Regioisomeric differentiation of 2,3- and 3,4-methylenedioxy ring-substituted phenylalkylamines by gas chromatography/tandem mass spectrometry

Numerous abused drugs of the 3,4-methylenedioxymetamphetamine (MDMA; Ecstasy; N-methyl-1-(3,4-methylenedioxyphenyl)-2-propaneamine) type and various alkyl chain- and aromatic ring-substituted isomers give very similar electron ionization (EI) mass spectra. This seriously affects the analysis of especially ring regioisomeric drug variants. Using collision-induced dissociation (CID) (argon) under EI and chemical ionization, the mass spectra of 18 2,3- and 3, 4-methylenedioxy ring-substituted phenylethylamines were recorded. These techniques permitted an unequivocal differentiation of all studied ring regioisomeric methylenedioxyphenylethylamines. CID mass spectrometry therefore appear to be a reliable tool to establish the kind of ring substitution pattern in regioisomeric methylenedioxyphenalkylamines. Copyright 2000 John Wiley & Sons, Ltd.     

High-resolution LC/MS for analysis of minor components in complex mixtures: negative ion ESI for identification of impurities and degradation products of a novel oligosaccharide antibiotic

High-resolution mass spectrometry has been routinely used for structural confirmation and identification; however, it has mostly been applied to relatively pure samples. Exact mass measurement of minor components such as impurities, degradation products or metabolites in complex mixtures has been difficult without prior separation and isolation. Here we report the utilization of on-line liquid chromatography in combination with high-resolution mass spectrometry for the identification of impurities and base degradation products of Sch 27899, a member of the everninomicin class of antibiotics. Nine Sch 27899-related impurities and degradation products were detected by negative ion electrospray ionization using a magnetic sector mass spectrometer. Exact mass measurements were obtained at a resolution of 5000 using polyethylene glycol (PEG) sulfates as internal standards. Corresponding elemental compositions were determined within a 2 ppm error tolerance and structures were proposed for all components.     

Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

Direct assay of Aplysia tissues and cells with laser desorption/ionization mass spectrometry on porous silicon.

Desorption/ionization on porous silicon (DIOS) is a form of laser desorption mass spectrometry that allows for the direct mass analysis of a variety of analytes without the addition of organic matrix. Protocols are described for the direct analysis of exocrine tissue and single neurons using DIOS-MS. The atrial gland of Aplysia californica was blotted on to porous silicon and analyzed with DIOS-MS in the range m/z 1000-4000. The ability to culture invertebrate neurons directly on porous silicon is also presented. Isolated bag cells regenerated neuronal processes in culture on porous silicon. DIOS-MS allowed the direct detection of the peptides contained in individual cultured neurons indicating that with appropriate protocols, DIOS can be used with biological samples with considerable thickness.     

High surface area of porous silicon drives desorption of intact molecules

The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si(n)(+) and OSiH(+)). A threshold laser energy for DIOS is observed (10 mJ/cm(2)), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption.     

DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY

An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3'-terminal phosphate monoester and 3'-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N(3) (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.     

Desorption sonic spray ionization for (high) voltage-free ambient mass spectrometry

Sonic spray ionization is shown to create a supersonic cloud of charged droplets able to promote efficient desorption and ionization of drugs directly from the surfaces of commercial drug tablets at ambient conditions. Compared with desorption electrospray ionization (DESI), desorption sonic spray ionization (DeSSI) is advantageous since it uses neither heating nor high voltages at the spray capillary. DeSSI therefore provides a more friendly environment in which to perform ambient mass spectrometry (MS). DeSSI-MS is herein evaluated for the analysis of drug tablets, and found to be, in general, as sensitive as DESI-MS. The (high) voltage-free DeSSI method provides, however, cleaner mass spectra with less abundant solvent cluster ions and with enough abundant analyte signal for tandem mass spectrometry (MS/MS). These features may therefore facilitate the DeSSI-MS detection of low molar mass components or impurities, or both. The higher-velocity supersonic DeSSI spray also facilitates matrix penetration thus providing more homogenous sampling and longer lasting ion signals.     

