Marzia Innocenti

Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method

Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D(12)-topiramate was linear in the concentration range of 0.0166-1.66mg/L (0.5-50mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5-50mg/L, the coefficients of variation in DBS were in the range 2.13-11.85% and the accuracy ranged from 93.93% to 110.67%. There was no significant differences between the concentrations (ranging 0.5-50mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples. The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.

Carotenoid profiles of yeasts belonging to the genera Rhodotorula, Rhodosporidium, Sporobolomyces, and Sporidiobolus

Eighteen yeast species of the genera Rhodotorula, Rhodosporidium, Sporobolomyces, and Sporidiobolus, each one represented by its type strain, were investigated with the objective of evaluating their carotenoid composition. The pigments were extracted from yeast cells, quantified by high pressure liquid chromatography diode array detector and the main compounds were confirmed by atmospheric pressure chemical ionization quadrupole mass spectrometry. Significant (P < 0.01) differences among several species and (or) genera were observed. Thirteen strains were seen to be able to produce carotenoids, from 16.4 to 184 microg/g cell dry mass and from 6.0 to 1993.4 microg/L culture. The main carotenoids produced were identified as torularhodin, torulene, gamma-carotene, and beta-carotene. The correlation matrix calculated on the basis of the carotenoid composition data matrix indicated significant (P < 0.01) relationships between torulene and torularhodin (r = 0.81), gamma-carotene and torulene (r = 0.49), beta-carotene and torulene (r = -0.72), as well as beta-carotene and gamma-carotene (r = 0.64). These significant correlation coefficients may suggest that species belonging to the genera Rhodosporidium, Sporobolomyces, and Sporidiobolus possess a carotenoid biosynthetic pathway analogous to that elsewhere postulated for Rhodotorula species.

Effect of cooking on the anthocyanins, phenolic acids, glycoalkaloids, and resistant starch content in two pigmented cultivars of Solanum tuberosum L.

HPLC/DAD/MS analysis of the phenolic acids and anthocyanin content of three cultivars of Solanum tuberosum L. (Vitelotte Noire, Highland Burgundy Red, with pigmented flesh, and Kennebec with white pulp) was performed. The analyses were carried out both on fresh tubers and after cooking treatments (boiling and microwaves). Starch digestibility and the % of resistant starch were also determined on cooked tubers by in vitro methods. For the pigmented potatoes, the heating treatment did not cause any changes in the phenolic acids content, while anthocyanins showed only a small decrement (16-29%). The cv. Highland Burgundy Red showed anthocyanins and phenolic acid concentrations close to 1 g/kg and more than 1.1 g/kg, respectively. Vitellotte Noire showed the highest amounts of resistant starch. Potato starch digestibility and % of resistant starch, considered as a component of dietary fiber, were affected both by cultivar and by heating/cooling treatments.

Study of the phenolic composition of spanish and italian monocultivar extra virgin olive oils: Distribution of lignans, secoiridoidic, simple phenols and flavonoids

The aim of the present study was to compare the phenolic content in selected monocultivar extra virgin olive oils. Analyses were carried out by HPLC/DAD/MS on Picual, Picuda, Arbequina and Hojiblanca oils from Spain and Seggianese and Taggiasca oils from Italy. Oils from cultivar Picual showed similar characteristics to those of Seggianese oils, with total amounts of secoiridoids of 498.7 and 619.2mg/L, respectively. The phenolic composition of Arbequina oils is close to that of the Taggiasca variety with lignans among the main compounds. The determination of free and linked OH-Tyr, by way of an acid hydrolysis, represents a rapid and suitable method, especially when standards are not available, to determine antioxidant potentialities in terms of MPC, particularly for fresh extra virgin olive oils rich in secoiridoidic derivatives.

Storage method, drying processes and extraction procedures strongly affect the phenolic fraction of rosemary leaves: an HPLC/DAD/MS study.

