Analysis of platelet-activating factor by GC-MS after direct derivatization with pentafluorobenzoyl chloride and heptafluorobutyric anhydride

Parallel analysis of platelet-activating factor (PAF) using chemical ionization gas chromatography-mass spectrometry after direct derivatization with pentafluorobenzoyl chloride (PFB) and heptafluorobutyric anhydride (HFB) provides a facile and highly sensitive means for detecting and elucidating the structure of the numerous alkyl-chain homologs of this acetylated phospholipid autacoid. In the present study, the PFB derivative was used for initial electron capture negative ion chemical ionization analysis of PAF candidate molecules in human PMN extracts of unknown composition. Subsequent pulsed positive ion/electron capture negative ion chemical ionization evaluation of the HFB derivative furnished a measure of the molecular weight from [MH]+ and yielded the required structural information from characteristic negative ions, in particular [M-(2HF + ketene)]- and [M-(HF + acetic acid)]-. These procedures easily permitted confirmation of the presence of C16:0-, C17:0-, C18:0-, and C18:1-AGEPC (acetyl glyceryl ether phosphocholine) in extracts of stimulated human PMN and also demonstrated that the C17:0- homolog was comprised of both straight-chain and branch-chain varieties.     

Isotopic exchange during derivatization of platelet activating factor for gas chromatography-mass spectrometry

One approach to the quantitative analysis of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; also referred to as AGEPC, alkyl glyceryl ether phosphocholine) is hydrolytic removal of the phosphocholine group and conversion to an electron-capturing derivative for gas chromatography-negative ion mass spectrometry. [2H3]Acetyl-AGEPC has been commonly employed as an internal standard. When 1-hexadecyl-2-[2H3]acetyl glycerol (obtained by enzymatic hydrolysis of [2H3]-C16:0 AGEPC) is treated with pentafluorobenzoyl chloride at 120 degrees C, the resulting 3-pentafluorobenzoate derivative shows extensive loss of the deuterium label. This exchange is evidently acid-catalyzed since derivatization of 1-hexadecyl-2-acetyl glycerol under the same conditions in the presence of a trace of 2HCl results in the incorporation of up to three deuterium atoms. Isotope exchange can be avoided if the reaction is carried out at low temperature in the presence of base. Direct derivatization of [2H3]-C16:0 AGEPC by treatment with pentafluorobenzoyl chloride or heptafluorobutyric anhydride also results in loss of the deuterium label. The use of [13C2]-C16:0 AGEPC as an internal standard is recommended for rigorous quantitative analysis.     

Determination of platelet-activating factor and alkyl-ether phospholipids by gas chromatography-mass spectrometry via direct derivatization

A mass spectrometric method has been developed for the quantitative analysis of platelet-activating factor (PAF) and lyso-platelet-activating factor (lyso-PAF) based on electron-capture gas chromatography-mass spectrometry using a stable-isotope dilution technique. The cleavage and derivatization was accomplished in a single step by direct reaction of phospholipid with pentafluorobenzoyl chloride at 150 degrees C. Spectroscopic and chromatographic data indicated that PAF and lyso-PAF were converted into derivatives containing a pentafluorobenzoyl group in place of the original phosphocholine group with 95 and 51% yield, respectively. Additionally, in the lyso-PAF derivative, the free hydroxyl group was found to be replaced by chlorine. Phosphatidylcholines containing an arachidonoyl group can be derivatized with a solution of PFBCl/chloroform at 120 degrees C for 18 h, producing 90% derivative. Analysis by GC/MS and LC/MS allowed the detection of 1 or 250 pg derivative, respectively, injected onto the column with S/N greater than 3. Newly available analogues of high isotopic purity containing either three or four deuterium atoms located in the 1-O-hexadecyl chain were used as internal standards. The developed GC/MS assay was used to quantitate PAF and lyso-PAF in rabbit leukocytes before and after stimulation with calcium ionophore. The levels of PAF in unstimulated cells were in the order of 2.27 pmol/10(6) cells and increased about 17-fold during 10-min stimulation with 2 microM ionophore A23187. The lyso-PAF levels in resting cells were in the order of 3.76 pmol/10(6) cells and increased 1.7-fold during stimulation. This assay exhibited satisfactory sensitivity, reproducibility, and accuracy.     

GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis

The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.     

Development of a mass spectrometric method to quantitate platelet activating factor in mouse urine

Platelet activating factor (PAF) is a lipid mediator of inflammation released by a variety of stimulated inflammatory cells. It may be involved in immune glomerulonephritis. Thus, its measurement in urine could give information on the mechanism of this disease. We present here a method to measure PAF in mouse urine, using gas-liquid chromatography-mass spectrometry (GLC-MS) in the selected ion recording (SIR) mode. Before instrumental analysis, the extracted and purified samples were hydrolyzed and derivatized with pentafluorobenzoyl chloride. Different experimental conditions are presented and discussed to corroborate the analytical findings. PAF levels in mouse urine were 2.08 +/- 0.46 ng/24 h. This procedure might represent a new experimental tool to establish the possible role of PAF as mediator of tissue damage in renal disease.     

