WO2006092689A1

WO2006092689A1 - Assay of sebum and meibum lipid components by mass spectrometry

Info

Publication number: WO2006092689A1
Application number: PCT/IB2006/000346
Authority: WO
Inventors: Kristian Erich Kindt; Gabriella Szekely-Klepser; Kimberly Jane Wade; Wenlin Li
Original assignee: Warner-Lambert Company Llc
Priority date: 2005-03-03
Filing date: 2006-02-20
Publication date: 2006-09-08

Abstract

The invention provides a method for identifying a compound that modulates sebum production or that modulates the amount of at least one lipid component of a sebum sample utilizing mass spectrometry.

Description

ASSAY OF SEBUM AND MEIBUM LIPID COMPONENTS BY MASS

SPECTROMETRY

FIELD OF THE INVENTION

The present invention relates to an assay for identifying compounds that modulate the lipid components of sebum or meibum.

BACKGROUND OF THE INVENTION

Human skin is composed of three primary layers, the stratum corneum, the epidermis, and the dermis. The outer layer of the skin, the stratum corneum, primarily functions as a barrier to the external environment preventing water loss and preventing the invasion of microorganisms. Lipids, secreted to the stratum corneum from the sebaceous glands, are the key components in maintaining this barrier. Abramovits et al, Dermatologic Clinics, Vol. 18, Number 4, Oct. 2000.

Sebum, a complex mixture of proteins and lipids, is produced by the sebaceous glands. At maturation, the acinar cells of the sebaceous glands lyse and release sebum into the lumenal duct, from which the sebum is secreted.

Squalene, cholesterol, cholesterol esters, wax esters, and triglycerides are the primary lipids found in human sebum. Wax esters and squalene are unique to sebum in that they are not synthesized by other cells in the body. During passage of sebum to the skin surface, bacterial enzymes hydrolyze some of the triglycerides, so that the lipid mixture reaching the skin surface also contains free fatty acids and small amounts of mono- and diglycerides.

The meibonian gland is a sebaceous gland that produces the tear film's lipid layer (meibum) and is critical in preventing the evaporation and maintaining the stability of the tear film. Polar and non-polar lipids are present in the meibum. The non-polar lipids include wax and sterol esters and hydrocarbons, while the main polar lipid components are phospholipids, sphingolipids and triglycerides.

A number of methods to isolate, separate and qualitatively and/or quantitatively analyze the lipid components of sebum and/or meibum have been reported (O'Neill and Gershbein, J. Chrom. ScL, 1976, (14) 28-36; Nordstrom et al., Journal of Investigative Dermatology, 1986; 86 (6), 700-5; Sullivan et al., Arch. Ophthalmol., 2002; 120:1689-1699). These methods are labor intensive, often require large amounts of sample and are not suitable for high throughput screening of samples. What is needed is an accurate method for identifying compounds that modulate sebum production and/or to qualitatively or quantitatively assess changes in the lipid components of sebum or meibum.

SUMMARY OF THE INVENTION

The invention provides a method for identifying a compound that modulates sebum or meibum production or that modulates the amount of at least one lipid component of a sebum or meibum sample, comprising contacting a sample from a treated subject with a mass spectrometer; generating an extracted ion or selected reaction monitoring chromatogram for at least one lipid component of a sample from a treated subject; and comparing the peak area under the chromatogram of said sample with the peak area of a control sample. The lipid component may be selected from the group consisting of mono-, di- or triglycerides, cholesterol, cholesterol esters, fatty acids, phospholipids, sphingolipids, and hydrocarbons, such as squalene.

In the method of the invention, a test compound is contacted with a source of sebum or meibum and, after an appropriate treatment period, the sebum, meibum or source of the sebum or meibum (collectively, hereinafter, "sebum") is collected and contacted with a mass spectrometer to analyze the presence and/or amount of at least one lipid. In one embodiment, the lipids may be extracted from the test sample prior to contacting with the mass spectrometer. The test sample may be directly subjected to mass spectrometry or may be subjected to HPLC chromatography prior to analysis by mass spectrometry. The mass spectrometer used in the present invention comprises an inlet, an ionization source, an analyzer, and an ion detector. The ionization source for said spectrometer may be selected from electrospray ionization, atmospheric pressure chemical ionization, electron impact, chemical ionization, atmospheric pressure photoioniozation and laser desorption ionization. The analyzer of the spectrometer may be selected from a quadrupole, a triple quadrupole, a Fourier-transform, a magnetic-sector, an electric sector, an ion trap and a time-of flight analyzer.

