HPLC Determination of Chlorpropamide in Human Serum by Fluorogenic Derivatization Based on the Suzuki Coupling Reaction with Phenylboronic Acid

A fluorogenic derivatization method for the determination of chlorpropamide in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. The Suzuki coupling reaction with a non-fluorescent reagent, phenylboronic acid (PBA), was employed to convert chlorpropamide into highly fluorescent biphenyl derivative. Chlorpropamide was extracted from human serum by liquid–liquid extraction with toluene after addition of hydrochloric acid, and subsequently reacted with PBA. Because the fluorogenic derivatization was highly selective for aryl halide, the proposed method allowed sensitive and selective detection of chlorpropamide with a detection limit (at a signal to noise ratio of 3) of 0.5 ng mL⁻¹. The sensitivity of our method was from 4 to 100 times better than HPLC–UV, gas chromatography, and LC-mass spectrometry.

     

Optimization of o‐phtaldialdehyde/2‐mercaptoethanol postcolumn reaction for the hydrophilic interaction liquid chromatography determination of memantine utilizing a silica hydride stationary phase

A rapid procedure for the determination of memantine based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o‐phtaldialdehyde/2‐mercaptoethanol was performed at excitation and emission wavelengths of 345 and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, and reagents concentration were studied due to steric hindrance of amino group of memantine. The derivatization reaction was applied for the hydrophilic interaction liquid chromatography method which was based on Cogent Silica‐C stationary phase with a mobile phase consisting of a mixture of 10 mmol/L citric acid and 10 mmol/L o‐phosphoric acid (pH 6.0) with acetonitrile using an isocratic composition of 2:8 v/v. The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, accuracy, precision, and selectivity according to the International Conference on Harmonisation guidelines. The developed method was successfully applied for the analysis of commercial memantine tablets.     

HPLC Determination of Chlorpropamide in Human Serum by Fluorogenic Derivatization Based on the Suzuki Coupling Reaction with Phenylboronic Acid

A fluorogenic derivatization method for the determination of chlorpropamide in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. The Suzuki coupling reaction with a non-fluorescent reagent, phenylboronic acid (PBA), was employed to convert chlorpropamide into highly fluorescent biphenyl derivative. Chlorpropamide was extracted from human serum by liquid–liquid extraction with toluene after addition of hydrochloric acid, and subsequently reacted with PBA. Because the fluorogenic derivatization was highly selective for aryl halide, the proposed method allowed sensitive and selective detection of chlorpropamide with a detection limit (at a signal to noise ratio of 3) of 0.5 ng mL^?1. The sensitivity of our method was from 4 to 100 times better than HPLC–UV, gas chromatography, and LC-mass spectrometry.     

Derivatization reaction of the mycotoxin moniliformin with 1,2-diamino-4,5-dichlorobenzene: structure elucidation of an unexpected reaction product by liquid chromatography/tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance spectroscopy

The derivatization reaction of the mycotoxin moniliformin with 1,2-diamino-4,5-dichlorobenzene was previously introduced to improve distinctly the sensitivity of an assay applying high-performance liquid chromatography prior to fluorescence detection. In the course of the implementation of this derivatization approach into a liquid chromatographic/mass spectrometric method, an unexpected derivatization product has now been discovered by mass spectrometry. In order to elucidate its structure, detailed investigations with liquid chromatography/tandem mass spectrometry and liquid chromatography coupled on-line with NMR spectroscopy were performed. These studies give evidence for a heterocyclic structure that has been formed by the loss of one water and one carbon monoxide molecule. A reasonable mechanism for this derivatization reaction is proposed.     

