Method of detecting unknown substances in body fluids of patients taking narcotic or psychoactive substances

FIELD: chemistry.

SUBSTANCE: disclosed is a method of detecting unknown substances in body fluids of patients taking narcotic or psychoactive substances. The method involves preparation of three body fluid samples - the first through extraction with re-solution, the second through acid hydrolysis and the third through enzymatic hydrolysis. The first sample undergoes GC/MS analysis at temperature gradient of 15°C/min and data are analysed by comparing with a data base from which features of the unknown substance are detected, specifically spectra with m/z values which coincide with basic ions of the narcotic or psychoactive substance or metabolites and content of the unknown substance in the sample. The second sample undergoes GC/MS analysis at temperature gradient of 25°C/min and the third sample undergoes GC/MS analysis also at temperature gradient of 15°C/min and, if content of the unknown substances in the last two samples is higher than the in the first, the narcotic or psychoactive substance undergoes GC/MS analysis for presence of the unknown substance also at temperature gradient of 15°C/min, and if also not present in the basic substance. Presence of the unknown substance in intact body fluid is also checked, for which a sample of the intact body fluid is prepared via acid hydrolysis and undergoes GC/MS analysis at temperature gradient of 15°C/min and 25°C/min, and if the unknown substance is detected in the intact body fluid, the substance is classified as endogenous, and in the absence of features, an aliquot of the first sample is mixed with the sample of intact body fluid. The sample is prepared via acid hydrolysis of the mixture. The sample undergoes GC/MS analysis at temperature gradient of 15°C/min and 25°C/min. Further, content of the unknown substance is determined from results of both analysis modes and then compared with content of the known substance in the first sample. If content values of the unknown substance in the said three samples coincide, the unknown substance is classified as a new, previously unknown product of metabolism of the basic narcotic or psychoactive substance.

EFFECT: possibility of unique identification of chemical compounds and their fragments in arbitrary combinations while increasing accuracy and rapidness of detection.

4 tbl

 

The invention relates to gas chromatography analysis of various chemical compounds and can be used in medicine, biology, ecology and doping control.

There is a method of analysis of liquid preparations based on vegetable raw material by chromatography, which involves removing the volatiles by distillation of the original sample steam, concentrated volatiles extraction of the low-boiling solvent followed by distillation of the solvent and, finally, the actual definition of the components by the methods of gas or liquid chromatography [1].

The disadvantage of this technical solution is the low information content of the data received, in particular chromatographic spectra, which prevents unambiguous identification of chemical compounds and their fragments in arbitrary combinations.

There is also known a method of chromatographic identification of components of complex mixtures of organic compounds, including passing the substance through a system of series-connected columns filled with different sorbent polarity, sampling after each column, detection at the detectors of various types and identification of the analyte by calculating the sensitivity coefficient and the relative retention data of two detectors [2].

The disadvantages of the criminal code of the above method include the complexity of the analysis procedure, as well as the probability of obtaining inaccurate results when calculating retention values of different columns.

There is also known a method for the identification of unknown substances by gas chromatography in combination with mass spectrometry. At the specified method record the chromatogram as a function of retention time and record the mass spectrum in the period of time corresponding to the release of substances from the column, and compared with the mass spectra of known substances in the database, then define the retention index and compare it with those from the database and identify the substance by the two parameters : the mass spectrum and retention index [3].

The disadvantage of this method, despite the attractiveness of using the retention index is high the probability of obtaining false results of the analysis, because the retention indices of the initially bound to a specific column with defined parameters and is often either not played or played back on other equipment with distortion, which can lead to inaccurate interpretation of results.

The closest analogue to the claimed technical solution is a way of performing mass spectral analysis of multicomponent systems, which consists in registration of mass spectra of the sample, de is analucia of mass spectra and their associated chromatograms of one substance, and recognition by comparison with the reference spectrum of a substance by comparing the intensities of peaks and retention time recognized substances with predefined parameters of the required substances confirming recognition [4].

