home / Success stories/ Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics. Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics

Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics. Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics

A validated HPLC-MS/MS method was developed to identify and quantify the novel 1,3,4-thiadiazole amino acid derivative LXT7-09. The maximum sensitivity of mass spectrometric detection of LHT7-09 was achieved in the positive ion detection mode at an electrospray voltage of 5500 V and a declustering potential of 36 V. The identified MRM transitions were confirmed chemical structure a new amino acid derivative of 1,3,4-thiadiazole. To effectively isolate LXT7-09 from multicomponent mixtures of thiadiazolylamides, a gradient mode of high-performance liquid chromatography was developed using a mixture of acetonitrile and deionized water in different ratios as an eluent. For these chromatographic conditions, the retention time of compound LHT7-09 was determined to be 11 minutes. For the quantitative determination of the LHT7-09 compound, a calibration solution was developed for the dependence of the chromatographic peak area on the solution concentration.

HPLC-ms/ms

chromatography

mass spectrometry

thiadiazole

1. Kazaishvili Yu.G., Popov N.S. Study of the anti-inflammatory activity of new thiadiazole derivatives in formalin-induced paw edema in rats / Yu.G. Kazaishvili, N.S. Popov // Contemporary issues science and education. – 2013. – No. 3. www..

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4. Popov N.S., Demidova M.A. Assessment of acute toxicity of a new amino acid derivative of thiadiazole when administered intraperitoneally to mice / N.S. Popov, M.A. Demidova // Upper Volga Medical Journal. – 2016. – T. 15, issue. 1. – pp. 9-12.

5. Popov N.S., Demidova M.A. Assessment of the ulcerogenicity of a new amino acid derivative of thiadiazole when administered intragastrically to rats / N.S. Popov, M.A. Demidova // Doctor-graduate student. – 2017. – No. 1(80). – pp. 71-78.

6. Synthesis and antimicrobial activity of amides of phenylthio- and benzylsulfonylacetic acids based on 2-amino-5-alkyl(arylalkyl)-1,3,4-thiadiazoles / S.A. Serkov [et al.] // Chemical-pharmaceutical journal. – 2014. – T. 48, No. 1. – P. 24-25.

7. Arpit K., Basavaraj M., Sarala P., Sujeet K., Satyaprakash K. Synthesis and pharmacological activity of imidazothiadiazole derivatives // Acta Poloniae Pharmaceutica, Drug Research. 2016. Vol. 73.No. 4. P. 937-947.

8. Eman M. Flefel, Wael A. El-Sayed, Ashraf M. Mohamed. Synthesis and Anticancer Activity of New 1-Thia-4-azaspirodecane, Their Derived Thiazolopyrimidine and 1,3,4-Thiadiazole Thioglycosides // Molecules. 2017. No. 22(1). P. 170.

9. Jorge R.A. Diaz, Gerardo Enrique Cami. Salts of 5-amino-2-sulfonamide-1,3,4-thiadiazole, a structural and analog of acetazolamide, show interesting carbonic anhydrase inhibitory properties, diuretic, and anticonvulsant action // Journal of Enzyme Inhibition and Medicinal Chemistry. 2016. Vol. 12.No. 6. P. 1102-1110.

10. Naiyuan Chen, Wengui D., Guishan L., Luzhi L. Synthesis and antifungal activity of dehydroabietic acid-based 1,3,4-thiadiazole-thiazolidinone compounds // Molecular Diversity. 2016. Vol. 20.No. 4. P. 897-905.

11. Yomna, I. El-Gazzar, Hanan H. Georgey, Shahenda M. El-Messery. Synthesis, biological evaluation and molecular modeling study of new (1,2,4-triazole or 1,3,4-thiadiazole)-methylthio-derivatives of quinazolin-4(3H)-one as DHFR inhibitors // Bioorganic Chemistry. 2017. Vol. 72. P. 282-292.

High-performance liquid chromatography with mass spectrometric detection is one of the most promising methods for the identification and quantitative determination of drugs in various biological objects. The method is characterized by high specificity, accuracy and the ability to determine substances in minimal concentrations, which allows it to be used for the quantitative determination of drugs during pharmacokinetic studies and drug monitoring, which is significant for clinical laboratory diagnostics. For this purpose, it is necessary to develop and validate methods for the quantitative determination of various medicinal substances, including innovative ones, based on the HPLC-MS/MS method.

The original drug from the group of non-steroidal anti-inflammatory drugs is acexazolamide, a new derivative of 1,3,4-thiadiazole amide and acexamic acid. Significant advantage of this connection is low toxicity and low ulcerogenicity. To conduct pharmacokinetic studies and assess the bioavailability of this drug through various routes of administration, it is necessary to develop a reliable method for its quantitative determination in biological fluids.

The purpose of this study was the development of a technique for the identification and quantitative determination of a new non-steroidal anti-inflammatory drug from the group of thiadiazole derivatives using HPLC-MS/MS.

