The present invention relates in general to the field of oncology, in particular to monitoring the effects of chemotherapeutic treatment of lung cancer, in particular non small cell lung carcinoma (NSCLC), by assessing the effectiveness status of a chemotherapy treatment with the use of a quantitative method for determining a panel of metabolic biomarkers.

Prior Art

Lung cancer is the most frequently diagnosed oncologic disease in Poland. It is also the most frequent cause of deaths resulting from an oncologic disease (31% cancer deaths in men and 16% in women). Operative procedures are the main and the most effective method for the treatment of lung cancer; however such therapy cannot be used in all patients. A contraindication may be the location or size of a tumour, the stage of a cancer disease or the general health condition of a patient. Among other therapeutic methods one may distinguish radiotherapy, chemotherapy, hormonotherapy or targeted molecular treatment. Nevertheless, the best effects are achieved by a combination of two methods - local treatment (operative treatment and radiotherapy) with systemic treatment

(chemotherapy, hormonotherapy, biological treatment).

Standard chemotherapeutic treatment of cancer patients consists in administration to the patients four to six chemotherapy cycles. Every two cycles, the assessment of effectiveness of treatment is performed, i.e. monitoring/control of response to treatment. At present, the effectiveness of a chemotherapeutic treatment applied is assessed by observing changes in images obtained by means of computer tomography (CT), positron emission tomography (PET) or magnetic resonance (MR). Unfortunately, numerous contraindications to performing imagining tests of this kind make it impossible to carry out imagining tests in a wide group of patients. Although such methods are non- invasive, they are limited by a number of contraindications, the existence of which may make it impossible to perform such tests. For example, contraindications to performing computer tomography include pregnancy, hyperthyroidism, impaired kidney function or hypersensivity to contrast medium. In the case of magnetic resonance, tests are not performed in patients in the first trimester of pregnancy, and another counterindication are electric and electronic devices implanted in patients; for example, a pacemaker, an insulin pump, an implanted hearing aid, neurostimulators, intracranial metal clips or metal endoprostheses. Additionally, tests based on imagining techniques belong to very expensive solutions, the access to which is still difficult and which at the same time are not without any effect on the patient’s condition (absorbed dose of radiation).

Lipids commonly occur in human blood; among them are phosphatidylethanolamine 16:0/20:4 (PE 16:0/20:4), phosphatidylethanolamine 18:0/22:6 (PE 18:0/22:6) and lysophosphatidylethanolamine 18:0 (LPE 18:0). They also play a major role in carcinogenesis as they constitute a basic structural component of cell membranes multiplying in the process of cell carcinogenesis. A chemotherapy based treatment causes a tumour shrinkage and thus destruction of cancer cells.

Routine methods for a total lipid content analysis are known. One of them is the enzymatic colorimetric method that enables determination of total cholesterol, triglycerides and High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL). However, the total level of lipids, or even fractions thereof, does not make it possible to obtain precise information because the result obtained is an averaged result which is the resultant concentration of all lipid compounds belonging to the same category. That is why it is so important to create a selective method that will allow the quantification of selected lipids only.

Therefore, there is a need to develop a non-invasive and patient-safe method and means for assessing the effectiveness of the chosen chemotherapeutic treatment of lung cancer. It is essential to indicate a method that will be also available to a wide group of people, including patients in the case of whom the use of commonly applied imagining techniques is excluded. At the same time, such a method must be distinguished by high sensitivity and specificity, because it may provide a basis for taking key diagnostic decisions.

The object of the invention is therefore to provide novel, effective, reliable, repeatable, sensitive and specific methods and means for assessing the effectiveness of a chemotherapeutic treatment in patients with lung cancer which can be applied in all patients in need thereof, particularly in in vitro diagnosis, especially in the diagnosis of lung cancer, such as non-small cell lung carcinoma.

The object of the invention is also do provide novel, effective, reliable, repeatable, sensitive and specific methods and means for the quantitative determination of selected lipids in a sample from a subject, which are suitable for use in clinical diagnosis in all patients.

Summary of the Invention

The above objects have been realized by the solutions claimed in the appended patent claims. Preferable variants of the invention are defined in the dependent claims.

The subject matter of the invention relates to a method for assessing the effectiveness of chemotherapeutic treatment in a subject with lung cancer, wherein a) a panel of metabolic biomarkers (panel of metabolites) comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 is determined quantitatively in a sample from a subject with lung cancer before the start of a chemotherapeutic treatment; b) the panel of metabolic biomarkers (panel of metabolites) comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 is determined quantitatively in a sample from the subject according to a) after at least two cycles of a chemotherapeutic treatment; c) the results obtained in step a) and in step b) are compared and the effectiveness status of the chemotherapeutic treatment in the subject is determined on the basis of obtained results of the comparison. In the method according to the invention an increase in the amount of phosphatidylethanolamine 18:0/22:6 preferably indicates stabilization of the treated lung cancer in a subject.

In the method according to the invention an increase in the amount of phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 preferably indicates a response of the subject to the chemotherapeutic treatment.

In the method according to the invention the metabolite panel preferably comprises also the following metabolite: lysophosphatidylethanolamine 18:0.

In the method according to the invention the quantitative determination of metabolites from the metabolic panel is preferably carried out using liquid chromatography coupled to a triple quadrupole mass spectrometer.

In the method according to the invention the metabolic panel preferably comprises also the following metabolite: uric acid.

More preferably, the quantitative determination of the above metabolite is carried out using a high-throughput solid-phase extraction system coupled to triple quadrupole mass spectrometry.

In the method according to the invention a sample from a subject is preferably a whole blood sample, more preferably a plasma sample.

In the method according to the invention the subject is preferably a human subject.

In the method according to the invention lung cancer is preferably non-small cell lung carcinoma.

The subject matter of the invention also relates to a panel of metabolic biomarkers comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6.

The panel according to the invention preferably comprises also the following metabolite: lysophosphatidylethanolamine 18:0.

The panel according to the invention preferably comprises also the following metabolite: uric acid. The subject matter of the invention also relates to a panel of metabolic biomarkers comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 for use for in vitro diagnosis.

The subject matter of the invention also relates to a panel of metabolic biomarkers comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 for use for the in vitro monitoring of the effectiveness of a chemotherapeutic treatment of lung cancer in a subject.

The panel for the uses according to the invention preferably comprises also the following metabolite: lysophosphatidylethanolamine 18:0.

The panel for the uses according to the invention preferably comprises also the following metabolite: uric acid.

The panel for the uses according to the invention is preferably used in the case of non-small cell lung carcinoma.

The panel for the uses according to the invention is preferably used in the case of a subject that is a human subject.

