Relative Retention Times Research Articles

QSRR model for identification and screening of emerging pollutants based on artificial intelligence algorithms

ABSTRACT It is urgent to identify and screen emerging pollutants (EPs), which have caused great harm to human health and the environment. In their detection of liquid chromatography-mass spectrometry (LC-MS), the quantitative structure–retention relationship (QSRR) model is simple and efficient to predict the retention behavior of compounds. In the present work, we collected more data with the relative retention time (RRT) of 490 compounds, and filtered the molecular descriptors with lasso regression and multiple linear regression analysis. Then ten important molecular descriptors were screened and applied the QSRR models with deep neural network (DNN), multiple linear regression (MLR), and support vector machine. The DNN model had the best accuracy which the correlation coefficient R2 reached 0.913. Finally, we determined the applicability of the DNN model through a descriptor value range to assist in the identification and screening of EPs.

Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography.

Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Hydrophilic interaction liquid chromatography (HILIC) is a mode of choice for separation of intact glycopeptides, which are inadequately resolved by reversed phase chromatography. In this work, we propose an easy-to-use model to predict retention time windows of glycopeptides in HILIC. We constructed this model based on the parameters derived from chromatographic separation of six differently glycosylated peptides obtained from tryptic digests of three plasma proteins: haptoglobin, hemopexin, and sex hormone-binding globulin. We calculated relative retention times of different glycoforms attached to the same peptide to the bi-antennary form and showed that the character of the peptide moiety did not significantly change the relative retention time differences between the glycoforms. To challenge the model, we assessed chromatographic behavior of fetuin glycopeptides experimentally, and their retention times all fell within the calculated retention time windows, which suggests that the retention time window prediction model in HILIC is sufficiently accurate. Relative retention time windows provide complementary information to mass spectrometric data, and we consider them useful for reliable determination of protein glycosylation in a site-specific manner.

Preparation of a zeolite imidazole skeleton-silica hybrid monolithic column for amino acid analysis via capillary electrochromatography.

We developed a novel, convenient and low-cost one-pot strategy for preparing a zeolitic imidazolate framework-8 (ZIF-8)-silica hybrid monolithic column by adding ZIF-8 directly to a polymer solution of the silica matrix. The simulated stationary phase and monolithic column prepared under optimal conditions were characterized using X-ray diffraction, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis nitrogen physisorption and zeta potential. The results obtained confirmed the successful introduction of ZIF-8 into the silica monolithic column, and the prepared monolithic column exhibited good permeability and physicochemical stability. A capillary electrochromatography method was developed based on a ZIF-8-silica hybrid monolithic column through which 15 mixed amino acids, 4 neutral compounds, 4 nipagin esters and 2 chlorinated fungicides were separated in 14, 5, 7 and 6min, respectively, under optimal conditions. The relative standard deviations retention times and column efficiencies in run-to-run, day-to-day and column-to-column varied in the ranges of 1.90%-2.21%, 2.13%-2.51% and 3.08%-6.65%, respectively, which demonstrated that ZIF-8-silica hybrid monolithic column exhibited satisfactory reproducibility and stability. The incorporation of ZIF-8 into a silica monolithic column is a promising method for preparing novel monolithic columns composed of a metal-organic framework.

Quality control of cannabis inflorescence and oil products: Response factors for the cost-efficient determination of ten cannabinoids by HPLC

