Lipids of human meibum: mass-spectrometric analysis and structural elucidation
2007; Elsevier BV; Volume: 48; Issue: 10 Linguagem: Inglês
10.1194/jlr.m700237-jlr200
ISSN1539-7262
AutoresIgor A. Butovich, Eduardo Uchiyama, James P. McCulley,
Tópico(s)Advancements in Transdermal Drug Delivery
ResumoThe purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MSn). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MSn detection of the analytes (HPLC API MSn). The major precursor ions were isolated and subjected to further sequential fragmentation in MSn experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M − H2O + H]+) and oleic acid (OA; m/z 283; [M + H]+) were found. The other major compounds of the general molecular formula CnH2n-2O2 were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas CnH2n-4O2 and CnH2nO2. Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MSn analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components. The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MSn). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MSn detection of the analytes (HPLC API MSn). The major precursor ions were isolated and subjected to further sequential fragmentation in MSn experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M − H2O + H]+) and oleic acid (OA; m/z 283; [M + H]+) were found. The other major compounds of the general molecular formula CnH2n-2O2 were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas CnH2n-4O2 and CnH2nO2. Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MSn analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components. atmospheric pressure chemical ionization atmospheric pressure ionization behenyl oleate ceramide cholesterol cholesteryl ester cholesteryl oleate diacylglycerol dry eye syndrome 1,2-dipalmitoylglycerol electrospray ionization n-hexane-propan-2-ol-acetic acid high-pressure liquid chromatography monoacylglycerol methanol-chloroform meibomian gland meibomian gland secretions mass spectrometry normal-phase HPLC oleic acid oleamide phosphatidylcholine phospholipid 1-palmitoyl-2-oleoyl-phosphatidic acid 1-palmitoyl-2-oleoyl-phosphatidylcholine 1-palmitoyl-2-oleoyl-phosphatidylglycerol 1,2-dipalmitoylphosphatidylethanolamine retention time 1-stearoyl-2-arachidonoyl-phosphatidylinositol 1-stearoyl-2-oleoylphosphatidylserine stearyl stearate 1,2-distearoyl-sn-glycero-3-phospho-[(CD3)3N+]-choline triacylglycerol tear film lipid layer trimyristin tripalmitin wax ester Meibomian gland (MG), found in the eyelids of humans and other mammalians (1.Jester J.V. Nicolaides N. Smith R.E. Meibomian gland studies: histologic and ultrastructural investigations.Invest. Ophthalmol. Vis. Sci. 1981; 20: 537-547PubMed Google Scholar), is a major source of various lipids that participate in formation of the tear film lipid layer (TFLL) (2.McCulley J.P. Shine W.E. A compositional based model for the tear film lipid layer.Trans. Am. Ophthalmol. Soc. 1997; 95: 79-88PubMed Google Scholar). The latter is believed to play a critical role in protecting the ocular surface from dehydration by creating a physical barrier, where lipids, due to their poor miscibility with water and lower density, tend to locate at the air/aqueous phase interface (2.McCulley J.P. Shine W.E. A compositional based model for the tear film lipid layer.Trans. Am. Ophthalmol. Soc. 1997; 95: 79-88PubMed Google Scholar, 3.McCulley J.P. Shine W.E. Tear film structure and dry eye.Contactologia. 