The subject matter of the invention also relates to a kit for monitoring the effectiveness of a chemotherapeutic treatment in a subject with lung cancer, characterized in that it comprises means for quantitative determination of the level of a panel of metabolic biomarkers comprising the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 as well as instructions for carrying out the method for monitoring the effectiveness of treatment.

The kit according to the invention preferably comprises means for quantitative determination of a panel of metabolic biomarkers comprising also the following metabolite: lysophosphatidylethanolamine 18:0.

The kit according to the invention more preferably comprises means for the quantitative determination by the liquid chromatography method coupled to a triple quadrupole mass spectrometer.

The kit according to the invention preferably comprises means for the quantitative determination of a panel of metabolic biomarkers comprising also the following metabolite: uric acid. The kit according to the invention more preferably comprises means for the quantitative determination using a high-throughput solid-phase extraction system coupled to triple quadrupole mass spectrometry.

Detailed Description of the Invention

Metabolic biomarkers, biomarker metabolites or bioindicators are metabolic indicators occurring in subjects under examination, which enable qualitative and quantitative assessment of various medical, pathologic conditions and diseases. In contemporary medicine biomarkers play an invaluable role as they allow to make a quick, precise, specific and sensitive diagnosis of various diseases and disorders and to assess the effectiveness of applied treatment, such as a chemotherapeutic treatment. Metabolite biomarkers are biochemical agents that are used for a precise and easy diagnosis of diseases, for example cancer diseases, as well as for the assessment of progression of such diseases or for monitoring the treatments thereof or for the assessment of the probability of occurrence of such diseases in a subject under examination. In order to obtain a precise, clear and credible assessment of effectiveness of such a treatment it is often necessary to determine more than one such metabolic biomarker, i.e. a panel of metabolic biomarkers or a panel of metabolites.

The present inventors have demonstrated that complex changes in the level of a panel of metabolic biomarkers comprising the following metabolites: PE 16:0/20:4, PE 18:0/22:6 and preferably LPE 18:0 as well as uric acid may be a useful indicator of patient response for the applied chemotherapy based treatment.

An increase in the concentration of the above indicated lipids (PE 16:0/20:4, PE 18:0/22:6 and preferably LPE 18:0) and preferably uric acid after at least the second chemotherapy cycle, determined by the method according to the invention which involves a quantitative analysis of these metabolites in the blood of patients with non-small cell lung carcinoma subjected to the treatment proves the effectiveness of the applied therapy. According to the invention, a method for measuring a panel of the above indicated metabolites has been developed which can preferably be carried out by means of ultra high-performance liquid chromatography - UHPLC (PE 16:0/20:4, PE 18:0/22:6 and LPE 18:0) or a high-throughput solid-phase extraction system (uric acid) coupled to a triple quadrupole mass spectrometer (e.g. UHPLC 1290 Infinity II, Rapid Fire 365, QQQ 6495 Agilent Technologies). According to the invention the selected lipids (PE 16:0/20:4, PE 18:0/22:6 and preferably LPE 18:0) and uric acid is determined quantitatively in a biological sample, preferably in a blood plasma sample. Fig. 1 shows a schematic workflow of the method according to the invention. More specifically, lipids are preferably determined by performing the following steps: la) provision of a biological sample from a subject, lb) alcohol extraction combined with protein precipitation, lc) centrifugation of precipitate formed residue (residue is discarded),

Ld) filtration of supernatant, le) measurement with the use of UHPLC technique coupled to a mass spectrometer (QQQ).

Uric acid is preferably determined in the following manner:

2a) provision of a biological sample from a subject,

2b) alcohol extraction with simultaneous filtration of extract and removal of lipids,

2c) centrifugation of samples,

2d) measurement with the use of a high-throughput solid-phase extraction (Rapid Fire) coupled to a mass spectrometer (QQQ).

According to the knowledge from literature the most efficient solvent used for the extraction of lipids is cold methanol (-20°C).

Since the determination of uric acid is not preceded by chromatographic separation (preferably a high-throughput solid-phase extraction is used), it is recommended to remove matrix components that could interfere with the measurement.

For this purpose, ready, commercially available kit enabling quick extraction combined with filtering and removal of lipids from the extract was used (Captiva ND Lipids kit (Agilent Technologies)).

The method according to the invention is adapted for use in the course of monitoring the effects of a chemotherapeutic treatment in patients with lung cancer, preferably non-small cell lung carcinoma. The use of the panel of metabolites according to the invention, which is composed of PE 16:0/20:4 and PE 18:0/22:6, and preferably LPE 18:0 as well as uric acid, for determining the effectiveness of applied chemotherapeutic treatment has not been described so far. The present inventors have demonstrated usefulness of the method according to the invention using a panel of metabolic biomarkers for monitoring/controlling the effects of a chemotherapeutic treatment in patients with lung cancer, which fact is confirmed in the examples included herein. The method according to the invention also makes it possible to confirm a stabilization status of a cancer disease.

The method for assessing the effectiveness of a chemotherapeutic treatment in a subject with lung cancer according to the invention is characterized by good sensitivity and specificity. Additionally, its realization generates significantly lower costs compared to currently used imagining techniques. At the same time, the fact that the panel of selected metabolites is measured in a blood sample collected from a patient makes such a test available to every patient, which is not the case with imagining techniques that cannot be carried out in a large group of persons due to numerous contraindications and limitations of such methods.

The subject matter of the invention also relates to a panel of metabolic biomarkers comprising the following metabolites: PE 16:0/20:4 and PE 18:0/22:6, as well as a panel that preferably comprises also the following metabolite: LPE 18:0 and a panel that preferably comprises also the following metabolite: uric acid. Such panels may be used for in vitro diagnosis, in particular for assessing the effectiveness of chemotherapeutic treatment of lung cancer, especially in patients with non-small cell lung carcinoma, and thus it is an effective diagnostic tool.

The present invention was invented on the basis of results of research works. The first step (initial selection of potential indicators) involved an untargeted metabolomic analysis of plasma derived from patients with diagnosed non-small cell lung carcinoma subjected to a chemotherapeutic treatment. Material for tests (blood samples) was taken from patients before the start of treatment and after they had received two cycles of chemotherapy. The analysis of samples was carried out using a liquid chromatography coupled to high resolution mass spectrometer (LC-QTOF-MS). This apparatus enables detection of a wide spectrum of small-particle components present in samples (metabolites). The data obtained were subjected to statistical analysis as a result of which a group of phosphatidylethanolamines and lysophosphatidylethanolamines as well as uric acid were selected as potential markers of a response to chemotherapeutic treatment. Complex changes of a group of compounds (panel) suggested that a diagnostic potential would be revealed by a metabolite panel and not a single compound. Potential usefulness was indicated by the fact that all compounds from the selected group demonstrated changes in a similar range (30-50%) and direction (the level of all the examined metabolites showed increase after administration of two cycles of chemotherapy in patients in whom the treatment proved to be effective. The method for quantitative determination of the indicated panel of metabolites has thus been developed, which can be preferably carried out using a mass spectrometry technology (triple quadrupole mass spectrometer - QQQ) combined with liquid chromatography (LC) (panel of lipids) high-throughput solid-phase extraction (Rapid Fire) (uric acid). The technology selected for the quantitative determination of the selected metabolites enables a very precise determination of the concentration of the examined analyte in samples, which enables obtaining more precise results of the tests. Methods based on QQQ detection have been validated in accordance with the guidelines of the European Medicine Agency (ΈMA) and the U.S. Food and Drug Administration (FDA). The material from 9 patients with partial response to the treatment and 14 patients in whom stabilization of the disease was observed after administration of chemotherapy was analyzed. Results obtained with the use of another analytical method (relative to the method according to the invention) confirmed the results obtained earlier.

