Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

Identification of reaction products from the pyridinium dichromate derivatization of prostaglandins by high-performance liquid chromatography and direct chemical ionization mass spectrometry

The reaction products from the oxidation of prostaglandins with pyridinium dichromate have been identified by direct chemical ionization mass spectrometry of the underivatized compounds after separation by reversed-phase high-performance liquid chromatography and ultraviolet diode-array detection. The thermal influence on the reproducibility of the dehydration patterns of the mass spectra was studied. The main products from the prostaglandins E1, E2, F1 alpha and F2 alpha were the corresponding 15-oxo derivatives. Minor amounts of the 9,11,15-trioxoprostaglandin were formed from PGE, while the oxidation of PGF was less selective, yielding additional dioxo derivatives. Addition of water to the reagent reduced the reactivity, but increased the selectivity in favour of the formation of 15-oxo-PGF during the oxidation of PGF.     

Derivatization procedures for detection of prostaglandins in biological matrices by liquid chromatography/electrochemistry

Conventional reversed-phase HPLC conditions have been optimized for resolution of a mixture containing prostaglandins PGE(1), PGE(2), PGF(1alpha), and PGF(2alpha). Electroactive derivative-forming reagents, such as p-nitrobenzyloxyamine, 2-bromo-2'-nitroacetophenone, and 2,4-dinitrophenylhydrazine have been evaluated for use as precolumn reagents for forming prostaglandin derivatives. The results indicate that detection limits of 120 pg are achievable with amperometric detection. The utility of the procedures developed is illustrated by the detection of prostaglandins in human urine and plasma.     

Gas chromatographic-mass spectrometric profiling with negative-ion chemical ionization detection of prostaglandins and their 15-keto and 15-keto-13,14-dihydro catabolites in rat blood

A method was set up in which the primary prostaglandins (PGF2 alpha, PGD2, PGE2, thromboxane B2, 6-keto-PGF1 alpha and 6-keto-PGE1) and their catabolites (15-keto and 15-keto-13,14-dihydro) could be analyzed in the same sample at the same time. The method makes use of long capillary columns (60 m) to resolve the complex mixture during gas chromatography and mass fragmentography to provide the specificity of detection of these products. Selectivity and sensitivity is provided through use of appropriate derivatives (pentafluorobenzyl esters) which allow detection by negative-ion chemical ionization in which high-abundance fragments in the high end of the mass spectrum (M-pentafluorobenzyl) are observed. A purification procedure of whole blood is described involving diethyl ether extraction, C18 Sep-Pak chromatography, derivatization into the pentafluorobenzyl-O-methyloxime, C18 Sep-Pak and silicic acid chromatography followed by final derivatization into trimethylsilyl ethers for gas chromatographic-mass spectrometric analysis. Recovery of added [3H]PGF2 alpha was 73.8 +/- 2.2% (n = 10). Sample workup and analysis takes ten days for six samples. The method is sufficiently sensitive for the profiling of a 10-ml sample of whole blood (limit approximately 1 pg/ml; 1-pg injection on column).     

Differentiation of Boc- alpha,beta- and beta,alpha-peptides and a pair of diastereomeric beta,alpha-dipeptides by positive and negative ion electrospray tandem mass spectrometry (ESI-MS/MS)