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

Analysis of impurities in pharmaceuticals by LC‐MS with cold electron ionization

Pharmaceuticals require careful and precise determination of their impurities that might harm the user upon consumption. Although today, the most common technique for impurities identification is liquid chromatography‐mass spectrometry (LC‐MS/MS), it has several downsides due to the nature of the ionization method. Also, the analyses in many cases are targeted thus despite being present, some of the compounds will not be revealed. In this paper, we propose and show a new method for untargeted analysis and identification of impurities in active pharmaceutical ingredients (APIs). The instrument used for these analyses is a novel electron ionization (EI) LC‐MS with supersonic molecular beams (SMB). The EI‐LC‐MS‐SMB was implemented for analyses of several drug samples spiked with an impurity. The instrument provides EI mass spectra with enhanced molecular ions, named Cold EI, which increases the identification probabilities when the compound is identified with the aid of an EI library like National Institute of Standards and Technology (NIST). We analyzed ibuprofen and its impurities, and both the API and the expected impurity were identified with names and structures by the NIST library. Moreover, other unexpected impurities were found and identified proving the ability of the EI‐LC‐MS‐SMB system for truly untargeted analysis. The results show a broad dynamic range of four orders of magnitude at the same run with a signal‐to‐noise ratio of over 10 000 for the API and almost uniform response.     

Separation,Characterization, and Quantification of Atorvastatin and Related Impurities by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

A method coupling high-performance liquid chromatography with diode-array detector and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MSⁿ) has been developed for the separation and characterization of atorvastatin and its related impurities. The results obtained using positive ion mode showed some diagnostic fragments that are useful for the identification of atorvastatin related impurities in real samples. Quantitative analysis of drug impurities was performed in the multiple reaction monitoring mode. Quantification limits for impurities were in the ranges 21.5–70.8 ng mL^?1. The method was successfully applied to the drug purity evaluation and quantitative determination of atorvastatin related impurities in bulk drugs and pharmaceutical formulations.     

Drug impurity profiling by capillary electrophoresis/mass spectrometry using various ionization techniques

Capillary electrophoresis/mass spectrometry (CE/MS) is predominantly carried out using electrospray ionization (ESI). Recently, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) have become available for CE/MS. With the VUV lamp turned off, the APPI source may also be used for CE/MS by thermospray ionization (TSI). In the present study the suitability of ESI, APCI, APPI and TSI for drug impurity profiling by CE/MS in the positive ion mode is evaluated. The drugs carbachol, lidocaine and proguanil and their potential impurities were used as test compounds, representing different molecular polarities. A background electrolyte of 100 mM acetic acid (pH 4.5) provided baseline separation of nearly all impurities from the respective drugs. APPI yielded both even‐ and odd‐electron ions, whereas the other ionization techniques produced even‐electron ions only. In‐source fragmentation was more pronounced with APCI and APPI than with ESI and TSI, which was most obvious for proguanil and its impurities. In general, ESI and TSI appeared the most efficient ionization techniques for impurities that are charged in solution achieving detection limits of 100 ng/mL (full‐scan mode). APPI and APCI showed a lower efficiency, but allowed ionization of low and high polarity analytes, although quaternary ammonium compounds (e.g. carbachol) could not be detected. Largely neutral compounds, such as the lidocaine impurity 2,6‐dimethylaniline, could not be detected by TSI, and yielded similar detection limits (500 ng/mL) for ESI, APPI and APCI. In many cases, impurity detection at the 0.1% (w/w) level was possible when 1 mg/mL of parent drug was injected with at least one of the CE/MS systems. Overall, the tested CE/MS systems provide complementary information as illustrated by the detection and identification of an unknown impurity in carbachol. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.     

Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.     

氯胺酮、羟亚胺及邻氯苯基环戊酮合成工艺的判别研究

氯胺酮通常以羟亚胺和邻氯苯基环戊酮为主要合成原料。该文采用气相色谱-质谱联用(GC-MS)检测缴获邻氯苯基环戊酮样品中的杂质,并筛选出邻氯苯甲腈以及格氏试剂合成过程中的副反应产物环戊酮、环戊醇、环戊基环戊烷、2-环戊基环戊酮和2-环戊基环戊醇作为特征杂质。通过邻氯苯基环戊酮合成实验,验证了6种特征杂质为邻氯苯甲腈与格氏试剂反应所产生。通过液-液萃取的前处理方法,从缴获羟亚胺样品检测出残留的4种特征杂质。采用顶空/气相色谱-质谱联用(HS/GC-MS)方法从氯胺酮样品中检出残留的2种特征杂质。结果表明,所有缴获的邻氯苯基环戊酮和羟亚胺样品,以及105份氯胺酮样品中的102份均检出2种以上特征杂质,由此推测,格氏试剂法是国内非法工厂生产邻氯苯基环戊酮普遍采用的工艺路线。     

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.     

