Torben Breindahl

Identification and characterization by LC-UV-MS/MS of melanotan II skin-tanning products sold illegally on the Internet.

New methods were developed and validated to determine the identity, contents, and purity of samples of melanotan II, a synthetic melanocortin receptor agonist, sold in vials as injectable skin-tanning products that were purchased from three online shops. Methods were based on liquid chromatography with ultra-violet detection (LC-UV) at wavelength 218 nm, and tandem mass spectrometric detection (MS/MS) after collision-induced fragmentation of the double charged [M+2H](2+) precursor ion (m/z 513). Identification of melanotan II was verified by correct chromatographic retention time, and relative abundance ratios of five qualifying fragment ions. LC-UV was used to quantify melanotan II as well as impurities. Method validation was performed with reference to guidelines for assessing active substances in authorized medicinal products to reach acceptable accuracy and precision. Vials from two shops contained unknown impurities ranging from 4.1 to 5.9%; impurities from one shop were below the quantification limit. The total amount of melanotan II in vials ranged between 4.32 and 8.84 mg, although each shop claimed that vials contained 10 mg melanotan II. A broad range of drugs used for enhancement purposes can be obtained from the illicit market. However, users of these drugs may be exposed to a range of potential harms, as shown in this study, given that these products are manufactured, distributed and supplied from an illicit market.

Identification of a New Metabolite of GHB: Gamma-Hydroxybutyric Acid Glucuronide

Gamma-hydroxybutyric acid (GHB) is an important analyte in clinical and forensic toxicology with a narrow detection window of 3-6 h. In the search of improved detection methods, the existence in vivo of a glucuronated GHB metabolite (GHB-GLUC) was hypothesized. Chemically pure standards of GHB-GLUC and a deuterated analogue for chromatography were synthesized. Liquid chromatography and tandem mass spectrometry were used for targeted analysis in anonymous clinical urine samples (n = 50). GHB-GLUC was found in concentrations ranging from 0.11 to 5.0 µg/mL (mean: 1.3 ± 1.2 µg/mL). Thus far, this is the first report of a GHB glucuronide detected in biological samples. Given that glucuronides generally have longer half-life values than their corresponding free drugs, GHB-GLUC should theoretically be a biomarker of GHB intoxication. It is also proposed that the hitherto unexplained reports of elevated GHB concentrations in some biological samples, which has caused the setting of a relatively high cutoff value (10 µg/mL), represent total GHB measurements (sum of free GHB and actively chemically hydrolyzed GHB-GLUC). To address these challenges, the present study must be followed by comprehensive pharmacokinetic and stability studies after the controlled administration of GHB.

Identification of a new psychoactive substance in seized material: the synthetic opioid N‐phenyl‐N‐[1‐(2‐phenethyl)piperidin‐4‐yl]prop‐2‐enamide (Acrylfentanyl)

Among the new psychoactive substances (NPS) that have recently emerged on the market, many of the new synthetic opioids have shown to be particularly harmful. A new synthetic analogue of fentanyl, N‐phenyl‐N‐[1‐(2‐phenethyl)piperidin‐4‐yl]prop‐2‐enamide (acrylfentanyl), was identified in powder from a seized capsule found at a forensic psychiatric ward in Denmark. Gas chromatography with mass spectrometry (GC‐MS) identified a precursor to synthetic fentanyls, N‐phenyl‐1‐(2‐phenylethyl)piperidin‐4‐amine; however, the precursor 1‐(2‐phenethyl)piperidin‐4‐one, was not detected. Analysis of the electron impact mass spectrum of the main, unknown chromatographic peak (GC) tentatively identified an acryloyl analogue of fentanyl. Further analyses by quadrupole time‐of‐flight high resolution mass spectrometry (QTOF‐MS), matrix‐assisted laser ionization Orbitrap mass spectrometry (MALDI‐Orbitrap‐MS), nuclear magnetic resonance spectroscopy (NMR), and infra‐red spectroscopy (IR) confirmed the presence of acrylfentanyl (also known as acryloylfentanyl). Quantitative analysis with liquid chromatography and triple quadrupole mass spectrometry (LC‐MS/MS) determined the content of acrylfentanyl in the powder, equal to 88.3 mass‐% acrylfentanyl hydrochloride. An impurity observed by NMR was identified as triethylamine hydrochloride. Acrylfentanyl is sold on the Internet as a ‘research chemical’. Like other synthetic fentanyls, such as acetylfentanyl, it poses a serious risk of fatal intoxication. Copyright

Online marketing of synthetic peptide hormones: poor manufacturing, user safety, and challenges to public health.

