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3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) tablets are widely used recreationally, and not only vary in appearance, but also in MDMA content. Recently, the prevalence of high-content tablets is of concern to public health authorities. To compare UK data with other countries, we evaluated MDMA content of 412 tablets collected from the UK, 2001-2018, and investigated within-batch content variability for a sub-set of these samples. In addition, we investigated dissolution profiles of tablets using pharmaceutical industry-standard dissolution experiments on 247 tablets. All analyses were carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data supported other studies, in that recent samples (2016-2018) tend to have higher MDMA content compared to earlier years. In 2018, the median MDMA content exceeded 100 mg free-base for the first time. Dramatic within-batch content variability (up to 136 mg difference) was also demonstrated. Statistical evaluation of dissolution profiles at 15-minutes allowed tablets to be categorized as fast-, intermediate-, or slow-releasing, but no tablet characteristics correlated with dissolution classification. Hence, there would be no way of users knowing a priori whether a tablet is more likely to be fast or slow-releasing. Further, within-batch variation in dissolution rate was observed. Rapid assessment of MDMA content alone provides important data for harm reduction, but does not account for variability in (a) the remainder of tablets in a batch, or (b) MDMA dissolution profiles. Clinical manifestations of MDMA toxicity, especially for high-content, slow-releasing tablets, may be delayed or prolonged, and there is a significant risk of users re-dosing if absorption is delayed.

Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent-protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-s-d-maltoside (sDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D2O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2-10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 A. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by X-ray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D2O provide information about the size and shape of the whole detergent-dPSIIcc. The maximum position of P(r) function increases to 68 A, i.e., it is about 6 A larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the sDM-dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 A. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II-detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the sDM-dPSIIcc.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 x 2.1 mm, 3 mu m), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 mu g/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. the plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP., Medical Research CouncilMedical Research Council UK (MRC) [MR/K025406/1]; Orphan Technologies, Ltd. [2016/22506-1]; Sao Paulo Research Foundation (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), This work was supported by the Medical Research Council [grant number MR/K025406/1] in partnership with Orphan Technologies, Ltd. N.J.A was in receipt of scholarship Grant n. 2016/22506-1, Sao Paulo Research Foundation (FAPESP).

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225 A and a diameter of approximately 100 A. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300 A (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28 A only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225 A can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28 A. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5 K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

This research focuses on retention mechanisms in a LC column with C18 stationary phase when novel eluent additives (HFIP, HFTB and TFE as well as NFTB and perfluoropinacol) are used. The retention factors between novel eluent additives and conventional ones like ammonium acetate and ammonium bicarbonate at different eluent pH values were compared. A simple set of drug-like molecules, widely spread over different logP values, containing protonated and deprotonated acids and bases was selected for this investigation. HFIP, HFTB, NFTB and PP demonstrated strong influence on basic polar analytes in basic medium. These additives drastically increased retention. A decrease in retention was observed for acidic analytes when novel eluent additives were used. Additionally, for the first time, the absolute pH (pHabs) scale was used for expressing the mobile phase pH.

Traditionally, bioanalytical laboratories do not report actual concentrations for samples with results below the LOQ (BLQ) in pharmacokinetic studies. BLQ values are outside the method calibration range established during validation and no data are available to support the reliability of these values. However, ignoring BLQ data can contribute to bias and imprecision in model-based pharmacokinetic analyses. From this perspective, routine use of BLQ data would be advantageous. We would like to initiate an interdisciplinary debate on this important topic by summarizing the current concepts and use of BLQ data by regulators, pharmacometricians and bioanalysts. Through introducing the limit of detection and evaluating its variability, BLQ data could be released and utilized appropriately for pharmacokinetic research.

Sample preparation for the analysis of clinical samples with the mass spectrometer (MS) can be extensive and expensive. Simplifying and speeding up the process would be very beneficial. This paper reports sponge spray—a novel sampling and direct MS analysis approach—attempting exactly that. It enables direct analysis without any sample preparation from dried blood, plasma, and urine. The tip of a volumetric absorptive microsampling device is used to collect an exact amount of sample and from that same tip an electrospray can be directed into a mass spectrometer. We demonstrate here that, although with significant matrix effects, quantitation of penicillin G, a common antimicrobial, is possible in plasma and in urine, with essentially no sample preparation.

The simultaneous quantification of protein concentrations via proteotypic peptides in human blood by liquid chromatography coupled to quadrupole MS/MS is an important field of bioanalytical research with a high potential for routine diagnostic applications. This review summarizes currently available sample preparation procedures and trends for absolute protein quantification in blood using LC-MS/MS. It discusses approaches of transferring established qualitative protocols to a quantitative analysis regarding their reliability and reproducibility. Techniques used to enhance method sensitivity such as the depletion of high-abundant proteins or the immunoaffinity enrichment of proteins and peptides are described. Furthermore, workflows for (i) protein denaturation, (ii) disulfide bridge reduction and (iii) thiol alkylation as well as (iv) enzymatic digestion for absolute protein quantification are presented. The main focus is on the tryptic digestion as a bottleneck of protein quantification via proteotypic peptides. Conclusively, requirements for a high-throughput application are discussed.

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on-line SPE and fast LC-MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within-run and between-day imprecisions below 10 and 15% (n = 10) and high recovery rates (94-131%) were obtained applying the high-throughput setup. High-quality porcine trypsin was used, which outperformed cost-effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC-MS/MS assay demonstrated good correlation (Pearson's R: 0.81-0.98). Further, requirements on sample quality concerning sampling, processing, and long-term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE-Adult-Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid-lowering medication. In conclusion, this novel highly standardized, high-throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.

Background A rapid liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. Method In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10 mg atherosclerotic plaques an additional liquid–liquid extraction was included. Free 7-keto-, 7-α/s-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3s,5α,6s-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. Results The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n = 5). Conclusion This rapid LC–MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.

In this paper a conductance method is presented for determining the concentration of salts in steam-plant water supplies. The method is applied to determine the purity of steam, of supplies used for boiler-feed purposes, and the concentration of boiler and evaporator water. The conductance data are refined by applying both a water and a CO2 correction. The method for establishing the CO2 correction is based on the preparation of a standard “synthetic” water, and eliminates the errors accompanying the chemical analysis of “pure” water. The conductance meter used may be calibrated in terms of dissolved salts. The relation of dissolved salts to conductance is discussed. Simple commercial equipment is used for measuring conductance. The data are obtained rapidly by the steam-plant personnel, and the results may be applied easily to the control of water conditions.