RU2410691C1 - Method of quantitative determination of manganese, lead and nickel in bile by method of atomic-absorption analysis with atomisation in flame - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to laboratory methods of analysis and deals with method of quantitative determination of manganese, lead and nickel in bile by method of atomic-absorption analysis with atomisation in flame. Essence of method lies in the following: sampling of bile is carried out during duodenal probing, after that it is frozen, and unfrozen at room temperature, homogenisation of bile by mixing being performed already at partial soft unfreezing. After that, sampling of homogenised bile is carried out for preparation for analysis, concentrated nitric acid is introduced into it with volume ratio 1:1, mixture is kept at room temperature, then heated and further mixture is kept for not less than 2.5 hours at room temperature. In order to obtain analyte, to obtained mixture added is concentrated hydrogen peroxide in volume ratio 1:1 to volume of bile sample volume, analyte is heated, after that cooled to room temperature. After that by method of atomic-absorption spectrometry, using graduated diagram, quantitative content of particular type of metal: manganese, lead and nickel is determined in analyte.

EFFECT: invention allows increasing accuracy of quantitative determination of manganese, lead and nickel in bile.

6 tb

Description

The invention relates to the field of medical chemistry, in particular to medical toxicological studies, and can be used in the diagnosis of environmentally caused pathologies caused by the presence of heavy metals in the human body.

The invention may find application in biochemistry laboratories, specialized institutions and clinical diagnostic laboratories of medical centers.

As a result of increasing anthropogenic impact, progressive environmental pollution by geochemical elements, including heavy metals, is observed. The accumulation of various pollutants in the atmosphere, soil and water is facilitated by industrial emissions, household and agricultural waste. These pollutants have toxic effects on living organisms. So the accumulation of heavy metals (for example, lead, manganese, nickel, zinc, cadmium, cobalt, chromium, etc.) in the body can lead to damage to the central nervous and cardiovascular systems, contribute to the occurrence of chronic diseases of the gastrointestinal tract, etc. .P. By the content of heavy metals in biological media (for example, in bile), symptoms of various morphological and functional disorders of the hepatobiliary system and stomach of a person living under conditions of high anthropogenic load can be detected.

Currently, a number of methods are known for determining specific types of heavy metals in biological media by atomic absorption analysis, namely:

- according to the patent of the Russian Federation No. 2342659 the “Method for determining the cadmium content in organs and muscle tissue of pigs” is known, according to which the cadmium content is determined by the level of manganese and potassium in the hoofed horn and the corresponding regression equations;

- according to RF patent No. 2009484, the “Method for the determination of cobalt in biological material” is known, according to which a biomaterial sample is mineralized, it is treated with picric acid, extracted with a chloroform solution - methylcyclohexene 1,1 - dicarboxylic acid, the extract is treated with ethyl alcohol, followed by atomic absorption analysis ;

- according to the patent of the Russian Federation No. 1257520 the “Method for the determination of heavy metals in ectodermal tissues” is known, according to which a hair or nail sample is crushed, 30% tetramethylammonium solution is added to them, heated, then diluted with bidistilled water, centrifuged, solution 1 is separated, and non-dissolved the precipitate is converted into a soluble form with nitric acid and perhydrol to obtain a solution of 2, then cobalt, nickel, copper, manganese, zinc are determined by atomic absorption analysis in the indicated solutions.

However, all of these known methods are complex, characterized by a long determination time and do not allow the use of bile as a biomaterial.

Closest to the proposed technical solution by technical nature is the Method for determining the content of iron, zinc, nickel in bile by atomic absorption method, given in section 4.1 of the Methodological Instructions of the MUK 4.1.775-99, approved on July 6, 1999 and developed by the Department of State Sanitary and Epidemiological Supervision of the Ministry of Health of Russia.

According to the known method, bile sampling is performed during duodenal sounding in a volume of 5 cm ³ and metal concentration is measured by atomic absorption analysis by direct determination, while the measurement results are recorded according to the readings of a device with digital indexing calibrated according to working standard solutions of the metal being determined and present in the form: X _cf. ± 0.25 · X _{cp., Μg} / cm ³ , where X _cf. - the average value of the measured concentrations.