The disadvantage of this method is the low threshold of the definition, namely, that it is not possible to determine the analyte at low concentrations, and the resulting high probability of obtaining false results of the analysis in this two-stage recognition.

The technical result, which directed the establishment of this invention is the provision of opportunities for unambiguous identification of chemical compounds and their fragments in arbitrary combinations, while improving the accuracy and timeliness of the definition.

The technical result is achieved by the fact that prepare three samples studied sample of biological fluid first by extraction with reconstitution, second by acid hydrolysis and the third by enzymatic hydrolysis, and the first sample is subjected to GC/MS analysis mode temperature gradient 15°C/min, and the data is analyzed by comparing with the database, which identifies the characteristics of an unknown substance (HB), namely spectra with mz, coinciding with basic ions acting on the patient's drug or substance and its metabolites and the content of HB in the sample, the second sample was subjected to GC/MS analysis mode temperature gradient 25°C/min and the third sample was subjected to GC/MS analysis mode temperature gradient 15°C/min and the increase in the content of HB in the last two samples in comparison with the first, is subjected to GC/MS analysis mode temperature gradient 15°C/min operating on a patient's drug or psychoactive substance in the presence of HB, and in its absence in the base substance, check the presence of HB in intact biological fluid, for which the sample it is prepared by acid hydrolysis and subjected to GC/MS analysis mode temperature gradient 15°C/min and 25°C/min and in case of detection of HB in intact biological fluid it qualify as endogenous, and in the absence of signs - an aliquot of the first sample, is mixed with a sample of intact biological fluid, prepare the sample by acid hydrolysis of the mixture, subjecting the sample GC/MS analysis mode temperature gradient 15°C/min and 25°C/min, define the content of HB on the results of both analysis modes and compare it with the content of HB in the first sample and the matching values of the content of HB in these three samples qualify HB ka is new, previously unknown product of the metabolism of narcotic drugs or psychotropic substances.

It should be noted that the inventive method can also be carried out using analytical system HPLC-MS/MS, because the principal provisions of the proposed technical solutions are sample preparation, analyses in different modes of climate parameters on the temperature gradient, the choice of objects for analysis and subsequent identification of the designated substances by comparing the results of the analysis of baseline data of the reference substance.

As a biological fluid using blood, urine, saliva, water extracts disintegrated organs and tissues, etc.

The analytical system GC-MS can be used, for example, gas chromatography-mass spectrometer with quadrupole analyzer Agilent-5973, coupled with a gas chromatograph model Agilent-6890.

As accessories can be used:

- automatic shaker firm Glas-Col®, USA - for JJA;

- centrifuge brand Rotina 46R company Hettich, Germany) to obtain the contrast of the boundary surface between the organic and aqueous phases;

- vacuum concentrator company Barnstead Inc. (USA) for evaporation of the organic extract;

- column HP-5MS, VF-5MS for chromatographic separation.

Of course, for having realizatsii of the claimed invention can be used, and other devices with characteristics similar to the above.

As the carrier gas can be used helium, nitrogen or hydrogen.

As internal standards (SU) use diphenylamine or other suitable connection.

The ionization is carried out by electron impact in a vacuum. Detection of the detected substances is carried out in the registration mode full scan or selected ions.

Data processing is carried out with application of the automatic processing AMDIS and databases.

In principle, search, and detection should be implemented in order to find new, previously unknown metabolites, which can be potentially active and therefore be responsible for the side effects of the drug such as ketamine, or in the case of the same ketamine should be the basis for the development of new anesthetics.