1998; 20: 145-149Google Scholar). The protective efficacy of the TFLL, therefore, should directly relate to the chemical composition of the lipid layer, and its thickness. There is evidence that irregularities in meibomian gland secretions (MGS) are one of the main causes of a pathological condition commonly known as dry eye syndrome (DES) [(4.Ohashi Y. Dogru M. Tsubota K. Laboratory findings in tear fluid analysis.Clin. Chim. Acta. 2006; 369: 17-28Crossref PubMed Scopus (163) Google Scholar, 5.McCulley J.P. Shine W. The lipid layer of tears: dependent on meibomian gland function.Exp. Eye Res. 2004; 78: 361-365Crossref PubMed Scopus (111) Google Scholar) and references cited therein]. The chemical composition of MGS was evaluated by a wide range of analytical methods, but surprisingly limited information was obtained using the current de facto standard of lipidomic analysis, mass spectrometry (MS) with direct infusion of the analytes or in combination with high-pressure liquid chromatography (HPLC) (6.Wenk M.R. The emerging field of lipidomics.Nat. Rev. Drug Discov. 2005; 4: 594-610Crossref PubMed Scopus (966) Google Scholar). In our recent studies, we implemented MS and HPLC with atmospheric pressure ionization MS (API MS) detection to evaluate the major lipid classes of MGS (7.Butovich I.A. Di Pascuale M.A. Uchiyama E. Aronowicz J.D. McCulley J.P. Mass Spectrometric Analysis of Polar and Nonpolar Lipid Species Found in Meibomian Gland Secretions (Abstract).Invest. Ophthalmol. Vis. Sci. 2006; 47 (ARVO E-Abstract 5605.)Google Scholar, 8.Butovich I.A. Uchiyama E. Agee S. Mendiola L. McCulley J.P. Structural Analysis of the Nonpolar Lipids Present in the Human Meibomian Gland Secretions Using Ion Trap Mass Spectrometry (Abstract).Invest. Ophthalmol. Vis. Sci. 2007; 48 (ARVO E-Abstract 441.)Google Scholar, 9.McCulley J.P. Uchiyama E. Mendiola L. Agee S. Butovich I.A. High Pressure Liquid Chromatographic Analysis of Lipids Present in the Human Meibomian Gland Secretions (Abstract).Invest. Ophthalmol. Vis. Sci. 2007; 48 (ARVO E-Abstract 442.)Google Scholar). The HPLC API MS experiments with human MGS produced clear evidence of the presence of very hydrophobic compounds similar to wax esters (WEs), cholesteryl esters (Chl-Es), free cholesterol (Chl), and possibly free fatty acids (FAs) and triacylglycerols (TAGs). To our surprise, no detectable amounts of compounds that would coelute with authentic monoacylglycerols (MAGs), diacylglycerols (DAGs), ceramides (Cers), and phospholipids (PLs) were detected (9.McCulley J.P. Uchiyama E. Mendiola L. Agee S. Butovich I.A. High Pressure Liquid Chromatographic Analysis of Lipids Present in the Human Meibomian Gland Secretions (Abstract).Invest. Ophthalmol. Vis. Sci. 2007; 48 (ARVO E-Abstract 442.)Google Scholar). Previously, DAG (10.Sullivan D.A. Sullivan B.D. Ullman M.D. Rocha E.M. Krenzer K.L. Cermak J.M. Toda I. Doane M.G. Evans J.E. Wickham L.A. Androgen influence on the meibomian gland.Invest. Ophthalmol. Vis. Sci. 2000; 41: 3732-3742PubMed Google Scholar, 11.Sullivan B.D. Evans J.E. Dana R.M. Sullivan D.A. Influence of aging on the polar and neutral lipid profiles in human meibomian gland secretions.Arch. Ophthalmol. 2006; 124: 1286-1292Crossref PubMed Scopus (127) Google Scholar), PL, and Cer (12.Shine W.E. McCulley J.P. Polar lipid in human meibomian gland secretions.Curr. Eye Res. 2003; 26: 89-94Crossref PubMed Scopus (119) Google Scholar) were reported to be present in human MGS, whereas in rabbits, increased levels of Cer and free Chl were indicative of MG dysfunction, apparently due to hyperkeratinization of their eyelids (13.Nicolaides N. Santos E.C. Smith R.E. Jester J.V. Meibomian gland dysfunction. III. Meibomian gland lipids.Invest. Ophthalmol. Vis. Sci. 1989; 30: 946-951PubMed Google Scholar). No structural evaluations of the intact lipids were performed in any of those studies. A decade ago, 31P-nuclear magnetic resonance spectroscopy (31P-NMR) was implemented to analyze the PL composition of homogenized tarsal plates of rabbits (14.Greiner J.V. Glonek T. Korb D.R. Booth R. Leahy C.D. Phospholipids in meibomian gland secretions.Ophthalmic Res. 1996; 28: 44-49Crossref PubMed Scopus (75) Google Scholar, 15.Greiner J.V. Glonek T. Korb D.R. Leahy C.D. Meibomian gland