The Rosmarinus officinalis L. is widely known for its numerous applications in the food field but also for the increasing interest in its pharmaceutical properties. Two groups of compounds are mainly responsible for the biological activities of the plant: the volatile fraction and the phenolic constituents. The latter group is mainly constituted by rosmarinic acid, by a flavonoidic fraction and by some diterpenoid compounds structurally derived from the carnosic acid. The aim of our work was to optimize the extractive and analytical procedure for the determination of all the phenolic constituents. Moreover the chemical stability of the main phenols, depending on the storage condition, the different drying procedures and the extraction solvent, have been evaluated. This method allowed to detect up to 29 different constituents at the same time in a relatively short time. The described procedure has the advantage to being able to detect and quantify several classes of compounds, among them numerous minor flavonoids, thus contributing to improving knowledge of the plant. The findings from this study have demonstrated that storing the raw fresh material in the freezer is not appropriate for rosemary, mainly due to the rapid disappearing of the rosmarinic acid during the freezing/thawing process. Regarding the flavonoidic fraction, consistent decrements, were highlighted in the dried samples at room temperature if compared with the fresh leaf. Rosmarinic acid, appeared very sensitive also to mild drying processes. The total diterpenoidic content undergoes to little changes when the leaves are freeze dried or frozen and limited losses are observed working on dried leaves at room temperature. Nevertheless it can be taken in account that this fraction is very sensitive to the water presence during the extraction that favors the conversion of carnosic acid toward it oxidized form carnosol. From our findings, it appear evident that when evaluating the phenolic content in rosemary leaves, several factors, mainly the type of storage, the drying process and the extraction methods, should be carefully taken into account because they can induce partial losses of the antioxidant components.

The inclusion of succinylacetone as marker for tyrosinemia type I in expanded newborn screening programs

In expanded newborn screening programs by liquid chromatography/tandem mass spectrometry false negatives for tyrosinemia type I are a significant problem. We describe a method for inclusion of succinylacetone in order to avoid false negatives. We studied spots from 13,000 neonates born in Tuscany (January-May 2007) and ten spots from six patients with tyrosinemia type I. The traditional screening method was modified by adding dioxooctanoid acid (or 13C2-succinylacetone) as an internal standard to the methanolic solution of deuterated acylcarnitines and amino acids. A hydrazine solution was added to the mixture. The times of extraction, butylation and drying were only slightly prolonged. Specific multiple reaction monitoring for derivatized and labelled succinylacetone and dioxooctanoic acid was carried out. The assays were linear up to 100 micromol/L for succinylacetone. Intra- and inter-day imprecision data were in the range of 1.34% to 7.09% and 3.50% to 4.49%. Limits of detection and of quantification were 0.2 micromol/L and 0.4 micromol/L, respectively. Recovery ranged from 97.02% to 100.29%. Succinylacetone levels in samples from unaffected neonates were very close to the detection limit. Of the 46 recalls, eight (17.4%) were for abnormal tyrosine levels and all these cases had succinylacetone levels within the normal range (<2.4 micromol/L). In ten spots from six affected patients succinylacetone values ranged from 3.3 to 35.0 micromol/L. Including succinylacetone in newborn screening programs for amino acids and acylcarnitines avoids false-negative results for tyrosinemia type I. Newborn screening laboratories should consider implementing these modifications.

Effects of elicitors on the production of resveratrol and viniferins in cell cultures of Vitis vinifera L. cv Italia.