The method according to the invention comprising quantitative determination of a panel of metabolites that includes PE 16:0/20:4, PE 18:0/22:6, and a panel of metabolites that also includes LPE 18:0, and a panel of metabolites that further comprises uric acid according to the invention is used for detecting and determining the above compounds in biological samples (e.g. blood plasma) derived from patients with lung cancer, e.g. with non-small cell lung carcinoma. The method can preferably be carried out using liquid chromatography and a solid state extraction system, such as Rapid Fire, coupled to a triple quadrupole mass spectrometer.

The method according to the invention comprising quantitative determination of a panel of metabolites that includes PE 16:0/20:4, PE 18:0/22:6, and a panel of metabolites that also includes LPE 18:0, and a panel of metabolites that further comprises uric acid according to the invention can be used for assessing the effectiveness of applied chemotherapeutic treatment, which preferably comprises:

- determining the level of PE 16:0/20:4, PE 18:0/22:6, LPE 18:0, uric acid in a biological sample derived from a patient with non-small cell lung carcinoma (a sample taken at the moment when a decision to apply a chemotherapeutic treatment was made);

- determining the level of PE 16:0/20:4, PE 18:0/22:6, LPE 18:0, uric acid in a biological sample taken from a patient after administration of every two cycles of chemotherapy and comparing the result with the level of the same metabolites measured before the beginning of the therapy. An increase indicates a positive response to the administered treatment.

The use of a sample for the examination that was obtained in a noninvasive way as a source of information concerning the progress in the chemotherapeutic treatment enables monitoring the effectiveness of the treatment in all patients with lung_cancer. So far, information concerning the effectiveness of the applied treatment has been collected by observing changes in the images obtained with the use of computer tomography, positron emission tomography or magnetic resonance. Unfortunately, a wide range of contraindications for performing such tests made it impossible to perform imagining in many patients.

The fact that the methods according to the invention are carried out by analysing a sample from a patient, preferably a whole blood sample collected from the patient, eliminates the risk of the patient being exposed to an additional dose of radiation but, first of all, it does eliminate the possibility of performing the measurement in all patients in need thereof. Due to their simplicity, speed, noninvasive nature, lack of necessity of performing a surgical procedure or biopsy, and a relatively low cost of such a measurement, the methods according to the invention provide an alternative for currently applied imagining techniques used for assessing the effectiveness of a chemotherapeutic treatment, which alternative makes it possible to monitor the effects of a chemotherapeutic treatment in all patients irrespective of the above mentioned factors excluding patients from imagining tests. Additionally, it should be noted that elimination of the necessity of using costly imagining methods for this purpose allows one to reduce the costs of the treatment and include a wider population in the study. Below is presented a schematic diagram of realization of the invention, including the recommended and preferable methods, procedures, reagents, conditions and devices for realization of the present invention. It should be noted that other equivalent means can be used for the same purpose, so the following preferable methods, procedures, reagents, conditions and devices that are used for carrying out the present invention shall not be interpreted in a limiting manner because they are not intended to limit the scope of the invention defined in the appended patent claims but to illustrate the exemplary embodiments of the present invention.

Exemplary realization scheme of the invention All procedures related to the preparation of solutions and samples for tests should be performed in gloves. Filtration should be performed using protective glasses. All used test tubes and disposable tips should be placed in containers marked as “Biohazard”.

A) Exemplary determination of phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 and lysophosphatidylethanolamine

18:0

1. Principles of determination

Analysis of phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 and lysophosphatidylethanolamine 18:0 in the blood is performed by means of alcohol precipitation of proteins from the obtained blood plasma, centrifuging the residue formed and filtering the supernatant obtained. A solution obtained in this manner is analyzed using liquid chromatography coupled to a triple quadrupole mass spectrometer.

2. Reagents and standard substances for realization of the invention 2.1. Reagents

2.1.1. Water (Purity level 1 )

2.1.2. Acetonitrile (LC-MS Ultra Chromosolv)

2.1.3. Methanol (LC-MS Ultra Chromosolv)

2.1.4. Isopropanol (LC-MS Ultra Chomosolv) 2.1.5. Formic acid (>98%)

2.1.6. Butylated hydroxytoluene

2.1.7. Chloroform Standard substances

2.2.1. Phosphatidylethanolamine 16:0/20:4 dissolved in chloroform at a concentration of lO mg/ml

2.2.2. Phosphatidylethanolamine 18:0/22:6 dissolved in chloroform at a concentration of 10 mg/ml

2.2.3. Lysophosphatidylethanolamine 18:0, powder Internal standard

2.3.1. Phosphatidylethanolamine 15:0/18:1 d7 dissolved in chloroform at a concentration of 1 mg/ml

2.3.2. Lysophosphatidylethanolamine 18:0 d7 dissolved in chloroform at a concentration of 1 mg/ml Other solutions

2.4.1. Butylated hydroxytoluene solution ( 1 mM)

Weight out 2.204 mg of butylated hydroxytoluene (2.1.6.). Then dissolve the weighed amount in 10 ml methanol (2.1.3.). Mix thoroughly.

2.4.2. Butylated hydroxytoluene solution (1 mM)

Prepare a working solution of butylated hydroxytoluene of concentration 1 mM. For this purpose measure up 100 pi of the solution of concentration 1 mM (2.4.1.) and make up to the volume of 100 ml by adding 99,9 ml of methanol (2.1.3.). Mix all the components thoroughly.

2.4.3. Solution of internal standards of concentration 10 pg/ml Prepare a working solution of phosphatidylethanolamine 15:0/18: 1 d7 and lysophosphatidylethanolamine 18:0 d7 of concentration 10 pg/ml (both). For this purpose measure 10 pi of phosphatidylethanolamine 15:0/18:1 d7 solution of concentration 1 mg/ml (2.3.1.) and lysophosphatidylethanolamine 18:0 d7 solution of concentration 1 mg/ml (2.3.2.) and make up to the volume of 1 ml by adding 980 pi of methanol (2.1.3.). Mix all the components thoroughly.

