High-performance Liquid Chromatographic Separation Research Articles

“Click” preparation of a chiral macrocycle-based stationary phase for both normal-phase and reversed-phase high performance liquid chromatography enantioseparation

Chiral polyimine macrocycles (CPMs) constitute a new family of organic macrocycles that have defined cavities, rigid shapes, inherent chirality and multiple cooperative binding sites, and have shown great potential in diverse areas. However, the application of CPMs for high performance liquid chromatography (HPLC) enantioseparation has rarely been reported. In this work, a novel chiral stationary phase (CSP) for HPLC was prepared by chemical bonding of a CPM (C54H72N6O3) onto thiolated silica via thiol-ene click reaction. The CSP exhibited good enantioselectivity in both normal- and reversed-phase HPLC. Chiral compounds included alcohols, diols, ketones, organic acids, esters, ethers, amines, and epoxides were enantioseparated on the column in normal-phase mode (17 compounds) and reversed-phase mode (20 compounds). Importantly, broader chiral resolution was observed with the column than that obtained using our previously studied chiral macrocycle H3L-based column, indicating the potential to significantly improve and broaden applicability of this novel macrocycle-type CSPs. Moreover, the CSP exhibited good complementary enantioseparation to Chiralpak AD-H and Chiralcel OD-H columns, enabling separation of some racemates that could not be separated by the two popular chiral HPLC columns. In addition, the fabricated column exhibited good stability and reproducibility. The relative standard deviations (RSDs) (n = 5) of retention time and resolution after multiple injections were < 0.20 % and < 0.39 %, respectively. The results demonstrated the great potential of this type of CPM for HPLC separation of enantiomers.

Preparation of Porous Silica Particles with a Controlled Mesopore Size by Ultrasonic Spray Drying for High-Performance Liquid Chromatography (HPLC)

Highly spherical porous silica particles (PSPs) with a controlled pore size were prepared by ultrasonic spray drying and were used as a packing material for high-performance liquid chromatography (HPLC). By using organic additives added to the silica sol as porogens instead of low-melting salts, choosing the size of the colloidal SiO2 particles, and varying the type and the ratio of additives, the pore size of the PSPs was finely tuned from 2.9 to 34.4 nm. Subsequently, PSPs having pore sizes of 7.4, 13.5, and 29.0 nm were modified by chlorodimethyloctadecylsilane (PSPs-C18), and HPLC columns packed with the different PSPs-C18 were evaluated systematically using aromatic compounds and alkylbenzene derivatives as probes. A plate number of 81,000/m and the relative standard deviation of less than 1% in the retention time were obtained for pentylbenzene over 100 injection and HPLC separation cycles for the 7.4-PSPs-C18 column. Five proteins were also separated within 25 min using the 29.0-PSPs-C18 column.

Determination of 32 oxidative dyes by high performance liquid chromatography and confirmation by high performance liquid chromatography-tandem mass spectrometry

