Quantitative profiling of LCB species in HeLa cells.

<p>Lysates of HeLa cells were spiked with internal standard C₁₇-sphingosine, subjected to lipid extraction followed by CD₃I-derivatization and PRM. Data are expressed as average of two replicate measurements indicated by black dots. Data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144817#pone.0144817.s008" target="_blank">S3 Table</a>.</p

Outline of the mass-tag strategy used for monitoring of LCB species.

<p>LCB analytes are chemically derivatized using CD₃I to produce TMLCB-like molecules having a precursor ion mass offset 9 Da. Upon fragmentation (MS/MS), derivatized LCBs ((CD₃)₃-LCB) produce TMLCB-like fragment ions also having a 9 Da mass offset. Note that fragmentation of derivatized 3-ketosphinganine (denoted LCB(keto)) also yields two structure-specific fragment ions.</p

Dynamic quantification range of the PRM assay for quantitative LCB profiling.

<p><b>(A)</b> Synthetic C₁₈-4-hydroxysphinganine (denoted LCB 18:0;3) was titrated relative to a constant amount of synthetic C₁₇-sphinganine (LCB 17:0;2), and spiked into <i>S</i>. <i>cerevisiae sur2</i>Δ cell lysates followed by lipid extraction. Lipid extracts were derivatized using CD₃I and analyzed by PRM. The upper x-axis shows the absolute spike amount of LCB 18:0;3 (1–2457 pmol). The lower x-axis shows the spike amount of LCB 18:0;3 relative to LCB 17:0;2. The y-axis shows the intensity ratio of the transition <i>m/z</i> 369.4→<i>m/z</i> 69.14 (monitoring (CD₃)₃-LCB 18:0;3) and <i>m/z</i> 339.4→<i>m/z</i> 69.14 (monitoring (CD₃)₃-LCB 17:0;2). Depicted values derive from two replicate analyses. The line indicates the linear function with slope 1. <b>(B)</b> Synthetic C₁₇-sphingosine (denoted LCB 17:1;2) was titrated relative to a constant amount synthetic C₁₇-4-hydroxysphinganine (LCB 17:0;3), and spiked into HeLa cell lysates followed by lipid extraction. Lipid extracts were derivatized using CD₃I and analyzed by PRM. The upper x-axis shows the absolute spike amount of LCB 17:1;2 (2–1833 pmol). The lower x-axis shows the spike amount of LCB 17:1;2 relative to LCB 17:0;3. The y-axis shows the intensity ratio of the transition <i>m/z</i> 337.4→<i>m/z</i> 69.14 (monitoring (CD₃)₃-LCB 17:1;2) and <i>m/z</i> 355.4→<i>m/z</i> 69.14 (monitoring (CD₃)₃-LCB 17:0;3). Depicted values are the average of two replicate analyses. The line indicates the linear function with slope 1.</p

Optimization of collision energy settings for PRM of derivatized LCB species.

<p><b>(A)</b> Relative intensity of fragment ion <i>m/z</i> 69.14 as function of collision energy for C₁₇-sphingosine (denoted (CD₃)₃-LCB 17:1;2), C₁₇-sphinganine ((CD₃)₃-LCB 17:0;2) and C₁₇-4-hydroxysphinganine ((CD₃)₃-LCB 17:0;3). <b>(B)</b> Relative intensity of fragment ion <i>m/z</i> 69.14 as function of collision energy for C₁₈-sphingosine ((CD₃)₃-LCB 18:1;2) and C₁₈-sphinganine ((CD₃)₃-LCB 18:0;2), and of fragment ion <i>m/z</i> 60.08 trimethyl-C₁₈-sphingosine (TMLCB 18:1;2). <b>(C)</b> Relative intensity of fragment ion <i>m/z</i> 69.14 as function of collision energy for C₂₀-sphingosine ((CD₃)₃-LCB 20:1;2) and C₂₀-sphinganine ((CD₃)₃-LCB 18:0;2). <b>(D)</b> Relative intensities of fragment ions <i>m/z</i> 66.12, <i>m/z</i> 68.13 and <i>m/z</i> 69.14 as function of collision energy for CD₃I-derivatized C₁₈-3-ketosphinganine.</p

Structural characterization of CD₃I-derivatized LCB species and trimethyl-C₁₈-sphingosine.

<p><b>(A)</b> TOF MS/MS spectrum of derivatized C₁₈-sphingosine ((CD₃)₃-LCB 18:1;2) using collision energy at 30 eV. <b>(B)</b> TOF MS/MS spectrum of trimethyl-C₁₈-sphingosine (TMLCB 18:1;2) using collision energy at 30 eV. <b>(C)</b> TOF MS/MS spectrum of derivatized C₁₈-sphinganine ((CD₃)₃-LCB 18:0;2) using collision energy at 35 eV. <b>(D)</b> TOF MS/MS spectrum of derivatized C₁₇-4-hydroxysphinganine ((CD₃)₃-LCB 17:0;3) using collision energy at 35 eV. <b>(E)</b> TOF MS/MS spectrum of derivatized C₁₈-3-ketosphinganine ((CD₃)₃-LCB 18:1;2(keto)) using collision energy at 30 eV. <b>(F)</b> FT MS/MS spectrum of derivatized C₁₈-3-ketosphinganine ((CD₃)₃-LCB 18:1;2(keto)) using a relative collision energy at 55%. The mass resolution of the fragment ion <i>m/z</i> 66.1153 is 434,420 (full width at half maximum). All TOF MS/MS spectra were acquired using ion enhancement at <i>m/z</i> 69.14.</p

Profiling LCB species composition of <i>S</i>. <i>cerevisiae</i>.

<p>Wild-type BY4742, <i>sur2</i>Δ and <i>elo3</i>Δ were cultured at 20°C, 30°C and 35°C. Lipid extracts were prepared, derivatized using CD₃I and analyzed by PRM. <b>(A)</b> Absolute levels of LCB species. <b>(B)</b> Molar abundances of sphinganine and 4-hydroxysphinganine species. <b>(C)</b> Molar abundances of 3-ketosphinganine and monounsaturated sphinganine species. <b>(D)</b> Combined molar abundances of C₁₆-, C₁₈- and C₂₀-LCB species. <b>(E)</b> Combined molar abundances of all LCB species with two or three oxygen atoms. Data are expressed as average of two biological replicates analyzed by two repeated injections. The averages of the repeated injections are shown by black dots. Data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144817#pone.0144817.s007" target="_blank">S2 Table</a>.</p

Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of <i>m</i>/<i>z</i> 141, 159, and 177, in positive mode and <i>m</i>/<i>z</i> 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50–300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed

Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated