PL241608B1 - Method for the evaluation of the effectiveness of chemotherapeutic treatment of lung cancer, panel of metabolic biomarkers and set for monitoring the effectiveness of chemiotherapeutic treatment of lung cancer - Google Patents

Abstract

The subject of the invention is a method for assessing the effectiveness of chemotherapeutic treatment in a subject with lung cancer, in which a panel of metabolic biomarkers is quantified in a sample from a subject with lung cancer before starting chemotherapeutic treatment, then in a sample from the same subject after at least two cycles of chemotherapeutic treatment this panel of metabolic biomarkers is quantified again, the results obtained are compared, and based on the result of the comparison obtained, the efficacy status of the chemotherapeutic treatment of the subject is determined. The subject of the invention is also a panel of metabolic biomarkers, a panel of metabolic biomarkers for use in in vitro diagnostics, a panel of metabolic biomarkers for use in in vitro monitoring of the effectiveness of chemotherapeutic treatment of lung cancer and a kit for monitoring the effectiveness of chemotherapeutic treatment of lung cancer.

Description

The subject of the invention is a method for assessing the effectiveness of chemotherapeutic treatment in a subject with lung cancer, diagnostic panels of metabolic biomarkers, the use of diagnostic panels of metabolic biomarkers for in vitro diagnostics of lung cancer, the use of diagnostic panels of metabolic biomarkers for monitoring in vitro the effectiveness of chemotherapeutic treatment of lung cancer in a subject, and a kit for monitoring the effectiveness of the chemotherapeutic treatment in a subject with lung cancer.

The field of invention

The present invention relates to the field of oncology in general, and in particular to controlling the effects of chemotherapeutic treatment of lung cancer, in particular non small cell lung carcinoma (NSCLC), by assessing the efficacy status of the chemotherapeutic treatment using a quantification panel of metabolic biomarkers.

state of the art

Lung cancer is the most frequently diagnosed oncological disease in Poland. It is also the most common cause of cancer-related deaths (31% of cancer deaths in men and 16% in women). Surgery is the main and most effective method of lung cancer treatment, however, not all patients are able to undergo such therapy. The contraindication may be the location or size of the tumor, the stage of the cancer or the general health of the patient. Other therapeutic methods include radiotherapy, chemotherapy, hormonal therapy or molecularly targeted treatment. Nevertheless, the best results are obtained by combining two methods - local treatment (surgical treatment or radiotherapy) and systemic treatment (chemotherapy, hormone therapy, biological treatment).

Chemotherapy treatment of cancer patients typically involves giving patients four to six cycles of chemotherapy. Every two cycles, an assessment of the effectiveness of the treatment is carried out, i.e. a control of the response to treatment.

At present, the effectiveness of chemotherapy treatment is assessed by observing changes in images obtained using computed tomography (CT), positron emission tomography (PET) or magnetic resonance imaging (MR). Unfortunately, a wide range of contraindications to this type of imaging makes it impossible to perform imaging in a wide group of patients. Although these methods are non-invasive, they are limited by a number of contraindications, the occurrence of which may make it impossible to perform such tests. For example, a contraindication to computed tomography is the patient's pregnancy, hyperthyroidism, impaired kidney function or hypersensitivity to the contrast agent. In the case of magnetic resonance imaging, examinations are not performed on patients in the first trimester of pregnancy, and electrical and electronic devices implanted in the patient, such as pacemakers, insulin pumps, implanted hearing aids, neurostimulators, metal intracranial clips or metal endoprostheses, are also contraindicated. Moreover, examinations based on imaging techniques are very expensive solutions, which are still difficult to access, and which at the same time have an impact on the patient's condition (receiving a dose of radiation).

Lipids are commonly found in human blood, such as 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 an important role in the process of cancer development, being the basic building blocks of cell membranes that multiply in the process of cell carcinogenesis. On the other hand, chemotherapy-based treatment causes the tumor to shrink, and thus the cancer cells to break down. In patients in whom chemotherapy treatment brought good results, an increase in the level of lipids is observed, which may come from disintegrating cancer cells.

Routine methods for total lipid determination are known. This is, for example, an enzymatic-colorimetric method that allows the determination of total cholesterol, triglycerides and high-density lipoprotein (HDL) and low-density lipoprotein (LDL). However, the total level of lipids, or even their fractions, does not allow for obtaining precise information, because the obtained result is an averaged result being the resultant of the concentration of all lipid compounds belonging to a given category. That is why it is so important to create a selective method that will allow for the quantification of only the indicated lipid compounds. However, no methods are known to date that allow the quantification of only the indicated lipids.

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Therefore, there is a need to develop a non-invasive and patient-safe method and means for assessing the effectiveness of the undertaken chemotherapeutic treatment of lung cancer. It is important to indicate a method that will also be available to a wide group of people, including patients for whom the use of imaging techniques is excluded. At the same time, such a method must be characterized by high sensitivity and specificity, because it can be the basis for making key diagnostic decisions.

Thus, there is also a need to develop a method for determining the level of selected lipids and compounds in a biological sample from an individual, such as a plasma or serum sample, with particular application in assessing the effects of chemotherapeutic treatment in patients with non-small cell lung cancer.

The aim of the invention is therefore to provide new, effective, reliable, reproducible, sensitive and specific methods and means for assessing the effectiveness of chemotherapeutic treatment in patients with lung cancer, which are applicable to all patients in need, in particular in in vitro diagnostics, especially of lung cancer such as non-small cell lung cancer.

It is also an object of the invention to provide new, effective, reliable, reproducible, sensitive and specific methods and means for the quantification of selected lipids in a sample from a subject, which are suitable for clinical diagnosis in all patients.

Brief description of the invention

The above objectives have been achieved thanks to the solutions claimed in the appended claims. Advantageous variants of the invention are defined in the dependent claims.

The present invention relates to a method for evaluating the effectiveness of a chemotherapeutic treatment in a subject with lung cancer in which

a) in a sample from an individual with lung cancer, a panel of metabolic biomarkers (metabolite panel) containing the following metabolites is quantified before starting chemotherapy treatment: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6,

(b) in a sample from the subject of (a) after at least two cycles of chemotherapeutic treatment, a panel of metabolic biomarkers (metabolite panel) containing the following metabolites is quantified: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6,

c) the results obtained in step a) and step b) are compared, and based on the obtained result of the comparison, the efficacy status of the chemotherapeutic treatment in the subject is determined, wherein an increase in the concentration of phosphatidylethanolamine 18:0/22:6 indicates stabilization of the treated lung cancer in the subject and an increase in phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 is indicative of the subject's response to chemotherapeutic treatment.

In the method of the invention, the metabolite panel preferably further comprises the following metabolite: lysophosphatidylethanolamine 18:0.

In the method of the invention, quantification of metabolic panel metabolites is preferably performed using liquid chromatography coupled to a triple quadrupole mass spectrometer.

In the method of the invention, the metabolite panel preferably further comprises the following metabolite: uric acid.

More preferably, the quantification of the above metabolite is performed using a high throughput solid phase extraction system coupled to triple quadrupole mass spectrometry.

