Gas chromatographic-mass fragmentographic determination of propiverine and its metabolites in plasma and urine

1-Methyl-4-piperidyl diphenylpropoxyacetate hydrochloride has been developed clinically for the therapy of urinary bladder dysfunction. A gas chromatographic-mass fragmentographic method was developed for the determination of this drug and its seven metabolites in plasma and urine. The sample was first treated with a Sep-Pak C18 cartridge, the methanol eluate was evaporated to dryness, and the resulting residue was redissolved in distilled water. This solution was then extracted with chloroform and adjusted to pH 9.0 with 0.1 M sodium borate solution. The acidified aqueous layers were extracted with ethyl acetate. The chloroform layer, which contained non-polar metabolites, was concentrated to dryness, then subjected to trifluoroacetylation, decomposition and methylation. The extract from the plasma sample was trimethylsilylated. The dried residue of the ethyl acetate layer, which contained polar metabolites, was subjected to methylation, trifluoroacetylation and decomposition. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer and analysed by the selected-ion monitoring method using an internal standard. Detection was limited to 1-2 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of 1-methyl-4-piperidyl diphenylpropoxyacetate hydrochloride and its metabolites in plasma and urine was thus established, and it should prove useful in basic and clinical pharmacological studies.     

Determination of clenbuterol in bovine plasma and tissues by gas chromatography-negative-ion chemical ionization mass spectrometry

A highly sensitive and specific assay was developed for the determination of clenbuterol in bovine plasma and tissues. Clenbuterol and the internal standard [2H9]clenbuterol were measured by gas chromatography-negative-ion chemical ionization mass spectrometry with methane as the reagent gas. Bovine tissues including muscle, liver, heart, kidney, lung, suet, brain, spinal cord and thymus were ground in a buffer of pH 7 and then extracted using ethyl acetate. After two subsequent purification steps, the cleaned-up organic extract was derivatized with pentafluoropropionic anhydride. The mass spectrometer was set to monitor the abundant ions m/z 368 and 377 of the perfluoroacyl derivatives. This assay was performed with 1 ml of plasma or 0.2 g of tissue. The feasibility of this method was demonstrated by the determination of clenbuterol residues as the femtomole level in a variety of tissues.     

Determination on the microscale of plasmatic lactic acid as its t-butyldimethylsilyl derivative by stable-isotope dilution using capillary gas chromatography/mass spectrometry.

A new sensitive and precise method for the determination of lactic acid in plasmatic microsamples (50 microL) has been developed. Lactic acid was directly extracted from plasma by ethyl acetate in acidic conditions, and analysed as its di-t-butyldimethylsilyl derivative by capillary gas chromatography/electron-impact mass spectrometry (GC/MS). The internal standard was a previously synthesized deuterated compound, 3-[2H]-(2R)-lactic acid. The method gives good reproductibility and precision, the overall standard deviation being better than 3%. The GC/MS assay was in good agreement with the enzymatic determination.     

Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Quantitative measurement of N-acetyl-L-aspartic acid in urine by gas chromatography with negative-ion chemical ionization mass spectrometry.

A highly specific and sensitive assay for N-acetyl-L-aspartic acid has been developed. The trideuterated compound was synthesized and used as an internal standard for gas chromatography with negative-ion chemical ionization mass spectrometry. Urine samples were acidified and extracted with ethyl acetate, and the compounds converted into their pentafluorobenzyl ester derivatives. Under these conditions, sub-picogram amounts of the pure derivatives could be detected. Thus, only microliter volumes of urine samples have to be processed to achieve reliable quantification of "basal" levels of N-acetyl-L-aspartic acid.     

Quantitation of SR 27417 in human plasma using electrospray liquid chromatography-tandem mass spectrometry: A study of ion suppression

The effect of coeluting matrix compounds on the quantitation of SR 27417 in human plasma using electrospray liquid chromatography-tandem mass spectrometry has been examined. During the method development stage of this assay, plasma samples spiked with the analyte at 100 pg/mL were extracted using three different procedures: a hexane liquid-liquid extraction, an ethyl acetate back-extraction, and a solid phase extraction. Ion intensity of the analyte was found to be related inversely to the percent ionization of coeluting matrix components as evidenced by full scan spectra. The ethyl acetate back-extraction, which contained the fewest coeluting components, resulted in the highest ion intensity for the analyte. An assay comparison was done by using the liquid-liquid hexane and the ethyl acetate back-extractions for sample preparation. Replicate 1-mL samples (n=5) at 11 concentrations from 5 to 2000 pg/mL were extracted and analyzed. The results for the ethyl acetate back-extracted samples were acceptable from 2000 to 5 pg/mL with accuracy ranging from ?11.6 to 2.61% of the nominal concentrations. In contrast, the hexane liquid-liquid method had poor accuracy and precision below 20 pg/mL. The difference is explained by suppression of analyte ion intensity. These results are consistent with the current theory of electrospray ionization.     