The disadvantage of this method is the instability of the analysis results in the study of bile samples of heterogeneous viscosity and composition, in addition, the influence of the organic component is not excluded. In cases of bile heterogeneity, the reliability of the obtained research results exceeds the permissible values of the operational accuracy control standards declared in the known mentioned technique.

The technical result achieved by the invention is to increase the accuracy of the quantitative determination of manganese, lead and nickel in bile by translating the bile sample into a homogeneous homogeneous state, decomposing the organic component and, thereby, reducing its effect while saving used biomaterial.

The specified technical result is achieved by the proposed method for the quantitative determination of manganese, lead and nickel in bile by atomic absorption analysis with atomization in a flame, including the selection of bile and determination of the quantitative content of heavy metals in it using atomic absorption spectrometry, while after selection of bile freezing it, then thawing, in which at the same time, starting with partial mild thawing, bile is homogenized by stirring After this, a homogenized bile sample is taken to prepare for analysis, then concentrated nitric acid is introduced into the resulting sample in a volume ratio of 1: 1, respectively, the mixture is kept at room temperature for 15-20 minutes, then heated for 5-10 minutes at a temperature of 120 ± 5 ° C, then incubated for at least 2.5 hours at room temperature, then to obtain the analyte, concentrated hydrogen peroxide was added to the mixture in a volume ratio of 1: 1 to the volume of the bile sample, the analyte was heated for 5-10 minutes at a temperature re of 120 ± 5 ° С, cooled to room temperature and then using the method of atomic absorption spectrometry and, using a calibration graph, the quantitative content of a specific type of metal — manganese, lead and nickel — is determined in the analyte, with concentrated nitric acid and concentrated peroxide hydrogen is taken in a volume ratio of 1: 1.

The specified technical result when implementing the proposed method for the quantitative determination of manganese, lead and nickel in bile by atomic absorption analysis with atomization in a flame is achieved due to the following.

It must be clarified that bile quite often is a heterogeneous biological environment. It contains the liquid phase, mucus, various clots, sediment inclusions that interfere with the analysis. Therefore, using natural, i.e. pre-unprepared, biological environment in studies there are large fluctuations in the definitions, the capillary of the spectrometer is often clogged.

Due to the fact that before research, bile is frozen and then thawed, starting to mix it even with partial soft thawing (already in the presence of ice "porridge"), the sample is homogenized, its viscosity decreases, because in ice "porridge" clots, large inclusions, mucus are crushed and mixed with the liquid phase. After that, it is possible to take a sample with a pipette or pipette to further prepare it for analysis.

To achieve sample homogeneity and destruction of the organic matrix, concentrated nitric acid and concentrated hydrogen peroxide are successively added to it, with heating after the introduction of each specified reagent for 5-10 minutes at a temperature of 120 ± 5 ° С to dissolve the residual forms of the precipitate.

By adding to the bile the indicated reagents (concentrated nitric acid and concentrated hydrogen peroxide) sequentially in their declared quantitative ratio between each other and with the sample volume, chemical modification of the analyzed material is ensured with the formation of soluble compounds and the quantitative transfer of the studied metals into solution, the sample is converted into a form suitable for analysis.

Heating and maintaining the mixture of bile samples with concentrated nitric acid at room temperature for at least 2.5 hours ensures the decomposition (mineralization) of the organic component of bile, helps to remove the formed gases.

Sample preparation in the proposed method takes place in almost one test tube, which reduces the risk of contamination of the sample from the dishes and possible losses if it is necessary to transfer the sample.

Thanks to the above operations, their sequence and the declared modes, it is possible to obtain an analyte solution from a bile sample, which can already be used to conduct a number of studies, the results of which will be guaranteed stable and accurate, which was proved experimentally.

The experiments showed that a deviation to a smaller side from the stated volume ratios of the bile sample and the volumes of concentrated nitric acid and concentrated hydrogen peroxide introduced into it can lead to incomplete oxidation of the organic component and overestimated results, apparently against the background of the matrix effect, and with deviations to a large side - the dilution of the sample increases, which can lead to a way out of the sensitivity range of the method, in addition, the acidity of the analyte should not exceed the norms specified in instructions p on the operation of the spectrophotometer.