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

In the method of the invention, the subject is preferably a human.

In the method of the invention, the lung cancer is preferably non-small cell lung cancer.

The subject of the invention is also a diagnostic panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 and uric acid.

The subject of the invention is also a diagnostic panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6, uric acid and lysophosphatidylethanolamine 18:0.

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The subject of the invention is also the use of a diagnostic panel of metabolic biomarkers containing the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 and uric acid for in vitro diagnostics of lung cancer.

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

Use of a metabolic biomarker diagnostic panel according to the invention preferably further comprising the following metabolite: lysophosphatidylethanolamine 18:0.

The use of the metabolic biomarker diagnostic panel of the invention preferably relates to non-small cell lung cancer. The use of the metabolic biomarker diagnostic panel of the invention preferably relates to a human subject.

The invention further provides a kit for monitoring the effectiveness of chemotherapeutic treatment in a subject with lung cancer, characterized in that it comprises means for quantifying the level of a panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/ 22:6, by liquid chromatography coupled to a triple quadrupole mass spectrometer, and uric acid, using a high throughput solid phase extraction system coupled to a triple quadrupole mass spectrometer, and instructions on how to monitor treatment efficacy.

The kit for monitoring the effectiveness of a chemotherapeutic treatment in a subject with lung cancer according to the invention preferably further comprises means for quantifying the level of the following metabolic biomarker: 18:0 lysophosphatidylethanolamine by liquid chromatography coupled to a triple quadrupole mass spectrometer.

Detailed description of the invention

Metabolic biomarkers, biomarker metabolites or bioindicators are metabolic indicators present in the tested individuals, which allow for qualitative and/or quantitative assessment of various medical, pathological and disease states. In modern medicine, biomarkers play an invaluable role, allowing, among others, for quick, precise, specific and sensitive diagnosis of various diseases or disorders, as well as assessment of the effectiveness of treatment, such as chemotherapeutic treatment. Metabolic biomarkers are biochemical factors used for precise and easy diagnosis of diseases, for example, cancer, as well as for assessing the progression of such diseases or monitoring their treatment, or assessing the likelihood of such diseases in the examined individual. Often, to obtain a precise, unambiguous and reliable assessment of the effectiveness of such treatment, it is necessary to determine more than one such metabolic biomarker, i.e. a panel of metabolic biomarkers or a panel of metabolites.

The inventors have shown that comprehensive changes in the level of a panel of metabolic biomarkers containing the following metabolites: PE 16:0/20:4, PE 18:0/22:6, and preferably LPE 18:0 as well as uric acid, can be a useful indicator patient's response to chemotherapy-based treatment.

An increase in the concentration of the indicated lipids (PE 16:0/20:4, PE 18:0/22:6 and preferably LPE 18:0) and preferably uric acid at least after the second cycle of chemotherapy determined using the method of the invention consisting in the quantitative analysis of these of metabolites in the blood of patients with non-small cell lung cancer undergoing treatment proves the effectiveness of the therapy. According to the invention, a method for measuring a panel of indicated metabolites has been developed, which can be carried out advantageously by means of high-performance liquid chromatography - UHPLC (PE 16:0/20:4, PE 18:0/22:6 and LPE 18:0) or a high-performance extraction system for solid phase (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, selected lipids (PE 16:0/20:4, PE 18:0/22:6 and preferably LPE 18:0) and uric acid are quantified in a biological sample, preferably a blood plasma sample. A schematic flow of the preferred method of the invention is shown in Fig. 1. More specifically, lipids are preferably determined by performing the following steps:

1a) providing a biological sample from the entity,

1b) alcohol extraction combined with protein precipitation,

1c) centrifugation of the resulting sludge (the sludge is discarded),

1d) filtering the supernatant,

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1e) measurement using the UHPLC technique coupled with a mass spectrometer (QQQ).

Uric acid is preferably determined as follows:

2a) providing a biological sample from the entity,

2b) alcohol extraction with simultaneous filtration of the extract and removal of lipids, 2c) centrifugation of samples,

2d) carrying out the measurement using the technique of high-performance solid phase extraction (Rapid Fire) coupled with a mass spectrometer (QQQ).

According to the literature, the most efficient solvent used for lipid extraction is cold (-20°C) methanol.

Due to the lack of chromatographic separation prior to the determination of uric acid (high-performance solid phase extraction technique is preferably used), it is advisable to remove matrix components that may interfere with the measurement. For this purpose, a ready-made, commercial kit enabling quick extraction combined with filtering and removal of lipids from the extract was used (Captiva ND Lipids kit (Agilent Technologies) was used).

The method according to the invention is adapted for use in the control of the effects of chemotherapy treatment in patients with lung cancer, preferably non-small cell lung cancer. The use of a diagnostic panel of metabolic biomarkers according to the invention, comprising 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 the undertaken chemotherapeutic treatment has not been described so far. The inventors demonstrated the usefulness of the method according to the invention using a panel of metabolic biomarkers in controlling the effects of chemotherapeutic treatment in patients with lung cancer, which is confirmed by the attached examples. The method according to the invention also makes it possible to determine the stabilization of the status of the neoplastic disease.

The method of assessing the effectiveness of a chemotherapeutic treatment in a subject with lung cancer according to the invention has good sensitivity and specificity. In addition, its implementation generates significantly lower costs than currently used imaging techniques. At the same time, the measurement of a panel of selected metabolites in a patient sample makes such a test available to every patient, which is not available in the case of imaging techniques that cannot be performed on a wide range of people due to numerous contraindications and limitations of such methods.

The subject of the invention is also a diagnostic panel of metabolic biomarkers consisting of the following metabolites: PE 16:0/20:4, PE 18:0/22:6 and uric acid. The subject of the invention is also a diagnostic panel of metabolic biomarkers consisting of the following metabolites: PE 16:0/20:4, PE 18:0/22:6, uric acid and LPE 18:0. Such panels can be used for in vitro diagnostics, in particular for assessing the effectiveness of chemotherapeutic treatment of lung cancer, especially in patients with non-small cell lung cancer, constituting an effective diagnostic tool.

The present invention was based on the results of scientific work. The first stage (preliminary selection of potential indicators) was a non-targeted metabolomic analysis of plasma from patients diagnosed with non-small cell lung cancer and treated with chemotherapy. Materials for the study (blood samples) were collected from patients before the start of treatment and after receiving two cycles of chemotherapy. A liquid chromatograph coupled with a high-resolution mass spectrometer (LC-QTOF-MS) was used to analyze these samples. This apparatus allows the detection of a wide spectrum of low molecular weight components present in the samples (metabolites). The obtained data were subjected to statistical analysis, which identified the group of phosphatidylethanolamines and lysophosphatidylethanolamines as well as uric acid as potential indicators of response to chemotherapeutic treatment. Comprehensive changes to the group of compounds (panel) suggested that only a panel of metabolites, and not a single compound, would have diagnostic potential. The potential usefulness was indicated by the fact that all compounds from the selected group showed changes in a similar range (30-50%) and direction (the level of all observed metabolites increased after taking two cycles of chemotherapy in patients in whom this treatment turned out to be effective). Therefore, a method for the quantification of a panel of indicated metabolites was developed, which can be advantageously implemented using mass spectrometry technology (triple quadrupole mass spectrometer - QQQ) combined with liquid chromatography (LC) (lipid panel) and high-performance solid phase extraction ( Rapid Fire) (uric acid). The technology chosen to quantify the selected metabolites allows for very accurate results

PL 241 608 B1 determination of the analyte concentration in the samples, which allows for more accurate test results. Methods based on the detection of QQQ have been validated according to the guidelines of the European Medicine Agency (EMA) and the U.S. Food and Drug Administration (EDA). Material from 9 patients with a partial response to treatment and 14 patients with stabilization of the disease after receiving chemotherapy was analyzed. The results obtained using a different analytical method (in relation to the method according to the invention) confirmed the previously obtained results.