Materials and methods

The object of the study was a new derivative of thiadiazole with the laboratory code LHT 7-09, synthesized at OJSC "VNTs BAV" (Staraya Kupavna) by prof. S.Ya. Skachilova (Fig. 1).

2-(5-ethyl-1,3,4-thiadiazolyl)amide 2-acetylaminohexanoic acid

Rice. 1. Chemical structure of LHT 7-09 (gross formula: C 12 H 20N 4 O 2S; molar mass 284,4 g/mol)

Connection LHT 7-09 appearance It is a white powder that is practically insoluble in water, soluble in alcohol, and easily soluble in acetonitrile.

A validated high-performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS) method was used to identify and quantify LCT 7-09.

Chromatography was carried out using an Agilent 1260 InfinityII high-performance liquid chromatograph (Agilent Technologies, Germany). An Agilent Poroshell 120 EC-C18 2.7 µm 2.1 x 10 mm analytical column was used in the study. To isolate the compound under study, we developed a gradient chromatography mode. Acetonitrile, deionized water and ammonium acetate in various ratios were used as eluents.

For mass spectrometry, an ABSciexQTrap 3200 MD triple quadrupole mass spectrometer (ABSciex, Singapore) with an electrospray ion source (TurboV with TurboIonSpray probe) was used. The mass spectrometer was calibrated using a reserpine test solution at a concentration of 6.1×10 -2 mg/l.

Mass spectrometric analysis of the studied samples was carried out in electrospray mode with direct injection of the sample and eluate supplied by the chromatograph. Direct injection of the test samples into the mass chromatograph was carried out using a syringe pump with a diameter of 4.61 mm at a speed of 10 μl/min.

When developing a method for identifying and quantifying a new thiadiazole derivative, optimal conditions for high-performance liquid chromatography and mass detection were selected. The time of release of the substance from the chromatographic column and the MRM transition were taken into account (registration was carried out m/z precursor ion on the first analytical quadrupole Q1 and m/z product ions on the second analytical quadrupole Q3). To quantify LCT 7-09, a calibration graph was constructed in the concentration range from 0.397 to 397 ng/ml.

As software AnalystMD 1.6.2.Software (ABSciex) was used.

Results and discussion

At the first stage of the experimental study, mass detection of the test sample was carried out by directly introducing it into the mass detector using a syringe pump. At the sample preparation stage, a solution of LHT 7-09 (500 ng/ml) was prepared in a mixture of acetonitrile and ionized water in a ratio of 2:1 with the addition of ammonium acetate (0.1%).

Preliminary experiments showed that in the mode of registration of positive ions, the sensitivity of determining LHT 7-09 was higher, and the mass spectrum was more intense and more informative than in the mode of registration of negative ions. In this regard, in further studies, only the positive ionization mode was used.

To obtain an intense peak, the following mass detection conditions were selected: : positive polarization, electrospray voltage 5500.0 V, declustering potential and injection potential - 36.0 and 6.5 V, respectively, with curtain gas pressure of 20.0 psi and atomization gas pressure of 40.0 psi, speed 10 μl/min. The scanning range was 270-300 Da.

Analysis of the obtained mass spectrum of the first analytical quadrupole Q1 showed that under these conditions, due to the addition of a hydrogen proton, a protonated molecule of the studied compound + is formed with the value m/ z 285.2 Yes (Fig. 2).

Rice. 2. Mass spectrum of the protonated molecule LHT 7-09 (in positive ion + scanning mode)

On the second analytical quadrupole Q3, product ions were recorded for the precursor ion with the value m/z 285.2 Yes. Analysis of the 2nd order mass spectrum showed the presence of many peaks, of which 3 were the most intense - m/z 114.2 Da, m/z 130.2 Da and m/z 75.1 Da (Fig. 3).

Rice. 3. Mass spectrum of product ions (in positive ion scanning mode, precursor ionm/ z285.2 Yes)

To obtain a high-intensity ion signal, the optimal energy values in the Q2 collision cell were selected (the energy range from 0 to 400 V was considered). For product ions with values m/ z 114.2 Da, 130.2 Da and 75.1 Da. The optimal energy in the collision cell was 27 V, respectively; 23 V and 49 V.

It is assumed that the product ion with the value m/ z 114.2 Da is a fragment of 5-amino-2-ethyl-1,3,4-thiadiazole, since fragmentation of other 1,3,4-thiadiazole derivatives also reveals a product ion with the same value m/ z. Product ion with meaning m/ z 130.2 Da is probably a protonated moiety of acexamic acid. Thus, the results of mass detection of the sample under study confirmed the chemical structure of the new 1,3,4-thiadiazole derivative.

At the next stage of the experimental study, the test compound was analyzed by HPLC-mass spectrometry.