With regard to the implementation of the proposed technical solution, the authors, based on their knowledge and experience, I think it advisable to search in the following sequence:

- search and discovery of the unknown substance (GC-MS analysis of the samples analyzed biological fluid at the temperature gradient of 15°C/min);

- identify the impact parameter analysis on the education of unknown substances (GC-MS analysis of the acid hydrolysate sample research which has been created biological fluid at the temperature gradient of 25°C/min);

- detection of conjugates (GC-MS analysis of enzymatic hydrolysate samples studied biological fluid at the temperature gradient of 15°C/min);

- identification of the unknown substance as an impurity or degradation product of narcotic or psychoactive substances (GC-MS analysis of samples of drugs or psychoactive substances at the temperature gradient of 15°C/min);

- identification of unknown substances in the intact liquid (GC-MS analysis of the acid hydrolysate samples of intact biological fluid at the temperature gradient of 15°C/min and 25°C/min);

confirmation of origin of an unknown substance from the study of biological fluid (GC-MS analysis of the acid hydrolysate of a mixture of samples analyzed and intact biological fluid at the temperature gradient of 15°C/min and 25°C/min).

The invention can be implemented as follows.

Previously, to create a library (database) prepare solutions of substances-standards (they are the same drugs and/or psychoactive substances) in organic solvents. Remove and register chromatographic and mass spectrometric characteristics of substances-standards (detects at least three characteristic ions of each reference substance, determine the retention time, molecular mass precursor ions, characteristic ions, the lower limit of the in detection).

Prepare the internal standard solution, then hold the sample preparation, in which a sample of biological fluid injected internal standard solution to bring the pH of the sample to 9.0 solid buffer, conduct liquid-liquid extraction with a mixture of organic solvents of different polarity, the organic layer is evaporated to dryness in a stream of nitrogen, pererastayut in ethyl acetate and then divide the sample into two analyte, the first of which is introduced into the system GC-MS. Remove and register chromatographic and mass spectrometric characteristics of the sample. The results of the analysis are compared with the database and to detect signs of unknown substances (HB). Next, prepare the second and third samples of the investigated biological fluid acid and enzymatic hydrolysis, respectively, and also enter them into the system GC-MS, shoot and record chromatographic and mass spectrometric characteristics of the samples. The results of the analysis are compared with the database, and signs HB determine its content in each sample. Then check the probability of the presence of HB in the drug or psychoactive substance, which is prepared sample of the specified substances by extraction and introduce it into the system GC-MS, shoot and record chromatographic and mass spectrometric characteristics of the sample and determine the presence of HB. Further, by acid hydrolysis to prepare a sample of intact biological fluid is injected into the system GC-MS and shoot and record chromatographic and mass spectrometric characteristics of the sample in two modes conditioning the temperature gradient, and then extracted a sample of intact biological fluid, mix it with the second analyte samples studied biological fluid mixture is subjected to acid hydrolysis and thus obtained sample is introduced into the system GC-MS, shoot and record chromatographic and mass spectrometric characteristics of the sample in two modes conditioning the temperature gradient. Next, compare, based on the results of the analyses, the HB content in all samples and is based on the ratio of the content of his qualify HB origin.

For a better understanding of the invention can be illustrated by, but is not exhausted by the following specific examples of its implementation.

Example 1.

The definition of the products of metabolism of ketamine.

A. Preparation of the reference sample and the analysis sample.

As an internal standard using diphenylamine, which is added to the analyzed samples to a concentration of 2 mg/DM3. To 1 ml of the investigated reference liquid add the internal standard to a concentration of 2 mg/l and extracted with 1 m is a mixture of hexane/diethyl ether 1:1. The extract evaporated, add 100 ál of ethyl acetate and 1 ál of sample injected.

The analysis is performed by gas chromatography-mass spectrometry system Agilent 6890/5973N mass-selective detector (record the mass spectrum of the quadrupole and the mass spectrum of the ion trap). The temperature of the node input of the sample - 280°C, analytical interface 240°C. the Separation is performed on silica capillary column HP-5MS length 30 m, internal diameter 0.25 mm, film thickness NF 0.25 μm. Temperature program: 70°C (1 min), 20°C/min, 280°C. the flow Rate of the carrier gas of 0.62 ml/min, the average linear velocity of carrier gas 29 cm/sec. The volume of injected sample 1 μl. The registration signal is carried out on total ion current (SCAN) in the mass range m/z 29-550 Amu Quantitative analysis is performed on the selected ions.

