Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.     

Selective and sensitive determination of pamidronate in human plasma and urine by gas chromatography with flame photometric detection

A rapid, selective and sensitive method for the determination of pamidronate in human plasma and urine samples by gas chromatography (GC) has been developed. After deproteinization of the sample with trichloroacetic acid, pamidronate was converted into its N-isobutoxycarbonyl methyl ester derivative and measured by GC with flame photometric detection (FPD), using a HP-1 capillary column. The derivative preparation and GC analysis were accomplished within 30 min. The derivative was sufficiently volatile and stable, giving a single and symmetrical peak, and provided an excellent FPD response. The detection limit of pamidronate, at a signal-to-noise ratio of 3, was ca. 100 pg injected, and the calibration curve for this compound in the range 20-1000 ng was linear and sufficiently reproducible for quantitative determination. This method could be successfully applied to plasma and urine samples without a preliminary clean-up except for deproteinization with trichloroacetic acid, and pamidronate could be measured without any influence from coexisting substances. Overall recoveries of pamidronate added to plasma and urine samples were 93-97%. The coefficients of variation for intra-assay and inter-assay of pamidronate in these samples were 1.0-7.9% (n = 3) and 4.1-8.3% (n = 6), respectively.     

Determination of free and total proline and hydroxyproline in plasma and tissue samples by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of free and total proline (Pro) and 4-hydroxyproline (Hyp) by gas chromatography (GC) was developed. For free Pro and Hyp analysis, plasma and tissue homogenate were extracted with methanol. For total Pro and Hyp analysis, these samples were hydrolysed in 6 M HCl. After removal of primary amino compounds by the reaction with o-phthaldialdehyde, Pro and Hyp in methanol extract and acid hydrolysate were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. This method was successfully applied to small samples without prior clean-up, and Pro and Hyp in these samples could be analysed without any influence from coexisting substances. Overall recoveries of Pro and Hyp added to plasma and tissue samples were 92-106%. The analytical results of free and total Pro and Hyp in human plasma and mouse tissue samples are presented.     

Capillary electrophoresis with laser-induced fluorescence: a routine method to determine moxifloxacin in human body fluids in very small sample volumes

Methods for the simultaneous determination of methamphetamine (MP) and its related compounds (MPs) using capillary electrophoresis (CE) with UV absorbance and laser-induced fluorescence (LIF) detection are described. In UV detection, MPs were applied to CE without any derivatization procedure and detected at 210 nm for a rapid and simple analysis. Capillary zone electrophoresis (CZE) and electrokinetic capillary electrophoresis (MEKC) were used. MP, amphetamine (AP), 1-phenylethylamine (1-PA as an I.S.), 2-phenylethylamine (2-PA), 4-hydroxymethamphetamine (4-HMP) and 4-hydroxyamphetamine (4-HAP) were separated within 15 min by both CZE and MEKC. Detection limits of MPs were in the range 48-72 fmol/injection for CZE and 85-191 fmol/injection for MEKC. MEKC was successfully applied to the determination of MPs in urine. For a highly sensitive analysis, LIF detection was also examined using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. By the method, in which MPs derivatives were separated within 45 min by MEKC, 22-40 amol/injection of primary amines (AP, 4-HAP and 2-PA) and 690 amol/injection of MP and 300 amol/injection of 4-HMP were detected. The concentration of MP and AP in 50 microliters urine from MP addicts were successfully determined. A comparison of the characteristics for both UV and LIF detections was also discussed.     

A sensitive method for detection of sympathomimetic amines in pentafluorobenzamide form in biological samples using gas chromatography

A toxicological method of sensitive and specific confirmation of methamphetamine and other primary and secondary amines in biological samples after extractive perfluorobenzoylation is described. The method is based on the principle of gas chromatography with parallel specific nitrogen detection and electron capture detection. The other alternative is the gas chromatography combined with mass spectrometry in chemical ionization mode. The method described allows the detection of methamphetamine and amphetamine in urine in concentrations below 10 ng/ml.     

