Elution Research Articles

A simple, cost-efficient stability-indicating RP-HPLC method for simultaneous estimation of embelin and piperine for routine analysis of marketed polyherbal capsules and tablets

Embelin (EMB) and Piperine (PIP) alkaloids are reported for ­antidiabetic, hypolipidemic, antioxidant, and anti-cancer properties. However, simultaneous analytical methods are scarce. A stability-indicating RP-HPLC method was developed with mobile phase MeOH: 0.1%TEA (pH 4.2 adjusted by OPA) (92:08 v/v ratio), CHEMSIL-ODS, C18 (4.6 × 250mm, 5 µm) column, and 1.2 mL/min ­flowrate. Retention times were 3.2, and 6.2 min for PIP and EMB respectively, at 289 nm, and validated as per Q2 (R1) ICH guidelines. Linearity ranging (2-10 µg/mL), with regression coefficients (r 2), LOD, and LOQ for PIP were r 2 = 0.9979, 0.793 µg/mL, 2.403 µg/mL, and r 2 = 0.9974, 0.851 µg/mL, 2.578 µg/mL for EMB. Developed method precision, robustness, and ruggedness were confirmed by <2%RSD. In tablet and capsule formulations, PIP %purity was 90.36%, 94.33%, and EMB 91.65, 92.85%. Additionally, forced-degradation studies ­indicated method stability through various stressed conditions. Hence, this ­validated method is specific, precise, linear, accurate, and robust, ­suitable for routine analysis of EMB and PIP in polyherbal ­formulations containing Embelia ribes Brum. and Piper nigrum Linn. extracts.

Novel RP-HPLC Method Development and Validation of Budesonide Suppository

In the current study, a simple and cost-effective stability-indicating RP-HPLC method was developed and validated to estimate Budesonide from bulk and pharmaceutical dosage forms.0.1% formic acid and methanol (15:85 v/v) were employed as the mobile phase with an injection volume of 20μl, and detection was carried out at 244 nm. The developed method was validated as per ICH Q2 guidelines. The RT of Budesonide was determined to be 4.3 ± 0.328 minutes, providing a reliable marker for its identification. The method was found to be linear between 2-12 μg/ml concentration with (R²) of 0.997. This demonstrates the method's ability to measure varying concentrations of Budesonide accurately. Additionally, the percentage recovery of Budesonide was approximately 100%, confirming the accuracy of the developed method. The parameters for the system's suitability have also been found to be within acceptable limits. Force degradation studies reinforced the method's selectivity and sensitivity in detecting Budesonide under various degradation scenarios. In conclusion, our proposed RP-HPLC method provides a sensitive, accurate, and precise way to analyze Budesonide in bulk and pharmaceutical dosage forms.

Method Validation of Crisaborole using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Quantitation of Crisaborole in Nanoemulsion Formulation for its Application in Ex Vivo Transdermal Permeation Study

Crisaborole is a boron containing phosphodiesterase 4 (PDE4) inhibitor which in this study was formulated in nanoemulsion form to treat atopic dermatitis. The study aims to develop and validate a selective analytical method for determining crisaborole in nanoemulsion formulations, facilitating its application in ex vivo transdermal permeation studies for the treatment of atopic dermatitis. Crisaborole was eluted using gradient elution method in reverse phase (C18) chromatography. The mobile phases were comprised of phosphate buffer (pH3) and acetonitrile, which increasing acetonitrile volume from 30 to 95 percent within 15 min with constant flow rate of 0.65 ml/min. Column temperature was set at 40 ºC and detection wavelength was at 252 nm. The method produced linear responses (R2=0.99) in concentrations ranges from 1.56-100 μg/ml (n=7). The developed method was sensitive with limit of detection (LOD) and limit of quantitation (LOQ) values of 1.84 μg/ml and 5.58 μg/ml; respectively. Crisaborole was stable in various working conditions which were within the acceptance value provided by guidelines (±15%). In ex vivo studies, the concentration of crisaborole was plotted in cumulative permeated graph and this quantitative method was proven to provide a selective and sensitive measurement for detection of crisaborole.