染发类产品中氧化型染料种类多,实际样品测定时干扰多,建立染发类产品中多种常用染料的测定方法,为该类产品的有效监管提供技术手段十分必要。该研究根据染料使用频率分组,采用能够屏蔽硅羟基和金属离子影响的C18柱,优化了《化妆品安全技术规范》(2015年版)中32种染料的高效液相色谱法(HPLC)并建立了高效液相色谱-串联质谱(HPLC-MS/MS)确证方法。样品以10 g/L亚硫酸氢钠水溶液为抗氧化剂,用无水乙醇-水(1∶1, v/v)混合溶液冰浴超声提取10 min。HPLC方法采用甲醇、乙腈和磷酸盐缓冲液为流动相分两个液相色谱条件进行梯度洗脱分离,于280 nm波长下检测,其中一个HPLC条件中的相互干扰组分均在另一个HPLC条件下完全分离,避免了实际样品检测时组分间的干扰,并排除了32种以外的其他15种常用染料的干扰。HPLC-MS/MS方法分别采用5 mmol/L乙酸铵水溶液-乙腈和5 mmol/L乙酸水溶液-乙腈为正离子和负离子模式下的流动相,电喷雾离子模式下用多反应监测(MRM)模式进行定性和定量分析。HPLC和HPLC-MS/MS两个方法中,日内精密度和48 h内稳定性的相对标准偏差(RSD)<10%,回收率为82.6%~114.9%(RSD<10%)。HPLC方法中32种染料在大约10~500 mg/L范围内线性关系良好(r2>0.99),检出限为9.7~40.1 μg/g; HPLC-MS/MS方法中氢醌线性范围为2.0~79.7 mg/L,检出限为8.0 μg/g,其他组分线性范围约为0.1~4 mg/L,检出限为0.01~0.4 μg/g。采用HPLC、HPLC-MS/MS两个方法和《化妆品安全技术规范》方法同时测定实际样品,共检出16种染料,检出含量范围为58~25160 μg/g。3个方法检测结果的RSD为1.9%~10.1%。该研究增加了HPLC-MS/MS确证方法,适应化妆品法定检验中的未知物确认程序;方法简便快速,结果准确,专属性强,具有较好的通用性和可操作性。

A discovery biotransformation strategy: combining in silico tools with high-resolution mass spectrometry and software-assisted data analysis for high-throughput metabolism

Understanding compound metabolism in early drug discovery aids medicinal chemistry in designing molecules with improved safety and ADME properties. While advancements in metabolite prediction brings increased confidence, structural decisions require experimental data. In vitro metabolism studies using liquid chromatography and high-resolution mass spectrometry (LC–MS) are generally resource intensive and performed on very few compounds, limiting the chemical space that can be examined. Here, we describe a novel metabolism strategy increasing compound throughput using residual in vitro clearance samples conducted at drug concentrations of 0.5 µM. Analysis by robust ultra high-performance liquid chromatography separation and accurate-mass MS detection ensures major metabolites are identified from a single injection. In silico prediction (parent cLogD) tailors chromatographic conditions, with data-dependent tandem mass spectroscopy targeting predicted metabolites. Software-assisted data mining, structure elucidation and automatic reporting are used. Confidence in the globally aligned workflow is demonstrated with 16 marketed drugs. The approach is now implemented routinely across our laboratories. To date, the success rate for identification of at least one major metabolite is 85%. The utility of these data has been demonstrated across multiple projects, allowing earlier medicinal chemistry decisions to increase efficiency and impact of the design–make–test cycle thus improving the translatability of early in vitro metabolism data.

The separation of several organophosphate pesticides on immobilized polysaccharide chiral stationary phases.

While not initially a focus or priority, in recent decades, an emphasis has been placed on the activity of individual enantiomers of widely used pesticides. Of particular note are organophosphorus‐based pesticides like fenamiphos and profenofos, as examples. This work explores the enantioselective high‐performance liquid chromatography (HPLC) separations of seven such organophosphorus pesticides (OP's) on the library of immobilized polysaccharide‐based chiral stationary phases (CSPs) with normal phase hexane/alcohol mixtures. Further exploration of the effect of mobile phase strength and temperature on several of the separations was performed using simple factorial design. Equivalent retention of the first eluting enantiomer of several combinations of temperature and mobile phase was compared for peak shape, selectivity, and resolution. Similarly, equivalent selectivity of several combinations of temperature and mobile phase was compared for peak shape, retention of the first eluting enantiomer, and resolution. The results of this study make available several new chiral separations of the OPs included in the work that were not previously documented, including separations on the three most recently commercialized phases, Chiralpak IH, IJ, and IK. Additionally, sufficient understanding was obtained to be able to predict the trade‐off of resolution, analysis time, peak sharpness (and thus improve limit‐of‐detection [LOD]/limit‐of‐quantification [LOQ]), robustness, and convenience of conditions for further application optimization.

