Isotopic analogs as internal standards for quantitative analyses by GC/MS--evaluation of cross-contribution to ions designated for the analyte and the isotopic internal standard

Isotopic analogs of the analytes are currently preferred internal standards (IS) for quantitative analyses of drugs and their metabolites in biological matrices by GC/MS procedures. Contributions of the analyte and the IS to the intensities of ions designated for the IS and the analyte, respectively--an undesirable phenomenon termed "cross-contribution"--greatly weakens the effectiveness of this approach. The cross-contribution phenomenon has been, in the past, evaluated by a "direct measurement" approach, in which intensities of interested ions were measured in two separate experiments using equal quantities of the analyte and the IS. Alternate procedures that may generate improved results are hereby studied. For the "improved direct measurement" approach, ion intensity data derived from the previously reported direct measurement procedure are first normalized before being used to calculate the extent of cross-contribution. An "internal standard" approach is also developed, in which a set amount of a third compound is incorporated into these two separate experiments, thus allowing corrections of ion intensity data that are imbedded with variations inherent to separate experiments. Finally, a "standard addition" approach, involving a series "addition" of "standards", generates multiple data points; thus, providing a mechanism to validate the resulting cross-contribution data. Secobarbital/(2)H(5)-secobarbital and secobarbital/(13)C(4)-secobarbital pairs are adapted as the exemplar systems for this study.     

Identification and characterization of 2,5-dimethoxy-4-nitro-β-phenethylamine (2C-N) - A new member of 2C-series of designer drug

The online sale of psychoactive substances, including hallucinogens, is becoming a serious problem in many countries. This paper presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5-dimethoxy-4-nitro-β-phenethylamine (2C-N), which was identified in a powder sample seized by the authorities in 2011 in Poland. The molecular mass of 2C-N (226.0954amu) was confirmed in the LC/ESI-QTOFMS experiment. The molecular ion was also observed in the GC-EI/MS spectrum. A characteristic set of ions for the parent substance was found using both chromatographic methods, and when derivatization with trifuluoroacetic anhydride (TFAA) was applied. Two broad dominant bands at 1520cm(-1) and 1342/1322cm(-1), observed in the FTIR spectrum of 2C-N, originated from the nitro group. NMR spectroscopy helped unequivocal elucidation of the structure. The applied identification procedure proved to be a powerful tool to determine the structure of a new designer drug.     

Liquid chromatography-mass spectrometric and liquid chromatography-tandem mass spectrometric determination of hallucinogenic indoles psilocin and psilocybin in "magic mushroom" samples

Accurate and sensitive analytical methods for psilocin (PC) and psilocybin (PB), tryptamine-type hallucinogens contained in "magic mushrooms," were investigated using liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The chromatographic separation on an ODS column and mass spectral information gave complete discrimination between PC and PB without derivatization. The mass spectrometric detection had a high sensitivity, and the tandem mass spectrometric detection provided more specificity and accuracy, as well as high sensitivity. The detection limits ranged from 1 to 25 pg by LC-MS in the selected ion monitoring mode, and the intra- and inter-day coefficients of variation were estimated to be 4.21-5.93% by LC-MS-MS in the selected reaction monitoring mode. By applying the present LC-MS-MS technique to four real samples, the contents of PC and PB were found to vary over a wide range (0.60-1.4 and 0.18-3.8 mg/g dry wt. for PC and PB, respectively) between samples.     

Hair analysis: self-reported use of "speed" and "ecstasy" compared with laboratory findings

Drug use histories were collected from 100 subjects recruited from the "dance scene" in and around Glasgow, Scotland. In addition, each subject donated a hair sample which was analyzed by gas chromatography/mass spectrometry (GC/MS) for amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MD MA) and 3,4-methylenedioxyethylamphetamine (MDEA). The hair samples were analyzed in two 6 cm segments or in full, ranging from 1.5 to 12 cm depending on the length of the hair. Approximately 10 mg of hair was ground to a fine powder before treatment with beta-glucuronidase/aryl sulfatase. A solid-phase extraction procedure was carried out followed by derivatization with pentafluoropropionic anhydride (PFPA). All extracts were analyzed by gas chromatography/mass spectrometry (GC/MS). Of the 139 segments analyzed, 77 (52.5%) were positive for at least one of the five amphetamines. The drug concentrations found in the hair were compared with the self-reported drug histories. A concordance of greater than 50% was found between the self-report data and levels detected in hair. However, no correlation was found between the reported number of "ecstasy" tablets consumed and the drug levels detected in hair. An increase in the average drug levels measured was observed from low to high use (number of "ecstasy" tablets/month). A large number of false negatives and a low number of false positives were observed.     

