regression method: i) a PLS-DA model contributing to the classification of such compounds into classes (relative to the signal), and ii) a PLS model for predicting the signal acquired. Validation was based on statistical metrics Q2ext(F3) for the test set and Roy's metrics r2m(Av) & r2m(δ), assessing both predictive stability and internal validity. Applied in tandem, those models can assist in the recognition of available compounds as potential enhancers or inspire the synthesis of novel analogues. The significance of some characteristics governing the compounds' behaviour are also discussed based on the molecular descriptors chosen.

A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening.

Background: Variants of fat mass and obesity associated gene (FTO) and melanocortin-4 receptor gene (MC4R) are related to obesity, overweight and type 2 diabetes. Objectives: To examine the presence of FTO and MC4R variants in Greek newborns. Subjects and methods: A total 1530 newborns of Greek origin were subjected to genetic testing for rs9939609 (FTO) and for rs17782313 (MC4R) variants using dried blood spot (DBS) analysis. Results: Some 20.2% of newborns carried none of the tested variants. FTO homozygotes and FTO heterozygotes correspond to 18.0% and 45.9% of neonates, respectively. MC4R homozygotes and MC4R heterozygotes were identified in 6.7% and 36.3% of neonates, respectively. Of the infants, 2.2% carried both variants in homozygosity, whereas heterozygotes for both variants correspond to 16.7% of the tested neonates. Conclusion: The results indicate high prevalence of homozygosity and heterozygosity for tested variants. Early screening via DBS may be beneficial in order to adopt a healthy lifestyle.

A novel Liquid Chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the quantitative determination of two potential genotoxic impurities (PGIs) in rabeprazole active pharmaceutical ingredient (API). In order to overcome the analytical challenges in the trace analysis of PGIs, a development procedure supported by Quality-by-Design (Q1313) principles was evaluated. The efficient separation between rabeprazole and the two PGIs in the shortest analysis time was set as the defined analytical target profile (ATP) and to this purpose utilization of a switching valve allowed the flow to be sent to waste when rabeprazole was eluted. The selected critical quality attributes (CQAs) were the separation criterion s between the critical peak pair and the capacity factor k of the last eluted compound. The effect of the following critical process parameters (CPPs) on the CQAs was studied: %ACN content, the pH and the concentration of the buffer salt in the mobile phase, as well as the stationary phase of the analytical column. D-Optimal design was implemented to set the plan of experiments with UV detector. In order to define the design space, Monte Carlo simulations with 5000 iterations were performed. Acceptance criteria were met for Cs column (50 x 4 mm, 5 p.m), and the region having probability pi >= 95% to achieve satisfactory values of all defined CQAs was computed. The working point was selected with the mobile phase consisting of ACN, ammonium formate 11 mM at a ratio 31/69 v/v with pH-6,8 for the water phase. The LC protocol was transferred to LC-MS/MS and validated according to ICH guidelines.

A sensitive Liquid Chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitative analysis of three potential genotoxic impurities (318BP, M9, S5) in meropenem Active Pharmaceutical Ingredient (API). Due to the requirement for LOD values in ppb range, a high concentration of meropenem API (30 mg/mL) had to be injected. Therefore, efficient determination of meropenem from its impurities became a critical aim of this study, in order to divert meropenem to waste, via a switching valve. ‎ After the selection of the important factors affecting analytes’ elution, a Box-Behnken design was utilized to set the plan of experiments conducted with UV detector. As responses, the separation factor s between the last eluting impurity and meropenem, as well as meropenem retention factor k were used. Grid point search methodology was implemented aiming to obtain the optimal conditions that simultaneously comply to the conflicted criteria. Optimal mobile phase consisted of ACN, methanol and 0.09% HCOOH at a ratio 71/3.5/15.5 v/v. All impurities and internal standard omeprazole were eluted before 7.5 min and at 8.0 min the eluents were directed to waste. The protocol was transferred to LC–MS/MS and validated according to ICH guidelines.