An increasing range of drugs that are being used to enhance the body have recently become available on the illicit market. Many of these products contain pharmacologically active substances that are untested or banned (after being licensed as a medicinal product) and they are widely subject to adulteration and misbranding. While the existence of illicitly manufactured drugs containing anabolic steroids as the active substance has been known for decades, new generation drugs for human enhancement include fibroblast growth factor 1, mechano growth factor analogues, and His-tagged Long-R3-IGF-I. Forensic analysis of drugs seized by Norwegian law enforcement revealed the presence of the synthetic growth hormone secretagogue CJC-1295, whereas analysis of drugs seized in Italy identified the synthetic growth-hormone-releasing peptide GHRP-2 as the active substance. In addition, GHRP-2 has also been found in a product sold as a ‘nutritional supplement’. Notable amongst novel drugs on the illicit market are the synthetic peptide hormones GHRP-6, taken to stimulate the secretion of natural growth hormone, as well as melanotan II, a synthetic analogue of the endogenous α-melanocyte stimulating hormone (α-MSH) which is predominantly used to achieve a skin tan. Both of these drugs have wide appeal for those who want to enhance their physical performance and improve body satisfaction. Synthetic peptide hormones like GHRP-6 and melanotan II can supposedly be bought from traditional ‘street dealers’ or specific venues associated with a particular lifestyle or behaviour; for example, muscular enhancement drugs (GHRP-6) from a gym or skin darkening drugs (melanotan II) from a tanning salon. These drugs also appear to be traded from unauthorized shops on the Internet offering at least some anonymity to consumers as well as protecting illicit suppliers from regulatory agencies. This is one of the barriers to obtaining a clear picture of what specific types of enhancement drugs are available online, along with their composition and quality.

Therapeutic drug monitoring of flecainide in serum using high-performance liquid chromatography and electrospray mass spectrometry.

High-performance liquid chromatography with electrospray mass spectrometry (LC-MS) was used for analysis of the drug flecainide in serum. The clean-up was performed by solid-phase extraction, and an aromatic ring positional isomer was used as internal standard. Results from method validation on spiked serum samples showed excellent reproducibility; intra- and inter-assay variations (C.V.% and %Bias) were less than 6% within the therapeutic concentration range of the drug (0.2-1.0 microg/ml). Linearity was demonstrated from 0.05 to 2.0 microg/ml. The limit of detection and quantification was 0.025 and 0.05 microg/ml, respectively. Due to the high selectivity of the mass spectrometric detection, no interferences were observed. Results from clinical samples (n=18) from patients in treatment with Tambocor (flecainide acetate) showed excellent correlation with parallel data obtained from a method based on high-performance liquid chromatography (HPLC) with fluorescence detection after liquid/liquid extraction. The chromatographic separation of flecainide and internal standard was improved compared to earlier HPLC methods. The methodology is simple, accurate and requires only 0.25 ml of sample. It is a well suited method for routine therapeutic drug monitoring in a hospital or clinical chemistry laboratory.

Synthesis and stability study of a new major metabolite of γ-hydroxybutyric acid

Summary γ-Hydroxybutanoic acid (GHB) is used as a date-rape drug, which renders the victims unconscious and defenceless. Intoxications are very difficult to detect for forensic scientists due to rapid metabolism to endogenous levels of GHB. We recently discovered a new major metabolite, 2, of GHB (1) that could potentially extend the analytical detection window for GHB intoxications. Herein we disclose synthetic procedures based on a Koenigs–Knorr glucuronidation approach that provides GHB glucuronide 2 and a deuterium-labelled analogue d 4-2 of high purity suitable for analytical chemistry. In addition, we have assessed the stability of GHB glucuronide 2 by mimicking the natural pH range for urine, which is of importance in the development of new analytical methods. Using NMR we show that GHB glucuronide 2 is highly stable towards aqueous hydrolysis within the pH range normally observed for urine even at elevated temperature.

Synthetic growth hormone releasers detected in seized drugs: new trends in the use of drugs for performance enhancement.

Keywords: Doping; drug monitoring; growth hormone releasers; high-performance tandem mass spectrometry; performance-enhancing drugs; synthetic peptide hormones; the illicit market

Validation of urine drug-of-abuse testing methods for ketobemidone using thin-layer chromatography and liquid chromatography-electrospray mass spectrometry.