Positive and negative ion electrospray ionization (ESI) tandem mass spectral study of a new series of hybrid peptides, viz, BocN-alpha,beta-peptides and BocN-beta,alpha-peptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and L-Ala has been carried out. The alpha,beta-peptides have been differentiated from beta,alpha-peptides by the collision-induced dissociation (CID) of [M + H]+ and [M - H]- ions in positive and negative ion ESI-MS respectively. The fragment ion [M + H - C(CH3)3 + H]+ formed from [M + H]+ ions by the loss of 2-methyl-prop-2-ene in alpha,beta-peptides with L-Ala at the N-terminus is insignificant or totally absent for beta,alpha-peptides which have the Caa (S) at N-terminus. The fragment ion [M - H-C(CH3)3OH - HNCO]- formed from [M - H]- of beta,alpha-peptide acids is totally absent for alpha,beta-peptide acids. This has been attributed to the absence of the beta-methylene group in alpha,beta-peptides, and the participation of the beta-methylene group in the loss of HNCO in beta,alpha-peptide acids is confirmed by the deuteration experiments. The CID of [M + H-Boc + H]+ ions of these peptides also produce characteristic fragmentation. In the CID spectra of alpha,beta-peptides, the b(n)+ ions and the resulting y(n)+ ions occur at a mass difference of 243 and 71 Da corresponding to the successive losses of Caa and L-Ala, whereas a mass difference of 71 and 243 Da is observed for beta,alpha-peptides. In contrast to the CID of protonated peptides, the CID of [M - H]- ions of the alpha,beta- and beta,alpha-peptide acids do not give b(n)- ions and show abundant z(n) (-) ions. Further, a pair of diastereomeric dipeptide esters and acids have been distinguished by the CID of [M + H]+ ions. The loss of 2-methyl-prop-2-ene is more pronounced for Boc-NH-Caa(R)-D-Ala-OCH3 (21) and Boc-NH-Caa(R)-D-Ala-OH (23) with Caa (R) at the N-terminus, whereas it is totally absent for Boc-NH-Caa (S)-D-Ala-OCH3 (22) and Boc-NH-Caa(S)-D-Ala-OH (24) peptides, which have Caa (S) at the N-terminus. Thus, on the basis of our previous and present studies, we propose that the CID of [M + H]+ ions provides a simple and useful method for distinguishing the configuration of Caa (S or R) at the N-terminus of BocN-carbo beta,alpha- and beta,beta-dipeptides.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.     

Gas phase synthesis and reactivity of Agn+ and Ag(n-1)H+ cluster cations

Multi-stage mass spectrometry (MSn) on [(M + Ag - H)x + Ag]+ precursor ions (where M = an amino acid such as glycine or N,N-dimethylglycine) results in the formation of stable silver (Ag3+, Ag5+ and Ag7+) and silver hydride (Ag2H+, Ag4H+ and Ag6H+) cluster cations in the gas phase. Deuterium labelling studies reveal that the source of the hydride can be either from the alpha carbon or from one of the heteroatoms. When M = glycine, the silver cyanide clusters Ag4CN+ and Ag5(H,C,N)+ are also observed. Collision induced dissociation (CID) and DFT calculations were carried out on each of these clusters to shed some light on their possible structures. CID of the Agn+ and Ag(n-1)H+ clusters generally results in the formation of the same Ag(n-2)+ product ions via the loss of Ag2 and AgH respectively. DFT calculations also reveal that the Agn+ and Ag(n-1)H+ clusters have similar structural features and that the Ag(n-1)H+ clusters are only slightly less stable than their all silver counterparts. In addition, Agn+ and Ag(n-1)H+ clusters react with 2-propanol and 2-butylamine via similar pathways, with multiple ligand addition occurring and a coupled deamination-dehydration reaction occurring upon condensation of a third (for Ag2H+) or a fourth (for all other silver clusters) 2-butylamine molecule onto the clusters. Taken together, these results suggest that the Agn+ and Ag(n-1)H+ clusters are structurally related via the replacement of a silver atom with a hydrogen atom. This replacement does not dramatically alter the cluster stability or its unimolecular or bimolecular chemistry with the 2-propanol and 2-butylamine reagents.     

混合二肽的场解吸质谱

为了探索生物化学、生物医学中混合寡肽的分析问题,对未衍生的混合二肽场解吸质谱进行了研究(图1和图2)。在图1图2中出现了所有二肽的准分子离子[M+H]~+,除此外还出现了[2M+H]~+、[M_(AB)+H]~+、[M_(AC)+H]~+等集聚离子,这些集聚离子能用于进一步确定各个肽的分子量。     

High-performance liquid chromatographic detection of myocardial prostaglandins and thromboxanes

Reperfusion of ischemic myocardium is associated with the breakdown of membrane phospholipids and a corresponding increase in arachidonic acid, ultimately resulting in the production of prostaglandins (PGs) and thromboxanes (TXs). However, quantification of these arachidonic acid metabolites has been limited to radioimmunoassay because of their presence in extremely low amounts. In this report, we describe a method suitable to detect sub-picogram levels of 6-keto-PGF1 alpha, PGF1 alpha, PGE2 and TXB2 in myocardial perfusates by high-performance liquid chromatography (HPLC) with a high-gain photomultiplier and a xenon-mercury are lamp. Strong Raleigh scatter of the lamp was eliminated by both interference and long-pass cut-off filters. Improved sample clean-up and HPLC separation were achieved by an HPLC system with an Ultrasphere 3-microns C18 column.     

