Targeted screening and quantification of synthetic cathinones and metabolites in hair by UHPLC-HRMS.

OBJECTIVE: Synthetic cathinones (SCs) are new psychoactive substances with sympathomimetic effects, which emerged into the illegal drug market to replace controlled stimulants. Since every year more powerful and toxic substances enter the illicit market, there is the need for analytical methodologies able to detect these new compounds in conventional and non-conventional biological matrices. We sought to develop and validate a targeted screening and quantification method for thirty-two parent SCs and two metabolites in hair samples by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). MATERIALS AND METHODS: 20 mg hair samples were soaked in 250 µL of 2 mM ammonium formate, methanol and acetonitrile mixture (50/25/25, v/v/v) and incubated overnight at 40°C. After incubation, the samples were evaporated to dryness under nitrogen stream and reconstituted with 100 µL of mobile phase mix (A:B, 80:20) and 10 µL were injected into UHPLC-HRMS. A Q ExactiveTM Focus Orbitrap Mass spectrometer with full scan and targeted data-dependent MS/MS scan acquisition was used for the screening and quantitation analysis. RESULTS: The assay was linear from 5 to 500 pg/mg hair for all the analytes under investigation. Intra-day and inter-day precision were always < 15% and matrix effect and analytical recovery were always within acceptable criteria (±25% and >50%, respectively). The developed method was applied to authentic hair samples from SCs consumers. The most prevalent found SCs were 3,4-Methylenedioxy-α-Pyrrolidinohexanophenone with a concentration range of 6.0-1,000.0 pg/mg along with α-Pyrrolidinohexiophenone (54.0 and 554.0 pg/mg, respectively), 3-Methylmetcathinone (556.0 and 5,000.0 pg/mg) and 4-Methylethcathinone (11.5 and 448.0 pg/mg) CONCLUSIONS: The developed method showed good selectivity, specificity, an easy and low-cost sample preparation and an analysis time compatible with a high throughput laboratory.

Pharmacokinetics of methylphenidate in oral fluid and sweat of a pediatric subject.

Methylphenidate (MPH) is a stimulant medication widely used for treating attention-deficit hyperactivity disorder (ADHD) in children and adolescents. Therapeutic monitoring for this drug is essentially lacking and alternative biological matrices, such as oral fluid and sweat, should be investigated for noninvasive assessment of short- and long-term history of drug use. We report the excretion profile of MHP and its metabolite ritalinic acid (RA) in oral fluid and sweat from a 12-year-old boy treated with the extended release drug formulation. Concentrations of MPH and RA in oral fluid, sweat and plasma were measured by liquid chromatography-mass spectrometry. Oral fluid-to-plasma ratio at each time interval was calculated at the start of the treatment and correlated with salivary pH. Excretion of MPH in sweat patches, collected up to 24h with PharmChek patches was also investigated. MPH and RA were both detected in oral fluid with a pharmacokinetic profile similar to that in plasma. Oral fluid peak concentrations of MPH ranged between 13.5 and 30.9 ng/mL at 3.0 h after drug intake. Oral fluid-to-plasma MPH ratio between 13.1 and 3.2 demonstrated an accumulation of the drug in oral fluid. Conversely, RA was found in oral fluid at peak concentration (23.4-62.9 ng/mL) equivalent to one-tenth of those found in plasma. Concentration profiles of MPH and RA in oral fluid were quite constant during the four weeks of drug administration. In sweat, MPH was detected for the first time at 5h after drug administration (range: 9.3-11.2 ng/patch) up to 24h (range: 29.8-38.7 ng/patch). RA was not detected in the sweat patches during the 24h time of collection. The results suggest that measurement of MPH in oral fluid can be used as a potential alternative to drug monitoring in plasma. Moreover, MPH measurement in sweat patches can be used for noninvasive monitoring of MPH consumption and misuse in situations where detection of recent abuse is of interest.

Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol.

This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother-infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography-tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography-mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2 nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother-infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs.

On-site screening and GC-MS analysis of cocaine and heroin metabolites in body-packers urine.

The illicit transportation of cocaine and heroin either swallowed or inserted into the rectum and/or vagina of individuals, defined as "body-packers", is becoming increasingly common. Assessment of smuggling by urinalysis from body-packers has been sparsely reported and on-site rapid screening methods are essentially lacking. We screened the presence of cocaine and heroin metabolites in urine from suspected body-packers by an on-site immunochromatographic test and confirmed the obtained results by gas chromatography-mass spectrometry and X-ray examination. Samples were collected from 64 individuals (45 men, 19 women) stopped at Fiumicino and Ciampino airports of Rome (Italy) for suspicion of internal concealment of cocaine and heroin between October 2006 and July 2007. Urine was immediately screened on-site by Cozart rapid urine test. Irrespective of test results, individuals underwent X-ray examination and urine samples were analyzed by gas chromatography-mass spectrometry (GC-MS). In 48 out of 64 cases (24 positives and 24 negatives) screening results were confirmed by GC-MS assay and X-ray examination. In 5 cases, positive to the on-site test and GC-MS analysis, abdominal radiography was negative and individuals resulted to be drug users. In 11 cases, negative to the on-site test and radiological investigation, GC-MS analysis found benzoylecgonine in 10 cases and morphine in one case. Concentration of both substances was in all cases lower than 50ng/ml and compatible with personal drug use. From obtained results, on-site detection of cocaine and heroin metabolites in the urine of suspected body-packers appears to be a reliable screening test to disclose internally concealed drugs and justify subsequent radiological investigations.

Development and validation of a liquid chromatography-mass spectrometry assay for hair analysis of methylphenidate.

Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of childhood attention-deficit hyperactivity disorder. MPH is biotransformed in the body by the hydrolysis of the methyl ester linkage to its metabolite, ritalinic acid. Whereas both compounds are usually measured in plasma and urine, preliminary observations show that only the parent compound is present in hair from treated individuals. Since in children hair samples can be easily collected without the need for special skills and exposing a patient to discomfort, hair testing of MPH should be an alternative to check compliance in a wider time-window than if using blood. A procedure based on liquid chromatography-mass spectrometry (LC-MS) has been developed for the determination of MPH in hair of treated children. After addition of 3,4-methylenedioxypropylamphetamine as internal standard, hair samples were overnight digested with 0.1M HCl at 37 degrees C. Then, after pH adjustment to 6 using 1N NaOH, and 0.1M phosphate buffer, the analyte was extracted with Bond-Elut Certify columns. Chromatographic separation was achieved at ambient temperature using a reverse phase column and a mobile phase of 80% 10mM ammonium acetate-20% acetonitrile with a 20 min gradient program. The mass spectrometer was operated in positive electrospray ionization and selected ion monitoring acquisition mode. The method was validated in the range 0.15-50 ng MPH/mg hair, using 20mg hair per assay. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 73.2 and 77.1%. First results show MPH hair concentration varying from 0.15 to 4.17 ng/mg hair, with decreasing drug concentration in distal hair segments, even in children treated with the same MPH dose during the period corresponding to different segments. This fact could be either attributed to sebum or sweat shunt with the most proximal hair segment or drug degradation by cosmetic treatments in more distal segments.

Application of a validated high-performance liquid chromatography-mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium.

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) assay, already validated for opiates and cocaine in meconium, has been re-applied for determination of m- and p-hydroxybenzoylecgonine, using nalorphine as the internal standard. Methodology included an initial extraction from the matrix by methanol and then a solid-phase extraction (SPE). A reversed-phase chromatography was used with a gradient of 1% acetic acid-acetonitrile coupled to atmospheric pressure ionization electrospray-mass spectrometry single ion monitoring mode. This method, validated in the range 0.005-1.00 microg analytes/g meconium, proved useful to identify and quantify these two metabolites in meconium samples, already tested for the presence of cocaine, benzoylecgonine and cocaethylene. A positivity of range of concentrations varied between 0.007 and 0.338 microg/g, confirming the importance of these two hydroxylated derivatives to monitor fetal exposure to cocaine.

New vesicular ampicillin-loaded delivery systems for topical application: characterization, in vitro permeation experiments and antimicrobial activity.

In this paper, the experimental conditions for preparing ampicillin-loaded surfactant vesicles (SVs) are described. Our studies are focused on the potential use of a vesicular polymeric dispersion as ampicillin delivery system for topical application. The main components of the formulation are uncharged and charged SVs loaded with ampicillin and dispersed in a gellan solution. The following issues are addressed: the drug encapsulation efficiency (e.e.), the kinetic of drug release from the delivery systems, the antimicrobial activity of vesicle-entrapped ampicillin. The in vitro permeation experiments through a synthetic lipophilic barrier (Silastic) and through porcine skin are carried out to evaluate the potential use as a dermal formulation. The use of both a synthetic and a biological membrane allows to discriminate between the effects related to variations of thermodynamic parameters and those correlated to biological factors. The release rate of ampicillin is increased by encapsulation in neutral and negatively charged SVs and the permeation rate was slowed by dispersion of drug-loaded SVs in gellan solution. Finally, studies of antimicrobial activity on prepared systems evidenced that ampicillin encapsulated in SVs exhibit a higher activity than the free drug.

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.

Assay of gamma-glutamylcysteine synthetase activity in Plasmodium berghei by liquid chromatography with electrochemical detection.

This work describes a high-performance liquid chromatography (HPLC) method to determine gamma-glutamylcysteine (gamma-GC), the intermediate product of glutathione biosynthesis. Separation relies on isocratic reversed-phase chromatography using a Symmetry C18 HPLC column, particle size 5 microm, 4.6 x 250 mm i.d. The mobile phase is methanol-dibasic sodium phosphate (pH 6.6; 2.8 mM) (10:90, v/v) at the flow-rate of 0.5 ml/min and detection is operated electrochemically (+200 and +550 mV) with a pre-column derivatisation reaction using ortho-phthalaldehyde (OPA) as reagent. Under these conditions the calibration range of gamma-GC was 0.3-10 microg/ml; the limit of quantification was 0.3 microg/ml; accuracy, expressed as %Bias, was <10 and precision (%CV) was <6. The proposed HPLC assay was used to quantitate the gamma-glutamylcysteine produced by the gamma-glutamylcysteine synthetase of the rodent malaria parasite Plasmodium berghei in an in vitro enzymatic assay.

Morphological analysis of the interaction of charged surfactant vesicles (SVs) with human cultured cells.

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.