Use of Ethoxy-Homologs as Internal Standards for Determination of Urinary Vanillylmandelic Acid and Normetanephrine in Man by High Performance Liquid Chromatography

Abstract

Synthesis of the ethoxy-homologs of vanillylmandelic acid (3-ethoxy-4-hydroxymandelic acid, EMA) and normetanephrine (3-ethoxy-4-hydroxyphenylethanolamine, EHPEA) and their use in minor modifications of existing assays on human urine are described. For analysis of vanillylmandelic acid, the homologous internal standard is added to an aliquot of urine, organic acids are then extracted into ethyl acetate and back extracted into 10% K₂CO₃, and finally extracted a second time into ethyl acetate. After evaporation of the ethyl acetate, the residue is redissolved in the mobile phase to be used in the chromatography, and injected onto a reverse phase column. Comparison of results with a gas chromatography-mass spectrometry (GCMS) assay gave almost identical results (96% ± 2%, mean ± SE, n=10). Analysis of normetanephrine is performed by addition of internal standard to an aliquot of urine before heat hydrolysis of amine conjugates. The amines are adsorbed onto columns of Bio Rex 70, and then eluted with a solution of 1 M NH₄OH, which is then evaporated to dryness. After residue is redissolved in borate buffer, the amines are extracted into toluene:isoamyl alcohol and back extracted into 0.1 M acetic acid before injection onto a reverse phase column. Correlation with a GCMS assay gave similar results (102% ± 4%, mean ± SE, n=9).     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Indolic urinary melanogens: separation and identification by gas chromatography with selected-ion monitoring mass spectrometry of 5-hydroxy-6-methoxyindole-2-carboxylic and 5-methoxy-6-hydroxyindole-2- carboxylic acids.

Two isomeric urinary melanogens, 5-hydroxy-6-methoxyindole-2-carboxylic acid and 5-methoxy-6-hydroxyindole-2-carboxylic acid, have been separated by gas chromatography with selected-ion monitoring mass spectrometry. After chemical synthesis of one of these two isomers, 5-methoxy-6-hydroxyindole-2-carboxylic acid, and the establishment of the mass spectrum of its trimethylsilylated derivative, a 30-ml sample of a melanotic 24-h urine was adjusted to pH 1 and extracted twice with 10 ml of ethyl acetate. The extract was evaporated to dryness and the residue derivatized with methyl-8, followed by Tri-Sil/TBT. Silylated derivatives were analysed by gas chromatography with selected-ion monitoring mass spectrometry. The mass spectrum of the 5-methoxy-6-hydroxyindole-2-carboxylic acid allowed the determination of the retention times of both isomers.     

Determination of a selective Na+/H+ exchanger inhibitor, 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of a selective Na(+)/H(+) exchanger inhibitor 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma. KR-33028 and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at neutral pH. The analytes were separated on an XBridge C(18) column with a mixture of methanol-0.1% formic acid (35:65, v/v) as mobile phase and detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.9998) over the concentration range of 2.0-1000 ng/mL. The coefficients of variation of intra- and inter-assay were 1.3-6.8% and the relative error was 0.8-5.0%. The recoveries of KR-33028 and linezolid were 70.5 and 84.6%, respectively. The lower limit of quantification for KR-33028 was 2.0 ng/mL using 50 microL plasma sample. This method was successfully applied to the pharmacokinetic study of KR-33028 in rats.     

Assay of trimethoprim,sulfamethoxazole and its N4-acetyl metabolite in biological fluids by high-pressure liquid chromatography

A reversed-phase HPLC assay method was developed to determine levels of the drugs trimethoprim, sulfamethoxazole and the N₄-acetyl metabolite of the latter in human blood plasma and urine. The organic substances are extracted with ethyl acetate by a single extraction at pH 6.8. The method is rapid, accurate, sensitive and precise and well suited to routine determination of these drugs on patients during clinical pharmacokinetic investigations.     