Column Screening and Development of HILIC and RPLC Methods Coupled to Tandem Mass Spectrometry for the Monitoring of Albumin on Cysteine 34 Exposed to Mustard Agents.

Adduction on protein nucleophile sites by mustard agents can be monitored to assess detection of retrospective exposure to these agents. Cysteine 34 (Cys34) on human serum albumin was selected as the target of choice. This work targets di- and tripeptides adducted on Cys34 by sulfur mustard, sesquimustard, and nitrogen mustards separated in hydrophilic liquid chromatography (HILIC) and Reversed-Phase (RP) mode. The effect of several mobile phase additives on the mass spectrometry (MS) and MS/MS signal and on LC retention profile was studied. A mix of ammonium acetate and acetic acid offered satisfactory results in terms of MS sensitivity. Screening of HILIC columns was performed, and ZIC-HILIC stationary phase was selected for HILIC mode, and C18 stationary phase was used for RP analysis. Negative ionization mode leads to a higher S/N ratio compared to positive ionization mode. Adducted tripeptides were selected for the monitoring of mustard agents' exposure, allowing better sensitivity than their dipeptide homologues. The two developed chromatographic methods have similar sensitivities with LOQs ranging from 1.9 to 20.5ng/mL for the reversed-phase liquid chromatography (RPLC)-ESI-(-)-MS/MS method and from 1.7 to 43.3ng/mL for the HILIC-ESI-(-)-MS/MS method. The monitoring method should be selected based on the targeted mustard agent, and the remaining method can be a confirmation tool.

Analysis of The Chemical Content of Paracetamol and Dexamethasone in Pegal Linu Herbal Medicine from "X" Herbal Store in Lamongan Regency

Chemical Drug Substances (BKO) are added to herbal medicines by BPOM because it can cause dangerous side effects. The purpose of this study is to quantify the amounts of dexamethasone and paracetamol in herbal remedies for aches and pains. In Lamongan District, Lamongan Regency, four samples of herbal remedies for aches and pains were employed in the study at herbal medicine shop X. The experiment was conducted using a combination of qualitative analysis and TLC testing. The stationary phase was a silica gel GF254 TLC plate, while the mobile phase was paracetamol made with a combination of ethyl acetate, N-Hexane, and dexamethasone with chloroform.Quantitative analysis uses HPLC with a stationary phase in the form of a Zorbax ODS column 4.6 ID×250 nm (5µm) and the mobile phase is polar, namely using water and methanol and can be detected at a wavelength of 242 nm. The TLC test results showed the presence of BKO paracetamol with Rf values ​​in sample A of 0,46, B=0,46, and C=0,53. Meanwhile, the Rf of dexamethasone for sample A was 0,32, B=0,53, and C=0,58. The results of quantitative analysis for sample A were 25,59 mg/50 mg, B=19,32 mg/50 mg, C=9,40 mg/50 mg, and D=29 mg/50 mg paracetamol in herbal medicine powder. Meanwhile, herbal medicine B contains 19,95 mg/50 mg of dexamethasone and C=24,64 mg/50 mg for herbal medicines A and D which do not contain dexamethasone. It was concluded that the herbal medicine samples containing BKO paracetamol were in herbal medicines A, B, C, and D, while dexamethasone was only in herbal samples B and C.

Highly Sensitive LC-MS/MS Method for Determination of Dexamethasone in Rat Plasma and Brain Tissue: An Application to Pharmacokinetic Study in Rats.

