Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Derivatization in Liquid Chromatography/Mass Spectrometric Analysis of Neurosteroids

1　Ｉｎｔｒｏｄｕｃｔｉｏｎ　Ｇａｓｃｈｒｏｍａｔｏｇｒａｐｈｙ/ｍａｓｓｓｐｅｃｔｒｏｍｅｔｒｙ (ＧＣ/ＭＳ)ａｎｄｌｉｑｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ/ｍａｓｓｓｐｅｃｔｒｏｍｅｔｒｙ (ＬＣ/ＭＳ) ,ｒｅｐｒｅｓｅｎｔａｔｉｖｅｓｏｆｔｈｅｈｙｐｈｅｎａｔｅｄｔｅｃｈｎｉｑｕｅｓ ,ａｒｅｓｏｍｅｏｆｔｈｅｍｏｓｔｒｅｌｉａｂｌｅａｎａｌｙｔｉｃａｌｍｅｔｈｏｄｓ ,ｗｈｉｃｈａｒｅｔｈｅｓｙｎｅｒｇｉｓｔｉｃｃｏｍｂｉｎａｔｉｏｎｏｆｔｗｏｐｏｗｅｒｆｕｌａｎａｌｙｔｉｃａｌｔｅｃｈｎｉｑｕｅｓ;…     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Derivatization in Liquid Chromatograhy/Mass Spectrometric Analysis of Neurosteroids

 Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview     

Characterization of volatile metabolites formed by molds on barley by mass and ion mobility spectrometry

The contamination of barley by molds on the field or in storage leads to the spoilage of grain and the production of mycotoxins, which causes major economic losses in malting facilities and breweries. Therefore, on‐site detection of hidden fungus contaminations in grain storages based on the detection of volatile marker compounds is of high interest. In this work, the volatile metabolites of 10 different fungus species are identified by gas chromatography (GC) combined with two complementary mass spectrometric methods, namely, electron impact (EI) and chemical ionization at atmospheric pressure (APCI)‐mass spectrometry (MS). The APCI source utilizes soft X‐radiation, which enables the selective protonation of the volatile metabolites largely without side reactions. Nearly 80 volatile or semivolatile compounds from different substance classes, namely, alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, alkenes, terpenes, oxidized terpenes, sesquiterpenes, and oxidized sesquiterpenes, could be identified. The profiles of volatile and semivolatile metabolites of the different fungus species are characteristic of them and allow their safe differentiation. The application of the same GC parameters and APCI source allows a simple method transfer from MS to ion mobility spectrometry (IMS), which permits on‐site analyses of grain stores. Characterization of IMS yields limits of detection very similar to those of APCI‐MS. Accordingly, more than 90% of the volatile metabolites found by APCI‐MS were also detected in IMS. In addition to different fungus genera, different species of one fungus genus could also be differentiated by GC‐IMS.     

Unexpected observation of ion suppression in a liquid chromatography/atmospheric pressure chemical ionization mass spectrometric bioanalytical method

Ion suppression is a well-known phenomenon in electrospray ionization (ESI) mass spectrometry. These suppression effects have been shown to adversely affect the accuracy and precision of quantitative bioanalytical methods using ion spray. Such suppression effects have not been as well defined in atmospheric pressure chemical ionization (APCI) and there is some debate whether these effects actually occur in the ionization process using APCI. Here an example is described where clear ion suppression was observed during studies on a model compound and three metabolites using APCI liquid chromatography/tandem mass spectrometry (LC/MS/MS).     

Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry

The phenomena of ionization suppression in electrospray ionization (ESI) and enhancement in atmospheric pressure chemical ionization (APCI) were investigated in selected-ion monitoring and selected-reaction monitoring modes for nine drugs and their corresponding stable-isotope-labeled internal standards (IS). The results showed that all investigated target drugs and their co-eluting isotope-labeled IS suppress each other's ionization responses in ESI. The factors affecting the extent of suppression in ESI were investigated, including structures and concentrations of drugs, matrix effects, and flow rate. In contrast to the ESI results, APCI caused seven of the nine investigated target drugs and their co-eluting isotope-labeled IS to enhance each other's ionization responses. The mutual ionization suppression or enhancement between drugs and their isotope-labeled IS could possibly influence assay sensitivity, reproducibility, accuracy and linearity in quantitative liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, calibration curves were linear if an appropriate IS concentration was selected for a desired calibration range to keep the response factors constant.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]+, 13C2 acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

