p-Tolyl isocyanate derivatization for analysis of CWC-related polar degradation products by mass spectrometry.

Most of the precursors and/or degradation products related to the Chemical Weapons Convention (CWC) are polar. Identification of these molecules in environmental samples provides clues regarding the alleged usage and/or synthesis of the parent toxic chemicals. Such polar compounds need to be derivatized in order to analyze them by gas chromatography-mass spectrometry (GC-MS). In this study, we developed a new derivatizing reagent, para-tolyl isocyanate (PTI), for derivatization of polar CWC-related compounds. The PTI reagent selectively derivatizes the -OH and/or-SH functional groups with high efficiency, but does not react with carboxylic acid (-COOH) or phosphonic acid (-(O)P(OH)2) groups. The PTI derivatives of dialkyl aminoethanols, dialkyl aminoethanol-N-oxides, and 3-quinuclidinol were successfully eluted through GC, and their electron ionization (EI) mass spectra were distinct and provided the structure information by which the isomeric compounds can be easily distinguished. We also calculated the GC-retention index values that can be used for further confirmation of the target compounds. All the studied PTI derivatives can be analyzed by EI-MS with direct insertion probe and/or by direct electrospray ionization mass spectrometry (ESI-MS) together with the MS-MS data; both sets of data provide full structure information. The PTI reagent was found to be better in some respects than the conventional bistrimethylsilyl trifluoroacetamide (BSTFA), a trimethyl silylating reagent. The PTI reagent is commercially available, and the PTI derivatives are highly stable for months and are not sensitive to moisture. The applicability of the PTI derivatization for trace-level determination of the target CWC-related polar compounds in environmental matrices and in human plasma samples is also evaluated.

Mass spectral characterization of the CWC-related isomeric dialkyl alkylphosphonothiolates/alkylphosphonothionates under gas chromatography/mass spectrometry conditions.

RATIONALE: The isomeric dialkyl alkylphosphonothiolates and dialkyl alkylphosphonothionates are listed as scheduled chemicals of the Chemical Weapons Convention (CWC) implemented by the OPCW. The P-S and P-R bond connectivity has to be correctly identified for the verification of the CWC. The present study demonstrates successful identification of the target isomers by selective fragmentation under electron ionization (EI) or chemical ionization (CI) conditions. METHODS: All the studied isomeric compounds (27 in total) were synthesized in our laboratory using established methods, then analyzed by EI and CI gas chromatography/mass spectrometry (GC/MS) using an Agilent 6890 gas chromatograph equipped with a HP-5MS capillary column and interfaced to a 5973 N mass-selective detector. The retention index (RI) values of all the compounds were calculated using Van den Dool's formula. GC/MS/MS and GC/HRMS experiments were also performed using a VG-Autospec (magnetic sector) and JEOL-AccuToF (time-of-flight) mass spectrometer, respectively. RESULTS: The EI mass spectra of all the compounds had an abundant molecular ion at m/z 182, except in the case of a few selected butyl-substituted compounds, where this ion was of low abundance. The EI fragmentation pathways include α-cleavage, McLafferty rearrangement, McLafferty + 1 rearrangement, O/S-alkyl radical loss, and an alkene loss with a hydrogen shift. The characteristic fragment ions and their relative abundances are significant in elucidating the alkyl group attached to the P/S/O-atoms as well as the P-S/P = S bond connectivity. The EI and CI mass spectra together with RI values enable unambiguous identification of all the studied isomeric compounds. CONCLUSIONS: The present study highlights the structural characterization of the isomeric phosphonothiolates and phosphonothionates based on their selective EI fragmentation. The assigned fragmentation pathway helps in the assignment of P-S and P-alkyl connectivity in phosphonothiolates and phosphonothionates, consequently the structure of the unknown compounds. The EI mass spectra (27 compounds) of isomeric compounds are immensely useful in the OPCW official proficiency tests and for off-site analysis.

Differentiation of enantiomeric drugs by iodo-substituted L-amino acid references under electrospray ionization mass spectrometric conditions.

