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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.

A fast and simple method based on LC with fluorescence detection has been developed for the determination of insulin in innovative formulations consisting of microparticles and inserts for oral and nasal drug administration respectively. A reversed-phase C8 column and a mobile phase composed of pH 3.7, 40 mM sodium sulphate solution and acetonitrile (24%, v/v) were employed. Using isocratic elution at 1.0 mL/min flow, analysis is completed within 7 min. Three different kinds of spray-dried microparticles were analysed, consisting of an insulin loaded core composed of chitosan salts (chitosan succinate, chitosan adipate or chitosan suberate) coated with stearic acid. Nasal inserts consisted of chitosan/hyaluronate polyelectrolyte complexes which were loaded with insulin and freeze-dried. Insulin was extracted from both the oral and nasal formulations using pH 7.4 phosphate buffer. The employment of fluorescence detection (λexc = 276 nm, λem = 306 nm) granted high selectivity, with no interference from the matrix. Full method validation was performed with good results in terms of linearity (insulin concentration range 0.10 - 30.0 µg/mL), LOD (0.03 µg/mL) and LOQ (0.10 µg/mL), precision (RSD% 90.0%). Insulin content in innovative formulations, expressed as percentage w/w, resulted to be between 0.90 and 0.97 for oral innovative formulations, while an average value of 342 μg of insulin was found in a single nasal insert, in good agreement with preparative protocols.

Benzodiazepines are often prescribed to schizophrenic or depressed patients, as a part of polypharmacy regimens. A high-performance liquid chromatographic method has been developed for the simultaneous determination of fifteen benzodiazepines in human plasma. Separation was obtained by using a C8 reversed-phase column and a mobile phase composed of 65% aqueous phosphate buffer containing triethylamine at pH 3.0 and 35% acetonitrile. The UV detector was set at 220 nm and clomipramine was used as the internal standard. A careful pre-treatment procedure of plasma samples was developed, using solid-phase extraction with C1 cartridges, which gives high extraction yields (> 93%).The limits of quantitation (LOQ) were always lower than 7.6 ng/mL and the limits of detection (LOD) always lower than 2.6 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed and schizophrenic patients undergoing polypharmacy with one or more benzodiazepines. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients undergoing therapy with one or more benzodiazepines.

I derivati dell’aloe hanno trovato negli ultimi anni ampio utilizzo, sia in campo cosmetico, sia in campo erboristico. In entrambi i casi si rende necessario effettuare controlli di qualità, allo scopo di prevenire possibili effetti indesiderati dovuti alla presenza di alcuni principi attivi, soprattutto di natura antrachinonica. Inoltre, è necessario poter distinguere gli estratti provenienti da aloe di diverse specie (ad esempio, Aloe vera ed Aloe del Capo), per evitare frodi merceologiche. Scopo di questo studio è, quindi, lo sviluppo di un metodo analitico per la separazione e la determinazione simultanea dei principali composti antrachinonici dell’aloe, come ad esempio le forme isomeriche di aloina, aloeresina, 5-idrossialoina, 7-idrossialoina e aloinoside. Il metodo sviluppato è basato sull’HPLC con rivelazione a serie di fotodiodi (DAD); utilizza una colonna a fase inversa C8 ed una fase mobile composta da acqua e metanolo. Le analisi su estratti vegetali di aloe sono effettuate dopo purificazione dei campioni mediante cromatografia preparativa su colonna di gel di silice. Un’aliquota di estratto polverizzato è applicata alla colonna e si raccolgono le frazioni ottenute dall’eluizione con etere di petrolio / etile acetato. Le frazioni sono poi oppportunamente diluite ed iniettate nel sistema HPLC. È stato possibile distinguere campioni provenienti da diverse specie di aloe in base alla presenza di composti caratteristici. Il metodo analitico è attualmente in fase di convalida.