A highly sensitive and rapid LC-MS/MS method was developed and validated for the quantification of dexamethasone in rat plasma and brain tissue. Protein precipitation method was used for sample preparation. The separation of dexamethasone and the IS (labetalol) was achieved on an Atlantis dC18 column using an isocratic mobile phase (10 mM ammonium formate and acetonitrile, 25/75, v/v) delivered at 0.7 mL/min flow-rate. Dexamethasone and the IS were eluted at 1.03 and 1.06 min, respectively. The MS/MS transitions monitored were m/z 393.100 → 373.100 (dexamethasone) and 329.100 → 91.100 (IS). Method validation was performed as per FDA guidelines and all parameters met the acceptance criteria. The assay was validated with a quantification range of 0.05-1046 ng/mL in both matrices. The intraday and interday precision for were in the range of 2.62-7.28 and 2.76%-6.98% and 2.24-6.85 and 2.97%-6.37%, in plasma and brain tissue, respectively. Dexamethasone was stable in a series of stability conditions in both matrices. Post-intravenous administration to rats, dexamethasone concentrations in plasma and brain tissue were quantifiable up to 24 and 10 h, respectively. Dexamethasone half-life was ~2.30 h. Dexamethasone exhibited low clearance and moderate volume of distribution in plasma but in brain tissue the clearance and volume of distribution were high.

Chromatographic Separation and Quantification of Nine Nitrosamine Genotoxic Impurities in a Single Method in Nebivolol Tablet by Using Validated Ultra-Sensitive Liquid Chromatography: Mass Spectrometry Analytical Method.

N-nitrosamine impurities have been detected in a vast variety of drug substances and drug products, showing concern for regulatory aspects. To meet the regulatory requirement for the concerned impurity, a sensitive analytical method capable of quantifying these impurities at a lower level with accuracy and precision is required. This article focuses on the development and validation of an analytical method for the simultaneous detection of nine nitrosamine impurities in a single method for nebivolol drug product using liquid chromatography-mass spectrometry/mass spectrometry-atmospheric pressure chemical ionization (LC-MS/MS-APCI). The chromatographic separation was performed using the LC-MS column Allure BiPh C18 (250 × 4.6mm), 5μm employed a gradient mode elution program using 0.002M Ammonium acetate buffer pH4.5 as mobile phase A and methanol as mobile phase B. The method was challenged for accuracy, precision and linearity in accordance with International Council for Harmonization guidelines to ensure its suitability for the intended usage. The developed method was specific, accurate and linear with square of correlation coefficient (r2) found to be greater than 0.99 (0.9970-0.9992). The LOQ obtained in the range of 9.85-19.62ppb for nine nitrosamines showed good sensitivity. The results demonstrated that method can be applied to quantify the nitrosamines in nebivolol drug products.

Improving process robustness of cation exchange chromatography with cationic buffers for the reduction of aggregates.

Cation exchange chromatography (CEX) is commonly used to separate aggregates from monomers during the industrial manufacturing of recombinant proteins. However, the similar isoelectric point of aggregates and monomers makes the stepwise elution CEX an unstable process. In this study, the performance robustness of sodium chloride stepwise elution and cationic buffers (histidine and Bis-Tris) stepwise elution were compared through Monte Carlo simulation. While all trials achieved acceptable levels of monomer purity, sodium chloride stepwise elution exhibited significant fluctuations in yield and elution volume due to variations in elution pH and eluent concentration. In contrast, histidine or Bis-Tris stepwise elution resulted in more consistent yield and elution volume across a broad operating range. The findings indicate that the dissociation behavior of cationic buffers mitigates the impact of pH variation on ion-exchange equilibrium and thus enables a more robust CEX process.

Analytical Quality by Design Based HPLC for Quantitative Analysis of Teneligliptin and Rosuvastatin Calcium Tablets in the Presence of Force Degradation Products

ABSTRACTThe application of analytical quality by design in developing an HPLC method ensures robust, reliable, and regulatory‐compliant pharmaceutical analysis. This study aimed to establish a stability‐indicating HPLC for the quantification of teneligliptin and rosuvastatin calcium in tablets using analytical quality by design principles. The analytical target profile was defined, and a risk assessment identified critical method attributes and critical method parameters. A Plackett–Burman design screened significant critical method parameters, followed by optimization using a central composite design. The optimized method utilized a Phenomenex Gemini C18 column with a mobile phase of phosphate buffer (pH 5.0) and ACN (64:36% v/v), achieving retention times of 4.27 ± 0.2 min for teneligliptin and 11.84 ± 0.2 min for rosuvastatin. The HPLC method was validated as per International Council for Harmonisation (ICH) guidelines, demonstrating good linearity across concentration ranges of 20–60 µg/mL of teneligliptin and 10–30 µg/mL of rosuvastatin, respectively. Forced degradation studies under various stress conditions confirmed the method's specificity, ensuring reliable detection of teneligliptin and rosuvastatin even in the presence of degradation products. This study offers an innovative and efficient solution for the analysis of teneligliptin and rosuvastatin in combined fixed‐dose tablet formulations.

