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Nutraceuticals are foodstuffs, which contain health-promoting substances and whose constant intake can contribute to the overall well-being of people. They are currently considered among the most promising prospective means of maintaining lasting conditions of healthiness, especially during old age. In fact, the constant increase of expected lifespan in developed countries does not currently correspond to a similar improvement in health conditions of elderly people, leading to a dramatic increase in the number of people who are not self-sufficient due to deteriorating health in the last stages of their life. One of the basic tenets of nutraceutics is that several micronutrients commonly contained in food can have healthy effects. As a consequence, it is of the most importance to simply and reliably determine the content of these substances in the selected foods. The Laboratory of Pharmaco-Toxicological analysis is involved since several years in the analysis of different, potentially beneficial compounds in alimentary matrices, be they conventional foodstuffs or dietary supplements. At the same time, attention has been paid to the determination of potentially harmful compounds or markers of adulteration. In the last few years, different analytical methods have been developed in the Laboratory, for example for the analysis of melatonin, other antioxidants and amino acids in grapes and related foodstuffs; of glycyrrhizin and glycyrrhetic acid in liquorice and confectionery; of aloin and related compounds in Aloe vera leaves and supplements; of antioxidants in chamomile and so on. These methods were based on reliable, widespread techniques such as HPLC (with different kinds of detection) and capillary electrophoresis (CE), in order to make them easily reproducible in other laboratories for quality control and nutritional purposes.

Le benzodiazepine sono farmaci utilizzati nella terapia degli attacchi d'ansia e dell'insonnia; sono anche prescritti come coadiuvanti nel trattamento della depressione maggiore, dello stato epilettico e nell'induzione dell'anestesia. Nonostante le benzodiazepine siano farmaci piuttosto sicuri, molto spesso si segnalano effetti collaterali ed il loro uso prolungato può causare tolleranza all'effetto terapeutico. In tutti questi casi, è opportuno effettuare un attento monitoraggio terapeutico (TDM) dei pazienti in trattamento con benzodiazepine. Lo scopo di questa ricerca è la messa a punto di un metodo cromatografico (HPLC) per la determinazione simultanea di composti a struttura benzodiazepinica in matrici complesse, come i fluidi biologici. Gli analiti presi in considerazione sono: alprazolam, bromazepam, brotizolam, clobazam, clonazepam, clordiazepossido, clotiazepam, delorazepam, diazepam, flunitrazepam, flurazepam, lorazepam, lormetazepam, oxazepam e triazolam. La separazione è ottenuta mediante una colonna a fase inversa C8 ed una fase mobile costituita da tampone fosfato a pH acido ed acetonitrile (65/35). La clomipramina è stata scelta come standard interno. La lunghezza d’onda utilizzata per la rivelazione spettrofotometrica è di 220 nm. Queste condizioni sperimentali hanno permesso di ottenere una buona separazione cromatografica dei 15 analiti entro 18 minuti ed una buona linearità nei range di concentrazioni plasmatiche attese. Per l'applicazione ai campioni biologici, si è scelto di utilizzare come procedura di pretrattamento l’estrazione in fase solida (SPE). Essa è stata applicata a campioni di plasma umano: si caricano 250 µL di plasma diluito e, dopo opportuni step di lavaggio, gli analiti vengono eluiti con 1 mL di metanolo. Il metodo analitico sviluppato fornisce buone rese d’estrazione (>90%) e garantisce un’adeguata purificazione della matrice biologica da interferenti endogeni ed esogeni. Il metodo analitico sviluppato sembra essere promettente per la determinazione di benzodiazepine in campioni di plasma di pazienti in terapia con questi farmaci. Sono in fase di svolgimento ulteriori prove per completare la convalida del metodo analitico ed estenderne l'applicazione ad altri tipi di matrici biologiche.

