WO2009134120A1

WO2009134120A1 - Mass spectrometric analysis of small molecule analytes

Info

Publication number: WO2009134120A1
Application number: PCT/NL2008/050267
Authority: WO
Inventors: Theo Marten Luider; Jeroen J.A. Van Kampen
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2008-04-29
Filing date: 2008-04-29
Publication date: 2009-11-05

Abstract

The present invention relates to a method for analyzing a biological sample for the presence of a small molecule analyte comprising loading said sample on a MALDI mass spectrometric sample matrix and subjecting said sample to MALDI mass spectrometric analysis, characterized in that the MALDI matrix comprises 7-hydroxy-4-(trifluoromethyl)coumarin (HFMC) as a MALDI matrix compound.

Description

Title: Mass spectrometric analysis of small molecule analytes

FIELD OF THE INVENTION

The present invention is in the field of analytical chemistry, more in particular to the monitoring of drugs in patients. The present invention provides methods for analyzing a biological sample for small molecule analytes, such as small molecule drugs, e.g. antiretroviral drugs, comprising MALDI mass spectrometric analysis of the sample. The present invention further provides a MALDI sample matrix comprising 7-hydroxy-4- (trifluoromethyl)coumarin (HFMC) as a MALDI matrix compound, a MALDI sample loading substrate comprising the MALDI sample matrix of the invention and the use of HFMC as a MALDI matrix compound.

BACKGROUND OF THE INVENTION

The treatment of viral infections has benefitted greatly from recent developments in the field of antiretroviral therapeutics. However for many infections like the Human Immunodeficiency Virus (HIV) a complete cure is still lacking. Treatment for HIV infection consists of highly active antiretroviral therapy, or HAART. HAART involves combination therapy with a cocktail of several (typically three or four) antiretroviral drugs preferably selected from different classes of antiretroviral drugs targeting different stages of the HIV life cycle. Typically, these classes include nucleoside or nucleoside analogue reverse transcriptase inhibitors (NRTIs or NARTIs), protease inhibitors and non-nucleoside reverse transcriptase inhibitors (NNRTI). HAART allows the stabilization of the patient's symptoms and viremia, but it neither cures the patient, nor alleviates the symptoms. High levels of HIV, often HAART resistant, return once treatment is stopped. Thus, patients require HAART medication for the rest of their live. Yet, many HIV-infected individuals have experienced remarkable improvements in their general health and quality of life, which has led to a large reduction in HIV-associated morbidity and mortality in the developed world.

For proper medication it is important to be able to regulate the drug levels in a patient. The drugs should be present in sufficient high concentrations for sufficient periods of time in order to be efficacious while avoiding the development of drug resistance by the virus. It is therefore desirable to be able to measure the drug levels in tissues of a patient during the treatment.

For detection of such low molecular weight drugs in biological samples mass spectrometry (MS) could be very useful. MS is a technique whereby the mass-to-charge ratio of charged particles is measured. Mass spectrometers consist of three basic parts: an ion source, a mass analyzer, and a detector system. Ions are produced from a sample, which are then separated on basis of their mass, the number of ions of each mass is detected and this data is collected to generate a mass spectrum representing the masses of sample components. The mass spectrum is measured by a mass spectrometer.

The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are extensively used as ionization techniques for mass spectrometric analysis of biological samples.

MALDI triggers ionization via a laser beam (normally nitrogen or YAG laser). A matrix is used to protect the biological sample/analyte from being destroyed by direct laser beam illumination and to facilitate vaporization and ionization. Analysis of small molecules with MALDI is often hampered by matrix-derived chemical noise in the low mass range. MALDI, however, does offer important advantages over ESI, namely, the much higher sample throughput (allowing high-throughput analysis) and its relative insensitivity to ion suppression. In addition, samples can be conveniently stored on the target plates for future (re)analysis. In MALDI experiments, the nature of the matrix compound has a crucial influence on the ionization efficiencies and the signal intensities of the analytes of interest.

Despite all the developments in MS technology it is still difficult to detect low concentrations of antiviral drugs in biological samples, that is, to detect the drugs at levels which in a patient results in effective inhibition of virus replication and that are safe.

SUMMARY OF THE INVENTION The present inventors have now developed a method for analyzing a biological sample for the presence of an analyte, in particular a small molecule analyte, preferably a small molecule drug, such as an antiretroviral drug, said method comprising comprising loading said sample on a MALDI mass spectrometric sample matrix and subjecting said sample to MALDI mass spectrometric analysis, characterized in that the MALDI matrix comprises 7- hydroxy-4-(trifluoromethyl)coumarin (HFMC) as a MALDI matrix compound.

In a preferred embodiment of the method of the invention, the small molecule analyte is a small molecule drug, more preferably an antiretroviral drug, in particular a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. The protease inhibitor is preferably a HIV protease inhibitor, more preferably selected from the group consisting of nelfinavir, saquinavir, indinavir, lopinavir or ritonavir. The non-nucleoside reverse transcriptase inhibitor is preferably a HIV inhibitor, more preferably nevirapine. In another preferred embodiment of a method of the invention, the

MALDI mass spectrometric sample matrix is provided on a MALDI target plate that is coated with a hydrophobic coating. Preferably said hydrophobic coating comprises a fluoropolymer. More preferably said coating consists of a fluoropolymer. Suitable fluoropolymers include, but are not limited to polytetrafluoroethylene (PTFE), perfluoroalkoxy polymer resin (PFA), fluorinated ethylene -propylene (FEP), polyethylenetetrafluoroethylene (ETFE), polyvinylfluoride (PVF), polyethylenechlorotrifluoroethylene (ECTFE), polyvinylidene fluoride (PVDF), and polychlorotrifluoroethylene (PCTFE). Very good results have been obtained with the compound PFC1601V, which is a commercially available fluoropolymer (Cytonix Corp., Beltsville, MD, USA).

