Measurement of acid-catalyzed isomerization of unsaturated aldehyde-2,4-dinitrophenylhydrazone derivatives by high-performance liquid chromatography analysis

Aldehyde-2,4-dinitrophenylhydrazones exist as (E)- and (Z)-geometrical isomers, and adventitious isomerization during sample preparation can cause analytical errors. Purified alkenal-2,4-dinitrophenylhydrazone derivatives comprise only the (E)-isomer. However, partial isomerization to the (Z)-isomer occurs upon addition of acid to attain an equilibrium isomer ratio. The UV-visible spectral properties of the isomers differ; the (Z)-isomer exhibiting a 6-10 nm lower absorption maximum compared to the (E)-isomer. Alkenal-2,4-dinitrophenylhydrazones having a CC double bond at the 2- or 3-position of the alkenal exhibited similar absorption maxima with an equilibrium isomer ratio (0.035) that was much lower than those of other alkenals. The CC double bond at the 3-position migrates to a position of conjugation with the CN double bond during hydrazone synthesis to form a stabilized molecular structure. Alkenal-2,4-dinitrophenylhydrazones having a double bond at the 4-position or greater exhibited a similar absorption maxima equilibrium isomer ratio (0.14) to alkanal-2,4-dinitrophenylhydrazones. The quantitative analysis of carbonyl compounds in air or water using DNPH is usually conducted in the presence of an acid catalyst. Consequently, the solution of the direct extract prepared for HPLC or GC analysis contains both (E)- and (Z)-isomers.     

Reductive amination of glutaraldehyde 2,4-dinitrophenylhydrazone using 2-picoline borane and high-performance liquid chromatographic analysis

A typical method for the measurement of glutaraldehyde (GLA) employs 2,4-dinitrophenylhydrazine (DNPH) to form GLA-DNPhydrazone derivatives. However, this method is subject to analytical errors because GLA-DNPhydrazone is a quaternary bis-derivative and forms three geometric isomers (E-E, E-Z and Z-Z) as a result of the two C[double bond, length as m-dash]N double bonds. To overcome this issue, a method for transforming the C[double bond, length as m-dash]N double bond into a C-N single bond, using reductive amination of DNPhydrazone derivatives, has been applied. The amination reaction of GLA-DNPhydrazones with 2-picoline borane is accelerated with catalytic amounts of acid and is completed within 10 minutes in the presence of 100 mmol L(-1) phosphoric acid. Reduction of GLA-DNPhydrazone by 2-picoline borane is unique and results in the formation of N-(2,4-dinitrophenyl)-1-piperidinamine (DNPPA). NMR and LC-APCI-MS data confirmed the product identification. DNPPA is very stable and did not change when stored for at least four weeks at room temperature. DNPPA has excellent solubility of 14.6 g L(-1) at 20 °C in acetonitrile. The absorption maximum wavelength and the molar absorptivity of DNPPA were 351 nm and 4.2 × 10(4) L mol(-1) cm(-1) respectively. Complete separation between the reduced forms of C1-C10 aldehyde DNPhydrazones, including DNPPA, can be achieved by operating the reversed-phase high-performance liquid chromatograph at 351 nm in gradient mode using a C18 amide column. The reductive amination method for GLA overcomes analytical errors caused by E-E, E-Z and Z-Z geometrical isomers.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation

The most important steps in the validation of high-performance liquid chromatographic (HPLC) methods are discussed. The establishment of system suitability data and the assessment of peak purity are demonstrated on the example of bisquaternary amino steroids. For the recognition of incomplete resolution of adjacent peak pairs, the absorbance-ratio method in which the ratio of absorbances at two preselected wavelengths are plotted as a function of time in combination with the separation of sample components subjected to various chemical and physico-chemical treatments (stress conditions) is applied. The separation power and performance of the HPLC systems are characterized by the system resolution (SR) and system selectivity (SS). The special demands of stability-indicating methods are summarized.     

Results from interlaboratory comparisons of aldehyde-2,4 dinitrophenylhydrazone analysis

Within the framework of the SMT "Aldehydes" project (SMT4-CT97-2910) three laboratory comparisons have been organized in which artificial aldehyde hydrazone samples have been distributed among participating laboratories. To these samples potentially interfering compounds have been added to simulate as closely as possible real air or air emission samples.In addition to the "Aldehydes" project partners external EU laboratories have participated. It may, therefore, be assumed that the results of these comparisons reflect the state-of-the-art in the analytical determination of aldehydes in air samples using the 2,4-dinitrophenylhydrazine (DNPH)-method. In this paper several aspects of the organization of these comparisons and the overall results obtained are presented. These demonstrate that results obtained using liquid chromatographic techniques are usually comparable between laboratories within +/-2 times the coefficient of variation (approximately 6-15%), indicating the robustness of the methodology. In addition, laboratory (liquid chromatography) mean results can be made to agree with preparation concentrations at the 95% confidence levels after optimization of separation and integration conditions; these are mainly related to the measurement of formaldehyde in the presence of interferents and the measurement of low concentrations of acrolein and acetone. Some problem areas still remain, however--namely the measurement of glutaraldehyde and the measurement of low levels of acetone.     

