ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY METHODS FOR THE DETERMINATION OF THE RESIDUAL QUANTITIES OF RAMIPRIL AND HYDROCHLOROTHIAZIDE FOR CONTROLLING THE CLEANING OF EQUIPMENT

ramipril and 0.35 mL/min for the determination of hydrochlorothiazide; column temperature 45 °C for the de - termination of ramipril and 40 °C for the determination of hydrochlorothiazide, ionisation mode – electric spray in positive mode; The detection parameters are the mode of registration of the daughter ion 417→234 m/z for the determination of ramipril and 298→281 m/z for the determination of hydrochlorothiazide. Results and discussion. Methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry have been developed. The developed methods have sufficient linearity, correctness and precision. The sensitivity of the techniques was confirmed at the level of 0.0026 μg/ml. The techniques can be used in the concentration range of 0.0026–0.0255 μg/ml Conclusions. Analytical methods for determining ramipril and hydrochlorothiazide in wash waters have been developed and validated.


Introduction
Equipment cleaning and control of the completeness of equipment cleaning is an important step in preventing contamination of the medicinal product with a previously manufactured medicinal product.Each manufacturer must ensure that after cleaning, no active ingredient or detergent residues beyond the permissible maximum remain on the surface of the equipment [1].The completeness of cleaning of the equipment is monitored by analysing the most recent washing water or by analysing the washes from the surface of the equipment.The methods used to analyse wash water should be highly selective and sensitive.For the development of such techniques, methods of high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) with spectrophotometric or mass spectrometric (MS) detection, gas chromatography, ion exchange chromatography, atomic absorption spectroscopy and UV spectroscopy are used.Due to insufficient sensitivity, UV spectroscopy cannot be used for highly active substances.Ion exchange chromatography and atomic absorption spectroscopy are usually used to control the residual content of detergents [2] or preparations containing metal ions [3].Gas chromatography can only be used to monitor volatile compounds.The methods of HPLC and UPLC with spectrophotometric detection are the most widespread; however, their use is limited for highly active compounds and compounds with weak chromatophores [4].Ramipril (2S,3aS,6aS)-1-[(2S)-2-[[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino]propanoyl]-3,3a,4,5,6,6a-hexahydro-2H-cyclopenta[b]pyrrole-2-carboxylic acid is a prodrug and non-sulfhydryl ACE inhibitor with antihypertensive effect.It is metabolised to ramiprilat in the liver and kidneys.Hydrochlorothiazide, 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, is a diuretic that is often used to treat hypertension and oedema caused by fluid retention.Ramipril and hydrochlorothiazide can be used as mono preparations or in combination.The combination of these drugs has been well studied and has not lost its relevance for more than containing 0.01 μg/ml of hydrochlorothiazide in 0.05 % phosphoric acid was prepared.
Test solution for the determination of ramipril: 1.1 ml of analysed washing water was adjusted to 20.0 ml with a mixture of acetonitrile: water (5:95).
Test solution for the determination of hydrochlorothiazide: 0.85 ml of analysed washing water was adjusted to 50.0 ml with 0.05 % phosphoric acid.
For validation using Agilent 6420, 10 model solutions were prepared for both substances in the concentration range of 0.0026-0.0255μg/ml (equivalent to 25-247 % of the lower limit of the range of acceptance criteria for ramipril and 25-253 % of the lower limit of the range acceptance criteria for hydrochlorothiazide).
To confirm the reproducibility of the technique using Waters Xevo TQD ACQUITY, 5 model solutions were prepared in the concentration range of ramipril 0.0026-0.0250μg/ml (equivalent to 25-242 % of the lower limit of the range of acceptance criteria) and hydrochlorothiazide 0.0026-0.0255μg/ml (equivalent to 25-250 % of the lower limit of the range of acceptance criteria).