Differentiation of the diastereomeric synthetic precursors of isofebrifugine and febrifugine by electrospray ionization tandem mass spectrometry

Febrifugine is an alkaloid with potent antimalarial activity isolated from Dichroa febrifuga and Hydrangea umbellate, and it exists naturally with its diastereomeric component, isofebrifugine. Here we report the differentiation of diastereomeric synthetic precursors of isofebrifugine (1, cis) and febrifugine (2, trans) and a structurally similar model diastereomeric pair without a halogen substituent (3 and 4) by electrospray ionization (ESI) tandem mass spectrometry. Compounds 1-4 contain a tert-butoxycarbonyl (BOC) substituent, and the collision-induced dissociation (CID) spectra of the [M+H](+), [M+Na](+) and [M+Li](+) ions of 1-4 include the expected product ions corresponding to the loss of C(4)H(8) (isobutene) and of C(5)H(8)O(2) (BOC-H). Loss of C(5)H(8)O(2) is dominant in cis isomers (1 and 3) and/or loss of C(4)H(8) ions is dominant in trans isomers (2 and 4). The decomposition of [M+H](+) ions shows stereoselectivity in the formation of the [M+H-(BOC-H)-C(3)H(5)OBr](+) and [M+H-(BOC-H)-C(6)H(5)CH(2)OH](+) ions. The [M+Cat](+) ions (where Cat = Na or Li) additionally show loss of NaBr and HBr from [M+Cat-(BOC-H)](+), and these product ions are constantly more abundant in cis isomers than in trans isomers. The stereoselectivity for the product ion corresponding to the loss of [(BOC-H)+C(3)H(5)OBr] from [M+H](+) ions differs from that from [M+Cat](+) ions.     

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.     

Field dependence of mobilities for gas-phase-protonated monomers and proton-bound dimers of ketones by planar field asymmetric waveform ion mobility spectrometer (PFAIMS)

The dependence of the mobilities of gas-phase ions on electric fields from 0 to 90 Td at ambient pressure was determined for protonated monomers [(MH+(H2O)n] and proton bound dimers [M2H+(H2O)n] for a homologous series of normal ketones, from acetone to decanone (M=C3H6O to C10H20O). This dependence was measured as the normalized function of mobility alpha (E/N) using a planar field asymmetric waveform ion mobility spectrometer (PFAIMS) and the ions were mass-identified using a PFAIMS drift tube coupled to a tandem mass spectrometer. Methods are described to obtain alpha (E/N) from the measurements of compensation voltage versus amplitude of an asymmetric waveform of any shape. Slopes of alpha for MH+ versus E/N were monotonic from 0 to 90 Td for acetone, butanone, and pentanone. Plots for ketones from hexanone to octanone exhibited plateaus at high fields. Nonanone and decanone showed plots with an inversion of slope above 70 Td. Proton bound dimers for ketones with carbon numbers greater than five exhibited slopes for alpha versus E/N, which decreased continuously with increasing E/N. These findings are the first alpha values for ions from a homologous series under atmosphere pressure and are preliminary to explanations of alpha (E/N) with ion structure.     

Electrospray tandem mass spectrometry of alkali-cationized BocN-carbo-alpha,beta- and -beta,alpha-peptides: Differentiation of positional isomers

Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-alpha,beta- and -beta,alpha-carbopeptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and alpha-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized alpha,beta- and beta,alpha-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x(n)+, y(n)+, z(n)+) and N-terminal (a(n)+, b(n)+, c(n)+) ions. Another noteworthy difference is cationized beta,alpha-peptides eliminate a molecule of ammonia whereas this pathway is absent for alpha,beta-peptides. This is useful for identifying the presence of a beta-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [b(n) + 17 + Cat]+ (n = 1 to n-1) ions which is diagnostic for distinguishing between alpha- and beta-amino acid at the C-terminus. MS(n) experiments of [b(n) + Li-H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [a(n) + Li-H]+ and cationized nitrile product ions which render support to earlier proposals that b(n)+ or [b(n) + Cat-H]+ ions have protonated or cationized oxazolinone structures, respectively.     

Silica capillary gas chromatography of prostaglandins with electron-capture detection and its application to the forensic investigation of sexual offences

Low picogram levels of the E series prostaglandins, PGE1, PGE2, 19-hydroxy PGE1 and 19-hydroxy PGE2, in human semen were analysed by silica capillary column gas chromatography with electron-capture detection after conversion to the methyl ester O-trimethylsilyl derivatives of the corresponding B series prostaglandins. The method was used to detect traces of semen on post-coital vaginal swabs, and on rectal, oral and skin swabs after simulated sexual acts. Semen was detectable on a vaginal swab taken 58 h after intercourse, and was readily detectable for at least 6 h on rectal and skin swabs. Preliminary results suggest that the ratios of prostaglandins on vaginal swabs may indicate how recently intercourse occurred.     

