Utility of "flip-flop" chromatography employing silica hydride stationary phases with simultaneous photodiode array ultraviolet and single quadrupole mass detection for the analysis of seized drugs.

For confirmation and/or screening purposes, rapid, selective, and precise chromatographic methods are required. In this vein, the utility of SiH columns (C18, UDC Cholesterol, and Diamond Hydride) with photodiode array UV absorption and single quadrupole MS detection for multi-modal separation of representative drugs from different drug classes on a single column using the same solvent reservoirs was investigated. For a conventional two column approach employing a combination of conventional C18 and silica columns operating in both the reversed phase chromatographic and hydrophilic interaction chromatographic modes, gradient analysis is required for the first column and there is a lack of retention on the second column for non-amine analytes. In comparison, all analytes are retained for two relatively rapid (< 10 min), precise (% RSD <0.4%), and non-correlated isocratic separations (R2=0.2115) when using a UDC Cholesterol column.

Recent forensic applications of enhanced chromatographic separation methods.

This study reviews the recent applications of enhanced separation methods employed in forensic analysis utilizing gas chromatography, liquid chromatography, and supercritical fluid chromatography published between 2015 to 2020, except papers previously covered in relevant review articles. Applications of enhanced chromatographic separation methods to arson investigations, environmental forensics, sexual assault investigations, drug analysis, and toxicology are discussed. Future directions for enhanced chromatographic separation methods in forensic science are also explored.

Use of micro, capillary, and nano liquid chromatography for forensic analysis.

The use of micro, capillary, and nano liquid chromatography systems for forensic analysis has excellent potential. In a field where sample size is often limited, several studies have presented the viability of capillary columns with microflow and nanoflow, and when using mass spectrometric analysis limits of detection can be improved. Reduction in flow rates result in significant reduction in operating costs. Recent advances in miniaturized liquid chromatography systems also aim at in-laboratory and on-site detection, which have already been applied to forensic drug cases. This critical review will discuss the advantages, disadvantages, and applicability of microflow and nano liquid chromatography. In this regard, included in this article is a discussion of some promising areas not yet applied to forensic research.

The separation and identification of synthetic cathinones by portable low microflow liquid chromatography with dual capillary columns in series and dual wavelength ultraviolet detection.

This study ascertained the viability of a portable liquid chromatograph, operating at low microliter per minute flow, for the analysis of seized drugs at remote sites as well as in laboratory settings. Synthetic cathinones were screened using dual capillary columns in series, C8 and biphenyl, with on-column ultraviolet detection at 255 and 275 nm. The relative retention times of the two columns in series and their peak area absorbance ratio were used to determine if the 16 synthetic cathinones investigated could be uniquely identified in these conditions. Based on these parameters all of the analytes could be distinguished. Representative mixtures of synthetic cathinones were used to determine the repeatability, linearity, and limits of detection of the method. This cost effective and green instrumentation has the potential to satisfy minimum international guidelines for the analysis of seized drugs.

The utility of silica hydride-based stationary phases for dual-mode ultra high performance liquid chromatography separation of synthetic cathinone positional isomers.

Emerging drugs usually mimic the effects of traditional drugs, but are not always controlled due to the chemically altered structures. Positional isomers of emerging drugs are difficult to analyze because they challenge the separation and detection techniques commonly employed by forensic laboratories. The utility of silica hydride stationary phases for the ultra-high performance liquid chromatography separation of synthetic cathinone positional isomers was studied in this manuscript. SiH phases are operable under both reversed phase and aqueous normal phase chromatographic conditions without the need to change solvent reservoirs. The separation of eight positional isomers of the synthetic cathinone, pentedrone, was investigated using five silica hydride phases, and compared to a classical dual column reversed phase, hydrophilic interaction chromatography system, and to a bimodal pentaflurophenyl column. Significant selectivity differences were observed using either a combination of a classical reversed phase C18 and NP Silica columns or the various bimodal columns. The silica hydride silica-C column, which contains no derivatized ligands attached to the silica hydride backbone, not only gave the most orthogonal separation of the bimodal columns, but provided a unique separation of all eight positional isomers (resolution ≥ 1) using the combination of reverse phase and aqueous normal phase chromatographic conditions.

Gas chromatography with tandem cold electron ionization mass spectrometric detection and vacuum ultraviolet detection for the comprehensive analysis of fentanyl analogues.

