Elution Research Articles

Comparative pharmacokinetic study of Anisodamine Hydrobromide tablets and injection in septic acute lung injury rats.

Aim: We aimed to establish a sensitive LC-MS/MS method to analyze the pharmacokinetics of Ani HBr tablets and injection. Methods: Around 10mmNH4Ac containing 0.1% formic acid and acetonitrile were used as the mobile phase. Acute lung injury in septic and normal rats, respectively, were administered Ani HBr tablets at doses of 12.5, 25 and 50mg/kg and injection at doses of 4, 8 and 16mg/kg, followed by extraction of the drugs from plasma using ethyl acetate for subsequent analysis. Results & conclusion: The method met the requirements for biological analysis. Ani HBr tablets absorbed slowly in rats with disease, tail vein administration was a more promising approach for treating septic acute lung injury.

A robust ultra performance liquid chromatography method for dacarbazine quantification in nanosponge formulation: A quality by design approach

A new, user-friendly Ultra Performance Liquid Chromatography (UPLC) method was developed using Analytical Quality by Design (QbD) for precise analysis of Dacarbazine in both bulk powder and Dacarbazine-loaded Nanosponges. This method follows ICH guidelines and uses a statistically designed approach to optimize the separation conditions for Dacarbazine. A central composite design was implemented to identify the ideal combination of organic solvent (mobile phase composition) and flow rate. The UPLC method was successfully optimized for rapid separation (1.3 minutes) for Dacarbazine using a mobile phase consisting of 70% Methanol: 30% Orthophosphoric acid (0.1%) at a flow rate of 1.0 mL/min, using a Hypersil C18 column. The method exhibited excellent linearity with a correlation coefficient value of 0.997 across a concentration range of 4-20 μg/mL, accuracy (drug recovery of 96-100%) and sensitivity (LOD 1.15 μg/mL, LOQ 3.50 μg/mL) and it successfully quantified the drug in the formulated Dacarbazine-loaded Nanosponges. This QbD-driven UPLC method offers a valuable tool for reliable Dacarbazine quality control in pharmaceuticals providing rapid analysis, high accuracy, and low detection limits.

DoE-assisted HPLC method development and validation of enzalutamide and repaglinide in rat plasma.

Aim: A simple and rapid HPLC technique was developed and validated to simultaneously estimate enzalutamide (ENZ) and repaglinide (REP) in rat plasma. Methods: In silico predictions using DDinter and DDI-Pred indicated possible drug-drug interactions between ENZ and REP. A central composite design was used to identify factors influencing the separation of the drugs. Interactions between chromatographic parameters were studied through 51 experiments, followed by illustration with three-dimensional response surface plots. The four factors optimized for the separation of the two drugs are column temperature (A), % organic strength (B), pH (C) and column type (D). Results: Plate count(R1), tailing factor (R2) and resolution (R3) responses in the experimental design were analyzed with the favorable chromatographic conditions predicted to be 0.1% formic acid and acetonitrile as mobile phases on a Phenomenex C18 LC column (250×4.6mm, 5μm). The method was applied to estimate the drugs in rat plasma using a simple protein-precipitation step and found to be linear, accurate and precise within the ranges of 0.5-16 and 5-50μg/ml for ENZ and REP, respectively. Conclusion: The optimized method can be used in future bioanalytical workflow for drug quantification and drug-drug compatible studies.

Stability indicating HPTLC method development and validation of evogliptin in bulk and tablet dosage form by using a QbD approach

This study explains the well-structured development and validation of the HPTLC method and the qualitative evaluation of Evogliptin in both bulk and tablet formulation using the quality by design (QbD) approach. Chromatographic separation was carried out on an aluminium-backed silica gel F254 plate using a mobile phase consisting of ethyl acetate :methanol: triethylamine (6:4:0.1 V/V/V). Densitometry was carried out at a wavelength of 267 nm. The band corresponding to the Evogliptin was obtained at Rf=0.39. The method was found to be linear, with r2=0.9961. The critical analytical attributes (CAAs) for the HPTLC method were identified. The saturation time, band length and solvent front were determined as critical method parameters (CMP) and extensively optimized employing Box-Behnken Design (BBD), with a focus on the retention factor (Rf) value as the CAA. The method described in this study exhibited linearity. The accuracy, precision, linearity, LOD, LOQ and robustness values were within the specified limits. The developed method was validated according to ICH guidelines and the results were found to be within the acceptance criteria. This study effectively demonstrates the value of the QbD approach for creating an incredibly accurate HPTLC method with improved system effectiveness.