The quality control of medicinal cannabis should include quantification of as many cannabinoids as practicable in a routine analytical lab, to accurately reflect the quality of the product. However, the cost and availability of some cannabinoid standards is an impediment to their routine use. This work seeks to overcome this obstacle by analysing samples using relative retention times (RRT) and relative response factors (RRF), relative to CBD and CBDA reference standards which are readily available. A high-performance liquid chromatography-photodiode array method was developed to quantify ten cannabinoids (Δ9-THC, Δ8-THC, THCA-A, CBN, CBD, CDBA, CBC, CBDV, CBG, and CBGA) in dried cannabis inflorescence and cannabis oil. This method was validated according to ICH guidelines. The proposed method has detection limits ranging from 20 to 78 µg/g, which provided sufficient sensitivity for the panel of cannabinoids. Non-cannabinoid surrogate matrices were used for spike recovery studies to determine method accuracy – analyte recoveries for the inflorescence and oil ranged from 90.1 to 109.3% (inflorescence mean, 100.9%; oil mean, 99.6%). The RRT and RRF values determined independently by three analysts were comparable, indicating the method is robust. The validity of analysis using RRT and RRF was further confirmed by testing six inflorescence samples, as it was found that concentrations above the order of magnitude of the LoQ agreed satisfactorily (range, 95.0 to 111.9%; mean, 100.0%) with the concentrations obtained through the conventional approach of multipoint calibration using pure standards. The proposed method is therefore suitable for the rapid and simple determination of a panel of ten cannabinoids without having to repeatedly purchase every expensive pure standard. Accordingly, analysts in the medicinal cannabis field may explore the use of RRF and RRT for their methods and instruments.

On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii

Aim: To reveal whether methanolic extract and essential oil from Origanum vulgare subsp. hirtum in doses causing even low levels of mortality in aphids, would have harmful effects on plants-genotoxic, mutagenic and/or DNA damaging. Materials and methods: Aerial parts of Origanum vulgare ssp. hirtum from the ex-situ collection of IBER, BAS during flowering were collected. Extraction and isolation procedures, as well as GC/MS analysis of essential oil and methanolic extract were performed by standard protocols. The components were identified by comparing their relative retention times to the retention times of authentic standards, and with mass spectra with the NIST. Test system: Chlamydomonas reinhardtii strain 137 C+ (WT). Endpoints: “clonal” assay, the test of “visible mutations”, constant field gel electrophoresis. Statistics: GraphPad Prism version 6.04 (San Diego, USA) and One-way Analysis of Variance ANOVA with multiple comparisons using the Tukey method. Results: A good correlation was observed between chemical composition of essential oil and methanolic extract, and their mode of action. Our genotoxic and double strand breaks results demonstrated mild genotoxic and statistically non-significant DNA damaging potential of methanolic extract and concentration-dependent well - expressed genotoxic and DNA damaging potential of essential oil. A good relationship between increased double strand breaks levels and decreased survival might be related to one of the main constituents of essential oil, suspected to be carvacrol. No mutagenic effect for ME and EO was found. Conclusion: Well-expressed toxic/genotoxic capacity of essential oil, as well as its capacity to damage DNA inducing double strand breaks, but the absence of mutagenic potential, could be considered as a good reason to recommend Origanum vulgare subsp. hirtum essential oil as a promising candidate for purposes of “green” technologies.

Machine Learning-Based Retention Time Prediction of Trimethylsilyl Derivatives of Metabolites.

In gas chromatography–mass spectrometry-based untargeted metabolomics, metabolites are identified by comparing mass spectra and chromatographic retention time with reference databases or standard materials. In that sense, machine learning has been used to predict the retention time of metabolites lacking reference data. However, the retention time prediction of trimethylsilyl derivatives of metabolites, typically analyzed in untargeted metabolomics using gas chromatography, has been poorly explored. Here, we provide a rationalized framework for machine learning-based retention time prediction of trimethylsilyl derivatives of metabolites in gas chromatography. We compared different machine learning paradigms, in addition to exploring the influence of the computational molecular structure representation to train the prediction models: fingerprint class and fingerprint calculation software. Our study challenged predicted retention time when using chemical ionization and electron impact ionization sources in simulated and real cases, demonstrating a good correct identity ranking capability by machine learning, despite observing a limited false identity filtering power in cases where a spectrum or a monoisotopic mass match to multiple candidates. Specifically, machine learning prediction yielded median absolute and relative retention index (relative retention time) errors of 37.1 retention index units and 2%, respectively. In addition, fingerprint class and fingerprint calculation software, as well as the molecular structural similarity between the training and test or real case sets, showed to be critical modulators of the prediction performance. Finally, we leveraged the structural similarity between the training and test or real case set to determine the probability that the prediction error is below a specific threshold. Overall, our study demonstrates that predicted retention time can provide insights into the true structure of unknown metabolites by ranking from the most to the least plausible molecular identity, and sets the guidelines to assess the confidence in metabolite identification using predicted retention time data.

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification. The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping. Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation. Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

A novel HPLC method developed for the estimation of bempedoic acid and ezetimibe in bempedoic acid and ezetimibe pharmaceutical dosage forms of tablets

A simple, accurate, precise method was developed for the estimation of Bempedoic acid and Ezetimibe in the tablet dosage form of Bempedoic acid and Ezetimibe. Optimized separation was achieved on an Ascentis C18 150x 4.6mm, 5 using mobile phase composition of phosphate buffer pH 4.0 and acetonitrile in the ratio of 700 mL:300 mL (v/v) at a flow rate of 1.0 mL/min; the injection volume is 10 μL and run time 6 minutes in isocratic elution. UV detection was carried out at a wavelength of 230 nm. The temperature was maintained at 30°C. Wellresolved peaks were observed with high numbers of theoretical plates, lower tailing factor and reproducible relative retention time. The method was validated and all the validation parameters were found to be within the acceptable limits.

Isolation and Structural Elucidation of Impurity in Sulfonamide Intermediate of Glyburide

A reproducible isolation method by Reverse Phase (RP) preparative HPLC technique for the isolation of one crucial impurity at 1.65 RRT (Relative Retention Time) in sulfonamide stage of Glyburide API (Active Pharmaceuticals Ingredient) was developed. Preparative chromatography was done on Luna C8, 10µm (250 mm x 21.2mm) preparative HPLC column with acetonitrile: water in 70:30 % v/v proportion as a mobile phase and 8 ml/min as a flow rate. This impurity was detected at 300 nm UV-wavelength maximum. This impurity was isolated from synthesized crude impurity of sulfonamide stage of Glyburide substance by preparative HPLC by injecting 50 mg/ml concentration over 5 ml fixed loop. Isolated impurity was elucidated as N-methyl impurity of sulfonamide intermediate of Glyburide API by means of chromatographic and spectral data. Structural elucidation carried out by spectral data was reviewed. This impurity was analyzed by reverse phase HPLC for purity analysis. A Inertsil C8 (250 x 4.6) mm, 5µ particle size was employed for separation. The mobile phase consisted of Water: Acetonitrile: Methanol in the ratio of 60:15:25 % v/v. The flow rate was set at 1 ml/min. Detection was carried out at 300 nm. 10µL of 2 mg/ml concentration of sample in methanol was injected. The column oven temperature was at 25°C.

Chemical synthesis and characterization of single-chain C18-chloroparaffin materials with defined degrees of chlorination

Technical chlorinated paraffins (CPs) are produced via radical chlorination of n-alkane feedstocks with different carbon chain-lengths (∼C10–C30). Short-chain CPs (SCCPs, C10–C13) are classified as persistent organic pollutants (POPs) under the Stockholm Convention. This regulation has induced a shift to use longer-chain CPs as substitutes. Consequently, medium-chain (MCCPs, C14–C17) and long-chain (LCCPs, C>17) CPs have become dominant homologues in recent environmental samples. However, no suitable LCCP-standard materials are available. Herein, we report on the chemical synthesis of single-chain C18-CP-materials, starting with a pure n-alkane and sulfuryl chloride (SO2Cl2). Fractionation of the crude product by normal-phase liquid-chromatography and pooling of suitable fractions yielded in four C18-CP-materials with different chlorination degrees (mCl,EA = 39–52%). In addition, polar side-products, tentatively identified as sulfite-, sulfate- and bis-sulfate-diesters, were separated from CPs. The new single-chain materials were characterized by LC-MS, 1H-NMR and EA. LC-MS provided Relative retention times for different C18-CP homologues and side-products. Mathematical deconvolution of full-scan mass spectra revealed the presence of chloroparaffins (57–93%) and chloroolefins (COs, 7–26%) in the four single-chain C18-CP-materials. Homologue distributions and chlorination degrees were deduced for CPs and COs. 1H-NMR revealed chemical shift ranges of mono-chlorinated (δ = 3.2–5.3 ppm) and non-chlorinated (δ = 1.0–3.2 ppm) hydrocarbon moieties. The synthesized C18-single-chain standard materials and respective spectroscopic data are useful to identify and quantify LCCPs in various materials and environmental samples. CP- and CO-distributions resemble the ones of existing SCCP and MCCP reference materials and technical mixtures. Furthermore, these materials now allow specific studies on the environmental fate and the transformation of long-chain chloroparaffins and chloroolefins.

