Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid

An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method.     

Determination of dimethylamine in biological samples by high-performance liquid chromatography

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 μmol/1. Recoveries from spiked serum samples were 99–107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3±1.5 and 29.2±12.1 μmol/1, respectively.     

Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl- -cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free -serine (0.22 μmol/g of tissue; + RATIO = 0.25) in the brain whereas -aspartate and -alanine were established to be at trace levels. These results further support the presence of -serine in adult brain tissues as demonstrated by recent work using gas chromatography.     

High-performance liquid chromatographic method for rapid and highly sensitive determination of histidine using postcolumn fluorescence detection with o-phthaldialdehyde

A high-performance liquid chromatographic method was developed for the rapid and sensitive determination of histidine. The method is based on separation by reversed-phase ion-pair chromatography followed by highly selective fluorescence derivatization of histidine with o-phthaldialdehyde. A linear calibration curve was obtained over the range of 0.25–200 pmol per injection (10 μl) with the coefficient of variation of 0.9% at 2 pmol (n=10) and with the detection limit (S/N=8) of 25 fmol. The method was applicable to the assay of histidine in human serum. Serum histidine values obtained by the present method were in good agreement with values obtained with an amino acid analyzer.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

A direct and sensitive determination of histamine in acid-deproteinized biological samples by high-performance liquid chromatography

A convenient method for the routine measurement of histamine (HA) in biological samples was developed. This method does not require any preliminary purification or concentration of HA, and features high sensitivity, specificity, and reliability. The method consists of the direct application of the acid-deproteinized sample to high-performance liquid chromatography on a sulfonated polystyrene column with detection by means of a postcolumn fluorogenic reaction with o-phthaladehyde. The detection limit was found to be 0.1 pmol (signal-to-noise ratio = 3). The coefficient of variation for measurements of 10 pmol of standard histamine was 1.1%. Each chromatography takes only 10 min and therefore more than 50 samples can be measured in a day. The high sensitivity of the method allows it to be applied even to samples of very low HA concentration such as human plasma without any procedure for concentration of the sample, and further, only 0.1 ml of the sample is necessary for determination. The method was applied to compare the HA levels of the whole blood and plasma of man and various animals. Applications of the method to the supernatant of rat peritoneal mast cell incubates and to extracts of mouse brain and stomach are also described.     

Determination of 5-aminolevulinic acid in biological samples by high-performance liquid chromatography

5-Aminolevulinic acid (ALA), the common precursor of all naturally occurring tetrapyrroles, forms a stable condensation product with 2-amino-3-hydroxynaphthalene which can be identified by its fluorescence. Separation of the compound by reversed-phase high-performance liquid chromatography on RPC-18 columns allows its detection down to the picomolar range and can be successfully applied for ALA analysis in small biological samples. The reaction product of ALA with 2-amino-3-hydroxynaphthalene has been synthesized and characterized.     

Determination of perfluorinated carboxylic acids in biological samples by high-performance liquid chromatography

This paper describes a method for the quantitative determination of perfluorinated carboxylic acids (PFCAs), perfluorohexanoic acid (C6-PFCA), perfluoroheptanoic acid (C7-PFCA), perfluorooctanoic acid (C8-PFCA), perfluorononanoic acid (C9-PFCA) and perfluorodecanoic acid (C10-PFCA), in biological samples. PFCA in liver homogenates was extracted as an ion pair with tetrabutylammonium (TBA) ion into organic solvent, then the PFCA was derivatized with 3-bromoacetyl-7-methoxycoumarin (BrAMC) and quantified by HPLC with fluorescence detection. This method is applicable for the studies on tissue accumulation and elimination of PFCAs in animals after the administration.     

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples

The ascorbic acid (AA)—dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.     

Determination of trovafloxacin,a new quinolone antibiotic,in biological samples by reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C₁₈ column with a mobile phase consisting of 0.04 M H₃PO₄-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 μg/ml and the calibration curves were linear over a concentration range of 0.1 to 20..0 μg/ml (r² = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 μg/ml. Human serum samples could be stored for up to 12 months at −20°C and urine samples could be stored up to 18 months at −80°C.     