Micro-solid phase extraction with in-lab-packed columns for cost-efficient drug extraction from urine samples

AbstractThis study introduces a miniaturised solid-phase extraction (µ-SPE) method coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous quantification of 23 compounds, including psychoactive drugs, narcotics and their metabolites, in urine samples. The µ-SPE method, using standard laboratory equipment such as a centrifuge, was developed and validated, offering a practical and economical alternative to more traditional SPE methods. The key extraction parameters: elution solvent composition, sample and elution volumes, and sorbent mass were optimized to enhance performance. The method demonstrated high sensitivity, with detection limits between 0.06 and 0.41 ng cm−3 and quantification limits from 0.19 to 1.24 ng cm−3. The precision was confirmed with an intra-day precision ranging from 0.2% to 7.5% and an inter-day precision from 1.2% to 13.4%. Recovery rates ranged from 84.2% to 109.4%, with minimal matrix effects observed for most analytes (|ME|< 10%). The relative error remained below 5%, indicating good accuracy. The method was also evaluated in terms of greenness, practical, and analytical aspects using the WAC model. Based on the validation results, it was concluded that the developed method could be a good confirmatory method for results obtained by immunochemical tests and could also be used in both clinical analyses and forensic toxicology. Graphical abstract

Quantitative analysis of mixed lipid nanostructures in rat skin by HPLC-MS

Abstract Liquid chromatography-mass spectrometry (LC-MS) is a very sensitive technique for determining small concentrations of drugs in fixed dose combinations or even those deposited in skin layers. Therefore, an LC-MS method was applied for determining the drugs under investigation, namely, clioquinol (CLIO), tolnaftate (TOL), and betamethasone (BETA) in Quadriderm® cream and mixed lipid nanostructures (MLNs) prepared in laboratory in the presence of potential interferents, and was applied as a dermato-kinetic study in rat’s skin. The separation was achieved within 4.5 min by using C18 column as a stationary phase and the mobile phase used were 20% phase A composed of 0.1% formic acid (v/v) and 80% phase B composed of 0.1% formic acid in acetonitrile (v/v), coupled with triple quadrupole mass spectrometer. MLNs were prepared and characterized to be compared with the conventional commercially available Quadriderm® cream. The proposed method was accurate and precise with a linearity range of 0.2–20.0 µg·mL−1 for BETA, and 0.5–400.0 µg·mL−1 for CLIO and TOL and a better bioavailability of the new formulation was obtained ensuring the capability of the nanoparticles to accumulate the drugs within the skin layers. In conclusion, the LC-MS method was accurate and precise for the determination of the three drugs under investigation in cream dosage form and skin tissues.

Stability Indicating UPLC Method Development and Validation for the Quantitative Estimation of Pazopanib in Pure form and Marketed Pharmaceutical Dosage form.

An analytical, accurate, precise, specific, efficient and simple Ultra-Performance Liquid Chromatography method has been developed and validated for the determination of Pazopanib in bulk and was applied on marketed Pharmaceutical Dosage form. The mobile phase used for the chromatographic runs consisted of 0.1% OPA Buffer and Acetonitrile in the ratio of 30:70% v/v. The separation was achieved on a BHEL UPLC column using isocratic mode. Pazopanib Drug peak were well separated and were detected by a PDA detector at 256 nm. The developed method was linear at the concentration range 6-14 μg/ml for Pazopanib. The method has been validated according to ICH guidelines with respect to system suitability, specificity, precision, accuracy and robustness. The LOD and LOQ for the Pazopanib were found to be 0.5853 µg/ml and 1.7738µg/ml respectively. The developed method is simple, precise, specific, accurate and rapid, making it suitable for estimation of Pazopanib in bulk and marketed pharmaceutical dosage form dosage form.

Analytical Method Development and Validation of Simultaneous Estimation of Pure and Tablet Dosage Form by RP-HPLC

The aim of this analytical research is to establish and validate the RP-HPLC (Reverse Phase High Performance Liquid Chromatography) method for concurrently quantifying Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline in a pharmaceutical formulation. The method utilized Rosagiline mesylate as an internal standard, employing a Shim-Pack Solar C18 Column (4.6 x 150 mm, 5 µm) as the stationary phase and a mobile phase composed of acetonitrile, methanol, and 10 mM Na2HPO4 buffer (50:30:20 % v/v/v, pH 5.5). The flow rate was maintained at 1.0 mL/min, and detection occurred at 210 nm. Validation followed ICH guidelines. The linear concentration ranges were 5-30 µg/ml, 10-60 µg/ml, and 10-100 µg/ml at 210 nm for Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Retention times were 7.643 min, 2.117 min, 3.863 min, and 3.050 min for Montelukast sodium, Fexofenadine Hydrochloride, Acebrophylline, and Rosagiline mesylate respectively. The RP-HPLC analysis of marketed formulations yielded concentrations within the ranges of 98.3–100.9%, 99.46–101.23%, and 99.82–101.74% for Montelukast sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Recovery fell within the range of 95.81-101.35% for both drugs. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of Montelukast, Fexofenadine and Acebrophylline in their combined tablet dosage form.

Determination of ivermectin in plasma and whole blood using LC-MS/MS

Background Ivermectin is a widely used drug for the treatment of helminthiasis and filariasis worldwide, and it has also shown promise for malaria elimination through its potent mosquito-lethal activity. The objective of this study was to develop and validate a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods Phospholipids were removed in patient whole blood (100 µl) and plasma (100 µl) samples using a 96-well plate Hybrid-solid phase extraction technique. Ivermectin and its isotope-labelled internal standard (ivermectin-D2) were separated on an Agilent Poroshell 120 EC-C18 50mm × 3.0mm I.D. 2.7µm, using a mobile phase of acetonitrile: ammonium formate 2 mM containing 0.5% formic acid (90: 10, v/v). Detection was performed using a triple quadrupole mass spectrometer in the positive ionization mode. Results The method was validated in the concentration range 0.970 - 384 ng/ml in both plasma and whole blood matrices. Intra- and inter-batch precisions during the validation were below 15%. There was no carryover or matrix effects detected. Ivermectin is a stable compound and results showed no degradation in the different stability tests. Conclusions The validated method proved to have high sensitivity and precision, good selectivity and to be suitable for clinical application or laboratory quantification of ivermectin in plasma or whole blood samples.

Establishment of an HPLC Fingerprint and Cluster Analysis for Miao Ethnic Medicine Osbeckia opipara

: Osbeckia opipara, a traditional Miao medicine, is commonly used by the renowned national-level Chinese Traditional Medicine practitioner Zhengshi Wu for the treatment of diarrhea due to its strong antioxidative, anti-inflammatory, and antidiarrheal effects. This study aimed to establish a high-performance liquid chromatography (HPLC) fingerprint for O. opipara to provide new evidence and technical means for the scientific evaluation and effective quality control of O. opipara. The procedure involved isolation with a Nano ChromCore C18 column (250 mm × 4.6 mm, 5 μm), using a gradient elution of 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phase, with a flow rate of 1.0 mL/min, a column temperature of 30°C, an injection volume of 10 μL, and detection at a wavelength of 254 nm. Under these chromatographic conditions, fingerprint analysis was conducted on 11 batches of O. opipara collected from different origins. The National Pharmacopoeia Committee developed the 'Chromatographic Fingerprint Similarity Evaluation System' (2004A version) for automated comparison, similarity computation, and analysis of chromatographic data. The results revealed 13 common peaks across the 11 batches of O. opipara samples, with a similarity to the automatically generated reference spectrum exceeding 0.9. SPSS 26.0 software was used to conduct cluster analysis on the peak areas of the 13 common peaks. The observations indicated that the reference spectrum generated from the 11 batches could serve as the standard fingerprint profile for O. opipara, providing sufficient characteristic information extraction.

