Ultra-high Performance Liquid Chromatography Method Research Articles

Determination of serum imatinib and its' metabolite in patients chronic myeloid leukemia

IntroductionImatinib has favorable pharmacokinetic properties, but primary and secondary resistance mechanisms may cause a decrease in clinical response over time. There is a positive correlation between serum imatinib concentrations and treatment response. Our aim was to develop a method for the measurement of imatinib and its' active metabolite N-desmethyl imatinib. MethodsSerum imatinib and N-desmethyl imatinib levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and validation studies were carried out according to CLSI (The Clinical & Laboratory Standards Institute) protocols. Serum samples were collected from 40 patients with chronic myeloid leukemia (CML) and analyzed with LC-MS/MS and ultra high-performance liquid chromatography (UHPLC) methods. ResultsThe linearity range and correlation coefficient were 12.2–12,500 ng/mL and 0.9987 for LC-MS/MS method, respectively. Limit of quantitation was determined as 24.4 ng/mL. The retention times of imatinib and N-desmethyl imatinib were 1.66 and 1.60 min, respectively. There was no statistically significant difference between the results of both methods. DiscussionThis LC-MS/MS method is cost-effective and has adavantages such as using low serum volumes, requiring simple pretreatment steps (only protein precipitation) and reduced turnaround times for analysis.

A UPLC/DAD method for simultaneous determination of empagliflozin and three related substances in spiked human plasma

A simple, rapid and sensitive ultrahigh performance liquid chromatographic method was developed for the determination of the anti-diabetic drug: empagliflozin (EMPA) and three related substances in spiked human plasma, using dapagliflozin (DAPA) as an internal standard and tetrahydrofuran as a plasma protein precipitating agent. The chromatographic separation was achieved on an Acquity “UPLC® BEH” C18 column (50 mm × 2.1 mm i.d, 1.7 µm particle size), and a mobile phase consisting of aqueous trifluoroacetic acid (0.1%, pH 2.5): acetonitrile (60:40, v/v) at a flow rate of 0.5 mL/min. Upon using the UPLC system, the run time could be reduced to less than 1.2 min, and the solvents consumption decreased to 0.36 mL of acetonitrile per run. The response was linear over a concentration range of 50–700 ng/mL and 40–200 ng/mL (r2 = 0.9994–0.9999) with lower limits of detection and quantification (LOD/LOQ) of 15/50, 11.5/40, 12/40 and 12.5/40 ng/mL for EMPA and the three related substances, respectively. Good accuracy was obtained with mean percentage recoveries ≥ 96.97% for the studied compounds. The method was validated according to the ICH guidelines and was found suitable for routine analysis of EMPA and its related substances in human plasma.

Forced degradation of tacrolimus and the development of a UHPLC method for impurities determination.

An ultra-high performance liquid chromatography method for simultaneous determination of tacrolimus impurities in pharmaceutical dosage forms has been developed. Appropriate chromatographic separation was achieved on a BEH C18 column using gradient elution with a total run time of 14 min. The method was applied to analyses of commercial samples and was validated in terms of linearity, precision, accuracy, sensitivity and specificity. It was found to be linear, precise and accurate in the range of 0.05 to 0.6 % of the impurities level in pharmaceutical dosage forms. Stability indicating power of the method was demonstrated by the results of forced degradation studies. The forced degradation study in solution revealed tacrolimus instability under stress alkaline, thermal, light and photolytic conditions and in the presence of a radical initiator or metal ions. The drug was stable at pH 3-5. Solid-state degradation studies conducted on amorphous tacrolimus demonstrated its sensitivity to light, elevated temperature, humidity and oxidation.

A rapid and efficient extraction method based on industrial MCM-41-miniaturized matrix solid-phase dispersion extraction with response surface methodology for simultaneous quantification of six flavonoids in Pollen typhae by ultra-high-performance liquid chromatography.