Fentanyl and its derivatives are amongst the ever-growing list of emerging drugs which are impinging on current traditional analytical techniques employed in forensic laboratories. To avoid current regulations, fentanyl analogues are being illicitly synthesized by slight alterations of functional groups to the fentanyl skeleton leading to inaccurate identifications, thus posing the greatest challenge to laboratories. In this study, a novel analytical technique is presented in which gas chromatography (GC) is interfaced with both cold electron ionization mass spectrometric (cold EI MS) and vacuum ultraviolet (VUV) detection by the means of a flow splitter for the simultaneous qualitative and quantitative analysis of twenty-four fentanyl analogues, including seven sets of positional isomers. For most of the twenty-four analogues, enhanced molecular ions were obtained with at least 1% intensity relative to base peak. In addition to enhanced molecular ions, the GC-cold EI MS maintained fragmentation pathways observed by GCMS with classical electron ionization. For the most part, VUV detection resulted in unique VUV spectra for fentanyl analogues including positional isomers. The combination of these two complementary detectors in tandem with the high resolving power of the gas chromatograph, allows for higher confidence in analyte identification by the combination of retention times, cold EI mass spectra and VUV spectra. The preferred method for quantitation was based on VUV detection and offered excellent determination coefficients (R2≥0.999) for most analytes over two orders of magnitude dynamic range, without the need for deuterated internal standards. For both run-to-run and day-to-day repeatability studies, at moderate solute concentrations, the correct fentanyl related compound was identified in almost every instance from a library containing all the fentanyl analogues plus hundreds of other analytes.

Analysis of fentanyl derivatives by ultra high performance liquid chromatography with diode array ultraviolet and single quadrupole mass spectrometric detection.

Fentanyl has become pervasive as a drug of abuse and as adulterant in seized drugs. Positional isomers analyzed by gas chromatography with mass spectrometry can follow the same fragmentation pathway and therefore may not be differentiated. Additionally, electron ionization leads to lack of discernible molecular ion for most fentanyl related compounds. Liquid chromatography may be used as an orthogonal identification technique with diode array ultraviolet and mass spectrometric detection. Here we provide a chromatographic method for the separation of 20 different fentanyl analogues, homologues and positional isomers using ultra high performance liquid chromatography with photodiode array ultraviolet and mass spectrometry detection. Five different columns were investigated utilizing reverse phase chromatography and hydrophilic interaction chromatography. Chromatographic systems were evaluated to determine which could separate the most compounds overall, as well as the most positional isomers. We found that isocratic elution, with a methanol modifier (35%) and formic acid (0.1%) as an additive, on a C18 column at a temperature of 25°C could resolve 10/20 compounds overall and 16/20 positional isomers. Using electrospray ionization, compounds with different masses could easily be distinguished based on their pseudo molecular ions. Ultraviolet detection facilitated differentiation of positional isomers that could not be distinguished by either electron ionization or electrospray ionization mass spectrometry alone.

A comparison of single quadrupole mass detection and diode array ultraviolet detection interfaced to ultra-high performance supercritical chromatography for the quantitative analysis of synthetic cathinones.

A comparison of single quadruple mass spectrometry and diode array-ultraviolet (PDA-UV) detection interfaced to ultra-high performance supercritical fluid chromatography was performed for the quantitative analysis of synthetic cathinones. Synthetic cathinones, also known as "bath salts", are derived from cathinone, a component of the khat plant. For 15 controlled solutes linearity, repeatability, and limits of detection were determined using both UV and MS detection. Quantitation studies were performed using the above detectors for 19 mixtures of up to 4 of the above compounds with an adulterant to simulate seized samples. MS detection provided approximately an order of magnitude greater linearity range and allowed for two to three orders of magnitude lower limits of detection than UV detection. Both detection techniques exhibited similar results of analysis and comparable repeatability. The latter detection mode which provided significantly high linearity correlation coefficients (0.9994 ≤ R2 ≤ 1.0000) would be preferred for quantitative analysis.

Comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones.

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite-5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.

The role of diode array ultraviolet detection for the identification of synthetic cathinones.

