Manuela Pellegrini

γ‐Hydroxybutyrate (GHB) in Humans

Abstract:  Despite γ‐hydroxybutyrate (GHB) therapeutic uses and the increasing concern about its toxicity, few studies have addressed GHB dose‐related effects under controlled administration and their relationship with its pharmacokinetics. The study design was double‐blind, randomized, crossover, and controlled. As a pilot pharmacology phase I study, increasing doses of GHB were given. Single oral sodium GHB doses (40, 50, 60, and 72 mg/kg) were administered to eight volunteers. Plasma and urine were analyzed for GHB by gas chromatography–mass spectrometry. Physiological effects, psychomotor performance, and subjective effects were examined simultaneously. GHB produced dose‐related changes in subjective effects as measured by questionnaires and VAS. GHB showed a mixed stimulant‐sedative pattern, with initially increased scores in subjective feeling of euphoria, high, and liking followed by mild‐moderate symptoms of sedation with impairment of performance and balance. Mean peak GHB plasma concentrations were 79.1, 83.1, 113.5, and 130.1 μg/L for 40, 50, 60, and 72 mg/kg, respectively. GHB‐mediated physiological and subjective effects were dose dependent and related to GHB plasma concentrations. GHB urinary excretion was mainly related to administered doses. GHB‐mediated subjective and physiological effects seem dose dependent and related to GHB plasma concentrations. Results suggest a high abuse liability of GHB in the range of dose usually consumed.

Hair analysis for nicotine and cotinine: evaluation of extraction procedures, hair treatments, and development of reference material

Analysis of nicotine and cotinine in human hair can provide information on nicotine intake and exposure to environmental tobacco smoke over a long period of time. Nonetheless, to better assess the usefulness of hair analysis to determine smoking habits or exposures, all procedures have to be standardized. Various solvents were tested as washing solvents to eliminate external contamination from nicotine. Dichloromethane was found effective when used for two washes prior to the extraction. Basic and acid digestion of hair followed by solid phase extraction with Extrelut-3 glass column using dichloromethane:isopropyl alcohol (9:1) as eluting mixture both gave good recoveries of nicotine and cotinine, when compared with extractions reported in the literature. The extraction method was free from substances, which could interfere in the chromatographic analysis. Furthermore, the addition of methanolic HCl to the eluting mixture prevented the loss of nicotine during the evaporation step before chromatography. Chromatography was performed using a reversed-phase column and a U.V. detection at 254 nm. Furthermore, hair treatments (dyes, permanent wave, hydrogen peroxide) caused a major decrease in the nicotine content in hair, and a smaller effect on cotinine levels. However, the effect of various treatments was not reproducible. Several attempts to produce reference materials were carried out. Nicotine and cotinine standard solutions at different concentrations were added to blank hair soaked in dimethylsulfoxide, methanol and water.

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 microl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C18 reversed-phase column with a mobile phase consisting of water-acetonitrile (71:29, v/v) containing 0.05 M Na2HPO4 and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1-500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.

Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast milk by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of frequently used licit (caffeine, nicotine and cotinine) and illicit drugs (opiates, cocaine, cannabinoids and amphetamines) in breast milk was developed and fully validated. Chromatography was performed on a reverse-phase column using a gradient of 2mM ammonium acetate, pH 6.6, and methyl alcohol as mobile phase at a flow rate of 0.35 mL/min. Separated analytes were quantified by electrospray ionization tandem mass spectrometry in positive ion mode using multiple reaction monitoring. Milk samples were kept at -20 °C until analysis and the compounds under investigation were extracted from the matrix by Bond Elut Certify cartridges. The concentration range covered was LOQ to 1000 ng/mL for all the investigated drugs. Intra- and inter-assay imprecision was less than 20%, analytical recovery ranged between 51.6% and 86.5%, matrix effect between 71.1% and 116.6% and process efficiency between 46.8% and 84.0%. Analytes were stable after three freeze-thaw cycles, after 6 months at -20 °C and after the pasteurization process (differences to the initial concentration always lower than 10%). matrix effect ranged from 77.6% to 116.6%, recovery from 51.6% to 86.5%, and process efficiency from 46.8% to 79.0%. This LC-MS-MS assay was applied to screen samples from the largest Spanish milk bank and samples coming from drug addicted mothers. The developed method provided adequate sensitivity and performance characteristics to prove the presence of only caffeine in a small percentage of samples from milk donating nursing mothers and the presence or absence of most commonly used illicit drugs in breast milk from addicted lactating mothers.

High performance liquid chromatography-diode array and electrospray-mass spectrometry analysis of vardenafil, sildenafil, tadalafil, testosterone and local anesthetics in cosmetic creams sold on the Internet web sites

A simple high-performance liquid chromatography (HPLC) method with ultraviolet diode array (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) detection has been developed for the determination of vardenafil, sildenafil, tadalafil, testosterone, procaine, lidocaine, prilocaine, and benzocaine in cosmetic creams sold as promising remedies for male erectile dysfunction and female genitals stimulation. The presence of these substances in commercial cosmetic samples is prohibited. Aliquots (1 g) of the cosmetic creams under investigation were diluted 1:100 in methanol, subjected to ultrasonic treatment, added with benzoic acid as internal standard, and analyzed by HPLC-DAD and HPLC-ESI-MS after a further 1:1000 dilution. The compounds were separated by reversed phase chromatography with water (0.02% trifluoroacetic acid) and acetonitrile gradient elution and detected by UV-DAD at 228, 255 and 290 nm and by ESI-MS positive ionisation mode. Benzoic acid was used as internal standard. Linearity was studied with UV-DAD detection from 2.5-7.8 to 250 microg/g range, depending on the different compounds and with ESI-MS in the 3.3-8.9 to 250 ng/g range. Good determination coefficients (r(2) > or = 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 microg/g for HPLC-UV-DAD assay and between 3.3 and 8.9 ng/g for HPLC-ESI-MS assay depending on different analyzed substances. At three concentrations spanning the linear dynamic ranges of both UV-DAD and ESI-MS assay, mean recoveries were always higher than 90% for the different analytes and intra-assay and inter-assay precision always better than 15% and 12%. This method was successfully applied to the analysis of substances under investigations present in cosmetic creams, freely sold on the Internet web-sites.