An industrial MCM-41-miniaturized matrix solid-phase dispersion extraction coupled with response surface methodology was explored to determine L-epicatechin, typhaneoside, isorhamnetin-3-O-neohespeidoside, naringenin, kaempferol, and isorhamnetin in Pollen typhae by ultra-high performance liquid chromatography connected to a photodiode array detection. Several variables were optimized in detail, including mesh number of sieve, type of adsorbent, mass ratio of sample to adsorbent, grinding time, methanol concentration, and elution volume. Central composite design was applied to optimize the best conditions for the maximum yields of the total flavonoids. The results displayed a good linear relationship (R>0.9992) and the recoveries ranged from 92.9 to 103% (RSD<4.53%) of the six flavonoids. The optimal method with high efficiency and low consumption was obviously better than heating reflux and ultrasonic extraction. It was proven that the developed industrial MCM-41-miniaturized matrix solid-phase dispersion extraction coupled with simple ultra-high performance liquid chromatography method could be a rapid and efficient tool for extraction and determination of flavonoids in natural products.

Riboflavin, niacin, folate and vitamin B12 in commercial microalgae powders

This study aimed to investigate the riboflavin, niacin, folate and B12 content in microalgae powders. Riboflavin was determined with an ultra-high-performance liquid chromatographic (UHPLC) method after extraction and a two-enzyme treatment. Niacin analysis involved mild acidic hydrolysis, with niacin quantitated as the sum of nicotinic acid and nicotinamide using a UHPLC method. Both a microbiological (MBA) and a UHPLC method was used for vitamin B12 quantification as a cyanocobalamin (CNCbl) and UHPLC-mass spectrometry was used to confirm the vitamin B12 form. Total folate was determined both with MBA and as a sum of the vitamers with a UHPLC method after extraction and tri-enzyme treatment.The riboflavin and niacin content varied from 21 to 41 μg/g and 0.13–0.28 mg/g, respectively, in Chlorella sp., Spirulina (Arthrospira sp.) and Nannochloropsis gaditana powders. Chlorella powders were, on average, richer in total folate (19.7 μg/g) than Spirulina powders were (3.5 μg/g). The sum of the folate vitamers determined with UHPLC matched better with the microbiological total folate content in Chlorella than in Spirulina powders. Pseudovitamin B12 was the predominant form over active vitamin B12 in Spirulina powders, whereas Chlorella sp. and N. gaditana powders solely contained active vitamin B12 up to 2.1 μg/g.

Determination of epoxide impurity in sarpogrelate hydrochloride intermediate by UHPLC and column-switching liquid chromatography

The determination of genotoxic impurities, which is closely related to toxicological concern and daily dose, plays a key role in drug quality control. Epoxide impurity is a kind of genotoxic impurity with an epoxy ring structure during the synthesis process of sarpogrelate hydrochloride. According to the sarpogrelate hydrochloride daily dose, epoxide impurity is limited to the under 5 ppm level. The liquid chromatographic-tandem mass spectrometric (LC/MS/MS) or the gas chromatography-mass spectrometric (GC/MS) method is commonly used to characterize the epoxide impurity of sarpogrelate hydrochloride intermediates. However, these methods are not simple or economical enough to detect epoxide impurity. In this study, we resolved the problem by using the most common UV method with two ideas: one was to improve the absolute sensitivity, and the other was to reduce matrix effects. Both ultra high-performance liquid chromatography (UHPLC with high sensitivity LightPipe™ flow cells) and column-switching liquid chromatography methods were developed and validated for the quantitative determination of epoxide impurity in sarpogrelate hydrochloride intermediates. The limits of detection (LODs) of the UHPLC and column-switching liquid chromatography methods were 0.09 ppm (0.09 μg/g) and 0.33 ppm (0.33 μg/g), and the recovery rates of both methods were 87.2%–132.1% and 97.4%–100.1%, respectively. Both methods established and provided guidance for analysts to develop procedures for impurity control, especially for structures of impurity with similar matrices.