Letter: A simple ion source set-up for desorption/ionization on silicon with ion mobility spectrometry and ion mobility spectrometry-mass spectrometry

Using a simple ion source set-up, laser desorption/ionization on silicon (DIOS) was demonstrated with the use of a custom-made drift tube ion mobility spectrometer (IMS), mounted on a commercial triple quadrupole mass spectrometer, and with an IMS equipped with a Faraday plate detector. DIOS was tested by mobility measurement of tetrapropylammonium iodide, tetrabutylammonium iodide and tetrapentylammonium iodide, whilst 2,6-di-tert- butylpyridine was used as a standard. The reduced mobilities measured for the test halides are in concordance with previously obtained ion mobility spectrometry-mass spectrometry data.     

Application of Mass Spectrometry and Chromatography–Mass Spectrometry in Drug Analysis

The review is devoted to the use of mass spectrometry and chromatography–mass spectrometry in various areas of pharmaceutical chemistry. The role of the above techniques in the structural identification of impurities in drug preparations and in the determination of the biotransformation behavior of pharmaceuticals in human and animal bodies is shown. The inactivation of drugs under the action of external factors (oxidation by atmospheric oxygen and the effects of moisture, heat, and light) is illustrated. The use of various ionization techniques and the spectra of metastable ions for determining the structures of components of biologically active substances are exemplified.     

Impurity profiling of acetylspiramycin by liquid chromatography–ion trap mass spectrometry

Investigation of acetylspiramycin (ASPM) and its related substances was carried out using a reversed-phase liquid chromatography/tandem mass spectrometry method. The identification of impurities in the ASPM complex was performed with a quadrupole ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode which provides MSⁿ capability. A total of 83 compounds were characterized in commercial samples, among which 31 impurities that had never been reported and 31 partially characterized impurities were deduced using the collision-induced dissociation (CID) spectra of major ASPM components as templates. Most of the major impurities arise from the starting materials and the synthesis process. This work provides very useful information for quality control of ASPM and evaluation of its synthesis process.     

Desorption electrospray ionization mass spectrometry: direct toxicological screening and analysis of illicit Ecstasy tablets

Desorption electrospray ionization mass spectrometry (DESI-MS) was used as a simple and rapid way to analyze drug tablets and powders without sample preparation. Experiments were performed with a home-made DESI source coupled to a triple-quadrupole linear-ion trap (QqQ(LIT)) mass spectrometer. Twenty-one commercial drugs as well as some illicit Ecstasy tablets and powders were analyzed. MS spectra almost exclusively showed the protonated or deprotonated ion of the drug after directing the pneumatically assisted electrospray onto the tablet's surface. With some tablets, inhomogeneity of the surface resulted in different spectra depending on the spot analyzed, thus showing that DESI could be used for imaging. Directly triggered MS/MS spectra were used for confirmatory analysis, with analysis times often below 10 s per tablet. For illicit Ecstasy tablets, DESI-MS, GC/MS and LC/MS analyses provided similar qualitative results for the main analytes. With MS/MS spectra library comparison or exact mass measurements, this technique could become very powerful for the rapid analysis of unknown tablets and shows the great potential of desorption techniques as an alternative to solution-based analysis.     

Application of liquid chromatography/ion trap mass spectrometry to the characterization of the related substances of clarithromycin

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method was developed for the characterization of components of the semi-synthetic macrolide clarithromycin. Mass spectral data were acquired online on a LCQ ion trap mass spectrometer equipped with an electrospray ionization source operated in the positive ion mode. One unknown compound was structurally elucidated and two other unknowns were characterized using the MS/MS and MS(n) collision-induced dissociation spectra of reference substances as interpretative templates, combined with knowledge of the nature of functional group fragmentation behaviour. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing clarithromycin.