Heating a bile sample mixture with concentrated nitric acid, and then with concentrated hydrogen peroxide for less than 5 minutes at a temperature of 120 ± 5 ° C, can lead to incomplete mineralization and insufficient degassing of the analyte, and when heated for more than 10 minutes, boiling, spraying and evaporation are too rapid samples, which leads to distortion of the results.

Holding the mixture of bile samples with concentrated nitric acid after the specified heating for at least 2.5 hours at room temperature ensures the destruction of the organic matrix. A decrease in this stage of sample preparation often leads to incomplete mineralization, which does not guarantee a quantitative transfer of the test element to the solution.

Sampling of bile for the preparation of one portion of the analyte is carried out in a volume of 1 cm ³ , but not more than 2 cm ³ , because the reaction can proceed quite rapidly and it is necessary to select other conditions for mineralization.

When implementing the proposed method carry out the following operations in the following sequence:

- produce the selection of bile with duodenal sounding in a volume of approximately 5 cm ³ (minimum volume 2 cm ³ );

- after selection of bile, it is frozen, i.e. get a solid phase;

- then it begins to thaw at room temperature;

- during thawing, even with partial mild thawing, bile is homogenized at the same time by stirring (stirring must be started already when an ice "porridge" is formed, that is, this characterizes partial, mild thawing, and then mix until completely thawed);

- then produce a homogenized bile sample in a clean tube, preferably in a volume of 1 cm ³ to prepare for analysis;

- then introduced into it in a volume ratio of 1: 1, i.e. in a volume of 1 cm ³ concentrated nitric acid with a concentration of 70%;

- the mixture is kept at room temperature for 15-20 minutes, then heated for 5-10 minutes in a test tube electric heater, for example, brand HACH COD REACTOR, at a temperature of 120 ± 5 ° C;

- and then the mixture can withstand at least 2.5 hours (in the preferred embodiment, 2.5-3 hours) at room temperature,

- then, to obtain the analyte, concentrated hydrogen peroxide with a concentration of 33% is added to the resulting mixture; in a volume ratio of 1: 1 (i.e. 1 cm ³ ) to the volume of the bile sample (it should be indicated that to obtain the analyte concentrated nitric acid and concentrated hydrogen peroxide are taken in a volume ratio of 1: 1, i.e., in this example, 1 cm ³ );

- the analyte is again heated in a test tube electric heater for 5-10 minutes at a temperature of 120 ± 5 ° C,

- cooled to room temperature,

- for each series of experiments put 2 blank samples, repeating the procedure for preparing the analyte, replacing the bile with purified bidistilled water. Measurement of blank samples is carried out together with real samples;

- the volumes of the obtained analyte and blank sample are fixed, in the case of evaporation is adjusted to 3 cm ³ (corresponding to the initial total volume of the sample of bile and added reagents) with purified bidistilled water,

- and further by atomic absorption spectrometry (using, for example, an atomic absorption spectrophotometer of the Perkin Elmer 3110 brand or others), using a calibration graph constructed by the absolute calibration method on certified mixtures of metal solutions, the analyte determines the quantitative content of a specific type of metal: lead, manganese and nickel.

Determination on an atomic absorption spectrophotometer begins with the installation of a spectral lamp in the device corresponding to the metal being determined, and is heated for at least 20 minutes (according to the operating instructions). For measurement, absorption is used with a wavelength equal to the absorption maximum of the metal being determined when passing through an air layer containing pairs of metal atoms: manganese — 248.3 nm; lead - 283.3 nm; Nickel - 232.2 nm. The monochromator is set to the desired wavelength, the width of the spectral slit is selected, the purified bidistilled water is placed for spraying, the necessary gas ratio (acetylene - air) is selected to maintain combustion, and the flame is ignited. The capillary supplying the solution to the flame is lowered into a 1% solution of nitric acid and the zero line is determined. Certified mixtures of the analyzed metal are sprayed into the flame to build a calibration curve, then the samples are introduced and the concentration values of the studied samples are recorded. The accuracy of the device settings is checked by introducing a certified mixture of a given concentration every five samples, if necessary, recalibrate. At a high content of the metal to be determined, the analyte and blank samples are diluted with a 1% solution of nitric acid, the dilution factor is taken into account when calculating the analysis result. The calculation of the content (X) of metals in bile is carried out according to the formula:

where C is the concentration determined by the calibration graph constructed by the absolute calibration method on certified mixtures of metal solutions, μg / cm ³ ;

C 'is the concentration value of the blank sample, μg / cm ³ ;

V is the total analyte volume, cm ³ ;

V '- the volume of the sample of bile taken for analysis, cm ³ .

