Enantiomeric separations of illicit drugs and controlled substances using cyclofructan-based (LARIHC) and cyclobond I 2000 RSP HPLC chiral stationary phases.

Recently a novel class of chiral stationary phases (CSPs) based on cyclofructan (CF) has been developed. Cyclofructans are cyclic oligosaccharides that possess a crown ether core and pendent fructofuranose moieties. Herein, we evaluate the applicability of these novel CSPs for the enantiomeric separation of chiral illicit drugs and controlled substances directly without any derivatization. A set of 20 racemic compounds were used to evaluate these columns including 8 primary amines, 5 secondary amines, and 7 tertiary amines. Of the new cyclofructan-based LARIHC columns, 14 enantiomeric separations were obtained including 7 baseline and 7 partial separations. The LARIHC CF6-P column proved to be the most useful in separating illicit drugs and controlled substances accounting for 11 of the 14 optimized separations. The polar organic mode containing small amounts of methanol in acetonitrile was the most useful solvent system for the LARIHC CF6-P CSP. Furthermore, the LARIHC CF7-DMP CSP proved to be valuable for the separation of the tested chiral drugs resulting in four of the optimized enantiomeric separations, whereas the CF6-RN did not yield any optimum separations. The broad selectivity of the LARIHC CF7-DMP CSP is evident as it separated primary, secondary and tertiary amine containing chiral drugs. The compounds that were partially or un-separated using the cyclofructan based columns were screened with a Cyclobond I 2000 RSP column. This CSP provided three baseline and six partial separations.

Metal cation detection in positive ion mode electrospray ionization mass spectrometry using a tetracationic salt as a gas-phase ion-pairing agent: evaluation of the effect of chelating agents on detection sensitivity.

RATIONALE: The detection of metal cations continues to be essential in many scientific and industrial areas of interest. The most common electrospray ionization mass spectrometry (ESI-MS) approach involves chelating the metal ions and detecting the organometallic complex in the negative ion mode. However, it is well known that negative ion mode ESI-MS is generally less sensitive than the positive ion mode. To achieve greater sensitivity, it is necessary to examine the feasibility of detecting the chelated metal cations in positive ion mode ESI-MS. METHODS: Since highly solvated native metal cations have relatively low ionization efficiency in ESI-MS, and can be difficult to detect in the positive ion mode, a tetracationic ion-pairing agent was added to form a complex with the negatively charged metal chelate. The use of the ion-pairing agent leads to the generation of an overall positively charged complex, which can be detected at higher m/z values in the positive ion mode by electrospray ionization linear quadrupole ion trap mass spectrometry. RESULTS: Thirteen chelating agents with diverse structures were evaluated in this study. The nature of the chelating agent played as important a role as was previously determined for cationic pairing agents. The detection limits of six metal cations reached sub-picogram levels and significant improvements were observed when compared to negative ion mode detection where the metal-chelates were monitored without adding the ion-pairing reagent (IPR). Also, selective reaction monitoring (SRM) analyses were performed on the ternary complexes, which improved detection limits by one to three orders of magnitude. CONCLUSIONS: With this method it was possible to analyze the metal cations in the positive ion mode ESI-MS with the advantage of speed, sensitivity and selectivity. The optimum solution pH for this type of analysis is 5-7. Tandem mass spectrometry (MS/MS) further increases the sensitivity. Speciation is straightforward making this a broadly useful approach for the analysis of metal ions.

Degradation study of carnosic acid, carnosol, rosmarinic acid, and rosemary extract (Rosmarinus officinalis L.) assessed using HPLC.

Rosemary, whose major caffeoyl-derived and diterpenoid ingredients are rosmarinic acid, carnosol, and carnosic acid, is an important source of natural antioxidants and is being recognized increasingly as a useful preservative, protectant, and even as a potential medicinal agent. Understanding the stability of these components and their mode of interaction in mixtures is important if they are to be utilized to greatest effect. A study of the degradation of rosmarinic acid, carnosol, carnosic acid, and a mixture of the three was conducted in ethanolic solutions at different temperatures and light exposure. As expected, degradation increased with temperature. Some unique degradation products were formed with exposure to light. Several degradation products were reported for the first time. The degradation products were identified by HPLC/MS/MS, UV, and NMR. The degradation of rosemary extract in fish oil also was investigated, and much slower rates of degradation were observed for carnosic acid. In the mixture of the three antioxidants, carnosic acid serves to maintain levels of carnosol, though it does so at least in part at the cost of its own degradation.

