De novo biosynthesis of dihydrosphingosine-1-phosphate by sphingosine kinase 1 in mammalian cells

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.     

Characterization and direct quantitation of sphingoid base-1-phosphates from lipid extracts: a shotgun lipidomics approach

Here, we have extended shotgun lipidomics for the characterization and quantitation of sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) in crude lipid extracts in the presence of ammonium hydroxide by using precursor ion scanning of m/z 79.0 (corresponding to [PO(3)](-)) in the negative-ion mode. It is demonstrated that a broad linear dynamic range for the quantitation of both S1P and DHS1P and a detection limit at low amol/mul concentration are achieved using this approach. The developed method for the quantitation of sphingoid base-1-phosphates is generally simpler and more efficient than other previously published methods. Multiple factors influencing the quantitation of sphingoid base-1-phosphates, including ion suppression, extraction efficiency, and potential overlapping with other molecular species, were examined extensively and/or are discussed. Mass levels of S1P and DHS1P in multiple biological samples, including human plasma, mouse plasma, and mouse brain tissues (e.g., cortex, cerebellum, spinal cord, and brain stem), were determined by the developed methodology. Accordingly, this technique, as a new addition to shotgun lipidomics technology, will be extremely useful for understanding the pathways of sphingolipid metabolism and for exploring the important roles of sphingoid base-1-phosphates in a wide range of physiological and pathological studies.     

Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5'-phosphate binding domain exposed to the cytosol

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S1P is degraded either by S1P lyase or by S1P phosphohydrolase. The gene encoding mammalian S1P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-1-phosphate (DHS1P) lyase activity in most tissues. However, liver and heart exhibited high DHS1P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S1P phosphohydrolase, indicating that the degradation of S1P by two S1P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.     

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography-tandem mass spectrometry

D-erythro-sphingosine (Sph) and its phosphorylated product, d-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the levels of Sph and S1P in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC-MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol-0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1-100 ng/mL for Sph and 0.1-10 ng/mL for S1P with correlation coefficient (r2) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36-89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α whereas decreased in cells treated with DMS. These results indicated that this new LC-MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and S1P levels in biological samples simultaneously.     

Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.     

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.     

A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry

D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI–MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO₃]^- in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay.     

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.     

The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.     

An enzymatic colorimetric assay for glucose-6-phosphate

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP⁺); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).     

Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.     

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of rasagiline in human plasma and urine. LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI(+)) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01-40 ng/mL in plasma and 0.025-40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.     

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.     

Quantitative determination of dehydroepiandrosterone fatty acyl esters in human female adipose tissue and serum using mass spectrometric methods

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) are naturally occurring water-insoluble metabolites of DHEA, which are transported in plasma exclusively by lipoproteins. To find out whether DHEA, like estradiol, might be stored in adipose tissue in FAE form, we set up a mass spectrometric method to quantify DHEA-FAE and free DHEA in human adipose tissue and serum. The method consists of chromatographic purification steps and final determination of hydrolyzed DHEA-FAE and free DHEA, which was carried out by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that no detectable amounts of DHEA-FAE could be found in adipose tissue although 32-178 pmol/g of free DHEA were determined by GC-MS and LC-MS/MS. The DHEA-FAE concentrations in serum quantified by GC-MS were 1.4±0.7 pmol/ml in premenopausal women (n=7), and 0.9±0.4 pmol/ml in postmenopausal women (n=5). Correspondingly, the free DHEA concentrations were 15.2±6.3 pmol/ml and 6.8±3.0 pmol/ml. In addition, the mean proportions of DHEA-FAE of total DHEA (DHEA-FAE+free DHEA) in serum were 8.6% and 11.2% in pre- and postmenopausal women, respectively. Serum DHEA-FAE concentration was below quantification limit for LC-MS/MS (signal-to-noise ratio, S/N=10), while free DHEA concentrations varied between 5.8 and 23.2 pmol/ml. In conclusion, the proportion of DHEA-FAE of total DHEA in serum was approximately 9%. However, in contrast to our previous findings for estradiol fatty acid esters in adipose tissue which constituted about 80% of total estradiol (esterified+free), the proportion of DHEA-FAE of total DHEA was below 5%. Four to ten times higher concentrations of free DHEA were quantified in adipose tissue compared to those in serum.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization–liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography–electrospray ionization–tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25 μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC₅₀ value of 52.4 μM.