Pre-Column Derivatization of Amines with 1,2-Benzo-3,4-Dihydrocarbazole-9-Isopropyl Chloroformate Followed by LC-Fluorescence and LC-APCI-MS

A pre-column derivatization method for the sensitive determination of amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate (BCIC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives is carried out by high performance liquid chromatography/atmospheric pressure chemical ionization (LC-APCI-MS-MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent is replaced by 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl functional group, which results in a sensitive fluorescence derivatizing reagent BCIC-Cl. BCIC-Cl can easily and quickly label amines. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography and show an intense protonated molecular ion corresponding m/z [MH]⁺ under APCI in positive-ion mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 260 corresponding to the cleavage of CH₂-OCO bond. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0–10.0. Maximal yields close to 100% are observed with a 3 to 4-fold molar reagent excess. In addition, the detection responses for BCIC derivatives are compared with those obtained using CEOC and FMOC as derivatization reagents. The ratios of I_BCIC/I_CEOC and I_BCIC/I_FMOC are, respectively, 1.23–3.14 and 1.25–3.08 for fluorescent (FL) responses (here, I is relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C₈ column. Detection limits are calculated from 1.0 pmol injection, at a signal-to-noise ratio of 3, are 10.6–37.8 fmol. The mean interday accuracy ranges from 94 to 105% for fluorescence detection with the largest mean %CV<7.5. The mean interday precision for all standards is < 6.0% of the expected concentration. Excellent linear responses are observed with coefficients of > 0.9997. Revised: 12 December 2005 and 13 Febrauary 2006     

A Versatile Dynamic Light Scattering Strategy for the Sensitive Detection of Plant MicroRNAs Based on Click-Chemistry-Amplified Aggregation of Gold Nanoparticles

Plant microRNAs (miRNAs) are naturally 2′-O-methylated at the 3′-terminal; as a consequence, they cannot be efficiently detected by traditional target-triggered polymerization reactions. Here, a simple but robust enzyme-free sensing strategy was developed for plant miRNA analysis by using dynamic light scattering (DLS) to monitor the crosslinking of gold nanoparticles (AuNPs) amplified by click chemical ligation. Combining the enzyme-free cycling chemical-ligation-mediated signal amplification, and the intrinsic outstanding ability of DLS for discriminating the extremely low level of particle aggregation in a large pool of monodisperse AuNPs, high sensitivity was achieved and as low as 78.6 fm plant miRNA could be easily detected.     

A computational model for non-conserved mature miRNAs from the rice genome

Several computational approaches employ the high complementarity of plant miRNAs to target mRNAs as a filter to recognize miRNA. Numerous non-conserved miRNAs are known with more recent evolutionary origin as a result of target gene duplication events. We present here a computational model with knowledge inputs from reported non-conserved mature miRNAs of Oryza sativa (rice). Sequence- and structure-based approaches were used to retrieve miRNA features based on rice Argonaute protein and develop a multiple linear regression (MLR) model (r² = 0.996, q²_cv = 0.989) which scored mature miRNAs as predicted by the MaturePred program. The model was validated by scoring test set (q² = 0.990) and computationally predicted mature miRNAs as external test set (q²_test = 0.895). This strategy successfully enhanced the confidence of retrieving most probable non-conserved miRNAs from the rice genome. We anticipate that this computational model would recognize unknown non-conserved miRNA candidates and nurture the current mechanistic understanding of miRNA sorting to unveil the role of non-conserved miRNAs in gene silencing.     

Response surface methodology for optimization of cyanamide analysis by in situ derivatization and dispersive liquid–liquid microextraction