In the last twenty years, several new antidepressant drugs have been introduced in therapy; however, the traditional tricyclic antidepressants (TCAs), which act by inhibiting the uptake of norepinephrine and serotonin, are still widely used by psychiatrists. TCAs are very effective against major depression. However, TCAs can cause several, potentially dangerous side effects, such as cardiovascular effects. Furthermore, the therapeutic window is quite narrow and even modest overdosing can cause severe toxic effects. Thus, in the last few years the practice of therapeutic drug monitoring (TDM) has been receiving increasing attention as a way of optimising therapy effectiveness and to increase patient compliance. Most TCAs have very long half-lives and generate active metabolites, which can complicate the TDM. Futhermore, the high structural similarity makes it quite difficult to discriminate between the different TCAs. To obtain reliable monitoring data, a new HPLC method has been developed, which allows the simultaneous determination of the plasma levels of seven TCAs (dibenzepine, amoxapine, protriptyline, imipramine, amitriptyline, maprotiline and clomipramine) and seven of their main active metabolites (8-hydroxyamoxapine, 8-hydroxyclomipramine, N-desmethylmaprotiline, nortriptyline, dinorclomipramine, amitriptyline N-oxide and norclomipramine) in a single chromatographic run. The method is based on the use of a Phenomenex C8 reversed-phase column as the stationary phase and a mixture of acetonitrile and a phosphate buffer as the mobile phase. Using this system, all fourteen analytes and the internal standard loxapine are baseline separated within 16 minutes. Spectrophotometric detection is carried out at 220 nm. The plasma sample pre-treatment is performed on C2 cartridges, using only 250 µL of plasma. The analytes are eluted with methanol and, when low levels are suspected, concentrated twice with respect to the original amount. This solid phase extraction (SPE) procedure eliminates all endogenous interference, while obtaining very satisfactory extraction yields for the analytes. In fact, extraction yield results range from 80% for protriptyline to 99% for amitriptyline. The preliminary results from its application to real samples from depressed patients are very encouraging, thus the method is promising for the TDM of TCAs in human plasma.

Duloxetine ((γS)-N-methyl-γ-(1-naphthalenyloxy)-2-thiophenepropanamine, DLX) is the most recent antidepressants introduced onto the Italian market. Like venlafaxine and milnacipran, it acts as a dual serotonin and norepinephrine reuptake inhibitor (SNRI), with approximately equal potency at both transporters; DLX seems to be very efficient and to have a fast onset of action. DLX (Cymbalta®, Xeristar®, Yentreve®, Ariclaim®) is administered as enteric-coated pellets in capsules containing 20, 30 or 60 mg of active principle. The most usual dose for the treatment of depression is 60 mg/day, with a maximum suggested dose of 120 mg/day. The most common side effects are nausea, dry mouth, fatigue, insomnia, sedation, dizziness, constipation, increased sweating, increased blood pressure, decreased appetite and body weight. Furthermore, DLX overdose can present worrisome effects, such as signs of altered mental status, cardiovascular alterations with hypotension, sinus bradycardia and prolonged QTc interval. It is evident that there is a need for having on hand new, reliable analytical methods for the determination of DLX plasma levels in depressed patients. An original HPLC method coupled to solid-phase extraction (SPE) for the determination of DLX plasma levels has been developed. It is based on the use of a Genesis C8 column (150×4.6 mm I.D., 5 μm) as the stationary phase and a mixture of acetonitrile and a pH 3.0 phosphate buffer containing triethylamine (40/60, v/v) as the mobile phase. UV detection is carried out at 230 nm. Using loxapine as the Internal Standard (IS), a chromatographic run lasts 5 minutes. The sample pre-treatment employs mixed mode reversed phase – cation exchange (MCX) cartridges (30 mg, 1 mL) and only 450 µL of human plasma are needed for a complete analysis. Cartridge elution is carried out with methanol, which is then dried and redissolved in 150 µL of mobile phase, thus obtaining a threefold concentration of the analytes. Extraction yields are satisfactory, always higher than 90%. Good linearity (r2 > 0.9990) has been found in the 2-200 ng/mL DLX concentration range. Precision assays are also satisfactory, with RSD% values always lower than 5%. The method seems to be suitable for the TDM of DLX in depressed patients' plasma.