In a method of the present invention the biological sample is preferably a body sample of a subject receiving small molecule drug therapy, preferably antiretroviral therapy. The body sample may be a blood sample or tissue sample, or a sample of peripheral blood mononuclear cells (PBMC) from a subject in which the presence of the small molecule analyte, such as a small molecule drug, preferably an antiretroviral drug, is to be detected.

The MALDI mass spectrometric analysis in a method of the present invention is preferably MALDI Triple Quadrupole mass spectrometry. In another aspect, the present invention provides a MALDI sample matrix comprising HFMC as a MALDI matrix compound.

In another aspect, the present invention provides a MALDI sample loading substrate comprising the MALDI sample matrix of the present invention. Preferably the sample loading substrate is coated with a fluoropolymer coating and said matrix is provided on said coating.

In another aspect, the present invention provides a MALDI sample loading substrate comprising a hydrophobic fluoropolymer coating as described above.

In yet another aspect, the present invention provides the use of HFMC as a MALDI matrix compound for the production of a MALDI matrix.

In yet another aspect, the present invention provides the use of a fluoropolymer as a hydrophobic coating for a MALDI sample loading substrate. Suitable fluoropolymers are as described above for the method. The advantages of using this hydrophobic coating are exemplified in great detail in the Examples below. DESCRIPTION OF THE DRAWINGS

Figure 1 shows a comparison of signal intensities for HIV-I protease inhibitors using various MALDI matrices. Seven protease inhibitors were spiked in a lysate of 1 x 10⁶ PBMC and analyzed using various matrices. The amount of protease inhibitor per spot was 5 picomole. Reported are the mean areas and standard deviations of the SRM traces for a single spot. The areas for saquinavir were divided by ten.

Figure 2. Spotting droplets often μL of a solution of the conventional matrix compound 2,5-dihydroxybenzoic acid (DHB) in various solvents. Upper left: DHB in water. Upper middle: DHB in ACN/water (1:1, v/v). Upper right: DHB in MeOH/water (1:1, v/v). Lower left: DHB in ACN. Lower middle: DHB in MeOH. Lower right: DHB in acetone.

Figure 3 shows the HFMC matrix spotted on to a stainless steel target plate and a fluoropolymer-coated target plate. Left picture: One μL

HFMC matrix spotted on to a stainless steel target plate (10 mg/mL in ACN / H2O (1:1, v/v)). Right picture: Ten μL HFMC matrix spotted on to a fluoropolymer-coated target plate (1 mg/mL in 100% acetonitrile) as described in the Examples. The dashed line represents the actual width of the laser beam (diameter of 110 μm). The diameter of the etched circle is 3.5 mm.

Figure 4 shows the result of SEM analysis of dried sample spots. Spot size reduction for dispensed DHB solutions (25/75 acetonitrile/water). (a) 1 mL on an uncoated stainless target; (b) 1 mL on a fluoropolymer coated plate as described in the Examples. The diameter of the dried spot on the coated plate is -200 mm.

Figure 5 shows selected reaction monitoring (SRM)-traces of 4 pmol indinavir (dots) and 1 pmol saquinavir (triangles) in a single spot using the novel matrix HFMC on a stainless steel target plate (Left) and on a fluoropolymer-coated target plate (Right) as described in the Examples. Figure 6 shows a comparison of background signals in the full-scan mode on uncoated and coated stainless steel plates (panel a) as described in the Examples. Panel b shows the further improvements by heat-curing the coated plates for 20 min at 200⁰C. Figure 7 shows a quantitative analysis of indinavir in PBMC lysates using the novel matrix HFMC on a stainless steel target plate and on a fluoropolymer-coated plate as described in the Examples.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms "MALDI mass spectrometry" and "MALDI-MS" refer to matrix-assisted laser desorption/ionization mass spectrometry, a spectrometric technique used to identify the mass of an analyte in a sample by firing a laser at a matrix-embedded sample in order to liberate the analytes in the sample from the matrix for subsequent analysis. MALDI refers to the ionization technique that ionizes the analyte/matrix mixture, allowing the analysis of analytes from biological samples. The ionization is triggered by a laser beam. Several mass spectrometry types can be used with MALDI. Examples are MALDI-TOF, MALDI-qTOF, MALDI-FTICR and MALDI-triple quadrupole mass spectrometry. The type of a mass spectrometer most widely used with MALDI is the Time-of-fiight (TOF) mass spectrometry. In TOF, ions are accelerated by an electric field of known strength. The velocity of the ion depends on the mass-to-charge ratio, and the mass of the ion thus determines the time to reach the detector. MALDI-FTICR-MS refers to Fourier Transform Ion Cyclotron Resonance mass spectrometry, a mass analyzer (or mass spectrometer) for determining the mass-to-charge ratio (m/z) of ions based on the "cyclotron frequency" of the ions in a fixed magnetic field. MALDI-triple quadrupole mass spectrometry is based on the use of oscillating electrical fields to selectively stabilize or destabilize ions passing through a radio frequency (RF) quadrupole field. The quadrupole mass analyzer uses four parallel rods with fixed direct current and alternating radio-frequency potentials to selectively focus ions for transmission through the rods according to their m/z ratio. Triple quadrupole (QqQ) and quadrupole-quadrupole-TOF (QqTOF) instruments are particularly well suited for MS/MS because they permit efficient ion selection by a quadrupole, fragmentation within the collision cell, and fragment ion detection by quadrupole or TOF mass analyzers. The mass accuracy of QqTOF instruments is typically higher than that of triple quadrupoles.