phospholipids.Curr. Eye Res. 1996; 15: 371-375Crossref PubMed Scopus (62) Google Scholar). Several PL species were detected, among which phosphatidylcholine (PC) and phosphatidylethanolamine comprised almost 50% of the overall PL pool. However, considering the very low sensitivity of the 31P-NMR technique, the duration of the experiments, and the method of collecting the sample material (dissection of eyelids), that approach seemed to be unsuitable for human studies. Moreover, it was suggested that animals (rabbits, in particular) are poor models of human TFLL and DES (16.Barabino S. Dana M.Reza Animal models of dry eye: a critical assessment of opportunities and limitations.Invest. Ophthalmol. Vis. Sci. 2004; 45: 1641-1646Crossref PubMed Scopus (117) Google Scholar, 17.Greiner J.V. Glonek T. Korb D.R. Hearn S.L. Whalen A.C. Esway J.E. Leahy C.D. Effect of meibomian gland occlusion on tear film layer thickness.Adv. Exp. Med. Biol. 1998; 438: 345-348Crossref PubMed Scopus (8) Google Scholar). Therefore, to 1) further evaluate the structures of the compounds detected in normal human MGS collected from individual subjects, and 2) corroborate our observation of the lack of DAG, Cer, and PL in human MGS, we conducted ion trap API MSn analyses with direct infusion of the samples, which allowed us to perform multiple sequential fragmentations of the analytes. This paper summarizes the results of our studies and presents structural data obtained with unmanipulated lipid species found in human MGS. HPLC-grade acetic acid, chloroform, n-hexane, methanol, propan-2-ol, and water were purchased from Aldrich (Milwaukee, WI) and/or from Burdick and Jackson (Muskegon, MI) through VWR (West Chester, PA). Lipid standards were products of Avanti Polar Lipids, Inc. (Alabaster, AL) and Sigma Chemical Co. (St. Louis, MO). 1,2-Distearoyl-sn-glycero-3-phospho-[(CD3)3N+]-choline (SSPC-D9) was also a product of Avanti. A Lichrosorb Si-60 silica gel column (3.2 × 125 mm, 5 μm) was a product of Supelco (Bellefonte, PA). A Lichrosphere Diol HPLC column (3.2 × 125 mm, 5 μm) was obtained from Phenomenex (Torrance, CA). An Alliance 2695 HPLC Separations Module was from Waters Corp. (Milford, MA). An LCQ Deca XP Max ion trap spectrometer equipped, depending on the application, with either an electrospray ionization (ESI) or an atmospheric pressure chemical ionization (APCI) ion source, operated under the XCalibur software, were products of Thermo Electron Corp. (San Jose, CA). Volunteers (five male and five female, median age 34 ± 3 years), who underwent standard clinical tests for DES and showed no signs of ocular diseases, participated in the study. Samples collected from different volunteers were analyzed individually. The study was approved by the Institutional Review Board. The samples were collected as follows. A dry glass vial preweighed on an analytical microbalance was filled with 1 ml of MS-grade chloroform. The MGS were expressed from a subject's four eyelids using a plastic conformer and a cotton swab. The lipid samples were harvested from the MG orifices using a platinum spatula. The samples were never in contact with the conformer or the swab. Samples collected from all four eyelids of an individual were pooled. The secretions that had been expressed from the glands immediately solidified at room temperature to assume a typical waxy texture. The sample was transferred into the vial with chloroform, dissolved, and the solvent was evaporated at room temperature under a stream of dry nitrogen. Then, the vial with the sample was reweighed to determine the weight of the dry lipid material. Depending on the donors, the dry samples were between 0.1 and 3 mg. The standard error of weighing was ≤5% for each of the dry samples. The average weight of the nine samples collected from ten volunteers was 0.45 mg. The samples were stored under nitrogen