Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.

An efficient HILIC-MS/MS method for the trace level determination of three potential genotoxic impurities in aripiprazole active drug substance

A sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for trace analysis of potential genotoxic impurities (PGIs): 2,3-dichloroaniline (PGI-1), bis(2-chloroethyl) amine (PGI-2), and 2-chloroethylamine (PGI-3), in aripiprazole (APZ) active drug substance. Separation of analytes was achieved on ACE HILIC–N Column (HILN-5-1046U, 100 × 4.6 mm, 5 μm) in gradient elution mode with mobile phase A [acetonitrile:ammonium formate buffer (95:5 v/v)] and mobile phase B [acetonitrile:ammonium formate buffer (50:50 v/v)] at a flow rate of 0.8 mL/min. Developed method was linear in the concentration range of 8–100 ppm for PGI-1, 11–100 ppm for PGI-2, and 12.5–100ppm for PGI-3 with R2 > 0.996. The developed method was accurate for quantification of each PGI with percent recoveries greater than 96% and RSD (%) not more than 5%. The developed method was precise for quantification of PGIs in aripiprazole with RSD (%) of not more than 4% for any of the PGIs. There was no interference of diluent peaks at the retention time of the PGIs and APZ in the method. All the PGIs and sample solutions were found to be stable at ambient laboratory temperature (25 ± 5 °C) and refrigerated condition (2–8 °C) for a period of 48 h. The developed HILIC-MS/MS method can be used for trace quantification of PGIs in aripiprazole drug in quality control laboratories of the pharmaceutical industry.

Combined Determination of Succinic Acid and Cetylpyridinium Chloride in Medicinal Films by Gradient High Performance Liquid Chromatography

A technique has been developed based on reversed-phase high-performance liquid chromatography with diode-matrix detection for the joint determination of succinic acid and cetylpyridinium chloride in complex action medicinal films. Efficient chromatographic separation of active drug components was achieved in a gradient elution mode on a Luna C18 (2) 100A column (4.6 × 250 mm, 5 µm) using a mobile phase consisting of a 0.1% solution of phosphoric acid and acetonitrile. The detection wavelength was 210 nm for both compounds. The developed method is validated in terms of specificity, linearity, precision, accuracy and can be used to determine the authenticity and quantitative content of succinic acid and cetylpyridinium chloride in the joint presence in assessing the quality of medicinal films.

HPLC Estimation of New Impurity Methyl Ezetimibe in Ezetimibe Drug

A new gradient elution mode HPLC method was developed and validated to detect and monitor the novel impurity namely methyl ezitimibe in ezetimibe drug substances. Chromatographic detection and analysis of methyl ezetimibe was performed on XBridge C18 column with mobile phase consisting of 0.02 M phosphate buffer (pH 5) and acetonitrile with 1 mL/min flow rate in gradient elution mode. Methyl ezetimibe was detected and monitored at 248 nm. The calibration curve was linear over range of 0.015 to 0.219% concentration. The limit of detection and quantification were computed as 0.005% (signal to noise ratio 3.60) and 0.015% (signal to noise ratio 15.96), respectively. The precision was 0.97% (%RSD) and accuracy was 93.2 to 98.2% (recovery). The developed method was proved suitable to detect and monitor methyl ezetimibe impurity in ezetimibe drug substances.

DEVELOPMENT OF HPLC–UV TECHNIQUES FOR QUANTITATIVE ANALYSIS OF 4-HYDROXYBENZOIC ACID ESTERS (PARABENS) IN FOODSTUFFS, COSMETICS AND PHARMACEUTICAL PRODUCTS

Four methods have been proposed for the determination of methyl-, ethyl- and propyl- esters of 4-hydroxybenzoic acid (parabens) in foodstuff, cosmetic products and pharmaceutical formulations by RF-HPLC. Electronic spectra parameters of parabens have been determined. It was found that in neutral solutions the absorption maximums were 195 and 254 nm for all analytes. Signal ratios have been calculated (S254/S230). Signal ratios were used as an additional criterion for identification. The conditions of chromatographic separation were common for all types of samples. A precolumn and an Acclaim (Thermo) column with a C-18 sorbent without any additional modifications (both structural and dynamic), a gradient elution mode with water-acetonitrile mixtures and a spectrophotometric method of detection at two wavelengths were used. The total time of chromatographic analysis was 45 min, including the stage of conditioning the system with a starting eluent (15 min). The retention times of parabens in the reversed-phase variant of separation increased with the increase in alkyl groups in the structure of molecules. The effectiveness of the removal of surfactants from cosmetics by the column chromatography method with silica gel as a polar sorbent and using ethylacetate (an ethylacetate extract of the sample) as an eluent has been shown. The necessity of applying gradient elution mode for reducing analysis time and increasing the amplitude of analytes signals was established. The calculated validation parameters (detection limit, limit of quantitation, limit of repeatability, recovery) show the compliance of the methods with the current regulatory documents (SanPiN 2.3.2.1293-03, SanPiN 2.3.2.2364-08).

