Franca Castagna

Therapeutic gamma-hydroxybutyric acid monitoring in plasma and urine by gas chromatography—mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) method for the determination of therapeutic levels of gamma-hydroxybutyric acid (GHB) in plasma and urine samples is described. GHB is converted to its lactonic form gamma-butyrolactone (GBL) which is extracted from biological fluids after the addition of the internal standard delta-valerolactone. Final GC-MS analysis is obtained under electron impact selected ion monitoring (SIM) conditions. Mean relative recoveries of GHB from plasma and urine are 75.5% (RSD% = 2.2) and 76.4% (RSD% = 2.4), respectively. The assay is linear over a plasma GHB range of 2-200 micrograms ml-1 (r = 0.999) and a urine GHB range of 2-150 micrograms ml-1 (r = 0.998). Intra- and inter-assay relative standard deviations (n = 5) determined at 10 and 100 micrograms ml-1 are below 5%. The method is simple, specific and accurate, and may be applied for analytical purposes related to pharmacokinetic studies and therapeutic drug monitoring.

Simultaneous identification of amphetamine and its derivatives in urine using HPLC-UV

SummaryAn HPLC-UV method for the simultaneous identification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine is described. It includes a rapid extraction procedure of the 4 analogs from urine using Extrelut 3 columns, derivatization with sodium 1,2-naphthoquinone-4-sulphonate (NQS) to obtain highly chromophoric UV-VIS derivatives, and a final HPLC analysis using an ion-pair reversed-phase technique with eluent monitoring at 480 nm. Structural characterization of the derivatives obtained by mass spectrometry is reported. Recoveries of the amphetamines were in the range 80–85% at concentrations of 300 ng/ml. Practical detection limits were 40–60 ng/ml (S/N ratio = 10) for all derivatives. The chromatographic peaks of the NQS derivatized amphetamines are fairly narrow and well resolved. The method is simple, rapid, quite sensitive, and specific for convenient confirmation of preliminary positive results obtained with immunoassays.ZusammenfassungEine HPLC-UV-Methode zur gleichzeitigen Bestimmung von Amphetamin, Methamphetamin, 3,4-Methylendioxyamphetamin (MDA) und 3,4-Methylendioxymethamphetamin (MDMA) im Urin wird beschrieben. Vorgestellt wird eine Schnellextraktion der 4 Amphetamine über Extrelut-3-Säulen und eine Derivatisierung mit Natrium-1,2-naphthochinon-4-sulfonat (NQS) um hochchromophore UV-VIS-Derivate zu erhalten. Abschließend erfolgt eine HPLC-Analyse mittels einer reversed phase Technik (Ionenpaar) mit Detektion des Eluats bei 480 nm. Die Struktur der NQS-Derivate wird durch Massenspektrometrie charakterisiert. Die Wiederfindungsraten der Amphetamine liegen bei Konzentrationen von 300 ng/ml bei 80–85%. Die Nachweisgrenze liegt für alle Derivate bei 40–60 ng/ml (S/N-Verhältnis = 10). Die Trennung der NQS-derivatisierten Amphetamine gelingt gut, die Methode ist einfach, schnell, empfindlich und spezifisch genug für die Bestätigung vorläufiger Screening-Resultate, wie sie mit Hilfe von Immunoassays erhalten werden.

Neonatal Hair Analysis by Liquid Chromatography-High-Resolution Mass Spectrometry to Reveal Gestational Exposure to Venlafaxine

Venlafaxine (VEN) is a second generation antidepressant drug, belonging to the class of selective serotonine and norepinephrine reuptake inhibitors, widely used in the treatment of depression and anxiety disorders. Though its pharmacological profile is considered safe, treatment with VEN can cause several nervous, gastrointestinal, cardiovascular and genitourinary adverse effects. Therapeutic drug monitoring for VEN could be useful in specific situations, including exposure to the drug during pregnancy. A liquid chromatography-high-resolution mass spectrometry method was developed and validated for the assay of VEN in 2.5-mg hair samples from 2 newborn identical twin sisters. The analyte was extracted by a rapid, simultaneous pulverization and extraction step, allowing analysis when tiny amounts of hair are available, such as in the case of a newborn. Gradient elution on an Atlantis T3 column was performed using nordiazepam-d5 as an internal standard. Positive ion electrospray ionization and high-resolution full scan determination were performed in an Orbitrap mass spectrometer. The method was linear range in the range 0.2-25 ng/mg, and had a quantification limit of 0.2 ng/mg, a relative standard deviation in the range 0.7%-1.4% (intra-assay) and 2.9%-5.9% (interassay), and was accurate (as % relative error) in the range −9% to + 2%, using a hair sample size as low as 2.5 mg. The utilization of high-resolution/high accuracy mass spectrometry in full-scan mode allowed both the quantitative determination of VEN in the hair of the 2 newborns and the straightforward identification of 4 VEN metabolites, namely O-desmethylvenlafaxine, N-desmethylvenlafaxine, N,N-didesmethylvenlafaxine, and N,O-didesmethylvenlafaxine, by means of retrospective screening, thus unequivocally documenting in utero exposure.