Direct derivatization of alprenolol and its 4-hydroxy metabolite in urine with phosgene and methanol prior to analysis by capillary column gas chromatography

A method for the determination of alprenolol and its 4-hydroxy metabolite has been developed. The urine sample is made alkaline with buffer (pH 12) and derivatized with 60 microliter of 2 M phosgene in toluene with vigorous shaking. In the presence of 2.5% methanol, an oxazolidineone methyl carbonate is formed from 4-hydroxy alprenolol. The now neutral derivatives are extracted with an equal volume of dichloromethane. After evaporation of the organic phase, the residue is taken up in a small volume of ethyl acetate and subjected to capillary column gas chromatography with CP-Sil 8 as the stationary phase. The precision was 2.1% at the 3.3 micrograms/ml level of the metabolite in urine (n = 8). The isopentylamino analogue was used as the internal standard.     

Rapid, high-sensitivity reversed-phase liquid chromatographic assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine and its oxo metabolite in plasma using fluorescence detection

A rapid, sensitive and specific assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine (I) and its oxo metabolite (II) in plasma was developed and validated employing reversed-phase high-performance liquid chromatography with fluorescence detection. Sample preparation was achieved by a simple ethyl acetate extraction from plasma buffered at pH 10 (0.1 M boric acid-0.1 M potassium chloride). Chromatographic analyses were performed isocratically on a C18 column, with a mobile phase consisting of methanol-0.2 M sodium acetate buffer, pH 5.0 (67:33, v/v). Chromatographic run time was less than 8 min. The assay was linear (r greater than 0.9998) over the concentration range 1.50-10,000 ng/ml for both I and II; for individual studies, curves covering a range of two orders of magnitude were generally employed. Limits of detection for I and II were 0.5 and 1.0 ng/ml, respectively. A preliminary investigation of the plasma concentrations of I and II in the rat following a single 30 mg/kg oral dose demonstrated the applicability of the method for pharmacokinetic studies.     

Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [2H11]pipecolic acid as internal standard.

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [2H11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH less than or equal to 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age greater than 1 week (n = 21, mean = 1.36 microM, range = 0.47-3.27 microM). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.     

Reactions of 3-[3-Chlorophenyl]-1-[4-(3,4-Methylenedioxybenzylidine) a Minophenyl]Prop-2-Enone with Some Nucleophiles

The action of 2-mercaptoacetic acid, thiosemicarbazide and hydrazine hydrate on the titled compound 1 is studied. On the other hand, the behaviour of the titled compound towards thiourea, ethyl acetoacetate and malononitrile under Michael's conditions is reported. The structure of the new heterocycles 2 - 8 is discussed in the light of IR and ¹HNMR spectra.     

Analysis of a new H2 receptor antagonist, 3-amino-5-[3-[4-(1-piperidinoindanyloxy)]propylamino]-1-methyl-1 H-1,2,4-triazole, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of a new H2 receptor antagonist, 3-amino-5-[3-[4-(piperidinoindanyloxy)]propylamino] -1-methyl-1H-1,2,4-triazole (I), in human plasma and urine was developed. The method employs liquid-liquid extraction of the analyte and an internal standard and chromatographic separation using an alkylphenyl-bonded HPLC column. The total time of chromatography was less than 10 min. Sensitivity was 10 ng/ml for the plasma analysis and 1 microgram/ml for the analysis of I from urine. The coefficients of variation, based on interpolated concentrations, were less than 10%. The method was used for more than 5000 samples during clinical pharmacokinetic studies.     

Determination of low plasma concentrations of 3-methoxy-4-hydroxyphenylethylene glycol using gas chromatography-negative-ion chemical ionization mass spectrometry

Gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICI-MS) allowed the detection of extremely low plasma concentrations of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG). Glucuronide and sulphate conjugates of MHPG were determined after enzymatic hydrolysis of plasma with beta-glucuronidase-arylsulphatase. A 1-ml plasma sample was extracted at the pH of the hydrolysis (pH 4.8) with ethyl acetate, and the dry extract was derivatized with pentafluoropropionic anhydride in ethyl acetate. After evaporation of the solvent, the residue was dissolved in benzene and an aliquot was analysed by GC-NICI-MS. A trideuterated analogue of MHPG was used as an internal standard. Negative-ion chemical ionization of the pentafluoropropionyl derivatives was carried out using ammonia. The ion-molecule adducts at m/e 766 and 785 (MHPG) and m/e 769 and 788 (internal standard) were formed from the pentafluoropropionyl derivatives with the ions of m/e 163 (CF3CF2COO-) and m/e 144 (loss of fluorine from m/e 163). The concentrations of the ions of m/e 163 and 144 play a major role in the sensitivity and precision of this technique, which allows the detection of free MHPG plasma concentrations as low as 100 pg/ml in routine analysis.     

