RU2719574C1 - Method of increasing signal-to-noise ratio when analyzing aqueous solutions using raman spectroscopy - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry and specifically to a method of increasing the signal-to-noise ratio when analyzing aqueous solutions using Raman spectroscopy and Raman spectroscopy, particularly analyzing the quality of medicinal preparations by Raman spectroscopy. Method for increasing the sensitivity of qualitative and quantitative analysis of aqueous solutions of active substances of medicinal preparations by Raman spectroscopy involves obtaining a fine powder of anhydrous salts of composition MxAy, capable of forming crystalline hydrates, where x and y=1–2; M is a cation of alkali or alkali-earth metals; A is an anion of mono- or dibasic acids; production of a tablet from said salts, one salt or a mixture of salts of said composition, by pressing under pressure of not less than 50 kg/cm², applying an aliquot of the analyzed aqueous solution onto the obtained tablet; analysis of the surface of the tablet by Raman spectroscopy and processing of spectral data using the corresponding software.

EFFECT: disclosed is a method for increasing the sensitivity of qualitative and quantitative analysis of aqueous solutions of active substances of medicinal preparations.

1 cl, 8 dwg, 6 tbl, 3 ex

Description

The invention relates to the field of analytical chemistry and Raman spectroscopy, in particular to the analysis of the quality of drugs by Raman spectroscopy.

The proposed method can be used for qualitative and quantitative analysis of aqueous solutions of organic substances, for example, to control the quality of drugs in testing laboratories and in production, as well as in any other areas where Raman spectroscopy is used.

The claimed method is based on the effect of water absorption during the formation of crystalline hydrates of salts, which leads to an increase in the local concentration of the dissolved substance. The analyzed solution is proposed to be applied to a tablet from compressed anhydrous or dried salts of a certain composition (for example, sodium sulfate, lithium sulfate and calcium chloride), and after water absorption time (at least 5 minutes), due to the formation of crystalline hydrates, spectrograms are recorded according to the standard method for example, calibration using methods based on Beer's law, CLS, SMLR, PCR or PLS, without further changes.

It is shown that salts that do not form crystalline hydrates do not cause the effect of amplification of the Raman signal. When using the proposed method for the analysis of drugs, a 16-fold increase in the signal-to-noise ratio was observed compared to the analysis according to the standard method, which is equivalent to a 256-fold reduction in the analysis time and the number of recorded spectra. A study was made of the concentration dependence of the response of the analytical signal and the linearity of the calibration curve for a 10-fold difference in the analyte concentration was shown. The calculation of the metrological parameters of the method proves the applicability of the proposed method for quantitative analysis.

The prior art patent US 16072393, priority date 06/21/2016, "Analysis method and analysis device" is known. The proposed method differs from the specified analogue in that this patent uses the direct concentration determination method by Raman spectroscopy based on the obtained calibration model describing the dependence the intensity of the analytical signal from the analyte concentration, without using any signal amplification methods. This approach has a limitation on the lower limit of the determined concentrations due to the low signal to noise ratio in the concentration range below 1-5%, which is a source of high measurement errors, for the reduction of which may require the use of Raman spectrometers of a higher accuracy class, or a significant increase in the analysis time . Our proposed method allows, without changing the analysis parameters, to significantly increase the signal-to-noise ratio and, as a result, reduce the limit of quantitative determination of substances in aqueous solutions to at least 0.25%.

The invention is known US 16022121, priority date 06/28/2018 "Reaction control in acetic acid processes", which describes the method for determining the components of the reaction mixture in a high concentration of 65.3% -70.5% with a water content of 2-14 %, which reduces its effect on the analysis result. Our proposed method will allow us to analyze more complex mixtures based on aqueous solutions with an active substance detection limit of at least 0.07%, while water, as a solvent, can occupy the bulk of the analyzed solution (up to 99.93%, respectively).

