Ion-pair High-performance Liquid Chromatographic Method Research Articles

Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

New Analytical Approach for The Determination of Calcium Phosphate Dibasic and Tribasic in Processed Food by Comparison of Ion Chromatography With High-Performance Liquid Chromatography.

An analytical method to measure solubilized orthophosphate ions (HPO42− and PO43− ) from the water-insoluble food additives calcium phosphate dibasic (DCP) and calcium phosphate tribasic (TCP) in processed foods was optimized by comparing ion chromatography (IC) coupled with DS6 conductivity detector (Cond.) and high-performance liquid chromatography (HPLC) with Evaporative light scattering detector (ELSD) methods. The ion-pairing HPLC method could analyze calcium and phosphate ions successively. However, this method exhibited low reproducibility after approximately 48 hours of measurements. The IC method was established as an effective method of measuring orthophosphate ions with high reproducibility using distilled water and KOH solution as the mobile phase with a Dionex column. Matrix-based limit of detections (LOD) and limit of quantifications (LOQ) for snacks and cereals were estimated in the range of 0.01–0.91 µg/mL and 0.21–2.74 µg/mL, respectively. In inter-day and intra-day tests, the calculated precision (%RSD) and accuracy (recovery %) ranged from 0.5% to 6.6% and 82% to 117%, respectively, in both food samples. The levels of DCP or TCP could be analyzed in various positive food samples, and the developed IC method demonstrated good applicability in the analysis of DCP and TCP in collected processed foods.

Ion-pair HPLC method for the quantification of metformin in human urine

Metformin is an oral antidiabetic drug that has been used in the treatment of non-insulin-dependent diabetes, which improves glycemic control by primarily inhibiting hepatic gluconeogenesis and glucogenolysis [1]. Metformin is slowly absorbed after oral administration, about 60% of an oral dose is excreted in the urine as unchanged drug within 24 h, and about 30% of the dose is nonabsorbed and eliminated unchanged in feces [2]. Previous studies have shown a plasma elimination half-life ranging from 2.0 to 6.0 h after oral administration of varying doses (0.5 to 1.5 g/dose) [3]. Urine provides a non-invasive sample collection method, and determination of drug levels in urine is comparatively less complex than plasma and other body fluids [4]. Several reports indicate that urinary excretion data can be used to arrive at bioequivalence decision of different drug formulations [4-6]. Chemically, it is 1,1-dimethyl biguanide (Figure 1). Metformin is a small highly polar molecule (pKa=2.8, 11.5, log P octanol:water= −2.6), which has great solubility in water and poor solubility in lipids; it is also possible to retain in reversed-phase (RP) columns using ion-pairing reagents. Numerous methods for the quantitation of metformin in urine have been utilized over years. They include capillary electrophoresis with contactless conductivity detection [7], voltammetric method [8], cation exchange high-performance liquid chromatography (HPLC) [9], reverse phase RP-HPLC [10-12] and liquid chromatography (LC)–mass spectrometry (MS) [13]. Most of the methods have been based on HPLC with spectrophotometric detection in the range of 230–240 nm (Table 1). Chemical derivatization of metformin has been used JOURNAL OF APPLIED BIOANALYSIS, January 2016, p. 16-24. http://dx.doi.org/10.17145/jab.16.004 (ISSN 2405-710X) Vol. 2, No. 1

Three-phase hollow-fiber microextraction combined with ion-pair high-performance liquid chromatography for the simultaneous determination of five components of compound α-ketoacid tablets in human urine.

The determination of α-ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound α-ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three-phase hollow-fiber liquid-phase microextraction combined with ion-pair high-performance liquid chromatography method was established for the determination of d,l-α-hydroxymethionine calcium, d,l-α-ketoisoleucine calcium, α-ketovaline calcium, α-ketoleucine calcium, and α-ketophenylalanine calcium of compound α-ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11-, 110-, 198-, 202-, and 50-fold, respectively. The calibration curves for these analytes were linear over the range of 0.1-10 mg/L for α-ketovaline calcium, d,l-α-ketoisoleucine calcium, and α-ketoleucine calcium, 0.5-10 mg/L for d,l-α-hydroxymethionine calcium, and α-ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 μg/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92-118%) were obtained. The proposed method demonstrated excellent sample clean-up and analytes enrichment to determine the five components in human urine.

