Chromatographic Conditions For Analysis Research Articles

Метод количественного определения кверцетина с помощью ВЭЖХ-МС/МС

INTRODUCTION: Human breast cancer resistance protein (BCRP, ABCG2) is a transport protein of ABC superfamily of transporters that uses ATP energy of for its work. BCRP plays an important role in the pharmacokinetics of drugs, therefore all new drugs are recommended to be tested for belonging to its substrates, inducers and inhibitors. The influence of substances on the activity of this transport protein in vitro is evaluated by a change of transmembrane transfer of its substrates, one of which being quercetin. This, in turn, requires the development and validation of a method for its quantitative analysis in an appropriate matrix. AIM: To develop a method for the quantitative determination of quercetin using high performance liquid chromatography (HPLC) with tandem mass selective detection (MS/MS) with full validation. MATERIALS AND METHODS: The method was developed on Ultimate 3000 HPLC equipped with TSQ Fortis (Thermo Fisher, USA) MS/MS detector. The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm*100 mm 5μm, 100 A, Selectra C18 Guard Cartridges SLC-18 GDC46-5UM. The analysis time was 11 min at separation temperature 35°С, flow rate 0.5 ml/min, injected sample volume 5 μl. Gradient elution regime was used: on 0th minute, the ratio of 0.1% formic acid solution and acetonitrile was 70% and 30%; 0.3 min — 30% and 70%; 4 min — 1% and 99%; 9 min — 70% and 30%. Quercetin retention time was 3.91 minutes. Detection conditions: negative ionization mode, 301 m/z → 150.9 m/z with collision energy 22 V, 301 m/z →178.9 m/z with collision energy 17 V, source fragmentation 5 V, electrospray voltage 3 000 V, CID gas pressure 1 mTorr, Sheath gas 50 Arb, Aux gas 10 Arb, Sweep gas 10 Arb, ion transfer tube temperature 300°C, vaporizer temperature 350°C. The matrix was transport medium after incubation for 3 hours with Caco-2 cells overexpressing BCRP. In this medium, the transport of quercetin through the cell monolayer was further evaluated in vitro. Precipitation of protein and isolation of quercetin from the transport medium was realized by a mixture of water and acetonitrile in a ratio of 1:1. RESULTS: The developed method was validated by the following parameters: selectivity, linearity, accuracy, precision, limit of quantification, sample transfer, matrix effect, sample stability. The analytical range of the technique was 5–500 nmol/l. In this case, the correlation coefficient was more than 0.99. The limit of detection and the lower limit of quantification of quercetin (LLQQ) were 1 and 5 nmol/l, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for other concentrations. The analyte demonstrated stability in triple freeze-defreeze cycle at -80°C, in storage at -80°C for 60 days, after sample preparation and being kept in the autosampler for 24 hours. There was no sample transfer and no matrix effect. CONCLUSION: A method for the quantitative determination of quercetin using HPLC-MS/MS in a transport medium was developed and fully validated.

Novel Bispidine-Monoterpene Conjugates-Synthesis and Application as Ligands for the Catalytic Ethylation of Chalcones.

A number of new chiral bispidines containing monoterpenoid fragments have been obtained. The bispidines were studied as ligands for Ni-catalyzed addition of diethylzinc to chalcones. The conditions for chromatographic analysis by HPLC-UV were developed, in which the peaks of the enantiomers of all synthesized chiral products were separated, which made it possible to determine the enantiomeric excess of the resulting mixture. It was demonstrated that bispidine-monoterpenoid conjugates can be used as the ligands for diethylzinc addition to chalcone C=C double bond but not as inducers of chirality. Besides products of ethylation, formation of products of formal hydrogenation of the chalcone C=C double bond was observed in all cases. Note, that this formation of hydrogenation products in significant amounts in the presence of such catalytic systems was found for the first time. A tentative scheme explaining the formation of all products was proposed.

