Derivatization Reaction Research Articles

Liquid Chromatography Analysis of Reactive Oxoammonium Cations

This study presents the first liquid chromatography method for the quantitative and qualitative analysis of highly reactive oxoammonium cations based on a simple derivatization reaction. Rapid 1,2-electrophilic addition reactions with olefins were used to transform these reactive species into analyzable derivates. Three model substances were chosen to represent each of the main application fields of oxoammonium cations and to demonstrate the versatility of the method. The measuring protocol was validated according to the ICH and USP guidelines. The method revealed an excellent linearity (R2 = 0.9980–0.9990) with a low limit of detection (0.16–0.14 mmol L−1) and a low limit of quantification (0.55–0.43 mmol L−1). The protocol was finally used to determine the oxoammonium cations in the presence of their corresponding radical, showing a robustness against impurity concentration of up to approx. 30%.

Parallel derivatization strategy for comprehensive profiling of unconjugated and glycine-conjugated bile acids using Ultra-high performance liquid chromatography-tandem mass spectrometry

Bile acids (BAs) are steroidal compounds that play important roles in the occurrence and development of liver injury. However, comprehensive characterization of BAs was rarely reported due to the limitations of both standards access and detection sensitivity. In this study, a parallel derivatization strategy was established for the sensitive and comprehensive profiling of unconjugated and glycine-conjugated BAs by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Two structural analogues 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP) were used as the parallel derivatization reagents for BAs labeling, facilitating the improvements of both detection sensitivities and chromatographic performances. The derivatization reactions can be completed in 20 min at room temperature, with derivatization efficacy higher than 99 %. Through derivatization, the sensitivity of BAs increased dozens or hundreds of times compared to their non-derivatized forms. Due to the structural similarities of derivatized BAs, general MS parameters can be forged for the analysis of DMP and DP labeled BAs. In addition, the DP labeled BAs were incorporated into the DMP derivatized biological samples for both the discovery and comprehensive characterization of BAs. Retention time shift (RTS) and peak area ratio (PAR) induced by the parallel DMP and DP labeled BAs were used for the rapid identification of BAs from complex biological samples. More than 200 BAs were profiled in rat serum using this parallel derivatization strategy. Further, the new strategy was successfully implemented in BAs profiling of serum samples from tripterysium glycosides-induced liver injury rat model. The disturbance of the BA metabolism network was further interpreted.

Separation and determination of 3-hydroxyaspartate by online concentration capillary electrophoresis/laser-induced fluorescence with microwave-assisted derivatization.

A chiral analytical method was proposed based on capillary electrophoresis with laser-induced fluorescence detection coupled with microwave-assisted derivatization for the simultaneous baseline separation and sensitive detection of four stereoisomers of 3-hydroxyaspartate. The derivatization reaction of 3-hydroxyaspartate with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole was greatly accelerated by microwave irradiation. Under the optimized conditions, the derivatization yield was increased by 20% and the derivatization time was shortened by 20 min when compared with those from conventional water bath heating. In addition, the sensitivity was improved by online sample concentration methods. The detection limit of l-threo-3-hydroxyaspartate obtained by large-volume sample stacking with polarity switching was 5.3nmol/L, which was around 1000-fold lower than that of the capillary electrophoresis/laser-induced fluorescence without stacking. The excellent analytical performance in terms of linearity and precision was also achieved. Furthermore, the developed method was successfully applied to the determination of 3-hydroxyaspartate in the spiked urine, and satisfactory recoveries were obtained ranging from 90.5 to 107.0%.

Derivatizing Reagents for Detection of Organic Compounds By HPLC

Derivatization is the process of chemically modifying a compound to develope a new compound which has properties that are suitable for analysis using HPLC. Derivatization improves the detectability of a target analyte by reaction with suitable derivatizing agent. Derivatization reactions are simple chemical modification of substance that make it compatible with the selected separation method or transforms substance with a low UV- absorption into highly sensitive product. Derivatization reactions in liquid chromatography modify the solutes adding a chomophore for easy UV detection or a fluorophore for sensitive fluorescent detection. The chemical structure of the compound remains same and just modifies the specific functional group for reacting compounds to derivative of deviating chemical and physical properties in order to make them detectable. Introduction of certain elements or groups through chemical derivatization may enhance the detector’s response helpful for the elucidation of structure of analytes. In conclusion, the present review describe various derivatization reagents for pre-column and post column derivatization process in HPLC by UV-visible and fluorescence detection are summarized along with reactions and some practical aspects. The commonly used derivatizing reagents in HPLC are 1-fluoro-2, 4-dinitrobenzene, ninhydrine, 4-N-N-dimethylaminoazobenzene-4’-sulfonyl chloride, benzoyl chloride, phenyl isocyanate for UV-visible detection and o-phthalaldehyde, fluorescamine, 1-dimethylaminonapthalene-5-sulfonyl chloride (DNS-Cl), 9-fluorenylmethyl chloroformate (Fmoc-Cl), benzofurans for Fluorescence detection.