двух параллельных определений X_max, X_min, расхождение между которыми r, не должно превышать предела повторяемости r_n:The measurement result is the arithmetic mean value

two parallel determinations X _max, X _min, divergence between which r, should not exceed the limit of repeatability r _n:

±Δ), мкг/см³, при доверительной вероятности (предельной ошибке для заданной надежности) Р=0,95, гдеThe result of a quantitative analysis in documents providing for its use is presented in the form: (

± Δ), μg / cm ³ , with confidence probability (marginal error for a given reliability) P = 0.95, where

- средний результат анализа, мкг/см³,

- the average result of the analysis, μg / cm ³ ,

;

;

Δ is the characteristic error, μg / cm ³ at P = 0.95

,

,

where δ is the relative value of the error characteristic,%.

The proposed method was tested in laboratory conditions. For its implementation, the following reagents and solutions were used:

Concentrated nitric acid, GOST 4461-77, osch .;

Acetylene, GOST 5457-75;

Hydrogen peroxide, GOST 177-88, 33%;

Nitric acid (HNO ₃ ), 1%;

Purified bidistilled water;

Nitric acid (HNO ₃ ), 1%;

Hydrogen Peroxide, 6%.

Studies of bile for the quantitative determination of nickel, manganese and lead in it were carried out by carrying out the above operations and modes.

Data on the quantitative content of nickel in bile, obtained during the implementation of the proposed method, are shown in table 1, by the quantitative content of manganese in table 2, by the quantitative content of lead in table 3.

Further, by calculating on the basis of the obtained data, the indicators of repeatability, reproducibility, and accuracy of the proposed method were determined. The following is the calculation procedure for the example of the quantitative nickel content determined in the bile sample by the proposed method.

1. Assessment of the repeatability of the proposed method

рассчитывают по формуле (данные для расчета взяты из таблицы 1):1.1. Arithmetic mean of parallel definitions

calculated by the formula (the data for the calculation are taken from table 1):

рассчитывают по формуле (таблица 1):1.2. Selective dispersion of the results of a single analysis of the nickel content obtained under repeatability conditions (parallel determinations),

calculated by the formula (table 1):

1.3. Testing the hypothesis of equality of general variances. The value of G _l (max) is calculated by the formula:

G ₁ (max) = 0.120, G _tab = 0.602 (with a confidence probability of P = 0.95). The dispersion is homogeneous (G ₁ <G _table ).

1.4. The calculation of the mean square deviation (RMS) S _{r is} performed according to the formula:

,

,

1.5. The repeatability index σ _{r of the} proposed method in the form of standard deviation:

The repeatability index of the proposed method in the form of a repeatability limit r _{n is} calculated by the formula:

r _n = Q (P, n) · σ _r, where n is the number of parallel definitions provided by the proposed method for obtaining the analysis result.

Q (P, n) = 2.77 for n = 2, P = 0.95

r _n = 2.77 · 1.94 · 10 ^-3 = 5.36 · 10 ^-3

1.6. The average value of the analysis results obtained under reproducibility conditions:

1.7. Показатель повторяемости σ_r≈S_r в относительных единицах:

1.7. The repeatability index σ _r ≈S _r in relative units:

The repeatability limit of the proposed method r in relative units:

2. Assessment of the reproducibility of the proposed method

по формуле:2.1. Calculate the variance characterizing the scatter of the arithmetic mean of the results of parallel determinations (X _l ) relative to the total mean

according to the formula:

2.2. The reproducibility index of the proposed method in the form of standard deviation during an experiment in one laboratory is calculated by the formula:

The reproducibility index of the proposed method in relative units:

рассчитывают по формуле:The reproducibility index of the proposed method in the form of a reproducibility limit

calculated by the formula:

при P=0,95

at P = 0.95

в относительных единицах:2.3. Reproducibility limit

in relative units:

3. Assessment of the correctness of the method

и пробе с добавкой (проба с добавкой определяемого металла) -

(см. таблицу 4).Get a series of L results of the metal being determined - Nickel in the sample without additives (working sample) -

and sample with additive (sample with the addition of a defined metal) -

(see table 4).