Development and validation of the method for the quantitative determination of methotrexate in a transport medium by HPLC-MS/MS

Relevance. BCRP is an efflux transporter protein that plays an important role in the pharmacokinetics of a wide range of drugs. The BCRP activity in vitro experiments is assessed by the transport of transporter protein substrates (methotrexate, etc.) across the bilipid membrane of cells overexpressingBCRP, for example, Caco-2 cells. The aim is to develop and validate a method for the quantitative determination of the BCRP substrate, methotrexate, in the transport medium of Caco-2 cells by HPLC-MS/MS. Methods. The work was performed on an Ultimate 3000 HPLC chromatograph (ThermoFisher, USA) with a TSQ Fortis tandem mass-selective detector (ThermoFisher, USA). The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm * 100 mm 5um, 100A, Selectra C18 Guard Cartridges SLC-18GDC46-5UM, separation temperature 35 °С, flow rate 0.3 ml/min, injected sample volume - 2 μl, analysis time - 10 min. Used a gradient elution: the ratio of the solution of 0.1 % formic acid and acetonitrile was at 0 min 75 and 25 %; 0.4 min 60 and 40 %; 6 minutes 20 and 80 %; 8 minutes 75 and 25 %. Under these conditions, the retention time of methotrexate is 3.11 minutes. Detection conditions: methotrexate - positive ionization mode, 455.15 m / z → 308.125 m / z, collision energy 22.99 V, source fragmentation 5, CID gas pressure 2 mTorr. The extraction of methotrexate from the transport medium (Hanks solution with 25 mM Hepes and 1% dimethyl sulfoxide) after incubation with Caco-2 cells for 3 h was carried out with a mixture of methanol + water in a ratio of 1: 1. Results. The developed method was validated according to the following parameters: selectivity, linearity, accuracy, precision, limit of quantitative determination, sample transfer, sample stability. The confirmed analytical range of the method was 60 -10,000 nmol / L in the transport medium. Conclusions: a method for the quantitative determination of methotrexate in the transport medium of Caco-2 cells by HPLC-MS / MS was developed and validated.

Use of High Performance Liquid Chromatography in the Analysis of a New Substance VMA-10-18

Introduction. Quinazoline derivatives have a wide range of pharmacological properties, which makes this group quite unique among other classes of heterocyclic compounds. Substance VMA-10-18, which has cerebrovasodilating, antidepressant, anxiolytic and nootropic properties, may become a promising new drug. In this regard, an urgent task is to develop methods for standardizing this substance.Aim. Development of a method for the quantitation of related impurities of a new biologically active substance VMA-10-18 (Quinazophene) by HPLC with subsequent statistical processing of the results.Materials and methods. To develop the conditions for chromatographic analysis, was used a highly purified substance 3-[2-(4-methoxyphenylamino)-2-oxoethyl]-quinazolin-4(3H)-one, as well as its related impurities: impurity I (unsubstituted quinazolin-4(3H)-one) and impurity II (4-methoxychloroacetanilide). Test solutions were prepared using volumetric glassware of accuracy class 1. Ethyl alcohol 95 % was used as a solvent. Chromatography was performed using a Dionex UltiMate 3000 system (Dionex, United States) with a spectrophotometric detector. The analysis was carried out at a wavelength of 231 nm. Data collection and processing was carried out using the Chromeleon v.7 system. A mixture of acetonitrile and orthophosphoric acid was used as a mobile phase. The analysis was performed in an isocratic mode. The validation of the developed method was carried out taking into account the requirements of the State Pharmacopeia of Russian Federation XIV edition and the recommendations of the ICH.Results and discussion. The optimal conditions for chromatography of the VMA-10-18 substance and its impurities have been developed. It was found that for a clear separation of the peaks of the substance and impurities among themselves, the mobile phase should contain acetonitrile and orthophosphoric acid in a ratio of 80 : 20. The specificity of the method was determined by chromatography of ethyl alcohol in order to exclude its influence on the analysis results. The linearity and correctness of the method were determined at 7 levels of concentration of impurities of the substance. The correlation coefficient has exceeded 0.99. Also, the free term of the linear dependence equation (a) for both impurities was less than its confidence interval (Δа), which proves the absence of a systematic error of the method. When determining the "Convergence" indicator, the calculated relative standard deviation did not exceed 2 %. When determining the intralaboratory precision, Student's t-test and Fisher's F-test were calculated. Both indicators met the stated requirements.Conclusion. A method for the quantitative determination of impurities in the VMA-10-18 substance by HPLC has been developed and validated.