GREEN HPLC DETERMINATION OF PHENYTOIN AND METHOD VALIDATION

Objective: In this study, the chromatographic behavior of the antiepileptic drug phenytoin was determined by the green HPLC method. The optimization of the developed method was based on the capacity factor values of phenytoin in varying water-ethanol binary mixtures and the ethanol concentration in the mobile phase where the compound was analyzed. Material and Method: Ethanol-water binary mixtures containing 35%, 40%, and 45% (v/v) ethanol were used in the optimization for the determination performed by the RPLC method. Retention times of the compound were determined with the Zorbax SB-CN (150x4.6 mm, 3.5 µm ID) column. Analyzes were performed at a constant flow rate (0.3 ml/min) and column temperature (37°C). The optimum condition for quantitative analysis was determined as an ethanol-water binary mixture containing 40% (v/v) ethanol with a pH of 6.5. Result and Discussion: In this study, the hydrophobicity of phenytoin was calculated using the logk-φ relationship. The optimum condition was determined using the obtained chromatographic data, and the quantitative determination of phenytoin in the commercial tablet formulation was made by the internal standard method. Under these conditions, excellent linearity (r>0.99) was obtained in the concentration range of 0.8-2.8 μg/ml. The detection limit of the developed method is 0.021 μg/ml; the limit of quantitation was calculated as 0.064 μg/ml. The recovery value of the method was determined as 99.61%. It was concluded that the parameters of precision, accuracy, and method robustness were appropriate for the validation procedures.

A dual methodology employing ion-pair chromatography and built-in UV spectrophotometry for quantifying recently approved combination of mometasone and indacaterol in a novel combined metered dose inhaler: assessing the greenness, carbon footprint, blueness, and whiteness

Developing analytical techniques that align with green and sustainable chemistry principles is crucial in today's scientific landscape. This work introduces two innovative approaches for the simultaneous quantification of indacaterol (IND) and mometasone (MOM), a recently approved combination therapy for chronic obstructive pulmonary disease. These methods—rapid isocratic ion pair chromatography (IPC) and UV–visible spectrophotometry—demonstrate improved environmental sustainability, cost-effectiveness, and versatility compared to existing techniques. The optimized 4-min IPC method achieved excellent resolution (retention times 2.18 ± 0.1 min for IND and 3.95 ± 0.1 min for MOM), peak symmetry, and sensitivity. It utilizes a low-cost ion pair mobile phase of acetonitrile and acidified water containing 0.025% sodium dodecyl sulfate (50:50% v/v), making it suitable for laboratories with standard chromatographic instruments. The spectrophotometric approach offers two procedures: first derivative and ratio derivative methods. These serve as simplified, low-cost alternatives for resource-limited laboratories without access to advanced instruments. Both techniques feature simplified protocols that minimize extraction and fractionation steps. Comprehensive validation confirmed outstanding accuracy (98–102%) and precision (%2 <). Sustainability assessments using ComplexGAPI, AGREE, carbon footprint, BAGI, and RGB12 tools demonstrated enhanced environmental performance compared to existing methods. The IPC and spectrophotometry methods achieved greenness scores of 0.81 and 0.85, respectively, surpassing the 0.63–0.67 range of reported techniques. Additionally, they showed lower carbon footprints of 0.035 and 0.022 kg CO2 equivalent emissions per sample, compared to 0.079–0.092 kg for conventional procedures. The application of novel "blueness" and "whiteness" concepts using BAGI and RGB12 algorithms further confirmed superior sustainability, with scores of 87.5 & 90 for blueness and 88.1 & 89.8 for whiteness. Successfully applied to quantify IND and MOM in combined capsules, this work provides a model for eco-friendly pharmaceutical analysis that maintains high analytical reliability while improving sustainability metrics.