Systematic Study on a Quantitative Analysis of Multicomponents by Single Marker (QAMS) Method for Simultaneous Determination of Eight Constituents in Pneumonia Mixture by UPLC-MS/MS.

Pneumonia mixture was formulated and is available to treat children acute pneumonia and acute bronchitis in our hospital for nearly forty years, but there are few studies of its quality evaluation or control. In this paper, a new strategy for quality evaluation of pneumonia mixture was explored and verified through qualitative and quantitative analyses of multicomponents by single marker (QAMS) by UPLC-MS/MS. Baicalein was selected as an internal reference, and the relative correction factors (RCFs) and the relative retention time (RRT) of (R, S)-goitrin, amygdalin, chlorogenic acid, pseudoephedrine hydrochloride, ephedrine hydrochloride, ammonium glycyrrhizinate, and baicalin were established. The robustness and durability of the QAMS method were investigated. RCF values calculated by the average (AVG) method and linear regression (LRG) method had good repeatability and were acceptable for quantitative analysis, and the RTT combined with the exact masses of precursor and fragment ions and their abundance could be adopted for accurately positioning the chromatographic peak of the eight constituents. The consistency and feasibility of the QAMS method were verified by comparing the contents of the seven components calculated by a classic and validated external standard method (ESM) with those of the QAMS method, which reduces analytical cost and time of detection and avoids the problem of the diversity and large quantity of reference standards. The results demonstrated that the QAMS method developed in this paper could provide a new, alternative, and promising method to comprehensively and effectively determine multicomponents and control the quality of pneumonia mixture or even a group of similar medicines.

Chromatographic determination of organohalogen compounds in swimming pool water

The purpose of the work was to search for a sorbent with good selective properties for water purification. For this purpose, we determined the organohalogen compounds in the water of a swimming pool. A method of gas-liquid chromatography was used, using packed and capillary chromatographic columns. To concentrate the impurities of the organohalogen compounds, an automatic vapour-phase dispenser was used. This established that the swimming pool water, taken in the evening, had a complex composition of components, including 33 organohalogens, which have different toxicities for the human body. These are classed as belonging to the first and second groups of hazards. Identification of the individual component composition was carried out by determining the relative retention times of the test compounds and standard samples of the organohalogen compounds dissolved in methanol. Out of the entire range of the organohalogen compounds contained in the swimming pool water it was determined that the highest concentration of chloroform was 0.024 mg/dm3 and the highest concentration of carbon tetrachloride was 0.018 mg/dm3. The total content of organohalogen substances in the swimming pool water was 0.370 mg/dm3, which increases the risk of human diseases while the swimming pool is in operation. It is proposed to clean the water in the swimming pools from toxic impurities by an adsorption method using rocks containing zeolite from the Tatar-Shatrashan deposit, which are characterized by a sufficiently high sorption capacity in relation to the organohalogen compounds.

A novel multidimensional strategy to evaluate Belamcanda chinensis (L) DC and Iris tectorum Maxim based on plant metabolomics, digital reference standard analyzer and biological activities evaluation