Fast GC program: 100°C, 1 min of exposure, (25°C/min); 300°C (15 min). The retention time of internal standard is to 4.52 min

Smooth GC program: 50°C; 0.5 min exposure; 99°C/min; 100°C (1 min); (15°C/min; 280°C (35 min). The retention time of internal standard is 9,27 minutes

B. sample Preparation and analysis of biological fluids (urine patient taking ketamine).

The urine sample (5 ml) add 5 ál of the internal standard solution containing diphenylamine (100 μg/ml), 0.1 g of the solid buffer, 5.0 g of ammonium sulfate and 5 ml of diethyl ether, the AC shall're asked for 2 minutes, further centrifuged at 1000 rpm for 5 min, the organic layer is separated, evaporated to dryness in a stream of nitrogen at 40°C and pererastayut dry residue in 50 ál of ethyl acetate, separated into four aliquots, the first of which is introduced into the system GC-MS. Remove and register chromatographic and mass spectrometric characteristics of the sample (detects at least three characteristic ions of each reference substance, determine the retention time, molecular mass precursor ions, characteristic ions, the lower limit of detection): take 5 µl of a solution and injected into the system GC-MS with ionization by electron impact in a vacuum. The analysis are as in part a smooth program (15°C/min). The results of the analysis are compared with the database (ketamine, norketamine) are presented in Table 1.

Table 1
The basic substanceTable of contents
in the sample
Retention timeThe mass spectrum of the quadrupoleThe mass spectrum of the ion trapMolecular weight
Ketamine-11,40237[M+]1 , 20923, 18099, 16611, 15217, 11513238[M+H]+25, 20912, 18099, 16640, 15220, 11515,237
Norketamine-11,10223[M+]1, 19522, 16699, 13814, 13116, 11513, 10212224[M+H]+50, 19520, 16699, 13132, 13816, 11518, 10240223
an unknown substance10 ng/ml9,94208[M+]1, 17399, 14510, 12997, 13829209[M+H]+80, 17399, 14525, 12940, 1385, 11535208

Next, a second aliquot of the sample is injected hydrochloric acid, hydrolyzing the mixture, and the hydrolysate is introduced into the system GC-MS. The analysis lead for fast program (25°C/min). A third aliquot of the sample is subjected to enzymatic hydrolysis, and the hydrolysate is introduced into the system GC-MS. The analysis lead to a smooth program (15°C/min). Next, prepare a sample of a basic substance ketamine. Dissolve ketamine in ethyl acetate, 1 ml of the solution add EXT is NNI standard to a concentration of 2 mg/l and extracted with 1 ml of a mixture of hexane/diethyl ether 1:1. The extract evaporated, add 100 ál of ethyl acetate and 1 ál of sample injected. The analysis lead to a smooth program (15°C/min). Next, prepare the sample of intact biological fluids (urine man, not taking ketamine) by acid hydrolysis, as described above, the hydrolysate analyze for smooth and fast programs (15°C/min and 25°C/min). Further non-hydrolyzed sample of intact biological fluid is mixed with the fourth aliquot samples of the investigated biological fluid, hydrolyzing with hydrochloric acid, as described above, and the hydrolysate analyze for smooth and fast programs (15°C/min and 25°C/min). The results of the analyses are presented in Table 2.

Table 2
№ p/pThe object of analysisThe analysis mode Gradient t-ture (°C/min)The content of the unknown substance (ng/ml)Note
1Reconstituted extract analyzed urine15°C/min10
2The acid hydrolysate of the extract analyzed urine 25°C/min1000
3Enzymatic hydrolysate of the extract analyzed urine15°C/min1000
4Sample of ketamine15°C/min0
5The acid hydrolysate of the extract of intact urine15°C/min0
6The acid hydrolysate of the extract of intact urine25°C/min0
7Acid hydrolysate of a mixture of extracts analyzed and intact urine15°C/min10
8Acid hydrolysate of a mixture of extracts analyzed and intact urine25°C/min10

According to the results of benchmarking the analysis of an unknown substance in the test biological fluid is a new metabolite of ketamine.