A new RP-HPLC method for the simultaneous detection and quantification of minocycline and ascorbic acid in pure forms and combined pharmaceutical dosage forms

We report the first reverse-phase, high performance liquid chromatography (RP-HPLC) method for the rapid and simple simultaneous detection and quantification of minocycline and ascorbic acid in raw and pharmaceutical dosage forms. Detection was performed on a Sunniest C-18 column (5 μm particle size, 150 mm × 4.6 m mi.d(, at UV wavelength of 255 nm. The mobile phase consisted of potassium dihydrogen phosphate pH 2.8: Acetonitrile: Methanol (85:10:5), with 0.6 v/v% TEA and adjusted with orthophosphoric acid to a pH of 2.8 in an isocratic elution mode, with a flow rate of 0.8 mL/min. Performed analysis on the method proved that the newly developed method was sensitive, selective, accurate, precise, and robust. The method demonstrated good linearity with r2 = 0.9999 (n = 7) for both drugs and was validated in the sample concentration ranges of 1–100 and 5–500 µg mL−1 for minocycline and ascorbic acid, respectively. Both intra- and inter-day precisions were less than 2 % for both drugs, with limits of detection of 1.59 and 7.89 µg/mL and limits of quantification of 4.83 and 23.89 µg/mL, for minocycline and ascorbic acid, respectively. The applicability of the method was demonstrated by accurately determining the drug content of two commercial pharmaceutical formulations.

Fast Determination of Eleven Food Additives in River Water Using C18 Functionalized Magnetic Organic Polymer Nanocomposite Followed by High-Performance Liquid Chromatography

A novel magnetic nanomaterial with Fe3O4 as the core, PS-DVB as the shell layer, and the surface modified with C18 (C18−PS−DVB−Fe3O4) had been synthesized by seeded emulsion polymerization. C18−PS−DVB−Fe3O4 retains the advantages of the chemical stability, large porosity, and uniform morphology of organic polymers and has the magnetic properties of Fe3O4. A simple, flexible, and efficient magnetic dispersive solid phase extraction (Mag-dSPE) method for the extraction of preservatives, sweeteners, and colorants in river water was established. C18−PS−DVB−Fe3O4 was used as an adsorbent for Mag-dSPE and was coupled with high-performance liquid chromatography (HPLC) to detect 11 food additives: acesulfame, amaranth, benzoic acid, tartrazine, saccharin sodium, sorbic acid, dehydroacetic acid, sunset yellow, allura red, brilliant blue, and erythrosine. Under the optimum extraction conditions, combined with ChromCoreTMAQC18 (5 μm, 4.6 × 250 mm), 20 mmol/L ammonium acetate aqueous solution and methanol were used as mobile phases, and the detection wavelengths were 240 nm and 410 nm. The limits of detection (LODs) of 11 food additives were 0.6–3.1 μg/L with satisfactory recoveries ranging from 86.53% to 106.32%. And the material could be reused for five cycles without much sacrifice of extraction efficiency. The proposed method has been used to determine food additives in river water samples, and results demonstrate the applicability of the proposed C18−PS−DVB−Fe3O4 Mag-dSPE coupled with the HPLC method to environment monitoring analysis.

New method for rapid detection and simultaneous determination of prohibited adrenergic drugs in sports using thin layer chromatography and image processing

This study presents a new method for simultaneous determination of fenoterol, procaterol, clenbuterol, terbutaline and isoprenaline drugs using thin-layer chromatography assisted by image analysis (HPTLC-IA) combined with sensitive detection modes. Separation of the selected drugs was obtained using HPTLC Silica gel 60 F254 plates and mixture of isopropyl alcohol, ethyl acetate and ammonia (20:25:1.5 v/v/v) as mobile phase. UV detection mode combined with the use of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2'-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS+•) radicals were applied for the first time for sensible detection of the separated drugs. Grey, green, red and blue scales respectively were selected for image analysis and accurate quantification of spot area. According to the obtained result, the use of DPPH• and ABTS+• reagents revealed a higher sensitivity in detection and accurate quantification of the analyzed drugs. Lower limit of detection (LOD = 0.07 ± 0.03 µg/spot; 0.09 ± 0.03 µg/spot and 0.10 ± 0.04 µg/spot) was obtained for fenoterol, procaterol and terbutaline using the ABTS+• radical and red scale selection for image processing. With the exception of isoprenaline, the proposed HPTLC-IA method showed a good separation of fenoterol, procaterol, clenbuterol and terbutaline drugs from other compounds in urine samples. Good recovery was obtained for these drugs in spiked urine samples.

