Determination of ethambutol hydrochloride in the combination tablets by precolumn derivatization

A new high-performance liquid chromatography method for the quantitative determination of ethambutol hydrochloride in combination tablets is presented. Ethambutol is derivatized with phenylethylisocyanatate at room temperature (22 +/- 2 degrees C) for 5 min. Separation is performed by a C(18) column using methanol-water-glacial acetic acid (70:30:0.2, v/v/v) as the mobile phase. The method is linear for drug concentrations in the range of 20-120 microg/mL (r=0.9995). The intra- and inter-day precisions are lower than 1.46% and 2.22%, respectively. The average recovery of the samples at three levels is 99.8%. The results show that derivatization of ethambutol is stable at 30 degrees C for 24 h. This method is simple, rapid, and stable in the presence of common excipients and antituberculosis drugs in the tablets.     

盐酸二甲双胍的毛细管电泳法快速测定

建立了毛细管电泳高频电导法快速测定片剂中盐酸二甲双胍的方法。考察了缓冲溶液、有机溶剂添加剂、毛细管长度以及分离电压和进样条件等因素对分离检测的影响。在最佳条件下5.0 min内即可实现盐酸二甲双胍分离检测,盐酸二甲双胍的线性范围为1.50μg/mL~130μg/mL,检出限为1.0μg/mL。该方法成功地测定了盐酸二甲双胍片剂中的盐酸二甲双胍。     

A method for the determination of histamine in wine by HPLC with precolumn derivatization with phenylisothiocyanate

Summary A method for determining histamine in wine by precolumn derivatization with PITC (phenylisothiocyanate) with reversed-phase HPLC and UV detection is reported. Histamine can be determined together with the 24 amino acids within 40 min, or separately in a shorter time (less than 4 min) if a prior solid phase extraction clean-up is used.     

An improved method for the determination of S-Carboxymethyl-L-cysteine in biological fluids with precolumn derivatization and on-line clean-up

Summary In order to follow levels of S-Carboxymethyl-L-cysteine in biological fluids for a period as long as three half-lives after drug administration during pharmacokinetic studies, an improved method for its determination had to be developed. Like the previous one, this method uses a protein precipitation step followed by an O-Phthalaldehyde derivatization step and then an HPLC on-line clean-up. This latter was obtained by means of a switching valve system, including a Nucleosil CN 5 m (3 cm × 4.6 mm i.d.) precolumn and a Spherisorb ODS 5 m (15 cm×4.6 mm i.d.) analytical column. The sensitivity limit was improved to 0.1 g/ml in plasma samples and 0.2 g/ml in urine samples.This method was applied in studies comparing single (0.75 g) and repeated (0.75 g tid) oral administration of the drug to 30 elderly patients and 20 healthy volunteers. Results showed that the half-life was 40% longer in elderly patients than in healthy volunteers, and that area under the plasma concentration versus time curve (AUC) values in elderly patients were twice those obtained with young subjects.     

盐酸二甲双胍药物中杂质测定方法研究进展

对近年来盐酸二甲双胍类药物中杂质的测定方法进行综述.盐酸二甲双胍类药物中的杂质主要有双氰胺、二甲胺以及由二甲胺形成的亚硝胺类基因毒性杂质等.测定方法主要有高效液相色谱(HPLC)法、离子色谱(IC)法、高效液相色谱–串联质谱(HPLC–MS/MS)法和气相色谱–串联质谱(GC–MS/MS)法,其中高效液相色谱法主要用于...     

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.     

High-performance liquid chromatographic method for determination of amino acids by precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

This work presents an high-performance liquid chromatography method for the determination of amino acids after precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) which can readily react with both primary and secondary amines. The precolumn derivatization conditions, including the CNBF concentration, reaction pH, temperature and reaction time were investigated for method optimization. In pH 9.0 borate buffer, the reaction of amino acids with CNBF was carried out at 60 °C for 30 min, the optimized concentration of CNBF was 70 mmol L⁻¹ and the molar ratio of amino acids to CNBF was 1:5.25. The chromatographic separation of 19 amino acids derivatives was performed on a Kromasil ODS C₁₈ column (250 mm × 4.6 mm, 5 μm) with good reproducibility, and ultraviolet detection was applied at 260 nm. The mobile phase was a mixture of phase A (acetonitrile) and phase B (acetate buffer, acetonitrile, triethylamine; 82.8:17:0.2, pH 4.9), and the flow rate was 0.4 mL min⁻¹. The separation of all the labeled amino acids was achieved within 45 min at room temperature by gradient elution mode. The method linearity, calculated for each amino acid, had a correlation coefficient higher than 0.9979, in concentrations ranging from 9.60 to 3330.00 μmol L⁻¹. The detection limits of amino acids were 2.40-6.50 μmol L⁻¹, at a signal-to-noise ratio of 3. The proposed method was applied for the determination of amino acids in beer with recoveries of 97.0-103.9% and relative standard deviations of 2.62-4.22%, respectively. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