BackgroundBelamcanda chinensis (L.) DC. (BC) belongs to the family of Iridaceae and is widely cultivated and used in many Chinese patent medicine and Chinese medicinal formulae. However, due to the high similarities in appearance such as color and shape to Iris tectorum Maxim (ITM), another plant from the same family, BC is often confused or even misused with ITM.MethodsTherefore, in order to distinguish the chemical constituents, qualities and biological activities of BC and ITM, multiple technologies including plant metabolomics, digital reference standard (DRS) analyzer and biological activities assay were employed to provide a sufficient basis for their practical applications.ResultsIn plant metabolomics, the PCA and OPLS-DA score plot indicated the obvious differences in chemical profiling between BC and ITM and 6 compounds were successfully identified to contribute to the differences. In DRS study, the fingerprints of 10 and 8 compounds in BC and ITM were developed based on DRS analyzer, respectively, involving relative retention time (RRT) method and linear calibration using two reference substances (LCTRS) technique. The DRS analyzer also accurately identified 10 and 8 compounds from BC and ITM, respectively, by using only two reference standards. In biological activities assay, BC had a better anticancer effect than ITM due to the high abundance of irigenin, while ITM showed stronger hepatoprotective activity than BC because of the high abundance of tectoridin.ConclusionsTherefore, due to the significant differences of B. chinensis and I. dichotoma in chemical composition and biological activities, the current studies strongly proved that these two medicinal plants could not be mixed in industrial production and clinical medication.

Triaromatic dinosteroids – Isomeric distributions and their geochemical significance

The distribution of triaromatic dinosteroids in a range of oils and some source rock bitumens was determined by GC–MS selected ion monitoring and GC–MS-MS in order to investigate possible ambiguity in the observed distribution of isomers, including those related to maturation and alteration processes commonly affecting oil accumulations. The results confirm that the eight possible isomers for the triaromatic dinosteroids and their 28-nor equivalents (triaromatic 23,24-dimethylcholesteroids) in post-Paleozoic samples are consistently represented by six peaks on commonly used GC stationary phases, although the fifth eluting is broadened and probably represents a pair of isomers. The presence of a seventh, early eluting, triaromatic dinosteroid isomer occasionally encountered in m/z 245 mass chromatograms in the literature was not substantiated. Closely co-eluting compounds can give the seemingly misleading impression that one or two triaromatic dinosteroid isomers (including the last eluting) are present at low levels in pre-Mesozoic samples and in younger oils from dominantly higher plant sources, so care is required in inferring source age for such samples. The relative abundance and retention time pattern observed for the triaromatic dinosteroids is duplicated by their 28-nor analogues. Triaromatic dinosteroid isomer distributions vary little throughout the oil window, based on data from Norwegian North Sea oils representing a range of mean maturities. Overall abundance of the series seems to decline in a similar way to other biomarkers towards the end of the oil window. In source rock bitumen corresponding to maturities of ≤0.5% vitrinite reflectance, when diasterenes are still abundant, the second eluting triaromatic dinosteroid peak was enhanced and the fifth depleted in the limited set of samples studied (Late Jurassic marine shales from the Norwegian continental shelf). Both dinosteroid series appear particularly resistant towards biodegradation and metamorphism, with no obvious change in isomeric distributions compared to unaltered oil. As a consequence, their relative abundances, as represented by age parameters that ratio each series to their 24-ethyl counterparts, increase when alteration is severe. Lacustrine oils can be difficult to date because post-Paleozoic samples do not always contain detectable dinosteroids and, when they are present, they can vary significantly in abundance. Unambiguous age resolution at the Period level was not possible for the Triassic-Tertiary oils studied, neither could depositional environments be distinguished with confidence.

Isotopic Peak Index, Relative Retention Time, and Tandem MS for Automated High Throughput IGF-1 Variants Identification in a Clinical Laboratory.

Measuring insulin-like growth factor-1 (IGF-1) is useful for assessing and managing growth-related disorders, such as acromegaly and growth hormone deficiency. High-resolution liquid chromatography-mass spectrometry (LC-MS) is used for measuring IGF-1 due to its molecular specificity, quantitative performance, well-characterized reference materials, and detailed age/sex-specific reference intervals. However, polymorphisms in the IGF1 gene may cause mass shifts in the polypeptide, which can impede quantitation and cause errors in clinical interpretation. We (1) developed a concept of "isotopic peak index", which allows simultaneous monitoring of 15 IGF-1 variants by using only four m/z ratios; (2) developed a "relative retention time" parameter that allows distinction of previously unresolved variants; and (3) utilized tandem mass spectrometry (MS/MS) to distinguish between the most common pair of variants: isobaric A67T and A70T. All methods were validated with DNA sequencing. This approach identified six variants from the ExAC database, P66A, A67S, S34N, A38 V, A67T, and A70T; two previously reported V44M and A67V variants; and discovered six unreported variants, Y31H, S33P, R50Q, R56K, T41I, and A62T. Major improvements in our workflow include enhanced automation, avoiding detailed manual calculations that are prone to human error, and the ability to monitor more, and discover new, IGF-1 variants. The workflow provides a profile of a patient's IGF-1 status and can be used to explore genotype-phenotype relationships in IGF-1 variants.