Inherently Chiral 6,7-Diphenyldibenzo[ e,g ][1,4]diazocine: Enantioselective Synthesis and Application as a Ligand Platform

Inherently Chiral 6,7-Diphenyldibenzo[ <i>e,g</i> ][1,4]diazocine: Enantioselective Synthesis and Application as a Ligand Platform

Characterization of Humulus lupulus glycosides with porous graphitic carbon and sequential high performance liquid chromatography quadrupole time-of-flight mass spectrometry and high performance liquid chromatography fractionation

Monoterpenes contribute to the characteristic aroma of several hop varieties and may occur as nonvolatile glycosides. Upon hydrolysis, the volatile terpenes are released from the glycoside precursors. Little is known, however, about the glycoside composition of hops. Seven pentose-hexose monoterpene alcohol glycosides from dried Humulus lupulus L. cv. Citra cones were isolated using high performance liquid chromatography separation and fractionation on a reverse phase phenyl-hexyl column. Further evaluation of each isolated fraction through HPLC qTOF MS with porous graphitic carbon (PGC) showed that the seven isolated monoterpenyl glycoside fractions could be further resolved into 20 isomers. Isolation on phenyl-hexyl followed by separation on PGC was needed to distinguish each isomer present. Additionally, the hop cones were screened for potential aroma glycosides. Using the PGC column combined with a database of over 900 potential glycosides, the identification of 21 additional monoterpene-polyol, norisoprenoid, volatile phenol, and aliphatic alcohol glycosides is reported.

Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

Urinary free-glycans are promising markers of disease. In this study, we attempted to identify novel tumor markers by focusing on neutral free-glycans in urine. Free-glycans extracted from the urine of normal subjects and cancer patients with gastric, colorectal, pancreatic and bile duct were fluorescently labeled with 2-aminopyridine. Profiles of these neutral free-glycans constructed using multidimensional high performance liquid chromatography separation were compared between normal controls and cancer patients. The analysis identified one glycan in the urine of cancer patients with a unique structure, which included a pentose residue. To reveal the glycan structure, the linkage fashion, monosaccharide species and enantiomer of the pentose were analyzed by high performance liquid chromatography and mass spectrometry combined with several chemical treatments. The backbone of the glycan was a monoantennary complex-type free-N-glycan containing β1,4-branch. The pentose residue was attached to the antennal GlcNAc and released by α1,3/4-l-fucosidase. Intriguingly, the pentose residue was consistent with d-arabinose. Collectively, this glycan structure was determined to be Galβ1-4(d-Araβ1-3)GlcNAcβ1-4Manα1-3Manβ1-4GlcNAc-PA. Elevation of d-arabinose-containing free-glycans in the urine of cancer patients was confirmed by selected reaction monitoring. This is the first study to unequivocally show the occurrence of a d-arabinose-containing oligosaccharide in human together with its detailed structure.

Enantioselective high-performance liquid chromatographic separation of fluorinated ß- phenylalanine derivatives utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases: Enantioselective separation of fluorinated ß-phenylalanine derivatives

The enantioselective separation of newly synthesized fluorine-substituted β-phenylalanines has been performed utilizing Cinchona alkaloid-based ion-exchanger chiral stationary phases. Experiments were designed to study the effect of eluent composition, counterion content, and temperature on the chromatographic properties in a systematic manner. Mobile phase systems containing methanol or mixtures of methanol and acetonitrile together with acid and base additives ensured highly efficient enantioseparations. Zwitterionic phases [Chiralpak ZWIX (+) and ZWIX(–)] were found to provide superior performance compared to that by the anion-exchangers (Chiralpak QN-AX and QD-AX). A detailed thermodynamic characterization was also performed by employing van't Hoff analysis. Using typical liquid chromatographic experimental conditions, no marked effect of the flow rate could be observed on the calculated thermodynamic parameters. In contrast, a clear tendency has been revealed about the effect of the eluent composition on the thermodynamics for the zwitterionic phases.