at −80°C. The samples were stable for at least 3 months. The study was approved by the Institutional Review Board of The University of Texas Southwestern Medical Center at Dallas. The informed consents were obtained from the human subjects. Mass spectra of the compounds were obtained in both positive- and negative-ion modes using both the ESI and the APCI ion sources. The m/z ratios were used to calculate molecular masses (M) of the parent compounds and their fragments. In the positive-ion mode experiments, the compounds were typically detected as either proton (M + H)+, or sodium (M + Na)+ adducts. In the negative-ion mode, acidic compounds were visible as (M − H)− or (M + Cl)− species. An interesting exclusion was a group of compounds of PC and SM families. To comply with current views, for zwitterionic compounds of PC and SM families with quaternary amino groups [−N+(CH3)3], M was assumed to be the mass of the species with dissociated phosphoric acid residues [−O−P(O)(O−)−O−], which resulted in electroneutral lipid species. To be visible in MS experiments, those electroneutral species must be further ionized either by losing a proton (to acquire an overall negative charge, [M − H]−), or forming various adducts with already-ionized particles (see below). The samples were dissolved in methanol-chloroform (MC) 2:1 (v/v) solvent mixture for the ESI experiments, and in the n-hexane-propan-2-ol-acetic acid (HPA) 95:5:0.1 (v/v/v) solvent mixture for the APCI analyses. The HPA solvent mixture was also used for HPLC separation of nonpolar lipids. The solvent compositions were chosen to ensure complete solubility of all the analyzed lipid samples in the indicated concentration ranges, as well as their effective ionization under the tested conditions. To minimize the chances of sample degradation due to hydrolysis or solvolysis, no modifiers (acids, bases, or water) were typically added to the MC solvent in the direct-infusion ESI experiments, unless stated otherwise. For APCI experiments in n-hexane-based solvent mixtures, between 0.1% and 1% (v/v) of acetic acid had to be added to achieve good ionization of the analytes. High-purity nitrogen was utilized as sheath gas throughout the experiments. To achieve the highest possible precision, the m/z ratios of the precursor compounds were routinely recorded in the zoom scan modes, in accordance with Thermo Electron's recommendations. To obtain more-detailed information on the structures, the major precursor ions were isolated and subjected to further sequential fragmentation in the MSn mode. The analyses were performed using the settings that were individually optimized for each analyte. Addition of metal ions (Na+ or Li+), often used to promote the formation of charged adducts (18.Ham B.M. Jacob J.T. Cole R.B. MALDI-TOF MS of phosphorylated lipids in biological fluids using immobilized metal affinity chromatography and solid ionic-crystal matrix.Anal. Chem. 2005; 77: 4439-4447Crossref PubMed Scopus (62) Google Scholar, 19.Ham B.M. Cole R.B. Jacob J.T. Identification and comparison of the polar phospholipids in normal and dry eye rabbit tears by MALDI-TOF mass spectrometry.Invest. Ophthalmol. Vis. Sci. 2006; 47: 3330-3338Crossref PubMed Scopus (35) Google Scholar), was avoided at this stage of initial characterization of MGS because, at the time, their effects on the resulting MS spectra could not be predicted. Often, such modifiers lead to more complex MS spectra, because they do not guarantee complete conversion of all the lipid species present in the mixture into one particular type of adduct (18.Ham B.M. Jacob J.T. Cole R.B. MALDI-TOF MS of phosphorylated lipids in biological fluids using immobilized metal affinity chromatography and solid ionic-crystal matrix.Anal. Chem. 2005; 77: 4439-4447Crossref PubMed Scopus (62) Google Scholar, 19.Ham B.M. Cole R.B. Jacob J.T. Identification