The classical stimulants amphetamine, methamphetamine, ethylamphetamine and the amphetamine-derived designer drugs MDA, MDMA ('ecstasy'), MDEA, BDB and MBDB have been widely abused for a relatively long time. In recent years, a number of newer designer drugs have entered the illicit drug market. 4-Methylthioamphetamine (MTA), p-methoxyamphetamine (PMA) and p-methoxymethamphetamine (PMMA) are also derived from amphetamine. Other designer drugs are derived from piperazine, such as benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP), trifluoromethylphenylpiperazine (TFMPP), m-chlorophenylpiperazine (mCPP) and p-methoxyphenylpiperazine (MeOPP). A number of severe or even fatal intoxications involving these newer substances, especially PMA, have been reported. This paper describes a method for screening for and simultaneous quantification of the above-mentioned compounds and the metabolites p-hydroxyamphetamine and p-hydroxymethamphetamine (pholedrine) in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. It was linear from 5 to 1000 micro g l(-1) for all analytes. Data for accuracy and precision were within required limits with the exception of those for MDBP. The limit of quantification was 5 micro g l(-1) for all analytes. The applicability of the assay was proven by analysis of authentic plasma samples and of a certified reference sample. This procedure should also be suitable for confirmation of immunoassay results positive for amphetamines and/or designer drugs of the ecstasy type.

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The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

A qualitative and quantitative analysis of erlotinib (RO0508231) and its metabolites was carried out on rat tissue sections from liver, spleen and muscle. Following oral administration at a dose of 5 mg/kg, samples were analyzed by matrix-assisted laser desorption ionization (MALDI) with mass spectrometry (MS) using an orthogonal quadrupole time-of-flight instrument. The parent compound was detected in all tissues analyzed. The metabolites following drug O-dealkylation could also be detected in liver sections. Sinapinic acid (SA) matrix combined with the dried-droplet method resulted in better conditions for our analysis on tissues. Drug quantitation was investigated by the standard addition method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the tissue extracts. The presence of the parent compound and of its O-demethylated metabolites was confirmed in all tissue types and their absolute amounts calculated. In liver the intact drug was found to be 3.76 ng/mg tissue, while in spleen and muscle 6- and 30-fold lower values, respectively, were estimated. These results were compared with drug quantitation obtained by whole-body autoradiography, which was found to be similar. The potential for direct quantitation on tissue sections in the presence of an internal standard was also investigated using MALDI-MS. The use of alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix resulted in better linearity for the calibration curves obtained with reference solutions of the drug when compared to SA, but on tissue samples no reliable quantitative analysis was possible owing to the large variability in the signal response. MS imaging experiments using MALDI in MS/MS mode allowed visualizing the distribution of the parent compound in liver and spleen tissues. By calculating the ratio between the total ion intensities of MS images for liver and spleen sections, a value of 6 : 1 was found, which is in good agreement with the quantitative data obtained by LC-MS/MS analysis.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Studies are described on the metabolism and toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that 2C-T-7 was metabolized by hydroxylation of the propyl side chain followed by N-acetylation and sulfoxidation and also by deamination followed by oxidation to the corresponding acid or by reduction to the corresponding alcohol. To a minor extent, 2C-T-7 was also metabolized by S-dealkylation followed by N-acetylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-7 in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-7 in human urine.

Designer drugs of the amphetamine type (eg, MDMA, MDEA, MDA), of the new benzyl or phenyl piperazine type (eg, BZP, MDBP, mCPP, TFMPP, MeOPP), or of the pyrrolidinophenone type (eg, PPP, MOPPP, MDPPP, MPPP, MPHP) have gained popularity and notoriety as rave drugs. These drugs produce feelings of euphoria and energy and a desire to socialize. Although in the corresponding drug scene designer drugs have the reputation of being safe, studies in rats and primates in combination with human epidemiologic investigations indicate potential risks to humans. Thus, a variety of adverse effects have been associated with the use/abuse of this class of drugs in humans, including a life-threatening serotonin syndrome, hepatotoxicity, neurotoxicity, and psychopathology. Metabolites were suspected to contribute to some of the toxic effects. Therefore, knowledge of the metabolism is a prerequisite for toxicologic risk assessment. The metabolic pathways, the involvement of cytochrome P450 isoenzymes in the main pathways, and their roles in hepatic clearance are described for designer drugs of different groups. In summary, polymorphically expressed CYP2D6 was the major enzyme catalyzing the major metabolic steps of the studied piperazine- and pyrrolidinophenone-derived designer drugs. However, it cannot be concluded at the moment whether this genetic polymorphism is of clinical relevance.

Abuse of designer drugs is widespread among young people, especially in the so-called "dance club scene" or "rave scene", worldwide. Severe and even fatal poisonings have been attributed to the consumption of such drugs of abuse. However, in contrast to new medicaments, which are extensively studied in controlled clinical studies concerning metabolism, including cytochrome P450 isoenzyme differentiation, and further pharmacokinetics, designer drugs are consumed without any safety testing. This paper reviews the metabolism of new designer drugs of abuse that have emerged on the black market during the last years. Para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA) and 4-methylthioamphetamine (4-MTA), were taken into consideration as new "classical" amphetamine-derived designer drugs. Furthermore, N-benzylpiperazine (BZP), 1-(3, 4-methylenedioxybenzyl)piperazine (MDBP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-methoxyphenyl)piperazine (MeOPP) were taken into consideration as derivatives of the class of piperazine-derived designer drugs, as well as alpha-pyr-rolidinopropiophenone (PPP), 4'-methoxy-alpha-pyrrolidinopropiophenone (MOPPP), 3', 4'-methylenedioxy-alpha-pyrrolidino-propiophenone (MDPPP), 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP), and 4'-methyl-alpha-pyrrolidinoexanophenone (MPHP) as derivatives of the class of alpha-pyrrolidinophenone-derived designer drugs. Papers describing identification of in vivo or in vitro human or animal metabolites and cytochrome P450 isoenzyme dependent metabolism have been considered and summarized.