Determination of Zoledronic Acid and Creatinine by Hydrophilic Chromatography

We propose a version of the simultaneous determination of zoledronic acid and creatinine in serum and plasma samples by hydrophilic chromatography (HILIC). Factors affecting the determination of zoledronic acid were found: the composition and concentration of the buffer solution, the nature of the stationary phase, the presence of tartaric acid in the eluent as a complexing agent to prevent the complexation of zoledronic acid with metal ions. We determined the linear concentration ranges for zoledronic acid and creatinine and their limits of detection. The following conditions for chromatographic analysis were found: amide stationary phase; a 5 mM phosphate buffer solution, 2 mM tartaric acid (pH 7.0)/CH3CN; gradient 90–70% of CH3CN.

Development of HPLC method for quantitative determination of epimidin - new perspective АPhI with anticonvulsive activity

The aim. Development of optimal, high-precision and reproducible methods for quantitative determination of the main substance in the substance Epimidin - 1-(4-methoxyphenyl)-5-[2-[4-(4-methoxyphenyl)piperazin-1-yl]-2-oxo-ethyl]pyrazolo[3,4-d]pyrimidin-4-one by high performance liquid chromatography.Materials and methods. High performance liquid chromatography (HPLC) was performed using a ShimadzuNexeraX2 LC-30AD system (Shimadzu, Japan) equipped with a SPD-M20A diode array detector (DAD). ACE C18 column, size 250 x 4.6 mm, YMC with pre-column, particle size 5 μm, filled with octylsilyl silica gel for chromatography P. During the work acetonitrile and trifluoroacetic acid of HPLC class (Sigma-AldrichGmbH, Switzerland) were used, other chemicals and solvents were of analytical grade. In the study an analytical ware class A were used that meet the requirements of SPhU.Results. The following optimal conditions of chromatographic distribution are established: column C18 (250*4.6 mm); the speed of the mobile phase 1 ml / min; column thermostat temperature 35 °С; injection volume 10 μl; mobile phase A - 0.1 % trifluoroacetic acid; mobile phase B - acetonitrile P; the detection wavelength is 270 nm, the retention time of the test compound is 7.22 minutes. The performance of the column was determined for its main indicators, such as the number of theoretical plates (more than 25410) and the coefficient of symmetry (about 1.00). The technique was tested for the influence of various factors, such as flow rate, mobile phase composition and column thermostat temperature. It was established that the influence of these factors is insignificant and does not affect the results obtained by this method. The method was validated in accordance with the recommendations of SPhU on the parameters of specificity, linearity, correctness, precision, robustness (stability).Conclusions. For the first time, a high-precision and reproducible method for quantitative determination of the main substance in the substance Epimidin with anticonvulsant activity by high-performance liquid chromatography was developed. Conditions for chromatographic analysis (HPLC) were standardized. The requirements for the test “System suitability test criteria for chromatographic methods” are set. Statistical processing of the experimental results shows that the relative uncertainty of the average result is within acceptable limits. The correctness of the method was confirmed by validation studies. The developed technique will be used for pharmaceutical development and standardization of dosage form

Development and validation of GLC/MS-procedure of doxylamine quantitative determination

Doxylamine is a hypnotic medicine used for treatment of minor sleep disorders and is a frequent cause of poisoning.Aim. To develop GLC/MS-procedure of doxylamine quantification and carry out step-by-step validation ofthe developed procedure in the variants of the method of calibration curve, method of standard and method of additions.Results. The chromatographic conditions have been chosen for doxylamine determination by the method of GLC with mass-spectrometry detection with temperature program changing during the analysis from 70 °C to 320 °C. Retention time for doxylamine is 14.53 min. To prove the possibility of the proposed procedure application in further analysis its validation has been carried out in the variants of the method of calibration curve, method of standard and method of additions. Such validation parameters as in process stability, linearity, accuracy, precision and limit of determination have been estimated by model solutions.Experimental part. Conditions of chromatographic analysis: Agilent 6890N/5973N/7683; НР-5MS ∅0.25 mm × 30 m, 0.25 μm; DB-17MS ∅0.25 mm × 30 m, 0.15 μm; columns are connected sequentially through Deans switch; thermostat – 70 CºС (2 min), 45 ºС/min to 210 ºС, 6 ºС/min to 320 ºС (12.56 min); transfer line – 280 ºС; ion source – 230 ºС; quadrupole – 150 ºС; electron impact, 70eV; 40 – 750 m/z; injector – 250 ºС; splitless mode; inlet carrier gas (helium) pressure: 1st column – 26.06 psi, 2nd column – 19.30 psi.Conclusions. New procedure of doxylamine quantitative determination by the method of GLC/MS has been developed. Its validation has been carried out and acceptability for application has been shown.