Optimization of the Separation and Determination of Nitidine and Chelerythrine in Zanthoxylum nitidum by High-Performance Liquid Chromatography with Fluorescence Detection

A new ion-pair high-performance liquid chromatography method with fluorescence detection was developed for the determination of nitidine and chelerythrine in Zanthoxylum nitidum. To optimize the separation of the two compounds in reversed-phase liquid chromatography, a response surface method (Box-Behnken designs) was used. Three important factors: concentration of ion pair agent, mobile phase composition and buffer pH, were studied for their contribution to the analytes' response, leading to a total of 17 experiments performed on a Kromasil C18 column. The experimental responses were fitted into a second-order polynomial to predict the best conditions. The optimal mobile phase conditions were predicted to be acetonitrile-sodium dodecyl sulphate (17.8 mM, 20 mM citric acid, pH 2.98, 57:43, v/v). The proposed method was validated according to International Conference on Harmonization guidelines and it is suggested to be appropriate for the routine quality control analysis of nitidine and chelerythrine in Zanthoxylum nitidum.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

Determination of Cyanuric Acid in Milk Powder and Swimming Pool Water by Ion-pair Reversed-phase Liquid Chromatography

An ion-pair reversed-phase high-performance liquid chromatographic method, using tetrabutylammonium bromide (TBAB) as ion-pair reagent, has been developed for the analysis of cyanuric acid (CA) in milk powder and swimming pool water. It was found that the effect of the concentrations of ion-pair reagent on the retention of cyanuric acid was different for standard solution and different real samples. The separation was carried out on a reversed-phase C18 column with 85:15 (V/V) water-acetonitrile (ACN) containing different concentration of TBAB as mobile phase for different samples. The linear range of the calibration curve for CA was 0.1–100 mg·L−1. The detection limits calculated at S/N=3 was 0.11 mg·L−1 for the analysis of milk powder and 0.31 mg·L−1 for the analysis of swimming pool water, respectively. The method was successfully applied to the analysis of CA in milk powder and swimming pool water.

Development of an Ion-Pair HPLC Method for Determination of Acebutolol in Pharmaceuticals

A simple and accurate ion-pair reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for determination of acebutolol (ACE) in tablet dosage forms. Both ACE and ambroxol (internal standard) were well separated using a reversed phase column and a mobile phase consisting of a mixture of methanol-0.05 M acetic acid (containing 8 mM sodium 1-heptanesulfanate) (65:35 v/v) with pH adjusted to 3.2 with triethylamine. The mobile phase was pumped at 0.80 mL min−1 flow rate and ACE was detected by diode-array detection at 240 nm. The retention times for ACE and internal standard (IS) were 4.574 and 8.236 min, respectively. A linear response (r = 0.9998) was observed in the range of 0.27–2.93 μg mL−1 in mobile phase. The limit of detection and limit of quantification were found as 0.07 and 0.23 μg mL−1 in the mobile phase. Validation parameters such as precision, accuracy, selectivity, reproducibility, and system suitability tests were also determined. The excipients did not interfere with the assay of ACE in tablet dosage forms. It is suggested that the proposed method can be used for routine quality control and dosage-form assay of ACE.

An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze–thaw degradation of ATP

Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe an ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze–thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results in a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen.

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C18 column using a mixture of 50mmolL−1 borate buffer pH 9.0 containing 0.25mmolL−1 tetrabutylammonium chloride and 0.5mmolL−1 EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440nm with excitation at 375nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmolL−1 level), accurate, specific, rapid (analysis time approximately 13min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05molL−1 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid.

Simultaneous determination of silicon and phosphorus in soil and plants by reversed-phase ion-pair chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method, using tetrabutylammonium bromide (TBABr) as ion-pair reagent, has been developed for the simultaneous analysis of silicon (Si) and phosphorus (P) as heteropoly acids in soil and plant samples. The effect of the concentrations of ion-pair reagent, acetate buffer and organic modifier as well as the pH of buffer on separation was made clear. The reaction conditions and stability of heteropoly acids were investigated. Furthermore, the phenomenon occurred in the optimized process was also further researched. The separation was performed on a reversed-phase C 18 column within 11 min with 40:60 (v/v) 0.1 M acetate buffer (pH 3.9)–acetonitrile (ACN) containing 0.8 mM TBABr as a mobile phase. The linear ranges of the peak area calibration curves for Si and P were 0.08–50 mg/L and 0.40–50 mg/L, respectively. The detection limits calculated at S/N = 3 were 0.0057 mg/L and 0.0280 mg/L for Si and P, respectively. The method was successfully applied to the analysis of soluble and total contents of Si and P in soil and plant samples.