Sensitive analysis of metal cations in positive ion mode electrospray ionization mass spectrometry using commercial chelating agents and cationic ion-pairing reagents.

Metals play a very important role in many scientific and environmental fields, and thus their detection and analysis is of great necessity. A simple and very sensitive method has been developed herein for the detection of metals in positive ion mode ESI-MS. Metal ions are positively charged, and as such they can potentially be detected in positive ion mode ESI-MS; however, their small mass-to-charge (m/z) ratio makes them fall in the low-mass region of the mass spectrum, which has the largest background noise. Therefore, their detection can become extremely difficult. A better and well-known way to detect metals by ESI-MS is by chelating them with complexation agents. In this study eleven different metals, Fe(II), Fe(III), Mg(II), Cu(II), Ru(III), Co(II), Ca(II), Ni(II), Mn(II), Sn(II), and Ag(I), were paired with two commercially available chelating agents: ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS). Since negative ion mode ESI-MS has many disadvantages compared to positive ion mode ESI-MS, it would be very beneficial if these negatively charged complex ions could be detected in the positive mode. Such a method is described in this paper and it is shown to achieve much lower sensitivities. Each of the negatively charged metal complexes is paired with six cationic ion-pairing reagents. The new positively charged ternary complexes are then analyzed by ESI-MS in the positive single ion monitoring (SIM) and single reaction monitoring (SRM) modes. The results clearly revealed that the presence of the cationic reagents significantly improved the sensitivity for these analytes, often by several orders of magnitude. This novel method developed herein for the detection of metals improved the limits of detection (LODs) significantly when compared to negative ion mode ESI-MS and shows great potential in future trace studies of these and many other species.

Coupling solid-phase microextraction and laser desorption ionization for rapid identification of biological material.

RATIONALE: Solid phase microextraction (SPME) use small fibers directly plunged in the solution under investigation to quickly extract and quantify by different techniques the amount of selected dissolved compounds. METHODS: Biological materials, peptides or proteins are accurately identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They are difficult to extract by SPME. This work looks for a chemical to be deposited onto fibers and able to act as a good SPME extractant as well as efficient matrix for MALDI detection. RESULTS: 3-Hydroxy-2-naphthoic acid (HNA) and 2-hydroxy-1-(2-hydroxy-4-sulfo-1-naphthylazo)-3-naphthoic acid (HHSNNA) were compared to two classical matrices: α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydrobenzoic acid (DHB). Bound to silica particles, DHB and HNA were found to be good MALDI matrices. Only the wide pore particles gave observable spectra. These particles were then attached in a thin layer onto wires to be used as fiber tips in SPME. Fibers loaded with peptides were introduced into the mass spectrometer to record fiber laser desorption ionization (FILDI) spectra. CONCLUSIONS: SPME-FILDI experiments could quickly identify peptides and proteins in solutions. More work is needed to find the best matrix and the way to fix it onto the fiber.

Use of ion pairing reagents for sensitive detection and separation of phospholipids in the positive ion mode LC-ESI-MS.

Phospholipids make up one of the more important classes of biological molecules. Because of their amphipathic nature and their charge state (e.g., negatively charged or zwitterionic) detection of trace levels of these compounds can be problematic. Electrospray ionization mass spectrometry (ESI-MS) is used in this study to detect very small amounts of these analytes by using the positive ion mode and pairing them with fifteen different cationic ion pairing reagents. The phospholipids used in this analysis were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), cardiolipin (CA) and sphingosyl phosphoethanolamine (SPE). The analysis of these molecules was carried out in the single ion monitoring (SIM) positive mode. In addition to their detection, a high performance liquid chromatography and mass spectrometry (HPLC-MS) method was developed in which the phospholipids were separated and detected simultaneously within a very short period of time. Separation of phospholipids was developed in the reverse phase mode and in the hydrophilic interaction liquid chromatography (HILIC) mode HPLC. Their differences and impact on the sensitivity of the analytes are compared and discussed further in the paper. With this technique, limits of detection (LODs) were very easily recorded at low ppt (ng L(-1)) levels with many of the cationic ion pairing reagents used in this study.

Mechanisms of ESI-MS selectivity and sensitivity enhancements when detecting anions in the positive mode using cationic pairing agents.