Determination of cysteamine and cystamine by gas chromatography with flame photometric detection

A gas chromatographic method for the determination of cysteamine and its disulphide cystamine is described. Cysteamine and cystamine are converted into N,S-diisobutoxycarbonyl and N,N-diisobutoxycarbonyl derivatives, respectively. The derivatives are analysed by gas chromatography with flame photometric detection, using a DB-210 capillary column. The calibration curves for cysteamine and cystamine in the range of 0.2-5.0 nmol are linear and sufficiently reproducible for quantitative analysis, and the detection limit is about 0.5 pmol injected. Cysteamine in mouse tissues is found in the free reduced, free oxidized and protein-bound forms. Free oxidized and protein-bound forms are reduced to free cysteamine by the use of sodium borohydride, and then derivatized. Cysteamine and cystamine in mouse tissues can be measured without any interference from coexisting substances by this method. The recoveries of cysteamine and cystamine added to the tissue samples are 91-106%, and their reproducibilities are found to be satisfactory. Analytical results for the determination of various forms of cysteamine in mouse tissues are presented.     

An analog technique for recording leg position learning in the cockroach

A liquid chromatographic method is described for the determination of biogenic amines found in dry sausages: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermidine, and spermine. Amines were extracted with perchloric acid solution and derivatized with dansyl chloride. After derivatization, ammonia was added to remove an interfering peak near cadaverine. Liquid chromatographic separations were performed by using a Spherisorb ODS2 column and an ammonium acetate-acetonitrile gradient elution program. The limits of determination of the individual amines were 1-5 mg/kg. This method is also applicable to detection of amines in other food samples.     

Phenol-degrading denitrifying bacteria in wastewater sediments

The simultaneous determination of amino and organic acids in plant tissue extracts using capillary gas chromatography is described. Plant leaves were extracted in 5% (w/v) perchloric acid and neutralized extracts were purified using C18 cartridges. The amino and organic acids in purified extracts were then converted to tert-butyldimethylsilyl (TBDMS) derivatives prior to separation and detection by capillary gas chromatography (GC) with flame ionization detection. Conditions required for optimal derivatization were investigated. Amino and organic acids were readily converted to their TBDMS derivatives using N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide in dimethylformamide solvent 1:6 (v/v) with an average recovery of 90% and a reproducibility of about 5%. The characteristic [M-57] and [M-159] fragment ion of the TBDMS derivatives was confirmed using GC-MS. The proposed method was demonstrated by the determination of amino and organic acids in extracts of Acacia and Eucalyptus leaves, where detection limits were 1-20 ng.     

Prenatal care in Pelotas, Rio Grande do Sul, Brazil, 1993]

The urinary excretion of 2-hydroxyphenylacetic acid (2HPAA) was studied in human volunteers after oral and parenteral doses of coumarin. The presence of 2HPAA in the urine was confirmed by gas chromatography mass spectroscopy (GC MS). Mass spectra of reference material and samples are presented. The determination of 2HPAA was carried out by GC with flame-ionization detection. Prior to analysis samples were extracted into ethyl ether and the analytes were derivatized with trimethlyphenylammonium hydroxide. A calibration range from 0.3 to 150 micrograms ml-1 was established using 3-hydroxyphenyl acetic acid (3HPAA) as an internal standard. On average less than 10% of the coumarin administered were excreted into the urine in the form of 2HPAA.     

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Analytical confirmation of error in false positive amphetamine immunoassays and results

Numerous urine samples were found to be positive when using a new amphetamine immunoassay (AxSYM). Confirmation analysis was carried out in a second laboratory at "reasonable prices" using a simple TLC-method with non specific ninhydrine detection and resulted in many "positive" confirmation findings. The GC/MS analysis clearly indicated the absence of amphetamine derivatives regularly encountered in forensic toxicology. The false-positive immunochemical findings may probably be caused by endogenous substances.     

Determination of trace amounts of urea by using flow injection with chemiluminescence detection

Reaction between urea and hypobromite in alkaline solution was found to produce chemiluminescence with a maximum wavelength at 510 nm. A simple chemiluminescence detection method was used for the determination of urea in human urine and natural aqueous samples, which combined this chemiluminescence reaction with a flow injection analysis system. The relative standard deviation for 5 x 10(-7) mol dm-3 urea is 1.9% (n = 6), and the detection limit is 9.0 x 10(-8) mol dm-3 (3Sr). As this chemiluminescence reaction is very fast, a double concentric tube mixer connected directly to the chemiluminescence cell was used to mix urea solution and hypobromite solution. Alkylamines, carboxylic acids and most amino acids do not interfere in the determination. Ammonium ion interferes, but the sensitivity for ammonium ion is only 1% of that for urea. The interference from ammonium ion was removed sufficiently by using an on-line cation-exchange column.     