Stability-indicating capillary electrophoresis method for the simultaneous determination of metformin hydrochloride,saxagliptin hydrochloride,and dapagliflozin in pharmaceutical tablets

A capillary electrophoretic method coupled to a diode array detector (CE-DAD) was developed and validated for the simultaneous determination of metformin hydrochloride (MET), the dipeptidyl peptidase-4 (DPP-4) inhibitor saxagliptin hydrochloride (SAX), and the sodium glucose co-transporter (SGLT 2) inhibitor dapagliflozin (DAP). The proposed method was used for the determination of these drugs in binary antidiabetic combinations namely, SAX/MET, combination I, DAP/MET, combination II, and SAX/DAP, combination III. CE separation was performed on a fused silica capillary with background electrolyte consisting of 30?mM phosphate buffer (pH 6.0) with a high voltage of 30?kV, a pressure of 20 mbar, and an injection time of 40?s. The compounds were detected at 203?nm for SAX/DAP and 250?nm for MET. The method was linear in the concentration range of 10–200?µ?g/mL (SAX), 1.25–50?µ?g/mL (DAP), and 7.5–1000?µ?g/mL (MET). Full validation of the proposed method was performed as per the ICH guidelines. The obtained errors and deviation values did not exceed 2% assessing good accuracy and precision, respectively. The stability-indicating potential of the proposed method was proved under different stress-degradation conditions. The proposed method was successfully applied to the analysis of the three binary combinations in their tablets.     

Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

For the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wave-length of 300 nm and an emission cut-off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 μl of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.     

HPLC determination of thiol drugs in pharmaceutical formulations using ethacrynic acid as a precolumn ultraviolet derivatization reagent

Summary A high performance liquid chromatographic method has been developed for the determination of aliphatic thiol drugs, such as N-acetyl-L-cysteine, captopril and mercaptopropionylglycine in pharmaceutical formulations. The procedure involves a precolumn derivatization of the thiol drug with ethacrynic acid followed by reversedphase HPLC separation and UV detection. The conditions for a rapid and selective reaction of the thiols with ethacrynic acid have been investigated. The method proved to be suitable for a reliable and selective quality control of commercial dosage forms of the examined thiol drugs.     

A precolumn derivatization high-performance liquid chromatographic method with improved sensitivity and specificity for the determination of astragaloside IV in Radix Astragali

A reversed-phase high-performance liquid chromatographic method is developed for the determination of astragaloside (AGS) IV, which is known as the active constituent of Radix Astragali. The method uses precolumn derivatization with benzoyl chloride to form the benzoyl ester of AGS IV quantitatively and is carried out with a wide-ranging concentration (0.004-0.080 mg/mL) of the derivatized AGS IV. The eluent consists of 90% methanol, 4% tetrahydrofuran, 6% water, and 0.2% triethylamine, with vitamin D3 added as the internal standard.     

Multiresidue analytical method for the determination of eight penicillin antibiotics in muscle tissue by ion-pair reversed-phase HPLC after precolumn derivatization.

A high-performance liquid chromatographic multiresidue method was developed for the determination of 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer pH 9 followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C for 5 min and with 1,2,4-triazole and mercury(II) chloride solution pH 9 at 65 degrees C for 10 min. The derivatized compounds are eluted on a C8 column with a mobile phase containing acetonitrile and phosphate buffer (pH 6; 0.1 mol/L) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The method detection limit is approximately 3-11 micrograms/kg and the limit of determination was evaluated down to 25 micrograms/kg in line with the criteria of the EU decision No. 93/256/EEC.     

HPLC fluorescence determination of nitrites in water using precolumn derivatization with 4-methyl-7-aminocoumarin

Summary A rapid, simple, and sensitive method is described for determination of nitrites in water. Nitrite (NO₂–) ions react with coumarin 120^® (4-methyl-7-aminocoumarin) in sulfuric acid medium to give the corresponding 7-diazo compound. After hydrolysis, this latter yields (95%) the highly fluorescent 4-methyl-7-hydroxycoumarin (4-methylumbelliferone) which is fluorimetrically detected at 380 nm after excitation at 325 nm.In order to avoid interference from both excess coumarin 120^® and the trace amounts of 4-methylumbelliferone which occurs in coumarin 120^® as an impurity, use of HPLC is mandatory; a satisfactory separation is obtained on a cyano stationary phase with apolar hexane-isopropanol (955, v/v) as eluent. Under these conditions, linearity of response is obtained from 1 to 30 g.L^–1 of NO₂–; the limit of detection is 0.5 g.L^–1. The repeatability and reproducibility, expressed as RSD %, are 2.5 and 4.7 % respectively, for n=6 and 5 g.L^–, analytical characteristics which demonstrate the reliability of the proposed method.     

