Giampietro Frison

Dose-dependent absorption and elimination of gamma-hydroxybutyric acid in healthy volunteers

SummaryGamma-hydroxybutyric acid (GHB) is effective in treatment of the alcohol and opiate withdrawal syndromes. Its absorption and disposition kinetics have been studied in 8 healthy male volunteers following oral administration of single doses of 12.5, 25 and 50 mg kg−1.The AUC increased disproportionately with the dose and so the apparent oral clearance decreased significantly as the dose was increased, whereas the terminal half-life and mean residence time increased. The peak plasma concentrations normalised to the lowest dose fell significantly with increasing doses, whilst the corresponding peak times increased.These findings suggest that both the oral absorption and the elimination of GHB are capacity-limited processes. GHB did not bind to significant extent to plasma proteins over the therapeutic concentration range.The pharmacokinetic parameters in healthy volunteers were not significantly different from those previously observed in alcohol-dependent patients with compensated alcoholic liver disease.

Fatality Due to Gamma-Hydroxybutyric Acid (GHB) and Heroin Intoxication

The first case of fatal intoxication due to ingestion of gamma-hydroxybutyric acid (GHB) and intravenous use of heroin is reported. A 42-year-old man, known to have been a heroin addict and to have taken other psychoactive substances, who had been in treatment with GHB for several months, was found dead. Anatomohistopathologic examination showed generalized visceral congestion, edema and pulmonary anthracosis, chronic bronchitis and chronic active hepatitis. Toxicological findings included fluid and tissue distributions of GHB, morphine and 6-monoacetylmorphine. GHB and morphine concentrations were respectively 11.5 and 0.77 micrograms/mL (blood), 84.3 and 0.3 micrograms/mL (vitreous humor), 258.3 and 1.35 micrograms/mL (urine), 57.0 and 14.3 micrograms/mL (bile), 40.0 and 0.43 micrograms/g (brain), 43.0 and 0.60 micrograms/g (liver), 47.0 and 0.68 micrograms/g (kidney). Blood and urine levels of 6-monoacetylmorphine were 28.5 and 12.1 ng/mL respectively. The presumed mechanism of action and pharmacokinetics of GHB are briefly reviewed, with reference to its therapeutic use and to reports of non-fatal GHB intoxication.

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.

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault.

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.

A case of β-carboline alkaloid intoxication following ingestion of Peganum harmala seed extract

Beta-carboline alkaloids harmine, harmaline, and tetrahydroharmine can stimulate the central nervous system by inhibiting the metabolism of amine neurotransmitters, or by direct interaction with specific receptors; they are found in numerous plants, including Peganum harmala, Passiflora incarnata and Banisteriopsis caapi, and in the entheogen preparation Ayahuasca, which is traditionally brewed using B. caapi to enhance the activity of amine hallucinogenic drugs. The ingestion of plant preparations containing beta-carboline alkaloids may result in toxic effects, namely visual and auditory hallucinations, locomotor ataxia, nausea, vomiting, confusion and agitation. We report a case of intoxication following intentional ingestion of P. harmala seed infusion; P. harmala seeds were bought over the Internet. The harmala alkaloids were identified by gas chromatography-mass spectrometry in the seed extract and the patients urine. This is, to our knowledge, the first case of P. harmala intoxication corroborated by toxicological findings.

Determination of γ‐hydroxybutyric acid (GHB) in plasma and urine by headspace solid‐phase microextraction and gas chromatography/positive ion chemical ionization mass spectrometry

A new method for the qualitative and quantitative analysis of γ-hydroxybutyric acid (GHB) in plasma and urine samples is described. It involves the conversion of GHB to γ-butyrolactone (GBL), its subsequent headspace solid-phase microextraction (SPME), and detection by gas chromatography/positive ion chemical ionization mass spectrometry (GC/PICI-MS), using D6-GBL as internal standard. The assay is linear over a plasma GHB range of 1–100 µg/mL (n = 5, r = 0.999) and a urine GHB range of 5–150 µg/mL (n = 5, r = 0.998). Relative intra- and inter-assay standard deviations, determined for plasma and urine samples at 5 and 50 µg/mL, are all below 5%. The method is simple, specific and reasonably fast. It may be applied for clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring. Copyright