Methyl jasmonate, jasmonic acid and chitosan were tested as elicitors on cell suspension cultures obtained from Vitis vinifera cv Italia to investigate their effect on stilbene production. Stilbene accumulation in the callus, grown under nonelicited conditions, was also investigated. Calli and cell suspensions were obtained in a B5 culture medium supplemented with 0.2 mg L(-1) NAA and 1 mg L(-1) KIN. Stilbene determination was achieved by HPLC/DAD/MS. Whereas callus biosynthesized only piceid, cell suspensions elicited with jasmonates produced several stilbenes, mainly viniferins. In suspended cells, methyl jasmonate and jasmonic acid were the most effective in stimulating stilbene biosynthesis, whereas chitosan was less effective; in fact, the amount of stilbenes obtained with this elicitor was not significantly different from that obtained for the control cells. The maximum production of total stilbenes was at day 20 of culture with 0.970 and 1.023 mg g(-1) DW for MeJA and JA, respectively.

Optimization of the chromatographic determination of polyphenols in the aerial parts ofCichorium intybus L.

SummaryThe aim of this work was to contribute to the phytochemical characterization ofCichorium intybus L. var.silvestre, chicory. Semi-preparative HPLC analysis was applied to an extract of fresh wild chicory leaves to separate and collect the main polyphenolic compounds.HPLC-diode-array detection (DAD), HPLC-MS, and NMR were used for the complete chemical characterization of all the compounds isolated. The molecules characterized were monocaffeoyl tartaric acid, chicoric acid, monocaffeolyp-hydroxycinnamoyl tartaric acid, caffeoyl feruloyl tartaric acid, chlorogenic acid, quercetin-3-O-glucuronide, luteolin-7-O-glucuronide, and quercetin-3-O-glucoside.The chromatographic behaviour of the main components of the extract of the leaves has been compared on three different stationary phases-LiChrosorb RP18, Luna C18, and Luna Phenyl-Hexyl.

Enhancement of viniferin production in Vitis vinifera L. cv. alphonse lavallée cell suspensions by low-energy ultrasound alone and in combination with methyl jasmonate

This study examined for the first time the effect of low-energy ultrasound (US), used alone or in combination with methyl jasmonate (MeJA), on viniferin production in cell cultures of Vitis vinifera L. cv Alphonse Lavallée. Cell suspensions were exposed for 2 min to US (power 30, 60, and 90 mW cm(-3)). The highest viniferin production was obtained at 30 mW cm(-3). When sonication was performed twice, the effect on viniferin production was negligible, whereas triple sonication slightly increased production. US treatment at 30 mW cm(-3) for 5 min decreased viniferin production and induced cellular death. The combined use of MeJA and US (2 min) increased the production of δ-viniferin, the dominant stilbene, more than each elicitor used alone. These results suggest that low-energy US, alone and in combination with MeJA, can act as a physical elicitor to stimulate viniferin production in V. vinifera cell cultures.

Rosmarinic acid prevents lipid peroxidation and increase in acetylcholinesterase activity in brain of streptozotocin-induced diabetic rats.

We investigated the efficacy of rosmarinic acid (RA) in preventing lipid peroxidation and increased activity of acetylcholinesterase (AChE) in the brain of streptozotocin‐induced diabetic rats. The animals were divided into six groups (n = 8): control, ethanol, RA 10 mg/kg, diabetic, diabetic/ethanol and diabetic/RA 10 mg/kg. After 21 days of treatment with RA, the cerebral structures (striatum, cortex and hippocampus) were removed for experimental assays. The results demonstrated that the treatment with RA (10 mg/kg) significantly reduced the level of lipid peroxidation in hippocampus (28%), cortex (38%) and striatum (47%) of diabetic rats when compared with the control. In addition, it was found that hyperglycaemia caused significant increased in the activity of AChE in hippocampus (58%), cortex (46%) and striatum (30%) in comparison with the control. On the other hand, the treatment with RA reversed this effect to the level of control after 3 weeks. In conclusion, the present findings showed that treatment with RA prevents the lipid peroxidation and consequently the increase in AChE activity in diabetic rats, demonstrating that this compound can modulate cholinergic neurotransmission and prevent damage oxidative in brain in the diabetic state. Thus, we can suggest that RA could be a promising compound in the complementary therapy in diabetes. Copyright