High-performance thin-layer chromatography (TLC) with visual detection (post-chromatographic derivatization) was used in screening for the drug ketobemidone in human urine samples. High-performance liquid chromatography with electrospray mass spectrometry (LC-ESI-MS) was used for final confirmation of the result. The clean-up was performed by mixed-mode solid-phase extraction, and nalorphine was used as internal standard. A screening cut-off for TLC was established at 0.2 microg/ml. The mean recovery for LC-MS was 91% (n=60) with coefficients of variation (C.V.) in the range of 7 to 16%. Qualifying fragment ions of ketobemidone (m/z 190, 201 and 230) were generated by up front collision-induced dissociation (CID) on a single quadrupole instrument. Relative ion intensities were within +/- 15% deviation compared with standards in the same batch. The limit of detection for LC-MS was 0.025 microg/ml. Positive clinical samples from drug abusers (n=10) had concentrations in the range 0.07 to 3.2 microg/ml, which could be determined by LC-MS without matrix interference. During screening of unknown clinical samples (n=27) the results from TLC was in agreement with LC-MS data. After acid hydrolysis of conjugates in clinical samples the analyte response of ketobemidone and norketobemidone was increased by a factor of approximately two and twelve, respectively. A qualitative GC-MS technique was demonstrated for the detection of the spasmolyticum A29 (N,N-dimethyl-4,4-diphenyl-3-buten-2-amine), which can be found in a preparation combined with ketobemidone (Ketogan).

NT-proBNP on Cobas h 232 in point-of-care testing: Performance in the primary health care versus in the hospital laboratory

BACKGROUND NT-proBNP may be useful for ruling out heart failure in primary health care. In this study we examined the analytical quality of NT-proBNP in primary health care on the Cobas h 232 point-of-care instrument compared with measurements performed in a hospital laboratory. MATERIALS AND METHODS Blood samples requested for NT-proBNP were collected in primary health care (n = 95) and in a hospital laboratory (n = 107). NT-proBNP was measured on-site on Cobas h 232 instruments both in primary health care centres and at the hospital laboratory and all samples were also analyzed with a comparison method at the hospital. Precision, trueness, accuracy, and lot-variation were determined at different concentration levels and evaluated according to acceptance criteria. Furthermore user-friendliness was assessed by questionnaires. RESULTS For Cobas h 232 repeatability CV was 8.5-10.7% in the hospital setting and 5.3-10.0% in the primary health care and within the analytical quality specifications, but higher than with the comparison method (< 4%). NT-proBNP results obtained in primary health care were significantly higher than by the hospital comparison method (bias ranged from 14.3-23.7%), whereas there was no significant bias when Cobas h 232 was used in the hospital setting (bias ranged from - 4.9 to 7.0%). User-friendliness of Cobas h 232 was overall acceptable. CONCLUSION Cobas h 232 point-of-care instrument for measurement of NT-proBNP performed satisfactorily with regard to precision, user-friendliness, and lot-variation. A decrease in NT-proBNP levels observed in samples transported to a central laboratory needs further attention and investigation.Abstract Background. NT-proBNP may be useful for ruling out heart failure in primary health care. In this study we examined the analytical quality of NT-proBNP in primary health care on the Cobas h 232 point-of-care instrument compared with measurements performed in a hospital laboratory. Materials and methods. Blood samples requested for NT-proBNP were collected in primary health care (n = 95) and in a hospital laboratory (n = 107). NT-proBNP was measured on-site on Cobas h 232 instruments both in primary health care centres and at the hospital laboratory and all samples were also analyzed with a comparison method at the hospital. Precision, trueness, accuracy, and lot-variation were determined at different concentration levels and evaluated according to acceptance criteria. Furthermore user-friendliness was assessed by questionnaires. Results. For Cobas h 232 repeatability CV was 8.5–10.7% in the hospital setting and 5.3–10.0% in the primary health care and within the analytical quality specifications, but higher than with the comparison method (< 4%). NT-proBNP results obtained in primary health care were significantly higher than by the hospital comparison method (bias ranged from 14.3–23.7%), whereas there was no significant bias when Cobas h 232 was used in the hospital setting (bias ranged from − 4.9 to 7.0%). User-friendliness of Cobas h 232 was overall acceptable. Conclusion. Cobas h 232 point-of-care instrument for measurement of NT-proBNP performed satisfactorily with regard to precision, user-friendliness, and lot-variation. A decrease in NT-proBNP levels observed in samples transported to a central laboratory needs further attention and investigation.

The composition of anabolic steroids from the illicit market is largely unknown: implications for clinical case reports

As Ammatuna and Nijziel note,1 obtaining an accurate and comprehensive drug history is crucial in the treatment of patients presenting with symptoms that can potentially be attributed to the use of anabolic steroids, including renal disorders. Most people who use anabolic steroids obtain these drugs from the illicit market.2 Here, many products are manufactured in ‘underground laboratories’ operating outside the formal parameters of the production of licensed medicinal products.3 In the UK, investigations by the Medicines and Healthcare …