The structure of a urinary metabolite of prostaglandin F2a in man

Studies on metabolism of prostaglandin E2 in man have led to the identification of a dicarboxylic acid as a major urinary metabolite. This study examiens the structure of urinary metabolites of PGF2 alpha in man. Female subjects were injected intravenously with radioactive PGF2 alpha (35 mcg, 200 mcCi/mcmole) and unlabeled PGF2 alpha. Urine samples were collected and processed as described. Gas-liquid chromatography and mass spectrometry were used to analyze the C values of the derivatives. The structures of the different derivatives are described. A metabolite of the PGF2 alpha is shown to correspond to a metabolite formed from PGE2. The metabolites were found to differ only in the functional group at C-5. Further studies on the structure of the remaining urinary metabolites of PGF2 alpha are being conducted in the authors' laboratory.     

Internal energy deposition in chemical ionization/tandem mass spectrometry

The efficiency of the collision-induced dissociation (CID) process as a function of the internal energy deposited into the ion during the ionization event was evaluated. (M + H)+ ions of pyrrole, pyrrolidine, pyridine and piperidine (five and six-membered ring heterocyclics) were generated by chemical ionization (CI). The internal energy of the ions was varied by using different reagent gases. Both high-energy (keV) and low-energy (eV) CID were performed on these ions. The experiments showed that the (M + H)+ ions of the five-membered ring compounds, pyrrole and pyrrolidine, have higher fragmentation efficiencies than the six-membered ring compounds, pyridine and piperidine. Fragmentation efficiencies in high-energy CID clearly correlate with the internal energy deposited by the ionization technique. Experiments showed that the low-energy CID process is more sensitive than high-energy CID to changes in internal energy.     

Plasma chromatography of heroin and cocaine with mass-identified mobility spectra.

Plasma chromatography detects and identifies compounds in trace quantities at atmospheric pressure through characteristic positive and negative mobility spectra. To facilitate use of the technique to detect gas chromatographic effluents, a number of reference mobility spectra for different classes of compounds have been reported. Reference spectra for two more compounds, heroin and cocaine, are presented in this study. The primary ions found in these mobility spectra were determined to be M+, (M - H2)+, and (M - CH3CO2)+ for heroin and M+, (M - C6H5CO2)+ and (M - C6H5CO2 - CO2CH3)+ for cocaine using a directly interfaced plasma chromatograph-mass spectrometer. The identified ions agree closely with those predicted in the ion mobility spectra using mass-mobility correlation data coupled with chemical ionization mass spectrometry data. Also, an independent check demonstrating the reliability of reduced mobility values reported in earlier reference spectra was made.     

Human liver dihydrodiol dehydrogenase 1-catalyzed reaction generating 9alpha,11beta-prostaglandin F2 from prostaglandin D2 followed by micellar electrokinetic chromatography

An enzyme reaction converting prostaglandin D2 (PGD2) into 9alpha,11beta-prostaglandin F2 (9alpha,11beta-PGF2) by a human liver-originated recombinant dihydrodiol dehydrogenase 1 (DD1) has been studied using CE. Four prostaglandins, viz. PGD2, 9alpha,11beta-PGF2, PGE2, and PGF2a (see Fig. 1, the latter two major PGs are possibly coexisting compounds in the assay mixtures), were completely separated by using SDS or PEG as buffer additives. Because analysis times in the SDS system were shorter than in the PEG system, SDS was employed in measurements of the enzyme activity of DD1. The pH dependence and the reaction temperature dependence of enzyme activity have been studied. The present method enabled us to detect all of the participants in the enzyme reaction: PGD2, 9alpha,11beta-PGF2, nicotinamide adenine dinucleotide phosphate (NADPH), and NADP+. Thus, direct, comprehensive, and reliable analysis of the enzyme reaction becomes possible. Enzyme activity has hitherto been estimated indirectly from the decrease of fluorescence derived from NADPH as an index of progress of the enzyme reactions in batch methods employed in conventional studies. In addition, the low sample consumption characteristic of CE should be a significant advantage of the present method in characterization of less commonly available enzymes such as the recombinant species used in this work.     

Simultaneous analysis of prostanoids using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.