Example 2

The definition of products of the metabolism of tramadol.

The analysis is performed as described in example 1, except that analyze biological fluid (blood sample) patient taking tramadol. The results of the analysis in comparison with the basic substances are presented in Table 3.

Table 3
Substance, mol. weightRetention time, minMass spectrum (ion trap)
Tramadol 263 u19.01263[M+H]+(8%); 58(100%)
Epoxiconazol, impurity substance (and potential metabolite) 261 u19.26261[M]+(8%); 202(15%); 189(50%); 135(20%); 121(35%); 73(40%); 58(100%)
O-desmethyl - tramadol 249 u19.19250[M+H]+(100%); 58(30%)
An unknown substance17.55246[M+H]+(5%); 58(100%)

During analysis as in Example 1 non-hydrolyzed sample of intact biological fluids (blood sample of the patient not taking tramado is) is mixed with the fourth aliquot samples of the investigated biological fluid, subjected to enzymatic hydrolysis, and the hydrolysate analyze for smooth and fast programs (15°C/min and 25°C/min).

The content of the unknown substance in the analyzed samples are presented in Table 4.

Table 4
№ p/pThe object of analysisThe analysis mode
Gradient t-ture (°C/min)
The content of the unknown substance (ng/ml)Note
1Reconstituted extract analyzed blood15°C/min750
2The acid hydrolysate of the extract of the investigated blood25°C/min0
3Enzymatic hydrolysate of the extract of the investigated blood15°C/min750
4Test tramadol15°C/min10
5The acid hydrolysate of the extract of intact blood15°C/min0
6The acid hydrolysate of the extract of intact blood25°C/min0
7enzymatic hydrolysate of a mixture of extracts analyzed and intact blood15°C/min750
8Enzymatic hydrolysate of a mixture of extracts analyzed and intact blood25°C/min750

Despite the fact that in the sample of tramadol (item 4 of Table 2) also detected an unknown substance, it, whereas the contents in each case, qualify as a new, previously unknown metabolite of tramadol.

As can be seen from the description and examples of the method of the claimed technical solution provides the ability to uniquely identify chemical compounds and their fragments in arbitrary combinations with odnovremenno improving the accuracy and timeliness of the definition.

Sources of information

1. EN 2093822 C1 Ál. G01N 30/04, publ. 1997

2. EN 2069363 C1 Ál. G01N 30/02, publ. 1996

3. WO 2004/104571, Ál. G01N 30/00, publ. 2004

4. EP 1846757 A2 Μl. G01N 30/86, publ. 2007 - the nearest equivalent.

How to identify unknown substances in biological fluids of patients taking narcotic drugs or psychotropic substances, which prepare the test sample, is subjected to its GC/MS analysis, record the mass spectra of the sample, and their associated performance and conduct recognition of the substance by comparison with a database of reference analytical characteristics of substances, characterized in that prepare three samples studied sample of biological fluid first by extraction with reconstitution, second by acid hydrolysis and the third by enzymatic hydrolysis, and the first sample is subjected to GC/MS analysis mode temperature gradient 15°C/min and data analyze by comparing with the database, which identifies the characteristics of an unknown substance (HB), namely spectra with m/z coinciding with basic ions narcotic drugs or psychotropic substances or metabolites, and content (HB) in the sample, the second sample was subjected to GC/MS analysis mode temperature gradient 25°C/min and the third sample was subjected to GC/MS analysis mode temperature gradient 15°C/min and increase with the holding NV in the last two samples in comparison with the first, subjected to GC/MS analysis mode temperature gradient 15°C/min basic drug or psychotropic substance for the presence of HB, and in its absence in the base substance, check the presence of HB in intact biological fluid, for which the sample it is prepared by acid hydrolysis and subjected to GC/MS analysis mode temperature gradient 15°C/min and 25°C/min, and in case of detection of HB in intact biological fluid it qualify as endogenous, and in the absence of signs of an aliquot of the first sample is mixed with a sample of intact biological fluid, prepare the sample by acid hydrolysis of the mixture, subjecting the sample GC/MS analysis mode temperature gradient 15°C/min and 25°C/min, determine the content of HB on the results of both analysis modes and compare it with the content of HB in the first sample and the matching values of the content of HB in these three samples qualify HB as new, previously unknown product of the metabolism of the basic narcotic drugs or psychotropic substances.