This paper presents a LC-MS/MS method for the determination of antiepileptic drug vigabatrin in dried plasma spots (DPS). Due to its zwitterionic chemical structure, a pre-column derivatization procedure was performed, aiming to yield enhanced ionization efficiency and improved chromatographic behaviour. Propyl chloroformate, in the presence of propanol, was selected as the best derivatization reagent, providing a strong signal along with reasonable run time. A relatively novel sample collection technique, DPS, was utilized, offering easy sample handling and analysis, using a sample in micro amount (similar to 5 mu L). Derivatized vigabatrin and its internal standard, 4-aminocyclohexanecarboxylic acid, were extracted by liquid-liquid extraction (LLE) and determined in positive ion mode by applying two SRM transitions per analyte. A Zorbax Eclipse XDB-C8 column (150 x 4.6 mm, 5 mu m particle size) maintained at 30 degrees C, was utilized with running mobile phase composed of acetonitrile: 0.15% formic acid (85:15, v/v). Flow rate was 550 mu L/min and total run time 4.5 min. The assay exhibited excellent linearity over the concentration range of 0.500-50.0 mu g/mL, which is suitable for the determination of vigabatrin level after per os administration in children and youths with epilepsy, who were on vigabatrin therapy, with or without co-medication. Specificity, accuracy, precision, recovery, matrix-effect and stability were also estimated and assessed within acceptance criteria.

Glucose-6-Phosphate Dehydrogenase (G6PD) gene is located at the X-chromosome at Xq28 and the disease is recessively inherited predominantly in males. More than 400 variants have been proposed based on clinical and enzymatic studies. The aim of the current study was to identify C563T mutation in G6PD-deficient newborns and to correlate the enzyme residual activity with the presence of the mutation. Some 1189 full-term neonates aged 3-5 days old were tested for G6PD activity in dried blood spots from Guthrie cards using a commercial kit. DNA extraction from Guthrie cards and mutation identification among the deficient samples were performed with current techniques. A total of 92 (7.7%) newborns were G6PD-deficient. In 46 (50%), the mutation C563T was identified. The residual activity in C563T hemizygote males (n = 28) was statistically significantly lower (1.23 ± 0.93 U/g Hb) than that in non-C563T G6PD-deficient males (n = 25) (4.01 ± 1.20 U/g Hb, p0.0001) and in controls (13.6 ± 2.9 U/g Hb, p0.0001). In C563T heterozygote females, the estimated enzyme activity was lower than that determined in non-C563T females. Male C563T hemizygotes suffer from G6PD deficiency and severe neonatal jaundice. G6PD activity showed statistically significant correlation with total bilirubin blood levels.

In this article, a step-by-step optimization procedure for improving analyte response with implementation of experimental design is described. Zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, were chosen as model compounds to undergo chloroformate-mediated derivatization followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Application of a planned stepwise optimization procedure allowed responses of analytes, expressed as areas and signal-to-noise ratios, to be improved, enabling achievement of lower limit of detection values. Results from the current study demonstrate that optimization of parameters such as scan time, geometry of ion source, sheath and auxiliary gas pressure, capillary temperature, collision pressure and mobile phase composition can have a positive impact on sensitivity of LC-MS/MS methods. Optimization of LC and MS parameters led to a total increment of 53.9%, 83.3% and 95.7% in areas of derivatized vigabatrin, pregabalin and gabapentin, respectively, while for signal-to-noise values, an improvement of 140.0%, 93.6% and 124.0% was achieved, compared to autotune settings. After defining the final optimal conditions, a time-segmented method was validated for the determination of mentioned drugs in plasma. The method proved to be accurate and precise with excellent linearity for the tested concentration range (40.0 ng ml−1–10.0 × 103 ng ml−1). Copyright © 2013 John Wiley & Sons, Ltd.