The method of the invention comprising the quantification of a panel of metabolites comprising PE 16:0/20:4, PE 18:0/22:6 and a panel of metabolites further comprising LPE 18:0 as well as a panel of metabolites further comprising uric acid according to the invention is applicable for the detection and determination of the above compounds in biological samples (e.g. blood plasma) from lung cancer patients, e.g. non-small cell lung cancer. This process can advantageously be carried out using liquid chromatography and a solid phase extraction system, such as, for example, Rapid Fire, coupled to a triple quadrupole mass spectrometer.

The method of the invention comprising the quantification of a panel of metabolites comprising PE 16:0/20:4, PE 18:0/22:6 and a panel of metabolites further comprising LPE 18:0 as well as a panel of metabolites further comprising uric acid according to the invention can be used to assess the effectiveness of the chemotherapeutic treatment undertaken, which preferably includes:

- determination of the level of PE 16:0/20:4, PE 18:0/22:6, LPE 18:0, uric acid in a biological sample from a patient with non-small cell lung cancer (sample taken at the time of the decision to use chemotherapy treatment );

- determination of the level of PE 16:0/20:4, PE 18:0/22:6, LPE 18:0, uric acid in a biological sample from the patient after each two cycles of chemotherapy and comparing the result with the level of the same metabolites measured before starting therapy. The increment indicates a positive response to the given treatment.

The fact that a sample collected in a non-invasive way is used as a source of information on the progress of chemotherapeutic treatment makes it possible to monitor the effectiveness of treatment in all patients with lung cancer. Until now, information on the effectiveness of the treatment was determined by observing changes in images obtained using computed tomography, positron emission tomography or magnetic resonance imaging. Unfortunately, a wide range of contraindications to perform such a test prevented imaging in many patients.

Implementation of the methods according to the invention by analyzing a sample from a patient, preferably a blood sample taken from a patient, does not expose the patient to an additional dose of radiation, but above all, it does not exclude the possibility of performing the measurement in all patients who require it. Due to their simplicity, speed, non-invasiveness, no need to perform surgery or biopsy, and the relatively low cost of the measurement, the methods according to the invention are an alternative to the currently used imaging techniques used to assess the effectiveness of chemotherapy treatment, allowing to control the effects of chemotherapy treatment in all patients, regardless of the above-mentioned disqualifying factors for imaging tests. In addition, it should be noted that by eliminating the need to use expensive imaging methods for this purpose, it allows to reduce treatment costs and to cover a wider patient population.

The following is an exemplary scheme for practicing the invention, including recommended and preferred methods, procedures, reagents, conditions and apparatus for practicing the present invention. It should be noted that equivalent means for the same purpose may be used, therefore the methods, procedures, reagents, conditions and apparatus for practicing the present invention set forth below and preferred are not to be construed as limiting, as they are not intended to limit the scope of the invention as defined in the appended claims, but illustrative of embodiments of the present invention.

An exemplary diagram of the implementation of the invention

All activities related to the preparation of solutions and samples for testing should be performed in gloves. Filtering should be done with protective glasses. All used tubes and disposable tips should be placed in containers labeled "Biohazard - Biohazard".

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A) Example determination of phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine

18:0/22:6 and lysophosphatidylethanolamine 18:0

1. Marking principle

Detection of phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 and lysophosphatidylethanolamine 18:0 from the blood of patients is performed by alcohol precipitating proteins from the blood plasma obtained, centrifuging the resulting precipitate and filtering the resulting supernatant. The solution thus obtained is analyzed by liquid chromatography coupled to a triple quadrupole mass spectrometer.

2. Reference reagents and substances for the implementation of the invention

2.1. Reagents

2.1.1. Water (1st degree of purity)

2.1.2. Acetonitrile (LC-MS Ultra Chromosolv)

2.1.3. Methanol (1C-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

2.2. Reference substances

2.2.1 Phosphatidylethanolamine 16:0/20:4 dissolved in chloroform at mg/ml

2.2.2. Phosphatidylethanolamine 18:0/22:6 dissolved in 10 mg/ml chloroform

2.2.3. Lysophosphatidylethanolamine 18:0, powder

2.3. Internal pattern

2.3.1. Phosphatidylethanolamine 15:0/18:1 d7 dissolved in 1 mg/ml chloroform

2.3.2. Lysophosphatidylethanolamine 18:0 d7 dissolved in 1 mg/ml chloroform

2.4. Other solutions

2.4.1. Butylated hydroxytoluene solution (1 mM)

Weigh out 2 204 mg of butylated hydroxytoluene (2.1.6). Then dissolve the test portion in 10 ml of methanol (2.1.3). Mix thoroughly.

2.4.2 Butylated hydroxytoluene solution (1 μM)

Make a 1 μM butylated hydroxytoluene working solution. For this purpose, measure 100 μl of the 1 mM solution (2.4.1) and make up to 100 ml by adding 99.9 ml of methanol (2.1.3). Mix everything thoroughly.

2.4.3. 10 μg/mL internal standard solutions

Prepare a 10 μg/mL 10 μg/mL lysophosphatidylethanolamine 18:0/d7 and lysophosphatidylethanolamine 18:0 d7 working solution. For this purpose, measure 10 μl of a solution of phosphatidylethanolamine 15:0/18:1 d7 at a concentration of 1 mg/ml (2.3.1.) and lysophosphatidylethanolamine 18:0 d7 at a concentration of 1 mg/ml (2.3.2.) and make up to 1 ml by adding 980 μl of methanol (2.1.3). Mix everything thoroughly.

2.4.4. Methanol/chloroform/water mixture (65/35/8 v/v/v)

Measure 1300 μl methanol (2.3.2), 700 μl chloroform (2.1.7) and 160 μl water (2.1.1). Mix everything thoroughly.

2.4.5. Lysophosphatidylethanolamine 18:0 (2.2.3) working solution at 1 mg/ml Weigh out 1 mg of lysophosphatidylethanolamine 18:0 (2.2.3). Then dissolve the test portion in 1 ml of methanol/chloroform/water (65/35/8 v/v/v) (2.4.4). Mix everything thoroughly.