Analysis of alprazolam by DART-TOF mass spectrometry in counterfeit and routine drug identification cases

The high prevalence of alprazolam abuse translates to an increased workload for crime laboratories in characterizing seized tablets. These tablets may originate as diverted pharmaceuticals or counterfeited mimics, so efficient analytical techniques should provide confirmatory data while minimizing destruction of evidence. We offer the first report of a validated forensic method for confirming alprazolam tablets by direct analysis in real time-time of flight (DART-TOF) mass spectrometric analysis. This technique provides rapid identification of target analytes with minimal sample preparation, allowing direct analysis in the atmospheric sample gap. Selectivity is achieved through high resolution and mass accuracy, unique ion fragments, and chlorine isotopic ratios. This method utilizes fragmentation in two separate voltage functions to observe the alprazolam pseudo molecular ion at 309.09070 using 40 V and major ion fragments of 281.07197 and 205.07657 at 120 V. These parameters allow our laboratory to confirm alprazolam tablets efficiently, without compromising quality forensic standards.     

Positive and negative ion mass spectrometry of antiepileptic hydantoins and their analogs

Positive ion electron impact (PIEI), positive ion chemical ionization (PICI), and negative ion chemical ionization (NICI) mass spectra of seven compounds of hydantoins and their analogs are presented; their probable fragmentation modes are also presented. In the PIEI mode, intensities of molecular ions differed according to different compounds. Cleavage at outsides of both carbonyl groups was commonly observed for all compounds. In the PICI mode, all compounds showed [M + 1]+ quasi-molecular ions constituting the base peaks. In the NICI mode, [M - 1]- quasi-molecular anions were the base peaks except for trimethadione and paramethadione. All negative spectra showed anions at m/z 42 due to [NCO]-; these peaks seem useful for screening of antiepileptics. An extraction procedure for the anti-epileptics from human urine or plasma, and their separation by gas chromatography (GC), are also presented to serve for their actual identification by GC/mass spectrometry.     

13C4-secobarbital as the internal standard for the quantitative determination of secobarbital--a critical evaluation

In this study, 13C4-secobarbital was used as an exemplar compound to illustrate the mechanism based on which the effectiveness of a proposed internal standard (IS) could be evaluated. A deuterated analog, 2H5-secobarbital, was also studied in parallel for comparison purposes. Well-established solid-phase extraction and methylation procedures were used prior to the GC/MS measurement step. The contribution of the intensity of an ion designated for the analyte (secobarbital) by the proposed IS, and similarly, the contribution of the intensity of an ion designated for the IS by the analyte--a phenomenon termed "cross-contribution"-were evaluated based on a "direct measurement" procedure in which equimolar amounts of the analyte and the IS were used to generate intensity data. These intensity data were then used as the basis for the calculation of "cross-contribution" (in percentages) of ions designated for the analyte and the IS. Cross-contribution data were compared with the linearity data resulting from two series of standards containing 25 to 9600 ng/mL secobarbital using two sets of quantitation ion pairs--m/z 196/200 and 195/199 with 13C4-secobarbital as the IS and m/z 196/201 and 195/200 with 2HS-secobarbital as the IS. 13C4-secobarbital was found to be much less problematic and thus can serve as a very effective IS. Cross-contribution data alone cannot fully explain the observed differences resulting from the use of these two ISs; further systematic study is needed to provide better understanding of the underlying interference mechanism.     