A Facile Method for the Quantification of Urinary Uracil Concentration by a Uracil-Specific Fluorescence Derivatization Reaction.

A facile and reliable fluorescence method for the quantification of urinary uracil concentration is proposed herein. The assay utilizes a specific fluorescence (FL) derivatization reaction for uracil using 3-methylbenzamidoxime as a fluorogenic reagent. Although the presence of urine inhibited the FL reaction, 10 µL of urine was sufficient for the detection of urinary uracil. The uracil derivative was successfully separated from other fluorescent impurities using simple reversed-phase LC with FL detection. Urinary uracil concentrations from 16 people were compared with the concentrations obtained by the traditional column-switching liquid chromatographic analysis with UV detection. The FL derivative of uracil appeared as a single peak in the chromatograms of all samples. However, several samples showed an additional peak overlapping the uracil peak when using the column-switching method because of UV-active impurities. These results indicated that that the present method is not affected by interfering substances in urine and affords a precise determination of urinary uracil. We expect the proposed method to be applicable for diagnosing dihydropyrimidine dehydrogenase deficiency in 5-fluorouracil chemotherapy.

Analysis of Organophosphorus-Based Nerve Agent Degradation Products by Gas Chromatography-Mass Spectrometry (GC-MS): Current Derivatization Reactions in the Analytical Chemist's Toolbox.

The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to stress the importance of developing methods for their rapid and unambiguous detection. Although the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must rely on the detection of the products arising from their degradation. To this end, derivatization reactions mainly in the form of silylations and alkylations employing a vast array of reagents have played a pivotal role in the efficient detection of these products that can be used retrospectively to identify the original OPNA.

Determination of hydrazine in prednisolone by derivatization-gas chromatography-triple quadrupole mass spectrometry

泼尼松龙是一种广泛用于临床治疗的肾上腺糖皮质激素药物,其中联氨的残留会直接影响用药安全,但目前国内外还没有出台相应的法律法规和标准来管控药物中联氨的残留限值。联氨具有强极性和强还原性,理化性质很不稳定,易被氧化,又因缺少发色团,相对分子质量太小,检测起来难度很大,需引入一种衍生化试剂,降低其极性,生成相对分子质量较大且理化性质稳定的衍生产物。该研究通过优化衍生化试剂、色谱-质谱条件、溶剂体系和衍生化条件,建立了衍生化-气相色谱-三重四极杆质谱法(GC-MS/MS)测定泼尼松龙中联氨残留的方法,并进行了方法学验证,结果满意。称取1 g泼尼松龙样品置于10 mL具塞离心管中,加入稀释溶剂(甲醇-二氯甲烷(14:23, v/v))至刻度线,涡旋振荡至样品完全溶解后,吸取100 μL置于进样小瓶中,再加入丙酮900 μL,涡旋振荡混匀,样品在丙酮-稀释溶剂(9:1, v/v)中同时完成稀释和衍生化反应后,再经GC-MS/MS检测分析。该研究的衍生化反应无需在添加冰乙酸和超声条件下进行,也无需再添加其他试剂进行萃取操作,联氨与丙酮可瞬间发生衍生化反应,直接实现泼尼松龙中联氨的快速测定。结果表明,联氨在1~12 μg/L范围内线性关系良好,线性相关系数(r2)为0.9999;检出限、定量限分别为0.03和0.10 mg/kg;进样精密度(relative standard deviation, RSD)为1.10%。加标回收率和重复性良好,加标水平分别为1、6、12 μg/L时的回收率为96.15%~96.46%,对应的RSD值为1.77%~2.12%。中间精密度良好,不同时间、不同人员在同一台仪器上测定结果的RSD值为1.77%。方法耐用性良好,通过改变色谱条件来研究检测结果受影响程度大小,在原条件、初始柱温±5 ℃、升温速率±2 ℃/min、柱流量±0.1 mL/min的条件下分别对加标6 μg/L的样品溶液中的联氨含量进行检测,检测结果的RSD值为2.58%。应用建立的方法测定泼尼松龙市售标准品和某药企提供的9个不同批次的泼尼松龙样品,均未检出联氨。该方法操作简便、准确可靠、灵敏度高、选择性好,可用于泼尼松龙中联氨的检测。

