Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H](+). The collision-induced dissociation of the molecular ion produced fragment ions at [MH - H2O](+), [MH - 2H(2)O](+), [MH - 3H(2)O](+). The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N - CO, O - CO, and C - OCC, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M + H]+ -> [MH - H2O](+), [MH - H2O](+), [MH - 2H(2)O](+), [MH-3H(2)O](+), 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was < 15% at broad linear dynamic ranges (0.0244-25nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Determination of five pharmacologically active compounds extracted from Rhodiola for natural product drug discovery with HPLC-APCI-MS

A rapid and sensitive liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS) assay for the determination of five pharmacologically active compounds (PAC) extracted from the traditional Chinese medicine, Rhodiola , namely salidroside, tyrosol, rhodionin, gallic acid, and ethyl gallate has been developed. In this method, PAC could be baseline separated and detected with DAD at 275 nm. The validation of the method, including sensitivity, linearity, repeatability, and recovery, was examined. The linear calibration curves were acquired with correlation coefficient >0.999 and the limits of detection LOD (at a signal-to-noise ratio=3:1) were between 0.058 and 1.500 mu mol/L. It was found, that the amounts of PAC varied with different species of Rhodiola . The established method is rapid and reproducible for the separation of five natural pharmacologically active compounds from extracts of Rhodiola with satisfactory results.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

Determination of sialic acids in the serum of cancer patients by capillary electrophoresis

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L-1 and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for

Alignment determination of symmetric top molecule using rotationally resolved LIF

This payer presents a concrete theoretical treatment which can be used for transforming the laser-induced fluorescence (LIF) intensity into the population and alignment parameters of a symmetric top molecule, The molecular population and alignment are described by molecular state multipoles. The results are presented in a general excitation-detection geometry and then specialized in some special geometries. The problem how to extract the initial molecular state multipoles from the rotationally resolved LIF intensity is discussed in detail. (C) 1999 Elsevier Science B.V. All rights reserved.

Determination of alcohols by high-performance liquid chromatography with fluorimetric detection after pre-column derivatisation with carbazole-9-N-acetic acid and chromatographic behaviour of alcoholic derivatives

Alcohols were derivatised to their carbazole-9-N-acetic acid (CRA) esters with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC . HCl) as the dehydrating agent. Studies on derivatisation conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives. The retention behaviour of alcohol derivatives was investigated by varying mobile phase compositions (ACN-water and MeOH-water). The parameters from the equation log k'=A-BX were evaluated by retention data of derivatives using an isocratic elution with different mobile phases. The results indicated that the parameters derived allowed computation of retention factors in good agreement with experiments. At the same time, a general equation was derived that makes possible predictions of partition coefficient in binary mobile phases with different proportions of organic solvent to water based on some simple regression analysis. The LC separation for the derivatised alcohols containing higher carbon alcohols showed good reproducibility on a reversed-phase C-18 column with gradient elution. The detection limits (excitation at 335 nm, emission at 360 nm) for derivatised alcohols (signal-to-noise ratio=3:1) were in the range of 0.1-0.4 pg per injection. (C) 2001 Elsevier Science B.V. All rights reserved.