In HPLC-MS/MS mode, the following ionization conditions were used: electrospray voltage 5500.0 V, mobile phase flow rate 400 μL/min, nitrogen temperature 400 °C, curtain gas and spray flow pressure 20.0 and 50.0 psi, respectively. The recording speed of single mass spectra was 100 spectra per second. To obtain the summarized mass spectrum, a time period of 10.5-11.5 minutes was selected on the chromatogram; Based on the intensity of the signal of product ions, curves of the time dependence of the ion current and the peak area of individual signals corresponding to the compound under study were constructed. The volume of the sample introduced into the analytical column was 10 μl.

To isolate the compound under study, a gradient mode of high-performance liquid chromatography was used, which was ensured by changing the composition of the eluent at the entrance to the analytical column. Acetonitrile, deionized water and ammonium acetate in various ratios were used as eluents. The choice of a gradient chromatography mode was due to the fact that under conditions of an isocratic elution mode (including when using different concentrations of acetonitrile), it was not possible to obtain a peak of the test substance of a symmetrical shape with a retention time suitable for analysis. According to the study, the optimal eluent supply mode was: from 0 to 4 min, acetonitrile concentration was 1%; from 4 to 8 minutes, a linear increase in the concentration of acetonitrile to 99%; from 8 to 12 minutes - isocratic section (1% acetonitrile). Upon completion of the study, the chromatographic column was washed with a 30% acetonitrile solution for 5 minutes.

When using the described chromatography mode, a symmetrical peak of sufficient intensity was obtained for the studied compound (Fig. 4).

Rice. 4. Chromatogram LHT 7-09 (analytical columnAgilentPoroshell 120 EC-C18 2.7 µm 2.1×10 mm; gradient chromatography mode)

Analysis of the obtained chromatograms for LCT 7-09 solutions of different concentrations showed that the retention time (tR) under these elution conditions was 11 minutes and did not depend on the concentration of the substance under study. In this regard, the retention time value can be used as an additional criterion for confirming the authenticity of LHT 7-09 in multicomponent mixtures. Noteworthy is the fact that these chromatography parameters can be used to identify LHT 7-09 not only using a mass detector, but also other detectors, including photometric.

To quantify the new thiadiazole derivative, a calibration graph was constructed in the concentration range from 0.397 ng/ml to 397 ng/ml (Fig. 5).

Rice. 5. Calibration graph for determining the concentration of LCT 7-09 (along the abscissa axis is the concentration of LCT 7-09 in ng/ml, along the ordinate axis is the peak area in pulses)

To develop a calibration solution, solutions of LHT 7-09 were used in concentrations of 0.397; 1.980; 3.970; 19.8; 39.7; 198.0; 397.0 ng/ml. The dependence of the peak area on the concentration of the studied compound was described by the following regression equation:

y= 8.9e 5 ·x 0.499, the value of the regression coefficient was r=0.9936.

It should be noted that the developed calibration solution allows for high-precision quantitative determination of the studied compound in a wide range of concentrations, which makes it possible to use this method for assessing the quality of a medicinal substance and for conducting pharmacokinetic studies.

Thus, the result of the study was the development of a method for the identification and quantification of a new amino acid derivative of thiadiazole using HPLC-MS/MS.

conclusions

HPLC-MS/MS enables the identification and quantification of a new amino acid derivative of thiadiazole with high accuracy.
Mass detection of the new thiadiazole derivative LHT 7-09 should be carried out in the positive ion scanning mode (MRM transition - precursor ion Q1 m/ z 285.2 Yes; product ions Q3 m/ z 114.2 Da, m/ z 130.2 Da and m/ z 75.1 Da).
To isolate LCT 7-09 from multicomponent mixtures, a high-performance liquid chromatography technique has been developed (analytical column Agilent Poroshell 120 EC-C18 2.7 μm 2.1×10 mm; eluent acetonitrile: deionized water: ammonium acetate; gradient mode).

Bibliographic link

Popov N.S., Malygin A.S., Demidova M.A. DEVELOPMENT OF HPLC-MS/MS METHOD FOR IDENTIFICATION AND QUANTITATIVE DETERMINATION OF A NEW THIADIAZOLE DERIVATIVE // Modern problems of science and education. – 2017. – No. 5.;
URL: http://science-education.ru/ru/article/view?id=26988 (access date: 02/01/2020). We bring to your attention magazines published by the publishing house "Academy of Natural Sciences"

Keywords

STEROIDS / LIPIDS / BLOOD SERUM / METABOLIC PROFILE / INDUSTRIAL WASTE/ STEROIDS / LIPIDS / BLOOD SERUM / METABOLIC PROFILE / INDUSTRIAL WASTES

annotation scientific article on veterinary sciences, author of the scientific work - Chakhovsky Pavel Anatolyevich, Yantsevich Alexey Viktorovich, Dmitrochenko Alesya Egorovna, Ivanchik Alexander Viktorovich

Impact anthropogenic factors has a multifaceted effect on the human and animal body. Due to their complex impact, identifying the negative effects of individual factors is a rather difficult task. Metabolomics methodology, which makes it possible to overcome these difficulties, was used to assess the nature and degree of influence of waste from the production of potash fertilizers on the lipid profiles of experimental animals during intranasal inoculation with waste from the production of potash fertilizers and consumption of drinking water obtained from sources located in the zone of potential action of potash production. The separation of lipids from serum was carried out using a specially developed technique based on solid-phase extraction, which makes it possible to remove cholesterol from the samples. Each sample was analyzed by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS), after which the resulting chromatograms were processed using principal component analysis (PCA) and cluster analysis. The developed technique makes it possible to effectively separate hydrophobic metabolites in blood serum. The lipid profile of the blood serum of experimental animals was established, in particular the content of phospholipids and oxysteroids, and differences in metabolic processes were found between experimental and control animals. In the blood serum of experimental animals, the concentration of oxysteroids was increased compared to the control group.