RATIONALE: In chiral differentiation by mass spectrometry, use of a single reference that differentiates various classes of compounds including drugs is ideal, but so far there are no such reports in the literature. We have successfully used iodo-substituted amino acids for the chiral differentiation of ten enantiomeric pairs of drugs. METHODS: To achieve the chiral differentiation, the trimeric Cu complex ion consisting of two chiral reference molecules and an analyte molecule was generated under positive ion electrospray ionization (ESI) conditions and subsequently subjected for collision- induced dissociation (CID) experiments using an LCQ ion trap mass spectrometer. The spectra were recorded under identical experimental conditions for both the enantiomers, and were averages of 30 scans. Cooks' kinetic method and chiral recognition ratio method (CR method) were used to arrive at the R(chiral) /CR values, respectively. RESULTS: The R(chiral) or CR values of the studied drugs are higher for 3,5-diiodo-L-tyrosine as the reference, than for 4-iodo-L-phenylalanine, except for isoproterenol and atenolol. Both the references show the same selectivity (R- or S-selectivity) towards all the studied drugs. With 3,5-diiodo-L-tyrosine as the reference, an R(chiral) value of 12.75 is obtained for DOPA and this is the highest reported value in the literature till now. The suitability of the current method in measuring enantiomeric excess is also demonstrated for DOPA. CONCLUSIONS: The use of 4-iodo-L-phenylalanine or 3,5-diiodo-L-tyrosine as a chiral reference for the chiral differentiation of ten enantiomeric pairs of pharmaceutically important drugs has been demonstrated. The chiral differentiation of pregabalin, tenofovir and pramipexole is reported for the first time. This study shows that it is possible to develop a single chiral reference compound for the differentiation of a group of chiral drugs having some similarities.

Mass spectrometry in India.

This review emphasizes the mass spectrometry research being performed at academic and established research institutions in India. It consists of three main parts covering the work done in organic, atomic and biological mass spectrometry. The review reveals that the use of mass spectrometry techniques started in the middle of the 20th century and was applied to research in the fields of organic, nuclear, geographical and atomic chemistry. Later, with the advent of soft and atmospheric ionization techniques it has been applied to pharmaceutical and biological research. In due course, several research centers with advanced mass spectrometry facilities have been established for specific areas of research such as gas-phase ion chemistry, ion-molecule reactions, proscribed chemicals, pesticide residues, pharmacokinetics, protein/peptide chemistry, nuclear chemistry, geochronological studies, archeology, petroleum industry, proteomics, lipidomics and metabolomics. Day-by-day the mass spectrometry centers/facilities in India have attracted young students for their doctoral research and other advanced research applications.

Mass spectral analysis of N-oxides of Chemical Weapons Convention related aminoethanols under electrospray ionization conditions.

N,N'-Dialkylaminoethanols are the hydrolyzed products or precursors of chemical warfare agents such as V-agents and nitrogen mustards, and they are prone to undergo oxidation in environmental matrices or during decontamination processes. Consequently, screening of the oxidized products of aminoethanols in aqueous samples is an important task in the verification of chemical weapons convention-related chemicals. Here we report the successful characterization of the N-oxides of N,N'-dialkylaminoethanols, alkyl diethanolamines, and triethanolamine using positive ion electrospray ionization mass spectrometry. The collision-induced dissociation (CID) spectra of the [M+H](+) and [M+Na](+) ions show diagnostic product ions that enable the unambiguous identification of the studied N-oxides, including those of isomeric compounds. The proposed fragmentation pathways are supported by high-resolution mass spectrometry data and product/precursor ion spectra. The CID spectra of [M+H](+) ions included [MH-CH(4)O(2)](+) as the key product ion, in addition to a distinctive alkene loss that allowed us to recognize the alkyl group attached to the nitrogen. The [M+Na](+) ions show characteristic product ions due to the loss of groups (R) attached to nitrogen either as a radical (R) or as a molecule [R+H or (R-H)] after hydrogen migration.

TGF-ß1-stimulation of NFATC2 and ATF3 proteins and their interaction for matrix metalloproteinase 13 expression in human breast cancer cells.

Activating transcription factor 3 (ATF3), an inducible stress gene, is stimulated by transforming growth factor-beta1 (TGF-ß1) in a protracted and relentless manner in human mammary cancer cells (hBC cells; MDA-MB231). The molecular mechanism behind this stable expression of ATF3 via TGF-ß1 in MDA-MB231 cells is unknown. This study found that TGF-ß1 stimulated the expression of the nuclear factor of activated T Cells 2 (NFATC2) in MDA-MB231 cells and provided evidence of its interaction with ATF3. The functional characterization of NFATC2 in association with ATF3 was determined by silencing of NFATC2 using siRNA. Knock-down of NFATC2 decreased the expression of both ATF3 and its target gene MMP13 (matrix metalloproteinase 13, a critical invasive gene) in hBC cells. Chromatin immunoprecipitation revealed that TGF-ß1 promoted NFATC2 binding and NFATC2-ATF3 complex binding at the MMP13 promoter region, whereas silencing of NFATC2 decreased their binding in hBC cells. Thus, we uncovered the mechanism of interaction between NFATC2 and ATF3 regulated by TGF-ß1, and NFATC2 acted as a pivotal factor in providing ATF3 stability and further drove MMP13 transcription. Targeting NFATC2 and blocking its association with ATF3 could therefore help to slow the progression of breast cancer.