Determination of acetaldehyde in tobacco smoke using N-methyl-4-hydrazino-7-nitrobenzofurazan and liquid chromatography/mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method for the determination of carbonyl compounds based on derivatization with N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) has been developed. Atmospheric pressure chemical ionization (APCI) in the positive mode proved the most versatile ionization technique for MNBD-hydrazones. APCI/MS spectra were recorded and the detection limits were determined for [M+H]⁺. ¹³C₂ acetaldehyde MNBD-hydrazone has been synthesized and characterized. It is applied as internal standard for the quantification of acetaldehyde. Tobacco smoke has been investigated concerning its carbonyl content. Acetaldehyde was identified as main product and quantified by LC/MS using internal standardization. The result is in good agreement to quantification data obtained with UV/vis detection.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Decomposition of N‐hydroxylated compounds during atmospheric pressure chemical ionization

N‐Hydroxylated polyamine derivatives were found to decompose during the ionization process of liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry (LC‐APCI‐MS) experiments. The phenomenon was studied with a model compound, a synthetic N‐hydroxylated tetraamine derivative. It was found that reduction, oxidation and water elimination occurred during APCI to generate the corresponding amine, N‐oxide, and imine. The investigation further revealed that decomposition of hydroxylamines during APCI depends upon the concentration of the analyte and on the acidity of the solution introduced into the ionization source. The pH‐dependence of decomposition was utilized for the development of an MS method that allows for the unambiguous identification of N? OH functionalities. This method was applied for the study of natural products including polyamine toxins from the venom of the spider Agelenopsis aperta and mayfoline, a cyclic polyamine derivative of the shrub Maytenus buxifolia. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Hydrogen/deuterium exchange on aromatic rings during atmospheric pressure chemical ionization mass spectrometry

It has been demonstrated that substituted indoles fully labelled with deuterium on the aromatic ring can undergo substantial exchange back to partial and even fully protonated forms during atmospheric pressure chemical ionisation (APCI) liquid chromatography/mass spectrometry (LC/MS). The degree of this exchange was strongly dependent on the absolute quantity of analyte, the APCI desolvation temperature, the nature of the mobile phase, the mobile phase flow rate and the instrument used. Hydrogen/deuterium (H/D) exchange on several other aromatic ring systems during APCI LC/MS was either undetectable (nitrobenzene, aniline) or extremely small (acetanilide) compared to the effect observed for substituted indoles. This observation has major implications for quantitative assays using deuterium‐labelled internal standards and for the detection of deuterium‐labelled products from isotopically labelled feeding experiments where there is a risk of back exchange to the protonated form during the analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Atmospheric pressure chemical ionization in gas chromatography-mass spectrometry for the analysis of persistent organic pollutants

Atmospheric pressure chemical ionization (APCI) was primarily applied as the ion source for liquid chromatography-mass spectrometry (LC–MS). While APCI started to be used in gas chromatography-mass spectrometry (GC–MS) in 1970s, GC-APCI-MS was not widely used until recently. As a soft ionization technique, APCI provides highly diagnostic molecular ions, which is favored for the wide-scope screening. With the capability of tandem mass spectrometry (MS/MS), GC-APCI-MS methods with high sensitivity and selectivity have been developed and applied in the analysis of persistent organic pollutants (POPs) in environment and biological samples at trace levels. The present review introduces the history of the APCI source, with emphasis on mechanisms of ionization processes under the positive and negative ionization modes. Comparison between GC-APCI-MS and GC–MS with traditional electron ionization (EI) and chemical ionization (CI) are provided and discussed for selectivity, sensitivity and stability for the analyses of POPs. Previous studies found that the GC-APCI-MS methods provided limits of detection (LODs) around 10–100 times lower than other methods. An overview of GC-APCI-MS applications is given with the discussions on the advantages and drawbacks of various analytical methods applied for the analyses of POPs.     