Bioactivities of serotonin transporter mediate antidepressant effects of Acorus tatarinowii Schott

Ethnopharmacological relevanceAcrorus tatarinowii Schott has been widely used in the treatments of neuropsychiatric and digestive disorders in clinical practices of traditional Chinese medicine for thousands of years. Both clinical and preclinical studies demonstrated antidepressant effects of A. tatarinowii. However, the possible action mechanisms of antidepressant effects of A. tatarinowii remain unraveled.Aim of the study: The present study aimed to investigate the roles of serotonin transporter (SERT) in antidepressant effects of A. tatarinowii. Materials and methodsAntidepressant effects of water extract of A. tatarinowii were evaluated by forced swimming test (FST), tail suspension test (TST) and locomotor activity test. The water extract was analyzed by ultra high performance liquid chromatography (UPLC) method. Two major fractions of A. tatarinowii, petroleum ether extract and water extract after petroleum ether processed, were prepared and analyzed by UPLC method. Further, volatile oil extracted by ether extraction, solid phase micro-extraction (SPME) and hydro-distillation were compared and analyzed by gas chromatography-mass spectrometer (GC-MS) method. Finally, major constituents of water extract of A. tatarinowii were isolated by preparative high performance liquid chromatography (HPLC) and identified by extensive spectroscopic analyses. Effects of all of the above mentioned samples on SERT activity were tested by a high content assay (HCA). ResultsResults of FST, TST and locomotor activity confirmed that water extract of A. tatarinowii significantly decreased mice immobility time but did not change mice locomotor activity. UPLC analysis results revealed that the water extract contained trace amount of β-asarone (0.0004206%) and α-asarone (0.0001918%). HCA results demonstrated that the water extract significantly enhanced SERT activity at 100 μg/mL. Further, GC-MS and UPLC analyses revealed that petroleum ether extract contained high content of β-asarone (45.63%) and α-asarone (12.50%). GC-MS analysis results demonstrated that the volatile oil extracted by ether extraction, SPME and hydro-distillation contained similar major components. HCA results verified that the petroleum ether extract significantly enhanced SERT activity at 1.56 μg/mL. Moreover, UPLC analysis of water extract after petroleum ether processed did not show any characteristic peaks. HCA results demonstrated that this extract significantly inhibited SERT activity at 50–100 μg/mL. Finally, phytochemistry investigation on the water extract of A. tatarinowii afforded seven constituents including veratric acid (9), anisic acid (7), 3,4,5-trimethoxybenzoic acid (3), trans-isoferulic acid (2), 2,4,5-trimethoxybenzoic acid (11), 4-hydroxybenzoic acid (6) and syringic acid (13). Their structures were established on the basis of nuclear magnetic resonance (NMR) and mass spectrometer (MS) data and comparative UPLC analyses. HCA results demonstrated the major components of the water extract of A. tatarinowii demonstrated SERT enhancement/inhibition activities. ConclusionsThis study first systematically demonstrated the roles of SERT activity in antidepressant effects of A. tatarinowii, including water extract, major fractions and main constituents. These results revealed that A. tatarinowii could regulate SERT activities in bidirectional ways.

Validated Stability Indicating HPTLC, UHPLC and UV-Spectrophotometric Techniques for the Determination of Bepotastine Besilate in Presence of Its Oxidative Degradate

The study is aimed at developing methods which have a complete validation as stipulated in the ICH guidelines and to be applied for the determination of Bepotastine besilate (BB) in pure form and in pharmaceutical formulations in the presence of its oxidative degradation product. High performance thin layer chromatography (HPTLC), Ultra high performance liquid chromatography (UHPLC) and different spectrophotometric methods (first derivative, first derivative of ratio spectra and ratio difference are developed for simultaneous determination of bepotastine besilate in laboratory-prepared mixtures of bepotastine besilate with its oxidative degradate and in pharmaceutical formulations were used in the study design. Firstly, HPTLC was performed and separation occurred on silica gel 60 F254 plates, with butanol: ammonia (8:2, v/v) as a developing system. UHPLC in which separation occurred on a Kinetex C 18 column using methanol- 0.1% O-phosphoric acid - acetonitrile (70:20:10, by volume) as mobile phase, followed. And lastly was UV/Vis spectrophotometry which included first derivative determination of the drug at 252.6 nm, first derivative of ratio of peak amplitudes at 233.4, 250 and 275.6 nm and the ratio difference with the amplitude difference between (240 nm and 260 nm). Result showed that HPTLC method was applicable over the concentration range of 0.5-5 μg / band, while UHPLC method was linear over the concentration 2- 12 μg/mL and spectrophotometric methods were linear over the concentration range 20-120 μg/mL for bepotastine besilate. The proposed three techniques are quite accurate and precise. They can be used for routine analysis of bepotastine besilate in pharmaceutical formulation and stability indicating methods.