2.4.4. Methanol/chloroform/water mixture (65/35/8 v/v/v) Measure 1300 mΐ of methanol (2.3.2.), 700 mΐ of chloroform (2.1.7.) and 160 mΐ of water (2.1.1.). Mix all the components thoroughly.

2.4.5. Working solution of lysophosphatidylethanolamine 18:0 (2.2.3.) of concentration 1 mg/ml

Weigh up 1 mg of lysophosphatidylethanolamine 18:0 (2.2.3.). Then dissolve the weighed amount in 1 ml of methanol/chloroform/water mixture (65/35/8 v/v/v) (2.4.4.). Mix all the components thoroughly.

2.4.6. Solution of phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0 of concentration 10 pg/ml Measure 1 mΐ of phosphatidylethanolamine 16:0/20:4 solution of concentration 10 mg/ml (2.2.1.) and 10 mΐ of lysophosphatidylethanolamine 18:0 solution of concentration 1 mg/ml (2.4.5.) and make up to 1 ml by adding 989 mΐ of methanol (2.1.3.). Mix all the components thoroughly.

2.4.7. Solution of phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0 of concentration 1 pg/ml Measure 100 mΐ of phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0 solutions of concentration 10 pg/ml (2.4.6.) and make up to 1 ml by adding 900 mΐ methanol (2.1.3.). Mix the components thoroughly.

2.4.8. Solution of phosphatidylethanolamine 18:0/22:6 of concentration 100 pg ml

Measure 10 mΐ of phosphatidylethanolamine 18:0/22:6 solution of concentration 10 mg/ml (2.2.2.) and make up to 1 ml by adding 990 mΐ of methanol (2.1.3.). Mix the components thoroughly.

2.4.9. Solution of phosphatidylethanolamine 18:0/22:6 of concentration 10 pg/ml

Measure 1 pi of solution of phosphatidylethanolamine 18:0/22:6 of concentration 10 mg/ml (2.2.2.) and make up to 1 ml by adding 999 pi of methanol (2.1.3.). Mix all the components thoroughly. The mobile phases 2.5.1. The mobile phase A

Prepare a solution consisting of 999 ml of water (2.1.1.) and 1 ml of formic acid (2.1.5.) in a 1 dm ³ volumetric flask. Mix thoroughly.

2.5.2. The mobile phase B

Prepare a solution consisting of 399.5 ml of isopropanol (2.1.4.), 599.5 ml of acetonitrile (2.1.2.) and 1 ml of formic acid (2.1.5.). Mix thoroughly. Laboratory apparatuses and equipment necessary to realize the invention.

3.1. High-performance liquid chromatograph coupled to a triple quadrupole mass spectrometer.

3.2. Chromatography column: Zorbax Eclipse Cl 8 RRHD (2.1 x 50 mm, particle size 1.8 pm).

3.3. Water purification system Milli-Q Integral 3

3.4. Automated pipettes and disposable tips dedicated therefor

3.5. Vortex

3.6.Laboratory centrifuge with a fixed-angle rotor for centrifugation of 1.5 ml test tubes.

3.7. Laboratory centrifuge with a horizontal swing-out rotor for centrifugation of 9 ml test tubes.

3.8. Laboratory glassware

3.9. 1.5 ml Safe-Lock tubes

3.10. Chromatographic vials

3.11. Chromatographic vial insert that decreases volume.

3.12. Insulin syringes

3.13. Nylon syringe filters having pore size 0.22 pm Preparation of samples

4.1. Analyzed samples

4.1.1. Venous blood should be drawn in sterile conditions into one 9 ml test tube-syringe containing EDTA. Mix the blood by inverting the test tube 8 to 10 times. The blood sample should be immediately transported in room temperature to a laboratory.

4.1.2. The test tube with blood should be centrifuged in a centrifuge with a horizontal swing-out rotor for 10 min, at 1900 xg and at 4°C. 4.1.3. Transfer the blood plasma to 1.5 ml Safe-Lock test tubes . Transfer each 0.5 ml of plasma to individual test tubes and leave about 0.3 ml of plasma over the buffy coat. The blood plasma should be pipetted carefully so as not to disturb the buffy coat and transfer the leukocyte/platelet fraction.

4.1.4. Properly marked test tubes with plasma should be preserved and placed in an ultra-low temperature freezer (-80°).

4.2. Plasma samples from healthy donors for preparing a calibration curve. The samples should be secured in the same way as the tested samples.

5. Procedure

5.1. Analytical sample

Thaw the samples (4.1.) by keeping them on ice for 60-120 min (until they are completely thawed), vortex them for 10 seconds.

5.2. Blank sample

Thaw the samples (4.2.) by keeping them on ice for 60-120 min (until they are completely thawed), vortex them for 10 seconds.

5.3. QC 4 enriched samples

Measure 145 pi of cold (-20°C) methanol solution containing butylated hydroxytoluene (1 mM) (2.4.2.) to a Safe-Lock test tube, add 5 mΐ of internal standards solution of concentration 10 pg/ml (2.4.3.), 5 pi of phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0 solution of concentration 10 pg/ml (2.4.6.) and 2 mΐ of phosphatidylethanolamine 18:0/22:6 solution of concentration 100 pg/ml (2.4.8.). Mix thoroughly. Carefully add 50 mΐ of plasma pool derived from healthy donors (4.2.). The analyte content corresponds to the following concentrations of both phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0: 250 ng/ml and phosphatidylethanolamine 18:0/22:6: 1 pg/ml.

5.4. Calibration sample - CAL

Add cold (-20°C) methanol solution containing butylated hydroxytoluene (1 mM) (2.4.2.) to Safe-Lock test tubes (the amounts are given in Table 1), add 5 mΐ of internal standards solution of concentration 10 pg/ml (2.4.3.) and the amounts of standard substances as given in Table 1. Then add 50 mΐ of plasma pool derived from healthy donors (4.2.) to each test tube.

Table 1.

Extraction

Thaw the samples (4.1.) by keeping them on ice for 60-120 minutes. When the samples are completely thawed vortex each sample for 10 seconds. Then precipitate proteins using cold methanol (-20°C). For this purpose add 145 mΐ of the internal standards solution containing butylated hydroxytoluene (ImM) (2.4.2.) and 5 mΐ of the internal standards solution of concentration 10 mg/ml (2.4.3.). Carefully add 50 mΐ of an analysed sample. Vortex for 1 minute. Then keep the samples on ice for 10 min. After that time centrifuge them for 10 minutes at 4°C at 21 OOOxg. Filter the supernatant through nylon syringe filters (0 0.22 pm) into inserts placed in chromatographic vials and conductJLC- MS/MS analysis. Conditions of analysis

7.1. Chromatographic conditions

7.1.1. Column: Zorbax Eclipse C18 RRHD (2.1 x 50 mm, particle size 1.8 pm)

Guard column: Zorbax Eclipse C18 (2.1 x 5 mm, 1.8 pm)

The column was thermostated at 40°C Mobile phase flow rate: 0.5 ml/min Injection volume: 2 pL

7.1.2. Mobile phases:

A: water + 0.1% formic acid (2.5.1.)