Supramolecular Nanohelix Fabricated by Pillararene-Based Host–Guest System for Chirality Amplification, Transfer, and Circularly Polarized Luminescence in Water

Supramolecular Nanohelix Fabricated by Pillararene-Based Host–Guest System for Chirality Amplification, Transfer, and Circularly Polarized Luminescence in Water

Isolation and structure elucidation of caryophyllane sesquiterpenoids from leaves of Eremophila spathulata

• This is the first phytochemical investigation of the leaves of Eremophila spathulata . • A combination of HPLC and HPLC-PDA-HRMS-SPE-NMR afforded seven compounds. • The structures were identified as new caryophyllane sesquiterpenoids by HRMS and 2D NMR. Crude extract of Eremophila spathulata leaves was investigated by semi-preparative scale high-performance liquid chromatography (HPLC), analytical scale HPLC, and hyphenated high-performance liquid chromatography-photodiode array-high-resolution mass spectrometry-nuclear magnetic resonance (HPLC-PDA-HRMS-SPE-NMR), which afforded seven previously unreported caryophyllane sesquiterpenoids. Semi-preparative scale separation of the crude extract afforded (1 R* ,4 R *,9 S* , E )-8-formyl-11,11-dimethylbicyclo[7.2.0]undec-7-ene-4-carboxylic acid ( 5 ) and analytical-scale HPLC separation afforded (1 R* ,4 S* ,7 S *,9 S* )-7-hydroxy-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 1 ), (1 S* ,6 R* ,9 R*,E )-10,10-dimethylbicyclo[7.2.0]undec-2-ene-2,6-dicarboxylic acid ( 2 ), (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 3 ), and (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 4 ). HPLC-PDA-HRMS-SPE-NMR afforded (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 6 ) and (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 7 ). The structures of all isolated compounds were established based on HRMS as well as extensive 1D and 2D NMR analysis. Relative configurations were determined by correlations in spectra from rotational Overhauser effect spectroscopy.

Disclosing the Antioxidant and Neuroprotective Activity of an Anthocyanin-Rich Extract from Sweet Cherry (Prunus avium L.) Using In Vitro and In Vivo Models.

In this study, an autochthonous variety of sweet cherry (Prunus avium L.), namely “Moretta di Vignola”, was processed to prepare extracts rich in polyphenols, which were characterized by high-performance liquid chromatography (HPLC) separation coupled to UV/DAD and ESI-MSn analysis. Then, a sweet cherry anthocyanin-rich extract (ACE) was prepared, fully characterized and tested for its activity against Parkinson’s disease (PD) in cellular (BV2 microglia and SH-SY5Y neuroblastoma) and in Drosophila melanogaster rotenone (ROT)-induced model. The extract was also evaluated for its antioxidant activity on Caenorhabditis elegans by assessing nematode resistance to thermal stress. In both cell lines, ACE reduced ROT-induced cell death and it decreased, alone, cellular reactive oxygen species (ROS) content while reinstating control-like ROS values after ROT-induced ROS rise, albeit at different concentrations of both compounds. Moreover, ACE mitigated SH-SY5Y cell cytotoxicity in a non-contact co-culture assay with cell-free supernatants from ROT-treated BV-2 cells. ACE, at 50 µg/mL, ameliorated ROT (250 μM)-provoked spontaneous (24 h duration) and induced (after 3 and 7 days) locomotor activity impairment in D. melanogaster and it also increased survival and counteracted the decrease in fly lifespan registered after exposure to the ROT. Moreover, heads from flies treated with ACE showed a non-significant decrease in ROS levels, while those exposed to ROT markedly increased ROS levels if compared to controls. ACE + ROT significantly placed the ROS content to intermediate values between those of controls and ROT alone. Finally, ACE at 25 µg/mL produced a significant increase in the survival rate of nematodes submitted to thermal stress (35 °C, 6–8 h), at the 2nd and 9th day of adulthood. All in all, ACE from Moretta cherries can be an attractive candidate to formulate a nutraceutical product to be used for the prevention of oxidative stress-induced disorders and related neurodegenerative diseases.