High-performance liquid chromatography of dihydroxyacetone as its bis-2,4-dinitrophenylhydrazone derivative

Summary A rapid and simple HPLC method for the identification and determination of dihydroxyacetone, a tanning agent widely used in a variety of preparations, is presented. The method consists of the analysis of the formulated products after a derivatization reaction which enhances the response and the detection limit of the chromatographic analysis. The characterization and the elucidation of the spacial geometry of the synthesized derivative are described.     

High-performance liquid chromatography of dihydroxyacetone as its bis-2,4-dinitrophenylhydrazone derivative

Summary A rapid and simple HPLC method for the identification and determination of dihydroxyacetone, a tanning agent widely used in a variety of preparations, is presented. The method consists of the analysis of the formulated products after a derivatization reaction which enhances the response and the detection limit of the chromatographic analysis. The characterization and the elucidation of the spacial geometry of the synthesized derivative are described.     

Ion-pair high-performance liquid chromatographic analysis of aspartame and related products

A simple and accurate quantitative determination of aspartame (L-alpha-aspartyl-L-phenylalanine methyl ester), a new artificial sweetener, is described. The method, which is based on ion-pair high-performance liquid chromatography, allows the determination of aspartame in finished bulk and dosage forms, and the detection of a few related products at levels down to 0.1%.     

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.     

Method development and validation of a high-performance liquid chromatographic method for pyrethrum extract.

A robust analytical methodology is developed for the quantitative determination of all six insecticidal components of pyrethrum extract: jasmolin I, cinerin I, pyrethrin I, jasmolin II, cinerin II, and pyrethrin II. This method, based on the separation technique normal-phase high-performance liquid chromatography, offers selectivity, accuracy, precision, linearity, range, ruggedness, and robustness as well as efficiency and ease.     

High-performance liquid chromatographic separation of racemic and diastereomeric mixtures of 2,4-pentadienoate-iron tricarbonyl derivatives

beta-Cyclodextrin chiral stationary phase facilitates the chiral separation of the (+/-)-methyl-5-formyl-2,4-pentadienoate-iron tricarbonyl (1) racemic mixture. The separation of oxazolidine derivatives 2 and 3 diastereomers were achieved with a C18 column but the compounds underwent in-column hydrolysis to give (-)- and (+)-1, respectively. This hydrolysis was exploited for the determination of 2 and 3 by the beta-cyclodextrin column, namely 2 and 3 were initially and completely hydrolyzed in the column to give (-)- and (+)-1 and this racemic mixture was then separated by this chiral column.     

alpha-Alkyl-alpha-arylacetic acid derivatives as fluorescence markers for thin-layer chromatographic and high-performance liquid chromatographic assay of amines and alcohols

Activated R,S-benoxaprofen is described as a new reagent for fluorescent derivatization of drugs with primary or secondary amino groups or with hydroxyl groups. Separation of the reaction products is demonstrated by thin-layer chromatography and high-performance liquid chromatography. The sensitivity of the detection is in the picomole range. Derivatization procedures can be easily and rapidly performed.     

Development and validation of a stability-indicating high-performance liquid chromatographic assay for ketoprofen topical penetrating gel

An isocratic RP-HPLC procedure has been developed and validated for the quantitative determination of ketoprofen in a topical gel. The HPLC procedure consist of a YMC ODS-AQ, 5-microm particle size analytical column (150 mm x 4.6 mm); Alltech Econosphere C18, 5-microm particle size guard column; detection at 233 nm; 1 ml/min flow rate; 20-microl injection volume. The mobile phase consisted of pH 3.5 phosphate buffer-water-acetonitrile (8:43:49, v/v). Sample preparation was a simple extraction of ketoprofen with mobile phase. The above conditions resolved and eluted ketoprofen, excipients, and potential degradants within 35 min, with ketoprofen eluting at about 6.5 min. The procedure was validated with respect to specificity, accuracy, precision, and linearity. The accuracy of the procedure, determined by spike recovery measurements, was 100.1-100.5%. The intra- and inter-day precisions were demonstrated by the relative standard deviations (RSD) of 0.3-0.6% and 0.5%, respectively. The intermediate precision was determined by comparing the results obtained with four independently prepared samples by two chemists using two columns on different days. The results indicate no significant difference (P = 0.17). The procedure showed linearity over the concentration range 4 x 10(-5) to 1 x 10(-1) mg/ml.     

茂铁基二氢吡唑类衍生物的液相色谱分离研究

本文利用梯度洗脱进行溶剂强度的优化,确定分离一组化合物的最佳二元流动相体系,即甲醇-水、四氢呋喃-水和乙腈-水,将这三种二元流动相的最佳组成作为溶剂选择性三角形的三个顶点,利用ORM法对流动相进行优化选择,得到流动相的最佳组成和配比为V乙腈:V四氢呋喃:V水=33.75:33:33.25,有效地分离了六种二茂铁取代二氢...     

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Synthesis and Crystal Structure of Salicylaldehyde-2,4-dinitrophenylhydrazone

Some phenylhydrazone derivatives have been shown to be potentially DNA-damaging and mutagenic agents.[1] In order to investigate the relationship of the biological activity and the molecular structure, a series of new phenylhydrazone derivatives have been synthesized and their structures have been determined in our laboratory.