Preparation of 4,4'-(1-[(2)H6]methylethylidene)bis-[2,3,5,6-(2)H4]phenol and its application to the measurement of bisphenol A in beverages by stable isotope dilution mass spectrometry

The preparation of 4,4'-(1-[(2)H6]methylethylidene)bis-[2,3,5,6-(2)H4]phenol, bisphenol A-d14, was achieved in excellent yield by reaction of [2H6]acetone with [2H6]phenol in the presence of deuterium chloride. The product thus obtained was shown by mass spectroscopy to be a mixture of the 2H14, 2H13 and 2H12 isotopomers in the relative proportions of 82.3:16.2:1.5, respectively. An isotope dilution gas chromatography-mass spectrometric method using bisphenol A-d14 as an internal standard was developed to measure the level of bisphenol A in beverages. The procedure involves extracting bisphenol A into dichloromethane, and then purifying the analyte by back extraction into dilute aqueous sodium hydroxide. Conversion of bisphenol A and its internal standard, bisphenol A-d14, to their corresponding O-bis(trifluoroacetyl) derivatives by treatment with trifluoroacetic anhydride gave compounds with good chromatographic properties and whose mass fragmentometry is such that loss of M-CH3 and M-C2H3 are the base peaks in the mass spectra of the analyte and internal standard, respectively. Quantification of bisphenol A was achieved by comparing the area of the M-15 ion to that of the corresponding ion of bisphenol A-d14. The characteristics of our assay are: an analyte recovery of better than 95%, a root mean square signal-to-noise ratio of 79:1 for 1.7 pg on column and an inter-assay RSD of better than 4% (n=5).     

Synthesis and characterization of novel 4-benzyloxyphenyl 4-[4-(n-dodecyloxy)benzoyloxy]benzoate liquid crystal

Liquid crystal (LC) compound 4-Benzyloxyphenyl 4-[4-( n -dodecyloxy)benzoyloxy]benzoate (BDBB) was prepared and characterized. Inverse gas chromatography (IGC) was to be a beneficial analysis method for the research of thermodynamic characteristics of the new LC. Acetate and alcohol isomers were used to examine LC selectivity via the IGC technique at temperatures between 333.2 K and 483.2 K. The retention diagrams of n -heptane, n -octane, n -nonane, n -decane, undecane, dodecane, tridecane, n -butyl acetate, isobutyl acetate, ethyl acetate, n -propylbenzene, isopropylbenzene, ethylbenzene, chlorobenzene, and toluene on BDBB were plotted with temperatures of 483.2–493.2 K. Flory–Huggins interaction parameter and weight fraction activity coefficient at infinite dilution were researched for BDBB.     

Quantitation of the antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide in plasma by high-performance liquid chromatography

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide is a new experimental antitumour agent which has excellent in vivo activity against the Lewis lung tumour in mice. A reversed-phase high-performance liquid chromatographic method is described for the measurement of this agent in plasma. The internal standard was N-[2-(diethylamino)ethyl]acridine-4-carboxamide. The compounds of interest were extracted from plasma (0.2 ml) with acetonitrile and further purified on C18 solid-phase extraction Bond Elut columns. After elution with acetonitrile-ammonium acetate buffer and evaporation, the final separation was carried out on a C18 muBondapak column with fluorimetric detection. Over the plasma concentration range 100-5000 nM, the intra- and inter-assay coefficients of variation were less than 4.1 and 7.7%, respectively. The accuracy of the method varied from 97 to 105% of the theoretical values. The lowest concentration which could be measured with acceptable accuracy (+/- 10%) and precision (coefficient of variation less than 10%) was 10 nM. The method was sufficiently sensitive to allow pharmacokinetic analyses of 30 mumol/kg doses for more than six half-lives (t1/2) in rabbits (t1/2 = 4) and mice (t1/2 = 1.3 h).