Risk of false decisions in chromatographic and spectrometric identification of chemical compounds

The reliability of qualitative analysis has been much less studied than quantitative analysis. Thus, the aim of the work was to propose a procedure to estimate the risk of false identification of chemical compounds using chromatographic and spectrophotometric analytical procedures. For the chromatography test, the Monte Carlo method was used to simulate the relative retention time of the tested substances considering the chromatographic parameters. For the UV spectrometry test, the spectra of test sample and standard were decomposed, and the simulation of each peak was obtained by using a normally distributed random generator. The probabilities of true positives and false positives were estimated using MS-Excel spreadsheets. The estimation of true positive and false positive results is relevant to guarantee the reliability of qualitative results. In addition, the likelihood ratio was calculated to support the evidence of a positive identification result, which is a relevant issue to distinguish between original and counterfeit or substandard medicines.

Substantiation of an approach to determination of ketoprofen macrogol 400 esters

The aim. The work is concerned with the substantiation of the approach to the identification and quantitative determination of ketoprofen macrogol 400 esters. Materials and methods. Ketoprofen, macrogol 400, ketoprofen macrogol 400 ester (KM400E), as well as model cream-gels were studied by the following methods: absorption spectrophotometry ultraviolet (UV) and visible, high-performance liquid chromatography (HPLC), gas chromatography (GC), GC / mass spectrometry, nuclear magnetic resonance (NMR) spectrometry and thermogravimetry. Results. It was found by GC and GC / mass spectrometry that the average molecular mass (Mr) of the test macrogol 400 is 383.50 and it contains oligomers with molecular masses from 150.17 to 546.65. KM400E, which is a mixture of esters of ketoprofen with macrogol oligomers, was synthesized. The formed esters were characterized by 1H NMR spectra. It was shown that the ratio of the average molecular mass of KM400E, calculated for monoesters, and the molecular mass of ketoprofen corresponds to the ratio of specific absorbances of solutions of ketoprofen and solutions of KM400E, this fact indicated the formation of monoesters. Taking into account the risk of variability of the fractional composition of macrogol 400 in different batches, it is advisable to quantify KM400E using ketoprofen reference standard (RS) and not KM400E RS. Using HPLC with diode array detection the peak of KM400E should be identified by the UV absorption spectrum with λmax≈255 nm, which is characteristic for ketoprofen, and the relative retention time (RRt) of the peak; KM400E should be quantified by the content of ketoprofen in this impurity. During storage of model cream-gels the content of KM400E impurity is significantly lower than the content of ketoprofen propylene glycol ester (mixture of isomers). Conclusions. The approach to the identification and quantitative determination of KM400E is substantiated. The analytical procedure for determination of KM400E impurity by HPLC with a diode array detection using ketoprofen RS was developed. Correctness of the procedure was proved by the results of the validation studies.

A Fast and Validated HPLC Method for the Simultaneous Analysis of Five 5-HT3 Receptor Antagonists via the Quantitative Analysis of Multicomponents by a Single Marker.