INTERNATIONAL CONFERENCE ON HARMONIZATION RECOMMENDED FORCED DEGRADATION STUDIES AND DEVELOPMENT OF A NEW VALIDATED ISOCRATIC REVERSE-PHASE ULTRA HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF TELMISARTAN AND AMLODIPINE I

Objective: The objective of this research work is to develop a simple, rapid, cost-effective, accurate, precise, and robust stability-indicating reversephase ultra-high performance liquid chromatography method for the quantitative estimation of telmisartan (TEL) and amlodipine (AMD) in combined tablet dosage form.&#x0D; Methods: The method uses Poroshell 120EC-C18 column (4.6 mm × 50 mm, 2.7 μm) with an optimized mobile phase containing acetonitrile:50 mM ammonium acetate buffer in the ratio (45:55 v/v), pH adjusted to 4.5 with acetic acid. The flow rate was 0.5 ml/min, column temperature at 25°C and detection was monitored by a PDA detector at a wavelength of 245 nm. International Conference on Harmonization (ICH) recommended stress degradation studies were performed on TEL, AMD standard drugs, and tablet formulations; further stressed samples were analyzed by the proposed method.&#x0D; Results: Major degradation of TEL and AMD was observed under acidic, alkali, hydrolytic and oxidation conditions. The described method was validated as per the ICH guideline and validation parameters such as system suitability, linearity, accuracy, precision, specificity, and robustness results were within acceptable limits.&#x0D; Conclusion: The method was found to be suitable and accurate for quantitative estimation and stability study of title drugs in pharmaceutical preparations.

UHPLC Method Development for Determining Sitagliptin and Dapagliflozin in Lipid-Based Self-Nanoemulsifying Systems as Combined Dose in Commercial Products and its Application to Pharmacokinetic Study of Dapagliflozin in Rats

Anew simple, precise, selective and reproducible reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) method has been developed and validated for the first time in simultaneous determination of two anti-diabetic drugs, sitagliptin (SN) and dapagliflozin (DN), in lipid-based self-nanoemulsifying formulations as a new combined preparation dose. The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol–acetonitrile–water mixture at 24/18/58 (%v/v/v) ratio as the mobile phase. The isocratic flow rate was 0.4 mL/min with a run time of 6 min. The UV detection of both SN and DN was performed at 210 nm. The method was validated over a concentration range of 100 – 10000 ng/mL (r2 = 0.9997 and 0.9999 for SN and DN, respectively). The selectivity, specificity, recovery, accuracy and precision for determining SN and DN in lipid-based formulation were validated. The lower limits of detection and quantitation of the method were 27 and 89 ng mL–1 for SN and 19 and 61 ng mL–1 for DN, respectively. The intra- and inter-day coefficients of variation were less than 4%. The validated method has been successfully applied to quantify both SN and DN in self-nanoemulsifying formulation of combined dosage form. In addition, the proposed method was also applied for in vivo pharmacokinetic study using an animal model (rat) to further illustrate the scope and application of the proposed method.

Development of a high resolution mass spectrometry method for the determination of danshensu and salvianolic acid B

In this work, a highly sensitive ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method has been developed and validated by the quantifications of danshensu (DSS) and salvianolic acid B (SAB) in Danhong injection (DHI) in human plasma, urine and feces for the pharmacokinetic and excretion studies. The DSS and SAB were extracted from biological matrix by direct protein precipitation with fivefold volume of methanol and separated by a Waters CORTECS C18+ chromatographic column (100 × 2.1 mm, 2.7 μm) with gradient elution (mobile phase: methanol–H2O, both containing 0.1% formic acid). The detection of analytes was achieved using negative ion ESI in Full MS/dd-MS2 (data-dependent MS2) scan mode. The human subjects received either a single intravenous injection (i.v.) dose (containing 0.331 mg/kg DSS and 0.117 mg/kg SAB) or a single intramuscular injection (i.m.) dose (containing 0.663 mg/kg DSS and 0.234 mg/kg SAB) of DHI. For i.v., the Cmax, half-life (t1/2) and AUC0−∞ were 906.2 ± 31.82 μg/L, 0.604 ± 0.103 h and 242.19 ± 18.24 μg/L × h for DSS and 1118.5 ± 35.64 μg/L, 0.893 ± 0.159 h and 277.63 ± 11.24 μg/L × h for SAB, respectively. For i.m., the Cmax, Tmax, t1/2 and AUC0−∞ were 243.0 ± 29.1 μg/L, 0.67 h, 0.71 ± 0.03 h and 318.98 ± 7.62 μg/L × h for DSS and 119.4 ± 6.51 μg/L, 0.50 h, 1.08 ± 0.18 h and 185.63 ± 4.23 μg/L × h for SAB, respectively. Based on the AUC0−∞ obtained from i.m. and i.v. administrations, the absolute bioavailabilities (F) were estimated to be 65.9% for DSS and 33.4% for SAB.

