Novel method for the determination of five carbamate pesticides in water samples by dispersive liquid-liquid microextraction combined with high performance liquid chromatography

A novel method for the determination of five carbamate pesticides （metolcarb, carbofuran, carbaryl, isoprocard and diethofencard） in water samples was developed by dispersive liquid-liquid microextraction （DLLME） coupled with high performance liquid chromatography-diode array detector （HPLC-DAD）. Some experimental parameters that influence the extraction efficiency were studied and optimized to obtain the best extraction results. Under the optimum conditions for the method, the calibration curve was linear in the concentration range from 5 to 1000 ng mL^-1 for all the five carbamate pesticides, with the correlation coefficients （r^2） varying from 0.9984 to 0.9994. Good enrichment factors were achieved ranging from 80 to 177- fold, depending on the compound. The limits of detection （LODs） （S/N = 3） were ranged from 0.1 to 0.5 ng mL^-1. The method has been successfully applied to the analysis of the pesticide residues in environmental water samples.

Rapid Determination of Dopamine and Its Metabolites During in vivo Cerebral Microdialysis by Routine High Performance Liquid Chromatography With Electrochemical Detection

To determine dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection. Methods Microdialysis probes were placed into the right striatum of Wistar rat brains and perfused with Ringer＇s solution at a rate of 1.5 pL/min. A reverse phase HPLC with electrochemistry was used to assay DA, DOPAC, and HVA after cerebral microdialysates were collected every 20 minutes from awake and freely moving rats. In order to identify the reliability of this method, its selectivity, linear range, precision and accuracy were tested and the contents of DA, DOPAC, and HVA in rat microdialysates were determined. Results The standard curve was in good linear at the concentration ranging from 74 nmol/L to 1.5 pmol/L for DOPAC （r^2= 0.9996）, from 66 nmol/L to 1.3 gmol/L for DA （r^2=l.0000） and from 69 nmol/L to 1.4 pmol/L for HVA （r^2=0.9992）. The recovery of DOPAC （0.30, 0.77, 1.49 gmol/L）, DA （0,26, 0.69, 1.32 gmol/L）, and HVA （0.27, 0.71, 1.37 gmol/L） was 82.00±1.70%, 104.00±4.00%, 98.70±3.10%; 92.30± 1.50%, 105.30±2.30%, 108.00±2.00%; 80.00±7.80%, 107.69±8.00%, and 108.66±3.10%, respectively at each concentration. Their intra-day RSD was 3.3%, 3.4%, and 2.5%, and inter-day RSD was 4.2%, 2.3%, and 5.6%, respectively. The mean extracellular concentrations of DOPAC, DA, and HVA in rat brain microdialysates were 10.7, 2.4, and 9.2 gmol/L （n=6）, respectively. Conclusion The findings of our study suggested that the simple, accurate and stable method can be applied to basic researches of diseases related to monoamines neurotransmitters by cerebral microdialysis in rats.

Trace Determination of Tamoxifen in Biological Fluids Using Hollow Fiber Liquid-Phase Microextraction Followed by High-Performance Liquid Chromatography-Ultraviolet Detection

The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a 15 mL aqueous sample (source phase;SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase;MP) followed by the back-extraction into a second aqueous solution (receiving phase;RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 - 500 ng?mL–1 and the limit of detection was found to be 0.5 ng?mL–1 in aqueous medium. A reasonable relative recovery (≥89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3) precision illustrated good performance of the analytical procedure in spiked human urine and plasma samples.

Comparison of protocatechuic aldchyde in Radix Salvia miitiorrhiza and corresponding pharmacological sera from normal and fibrotic rats by high performance liquid chromatography