The ion detector of the spectrometer may be selected from the group consisting of an electron multiplier, a photomultiplier, multichannel plate, a channel electron multiplier and a penning electrode.

The sample may be subjected to chromatography prior to analysis by mass spectrometry. The chromatography may be selected from the group consisting of liquid chromatography and gas chromatography.

-A-

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Depiction of a Typical Mass Spectrometer

Figure 2. Relative amounts of selected triglycerides and Cholesterol esters in human meibum extracts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for identifying compounds that modulate the production of sebum by quantitatively detecting one or more sebum lipids in a sample by mass spectrometry. The assay of the present invention may be utilized to assess whether a test compound modulates the production, composition, and/or secretion of sebum or meibum. It may be used to quantitate the amount of sebum produced, to determine if a test compound modulates (increases or decreases) the amount of at least one individual sebum lipid component and to correlate the relative amounts of the sebum lipid components.

The method of the present invention can rapidly and accurately quantitate the amount of sebum or meibum components and compare the effectiveness of a treatment to modulate the production and/or secretion of sebum or meibum. Briefly, a test agent that potentially modulates sebum and/or meibum production is applied to a subject or to cells. After an appropriate treatment period, a test sample from a treated subject (a subject exposed to a test compound) and a sample from an untreated subject (control sample) are harvested, the lipids are extracted from the test samples and at least one or more of the lipids are analyzed by mass spectrometry. A compound inhibits the production of sebum or the amount of a sebum lipid component if the peak area under the chromatogram for said lipid component in said treated sample is less than the peak area under the chromatogram in a control sample. The test compound increases the amount of a lipid if the peak area under the chromatogram for said lipid component in a treated sample is higher than the peak area under the chromatogram in a control sample.

The term "subject" refers to a human, an animal or cells from a human or animal source that can produce a sample containing sebum or meibum lipids.

The term "sample" refers to any biological sample that may be a source of sebum or meibum (hereinafter, collectively "sebum"), including, but not limited to, sebaceous glands, meibomian glands, normal cells, tumors, skin, hair, specific layers of skin, i.e. the dermis, epidermis, stratum corneum or tear film or fluid. The tissue may be of animal origin. Thus, it may be derived from fur, feathers, animal skin or other tissues. For instance, the "sample" may be a skin surface sample collected from the skin surface by, for instance blotting, wiping the surface of the skin or by absorbing skin surface materials on to absorbent papers or absorbent tapes. One such absorbent tape is described in US Patent No.

4,532,937, which is incorporated herein by reference. Skin surface samples may include samples from which all of part of the stratum corneum has been removed such as that described by Cotterill et al. (Br. J. Derm. (1972) Vol. 86, pp 356-360) or may include cells of the stratum corneum.

The method of the present invention may be used to identify compounds that modulate the amount of one or more lipids selected from the group consisting of cholesterol, cholesterol esters, glycerides, fatty acids, phospholipids, sphingolipids, hydrocarbons and ceramides in a sample comprising contacting said sample with a mass spectrometer.

Mass spectrometry (MS) possesses a number of advantages for the analysis and quantitation of sebum or meibum components compared to previously described assays. It allows the analysis to be conducted with significantly smaller quantities of test samples, a significant advantage when the assay is being conducted on human subjects.

Mass spectrometers traditionally generate ions from neutral molecules by bombarding them with high-energy electrons (electron impact), atoms or ions leading to the subsequent ionization and fragmentation of the substance under analysis (chemical ionization). More modern instruments use alternative ionization sources such as electrospray ionization, atmospheric pressure chemical ionization or laser desorption ionization to generate charged species.

The various ionization methods and their applicability are well known to those skilled in the art.

Figure 1 depicts the main components of a typical mass spectrometer. As shown in Figure 1 , all mass spectrometers have an inlet (a), in which the test sample is introduced into the spectrometer. In the inlet, the test sample passes from atmospheric pressure to a lower pressure. After the inlet, the test sample is contacted with an ion source (b). In the ion source, the sample is converted into gas phase ions. A variety of different ionization methods are available in the field of mass spectrometry. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are particularly useful in conjunction with HPLC. The ions can be subsequently fragmented in a collision cell. The ions are then directed to a mass analyzer (c). In the mass analyzer, the ions are separated based upon their mass-to-charge ratio (m/z) values. Only the ions matching the mass analyzer parameters pass through the mass filter at a given time. Once inside the detector (d), the ions generate a signal that can be displayed and recorded by the data system. The detector counts the ions and a signal is generated that is proportional to the total number of ions.