Development and Validation of a UHPLC-DAD Method for the Quantitative Analysis of Major Dihydrochalcone Glucosides from Thonningia sanguinea VAHL.

Thonnigia sanguinea is a plant widely used in traditional African medicine against a variety of diseases. The obligate parasite is growing throughout tropical African forests and utilizes a large variety of hosts. Dihydrochalcone glucoside derivatives isolated from the subaerial parts of this plant were identified as potential antidiabetic lead compounds. In this study, an ultrahigh-performance liquid chromatographic method coupled with a photodiode array detector was developed for the quantitation of six major dihydrochalcone derivatives. The analytes were baseline separated in complex samples within 14 minutes on a Phenomenex Luna Omega 1.6 µm C18 column using a mobile phase consisting of water and acetonitrile (each + 0.01% trifluoroacetic acid) in gradient elution. Method validation confirmed the selectivity, linearity (R2 ≥ 0.9992), precision (inter-day ≤ 1.98%, intraday ≤ 2.00%), and accuracy (recovery rates of 97.4 - 106.3% for all analytes). At 280 nm, the LODs and LOQs were found to be lower than 1.42 and 4.30 µg/mL, respectively. Eight plant batches from the northern Angolan province of Uíge (collected in the wild or bought on markets) were extracted with methanol using an ultrasound-assisted extraction protocol and subsequently analyzed with the validated method. Results indicated high contents of dihydrochalcone glucosides in all eight samples. Most notably, the two bioactive constituents thonningianin A and B were present in fairly large amounts (2.42 - 5.35 w%).

The content of selected vitamins and iodine in mare’s milk

The aim of the study was to contribute to scarce literature data on the content of selected vitamins and iodine in mare’s milk. The study focused on the content of selected lipophilic vitamins (A, E), hydrophilic vitamins B1, B2, B6) and iodine in milk samples obtained from 8 mares during 6 months of lactation. The content of micronutrients was evaluated according to the stage of lactation. Vitamins B2 and B6 were determined using ion-pair reversed phase high performance liquid chromatography with fluorescence detection, vitamin B1 by fluorescence detection by reversed-phase liquid chromatography, vitamins A and E by the ultra-high performance liquid chromatography method with ultraviolet and fluorescence detection and iodine by the spectrophotometric method. Mean concentrations of vitamins A (0.06 ± 0.08 mg/l), E (0.083 ± 0.14 mg/l), B1 (256.24 ± 44.19 µg/l), B2 (111.40 ± 81.88 µg/l), B6 (0.30 ± 0.12 mg/l) reached lower values in mare’s milk compared to cow’s milk. The mean value of iodine in mare’s milk reached 44.48 ± 54.45 µg/l. Lactation stage proved to be a significant factor for vitamin B6 and iodine.

UHPLC analysis of major functional components in six types of Chinese teas: Constituent profile and origin consideration

In this study, an effective ultra-high performance liquid chromatography (UHPLC) method for simultaneous determination of 15 constituents of catechins, caffeine, gallic acid and theaflavins was developed. Employing the developed UHPLC method, the contents of the 15 constituents were analyzed in 245 samples of 6 types of Chinese teas collected from different places. The catechin contents in these samples ranged from 16.50 to 94.35 mg/g. Theaflavins were not detected in green tea and white tea, and caffeine did not strongly relate to germplasm resource or fermentation degree. Principal component analysis (PCA) of UHPLC data revealed that white, oolong and green tea samples could be clustered closely, demonstrating notable difference of functional components between the three types of teas resulting from preparation process and varieties. The constituent profile could be primarily attributed to the structural transformation of catechins and pigments during tea processing. This study would advance our understanding of chemical profile of Chinese teas afforded by different processing technologies.