Development and evaluation of a hydrogenated rosin (β-acryloxyl ethyl) ester–bonded silica stationary phase for high-performance liquid chromatography separation of paclitaxel from yew bark

Paclitaxel (PTX) is a complex diterpenoid anticancer drug whose separation from yew biomass poses a significant challenge. In this study, a new stationary phase comprising hydrogenated rosin (β-acryloxyl ethyl) ester (HRE)–bonded silica (HRE@SiO2) is developed to separate and purify PTX from crude yew-bark extract using high-performance liquid chromatography. In HRE@SiO2, HRE molecules, which are functional ligands, are bonded to the surface of a silica gel matrix using a coupling agent, (3-mercaptopropyl)trimethoxysilane. The proposed HRE@SiO2 stationary phase was characterized by Fourier-transform infrared spectroscopy, elemental analysis, thermogravimetric analysis, scanning electron microscopy, laser diffraction granulometry, and nitrogen gas adsorption. The HRE@SiO2 column exhibited excellent chromatographic performance, satisfactory performance reproducibility, and typical reversed-phase chromatographic behavior. An HRE@SiO2 column was used to separate PTX and its analogs, achieving resolutions exceeding 7.43 for consecutively eluted species. Stoichiometric displacement theory for retention (SDT-R), the van Deemter equation, and van 't Hoff plots were used to analyze the separation mechanism and properties of the HRE@SiO2 column. The results showed that hydrophobic interactions determine the analyte retention and the separation of PTX and its analogs on an HRE@SiO2 column is an exothermic process driven by enthalpy. Furthermore, an HRE@SiO2 column was employed to separate and purify PTX from crude yew-bark extract, increasing PTX purity from 6% to 82%. The findings of this study provide insights for developing rosin-based stationary phases for the separation of natural products.

Single-Crystalline Covalent Organic Frameworks as High-Performance Liquid Chromatographic Stationary Phases for Positional Isomer Separation

Vigorously developing new stationary phases to meet the requirements for the separation of positional isomers that have similar physicochemical properties is still an urgent topic in separation science. Herein, a single-crystalline covalent organic framework (COF-300) packed column for the separation of positional isomers in high-performance liquid chromatography (HPLC) was reported for the first time. Benefitting from its regular shape, excellent chemical and thermal stability, microporous feature, and strong hydrophobicity of single-crystalline COF-300, the single-crystalline COF-300-packed column showed excellent resolution for the separation of positional isomers, including nitroaniline, dichlorobenzene, dibromobenzene, diiodobenzene, diethylbenzene, chloronitrobenzene, bromonitrobenzene, and iodonitrobenzene isomers, which cannot be all separated on commercial columns and a polycrystalline COF-300-packed column. Especially, the resolution values for m-/p-diiodobenzene and o-/m-diiodobenzene were 4.45 and 2.53. Moreover, the alkylbenzene, monosubstituted aromatics, polycyclic aromatic hydrocarbons, and the mixture of ethylbenzene and styrene were also baseline separated on the single-crystalline COF-300-packed column. This successful application not only confirmed the great potential of single-crystalline COFs in HPLC separation of positional isomers but also pioneered the utilization of single-crystalline COFs in separation science.