2.4.6. Phosphatidylethanolamine 16:0/20:4 and Lysophosphatidylethanolamine 18:0 solution 10 μg/ml

Measure 1 μl of the 10 mg/ml 16:0/20:4 phosphatidylethanolamine solution (2.2.1) and 10 μl of the 1 mg/ml lysophosphatidylethanolamine 18:0 1 mg/ml solution (2.4.5) and make up to 1 ml by adding 989 μl methanol (2.1.3). Mix everything thoroughly.

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2.4.7. Phosphatidylethanolamine 16:0/20:4 and Lysophosphatidylethanolamine 18:0 1 μg/ml solution Measure 100 μl of the Phosphatidylethanolamine 16:0/20:4 and Lysophosphatidylethanolamine 18:0 10 μg/ml solution (2.4.6) and make up to 1 ml with 900 μl of methanol (2.1.3). Mix everything thoroughly.

2.4.8. Phosphatidylethanolamine solution 18:0/22:6 at 100 μg/ml

Measure 10 μl of the 10 mg/ml phosphatidylethanolamine 18:0/22:6 solution (2.2.2) and make up to 1 ml with 990 μl methanol (2.1.3). Mix everything thoroughly.

2.4.9. Phosphatidylethanolamine 18:0/22:6 10 μg/ml solution Measure 1 μl of the phosphatidylethanolamine 18:0/22:6 10 mg/ml solution (2.2.2) and make up to 1 ml with 999 μl methanol (2.1 .3). Mix everything thoroughly.

2.5. Mobile phases

2.5.1 Mobile phase A

In a 1-dm3 graduated flask, prepare a solution of 999 ml of water (2.1.1) and 1 ml of formic acid (2.1.5). Mix thoroughly.

2.5.2. Mobile phase B

In a 1 liter volumetric flask, 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.

3. Apparatus and laboratory equipment necessary to implement the invention

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

3.2. Chromatography column: Zorbax Eclipse C18 RRHD (2.1 x 50 mm, particle size 1.8 μm).

3.3. Milli-Q Integral 3 water purification system

3.4. Automatic pipettes and dedicated disposable tips

3.5. Vortex centrifuge

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

3.7. Laboratory centrifuge with horizontal deflector rotor for centrifugation of 9 ml tubes

3.8. Laboratory glassware

3.9. 1.5 ml Safe-Lock tubes

3.10. Chromatographic vials

3.11. Volume-reducing chromatography vial inserts

3.12. Insulin syringes

3.13. Nylon syringe filters with a pore size of 0.22 μm

4. Sample preparation

4.1. Samples analyzed

4.1.1. Venous blood should be collected under sterile conditions into one 9 ml tube-syringe containing EDTA. Mix the blood by inverting the tube 8-10 times. The blood tube should be immediately transported at room temperature to the laboratory.

4.1.2. The blood tube should be centrifuged in a horizontal deflector centrifuge for 10 min at 1900 xg at 4°C.

4.1.3. Transfer blood plasma to 1.5 ml Safe-Lock tubes. Transfer 0.5 ml of plasma to each tube, leaving approximately 0.3 ml of plasma above the buffy coat. Carefully pipet the blood plasma to avoid disturbing the buffy coat and transferring the leucocyte-platelet fraction.

4.1.4. Properly labeled plasma tubes should be sealed and placed in a low-temperature freezer (-80°).

4.2. Plasma samples from healthy donors for calibration curve preparation. These samples should be secured in the same way as the analyzed samples.

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5. Procedure

5.1. Analytical sample

Thaw the samples (4.1.) by keeping them on ice for 60-120 minutes (until they are completely thawed), mix using a vortex centrifuge for 10 sec.

5.2. Blank sample

Thaw the samples (4.2.) by keeping them on ice for 60-120 minutes (until they are completely thawed), mix using a vortex centrifuge for 10 sec.

5.3. QC spiked sample 4

Measure 145 μl of cold (-20°C) methanol solution containing butylated hydroxytoluene (1 μΜ) (2.4.2) into a Safe-Lock tube (2.4.2.), add 5 μl of 10 μg/ml internal standard solution (2.4.3.) 5 μl of phosphatidylethanolamine 16:0/20:4 and 10 μg/ml lysophosphatidylethanolamine 18:0 solution (2.4.6) and 2 μl of phosphatidylethanolamine 18:0/22:6 100 μg/ml solution (2.4. 8.). Mix thoroughly. Carefully add 50 μl of the plasma mixture from healthy donors (4.2). The content of the analytes corresponds to the following concentrations: phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0 250 ng/ml and phosphatidylethanolamine 18:0/22:6 1 μg/ml.

5.4. Calibration sample - CAL

Into Safe-Lock tubes, measure cold (-20°C) methanol solution containing butylated hydroxytoluene (1 μM) (2.4.2.) (quantities given in table 1), add 5 μl of 10 μg/ml internal standard solution ( 2.4.3.) as well as the amounts of standard substances with the amounts given in table 1. Then add 50 μl of the plasma mixture from healthy donors (4.2.) to each of the test tubes.

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Table 1.

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500 2000 131 ABOUT Phosphatidylethanolamine solution 16:0/20:4 and lysophosphatidylethanolamine 18:0 at a concentration of 10 μ gdul (2.4.6.) Phosphatidylethanolamine solution 18:0/22:6 at 100 pg/ml (2.4.8.)

ABOUT ^! 5000 120 LT1 st Phosphatidylethanolamine solution 16:0/20:4 and lysophosphatidylethanolamine 18:0 at a concentration of 10 pg/ml (2.4.6.) Phosphatidylethanolamine solution 18:0/22:6 at 100pg/ml (2.4.8.) kO

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6. Extraction

Thaw the samples by keeping them on ice for 60-120 minutes. Once the samples are completely thawed, vortex each sample for 10 seconds. Then precipitate proteins using cold (-20°C) methanol. For this purpose, measure 145 μΙ of methanol containing butylated hydroxytoluene (1 mM) (2.4.2.) and 5 μΙ of the internal standard solution at a concentration of 10 μg/ml (2.4.3.) in a Safe-Lock tube. Carefully add 50 μΙ of the sample to be analyzed. Mix by vortexing for 1 min. Then, keep the samples on ice for 10 minutes. After this time, centrifuge for 10 min at 4°C at 21,000 xg. Filter the supernatant using nylon syringe filters (0.22 μm) into cartridges placed in chromatographic vials and analyze by LC-MS/MS.

7. Analysis conditions

7.1. Chromatography conditions

7.1.1. Column: Zorbax Eclipse 08 RRHD (2.1 x 50 mm, particle size 1.8 μm) Pre-column: Zorbax Eclipse C18 (2.1 x 5 mm, 1.8 μm) Column was thermostated at 40°C Mobile phase flow: 0.5 ml/ min 2 μι of sample was dispensed

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

time [min] %A %B 0.0 95 5 1.0 40 60 5.0 35 65 8.0 15 85 12.0 0 100 13.0 0 100

After each dosing, 2 minutes of stabilization of the system in the initial conditions of the analysis were set.

