Antitrypanosomal structure–activity-relationship study of synthetic cynaropicrin derivatives

Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four exo-olefins, and two hydroxyl groups. Recently, it was found that the compound is a potent in vitro and in vivo inhibitor of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT; sleeping sickness). In this Letter, chemical derivatization of cynaropicrin and the structure–activity-relationship (SAR) study against T. brucei is described.     

Sensitive high-performance liquid chromatographic assay for pilocarpine in biological fluids using fluorescence derivatisation

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogeneous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.     

Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2

The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Confirmation of cocaine exposure by gas chromatography-mass spectrometry of urine extracts after methylation of benzoylecgonine

Volatility and thermal stability are necessary physical-chemical properties for analysing a substance by gas chromatography. A derivatization step is required before gas chromatography of benzoylecgonine (the main metabolite of cocaine). In the literature, reactions such as silylation, perfluoroalkylation or alkylation are the most frequently used to derivatize benzoylecgonine. However, they allow the formation of products sensitive to moisture or require a purification step. So, a procedure to derivatize benzoylecgonine with diazomethane before gas chromatographic analysis was evaluated. A study was performed to evaluate the efficiency of conversion of benzoylecgonine in cocaine, the necessary time for reaction and the stability of ethereal solution of diazomethane. The reaction was shown to be very fast in mild conditions and there was no need for a further purification step. When benzoylecgonine was extracted from urine by solid-phase extraction and derivatized with diazomethane, concentrations as low as 25 ng/ml could be detected using GC-MS in the full scan mode.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

Discriminative determination of alkyl methylphosphonates and methylphosphonate in blood plasma and urine by gas chromatography-mass spectrometry after tert.-butyldimethylsilylation

A method for determining two nerve gas hydrolysis products, alkyl (ethyl, isopropyl and pinacolyl) methylphosphonates (RMPAs) and methylphosphonate (MPA), separately, in human plasma and urine samples was developed, using two different deproteinization procedures. In the first method, the plasma sample was deproteinized by adding a fourfold volume of acetonitrile, followed by passing the supernatant through a Bond Elut strong anion-exchange (SAX) cartridge [fluoride (F(-)) form]. After washing the cartridge with water and methanol, the RMPAs were eluted with a 3% (v/v) solution of methanolic ammonia, and analyzed by gas chromatography-mass spectrometry (GC-MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. The detection yields of TBDMS derivatives of RMPAs were in the range of 69 to 99%, in contrast to the poor yields obtained when only acetonitrile deproteinization pretreatment was used (yield: 13-26%). The yield of the TBDMS derivative of MPA was very low (8%), however. In a the second method, a plasma sample was deproteinized by adding a half volume of 10% (w/v) trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA, the aqueous fraction was then passed through a Bond Elut SAX cartridge. After washing the cartridge with 0.5% (v/v) methanolic ammonia, MPA was eluted with 3% (v/v) methanolic ammonia. The detection yield of the TBDMS derivative of MPA was nearly quantitative. A pretreatment method using SAX solid-phase extraction was also developed for the cleanup of a urine sample, in which the sample was directly applied to a Bond Elut SAX cartridge, followed by elution of the RMPAs and MPA with 3% (v/v) methanolic ammonia, which were then derivatized and analyzed by GC-MS. The detection yields of TBDMS derivatives of RMPAs and MPA were in the range of 61 to 97%.     

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.