Il trazodone (2-[3-[4-(3-clorofenil)-1-piperazinil]propil]-1,2,4-triazolo[4,3-a]piridin-3(2H)-one, TRZ) è stato approvato nel 1998 per il trattamento della depressione maggiore. TRZ è un debole inibitore del reuptake delle monoammine ed è un antagonista dei recettori della serotonina. Il metabolismo epatico produce il principale metabolita attivo del trazodone, l'1-(3-cloro-fenil)piperazina (mCPP), agonista serotoninergico con una lunga emivita plasmatica. I principali effetti collaterali del trattamento con TRZ sono: sedazione, ipotensione ortostatica, emicrania, capogiri e nausea. Alcuni di questi effetti collaterali (come nausea ed emicrania) sono stati attribuiti anche ad mCPP. Quindi è evidente l'importanza di effetturare il Monitoraggio Terapeutico (TDM) per la determinazione di TRZ e del suo principale metabolita mCPP. Lo scopo di questo lavoro è lo sviluppo di un metodo analitico semplice ed affidabile, basato sull'HPLC-UV, per la determinazione di questi composti in plasma umano. Una colonna a fase inversa C8 è usata come fase stazionaria ed una miscela di tampone fosfato acido (70%) ed acetonitrile (30%) come fase mobile. La rivelazione spettrofotometrica è effettuata alla lunghezza d'onda di 255 nm. Il campione di plasma (125 µL) viene sottoposto ad estrazione in fase solida (SPE) su cartucce C8 e l'eluizione viene effettuata con metanolo. La procedura SPE garantisce soddisfacenti rese d'estrazione (> 90%) ed una buona purificazione della matrice biologica. Il metodo è attualmente in fase di convalida e si è dimostrato promettente per il TDM di TRZ e mCPP in plasma di pazienti depressi.

Cocaine (3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester, COC) is one of the most widespread abuse drugs in the world. In the last few years, both the number of consumers and the total amount of COC consumed have risen dramatically in most countries, thus aggravating several health and social problems. In fact, acute cocaine use produces very pleasant feelings of well-being, lack of weariness and euphoria, but it is powerfully addictive and can cause severe acute and chronic health effects, such as infarction, brain stroke, disphoria, depressive syndrome and psychoses. For these reasons, it is important to detect COC abusers. Hair analysis is perfectly suitable for this purpose: in fact, the hair matrix absorbs COC and traps it into its structure. The resulting hair concentrations can also be related to the time elapsed since the drug intake, as hair has an almost constant growth rate. This allows a complete and constant monitoring of drug use to be carried out with few, infrequent and non-invasive samplings. Of course, to perform this kind of monitoring, reliable and easily applicable analytical methods are needed. An original HPLC method coupled to spectrofluorimetric detection has been developed for the analysis of COC in human hair. It is sensitive, reliable and uses easily available instrumentation. The analyte and the Internal Standard mirtazapine are separated on a reversed-phase C18 column, using a mixture of methanol, acetonitrile and an acidic phosphate buffer (10/15/75, v/v/v) as the mobile phase. Fluorimetric detection is carried out at excitation wavelength = 230 nm, emission wavelength = 315 nm. The hair sample pre-treatment is carried out by solid-phase extraction (SPE) using C2 cartridges, after digestion of the finely cut hairs (1-mm length) by extractive incubation in HCl at controlled temperature. This procedure gives good extraction yield values (> 80%). From these preliminary results, the method for the monitoring of COC intake, using human hair as the sample matrix, seems to be promising.

Omeprazole (6-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazo-le, OMP) and lansoprazole (2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridinyl]methyl]sulfinyl]-1H-benzimidazole, LNP) are two of the most widely used anti-ulcer agents which act on the gastric H+-K+-ATPase pump, irreversibly inhibiting it and thus effectively reducing the acid secretion. They are used in the treatment of peptic ulcer of any origin and also for the treatment of gastro-esophageal reflux disease and as gastroprotectives during prolonged anti-inflammatory therapy cycles. OMP is administered at doses ranging from 10 to 40 mg/day, while LNP usual daily doses are in the 15-30 mg/day range. The typical therapy cycle lasts 8 weeks, but OMP and LNP are often prescribed by physicians for the occasional, acute treatment of gastric pain. However, clear dose/plasma level correlations have not been established. The main side effects are cephalea, abdominal pain, diarrhea, nausea, vomit, dry mouth, dermatitis, insomnia and rarely hepatitis, leukopenia, agitation, broncoconstriction. For these reasons, it is clear that a suitable therapeutic drug monitoring can be helpful in establishing chemical-clinical correlations leading to a safer and more effective use of these drugs. Feasible and relatively inexpensive analytical methods are obviously needed for the determination of OMP and LNP in human plasma. The methods should be capable of discriminating the two drugs: physicians often switch from OMP to LNP and vice versa, perceiving them as mostly equivalent, thus their simultaneous analysis is necessary. The aim of this study is the development of a simple and reliable HPLC method coupled to UV detection for the separation and the simultaneous determination of OMP and LNP in plasma. The developed method uses a C8 column (150×4.6 mm I.D., 5 μm) as the stationary phase and a mixture of acetonitrile and a pH 6.0 phosphate buffer containing triethylamine (30/70, v/v) as the mobile phase. UV detection is carried out at 220 nm. Indomethacin was chosen as the Internal Standard (IS). The sample pre-treatment is carried out by solid-phase extraction (SPE) using C8 cartridges; 250 µL of human plasma are sufficient for a complete analysis. The SPE procedure allows concentration of the analytes, while eliminating most endogenous and hexogenous interference. Extraction yield assays gave good results, with absolute recovery values always higher than 92%. Studies are in progress to complete the method validation.