Mass spectrometers can be further categorized based on the type of mass analyzer they use. The most common types of mass spectrometers are magnetic, quadrupole, time-of-flight, ion trap and ion cyclotron resonance. A magnetic sector mass spectrometer uses electric and magnetic fields to disperse and focus the ion beam based on the m/z values of the ions. The quadrupole mass analyzer employs a combination of direct-current (DC) and radio-frequency (RF) potentials as a mass filter. The ion trap mass spectrometer uses appropriately selected RF and DC voltages to trap ions over a specified m/z range. A time-of-flight mass spectrometer measures the time for an ion to travel from the ion source to the detector. The ion cyclotron resonance (ICR) mass spectrometer operates on the principle that ions in a magnetic field move in circular orbits at frequencies characteristic of their m/z values. Mass analyzers and their operating principles are well known to a person skilled in the art. In addition to detecting intact molecular ions to obtain molecular weight information, structure specific fragment ions can be produced and detected in the mass spectrometer. The fragmentation pattern detected for a molecule is very distinctive and provides structural information allowing not only the identification of unknown components but also their selective quantitation in a sample.

Any ion detectors known in the art may be used in the present invention. Examples of such ionization detectors include, but are not limited to, an election multiplier, a photo multiplier, a multichannel plate, a channel electron multiplier and a penning electrode.

Mass spectrometers are available from a number of commercial sources. These include but are not limited to Applied Biosystems (MDS Sciex, Ontario, Canada), Waters, (Beverly, Massachusetts), Thermofinnigan, (San Jose,

California), and Bruker, (Billerica, Massachusetts). Any commercially available or home built mass spectrometer may be used in the assay of the present invention.

Typically, the assay used in the present invention is carried out in the following manner. A sample from a subject that has been treated with a test compound is collected and contacted with a mass spectrometer. The sample can be introduced into the MS inlet as a solution, solid or a gas. Typically, the sample is in a solvent that is compatible with mass spectrometry. Solvents such as water, propanol, methanol, isopropanol, acetonitrile or a dilute solution of sodium or ammonium acetate or sodium or ammonium-formate or any suitable volatile buffer system compatible with mass spectrometry may be used. In one embodiment, the inlet will typically be connected to a chromatography column.

The column may be suitable for either liquid or gas.

Once the sample is introduced into the inlet, it is transported to the ion source. The ion source transforms the sample into a gas where it is ionized, fragmented and forwarded to the mass analyzer. The method of the present invention can measure changes in one or more sebum lipids brought about by treatment with a test compound. The method of the present invention can detect qualitative or quantitative changes in the molecular ion of a lipid in any ion form. The ion form may be selected from a positive protonated [M+H]⁺, a negative, deprotonated, [M-H]^" pseudo-molecular ion or any metal and/or solvent ion adducts or ammonium ion adducts, e.g. [M+Na]⁺, [M+NH₄]⁺ or their combinations, e.g. sodiated propanol adduct [M+Na]⁺[CH₃CH₂CH₂OH]. Furthermore, glycerides and cholesterol esters can also be detected in the form of their decomposition products that are produced in the mass spectrometer ion source, such as [MNa- fatty acid]⁺ . The mass spectrometer can be programmed to detect any of these ion forms.

Although, it is possible to form more than one type of glyceride or cholesterol-ester related ion, the dominant ion form of these lipids used in the invention is the sodium adduct molecular ion, [M+Na]⁺. Once the lipid specific molecular ions are determined, samples can be analyzed and extracted ion chromatograms of these lipid specific molecular ions can be obtained.

The molecular ion can also be selectively fragmented in the mass spectrometer to produce fragment (or product) ions collected in the MS/MS spectrum in order to determine structure specific fragment ions that are unique to the lipid of interest. After the selection of an abundant and lipid specific fragment/product ion from the MS/MS spectrum a selected reaction monitoring

(SRM) transition can be identified that can be used to monitor the fragmentation reaction of the lipid molecular ion to the fragment of choice and is very specific to the lipid of interest.