The utility of diode array ultraviolet (UV) detection for aiding in the identification of synthetic cathinones, including different sub-classes and positional isomers is presented. For 35 synthetic cathinones, unique UV spectra are obtained for seven sub-classes, including mostly beta ketones, where position and type of substitution on benzene rings give rise to differences in UV maxima and relative intensity of the spectral bands. This aspect is key to distinguishing positional isomers that contain differences in R substitution (mono and di) around the benzene ring, which provides complementary information to electron ionization mass spectrometry, where the latter technique cannot distinguish between these types of positional isomers. In addition, it is possible to ascertain the substitution position based on the UV spectra. For ten sets of positional isomers, it was possible to distinguish most of the positional isomers within a set. For ultra-high performance supercritical fluid chromatography (UHPSFC) versus reversed phase ultra-high performance liquid chromatography (UHPLC), there was at least a 10 nm blue shift in UV maximum (shift to shorter wavelengths). This highlights the importance of taking in account the effect of mobile phase on the UV maximum when performing method development in UHPSFC. Copyright © 2017 John Wiley & Sons, Ltd.

Isolation and characterization of a newly identified impurity in methamphetamine synthesized via reductive amination of 1-phenyl-2-propanone (P2P) made from phenylacetic acid/lead (II) acetate.

A trace processing impurity found in certain methamphetamine exhibits was isolated and identified as trans-N-methyl-4-methyl-5-phenyl-4-penten-2-amine hydrochloride (1). It was determined that this impurity was produced via reductive amination of trans-4-methyl-5-phenyl-4-penten-2-one (4), which was one of a cluster of related ketones generated during the synthesis of 1-phenyl-2-propanone (P2P) from phenylacetic acid and lead (II) acetate. This two-step sequence resulted in methamphetamine containing elevated levels of 1. In contrast, methamphetamine produced from P2P made by other methods produced insignificant (ultra-trace or undetectable) amounts of 1. These results confirm that 1 is a synthetic marker compound for the phenylacetic acid and lead (II) acetate method. Analytical data for 1 and 4, and a postulated mechanism for the production of 4, are presented. Copyright © 2015 John Wiley & Sons, Ltd.

Assessment of ultra high performance supercritical fluid chromatography as a separation technique for the analysis of seized drugs: Applicability to synthetic cannabinoids.

The recent development of modern methods for ultra high performance supercritical fluid chromatography (UHPSFC) has great potential for impacting the analysis of seized drugs. In the separation of synthetic cannabinoids the technique has the potential to produce superior resolution of positional isomers and diastereomers. To demonstrate this potential we have examined the capability of UHPSFC for the analysis of two different groups of synthetic cannabinoids. The first group was a mixture of 22 controlled synthetic cannabinoids, and the second group included JWH018 and nine of its non-controlled positional isomers The clear superiority of UHPSFC over other separation techniques was demonstrated, in that it was capable of near baseline separation of all 10 positional isomers using a chiral column. In total we examined four achiral columns, including Acquity UPC(2) Torus 2-PIC, Acquity UPC(2) Torus Diol, Acquity UPC(2) Torus DEA and Acquity UPC(2) Torus 1-AA (1.7µm 3.0×100mm), and three chiral columns, including Acquity UPC(2) Trefoil AMY1, Acquity UPC(2) Trefoil CEL1 and Acquity UPC(2) Trefoil CEL2 (2.5µm 3.0×150mm), using mobile phase compositions that combined carbon dioxide with methanol, acetonitrile, ethanol or isopropanol modifier gradients. Detection was performed using simultaneous PDA UV detection and quadrupole mass spectrometry. The orthogonality of UHPSFC, GC and UHPLC for the analysis of these compounds was demonstrated using principal component analysis. Overall we feel that this new technique should prove useful in the analysis and detection of seized drug samples, and will be a useful addition to the compendium of methods for drug analysis.

Regioisomeric and enantiomeric analyses of 24 designer cathinones and phenethylamines using ultra high performance liquid chromatography and capillary electrophoresis with added cyclodextrins.

DESIGNER: phenethylamines (PEAs) and cathinones have been encountered worldwide. Complete characterization of these substances can be challenging due to their chirality and variably substituted phenyl rings. In this study, 24 PEAs and cathinones were analyzed by ultra high performance liquid chromatography with photo diode array detection (UHPLC-PDA) on a variety of stationary phases, and by capillary electrophoresis on a dynamically coated capillary with PDA detection (CE-PDA). In the UHPLC-PDA study, a BEH Phenyl column resolved 18 of the 24 regioisomers in 8min, with good discrimination of the PEAs. In contrast, capillary zone electrophoresis (CZE) on a dynamically coated capillary partially or baseline resolved only 10 of the 24 regioisomers, but with improved discrimination of mono-substituted cathinones. A second series of CE-PDA experiments using 80mM (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD) in the run buffer resolved all 24 regioisomers and all but two sets of enantiomers within 18min. Five illicit samples were successfully analyzed using the described methods.

