Method for determining metronidazole in biosubstrate

FIELD: medicine.

SUBSTANCE: sample is applied on a paper filter, and standard calibration solutions of metronidazole in the concentration range of 10-100 mcl are radially applied on the same filter. The filter is processed with a developing agent containing 5% potassium hydroxide and acetone taken in ratio 2:1 and thermostated at 100°C until coloured spots are visible. A colour intensity of the sample spot is compared to the colour of the spot colour of the reference calibration solutions to determine the metronidazole concentration in the analysed sample.

EFFECT: method enables the fast and reliable monitoring of the metronidazole content and the timely correction of the therapeutic process.

3 ex

 

The present invention relates to medicine, namely to therapy, and can be used to control the content of metronidazole in liquid media (blood, saliva, lymph, cerebrospinal fluid, urine, pus) and the tissues of the body.

Metronidazole is used to treat clostridial anaerobic infections. Control over the level of this drug is necessary, since at lower concentrations below the minimum inhibitory growth of anaerobic bacteria in the septic focus, not suppressed. The excess concentration of the drug is associated with the risk of undesirable side effects, such as nausea, vomiting, diarrhea, urticaria, headache, weakness, drowsiness (Reference Vidal drugs in Russia: a Handbook. M: Estrofem Service, 1998, S. 148).

There is a method of determining the concentration of metronidazole in the blood, including extraction of sample (blood, tissue and other) ethanol exposure during the day at zero temperature, removing the spectrum of the resulting supernatant on a spectrophotometer at a wavelength of 320 nm and calculating the concentration of metronidazole using the calibration curve. (Baisogala D., Burdov B. A., Konoplyannikov A., and other Direct results of radiation and combined treatment of patients with malignant tumors with the use of metronidazole. J. Med. Radiola. - 1983, No. 2, S. 7-12).

The disadvantages of this method include the duration of the analysis (24-30 hours).

The closest to the technical nature of the claimed and adopted for the prototype is a method of determining the content of metronidazole in biosubstrate by conducting chromatographic analysis.

For the implementation of this method take 1 ml or 1 g of sediment sample (blood, saliva, cerebrospinal fluid, pus, tissue sampling), to which was added 1 ml of acetonitrile to precipitate high molecular weight fraction. The resulting mixture was centrifuged for 20 minutes at 3000 rpm and the supernatant evaporated to dryness in a stream of nitrogen at a temperature of 80-90°C. the Dry residue is completely dissolved in eluent acetonitrile-water, taken in the ratio 10:90, and then centrifuged for 20 minutes. The obtained supernatant injected with mobile phase: acetonitrile-water (10:90), a flow rate of 100 µl/min eluent Degassing with helium within 1-2 minutes quantification of metronidazole is carried out at a wavelength of 318 nm. The analysis, including sample preparation, an average of 2 hours (Kuznetsova, E. E., Gorokhova C. G., Andreeva T. A. and other high-performance liquid chromatography prolonged metronidazole - back. Chem-Pharm. J.-L. 2000, No. 8, S. 46-47).

The disadvantages of this method include the use of Dor is goteamsgo device, toxic solvents and multi-stage operations.

The objective of the proposed technical solution is the advanced diagnostic capabilities of the laboratory of control over concentration of metronidazole contained in the patient's blood.

The technical result of the proposed method is to create a simple, affordable rapid method of determining the concentration of metronidazole in fluids and tissues of the body, allowing the clock to be monitored.

The technical result is achieved in that the method of determining the content of metronidazole in biosubstrate carried out by chromatographic analysis of the sample.

Distinctive techniques of the proposed method lies in the fact that the sample assay applied on filter paper and on the same filter, radial, put the standard calibration solutions of metronidazole in the concentration range 10-100 μl. After that, the filter is treated with a reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio 2:1, and thermostatic at 100°C until the appearance of colored spots. The concentration of metronidazole in the analyzed sample set by matching the intensity of staining spot sampling spot one of the standard calibration solutions.

A comparative analysis was performed with a prototype of the m showed the proposed method differs from the known above-mentioned methods, and therefore meets the criterion of the invention of "novelty."

Comparison of the proposed technical solution is not only the prototype, but also with other technical solutions in medicine failed to reveal any sign of the claimed invention. In the available literature, the authors found no way of determining the content of metronidazole in biosubstrate using paper chromatography.

The authors of the proposed method experimentally established that the colour intensity of the analyzed sample is directly proportional with the concentration of metronidazole 10-100 ál.