The value of the additive to the working sample is C = 0.100 μg / cm ³ , the error in the preparation of the certified mixture is ± Δ _co = ± 0.001 μg / cm ³ (1%) (for the calibration curve, certified mixtures with specified nickel concentrations in a 1% nitric acid solution are prepared )

3.1. The average value of the result of the analysis of the sample without additives (working samples):

RMS, characterizing the random spread of the results of the analysis of the sample without additives:

3.2. The average value of the result of the analysis of the sample with the additive (see table 4):

RMS, characterizing the random variation of the results of the analysis of the sample with the addition of:

,

,

3.3. The value of the experimentally found value of the additive:

3.4. The systematic error of the proposed method θ _m :

3.5. The significance of the calculated values of θ _{m is} checked according to Student's criterion (t):

The value of t is calculated:

where S ₁ and S ₂ - standard deviation characterizing the random variation of the results of the analysis of the sample without additives and samples with additives;

Δ _co - the error of the certified value of the additive to the sample.

The value of t is compared with t _table. With the number of degrees of freedom f = L-1 for a confidence probability of P = 0.95 t _tab. = 2.26.

Since t <t _tab. (0.27 <2.26), the estimate of the systematic error is zero, θ _m = 0.

3.6. The correctness indicator of the proposed method is calculated by the formula:

3.7. The upper (Δ _CB ) and lower (Δ _CH ) boundaries of the non-excluded systematic error with the accepted probability P = 0.95 are found by the formula:

In relative units:

4. Evaluation of the accuracy indicator of the proposed method.

4.1. The standard deviation σ (Δ) of the error of the analysis results is equal to:

The upper and lower boundaries (Δ _B , Δ _H ), in which the error of any of the set of analysis results is with an accepted probability of P = 0.95, is calculated by the formula:

In relative units:

The calculation of repeatability, reproducibility, accuracy of the proposed method for manganese and lead is carried out in a similar way.

Data on the measurement range in the bile of nickel, lead and manganese by the proposed method, as well as on the values of repeatability, reproducibility and accuracy are shown in tables 5 and 6.

Known for the prototype, a method for the direct determination of metals in bile provides measurements with a total error of the result of up to 25%, while the proposed method allows to reduce the error in the determination of nickel to 5%, lead to 10%, manganese to 14.85% (see table 5).

Due to the specified sequence of operations and the use of certain modes, the proposed method improves the accuracy of determination of nickel, manganese and lead in a bile sample.

Table 1 Data on the quantitative nickel content in the bile sample obtained during the implementation of the proposed method, μg / cm ³ L, result number, l = 1, L N, number of parallel definitions, n = 1, N X _n , result of parallel determination

,среднее арифметическое параллельных определений, N=2

, arithmetic mean of parallel definitions, N = 2

, values of sample variances one one 0.150 0.1485 4,5 · 10 ^-6 2 0.147 2 one 0.151 0.1520 2.0 · 10 ^-6 2 0.153 3 one 0.149 0.1475 4,5 · 10 ^-6 2 0.146 four one 0.155 0.1540 2.0 · 10 ^-6 2 0.153 5 one 0.148 0.1495 4,5 · 10 ^-6 2 0.151 6 one 0.149 0.1475 4,5 · 10 ^-6 2 0.146 7 one 0.153 0.1540 2.0 · 10 ^-6 2 0.155 8 one 0.153 0.1545 4,5 · 10 ^-6 2 0.156 9 one 0.150 0.1485 4,5 · 10 ^-6 2 0.147 10 one 0.152 0.1535 4,5 · 10 ^-6 2 0.155

table 2 Data on the quantitative content of manganese in the bile sample obtained during the implementation of the proposed method, μg / cm ³ L, result number, l = 1, L N, number of parallel definitions, n = 1, N X _n , the result of parallel determination