In one embodiment, the assay is performed using the above described selected reaction monitoring (SRM) method performed on a triple quadrupole tandem mass spectrometer operated in the positive ion electrospray ionization mode, in conjunction with HPLC. Triple quadrupole mass spectrometers and their operation as described here is well known to those skilled in the art.

The amount or modulation of sebum lipids in a sample from a subject treated with a compound (treated subject) is determined by generating the extracted ion chromatogram (XIC) or selected reaction monitoring chromatogram

(SRM) for one or more lipids and comparing the peak area under the chromatogram of said lipids to those obtained for standard curves for selected lipids either alone or in a mixture and comparing said XIC or SRM chromatograms with that produced by samples from subjects that were not exposed to the test compound (control subjects). The method of the invention allows the determination of changes in the amount of at least one of the lipids selected from the group consisting of squalene, cholesterol esters, glycerides, phospholipids, spingolipids, ceramides and free fatty acids in a test sample by comparison of the chromatogram of the lipids in the test sample with the chromatogram of standards prepared for said lipids or by comparison with chromatograms of samples from untreated subjects.

While the test sample may be directly introduced into the mass spectrometer, the test sample may also be subjected to a chromatographic separation prior to application to the mass spectrometer. Suitable chromatographic systems are gas-chromatography (GC), liquid chromatography

(LC) including high performance as well as ultra-high pressure liquid chromatography (HPLC). Any type of chromatographic separation that can be coupled to the mass spectrometer may be used in the present invention.

Chromatography is a method of separating components in a sample based on differences in partitioning behavior between a mobile phase and a stationary phase. A column holds the stationary phase and the mobile phase carries the sample through the column. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column, they can be quantified directly or can be combined with other analytical devices such as a mass spectrometer. The various types of chromatographic methods and equipment are well known to those skilled in the art.

Liquid chromatography is used to separate ions or molecules that are in solution. The mobile phase is a solvent and the stationary phase may be a liquid on a solid support, a solid, or an ion-exchange resin. This method allows the separation of molecules or ions based on differences in size, ion-exchange, partitioning or adsorption. The differences in the partitioning behavior of components between the liquid and the stationary phases will cause them to pass through the column at different rates. Thus, the mobile phase and the stationary phases of a column may be optimized to separate components that may normally be very similar in transit time through a column. High performance liquid chromatography utilizes high-pressure pumps and smaller stationary phase particles sizes (3-5 μm) to increase the efficiency of the separation, provide higher resolution and faster analysis time. Ultra high pressure chromatography (UPLC) has increased the efficiency of separations even further by reducing the particles size to around 1.7 μm. Other chromatographic methods known to those skilled in the art and useful in the present invention include gas chromatography, size-exclusion chromatography and thin layer chromatography.

In one embodiment of the invention, the column is a high performance liquid chromatography column (HPLC). HPLC columns are typically packed with small particles of a stationary phase such as silica, functionalized silica, ion exchange resins or cross-linked functionalized organic polymers. Examples of suitable columns include, but are not limited to, columns such as Varian Metasil AQ, Atlantis HIILC, Supelcosil LC-18, Phenomenex Synergy Polar RP. Columns containing C-18 (octadecyl-silica) particles generally provide good retention for these lipids and facilitate their separation from matrix components and from each other. Columns that possess bound polar end-capping groups in their stationary phase, such as the Synergy Polar RP, provide good retention and separation from background but offer sharper peaks and shorter analytical run times. One embodiment of the invention uses of a Phenomenex, Synergy Polar RP column which provides a suitable peak shape for the cholesterol esters and triglycerides while affording a retention time away from the solvent front.

One skilled in the art, based upon the teachings of this application, could carry out the assay with a wide variety of mass spectrometers with and without chromatographic separation.

In general, the separation itself can be carried out on any chromatoghrapic column providing sufficient retention and separation of the analytes. HPLC with normal and reverse-phase columns and appropriately selected solvent systems can provide extensive resolution for tri-glycerides, cholesterol esters, ceramides and phospholipids. Such columns can include, C-4 through C-18 silica based columns with and without polar on non-polar endcaps, depending on the polar or non-polar nature of the lipids. The columns suitable for these separations are well known to people skilled in the art. Elution conditions can also vary for gradient conditions depending on the lipids of interest and various solvent systems can be used (e.g. propanol in hexane, chloroform-methanol- ammonium hydroxide, hexane-isopropanol-water with normal phase and acetone:acetonitrile or isopropanohacetonritrile systems for reverse phase separations. For polar lipids, the HPLC mobile phase for reverse phase separation can consist of an aquaeous component with organic modifiers, e.g. methanol-water-acetonitrile or propanol-water mixtures with ammonium acetate.