In this study, a new strategy for the simultaneous quantization of five serotonin 5-hydroxytryptamine receptor antagonists—ondansetron, azasetron, ramosetron, granisetron, and tropisetron—either in infusion samples or in injection dosage form was first established based on high-performance liquid chromatography combined with a quantitative analysis of multiple components by a single marker. The quantitative analysis of multicomponents by a single marker method was conducted with ondansetron as an internal reference substance and performed using relative retention time and ultraviolet spectral similarity as the double indicator. The quantitative analysis of the 5-HT3 receptor antagonists was calculated and investigated based on the relative correction factors. Chromatographic separation was achieved using a C18 column (150 mm × 4.6 mm, 5.0 μm), and the mobile phase was composed of acetonitrile-0.05 mol·L−1 potassium dihydrogen phosphate (pH 4.0) (25 : 75) at a flow rate of 1.0 mL·min−1 and detection wavelengths of 307 nm (ondansetron, azasetron, ramosetron), 302 nm (granisetron), and 285 nm (tropisetron). In addition, the accuracy of the quantitative analysis of multicomponents by a single marker method was compared with an external standard method, and no significant difference was observed between the two methods. The established method is rapid, is easy, and does not require many reference substances, and it can been successfully applied as part of the quality control of the five 5-HT3 receptor antagonists in their injection dosage form and infusion sample drugs in hospitals.

Simultaneous determination of five diuretic drugs using quantitative analysis of multiple components by a single marker

BackgroundLoop diuretics are commonly used in clinical practice to manage high fluid loads and to control fluid balance. In this paper, a novel quantitative analysis method for multiple components with a single marker (QAMS) was developed for the simultaneous determination of 5 diuretic drugs furosemide, torasemide, azosemide, etacrynic acid, and bumetanide, by HPLC. Qualitative analysis was performed using relative retention time and ultraviolet (UV) spectral similarity as the double indicator. The QAMS method was conducted with etacrynic acid as an internal reference substance. The quantities of the other four diuretics were calculated by using the relative correction factors for etacrynic acid. The quantities of the 5 diuretic drugs were also determined by the external standard method (ESM). Chromatographic separation was achieved on a Shimadzu HC-C18 column (150 mm × 4.6 mm, 5 µm) using 50 mM potassium dihydrogen phosphate (pH adjusted to 4.0 with phosphoric acid) with acetonitrile (64:36, v/v) as the mobile phase at a flow rate of 1.0 mL/min and a column temperature of 30 ℃.ResultsUnder these conditions, the 5 diuretic drugs were well separated, showing linear relationships within certain ranges. The quantitative results showed that there was no significant difference between the QAMS and ESM methods.ConclusionsOverall, the HPLC-QAMS analytical scheme established in this study is a simple, efficient, economical, and accurate method for the quantitative evaluation of 5 diuretic drugs.

A RP-HPLC Method for the Analysis of Neostigmine Methylsulfate and Process-Related Impurities, Forced Degradation Studies, in the Injection Formulation.

Neostigmine methylsulfate is an anticholinesterase agent and is clinically used for treating myasthenia gravis. It is also used for reversing nondepolarising neuromuscular blocking agents. Neostigmine methylsulfate may be administered by intravenous, intramuscular, or subcutaneous injection. In this research paper, a distinct stability-indicating reverse phase HPLC method was developed and validated for the quantitative determination of related impurities and degradation impurities in neostigmine methylsulfate API and injection formulation. The specific objective was to improve the resolution between European Pharmacopoeia listed impurity A and impurity B and degradation impurity of neostigmine methylsulfate API and injection formulation. The analysis was performed using Kromasil C18 column at 30°C of column oven temperature with phosphate-buffer/acetonitrile in a gradient mode. The RP-HPLC method was developed and validated for in-house neostigmine methylsulfate synthesis process sample and injection formulation. The injection formulation sample was studied for accelerated stability, temperature cycling stability, and photostability. The validation studies for neostigmine methylsulfate synthesis process API were studied using impurity A, impurity B, and impurity C. The analytical method validation parameters studied were specificity, precision, linearity, limit of detection, limit of quantitation, accuracy, and robustness. The API and the injection formulation were subjected to forced degradation under acid, alkali, oxidation, and photolytic and thermal conditions. The proposed method showed a significantly improved RRT (Relative Retention Time) of impurity A and impurity B with a resolution greater than 1.5. The developed method eliminates the use of an ion-pairing agent and thereby a good performance of column was established.