Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, 5‐hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λ_ex/λ_em = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005–1.0 μg/mL for tryptamine, 2‐phenethylamine and spermidine, 0.025–1.0 μg/mL for putrescine, 0.001–1.0 μg/mL for cadaverine, 0.25–20 μg/mL for histamine, 0.25–10 μg/mL for 5–hydroxytryptamine and dopamine, and 0.01–1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3–75.0 ng/mL and 1.0–250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra‐day and ≤6.58% for inter‐day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.     

Determination of polyamines in hydrolysates of uremic plasma by high-performance cation-exchange column chromatography

The levels of putrescine, cadaverine, spermidine and spermine in uremic plasma were determined with an automatic polyamine analyzer with a 7.5 X 0.2 cm I.D. cation-exchange column using a stepwise sodium chloride gradient. All four polyamines were higher in ten patients with chronic renal failure than in eight normal subjects. The total polyamine content was also measured in the patients' plasma before and after maintenance dialysis; putrescine and spermidine levels were significantly lowered by the procedure.     

High performance liquid chromatography of biological polyamines using immobilized enzyme as post-column reactor followed by electrochemical detection

A novel analytical method for biological polyamines (putrescine, spermidine and spermine) was developed. Polyamines were separated by ion-pair reversed phase chromatography using a polymer-based octadecyl bonded column. A polyamine oxidase immobilized column worked effectively as a post-column reactor to convert polyamines to hydrogen peroxide which was eventually detected by electrochemical oxidation on platinum electrode. This method required neither tedious derivatization nor gradient elution, permitting us to perform simple and rapid analysis of polyamines. The detection limits were 0.3, 0.6, and 4 pmol injected for putrescine, spermidine, and spermine, respectively with a linear range of two to three orders of magnitude. Chromatograms obtained with samples from human urine and rat brain homogenates demonstrated the high sensitivity and selectivity of the method.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

The polyamines of Xanthium strumarium and their response to photoperiod

Flowering plants of Xanthium strumarium L., grown in 8 h photoperiods, were analysed for polyamines. Putrescine, spermidine and spermine were found throughout the plant in three forms: (a) as free polyamines; (b) conjugates soluble in 5% trichloracetic acid (TCA); and (c) bound to the TCA-insoluble precipitate. On a fresh weight basis, total polyamines are most abundant in young leaves and buds, especially flower buds. Spermidine predominates in the free polyamine fractions, while spermine is dominant in the conjugated fraction. Transfer of vegetative plants from 16 h photoperiods to 1, 2, 3, or 4 inductive cycles (8 h light + 16 h uninterrupted dark) caused rapid and marked changes in the polyamine titer of the leaves and ultimately, floral initiation. The titer of free putrescine per mg protein declined progressively with induction in all leaf sizes, while the titers of free spermidine and spermine rose during days 2 and 3 in small and expanding leaves. Conjugated putrescine, spermidine and spermine rose sharply after only 1 inductive cycle, especially in small and expanding leaves, and maintained the higher level for at least several cycles. In plants given 4 inductive cycles, buds harvested after 4 additional days had sharply elevated levels of conjugated polyamines, especially spermine, on a protein basis.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.     

HPLC analysis of polyamines and their acetylated derivatives in the picomole range using benzoyl chloride and 3,5-dinitrobenzoyl chloride as derivatizing agent

Analytical methods are described for the quantitative determination of putrescine, cadaverine, spermidine, spermine and the acetylated derivatives of spermidine and spermine in biological fluids using pre-column derivatization with either benzoyl chloride or 3,5-dinitrobenzoyl chloride, which were added to each sample as solutions in diethyl ether. Putrescine, spermidine and spermine can be analysed in seminal plasma at nanogram levels when benzoyl chloride is used as derivatizing agent. In the analysis of putrescine, cadaverine, spermidine and acetyl derivatives of spermidine and spermine, higher sensitivity is obtained with 3,5-dinitrobenzoyl-chloride. This method can readily be used in the determination of acetylated polyamines in urine samples.     