Femtomole detection of amino acids and dipeptides by gas chromatography—negative-ion chemical ionization mass spectrometry following alkylation with pentafluorobenzyl bromide

Amino acids and di- and tripeptides were derivatized by extractive alkylation using pentafluorobenzyl bromide (PFBBr) followed by reaction with heptafluorobutyric anhydride (HFBA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Good chromatographic separation and the formation of intense diagnostic ions were observed for the derivative when examined using gas chromatography—negative-ion chemical ionization mass spectrometry (GC—NICI-MS). Of the 20 amino acids investigated, only Arg and Glu could not be detected by this method. Also, dipeptides which included neutral amino acid residues were derivatized with more success than those containing either acidic or basic residues. Each of the amino acids or dipeptides formed one major derivative with the exception of Asn which formed two derivatives with either one or two HFB groups. This derivatization method was optimized with respect to the reaction temperature, reaction time, and choice of derivatizing reagents. Recoveries of derivatized [³H]-labeled Phe, Lys, and Thr were 76, 55, and 34%, respectively. Linearity was observed from 10 to 2000 pg of Ala per vial; selected-ion monitoring provided a detection limit of less than 150 fg with a signal-to-noise (S/N) ratio of 80 to 1. This method has proven to work well with urine samples and shows great promise for the detection of small peptides at low levels.     

Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography-electrospray ionization-mass spectrometry

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).     

Electron-capture gas chromatographic assay for primaquine in blood

A sensitive gas chromatographic method for the quantitative determination of the anti-malarial drug primaquine is described. The method involves derivatization with heptafluorobutyric anhydride to form the diheptafluorobutyramide derivative after a single extraction at alkaline pH. The derivatives are quantitated by electron-capture gas chromatography. Blood levels of primaquine as low as 8 ng/ml can be measured with good precision.     

Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry

Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

New flourinated chitin derivatives: synthesis, characterization and cytotoxicity assessment

New fluorinated chitin derivatives have been synthesized and characterized. Fluorination of chitin was achieved by facile homogenous reaction of chitin solution with diethyl amino sulfur trifluoride (C₄H₁₀NSF₃). The degree of substitution of the C6-hydroxyl functionality of N-acetyl-glucosamine repeat unit ranged from 50 to 98%, achieved by varying the reaction time from 1 to 144 h at room temperature. The use of pentafluoropropionic anhydride, trifluoromethylbenzoyl chloride and pentafluorobenzoyl chloride gave fluoro-chitin derivatives with 40, 10 and 5% substitution, respectively. Solid-state nuclear magnetic resonance and Fourier-transform infrared spectroscopy, powder X-ray diffraction, and elemental analysis support the identity of all fluorinated chitin derivatives. The fluorinated chitin derivatives were subjected to MTT assay using human (ATCC CCL-186) and mouse (ATCC CCL-1) fibroblast cell lines. Fluorinated chitin derivatives prepared from C₄H₁₀NSF₃ at 1, 6, 12, 72 and 96 h showed good cell viability of 80–100% for human fibroblast and 60–70% for mouse fibroblast. The % cell viability for the other fluorinated chitin derivatives were above 60% for both cell lines.     

Pentafluorobenzoyl derivatives of doping agents : I. Extractive benzoylation and gas chromatography with electron-capture detection of primary and secondary amines

A sensitive and rapid method for the gas chromatographic (with electron-capture detection) confirmation of derivatizable sympathomimetic amines is described. Extractive derivatization with pentafluorobenzoyl chloride is performed on 2-ml urine or plasma samples. Especially for primary amines, the method appears to be very sensitive. Mass spectral data allowed confirmation of the monobenzoylation of all congeners.     

Bis(trifluoromethyl)aryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of low levels of clonidine in plasma

A gas chromatographic mass spectrometric assay for clonidine in plasma with a detection limit of a few picograms per ml was required. The p-trifluoromethylbenzyl, pentafluorobenzyl and pentafluorobenzoyl derivatives of clonidine were synthesized and the electron capture negative ion chemical ionization mass spectra of these compounds show extensive fragmentation with prominent ions at m/z 35 and 37 due to the two chlorine atoms in the clonidine molecule. Selected ion monitoring of specific high mass ions in these mass spectra indicated that the required sensitivity could not be obtained with these derivatives. Several bis(trifluoromethyl)pyrimidines were synthesized and these compounds were found to give an intense negative ion current under conditions of resonance electron capture. Consequently, a derivative of clonidine containing a bis(trifluoromethyl)aryl group was synthesized by reacting the drug with 3,5-bis(trifluoromethyl)benzoyl chloride. The negative ion mass spectrum of the reaction product has a base peak at m/z 673 and, when this ion is specifically monitored, an amount of derivative equivalent to 1 picogram of clonidine can be detected. This allowed the development of an assay for clonidine in plasma with a precision of 8% (SD) at 50 pg ml-1, 22% (SD) at 20 pg ml-1 and a lower limit for quantitative determination of 10 pg ml-1. Plasma concentrations of clonidine in 10 subjects given a single 50 micrograms oral dose are reported.     