B: acetonitrile/isopropanol (60/40 v/v) + 0.1% formic acid (2.5.2.)

7.1.3. Gradient:

Table 2. After each run, system stabilization in the initial conditions of analysis was set for 2 minutes. Mass spectrometer parameters

7.2.1. Ion source: ESI AJS Scanning mode: dMRM Quadrupole resolution Q1 and Q3: unit Ion mode: positive

Delta EMV (positive): +100 (0.0 - 7.0 min)

+400 (7.0 - 13.0 min)

7.2.2. Ion source parameters:

Gas temperature (°C): 200 Gas flow rate (1/min): 11 Nebulizer pressure (psi): 40 Sheath gas temperature (°C): 250 Sheath gas flow rate (1/min): 8 Capillary voltage (V): 4000 iFunnel parameters:

High pressure RF (V): 160 Low pressure RF (V): 100 Fragmentor: constant (380V)

7.2.3. Monitoring of fragmentation reactions (dMRM)

Table 3.

8. Sample analyzing system 8.1. Blank sample (5.2.) 8.2. Calibration sample level 1 (5.4.)

8.3. Calibration sample level 2 (5.4.)

8.4. Calibration sample level 3 (5.4.)

8.5. Calibration sample level 4 (5.4.)

8.6. Calibration sample level 5 (5.4.) 8.7. Calibration sample level 6 (5.4.)

8.8. QC 4 enriched sample (5.3.)

Analytical sample (a series of 10 samples) (5.1.) A) Exemplary determination of uric acid 1. Principles of determination

Analysis of uric acid in the patients’ blood is performed by alcohol precipitation of proteins from the obtained blood plasma, filtering the obtained mixture through a filer that retains the residue formed and removes lipids from the filtrate. A solution obtained in this manner is analyzed using a high- throughput solid-phase extraction coupled to a triple quadrupole mass spectrometer. 2. Reagents and standard substances for realization of the invention

2.1. Reagents

2.1.1. Water (Purity level 1)

2.1.2. Acetonitrile (LC-MS Ultra Chromosolv)

2.1.3. Methanol (LC-MS Ultra Chromosolv) 2.1.4. Ethanol (99,8% do HPLC)

2.1.5. Formic acid (>98%)

2.1.6. Sodium hydroxide

2.2. Standard substances

2.2.1. Uric acid (powder) 2.3. Internal standard

2.3.1. Uric acid ¹⁵ N (powder)

2.4. Other solutions

2.4.1. Methanol/ethanol mixture (1/1 v/v)

Measure 30 ml of methanol (2.1.3.) and 30 ml of ethanol (2.1.4.) to a 100 ml glass bottle. Mix the components thoroughly.

2.4.2. 16 mg/ml sodium hydroxide solution

Weigh up 128 mg of sodium hydroxide (2.1.6.). Then dissolve the weighed amount in 8 ml of water (2.1.1.). Mix thoroughly.

2.4.3. Sodium hydroxide solution 0,1 M Prepare a working solution of a sodium hydroxide of concentration 1 mM.

For this purpose measure up 6 mΐ of the solution of concentration 16 mg/ml (2.4.2.) and make up to the volume of 24 ml by adding 18 ml of water (2.1.1.). Mix all the components thoroughly. 2.4.4. Internal standard solution of concentration 1 mg/ml

Prepare a working solution of uric acid ¹⁵ N. To this end weigh up 1 mg of uric acid ¹⁵ N (2.3.1.) and dissolve it in 1 ml 0.1 M solution of sodium hydroxide (2.4.3.). Mix all the components thoroughly.

2.4.5. Internal standard solution in the methanol/ethanol mixture of concentration 9 pg/ml

Prepare ¹⁵ N uric acid solution in methanol/ethanol mixture (1:1 v/v). For this purpose, measure 272.7 mΐ of the internal standard solution of concentration 1 mg/ml (2.4.4.) and make up to 30 ml by adding methanol/ethanol (1/1 v/v) mixture (2.4.1.). Mix all the components thoroughly.

2.4.6. Working solution of uric acid of concentration 100 pg/ml

Weigh 1 mg of uric acid (2.2.1.). Then dissolve the weighed amount in 10 ml of sodium hydroxide solution 0.1 M (2.4.2.). Mix the components thoroughly.

2.5. The mobile phases

2.5.1. The mobile phase PI A and P2A

Prepare a solution consisting of 999 ml of water (2.1.1.) and 1 ml of formic acid (2.1.5.) in a 1 dm ³ volumetric flask. Mix thoroughly.

2.5.2. The mobile phase P1B and P3A

Prepare a solution consisting of 999 ml of acetonitrile (2.1.1.) and 1 ml of formic acid (2.1.5.) in a 1 dm ³ volumetric flask. Mix thoroughly. Laboratory apparatuses and equipment necessary to realize the invention.

3.1. High-throughput solid-phase extraction coupled to a triple quadrupole mass spectrometer

3.2. A-type (C4) cartridge

3.3. Water purification system Milli-Q Integral 3

3.4. Automated pipettes and disposable tips dedicated therefor

3.5. Vortex

3.6. Laboratory centrifuge with a horizontal swing-out rotor for centrifugation of 9 ml test tubes

3.7. Laboratory glassware

3.8. 1.5 ml Safe-Lock test tubes

3.9. 96-well polypropylene plate, well capacity: 500 pi 3.10. Vacuum- assisted filtration system (Captiva ND Lipids, Agilent Technology). Preparation of samples

4.1. Analysed samples

4.1.1. Venous blood should be drawn in sterile conditions into one 9 ml test tube- syringe containing EDTA. Mix the blood by inverting the test tube 8 to 10 times. The blood sample should be immediately transported in room temperature to a laboratory.

4.1.2. The test tube with blood should be centrifuged in a centrifuge with a horizontal swing-out rotor for 10 min, at 1900 xg and at 4°C.

4.1.3. Transfer the blood plasma to 1.5 ml Safe-Lock test tubes. Transfer each 10 ml of plasma to individual test tubes and leave about 0.3 ml of plasma over the buffy coat. The blood plasma should be pipetted carefully so as not to disturb the buffy coat and transfer the leukocyte/platelet fraction.

4.1.4. Properly marked test tubes with plasma should be preserved and placed in an ultra-low temperature freezer (-80°).

4.2. Plasma samples from healthy donors for preparing a calibration curve.

The samples should be secured in the same way as the tested samples. Procedure

5.1. Analytical sample

Thaw the samples (4.1.) by keeping them on ice for 60-120 min (until they are completely thawed), vortex them for 10 seconds.