ANALYSIS OF SERUM LIPID PROFILES IN GUINEA PIGS FOR EARLY DETECTION OF CHANGES IN METABOLISM UNDER EXPOSURE TO ENVIRONMENTAL CONTAMINANTS

The exposure to anthropogenic factors has a multifaceted impact on the body of humans and animals. Due to their complex influence the detection of negative effects of the certain factors is a rather complicated task. Metabolomic methodology which permits to overcome these difficulties, has been applied in the evaluation of the nature and degree of the impact of potash fertilizers production waste on lipid profiles of experimental animals after intranasal inoculation with potassic fertilizer production waste and consumption of drinking water obtained from sources located in the zone of potential action of potassic fertilizer production. Isolation of lipids from serum was performed with the help of specially developed technique based on solid-phase extraction of samples which allows to remove cholesterin from the samples. Each sample was subjected to HPLC-MS analysis, after which the obtained chromatograms were treated with the use of the method of principal component analysis and cluster analysis. The developed technique allows to efficiently separate hydrophobic metabolites in blood serum. There was an established serum lipid profile of experimental animals, in particular the content of phospholipids and oxysteroids, and there were differences found in the metabolic processes of the test and control animals. It is shown that in the serum of experimental animals, there is observed an increased concentration of hydroxysteroid as compared with the control group,.

Text of scientific work on the topic "HPLC-MS method for analyzing lipid profiles of guinea pig blood serum to identify early changes in metabolism when exposed to environmental pollutants"

[hyena and sanitation 3/2014

Chakhovsky P.A.1, Yantsevich A.V.2, Dmitrochenko A.E.2, Ivanchik A.V.2

HPLC-MS-TECHNIQUE FOR ANALYSIS OF SERUM LIPID PROFILES

guinea pigs to detect early metabolic changes when exposed to pollutants environment

TU "Republican Scientific and Practical Center of Hygiene", 220012, Minsk, Republic of Belarus; ^Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, 220141, Minsk, Republic of Belarus

The impact of anthropogenic factors has a multifaceted effect on the human and animal body. Due to their complex impact, identifying the negative effects of individual factors is a rather difficult task. Metabolomics methodology, which makes it possible to overcome these difficulties, was used to assess the nature and degree of influence of waste from the production of potash fertilizers on the lipid profiles of experimental animals during intranasal inoculation with waste from the production of potash fertilizers and consumption of drinking water obtained from sources located in the zone of potential action of potash production. The separation of lipids from serum was carried out using a specially developed technique based on solid-phase extraction, which makes it possible to remove cholesterol from the samples. Each sample was analyzed by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS), after which the resulting chromatograms were processed using principal component analysis (PCA) and cluster analysis. The developed technique makes it possible to effectively separate hydrophobic metabolites in blood serum. The lipid profile of the blood serum of experimental animals was established, in particular the content of phospholipids and oxysteroids, and differences in metabolic processes were found between experimental and control animals. In the blood serum of experimental animals, the concentration of oxysteroids was increased compared to the control group.

Key words: steroids; lipids; blood serum; metabolic profile; industrial waste.

P. A. Chakhovskiy1, A.V Yantsevich2, A. E. Dmitrochenko2, A. V. Ivanchik2 - ANALYSIS OF SERUM LIPID PROFILES IN GUINEA PIGS FOR EARLY DETECTION OF CHANGES IN METABOLISM UNDER EXPOSURE TO ENVIRONMENTAL CONTAMINANTS

1The Republican Scientific and Practical Center of Hygiene, Minsk, Republic of Belarus, 220012; 2The Institute of Bioorganic Chemistry, Minsk, Republic of Belarus, 220141

for correspondence: Chakhovsky Pavel Anatolyevich, chahovsky@gmail. com

The exposure to anthropogenic factors has a multifaceted impact on the body of humans and animals. Due to their complex influence the detection of negative effects of the certain factors is a rather complicated task. Metabolomic methodology which permits to overcome these difficulties, has been applied in the evaluation of the nature and degree of the impact of potash fertilizers production waste on lipid profiles of experimental animals after intranasal inoculation with potassic fertilizer production waste and consumption of drinking water obtained from sources located in the zone ofpotential action ofpotassic fertilizer production. Isolation of lipids from serum was performed with the help of specially developed technique based on solid-phase extraction of samples which allows to remove cholesterin from the samples. Each sample was subjected to HPLC -MS analysis, after which the obtained chromatograms were treated with the use of the method of principal component analysis and cluster analysis. The developed technique allows to efficiently separate hydrophobic metabolites in blood serum. There was an established serum lipid profile of experimental animals, in particular the content of phospholipids and oxysteroids, and there were differences found in the metabolic processes of the test and control animals. It is shown that in the serum of experimental animals, there is observed an increased concentration of hydroxysteroid as compared with the control group,.