Recently, we have shown that dilute multivalent cationic reagents can be paired with analyte anions in ESI-MS, thereby allowing them to be detected in the positive mode at very low limits of detection. However, there can be differences in the efficiency of this technique depending on the nature of the cationic pairing agent and the anion being analyzed. In this study, three dicationic ion-pairing agents and four singly charged anionic species were examined in a series of experiments to elucidate the mechanism of action that allows for such sensitive detection and the profound differences in the selectivity of this ion-pairing method. The binding constants for the dication/anion complexes were determined by NMR and ESI-MS. The results indicated that the binding of these species is greatly enhanced as they move from the solution phase to the gas phase. Furthermore, surface tension measurements for the complexes were performed. This test revealed that, as the dication pairs with the anion, it creates a surface-active species within the ESI droplet. This is determined to be one of the major factors that leads to the overall sensitivity enhancement. This has led to a better understanding of how this ion-pairing technique produces unprecedented limits of detection for anions and why there are selectivity differences in pairing agents of different structures.

Detection of nucleotides in positive-mode electrospray ionization mass spectrometry using multiply-charged cationic ion-pairing reagents.

Nucleotides are a class of molecules that play an essential role in biological systems. A new method has been developed in the detection of nucleotides. These molecules can exist as monomers or constituents of oligomers and polymers. As such, they carry from one to several negative charges. In this study, different cationic ion-pairing reagents were used to complex with each of the 28 nucleotide monomers and nucleotide containing compounds. By using this method, this discrete set of anions was able to be detected in the positive-mode electrospray ionization mass spectrometry, as positively charged complexes. Tandem mass spectrometry experiments were also completed on the ion-pairing reagents that performed the best in the single ion monitoring (SIM) ion mode, and the sensitivity was lowered even further for most of the anions. Limits of detection for compounds such as thymidine diphosphate were improved as much as 100 times compared to the positive SIM mode, and 750 times when compared to the negative mode. A few nucleotides did not show a significant increase in sensitivity when analyzed in the positive ion mode, but in general the new method developed herein resulted in a much greater sensitivity than traditional detection in the negative mode.

Evaluation of tetracationic salts as gas-phase ion-pairing agents for the detection of trivalent anions in positive mode electrospray ionization mass spectrometry.

In previous studies, new electrospray ionization mass spectrometry (ESI-MS) approaches were developed for the highly sensitive detection of singly and doubly charged anions in positive mode ESI-MS by using specially synthesized dicationic and tricationic ion-pairing agents, respectively. By detecting the positively charged ion complex in the positive mode, limits of detection (LODs) for the anions can be lowered by several magnitudes. In this work, we used eighteen newly synthesized tetracationic ion-pairing agents, constructed with different geometries, linkages and cation moieties, for the detection of eighteen triply charged anions of different structural motifs. The LODs for these anions were from ten to several thousand times lower in the positive selective ion monitoring (SIM) mode than in the negative mode. These tetracationic agents also were shown to be useful for the detection of -1 and -2 anions. In addition, the LODs for -3 anions can be further lowered by monitoring the product fragments of the ion-pairing complexes in the single reaction monitoring (SRM) mode.

Positive mode electrospray ionization mass spectrometry of bisphosphonates using dicationic and tricationic ion-pairing agents.

A general method for detecting bisphosphonate drugs by ESI-MS and LC-ESI-MS as positive ions has been developed. Bisphosphonates can have multiple negative charges in solution. Tricationic ion-pairing reagents were paired with bisphosphonates to form a positively charged complex. It was clear that this facile pairing method worked. However, an appreciable presence of -1 bisphosphonate species were observed in positive mode ESI-MS (i.e. as the +2 complex with tricationic reagents). This led to an extended investigation on the use of dicationic pairing agents. The use of dicationic reagents improved the detection sensitivity for all of the bisphosphonates. Tandem mass spectrometry also improved the limits of detection for most of the bisphosphonates using both the tricationic and dicationic pairing reagents. A tricationic reagent also was used as an ion-pairing reagent in chromatography experiments. Thus the addition of a single reagent produced benefits in that it increased chromatographic retention and enhanced the ESI-MS detection of bisphosphonates.

The evaluation and comparison of trigonal and linear tricationic ion-pairing reagents for the detection of anions in positive mode ESI-MS.

A general and sensitive method for detecting divalent anions by ESI-MS and LC/ESI-MS as positive ions has been developed. The anions are paired with tricationic reagents to form positively charged complexes. In this study, four tricationic reagents, 2 trigonal and 2 linear, were used to study a wide variety of anions, such as disulfonates, dicarboxylates, and inorganic anions. The limits of detection for many of the anions studied were often improved by tandem mass spectrometry. Tricationic pairing agents can also be used with chromatography to enhance the detection of anions. The tricationic reagents were also used to detect monovalent anions by monitoring the doubly charged positive complex. The limits of detection for some of the divalent anions by this method are substantially lower than by other current analytical techniques.