Determination of polyamines in human plasma by high‐performance liquid chromatography coupled with Q‐TOF mass spectrometry

A high‐performance liquid chromatography coupled with Q‐time of flight mass spectrometry (HPLC/Q‐TOF MS) method was developed and validated for the determination of 1, 3‐diaminopropane, putrescine, cadaverine, spermidine and spermine in human plasma. The plasma samples were first pretreated by 10% HClO₄ and then derived by benzoyl chloride with 1, 6‐diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column using a gradient program. The detection was performed on a Q‐TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentration range of 0.4 ~ 200.0 ng ? ml^?1, with limit of detection of 0.02 ~ 0.1 ng ? ml^?1. The intra‐ and inter‐day RSD for all polyamines were 2.5–14.0% and 2.9 ~ 13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancer patients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higher than that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicators in clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Monitoring of biologically active amines in cereals and cereal based food products by HPLC

Summary Biologically active amines (putreanine sulphate, N-acetyl putrescine, putrescine, cadaverine, histamine, agmatine, N-acetyl spermidine, spermidine, spermine) were separated and quantified in cereal flour and cereal products by a liquid chromatographic method. The method consists of the separation of ion pairs formed between biologically active amines and octanesulphonic acid on a reversed-phase column, postcolumn derivatization with o-phtalaldehyde-2-mercapthoethanol and spectrofluorometric detection. Results of the reliability study were satisfactory. The method was linear for each amine at 1–10 mg L⁻¹. Putrescine and spermidine were the only amines always detected in cereal flour and cereal products, ranging from 2.45 to 47.83 mg kg⁻¹ for putrescine and 3.27 to 37.14 mg kg⁻¹ for spermidine. The most important differences among types of samples were found in polyamine derivatives. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

An Ion-Pairing High-Pressure Liquid Chromatography Assay for the Determination of Cefoperazone in Plasma and Urine

Abstract

A high-performance liquid chromatographic assay for cefoperazone (cfp) in plasma and urine is described. For analysis, the internal standard ticarcillin (ticar) is solvated in acetonitrile, which is then added to plasma or urine. The supernatant is drawn off of the resulting protein precipitate and injected directly onto the reverse-phase C₁₈ column, with detection at 254 nm. The mobile phase is composed of phosphate-acetonitrile-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were less than 9% for extra-low, low, medium, and high controls. Limits of detection were 0.5 μG/mL for plasma and 1 μG/mL for urine. No interference from other cephalosporins or other antibiotics was found. This high-pressure liquid chromatographic ion-pairing assay is simple, accurate, inexpensive, and requires no extraction.     

High-speed analysis of dansyl derivatives of polyamines

Summary Analytical conditions for the high-speed, reversed-phase, liquid chromatographic of a diamine (putrescine) and polyamines (spermine and spermidine) were determined. Various elution modes were employed using the same mobile phase constituents: 20mM sodium heptane sulfonate and 20mM acetic acid as solvent A and, pure acetonitrile as solvent B. Samples were derivatized with dansyl chloride before injection.Under isocratic conditions, the separation of the three polyamines was achieved in 7 min. The use of a linear elution gradient led to the same analytical time but with a better resolution of the putrescine peak from non-polyamine frontal peaks. For these measurements, the sample size was 5l. This volume was increased to 20l and the use of a steep gradient combined with the peak-compression technique allowed a fast analysis in 2–3 minutes, which may be compared with a 30 min run time necessary when a conventional column is used.     

Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer

To evaluate the potential relationship between cancer and polyamine metabolome, a UHPLC–MS/MS method has been developed and validated for simultaneous determination of polyamine precursors, polyamines, polyamine catabolite in human plasma and urine. Polyamine precursors including l-ornithine, lysine, l-arginine and S-adenosyl-l-methionine; polyamines including 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, agmatine, N-acetylputrescine, N-acetylspermine and N-acetylspermidine; polyamine catabolite including γ-aminobutyric acid had been determined. The analytes were extracted from plasma and urine samples by protein precipitation procedure, and then separated on a Shim-pack XR-ODS column with 0.05% heptafluorobutyric acid (HFBA) in methanol and 0.05% HFBA in water. The detection was performed on UHPLC–MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The limits of quantitation for all analytes were within 0.125–31.25 ng mL⁻¹ in plasma and urine. The absolute recoveries of analytes from plasma and urine were all more than 50%. By means of the method developed, the plasma and urine samples from hepatic cancer patients and healthy age-matched volunteers had been successfully determined. Results showed that putrescine and spermidine in hepatic cancerous plasma were significant higher than those in healthy ones, while spermidine, spermine and N-acetylspermidine in hepatic cancerous urine were significant higher than those in healthy ones. The methods demonstrated the changes of polyamine metabolome occurring in plasma and urine from human subjects with hepatic cancer. It could be a powerful manner to indicate and treat hepatic cancer in its earliest indicative stages.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