Key words: steroids, lipids, blood serum, metabolic profile, industrial wastes.

Introduction

One of the most important tasks systems biology and functional genetics - integration of proteomics, transcriptomics and information about metabolic processes occurring in the body. Any disease or pathological process occurring in the body is reflected in the content of low molecular weight metabolites in tissues and blood. For the integral characteristics of low molecular weight metabolites in blood plasma, the term “metabolic profile” was introduced in 1971. Because simultaneous analysis of multiple classes of metabolites is extremely difficult and impractical, a series of techniques are commonly used to study metabolic profiles, including high-resolution nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry.

As a rule, when conducting metabolomic studies, they are limited to a certain group of substances that are separated from other components during sample preparation. The resulting group data is easier to interpret.

Metabolic profiles (particularly urine and plasma) can be used to determine the nature of physiological changes caused by the entry of toxic compounds into the body. In many cases, the observed changes can provide additional characterization of specific lesions, such as liver and adipose tissue.

Analysis of steroids and lipids in blood serum has great diagnostic potential. The lipid composition of blood serum, steroid hormones, their precursors and the products of their metabolic transformations characterize many functional parameters of the body. These substances play an important coordinating role in the regulation of metabolism and cardiovascular function and are involved in the body's response to acute and chronic stress.

The steroid profile is a unique diagnostic criterion for a number of gynecological and oncological diseases associated with impaired synthesis and metabolism of steroid hormones, while some of them can be diagnosed only by the steroid profile. In profile analysis, the possibility of using absolute values as simple variables and comparing them with the norm. However, changing the ratio of quantities may be more important. In addition, the steroid profile provides information about a large number of steroids at the same time.

Determination of serum steroid profile - effective method identifying almost all disorders of steroid metabolism, which makes it possible to diagnose

accurate diagnosis in many clinical situations, for example, with congenital adrenal hyperplasia, type I hyperaldosteronism, primary hyperaldosteronism, Itsenko-Cushing's disease, adrenal insufficiency, etc. The steroid profile is important in the diagnosis of disorders of sexual differentiation and gonadal function, as well as hypothalamic pituitary-adrenal insufficiency.

Excessive intake of salt, which is the predominant component of potassium fertilizer waste, into the body of experimental rats with excess body weight leads to excessive activation of aldosterone synthesis and causes hypertension and kidney damage with metabolic syndrome.

In regions of industrial production with a high degree of environmental contamination, the level of morbidity among the population is usually higher than in relatively “clean” regions. The object of our research was the city of Soligorsk, located in the zone of large-scale mining and processing of potash ores. In the areas of salt dumps and sludge storage facilities of potash plants, a zone of chloride-sodium salinity has formed, which covers groundwater to a depth of more than 100 m, which can affect the pollution of sources of drinking water supply and atmospheric air.

To assess the impact of pollution of individual environmental components in the region of industrial production of potash fertilizers, we analyzed the lipid profiles of blood serum as an indicator of early metabolic disorders under the influence of a mixture of chemicals.

The purpose of the work is to identify metabolic changes in laboratory animals with exposure to waste from the production of potassium fertilizers and consumption of drinking water obtained from sources potentially affected by production waste, using the method of high-performance liquid chromatography with mass spectrometric detection (HPLC-MS).

The object of the study was the blood serum of experimental animals (guinea pigs) of the experimental and control groups.

Materials and methods

Experimental studies carried out on 35 guinea pigs (17 females and 18 males) weighing 305-347 g.

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Experimental group (priming with waste from industrial production of potassium fertilizers and drinking water from the water supply system of Soligorsk), 20 individuals (10 females and 10 males);

Control group (priming isotonic solution sodium chloride to exclude the effects of the stress factor caused by the priming procedure), 15 individuals (8 males and 7 females).

During the experiment, the general condition of the animals, food and water consumption were monitored daily.

To model chronic inhalation exposure (12 weeks) to waste from the production of potassium fertilizers, we were guided by MU.No. 11-11-10-2002 “Requirements for conducting toxicological and allergological studies during hygienic regulation of protein-containing aerosols in the air of the working area,” including the determination of the priming dose. Samples from salt dumps were ground in a marble mortar to a homogeneous dusty state, dissolved in distilled water to the required concentration, taking into account the body weight of experimental animals (body weight was monitored weekly to adjust the dose). The calculated doses were: at the beginning of the experiment - 2.028 mg/0.1 cm3, after 4 weeks - 2.85 mg/0.1 cm3, after 6 weeks - 3.17 mg/0.1 cm3, after 8 weeks and until the end experiment 3.8 mg/0.1 cm3.