Feature of the proposed method is that for the determination of metronidazole in samples no prior sample preparation and special laboratory equipment.

Clinical studies of the authors of the proposed method show that the proposed technical solution is available, simply and quickly to monitor drug levels in biosubstrate and make appropriate adjustments in the process protivoavariynogo treatment of severely ill patients.

The above can conclude that the technical solutions according to the criterion "and oprettelse level.

The method constituting the invention, intended for use in medicine, namely in clinical practice (therapy of purulent surgery, proctology, Oncology, and others). The possibility of its fulfillment is confirmed as described in the application techniques and, consequently, the proposed solution meets the criteria of the invention "industrial applicability".

The inventive method is carried out as follows.

On filter paper by using the pipettor put the sample assay (serum, saliva, urine, lymph, cerebrospinal fluid, pus, exudate). On the same radial filter is applied and 10 standard calibration solutions of metronidazole in the concentration range 10-100 μl. After that, the filter is carefully sprayed pre-prepared reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio of 2:1. Then the filter is placed in a thermostat with a temperature of 100°C. and dried until the appearance of colored spots. The intensity of yellow spots appeared depends on the concentration of metronidazole. The concentration of metronidazole in the analyzed sample set visually by matching the intensity of staining spot sampling spot one of the standard calibration solutions. The total analysis time is 20-30 minutes.

The proposed method is illustrated in the examples of the specific implementation.

Example 1. Patient Century, history No. 19140, was admitted to the intensive care unit of septic center Regional hospital with the diagnosis of widespread purulent peritonitis. To inhibit the growth of anaerobic clostridial infection, isolated from the patient, was appointed metronidazole in a dose of 500 mg intravenously. After 3 and 6 hours after administration of metronidazole carried out a determination of its content in the serum of this patient by the present method.

After 3 hours, the concentration of the administered drug was 4,34 µg/ml after 6 hours, the concentration of metronidazole in the serum of this patient was 3.24 µg/ml.

Identified a number of metronidazole was below the minimum bactericidal concentration that caused a subsequent correction dose.

Example 2. The patient Was, the case history No. 17390, enrolled in septic Department Bureau diagnosed with widespread purulent peritonitis, anaerobic clostridial infection. In complex drug therapy was included metronidazole endolymphatic at a dose of 500 mg

After 3, 6 and 9 hours after administration of metronidazole was definitely its content in the lymph offer this sick way: 3 hours - 35,3 µg/ml after 6 hours - 29,7 µg/ml and 9 hours - to 25.3 mg/ml

The obtained data testify that in sidemy period, the contents of metronidazole exceeded its minimum bactericidal concentration, that did not require changes in the tactics of antimicrobial therapy.

Example 3. Patient K., case history No. 21019, enrolled in septic Department Bureau diagnosed with cellulitis of thigh. From the wound was selected anaerobic infection (Bac. fragilis).

The patient was assigned metronidazole intravenous dose of 500 mg After 3 and 6 hours after administration conducted the determination of metronidazole in purulent discharge from the wound. Determined after 3 hours of 3.8 µg/ml after 6 hours and 2.9 µg/ml.

Installed the concentration of metronidazole was below the minimum bactericidal, which required an increase in dose.

Clinical studies of the proposed method was conducted at the clinic FBGU "NCRV" SB RAMS in the Regional clinical hospital. We examined 39 patients, including: widespread purulent peritonitis - 20, pancreonecrosis - 9, cellulitis of thigh - 5, ulcerative colitis - 5.

For comparison, the concentration of metronidazole in the serum of these patients was also determined and by spectrophotometry. When comparing results, the error definitions are not exceeded 3.2%. Thus the sensitivity of the proposed method is 89,8%, a specificity of 87.8%.

Thus, the proposed method is simple, fast and reliably determine the content of metronidazole in biosubstrate that is allows the opportunity for rapid analysis and thereby, allows timely adjustments to the treatment process. The method does not require the preliminary sample preparation and does not require special instrumentation.

The method of determining the content of metronidazole in biosubstrate by chromatographic analysis of a sample, wherein the sample assay applied on filter paper and on the same radial filter applied standard calibration solutions of metronidazole in the concentration range 10-100 μl, after which the filter is treated with a reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio 2:1, and thermostatic at 100°C until the appearance of colored spots, the color intensity of the spots of the sample is compared with the color spots of standard calibration solutions, which determine the concentration of metronidazole in the analyzed sample.



 

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