Green Extraction and Optimization of Lutein From Tagetes erecta by Ultrasound‐Assisted Extraction Method and Identification by High‐Performance Liquid Chromatography Method

ABSTRACTLutein, an essential nutrient, is present (0.01%–0.5%) in marigold flowers (Tagetes erecta L.) and has been associated with benefits for eye health, including protection against age‐related macular degeneration. Green extraction methods and green solvents have been reported to extract high yields of lutein, maintain purity, and reduce environmental impact compared to conventional methods. This study aims to extract lutein from marigold flowers using green extraction methods (maceration, soxhlet, and ultrasound‐assisted extraction [UAE]) and green solvents (isopropyl alcohol [IPA], ethanol, 2‐methyltetrahydrofuran [2‐MeTHF], dimethyl carbonate [DC], and cyclopentyl methyl ether [CPME]). Using the Box–Behnken design (BBD), the selected method was optimized and evaluated for lutein extraction and antioxidant activity. The optimal conditions for the ultrasound extraction method are ultrasonic power (100 W), extraction time (2.5 min), and a solid‐to‐solvent ratio (5%). Lutein was quantified by HPLC with a mobile phases of acetonitrile and methanol (40:60) at 450 nm. The UAE method using 2‐MeTHF showed the highest lutein yield (41.5 ± 1.4 µg/mL), surpassing hexane (25.8 ± 1.9 µg/mL) and other green solvents. By optimizing the extraction method, significant improvements were achieved in both lutein extraction yield (10%) and antioxidant activity (93.78%). The lutein yield increased 240.96‐fold, as compared to reported values.

Stability-Indicating Liquid Chromatographic Method Development for the Simultaneous Determination of Amitriptyline Hydrochloride and Propranolol Hydrochloride in Tablet Dosage Form.

The combination of the tricyclic antidepressant amitriptyline hydrochloride (AMH) and the non-selective beta-adrenergic blocker propranolol hydrochloride (PPH) is used for migraine prophylaxis. Higher doses of AMH trigger cardiac arrhythmias, anxiety, tachycardia, convulsions, hyperglycemia and anticholinergic side effects. The combined dosage formulation of AMH and PPH leads to drug-drug interactions; causes sedation, xerostomia, dysuria, insomnia and bradycardia; and results in patient non-compliance. The quantification of AMH and PPN becomes essential, especially for combination formulations, in addition to regular quality control to avoid clinical issues. Considering these facts into account, the reverse-phase -high-performance liquid chromatography (RP-HPLC) method was developed in accordance with International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) guidelines for the simultaneous determination of AMH and PPH. The HPLC separation was performed on an HPLC system (Shimadzu, Japan, Prominence I series 2030C) using a Shimadzu Shim-Pack GIST C18 column (100mm × 4.6mm, 5μ), which was equipped with an ultraviolet detector at the isosbestic point 238nm. The mixture of acetonitrile and orthophosphoric acid (pH3.5) in a ratio of 35:65v/v with a flow rate of 0.75mL/min was used as the mobile phase. The regression coefficients of AMH (r2 > 0.998) and PPH (r2 > 0.999) show good linearity between peak areas and drug concentration ranges. The limits of detection (AMH = 0.67μg/mL, PPH = 0.67μg/mL) and limits of quantification (AMH = 2.04μg/mL, PPH = 2.05μg/mL) demonstrated the higher detection sensitivity of the proposed method.