Cholinesterase inhibitors assessment of aporphine alkaloids from Annona crassiflora and molecular docking studies

Annona crassiflora Mart. is an endemic plant from Brazilian Cerrado (savanna) biome, commonly employed in traditional medicine to treat wounds, diarrhea, and scalp infections. The pulp of the fruits is edible and has a characteristic taste, being employed to prepare sweets like jam, cakes, and ice cream by the people who live in the region of the Cerrado, although the peels are discarded. In this way, the A. crassiflora fruit peels ethanol extract was prepared and subjected to liquid–liquid extraction, which yielded the alkaloidal fraction (CH2Cl2). Subjected to high-performance liquid chromatography separations, this fraction allowed the purification of the aporphine alkaloids stephalagine (1), liriodenine (2), and atherospermidine (3), that were structurally characterized by high-resolution mass spectrometry with electrospray ionization, and nuclear magnetic resonance spectroscopy analyses. Aporphine alkaloids are recognized for their acetylcholinesterase (AChE) inhibitory activity, an important target in Alzheimer's disease therapy. Thus, the ethanol extract, alkaloidal fraction, and compounds1,2,and3were evaluated for acetyl- and butyrylcholinesterase (BChE) inhibitory activities. Compound1(IC50 104.2 µmol L–1) exhibited better BChE inhibitory activity than the standard compound galanthamine (IC50 162.7 µmol L–1), while2had a comparable result(and IC50 167.3 µmol L–1). Furthermore, molecular docking was performed to predict the compound's binding mode to the human AChE at a molecular level. Semiempirical calculation results show that the enthalpy interaction energy (ΔHint) between AChE and BChE active sites and all ligands were favorable for both enzymes, with the ligands interacting even more strongly with AChE, corroborating with IC50 results.

HPLC analysis of carotenoids from bacteria.

Bacteria synthesize different carotenoid structures. A powerful method for the determination of carotenoid profiles of bacteria, including quantification, is high performance liquid chromatography (HPLC). This chapter explains the basics of this method and its application, and provides instruction on how to proceed with HPLC. A basic HPLC system for bacterial carotenoids is presented which allows tentative identification and quantification. The individual analysis steps including extraction, sample preparation and HPLC separation are described in detail. HPLC examples of structurally diverse carotenoids from different bacterial phyla are displayed demonstrating the applicability of this method. In addition, information on spectroscopic properties useful for identification is included. Finally, a procedure for carotenoid quantification from the HPLC chromatogram is described.

Synthesis of the Magnetically Nanoporous Organic Polymer Fe3O4@SiO2-NH2-COP and Its Application in the Determination of Sulfonamide Residues in Surface Water Surrounding a Cattle Farm.

Efficient extractions of trace antibiotic residues in the environment are a key factor for accurate quantification of the residues. A new nanoporous material, namely, magnetically covalent organic polymer (MCOP, Fe3O4@SiO2-NH2-COP) was synthesized in this work and was used for magnetic solid-phase extraction (MSPE). The combination of MSPE with high-performance liquid chromatography separation together with ultraviolet detection (HPLC-UV) was established as an effective method for the determination of four sulfonamide (SA) residues in surface water surrounding a cattle farm. The synthesized magnetic material was characterized by SEM, TEM, FT-IR, magnetic properties measurement system (MPMS), and nitrogen gas porosimetry. The material possessed many attractive features, such as a unique microporous structure, a larger specific surface area (137.93 m2·g−1) than bare Fe3O4 (24.84 m2·g−1), high saturation magnetization (50.5 emu·g−1), open adsorption sites, and high stability. The influencing parameters, including pH, the used amount of MCOPs, the type of eluent, adsorption solution, and desorption time, were optimized. Under the optimized conditions, the method conferred good linearity ranges (R2 ≥ 0.9990), low detection limits (S/N = 3, LOD, 0.10–0.25 μg·L−1), and satisfactory recoveries (79.7% to 92.2%). The enrichment factor (EF) for the four SAs was 34.13–38.86. The relative standard deviations of intraday (n = 5) and of interday (n = 3) were less than 4.8% and 8.9%, respectively. The equilibria between extraction and desorption for SAs could be reached within 150 s. The proposed method was sensitive and convenient for detecting SA residues in complex environmental matrices, and the successful application of the new MCOPs as an adsorbent was demonstrated.

