Synthesis of 13C215N2‐labeled anti‐inflammatory and cytoprotective tricyclic bis(cyanoenone) ([13C215N2]‐TBE‐31) as an internal standard for quantification by stable isotope dilution LC‐MS method

Tricyclic bis(cyanoenone), TBE‐31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti‐inflammatory and cytoprotective agent. ¹³C₂¹⁵N₂‐labeled TBE‐31 ([¹³C₂¹⁵N₂]‐TBE‐31), which has two ¹³C and two ¹⁵N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE‐31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope‐labeled internal standard. [¹³C₂¹⁵N₂]‐TBE‐31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two ¹³C atoms with ethyl [¹³C]formate and two ¹⁵N atoms with hydroxyl[¹⁵N]amine. The stable isotope dilution liquid chromatography–mass spectrometry method for quantification of TBE‐31 was successfully developed using [¹³C₂¹⁵N₂]‐TBE‐31 to compensate for any variables encountered during sample processing and analysis.     

The preparation of genistein and LC‐MS/MS on‐line analysis

Genistein, a promising agent for cancer chemoprevention or treatment, can be obtained from soybean seeds, via a complex isolation procedure. The objective of this study was to investigate whether natural and high purity genistein could be acquired from Sophora japonica L. via a simple approach. High‐performance liquid chromatography electrospray ionization combined with tandem mass spectrometry (LC‐ESI‐MS/MS) was used to monitor the whole process of genistein preparation. The results showed there were at least 13 types of flavonoid in the crude extracts from sophora fruits with sophoricoside being the predominant component. The products obtained from our preparation were unambiguously identified as genistein based on molecular ion [M+H]⁺, UV spectra, retention time, IR spectra, ¹HNMR, ¹⁴CNMR spectra, and tandem mass spectrometric analysis. It demonstrated that 99.6% (w/w) genistein could be obtained via our preparation protocol. Drug Dev. Res. 61: 6–12, 2004. © 2004 Wiley‐Liss, Inc.     

Synthesis and purification of [1,2‐13C2]coniferin

The double labelled lignan precursors [1,2‐¹³C₂]coniferin and the glucoside of [1,2‐¹³C₂]ferulic acid were prepared by classical synthetic methods. Pure double labelled lignan precursors could only be obtained after separation from their contaminating Z‐isomers and dihydro by‐products by high‐performance liquid chromatography. Copyright © 2006 John Wiley & Sons, Ltd.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Regioselective syntheses of [13C]4‐labelled sodium 1‐carboxy‐2‐(2‐ethylhexyloxycarbonyl)ethanesulfonate and sodium 2‐carboxy‐1‐(2‐ethylhexyloxycarbonyl)ethanesulfonate from [13C]4‐maleic anhydride

The entitled monohydrolysis products, also known as α‐ethylhexyl and β‐ethylhexyl sulfosuccinate (EHSS), of the surfactant diisooctyl sulfosuccinate (DOSS) were synthesized in stable isotope‐labelled form from [¹³C]₄‐maleic anhydride. Sodium [¹³C]₄‐1‐carboxy‐2‐(2‐ethylhexyloxycarbonyl)ethanesulfonate (α‐EHSS) was prepared by the method of Larpent by reaction of 2‐ethylhexan‐1‐ol with [¹³C]₄‐maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of ¹³C/¹³C (incredible natural abundance double quantum transfer) and ¹H/¹³C (heteronuclear multiple‐bond correlation (HMBC)) NMR spectral correlation experiments. Sodium [¹³C]₄‐2‐carboxy‐1‐(2‐ethylhexyloxycarbonyl)ethanesulfonate (β‐EHSS) was prepared in four steps by reaction of 4‐methoxybenzyl alcohol with [¹³C]₄‐maleic anhydride, regioselective sodium bisulfite addition, N,N′‐dicyclohexylcarbodiimide‐mediated esterification with 2‐ethylhexan‐1‐ol, and p‐methoxybenzyl ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of ¹J_CC scalar coupling constant analysis and ¹H/¹³C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the liquid chromatography–mass spectrometry (LC‐MS)/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. Copyright © 2014 John Wiley & Sons, Ltd.     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of a selection of synthetic cannabinoids and metabolites in urine by UHPSFC‐MS/MS and by UHPLC‐MS/MS