5.2. Blank sample

Thaw the samples (4.2.) by keeping them on ice for 60-120 min (until they are completely thawed), vortex them for 10 seconds.

5.3. QC 5 enriched sample

Measure off and place in a well 165 mΐ of cold (-20°C) methanol/ethanol (1/1 v/v) mixture containing internal standard (2.4.5.), add 15 mΐ of standard solution of concentration 100 mg/ml (2.4.6.) and 70 mΐ of water (2.1.1.). Carefully add 50 mΐ of a mixture of plasma taken from healthy donors (4.2.) and mix three times in the pipette tip. Vacuum-filter the solution into a receiver plate. Transfer filtrate to a 96-well polypropylene plate. (3.9.).

5.4. Calibration sample - CAL Measure off and place in a well 165 mΐ of cold (-20°C) methanol/ethanol ( 1/1 v/v) mixture containing internal standard (2.4.5.) _» add suitable amount of standard solution of concentration 10G pg/ml (2.4.6.) and water (2.1.1.) (Table 4.). Carefully add 50 mΐ of a plasma pool derived from healthy donors (4.2.) and mix three times in the pipette tip. Transfer filtrate to a 96-well polypropylene plate

(3.9.).

Table 4. action

Thaw the samples by keeping them on ice for 60-120 minutes. When the samples are completely thawed, vortex each sample for 10 seconds. Then place 165 mΐ of cold (-20°C) methanol/ethanol (1/1 v/v) mixture containing 9 pg/ml of the internal standard (2.4.5.) and 80 mΐ of water (2.1.1.) each in the well on the filtration plate (3.10). Carefully transfer 50 mΐ of the analysed samples to individual filtration wells and mix three times in the pipette tip. Vacuum-filter the mixture into the receiver plate. Transfer the material from the receiver plate to a 96-well polypropylene plate (3.9.) and carry out Rapid Fire-MS/MS analysis. ditions of analysis Parameters of Rapid Fire analysis

7.1.1. Cartridge: A (C4) Table 5 shows duration times of Rapid Fire cycles. Table 5.

7.1.2. Pumps:

7.1.2.1. Pump 1:

Flow: 1.5 ml/min Composition: 80%P1A. 20% P1B

7.1.2.2. Pump 2:

Flow: 1.25 ml/min Composition: 100% P2A

7.1.2.3. Pump 3:

Flow: 1.25 ml/min Composition: 100% P3A Mass spectrometer parameters

7.2.1. Ion source: ESI AJS

Scanning mode: MRM Quadrupole resolution Q1 and Q3: unit Ion mode: negative Delta EMV (positive): -400

7.2.2. Source operation parameters:

Gas temperature (°C): 300 Gas flow rate (1/min): 11 Nebulizer pressure (psi): 40 Sheath gas temperature (°C): 250 Sheath gas flow rate (1/min): 8 Capillary voltage (V): 4000 iFunnel parameters:

High pressure RF (V): 140 Low pressure RF (V): 90 Fragmentor: constant (380V)

7.2.3. Monitoring of fragmentation reactions (dMRM)

Table 6. Sample analyzing system 8.1. Blank sample (5.2.)

8.2. Calibration sample level 1 (5.4.)

8.3. Calibration sample level 2 (5.4.)

8.4. Calibration sample level 3 (5.4.)

8.5. Calibration sample level 4 (5.4.) 8.6. Calibration sample level 5 (5.4.)

8.7. Calibration sample level 6 (5.4.)

8.8. QC 4 enriched sample (5.3.)

8.9. Analytical sample (a series of 10 samples) (5.1.) Calculation of results (common for part A and B) 9.1. Calibration

The internal standard addition method should be used to determine a linear calibration curve. For calibration levels 2-6 standard deviation of concentration determined on the basis of on calibration curve should not exceed ±20% of the of the predicted value of metabolite concentration in a sample. For the calibration level 1 it is necessary to assess a signal/noise ratio, which should be equal or higher than 10 for the ion used for quantification.

9.2. Calculating the quantities of metabolites. The content of the selected metabolites in the analyzed samples should be calculated on the basis of the calibration curve equation. Deviation of concentration in the enriched sample - QC (5.3.) should not exceed ±20% of the value of nominal concentration in the sample. If said deviation is greater than ±20% of the value of nominal concentration in the sample, calibration should be performed again.

Brief Description of Figures

Fig. 1 shows a workflow diagram of the preferable method according to the invention.

Fig. 2 shows an exemplary chromatogram obtained from the test sample. Fig. 3. shows a comparison between a sample obtained from healthy patients

(red (1)) and a sample enriched with the standards (blue (2)).

Fig. 4. shows results of a measurement obtained for solvents used for preparing samples.

Fig. 5. shows a comparison between a sample obtained from healthy patients (green (1)) and a sample enriched with the standard (red (2)).

Fig. 6. shows the results of a measurement obtained for mobile phases used for the analysis of samples.

Fig. 7. shows an analysis of ROC curve carried out for experimental data for a panel of metabolite biomarkers comprising all four metabolites. The present invention will be illustrated below by means of examples and figures which, however, are not intended to limit in any manner the scope of protection of the invention as defined in the patent claims. Unless indicated otherwise, all methods and parameters are such as commonly used in the field of the present invention and the devices and reagent used are applied in a manner recommended by the manufacturers thereof. Example - Quantitative determination of a two-component, three-component and four-component panel of metabolite biomarkers and assessment of the effectiveness of a chemotherapeutic treatment of lung cancer with the use of such panels.

Below is presented an example of conducted studies comprising a targeted analysis and its results leading to the confirmation of panels of metabolites comprising phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 and also, optionally, lysophosphatidylethanolamine 18:0, and, optionally, additionally, uric acid.

1. Collection of biological material

Blood samples were collected in the Clinical Hospital of the Medical University of Bialystok. The examined group consisted of patients with diagnosed non-small cell lung carcinoma who were subjected to chemotherapeutic treatment. Blood samples were taken before the beginning of the therapy and after administration of two cycles of chemotherapy. For tests 9 ml of peripheral blood was collected into a test tube containing EDTA. Then the blood was centrifuged (10 min., 1900 xg. 4°C). The blood plasma obtained was transferred to properly marked cryovials. The plasma samples taken from patients were kept at -80°C until the day on which day were analyzed.

2. Characteristics of the tested group

The experiment covered two groups of patients. The first group (n=9) consisted of persons in whom a panel of experts confirmed a partial response after two cycles of the treatment that had been administered. The second group (n=14) was made up of patients in whom merely a stabilization of the disease was confirmed after the administration of the treatment.