,среднее арифметическое параллельных определений, N=2

, arithmetic mean of parallel definitions, N = 2

, values of sample variances one one 0,052 0,0525 0.50 · 10 ^-6 2 0,053 2 one 0,046 0.0470 2.0 · 10 ^-6 2 0,048 3 one 0,049 0,0500 2.0 · 10 ^-6 2 0.051 four one 0,047 0,0460 2.0 · 10 ^-6 2 0,045 5 one 0,044 0,0455 4,5 · 10 ^-6 2 0,047 6 one 0,055 0,0535 4,5 · 10 ^-6 2 0,052 7 one 0,048 0,0500 8.0 · 10 ^-6 2 0,052 8 one 0.051 0,0545 0.5 10 ^-6 2 0,050 9 one 0,046 0.0440 8.0 · 10 ^-6 2 0,050 10 one 0,047 0,0455 4,5 · 10 ^-6 2 0,044

Table 3 Data on the quantitative content of lead in a bile sample obtained during the implementation of the proposed method, μg / cm ³ L, result number, l = 1, L N, number of parallel definitions, n = 1, N X _n , the result
parallel definition

,среднее арифметическое параллельных определений, N=2

, arithmetic mean of parallel definitions, N = 2

, values of sample variances one one 0.156 0.1525 2.45 · 10 ^-5 2 0.149 2 one 0.141 0.1435 1.25 · 10 ^-5 2 0.146 3 one 0.135 0.1385 2.45 · 10 ^-5 2 0.142 four one 0.137 0.1395 1.25 · 10 ^-5 2 0.142 5 one 0.152 0.1490 1.8 · 10 ^-5 2 0.146 6 one 0.134 0.1360 0.8 · 10 ^-5 2 0.138 7 one 0.142 0.1450 1.8 · 10 ^-5 2 0.148 8 one 0.154 0.1510 1.8 · 10 ^-5 2 0.148 9 one 0.138 0.1410 1.8 · 10 ^-5 2 0.144 10 one 0.151 0.1485 1.25 · 10 ^-5 2 0.146

Table 5 Data on the measurement range in the bile of nickel, lead and manganese by the proposed method, as well as on the values of its repeatability, reproducibility and accuracy The name of the determined component and the measurement range, μg / cm ³ Repeatability index (relative standard deviation of repeatability) σ _r ,% Reproducibility index (relative standard deviation of reproducibility) σ _R ,% Accuracy indicator (boundaries of relative error with probability Р = 0.95), ± δ,% Nickel,
from 0.10 to 1.00 incl. 1.28 2,32 4.94 Lead,
from 0.10 to 1.00 incl. 2.82 4.71 10.03 Manganese,
from 0.025 to 0.250 incl. 3.91 7.10 14.85

Table 6 The values of the limits of repeatability and reproducibility of the proposed method with a confidence probability of P = 0.95 The name of the determined component and the measurement range, μg / cm ³ Repeatability limit (relative value of the permissible discrepancy between two results of parallel determinations), r _n, %

, %The limit of internal laboratory reproducibility (the relative value of the permissible discrepancy between the two measurement results obtained in the same laboratory, but under different conditions),

% Nickel,
from 0.10 to 1.00 incl. 3,55 6.42 Lead,
from 0.10 to 1.00 incl. 7.82 13.04 Manganese,
from 0.025 to 0.250 incl. 10.83 19.68

Claims (1)

A method for the quantitative determination of manganese, lead and nickel in bile by atomic absorption analysis with atomization in a flame, including the selection of bile and determination of the quantitative content of heavy metals in it using atomic absorption spectrometry, characterized in that after selection of bile it is frozen, then thawing, in which at the same time, starting with partial mild thawing, bile is homogenized by stirring, then the selection is homogenized th bile sample to prepare for analysis, then concentrated nitric acid is introduced into the resulting sample in a volume ratio of 1: 1, respectively, the mixture is kept at room temperature for 15-20 minutes, then heated for 5-10 minutes at a temperature of 120 ± 5 ° С, then kept at least 2.5 hours at room temperature, then to obtain the analyte, concentrated hydrogen peroxide in a volume ratio of 1: 1 to the volume of the bile sample is added to the resulting mixture, the analyte is heated for 5-10 minutes at a temperature of 120 ± 5 ° C, cooled to room temperature and further with help Strongly method of atomic absorption spectrometry using a calibration graph produced in the analyte determination of the quantitative content of a particular kind of metal - manganese, lead and nickel, with concentrated nitric acid and concentrated hydrogen peroxide taken in a volume ratio of 1: 1.