Development of method of quantitative determination of cardiazol substance with using highly efficient liquid chromatography

The aim. Development of methods of quantitative determination of Cardiazol substance using high-performance liquid chromatography.Materials and methods. The method of high-performance liquid chromato-graphy (HPLC) was to determine of quantitative determination of Cardiazol substance ([3-Allil-4-(41-methoxyphenyl)-3H-thiazole-2-ylidene]-(32-trifluoro-methylphenyl)amine hydrobromide) using Shimadzu Nexera X2 LC -30AD (Shimad-zu, Japan). The acetonitrile of the HPLC grade (Sigma-Aldrich GmbH, Switzerland) was used in the work and other chemicals and solvents were of analytical grade. The test substance was diluted in acetonitrile at a final concentration of 400 μg/ml.Results and discussion. The method of quantitative determination of Cardiazol substance with the help of highly effective liquid chromatography is developed. The developed conditions of testing are selected experimentally. The following optimal conditions for the chromatographic distribution were found: column C8 (250 *4.6 mm; speed of the mobile phase 1 ml/min; thermostat temperature of the column 35 ° C; detecting wavelength 300 nm; holding time of the test compound is 13.9 min. Suitability of determination methods.The following optimal conditions for chromatographic separation were revealed: a C8 column (250*4.6 mm, a mobile phase speed of 1 ml/min, a column thermostat temperature of 35 °C, a detection wavelength of 300 nm. Under the proposed conditions, the retention time of the tested component is 13.9 minutes. The performance of the column was determined for its main indicators, such as the theoretical number of plates (over 65000) and the coefficient of symmetry (about 1.00). The method of quantitative determination was tested in accordance with the recommendations of the Ukrainian and European Pharmacopoeia. The proposed method meets all requirements. The method has been tested for the effects of various factors such as flow rate, the composition of the mobile phase and the temperature of the column thermostat. It is established that the influence of these factors is insignificant and does not affect the results obtained by this method.Conclusions. An analytical method for quantitative determination of Сardiazole substance with cardioprotective action has been developed on the basis of the high-performance liquid chromatography method. The conditions for chromatographic analysis (HPLC) were standardized. Requirements for the test Checking the suitability of the chromatographic system were established. The statistical processing of the results of the experiment shows that the relative uncertainty of the average result was within the permissible limits. The developed method for the determination of Сardiazole will be used for further study of substance as a component of various dosage forms