The closest analogue of the claimed invention is the application CN 102353665 (A), priority date 02.15.2012 "Surface enhanced raman spectroscopy detection method for sulfanilamide medicines". The claimed method differs from the specified analogue by a number of important technological details. In our proposed method, there is no need for a long and multi-stage synthesis of precious metal nanoparticles (for example, silver, gold, platinum, etc.), since the signal amplification effect is achieved not due to plasmon resonance of the nanoparticle with the analyte molecule, but as a result of water absorption due to the formation of crystalline hydrates of salts, resulting in an increase in the local concentration of analyte on the surface of the salt tablets. This significantly reduces the cost of analysis, and there is no need for the preliminary manufacture of nanoparticles and systems based on them whose shelf life is limited.

The technical result of the claimed invention is to increase the sensitivity of the analysis of aqueous solutions of substances, by increasing the signal-to-noise ratio; reducing the limit of quantification and the detection limit of substances in aqueous solution using Raman spectroscopy.

The proposed method allows to reduce the time for analysis by reducing the number of scans to obtain an acceptable signal to noise ratio, and also allows the analysis of aqueous solutions of unstable substances that are prone to destruction, chemical modification or any other type of change in physicochemical and / or chemical properties under the influence of laser radiation in the conditions of analysis.

The technical result is achieved by performing the following stages of sample preparation:

1. Obtaining a fine powder of anhydrous (or dried) salts of the composition MxAy,

where x and y = 1-2;

M - cation of alkali or alkaline earth metals;

A is the anion of mono- or dibasic acids;

2. The manufacture of tablets from these salts (one salt or a mixture of salts of the specified composition) by pressing under a pressure of at least 50 kg / cm ² ;

3. Application of an aliquot of the analyzed aqueous solution to the resulting tablet;

4. Analysis of the tablet surface by Raman spectroscopy and processing of spectral data using appropriate software (for example, TQ Analist).

For the implementation of the invention, the following preparations were used as a model preparation:

1. “Ketoject” (organization-developer: LLC “API-SAN”, Moscow). Anti-inflammatory, antipyretic and analgesic drug for veterinary use. As an active ingredient it contains ketoprofen - 100 mg / ml and excipients: L-arginine, benzyl alcohol, sodium hydroxide and water for injection.

2. Enroflon (development organization: VIK group of companies). Antibacterial injection drug. Contains 10% enrofloxacin as an active ingredient, as well as excipients: sodium hydroxide and water for injection.

For work, we used an IR-Fourier spectrometer with a Fourier-Raman module Thermo Scientific Nicolet iS50, USA (hereinafter KR spectrometer).

HPLC analysis of the preparations was carried out on an Agilent 1260 Infinity chromatograph with a 1260 TTC DAD detector, Germany.

For analysis, powders of anhydrous salts were prepared — calcium chloride (Honeywell, USA), lithium sulfate (Acros, Belgium) and sodium sulfate (Acros, Belgium), by grinding them in an agate mortar. Then, tablets with a diameter of 3 mm were made from the obtained powders by pressing 20-100 mg of the powder using a hand press (IS50 RMNZ pellet sample press, Thermo Scientific, USA) with a force of at least 50 kg / cm ² . 0.5 μl of the analyzed solution was immediately applied to the resulting tablet. After keeping at least 5 min at room temperature, to complete the crystallization process, the tablet was placed in a Raman spectrometer and Raman spectra were recorded at the following instrument settings: Resolution 16 cm ^-1 , measurement range 3500-700 cm ^-1 , detector - InGaAs, beam splitter - CaF ₂ , laser wavelength λ = 1064 nm, laser power 0.50 W, number of scans - 100, analysis time - 2 min.

Example No. 1. To determine the linearity range of the response of the analytical signal, we prepared a series of dilutions of the Ketoject preparation in water to obtain solutions with ketoprofen concentrations: 10, 7, 5, 3, 2, 1%. The Raman spectra of the resulting solutions were recorded under the described conditions on a tablet of sodium sulfate. To obtain the calibration curve, we used the dependence of the peak height at a frequency of 1598 cm ^-1 (corresponding to C = C stretching vibrations of the aromatic cycle) on the concentration of ketoprofen in solution. The linearity of the response of the analytical signal in the concentration range of ketoprofen from 1 to 10% was shown. The correlation coefficient (R) in this range was 0.9970, which corresponds to a good level of approximation and proves the applicability of the proposed method for quantitative analysis in this concentration range. The Raman spectra of the resulting solutions were recorded under the described conditions on a tablet of sodium sulfate. The calibration curve of ketoprofen concentrated on a tablet of sodium sulfate is shown in Fig. 1.