and comparison of the polar phospholipids in normal and dry eye rabbit tears by MALDI-TOF mass spectrometry.Invest. Ophthalmol. Vis. Sci. 2006; 47: 3330-3338Crossref PubMed Scopus (35) Google Scholar). In our preliminary experiments, tested lipids readily ionized in either of the solvents (MC or HPA), and the MS spectra of the major components obtained in the two solvents were quite similar. The great variability of lipids that were reported to be present in MGS and a need to separate lipid classes in HPLC experiments necessitated the use of two different API ionization methods, ESI and APCI. First, standard lipids and their mixtures were analyzed by ESI MSn in the positive-ion mode, and the analytes were detected as proton and/or sodium adducts. A sample of a lipid (0.1–10 μg/ml, depending on the analyte) dissolved in the MC solvent mixture was infused at a flow rate of 3–5 μl/min. The MS parameters were as follows: source current 3 μA; source voltage 4–5 kV; sheath gas flow of 6 to 10 arbitrary units; auxiliary/sweep gas 0; capillary temperature 250°C for PLs and 325°C for nonpolar lipids; capillary voltage 10–45 V. For the negative-ion mode ESI experiments, the capillary voltage was maintained between −30 and −45 V. The rest of the parameters were optimized for each individual analyte or their mixtures using the automatic tuning procedure built into the XCalibur software (Thermo Electron). PLs were analyzed with and without 0.1% aqueous ammonium formate. The full MS spectra were collected typically between m/z values of 150 and 2,000 for a period of at least 1 min and then were averaged using the Excalibur Qual Browser built-in routine. Monoisotopic molecular masses of the analytes were determined in the zoom scan mode in the range of m/z ±10. Second, the lipids were analyzed using the APCI ion source. For the APCI type of experiments, the source voltage was 4.4 kV (positive-ion mode) and 1.5 kV (negative-ion mode), source current was 5 μA for the positive- and 6 μA for the negative-ion modes; vaporizer and capillary temperatures were set at 375°C and 300°C, and the capillary voltage was 12 V (for the positive-ion mode) and −15 V (for the negative one). The lipids were dissolved either in the MC (for ESI experiments) or in the HPA solvent mixtures (for APCI experiments; see legends to the corresponding figures). Monoisotopic molecular masses of the analytes were determined in the zoom scan mode in the range of m/z ± 10. Once the m/z ratios had been measured, the individual components of the sample were subjected to fragmentation in a collision-induced dissociation experiment at the collision energy of 25–100 V (see legends to the corresponding figures). Then, nonpooled samples of MGS collected from 10 normal subjects were analyzed individually. The initial characterization of the lipid samples was performed as described above for lipid standards. A sample was dissolved in either MC solvent (for ESI experiments) or the HPA solvent mixture (for APCI experiments) to make, depending on the application, a solution of ∼0.05 mg to 0.3 mg dry sample/ml, and then directly infused into the mass spectrometer at a flow rate between 2 μl/min and 10 μl/min. The samples were analyzed in both the negative- and the positive-ion modes as described above for standard lipids. The standard nonpolar lipids and MGS samples were analyzed by normal-phase HPLC (NP-HPLC) on the Lichrosphere Diol column. Lipids were isocratically eluted from the column with the HPA solvent mixture at 30°C and a flow rate of 0.3 ml/min. The entire flow of the effluent was directed to the APCI ion source and analyzed in either the positive- or the negative-ion modes. The lipid standards (1–20 μg of individual lipid/ml) and MGS samples (0.1–3 mg/ml) were dissolved in the mobile phase. Between 1 μl and 20 μl of the sample solutions were injected. The