Vigabatrin (acido 4-ammino-5-esenoico) e gabapentin (acido 2-[1-(amminometil) cicloesil] acetico) sono farmaci antiepilettici commercializ-zati rispettivamente con il nome di Sabril® e Neurontin®. Il vigabatrin agisce probabilmente mediante inibizione della GABA transaminasi e viene somministrato in dosi da 1,5 a 4 g/die; i livelli plasmatici terapeutici sono compresi fra 20 e 60 µg/mL. Il gabapentin, invece, sembra agire sui canali del calcio voltaggio-dipendenti ed è somministrato in dosi comprese fra 0,3 e 3,4 g/die, con livelli plasmatici terapeutici nel range 2-10 µg/mL. Data la loro struttura chimica, entrambe le molecole non possono essere direttamente rivelate con i normali detector UV o a fluorescenza. Per la determinazione dei loro livelli plasmatici ai fini del monitoraggio terapeutico, sono quindi state impiegate reazioni di derivatizzazione in modo da rendere le molecole fluorescenti. Dopo procedura di estrazione in fase solida con opportune cartucce per la rimozione delle interferenze plasmatiche e derivatizzazione con dansil cloruro in tampone carbonato a pH basico, vigabatrin e gabapentin possono essere analizzati su plasma mediante HPLC (colonna a fase inversa C8, fase mobile costituita da tampone fosfato e acetonitrile); gli analiti sono rivelati con un detector a fluorescenza (lambda-ecc = 318 nm; lambda-em = 510 nm). I tempi di ritenzione sono pari a 5,4 min per vigabatrin, 6,7 min per gabapentin e 7,6 min per lo standard interno. Il metodo è stato validato in termini di linearità (2-100 µg/mL per Vigabatrin, 0,2-30 µg/mL per Gabapentin), resa di estrazione, precisione ed accuratezza. CFSE (6-carbossifluoresceina succinimidil estere) può essere invece utilizzato per derivatizzare il vigabatrin, in tampone borato a pH 9,5, ed ottenere una molecola rivelabile con detector a fluorescenza laser indotta (LIF), con laser a 488 nm. In queste condizioni è possibile operare una derivatizzazione in tempi molto più rapidi (30 min) rispetto a quelli necessari (diverse ore) qualora si utilizzi quale derivatizzante FITC. L’analisi può essere quindi condotta mediante CE-LIF utilizzando capillari di silice non ricoperti (e.l. 45 cm, i.d. 50 µm), come BGE un tampone borato a pH 9, addizionato di N-metil glucammina (100 mM). Il metodo è stato applicato a campioni biologici, dopo procedura SPE per la purificazione della matrice biologica

Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is one of the most widely used anxiolytic-hypnotic drugs. It is a long-acting benzodiazepine with anxiolytic, sedative, hypnotic, anticonvulsant, muscle relaxant and amnestic properties. Diazepam is also frequently used for the relief of anxiety during the first period of treatment with selective serotonin reuptake inhibitors (SSRIs) and generally as a coadjuvant during antidepressive therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. In order to clarify the matter, some studies are currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significative set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam, which are the demethylated, hydroxylated and demethylated hydroxylated analogues of diazepam, respectively. All of these compounds are pharmacologically active, have a long half-life and thus significantly contribute to the therapeutic effects of diazepam administration. Their determination can also give important insight into the respective balance of different metabolic pathways. Following our recent studies on benzodiazepine determination, we are developing a feasible and reliable HPLC method for the simultaneous determination of diazepam and its three main metabolites in rat brain and plasma. The method will be applied to “normal” rats and to genetic rat models of depression in order to estimate the drug metabolism in the different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer / acetonitrile mixture as the mobile phase. The detection wavelength is 238 nm. An accurate and innovative sample pre-treatment, based on microextraction by packed sorbent (MEPS) was developed in order to suitably eliminate endogenous interferences, using only 250 µL of matrix (brain homogenate or plasma) for a complete analysis. The analytes are eluted from the cartridge with methanol; the eluate is then dried under vacuum and redissolved in the mobile phase. The results obtained with the MEPS procedure were confirmed by comparison with those obtained with a solid-phase extraction (SPE) procedure. The method has been validated with good results in terms of precision, extraction yield, accuracy, sensitivity and selectivity on both matrices and the determination of diazepam and metabolite levels in rats is currently under way. The results obtained until now are satisfactory form an analytical point of view and will hopefully contribute to the clarification of some metabolic differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

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.

Il fenomeno delle tossicodipendenze sta assumendo sempre più importanza e diffusione. Purtroppo, però, gli esatti meccanismi neurobiologici dell'instaurazione e del mantenimento della dipendenza non sono ancora chiariti. Si può però affermare con sicurezza che diversi sistemi neurorecettoriali siano implicati in questi fenomeni: ad esempio, la serotonina (5-idrossitriptamina) è profondamente coinvolta nelle modificazioni del tono dell'umore. Pertanto, il monitoraggio dei neurotrasmettitori e dei loro prodotti di biotrasformazione permette di ottenere utili informazioni sui meccanismi delle dipendenze. Il metodo HPLC ora in fase di convalida consente di analizzare la serotonina ed il suo principale metabolita, l'acido 5-idrossiindolacetico (5-HIAA) in plasma umano. Il metodo utilizza una colonna C18, una fase mobile costituita da tampone fosfato acido e metanolo nel rapporto 85/15. La rivelazione di fluorescenza viene effettuata a lambda = 285 nm, eccitando a lambda = 325 nm. Il pretrattamento dei campioni di plasma è effettuato mediante estrazione in fase solida (SPE) con cartucce MAX (modalità mista: fase inversa – scambio anionico), permettendo di ottenere buone rese d'estrazione degli analiti (>90%). Il metodo sembra essere promettente per la determinazione di serotonina ed HIAA nel plasma sia di soggetti "drug- free" sia di soggetti tossicodipendenti.

A method has been developed for the analysis of the antidepressant drug sertraline together with its main metabolite N-desmethylsertraline in human plasma. It is based on capillary electrophoresis with laser-induced fluorescence (LIF) detection (lambda = 488 nm). A solid phase extraction procedure is employed for biological sample pre-treatment, followed by a derivatisation step with fluorescein isothiocyanate; reboxetine was the internal standard. The effect of cyclodextrin, acetone and N-methyl-glucamine as constituents of the background electrolyte for analyte separation was investigated. The final background electrolyte consisted of 20 mM carbonate buffer, pH 9.0, with 2.5 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin, 50 mM N-methylglucamine and 20%, v/v acetone. With 30 kV applied voltage the electrophoretic run is completed in 7.5 min. Linearity was observed in the plasma concentration range from 3.0 to 500 ng/mL for sertraline and 4.0 to 500 ng/mL for desmethylsertraline. Extraction yield was >97.1%, precision - expressed as RSD% - was 95.6%. Due to its sensitivity and selectivity the method was suited for the analysis of plasma samples from patients undergoing therapy with sertraline.

Pramipexolo (N'-propil-4,5,6,7-tetraidrobenzotiazolo-2,6-diammina) e ropinirolo (4-(2-dipropilamminoetil)-1,3-diidroindol-2-one) sono due agonisti dopaminergici utilizzati nella terapia del parkinsonismo, o in monoterapia o in associazione alla levodopa. I numerosi effetti collaterali associati alla terapia, come nausea, sintomi psicotici (allucinazioni), discinesie, tachicardia, consigliano un attento monitoraggio dei pazienti che assumono questi farmaci. Sono attualmente in fase di sviluppo due distinti metodi per la determinazione di pramipexolo e ropinirolo in urine, basati rispettivamente sull'HPLC con rivelazione UV e sull'elettroforesi capillare (CE) con rivelazione a fluorescenza laser-indotta (LIF). Il metodo HPLC prevede l'utilizzo di una colonna C8 e di una fase mobile composta da tampone fosfato ed acetonitrile. Il metodo CE prevede la derivatizzazione mediante fluoresceina isotiocianato e l'eccitazione dell'addotto così formato con laser alla lunghezza d'onda di 488 nm. Il pre-trattamento del campione biologico è effettuato mediante estrazione liquido-liquido per entrambi i metodi analitici. I risultati preliminari sono soddisfacenti e sembrano promettenti per l'applicazione a campioni di urine di pazienti.

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.

Nella soia e nei suoi estratti sono presenti numerosi composti a struttura isoflavonica che possono esercitare un'azione ormono-simile ed in particolare estrogeno-simile. Pertanto, la soia viene spesso utilizzata come integratore alimentare allo scopo di eliminare o ridurre i sintomi spiacevoli della menopausa: vampate di calore, insonnia, affaticabilità, nervosismo, iperforesi e aumento di peso. Recentemente, è stata introdotta in commercio una nuova formulazione (Soymen GN) comprendente due tipi di capsule di gelatina molle: per la somministrazione diurna, contenenti estratto di soia e per la somministrazione serale, contenenti melatonina oltre all'estratto di soia. In questo modo si potrebbe migliorare il controllo dell'insonnia, che risulta essere uno dei sintomi più fastidiosi e più difficilmente gestibili. Sono attualmente in fase di sviluppo due metodi HPLC rispettivamente con rivelazione UV e con rivelazione ED (di tipo amperometrico), che permettono di dosare contemporaneamente la melatonina, i principali isoflavoni della soia (genisteina, daidzeina e gliciteina) e le loro forme glicosilate (genistina e daidzina) nel plasma di soggetti che assumono Soymen GN®. I metodi utilizzano una colonna C18 a fase inversa ed una fase mobile composta da tampone fosfato a pH acido ed acetonitrile. Il pre-trattamento dei campioni biologici è effettuato mediante estrazione in fase solida con cartucce a bilanciamento idrofilo-lipofilo e pochi µL di plasma sono sufficienti per un'analisi completa. I risultati preliminari sono soddisfacenti, sia in termini di rese d'estrazione, sia in termini di purificazione dei campioni.