GC–MS analysis of eight aminoindanes using three derivatization reagents

Aminoindanes are a class of novel psychoactive substances (NPSs) that have become more prevalent over the past decade. GC–MS is often utilized for identifying seized drugs and is well regarded for its ability to separate mixtures. However, certain aminoindanes have similar mass spectral data and require specific gas chromatographic stationary phases for separation. Derivatization is an alternative method that can be applied to GC–MS to enhance chromatographic results, providing more selective analysis in seized-drug identification. This study investigates derivatization techniques to provide options for forensic science laboratories in accurately identifying aminoindanes. Three derivatization reagents, N-methyl-bis(trifluoroacetamide) (MBTFA), heptafluorobutyric anhydride (HFBA), and ethyl chloroformate (ECF) were evaluated for the analysis of eight aminoindanes by GC–MS using two common gas chromatographic stationary phases, Rxi®-5Sil MS and Rxi®-1Sil MS. All three derivatization methods successfully isolated eight aminoindanes, including the isomers 4,5-methylenedioxy-2-aminoindane (4,5-MDAI), and 5,6-methylenedioxy-2-aminoindane (5,6-MDAI) that could not be differentiated prior to derivatization. Reduced peak tailing and increased abundance were observed after derivatization for all the compounds, and mass spectra of the derivatives contained individualizing fragment ions that allowed for further characterization of the aminoindanes. This excluded 4,5-MDAI and 5,6-MDAI as they shared the same characteristic ions and were only distinguishable by their retention times. All three derivatization techniques used in this study allow for successful characterization of the aminoindanes and give forensic science laboratories flexibility in their analysis approach when they encounter these compounds.     

Spectroscopic characterization of 3,4-methylenedioxypyrrolidinobutyrophenone: a new designer drug with α-pyrrolidinophenone structure

This study presents and discusses the infrared spectroscopic, the nuclear magnetic resonance spectroscopic and mass spectrometric data of the designer drug 3,4 methylenedioxypyrrolidinobutyrophenone (MDPBP), a homolog of 3,4 methylenedioxypyrovalerone (MDPV). MDPBP was first seized in Germany in the year 2009. The structure elucidation of the aliphatic part of MDPBP was carried out by product ion spectrometry of the immonium ion with m/z=112 formed after electron ionization, and by one- and two-dimensional (1)H- and (13)C NMR spectroscopy.     

Hair analysis for drugs of abuse. Hair color and race differentials or systematic differences in drug preferences?

There is currently a debate in the literature on chemical drug analysis concerning the contribution of biophysical attributes associated with specimens and specimen donors to assay outcome. In recent years this debate has focused on hair analysis, but has in the past also been raised in urinalysis interpretation. In this article we examine several aspects of that controversy. First, we present data regarding the effects of hair color on the distribution of positive hair testing results for three drug classes. We compare these results to negative hair samples from comparable donors. This data is derived from head hair from preemployment donors that was classified according to seven visual color categories. We determined the distribution of colors for hair samples devoid of any of three assayed drugs (amphetamines, cocaine, and cannabinoids). Subsequently, this distribution was compared with the distributions for hairs that had tested positive for amphetamines, cocaine or cannabinoids. We examined a total of 2000 randomly selected samples; 500 negative hair samples and 500 positive samples for each of three drugs: cannabinoids, cocaine, and amphetamine. We also evaluated ethnic/racial factors in relation to positive urinalyses for various ethnic/racial groups. We examined approximately 4000 urine specimens from two different groups, each constituting around 2000 specimens. In addition to ethnicity/race and urinalysis outcome, we also examined the relationship between the hair color distributions of urine donors and the corresponding urinalysis results for the three drug classes. We also compared them to drug-negative samples. Our summary impression is that the observed outcome patterns were largely consistent with differences in drug preferences among the various societal groups. There was little evidence of a pattern attributable to hair color bias alone or selective binding of drugs to hair of a particular color. Likewise, there was no discernible pattern associated with race or ethnicity that would lend support to a "race effect" in drug analysis.     

Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.     

Lifestyle illicit drug seizures: A routine ESI-LC-MS method for the identification of sildenafil and vardenafil

For street samples suspected of containing the phosphodiesterase-5 inhibitors sildenafil (Viagra(?)) and/or vardenafil (Levitra(?)), including powders or adulterated herbal supplements, a chemical analysis is needed to provide confirmatory identification of these illegally procured substances. Sildenafil and vardenafil are structurally similar and it is difficult to differentiate between them, as previous mass spectrometric studies have shown the two drugs to produce similar fragmentation patterns. The use of tandem mass spectrometry can produce confirmatory data, but the technique requires a high level of technical expertise. We have developed an electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC-MS) method that allows differentiation between these two structurally similar molecules via in-source fragmentation in combination with an ion trap mass spectrometer. A very stable gas phase ion is formed during in-source fragmentation of vardenafil; the combination of the stability of this ion and the longer residence time for the ion in the ion trap results in a very strong signal. This feature results in a method that can provide clear differentiation between sildenafil and vardenafil while at the same time requiring less expertise from the routine analyst to confirm the presence or absence of the two compounds.     