3. Validation of analytical methods. Every instrumental method of analysis that enables quantitative determination must be validated; such tests were also carried out in the presented experiment. For this purpose a series of calibration samples was prepared in triplicate. Due to the significant importance of the presence of a matrix (the so called matrix effect) in the case of quantitative analyses based on mass spectrometry using ESI source, mixed (averaged) plasma obtained from 40 healthy donors was used for tests. The material constituted a basis for the tests used to validate the method. Validation was carried out with respect to both the method enabling determination of lipids (using liquid chromatography) and the method determining the concentration of uric acid (based on measurement by Rapid Fire technique).

In both cases plasma was thawed on ice for 60-120 min. After thawing it was mixed using a laboratory shaker (10 seconds).

3.1. V alidation of a method for determining lipids

Aliquots of cold (-20°C) methanol with 0.1 mM of butylated hydroxytoluene (used as antioxidant), aliquots of internal standards and a series of standard substances were measured off and placed in Safe-Lock test tubes. Each aliquot was also supplemented with thawed plasma (as described above). A series of samples was prepared in the concentration range of 10-750 ng/ml (for phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0) and 0.1-5 pg/ml for phosphatidylethanolamine 18:0/22:6). The concentration of internal standards was constant in all samples and amounted to 250 ng/ml (for both phosphatidylethanolamine 15:0/18:1 d7 and lysophosphatidylethanolamine 18:1 d7). The series was prepared in triplicate. A blank sample without the addition of standard substances was also prepared. After adding plasma all samples were mixed for 1 minute and then they were kept on ice for 10 minutes. After that time the samples were centrifuged at 4°C and at 21 000 x g centrifugation speed for 10 minutes. Then the supernatant was filtered using nylon syringe filters (0.22 pm) directly to the inserts of chromatographic vials. A sample which had no standards (either of the substances being determined or internal standards) added before the extraction procedure was also prepared. Such sample was prepared according to the above described procedure and all standard substances were added to the filtered supernatant at the final step. All the components were thoroughly mixed and subjected to LC-MS/MS analysis.

3.2. Validation of the method for determining uric acid.

An aliquot of methanol/ethanol (1/1 v/v) mixture was placed in a well of the filtration plate, the following aliquots were added: internal standard, standard substance and water. Each aliquot was also supplemented with thawed plasma. The series of samples was prepared within the concentration rage of 0.5-10 pg/ml. The series was prepared in triplicate. A blank sample without the addition of standard substances was also prepared. All samples were filtered using a kit dedicated to simultaneous precipitation of proteins and removal of the lipid fraction from the filtrate. A sample which had no internal standard or a standard of analyte added before the extraction procedure was also prepared. That sample was also filtered in accordance with the above procedure (without addition of standards). After filtering both standards (the standard of the analyte and the internal standard) in a strictly defined concentration were added. All samples were analysed by means of Rapid Fire system coupled to a triple quadrupole mass spectrometer.

4. Analytical samples

4.1. Analysis of lipids

The samples were thawed on ice for 60-120 min (until they were completely thawed). Subsequently each sample was vortexed for 10 seconds. Then precipitation of proteins and extraction was performed. For this purpose, cold methanol (-20°C) with 0.1 mM butylated hydroxytoluene (as antioxidant) was used. A solution of internal standard was also added. Precipitation of proteins and extraction was carried out by adding 3 aliquots of methanol to 1 aliquot of plasma (samples). The sample prepared in this manner was vortexed by 1 minute. After that time the sample was placed on ice (10 min.). Then it was centrifuged at 4°C at a speed of 21 000 xg for 10 min. The supernatant was filtered into the inserts of chromatographic vials using nylon syringe filters.

The calibration curve and an enriched sample (QC) were made by preparing series of samples of various standard concentrations and taking into account the matrix. The samples were prepared in the same manner as the samples of the validation series.

4.2. Analysis of uric acid

The samples were thawed on ice for 60 min (until they were completely thawed). Then each sample was vortexed for 10 seconds. A filtration plate was prepared by placing an aliquot of cold (-20°C) methanol/ethanol mixture with an addition of the internal standard as well as an aliquot of water in each filtration well. Further filtration wells prepared in such a way were supplemented with 50 mΐ of a sample (the ratio of 5 aliquots of the solvent to 1 aliquot of a sample was obtained). The samples were filtrated through the plate using a vacuum pump and then transferred from the receiver plate to a classic 96-well plate.

The calibration curve and the enriched sample (QC) were generated by preparing a series of samples with different concentrations of the standards taking into account the matrix. The samples were prepared in the same way as the samples of the validation series. 5. Analysis of samples

5.1. Analysis of lipids by means of liquid chromatograph coupled to a mass spectrometer.

The samples were analysed by means of liquid chromatography (LC) coupled to a triple quadrupole mass spectrometer (6495 iFunnel Triple Quad Agilent Technologies) (QQQ). ESI ion source with JetStream was used. Chromatographic separation was carried out by means of thermostated (40°C) column Zorbax Eclipse C18 RRHD (2.1 x 50 mm, particle size 1.8 pm) equipped with a pre-column (Zorbax Eclipse Cl 8 2.1 x 5 mm, 1.8 pm). The mobile phases having the flow rate of 0.5 ml/min were: water with addition of 0.1% formic acid (Phase A) and acetonitrile/isopropanol (60/40) mixture with addition of 0.1% formic acid (Phase B). Their mutual ratio was changing over time in accordance with the following gradient: 0.0 min 95% A, 1.0 min 40% A, 5.0 min 35% A, 8.0 min 15% A, 12.0 min to 13.0 min 0% A. Afterwards, it was left for 2 minutes so that the initial conditions of the system could stabilize before the subsequent analysis.

The spectrometer operated in dynamic MRM mode in positive mode. The observed dwell times are presented in Table 3.

Between analyzed samples, a solvent (methanol) was dosed. The analysis of samples started from the measurement of the reagent sample and the blank sample. Then the series of calibration samples was analyzed, which was repeated after every 20 samples. Between analysis of calibration samples, after every 7 analytical samples, enriched (QC) samples were analysed.

5.2.Analysis of uric acid by means of Rapid Fire system coupled to a mass spectrometer.

The samples were analysed by means of high-throughput solid-phase extraction (Rapid Fire 365) with a triple quadrupole mass spectrometer (6495 iFunnel Triple Quad Agilent Technologies). ESI source equipped with JetStream was also used. Cartridge A (C4 Agilent Technologies) was used for the analyses. The following mobile phases were used: in pump No. 1 (flow rate: 1.5 ml/min) - water (90%) and acetonitrile (10%), both with addition of 0.1% formic acid, in pump No. 2. (flow rate: 1.25 ml/min) - water with of 0.1% formic acid, and in pump No. 3 (flow rate: 1.25 ml/min) - acetonirile with 0.1% formic acid.