AIM： To observe the effect of protocatechuic aldchyde on the proliferation of hepatic stellate cells （HSCs）. METHODS： Liver fibrosis was induced in rats by carbon tetrachloride （CCh）. Then normal and fibrotic drug sera were extracted from rats. The effects of protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza on HSC growth were determined by CCKoS. The protocatechuic aldchyde was separated by high performance liquid chromatography （HPLC） in a AIItima C18 column （250 mm × 4.6 mm, 5 μm） with a mobile phase of acetonitrile-4% glacial acetic acid solution （gradient elution） at the wavelength of 281 nm. RESULTS： Protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza were found to have inhibitory effects on proliferation of rat HSCs. Raw Radix Salvia miltiorrhiza had a stronger inhibitory effect than the drug sera. The fibrotic drug sera showed a higher suppressive effect than the normal drug sera （P 〈 0.05）. Protocatechuic aldchyde was found in crude materials of both Radix Salvia miltiorrhiza and its corresponding drug sera. The average recovery （n = 6） was 110.5% for raw Salvia miltiorrhiza Bge, 102% for normal drug sera and 105.2% for fibrotic drug sera. The relative standard devitation （RSD） was 0.37%, 1.96% and 1.51%, respectively （n=6）. The contents of protocatechuic aldchyde were 0.22%, 0.15% and 0.19%, respectively （n = 6） （P〈 0.05）. The RSD was 0.33%, 0.75% and 1.24% （n=6） for raw material of Radix Salvia miltiorrhiza, normal drug sera and fibrotic drug sera, respectively. The samples were stable for 6 d. CONCLUSION： Protocatechuic aldchyde can inhibit the growth of HSCs. HPLC is suitable for the determination of virtual bioactive components of Chinese herbal medicines in vitro.

Development of a Fast and Facile Analytical Approach to Quantify Radiometabolites in Human Plasma Samples Using Ultra High Performance Liquid Chromatography

Introduction: Conventional metabolite analyses often require manual sample preparation, generating variability of measurements. This study describes a new method to quantify radiometabolites in blood, combining ultra high performance liquid chromatography (UHPLC) and turbulent flow chromatography, an alternative fully automated process allowing analyte’s extraction. Methods: A new radiotracer for dopamine transporter imaging, namely LBT-999, was used to demonstrate the method’s robustness. Matrix effect, Turboflow column loading, linearity, specificity and precision were evaluated with in vitro samples of LBT-999 in human plasma. Radiodetector sensitivity and preliminary evaluation were respectively determined by analysis of calibrated samples of [18F]LBT-999 and blood samples from 4 healthy subjects injected with [18F]LBT-999, withdrawn at 5, 15, 30 and 45 min pi. Results: With three sequential loadings (3 × 100 μL) of the Turboflow column, mean coefficients of variation were 1%, below 2%, 2% and 30.9% for matrix effect, specificity, repeatability and intermediate precision, respectively. Correlation coefficients for linearity were superior to 0.97. Limits of detection and quantification of the radiodetector were fixed at 3 and 9 c/s. Retention times for [18F]LBT-999 and the two radiometabolites detected by radio-UHPLC were 6.5, 4.8 and 9.6 min. Forty-five min after the injection, parent fraction was still predominant with 57.8% ± 25% of the total radioactivity. Conclusions: An innovative approach, allying UHPLC and Turboflow column, was developed and its sensitivity, linearity, specificity and repeatability validated. Preliminary results of the clinical trial are in accordance with literature data, demonstrating its efficiency in radiometabolites quantification.

Advancements in the preparation of high-performance liquid chromatographic organic polymer monoliths for the separation of small-molecule drugs

The various advantages of organic polymer monoliths, including relatively simple preparation processes,abundant monomer availability, and a wide application range of pH, have attracted the attention of chromatographers. Organic polymer monoliths prepared by traditional methods only have macropores and mesopores, and micropores of less than 50 nm are not commonly available. These typical monoliths are suitable for the separation of biological macromolecules such as proteins and nucleic acids, but their ability to separate small molecular compounds is poor. In recent years, researchers have successfully modified polymer monoliths to achieve uniform compact pore structures. In particular, microporous materials with pores of 50 nm or less that can provide a large enough surface area are the key to the separation of small molecules. In this review, preparation methods of polymer monoliths for high-performance liquid chromatography, including ultra-high cross-linking technology, post-surface modification, and the addition of nanomaterials, are discussed. Modified monolithic columns have been used successfully to separate small molecules with obvious improvements in column efficiency.

A high-performance liquid chromatography with fluorescence detection method for the simultaneous quantitation of monoamine neurotransmitters and their metabolites in subregions of rat brain

Abstract： In the presem study, we simultaneously quantified the levels of monoamine neurotransmitters （MANTs） and their metabolites （levodopa, norepinephrine, epinephrine, dopamine, 5-HT, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid） in different brain subregions of rats using a newly developed simple, sensitive and selective high-performance liquid chromatography with fluorescence detection （HPLC-FLD） method. In this new HPLC-FLD method, analytes were directly extracted and separated without deriveatization step within 20 min. The FLD wavelength was set at 280 nm and 330 nm for excitation and emission, respectively. The analytes were separated on an Agilent Eclipse Plus Cls column （4.6 mm×150 mm, 5.0 μm） equipped with an Agilent XDB-C18 security guard column （4.6 mm×12.5 mm, 5.0 lam）, and the column temperature was maintained at 35 ℃. The mobile phase for elution was isocratic. The mobile phase consisted of citric acid buffer （50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L octane sulfonic acid sodium salt, 0.5 mmol/L Na2EDTA and 5 mmol/L triethylamine, pH 3.8） and methanol （90：10, v/v） at a flow rate of 1.0 mL/min. The detection limit （DL） was 0.9-23 nM for all the MANTs and their metabolites with a sample volume of 50 μL. The method was shown to be highly reproducible in terms of peak area （intraday, 0.08%-1.85% RSD, n = 5）. The simultaneous measurement of these MANTs and their metabolites improved our understanding of the neurochemistry in the central nervous system （CNS） in relation to different addictive drugs （methamphetamine, heroin and their mixture） in drug-addicted rat models.

Imidazolium ionic liquid as the background ultraviolet absorption reagent for determination of morpholinium cations by high performance liquid chromatography-indirect ultraviolet detection

A novel analytical method was developed for determining morpholinium cations lacking ultraviolet absorption groups.This determination was carried out by high performance liquid chromatographyindirect ultraviolet（HPLC-1UV） detection using imidazolium ionic liquid as background absorption reagents,and imidazolium ionic liquid aq.soln.-organic solvent as mobile phase by a reversed-phase C18 column.The background ultraviolet absorption reagents,imidazolium ionic liquids and organic solvents were investigated.The imidazolium ionic liquid in the mobile phase is not only the background ultraviolet absorption reagent for IUV,but also an active component to improve the separation of morpholinium cations.It was found that morpholinium cations could be adequately determined when0.5 mmol/L 1-ethyl-3-methylimidazolium tetrafluoroborate aq.soln./methanol（80：20,v/v） was used as mobile phase with an IUV detection wavelength of 210 nm.In this study,the baseline separation of Nmethyl,ethylmorpholinium cations（MEMo） and N-methyl.propylmorpholinium cations（MPMo） was successfully achieved in 8.5 min.The detection limits（S/N = 3） for MEMo and MPMo were 0.15 and0.29 mg/L,respectively.This simple and practical method has been successfully applied to the determination of two morpholinium ionic liquids synthesized by the chemistry laboratory.

Separation and identification of moxifloxacin impurities in drug substance by high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform ion cyclotron resonance mass spectrometry

In this paper, a high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform-ion cyclotron resonance mass spectrometry （HPLC-UV/FrICRMS） method was described for the investigation of impurity profile in moxifloxacin （MOX） drug substance and chemical reference substance. Ten impurities were detected by HPLC-UV, while eight impurities were identified by using the high accurate molecular mass combined with multiple-stage mass spectrometric data and fragmentation rules. In addition, to our knowledge, five impurities were founded for the first time in MOX drug substance.

Development and Validation of a Method for Simultaneous Determination of Metformin Hydrochloride and Sitagliptin Phosphate in a Formulation by RP-HPLC

Present study was aimed to develop and validate a reverse-phase high-performance liquid chromatography method for simultaneous determination of sitagliptin phosphate and metformin hy-drochloride in a marketed formulation. The drug separation was performed on Hibar-240, Li-chrosphere-100 C18 ODS (250 × 4.6 mm, 5 μm) column, at a flow rate of 1 mL/min. The mobile phase used was a mixture of methanol: potassium di-hydrogen phosphate buffer at a ratio of 70:30 v/v. The detection was carried out at a wavelength of 266 nm. The retention times of sitagliptin phosphate and metformin hydrochloride were found as 6.1 and 4.9 min respectively. Linear calibration curves with good correlation coefficients were obtained over the concentration ranges of 10 - 50 μg/mL for sitagliptin and 20 - 100 μg/mL for metformin. The limit of detection was 0.016 and 0.14 μg/mL and the limit of quantification was 0.048 and 0.42 μg/mL for sitagliptin phosphate and metformin hydrochloride respectively. Validation of the method demonstrated system selectivity, specificity, linearity, accuracy and precision. The developed method was found useful in the simultaneous analysis of sitagliptin phosphate and metformin hydrochloride in formulation.

Qualitative and Quantitative Analysis of Five Bioactive Flavonoids in <i>Salix bordensis</i>Turcz. by HPLC-DAD and HPLC-ESI-MS

The qualitative characterization and quantitative analysis of five bioactive flavonoids in Salix bordensis Turcz. were achieved via reversed-phase high-performance liquid chromatography coupled with diode array detection and tandem mass spectrometry, by using an Agilent ZORBAX SB-C18 HPLC column with a gradient elution of 0.3% (v/v) formic acid in water and methanol as the mobile phase. The compounds in the mixture were clearly identified by comparing their HPLC-DAD ultraviolet spectra, retention times, and MS data with those of corresponding reference compounds. All calibration curves showed good linearity (r2 > 0.9998) within the test ranges. The LOD, LOQ, specificity, precision, and accuracy for the method were validated. The results demonstrated that this analytical approach is ideal for the determination of bioactive compounds, such as flavonoids, and that it constructed a basis for the comprehensive evaluation of the quality of Salix bordensis Turcz.

High performance liquid chromatography for the determination of flavonoids

Due to their biological and physiological importance,flavonoids receive considerable attention in the literature. Nowadays,high performance liquid chromatography（HPLC）is the most widely used analytical method.In this review,we summarize the principle of the choice of HPLC column and mobile phase,discuss and compare the features of various detections such as UV,fluorescence detection,electrochemical detection,chemilummescence detection,UV-MS etc.Recent developments in HPLC including ultra-LC and miniaturization of LC（micro-LC,capillary-LC,and nano-LC）,are also discussed.

Investigation of retention and chiral recognition mechanism using quantitative structure-enantioselectivity retention relationship in high performance liquid chromatography

The enantiomers of a series of fourteen O-ethyl O-(substituted) phenyl N-isopropyl-phosphoroamidothioates have been separated by high performance liquid chromatography (HPLC) on the Pirkle-type chiral stationary phase. Seven molecular descriptors were calculated and four significant descriptors were chosen to correlate against the experimental lnk' values in order to form the quantitative structure-enantioselectivity retention relationships (QSERRs). Through the QSERRs, the retention and enantioselectivity mechanism were examined.

Establishment of a Method for Determination of Anemoside B4 Content in Pulsatilla Water Extract

[Objective] This study aimed to establish a new method for determination of anemoside B4 content in pulsatilla water extract. [Method] Using acetonitrile-water （28：72） as the mobile phase, the high performance liquid chromatography, equipped with UV detector, was used to determine the anemoside B4 content in pulsatilla water extract. [Result] In the concentration range of 300-800 μg/ml, anemoside B4 content showed a good linear relationship with peak area. The average recovery of anemoside B4 was 98.12% （n=-6; RSD=-1.37%）. [Conclusion] The established method meets the requirements by methodology, and it can be used to determine the anemoside B4 content in pulsatilla water extract.

Analysis of Sucrose Esters with Long Acyl Chain by Coupling of HPLC-ELSD with ESI-MS System

The analysis of sucrose esters with long acyl chain by improved high performance liquid chromatographic method with evaporative light scattering detection （HPLC-ELSD） and electrospray ionization mass spectrum （ESI-MS） is investigated. The improved HPLC-ELSD method for the separation and quantitation of commercial and synthesized sucrose esters is described. Samples are analyzed by means of a reversed-phase （RP） HPLC using a Hypersil C8 column （250 mm× 4.6 mm, 5 μm particle size） with methanol-tetrahydrofuran （vo）ume ratio of 90 ： 10） and water under gradientcondition as the mobile phase, in which the flow rate is 1.0 ml·min^-1 and the column temperature is set at 40℃. This procedure provides a complete separation and determination ot monoester, diester, triester and higher esters with different acyl chain lengths in each fraction by a single run, in combination with the ESI-MS technology. With this method, it is possible to determine the approximate compositions of monoto polyesters in one analysis and quantitate pure positional isomers precisely using an external standard method. It is found that the method of ESI-MS coupling with HPLC system for the analysis of sucrose esters is straight forward, rapid and inexpensive, and can be readily applied in synthesis, purification and structure studies.

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Rucaparib in Bulk and Pharmaceutical Dosage Form

The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min-1 at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.

Qualitative and Quantitative Analysis of Linoleic Acid in Polygonati Rhizoma

Objective To explore the major compound in Polygonati Rhizoma(Huang Jing,黄精)for quality control.Methods The major compound was isolated and analyzed by liquid chromatography-mass spectrometry(LC-MS),and subsequently further identified by nuclear magnetic resonance(NMR).Thin layer chromatography(TLC)was optimized based on the previous methods reported in the Chinese Pharmacopeia(2015 edition).Results The major compound was isolated from the natural material and identified as linoleic acid.A high performance liquid chromatography(HPLC)method with robust linearity(R2=0.9997),specificity,precision,stability,repeatability and recovery was developed for linoleic acid determination.TLC chromatogram was improved significantly after optimization for qualitative analysis.Conclusions The optimized TLC method is practical and can be adopted for quality control of Polygonati Rhizoma(Huang Jing,黄精).The levels of linoleic acid vary between species of Polygonati Rhizoma(Huang Jing,黄精),with Polygonatum cyrtonema Hua(Jiang Xing Huang Jing,姜型黄精)showing the highest contents.This study provides valuable information for quality control of Polygonati Rhizoma(Huang Jing,黄精).

A Stability Indicating Reverse Phase-HPLC Method Development and Validation for the Estimation of Bimatoprost 0.3% &Timolol 0.5% Pharmaceutical Ophthalmic Dosage Form

The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min-1 at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.

Retention and chiral recognition mechanism of organo-phosphorus compounds in high-performance liquid chromatography

In normal phase condition, a series of chiral phosphorus organic compounds have been separated by high-performance liquid chromatography. In order to study the retention and chiral recognition mechanism, the method of quantitative structure-enantioselectivity retention relationships (QSERRs) has been investigated from the quantitative equations established between the chromatographic retention of enantiomers and their molecular descriptors of physicochemical properties. The results show that on the Pirkle-type chiral stationary phase (CSP) of Sumichiral OA4700, it is the parameter of LUMO that gives the most contribution to the chromatographic retention of O-ethyl O-(substituted) phenyl N-isopropyl phosphoroamidothioates resulting from the interaction of hydrogen bond and (or) π-π interaction. Meanwhile, the chiral recognition is formed from the contribution of logP and LUMO.

Enantioseparation of Palonosetron Hydrochloride and Its Related Enantiomeric Impurities by Computer Simulation and Validation

A rapid, simple and single stereo selective high-performance liquid chromatographic (HPLC) method was developed and validated for enantiomers of palonosetron hydrochloride (PALO) and its process related chiral impurities. A computer simulating software was used for the development of chiral method. The developed method was able to separate not only the enantiomers of palonosetron hydrochloride but also its process related chiral impurities within 12 min. The chromatographic separation was carried out by normal phase chromatography using a 3 μm column of cellulose based chiral stationary phase (Chiralcel-OD 250mm × 4.6mm) with a mobile phase comprised of n-hexane: ethanol: methanol: heptafluoro butyric acid: diethyl amine (70:15:15:0.05:0.1, v/v) at a flow rate of 1.0 mL/min. The effects of additive concentration as well as nature of polar organic modifier, flow rate, and temperature on enantioselectivity were investigated. The limit of detection (LOD) and limit of quantification (LOQ) of the palonosetron isomers and its related chiral impurities were found to be in the range 0.06-0.10 μg/mL and 0.14 - 0.24 μg/mL respectively. The method showed excellent linearity (R2 > 0.998) over a range of 0.14 to 1.125 μg/mL. The percentage recovery of the isomers in bulk drug samples ranged from 87.0 to 116.0.