METHODICAL SUPPORT FOR CONTROL OVER THE CONCENTRATION OF PERSISTENT ORGANIC POLLUTANT HEXACHLOROBENZENE IN BLOOD

Introduction. Persistent organic pollutants (POP) are extremely dangerous compounds contaminating the environment and exerting the negative impact on human health. Organochlorine pesticides, including hexachlorobenzene, are a specific group among POP. Our research goal is methodological support for the hexachlorobenzene content control in human biological environments. Material and methods. We developed highly both sensitive and selective procedure for determining hexachlorobenzene in blood. Hexachlorobenzene determination in blood is based on the application of capillary gas chromatography with linear programming of a column temperature, electron capture detector (ECD), and extraction with an organic solvent at a sample preparation stage. When implementing the developed methodology, the following indices were evaluated: accuracy and error characteristics of the analysis results. The lower limit of hexachlorobenzene quantification (LOQ) in blood amounted to 0.000024 µg/cm3, with the range of detectable concentrations from 0.00015 to 0.005 (determination method error ≥ 20%), which allows to adequately diagnose chemical burden with a highly toxic persistent organic pollutant in blood. Results. The results of quantitative chemical analysis of hexachlorobenzene contents in blood had the following quality indices: precision and correctness amounted to 15.621%; repeatability - 3.45%; reproducibility - 4.65%. In the process of testing the method, concentrations of hexachlorobenzene in the range of 0.0003 ± 0.0001 ÷ 0.0007 ± 0.0001 μg/cm3 were detected in the blood of the examined group. Discussion. The high efficiency of gas chromatography determination of hexachlorobenzene in blood was achieved via selection of optimal conditions for chromatographic analysis: capillary column HP-1- 35m∙0.32mm∙0.25µm with linear programming of column temperature, carrier gas (nitrogen), electron capture detector (ECD). The developed method of the preparing a blood sample for analysis, including liquid extraction with toluene, protein denaturation by centrifuging the biological medium at 7000 rpm for 15 min at pH of 6-7 in combination with gas chromatographic analysis and an electron capture detector (ECD) made it possible to complete extraction of hexachlorobenzene from blood in an amount of 99.7%.

Hazards associated with municipal waste storage. Vol. I

Depositing municipal waste in a responsible and controlled manner in landfills allows their decomposition to stabilized material. However, there are many environmental risks during operation and stabilization after landfill closure. These include: dusts, odors, potential fires associated with the presence of landfill gas and it is microbiological hazards and leachates. The latter are also generated many years after closure and reclamation of the landfill. In the event of a leak trough the anti-filtration shutter, toxic compounds found in the leachates can migrate and contaminate to groundwater. The article presents the quantitative and qualitative analysis of leachates in the final operational period of the landfill and after its closure. In both cases, the chromatographic analysis was carried out using the same conditions, i.e. solvent, extraction time, chromatograph and conditions for chromatographic analysis of samples. Physical and chemical leachate examinations were performed on the basis of valid standards. Their results show that the waste deposit is subject to increasingly advanced biodegradation processes of organic compounds. The values of such indicators as COD and BZT5 are decreasing. However, they remain quite significant, which indicates the presence of hard-to decompose and newly compounds in the leachates.

Generation of urinary steroid profiles in patients with adrenal incidentaloma using gas chromatography–mass spectrometry

A method is developed for the determination of steroid hormones in urine using gas chromatography with mass-spectrometric detection. Sample preparation procedure and the conditions of chromatographic analysis were optimized, and characteristic urinary steroid profiles of patients with adrenocortical cancer were obtained. Criteria for the evaluation of the deficiency of 21-hydroxylase and 11β-hydroxylase in patients with adrenal incidentaloma are proposed.

DENSITOMETRIC ANALYSIS OF SELECTED FLUOROQUINOLONES

Fluoroquinolones are the group of very important synthetic drugs, especially in the treatment of urinary tracks infections. The aim of this work was to find the best chromatographic conditions for analysis of ofloxacin and pefloxacin, as well as using the chromatograms for their densitometric analysis in drugs. The calibration curves were prepared for that purpose and the curves were described by mathematic functions. It permitted calculating the contents of fluoroquinolones in drugs investigated. The densitometric scanning was carried out for ofloxacin and pefloxacin. The quite similar values of quantity of spotted and determined ofloxacin and pefloxacin were stated.

Simultaneous chromatographic identification of a few components in mixtures based on a correlation between their absolute or relative retention times under different conditions

A correlation was established between the absolute and relative retention parameters of various compounds under different conditions of chromatographic analysis (isothermal and temperature programming conditions in gas chromatography or isocratic and gradient elution in high-performance liquid chromatography). This correlation is described by the linear regression equations t R (II) = = at R (I) + b with a high degree of accuracy. This property of chromatographic retention parameters allowed us not only to recalculate the values of these parameters determined under different conditions, but also to propose an algorithm for the simultaneous identification of a few components in complex mixtures with the use of published data on retention parameters found under other conditions (even presented as the drawings of chromatograms).