Amongst the most prescribed Central Nervous System agents are second-generation antidepressant drugs, which include SNRIs (serotonin and norepinephrine reuptake inhibitors), such as venlafaxine and duloxetine, SSRIs (selective serotonin reuptake inhibitors), such as fluoxetine, paroxetine, fluvoxamine, citalopram and sertraline, NRIs (norepinephrine reuptake inhibitors), such as reboxetine, and NaSSAs (noradrenergic and specific serotonergic antidepressants), such as mirtazapine. Over the last few years, our Pharmaco-Toxicological Laboratory has developed several methods for the TDM (Therapeutic Drug Monitoring) of depressed patients subjected to therapy with second generation antidepressants. Most recently, new methods have been developed for the analysis of sertraline, paroxetine and duloxetine in human plasma. Duloxetine is the most recent antidepressant introduced onto the market and thus its chemical-clinical correlations are not completely known. We have developed a feasible and selective liquid chromatographic method for the determination of duloxetine plasma levels. The method is coupled to an original sample pre-treatment procedure based on solid-phase extraction (SPE). It has recently been successfully applied to the analysis of biological fluids during a suspect suicide attempt with duloxetine overdose. Sertraline is a well-established antidepressant, frequently prescribed by psychiatrists worldwide. We have implemented a sensitive method for its determination, based on capillary electrophoresis coupled to laser-induced fluorescence (LIF) detection. Since sertraline is not natively fluorescent, a derivatisation procedure has been developed, based on the reaction of the analytes (sertaline and its major active metabolite N-desmethyl-sertraline) with fluorescein isothiocyanate. Finally, an HPLC method has been developed for the analysis of paroxetine and its three main metabolites in human plasma, which takes advantage of the native fluorescence of the four analytes to obtain suitable sensitivity and selectivity. It appears to be suitable for the TDM of patients taking paroxetine and also for pharmacokinetic studies.

Soymen GN is a new commercial preparation, containing soy extract and melatonin for the treatment of menopausal symptoms such as insomnia, hot flushes, tachycardia and osteoporosis. The commercial package contains two kinds of soft gelatin capsules: the “light” capsules for morning administration contain soybean extract (40 mg), while the “dark” capsules for evening administration contain soy extract (40 mg) and melatonin (5 mg). The main active compounds are the isoflavones, which are present both as aglycones and as glycosides: genistein, daidzein, glycitein, genistin and daidzin. These compounds are of particular interest for their possible estrogenic effects on the symptoms of menopause, in fact they have been used in therapy as a replacement to estrogen deficiency as a consequence of menopause. Melatonin is a hormone whose main function is the regulation of circadian rhythms. Thus, it can be helpful for the relief of insomnia and other sleep disturbances. The aim of this study is to develop a quality control system for the Soymen GN formulation. For this purpose, it is important to have access to reliable analytical methods for the determination of melatonin and of the main soy isoflavones, both aglycones and glycosides. Two original HPLC methods coupled to spectrophotometric and amperometric detection, respectively, have been developed for the quality control of Soymen GN capsules. UV detection is carried out at 260 nm, while amperometric detection is carried out at an oxidation potential of + 800 mV. The chromatographic separation is obtained using a reversed-phase C8 column as the stationary phase and a mixture of acetonitrile and an acidic phosphate buffer (30/70, v/v) as the mobile phase. A third analytical method based on micellar electrokinetic chromatography is currently undergoing validation. The BGE is composed of a 25 mM, pH 10.3 carbonate buffer, containing 55 mM SDS and 5% methanol. The analytes are quantitatively extracted from both “light” and “dark” capsules by agitation in a methanol/water mixture. From these preliminary results, all three methods seem to be promising for the quality control of the new formulation.