Mechanochemical-Assisted Extraction of Active Alkaloids from Plant with Solid Acids

In this work, a novel mechanochemical-assisted extraction (MCAE) coupled with the ultra-high performance liquid chromatography (UHPLC) method was developed to extract two alkaloids with solid acids from Stephania tetrandra S. Moore. To further understand the influence of extraction parameters on the MCAE process, the response surface methodology was applied for systematic investigation. The results showed that the highest yield of alkaloids was obtained within 15 min of the total extraction time under the optimal conditions: oxalic acid (20 wt %), grinding plant materials for 5 min, and the liquid/solid ratio of 20 mL/g. Mechanochemical treatment resulted in the destruction of cell walls and increased the total contact surface area due to the smooth surface changing to an open porous structure. Accordingly, the developed extraction method showed advantages of being more ecofriendly, faster, and more efficient than conventional extraction methods. Meanwhile, the extractant was analyzed by UHPLC. The obtain...

Employing a PLGA-TPGS based nanoparticle to improve the ocular delivery of Acyclovir

Delivering drugs via the ocular route has always been a challenge for poorly soluble drugs. The various anatomical and physiological barriers in the eye cavity hinder the residence of drugs within the corneal and precorneal regions. In this study, the nanosystem that could sufficiently deliver the poorly soluble Acyclovir topically via ocular route. Our nanosystem is composed of the biocompatible PLGA polymer stabilized with TPGS which possess a high emulsifying capacity and is also known as P-gp inhibitor. The optimized nanoparticles were prepared with 0.3% TPGS and had particle-size of 262.3 nm, zeta-potential of +15.14 mV. The physicochemical-characterization, ex vivo transcorneal permeation, ocular-irritation and Acyclovir ocular-availability, following topical ocular application of PLGA-NPs in rabbit eyes, were performed. The tested parameters and irritation by Draize’s test suggested the suitability and safety of PLGA-NPs for ocular use. An ultrahigh performance liquid chromatographic method was developed, validated, and applied to quantify Acyclovir in aqueous humor which was shown to be significantly higher (p < 0.05) using the developed nanoparticles as compared to Acyclovir-aqueous suspension following their single topical ocular administration. Noticeable 2.78-, 1.71- and 2.2-times increased values of AUC0–24h, t1/2 (h) and MRT0–24h were found, respectively, with the PLGA-TPGS-NPs as compared to ACY-AqS. These results demonstrate the superiority of delivering Acyclovir using a nanosystem compared to conventional methods.

Effect of Pentacyclic Triterpenoids-Rich Callus Extract of Chaenomeles japonica (Thunb.) Lindl. ex Spach on Viability, Morphology, and Proliferation of Normal Human Skin Fibroblasts.

The effect of the well-characterized callus extract of Chaenomeles japonica on viability, morphology, and proliferation of normal human skin fibroblasts was investigated. The phytochemical analysis was performed using the ultra-high performance liquid chromatography method. The total phenolic, phenolic acid, and flavonoid contents were determined spectrophotometrically. The antioxidant activity was investigated using the DPPH (1,1-Diphenyl-1-picrylhydrazyl Radical Scavenging), FRAP (Ferric Reducing Antioxidant Power), and CUPRAC (CUPric Reducing Antioxidant Capacity) assays. The callus growth index during passages was high as well as the content of pentacyclic triterpenoids. The microscopic observations of the fibroblast viability, morphology and the evaluation of the proliferation ratio (xCELLigence system) proved that the influence of callus extract on the fibroblasts was dose-dependent. The evaluated level of fibroblasts proliferation rate after 72 h of incubation with callus extract at concentration 12.5 µg L−1 was the highest compared to all the analyzed ligands. Moreover, callus extract administrated for 72 h caused a significant increase in the proliferation rate in comparison with the control group (5.7 ± 0.1 vs. 4.4 ± 0.9; p < 0.01). The preliminary studies carried out may suggest that the callus extract rich in triterpenoids may be a potential source of cosmetic ingredients with a beneficial effect on human skin.