Liquid chromatography-tandem mass spectrometry for fatty acid ethyl esters in meconium : Assessment of prenatal exposure to alcohol in two European cohorts

Fatty acid ethyl esters (FAEEs) in meconium emerged as a reliable, direct biological marker for establishing fetal exposure to ethanol. We developed an LC-MS/MS method for ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate using ethyl heptadecanoate as the internal standard. The analytes were extracted from meconium with hexane, followed by solid-phase extraction with aminopropyl-silica columns. Chromatography was performed on a C(8) reversed-phase column using water/isopropanol/acetonitrile (20:40:40, v/v/v) as a mobile phase. A triple quadrupole mass spectrometer that monitored the transitions in multiple reaction-monitoring mode was used for the detection of the analytes. Limits of quantification (LOQs) varied between 0.12 and 0.20 nmol/g. Calibration curves were linear from LOQs to 50 nmol/g for all analytes, with a minimum r(2)>0.99. At three concentrations spanning the linear dynamic range, mean recoveries ranged between 53.6 and 86.7% for the different analytes. The validated method was applied to analysis of meconium in newborns of two European cities. The two cohorts presented with different prevalence of gestational ethanol consumption during pregnancy.

Disposition of gamma-hydroxybutyric acid in conventional and nonconventional biologic fluids after single drug administration: issues in methodology and drug monitoring.

Little controlled drug administration data are available to aid in the interpretation of gamma-hydroxybutyric acid (GHB) distribution in conventional and nonconventional fluids and the potential correlation between the pharmacokinetics of GHB and drug effects. Single oral sodium GHB doses of 50 mg/kg were administered to five volunteers. Plasma, oral fluid, urine, and sweat were analyzed for GHB by gas chromatography-mass spectrometry. GHB stability in plasma was studied at different storage temperatures. Subjective effects were measured using a set of 13 different visual analog scales. Mean peak GHB plasma concentrations at 30 minutes were 83.1 μg/mL. After the absorption phase, concentrations declined to mean values of 0.9 μg/mL at 6 hours. GHB was found in oral fluid at peak value concentrations equivalent to one third to one fourth of those found in plasma. The oral fluid-to-plasma ratio varied two fold in the 1- to 6-hour time range but always was lower than unit. The mean half-life (t1/2) of GHB was approximately 0.7 hour in plasma and approximately 1.2 hours in oral fluid. GHB urinary excretion is less than 2% of the dose administered. GHB was also detected in sweat at low concentrations. GHB showed a mixed sedative-stimulant pattern with subjective effects peaking between 1 and 1.5 hours after drug administration and lasting for 2 hours. Oral fluid and sweat appeared not to be suitable biologic matrices for monitoring GHB consumption. GHB -mediated subjective effects are related to GHB plasma concentrations.

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of opiates and cocaine in meconium.

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C(8) reversed-phase column using a gradient of acetic acid 1%-acetonitrile as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.

The role of liquid chromatography–mass spectrometry in the determination of heroin and related opioids in biological fluids

The opioid most commonly sold in the illicit market is heroin. This substance, classified as an analgesic narcotic drug, has an extremely short half-life, and it is rapidly metabolized to 6-monoacetyl-morphine and further to morphine. Morphine is principally metabolized by conjugation to morphine-3 and morphine-6 glucuronides. Morphine itself is a potent analgesic that is frequently used in the pharmacological intervention of cancer pain. The toxicological and clinical evaluation of heroin and morphine have stimulated pharmacokinetic studies in human and animal models. Although a number of methods exist to determine opiates and their metabolites, liquid chromatography (LC) appears to be the technique that can separate without any pretreatment the lipophilic and the hydrophilic analytes of the complete metabolic profile of heroin and/or morphine. Moreover, mass spectrometry (MS) used as a detector for liquid chromatography is unique, because it offers universality and selectivity. Furthermore, efforts have been made to develop LC/MS interfaces that could overcome the previous problem of poor sensitivity. For this reason, in recent years LC combined with MS has been applied to the analysis of opiates--parent drugs and metabolites--in biological fluids. This article reviews the existing literature on the determination, using liquid chromatography coupled to mass spectrometry, of opiate metabolites found in different biological matrices after the administration of the parent compounds.

The analysis of nicotine in infants' hair for measuring exposure to environmental tobacco smoke.

Hair samples were collected from 24 infants (3-36 months) exposed and non-exposed to environmental tobacco smoke. Hair was washed in dichloromethane, digested in NaOH, extracted by solid-phase extraction and analyzed by high-performance liquid chromatography to determine the content of nicotine and cotinine. Nicotine concentration in non-exposed infants (1.3 +/- 1.7 ng/mg hair) was significantly different from that in occasionally exposed infants (6.8 +/- 2.1 ng/mg hair) and that in infants passively exposed to smoke (15.4 +/- 6.7 ng/mg hair). Cotinine could be measured only in passive smokers infants. These findings suggest the possibility of monitoring exposure to environmental tobacco smoke in infants by using nicotine measurement in hair rather than in urine, blood or saliva.