The application of LC/MS/MS provides an added degree of selectivity, since in addition to the retention time, the molecular weight of the analyte, as well as the molecular weights of one, or more, of its structure specific fragment ions are being used to confirm the identity.

SAMPLE PREPARATION

The lipids may be extracted from the sample by contacting the sample with an extraction medium, extracting the lipids from said test sample and analyzing the extracted lipids by mass spectrometry. The term "extraction medium" is meant to include any solution with which the test sample is contacted prior to subjecting said test sample to mass spectrometric analysis. The extraction media may be composed of polar and/or non-polar organic solvents such as chloroform, methanol, propanol, isopropanol, di-chloromethane, tri- methyl-pentene, hexane, or heptane or their combinations and may contain an aqueous phase with or without modifiers (such as acids or bases). The type of extraction media used depends on the lipids of interest. As a general rule of thumb, polar lipids require polar extraction media, while non-polar lipids are better extracted in non-polar solvents. The extraction can be carried out in any manner that extracts the lipid components of sebum and any single solvent or combination of solvents can be used in which the lipids of interest are soluble.

In one embodiment, the sebum is extracted from the test sample by contacting the test sample with an extraction medium comprising at least one volatile organic solvent for a sufficient period of time to sufficiently release the lipid components, so that they may be detected by mass spectrometry. The extraction medium may also comprise an aqueous phase in addition to the organic solvents. Any of these methods may be used in conjunction with the mass spectrometric analysis of the present invention. The extraction can be carried out in any manner that extracts the lipid components of sebum. Several methods for extracting the sebum components have been reported. Sebum lipids are most commonly extracted using a mixture of chloroform:methanol (2:1 v/v; "Folch solvent") or ether [J. of Dermatological Science, 1 (1990) 269-276, Invest. Opthalmol. Vis. Sci. 20, 4, 1981 , 522-536]. The volume of extraction medium used to extract the lipids from a sebum sample is not critical. It is only required that the volume of the extraction medium exceed the volume of the lipid in the sample. Typically, the sample is solubilized in a volume of solvent that is from about 5 to about 10 times the volume of the lipid of the sample. However, smaller or larger amounts of solvent may be used. One of skill in the art will readily be able to determine the amount of solvent suitable for their particular sample. The extracted test sample may be applied directly to the mass spectrometer. Alternately, the extraction medium may be removed by evaporation and the sample comprising the lipids stored for later analysis. The test sample may then be reconstituted in a solvent compatible with the mass spectrometer.

Sebaceous Glands

A test compound is applied to the sebaceous gland of a subject (treated subject). After a suitable time, the sebaceous glands are isolated and the lipids extracted by contacting the glands with an extraction medium. Typically, such samples are homogenized and extracted in an extraction medium. The homogenates are centrifuged and the lipid containing layer can be used directly for mass spectrometric analysis. Alternatively, the lipid containing extract can also be dried down and reconstituted in a suitable solvent mixture for direct injection or for chromatographic separation prior to mass spectrometric analysis. The sebaceous glands from any species may be utilized. Sebaceous glands from the ears of hamsters are used as a model to study the effect of compounds on the modulation of sebum production.

Preparation of Sebum or Meibum Samples In one embodiment of the invention, the test sample is selected from human sebum. The sebum samples are collected, placed in a suitable solvent and subjected to analysis by mass spectrometry. The samples may be collected by any means known in the art. For instance, samples can be collected using absorbents, such as Sebutape® (CuDerm Corporation, Dallas, TX) (lipid absorbing polymeric film), cigarette paper, clean tissue or filter paper that can absorb lipid components from tissue surfaces. Absorbents can be soaked in organic solvents (e.g. ether) prior to application to aid the absorption of lipids from the surface of the tissues. Bentonite clay patches have also used to collect surface lipids. (Clarys et al. in Clinics in Dermatology 1995, 13, 307-321) Meibum samples can also be collected by touching microcapillaries or cotton tipped applicators to the outer surface of the eye and collecting the expelled fluid with a chalazion curette (surgical stainless steel) (Sullivan et al. The Journal of Clinical and Endocrinology & Metabolism Vol. 85, No. 12. p. 4866-4872).