Effect of moderate or severe liver dysfunction on the pharmacokinetics of γ-hydroxybutyric acid

Objectives: To assess the effect of moderate or severe liver dysfunction on the pharmacokinetics of γ-hydroxybutyric acid (GHB).Methods: The absorption and disposition kinetics of GHB were studied in eight cirrhotic patients without ascites (Child’s class A) and eight cirrhotic patients with ascites (Child’s class C), after administration of a single oral dose of 25 mg⋅kg−1. The liver metabolic function of each patient was evaluated by measuring antipyrine clearance and the formation rate of the lidocaine metabolite monoethylglycinexylidide (MEGX).Results:Compared to those previously determined in eight healthy control subjects given the same GHB dose, mean AUC values were double or greater in the cirrhotic patients. Accordingly, apparent oral clearance was markedly reduced (from 9.1 to 4.5 and 4.1 ml⋅min−1⋅kg−1 in nonascitic and ascitic patients, respectively). Terminal half-life (t1/2), was significantly longer in nonascitic patients than in control subjects (32 vs 22 min). A further significant prolongation of t1/2, most likely due to an increased distribution volume, was observed in patients with ascites (56 min). Nonetheless, GHB plasma concentrations fell to either undetectable or negligible levels by the end of the usual dosing intervals (6–8 h).More limited changes were noted in the absorption parameters. The peak level (Cmax) increased only in nonascitic patients, but not proportionally to the increase in AUC. The time to Cmax increased from 30 to 45 min in both cirrhotic groups. These findings are consistent with a slowed rate of GHB absorption in cirrhotic patients. Adverse effects were similar, for intensity and duration, to those recorded in healthy volunteers, i.e., mild and transient.Conclusions:Although liver cirrhosis causes significant modifications of GHB disposition kinetics, the increase in t1/2 is not such as to cause drug accumulation on repetitive dosing. However, in consideration of the higher mean plasma levels observed in cirrhotic patients, it appears wise to keep the initial GHB daily dose at the lower end of the therapeutic range and to carefully monitor the patients if upward dose adjustments are required.

Quantification of Citalopram or Escitalopram and Their Demethylated Metabolites in Neonatal Hair Samples by Liquid Chromatography-Tandem Mass Spectrometry

Citalopram and escitalopram are highly selective serotonin reuptake inhibitors widely used in the treatment of depression. They exhibit adverse drug reactions and side effects, however, and the development of specific methods for their determination is of great interest in clinical and forensic toxicology. A liquid chromatography-tandem mass spectrometry method has been developed and validated for the assay of citalopram, escitalopram, and their demethylated metabolites in 10-mg hair samples. The analytes were extracted by incubation in methanol and liquid/liquid extraction with diethyl ether/dichloromethane. Gradient elution on a narrow bore C18 column was realized using clomipramine-d3 as an internal standard. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method exhibited a linear range of 25 to 2000 pg/mg, a quantification limit of 25 pg/mg for all analytes, relative standard deviations in the range of 12.10 to 9.80 (intraassay), and 13.80 to 11.78 (interassay), and accuracies (as percent recovery of the spiked standards) in the range of 90% to 110%; it was applied to the determination of citalopram and escitalopram and their metabolites in hair samples of two newborns to document their in utero exposure to the drugs. The method proved suitable for neonatal hair analysis of citalopram or escitalopram and was applied to two real cases of gestational exposure.

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.

Gas chromatography/mass spectrometry determination of mephedrone in drug seizures after derivatization with 2,2,2-trichloroethyl chloroformate.