The spectrometer operated in the negative ion mode. Observed dwell times are presented in Table 6. The analysis of samples started with the measurement of a reagent sample and a blank sample. Then a series of calibration samples was analysed, which was repeated after every 27 samples. Between the analysis of calibration samples, after every 8 analytic samples, the enriched samples (QC) were analysed.

6. Obtained results

6.1. Validation of the method for determining lipids

In order to validate the proposed analytical method the signal of the tested metabolites was measured in the series of calibration solutions in triplicate. On the basis of the results obtained in this manner a linear calibration curve was generated by the internal standard addition method. Deviation of concentration for the tested metabolite in a sample, determined on the basis of the calibration curve equation, did not exceed ±20% of the nominal concentration value. Reproducibility of results on each calibration level was also measured. The relative standard deviation (RSD) was within the following ranges: 2.58-14.15% for lysophosphatidylethanolamine 18:0, 2.68-1.35% for phosphatidylethanolamine 18:0/22:6 and 10.34-17.94% for phosphatidylethanolamine 16:0/20:4. On this basis the following limits of determination were specified: 50 ng/ml for lysophosphatidylethanolamine 18:0, 100 ng/ml for phosphatidylethanolamine 18:0/22:6 and 50 ng/ml for phosphatidylethanolamine 16:0/20:4.

The effectiveness of the proposed method for extracting a panel of the studied metabolites from plasma was also verified. This was done by comparing the result obtained for a sample enriched before extraction with the result from the sample enriched with standards after the process of extraction. Recovery amounted to 111% for lysophosphatidylethanolamine 18:0, 120% for phosphatidylethanolamine 18:0/22:6 and 165% for phosphatidylethanolamine 16:0/20:4.

The selectivity of the method was also examined. For this purpose, the mobile phases, solvents used to precipitate proteins and the plasma alone (derived from healthy donors) were measured. The results obtained in this manner were compared with the signal from a sample enriched with the standard. The results of the selectivity assessment of the method are presented in Figures 2-4. Based on these results, the method was found to be selective with respect to the chosen metabolites extracted from the plasma. Fig. 2 shows exemplary chromatogram obtained for the tested sample. Fig. 3 presents a comparison of the sample obtained from healthy patients (red (1)) with the sample enriched with standards (blue (2)). Differences between the samples can be clearly seen. Fig. 4 shows the results of a measurement obtained for solvents that are used for the preparation of samples.

6.2. Validation of the method for determining uric acid.

Validation of a method that enables measurement of uric acid was carried out in an analogical way as that described in 6.1. Deviation of concentration for the tested metabolite in a sample, determined on the basis of the calibration curve equation, did not exceed ±20% of the nominal concentration value. Reproducibility of results on each calibration level was also measured. The relative standard deviation (RSD) was within the range of: 1.99-6.06%. On that basis the limits of determination were defined to be at the level of 1.5 pg/ml.

In this case also the effectiveness of uric acid extraction from the plasma was verified. This was done in the same way as described in 6.1. Recovery for uric acid was 151%. An important parameter that should be specified in context of analyses based on Rapid Fire technology is the matrix effect. Despite the fact that there was no chromatographic separation preceding ionization, the influence of the matrix in the case of the proposed analysis is not so great as one would expect and amounts to 72%.

The selectivity of the method was examined in the same way as described above. The obtained results allow to define the method as selective (Figures 5-6). Fig. 5 presents a comparison between a sample obtained from healthy patients (green (1)) and a sample enriched with the standard (red (2)). Differences between the samples can be clearly seen. Fig. 6 shows the results of measurement obtained for the mobile phases used for the analysis of samples.

7. Results for analytical samples

The results, irrespective of the measurement technique used, i.e. chromatography or high-throughput solid-phase extraction, were analysed in an identical way. For this purpose Mass Hunter Quantitative Analysis software (version B.07.01 for QQQ) was used. A calibration curve was generated by the internal standard addition method. On the basis of this method the concentrations of each analyte were determined. Then the results obtained were subjected to a statistical analysis.

The metabolites which significantly change in patients who received two cycles of chemotherapy (the group of patients in whom partial response to the treatments was reported) are phosphatidylethanolamine 18:0/22:6 (increase by 24.6%, p value of 0.009) and phosphatidylethanolamine 16:0/20:4 (increase by 44%, p value of 0.045). Moreover, phosphatidylethanolamine 18:0/22:6 also increases significantly (by 25.3%, p value of 0.048) in patients in whom a stabilization of the disease was only reported.

8. Analysis of ROC curves

In order to assess the diagnostics usefulness of the panels of metabolites according to the invention an analysis of Receiver Operating Characteristic (ROC) curves was carried out using for this purpose Med Calc software. It was observed that the use of the other metabolites in addition to the two above indicated metabolites that significantly change as a result of the treatment, i.e. lysophosphatidylethanolamine 18:0 as well as uric acid, significantly improve the parameters of sensitivity and selectivity of such a panel (Fig. 7). As a result of the analysis of the panel of all four metabolites an Area Under ROC curve AUC = 0.762 (p value of 0.0189) was obtained with the sensitivity of 66.67% and specificity of 92.86%. These results indicate that a precise diagnostic tool has been developed which would facilitate the assessment of effectiveness of a chemotherapeutic treatment in all patients in need thereof and which would lower the costs of such examination.

The performed studies showed that a panel of metabolic biomarkers comprising phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 significantly changed after the start of a chemotherapeutic treatment of lung cancer which indicates that a response to the chemotherapeutic treatment was obtained (a significant increase of the level of said metabolite biomarkers).

The above metabolic panel comprising two metabolite biomarkers, i.e. phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, proved to be particularly important because these two biomarkers significantly changed (an increase by 44% (p value = 0.045) and an increase by 24.6% (p value = 0.009), respectively) after the administration of two chemotherapy cycles to patients, who subsequently showed partial response to such treatment.

Moreover, phosphatidylethanolamine 18:0/22:6 also changes significantly (an increase by 25.3% (p value = 0.048)) in patients in whom a stabilisation of the disease is only reported. The panel of metabolites comprising the two metabolic biomarkers, i.e. phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, allows effectively assessing the effectiveness of a chemotherapeutic treatment of lung cancer. The panel comprising phosphatidylethanolamine 18:0/22:6 allows to confirm stabilisation of the disease.

The performed studies showed also that the panel of metabolic biomarkers comprising phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 and lysophosphatidylethanolamine 18:0 as well as uric acid significantly increases the parameters of the ROC curves obtained, and thus it additionally increases sensitivity and selectivity and consequently preciseness of the method for assessing the effectiveness of a chemotherapeutic treatment in the examined patients with lung cancer compared to the above described two-component and three-component panel of metabolic biomarkers.