Determination of nicotine and two major metabolites in serum by solid-phase extraction and high-performance liquid chromatography, and high-performance liquid chromatography—particle beam mass spectrometry

A rapid and selective assay of nicotine, cotinine and rans-3'-hydroxycotinine in human serum, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries were ca. 95% for nicotine, 90% for cotinine and 50–55% for trans-3'-hydroxycotinine. The limit of quantitation observed with this method was 10 ng/ml for nicotine and 5 ng/ml for each of the metabolites. The compounds were also identified using high-performance liquid chromatography with particle beam mass spectrometry, to confirm their presence in human serum.     

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.     

Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), may be related to reduced biosynthesis of nitric oxide in diseases associated with accelerated atherosclerosis. The closely related compound symmetric dimethylarginine (SDMA) does not inhibit NOS, but may compete with arginine for cellular uptake, thereby limiting substrate availability for NOS. We report on a method for the simultaneous measurement of arginine, ADMA, and SDMA as a tool to gain insight in the role of these compounds in the regulation of NOS activity. Sample cleanup was performed by solid-phase extraction on polymeric cation-exchange columns using monomethylarginine as internal standard. After derivatization with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. The stable derivatives were separated with near baseline resolution. Using a sample volume of 0.2 ml, linear calibration curves were obtained with limits of quantification of 0.08 microM for arginine and 0.01 microM for ADMA and SDMA. Analytical recovery was 98-102%, and interassay CV was better than 3%. Plasma from healthy volunteers (n = 53) contained 94 +/- 26 microM arginine, 0.42 +/- 0.06 microM ADMA, and 0.47 +/- 0.08 microM SDMA. Due to its high precision and sensitivity this method is a valuable tool in research on the metabolism of dimethylated arginines and their role in the regulation of NOS activity.     

Determination of furosine in biomedical samples employing an improved hydrolysis and high-performance liquid chromatographic technique.

Traditionally, the most sensitive and specific determination of non-enzymatic protein glycation has involved an 18-24-h acid hydrolysis in order to generate the compound furosine, which has been detected employing reversed-phase h.p.l.c. In this study, we have reported that significant quantities of furosine can be generated with much shorter hydrolysis times employing a 90-min vapor-phase acid hydrolysis procedure. The furosine generated by vapor-phase hydrolysis is then quantitated by pulsed amperometric detection using anion-exchange high-performance liquid chromatography. Employing this method, we were able to show that furosine generated from acid hydrolysis of purified hepatic membranes in a diabetic and non-diabetic animal model agreed with traditional methods assessing total glycated protein (i.e., boronate affinity methods).     

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.     

Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normal-phase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providing nearly quantitative extraction (>95%) and a sensitivity limit of 0.01 to 0.02 μg/ml blood or plasma, without interference from sulfonamides.     

A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Extraction, pre-high-performance liquid chromatographic purification, and high-performance liquid chromatographic analysis of plant nucleotides

Problems encountered in obtaining reliable analytical data by HPLC for the free nucleotide constituents of plant tissues are considered and methods of overcoming them experimentally assessed. Major problems include suppression of residual phosphatase activity during extraction, and removal of pigments, phenolics, alkaloids, and other uv-absorbing nonnucleotides, prior to HPLC. An optimal combination of extraction and pre-HPLC purification techniques is discussed which, in combination with HPLC by anion exchange, yields quantitatively reliable data. The optimized procedure involves extraction with a monophasic mixture of methanol: chloroform:formic acid:water and purification of the nucleotide extract by a batch treatment with poly-N-vinylpyrrolidone, followed by ligand-exchange chromatography. The main HPLC separation uses mu Bondapak NH2 in a linear phosphate gradient and gives good resolution of all the commonly occurring plant nucleotides in a single chromatographic run.