Postcolumn derivatization liquid chromatography/mass spectrometry for detection of chemical-weapons-related compounds

Postcolumn derivatization for liquid chromatography/mass spectrometry (LC/MS) analysis was characterized for detection of some compounds related to chemical-weapons (CW) agents using an Atmospheric Pressure Chemical Ionization (APCI) source. The derivatizing reagents were added directly to the LC eluent flow, and the derivatization reactions occurred in the APCI source under typical operating conditions. The compound S-[2-(diisopropylamino)ethyl] methylphosphonothioic acid was methylated using the derivatizing reagent trimethylphenyl ammonium hydroxide (TMPAH). Methylphosphonic acid was doubly derivatized to form dimethyl methylphosphonate, although the signal for the derivatization product was very sensitive to the amount of TMPAH. Arsenic compounds related to the CW agent lewisite, including chlorovinyl arsonous acid and arsenic (III) oxide, were derivatized using 2-mercaptopyridine. The thiol group reacted readily with the arsenic (III) center and provided a significant improvement in sensitivity relative to the underivatized signal using APCI or electrospray ionization. Triethanolamine and ethyl diethanolamine were derivatized with benzoyl chloride, a commonly used LC derivatizing reagent for alcohols, to modify their mass spectra. Postcolumn derivatization using an APCI source gives an alternative for detecting some difficult-to-ionize compounds. It has the limitations that sensitivity was not always improved even though the major mass spectral peaks can be shifted; it is necessary to carefully select the reagent; and some reagents introduced strong interference peaks at specific masses in the spectrum and may suppress the ionization of some derivatized analyte ions. The reagent also produced contamination in the source, which had to be cleaned daily.     

Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry

Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI‐MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,‐trimethoxyphenyl)phosphonium acetic acid (TMPP‐AA) to improve liquid chromatography (LC)/ESI‐MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl‐sn‐glycerol, dihydrotachysterol, octadecanol, and alpha‐tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI‐MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.     

Detection of volatile organic compounds in the headspace above mold fungi by GC‐soft X‐radiation–based APCI‐MS

Mold fungi on malting barley grains cause major economic loss in malting and brewery facilities. Possible proxies for their detection are volatile and semivolatile metabolites. Among those substances, characteristic marker compounds have to be identified for a confident detection of mold fungi in varying surroundings. The analytical determination is usually performed through passive sampling with solid phase microextraction, gas chromatographic separation, and detection by electron ionization mass spectrometry (EI‐MS), which often does not allow a confident determination due to the absence of molecular ions. An alternative is GC‐APCI‐MS, generally, allowing the determination of protonated molecular ions. Commercial atmospheric pressure chemical ionization (APCI) sources are based on corona discharges, which are often unspecific due to the occurrence of several side reactions and produce complex product ion spectra. To overcome this issue, an APCI source based on soft X‐radiation is used here. This source facilitates a more specific ionization by proton transfer reactions only. In the first part, the APCI source is characterized with representative volatile fungus metabolites. Depending on the proton affinity of the metabolites, the limits of detection are up to 2 orders of magnitude below those of EI‐MS. In the second part, the volatile metabolites of the mold fungus species Aspergillus, Alternaria, Fusarium, and Penicillium are investigated. In total, 86 compounds were found with GC‐EI/APCI‐MS. The metabolites identified belong to the substance classes of alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, terpenes, and sesquiterpenes. In addition to substances unspecific for the individual fungus species, characteristic patterns of metabolites, allowing their confident discrimination, were found for each of the 4 fungus species. Sixty‐seven of the 86 metabolites are detected by X‐ray–based APCI‐MS alone. The discrimination of the fungus species based on these metabolites alone was possible. Therefore, APCI‐MS in combination with collision induced dissociation alone could be used as a supervision method for the detection of mold fungi.     

The application of gas chromatography/atmospheric pressure chemical ionisation time‐of‐flight mass spectrometry to impurity identification in Pharmaceutical Development

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time‐of‐flight (TOF) MS. To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI‐TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H]⁺) and radical cations (M^+.) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O]⁺. The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1–2 mm/zunits (m/z 80–500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)‐MS and GC/chemical ionisation (CI)‐MS to understand the capability of GC/APCI‐MS relative to these two firmly established techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry

Nandrolone (19‐nortestosterone) is an androgenic anabolic steroid illegally used as a growth‐promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19‐norandrosterone, 19‐norethiocolanolone and 19‐norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid‐phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of triclosan metabolites by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry

Triclosan is a widely used broad‐spectrum antibacterial agent that acts by specifically inhibiting enoyl–acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague‐Dawley (SD) rat liver S9 and microsome, while the in vivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI‐ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in‐source fragmentation under APCI provided intensive fragmentations for the structural identifications. The in vitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI‐ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd.