Symmetrical Peak Shapes Research Articles

A new method development and validation for simultaneous estimation of ramipril and telmisartan by RP-HPLC method in combined dosage form

The present research work was development and validation of new RP-HPLC method for simultaneous estimation of Ramipril and Telmisartan in combined dosage form. In simultaneous RP-HPLC method development, Waters 2695 Separations Module with PDA Detector and column used is C8 SB ZORBAX (150 X 4.6mm) column with 3.5-micron particle size. Injection volume of 10 µL is injected and eluted with the mobile phase selected after optimization was Phosphate buffer and Acetonitrile in the ratio of 70:30 was found to be ideal. The optimized flow rate was at 1.0 mL/min. Detection was carried out at 230 nm. This system produced symmetric peak shape, good resolution and reasonable retention times of Ramipril and Telmisartan were found to be 2.275 and 4.261 minutes respectively. The Ramipril and Telmisartan showed linearity in the range of 20-60 µg/mL and 160-480 µg/mL respectively. The %RSD values for precision was found to be within the acceptable limit, which revealed that the developed method was precise. The developed method was found to be robust and the %RSD value for percentage recovery of Ramipril and Telmisartan was found to be within the acceptance criteria. The results indicate satisfactory accuracy of method for simultaneous estimation of the Ramipril and Telmisartan.

Determination of multiple restricted and prohibited ingredients in hair dyes using a novel 3D stationary phase coupled with gas chromatography-mass spectrometry

Hair dyes (HDs) are mainly composed of various benzene series, amines, and phenolic compounds. These ingredients are well known to have allergenic, teratogenic, and carcinogenic properties. As such, the presence of these ingredients in HDs has received increased attention in recent years. At present, the applications of traditional analytical and detection methods and commercial chromatographic columns are limited by problems such as poor qualitative analysis and inaccurate quantification. Thus, the development of new analytical and detection technologies and stationary phases is an urgent endeavor. Moreover, HDs contain complex compounds and exhibit significant matrix interference. Hence, appropriate sample pretreatment methods are necessary to analyze HDs. In this study, the 3D nonpolar rigid structure of triptycene (TP) was combined with the polar flexible chains of polyethylene glycol (PEG) to design and synthesize a TP derivative, TP-PEG, as a stationary phase for chromatographic columns. The stationary phase enabled the expansion of the selection range for polar and nonpolar analytes. Subsequently, gas chromatography-mass spectrometry (GC-MS) was used to quantitatively analyze 22 ingredients in HDs. The experimental results demonstrated that analytes with different polarities exhibited sharp and symmetrical peak shapes on the stationary phase, and all 22 analytes achieved baseline separation on the chromatographic column. The 22 ingredients in HDs showed good linear relationships within their respective ranges, with correlation coefficients greater than 0.9985. The average recovery rates at three spiked levels were in the range of 89.2%-103.2%, and RSDs were less than 5%. Compared with traditional methods, the proposed method has higher efficiency and better accuracy, thus verifying the excellent separation performance of the new stationary phase and the effectiveness of the established GC-MS detection method. The findings indicated the applicability of the developed method to the detection and analysis of various compounds in HDs.

Chromatographic evaluation and application of nitrogenous heterocyclic ring-bonded stationary phase in hydrophilic interaction liquid chromatography

Hydrophilic interaction liquid chromatography (HILIC) based on polar stationary phases has vital research significance in the separation of polar compounds. Numerous HILIC stationary phases with different structures have been developed, which do not have universal properties and broad selectivity, making it a challenge to select the suitable column based on the properties of the samples. Consequently, it is particularly important to develop a bonded phase capable of separating a wide variety of samples, while having enhanced retention, improved selectivity, symmetric peak shape and good stability. Herein, a novel nitrogen-containing heterocyclic bonded phase with multiple functionalities, such as thioether, amino and hydroxyl groups (named AMTA) was employed as HILIC stationary phase. Detailed chromatographic evaluations were carried out, and the results showed that it was superior to other hydrophilic chromatographic columns in terms of selectivity, peak shapes and practical sample separation. Lastly, it has been verified that AMTA exhibited high orthogonality with the XBridge C18 column of reversed-phase liquid chromatography (RPLC) mode. In summary, we anticipate our assay to be instructive to other researchers in developing the HILIC stationary phase.

Removal of ceftriaxone and cefotaxime antibiotics from industrial wastewater via complexation with Cu(II).

A high-performance liquid chromatographic method (HPLC) with UV detection is described for determination of ceftriaxone sodium (CFX) and cefotaxime sodium (CFM) content in pharmaceutical industrial wastewater. These methods are based on the detection of these antibiotics via the formation of chelate complexes with Cu(II). The developed Liquid Chromatographic method offers symmetric peak shape, good resolution and reasonable retention time for both drugs. The removal percentage reached about 100 and 92.1% at pH 7.2 for CFX and CFM, respectively. In UV detection, the removal of the chelating antibiotics were based on forming of chelate complexes with Cu(II) which detected at λmax = 253 and 244nm for CFX and CFM, respectively. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5.99-59.86µgmL-1 for CFX and 14.33-71.63 µgmL-1 for CFM. The proposed method can be used for the quality control of industrial wastewater containing CFX and CFM.

Hybrid organic monolithic column containing MIL-68(Al) for the separation of small molecules by capillary HPLC

A hybrid organic monolithic column made of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) and the metal-organic framework MIL-68(Al) was prepared for the first time. The column was used in capillary liquid chromatography, both in isocratic and gradient elution modes. Separation performance towards small molecules of different chemical nature (polycyclic aromatic hydrocarbons, alkylbenzenes, phenols, etc.) was studied. Monte Carlo simulations were made to both select the proper precursors to obtain empty metal-organic framework micropores in the monolithic polymer and also, to analyze the potential free access of the studied analytes into the micropores (necessary to improve mass transfer and column efficiency). The hereby synthesized metal-organic framework microcrystals allowed obtaining homogeneous hybrid monolithic columns. Adding of MIL-68(Al) (1030 m2 g-1 BET specific surface area) increased the surface area from 3.9 m2 g-1 for the parent monolith to 18.2 m2 g-1 for the hybrid column containing 8 mg mL-1 of the microcrystals. Chromatographic performance of this new column was evaluated by studying retention factors, resolution, and plate counts at room temperature. Different compounds, not completely resolved in the parent monolith, were partially or completely separated after metal-organic framework addition. Using the monolithic column with only 2 mg mL-1 of MIL-68(Al), five alkylbenzenes were completely separated with very symmetrical peak shapes, resolution factors up to 3.60 and plate counts of 4300 plates m-1 for n-hexylbenzene. This value is higher than those obtained by other authors who used organic monolithic columns with embedded metal-organic frameworks to perform separations at room temperature. Additionally, nine polycyclic aromatic hydrocarbons were partially or completely resolved in gradient elution mode. The hybrid monolithic columns exhibited very good intra-day (%RSD=1.9), inter-day (%RSD=2.6), and column-to-column (%RSD=4.3) reproducibility values. Easy and fast column preparation, and versatility to efficiently separate several compounds of different chemical nature in isocratic and gradient mode, makes this new hybrid column a very good option for the analysis of small molecules in capillary (or nano) HPLC.

Penetapan Kadar Kuersetin pada Obat Herbal yang Mengandung Ekstrak Tempuyung (Sonchus arvensis) Menggunakan KLT Densitometri

Quercetin are the main marker compounds in extracts of Sonchus arvensis which show nephrolithiasis activity. For quality control of herbal medicines, Densitometric Thin Layer Chromatography (TLC) method has been developed in this study using quercetin as phytochemical markers. Objective: Determination for quercetin in herbal medicines. Determination of quercetin by determining the composition of the mobile phase, the wavelength of the analysis, the saturation time, the elution distance and the volume of the spot. The parameters observed in the optimization method and stability test are area, retardation factor (Rf), peak shape and resolution (Rs). The composition of selected mobile phase which results good separation and symmetrical peak shape is chloroform: methanol: dichloromethane: acetonitrile: formic acid (6: 2: 2: 0.05: 0.05 v/v/v/v/v) with a wavelength of 335 nm, time of saturation is 30 minutes, elution distance is 8 cm, volume of spots is 10 µL and Rf 0.48. In this study, it was shown that there were no factors that had a significant effect so that the method could be applied for the determination of quercetin levels in herbal medicines.The research data showed that the range of levels on quercetin 3.26%.

The Impact of Water as an Additive on the Elution of Some Basic Organic Compounds in Supercritical Fluid Chromatography.

In this study, water was used as an additive in the methanol-modified carbon dioxide-based eluent for the elution of some basic organic compounds from a hybrid silica column via supercritical fluid chromatography (SFC). The experiments were applied to sulfonamides, propranolol, and other organic nitrogen compounds involving aromatic rings from different classes of amine, pyrimidine, and purine with different pKa values (the pKa values for the studied analytes range from 4.6 to 10.4). The results revealed different responses to the different percentages of water addition. Adding 1~2% of water to the modifier (methanol) led to a positive effect manifested by more symmetrical peak shapes and reduced retention times for most compounds. The key factor for this improvement in the properties of chromatographic peaks is due to the adsorption of water on the silanol groups of the stationary phase, consequently resembling the phenomena observed in hydrophilic interaction liquid chromatography (HILIC). Moreover, the availability of hydrogen bond acceptor and donor sites in the analyte structure is an important factor to be considered when adding water as an additive to the modifier for improving the chromatographic peaks. However, introducing water in an amount higher than 3% resulted in perturbed chromatographic signals. It was also found that water as an additive alone could not successfully elute propranolol from the hybrid silica column with an acceptable peak shape; thus, the addition of a strong base such as amine salts was also necessary. The proposed use of a particular amount of water in the mobile phase could have a positive effect compared to the same mobile phase without water, improving the chromatographic peak properties of the elution of some basic organic compounds from the hybrid silica column.

Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (ERLIC) for the Quantitative Analysis of Polyamines

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules.

A nitrogenous heterocyclic ring-bonded stationary phase for separating alkaloids in supercritical fluid chromatography

A novel silica stationary phase was designed and prepared through thiol-epoxy click chemistry for supercritical fluid chromatography (SFC). The developed stationary phase was characterized by elemental analysis, Fourier transform infrared spectrometry and solid-state 13C/CP MAS NMR spectroscopy. In order to evaluate the chromatographic performance and retention mechanisms of the prepared column, a variety of alkaloids were used, including indoles, isoquinolines, pyrrolidines, piperidines, quinolizidines and organic amines. The stationary phase showed more symmetrical peak shapes and better performance for these compounds compared to the conventional SFC stationary phases. The investigations on the effects of pressure and temperature on retention provided information that the selectivity of the compounds can be improved by changing the density of the supercritical fluids. Moreover, it shows improved separation efficiency of three natural products with alkaloids as the main components at high sample loading. In conclusion, the developed stationary phase could offer flexible selectivity toward alkaloids and complex samples.

High Selectivity of A Novel Pillar[5]arene with Ester Units as a Gas Chromatographic Stationary Phase toward Aromatic Isomers.

This work reports the first example of employing ester-functionalized pillar[5]arene (P5A-C10-OAc) stationary phase for gas chromatography (GC) separations. The as-fabricated P5A-C10-OAc column achieved improved column efficiency of 4270 plates/m and separation performance in contrast to the P5-C10-Br column. The P5A-C10-OAc column showed good separation performance for a wide range of analytes such as alkanes, bromoalkanes, ketones, fatty acid methyl esters, aldehydes, alcohols, halobenzenes, anilines, phenols, naphthalenes, and showed sharp and symmetrical peak shapes for analytes that are liable to peak-tailing in GC analysis. As testified by the challenging isomer mixtures (bromonitrobenzene, chloronitrobenzene, bromobenzaldehyde, chlorobenzaldehyde, nitrobenzaldehyde), the P5A-C10-OAc column exhibited comprehensively higher separation capability than the P5A-C10-Br, P5A-C10 and commercial HP-35 columns. This work demonstrates the great potential of pillararene-based stationary phases as a new type of stationary phases for GC separations.

Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples

IntroductionMitotane (o,p’–DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. ObjectiveTo develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. MethodDrug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p’-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. ResultsThe present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4’-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00–50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). ConclusionDDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Concepts and principles for new rapid simple liquid chromatography method for quantification of antioxidants resveratrol, vitamin E, and coenzyme Q10 in capsules with high-performance liquid chromatography with a photo-diode array detector.

Principles and problems in the development of simultaneous liquid chromatography (LC) analytical methods for potent antioxidative molecules resveratrol, tocopherol, and coenzyme Q10 in capsules, have been investigated and systematically compared and summarized. For these purposes, experiments within the full polarity spectrum of LC techniques. were tested and recorded. The whole range of polarities included: Alkyl C18 bonded reversed phase, phenyl, cyanopropyl, diol, and the most polar base silica-filled column matrixes have been used. The summarized results concluded that all mentioned LC techniques could be used for the determination of the mentioned group of the three analytes with different run characteristics and efficiency. These successes could be achieved after careful analyses of molecular physicochemical data of analytes. They are especially organic solubilities. The ultraviolet spectral absorption characteristics of each analyte and the mobile phase constituents for appropriate separation were very important to be known. The ultimate targets were the development method with the isocratic mode of separation yielding symmetrical peak shapes for the best sensitivity and accuracy, with the shortest run time and best reproducibility. From an analytical point of view important for LC, the three analytes have quite distinct characteristics that contribute to successful method development. These features are their organic solvent and water solubility, molecular polarities, and ultraviolet-absorption characteristics, like spectra and absorptivities. All these mentioned parameters were taken into account for solving complications appearing in the development of rapid LC methods for the simultaneous determination of three antioxidant molecules.

GC-FID and GC-MS detection of Ethyl Isobutyryl Acetate as regulatory starting material

We developed GC-FID and GC-MS methods to determine the assay and chromatographic purity of Ethyl isobutyryl acetate (EIBA). The symmetric peak shape was on a DB-Wax stationary phase with the dimensions of 30 m column length, 0.25 mm as internal diameter, 0.50 microns film thickness with the mobile phase as helium gas in temperature ramping mode at a flow rate of 1.5 mL/min using flame ionization detector and mass spectrometer. The USP resolution between EIBA and its unknown impurities was more than 2.0, indicating a significant separation. Regression analysis shows a correlation coefficient greater than 0.999 for EIBA assay and chromatographic purity techniques. The detection and quantitation limits of EIBA are 0.01% and 0.03% for the chromatographic purity procedure. This method indicates that the recovery at different levels is 90 to 110% accurate. The test solution was stable in the diluent for 48 h and subjected to stress conditions. The mass balance was close to 99.5%.

Development of a Novel LC-MS/MS Multi-Method for the Determination of Regulated and Emerging Food Contaminants Including Tenuazonic Acid, a Chromatographically Challenging Alternaria Toxin.

The regulation of food contaminants in the European Union (EU) is comprehensive, and there are several compounds in the register or being added to the recommendation list. Recently, European standard methods for analysis have also been issued. The quick analysis of different groups of analytes in one sample requires a number of methods and the simultaneous use of various instruments. The aim of the present study was to develop a method that could analyze several groups of food contaminants: in this case, 266 pesticides, 12 mycotoxins, 14 alkaloid toxins, and 3 Alternaria toxins. The main advantage of the herein described approach over other methods is the simultaneous analysis of tenuazonic acid (TEA) and other relevant food contaminants. The developed method unites the newly published standard methods such as EN 15662:2018, EN 17194:2019, EN 17256:2019, EN 17425:2021, EN 17521:2021, which describes the analysis of both regulated and emerging contaminants. The developed method is based on a QuEChERS sample preparation, followed by LC-MS/MS analysis under alkaline mobile phase conditions. The pH of the aqueous eluent was set to 8.3, which resulted in baseline separation among ergot alkaloids and their corresponding epimers, a symmetric chromatographic peak shape for analyzing TEA and fit-for-purpose sensitivity for MS/MS detection in both positive and negative ionization modes. Those compounds, which possess the corresponding isotopically labeled internal standards (ISTD), allowed for direct quantification by the developed method and no further confirmation was necessary. This was proven by satisfactory analyses of a number of quality control (QC), proficiency test (PT), and validation samples.

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

PSAT096 Development and Validation of Mitotane Determination in Plasma Samples by LC-DAD

Abstract Introduction Mitotane (o,p'–DDD) is the drug of choice for adrenocortical carcinomas (ACC) and its measurement in plasma is essential to control drug administration. Objective To develop and validate a simple, reliable and straightforward method for Mitotane determination in plasma samples. Method Drug free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of DDD. p,p´-DDD was used as internal standard and was added at 25.0 µg /mL to each sample, standards and controls. Samples were submitted to protein precipitation with acetonitrile and than centrifuged and 50 uL sobrenadant was injecting into a 1260 Infinity II liquid chromatography system (Agilent Technologies) and DDD and IS were detected at 230 nm by diodo array detector (DAD) in a 12 minutes isocratic mode with a solvent mixture of 60% acetonitrile and 40% formic acid in water 0.1% pump mixed, at 0.6 mL/min flow rate. Chromatography column Waters Acquity HSS T3 was kept at 28°C. We evaluated sensitivity, precision, presence of carry-over, recovery, linearity and the accuracy of this method. Results Our method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4´-DDD (internal standard) with retention times of 6.0min, 6.4mim, respectively, with resolution higher than 1.5. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.0 to 50.0 µg/mL for DDD (R2 > 0.9987 and a 97.8% - 105.5% extraction efficiency). Analytical sensitivity was 0.98 µg/mL, functional sensitivity was 1.00 µg/mL, intra-assay and inter-assay coefficient of variation were less than 9. 98%, carry-over was not detected, recovery ranged from 98% to 117% and high accuracy was observed (89.4% to 105.9%) with no matrix effects for mitotane measurements. Patient's samples results were compared with previous measurement by GC-MS method with a high correlation (r=0.88 and bias=-10.2%) showed that our method is usefull for routine mitotane measurement. Conclusion DDD determination in plasma samples by our developed and validated method is simple, robust, efficient and sensitive for therapeutic drug monitoring and dose management in order to achieve therapeutic index of mitotane in patients with adrenocortical cancer. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

An LC-ESI-MS/MS assay for the therapeutic drug monitoring of 15 antiseizure medications in plasma of children with epilepsy.

Oral antiseizure medications are the preferred option for the clinical treatment of epilepsy. Therapeutic drug monitoring has become an important means of achieving individualized treatment of epilepsy. A sensitive, accurate and rapid LC-ESI-MS/MS method was developed and validated for the simultaneous determination of 15 antiseizure medications in human plasma (carbamazepine, gabapentin, pregabalin, phenytoin, zonisamide, oxcarbazepine, tiagabine, lamotrigine, topiramate, phenobarbital, lacosamide, primidone, 10,11-Dihydro-10-hydroxy carbamazepine, ethosuximide, and levetiracetam). The sample preparation procedure was an one-step protein precipitation with methanol. Mass detection was performed in ionization polarity switching mode (positive-negative-positive) using multiple reaction monitoring mode. A "boot-shaped" gradient elution program was applied to separate and concentrate those target analytes, resulting in symmetrical peak shapes within 10min, without endogenous interference. The method showed great linearity over the concentration ranges with acceptable correlation coefficients (0.9966-0.9996). The precision and accuracy values for intra- and inter-assays were within ±15%. Consequently, the method was successfully implemented on pediatric patients undergoing mono- or polytherapy for epilepsy and provided timely concentration results to ordering clinicians.

Stability-indicating HPLC method for the determination of related substances in lansoprazole sulphide

Symmetric peak shape was on a C18 stationary phase with the dimensions of 250 mm column length, 4.6 mm as internal diameter, 5 microns particles with an economical and straightforward mass-compatible mobile phase combination of formic acid and acetonitrile delivered in gradient mode at a flow rate of 1.0 mL/min at 260 nm. The resolution between Lansoprazole sulphide (LAN30) and its impurities (LAN30-I & LAN30-II) in the developed method was more than 2.0, indicating a significant separation. Regression analysis shows a correlation coefficient greater than 0.999 for Lansoprazole sulphide and its related substances. The detection limits of Lansoprazole sulphide and its impurities are 0.003%, 0.01%, and 0.01%; the quantitation limits are 0.01%, 0.03%, and 0.03% for LAN30, LAN30-I, and LAN30-II. This method indicates that the recovery at different levels is 95 to 105% accurate. The test solution was stable in the diluent for 48 h at refrigerator temperature and subjected to stress conditions. The mass balance was close to 99.5%.

STABILITY-INDICATING HPLC METHOD FOR THE DETERMINATION OF RELATED SUBSTANCES IN LANSOPRAZOLE INTERMEDIATE

A novel, reversed-phase liquid chromatographic method was developed and validated for the determination of related substances in lansoprazole intermediate. Symmetric peak shape was on a C18 stationary phase with the dimensions of 250 mm column length, 4.6 mm as internal diameter, 5 microns particles with an economical and straightforward mass-compatible mobile phase combination of formic acid/triethylamine and acetonitrile delivered in gradient mode at a flow rate of 1.0 mL/min at 260 nm. The resolution between lansoprazole intermediate (LAN20) and its impurities (LAN20-I & LAN20-II) in the developed method was more than 2.0, indicating a significant separation. Regression analysis shows a correlation coefficient greater than 0.999 for lansoprazole intermediate and its related substances. The detection and quantitation limits of lansoprazole intermediate and its impurities are 0.01% and 0.005%. This method indicates that the recovery at different levels is 90 to 110% accurate. The test solution was stable in the diluent for 48 h and subjected to stress conditions. The mass balance was close to 99.5%.

Optimization and validation of direct gas chromatography-mass spectrometry method for simultaneous quantification of ten short-chain fatty acids in rat feces

Short-chain fatty acids (SCFAs) play key roles in maintaining health and treating disease. Quantification of important fecal SCFAs is necessary to facilitate the clarification of their biological roles. However, the existing quantifying methods mainly depend on complicated precolumn derivatization, and/or are unable to determine formic acid, a SCFA commonly associated with toxicity. In this study, a direct gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of ten SCFAs including formic acid in rat feces was developed. The approach was optimized in terms of chromatographic and spectrometric conditions as well as sample preparation. DB-FFAP capillary column with temperature programming was used to get baseline separation and symmetrical peak shape of SCFAs without precolumn derivatization in a relatively short running time (8 min). Multiple reaction monitoring (MRM) scan mode was employed to enhance the sensitivity and selectivity of SCFAs. Acidification with 50% HCl and immediate extraction with diethyl ether were utilized to achieve sample preparation of ten SCFAs from feces. Furthermore, the developed method was validated with wide linear range, high sensitivity and precision, low matrix effect and acceptable accuracy. The established method was successfully applied to compare the contents of fecal SCFAs between normal and immunosuppressed animal models.