samples of standard PLs were analyzed by NP-HPLC using the Lichrosorb Si-60 HPLC column. HPLC analyses of the lipids were performed using gradient elution by n-hexane-propan-2-ol 5 mM aqueous ammonium formate mixtures at 30°C according to the protocol presented in Table 1 . The entire flow of the effluent was directed to the APCI ion source and analyzed in either the positive- or the negative-ion modes as described above. The sample (less than 0.1 mg dry sample/ml) was dissolved in a hexane-propan-2-ol 95:5 (v/v) solvent mixture. Between 0.5 and 10 μl of the sample solutions were injected. The same procedure was performed on the samples of MGS.TABLE 1.Gradient HPLC analysis of phospholipids on a silica gel Lichrosorb Si-60 columnStepTimeFlowPropan-2-ol5 mM NH4+COO−in Watern-Hexaneminml/min%%%100.540258250.5402583250.5405554400.5405555411405556601405557610.5402588700.540258HPLC, high-pressure liquid chromatography. Open table in a new tab HPLC, high-pressure liquid chromatography. A model mixture of nonpolar lipids composed of several standard lipids whose analogs had been reported to be present in human meibum (10.Sullivan D.A. Sullivan B.D. Ullman M.D. Rocha E.M. Krenzer K.L. Cermak J.M. Toda I. Doane M.G. Evans J.E. Wickham L.A. Androgen influence on the meibomian gland.Invest. Ophthalmol. Vis. Sci. 2000; 41: 3732-3742PubMed Google Scholar, 11.Sullivan B.D. Evans J.E. Dana R.M. Sullivan D.A. Influence of aging on the polar and neutral lipid profiles in human meibomian gland secretions.Arch. Ophthalmol. 2006; 124: 1286-1292Crossref PubMed Scopus (127) Google Scholar, 12.Shine W.E. McCulley J.P. Polar lipid in human meibomian gland secretions.Curr. Eye Res. 2003; 26: 89-94Crossref PubMed Scopus (119) Google Scholar, 13.Nicolaides N. Santos E.C. Smith R.E. Jester J.V. Meibomian gland dysfunction. III. Meibomian gland lipids.Invest. Ophthalmol. Vis. Sci. 1989; 30: 946-951PubMed Google Scholar, 14.Greiner J.V. Glonek T. Korb D.R. Booth R. Leahy C.D. Phospholipids in meibomian gland secretions.Ophthalmic Res. 1996; 28: 44-49Crossref PubMed Scopus (75) Google Scholar, 15.Greiner J.V. Glonek T. Korb D.R. Leahy C.D. Meibomian gland phospholipids.Curr. Eye Res. 1996; 15: 371-375Crossref PubMed Scopus (62) Google Scholar, 20.Tiffany J.M. Individual variations in human meibomian lipid composition.Exp. Eye Res. 1978; 27: 289-300Crossref PubMed Scopus (119) Google Scholar, 21.McCulley J.P. Shine W.E. The lipid layer: the outer surface of the ocular surface tear film.Biosci. Rep. 2002; 21: 407-418Crossref Scopus (69) Google Scholar) was analyzed using NP-HPLC with APCI detection of the analytes in the positive-ion mode (Fig. 1). The tested compounds included cholesteryl oleate (Chl-O), Chl, tripalmitin (TP), behenyl oleate (BO), 1,2-dipalmitoylglycerol (DP), C18-ceramide (C18-Cer), 1-miristoyl glycerol (MG), and oleamide (OAm). The lipid mixture was separated on a Lichrosphere Diol HPLC column (3.2 × 150 mm, 5 μm). The retention times (RTs) of the lipid standards were used to map the corresponding lipid classes that were expected to be observed in MGS. Surprisingly, MGS produced only one major elution peak whose RT was identical to those of standard WE, Chl-E, and TAG (Fig. 2) The peak showed a wide range of MS signals (Fig. 2, insert A), which were further analyzed using the direct infusion method (see below). A small peak of free Chl (m/z 369.4, [M − H2O + H]+, visible only under the conditions of a single-ion monitoring experiment or in extracted chromatograms; Fig. 2, insert B), along with an equally small peak of OAm (m/z 282.1, [M + H]+; see below) were detected. No perceptible peaks that would coelute with MAG, DAG, and Cer were observed. At the same time, we cannot rule out the possible existence of very small pools of other lipids in meibum, whose presence was too small to be detected without optimizing the conditions of the analyses for those particular compounds.Fig. 2.Total ion chromatogram of nonpolar lipids present in human meibomian gland (MG) secretions with the positive-ion mode APCI mass spectrometry (MS) detection. Insert A: Mass spectrum of the peak with RT 3.3 min. Insert B: Reconstructed ion chromatogram of ion m/z 369.3 (Chl − H2O + H)+. HPA solvent mixture was used as HPLC solvent.