Determination of Lisinopril in Pure and Tablet form by Using 2-Hydroxynaphthaldehyde as Derivatizing Reagent

An easy, sensitive and accurate spectrophotometric method has been developed for the determination of Lisinopril (LNP) in pure and tablet formulations based on derivatization reaction with 2-hydroxynaphthaldehyde (2HNA). The derivatization reaction was carried out in methanol solvent at pH-5.5 at 95±2C for 15 min. The linear calibration curve was obtained that obeyed the Beer’s law within the concentration range 5-50 μgmL-1 of LNP at 433 nm with a coefficient of determination R²=0.996. The recovery was in the range from 98.25-101.82 with molar absorptivity of drug 9×103 mole-1cm-1. The method was accurate and precise (intra-day variation 0.05-0.97% and inter-day 0.07-1.6%), with limit of detection (LOD) and limit of quantification (LOQ) 0.264 μgmL-1 and 0.8 μgmL-1, respectively. No interferences from the excipients were detected. The method was applied for the rapid analysis of LNP in pharmaceutical products.

A comparative study of two HPLC methods for dissolved monosaccharide analysis in seawater using 2-amino benzamide and 2-amino pyrazine as pre-column derivatization reagents

Two reversed-phase HPLC methods for molecular analysis of dissolved free monosaccharides in seawater were investigated comparatively by optimizing chromatographic separations and pre-column derivatization reactions. Monosaccharides derivatized with 2-amino benzamide or 2-amino pyrazine, and reduced with 2-picoline borane or 2-dimethylamino borane. According to results of optimization, separation performance, and detection limits, 2-amino benzamide method gave better results than 2-amino pyrazine method. Among 12 monosaccharides tested, it was possible to quantify glucose + galactose, galacturonic acid, glucuronic acid, xylose + arabinose, ribose, mannose, and N-acetyl-d-glucosamine with detection limits between 1.2 and 11 nM with intra-day repeatability of 2–9% and inter-day repeatability of 3–9%. The optimized method has the same level of detection limit with a widely used anion exchange chromatography method. Besides the preliminary results reported in this study, it may be possible to achieve higher sensitivity and to detect more monosaccharides by the use of shorter and narrow-bore columns at different polarities in further studies.

Characterization of benzenemethanethiol in sesame-flavour baijiu by high-performance liquid chromatography-mass spectrometry and sensory science

Chinese sesame-flavour baijiu (CSB) is one of aroma types discovered after the founding of China. Sulfur-containing compounds have low content in CSB but play an important role. This study developed a quantitative method by derivatization reaction combined LC-MS/MS for the determination of benzenemethanethiol (BM), which has a roasted aroma in CSB. Under the optimized conditions, the LOD and LOQ of BM were 11.73 ng L−1 and 39.09 ng L−1, respectively. The recovery yield of the derivatization product ranged from 82.12% to 93.99%. The content of BM in 4 CSBs were 150.22–860.40 ng L−1. To confirm the contribution of BM to the overall aroma of CSB, aroma recombination experiments and omission experiments were carried out. Compared with that of the simulated baijiu sample without BM, the roasted aroma of the simulated baijiu sample containing BM was enhanced. Omission experiments showed that BM made important contributions to the overall aroma of CSB.

Synthesis of γ-Oxo-α-amino Acids via Radical Acylation with Carboxylic Acids.

Herein we present a highly efficient, light-mediated, deoxygenative protocol to access γ-oxo-α-amino acid derivatives. This radical methodology employs photoredox catalysis, in combination with triphenylphosphine, to generate acyl radicals from readily available (hetero)aromatic and vinylic carboxylic acids. This approach allows for the straightforward synthesis of γ-oxo-α-amino acids bearing a wide range of functional groups (e.g., Cl, CN, furan, thiophene, Bpin) in synthetically useful yields (∼60% average yield). To further highlight the utility of the methodology, several deprotection and derivatization reactions were carried out.

Evaluation of Different Brands of Cefixime Available in Pakistan by Newly Developed Spectrophotometric Method

A fast, simple and valued method is developed to observe the quality and quantity of different pharmaceutical brands of cefixime. A spectrophotometric method has been developed for analysis of cefixime (CFX) by reacting with 4-dimethylamino benzaldehyde (DAB) as derivatizing agent. The molar absorptivity of CFX-DAB, newly synthesized derivative was calculated as 3.2 x 105 L.mole-1cm-1 and λ maximum was 393 nm. The calibration curve was developed in range of 5-25 µg.mL-1as this concentration followed beers law. The derivatization reaction is stable and didn’t show any difference in absorbance with radiation interaction for up to one day. The percentage recovery of CFX was checked and calculated in different pharmaceuticals was within 95 to 99.5% with RSD value calculated in between RSD 0.69-0.96% (n=3), respectively. This newly developed and validated procedure was proved to be accurate and precise for the analysis of CFX. This method was successfully applied to check amount of CFX from 7 different brands of pharmaceutical preparations commercially available in Pakistan.

Synthesis of cyclodextrin‐based radial polycatenane cyclized by amide bond and subsequent fabrication of water‐soluble derivatives

In recent years, functional materials using cyclodextrin (CD)-based polyrotaxanes have attracted considerable attention, and derivatization reactions are often performed to develop them because of the insolubility of the parent polyrotaxane in water. Recently, we prepared CD-based radial polycatenanes in one-pot through polypseudorotaxane formation between β-CD and thiolated pluronic and subsequent cyclization via disulfide bonds. However, no reports are available on syntheses of CD polycatenane derivatives. The disulfide bonds are not stable enough to synthesize CD polycatenane derivatives; therefore, we herein prepared a chemically stable β-CD polycatenane via amide bond through cyclization of β-CD/aminated pluronic polypseudorotaxane with terephthaloyl chloride. This allowed the derivatization of the polycatenane owing to its stable chemical structure, and resulted in the syntheses of three different water soluble polycatenane derivatives: aminated, succinylated, and hydroxypropylated polycatenanes. These findings may provide useful information for the fabrication of polycatenane-based materials such as slide-ring materials, drug carriers, and biomaterials.

Application of the TLC image analysis technique for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement

A simple and sensitive thin-layer chromatography (TLC) method coupled with an image analysis technique was developed for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement with good precision and accuracy. Separation was performed on silica gel plates using ethanol‒toluene (2:3, V/V) as the mobile phase. The visualization of chromatograms was based on iodine–azide reaction; therefore, pre-chromatographic derivatization reaction of amino acids with phenyl isothiocyanate was performed. Digital images of TLC plate chromatograms were converted into peak chromatograms, and quantitative analysis was conducted using TLSee software.

A novel approach for the detection and identification of sulfur mustard using liquid chromatography-electrospray ionization-tandem mass spectrometry based on its selective oxidation to sulfur mustard monoxide with N-iodosuccinimide.

A new derivatization strategy for the detection and identification of sulfur mustard (HD) via liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is developed. The method incorporates selective oxidation of the sulfide group by the electrophilic iodine reagent N-iodosuccinimide (NIS) to produce sulfur mustard monoxide (HDSO). The derivatization reaction efficiencies were evaluated with acetonitrile extracts of soil, asphalt, cloth, Formica, and linoleum spiked with HD at concentrations of 50-5000 pg/ml and found to be similar to that with pure acetonitrile. The current derivatization approach is the first to preserve the identity of chloride groups and support HD regulation and evidentiary findings.

Rhodium(II)‐Catalyzed [4+2] Annulation of Ester‐Tethered 1‐Sulfonyl‐1,2,3‐Triazoles and Hexahydro‐1,3,5‐Triazines

AbstractA rhodium(II)‐catalyzed [4+2] annulation of ester‐tethered 1‐sulfonyl‐1,2,3‐triazoles and hexahydro‐1,3,5‐triazines, which features a chemoselective 1,2‐ester migration of α‐imine rhodium carbene intermediates, has been developed. Most of the valuable 1,2,3,4‐tetrahydropyrimidine derivatives were generated in good to excellent yields under mild conditions. Several important derivatization reactions have been performed.

Development and Validation of Two New Methods for the Determination of Rilmenidine in Bulk and Pharmaceutical Preparation.

In the present study, two new methods were developed and validated for the determination of rilmenidine in bulk and pharmaceutical preparation. Both methods are based on a derivatization reaction using 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) as a fluorogenic substance. The drug reagent derivatives were formed by the reaction of rilmenidine with NBD-Cl at pH 9.0 at 70°C for 40 min. The reaction mixtures were analyzed by spectrofluorimetry in the first method and high performance liquid chromatography (HPLC) in the second method. Derivatives were determined at λex 493 nm and at λem 536 nm in the spectrofluorimetric method. The separation was performed place on a Phenomenex, C18 column (250 × 4.6 mm, 5 μm i.d) using a mobile phase comprising 0.2% formic acid and acetonitrile gradient elution mode in the HPLC method. Analytes were detected by a fluorescence detector at the same wavelength. The methods were validated for limit of quantitation, linearity, robustness, recovery, limit of detection, precision and accuracy. Calibration curves for the first and second methods were found to be linear in the range of 2.0-12.0 and 250-2000 ng/mL, respectively. Detection limits for the spectrofluorimetric and HPLC methods were calculated as 0.16 and 18.28 ng/mL, respectively. The validated methods were applied successfully to the determination of rilmenidine in bulk and pharmaceutical preparation.