Development and validation of stability indicating HPLC-DAD assay of adapalene, methylparaben and propylparaben in a cosmeceutical gel matrix with blueness, greenness and whiteness assessment via relative ranking: An ab initio-sub finem juxtaposed non-traditional approaches

Adapalene (ADA) is primarily used as a cosmeceutical gel in the treatment of mild-moderate acne and is also used off-label to treat keratosis pilaris as well as other skin conditions. A stability-indicating fit-for-purpose (FFP) method has been developed for co-determination of ADA, methylparaben (MPN), and propylparaben (PPN) combined in a cosmeceutical gel dosage form. This method was based on a reversed-phase RP-HPLC separation of the cited analytes. It avoided the use of tetrahydrofuran (THF), which is commonly used by both compendial and published procedures for determination of ADA, thus improving method blueness (applicability), greenness and whiteness. The method development and validation followed non-traditional eco-friendly approaches including chemical simulations and empirical modelling. The HPLC separation was performed on an RP Zorbax Eclipse XDB-C8 analytical column [150 × 4.6 mm, 5 µm (80 Å)] with gradient elution system of HPLC grade acetonitrile and 50 mM aqueous sodium dihydrogen phosphate buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA) adjusted to pH 8.0 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 25 °C. EDTA played an important role for reproducibility of retention time of the three analytes especially for ADA. It controlled control deprotonation of the three acidic analytes under study and hence reproduce their ionization, hydrophobicity, and hence retention time. Quantitation was achieved with the photodiode array detection (DAD) at wavelengths of 258 nm for both MPN and PPN and 312 nm for ADA. Target Measurement Uncertainty (TMU) for the three analytes was determined via two approaches; misclassification rates approach employing 100,000 Monte Carlo simulations (MCS) and in-specification probability (ISP) to ascertain fitness-for-purpose of the proposed method. Microsoft Excel workbooks were developed for both TMU calculation approaches in addition to calculation of procedure capability index (Ppk) and sigma level for each analyte and attached in the Supplementary material. Moreover, linearity of the method underwent additional assessment using the previously mentioned TMU and procedure capability determination techniques. The robustness of the method was checked using Plackett-Burman experimental design (PBD) as an empirical modelling technique which was examined thoroughly to detect some interactions. The calibration graphs for each drug were rectilinear in the range of 0.34–7.08, 0.38–7.00 and 0.08–1.75 µg mL−1 for ADA, MPN, and PPN, respectively. The stress testing revealed photostability of the three components and variable degrees of degradation in the other conditions. The proposed HPLC-DAD method was successfully validated and applied for the determination of the investigated analytes in a gel matrix. In addition, method blueness and greenness were comparatively evaluated via very recent approaches of Blue Applicability Grade Index (BAGI), Green Solvents Selection Tool (GSST), Spider Diagram for Assessment of the Green Analytical Index (SDAGI), Analytical Method Greenness Score (AMGS), Carbon Footprint (CF), Modified Complementary Green Analytical Procedure Index (ComplexMoGAPI), and Analytical GREEnness Metric Approach (AGREE). Furthermore, the method whiteness was assessed via Red Green Blue Twelve (RGB 12) model following a relative ranking approach.