The term "MALDI matrix" or "sample matrix" refers to a solid structure consisting of crystallized molecules of a matrix compound in which the sample or analyte is embedded. The matrix is used to protect the sample from being destroyed by direct laser beam illumination and to facilitate vaporization and ionization of the sample compounds. Prior to MALDI-MS analysis, the sample or analyte molecules is embedded in the MALDI matrix, which process is also referred to herein as "loading" of the matrix, usually by co-precipitation or co -crystallization from solution on a MALDI matrix substrate. This is achieved by spotting droplets of a fluid comprising matrix molecules, analyte molecules and an optional solvent, and allowing the solvent to evaporate, or the matrix compound to crystallize out of the fluid. This results in a co-precipitate of the sample or analyte molecules with the matrix compound wherein sample or analyte molecules are embedded in matrix compound crystals on the MALDI matrix substrate. The matrix can be optimized with respect to: the ability to embed and isolate analytes (e.g., by co- crystallization); the ability to be vacuum stable; the ability absorb the laser wavelength; the ability to cause co-desorption of the analyte upon laser irradiation; and the ability to promote analyte ionization.

The term "MALDI matrix compound" refers to the substance which is crystallized to form the MALDI matrix (the matrix-forming compound).

The term "MALDI matrix substrate" as defined herein refers to the MALDI-MS target plate that holds the MALDI matrix-embedded sample. The MALDI matrix substrate is usually a stainless steel plate which is optionally coated.

The term "7-hydroxy-4-(trifluoromethyl)coumarin" abbreviated as "HFMC" refers to a compound having the following formula:

The term "biological sample" is herein used to refer to samples taken from a subject, preferably a human, more preferably a human receiving treatment with a small molecule drugs, still more preferably receiving treatment with antiretroviral drugs. Said sample can be a tissue sample, or a sample of urine, feces, saliva, tears, mucous, sputum, semen, cervical secretion, cerebrospinal fluid, vomit, nasal secretions, sweat, amniotic fluid, breast milk or preferably blood (blood plasma, or blood serum). The skilled person will understand that the method of the present invention can in principle be used to detect or quantify antiretroviral drugs from any sample, not necessarily derived from a (mammalian) subject, all of which are collectively referred to as "biological samples".

The term "analyte" is herein defined as the compound in the biological sample that is to be detected by MALDI-MS, for instance, the small molecule drug.

The term "small molecule drug" is used herein in its art-recognized sense, meaning a medicinal drug compound having a molecular weight of less than 2000 Daltons, preferably less than 1200 or 1000 Daltons and typically between 100 and 3000 Daltons, such as between 300 and 700 Daltons. Particularly preferred small molecule drugs include protein inhibitors, such as antiretro viral drugs.

The term "antiretroviral drugs" is herein used to refer a medicament for the treatment of infection by retroviruses, primarily HIV, which when administered to a host or host cell inhibits the proliferation of the retrovirus in the host or host cell by inhibiting at least one phase of the retrovirus life -cycle. Antiretroviral drugs include, but are not limited to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), nucleoside analogue reverse transcriptase inhibitors (nNARTIs), non-nucleoside reverse transcriptase inhibitors (nNRTIs), Integrase inhibitors (e.g. raltegravir and elvitegravir), Entry inhibitors (e.g. maraviroc, vicriviroc, aplaviroc, and enfuvirtide) and Maturation inhibitors (such as bevirimat and Vivecon™). Preferably the antiretroviral drugs are protease inhibitors. A protease inhibitor is a small molecule or compound capable of blocking the protease active site (e.g. binding to or interfering with the substrate cleft of the protease). Protease inhibitors prevent viral replication by inhibiting the retroviral protease, an enzyme used by the viruses to cleave nascent proteins for final assembly of new virions. nNRTIs inhibit reverse transcriptase directly by binding to the enzyme reverse transcriptase and interfering with its function. When a cell is infected by a retrovirus, reverse transcriptase copies the viral single stranded RNA genome into a double stranded viral DNA after which the viral DNA can integrate into the host chromosomal DNA allowing host mediated transcription and translation necessary for viral replication. Blocking reverse transcriptase prevents the synthesis of double -stranded viral DNA and thus prevents viral multiplication.