Example No. 2. A quantitative determination of enrofloxacin in the preparation "Enroflon" was carried out using the proposed method. Sodium sulfate, pressed into a tablet according to the described procedure, was used as a water absorber. The concentration of enrofloxacin in the preparation determined by HPLC (according to TU BY 811000025.029-2007, notification of changes No. 1-5) was 106.4 mg / ml with an uncertainty of 4.4 mg / ml. This value was adopted as a certification value for determining the validation parameters of the proposed methodology. For calibration, a solution of enrofloxacin (Fluka) in water with a concentration of 101.97 mg / ml was prepared, the pH was adjusted with 1M NaOH to 10.0, which is similar to the acidity of the medium in the analyzed preparation. The measurement results obtained by Raman spectroscopy on days 1 and 2 of the analysis are presented in Table 1. To calculate the metrological parameters, the analysis was carried out with a variation of the factor “analysis time and operator”. On the first day, operator No. 1 made 4 sample preparations according to the indicated method (sodium sulfate was used as a desiccant salt) and twice measured the concentration for each sample preparation. On day 2, operator No. 2 conducted the same stages of sample preparation and analysis. The data are presented in table. 1.

To evaluate the technique for gross errors (misses) of dubious values of a sample from a random variable with a normal distribution, the Grubbs criterion was applied. In this case, the calculated value of the criterion Ucalc was determined by the formula

Here, xsr is the average value of the sample, xs is the doubtful value (maximum or minimum in the sample), s is the sample standard deviation (SD), determined by the formula

where xi are the sample elements, n is the sample size.

Since Ucalc <Utable, the obtained values do not contain gross errors and not one of them was excluded from the sample. The discrepancy between the variances was evaluated by the Cochren criterion. The data are presented in table 2.

The standard uncertainty under repeatability conditions and the relative standard uncertainty under intermediate precision conditions are presented in Table 3.

The bias is a systematic error as opposed to a random error. A larger systematic deviation from the true value corresponds to a larger bias value. Table 4 presents the values of the estimated bias of the concentration measurement results by the proposed methodology with respect to the arbitration value. So the values of the relative total standard uncertainty and total standard uncertainty are at the level of 2.6-2.7%.

Repeatability limit - a value that with a confidence level of 95% does not exceed the absolute value of the difference between the results of two measurements (or tests), obtained under conditions of repeatability (convergence). Precision is a measure of the similarity of the independent measurement results obtained by Raman spectroscopy. The limit of intermediate precision of the proposed methodology is less than 3%. The data are presented in table 5.

The results of the presented metrological evaluation of the method for determining the quantitative content of ketoprofen in a drug by Raman spectroscopy prove the applicability of this method according to the standards set forth in GOST 8.563-2009, GOST R 5725-2002.

Example No. 3. To prove the effectiveness of the proposed method for signal amplification compared with the standard analysis procedure, the signal-to-noise ratio, the quantification limit and the detection limit were measured by analyzing Enroflon and Ketoject preparations according to the proposed method using sodium sulfate, lithium sulfate, and chloride calcium and potassium bromide. For this, drug solutions were applied to tablets from powders of these salts and Raman spectra were recorded under the described conditions. In the case of the Ketoject preparation, a peak height of 1598 cm ^-1 was recorded (corresponds to C = C stretching vibrations of the aromatic cycle); for Enroflon, a peak height of 1388 cm ^{-1 was} recorded (corresponds to deformation vibrations of the CH ₃ group).