Natural Eutectic Solvent-Based Temperature-Controlled Liquid–Liquid Microextraction and Nano-Liquid Chromatography for the Analysis of Herbal Aqueous Samples

In this work, two novel (-)-menthol-based hydrophobic natural eutectic solvents with vanillin and cinnamic acid were prepared and applied as extraction solvents. In this regard, 12 endocrine disruptors, including phenol, 2,4-dimethylphenol, 2,3,6-trimethylphenol, 4-tert-butylphenol, 4-sec-butylphenol, 4-tert-amylphenol, 4-n-hexylphenol, 4-tert-octylphenol, 4-n-heptylphenol, 4-n-octylphenol, and 4-n-nonylphenol and bisphenol A, were studied in a green tea drink. A temperature-controlled liquid–liquid microextraction was used as the extraction method, and nano-liquid chromatography–ultraviolet detection was used as the separation and determination system. Different parameters affecting the compatibility of the non-ionic eutectic solvents with water-polar organic solvent mixtures and chromatographic and detection systems were optimized, including injection/dilution solvent, injection mode, mobile phase composition, and step gradient. With the same purpose, two stationary phases were tested, including XBridge® C18 and a mixed-phase Cogent C30-XBridge® C18. Finally, the greenness and blueness of the methodology were assessed to evaluate the environmental profile and usability of the procedure.

Quantitative Determination of a Series of Oxysterols by an Optimized LC-MS/MS Analysis in Different Tissue Types.

Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.

A Highly Sensitive RP HPLC‐PDA Analytical Method for Detection and Quantification of a Newly Synthesized (E‐2‐((E)‐4‐(5‐Ethoxy‐3‐Methyl‐1‐Phenyl‐1H‐Pyrazole‐4‐yl)but‐3‐en‐2‐Ylidene)) Hydrazine‐1‐Carbothioamide in Nanosuspension

ABSTRACTAnalytical methods development and validation are vital for the precise detection, quantification, and characterization of novel therapeutic compounds, especially those with poor aqueous solubility, such as pyrazolone derivatives. This study aimed to develop and validate a sensitive, accurate, and efficient RP HPLC‐PDA method for the detection and quantification of novel (E‐2‐((E)‐4‐(5‐ethoxy‐3‐methyl‐1‐phenyl‐1H‐pyrazole‐4‐yl)but‐3‐en‐2‐ylidene) hydrazine‐1‐carbothioamide in nanosuspension. The method was optimized for high sensitivity and specificity using a Shim‐pack GIST C18 (5 µm, 150 × 4.6 mm) column, with an isocratic mobile phase of ACN and 0.1% TFA in water (75:25 v/v). It employed a 0.5 mL/min flow rate, a 20 µL injection volume, and detected the compound at 333 nm. The method showed excellent linearity (R2 = 0.9994) over a concentration range of 2.5–50 µg/mL, with high precision, accuracy, and reproducibility, in compliance with ICH Q2 (R1) guidelines. The LOD and LOQ were 2.43 and 7.38 µg/mL, respectively. Recovery rates ranged from 110% to 112%, with RSD below 2%. The validated RP HPLC‐PDA method was effectively applied to detect, characterize, and quantify the novel compound in its nanosuspension form. This method offers a reliable analytical tool for the quality control of this novel compound, both in raw material and finished product forms, as well as for impurity profiling, drug release, and stability testing, which will, in turn, facilitate new drug development.

Separation, Identification, and Characterization of Transformation Products Related to Relugolix Upon Subjecting to In Vitro Gastrointestinal Fluids Utilizing Liquid Chromatography, Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy

ABSTRACTThis study investigated the gastrointestinal stability of relugolix, a biopharmaceutical classification system Class IV drug. Relugolix was found to be unstable in simulated gastric fluid but stable in simulated intestinal fluid and fed‐state–simulated gastric fluid. The high‐performance liquid chromatography method was developed and validated for separating the drug and its transformation products. The optimized method comprises mobile phase ammonium acetate (pH 4.75) as Solvent A and acetonitrile as Solvent B. A Phenomenex Gemini C18 (250 × 4.6 mm, 5 µm) column was used as the stationary phase. The transformation product, desmethyl relugolix, was identified and characterized using high‐resolution mass spectrometric and nuclear magnetic spectroscopic studies by enriching the compound using forced degradation studies under acidic stress hydrolytic conditions. Sodium alginate microspheres were prepared and evaluated to improve relugolix stability in the stomach. Additionally, gastrointestinal stability studies demonstrated that the microspheres protected relugolix from degradation in simulated gastric fluid. In silico analysis predicted minimal toxicity risks for both relugolix and desmethyl relugolix. These findings suggest that relugolix sodium alginate microspheres could be a promising strategy to mitigate the formation of desmethyl relugolix by protecting it from degradation in the stomach's acidic environment.

Green and High Throughput HPTLC Method for Simultaneous Estimation of Celecoxib and Tramadol Hydrochloride in their Newly Approved Analgesic Combination and Spiked Plasma with Dichromic Green and Blue Assessments.

The FDA "Food and Drug Administration" recently approved a novel co-crystal formulation of Celecoxib (CEX) and Tramadol (TRM) for the treatment of adults suffering from moderate to severe pain in several conditions. This novel combination has advantages over co-administration of the two drugs individually as better patient compliance, synergism and lower therapeutic cost. This work presents the first "High performance Thin Layer Chromatographic" (HPTLC) quantitative analytical technique for CEX and TRM simultaneous assay in bulk, their new dosage form and plasma.The proposed HPTLC assay is based on separation of CEX and TRM on silica gel 60 F254 sheets followed by densitometric scanning at 270nm. The plates' development was carried out using a mobile phase of ethyl acetate-methanol-ammonia (5:5:0.05, v/v). The two drugs showed linearity of 0.025-1μg.band-1& for plasma analysis linearity range was 0.2-10μg.mL-1plasma. The proposed chromatographic technique was validated and showed satisfying validation characteristics of linearity, selectivity, precision and accuracy. In addition, the assay was evaluated by AGREE, AGREEprep, ComplexMoGAPI and BAGI for greenness and blueness to ensure the method's environmental sustainability and its safety for routine quality control assay of this novel combination.

Mangiferin Content in N-Hexane and Diethyl Eter Fractions of Mango (Mangifera indica L.) Leaves Var. Gedong Gincu

Mango (Mangifera indica L.) is a tropical fruit plant with high economic value and potential as a source of phytopharmaceuticals. One well-known variety of mango is Gedong Gincu from Majalengka Regency, West Java, which contains active compounds such as mangiferin, flavonoids, saponins, and tannins, all of which have various therapeutic effects. This study aims to compare the mangiferin content in the n-hexane and ethyl acetate fractions of Gedong Gincu mango leaf extract using the High Performance Liquid Chromatography (HPLC) method. Mango leaves were extracted using ethanol, followed by fractionation with n-hexane and ethyl acetate. After obtaining the fractions, the mangiferin content was analyzed using the HPLC method. The weights of the n-hexane and ethyl acetate fractions were found to be 13.418 grams and 8.21 grams, respectively. Phytochemical screening revealed the presence of flavonoids, alkaloids, phenols, tannins, and saponins. The HPLC results showed that the two fractions had different mangiferin contents, with retention times (RT) of 1.599 minutes for the n-hexane fraction and 1.473 minutes for the ethyl acetate fraction, indicating differences in solubility and interactions with the mobile phase. Mangiferin content analysis using HPLC revealed that the n-hexane fraction contained 690.8 mg/g of mangiferin, while the ethyl acetate fraction contained 547.3mg/g . This study provides important information regarding the comparison of extraction solvents for mangiferin, which can be developed for herbal preparation production.

Umbrella Sampling MD Simulations for Retention Prediction in Peptide Reversed-phase Liquid Chromatography.