The fluoropolymer coating refers to a substrate modification, generating a very hydrophobic surface that allows efficient focusing of liquid droplets deposited on the surface, eventually leading to small dried spots. The general goal is to apply a coating material with a surface energy lower than that of the liquid dispensed on it, to avoid "wetting" or spreading of the liquid while the droplet dries. The fluoropolymer coating focuses the MALDI matrix solutions, e.g. the solutions of HFMC and HIV drugs, but is more generally applicable. It also efficiently focuses solutions of the MALDI matrix DHB and the molecules (usually petides or proteins) dissolved therein. In addition, other surface coating materials with low surface energies may be used. The skilled person will understand that other low surface energy coatings can also be used. The goal is to create hydrophobic surfaces (low surface energy, for example Teflon) for MALDI samples, so water or other solvent do not spread but instead form a spherical cap resting on the substrate. During the drying process, this spherical shape is preserved, allowing to reduce the size of the droplet, until a very small dry crystal spot is formed from the solutions. Generally suitable hydrophobic coatings include those of materials with surface energies of 18 dynes/cm or less. The fluoropolymer surface used in the Examples, i.e. PFC1601V, exhibits a surface energy of about 14 dynes/cm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors show herein that when using MALDI-MS in combination with a specific matrix, sensitive quantitative detection of analytes, in particular analysis of small molecule drugs, such as antiretroviral drugs in biological samples is possible. The present inventors have found an entirely new matrix compound: HFMC. This matrix even further improved the analysis of the small molecule drugs in biological samples compared to for instance conventional matrices. The limits of quantification readily allow detection of the drugs that are present at a low concentration, thereby allowing monitoring of drug levels in a patient. Typically, the low concentration is a concentration around 1 fmole of drug per milliliter, preferably below 1 fmole of drug per milliliter, typically above 0.0001 fmole per milliliter.

The present invention discloses a method for detecting analytes such as small molecule drugs in a biological sample comprising MALDI mass spectrometric analysis of said sample wherein a MALDI matrix is used in which HFMC is the matrix-forming compound. It is a particular advantage of the use of HFMC that it provides for very sensitive detection of small molecule drugs, that is, it provides for a matrix that allows for the detection of a small molecule drug at a lower limit of quantitation of less than 0.1 picomole per spot on the target plate, preferably less than 0.05 picomole. Usually the lower limit of detection is above 0.0001 picomole per spot. It was found that compared to other matrices, the HFMC matrix provides for a significantly improved reproducibility.

Prior to MALDI analysis, samples can be treated according to any protocol know in the art. A method for analyzing the drugs in a tissue sample of a subject may for instance be adapted based on a method described below in the Experimental part (In particular the Section headed "Sample preparation Peripheral Blood Mononuclear Cells PBMC"). Briefly the Example below describes how PBMC can be isolated from a biological sample (blood) using a standard Ficoll density gradient protocol. The PBMC are subsequently lysed (in this Example in 100% methanol), and the supernatant of these cells that is collected by centrifugation, and cleaned via a solid phase extraction (SPE) assay. The present inventors demonstrate that small molecule drugs spiked to the cleaned, vacuum dried and subsequently reconstituted supernatants could be easily detected in those samples by the method of the present invention. Thus, normal cell constituents of tissues proved not to interfere with the analysis method herein provided.

A protease inhibitor that can be analyzed by a method of the present invention is preferably a Human Immunodeficiency Virus (HIV) protease inhibitor, most preferably the protease inhibitor that is selected from the group consisting of:

- atazanavir (Reyataz™),

- amprenavir (Agenerase™),

- darunavir (Prezista™) - nelfinavir (Viracept™), - saquinavir (Invirase™ or Fortovase™),

- indinavir (Crixivan™),

- fosamprenavir (Lexiva™ or Telzir™)

- lopinavir (Aluvia™), - ritonavir (Norvir™),

- tipranavir (Aptivus™)

- functional derivatives of these drugs, and combinations thereof, such as:

- lopinavir + ritonavir (Kaletra™). Other antiretroviral drugs that can be measured very suitably by a method of the invention are non-nucleoside reverse transcriptase inhibitors (nNRTI) such as:

- efavirenz (Stocrin™) and

- nevirapine (Viramune™), - etravirine (Intelence™),

- rilpivirine (TMC-278),

- loviride (R89439),

- delavirdine (Re scrip tor™),

- functional derivatives of these drugs and combinations thereof. Preferably said nNRTI is a HIV nNRTI, most preferably nevirapine.

Other antiretroviral drugs that can be measured very suitably by a method of the invention are nucleoside reverse transcriptase inhibitors (NRTIs) or nucleoside analogue reverse transcriptase inhibitors (NARTIs) such as: - lamivudine (3TC or Epivir™),

- abacavir (Ziagen™),

- zidovudine (AZT or Retrovir AZT™),

- stavudine (d4T or Zerit™),

- zalcitabine (ddC or Hivid™), - didanosine (ddl or Videx™), - emtricitabine (FTC or Emtriva™),

- tenofovir (Viread™),

- apricitabine (AVX754)

- stampidine - elvucitabine (L-Fd4C)

- racivir

- amdoxovir

- functional derivatives of these drugs, and combinations thereof, such as: - emtricitabine + tenofovir (Truvada™)

- zidovudine + lamivudine (Combivir™), and

- abacavir + lamivudine + zidovudine (Trizivir™)

In addition to the above-mentioned antiretroviral drugs, the present invention may be used to analyze combinations of various classes of antiretroviral drugs listed above, such as the combinations:

- efavirenz + zidovudine + lamivudine,

- efavirenz + tenofovir + emtricitabine,

- lopinavir boosted with ritonavir + zidovudine + lamivudine, and

- lopinavir boosted with ritonavir + tenofovir + emtricitabine. A method of the invention uses at least one MALDI matrix comprising crystallized molecules of HFMC. The MALDI matrix may be prepared by first providing a solution of the matrix compound and subsequently allowing the MALDI matrix compound to crystallize out of this solution. Suitably, the matrix solution comprises the MALDI matrix compound dissolved in a mixture of water and an organic solvent (for example, acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A suitable example of a matrix- solution is for instance 10 mg/ml of HFMC in ACN:water:TFA (500:500:1), or when the HFMC matrix is used in combination with the hydrophobic target plate as described herein: 1 mg/mL HFMC in ACN/formic acid (200:1; v/v). The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the same. Additionally, salts can be added to the solution. It will be appreciated by a person skilled in the art, that variations of the composition of the above-described solutions may occur. MALDI matrix solutions can be prepared by simply dissolving the MALDI matrix compound in a suitable solvent as described above.

In order to embed the sample or analyte in the matrix, the MALDI matrix solution may be mixed with the analyte. Mixing can optionally be done under a protective atmosphere if required. This analyte/ MALDI matrix solution mixture is then spotted onto a MALDI substrate, usually a plate (usually a metal plate designed for this purpose but any other plate applicable for MALDI can be used). Preferable repetitive spots are used, and preferably an amount of about 1-10 μl mixture is spotted. The procedures used here are in principle standard MALDI-MS techniques. The solids in the mixture are allowed to crystallize out of the solution resulting in a crystallized matrix embedding the analyte molecules, also referred to as a MALDI spot.

For analysis of the sample, a laser is fired at the crystals in the MALDI spot in order to desorb/ionize the sample molecules in the matrix. Also, the procedures used here are in principle standard MALDI-MS techniques. The matrix absorbs the laser energy and without wishing to be bound to theory it is thought that primarily the matrix is ionized by this event. The matrix is then thought to transfer part of its charge to the analyte molecules (e.g. the small molecule drugs), thus ionizing them while still protecting them from the disruptive energy of the laser. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI generally produces singly-charged ions, but multiply charged ions ([M+nH]n+) can also be observed, usually as a function of the matrix, the laser intensity and/or the voltage used. Several lasers can be used and are known from the state of the art. In methods of the present invention the use of a 1,000 Hz Nd:YAG laser (355 nm) (for instance available from PowerChip NanoLaser, JDS Uniphase Inc, San Jose, CA, USA) is preferred.

The ions generated in the above described matrix-assisted laser desorption/ionization or MALDI procedure may then be detected by any mass analyzer available. Very suitably, a triple quadrupole mass analyzing system is used in the methods of the present invention. As stated earlier, in a quadrupole mass spectrometer the quadrupole mass analyzer is the component of the instrument responsible for filtering sample ions, based on their mass-to- charge ratio (m/z). A quadrupole mass analyzer is essentially a mass filter that is capable of transmitting only the ion of choice. Furthermore, the invention provides a method to analyze a body (fluid) sample of a patient for small molecule analytes comprising taking a body (fluid) sample of a patient and analyzing the presence of small molecule analytes in said body (fluid) sample using the methods of the invention as described above. Said patient is preferably a patient receiving small molecule drug therapy. The method is particularly suitable for detecting antiretroviral drugs in for instance patients infected with HIV. The body (fluid) sample can be taken from the patients in any suitable way known from in the art.

The invention further provides a MALDI matrix wherein the matrix material or matrix compound is HFMC. For as far as the inventors are aware, HFMC has never before been proposed for the use as the matrix-forming compound in MALDI-MS analysis.

In preferred embodiments of this aspect of the invention, HFMC is used as a MALDI matrix compound for analysis of small molecule analytes, more preferably drugs, such as antiretroviral drugs, in a biological sample. However, it should be noted that the use of HFMC as a MALDI matrix compound is not limited herein to the analysis of any specific analyte. As noted earlier, the choice of any MALDI matrix is usually optimized with respect to, inter alia, the ability to embed and isolate the analyte (e.g., by co- crystallization); the ability to cause co-desorption of the analyte upon laser irradiation; and the ability to promote analyte ionization. Thus, the suitability of a certain matrix/analyte combination for use in MALDI-MS is not determined solely by the type of analyte, but mostly by the interaction between the matrix and the analyte. Hence, it is an aspect of the present invention that HFMC is proposed as a matrix compound for MALDI in general. HFMC is a photo-luminescent molecule with a molecular mass of

230.02 Da. HFMC exhibits maximum light absorption at 355 nm, which ideally matches the wavelength of the frequency-tripled solid-state Nd: YAG laser of the MALDI-QqQ system (wavelength =355 nm). Upon excitation, HFMC emits fluorescent light at a wavelength of 498 nm. In acidic solutions, HFMC is colourless, while it is bright yellow in basic solutions. HFMC is preferably used in a MALDI matrix solution as described herein in a concentration of about 1- 100 mg/ml HFMC, preferably 1-10 mg/ml.