Sustainable Eco-Friendly Ultra-High-Performance Liquid Chromatographic Method for Simultaneous Determination of Caffeine and Theobromine in Commercial Teas: Evaluation of Greenness Profile Using NEMI and Eco-Scale Assessment Tools.

Background: Green analytical chemistry (GAC) aims to eliminate or minimize the amount of hazardous solvents consumed and generated daily worldwide. Considering the environmental impact of all analytical procedures and replacing the polluting methodologies with clean ones is of a paramount interest. Objective: This work aims to develop and validate a sustainable, fast, and economic ultrahigh-performance liquid chromatography method for simultaneous determination of methylxanthines in commercial tea samples as well as to evaluate the greenness profile of the proposed method using two greenness assessment tools: National Environmental Methods Index (NEMI) and analytical Eco-scale. Methods: The method was designed based on applying GAC principles in method development. The green chromatography approach was applied by using benign mobile phases. The chromatographic separation was optimized to minimize sample preparation, achieve short analysis time with low solvent consumption, and minimize waste generation. Results: All the studied analytes were separated in only 1.7 min. The detection limits of the studied analytes ranged from 0.015 to 0.03 mg/L, while LOQs were in the range of 0.05 to 0.1 μg/L with UV detection. The proposed method neither uses nor generates harmful chemicals, it passes the four quadrants of the NEMI greenness profile, and it has a high Eco-scale score. Conclusions: Compared with the reported methods, the proposed method is greener, more economical, and faster; therefore, it can be used as a green alternative to the existing conventional methods for routine analysis of the studied analytes without harming the environment.

A Novel Ultra-High Performance Liquid Chromatography Method for the Determination of Erdosteine, Related Impurities and Degradation Products in New Effervescent Tablets

A novel and automated, stability-indicating, reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantitative determination of erdosteine, its known impurities and two novel degradation products in a new pharmaceutical dosage form (effervescent tablets). The chromatographic separations were performed on a Waters Acquity UPLC HSS T3, 1.8 µm (2.1 mm × 150 mm, I.D.) stainless steel column. The mobile phase consisted of 0.1% TFA in water and methanol under gradient elution conditions, at a flow rate of 0.29 mL/min, for the assay and impurities analysis. UV detection was set at a wavelength of 238 nm. Erdosteine raw material, placebo and effervescent tablets were subjected to forced degradation. The new degradation products (labeled OX1 and OX2) were found after oxidative treatment and characterized by ultra performance liquid chromatography mass spectrometry. The validation parameters such as linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision, specificity and robustness were highly satisfactory for all analyzed compounds. LOD (0.020 and 0.011–0.385 µg/mL for erdosteine and impurities, respectively) and LOQ values show the high sensibility of the method. Specificity of the method was confirmed by testing the matrix components. The validated method demonstrated to be suitable for routine quality control purposes and for routine stability studies of erdosteine in effervescent formulations.

Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry

Complex biomolecules present in their natural sources have been difficult to analyze using traditional analytical approaches. Ultrahigh-performance liquid chromatography (UHPLC-MS/MS) methods have the potential to enhance the discovery of a less well characterized and challenging class of biomolecules in plants, the ellagitannins. We present an approach that allows for the screening of ellagitannins by employing higher energy collision dissociation (HCD) to generate reporter ions for classification and collision-induced dissociation (CID) to generate unique fragmentation spectra for isomeric variants of previously unreported species. Ellagitannin anions efficiently form three characteristic reporter ions after HCD fragmentation that allows for the classification of unknown precursors that we call targeted reporter ion triggering (TRT). We demonstrate how a tandem HCD-CID experiment might be used to screen natural sources using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) emerged. The method was verified not to yield false-positive results in complex plant matrices. We were able to identify 154 non-isomeric ellagitannins from strawberry leaves, which is 17 times higher than previously reported in the same matrix. The systematic inclusion of CID spectra for isomers of each species classified as an ellagitannin has never been possible before the development of this approach.