Cyanamide is widely used for agricultural purposes; therefore, its residues can be found in water. A new method was developed for its quantification using in situ derivatization with 2,6‐dimethyl‐4‐quinolinecarboxylic acid N‐hydroxysuccinimide ester followed by dispersive liquid–liquid microextraction (DLLME) and high‐performance liquid chromatography/fluorescence analysis. Multivariate chemometric techniques were successfully used to obtain the optimum conditions for direct derivatization and DLLME extraction. Derivatization parameters and DLLME extraction conditions were optimized by a two‐step design, 2^k factorial design for screening, and central composite design for optimization. Best derivatization conditions were addition of 600 μL of derivatizing reagent, a temperature of 4 ºC, and pH 8.5, whereas for optimum extraction 800 μL of solvent, 30% NaCl conc. w/v, and pH 3.8 were chosen. The analytical performance of the method for routine analysis was evaluated. Excellent linearity was achieved from 10 to 200 µg L⁻¹ with a correlation factor of 0.9996. Precision ranged from 3.5% to 5.5% for intraday assays and 8.5% to 8.6% for interday assays. The mean recoveries performed on water from different origins (ground, river, sea, tap, and mineral) at three levels of concentration (20, 75, and 200 µg L⁻¹) ranged from 90.2% to 110.2%. Copyright © 2014 John Wiley & Sons, Ltd.     

固相萃取-高效液相色谱荧光检测法测定人血浆3-硝基酪氨酸含量

建立了人血浆中痕量3-硝基酪氨酸的高效液相色谱-荧光检测法. 血浆经乙腈沉淀蛋白后,上清液用氮气吹干,残渣复溶后过C18固相萃取小柱净化浓缩,洗脱液再用氮气吹干,残渣进行荧光衍生化反应.采用高效液相色谱分离,荧光检测器检测,外标法定量.3-硝基酪氨酸在0.50～50.0 nmol/L范围内线性关系良好,相关系数为0.9999;样品加标绝对回收率为89.3%～91.9%,相对回收率为99.2%～107.9%;日内相对标准偏差为1.46%～4.79%,日间相对标准偏差为2.75%～9.20%;在血浆中检出限为0.25 nmol/L;测定正常人血浆中3-硝基酪氨酸平均浓度为118 nmol/L(n=21),Ⅱ型糖尿病病人血浆中3-NT平均浓度为3.63 nmol/L(n=23).本方法灵敏度高,重现性好,适用于临床研究中大样本量的测定.     

Tetrafluorination of Aromatic Azide Yields a Highly Efficient Staudinger Reaction: Kinetics and Biolabeling

The development of highly efficient bioorthogonal reactions is of paramount importance for the research fields of biomaterials and chemical biology. We found that the o,o′‐difluorinated aromatic azide was able to react with triphenylphosphine to produce water‐stable phosphanimine. To further improve the efficiency of this kind of nonhydrolysis Staudinger reaction, a tetrafluorinated aromatic azide was employed to develop a faster nonhydrolysis Staudinger reaction with a rate of up to 51 m ⁻¹ s⁻¹, as revealed by high‐performance liquid chromatography (HPLC) analysis and fluorescence kinetics. As a proof‐of‐concept study, the highly efficient Staudinger reaction was successfully used for chemoselective fluorescence labeling of proteins and nucleic acids (DNA and RNA) as well as for protein polyethyleneglycol (PEG)ylation. We believe that this bioorthogonal reaction can provide a broadly useful tool for various bioconjugations.     

Simultaneous Determination of Safingol and d-erythro-Sphinganine in Human Plasma by LC with Fluorescence Detection

l-threo-Sphinganine (safingol) is a putative synthetic sphingosine kinase inhibitor currently being tested in clinical trials as an anticancer agent. To enable defining the pharmacokinetic properties of safingol in humans, we developed a sensitive analytical method to simultaneously quantitate safingol and its naturally-occurring diastereomer, d-erythro-sphinganine in human plasma. Of the two different fluorogenic derivatization agents (NDA and OPA) and several pH conditions compared for the derivatization, we found that NDA derivatization achieved more than 20 times greater sensitivity compared with OPA derivatization, and pH 9.0 showed the highest sensitivity for both compounds. An analytical method for liquid chromatography (LC) with a fluorescence detector (FLD) was developed and validated with calibration curve ranges of 20–1,000 ng mL⁻¹ for safingol and d-erythro-sphinganine. Our LC-FLD method using NDA-derivatization enabled simultaneous quantification of safingol and its naturally-occurring diastereomer, d-erythro-sphinganine with satisfactory sensitivity in human plasma.

     

In situ derivatization–liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl‐4‐hydroxyproline by liquid chromatography–tandem mass spectrometry

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography–mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water‐immiscible organic phase. In the present work, the derivatization–liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker l ‐prolyl‐4‐l ‐hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N‐isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP‐C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7‐PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization–liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography–tandem mass spectrometry in the complex urine matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

A Simple and Rapid High Performance Liquid Chromatographic Method with Fluorescence Detection for the Estimation of Amikacin in Plasma ‐ Application to Preclinical Pharmacokinetics

A simple and sensitive reversed‐phase liquid chromatographic method has been developed for the determination of amikacin by derivatization. The method is based on the pre‐column derivatization of amikacin with 9‐fluorenylmethyl chloroformate (FMOC‐Cl). Isepamicin was used as the internal standard. The derivatization reaction proceeds in aqueous solution at room temperature with a borate buffer of pH 7.3. The formation of the corresponding derivative of amikacin is instantaneous and it is stable for more than 48 h. Detection was performed by fluorescence. Several factors influencing the derivatization reaction yields were studied and optimized. The system offered the following analytical parameters: limit of detection (LOD) of 90 ng mL^?1 (3σ), linear correlation coefficient of 0.9998 and linear range response from 0.45 to 21.60 μg‐mL^?1. The precision of the method was < 6%. As a preliminary application, the method has been successfully applied to the amikacin determination in parenteral pharmaceutical formulations.     

Reversed‐phase high‐performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors

Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of l ‐[³H]‐citrulline produced from l ‐[³H]‐arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed‐phase high‐performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of l ‐citrulline with o‐phthaldialdehyde/N‐acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N‐[3‐(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well‐established radiometric assay.     

Simple and sensitive determination of malondialdehyde in human urine and saliva using UHPLC–MS/MS after derivatization with 3,4‐diaminobenzophenone

Malondialdehyde has been used as a biomarker for lipid peroxidation in biological samples. An ultra‐high performance liquid chromatography with tandem mass spectrometry method was developed to determine the levels of malondialdehyde in human urine and saliva samples. To select the optimum derivatization reagent from four diamino compounds, the reactivity and sensitivity of their derivatives were compared, and 3,4‐diaminobenzophenone was selected. The optimum reaction conditions for malondialdehyde with 3,4‐diaminobenzophenone were as follows: a reagent dosage of 50 mg/L, pH of 4, and reaction for 30 min at 50°C. The formed derivative product was analyzed using ultra‐high performance liquid chromatography with tandem mass spectrometry without additional extraction or concentration steps. In the optimal conditions, the method was used to determine malondialdehyde concentration in human urine and saliva samples. The limits of quantification for malondialdehyde in biological samples were over a concentration range of 0.1–0.3 μg/L. Additionally, the calibration curve showed a linearity greater than r ² = 0.997. The method was used to analyze 14 human urine and saliva samples from healthy volunteers. Malondialdehyde was detected in the concentration range of 1.7–33.6 μg/g creatinine in all human urine samples and 0.1–1.3 μg/L in all human saliva samples.     

新型荧光衍生试剂用于脂肪胺的反相高效液相色谱-荧光检测分析

利用新型荧光试剂4-(1H-菲并[9,10-d]咪唑-2-)苯甲酸(PIBA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性。激发和发射波长分别为ex=261nm,em=443nm。80℃下在吡啶溶剂中用N-乙基-N’-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)做催化剂,衍生反应10min后获得稳定的荧光产物。在EclipseXDB-C8色谱柱(4.6150mm,5mm)上,梯度洗脱对12种游离脂肪胺衍生物进行了优化分离。采用大气压化学电离源(APCI)正离子模式,实现了各种脂肪胺衍生物的测定。多数脂肪胺的线性回归系数大于0.9999,检测限为10.5～53.4fmol。     