The invention further provides a MALDI substrate, in particular a MALDI-MS target plate, comprising the HFMC MALDI matrix. This aspect of the invention may be prepared by preparing a MALDI matrix solution as described above, and spotting droplets in a range of 0.1-100 μl on the optionally coated substrate. The MALDI matrix solution preferably comprises the analyte for co-precipitation or co-crystallization with HFMC on the substrate. Another aspect of the present invention includes a solution for preparing a MALDI matrix comprising HFMC dissolved to a concentration of about 0.1-1000 mg/ml, preferably 1-100 mg/ml of HFMA, in a liquid carrier, said liquid carrier comprising a mixture of an organic solvent and , optionally in combination with water. The obtained lower limits of quantitation readily allow clinical applications using just one million PBMC from HIV infected patients under therapy. The new matrix HFMC was used for quantitative analysis of the HIV protease inhibitor indinavir using a stainless steel target plate as well as a target plate with a novel strongly hydrophobic fluoropolymer coating. Using the coated target plate, the mean relative error improved from 10.1 % to 4.6 %, the mean precision improved 33.9 %CV to 9.9 %CV, the lower limit of quantitation went down from 16 fmol to 1 fmol, and the measurement time for one spot was reduced from 6 seconds to 2.5 seconds. The new matrix compound was used in conjunction with a novel substrate for MALDI analysis, readily allowing to spot HFMC solutions containing a large content of organic solvent, which is required for HFMC to be dissolved. Substrate selection is a parameter that significantly affects spot size and sensitivity, as well as crystal growth. In particular, surface modifications made to the plate material have a profound effect on the quality of the obtained sample spots. The novel fluoropolymer coating offers several advantages over standard stainless steel MALDI plates: (1) accurate sample deposition of spots or whole arrays of spots which is crucial for automated, unattended analyses as the laser beam does not need to be re-focused on specific crystal areas on the plate for every spot, (2) spot size reduction to increase sensitivity and stability of the ion signal, (3) deposition of solutions containing significant amounts of organic solvents, which is not possible on uncoated targets as excessive sample spreading occurs, (4) the possibility to remove the coating with methyl-tert- butyl ether to avoid carry-over effects in subsequent analyses. After removal of the coating, the plate can be rigorously cleaned without worry about damaging the hydrophobic surface, which is reapplied, (5) the efficient focusing of dihydroxy benzoic acid (DHB) MALDI matrix solution, which is the most important MALDI matrix for analysis of peptides and proteins, (6) the possibility of focusing most other organic molecules dissolved in aqueous/organic solutions in addition to the described HIV drugs, (7) the possibility to focus much larger volumes of sample in comparison to uncoated plates without spreading of the sample solution, (8) the reduction of chemical noise signals in the analyses caused by impurities present on the plate material, thus potentially lowering detection limits. The chemically inert fluoropolymer coating effectively seals the surface and does not generate analytical ion signals itself, (9) the generation of a long-lasting stable ion signal from a MALDI sample spot because of the concentrated sample spot, allowing a large number of experiments to be conducted from a single spot. Usually, MALDI signals last on the order of a few seconds. It is expected, that the spots obtained on the fluoropolymer coated substrates generate ion signals lasting for more than 1 minute.

The invention will be explained in more detail in the following, non- limiting examples (and experimental part).

EXAMPLES

Materials and methods

Chemicals

7-hydroxy-4-(trifluoromethyl)coumarin (HFMC), 2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 3- hydroxypicolinic acid (HPA), and α-cyano-4-hydroxycinnamic acid (HCCA) were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie Gmbh, Munich, Germany).

Pharmaceutical compounds

The investigated drug compounds were kindly donated by the following companies: lopinavir and ritonavir, Abbott Laboratories (Illinois, IL, USA); saquinavir, F. Hoffmann-La Roche (Basel, Switzerland); nelfinavir, Pfizer

(Groton, CT, USA); indinavir Merck (Rahway, NJ, USA); nevirapine and tipranavir, Boehringer Ingelheim (Ingelheim am Rhein, Germany); amprenavir, GlaxoSmithKline (Middlesex, United Kingdom). Carbamazepine was purchased from Sigma-Aldrich.

Mass spectrometry An API 3000 MALDI-triple quadrupole mass spectrometer (MDS

Analytical Technologies, Concord, ON, Canada) equipped with a prototype 1,000 Hz Nd:YAG laser (355 run) (PowerChip NanoLaser, JDS Uniphase, San Jose, CA, USA) MALDI source was operated in the positive ion mode. The pressure in the MALDI source was ~ 5 mTorr. During laser firing, the target plate was moved horizontally at a constant speed (~ 0.06 cm/s) and the selected reaction monitoring (SRM) traces of the analyte(s) and internal standard(s) were recorded (dwell time of 10 ms each). Analyst software version 1.1 (MDS Analytical Technologies) was used for data acquisition.

Sample preparation Peripheral Blood Mononuclear Cells (PBMC) were isolated from a buffy coat (Sanquin, Rotterdam, the Netherlands) using a standard Ficoll density gradient. PBMC pellets were lysed in 100% methanol overnight at 5 °C (100 μL MeOH per 1x106 PBMC). The lysed PBMC were spun down at 14,000 rpm for 5 minutes. Supernatants of 1x106 PBMC were collected in 1.5 mL vials and 400 μL water was added to each vial. Subsequently, the supernatants were cleaned using 96-well solid phase extraction (SPE) plates (Oasis HLB μelution plate, Waters, USA). Methanol and water (200 μL each) were drawn through each well using a vacuum manifold according to the manufacturer's protocol. Subsequently, the samples were loaded onto the SPE-plate and washed twice with methanol/water (1:4 v/v). Finally, the samples were eluted from the plate with 100 μL of methanol. Drug standards were added to the eluates and were subsequently dried using a SpeedVac (Savant, USA).