Melatonin (MT) is a chronobiotic hormone synthesized by the pineal gland and has a significant role in the regulation of the circadian biological clock. In order to be administered as a drug, the time of melatonin release from its formulation is of great significance. To this purpose, controlled release matrix tablets (200 mg) of MT were developed by using D-optimal experimental design, including 1 categorical and 2 numerical factors, aiming at effecting its sustained release using a USP XXIII dissolution apparatus II at pH 1.2 and 7.4 media. The excipients selected are polyvinyl pyrrolidone (M.W.: 10.000 and 55.000), hydroxylpropyl Methylcellulose K15M and lactose monohydrate. The in vitro release data were fitted to the Korsmeyer-Peppas empirical equation; the n exponent, which refers to release kinetics, was evaluated. The optimal composition was reached via the desirability function, as a compromise to the different responses (the time for 50% drug dissolution at pH=1.2 and the diffusional exponent (n) at pH values 1.2 and 7.4). As the differences in the n values amongst the formulations are closely related to variation in water penetration into the different excipients used, excipients with different physicochemical characteristics were used so that the desired formulation was produced. Overall, this procedure resulted in a suitable excipients’ composition for the controlled release of melatonin with the minimal number of experiments.

Background Hawkinsinuria is a rare inborn error of tyrosine metabolism. Objectives To study novel hawkinsinuria cases by monitoring their biochemical profile and conducting a mutation analysis. Subjects and methods Among 92,519 newborns that underwent expanded newborn screening, two unrelated cases with high tyrosine blood levels were further investigated by chromatographic techniques and via genetic testing for 4-hydroxyphenylpyruvate dioxygenase (HPD) gene. Results Elevated levels were monitored for blood/plasma tyrosine and for the specific diagnostic markers in urine. The two newborns were put on a special low tyrosine diet. Till completion of the 1st year of their life, liver function tests and brain MRI were normal. The mutation A33T was identified in both cases, while one neonate carried an additional novel mutation of HPD gene (V212M). Conclusions Two mutations of HPD gene, A33T, which are associated with hawkinsinuria and a novel one (V212M) were detected for the 1st time in Greek newborns.

Objectives The purpose of the current study was to screen newborns in Greece and to identify the responsible mutations for Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD). Design and methods 47.812 neonates were screened for the potential presence of MCADD in Greece, via a LC–MS/MS protocol. The “suspected” samples were subjected to genetic testing via PCR–RFLP and sequencing of the coding region of the ACADM gene. Urine samples were collected and then analyzed with a GC/MS method. Results The MCADD prevalence is 1 in 15,937 births. The alleles c.985A > G and c.245insT were detected in the 29.2% and 20.8% of the “suspected” cohort, respectively. A novel variant with potential pathogenicity was identified. Conclusions The c.245insT allele seems to prevail in the Greek cohort of “suspected” specimens. Therefore, this variant along with the c.985A > G allele could constitute a panel for both prenatal and neonatal MCADD screening in the Greek population.

In the current paper the application of multiobjective optimization (MOOP) technique, via Derringer's desirability function, to a microemulsion liquid chromatographic (MELC) method is described. Chromatographic separation of perindopril tert -butylamine and its four impurities was selected as the case study. Central composite design (CCD) with fractional factorial design, ± 0.5 α star design and four replications in central point was applied for a response surface study, in order to examine in depth the effects of the most important factors. As factors that influence the system mostly (i) content of ethyl acetate and (ii) butyl acetate in composite internal phase, (iii) content of sodium dodecyl sulfate (surfactant) and (iv) n -butanol (co-surfactant), as well as (v) pH of the mobile phase were selected. Retention factor of (a) perindoprilat and (b) impurity Y 31 and (c) resolution factor for impurities Y 32 and 33 were chosen for simultaneous optimization. By adjustment of the importance coefficients and weights, according to defined objectives, the optimal mobile phase composition was predicted to be: 0.24% w/v butyl acetate, 0.3% w/v ethyl acetate, 2% w/v SDS, 7.75% w/v n -butanol and pH of the mobile phase 3.7. The sensitivity analysis of desirability function for these optimal conditions was conducted for the first time in LC separations, by applying a sensitivity procedure. The performed sensitivity analysis confirmed that the higher overall desirability does not necessarily mean a better solution. The accuracy of prediction might be affected if the optimal levels of input variables, achieved from several design points, end up with equal settings and different corresponding overall desirability. In our study this was not the issue, which confirmed the adequacy of predicted optimum.