In order to provide greater speed and higher sample throughput, the extracted sample can be directly injected into the LC/MS/MS instrument without further processing. In one embodiment, the present invention allows the direct introduction of the extracted sample into the mass spectrometer by using a volatile extracting medium that allows removal of the extraction medium by evaporation without requiring further manipulation of the sample. The term "volatile" refers to any reagent that has a boiling point equal to or less than about 150° C at atmospheric pressure.

The sebum may be collected on Sebutape®. Typically, 0.1 -10 mg of sebum will be analyzed. The amount of extraction medium used for each sample may vary from, for instance, about 0.5 mL to about 5ml_. Typically, 1 -2 mL of the extraction medium will be used per Sebutape® test sample. In one embodiment, the samples are extracted in an extraction medium prepared by mixing 40 ul of propanol and 2 mL of dichloromethane. The extraction is carried out for a sufficient period of time at a temperature sufficient to extract the lipids in the test sample. Typically, this will range from about 5 min to 0.5 hrs. Typically, the sample is shaken or vortexed several times during the extraction period. The extraction may be carried out at any temperature that is compatible with the integrity of the lipids to be analyzed. Typically, the extraction is carried out at a temperature ranging from about 20° C to about 60⁰C. In one embodiment, the extraction is carried out at approximately 2O⁰C to about 25⁰C. After a sufficient period of time, the extracted lipids may be directly contacted with the mass spectrometer. Alternately, the extraction medium may be removed, for instance, by evaporation, and the lipid sample may be directly subjected to mass spectrometric analysis or it may be stored until subjected to mass spectrometric analysis. Typically, the extracted test samples are transferred to a microtiter plate and the extraction medium is removed by evaporation under nitrogen gas stream and the remaining residue is either used directly or stored frozen until subjected to mass spectrometric analysis.

Prior to application to the mass spectrometer, the test sample is reconstituted in a medium compatible with the mass spectrometer. The test sample may be directly applied to the mass spectrometer without further manipulation.

Quantification of Sebum Components

In order to quantitate the selected lipids of a test sample, for instance, the cholesterol-ester or triglyceride content of a sebum sample, the empirical relationship between the response detected by the mass spectrometer from selected lipid components of a test sample is determined by comparison to a calibration curve of known lipids using standard solutions. The calibration curve is typically set up by preparing at least five standard solutions in propanol of the cholesterol ester or triglyceride of interest ranging from 5 ng/mL to 1000 ng/mL Trileucin internal standard solution was also prepared at 400ng/mL in propanol.

The test samples may be extracted and analyzed in parallel with the standards of the calibration curve using identical conditions. Alternatively, calibration curve may have been previously run and the test sample data compared to the calibration curve for the specific lipid component of interest. The test sample content of a specific lipid component of interest, for instance a cholesterol ester or triglyceride, is calculated using linear regression analysis of the peak area responses from the cholesterol ester or triglyceride content of the test samples and correlating it to the cholesterol ester or triglyceride concentrations from the calibration curve. Quantification may be performed, for instance, by electrospray LC/MS/MS in the selected reaction monitoring (SRM) mode on a Waters Micromass Ultima Il (Beverly, Mass.) tandem quadrupole mass spectrometer with MassLynx version 3.5 controlling software or on a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) with Analyst 1.2 operating software. EXAMPLES

Example 1

Determination of Effect of Flutamide on Hamster ear Sebum by Mass Spectrometry

Male Syrian Hamsters approximately 8-10 weeks old were housed in individual cages. The animals were acclimated to16 hour light cycles for 2 weeks prior to dosing. For each treatment, test animals are anesthetized using isoflurane gas. Each treatment group consisted of five animals. Twenty-five microliters of a test compound was applied topically to the ventral side of each ear using a positive displacement pipette. The subjects were treated twice a day, 5 days a week, for four weeks with at least 6 hours between treatments. The test compound, 1.2% flutamide, was prepared in 70:30 (v/v) ethanohpropylene glycol (EtOHPG). Control animals were treated with 70:30 (v/v) EtOH:PG.