3D-poly (lactic acid) scaffolds coated with gelatin and mucic acid for bone tissue engineering.

Three-dimensional (3D) printing is a promising technology to fabricate the intricate biomimetic structure. The primary focus of this study was to develop the bioactive 3D-scaffolds to enhance bone regeneration. The 3D-poly (lactic acid) (PLA) scaffolds were extruded based on a computer-aided design (CAD) model and coated with gelatin (Gel) containing different concentrations of mucic acid (MA) and were investigated for their osteogenic potential. Coating the PLA scaffolds with Gel/MA improved their physicochemical properties, and the addition of MA did not alter these properties. The viability of mouse mesenchymal stem cells (mMSCs, C3H10T1/2) seeded onto the PLA/Gel/MA scaffolds remained unaffected both at metabolic and cell membrane integrity levels. Alkaline phosphatase and von Kossa staining indicated the promotion of osteoblast differentiation of mMSCs by MA in the PLA/Gel scaffolds. Inclusion of MA in PLA/Gel scaffolds also increased the expression of the master bone transcription factor, Runx2, and other osteoblastic differentiation marker genes in mMSCs. Thus, our results suggested that the 3D-printed PLA scaffolds coated with Gel/MA favor osteoblast differentiation and have potential applications in bone tissue engineering.

Osteostimulatory effect of biocomposite scaffold containing phytomolecule diosmin by Integrin/FAK/ERK signaling pathway in mouse mesenchymal stem cells.

Non-availability of an ideal alternative for autografts in treating critical-size bone defects is a major challenge in orthopedics. Phytocompounds have been proven to enhance osteogenesis via various osteogenic signaling pathways, but its decreased bioavailability and increased renal clearance limit its application. In this study, we designed a biocomposite scaffold comprising gelatin (Gel) and nanohydroxyapatite (nHAp) incorporated with diosmin (DM) and we investigated its bone forming potential in vitro and in vivo. Physiochemical characterization of the scaffold showed that DM had no effect on altering the material characteristics of the scaffold. The addition of DM enhanced the osteoblast differentiation potential of the scaffold in mouse mesenchymal stem cells at both cellular and molecular levels, possibly via the integrin-mediated activation of FAK and ERK signaling components. Using the rat tibial bone defective model, we identified the effect of DM in Gel/nHAp scaffold on enhancing bone formation in vivo. Based on our results, we suggest that Gel/nHAp/DM can be a potential therapeutic agent in scaffold-mediated bone regeneration.

Stimulation of ATF3 interaction with Smad4 via TGF-ß1 for matrix metalloproteinase 13 gene activation in human breast cancer cells.

We previously reported that transforming growth factor-ß1 (TGF-ß1) stimulated the sustained and prolonged expression of activating transcription factor 3 (ATF3) in highly metastatic and invasive human breast cancer cells (MDA-MB231), in contrast to normal human mammary epithelial cells. However, the mechanism behind the stability of ATF3 expression is not yet known. Based on an in silico approach with co-immunoprecipitation and mass spectrometric analyses, we identified a number of proteins, including Smad4, that interacted with ATF3 after TGF-ß1 treatment in MDA-MB231 cells. The knockdown of Smad4 using the siRNA technique resulted in a significant loss of ATF3 expression in these cells. Chromatin immunoprecipitation was then used to identify the formation of an ATF3 and Smad4 complex at the matrix metalloproteinase 13 (MMP13) promoter upon TGF-ß1-treatment, and the knockdown of Smad4 decreased MMP13 promoter activity in MDA-MB231 cells. Our findings indicate that Smad4 is a pre-requisite for providing stability to ATF3 via TGF-ß1 in human breast cancer cells. The targeting of Smad4 may thus provide the sustainable loss of ATF3 expression that is needed to control breast cancer progression.