Evaluation of a new reagent: anthraquinone-2-sulfonyl chloride for the determination of phenol in water by liquid chromatography using precolumn phase-transfer catalyzed derivatization

A new reagent, anthraquinone-2-sulfonyl chloride, is used for the derivatizaton of phenols. Several compounds with different polarities are selected to evaluate the new reagent and derivatives of these phenols that are prepared via a facile pathway. The optimal conditions for analytical derivatization and mechanism of the derivatization reaction are discussed. The derivatization procedure involves an ion-pair extraction of the deprotonated phenols with a tetrabutylammonium counter ion in the organic phase. At the interface of two phases, the derivatization reaction occurs quantitatively at room temperature within 3 min. The derivatives are stable and readily amenable to analysis by normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatography (HPLC). Excellent linearity response was demonstrated over the concentration range of 0.2-200 micromol/L at 320 nm for NP-HPLC and at 256 nm for RP-HPLC. Combined with preconcentration using a Waters Sep-Pak Plus C(18) cartridge, detection limits of phenols for water-sample analysis are as low as 1 x 10(-9) mol/L (approximately 0.1 microg/mL).     

Ion-pairing high-performance liquid chromatography determination of amitrole in apple after solid-phase extraction and precolumn derivatization

A novel method based on solid-phase extraction was studied for the extraction of amitrole (3-amino-1,2,4-triazole), and its residue determination in apples has been developed. The samples were derivatized with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). The derivatization conditions and the influence of elution composition on the separation were investigated. In pH 9.5 H₃BO₃–Na₂B₄O₇ media, the reaction of amitrole with CNBF was complete at 60°C after 30 min. The separation of derivatized amitrole was achieved at room temperature within 13 min by gradient elution mode with cetyltrimethylammonium bromide in mobile phase as ion-pair reagent. The method correlation coefficient was 0.9996, in concentrations ranging from 1.66 to 415 mg L⁻¹. The calculated recoveries of the proposed method were from 94.17% to 105.67%, and relative standard deviations were 1.57% to 6.44% in the application to the quantitative determination of amitrole in apples. The detection limit of amitrole was 0.10 mg L⁻¹ with a signal-to-noise ratio of 3. Figure Residue determination of amitrole in apple by ion-pairing high-performance liquid chromatography     

Development of a precolumn derivatization HPLC method with diode‐array detection for the determination of amino sugars in peat and soil humic acids

The work is focused on the development of a high‐performance liquid chromatography method with diode‐array detection for the separation and quantitation of the three most abundant amino sugars; d ‐glucosamine, d ‐galactosamine, and d ‐mannosamine. The high‐performance liquid chromatography separation was carried out by reversed‐phase chromatography on Chromolith Performance RP‐18e monolithic column after acid hydrolysis (5 M HCl) and precolumn derivatization of samples using diethyl ethoxymethylenemalonate. Gradient elution and a mobile phase composed of ammonium formate buffer solution (10 mmol/L, pH 3.60) and methanol with flow rate of 1.0 mL/min were used. The monitoring wavelength was set at 280 nm. The limits of detection and quantitation for analytes ranged from 0.017 to 0.122 mg/L and from 0.057 to 0.407 mg/L, respectively. The proposed method was successfully applied for the determination of amino sugars in samples of humic acids isolated from different soils and peat.     

RP-HPLC在线衍生法测定红景天中痕量铅的研究

铅在人体和动植物组织中能够蓄积,人若每天摄入1mg铅,长此则有中毒危险;过量摄入铅可以引起贫血症、神经功能失调及肾损伤,因此对食品中铅的含量必须严格控制。在环境、食品、化妆品的检测中,铅都是必须检测的指标。在铅矿的开采、冶炼和使用过程中,不可避免地会造成对环境的污染,影响人类和动植物的生命安全。因此,研究样品中铅含量的分析方法,对指导工农业生产和保护环境都有重要意义。目前常用铅的分析方法有原子吸收光谱法、极谱法哺、电感耦合等离子体-质谱法等。