1. Method performed on Agilent 6420 MSdetector
The development and validation of the method were carried out using a liquid chromatograph with an Agilent 6420 MS-detector.The content of acetonitrile in the mobile phase was varied in such a way that the retention time of hydrochlorothiazide was from 0.5 to 1.5 min.The mass of 417 m/z for ramipril corresponds to the [M+] molecular ion.The daughter ion (234 m/z) is formed by cleavage from the parent CO ion and part of the molecule with the gross formula C 8 H 13 NO 2 [8].The mass of 298 m/z for hydrochlorothiazide corresponds to the molecular ion [M+].The daughter ion (281 m/z) was formed by cleavage of the amino group.The lowest permissible content of ramipril in the washing solution was 0.188 μg/ ml, hydrochlorothiazide -0.592 μg/ml.The dilution of the washing solutions was chosen in such a way that the nominal concentration of the analyte in the tested solution was 0.010 μg/ml.To investigate the specificity of the method for determining ramipril, the solvent (mixture acetonitrile:water (5:95)) and the comparison solution were analysed.Typical chromatograms are shown in Fig. 1.The solvent (0.05 % phosphoric acid) and the reference solution were analysed to study the specificity of the hydrochlorothiazide determination method.Typical chromatograms are shown in Fig. 2.
The chromatogram of the solvent did not reveal peaks that could interfere with the peak of ramipril or hydrochlorothiazide.Linearity, accuracy and precision were investigated by a combined experiment in the concentration range of 0.0026 -0.0255 μg/ml (for both ramipril and hydrochlorothiazide). Linear regression parameters were calculated.According to the obtained regression equation, the "found" value of the peak areas was calculated according to the formula: where s -the value of the peak areas, calculated according to the regression equation; C i -analyte concentration in the corresponding solution for linearity research, μg/ml; a -slope of the regression line; b -y-intercept.
The calculation of the normalised value of the slope of the regression line (as a percentage of the signal of the equipment corresponding to the nominal concentration of the analyte in the solution) was carried out according to the formula: where S 0 -the area of the analyte peak in the comparison solution; C 0 -analyte concentration in the comparison solution (0.01 μg/ml); C nom -the analyte concentration equivalent to the smallest content criterion in the washing solution (0.01 μg/ml).
The found analyte concentration in model solutions was calculated using the formula: where S i -the analyte area in the corresponding model solution.
The ratio "Found"/"Put" was calculated according to the formula: where C t -the given concentration in model solutions.
Chromatographic results and calculations for ramipril are given in Table 1, for hydrochlorothiazide -in Table 2.The graph of the dependence of the response of the signal on the concentration of ramipril in model solutions is shown in Fig. 3, hydrochlorothiazide -in Fig. 4. For ramipril, the correlation coefficient of the calibration line was 0.99986, which satisfies the acceptance criterion (not less than 0.995).The ratio of the slope of the regression line to the nominal concentration of ramipril was 4.041 %, which satisfies the acceptance criterion (no more than 5.0 %).The residuals are randomly scattered around zero.That is, the method was linear in the studied range.For hydrochlorothiazide, the correlation coefficient of the calibration line was 0.99961, which satisfies the acceptance criterion (not less than 0.995).The ratio of the slope of the regression line to the nominal concentration of hydrochlorothiazide was 1.198 %, which satisfies the acceptance criterion (no more than 5.0 %).The residuals are randomly scattered around zero.That is, the method is linear in the studied range.Individual values of ramipril recovery The average value of the recovery rate was 100.0 %, which satisfies the acceptance criterion (95.0-105.0%).The accuracy of the method was sufficient.The relative standard deviation of the recovery of ramipril was 2.191 %, which satisfies the acceptance criterion (not more than 5.0 %).The relative standard deviation of the recovery of hydrochlorothiazide was 1.505 %, which satisfies the acceptance criterion (not more than 5.0 %).That is, the precision of the method was sufficient.The limit of detection (DL) and the limit of quantification (QL) were calculated based on the parameters of the calibration line in the concentration range (0.0026-0.0128 μg/ml) according to the formula: where S a -the deviation of the slope of the regression equation.
The results of the calculations are given in Table 3.
To confirm the QL, the signal-to-noise ratio, the degree of recovery, and the RSD between the degrees of recovery were calculated for model solution M1.Chromatographic results and calculations for ramipril are given in Table 4, for hydrochlorothiazide -in Table 5. QL of ramipril was confirmed at the concentration level of 0.0026 μg/ml, which was 25 % of the smallest criterion for the content of ramipril in the washing solution.The calculated value was almost three times less than the confirmed concentration; that is, at the lower limit of the studied range, the method was reproduced with a sufficient level of reliability.QL of hydrochlorothiazide was confirmed at the concentration level of 0.0026 μg/ml, which was 25 % of the smallest criterion for the content of hydrochlorothiazide in the washing solution.The calculated value was half the confirmed concentration; that is, at the lower limit of the studied range, the method was reproduced with a sufficient level of reliability.

2. The method performed on Waters Xevo TQD ACQUITY MS-detector
We investigated the possibility of reproducing the methods on another equipment (a liquid chromatograph with a Waters Xevo TQD ACQUITY MS-detector).Typical chromatograms of the solvent and reference solution for ramipril are shown in Fig. 5, for hydrochlorothiazidein Fig. 6.
The chromatogram of the solvent did not reveal peaks that could interfere with the peak of ramipril or hydrochlorothiazide.Linearity, accuracy and precision were investigated by a combined experiment in the concentration range of ramipril 0.0026-0.0250μg/ml and hydrochlorothiazide 0.0026-0.0252μg/ml.Chromatographic results and calculations for ramipril are shown in Table 6, for hydrochlorothiazide -in Table 7.The graph of the dependence of the response of the signal on the concentration of ramipril in model solutions is shown in Fig. 7, hydrochlorothiazide -in Fig. 8.For ramipril, the correlation coefficient of the calibration line was 0.99983, which satisfies the acceptance criterion (at least 0.995).The ratio of the slope of the regression line to the nominal concentration of hydrochlorothiazide was 1.121 %, which the acceptance criterion (no more than 5.0 %).For hydrochlorothiazide, the correlation coefficient of the calibration line was 0.99954, which satisfies the acceptance criterion (not less than 0.995).The ratio of the slope of the regression equation to the nominal concentration of hydrochlorothiazide was 1.567 %, which satisfies the acceptance criterion (no more than 5.0 %).The residuals were randomly scattered around zero.That is, the method was linear in the studied range.
Individual values of ramipril recovery were in the range of 97.1-101.4%, which satisfies the criterion of acceptability (90.0-110.0%).The average value of the degree of recovery was 99.4 %, which satisfies the acceptance criterion (95.0-105.0%).Individual values of hydrochlorothiazide recovery were in the range of 97.6-103.5 %, which satisfies the acceptance criterion (90.0-110.0%).The average value of the degree of recovery was 99.9 %, which satisfies the acceptance criterion (95.0-105.0%).That is, the accuracy of the method was sufficient.The relative standard deviation of the recovery rates of ramipril was 1.275 %, which satisfies the acceptance criterion (not more than 5.0 %).The relative standard deviation of the recovery rates of hydrochlorothiazide was 2.294 %, which satisfies the acceptance criterion (not more than 5.0 %).That is, the precision of the method is sufficient.To confirm QL, the signal-to-noise ratio, the degree of recovery, and the RSD between the degrees of recovery were calculated for model solution M1.Chro-matographic results and calculations for ramipril are given in Table 8, for hydrochlorothiazide -in Table 9. QL at the concentration level of 0.0026 μg/ml was confirmed.