Two different analytical techniques, ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS) and reversed phase ultra‐high performance liquid chromatography‐tandem mass spectrometry (UHPLC‐MS/MS), were used for the determination of two synthetic cannabinoids and eleven metabolites in urine; AM‐2201 N‐4‐OH‐pentyl, AM‐2233, JWH‐018 N‐5‐OH‐pentyl, JWH‐018 N‐pentanoic acid, JWH‐073 N‐4‐OH‐butyl, JWH‐073 N‐butanoic acid, JWH‐122 N‐5‐OH‐pentyl, MAM‐2201, MAM‐2201 N‐4‐OH‐pentyl, RCS‐4 N‐5‐OH‐pentyl, UR‐144 degradant N‐pentanoic acid, UR‐144 N‐4‐OH‐pentyl, and UR‐144 N‐pentanoic acid. Sample preparation included a liquid‐liquid extraction after deconjugation with ß‐glucuronidase. The UHPSFC‐MS/MS method used an Acquity UPC^{2 TM} BEH column with a mobile phase consisting of CO₂ and 0.3% ammonia in methanol, while the UHPLC‐MS/MS method used an Acquity UPLC® BEH C₁₈ column with a mobile phase consisting of 5 mM ammonium formate (pH 10.2) and methanol. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions. Deuterated internal standards were used for six of the compounds. Limits of quantification (LOQs) were between 0.04 and 0.4 µg/L. Between‐day relative standard deviations at concentrations ≥ LOQ were ≤20%, with biases within ±19%. Recoveries ranged from 40 to 90%. Corrected matrix effects were within 100 ± 10%, except for MAM‐2201 with UHPSFC‐MS/MS, and for UR‐144 N‐pentanoic acid and MAM‐2201 N‐4‐OH‐pentyl with UHPLC‐MS/MS. Elution order obtained by UHPSFC‐MS/MS was almost opposite to that obtained by UHPLC‐MS/MS, making this instrument setup an interesting combination for screening and confirmation analyses in forensic cases. The UHPLC‐MS/MS method has, since August 2014, been successfully used for confirmation of synthetic cannabinoids in urine samples revealing a positive immunoassay screening result. Copyright © 2015 John Wiley & Sons, Ltd.     

A gram‐scale synthesis of [3,4‐13C2,1α,7‐2H2]cortisone

A gram‐scale synthesis of [3,4‐¹³C₂,1α,7‐²H₂]cortisone from prednisone was developed. The deuterium atom at the C‐1 position was introduced through a regioselective and stereoselective deuteration of the 1,2‐double bond of the 1,4‐diene‐3‐one using Wilkinson's catalyst. After the oxidative cleavage of the A‐ring, two carbon‐13 atoms were introduced via acetylation of an A‐ring enol lactone with [1,2‐¹³C₂]acetyl chloride. The steroidal A‐ring was then reconstructed to incorporate the carbon‐13 atoms into the C‐3 and C‐4 positions. The deuterium atom at C‐7 was introduced through a regioselective deuteration of the 6,7‐double bond of a 4,6‐diene‐3‐one intermediate using palladium on strontium carbonate. The M + 4 stable isotope labeled cortisone was thus prepared in ca. 4% overall yield. In addition, [3,4‐¹³C₂,1α,7‐²H₂]‐11‐dehydrocorticosterone, [3,4‐¹³C₂,1α,7‐²H₂]cortisol, and [3,4‐¹³C₂,1α,7‐²H₂]corticosterone were also prepared. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of deuterium and C‐13‐labelled ethyl glycolate and their subsequent use in the synthesis of labelled analogues of the DNA adduct O6‐carboxymethyl‐2′‐deoxyguanosine