The role of ultra high performance liquid chromatography with time of flight detection for the identification of synthetic cannabinoids in seized drugs.

Separation and mass spectrometric techniques are integral parts of forensic drug analysis for both screening and confirmation. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG), which is responsible for setting standards for drug analysis, requires for drug identification a Category A test such as mass spectrometry with an additional test from either Category B or C. If a Category A method is not used at least two uncorrelated tests from Category B must be included, for which separation techniques such as gas chromatography and liquid chromatography would qualify. The utility and validity of using ultra high performance liquid chromatography (UHPLC) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of synthetic cannabinoids is presented. The separation of 32 solutes, including 23 controlled substances and nine non-controlled positional isomers of JWH-018, are compared using UHPLC with TOF detection and capillary GC with electron ionization (EI). For these solutes, the reversed phase UHPLC separation on three different 2.1 mm × 150 mm × 2.7 µm superficially porous (SPP) columns (C18, Phenyl-Hexyl and Dimethylpentafluorophenylpropyl (PFP)) compared favorably with the capillary gas chromatography (GC) separation using an Elite-5MS column 0.25 mm × 30 m × 0.25 µm. Principal component analysis revealed that all three UHPLC separations for the separation of the controlled substances are orthogonal to the capillary GC separation. It was also revealed by principal component analysis that the separation of JWH-018 and the nine non-controlled positional isomers for the various techniques were significantly more correlated than the separation of the controlled substances. Although most of the controlled synthetic cannabinoids gave unique TOF in-source collision-induced dissociation MS spectra and EI spectra, it was not possible to discriminate among the geometric isomers (CP47, 497, Epi CP47, 497; Cp47, 497 C8 homologue, Epi CP47, 497 C8 homologue). JWH-018 could be distinguished from the non-controlled isomers based on its EI spectra. In contrast, several of the non-controlled JWH-018 isomers give identical TOF in-source collision-induced dissociation MS spectra to JWH-018.

Screening of seized emerging drugs by ultra-high performance liquid chromatography with photodiode array ultraviolet and mass spectrometric detection.

The use of psychoactive "designer drugs" has increased rapidly due to their varying and sometimes ambiguous legal status and their ready access via the Internet and at local "headshops." A quick screening method for samples containing these substances, using ultra-high performance liquid chromatography with photodiode array UV and mass spectrometric detection (UHPLC-PDA/UV-MS), is presented. The method enables the screening of a variety of samples containing emerging/reemerging drugs, including ß-keto phenethylamines (cathinone derivatives), synthetic cannabinoids/cannabimimetics, and phenethylamine derivatives. The use of dual detectors not only provides molecular weight information but also differentiates the drugs by their categories and in some cases even their sub-categories. Moreover, ring positional isomers of cathinone and phenethylamine derivatives can be easily differentiated by their retention times and UV spectra.

Determination of heroin and basic impurities for drug profiling by ultra-high-pressure liquid chromatography.

Rapid, precise, accurate, and reproducible methodology using ultra-high-pressure liquid chromatography (UHPLC) for the analysis of heroin and basic impurities is described. The determination of heroin, morphine, O3-monoacetylmorphine, O6-monoacetylmorphine, codeine, acetylcodeine, noscapine, and papaverine is accomplished using reversed-phase chromatography (RPC), employing a 1.7 µm Acquity UPLC BEH C18 column (2.1 mm × 150 mm) with a phosphate buffer (pH 1.6)-acetonitrile gradient and PDA detection. The target analytes are well resolved from each other and most adulterants in less than 20 min. For the few instances when adulterants interfere with target analytes, a 1.7 µm Acquity CSH Fluoro-Phenyl (2.1 mm × 150 mm) column is utilized with the same gradient conditions. The reported methodology can detect impurities as low as 0.02% relative to heroin, and is well suited for heroin profiling.

Profiling of illicit fentanyl using UHPLC-MS/MS.