Rapid determination of nine preservatives in tobacco flavor by three phase-hollow fiber-liquid phase microextraction-high performance liquid chromatography

Tobacco flavors are extensively utilized in traditional tobacco products, electronic nicotine, heated tobacco products, and snuff. To inhibit fungal growth arising from high moisture content, preservatives such as benzoic acid (BA), sorbic acid (SA), and parabens are often incorporated into tobacco flavors. Nonetheless, consuming preservatives beyond safety thresholds may pose health risks. Therefore, analytical determination of these preservatives is crucial for both quality assurance and consumer protection. For example, BA and SA can induce adverse reactions in susceptible individuals, including asthma, urticaria, metabolic acidosis, and convulsions. Parabens, because of their endocrine activity, are classified as endocrine-disrupting chemicals. Despite extensive research, the concurrent quantification of trace-level hydrophilic (BA and SA) and hydrophobic (methylparaben, ethylparaben, isopropylparaben, propylparaben, butylparaben, isobutylparaben, and benzylparaben) preservatives in tobacco flavors remains challenging. Traditional liquid phase extraction coupled with high performance liquid chromatography (HPLC) often results in high false positive rates and inadequate sensitivity. In contrast, tandem mass spectrometry offers high sensitivity and specificity; however, its widespread application is limited by laborious sample preparation and significant operational costs. Therefore, it is crucial to establish a fast and sensitive sample pretreatment and analysis method for the nine preservatives in tobacco flavors. In this study, a method for the simultaneous determination of the nine preservatives (SA, BA and seven parabens) in tobacco flavor was established based on three phase-hollow fiber-liquid phase microextraction (3P-HF-LPME) technology combined with HPLC. To obtain the optimal pretreatment conditions, extraction solvent type, sample phase pH, acceptor phase pH, sample phase volume, extraction time, and mass fraction of sodium chloride, were examined. Additionally, the HPLC parameters, including UV detection wavelength and mobile phase composition, were refined. The optimal extraction conditions were as follows: dihexyl ether was used as extraction solvent, 15 mL sample solution (pH 4) was used as sample phase, sodium hydroxide aqueous solution (pH 12) was used as acceptor phase, and the extraction was carried out at 800 r/min for 30 min. Chromatographic separation was accomplished using an Agilent Poroshell 120 EC-C18 column (100 mm×3 mm, 2.7 μm) and a mobile phase comprising methanol, 0.02 mol/L ammonium acetate aqueous solution (containing 0.5% acetic acid), and acetonitrile for gradient elution. Under the optimized conditions, the nine target analytes showed good linear relationships in their respective linear ranges, the correlation coefficients (r) were ≥0.9967, limits of detection (LODs) and quantification (LOQs) were 0.02-0.07 mg/kg and 0.08-0.24 mg/kg, respectively. Under two spiked levels, the enrichment factors (EFs) and extraction recoveries (ERs) of the nine target analytes were 30.6-91.1 and 6.1%-18.2%, respectively. The recoveries of the nine target analytes ranged from 82.2% to 115.7% and the relative standard deviations (RSDs) (n=5) were less than 14.5% at low, medium and high levels. The developed method is straightforward, precise, sensitive, and well-suited for the rapid screening of preservatives in tobacco flavor samples.

Simultaneous determination of 10 quaternary ammonium salt bactericides in oral care products by high performance liquid chromatography-evaporative light-scattering detection