On the day of analysis, the dried samples were reconstituted in 10 μL matrix solution and replicates of 1 μL were spotted onto 10 x 10 stainless steel target plates (Perseptive Biosystems, Framingham, MA, USA).

The following matrices were used: 4 mg/mL HCCA, 50 mg/mL DHB, 10 mg/mL HFMC, 50 mg/mL HPA, and 4 mg/mL SA. AU matrices were dissolved in acetonitrile/water/formic acid (500:500:1; v/v/v).

Scanning electron microscope (SEM)

SEM photos were obtained on a Hitachi S-3000N SEM (Hitachi Science Systems, Hitachinaka, Japan). Various levels of magnification were used at 2.00 kV electron energy. Spot areas were determined using SimplePCI software (version 4.0.0, Compix Inc. Imaging Systems, Cranberry Township, PA, USA).

Preparation of target plate with hydrophobic coating The target plates were spin-coated with a hydrophobic coating using a modified centrifuge with a small hole in the top lid and a custom-fitted Teflon rotor for the MALDI plates. During spinning of the target plate at 1000 rpm, 500 μL PFC1601V coating (Cytonix Corp., Beltsville, MD, USA) was slowly applied through the hole to the center of the target plate. Subsequently, the target plate was spun for one minute to evenly distribute the coating solution. The coated plate was then heated for 20 minutes at ~ 200 °C. The coating material can also be applied onto the target plate by simply using a brush, with a slight compromise in coating homogeneity.

Quantitative analysis The following transitions were monitored for SRM: 630.5 → 183 for lopinavir (collision energy (CE), 30 eV) , 722 → 296 for ritonavir (CE, 30 eV), 671.5 → 570.5 for saquinavir (CE, 40 eV), 506.5 → 245.5 for amprenavir (CE, 30 eV), 614.5 → 421.5 for indinavir (CE, 40 eV), 569 → 331 for nelfinavir (CE, 40 eV), 603.5 → 411 for tipranavir (CE, 30 eV), 267 → 226 for nevirapine (CE, 30 eV), 237 → 194 for carbamazepine (CE, 30 eV). As internal standard carbamazepine for nevirapine analysis, indinavir for amprenavir analysis, lopinavir for tipranavir analysis, indinavir for nelfinavir analysis, saquinavir for ritonavir analysis, nelfinavir for indinavir analysis, and tipranavir for lopinavir analysis were used. Two data sets, A and B, were created, each containing five replicate spots per analyte. Data set A was used to construct the calibration curve while data set B served as quality control, and vice versa. For each spot, the peak area of the analyte was divided by the peak area of the internal standard. Subsequently, the average analyte/internal standard ratio for the replicate analyses was calculated, and used for the calibration curves and for quality control purposes. Linear 1/x-weighed curves were used for calibration, unless otherwise stated.

Results Comparison of matrices In the present study, we have compared the use of the α-cyano-4- hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 3,5- dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 3-hydroxypicolinic acid (HPA), and a new matrix, HFMC, for the analysis of seven HIV-I protease inhibitors by MALDI-QqQ (see Figure 1). SA and HPA yielded very poor signal intensities in comparison to HCCA, DHB and HFMC. HCCA exhibited intense signals only for nelfinavir, indinavir and saquinavir, but relatively weak signals for lopinavir, ritonavir, amprenavir and tipranavir. DHB and the new matrix HFMC on the other hand generated very strong signals for all seven protease inhibitors tested.

Coumarin matrix & fluoropolymer-coated target plates

We also explored the use of the new matrix compound HFMC for quantitative analysis of indinavir in PBMC lysates. HFMC is a photo- luminescent molecule with a molecular mass of 230.02 Da. HFMC exhibits maximum light absorption at 355 nm, which ideally matches the wavelength of the frequency-tripled solid-state Nd: YAG laser of the MALDI-QqQ system (λ=355 nm). Upon excitation, HFMC emits fluorescent light at λ = 498 nm. In acidic solutions, HFMC is colourless, while it is bright yellow in basic solutions. When the HFMC matrix was spotted on a stainless steel target plate using the dried droplet technique, the quantitative precisions and quantitative accuracies were poor. Inspection of the SRM traces showed differential incorporation of analyte and internal standard in the HFMC crystals. A fast evaporation approach, i.e. spotting from 100% organic solvents, was used to obtain a more homogenous distribution of analyte and internal standard in the HFMC crystals. However, extensive spreading of the sample was observed when the fast evaporation protocol was applied in combination with stainless steel target plates. We have previously shown that hydrophobic coatings can significantly improve crystallization behaviour on stainless steel plates. In this study, we coated the target plate with an inexpensive strongly hydrophobic fluoropolymer. The coated target plates allowed spotting of large volumes of sample, even for 100% organic solutions of up to 10 μL (see Figure 2). Still, such large volumes of sample dry on a small area of the target plate, due to the remarkable focussing abilities of the coating (see Figure 3). The reduction factor in the area of the crystal layers was further investigated by spotting droplets of 1 μL DHB solutions in acetonitrile/water mixtures of varying composition on coated as well as uncoated target plates (see Figure 4). Using the coated target plates, the area of the dried spots reduced by a factor 18.2 (acetonitrile content of 75%), 23.3 (50% ACN), and 31.5 (25% ACN). Figure 5 illustrates that the coated target plates significantly improve the co- crystallization of analyte and internal standard for indinavir when HFMC is used as matrix. In addition, the measurement times for one spot were reduced from 6 seconds to 2.5 seconds. Another feature of the coating is a significant reduction in background signals from contaminants on the target plate (see Figure 6). Figure 7 illustrates the improvement in the concentration-response curve for indinavir in PBMC lysates as well as improvement in precisions when the fluoropolymer-coated plate was used in combination with the HFMC matrix. Using the fluoropolymer-coated plate, the average precision improved from 34 %CV (sd 17.6) to 9.9 %CV (sd 5.7) and the relative error went down from 10.1 % (sd 4.8) to 4.6 % (sd 3.6). In addition, the lower limit of quantitation improved from 16 femtomole to 1 femtomole per spot on the target plate using a 1/x²- weighed linear curve.