Methodology is presented for the profiling of fentanyl in seized drugs using ultra high performance liquid chromatography in combination with tandem mass spectrometry (UHPLC-MS/MS). Target analysis was performed for 40 fentanyl processing impurities, several of which are markers for a specific synthetic route (Siegfried or Janssen). For the separation of these solutes, an Acquity BEH C18 1.7 µm particle column (150 mm × 2.1 mm) with a binary 1% formic acid (pH 2.0)/acetonitrile gradient was used. For MS/MS detection, an atmospheric pressure positive electrospray source was employed with selected reaction monitoring (SRM). The coupling of the high separation power of UHPLC with the highly selective and sensitive detection of MS/MS is amenable to the determination of synthetic route and linking of drug seizures. The technology is also applicable to exhibits containing trace levels of fentanyl in the presence of significantly excess amounts of heroin and/or adulterants.

Hydrophilic interaction chromatography of seized drugs and related compounds with sub 2 µm particle columns.

The use of hydrophilic interaction chromatography (HILIC) with sub 2 µm particle columns for the analysis of drugs and related compounds of forensic interest is described. This technique uses a high organic/low aqueous buffered mobile phase with a polar stationary phase, and is excellent for the separation of many of the charged solutes that are found in forensic drug exhibits. In this study, HILIC is investigated for 11 solutes of forensic interest, including weak bases, weak acids, and a neutral solute. In addition, for columns containing either ethylene bridged hybrid particles with or without an amide bonded phase, the effects of acetonitrile concentration, buffer type, buffer concentration, linear velocity, and sample concentration were studied. Based on these studies, HILIC with sub 2 µm particle columns can offer highly efficient, selective, and rapid isocratic separations of drugs and related compounds of forensic interest, with excellent peak shapes and low back pressures. This is in contrast to reverse phase chromatography (RPLC), where gradient elution is usually required, which can result in extensive overlap between acidic, neutral, and basic solutes. In addition, since HILIC exhibits a much greater loading capacity than RPLC, it could be a preferred technique for drug profiling. Furthermore, because high organic content mobile phases are highly amenable to mass spectrometric detection, the use of HILIC with tandem mass spectrometric detection for the analysis of seized drugs is described.

Use of multiple-reaction monitoring ratios for identifying incompletely resolved fentanyl homologs and analogs via ultra-high-pressure liquid chromatography-tandem mass spectrometry.

Fentanyl and 16 of its corresponding homologs and analogs were distinguished using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A 1.7 microm Acquity BEH C18 column (150 mm x 2.1 mm) was used with a 1% formic acid (pH 2.2), methanol gradient. Multiple-reaction monitoring (MRM) was employed for MS/MS detection. All selected fentanyl-related compounds, including incompletely resolved compounds, were uniquely identified using retention times and dual MRMs.

Applicability of ultra-performance liquid chromatography-tandem mass spectrometry for heroin profiling.

The applicability of ultra- performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) for heroin profiling is described. The coupling of the high separation power of UPLC with the highly selective and sensitive detection of MS/MS is well suited for heroin profiling. An Acquity UPLC BEH C18 1.7 microm particle column (100 mm x 2.1mm) with binary gradients containing 1% formic acid (pH 2.0) or 10 mM ammonium bicarbonate (pH 10.0)/acetonitrile mixtures was investigated for the profiling. For MS/MS detection, an atmospheric pressure positive electrospray source was employed with multiple reaction monitoring (MRM). MRMs for individual basic impurities were generated for heroin profiling using low and high pH mobile phases, while MRMs for neutral impurities were generated using a high pH mobile phase. Compared to a pH 2.2 mobile phase, the use of a pH 10 mobile phase allowed for significantly greater sample loading, major selectivity differences, and lower MRM sensitivity. UPLC-MS/MS allowed for the highly selective and sensitive detection of many of the targeted solutes in seized heroin exhibits. Basic impurities detected included morphine, codeine, noscapine, papaverine and the previously unreported solutes reticuline, reticuline monoacetate (2 products), reticuline diacetate, narceine, codamine, laudanidine, cryptopine, laudanosine, and norlaudanosine. Neutral impurities found included N,3,6-triacetylnormorphine, N-acetylnorcodeine, N-acetylnornarcotine, 3,6-dimethoxy-4-acetyloxy-5-[2-(N-methylacetamido)]-ethylphenanthrene, and cis-n-acetylanhydronornarceine. The detection of these impurities, at levels as low as 10(-6)% w/w should allow for greatly enhanced heroin profiles.