7.2. Mass spectrometer parameters

7.2.1. Ion source: ESI AJS

Scan mode: dMRM

Quadrupole resolution Q1 and Q3: unit

Ion charge: positive

Delta EMV (positive): +100 (0.0-7.0 min) +400 (7.0-13.0 min)

7.2.2. Source operating parameters:

Gas temperature (°C): 200

Gas flow rate (l/min): 11

Nebulizer pressure (psi): 40

Shielding gas temperature (°C): 250

Shielding gas flow rate (l/min): 8

Capillary voltage (V): 4000

iFunnel Parameters:

High pressure RF (V): 160

Low pressure RF (V): 100

Slicer: fixed (380V)

7.2.3. Monitored Fragmentation Reactions (dMRM)

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Table 3

Relationship Retention time [min] Parent ion Product Transition observation time Collision energy [V] I Time Segment (0.0 - 7.0 min) Lysophosphatidylethanolamine 18:0 d7 3.64 487.3 346.3 150 twenty Lysophosphatidylethanolamine 18:0 5.04 482.2 341.0 150 twenty 482.2 310.0 150 35 1st time segment (7.0 - 13.0 min) Phosphatidylethanolamine 16:0/20:4 9.99 740.5 599.3 125 twenty 740.5 313.4 125 35 Phosphatidylethanolamine 15:0/18:1 d7 10.27 711.4 429.3 125 twenty 711.4 354.8 125 twenty Phosphatidylethanolamine 18:0/22:6 10.39 792.4 651.4 125 twenty 792.4 34.5 125 twenty

8. Sample analysis system

8.1. Blank sample (5.2.)

8.2. Level 1 calibration sample (5.4.)

8.3. Level 2 calibration sample (5.4.)

8.4. Level 3 calibration sample (5.4.)

8.5. Level 4 calibration sample (5.4.)

8.6. Level 5 calibration sample (5.4.)

8.7. Level 6 calibration sample (5.4.)

8.8. Spiked sample QC 4 (5.3.)

Analytical sample (in series of 10 samples) (5.1.)

B) Exemplary determination of uric acid

1. Marking principle

The detection of uric acid from the blood of patients is carried out by precipitating proteins from the obtained blood plasma with alcohol, filtering the resulting mixture through a filter that retains the formed precipitate and removes lipids from the filtrate. The solution thus obtained is analyzed using a high performance solid phase extraction system coupled to a triple quadrupole mass spectrometer.

2. Reference reagents and substances for the implementation of the invention

2.1. Reagents

2.1.1 Water (1st degree of purity)

2.1.2. Acetonitrile (LC-MS Ultra Chromosolv)

2.1.3. Methanol (LC-MS Ultra Chromosolv)

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2.1.4. Ethanol (99.8% for HPLC)

2.1.5. Formic acid (>98%)

2.1.6. Sodium hydroxide

2.2. Reference substance

2.2.1. Uric acid (powder)

2.3. Internal pattern

2.3.1. Uric acid ^15N (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) into a 100 ml glass bottle. Mix everything thoroughly.

2.4.2. 16 mg/ml sodium hydroxide solution

Weigh out 128 mg of sodium hydroxide (2.1.6). Then dissolve the test portion in 8 ml of water (2.1,1). Mix thoroughly.

2.4.3. Sodium hydroxide solution 0.1M

Prepare a 0.1 M sodium hydroxide working solution. To do this, measure 6 ml of the 16 mg/ml solution (2.4.2) and make up to 24 ml by adding 18 ml of water (2.1.1). Mix everything thoroughly.

2.4.4. 1 mg/mL internal standard solution

Prepare a working solution of uric acid ¹⁵ N. To do this, weigh out 1 mg of uric acid 15 N (2.3.1.) and dissolve in 1 ml of 0.1 M sodium hydroxide solution (2.4.3.) Mix thoroughly.

2.4.5. 9 μg/ml methanol:ethanol internal standard solution. Prepare a ¹⁵ N uric acid solution in methanol:ethanol (1:1 v/v). For this purpose, measure 272.7 μl of the internal standard solution with a concentration of 1 mg/ml (2.4.4.) and make up to 30 ml with methanol:ethanol (1/1 v/v) (2.4.1.). Mix everything thoroughly.

2.4.6. 100 μg/ml uric acid working solution

Weigh out 1 mg of uric acid (2.2.1). Then dissolve the test portion in 10 ml of the 0,1 M sodium hydroxide solution (2.4.2). Mix everything thoroughly.

2.5. Mobile phases

2.5.1. Mobile phase P1A and P2A

In a ¹ dm3 volumetric flask, prepare a solution of 999 ml of water (2.1.1) and 1 ml of formic acid (2.1.5). Mix thoroughly.

2.5.2. Mobile phase P1B and P3A

In a ¹ liter volumetric flask, prepare a solution consisting of 999 ml of acetonitrile (2.1.2) and 1 ml of formic acid (2.1.5). Mix thoroughly.

3. Apparatus and laboratory equipment necessary to implement the invention

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

3.2. Type A cartridge (C4).

3.3. Milli-Q Integral 3 water purification system

3.4. Automatic pipettes and dedicated disposable tips

3.5. Vortex centrifuge

3.6. Laboratory centrifuge with horizontal deflector rotor for centrifugation of 9 ml tubes

3.7. Laboratory glassware

3.8. 1.5 ml Safe-Lock tubes

3.9. 96-well polypropylene plate, 500 μl well capacity

3.10. Vacuum assisted filtration system (Captiva ND Lipids, Agilent Technology).

4. Sample preparation

4.1. Samples analyzed

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4.1.1. Venous blood should be collected under sterile conditions into one 9 ml tube-syringe containing EDTA. Mix the blood by inverting the tube 8-10 times. The blood tube should be immediately transported at room temperature to the laboratory.

4.1.2. The blood tube should be centrifuged in a horizontal deflector centrifuge for 10 min at 1900 xg at 4°C.

4.1.3. Transfer blood plasma to 1.5 ml Safe-Lock tubes. Transfer 0.5 ml of plasma to each tube, leaving approximately 0.3 ml of plasma above the buffy coat. Carefully pipet the blood plasma to avoid disturbing the buffy coat and transferring the leucocyte-platelet fraction.

4.1.4. Properly labeled plasma tubes should be sealed and placed in a low-temperature freezer (-80°).

4.2. Plasma samples from healthy donors for calibration curve and blank.

These samples should be secured in the same way as the analyzed samples.

5. Procedure

5.1. Analytical sample

Thaw the samples (4.1.) by keeping them on ice for 60-120 minutes (until they are completely thawed), mix using a vortex centrifuge for 10 sec.

5.2. Blank sample

Thaw the samples (4.2.) by keeping them on ice for 60-120 minutes (until they are completely thawed), mix using a vortex centrifuge for 10 sec.

5.3. Sample enriched QC 5

Measure 165 μΙ of the cold (-20°C) methanol:ethanol mixture (1/1 v/v) containing the internal standard (2.4.5) into the well, add 15 μΙ of the 100 pg/ml standard solution (2.4.6. ) and 70 μΙ of water (2.1.1.). Carefully add 50 µΙ of the healthy donor plasma mixture (4.2) and mix three times with the pipette tip. Applying a vacuum, filter the solution into a receiving plate. Transfer everything to a 96-well polypropylene plate (3.9).