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.

A HPLC method with UV detection has been developed for the simultaneous determination of levomepromazine, clozapine and their main metabolites: N-desmethyl-levomepromazine, levomepromazine sulfoxide, O-desmethyl-levomepromazine, N-desmethylclozapine and clozapine N-oxide. The analytes were separated on a C8 reversed-phase column using a mobile phase composed of acetonitrile and a pH 2.0, 34 mM phosphate buffer containing 0.3% triethylamine (29:71, v/v). Loxapine was used as the internal standard. A reliable biological sample pre-treatment procedure by means of solid-phase extraction on C1 cartridges was implemented, which allows to obtain good extraction yields (>91%) for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy. These assays gave R.S.D.% values for precision always lower than 4.9% and mean accuracy values higher than 93%. The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine.

Gabapentin (acido 2-[1-(amminometil)cicloesil]acetico) e vigabatrin (acido 4-ammino-5-esenoico) sono due farmaci antiepilettici a struttura aminoacidica. Mentre il preciso meccanismo d'azione del gabapentin è ancora ignoto, si ritiene che il vigabatrin agisca inibendo la GABA transaminasi, con effetti inbitori sulla trasmissione neuronale. Come per molti altri antiepilettici, anche per gabapentin e vigabatrin è necessario personalizzare la terapia, effettuando un accurato monitoraggio terapeutico dei pazienti. È stato quindi sviluppato un metodo, basato sulla cromatografia liquida, per l’analisi simultanea di questi due farmaci in plasma umano. A tale scopo si è utilizzata una colonna a fase inversa C8 ed una fase mobile composta da tampone fosfato acido ed acetonitrile. Poiché le molecole non presentano cromofori significativi, la rivelazione spettrofotometrica non è direttamente applicabile. Pertanto, si è deciso di accoppiare all'HPLC una rivelazione spettrofluorimetrica dopo derivatizzazione degli analiti con dansil cloruro (eccitazione = 318 nm, emissione = 510 nm). Prima della derivatizzazione, i campioni biologici vengono purificati mediante estrazione in fase solida (SPE) con cartucce MCX (modalità mista: fase inversa – scambio cationico). Il metodo è ora in fase di convalida, ma i risultati preliminari sono promettenti: le rese d'estrazione sono maggiori di 80% e si è ottenuta una buona linearità nei range attesi di concentrazioni plasmatiche dei farmaci.

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.

È attualmente in corso uno studio in collaborazione con i Servizi Tossicodipendenze allo scopo di evidenziare gli effetti neuroendocrini dell'uso cronico di alcune sostanze psicoattive (alcool, cocaina, eroina). Lo studio si propone di individuare le correlazioni tra fenomeni comportamentali (es. aggressività) o socio-epidemiologici (es. vulnerabilità all'uso di droghe) e indicatori dello stato neuroendocrino dell'organismo. Infatti, l'individuazione di correlazioni statisticamente valide tra stato neuroendocrino ed esito della terapia di disintossicazione permetterebbe di prendere misure atte ad aumentare l'efficacia dei trattamenti e a diminuire il numero di soggetti che escono dal programma di reinserimento nella società. Inoltre, queste conoscenze potrebbero anche permettere di individuare i soggetti "a rischio", maggiormente vulnerabili all'abuso di sostanze psicoattive, e quindi di impostare azioni sempre più mirate alla prevenzione e cura delle tossicodipendenze. Da recenti studi risulta che i livelli plasmatici di catecolammine (adrenalina, noradrenalina, dopamina) possono essere considerati importanti markers neuroendocrini, pertanto utili per stabilire correlazioni significative con le caratteristiche psicosociali degli individui. Inoltre, la dopamina ed il suo principale metabolita acido omovanillico (HVA) sono coinvolti nei sistemi di gratificazione cerebrali che causano il craving (ricerca compulsiva dello stimolo che gratifica o che allevia lo stato di malessere generato dall’astinenza) caratteristico dello stato di dipendenza. Si è pertanto scelto di effettuare la determinazione plasmatica di questi composti in soggetti ex-tossicodipendenti, attualmente sottoposti a terapia di disintossicazione (psicoterapia, trattamento con metadone o buprenorfina, comunità terapeutica) e che non usano contestualmente sostanze stupefacenti. I campioni di plasma, dopo opportuno pretrattamento mediante procedura di estrazione in fase solida, vengono analizzati in cromatografia liquida (HPLC) accoppiata a rivelazione elettrochimica di tipo coulombometrico, che garantisce prestazioni ottimali in termini di sensibilità e selettività. Dai risultati finora ottenuti, sembra che i metodi analitici utilizzati consentano la determinazione riproducibile ed accurata degli analiti, pur se presenti a bassissime concentrazioni (a livello di parti per trilione) ed in una matrice altamente complessa come quella plasmatica. In un secondo momento, si prevede di ampliare la ricerca, sia studiando un numero maggiore di soggetti ex-tossicodipendenti, sia prendendo in considerazione differenti sostanze d'abuso (amfetamine, allucinogeni).

Homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) is one of the main metabolites of dopamine and can thus be used as an index of dopamine metabolism which is involved in reward systems, emotional responses, personality trait expression and psychopathological phenomena. The changes occurring in the levels of HVA can be of great importance for the diagnosis of behavioural disorders. Therefore, it is fundamental that reliable analytical methods are available for the determination of homovanillic acid in human plasma. A HPLC method with amperometric detection is currently under development for this purpose and preliminary results seem to be quite promising. Homovanillic acid is analysed on a C8 column (250×4.6 mm I.D., 5 µm) with a mobile phase composed of MeOH and a pH 4.8 citrate buffer (10:90) also containing octanesulfonic acid and EDTA. Electrochemical detection is carried out using an Antec Decade amperometric detector (working electrode: glassy carbon; reference electrode: Ag/AgCl; auxiliary electrode: stainless steel) set at + 0.800 V. Solid-phase extraction (SPE) on strong anion exchange (SAX) cartridges was chosen for sample purification and pre-concentration. After conditioning, 500 µL of plasma are loaded onto the cartridge, followed by suitable washing and elution, obtaining a final sample volume of 250 µL (1:2 pre-concentration). Assays are currently underway to validate the method and to apply it to plasma samples from "at risk" adolescents for studies regarding neuroendocrine responses.

Introduction: Amphetamine (alpha-methylphenylethylamine) and its analogues such as methamphetamine (N,alpha-dimethylphenylethylamine) and MDMA (N,alpha-dimethyl-3,4-methylenedioxyphenylethylamine) are currently among the most well-known abuse drugs. They are used especially by young people as stimulants during the week-end (e.g. in discos), and are thus often referred to as "recreational drugs". Their mechanism of action is complex, but surely involves the release of catecholamines and serotonin from synaptic vesicles. Aim of this study is the development of a reliable analytical method for the simultaneous determination of amphetamine, methamphetamine and MDMA, and its application to the analysis of these compounds in human biological fluids, such as plasma and urine. Materials and Methods: Since the analytes show native fluorescence, liquid chromatography (HPLC) with fluorimetric detection was chosen for the purpose of separating and quantitating them. Separation was achieved on a C8 reversed-phase column using a phosphate buffer/acetonitrile (88:12) mixture (apparent pH = 2.8) as the mobile phase, flowing at 1 mL/min. Quinine was chosen as the Internal Standard (IS). Detection wavelengths were: lambda(exc) = 210 nm; lambda(em) = 300 nm for the analytes; lambda(exc) = 340 nm; lambda(em) = 420 nm for the IS. The sample pre-treatment procedure was carried out by means of solid-phase extraction (SPE) on hydrophilic-lipophilic balance cartridges. The cartridges were loaded with 250 µL of plasma or urine; the analytes were then eluted, dried and redissolved with 250 µL of mobile phase. Results: Under the described leading conditions, all analytes are baseline separated and detected within 13 minutes. Good linearity was obtained over different concentration ranges, according to the drug and the matrix. The sample pre-treatment procedure gave good extraction yields for all analytes, with mean recovery values ranging from 85 to 95% in plasma. Furthermore, the samples are devoid of interference from the biological matrices, thus good purification has been achieved. Conclusion: The method thus developed seems to be suitable for the determination of amphetamine, methamphetamine and MDMA in human plasma and urine.