Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.     

Optimization of the separation of lysergic acid diethylamide in urine by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentrations of lysergic acid diethylamide (LSD), iso-lysergic acid diethylamide (iso-LSD) and lysergic acid N,N-methylpropylamide (LAMPA) in human urine were investigated by capillary electrophoresis-fluorescence spectroscopy using sodium dodecyl sulfate (SDS) as an anionic surfactant. A number of parameters such as buffer pH, SDS concentration, Brij-30 concentration and the content of organic solvent used in separation, were optimized. The techniques of sweeping-micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used for determining on-line concentrations. The advantages and disadvantages of this procedure with respect to sensitivity, precision and simplicity are discussed and compared.     

Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis

The determination of caffeine and its analogues is important for a wide variety of analyses and is performed in an assortment of matrices ranging from food to clinical samples. While reversed-phase HPLC has become the standard analysis protocol in most laboratories, capillary electrophoresis has the advantages of higher separation efficiency and shorter separation time. The micellar capillary electrophoresis (MECC) separation of caffeine and its metabolites, theobromine, paraxanthine, theophylline and 1,3,7-trimethyluric acid was investigated using sodium dodecyl sulphate (SDS) as the micellar phase. The effects of pH, micelle concentration, buffer concentration, ionic strength, buffer salts, applied voltage and injection time were studied to select the optimum conditions for the determination of caffeine and its four analogues in drugs, foods and body fluids. Caffeine and its three analogues were resolved within 120 s with detection limits less than 1 μg/ml. Samples could be analyzed utilizing direct injection with satisfactory resolution and reproducibility.     

Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100mM boric acid buffer containing 10mM SDS and 10mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0 x 10(-7) to 9.0 x 10(-7) M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.     

Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM beta-cyclodextrin, 5 mM 2-hydroxypropyl-beta-cyclodextrin, 50 mM SDS and sodium borate-dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C(18)-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 microM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.     

Simultaneous determination of theophylline and its major metabolites in urine by reversed-phase ion-pair high-performance liquid chromatography

A new, highly selective high-performance liquid-chromatographic (HPLC) assay for theophylline and its major metabolites in urine is described. The method utilizes an ion-pair extraction followed by separation and quantitation by reversed-phase ion-pair gradient-elution HPLC. Comparison with several other methods showed that interferences were present in too many blank urine samples to allow for the accurate quantitation of the metabolites of theophylline by direct injection-isocratic HPLC assays. Sample processing involving ion-pair complexing and extraction together with gradient-elution systems is recommended for accurate pharmacokinetic studies.     

Simultaneous determination of GTS-21 and its metabolite in rat plasma by high-performance liquid chromatography using solid-phase extraction

It has been suggested that GTS-21 can improve the learning deficits and inhibit the neuro-degeneration in patients with Alzheimer's disease. This paper describes a reversed-phase high-performance liquid chromatographic assay with visible detection at 405 nm for determination of GTS-21 and its metabolite, 4-hydroxy-GTS-21 in rat plasma. The method uses solid-phase extraction with a Bond Elut C₁₈ column. A quantitation limit of 1.0 ng/ml was achieved using 0.5 ml of rat plasma. In the validation study, the coefficients of variation and the relative errors of each compound were less than 10%. Also freeze-thaw and storage stability were confirmed. This method has proved to be applicable to the pharmacokinetic study of GTS-21 in rats.     

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 μL) were subjected to deproteinization with acetonitrile (200 μL) and then, after centrifugation, 150 μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb^® 5 μm CNRP 4.6 × 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.     

Simultaneous determination of trans-resveratrol-3-O-glucoside and its two metabolites in rat plasma using liquid chromatography with ultraviolet detection

A sensitive and selective high-performance liquid chromatographic method was developed for simultaneous determination of trans-resveratrol-3-O-glucoside (TRG) and its metabolites, trans-resveratrol-3-O-glucuronide (TRN) and trans-resveratrol (TR) in rat plasma. The plasma proteins were precipitated with acetonitrile and supernatant was evaporated to dryness. The analytes and internal standard baicalin were chromatographed on a C(18) column. The mobile phase consisted of 25% acetonitrile and 75% H(2)O adjusted with formic acid to pH 3.5. The flow-rate was 1.0 ml/min and ultraviolet detection was set at 320 nm. Standard curves were linear over the concentration range of 0.04-40 microg/ml for TRG and TRN, and 0.04-10 microg/ml for TR, respectively. The precision, expressed as the intra-day R.S.D. and inter-day R.S.D., was below 9.3% for TRG, TRN and TR. The accuracy, expressed as the relative error (RE) was within +/-7.4% for all analytes. The mean recoveries of TRG, TRN, TR and I.S. were 93.6%, 93.1%, 91.0% and 87.9%, respectively. This method was successfully applied to a pharmacokinetic study of TRG after an oral dose of 150 mg/kg to Wistar rats.     

Simultaneous quantification of amoxycillin and metronidazole in plasma using high-performance liquid chromatography with photodiode array detection

A simple, robust HPLC method was developed to measure simultaneously the plasma concentrations of amoxycillin and metronidazole in order to assess their disposition in the eradication of Helicobacter pylori. Plasma samples were protein precipitated, pH adjusted and the supernatant injected onto the HPLC system which used a C₁₈ column, paired-ion aqueous mobile phase and photodiode array detection of amoxycillin at 230 nm and metronidazole at 313 nm. Intra- and inter-day precision and inaccuracy were less than 10% for concentrations between 5 and 20 mg/l. The limit of quantification was 1 mg/l. Samples were stable on the HPLC injector for 48 h at room temperature and multiple freeze–thaw cycles led to no decomposition.     

Inhibition of human P450 enzymes by nicotinic acid and nicotinamide

Nicotinic acid has been used as a cholesterol-lowering agent for a few decades already, whereas the cytoprotective and antiviral properties of nicotinamide are slowly gaining attention. In both cases however, very high doses are needed to achieve a therapeutic effect, resulting in blood concentrations sometimes as high as 15 mM. Based on their common pyridine functionality, we hypothesized that these two molecules could inhibit human P450 enzymes. In vitro inhibition studies demonstrate that, at their therapeutic concentrations, both nicotinic acid and nicotinamide inhibit CYP2D6 (Ki = 3.8 +/- 0.3 and 19 +/- 4 mM, respectively). Nicotinamide also inhibits CYP3A4 (Ki = 13 +/- 3 mM) and CYP2E1 (Ki = 13 +/- 8 mM). As expected for nitrogen-containing heteroaromatic molecules, spectrophotometric analysis indicates that the inhibition occurs via coordination of the pyridine nitrogen atom to the heme iron.     

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH₂PO₄–K₂HPO₄ buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide–acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40°C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.     

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography—mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of isoflavones and bisphenol A in rat serum by high-performance liquid chromatography coupled with coulometric array detection

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.