Osteogenic stimulatory effect of heraclenin purified from bael in mouse mesenchymal stem cells in vitro.

Osteoporosis is a major health concern occurring to the aging adult population across the globe. Currently, there is an increasing demand for treatment of osteoporosis with plant-based medicines. In the present study, we report that heraclenin was extracted and purified from unripe fruit portion of Bael (Aegle marmelos Corr.) using silica gel column chromatography. The identification and characterization of heraclenin were carried out by UV-Vis, HPLC, LC-MS, NMR, FT-IR, and XRD analyses. The standardized purification method recorded a yield efficiency of 42% heraclenin microcrystals with 99% purity from bael fruit. SEM image revealed the shape of the purified compound to be an orthorhombic-sphenoid prism. Cytotoxicity studies indicated that heraclenin-treatment did not alter cell viability in mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The mRNA expression of Runx2, a bone transcription factor was found to be stimulated by heraclenin in these cells. At the cellular level, heraclenin-treatment enhanced osteoblast differentiation and mineralization in mMSCs. Thus, these results suggested that heraclenin purified from bael fruit has an osteogenic effect, indicating its potential towards bone regeneration.

Proliferation and differentiation of mesenchymal stem cells on scaffolds containing chitosan, calcium polyphosphate and pigeonite for bone tissue engineering.

OBJECTIVES: Treatment of critical-sized bone defects with cells and biomaterials offers an efficient alternative to traditional bone grafts. Chitosan (CS) is a natural biopolymer that acts as a scaffold in bone tissue engineering (BTE). Polyphosphate (PolyP), recently identified as an inorganic polymer, acts as a potential bone morphogenetic material, whereas pigeonite (Pg) is a novel iron-containing ceramic. In this study, we prepared and characterized scaffolds containing CS, calcium polyphosphate (CaPP) and Pg particles for bone formation in vitro and in vivo. MATERIALS AND METHODS: Chitosan/CaPP scaffolds and CS/CaPP scaffolds containing varied concentrations of Pg particles (0.25%, 0.5%, 0.75% and 1%) were prepared and characterized by SEM, XRD, EDAX, FT-IR, degradation, protein adsorption, mechanical strength and biomineralization studies. The cytocompatibility of these scaffolds with mouse mesenchymal stem cells (mMSCs, C3H10T1/2) was determined by MTT assay and fluorescence staining. Cell proliferation on scaffolds was assessed using MUSE™ (Merck-Millipore, Germany) cell analyser. The effect of scaffolds on osteoblast differentiation at the cellular level was evaluated by Alizarin red (AR) and alkaline phosphatase (ALP) staining. At the molecular level, the expression of osteoblast differentiation marker genes such as Runt-related transcription factor-2 (Runx2), ALP, type I collagen-1 (Col-I) and osteocalcin (OC) was determined by real-time reverse transcriptase (RT-PCR) analysis. Bone regeneration was assessed by X-ray radiographs, SEM and EDAX analyses, and histological staining such as haematoxylin and eosin staining and Masson's trichrome staining (MTS) in a rat critical-sized tibial defect model system. RESULTS: The inclusion of iron-containing Pg particles at 0.25% concentration in CS/CaPP scaffolds showed enhanced bioactivity by protein adsorption and biomineralization, compared with that shown by CS/CaPP scaffolds alone. Increased proliferation of mMSCs was observed with CS/CaPP/Pg scaffolds compared with control and CS/CaPP scaffolds. Increase in cell proliferation was accompanied by G0/G1 to G2/M phase transition with increased levels of cyclin(s) A, B and C. Pg particles in CS/CaPP scaffolds enhanced osteoblast differentiation at the cellular and molecular levels, as evidenced by increased calcium deposits, ALP activity and expression of osteoblast marker genes. In vivo implantation of scaffolds in rat critical-sized tibial defects displayed accelerated bone formation after 8 weeks. CONCLUSION: The current findings indicate that CS/CaPP scaffolds containing iron-containing Pg particles serve as an appropriate template to support proliferation and differentiation of MSCs to osteoblasts in vitro and bone formation in vivo and thus support their candidature for BTE applications.

Mass spectral studies of meso-dialkyl, alkyl aryl and cycloalkyl calix(4)pyrroles under positive and negative ion electrospray ionization conditions.