Simultaneous Identification and Quantification of Three Xanthones and Two Polyisoprenylated Benzophenones in Eight Indian Garcinia Species Using a Validated UHPLC-PDA Method

Background: Xanthones and polyisoprenylated benzophenones (PIBs) are two important classes of plant secondary metabolites with a wide range of bioactivities. Garcinia species synthesize numerous xanthones and PIBs. As per the literature, no data claiming simultaneous identification and quantification of three xanthones, α-mangostin, β-mangostin, γ-mangostin, and two PIBs, xanthochymol, isoxanthochymol, were found. Methods: A validated ultra-HPLC (UHPLC)-photodiode array (PDA) method for the simultaneous identification and quantification of five compounds in different extracts of eight Indian Garcinia species was developed. The compounds were separated on a Waters ACQUITY™ UPLC H-Class column using a mobile phase consisting of solvents 0.1% formic acid in water (A) and methanol (B) in gradient elution mode. The total run time was 9 min. Results: From fruit rinds of eight Indian Garcinia species, namely Garcinia cambogia, G. cowa, G. indica, G. loniceroides, G. mangostana, G. morella, G. pedunculata, and G. xanthochymus, extracts were prepared using solvents of varying polarity. These extracts were analyzed for five biologically important compounds, namely α-mangostin, β-mangostin, γ-mangostin, xanthochymol, and isoxanthochymol. The results revealed that there is a wide variation in concentration of these compounds in extracts of Garcinia species. Conclusions: The developed and validated UHPLC-PDA method could be used for simultaneous identification and quantification of these five compounds for bioprospection of other Garcinia species.

NEW UNIVERSAL METHOD FOR DETERMINATION OF ANTICOAGULANTS IN RODENTICIDES

Solid paraffin briquettes are one of the most modern types of rodenticide baits, because they protect it from the environmental exposure and do not reduce palatability of rodents. This paper proposes a simple universal method for the determination of brodifacoum, bromadiolone and difenacoum in solid briquettes, which are a mixture of paraffin, poison and filler, which can be used as grain, flour and the like. The difficulty in the analysis of baits containing paraffin, is to get rid of it. For this, we propose to use hexane-acetonitrile extraction in the system. Paraffin dissolves in hexane, while brodifacoum, bromadiolone and difenacoum do not. The resulting two-phase system was separated using a separatory funnel and the bottom fraction was collected. If any fillers were present in the sample, they were filtered until the mixture was separated, the residue was washed with acetonitrile. Before introducing the sample into the chromatograph, it was filtered twice with PTFE filters with a porosity of 0.45 µm. The best separation was achieved using a Thermo Acclaim® Surfactant column using acetonitrile and 0.1 M aqueous ammonium acetate solution (pH 5.4) as the mobile phase in a gradient elution mode. All three poisons were detected at a wavelength of 264 nm. This method has a sensitivity of 0.028 mg. The linearity range is from 0.00067 to 0.01%. The recovery rate for bromadiolone is 94%, for brodifacoum, 98%, and for difenacoum, 90%.

Development and Validation of HPLC/UV-Spectrophotometric Procedures for Metronidazole Quantitative Determination