 

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2 cl, 1 dwg, 1 ex

FIELD: analytical methods.

SUBSTANCE: to determine methyl alcohol in water, sample to be assayed is preliminarily subjected to distillation with sulfuric acid added in amount required to provide its concentration in mixture to be distilled c(1/2 H2SO4) = 0.002 M, while strippings constitute 6-7% of the volume of sample. Stripped liquid is thrice rinsed with hexane or Nefras at 1:1 hexane (Nefras)-to-strippings ratio. Rinsed material is then introduced into packed column filled with diatomite modified with 1,2,3-tris(β-cyanoethoxy)propane having deposited fixed phase thereon, which phase is prepared by way of consecutively keeping glycerol each time for 4 h at ambient temperature, 100°C, 130°C, 160°C, and 200°C, and then for 8 h at 230°C and for 40 h at 200°C under nitrogen bubbling conditions. Calculation of methanol content is performed taking into consideration calibrating coefficient.

EFFECT: enabled determination of small concentrations of methyl alcohol in water with sufficient selectivity and reliability.

2 cl, 2 tbl, 6 ex

FIELD: analytical chemistry.

SUBSTANCE: invention relates to method for quantitative determination of thiotriazoline and pyracetam in complex drugs by high performance chromatography, wherein silicagel with grafted 3-(chlorodimethyl)-propyl-N-dodecylcarbamate having particle size of 5 mum is used as sorbent; and degassed 0.05 M aqueous solution of potassium dihydrophosphate is used as mobile phase. Mobile phase velocity is 1 ml/min, and column temperature is 30°C. Method of present invention makes it possible to determine content of two abovementioned active ingredients simultaneously.

EFFECT: simplified process of sample preparation.

3 ex, 3 tbl

FIELD: biotechnology, in particular content determination of polymer chitosan molecules, chitosan-chitine polymer molecules and molecules of chitosan-protein complex in finished form of chitosan.

SUBSTANCE: claimed method includes application of high performance chromatography column filled with polyvinylbenzene sorbent with refractometer detector. As eluent and for dissolving of chitosan preparation samples acetic acid aqueous solution is used. Chain-length distribution is determined on the base of first chromatography peak, and polymer molecular content is calculated on the base of area of first, second and third chromatography peaks, divided up to zero line and belonging to polymer chitosan molecules, chitosan-chitine polymer molecules and molecules of chitosan-protein complex, respectively. To calculate chain-length distribution of polymer chitosan molecules separately calibration curve is plotted using dextran polymer standards.

EFFECT: new effective method for determination of polymer chitosan molecules in chitosan preparations.

4 cl, 3 dwg

Express-chromatron // 2300764

FIELD: the invention refers to laboratory chromatographic devices for conducting high-speed chromatographic analysis.

SUBSTANCE: the express-chromatron has an injector, a chromatographic column located in a thermostat, a detector, an amplifier of the signal of the detector, an analog-digital converter, a control system, a pneumatic system. The column is fulfilled either in the shape of a short capillary column or either in the shape of a polycapillary column. The injector is fulfilled with possibility of introduction of the test for the time of 5-50 ms. The detector and the amplifier of its signal are fulfilled with possibility of ensuring constant time of no worse then 10-3 sec. The analog-digital converter is fulfilled with possibility of ensuring speed of no less then 200 measurements in a second.

EFFECT: ensures conducting high-speed chromatographic analysis.

11 cl, 2 dwg

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