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To verify the recent findings of Nichols et al. (22.Nichols K.K. Ham B.M. Nichols J.J. Ziegler C. Green-Church K.B. Identification of fatty acids and fatty acid amides in human meibomian gland secretions.Invest. Ophthalmol. Vis. Sci. 2007; 48: 34-39Crossref PubMed Scopus (74) Google Scholar) regarding OAm in meibum, we also conducted appropriate HPLC MS studies of the human meibum with selective ion monitoring at m/z 282 (Fig. 3). Compared with the other lipids present in meibum, very little, if any, OAm was detected in all tested MGS samples. By using a standard curve for authentic OAm (not shown), its presence in MGS was found to be below 0.1% (w/w, dry weight). In the negative-ion mode, three major compounds were detected, which possessed m/z values of 729.8 ± 0.3, 757.8 ± 0.3, and 785.8 ± 0.2 (n = 44) (Fig. 4). The compounds eluted as one peak, and their RTs were between those of WE and Chl. A model mixture of standard PLs was analyzed on a Lichrosorb Si-60 column using a solvent gradient (Table 1). RTs of several standard PLs are presented in Table 2 . When MGS were analyzed by HPLC MS using the conditions optimized for PL analysis, they showed no noticeable HPLC peaks that would be indicative of PL (data not shown). The compounds with m/z ratios of 729.8, 757.8, and 785.8 eluted very quickly (RT ∼1.7 min) and did not coelute with any of the PL standards. Such short RTs put the analytes in the category of rather nonpolar compounds, and ruled out a possibility for them to be standard PLs.TABLE 2.NP-HPLC retention times of phospholipidsPhospholipidRetention Timemin1-palmitoyl-2-oleoyl-phosphatidylglycerol20–211,2-dipalmitoylphosphatidylethanolamine25–261-palmitoyl-2-arachidonoyl-phosphatidylinositol29–301-palmitoyl-2-oleoyl-phosphatidic acid29–34 (broad peak)1-stearoyl-2-oleoyl-phosphatidylserine34–361-palmitoyl-2-oleoyl-phosphatidylcholine37–38C16:0-sphingomyelin39–40NP-HPLC, normal-phase HPLC. Open table in a new tab NP-HPLC, normal-phase HPLC. A typical MS spectrum of MGS in the positive-ion mode is presented in Fig. 5 . Note a striking resemblance of this ESI spectrum to the APCI spectrum presented in Fig. 2. This means that 1) most of the ions detected in the direct infusion ESI experiments eluted from the column very quickly as a major HPLC peak with an RT of ∼3.3 min (Fig. 2) and, therefore, were nonpolar lipids; and 2) the type of ionization procedure did not explicitly affect the major detected MS signals. Among the prominent MS signals in the peak with RT 3.3 min, there were groups of ions with m/z values of 283.1; 369.3; 535.4; 549.5; 561.5, 563.6, and 565.5; 575.6, 577.6, and 579.4; 589.6, 591.6, and 593.5; 603.6, 605.6, and 607.4; 617.5, 619.6, and 621.5; 631.5, 633.6, and 635.6; 645.5 and 647.5; 659.7, 661.6, and 663.5; 673.6 and 675.5; and 701.6. The ion with m/z 283.2 was found to be similar to authentic OA (monoisotopic mass 283.2, [M + H]+). The ion was fragmented in an MSn mode to give the following ions: 283 (MS1)→265 (MS2)→247 (MS3) (sequential loss of two H2O). Subsequent fragmentation of ion m/z 247 (MS4) produced prominent product ions that were identical to the fragments of authentic OA (Fig. 6). Occasionally observed ions with m/z 282.3 and 304.4 (see Fig. 3) were similar to authentic OAm (monoisotopic mass 282.3, [M + H]+) and its sodiated derivative (monoisotopic mass 304.5, [M + Na]+), although the low a
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