Metronidazole is the most popular representative of antiprotozoal medicines from the group of 5-nitroimidazoles. Metronidazole blocks the enzymes of alcohol dehydrogenase and acetaldehyde dehydrogenase, therefore when its joint taking with alcohol it is observed the strong intoxication syndrome and fatal poisonings too. Therefore metronidazole can be a potential object of chemical toxicological investigations. The purpose of our paper is to develop HPLC/UV-procedure of metronidazole quantification with application of the system of HPLC-analyzer MiLiChrome® A-0230 implemented in practice of forensic medical laboratories in Russia and Ukraine and carry out step-by-step validation of the developed procedure. Chromatographic conditions: Eluent A (0.2 M LiClO4 – 0.005 M HClO4) and Eluent B (acetonitrile) wereused as the mobile phase components; HPLC microcolumn Ø2×75 mm and ProntoSIL 120-5-C18 AQ, 5 μm were used as an analytical column; temperature was 40°С; flow rate was 100 μl/min; gradient elution mode was from 5% to 100% Eluent B for 40 min, then 100% Eluent B for 3 min; detection was performed at 277 nm. Retention time for metronidazole is 5.95 min. Since metronidazole is easy soluble and stable enough in the solutions of diluted alkalis 0.001 M sodium hydroxide solution has been proposed for preparation of the solutions in developing HPLC/UV-procedure of metronidazole quantification. Validation of the procedure has been carried out in the variants of the method of calibration curve and method of standard by such parameters as in process stability, linearity/calibration model, accuracy and precision within 3 different analytical runs using different batches of reagents and different glassware; experiments have been performed by three different analysts. New procedure of metronidazole quantitative determination by the method of HPLC/UV has been developed. Its validation has been carried out and acceptability for application has been shown.

Evaluasi Pemisahan Alkilbenzena Menggunakan Kolom Monolith Berbasis Polimer Organik secara Kromatografi Cair Kinerja Tinggi

<p>Kolom monolith berbasis polimer organik poli-(lauril metakrilat-co-etilen dimetakrilat) disintesis secara in situ kopolimerisasi dalam kolom <em>silicosteel </em>dengan ukuran panjang 10 cm dan diameter dalam 1,02 mm. Kolom monolith ini digunakan untuk pemisahan alkilbenzena secara Kromatografi Cair Kinerja Tinggi (KCKT) fasa terbalik. Pada penelitian ini, efisiensi pemisahan ditingkatkan dengan menggunakan kolom monolith poli-(LMA-co-EDMA) untuk memisahkan senyawa alkilbenzena melalui tiga parameter, yakni temperatur kolom, pemisahan secara isokratik dan pemisahan secara gradien. Temperatur kolom yang digunakan berkisar antara 27–50 °C. Hasil yang diperoleh menunjukkan bahwa temperatur optimum untuk pemisahan alkilbenzena secara isokratik yaitu 27 °C yang setara dengan temperatur ruang dengan fasa gerak asetonitril-air (50:50 w/w). Pemisahan alkilbenzena yang lebih efisien ditunjukkan dari penggunaan mode gradien ditandai dengan nilai <em>peak capacity</em>, faktor retensi dan jumlah plat teoritis yang lebih baik. Fasa gerak yang digunakan pada pemisahan secara gradien yaitu pelarut A yang terdiri atas asetonitril-air (40:60 w/w) dan pelarut B yang terdiri atas asetonitril-air (60:40 w/w) dengan waktu gradien 20–40 menit 0–100% B. Perubahan waktu gradien berpengaruh terhadap faktor retensi dan <em>peak capacity</em>.</p><p><strong>Evaluation of Organic Polymer-Based Monolithic Column by High Performance Liquid Chromatography for The Separation of Alkyl Benzenes</strong><strong>. </strong>Organic polymer-based monolithic column of poly(lauryl methacrylate-co-ethylene dimethacrylate) has been prepared by in- situ copolymerization inside of <em>silicosteel</em> column with the size of 100 mm long x 1.02 mm i.d. This kind of monolith column used for separation of alkylbenzenes using reversed-phase high performance liquid chromatography (HPLC). The efficiency separation on this research is improved by using poly-(LMA-co-EDMA) monolithic column for separation of alkyl benzene compounds using three strategies involving optimization column temperature, isocratic elution mode, and gradient elution mode. The applied column temperatures were varied in the range of 27–50 °C. It was found that room temperature in isocratic mode with the mobile phase of acetonitrile-water (50:50 w/w) showed the excellent efficiency indicated by baseline-resolved of each peak of alkyl benzenes. The resulted separation efficiency by employing gradient elution mode exceeded its counterpart (isocratic mode), which is indicated by better in peak capacity, retention factor, and number theoritical plate. Two different mobile phases for gradient elution mode, composed of A that contain of acetonitrile-water (40/60 w/w) and B that contain of acetonitrile-water (60/40 w/w) were utilized in the range of 20-40 min for 0-100% B. It was found that increasing gradient time strongly affect to the retention factor and peak capacity.<strong></strong></p>