The adduct O⁶‐carboxymethyl‐2′‐deoxyguanosine (O⁶CMdG) is of importance as it has been previously linked to high red meat diet in humans, and as yet, a liquid chromatography‐mass spectrometry (LC‐MS) method has not been developed due to lack of appropriate standards. The synthesis of the deuterated and C‐13 analogues required the use of [²H₂]‐ and [¹³C₂]ethyl glycolate to label the carboxymethyl moiety of O⁶CMdG. [²H₂]Ethyl glycolate was synthesised via acid hydrolysis of ethyl diazoacetate using deuterated solvents (59% yield), whilst [¹³C₂]ethyl glycolate was synthesised from [¹³C₂]glycine in a three‐step procedure (35% yield). The labelled ethyl glycolates were then used to synthesise [²H₂]‐ and [¹³C₂]O⁶CMdG for future use as internal standards in the LC‐MS analysis of biological samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification and characterization of 2,5‐dimethoxy‐3,4‐dimethyl‐β‐phenethylamine (2C‐G) – A new designer drug

This study presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5‐dimethoxy‐3,4‐dimethyl‐β‐phenethylamine (2C‐G), a new designer drug. A powder sample containing 2C‐G was seized in Poland in 2011. The paper focuses on a comparison of the analytical features of 2C‐G and other members of the 2C‐series, in order to assess the possibility of unequivocal identification. The occurrence of intense peak at m/z = 178 and different intensities of the ions at m/z = 165 and 180 in the gas chromatography‐electron impact‐mass spectrometry (GC‐EI/MS) spectrum of 2C‐G made it possible to distinguish it from 2C‐E. Differences in relative intensities of the ions at m/z = 192, 179 and 177 were observed for GC‐EI/MS spectra of TFAA derivatives of 2C‐G and 2C‐E. An identical set of ions was recorded for these substances using the liquid chromatography‐electrospray ionization/quadrupole time of flight mass spectrometry (LC‐ESI/QTOFMS) method in both MS and tandem mass spectrometry (MS/MS) mode, but the distinction was possible based on differences in the ion intensities at m/z = 193.1223 and 178.0988. The Fourier transform infrared (FTIR) spectrum of 2C‐G was significantly different from other members of the 2C‐series, with a characteristic doublets at 993–1014 cm^‐1 and 1099–1124 cm^‐1, and the ratio of bands at higher wavenumbers. Final elucidation of the structure of 2C‐G was carried out by ¹H and ¹³C NMR spectroscopy. The study indicated that the marketing of analogues of controlled substances poses a real analytical challenge for forensic laboratories, and the application of sophisticated methods is often required for unequivocal identification of a new substance. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of C‐13‐labeled atrazine

Atrazine is a long‐lasting herbicide that has been shown to affect hormone levels in amphibians. Using the C‐13 labeled atrazine to detect its residue is effective and essential. This study presents three steps for the synthesis of [¹³C₃]atrazine, which starts from [¹³C]urea, and results in the incorporation of C‐13 atoms at the 1, 3 and 5 positions of the S‐triazine ring of atrazine. The method prepares the product in an overall yield of 57.6% and chemical purity of 98.6%, for use as an internal standard. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis of 13C‐labelled cutin and suberin monomeric dicarboxylic acids of the general formula HO213C‐(CH2)n‐13CO2H (n = 10, 12, 14, 16, 18, 20, 22, 24, 26, 28)

¹³C‐labeled dicarboxylic acids HO₂¹³C‐(CH₂)_n‐¹³CO₂H (n = 10, 12, 14, 16, 18, 20, 22, 24, 26, 28) have been synthesized as internal standards for LC‐MS and GC‐MS analysis of cutin and suberin monomer degradation by soil‐based microorganisms. Different synthetic strategies had to be applied depending on the chain length of the respective synthetic target and because of economic considerations. ¹³C‐labels were introduced by nucleophilic substitution of a suitable leaving group with labelled potassium cyanide and subsequent hydrolysis of the nitriles to produce the corresponding dicarboxylic acids. All new compounds are characterized by GC/MS, IR, and NMR methods as well as by elemental analysis.     