Immersed sol‐gel based amino‐functionalized SPME fiber and HPLC combined with post‐column photochemically induced fluorimetry derivatization and fluorescence detection of pyrethroid insecticides from water samples

A method based on direct immersion solid‐phase microextraction (DI‐SPME) coupled with high performance liquid chromatography combined with post‐column photochemically induced fluorimetry derivatization and fluorescence detection (HPLC‐PIF‐FD) was developed to extract three pyrethroid insecticides, i.e. cyfluthrin, cypermethrin, and flumethrin from water samples. A sol‐gel based coating fiber using 3‐(trimethoxysilyl propyl) amine as precursor was prepared and used for the extraction of the pyrethroids from groundwater samples. A post‐column photochemical reactor was designed and constructed for the derivatization of these environmentally important pollutants to increase their fluorescence sensitivity and determination in HPLC. The parameters affecting extraction process (extraction time and temperature, pH, salt addition, and co‐solvent) and desorption step (solvent, desorption time, and temperature) of the analytes from the sol‐gel‐based fiber, along with photochemical reaction conditions were investigated. The developed method proved to be relatively rapid, simple, and easy and offers high sensitivity and reproducibility. Linear dynamic ranges (LDR) for these insecticides were ranged between 0.25 to 50 μg/L. The regression coefficients were satisfactory (R² > 0.984) for these pyrethroids. The limits of detection and limits of quantification varied between 0.09 and 0.35 μg/L and 0.25 and 1.00 μg/L, respectively. Relative standard deviation RSDs values varied between 4.41% and 6.20%. Relative recoveries obtained from analysis of Jajroud river water sample ranged between 94% and 104%.     

Resolution of amine enantiomers using precolumn derivatization with dansyl-L-proline and reversed-phase liquid chromatography

A derivatization procedure was developed for converting enantiomeric amino acids, amino alcohols and amines into diastereoisomers for resolution by liquid chromatography. Dansyl-L-proline (DLP) was used as a chiral reagent for the precolumn derivatization of many enantiomeric compounds bearing primary and secondary amino groups. The diastereoisomeric amides that were formed in the presence of diethyl phosphorocyanidate can be resolved efficiently on a conventional reversed-phase column. The resolution was optimized by varying the mobile phase. Intramolecular hydrogen bonding of the diastereoisomeric amide is shown to be very important for the efficient resolution of enantiomers. The detection limits for DLP derivatives were 4 × 10^?14?5 × 10^?14 mol.     

Rapid determination of trace semicarbazide in flour products by high‐performance liquid chromatography based on a nucleophilic substitution reaction

Semicarbazide, a toxic food contaminant, widely exists in food products and it originates from the thermal degradation of a food additive of azodicarbonamide or a metabolite of nitrofurazone abused in meat specimens. Many previous methods for semicarbazide determination usually required expensive instruments, difficult‐to‐prepare monoclonal antibodies, and a long operation time. In this study, a high‐performance liquid chromatography method was developed for the rapid determination of trace semicarbazide coupling with a nucleophilic substitution reaction firstly using 4‐nitrobenzoyl chloride as derivatization reagent. The derivatization reaction was mild at room temperature for 1 min in neutral solution. Then, semicarbazide derivative was separated and quantified by high‐performance liquid chromatography with ultraviolet detection under optimal separation conditions at λ _max = 261 nm. The proposed method offered the detection limit of 1.8 μg/L and was successfully applied for the rapid determination of trace semicarbazide in flour products. Semicarbazide in positive real samples could be actually found and quantified in the range of 0.47−7.53 mg/kg. The recoveries were 76.6−119% with relative standard deviations of 0.5–9.1% (n = 3). This developed method was rapid, reliable, and convenient for the determination of trace semicarbazide in food.