Reversed-phase liquid chromatography (RPLC) is an essential tool for separating complex mixtures such as proteolytic digests in bottom-up proteomics. There is growing interest in methods that can predict the RPLC retention behavior of peptides and other analytes. Already, existing algorithms provide excellent performance based on empirical rules or large sets of RPLC training data. Here we explored a new type of retention prediction strategy that relies on first-principles modeling of peptide interactions with a C18 stationary phase. We recently demonstrated that molecular dynamics (MD) simulations can provide atomistic insights into the behavior of peptides under RPLC conditions (Anal. Chem. 2023, 95, 3892). However, the current work found that it is problematic to use conventional MD data for retention prediction, evident from a poor correlation between experimental retention times and MD-generated "fraction bound" values. We thus turned to umbrella sampling MD, a complementary technique that has previously been applied to probe noncovalent contacts in other types of systems. By restraining the peptide dynamic motions at various positions inside a C18-lined pore, we determined the free energy of the system as a function of peptide-stationary phase distance. ΔGbinding values determined in this way under various mobile phase conditions were linearly correlated with experimental retention times of tryptic test peptides. This work opens retention prediction avenues for novel types of stationary and mobile phases, and for peptides (or other analytes) having arbitrary chemical properties, without the need for RPLC reference data. Umbrella sampling can be used as a stand-alone tool, or it may serve to enhance existing retention prediction algorithms.

Isocratic liquid chromatography technique for the analysis of cyanocobalamin from beef liver and heart muscle extracts

Reverse-phase high-performance liquid chromatography (RP-HPLC) is among the most widely recommended techniques for analyzing water-soluble vitamins such as vitamins C and B-complexes. The research study was conducted to detect and quantify cyanocobalamin (vitamin B12) from the beef liver and heart muscle extracts using a validated isocratic RP-HPLC procedure. The analytical column was Luna® Phenomenex 5 µm C18 (2) 100 A LC-column (150 × 4.6 mm). The mobile phase consisted of water/ethanol in a ratio of 60:40 (v/v). An enzymatic digestion with 1% potassium cyanide was used for samples (beef liver and heart muscle) extraction. The validated method showed to be linear, R = 0.9977; fast, with a retention time of less than 6.00 min; precise, %RSD of 1.16 and 1.74%; and sensitive, with limits of detection and quantification of 0.00033 and 0.00100 µg/mL. The detected values of cyanocobalamin from the beef liver (BLV) and heart muscle (HRM) extracts were 52.04 ± 0.13 and 42.04 ± 0.29 µg/mL, respectively. BLV extract indicated a higher level of cyanocobalamin. Hence, the validated isocratic RP-HPLC technique can be recommended to analyze cyanocobalamin and related compounds in research laboratories such as diagnostics, foods, and pharmaceuticals.

High-performance liquid chromatography method for measuring Captopril: an empirical study on hydrogel film permeability test.

High-performance liquid chromatography (HPLC) has emerged as a highly sensitive and versatile analytical technique for quantifying antihypertensive drugs, such as Captopril (CAP). This study focused on the optimization and validation of an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. The main objective of this study was to develop and validate an HPLC method for quantifying CAP in an in vitro hydrogel permeability test. The HPLC method employed a C18 column (Waters, Sunfire, 5μm; 250 × 4.6mm) and a mobile phase consisting of methanol-water (85% v/v orthophosphoric acid) in a 55:45 (v/v) ratio at a flow rate of 0.5 mL/min. The UV-Vis detector was configured to detect CAP at a wavelength of 220nm. The hydrogel film used in the permeability test was prepared using poly (vinyl alcohol)/poly (vinyl caprolactam) (PVA/PNVCL) with citric acid as a crosslinking agent. All results met the validation parameters according to ICH Guideline. The HPLC method showed consistent retention time (4.7-4.9min), linearity (1-50µg/mL; r = 0.9995), accuracy (98.11-101.78%), precision (RSD ≤ 2%), and LoD/LoQ (0.19/0.62µg/mL). The developed HPLC method was successfully applied to an in vitro permeability test using horizontal diffusion cells. The results demonstrated that CAP permeated through the swollen hydrogel film, with a cumulative drug permeation exceeding 30%. This highlighted the method's utility in assessing drug transport properties through hydrogels. The validated HPLC method demonstrates robustness and reliability for quantifying CAP in the hydrogel permeability test.