At the end of the treatment period, one 8mm distal biopsy punch was taken, just above the anatomical "V" mark in the ear to normalize the sample area. The punch was pulled apart. The ventral biopsy surface (the area where the topical dose was directly applied to the sebaceous glands) was retained for testing and the dorsal surface of the biopsy punch was discarded.

Tissue samples were dried using nitrogen gas and stored at -8O⁰C under nitrogen until analysis.

Prior to HPLC, tissue samples were contacted with 3ml of solvent (a 4:1 v/v mixture of 2,2,4-trimethylpentane and isopropyl alcohol). The mixture was shaken for 15 minutes and stored overnight at room temperature, protected from light. One milliliter of water was added to the sample and the sample was shaken for 15 minutes. The sample was then centrif uged at approximately 1500rpm for 15 minutes. Two ml of the organic phase (top layer) was transferred to a glass vial, dried at 37⁰C, under nitrogen, for approximately 1 hour, and then lyophilized for approximately 48 hours. The samples were then removed from the lyophilizer and each vial was reconstituted with 250μl of 4:1 propanohchloroform (v/v), vortexed for 2 min and then were transferred to the wells of a 2mL 96 well plates. The samples were then sealed and vortexed for another 2 minutes. The 96 well plate was then transferred to the plate holder of a LEAP CTC PAL autosampler that was connected with two Schimadzu LC-10Advp HPLC pumps equipped with an SLC-10Avp controller. The autosampler HPLC system was interfaced with a Sciex API 4000 triple quadrupole mass spectrometer.

Three μl_ of each sample were injected onto a Phenomenex, C-18, 2.0 x 50 mm, 3 μm column. Two solvents were used for the solvent gradient. Solvent A was a 1 mM solution of Sodium Formate. Solvent B was propanol.

The gradient utilized is described in Table 1 below:

Table 1

The mass spectrometer was operated in the positive ion electrospray ionization mode using selected reaction monitoring (SRM). The main source parameter were as follows: curtain gas was at 10, GS 1 and GS 2 at 50, source temperature 450 ⁰C, CEM was at 2000, Q1 and Q3 mass resolution settings were at low. Table 2 summarizes the SRM mass transitions, declustering potential (DP) and collision energy (CE) settings used for the detection of select cholesterol ester components.

Table 2. Selected Reaction Monitoring Transitions and the Corresponding Declustering Potential and Collision Energy values used for the Hamster Ear

Sebum Analysis Table 2

* The sodium adduct of each analyte was selected for the parent mass setting.

The concentration of the selected cholesterol esters in the hamster ear sebum sample extracts (250 μl_ volume each) of untreated subjects and subjects (n=5 subjects/group) treated with 1.2 % Flutamide are summarized in Table 3 below.

Table 3

^* Concentration was determined based on the calibration curve of cholesteryl linoleate.

Table 3 shows that treatment of a subject with 1.2 % Flutamide decreases the cholesteryl esters by about from 27 to about 48% as measured by the method of the present invention.

Table 3 demonstrates that the method of the present invention is suitable for the detection of changes in the concentration of the selected sebum lipids as a response to drug treatment.

EXAMPLE 2

Determination of meibum components in human tear films collected on

Sebutape® by mass spectrometry

The liquid chromatography/mass spectrometry method of the current invention was used to determine the concentration profile of meibum lipids in human subjects.

Meibum samples were collected from the right and left eye (RE, LE) of an oily and normal male and female subject on three separate days and analyzed for triglyceride and cholesterol ester content. Briefly, prior to sample collection, the skin adjacent to the eye was cleansed with a 70% alcohol wipe and samples were collected 5 minutes after cleansing using Sebutape® from bilateral (left and right) lower eyelids (ciliary line) by touching the Sebutape® to the inner side of the eyelids for 5-10 seconds. The samples were stored at - 70 ⁰C until sample extraction and analysis.

The lipid content of the meibum collected on the Sebutape® membranes was extracted by inserting each membrane into a 5mL glass scintillation vial followed by the addition of 40μL of propanol and 2mL dichloromethane to each vial. The samples were vortexed for 5 minutes. The dichloromethane from each glass scintillation vial was transferred to separate wells of a 2ml_ polypropylene extraction plate (96-well format). The wells were evaporated to dryness at room temperature under nitrogen. Each well was reconstituted with 400μL of propanol.