Resolution limits for optical transillumination of abnormalities deeply embedded in tissues

For the quantification of nitrite and nitrate, the stable metabolites of L-arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic-mass spectrometric (GC-MS) method in which [15N]nitrite and [15N]nitrate were used as internal standards. Endogenous nitrite and [15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [15N]nitrate they were reduced to nitrite and [15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-microliter aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [15N]nitrite ([15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 +/- 0.25 of nitrite and 109.5 +/- 61.7 of nitrate (mean +/- S.D., mumol/mmol creatinine) with a mean 24-h output of 5.7 mumol for nitrite and 1226 mumol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean +/- S.D., mumol/l) 3.6 +/- 0.8 and 68 +/- 17, respectively.     

Rapid MRI and velocimetry of cylindrical Couette flow

A quantitative method was developed for determination of alpha2u-globulin in urine and kidney samples collected from male rats using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). Samples prepared from urine and kidney homogenates using size exclusion filters were subject to reversed-phase liquid chromatography and the effluent passed into an electrospray ionization source. Quantitative analysis using external standard calibration was based upon selected ion monitoring of protonated molecular ions by the mass spectrometer. Linear calibration curves were developed over the range of approximately 4. 6-370 microg of alpha2u-globulin/microL for spiked urine standards and over the range of approximately 4.6-550 microg of alpha2u-globulin/microL for spiked kidney standards. The precision (relative standard deviation) for repeated injection (using urine samples) and intra-assay precision (using both urine and kidney samples) were within +/-10.4% and +/-13.2%, respectively. Using spiked urine standards, inter-assay precision, intra-assay accuracy, and inter-assay accuracy were within +/-20%, +/-20%, and +/-15%, respectively. Using spiked kidney standards, intra-assay accuracy was within +/-15%. The limits of detection (LOD) for the determination of alpha2u-globulin in urine and kidney samples were approximately 0.41 pg/nL (1.0 fmol injected) and 25 pg/nL ( approximately 13 fmol injected), respectively. The limits of quantitation (LOQ) for determination of alpha2u-globulin in urine and kidney samples were calculated as 1.4 pg/nL (3.7 fmol injected) and 83 pg/nL (45 fmol injected), respectively. Applicability of the LC-ESI/MS method was demonstrated by determination of alpha2u-globulin in both urine and kidney samples collected from male Fischer 344/N rats dosed intravenously with cis-Decalin at concentrations of 0, 2.5, 5.0, 10, and 20 mg/kg. A dose-dependent relationship was found between the amount of cis-Decalin administered and alpha2u-globulin accumulation in kidney samples, whereas no significant change in the urinary levels of alpha2u-globulin occurred. These observations are consistent with excessive accumulation of alpha2u-globulin occurring in protein droplets in renal proximal tubule epithelial cells as a result of decreased catabolic activity due to formation of ligand-protein complexs with Decalin and its metabolite(s). This report demonstrates that LC-ESI/MS may be routinely applied for quantitative analysis of alpha2u-globulin in rat urine and kidney samples to address alpha2u-globulin accumulation and its role in the development of nephrotoxicity associated with chemical exposures.     

Determination of norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

A method for the determination of norfloxacin in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5-ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from +/- 0.37 to +/- 4.14% in plasma (concentration range: 70.3-2109.2 ng/ml) and from +/- 0.51 to +/- 1.56% in urine (concentration range: 7.5-299.4 micrograms/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18% to -9.47% in plasma and from -10.56% to - 5.91% in urine. The assay has been used to support human pharmacokinetic studies.     