Synthesis of [13C4]‐labeled 2′‐deoxymugineic acid

The phytosiderophore 2′‐deoxymugineic acid (DMA) is exuded via the root system by all grasses (including important crop plants like rice, wheat and barley) to mobilize Fe(III) from soil and improve plant Fe nutrition, crucial for high crop yields. Elucidation of the biogeochemistry of 2′‐deoxymugineic acid in the rhizosphere requires its quantification in minute amounts. To this end, ¹³C₄‐DMA was synthesized for the first time, from cheap isotopically labeled starting materials. The synthetic route utilizes l ‐allyl(¹³C₂)glycine and l ‐(2‐¹³C)azetidine (¹³C)carboxylic acid as versatile labeled building blocks. The title compound was recently used as an internal standard for analysis of soil and plant samples allowing the first accurate quantification of DMA in these matrices by means of LC‐MS/MS. It is furthermore used in tracer experiments investigating biodegradation of DMA in soil.     

The synthesis of [3,4,1′‐13C3]genistein

A facile synthesis is described for [3,4,1′‐¹³C₃]genistein for use as an internal standard in isoflavone analysis by mass spectrometric methods. Ethyl 4‐hydroxy[1‐¹³C]benzoate was first prepared from the reaction of diethyl [2‐¹³C]malonate and 4H‐pyran‐4‐one. Two further ¹³C atoms were incorporated using potassium [¹³C]cyanide as the source to give 4′‐benzyloxy‐[1,2,1′‐¹³C₃]phenylacetonitrile. [3,4,1′‐¹³C₃]Genistein was then constructed through coupling of the isotopically labelled phenylacetonitrile with phloroglucinol under Hoesch conditions, followed by formylation and cyclization. Copyright © 2007 John Wiley & Sons, Ltd.     

Quantitative determination of fentanyl in newborn pig plasma and cerebrospinal fluid samples by HPLC‐MS/MS

In this study, a selective and sensitive high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method requiring low sample volume (≤100 μL) was developed and validated for the quantitative determination of the opioid drug fentanyl in plasma and cerebrospinal fluid (CSF). A protein precipitation extraction with acetonitrile was used for plasma samples whereas CSF samples were injected directly on the HPLC column. Fentanyl and ¹³C₆‐fentanyl (Internal Standard) were analyzed in an electrospray ionization source in positive mode, with multiple reaction monitoring (MRM) of the transitions m/z 337.0/188.0 and m/z 337.0/105.0 for quantification and confirmation of fentanyl, and m/z 343.0/188.0 for ¹³C₆‐fentanyl. The respective lowest limits of quantification for plasma and CSF were 0.2 and 0.25 ng/mL. Intra‐ and inter‐assay precision and accuracy did not exceed 15%, in accordance with bioanalytical validation guidelines. The described analytical method was proven to be robust and was successfully applied to the determination of fentanyl in plasma and CSF samples from a pharmacokinetic and pharmacodynamic study in newborn piglets receiving intravenous fentanyl (5 µg/kg bolus immediately followed by a 90‐min infusion of 3 µg/kg/h). Copyright © 2015 John Wiley & Sons, Ltd.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Lefetamine‐derived designer drugs N‐ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA): Metabolism and detectability in rat urine using GC‐MS,LC‐MSn and LC‐HR‐MS/MS

N‐Ethyl‐1,2‐diphenylethylamine (NEDPA) and N‐iso‐propyl‐1,2‐diphenylethylamine (NPDPA) are two designer drugs, which were confiscated in Germany in 2008. Lefetamine (N,N‐dimethyl‐1,2‐diphenylethylamine, also named L‐SPA), the pharmaceutical lead of these designer drugs, is a controlled substance in many countries. The aim of the present work was to study the phase I and phase II metabolism of these drugs in rats and to check for their detectability in urine using the authors’ standard urine screening approaches (SUSA). For the elucidation of the metabolism, rat urine samples were worked up with and without enzymatic cleavage, separated and analyzed by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). According to the identified metabolites, the following metabolic pathways for NEDPA and NPDPA could be proposed: N‐dealkylation, mono‐ and bis‐hydroxylation of the benzyl ring followed by methylation of one of the two hydroxy groups, combinations of these steps, hydroxylation of the phenyl ring after N‐dealkylation, glucuronidation and sulfation of all hydroxylated metabolites. Application of a 0.3 mg/kg BW dose of NEDPA or NPDPA, corresponding to a common lefetamine single dose, could be monitored in rat urine using the authors’ GC‐MS and LC‐MSⁿ SUSA. However, only the metabolites could be detected, namely N‐deethyl‐NEDPA, N‐deethyl‐hydroxy‐NEDPA, hydroxy‐NEDPA, and hydroxy‐methoxy‐NEDPA or N‐de‐iso‐propyl‐NPDPA, N‐de‐iso‐propyl‐hydroxy‐NPDPA, and hydroxy‐NPDPA. Assuming similar kinetics, an intake of these drugs should also be detectable in human urine. Copyright © 2014 John Wiley & Sons, Ltd.     