Blood levels of 3-methylfentanyl in 3 fatal poisoning cases

Three fatal poisoning cases due to 3-methylfentanyl are described. In each case, the death was accidental and occurred after injection of the opioid combined with amphetamine, heroin, or other drugs. The victims' ages, ranging from 30 to 41 years, were higher than those typically found in heroin poisonings in Finland. The blood concentrations of cis-3-methylfentanyl, measured here for the first time by a specific tandem mass spectrometric method, ranged from 0.3 to 0.9 microg/L (mean 0.5 microg/L). These values are significantly lower than the levels reported for alpha-methylfentanyl and fentanyl in fatal poisonings. Repeated seizures of fentanyl and its analogs have been reported in Europe close to the Russian border.     

Electrospray-ionization MS/MS library of drugs as database for method development and drug identification

An ESI MS/MS library of 800 compounds has been developed and a collection of data is now available for Analyst 1.4 and higher. Compounds include forensically important drugs, such as illegal drugs, some deuterated analogues, hypnotics, amphetamines, benzodiazepines, neuroleptics, antidepressants and many others. For setting up the library of product ion spectra, 20-200 ng of the compounds have been injected either by flow injection or via a short LC-column, the precursor ions were chosen from the Q1 scan spectra, and product ion spectra were generated by CID in the collision cell using three different collision energies (20, 35 and 50 eV). Three spectra of each compound have been collected and compound names, CAS numbers, formulas and molecular weights have been added in the database, which has been generated by the Analyst software. The library can be used for compound identification during general unknown screening analysis by combination of Q1 scan techniques and subsequent MS/MS analysis in a second analytical run. Quantitative procedures for multi drug analysis using Multiple Reaction Monitoring can be established by selection of product ions and suitable collision energies from the library. For publication of the spectra, PDF-files have been generated and can be viewed on-line as supplementary data or from the website in alphabetical order: (supplementary data, should be made available via ELSEVIER-WEBSITE or via ).     

Signature Profiling and Classification of Illicit Heroin by GC-MS Analysis of Acidic and Neutral Manufacturing Impurities

Abstract:  The illicit manufacture of heroin results in the formation of trace level acidic and neutral impurities. These impurities are detectable in illicit heroin and provide valuable information about the manufacturing process used. The isolation, derivatization, and semiquantitative analysis of neutral and acidic heroin manufacturing impurities by programmed temperature vaporizing injector-gas chromatography-mass spectrometry (PTV-GC-MS) is described. Trace acidic and neutral heroin impurities were isolated from basic fractions using liquid–liquid extraction. Extracted impurities were treated with N -Methyl- N -trimethylsilyltrifluoroacetamide followed by PTV-GC-MS analyses. Semiquantitative data were obtained using full scan mass spectrometry utilizing unique ions or ion combinations for 36 trace impurities found in crude and/or highly refined heroin samples. Minimum detection limits for acidic and neutral impurities were estimated to be at the 10⁻⁷ level relative to total morphine. Over 500 authentic heroin samples from South America, Mexico, Southwest Asia, and Southeast Asia were analyzed. Classification of illicit heroin based on the presence or absence and relative amounts of acidic and neutral impurities is presented.     

Characterization and Differentiation of Geometric Isomers of 3‐methylfentanyl Analogs by Gas Chromatography/Mass Spectrometry,Liquid Chromatography/Mass Spectrometry,and Nuclear Magnetic Resonance Spectroscopy

The cis and trans isomers of 3‐methylfentanyl and its three analogs were chemically synthesized, and these compounds were characterized and differentiated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and nuclear magnetic resonance (NMR) spectroscopy. The cis and trans isomers of the 3‐methylfentanyl analogs were completely separated by GC/MS. Although the high temperature of the GC injection port caused thermal degradation of β‐hydroxy‐3‐methylfentanyl, the degradation was completely suppressed by trimethylsilyl derivatization. The isomers were also well separated by LC/MS on an octadecylsilyl column with 10 mM ammonium acetate and methanol as the mobile phase. The proton NMR signals were split when the hydrochloride salts of the 3‐methylfentanyl analogs were dissolved in deuterated chloroform because stereoisomers were formed by the coordination of the hydrochloride proton to the nitrogen of the piperidine ring of the 3‐methylfentanyl analogs.     