Enantiospecific drug analysis via the ortho-phthalaldehyde/homochiral thiol derivatization method

Pre-column derivatization with o-phthaldialdehyde and an optically active thiol has hitherto been used mainly for liquid-chromatographic chiral separation of amino acids. Chiral separation of non-amino-acid primary amines, especially of pharmaceuticals, via this approach has been largely ignored. We have therefore examined the applicability of the method to the chiral resolution of several pharmaceutical amines. o-Phthaldialdehyde and four commercially available homochiral thiols were used to study the separation of the enantiomers of amphetamine, p-hydroxyamphetamine, p-chloroamphetamine, 3-amino- 1-phenylbutane, 3-amino-1-(4-hydroxyphenyl)-butane, mexiletine, tocainide, tranylcypromine and rimantadine. The resulting highly fluorescent isoindole derivatives were resolved on a Waters Nova-Pak C18 column using mobile phases consisting of mixtures of methanol, a sodium acetate buffer and acetonitrile, and the column effluent was monitored using fluorescence or UV detection. In some cases the fluorescence and/or the UV absorbance of the two diastereomers were unequal. It was found that the resolution of most of the amines could be optimized by varying the homochiral thiol in the derivatization step. This method of chiral separation may have wide applicability in enantiospecific drug analysis of non-amino-acid primary amines due to its simplicity and the high sensitivity it provides.     

Monitoring aminopentamide urinary excretion by means of multiple 'microphase' extraction - a rapid method for the extraction and concentration of small amounts of lipophilic drugs from large volumes of biological fluids without distillation

It has been demonstrated that low concentrations of basic lipophilic drugs in biological fluids may be extracted and concentrated 10(4)-10(6) times by a series of extraction procedures in which the ratio of the extracting solvent to that of the solution to be extracted is of the order of 1:100 (microphase extraction procedure). Typically, basic drugs (atropine, aminopentamide, hyoscine, chloroquine, pyrimethamine) were extracted and concentrated sufficiently for direct GC analysis from 24-hour urine samples by a procedure involving three simple consecutive extraction steps. Using this procedure, it was demonstrated that after administration of aminopentamide (300 micrograms) to patients in the form of anti-diarrhoeal tablets, measurable quantities of the free, unchanged drug can be demonstrated in 24-hour urine samples. The main advantages of the method are simplicity, rapidity and sensitivity due to the low background interference in the GC separations. The principle involved can be extended to the analysis of acidic drugs with suitable solubility properties.     

Simple method for the routine determination of betaine and N,N-dimethylglycine in blood and urine

A simple and convenient method using commercially available derivatization reagents is described for the measurement of betaine and N,N-dimethylglycine (DMG) in blood and urine. Precolumn derivatization of plasma or urine is performed directly in acetonitrile without extraction with p-bromophenacyl bromide and crown ether as catalyst. The p-bromophenacyl ester derivatives are then separated by high-performance liquid chromatography, using an isocratic system of acetonitrile and water containing choline. Effluent was monitored at 254 nm. The limit of detection was 5 micromol/L for betaine and 2 micromol/L for DMG. Analytical recovery was >97% for both analytes. Total and within-day CVs were 2.0-4.4% and 0.9-2.2% for DMG. For betaine, the total and within-day CVs were 1.3-5.3% and 0.4-3.8%, respectively. The method is precise and cost-effective and has been used successfully to determine the concentrations of DMG and betaine in human plasma and urine.     

Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection

A convenient, reliable and rapid method for determination of total cysteamine in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves reduction of samples with dithiothreitol, derivatization of total cysteamine by addition of monobromobimane and protein precipitation by perchloric acid. The calibration curve was linear in the range 2-150 nmol ml-1 and the detection limit was 0.5 nmol ml-1. This method was successfully applied for a pharmacokinetic study of three cysteamine derivatives in healthy volunteers without any interference from coexisting substances.     

Fully automated determination of pesticides in wine

A fully automated solid-phase extraction gas chromatographic/mass spectrometric (SPE/GC/MS) method was developed for determination of pesticides in wine. All steps from aspiration of infiltrated wine to printout of the integrated chromatogram were performed without human interaction. A dedicated robot performed addition of internal standard, application of wine onto the SPE cartridge, elution of analytes, drying and concentrating of eluate, and passing of concentrate to the GC sampler. All steps were performed in standard liquid chromatography/GC vials, using a minimum of organic solvent. The method permits determination of 21 different pesticides. Individual detection limits were 0.005-0.01 mg/L. The regression coefficients relating to linearity were > 0.99; only 4,4-dichloro-benzphenone and dicofol showed lower coefficients. The recoveries for 17 pesticides ranged from 80 to 115%.