The concentration of each component in the sample extract was determined by spiking 10OμL of the reconstituted meibum extracts with 50 μl_ of Trileucin as an internal standard, vortexing the samples for 1 min and injecting 5 μl_ onto the LC/MS/MS system using a LEAP CTC PAL autosampler system connected with two Shimadzu LC-1 OAdvP pumps with an SLC-IOAvP controller. A Phenomenex, Synergi Polar RP, 2.0 x 50mm, 4μm HPLC column was used to separate the components using 1 mM Sodium Formate (Mobile Phase A) and Propanol (Mobile Phase B) as eluents at a flow rate of 0.18mL/minute. Gradient elution conditions were applied using the following program: 0-0.7 minutes (45% A), 1.2 minutes (2% A), 3.8 minutes (2% A), 4 minutes (45% A). At the end of each run the column was equilibrated with mobile phase A for 0.5 minutes. Acetone/lsopropanol (50/50, v/v) was used as autosampler wash solvent, using 3 syringes and 3 valve washes post-injection. The HPLC eluent was introduced into a Waters/Micromass Ultima Il mass spectrometer with Masslynx 3.5 controlling software. The MS was operated in the electrospray positive ionization mode; using selected reaction monitoring. The ion source parameters were as follows: capillary voltage 3.6kV; cone voltage 45V; source temperature 100⁰C; desolvation temperature 250⁰C, multiplier 650V; low and high mass resolution lens settings at13.0. Table 4 summarizes the SRM parameters used for the detection of the selected lipid components on the Ultima Il mass spectrometer. Table 4

* The sodium adduct of each analyte was selected for the parent mass setting.

The sum of all triglycerides concentration was divided by the sum of all cholesterol ester concentrations and the ratio was multiplied by 100 in order to express the relative ratio of these lipid components. The relative ratio is shown in Figure 2.

The data indicates that there is no significant difference in the TG/CE ratio between normal and oily females, but a significant difference can be detected between the TG/CE ratio of normal and oily male subjects.

Claims

CLAIMSWhat is claimed is:

1. A method for identifying a compound that modulates sebum production, the method comprising: a. contacting a sample from a treated subject with a mass spectrometer; b. generating a selected reaction monitoring chromatogram or an extracted ion chromatogram for at least one lipid component of said sample; and c. comparing the peak area under the chromatogram generated in step b with the peak area of a control sample.

2. The method of claim 1 wherein said sample is selected from a sebocyte cell sample, a sebum tissue sample, a skin surface sebum sample or a tear fluid sample.

3. The method of claim 2 wherein said tissue sample is selected from a human tissue sample and a hamster sebaceous gland sample.

4. The method of claim 1 wherein said lipid component is selected from the group consisting of triglycerides, cholesterol, cholesterol esters, fatty acids, phospholipids, sphingolipids, and hydrocarbons.

5. The method of claim 4 wherein said hydrocarbon is squalene.

6. A method for identifying a compound that modulates the amount of at least one individual lipid component of a sebum sample, the method comprising: a. generating a selected reaction monitoring chromatogram or an extracted ion chromatogram for at least one lipid component of a sample from a treated subject; and b. comparing the peak area under the chromatogram of said sample with the peak area of a control sample.

7. The method according to claim 1 wherein the ionization source for said spectrometer is selected from electrospray ionization, atmospheric pressure chemical ionization, electron impact, chemical ionization, atmospheric pressure photoioniozation and laser desorption ionization.

8. The method according to claim 8 in which said analyzer of said spectrometer is triple quadrupole.

9. The method according to claim 1 wherein said ion detector of said spectrometer is selected from the group consisting of an electron multiplier, a photomultiplier, multichannel plate, a channel electron multiplier and a penning electrode.

10. The method according to claim 1 in which said sample is subjected to chromatography prior to analysis by mass spectrometry.

11. The method according to claim 10 in which said chromatography is selected from the group consisting of liquid chromatography selected from the group consisting of high performance liquid chromatography column or an ultrahigh pressure liquid chromatography column, and gas chromatography.

12. The method according to claim 10 wherein is said mass spectrometer is connected to said chromatography column.

13. The method of claim 1 or claim 5 wherein said chromatograms are generated by analyzing said samples using selected reaction monitoring on a triple quadrupole tandem mass spectrometer.