5.4. Calibration sample - CAL

Measure 165 μΙ of the cold (-20°C) methane ethanol (1/1 v/v) mixture containing the internal standard (2.4.5) into the well, add the appropriate amount of the 100 pg/ml standard solution (2.4.6.) and water (2.1.1.) (Table 4). Carefully add 50 µΙ of the healthy donor plasma mixture (4.2) and mix three times with the pipette tip. Applying a vacuum, filter the solution into a receiving plate. Transfer everything to a 96-well polypropylene plate (3.9).

Table 4

calibration level Amount of added standard solution (2,4,6) [μΐ] Amount of added water (2,1,1,) [μΐ] Final concentration obtained [μg/ml] 1 1.5 83.5 0.5 2 3 82.0 1.0 3 4.5 80.5 1.5 4 6 79.0 2.0 5 15 70.0 5.0 6 thirty 55.0 10.0

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6. Extraction

Thaw the samples by keeping them on ice for 60-120 minutes. Once the samples are completely thawed, vortex each sample for 10 seconds. Then apply to the filter plate (3.10) 165 μΙ of a cold (-20 °C) mixture of methanol:ethanol (1/1 v/v) containing 9 pg/ml of the internal standard (2.4.5) and 80 μΙ of water (2.1 .1). Carefully transfer 50 μΙ of the analyzed samples to the individual filter wells and mix three times using the pipette tip. Applying a vacuum, carefully filter the mixtures into a collecting plate. From this, transfer the material to a polypropylene 96-well plate (3.9.) and analyze with Rapid Fire-MS/MS.

7. Analysis conditions

7.1. Rapid Fire analysis conditions

7.1.1. Cartridge: A (C4)

The duration of Rapid Fire cycles is shown in table 5.

Table 5

time [ms] download 600 Loading/Rinsing 2000 Additional washing 0 elution 5000 Conditioning 12000

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

7.2. Mass spectrometer parameters

7.2.1. Ion source: ESI AJS

Scan mode: MRM

Quadrupole resolution Q1 and Q3: unit

Ion charge: negative

Delta EMV (positive): -400

7.2.2. Source operating parameters:

Gas temperature (°C): 300

Gas flow rate (l/min): 11

Nebulizer pressure (psi): 40

Shielding gas temperature (°C): 250

Shielding gas flow rate (l/min): 8

Capillary voltage (V): 4000

iFunnel Parameters:

High pressure RF (V): 140

RF low pressure (V): 90

Slicer: fixed (380V)

7.2.3. Monitored Fragmentation Reactions (dMRM)

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Table 6

Relationship Parent ion Product Transition observation time Collision energy [V] uric acid 167.1 124.2 150 twenty 167.1 96.0 125 twenty uric acid ¹⁵ n 169.0 125.2 125 twenty 169.0 97.2 125 twenty

8. Sample analysis system

8.1. Blank sample (5.2.)

8.2. Level 1 calibration sample (5.4.)

8.3. Level 2 calibration sample (5.4.)

8.4. Level 3 calibration sample (5.4.)

8.5. Level 4 calibration sample (5.4.)

8.6. Level 5 calibration sample (5.4.)

8.7. Level 6 calibration sample (5.4.)

8.8. Spiked sample QC 4 (5.3.)

8.9. Analytical sample (in series of 10 samples) (5.1.)

9. Calculation of results (common to parts A and B)

9.1. Calibration

Using the internal standard addition method, a linear calibration curve should be determined. For calibration levels 2-6, the standard deviation of the concentration based on the calibration curve should not exceed ±20% of the predicted concentration of the metabolite in the sample. Estimate the signal-to-noise ratio for calibration level 1, which should be equal to or greater than 10 for the ion used for quantification.

9.2. Calculation of the amount of metabolites

The content of selected metabolites in the analyzed samples should be calculated from the equation of the calibration curve. The concentration deviation in the spiked sample - QC (5.3.) should not exceed ±20% of the nominal concentration in the sample. If this concentration deviation is greater than ±20% of the nominal concentration of the metabolite in the sample, the calibration should be re-calibrated.

Brief description of the figures

Fig. 1 is a schematic diagram of an embodiment of the preferred method of the invention.

Fig. 2 shows an exemplary chromatogram obtained for the test sample.

Fig. 3 shows a comparison of a sample obtained from healthy subjects (red (1)) with a sample spiked with standards (blue (2)).

Fig. 4 shows the measurement results obtained for the solvents used for sample preparation.

Fig. 5 shows a comparison of a sample obtained from healthy subjects (green (1)) with a standard spiked sample (red (2)).

Fig. 6 shows the measurement results obtained for the mobile phases used to analyze the samples.

Figure 7 shows the ROC curve analysis performed on experimental data for a panel of metabolic biomarkers containing all four metabolites.

The present invention will be illustrated below by means of examples and figures, which are not intended to limit in any way the scope of protection of the invention as defined in

PL 241 608 B1 patent claims. Unless otherwise indicated, all methods and parameters are those commonly used in the art to which this invention belongs, and the equipment and reagents used are used as recommended by their manufacturers.

Example - Quantification of a two-component, three-component and quaternary panel of metabolic biomarkers and evaluation of the effectiveness of chemotherapeutic treatment of lung cancer using such panels.

Below is an example of the conducted research involving the targeted analysis and its results leading to the recognition of panels of metabolites, which consist of phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, and optionally also lysophosphatidylethanolamine 18:0 and optionally acid uric.

1. Collection of biological material

Blood samples were collected at the University Clinical Hospital in Bialystok. The study group consisted of patients diagnosed with non-small cell lung cancer who were treated with chemotherapy. Blood samples were taken before treatment and after two cycles of chemotherapy. For the tests, 9 ml of peripheral blood were collected into a test tube containing EDTA. The blood was then centrifuged (10 min, 1900 xg, 4°C). The obtained blood plasma was transferred to appropriately marked cryotubes. Patient plasma samples were stored at -80°C until the day of analysis.

2. Characteristics of the study group

The experiment involved two groups of patients. The first of them (n=9) were people in whom the medical council, after administering two cycles of chemotherapy, found a partial response to the given treatment. The second group (n=14) consisted of patients with only disease stabilization after treatment.

3. Validation of analytical methods

Each instrumental analytical method enabling quantitative determinations requires validation, also in the presented experiment such tests were carried out. For this purpose, a series of calibration samples was prepared in triplicate. Due to the great importance of the presence of the matrix (the so-called matrix effect), in the case of quantitative determinations based on mass spectrometry using an ESI source, mixed (averaged) plasma from 40 healthy donors was used for testing. This material was the basis for the tests that comprised the validation of the method. Validation was carried out for both the lipid determination method (using liquid chromatography) and the method for determining the concentration of uric acid (based on the measurement with the Rapid Fire technology).