Quaternary ammonium salt bactericides are broad-spectrum bactericides often used in oral care products because of their high antibacterial efficacy, strong penetration, and low toxicity. However, the excessive use of quaternary ammonium salt bactericides may cause contact dermatitis, scalding poisoning, and even death. Existing methods to determine quaternary ammonium salt bactericides are unable to meet current requirements owing to the lack of determination components. Therefore, establishing a simple and accurate method for the simultaneous detection of more quaternary ammonium salt bactericides is necessary. In this study, a method that couples sample pretreatment with high performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) was developed for the simultaneous determination of quaternary ammonium salt bactericides in oral care products, including dodecyltrimethylammonium chloride, dodecyldimethylbenzylammonium chloride, benzethonium chloride, tetradecyl trimethyl ammonium chloride, tetradecyldimethylbenzylammonium chloride, N-hexadecyltrimethylammonium chloride, benzyldimethylhexadecylammonium chloride, trimethylstearylammonium chloride, stearyldimethylbenzylammonium chloride, and docosyltrimethylammonium chloride. Some of these bactericides do not absorb ultraviolet light, so a universal evaporative light-scattering detector was used owing to testing cost and stability concerns. The paste samples contained thickening agents, which are highly soluble in water but insoluble in organic solvents; these agents can seriously affect the results of sample pretreatment and damage the chromatographic column. Hence, sample dehydration was necessary. In this study, four dehydration methods were compared. Anhydrous sodium sulfate (Na2SO4) was selected, and the amount of Na2SO4 was optimized. Based on the solubility of the 10 target compounds and extraction efficiency, three extraction solvents were compared, and ethanol was selected. Ultrasonic extraction was the primary extraction process used in this study. The effects of different ultrasonication times, temperatures, and powers on the extraction recoveries were also investigated. Ultimately, the optimized conditions were as follows: extraction of the dehydrated paste and powder samples using ethanol at room temperature (25 ℃) for 20 min under 100 W ultrasound power, and dilution of the liquid sample with ethanol. After extraction, the samples were separated on an Acclaim Surfactant column (150 mm×4.6 mm, 5 μm) with 50 mmol/L ammonium acetate aqueous solution (pH=5.5) (A) and acetonitrile (B) as mobile phases. The gradient elution program were as follows: 0-5.0 min, 75%A-35%A, 5.0-15.0 min, 35%A-20%A, 15.0-20.0 min, 20%A, 20.0-21.0 min, 20%A-75%A, 21.0-25.0 min, 75%A. An external standard method was used for quantitative determination. The 10 compounds were analyzed within 25 min. Linear equations, correlation coefficients, and linear ranges were obtained by analyzing a series of mixed standard working solutions. The limits of detection (LODs, S/N=3) and quantification (LOQs, S/N=10) of the 10 components were determined. Stearyldimethylbenzylammonium chloride and docosyltrimethylammonium chloride showed good linear relationships in the range of 10-200 mg/L, while the other compounds demonstrated good linear relationships in the range of 5-100 mg/L. In all cases, correlation coefficients (R2) of no less than 0.9992 were obtained. The LODs and LOQs were in the range of 1.42-3.31 mg/L and 4.25-9.94 mg/L, respectively. Ten analytes were spiked in blank matrices, such as toothpaste (paste), mouthwash (liquid), and dentifrice powder (powder) at three levels, and the recoveries and precisions were calculated. The average recoveries were 87.9%-103.1%, and the corresponding relative standard deviations (RSDs) did not exceed 5.5% (n=6). The developed method was used to detect 109 oral care products. Benzyldimethylhexadecylammonium chloride and stearyldimethylbenzylammonium chloride revealed high detection rates. Moreover, the amount of stearyldimethylbenzylammonium chloride in one toothpaste sample exceeded regulatory requirements. Given its advantages of good precision and accuracy, the developed method is suitable for the quantitative analysis of the 10 aforementioned compounds in typical oral care products. The study findings can serve as a reference for the quality and safety monitoring of oral care products.

Selective Segregation of Thermo-Responsive Microgels via Microfluidic Technology.

Separation of equally sized particles distinguished solely by material properties remains still a very challenging task. Here a simple separation of differently charged, thermo-responsive polymeric particles (for example microgels) but equal in size, via the combination of pressure-driven microfluidic flow and precise temperature control is proposed. The separation principle relies on forcing thermo-responsive microgels to undergo the volume phase transition during heating and therefore changing its size and correspondingly the change in drift along a pressure driven shear flow. Different thermo-responsive particle types such as different grades of ionizable groups inside the polymer matrix have different temperature regions of volume phase transition temperature (VPTT). This enables selective control of collapsed versus swollen microgels, and accordingly, this physical principle provides a simple method for fractioning a binary mixture with at least one thermo-responsive particle, which is achieved by elution times in the sense of particle chromatography. The concepts are visualized in experimental studies, with an intend to improve the purification strategy of the broad distribution of charged microgels into fractioning to more narrow distribution microgels distinguished solely by slight differences in net charge.

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.