A guinea pig without anesthesia was fixed in a supine position with its head raised, and a dose of warm solution was injected alternately into the nostrils (fractionally) using a pipette dispenser (over 2-3 minutes) in such a way as to prevent sneezing. The resulting “squelching” sounds confirmed the penetration of the solution into the respiratory tract.

The experimental group of animals “inhaled” the mixture once daily, 5 days a week for 12 weeks. Animals in the control group “inhaled” saline solution (0.9% NaCl).

To collect biological material, the animals were anesthetized (ether anesthesia), and after decapitation, blood was collected. The serum was obtained by centrifugation at 3000 rpm for 15 minutes and stored at -20 C for further studies. Phospholipids, oxysteroids, and fatty acids were analyzed in blood serum.

Sample preparation. An internal standard, progesterone, was added to the blood serum to achieve a concentration of 10-5 M (10 μl per 1 ml of sample). Then, to precipitate the proteins contained in the sample and extract the steroids, methanol was added to a final concentration of 70% (2.33 ml of methanol per 1 ml of sample), followed by centrifugation for 15 min at 10,000 g. The proteins contained in the sample formed a dense sediment. The supernatant was separated from the precipitate and passed through a preconditioned solid phase extraction (SPE) column containing 100 mg of octadecylsilyl silica gel. The SPE column was conditioned by successively passing 2 mL of methanol, 2 mL of water, and 2 mL of 70% methanol. In the first stage, cholesterol, the content of which in blood plasma and other biological fluids is quite high, as well as a number of other highly hydrophobic lipids, binds to the column. After cholesterol binding, the column was further washed with 2 ml of 70% methanol. If there is a high cholesterol content in the sample or a large sample volume, use

We used a SPE column with a high sorbent content. The eluates were combined and evaporated. Evaporation was carried out at 50°C in a stream of inert gas. The dry residue was dissolved in 500 μl of methanol and centrifuged for 10 min at 10,000 g. In this case, polar compounds insoluble in methanol precipitated. The supernanant was separated from the sediment and diluted with water to a methanol concentration of 10%. The resulting solution was passed through a pre-conditioned SPE column (passed 2 ml of methanol, 2 ml of water and 2 ml of 10% methanol) and washed with 2 ml of 10% methanol. Steroids bound to the column were eluted with 3 mL of 80% methanol. The solution was evaporated and the dry residue was dissolved in 100 μl of methanol. The resulting solution was analyzed using HPLC-MS.

HPLC analysis. The analysis was carried out on an Accela chromatograph equipped with an LCQ-Fleet mass spectrometric detector. Separation was performed on a Cosmosil 5C18-MS-II column with geometric parameters 4.6*150 mm (Nacalai Tesque, Japan).

Separation program (solvent A - water, solvent B - methanol, flow rate 500 μl/min): for 5 min 60% B, 12 min - linear gradient 60-95% B, 10 min - 95% B, 8 min - linear gradient 95-100% B, 5 min - 100% B, 5 min - 60% B.

An atmospheric pressure chemical ionization (APCI) source was used for mass spectrometric analysis. Ionization source parameters: evaporator temperature - 350°C, drying gas flow - 35 units, auxiliary gas flow - 5 units, capillary temperature - 275°C, capillary voltage - 18 V, ion lens voltage - 80 V. Used Data Dependent™ scanning mode using an ion trap in the scanning range 50-1000 m/z.

Chromatograms obtained using a mass spectrometric detector in the chemical ionization mode (total ion current) were converted into text format using the Qual Browser program from the Xcalibur package (Thermo Sci, USA). The obtained information was processed using the principal component method implemented in the Statistica 10 package and tools for cluster analysis and dendrogram construction. The reference book was used to interpret the mass spectra and identify individual compounds.

Results and discussion

The initial method for adaptation was the method of solid phase extraction of steroids from serum and blood plasma, described in the Macherey-Nagel Solid phase extraction manual. Application guide, which provides recommendations for the use of solid phase extraction columns. An adapted sample preparation technique with solid-phase extraction made it possible to effectively isolate phospholipids, oxysteroids and fatty acids from the blood serum of guinea pigs.

The described chromatographic separation technique makes it possible to effectively separate both steroid hormones and lipids present in serum.

Samples were analyzed according to the described methods. In Fig. 1 (see page 2 of the cover) shows, as an example, superimposed chromatograms obtained from the analysis of 3 samples from the experimental group (highlighted

Rice. 4. Mass spectra of a substance with a retention time of 21.5 minutes: a - chemical ionization at atmospheric pressure in negative mode; b - chemical ionization at atmospheric pressure in positive mode.

in red) and 3 samples from the control group (in blue). A similar picture was observed in other cases.