For comparison, spectrograms of the preparations were recorded without applying salt tablets to each tablet - each drug in an amount of 500 μl was placed in the well of the tablet and the spectrogram was measured under the same conditions as in the case of analysis on the surface of a salt tablet. The data are presented in Table 6. The spectra of preparations applied to the corresponding salts are presented in Figures 2-8 (Ketoject / calcium chloride - Figure 2, Ketoject / sodium sulfate - Figure 3, Enroflon / sodium sulfate - Figure 4, Enroflon / calcium chloride - Figure 5, Enroflon / lithium sulfate - Figure 6, Enroflon / potassium bromide - Figure 7, Ketoject / potassium bromide - Figure 8).

From the above data it is seen that when using the proposed method, an increase in the signal-to-noise ratio was observed compared to the standard analysis procedure. Calcium chloride showed the greatest signal amplification effect (16.09 times) compared with lithium sulfates (2.35 times) and sodium (1.72 times), which has a correlation with the difference in the solubility of these salts in water (19, 2 g / 100 ml — sodium sulfate, 34.3 g / 100 ml — lithium sulfate and 74.5 g / 100 ml — calcium chloride) and, as a result, the ability to absorb water with the formation of crystalline hydrates. The maximum gain in the signal-to-noise ratio (16.09 times) was observed in the case of the analysis of the Enroflon preparation on a tablet of calcium chloride. Moreover, for the preparation “Ketoject”, analysis under the same conditions showed an increase in signal by only 3.13 times. A possible reason for this experimental fact may be the presence of benzyl alcohol in this preparation, which can impede the complete absorption of water due to the formation of a hydrophobic layer on the surface of calcium chloride.

Calculations show that an increase in the signal-to-noise ratio by 16 times is equivalent to an increase in the number of scans and analysis time by 256 times, which under these conditions corresponds to recording about 25600 spectra (approximately 8.5 hours of continuous operation of the Raman spectrometer) according to the standard method, whereas while using the proposed method requires registration of only 100 spectra and the entire analysis takes 2 minutes. In addition, with a long analysis time, the sample will inevitably undergo physico-chemical and chemical changes due to heating under the influence of laser radiation, which will lead to incorrect results. Thus, the proposed method allows you to study aqueous solutions of substances by Raman spectroscopy in the low concentration region that are not available for the standard analysis procedure.

It was shown that only salts capable of forming crystalline crystalline hydrates (for example, calcium chloride, lithium sulfates and sodium) lead to amplification of the Raman signal under the described analysis conditions. So, for example, in the manufacture of a compressed tablet from potassium bromide and application of Enroflon and Ketoject preparations, no signal amplification was observed, moreover, a decrease in the intensity of characteristic lines was recorded by 7 and 6 times, respectively, compared to the analysis of solutions of these drugs (see Table 6). Since potassium bromide is not known to form crystalline hydrates, the obtained data indicate that, to enhance the signal, it is not the degree of solubility of salts in water that is decisive, but the ability to form crystalline hydrates, while the use of salts without this property leads to a decrease in local concentration dissolved substance on the surface of the tablet and, as a consequence, a decrease in signal intensity.

The claimed method allows to increase the sensitivity of the qualitative analysis, as well as the limit of quantification and detection limit of substances in aqueous solution using Raman spectroscopy. Using the claimed method allows to reduce the analysis time, and also provides the opportunity to study solutions of unstable substances under conditions of prolonged exposure to laser radiation.

Claims (5)

A method for increasing the sensitivity of qualitative and quantitative analysis of aqueous solutions of active ingredients of drugs by Raman spectroscopy, including the preparation of a fine powder of anhydrous salts of the composition MxAy, capable of forming crystalline hydrates, where x and y = 1-2; M - cation of alkali or alkaline earth metals; A is the anion of mono- or dibasic acids; the manufacture of a tablet from these salts, one salt or a mixture of salts of the specified composition, by pressing under a pressure of at least 50 kg / cm ² , applying an aliquot of the analyzed aqueous solution to the resulting tablet; analysis of the tablet surface by Raman spectroscopy and processing of spectral data using appropriate software.

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