Structural elucidation of tramadol, its derivatives, and metabolites using chemical derivatization and liquid chromatography-high-resolution tandem mass spectrometry.

RATIONALE: Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information-limited mass spectra with one prominent peak representing the amine-containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI-MS) and ESI-MS/MS spectra could be misleading. METHODS: A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N-desmethyltramadol and O-desmethyltramadol) was developed using chemical modification and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) with Orbitrap technology. RESULTS: After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N-desmethyltramadol metabolite in urine samples at low ng/mL levels. CONCLUSIONS: An efficient derivatization-aided strategy was developed for rapidly elucidating the structure of tramadol-like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories.

Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.

RATIONALE: Nitrogen mustards (NMs) are blistering chemical warfare agents. The ability to detect NMs in environmental samples is very important for obtaining forensic evidence. The most common analytical techniques for NM detection are gas chromatography-mass spectrometry, which detects NMs in their intact form but is disadvantaged by high limits of detection (LODs), and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) of their hydrolysis products, which do not provide robust evidence to support NM use. METHODS: We developed a novel approach to detect and identify NMs using LC/ESI-MS/MS after chemical derivatization. The method is based on ethoxide-promoted ethanolysis prior to analysis. The effects of reaction time, temperature, ethoxide concentration and chromatography behavior were studied and optimized. In the developed procedure, 0.1% (v/v) sodium ethoxide solution is added to the NMs in ethanol and agitated for 2 h at 50°C, followed by LC/ESI-MS/MS, without any other pretreatment. RESULTS: The ethanolysis reaction efficiencies were evaluated in ethanolic extracts from soil, asphalt, and ethanol contaminated with 0.5% (v/v) diesel fortified with NMs at a five-point calibration curve. The calibration curves showed good linearity in the range of 0.05-1 ng/mL, with an R2 value of 0.99, and were similar to those of LC/MS-grade ethanol, with almost no observable matrix effects. The derivatization products were stable at room temperature, with LODs of 10 pg/mL, in all investigated extracts. CONCLUSIONS: Through this newly developed strategy, the derivatization of active NMs by ethanolysis was achieved for the first time, and these derivatization products can serve as specific indicators for the use and presence of NMs. The methodology can also verify trace levels of NM chemical warfare agents collected in war or terror scenarios in forensic investigations.

Rapid and simple identification of trace amounts of sodium azide in beverages and bodily fluids followed by derivatization and liquid chromatography-electrospray ionization tandem mass spectrometry.

RATIONALE: Sodium azide (NaN3 ) is a toxic chemical agent to humans by ingestion and inhalation with a growing number of intentional exposures and accidental cases over the last few decades. Due to its low molecular weight and lack of any chromophore, its retention and detection by reverse-phase liquid chromatography-ultraviolet-mass spectrometry methods are a challenging task. METHODS: To be able to confirm azide exposure, we have developed a method to identify azide in both beverages and bodily fluids. The identification of azide (N3 - ) is based on derivatization with N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) at 25°C for 15 min followed by LC/ESI-MS/MS analysis, with no other sample preparation. RESULTS: The azide after derivatization (CAX-N3 ) was stable, retainable by LC and sensitively detected by selected reaction monitoring. The ESI-MS/MS fragmentation of the M+ precursor ion produced characteristic product ions at m/z 118, 100, 91 and 86. The calibration curves for CAX-N3 showed linearity over two orders of magnitude with R2 value of 0.99. Low limits of identification of 0.1-0.5 ng/mL were obtained in all investigated matrices (drinking water, tea, orange juice, plasma and urine). CONCLUSIONS: Compared with previously reported chromatography-based methods, this method that was based on derivatization and LC/ESI-MS/MS analysis was substantially more sensitive, simpler and faster. The method can be used for forensic investigation to confirm azide exposure from fatal use to much smaller intoxication dose.

Extended retrospective detection of regenerated sarin (GB) in rabbit blood and the IMPA metabolite in urine: a pharmacokinetics study.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70 days for 0.1 LD50 and 0.5 LD50 and 77 days for 0.9 LD50, where rapid elimination occurred in the first 8 days with an initial average half-life of 1.2 days, followed by a second, slower elimination, with a terminal average half-life of 8.4 days. The time frame for monitoring IMPA in urine was 7, 15 and 16 days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8 days on days 2-6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4 days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

Retrospective determination of regenerated nerve agent sarin in human blood by liquid chromatography-mass spectrometry and in vivo implementation in rabbit.

The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography-mass spectrometry (LC-MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC-ESI-MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5-1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC-MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC-MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC-MS/MS trace analysis of regenerated GB from biological matrices.

Highly sensitive retrospective determination of organophosphorous nerve agent biomarkers in human urine implemented in vivo in rabbit.

Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO2 cartridge was accomplished with a 1% ammonium hydroxide (NH4OH) solution and was followed by analysis via liquid chromatography-mass spectrometry (LC-MS). The limits of quantitation (LOQs) were in the range of 10-100 pg/mL with recoveries of 64-71% (± 5-19%) after fast sample preparation and a total LC-MS analysis cycle time of 15 min and 13 min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD50 (7.5 µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15 days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC-MS/MS analysis.

Structural elucidation of phenidate analogues via the ESI-MS/MS spectra of their sodium adduct ions.

The identification of phenidate new psychoactive substances (NPS) by implementing MS (Mass spectrometry) techniques is a challenging task. Phenidate analogues present information-poor mass spectra, both in GC-EI-MS and LC-ESI-MS/MS of the protonated molecules [M+H]+, with a high abundance fragment/product ion representing the secondary amine-containing residue. This lack of EI-MS and ESI-MS/MS information is attributed to the strong tendency of the amine residue to stabilize the positive charge and leads to unavoidable ambiguity in the identification process. Moreover, thermal decomposition of these compounds occurs in the injection port and/or on the column under standard GC conditions. Herein, we demonstrate how structural information can be attained instantaneously through the LC-ESI-MS/MS fragmentation of the accompanied sodium adducts [M+Na]+. The sodium cation alters the charge distribution during ESI-MS/MS fragmentation, generating a major product ion corresponding to the Na+ adduction of the carbonyl group, providing new structural information of the main core of phenidate derivatives (alkylaryl acetate/acetic acid), enabling their reliable structural elucidation. This quick, simple and easy technique can be implemented to confirm the identity or identify various structurally related phenidate analogues in forensic toxicology and doping analysis without the need for sample handling.

Oxidation-assisted structural elucidation of compounds containing a tertiary amine side chain using liquid chromatography mass spectrometry.

A novel analytical technique for the structural elucidation of compounds bearing a tertiary amine side chain via "in vial" instantaneous oxidation and liquid chromatography mass spectrometry (LC-MS) was developed. A series of lidocaine homologs and benzimidazole derivatives with a major/single amine representative base peak in both their EI-MS and ESI-MS/MS spectra were subjected to oxidation by a 0.1% solution of hydrogen peroxide (including several 16 O/18 O exchange experiments), followed by LC-ESI-MS/MS analysis. The N-oxide counterparts promoted extensive fragmentation with complete coverage of all parts of the molecule, enabling detailed structural elucidation and unambiguous identification of the unoxidized analytes at low nanogram per milliliter levels.