Solid-phase extraction of nitro-PAH from aquatic samples and its separation by reverse-phase capillary liquid chromatography

In this work, an analytical methodology to extract 1-nitronaphthalene, 2-nitrofluorene, 9-nitroanthracene, 3-nitrofluoranthene, and 1-nitropyrene aquatic samples is proposed. Capillary liquid chromatography with on-column UV detection (µLC-UV) was employed to separate and quantitate all five nitrated polycyclic aromatic hydrocarbons (nitro-PAH) extracted from water samples. Two different extraction techniques were tested and compared: liquid-liquid extraction (LLE) and solid phase extraction (SPE) with two different sorbents, octadecylsilica (C18) and styrene divinylbenzene (XAD-2) particles. The elution of analytes was tested with both acetonitrile and methylene chloride. The best recovery results were obtained for SPE-C18 extraction with the methylene chloride with values ranging from 76 to 97% with RSD values smaller than 4%. The chromatographic conditions for analysis was carried out using a 250 × 0.3 mm i.d. packed capillary column with 5 µm C18 particles. The elution was isocratic with 65% acetonitrile in water (v/v) as mobile phase with a flow rate of 7 µL min-1. The detection limits due to on-column UV detection (ranging ~3-30 mmol L-1) are limiting for environmental applications, however, they are suited for toxicological studies.

Improvement in the Multiresidue Liquid Chromatographic Analysis of Residues of Mono- and Dibasic Penicillins in Bovine Muscle Tissues

A recently developed multiresidue method using gradient liquid chromatographic conditions for analysis of residues of beta-lactam drugs in bovine muscle tissues after precolumn derivatization with acetic anhydride was modified to permit isocratic analysis. The modification included replacing the acylating reagent, acetic anhydride, with benzoic anhydride and increasing sample size for extraction from 2 to 3 g. The modifications have reduced analysis time per sample from > 1 h to about 22 min without loss in detection sensitivities for the beta-lactams, resulting in a significant increase in the number of samples that can be analyzed in 1 day from 1 to 8. The modifications make the multiresidue LC method more suitable than the original method for use in regulatory programs for routine analysis of these veterinary drug residues in bovine muscle tissues.

Application of gas chromatography in analysis of industrial wastes

One of the important ecological problems is how to monitor the content of volatile substances in the air of industrial buildings and installations, as thi~" is a major factor in providing safe working conditions; another important problem is the determination of the toxicity class of industrial wastes. Gas chromatography is widely used in solving these problems. For the Novokuibyshevsk Insulating Materials Plant and the Plastik Production Association (in the city of Syzran'), a procedure has been developed for the determination of ethyl and butyl acetate in workplace atmospheres. The method now used to determine acetates -- a colorimetric method -- does not meet the modern requirements of analytical chemistry with respect to accuracy and speed [1]. Gas chromatography, with preconcentration of the acetates in ethanol, is also inadequate in accuracy, owing to the volatility of the absorbing solution [2]. In developing a procedure for this purpose, we selected a method of preconcentrating the analyte substances and the conditions for chromatographic analysis of the concentrate. We chose to absorb the acetates in water, as this is the most easily available absorbing agent, and it does not require any special storage conditions. For the chromatographic analysis, conditions were selected so as to meet the requirement of quantitative determination of individual acetates in aqueous solution. The internal standard method was preferred for quantitative interpretation of the chromatograms [3]. As a comparison sample we took butanol, the solubility of which in water is higher than that of the acetates [4]. In view of the appreciable polarity of oxygen-containing compounds, we selected as a stationary phase XE-60, supported in amount of 5 % by weight on Inerton AW. The analysis was performed in a Khrom-5 chromatograph with flame ionization detectors and a packed column with a length of 2.5 m; the determinations were performed at 65-70~ Complete separation of each of the acetates and the butanol comparison standard was achieved in 6-8 rain (Fig. 1). The water (absorbing agent) does not affect the separation.

Principles of the retention of 1,4-dihydropyridine derivatives and selection of the conditions of chromatographic analysis based on them

Principles of the retention of 1,4-dihydropyridine derivatives and selection of the conditions of chromatographic analysis based on them