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

3.1. Validation of the lipid determination method

Portions of cold (-20°C) methanol with 0.1 μM butylated hydroxytoluene (as an antioxidant), a portion of internal standards and a series of standard substances were measured into Safe-Lock tubes. Thawed (as described above) plasma was also added to each batch. A series of samples was prepared in the concentration range of 10-750 ng/mL (phosphatidylethanolamine 16:0/20:4 and lysophosphatidylethanolamine 18:0) and 0.1-5 μg/mL for phosphatidylethanolamine 18:0/22:6). The concentration of the internal standards was constant in all samples at 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 reference substances was also prepared. All samples after plasma addition were mixed for 1 min and then kept on ice for 10 min. After this time, they were centrifuged for 10 min at 4°C at a speed of 21,000 xg. The supernatant was then filtered using nylon syringe filters (0.22 μm) directly into chromatography vial cartridges. A sample was also prepared in which no standards (neither the substances to be determined nor internal standards) were added before the extraction procedure. Such a sample was prepared according to the procedure described above and all standard substances were added at the final stage to the filtered supernatant. The whole was thoroughly mixed and analyzed by LC-MS/MS.

3.2. Validation of the uric acid determination method

A portion of the methanol mixture (1/1 v/v) was placed in a well on the filter plate, portions of: internal standard, standard substance and water were added. Thawed plasma was also added to each serving. A series of samples was prepared in the concentration range of 0.5-10 μg/ml. The series was prepared in three

PL 241 608 B1 in repetitions. A blank sample without the addition of reference substances was also prepared. All samples were filtered through a set dedicated to the simultaneous precipitation of proteins along with the removal of the lipid fraction from the filtrate. A sample was also prepared in which neither the internal standard nor the standard of the analyte was added before the extraction procedure. This sample was also filtered according to the above procedure (excluding the addition of standards). After filtering, both standards (the standard of the test substance and the internal standard) were added to it in a strictly defined concentration. All samples were analyzed using a Rapid Fire system coupled to a triple quadrupole mass spectrometer.

4. Analytical samples

4.1. Lipid analysis

The samples were thawed on ice for 60-120 min (until completely thawed). Each sample was then mixed for 10 seconds using a vortex shaker. After that, protein precipitation and extraction were started. For this purpose, cold (-20°C) methanol with 0.1 μM butylated hydroxytoluene (as an antioxidant) was used. An internal standard solution was also added. Protein precipitation and extraction were performed by adding 3 portions of methanol to 1 portion of plasma (sample). The sample prepared in this way was mixed using a vortex shaker for 1 min. After this time, she was placed on ice (10 min). It was then centrifuged at 4°C at 21,000 xg for 10 min. The supernatant was filtered using nylon syringe filters into chromatography vial cartridges.

A calibration curve and spiked sample (QC) were created by making a series of samples with different concentrations of standards including the matrix. They were prepared in the same way as the samples from the validation series.

4.2. Uric acid analysis

The samples were thawed on ice for 60 min (until completely thawed). Each sample was then mixed for 10 seconds using a vortex shaker. The filter plate was prepared by placing an aliquot of cold (-20°C) methanol:ethanol mixture with an internal standard and an aliquot of water in each well. 50 μl of the sample were added to the next filter wells prepared in this way (the ratio was 5 parts of the solvent to 1 part of the sample). Using a vacuum pump, the samples were filtered through the plate and then transferred from the collection plate to a conventional 96-well plate.

A calibration curve and spiked sample (QC) were created by making a series of samples with different concentrations of standards including the matrix. They were prepared in the same way as the samples from the validation series.

5. Sample analysis

5.1. Lipid analysis by liquid chromatograph coupled to a mass spectrometer. Samples were analyzed by liquid chromatography (LC) coupled to a triple quadrupole mass spectrometer (6495 iFunnel Triple Quad Agilent Technologies) (QQQ). An ESI ion source with the JetStream option was used. The chromatographic separation was carried out on a thermostated (40°C) Zorbax Eclipse C18 RRHD column (2.1 x 50 mm, 1.8 μm particle size) equipped with a pre-column (Zorbax Eclipse C18 2.1 x 5 mm, 1.8 μm). The mobile phases at 0.5 ml/min were water with 0.1% formic acid (phase A) and acetonitrile/isopropanol (60/40) with 0.1% formic acid (phase B). Their relationship to each other changed over time according to 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. After this, 2 min was allowed to stabilize the initial conditions of the system before the next analysis.

The spectrometer worked in the dMRM mode recording positive ion charge.

The observed transitions are listed in Table 3.

Solvent dosing (methanol) was used between samples. The analysis of the samples started with the measurement of the reagent and the blank sample. A series of calibration samples was then analyzed and repeated after every 20 samples. Between the analysis of the calibration samples, spiked (QC) samples were analyzed every 7 analytical samples.

5.2. Uric acid analysis using a Rapid Fire system coupled to a mass spectrometer

Samples were analyzed by high-throughput solid phase extraction (Rapid Fire 365) with a triple quad mass spectrometer (6495 iFunnel Triple Quad Agilent Technologies). An ESI source equipped with JetStream was also used. Cartridge A (C4 Agilent

PL 241 608 B1

technologies). The following mobile phases were used: on pump No. 1 (flow 1.5 ml/min) water (90%) and acetonitrile (10%), both with the addition of 0.1% formic acid, on pump No. 2 (flow 1.25 ml /min) water with 0.1% formic acid, on pump #3 (flow 1.25 ml/min) acetonitrile with 0.1% formic acid.

The spectrometer was operated in the negative ion mode. The observed transitions are listed in Table 6.

The analysis started with the measurement of the reagent and the blank sample. A series of calibration samples was then analyzed and repeated every 27 samples. Between the analysis of calibration samples, spiked (QC) samples were analyzed every 8 analytical samples.

6. Results obtained

6.1. Validation of the lipid determination method

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

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

The selectivity of the method was also tested. For this purpose, measurements were made for mobile phases, solvents used for protein precipitation and plasma (from healthy donors). The results obtained in this way were compared with the signal from the sample enriched with the standard. The results of the method selectivity evaluation are presented in Figures 2-4. Based on these, the method was considered selective for selected metabolites extracted from plasma. Fig. 2 shows an example chromatogram obtained for the test sample, Fig. 3 shows a comparison of a sample obtained from healthy patients (red (1)) with a sample enriched with standards (blue (2)). The differences between the samples are clearly visible. Fig. 4 shows the measurement results obtained for the solvents used for sample preparation.

6.2. Validation of the uric acid determination method

The validation of the method enabling the measurement of uric acid was carried out in the same way as described in section 6.1. The concentration deviation for the tested metabolite in the sample, determined on the basis of the equation of the calibration curve, did not exceed ±20% of the nominal concentration value. The repeatability of the results at each of the calibration levels was also measured. The relative standard deviation (RSD) was in the range: 1.99-6.06%. On this basis, the limit of quantification was set at 1.5 μg/ml.

Also in this case, the efficiency of uric acid extraction from plasma was checked. This was done in the same way as described in section 6.1. The recovery for uric acid was 151%. An important parameter that should be specified in the context of analyzes based on the Rapid Fire technology is the influence of the matrix. Despite the lack of chromatographic separation prior to ionization, the influence of the matrix in the case of the proposed analysis is not as high as one might expect and amounts to 72%.