Discussion of research results
Methods UPLC-MS detection began to gain more and more The use of a highly selective detector and chromatographic columns allows the development of rapid methods using a small volume of low-toxicity mobile phases (a mixture of 0.1 % formic acid and acetonitrile).Ramipril is non-sulfhydryl ACE inhibitor with antihypertensive effect.Hydrochlorothiazide is a diuretic that is often used to treat hypertension and oedema caused by fluid retention.Both ramipril and hydrochlorothiazide are resistant to heating in solution [5,6], so it does not destroy when cleaning the equipment and can be determined in washing solutions.In the scientific literature, analytical methods for the determination of ramipril and hydrochlorothiazide in dosage forms are presented; however, no analytical method for controlling the cleaning of equipment is described.Previously, our scientific group had developed an HPLC method for the determination of ramipril in tablets, and we already had some developments in this direction, so we were interested in developing methods for monitoring the cleaning of equipment, which is important for routine pharmaceutical analysis [27].The use of the MS detector was justified in this case for the purposes that we have planned.We have proposed column C18 Kinetex 2.1 mm×30 mm×1.7 μm) and mobile phase 0.1 % solution of formic acid in deionised water -acetonitrile (73:27) with flow rate 0.4 ml/min and column temperature 45 °C for determination of ramipril and mobile phase 0.1 % solution of formic acid in deionised water -acetonitrile (91.5:8.5) with flow rate 0.35 ml/min and column temperature 40 °C for determination of hydrochlorothiazide.In order to study the robustness in more detail, we performed validation on 2 detectors from different manufacturers (Agilent 6420 MS-detector and Waters Xevo TQD ACQUITY MS-detector).The proposed analytical methods have sufficient linearity, accuracy and precision.The sensitivity of the techniques was confirmed at the level of 0.0026 μg/ml.The techniques can be used in the concentration range of 0.0026-0.0255μg/ml.We have fully presented the results of validation with calculation formulas, described its procedure and showed a new application of the technique for completely different purposes than determination in dosage forms, which is also no less important and cannot be ignored.
Practical Relevance.The proposed analytical methods can be used to determine the the residual quantities of ramipril and hydrochlorothiazide for controlling the cleaning of equipment.
Study limitations.The proposed methods can not be used to determine ramipril and hydrochlorothiazide in one single run.
Prospects for further research.The next research stage is planned to investigate the problems in the method development of the residual quantities of ramipril and hydrochlorothiazide for controlling the cleaning of equipment in the presence of other non-sulfhydryl ACE inhibitors.

Conclusions
Analytical methods for determining ramipril and hydrochlorothiazide in washing waters by UPLC-MS were developed and validated.The proposed analytical methods have sufficient linearity, accuracy, and precision.The developed methods are suitable for determining the residual amounts of ramipril and hydrochlorothiazide in the washing solution in the concentration range of 0.0026-0.0255μg/ml.The methods can be used to control equipment cleaning for mono preparations and combined preparations.

Fig. 3 .Fig. 4 .
Fig. 3. Graph of dependence of signal response on ramipril concentration: a -regression graph; b -residual graph a b

Fig. 7 .Fig. 8 .
Fig. 7. Graph of dependence of signal response on ramipril concentration: a -regression graph; b -residual graph a b

Table 1
Study results for ramipril

Table 3
Calculation results of DL and QL a DL, μg/ml QL, μg/ml

Table 4
Confirmation of QL of ramipril

Table 5
Confirmation of QL of hydrochlorothiazide

Table 6
Study results for ramiprilSolution C t , μg/ml S i RSD S s i S i -s i C m , μg/ml Recovery

Table 7
Study results for hydrochlorothiazideSolution C t , μg/ml S i RSD S s i S i -s i C m , μg/ml Recovery

Table 8
Confirmation of QL of ramipril

Table 9
Confirmation of QL of hydrochlorothiazide