UHPLC–MS/MS quantitation of porphyroxine in opium and application of porphyroxine‐acetylated products as signature markers for heroin

Porphyroxine, a trace alkaloid in opium, was identified in the early 1800s and isolated/characterized in the 1960s. Recently, two significant porphyroxine‐related byproducts found in the acidic and neutral extracts of illicit heroin were characterized by this laboratory as the N‐acetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( B ) and N,O⁸‐diacetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( C ). The prevalence of the B and C compounds has been consistent in the following order of abundance for the thousands of authentic heroin samples analyzed: Southwest Asia (SWA) > South America (SA) > Southeast Asia (SEA) > Mexico (MEX). In this research, a rapid and efficient ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the content of porphyroxine and five primary alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) in opium after extraction with methanol/water (50/50). The method was validated in terms of linearity, accuracy, recovery, and precision for porphyroxine. The limit of quantitation (LOQ) for porphyroxine was 2.5 ng/mL. The developed method was successfully applied to a total of 114 authentic opium samples from the major poppy‐growing regions. The amount of porphyroxine was determined at the level of part per thousand (‰) and the relative concentrations to morphine were in the range of 1x10^?4 and 1x10^?2 with an order of SWA > SEA, SA > MEX for its average abundance, which is consistent with the order of the average abundance of its acetylated products ( B , C ) in illicit heroin. This study reveals the significance of porphyroxine and its acylated compounds in classifying heroin and opium samples to major geographical regions of production.     

Syntheses of AZ12320927 labeled with H‐3, C‐14, and H‐2

In support of a program to develop a treatment for depression, three isotopically labeled forms of the 5‐HT_1B antagonist AZ12320927 were synthesized. A tritium labeled version was synthesized for autoradiography using Ir‐catalyzed hydrogen–tritium exchange. A C‐14 labeled version was prepared for use in metabolism studies in four‐steps from [u‐¹⁴C]p‐nitrophenol. A stable isotope labeled version was synthesized for use as an internal standard for LC/MS/MS quantitation in two steps from chromenone 1. Copyright © 2008 John Wiley & Sons, Ltd.     

Production and application of high quality stable isotope‐labeled human immunoglobulin G1 for mass spectrometry analysis

Here, we describe the production of stable isotope‐labeled human immunoglobulin G1 ([¹³C]‐hIgG1) using [¹³C]‐L‐lysine/arginine–labeled hIgG1. The fermentation process was run in shake flasks containing labeled arginine and lysinethat were incorporated into the produced recombinant hIgG1. The [¹³C]‐hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [¹³C]‐hIgG1 was then used as an internal standard (IS) for the development of a liquid chromatography–tandem mass spectrometry method applicable to the quantitative analysis of all human types of hIgG1 in rat serum. Four conserved peptides, namely, GPSVFPLAPSSK, TTPPVLDSDGSFFLYSK, VVSVLTVLHQDWLNGK, and FNWYVDGVEVHNAK, originating from different parts of the fraction crystallizable region of hIgG1, were used for quantitation of hIgG1 in rat serum. The calibration curves with a coefficient of determination (r²) between 0.9950 and 0.9962 resulting from the peak area ratio of each peptide to its respective labeled IS were reproducible. A mean bias within ±20.0% of the nominal values and a precision of ≤20.0 % were obtained for the calibration standards and quality control samples for each peptide. [¹³C]‐hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC‐MS/MS.