In the same way as described above, the selectivity of the method was tested. The obtained results allow to define the method as selective (Figures 5-6). Fig. 5 shows a comparison of a sample obtained from healthy patients (green (1)) with a sample spiked with a standard (red (2)). The differences between the samples are clearly visible. Fig. 6 shows the measurement results obtained for the mobile phases used to analyze the samples.

PL 241 608 B1

7. Results of analytical samples

The results, regardless of the measurement technique used, chromatography or high-performance solid phase extraction, were analyzed in the same way. Mass Hunter Quantitative Analysis software (version B.07.01 for QQQ) was used for this. A calibration curve was determined using the internal standard addition method. On its basis, the concentrations of each of the analytes were determined. Then the obtained results were subjected to statistical analysis. Metabolites that significantly change in patients after two cycles of chemotherapy (partial responder group) should be indicated phosphatidylethanolamine 18:0/22:6 (44% increase p-value 0.009) and phosphatidylethanolamine 16:0/ 20:4 (24.6% increase, p-value 0.045). Moreover, phosphatidylethanolamine 18:0/22:6 also increases (by 25.3%) significantly (p-value 0.048) also in patients with only stable disease.

8. Analysis of ROC curves

To assess the diagnostic usefulness of the metabolite panels according to the invention, ROC (Receiver Operating Characteristics) curves were analyzed using the MedCalc software. It was observed that using, in addition to the two indicated metabolites significantly changing as a result of treatment, also the following ones, i.e. 18:0 lysophosphatidylethanolamine, but also uric acid, the parameters of sensitivity and selectivity of such a panel significantly improved (Fig. 7). The analysis for a panel of all four metabolites resulted in an area under the ROC curve, AUC = 0.762 (p-value 0.0189) with a sensitivity of 66.67% and a specificity of 92.86%. These results indicate that a precise diagnostic tool will be obtained that will facilitate the assessment of the effectiveness of chemotherapy treatment in all patients who need it, while reducing the cost of such examination.

The study showed that the panel of metabolic biomarkers, containing phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6: changes significantly after the start of chemotherapeutic treatment of lung cancer and indicates a response to chemotherapeutic treatment (significant increase in these metabolic biomarkers).

The above-mentioned metabolic panel containing two metabolic biomarkers, i.e. phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, turned out to be particularly important, because it is these two biomarkers that change significantly (an increase of 24.6 % (p-value = 0.045) and an increase of 44% (p-value = 0.009) respectively) after receiving two cycles of chemotherapy in patients who showed a partial response after such treatment.

Moreover, phosphatidylethanolamine 18:0/22:6 is also significantly changed (increase of 25.3% (p value = 0.048)) in patients with only stable disease. The panel of metabolites containing two metabolic biomarkers, i.e. phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, allows for effective assessment of the effectiveness of chemotherapeutic treatment in lung cancer. The panel containing phosphatidylethanolamine 18:0/22:6 allows to determine the stabilization of the disease.

The conducted studies have also shown that a panel of metabolic biomarkers containing 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 obtained ROC curves, and thus additionally increases the sensitivity and selectivity and thus the accuracy of the method of assessing the effectiveness of chemotherapeutic treatment in the studied patients with lung cancer in relation to the two-component and three-component panel of metabolic biomarkers described above.

Claims (17)

1. A method for evaluating the effectiveness of a chemotherapeutic treatment in a subject with lung cancer in which a) a panel of metabolic biomarkers containing the following metabolites is quantified in a sample from an individual with lung cancer prior to the start of chemotherapy treatment: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, b) in a sample from a subject according to a) after at least two cycles of chemotherapeutic treatment, a panel of metabolic biomarkers containing the following metabolites is quantified: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6, PL 241 608 B1 c) the results obtained in step a) and step b) are compared, and based on the obtained result of the comparison, the efficacy status of the chemotherapeutic treatment in the subject is determined, wherein an increase in the concentration of phosphatidylethanolamine 18:0/22:6 indicates stabilization of the treated lung cancer in the subject and an increase in phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 is indicative of the subject's response to chemotherapeutic treatment. 2. The method of evaluating the effectiveness of treatment according to claim The method of claim 1, wherein the metabolite panel further comprises the following metabolite: lysophosphatidylethanolamine 18:0. 3. The method of claim The method of claim 1 or 2, characterized in that the quantification of the metabolic panel metabolites is performed using liquid chromatography coupled to a triple quadrupole mass spectrometer. 4. A method for evaluating the effectiveness of treatment according to one of claims 1 to 3. The use of any one of claims 1 to 3, wherein the metabolite panel further comprises the following metabolite: uric acid. 5. The method of claim The method of claim 4, wherein the quantification of the metabolite is performed using a high throughput solid phase extraction system coupled with triple quadrupole mass spectrometry. 6. The method according to one of the claims. The method of any one of claims 1 to 5, wherein the sample from the subject is a whole blood sample, preferably a plasma sample. 7. The method according to one of the claims. 1 to 6, wherein the subject is a human. 8. The method according to one of the claims. The use of any one of claims 1 to 7, wherein the lung cancer is non-small cell lung cancer. 9. Diagnostic panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 and uric acid. 10. Diagnostic panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6, uric acid and lysophosphatidylethanolamine 18:0. 11. The use of a diagnostic panel of metabolic biomarkers containing the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 and uric acid for in vitro diagnosis of lung cancer. 12. Use of a metabolic biomarker diagnostic panel containing the following metabolites: phosphatidylethanolamine 16:0/20:4 and phosphatidylethanolamine 18:0/22:6 and uric acid for in vitro monitoring of the effectiveness of chemotherapeutic treatment of lung cancer in a subject. 13. Use of a diagnostic panel of metabolic biomarkers according to claim The use of claim 11 or 12, wherein the panel further comprises the following metabolite: lysophosphatidylethanolamine 18:0. 14. Use of a diagnostic panel of metabolic biomarkers according to one of claims 1 to 5. Use according to any one of claims 12 to 13, wherein the lung cancer is non-small cell lung cancer. 15. Use of a diagnostic panel of metabolic biomarkers according to one of claims 1 to 5. 12 to 14, wherein the subject is a human. 16. A kit for monitoring the effectiveness of a chemotherapeutic treatment in a subject with lung cancer, comprising means for quantifying the level of a panel of metabolic biomarkers consisting of the following metabolites: phosphatidylethanolamine 16:0/20:4, phosphatidylethanolamine 18:0/22:6 , by liquid chromatography coupled to a triple quadrupole mass spectrometer, and uric acid, using a high throughput solid phase extraction system coupled to a triple quadrupole mass spectrometer, and instructions on how to monitor the effectiveness of the treatment. 17. A kit for monitoring the effectiveness of a chemotherapeutic treatment in a subject with lung cancer according to claim 1. The method of claim 16, further comprising means for quantifying the level of the following metabolic biomarker: lysophosphalidylethanolamine 18:0 by liquid chromatography coupled to a triple quadrupole mass spectrometer.