High Pressure Liquid Chromatographic Determination of Mecillinam in Human Plasma and Urine

Abstract

A simple, specific, rapid and sensitive method for the analysis of mecillinam in plasma and urine using high pressure liquid chromatography is described. The assay is performed by direct injection of a plasma protein free supernatant or a dilution of urine. A μBondapak phenyl column with an eluting solvent of 16% CH₃CN-0.2% H₃PO₄ was used, with UV detection of the effluent at 220 nm. Desacetyl-cephalothin was used as the internal standard and quantitation was based on peak height ratio of mecillinam to that of the internal standard. The lowest concentration detectable without extraction was 0.25 μg/ml for plasma and 8.9 μg/ml for urine. No interference from plasma and urine was noted.     

A Simple and Sensitive HPLC Method for Antipyrine in Plasma

Abstract

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of antipyrine in small volume (50 μl) of plasma samples. Aminopyrine was employed as the internal standard. The sample preparation is a direct plasma protein precipitation procedure so is less tedious and rapid. The assay employs a column packed with a C₁₈ reversed-phase material (5 μm Nucleosil) with an isocratic mixture of acetonitrile and water (25:75, v/v) as the mobile phase. The eluant was detected at 254 nm. The assay achieves the level of sensitivity (0.5 μg/ml) and accuracy required to obtain meaningful data about the single-dose pharmacokinetics of antipyrine in guinea pig and rat. The method gave high reproducibility with coefficients of variation less than 5%.     

Simultaneous Determination of Food-Related Amines by High-Performance Liquid Chromatography

Abstract

Ten amines found in marine foods, dimethylamine, trimethylamine, trimethylamine oxide, ammonia, urea, histamine, cadaverine, putrescine, spermine and spermidine were separated by HPLC using an ion-moderated partition column. Optimum resolution and sensitivity were obtained using 0.003N sodium hydroxide as the mobile phase and UV detection at 208 nm.     

Automated Determination of Urinary Polyamines

Abstract

A system for the determination of urinary polyamines by ion exchange chromatography with fluorescence detection is presented. It provides a sensitive and specific assay of putrescine, cadaverine, spermidine and spermine in urine hydrolyzates. An internal standard is used, which makes the automated determination of series of samples easy.     

Liquid chromatography–tandem mass spectrometric assay for aliskiren,a novel renin inhibitor in micro‐volumes of human plasma: a pharmacokinetic application in healthy South Indian male subjects

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of aliskiren in human plasma using nevirapine as an internal standard. Analyte and the internal standard were extracted from 100 μL of human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a C₁₈ column using a mixture of acetonitrile and 0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.10–1013 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 2.2 min for each sample made it possible to analyze a greater number of samples in a short time, thus increasing the productivity. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous Analysis of Cimetidine and Ranitidine in Human Plasma by HPLC

Abstract

A rapid method for the simultaneous quantitation of the H₂-receptor antagonist drugs cimetidine and ranitidine in human plasma by isocratic ion-pair reverse-phase HPLC is described. The method involves a simple organic extraction step of the alkalinized plasma containing added internal standard followed by back extraction of the extract with dilute acetic acid and subsequent analysis of the aqueous acidic phase on a reverse-phase (C18) column. The eluting solvent was acetonitrile-water (20:80 v/v) containing 0.005 mole/litre octanesulphonic acid and was monitored at 229 nm. The run time for the assay was 12.5 minutes, with a detection limit for cimetidine of 50 ng/m1/(0.2 μmole/1) and that for ranitidine was 20 ng/ml (0.06 umole/1).