Long-chain syn-1-phenylalkane-1,3-diyl diacetates, related phenylalkane derivatives, and sec-alcohols, all possessing dominantly iso-branched chain termini, and 2/3-methyl-branched fatty acids from Primula veris L. (Primulaceae) wax

Herein, the results of the first study of non-flavonoid constituents of aboveground surface-wax washings of Primula veris L. (Primulaceae) are presented. Chromatography of the washings yielded a minor fraction composed of n-, iso-, and anteiso-series of long-chained syn-1-phenylalkane-1,3-diyl diacetates, 3-oxo-1-phenylalkan-1-yl acetates, 1-phenylalkane-1,3-diones, 1-hydroxy-1-phenylalkan-3-ones, sec-alcohols (2- to 10-alkanols), and n-, iso-, anteiso-, 2-methylalkanoic and 3-methylalkanoic acids; 118 of these constituents represent up to now unreported natural compounds. The structural/stereochemical elucidation was accomplished by the synthesis of authentic standards, derivatization reactions, the use of gas chromatographic retention data and detailed 1D and 2D-NMR analyses of the obtained complex chromatographic fraction. Primula veris produces unusually high amounts of branched long-chained metabolites (>60%) except for the fatty acids where the percentage of branched isomers is comparable to the ones with n-chains. Noteworthy is the fact that long-chained α- and β-methyl substituted fatty acids were detected herein for the first time in the kingdom Plantae.

Simple and rapid determination of biogenic amines in fish and fish products by liquid chromatography–tandem mass spectrometry using 2,4,6-triethyl-3,5-dimethyl pyrylium trifluoromethanesulfonate as a derivatization reagent

A simple and rapid analytical method was developed for determination of four biogenic amines [histamine (Him), cadaverine (Cad), tyramine (Tym), 2-phenylethylamine (Pea)] in fish and fish products. This method uses a new derivatization reagent, 2,4,6-triethyl-3,5-dimethyl pyrylium trifluoromethanesulfonate (Py-Tag). The four biogenic amines in the samples were extracted with trichloroacetic acid. The diluted extract was derivatized with Py-Tag (15 min at 50°C) and then subjected to liquid chromatography–tandem mass spectrometry (LC–MS/MS). The limits of quantification for the method were 2 mg/kg for Him, Tym, and Pea and 10 mg/kg for Cad. The matrix effects derived from the tested fish and fish products were negligible in the LC–MS/MS analysis. The impact of the sample matrices on the Py-Tag derivatization was also negligible. The trueness and repeatability of the method were assessed by performing replicate analyses (n = 5) of five samples of fish and fish products, each spiked with the four biogenic amines at three different concentration levels. Analysis of the samples found 87%–104% of the spiked concentrations and the relative standard deviations were <6.1%. A reference sample and quality control canned fish samples were analyzed by the method, and the concentrations of the Him were within acceptable limits. The developed method was successfully used to determine concentrations of the four biogenic amines in 48 fish and fish products on the Japanese market. The developed method does not require cleanup using a solid-phase extraction column or similar, and the derivatization reaction time was only 15 min. The results suggested that the present method is reliable and suitable for rapid analysis of the four biogenic amines in fish and fish products.

Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the determination of glyphosate in human urine after pre-column derivatization with 9-fluorenylmethoxycarbonyl chloride

In 2015, glyphosate was classified as “Group 2A – probably carcinogenic to humans” by the International Agency for Research on Cancer (IARC). Therefore, public concerns about the environmental and health risks of this substance have rapidly increased. Considering its toxicokinetic characteristics, urinary levels of glyphosate could be a powerful tool for human biomonitoring. Nevertheless, the physicochemical properties of this molecule and the complexity of the matrix make this purpose particularly challenging. In order to solve this problem, the presented study describes a simple LC-MS/MS method for the quantification of glyphosate in human urine after pre-column derivatization with FMOC-Cl. Method development was focused on the optimization of the derivatization reaction in human urine, adjusting critical variables such as pH of borate buffer, FMOC-Cl concentration and derivatization time. Besides, chromatographic separation and spectrometric parameters were also established. The analytical method was fully validated according international guidelines for selectivity, carry over, linearity, accuracy, precision, lower limit of quantitation, matrix effect and stability under different conditions. All performance parameters were within the acceptance criteria. In addition, the method was successfully applied to 52 urine samples obtained from exposed subjects from northern Argentina, laying the foundation for future epidemiological studies.