A series of meso-dialkyl, alkyl aryl and cycloalkyl calix(4)pyrroles (1-15) are studied under positive and negative ion electrospray ionization (ESI) conditions. The positive ion spectra show abundant [M + H](+) and [M + Na](+) ions and the negative ion spectra show the [M + Cl](-) (the Cl(-) ions from the solvent) and [M - H](-) ions. The collision induced dissociation (CID) spectra of [M + H](+), [M + Na](+), [M + Cl](-) and [M - H](-) ions are studied to understand their dissociation pathway and compared to that reported for M(+) under electron ionization (EI) conditions. The beta-cleavage process that was diagnostic to M(+) is absent in all the CID spectra of the ions studied under ESI. Dissociation of all the studied ions resulted in the fragment ions formed by sequential elimination of pyrrole (A) and/or dialkyl/alkyl aryl/cycloalkyl (B) groups involving hydrogen migration to pyrrole ring at each cleavage of A--B bond, which clearly reveals the arrangement of A and B groups in the calix(4)pyrroles. The source of hydrogen that migrates to pyrrole ring during A--B bond cleavage is investigated by the experiments on deuterated compounds and [M + D](+) ions; and confirmed that the hydrogen attached to pyrrole nitrogen, hydrogen on alpha-carbon of alkyl group and the H(+)/Na(+) ion that added during ESI process to generate [M + H](+)/[M + Na](+) ions involve in the migration. The yields of [M + Na](+) ions are found to be different for the isomeric meso-cycloalkyl compounds (cycloheptyl, and 2-, 3- and 4-methyl cyclohexyl) and for normal and N-confused calix(4)pyrroles. The isomeric methyl and 3-hydroxy/4-hydroxy phenyl calix(4)pyrroles show specific fragmentation pattern during the dissociation of their [M - H](-) ions.

Alginate/Gelatin scaffolds incorporated with Silibinin-loaded Chitosan nanoparticles for bone formation in vitro.

Silibinin is a plant derived flavonolignan known for its multiple biological properties, but its role in the promotion of bone formation has not yet been well studied. Moreover, the delivery of Silibinin is hindered by its complex hydrophobic nature, which limits its bioavailability. Hence, in this study, we fabricated a drug delivery system using chitosan nanoparticles loaded with Silibinin at different concentrations (20µM, 50µM, and 100µM). They were then incorporated into scaffolds containing Alginate and Gelatin (Alg/Gel) for the sustained and prolonged release of Silibinin. The Silibinin-loaded chitosan nanoparticles (SCN) were prepared using the ionic gelation technique, and the scaffolds (Alg/Gel-SCN) were synthesized by the conventional method of freeze drying. The scaffolds were subjected to physicochemical and material characterization studies. The addition of SCN did not affect the porosity of the scaffolds, yet increased the protein adsorption, degradation rates, and bio-mineralization. These scaffolds were biocompatible with mouse mesenchymal stem cells. The scaffolds loaded with 50µM Silibinin promoted osteoblast differentiation, which was determined at cellular and molecular levels. Recent studies indicated the role of microRNAs (miRNAs) in osteogenesis and we found that the Silibinin released from scaffolds regulated miRNAs that control the bone morphogenetic protein pathway. Hence, our results suggest the potential for sustained and prolonged release of Silibinin to promote bone formation and, thus, these Alg/Gel-SCN scaffolds may be candidates for bone tissue engineering applications.

Scaffolds containing chitosan, gelatin and graphene oxide for bone tissue regeneration in vitro and in vivo.

Critical-sized bone defects are augmented with cell free and cell loaded constructs to bridge bone defects. Improving the properties of three-dimensional scaffolds with multiple polymers and others is of growing interest in recent decades. Chitosan (CS), a natural biopolymer has limitations for its use in bone regeneration, and its properties can be enhanced with other materials. In the present study, the composite scaffolds containing CS, gelatin (Gn) and graphene oxide (GO) were fabricated through freeze-drying. These scaffolds (GO/CS/Gn) were characterized by the SEM, Raman spectra, FT-IR, EDS, swelling, biodegradation, protein adsorption and biomineralization studies. The inclusion of GO in the CS/Gn scaffolds showed better physico-chemical properties. The GO/CS/Gn scaffolds were cyto-friendly to rat osteoprogenitor cells, and they promoted differentiation of mouse mesenchymal stem cells into osteoblasts. The scaffolds also accelerated bridging of the rat tibial bone defect with increased collagen deposition in vivo. Hence, these results strongly suggested the potential nature of GO/CS/Gn scaffolds for their application in bone tissue regeneration.