Within the framework of our forensic toxicology activities on both non-biological and biological samples, we were asked to analyze two off-white powders and a white tablet marked with a ’dolphin’ logo, seized by police. These seizures produced a positive result for ’amphetamines’ with the color tests performed in the field by police officers, but laboratory gas chromatographic analyses, both with flame ionization or mass spectrometric detectors (GC/FID, GC/MS), failed to identify in all specimens the presence of amphetamine, methamphetamine, ephedrines, methylenedioxyamphetamines, methylenedioxyphenylbutanamines, and methoxyamphetamines. Instead, both powders and the tablet showed the presence, as the active ingredient, of mephedrone (4-methyl-methcathinone, 2-methylamino-1-p-tolylpropane1-one, MW 177), whose chemical structure is depicted in Fig. 1. The concentrations ranged from 41% (tablet) to 50% (powders). Mephedrone is one of the most important members of a new class of designer drugs that have recently appeared on the recreational drug market, such as methylone, butylone, ethylone, methylenedioxypyrovalerone (the so-called betaketo amphetamines). Mephedrone is sold under various trade names such as ’meow meow, 4-M, MCAT, 4-MMC’, ’plant food’, and ’bubbles, subcoca-1’, terms derived from its chemical structure, commercial uses and subjective effects, respectively. It is readily available from ’smart shops’ and can be purchased over the internet, through many websites which sell it cheaply as a ’legal’ alternative to amphetamines. In many countries it is not yet illegal, although an increasing number of them are including it in their controlled substance legislations because of its potential for addiction and the associated health risks. In Italy mephedrone was scheduled as a controlled substance on July 16, 2010. Mephedrone is chemically similar to amphetamines and cathinones, and similar stimulant effects, with euphoric, entactogenic and hallucinogenic properties, have been described. It was also recently associated with non-fatal and fatal overdoses. Figure 2 shows typical electron ionization (EI) full scan GC/MS traces obtained from the analysis of methanolic solutions of the mephedrone-containing seized drugs. The analyses were performed on an Agilent 7890 series II/5975 GC/MS quadrupole system in full-scan (40–450 u) mode (Agilent Technologies, Cernusco sul Naviglio, Italy). Injection (1mL, 2508C)was in split (1:20) or splitless (1min) mode, with a carrier gas (He) flow rate of 1mL/min. An Agilent HP-5MS UI (ultra inert, 30m x 0.25mm, 0.25mm film thickness) capillary column was used and the oven temperature was programmed from 508C (0.5min) to 2008C at 308C/min, then to 3008C (5min) at 108C/min. The interface temperature was 2808C. The electron multiplier was set at þ300V with respect to the autotune value. A standard solution of mephedrone was obtained from LGC Standards, Milan, Italy. As expected, due to the low molecular weight and relatively high polarity of mephedrone, the obtained GC peak is characterized by a fairly short retention time (about 6.3min) and severe widening and tailing, thus hindering GC resolution. In addition to methanol (b.p. 64.58C), two other polar organic solvents with higher boiling points, acetonitrile (b.p. 828C) and n-butanol (b.p. 1178C), were tested to dissolve seized drugs, in order to improve the focusing of the analyte at the beginning of the column (solvent effect), and thus obtain better mephedrone peak shapes. Compared with methanol, no significant improvements, even in split injection mode, were obtained with the above solvents. Furthermore, the EI mass spectrum of mephedrone is quite non-specific due to the barely visible molecular ion atm/z 177 and the base peak at m/z 58, caused by the well-known formation of the immonium ion via the amine-initiated alpha-cleavage of the benzylic bond, characteristic of all N-methyl phenethylamines. Less abundant fragment ions are observed at m/z 119 (4-methylbenzoyl cation), 91 (CO loss from the preceding species to form the tropylium ion), 65 (acetylene loss from the tropylium ion), 56 (H2 loss from the immonium ion), and 162 (alpha-methyl loss from the molecular ion). If it was not for the presence of the fragment ion at m/z 119, the EI mass spectrum of mephedrone could be at first sight confused with that of methamphetamine, shown in Fig. 3. Pursuing our interest in the analysis of amphetamine-related drugs after derivatization with 2,2,2-trichloroethyl chloroformate, and with the aim of improving the potential of identification and quantitation of mephedrone in seized materials, we thought it of interest to employ the above derivatization agent also for mephedrone. As previously described for amphetamine-related drugs, the 2,2,2-trichloroethyl carbamate derivative of mephedrone was prepared by adding 50mL of a 3:7 mixture of 2,2,2-trichloroethyl chloroformate/ethyl acetate to residues obtained from methanolic solutions of mephedronecontaining seized drugs or methanolic solutions of pure mephedrone, heating at 808C for 15min, taking the mixtures to dryness under a gentle stream of nitrogen at 358C, and Rapid Commun. Mass Spectrom. 2011, 25, 387–390 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4842 Letter to the Editor