Conclusions There is an increasing interest in the use of MALDI-MS for quantitative analysis of small molecules due to its very high sample throughput capability, its insensitivity to ion suppression, and the possibility to store samples on target plates for future (re)analysis. Using a prototype MALDI-QqQ mass spectrometer, we have shown here that this technique can be used for accurate quantitative analysis of HIV protease inhibitors and a non-nucleoside reverse transcriptase inhibitor in lysates of one million peripheral blood mononuclear cells. The limits of quantitation readily allow clinical applications of the technique. In this study, we had to resort to chemical analogues as internal standards, because stable isotope labeled internal standards were not commercially available. Stable isotopes are generally very expensive, are frequently not commercially available, and custom synthesis of these compounds is laborious. While using chemical analogues as internal standards may at first not seem the ideal choice, it is frequently the only practical option for quantitative analysis using LC-MS or MALDI-MS. In our study, we showed that replicate analysis improved the precisions and accuracies, and that this approach is a simple and practical alternative to using chemical analogues as internal standards. In addition, we have shown that analyte and internal standard can be made to co-crystallize by using a fluoropolymer-coated target plate, thus further improving precisions and accuracies.

Claims

1. Method for analyzing a biological sample for the presence of a small molecule analyte comprising loading said sample on a MALDI mass spectrometric sample matrix and subjecting said sample to MALDI mass spectrometric analysis, characterized in that the MALDI matrix comprises 7-hydroxy-4-(trifluoromethyl)coumarin (HFMC) as a MALDI matrix compound.

2. Method according to claim 1, wherein said small molecule analyte is a small molecule drug.

3. Method according to claim 1 or 2, wherein said small molecule drug is an antiretroviral drug.

4. Method according to claim 3, wherein said antiretroviral drug is a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor.

5. Method according to claim 4, wherein said protease inhibitor is a HIV protease inhibitor selected from the group consisting of nelfinavir, saquinavir, indinavir, lopinavir or ritonavir.

6. Method according to claim 4, wherein said non-nucleoside reverse transcriptase inhibitor is nevirapine.

7. Method according to any one of claims 1-6, wherein said biological sample is a body sample of a subject receiving small molecule drug therapy.

8. Method according to any one of the preceding claims, wherein said biological sample is a blood sample or tissue sample.

9. Method according to any one of the preceding claims, wherein said

MALDI mass spectrometric sample matrix is provided on a MALDI target plate that is coated with a hydrophobic coating.

10. Method according to claim 9, wherein said coating comprises a fluoropolymer.

11. Method according to claim 10, wherein said fluoropolymer is PFC1601V.

12. Method according to any one of the preceding claims, wherein said MALDI mass spectrometric analysis involves MALDI Triple Quadrupole mass spectrometry.

13. A MALDI sample matrix comprising 7-hydroxy-4-(trifluoromethyl) coumarin (HFMC) as a MALDI matrix compound.

14. A MALDI sample loading substrate comprising the MALDI sample matrix of claim 13.

15. MALDI sample loading substrate according to claim 14, wherein said substrate is coated with a hydrophobic fluoropolymer coating and wherein said matrix is provided on said coating.

16. MALDI sample loading substrate according to claim 15, wherein the hydrophobic polymer in said hydrophobic fluoropolymer coating is PFC1601V.

17. A MALDI sample loading substrate comprising a hydrophobic fluoropolymer coating.

18. A MALDI sample loading substrate comprising a hydrophobic fluoropolymer coating with a surface energy of 18 dynes/cm or less.

19. MALDI sample loading substrate according to claim 17 or 18, wherein the hydrophobic polymer in said hydrophobic fluoropolymer coating is PFC 160 IV.

20. Use of 7-hydroxy-4-(trifluoromethyl)coumarin (HFMC) as a MALDI matrix compound for the production of a MALDI matrix.

21. Use of a fluoropolymer as a hydrophobic coating for a MALDI sample loading substrate.

22. Use of a fluoropolymer with a surface energy of 18 dynes/cm or less as a hydrophobic coating for a MALDI sample loading substrate.

23. Use of PFC1601V as a hydrophobic coating for a MALDI sample loading substrate.