Synthesis of 1-acyl-2-[3H]acetyl-SN-glycero-3-phosphocholine, a structural analog of platelet activating factor, by vascular endothelial cells

Human umbilical vein endothelial cells (HUVECS) were challenged with thrombin in the presence of [3H]acetate to stimulate the production of radiolabeled platelet activating factor (PAF, 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine, 1-O-alkyl-2-[3H]acetyl-GPC). The 3H-product was isolated by thin-layer chromatography, and 1-radyl-2[3H],3- diacetylglycerols were prepared by phospholipase C digestion and subsequent acetylation at the sn-3 position. When the 1-radyl-2[3H],3-diacetylglycerols were analyzed by zonal thin-layer chromatography, 96-97% of the radiolabeled derivative migrated with 1-acyl-2,3-diacetylglycerol standard. Only minor amounts (3-4%) of 1-alkyl-2[3H],3-diacetylglycerol were observed, demonstrating that the predominant acetylated product synthesized by thrombin-stimulated HUVECS was 1-acyl-2-[3H]acetyl-GPC. This relative abundance of 1-acyl-2-[3H]-acetyl-GPC was not significantly affected by thrombin dose, incubation time, or cell passage, and was also observed in HUVECS challenged with ionophore A23187. In addition, the acetylated product from ionophore A23187- or bradykinin-stimulated bovine aortic endothelial cells contained 90% 1-acyl-2-[3H]acetyl-GPC, suggesting that the synthesis of the 1-acyl PAF analog is not unique to HUVECS. These findings demonstrate that PAF is a minor synthetic component of HUVECS and bovine aortic endothelial cells. In light of the integral role which the vascular endothelial cell plays in the regulation of thrombosis, these findings also suggest that the production of 1-acyl-2-acetyl-GPC may be biologically important.     

A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides.

A rapid and quantitative procedure is described for the re-N-acetylation of amino sugar methyl glycosides prior to their analysis by gas-liquid chromatography. Two equivalents of pyridine are added to acidic methanolysates containing amino sugars, serving both to neutralize the acid and to act as a catalyst for the subsequent N-acetylation reaction with acetic anhydride. The N-acetylation is quantitative and complete within 10 min at ambient temperature. Excess acetic anhydride is destroyed by solvolysis with the methanolic solvent. The procedure has been used effectively for methanolysates containing 0.01–2.0 mg/ml glucosamine. The procedures utilizing ion-exchange columns and insoluble salts are thus circumvented and all reaction byproducts are volatile. The procedure is therefore ideally suited for the simultaneous workup of numerous samples for analytical procedures such as gas-liquid chromatography.     

Determination of low levels of poly(ethylene glycol) 400 in plasma and urine by capillary gas chromatography-selected ion-monitoring mass spectrometry after solid-phase extraction

A convenient and sensitive method for the quantitative determination of poly(ethylene glycol) 400 in plasma and urine with capillary gas chromatography-mass spectrometry has been developed. The sample preparation involves solid-phase extraction with subsequent derivatization with heptafluorobutyric anhydride, which proved to give the most stable derivative. The derivatization procedure was optimized using experimental design, and different solid-phase extraction columns were evaluated. The limit of quantitation was 1 μmol/l (0.4 μg/ml) for both plasma and urine.     

The preparation of bile acid amides and oxazolines, II, the synthesis of the amides and oxazolines of ursodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid and cholic acid

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases $\text{in vitro}$, and inhibit the growth of certain anaerobic bacteria in pure culture.     

Detection of methylated asparagine and glutamine residues in polypeptides

A residue of gamma-N-methylasparagine (gamma-NMA) is found at position beta-72 of many phycobiliproteins. delta-N-Methylglutamine is present in some bacterial ribosomal proteins. gamma-NMA was synthesized by reacting the omega-methyl ester of aspartate with methylamine and delta-N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the omega-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The gamma-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-gamma-NMA co-eluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic gamma-NMA to prepare the PTH derivative. The PTH-delta-N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.     

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.     

Quantitative assay of conjugated and free bile acids as heptafluorobutyrate derivatives by gas-liquid chromatography.

Quantitative analyses of individual bile acids in biological samples are limited by the lengthy multistep preparations necessary. Using heptafluorobutyric acid anhydride in pyridine as derivatizing agent, we reduced several steps to one. Bile acids and their glycine and taurine conjugates form stable heptafluorobutyrate derivatives, climinating the need for deconjugation and preparation of methyl esters. The derivatives have been characterized by mass spectrometry, and optimum reaction yields have been determined. Operating conditions for analyzing the bile acid heptafluorobutyrates by gas-liquid chromatography on various column packings were investigated, and 0.5% QF-1 or 3% OV-255 was found suitable. The bile acid derivatives were identical whether starting with the bile acid or the glycine or taurine conjugates. The procedure was applied to a quantitative analysis of artificial mixtures of bile acids and bile conjugates, and also of human bile. The results compared favorably to those obtained with a 3 alpha- and 7 alpha-hydroxysteroid dehydrogenase fluorimetric method.