To process these data sets, we used the principal component method (PCA) and cluster analysis, which made it possible to identify differences in lipid profiles between the control and experimental groups. PCA plot of the 1st and 2nd principal components obtained

when reducing the data dimension, is shown in Fig. 2 (see 2nd cover page). On the graph it is easy to notice that the points are combined into 2 groups, localized in quadrants 1, 4 and 2, 3, respectively. In this case, the points corresponding to the prototypes predominantly fall into the 1st and 4th quadrants, the points corresponding to the control samples are localized in the 2nd and 3rd quadrants. The dendrogram obtained by placing

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Identification of peaks present in the chromatogram

Retention time, min Main peaks in “+” mode Main peaks in “-” mode

19,15 393,7 446,8

448,7 493,5 623,4 524,4

19,35 87 227 271 335,5 353,3 371,2 389.1 448.2 493.3 405,4

21,50 316,1 390,0

430.3 448.4 779,1

23,8 319.4 391,6 429.5 783,2

24,35 414,8 448,8

31,73 313,3 330,9

33,9 231.5 245.5 263,3 281,1 295.1 305.2 371.3 521.0 663.1 279,4

Substance

Phosphatidylcholine

Arachidic acid

Phosphatidic acid 42:4

Arachidic and docosate-traenoic acids

Docosapentaenoic

Linoleic acid

Dihydroxycholesterol

sternum analysis, shown in Fig. 3. Thus, statistical analysis chromatographic data indicates differences in the metabolic processes occurring in the organisms of experimental animals belonging to the control and experimental groups.

To identify specific metabolic changes, mass spectra were deciphered and identified.

individual connections are specified (see table). The sample volume was insufficient for analysis and did not allow us to detect changes in the steroid hormone profile in the serum. However, lipids with intermediate polarity were detected in the chromatogram.

Individual compounds were identified by analyzing the mass spectra of substances recorded under different ionization modes. Thus, the mass spectrum of the substance in two ionization modes with a retention time of 21.5 min is presented in Fig. 4. Analysis of this spectrum showed that the substance is diacyl-sn-glycerophosphate with molecular weight 780 (R1(311) = 20:0 fatty acid (arachidic acid), R2(331) = 22:4 fatty acid (docosatetraenoic acid)).

It was found that the chromatographic peak with a retention time of 42.52 min corresponds to dihydroxycholesterol, presumably one of the precursors in the biosynthesis of bile acids. Differences in the content of oxysteroids in blood serum indicate a possible disorder in the metabolism of bile acids. It can be noted that in the chromatograms presented in Fig. 1, in the blood serum of experimental animals there is an increased concentration of oxysteroids compared to the control (peaks with a retention time of 35-45 minutes).

conclusion. The technique used in the work makes it possible to detect early disorders of lipid metabolism when exposed to environmental pollutants with a high degree of efficiency. The results obtained indicate that intranasal administration aqueous solutions salt dumps in experimental animals Cavia porcellus leads to changes in the metabolism of lipids and oxysteroids. In particular, the observed increased levels of bile acid precursors (hydroxysteroids) in animals may be associated with dysfunction of the liver and enzymes involved in the biosynthesis of bile acids. Thus, the described approach can be used to identify disorders of lipid metabolism in residents of regions with technogenic pollution.

Literature

1. Horning E.C., Horning M.G. Metabolic profiles: gas-phase methods for analysis of metabolites. Clin. Chem. 1971; 17(8): 802-9.

2. Constantinou M.A., Tsantili-Kakoulidou A., Andreadou I., Iliodro-mitis E.K., Kremastinos D.T., Mikros E. Application of NMR-based metabonomics in the investigation of myocardial ischemia-reperfusion, ischemic preconditioning and antioxidant intervention in rabbits. Eur. J. Pharm. Sci. 2007; 30(3-4): 303-14.

3. Lu W., Bennett B.D., Rabinowitz J.D. Analytical strategies for LC-MS-based targeted metabolomics. J. Chromatogr. B.Analyt. Technol. Biomed. Life Sci. 2008; 871(2): 236-42.

4. Novotny M.V., Soini H.A., Mechref Y. Biochemical individuality reflected in chromatographic, electrophoretic and mass-spectro-metric profiles. J. Chromatogr. B.Analyt. Technol. Biomed. Life Sci. 2008; 866(1-2): 26-47.

5. German J.B., Gillies L.A., Smilowitz J.T., Zivkovic A.M., Watkins S.M. Lipidomics and lipid profiling in metabolomics. Curr. Opin. Lipidol. 2007; 18(1): 66-71.

6. Schwarz E., Liu A., Randall H., Haslip C., Keune F., Murray M. et al. Use of steroid profiling by UPLC-MS/MS as a second tier test in newborn screening for congenital adrenal hyperplasia: the Utah experience. Pediatr. Res. 2009; 66(2): 230-5.

7. Rauh M. Steroid measurement with LC-MS/MS. Application

To Art. Chakhovsky et al.