Stability indicating RP-HPLC method for the determination of Tenofovir in pharmaceutical formulation

Stability indicating high performance liquid chromatography (HPLC) method was developed for the assay of Tenofovir in bulk and solid dose formulation. The HPLC separation was achieved on kromasil C18 (100mm × 4.6mm, 5 μm) column using a mobile phase of Methanol: Potassium dihydrogen orthophosphate buffer (30:70,v/v) at a flow rate of 1 ml min-1 and UV detection at 260 nm. Peak elutes at 7.33 appropriate. The method was validated for linearity, repeatability, accuracy, precision, robustness, limit of detection and limit of quantification. The accuracy was between 99.14 - 99.97%. The highest R.S.D. amongst interday and Intraday precision was found 0.808 and 0.473 respectively.The assay was linear over the concentration range of 10-50 μg/ml (R≈0. 999). The method was robust as no significant change in chromatographic parameters. LOD and LOQ was found to be 0.90 and 2.71 respectively. The stress studies were performed per ICH guidelines to confirm its Stress testing was carried out in presence of acid, base, hydrogen peroxide, heat and light to demonstrate specificity of the method as per ICH guidelines. The developed method could separate the potential degradation products from the Tenofovir peak. It was concluded that highest degradation occurs in basic condition. This proposed method was suitable and practical for analysis the content of Tenofovir in pharmaceutical products and could be of benefit for the prediction shelf life of Tenofovir in marketed formulations.

Bis-Rhodamines Bridged with a Diazoketone Linker: Synthesis, Structure, and Photolysis.

Two fluorophores bound with a short photoreactive bridge are fascinating structures and remained unexplored. To investigate the synthesis and photolysis of such dyes, we linked two rhodamine dyes via a diazoketone bridge (−COCN2−) attached to position 5′ or 6′ of the pendant phenyl rings. For that, the mixture of 5′- or 6′-bromo derivatives of the parent dye was prepared, transformed into 1,2-diarylacetylenes, hydrated to 1,2-diarylethanones, and converted to diazoketones Ar1COCN2Ar2. The high performance liquid chromatography (HPLC) separation gave four individual regioisomers of Ar1COCN2Ar2. Photolysis of the model compound—C6H5COCN2C6H5—in aqueous acetonitrile at pH 7.3 and under irradiation with 365 nm light provided diphenylacetic acid amide (Wolff rearrangement). However, under the same conditions, Ar1COCN2Ar2 gave mainly α-diketones Ar1COCOAr2. The migration ability of the very bulky dye residues was low, and the Wolff rearrangement did not occur. We observed only moderate fluorescence increase, which may be explained by the insufficient quenching ability of diazoketone bridge (−COCN2−) and its transformation into another (weaker) quencher, 1,2-diarylethane-1,2-dione.

Simultaneous HPLC estimation of Amphetamine and Caffeine abuse drugs in Iraqi human addicts

The main aim of this research is to establish and validate a high performance liquid chromatography (HPLC) process for the separation and estimation of Amphetamine (AM) and Caffeine (CAF) in its illegal formula and in sera of addicts. This method is established on the HPLC separation of the two drugs on the ZORBAX ODS column (250×4.6×5µm particle size). The mobile phase contained 1% ortho-phosphoric acid 85% and 1%of diethyl amine 99%, acetonitrile and methanol ratio was 85:10:5 v/v/v. The flow rate is 1.2 mL.min-1, buffer value pH of 2.5 via isocratic elution also UV detection at 210 nm. The retention times for the two drugs AM and CAF were obtained at 4.425 and 6.456 min, respectively. The calibration curves founded that the linear regression analysis data gave a good linear relationship for the concentration range 1 to 100 µg.mL-1 for AM and CAF. The values achieved for correlation coefficient, slope and intercept were 9999, 8104.2 and 5012 for AM and 0.9999, 9698.5 and 6342.9 for CAF, whereas the LOD and LOQ was 0.51, 1.64 µg.mL-1 for AM and 0.60, 1.32 µg.mL-1 for CAF