Mass spectral studies of a series of N,N-dialkyl aminoethyl-2-chlorides and trimethyl silyl ethers of N,N-dialkyl aminoethane-2-ols under electron impact conditions.

The electron impact (EI) mass spectra of a series of N,N-dialkyl-aminoethyl-2-chlorides, N(R(1))(R(2))-CH(2)-CH(2)Cl and trimethylsilyl ethers of N,N-dialkyl aminoethane-2-ols, N(R(1))(R(2))-CH(2)-CH(2)-O-Si(CH(3))(3), where R(1) and R(2) = methyl, ethyl, propyl and isopropyl, which are precursors of VX type of compounds, are studied. All the compounds (1-20) show abundant molecular ions, in addition to a weak [M - H](+) ion, except the N,N-diisopropyl group containing compounds (8 and 18). A general EI fragmentation pattern for the above two series of compounds is discussed. The observed fragment ions are due to simple homolytic cleavages, and they are distinct to allow the identification of the compounds unequivocally including those of isomeric compounds. The primary fragmentation of compounds 1-20 is beta-cleavage, i.e. homolytic cleavage of C-C bond, which is linked to the nitrogen atom. Three types of beta-cleavages are possible for these compounds, in which the abundance of beta-cleavage product ions is found to depend on the size and structure of the alkyl group attached to nitrogen. The alpha-cleavage fragment ions are found only for N,N-dialkyl aminoethyl-2-chlorides but are absent in the corresponding trimethylsilyl ethers of N,N-dialkyl aminoethane-2-ols. The retention indices are calculated for all the studied compounds (1-20) and are in the ranges of 750.38-1079.24 for 1-10 and 905.23-1190.25 for 11-20.

Characterisation of a series of acetals/ketals of bis(2-nitrophenyl) ethanediol and bis(4,5-dimethoxy-2-nitrophenyl) ethanediol under APCI mass spectrometric conditions.

A series acetals/ketals of aldehydes and ketones formed by the reaction of two photolabile protecting groups, bis(2-nitrophenyl) ethanediol and bis(4,5-dimethoxy-2-nitrophenyl) ethanediol (I and II, respectively), were analysed under EI, LSIMS, ESI and APCI conditions to obtain molecular weights as well as structural information. The EI and LSIMS techniques failed to give molecular weight information. The positive ESI yielded [M + H](+) ions only for I; however, with added Na(+) both I and II formed [M + Na](+) adducts. But upon decomposition, the [M + Na](+) ions yielded Na(+) ion as the only product ion. Similarly, under negative ion ESI conditions both I and II gave molecular weight information by forming adduct ions with halide anions (F(-), Cl(-), Br(-) and I(-)); however, they did not give structural information as they resulted in only the halide anion as the abundant fragment ion upon dissociation. All the compounds formed abundant M(-*) ions under negative ion APCI conditions, and their MS/MS spectra showed characteristic fragment ions; hence the acetals/ketals of I and II could be successfully characterized under negative ion APCI conditions.

Ion-pair solid-phase extraction and gas chromatography-mass spectrometric determination of acidic hydrolysis products of chemical warfare agents from aqueous samples.

The chemical warfare agents (CWA) degrade rapidly in aqueous samples and convert to acidic degradation products. Extraction and identification of the degradation products from complex matrices using simple sample preparation and sensitive detection and identification is the most important step in the off-site analysis of samples. In this present study, we report a simple sample preparation step based on ion-pair (IP) solid-phase extraction (SPE) for the extraction of acidic degradation products of CWA namely methyl, ethyl, propyl phosphonic acids, thiodiglycolic acid and benzilic acid. The analysis was performed on GC-MS in electron impact ionization mode. Three IP reagents triethylamine (TEA), tetrabutylammonium bromide (TBAB) and cetyltrimethyl ammonium bromide (CTAB) were used. The recoveries were estimated using the internal and external standard methods. The recovery of the compounds was almost negligible when TEA was used as IP reagent. The recoveries obtained when TBAB and CTAB were used as IP reagents were high and reproducible. The recovery of test chemicals is above 90%, except for methyl phosphonic acid and ethylphosphonic acid (20.6 +/- 3.2% and 35.8 +/- 2.5%, respectively). The minimum detection limits of the method were calculated for all chemicals in both full scan and selected ion monitoring modes. The test chemicals could be detected in microgram per litre quantities by the IP-SPE method.