Rice. 3. Dendrogram constructed on the basis of cluster analysis and illustrating the clustering of samples into groups.

To Art. Chakhovsky et al.

Rice. 1. Combined chromatograms of samples 1-3 from the control group (highlighted in red) and 15-17 from the experimental group (highlighted in blue).

Projection of the cases on the factor-plane (1 x 2) Cases with sum of cosine square >= 0.00

18/3 9/z 21/1 ■ O

1 loan "Sh o 28/1" 22/10 41/1 p

Factor 1: 65.71%

Rice. 2. Graph obtained by processing chromatograms using PCA. The control group is highlighted in blue, the experimental group in red.

High-performance liquid chromatography (HPLC) is a column chromatography method in which the mobile phase (MP) is a liquid moving through a chromatography column filled with a stationary phase (sorbent). HPLC columns are characterized by high hydraulic pressure at the column inlet, which is why HPLC is sometimes called "high pressure liquid chromatography".

Depending on the mechanism of separation of substances, the following HPLC options are distinguished: adsorption, partition, ion exchange, size exclusion, chiral, etc.

In adsorption chromatography, the separation of substances occurs due to their different abilities to adsorb and desorb from the surface of an adsorbent with a developed surface, for example, silica gel.

In partition HPLC, separation occurs due to the difference in the distribution coefficients of the substances being separated between the stationary phase (usually chemically grafted to the surface of a stationary carrier) and the mobile phase.

Based on polarity, PF and NP HPLC are divided into normal-phase and reverse-phase.

Normal-phase is a variant of chromatography that uses a polar sorbent (for example, silica gel or silica gel with grafted NH 2 or CN groups) and a non-polar PF (for example, hexane with various additives). In the reverse-phase version of chromatography, non-polar chemically modified sorbents (for example, non-polar alkyl radical C 18) and polar mobile phases (for example, methanol, acetonitrile) are used.

In ion exchange chromatography, the molecules of a mixture of substances, dissociated in solution into cations and anions, are separated when moving through a sorbent (cation exchanger or anion exchanger) due to their different rates of exchange with the ionic groups of the sorbent.

In size exclusion (sieve, gel permeation, gel filtration) chromatography, molecules of substances are separated by size due to their different ability to penetrate the pores of the stationary phase. In this case, the largest molecules (with the highest molecular weight) capable of penetrating into the minimum number of pores of the stationary phase are the first to leave the column, and substances with small molecular sizes are the last to leave.

often separation occurs not through one, but through several mechanisms simultaneously.

The HPLC method can be used to control the quality of any non-gaseous analyte. To carry out the analysis, appropriate instruments are used - liquid chromatographs.

A liquid chromatograph usually includes the following main components:

– PF preparation unit, including a container with the mobile phase (or containers with individual solvents included in the mobile phase) and a PF degassing system;

– pumping system;

– mobile phase mixer (if necessary);

– sample introduction system (injector);

– chromatographic column (can be installed in a thermostat);

– detector;

– data collection and processing system.

Pumping system

Pumps supply PF to the column at a given constant speed. The composition of the mobile phase may be constant or vary during analysis. In the first case, the process is called isocratic, and in the second - gradient. Filters with a pore diameter of 0.45 µm are sometimes installed in front of the pumping system to filter the mobile phase. A modern liquid chromatograph pumping system consists of one or more computer-controlled pumps. This allows you to change the composition of the PF according to a specific program during gradient elution. Mixing of PF components in a mixer can occur both at low pressure (before the pumps) and at high pressure (after the pumps). The mixer can be used to prepare the PF and during isocratic elution, however, a more accurate ratio of components is achieved by pre-mixing the PF components for the isocratic process. Pumps for analytical HPLC make it possible to maintain a constant flow rate of PF into the column in the range from 0.1 to 10 ml/min at a pressure at the column inlet of up to 50 MPa. It is advisable, however, that this value should not exceed 20 MPa. Pressure pulsations are minimized by special damper systems included in the design of the pumps. The working parts of the pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the PF composition.

Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics. Application of tandem mass spectrometry (HPLC-MSMS) in clinical diagnostics

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annotation scientific article on veterinary sciences, author of the scientific work - Chakhovsky Pavel Anatolyevich, Yantsevich Alexey Viktorovich, Dmitrochenko Alesya Egorovna, Ivanchik Alexander Viktorovich

Related topics scientific works on veterinary sciences, the author of the scientific work is Chakhovsky Pavel Anatolyevich, Yantsevich Alexey Viktorovich, Dmitrochenko Alesya Egorovna, Ivanchik Alexander Viktorovich

ANALYSIS OF SERUM LIPID PROFILES IN GUINEA PIGS FOR EARLY DETECTION OF CHANGES IN METABOLISM UNDER EXPOSURE TO ENVIRONMENTAL CONTAMINANTS

Text of scientific work on the topic "HPLC-MS method for analyzing lipid profiles of guinea pig blood serum to identify early changes in metabolism when exposed to environmental pollutants"

Pumping system

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