Mass, NMR and IR spectroscopic characterization of pentedrone and pentylone and identification of their isocathinone by-products

This study presents and discusses the mass spectrometric, nuclear magnetic resonance spectroscopic and infrared spectroscopic data of the designer drugs pentedrone (2-methylamino-1-phenylpentan-1-one) and its methylenedioxy analog pentylone (2-methylamino-1-(3,4-methylenedioxyphenyl)pentan-1-one). The structure elucidation of the aliphatic parts was carried out by product ion spectroscopy of the immonium ion with m/z=86 formed after electron ionization, and by one- and two-dimensional (1)H- and (13)C-NMR spectroscopy on the hydrochloride salts to verify the structure of the alkyl side chain and to determine the methylenedioxy position in the aromatic ring of pentylone. Furthermore, two typical cathinone synthesis by-products were detected besides the main compounds. Their mass spectra are discussed and for one of them (1-methylamino-1-phenylpentan-2-one (isopentedrone)) a NMR assignment was possible in the existing mixture.     

苯丙胺类兴奋剂β酮策划药代谢途径的研究进展

近年来,苯丙胺类兴奋剂的β酮(bK)策划药相继在许多国家的毒品市场中出现,由于该类物质潜在的依赖性和已经导致的死亡事件,许多国家已经将其列为管制的物质。本文介绍了通过GC/MS和LC/MS方法对苯丙胺类兴奋剂的衍生物4-甲基甲卡西酮、bk-MDMA、bk-MBDB和bk-MDEA检测的相关研究结果,以说明其主要代谢途径。以期为临床医学、法庭毒理学以及禁毒机构监控该类物质提供参考。     

Differentiation of regioisomeric ring-substituted fluorophenethylamines with product ion spectrometry

The electron ionization (EI) of aromatic ring-substituted isomers gives virtual identical mass spectra which seriously affects their analysis. Especially regioisomeric meta- and para-ring-substituted compounds cannot show any ortho-effect reactions making their differentiation by mass spectrometry impossible. Furthermore o-, m- and p-substituted compounds can only be separated insufficiently by chromatography due to their very similar retention that do not allow univocal identification. Product ion mass spectrometry has proved to be a useful tool to differentiate structurally closely related fluorophenethylamines even in the case of the meta- and para-isomers. A series of N-alkylated o-, m- and p-fluoroamphetamines and 1-(4-fluorophenyl)butan-2-amines have been synthesized in microscale and studied by product ion spectrometry. The combination of chemical ionization (CI) and product ion spectrometry of hydrogen fluoride loss ions [M+H?HF]⁺ allows a univocal differentiation of all studied fluoro-substituted phenethylamines without prior derivatization. This method with submicrogram detection limits provides great advantages for the differentiation between aromatic regioisomeric fluorophenethylamine designer drugs where other methods such as nuclear magnetic resonance (NMR) spectrometry lack sufficient sensitivity or might fail because complex mixtures have to be analyzed.     

Mass spectral and NMR spectral data of two new designer drugs with an alpha-aminophenone structure: 4'-methyl-alpha-pyrrolidinohexanophenone and 4'-methyl-alpha-pyrrolidinobutyrophenone

This study presents and discusses the nuclear magnetic resonance (NMR) spectroscopic and mass spectroscopic data of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) and its homolog 4'-methyl-alpha-pyrrolidinohexanophenone (MPHP) which were seized in 2004 and 2000 in Germany for the first time. The structure elucidation of the aliphatic part of MPBP was carried out by product ion spectroscopy of the immonium ion formed after electron ionization as well as with 1H and 13C NMR. Product ion spectroscopy of immonium ions again proved to be a powerful tool to determine the structure of designer drugs and to distinguish between isobaric structures of the alkyl-amino moiety.