Cocaine adulterants used as marker compounds

Cocaine has become a widely employed drug of abuse. In Italy its use may be estimated in the order of 9000 kg/year. In fact, considering that seizures in the last year in Italy were 3900 kg (Italian Minister of the Interior)[1] and that the worldwide interception rate has been estimated in the order of 42%,[2] it follows that the circulating cocaine is in the order of magnitude mentioned earlier. Large amounts of seized drug samples have been analyzed in order to identify, on the basis of the adulterants contained therein, their possible production area and the mechanisms by which they have been placed on the market. In fact the determination of cocaine content and adulterants in street samples is important not only from the clinical and toxicological points of view, but also for forensic purposes, related to the determination of geographical origin, comparison of various street samples for intelligence purposes and route of synthesis of the drugs. Nowadays illicit cocaine powders contain purposely a myriad of ‘new’ adulterants, having or not having any pharmacological effect, with the main aim to increase the amount of product available for sale with profit maximization. Although the risks to human health from cocaine use are known, the consequences from assumption of cocaine adulterants are not fully explored.[3] The cocaine adulterants most frequently employed are listed in Table 1.[4] A great number of adulterants have significant toxicological effects that may, in particular at high concentrations, amplify those of cocaine. The main categories of adulterants with pharmacological effects are: stimulants and local anesthetics (which are absorbed across mucosal surface and simulate the assumption of Cocaine) and sedatives (which are often assumed with cocaine to reduce the intensity of its unpleasant side effects).[5] The presence of adulterants could reflect on another aspect. In fact, as usually observed in pharmaceutical preparation, the impurities present at trace level (usually intermediates of the active product synthesis) are an effective marker of the chemical process used for production and covered by the related patent. Their presence is an unequivocal proof of the production process. Nowadays the street drug production can be considered due to, unfortunately, highly active industrial structures which necessarily, at marketing level, must define their sale regions. An effective method to control the presence of drugs from concurrent producers in the reserved area could be monitored on the basis of specific adulterants. In the Padova area (North-East of Italy) in the period March, 2006 – September, 2007 among the seized cocaine samples (259, for a

Concentrations of Phenobarbital, Flurazepam, and Flurazepam Metabolites in Autopsy Cases

In five cases of death resulting from acute intoxication with phenobarbital and flurazepam, the blood, urine, brain, lung, liver, and kidney levels of these drugs as well as the levels of N-1 hydroxyethyl, N-1 desalkyl, and N-1 desalkyl-3-hydroxy flurazepam metabolites were determined. Concentration of flurazepam and its metabolites was determined by using new gas chromatographic conditions employing a selective detector for nitrogen-containing substances and a column of 1% SP-1000. In addition, the EMIT technique was also employed on blood and urine samples and the results compared with GLC data.

Comparison of GLC-EMIT analysis for the assay of methadone and its major metabolite in urine

The enzyme-multiplied immunoassay technique (EMIT) was compared with the gas-liquid chromatographic (GLC) method in the analysis of 130 urine samples from subjects receiving methadone treatment and non-methadone controls. The GC method allowed the quantitation of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and major metabolite of methadone. A good correlation was found between the two methods. The advantages of using the EMIT system are discussed.

Proficiency testing for psychoactive substances in Italy.

Abstract This paper describes the general design and main results of the Italian proficiency testing program for the analysis of psychoactive substances in urine, a long-term initiative created in 1995 on an educational basis and characterized by an innovative internet-based service for data exchange between laboratories and the organizing body. Batches of six urine samples, validated by reference laboratories, are sent every 3 months to participating laboratories, which may choose which classes of substances to test from those planned by the program panel and, within those classes, which type of analytical commitment to work on: identification of just one class (Option 1), identification of single substances (Option 2), or identification and quantification of single substances (Option 3). Comprehensive periodical reports and annual reports are provided to participants with evaluation of their performance and an annual workshop is organized to discuss technical-scientific topics related to clinical, forensic and analytical toxicology. About 200 laboratories currently participate in the program and a total of 67,059 analyses have been carried out since 1995. The mean percentage of correct results was 96.8%, with a yearly improvement of about 0.4%. The best average false positive and false negative rates were obtained for methadone (0.2% and 2.1% respectively) and cocaine (0.3% and 2.2%). The worst average false positive rates were obtained for amphetamines and opiates (3.2% and 5.0%) and worst average false negative rates for amphetamines, barbiturates and cannabinoids (17.4%, 30.7% and 19.9%).

When color fails: illicit blue tablets containing anabolic androgen steroids.

The necessity of specific, confirmatory tests in the identification of seized illicit products was highlighted by the analysis of eighteen heart shaped, blue tablets confiscated by Police at a street control in the North East of Italy. The tablets responded as amphetamines to a preliminary color test (Marquis); a subsequent, confirmatory assay by gas chromatography-mass spectrometry revealed the presence of two anabolic androgen steroids (AAS), methandienone and methyltestosterone, in concentration of 1.7 and 1.5mg respectively per tablet; no trace of amphetamine-like or nitrogen containing compounds was found. The observed orange coloration was due to the reaction of concentrated sulphuric acid, contained in the Marquis reagent, with the Δ(4) C-3 keto group of steroids. The two AAS, banned under the world antidoping code, are not considered as psychoactive drugs of abuse in most countries, although their trafficking may entangle severe public health concerns.

Amphetamines and 3,4-methylendioxymetamphetamine (MDMA): evaluation of KIMS (kinetic interaction of microparticles in solution) assay at two cut-off levels.

Abstract Two screening methods for the assay of amphetamines and their derivatives have been applied to the same analytical instrument for their evaluation. In addition to an assay at a cut-off of 1000 μg/l, a new specific reagent was evaluated for an ultra-sensitive assay of amphetamines and 3,4-methylendioxyme-tamphetamine with a cut-off of 300 μg/l. The assay confirmation was performed using high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results were positive for both screening methods, confirming the efficacy of two simultaneous methods with different cut-off levels.

The EZ-SCREEN and RapidTest Devices for Drugs of Abuse

Many on-site drug-testing devices (OTD) have been described in the literature (1–7). Although the first of these (KDI Quick Test), marketed at the end of the 1980s, turned out to be inaccurate and unacceptable (8),improved devices have been developed and are now in use for analysis of drugs in urine (9–11).