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C18 column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min−1. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL−1 for baicalin, 5.05–101.0 μg*mL−1 for baicalein, 0.95–19.0 μg*mL−1 for wogonin, 2.75–55.0 μg*mL−1 for oxysophocarpin, 2.75–55.0 μg*mL−1 for oxymatrine and 4.90–98.0 μg*mL−1 for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C 18 analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min −1. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL −1, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL −1, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, % E r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Ion-Pair RP-LC of Tegaserod Maleate and its Impurities in Pharmaceutical Formulations and in Dissolution Studies

A simple ion-pair reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of tegaserod maleate and related impurities in tablet dosage forms. The mobile phase was 60:40 (v/v) acetonitrile-25 mmol L−1 sodium dodecyl sulfate, adjusted to pH 2.6 with glacial acetic acid. A C18 column was used as stationary phase and UV detection was at 314 nm. The method was optimized and validated. Response was linearly dependent on concentration between 0.1 and 100 µg mL−1 with a limit of quantification (LOQ) of 0.1 µg mL−1 for tegaserod maleate (S/N = 10). Under optimum conditions, tegaserod maleate was successfully separated from related substances, including 5-methoxyindole-3-carboxaldehyde remaining after synthesis and other impurities possibly resulting from oxidization and decomposition. The excipients did not interfere with assay of tegaserod maleate in tablet dosage forms. It is suggested that the proposed method can be used for routine quality control and dosage-form assay of tegaserod maleate.

Development and validation of a reversed-phase ion-pair liquid chromatography method for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams

A reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams. The determination was performed on a BDS C 18 analytical column (250 × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.020 M tetrabutylammonium hydroxide and 0.025 M potassium dihydrogen phosphate (pH 6.8) mixed with acetonitrile in a ratio (77:23, v/v) and pumped at a flow rate 1.00 ml min −1. The UV detector was operated at 260 nm. The retention times of the magnesium ascorbyl phosphate, melatonin and chlorthalidone that was used as internal standard, were 6.55, 9.18 and 11.07 min, respectively. Calibration graphs are linear ( r better than 0.9990, n = 6), in concentration range 1.00–10.00 μg ml −1 for magnesium ascorbyl phosphate and 0.63–6.25 μg ml −1 for melatonin. The intra- and inter-day R.S.D. values were less than 6.0%, while the relative percentage error E r was less than 3.5% ( n = 5). The quantitation limits were 0.69 and 0.47 μg ml −1, for magnesium ascorbyl phosphate and melatonin, respectively. The method was applied to the analysis of a cosmetic cream and proved to be suitable for rapid and reliable quality control.

Simple reversed-phase ion-pair liquid chromatography assay for the simultaneous determination of mycophenolic acid and its glucuronide metabolite in human plasma and urine

A simple and reproducible reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method using isocratic elution with UV absorbance detection is presented for the simultaneous quantitation of mycophenolic acid (MPA) and MPA-glucuronide (MPAG) in human plasma and urine. The sample preparation procedures involved simple protein precipitation for plasma and 10-fold dilution for urine. Each analytical run was completed within 15 min, with MPAG and MPA being eluted at 3.8 and 11.4 min, respectively. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.

Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K 2CO 3 just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C 18 3.5 μm (150×4.6 mm) column (Waters) equipped with a NovaPak C 18 Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 °C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A–B (60:40) at 0 min→(40:60) at 30 min→(40:60) at 60 min. Solvent A contained 10 m M tetrabutylammonium hydroxide, 10 m M KH 2PO 4 and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 m M tetrabutylammonium hydroxide, 50 m M KH 2PO 4 and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves ( r>0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases.

Scale-up potential of ion-pair high-performance liquid chromatography method to produce biologically active inositol phosphates

This study was undertaken to evaluate the possibility that an analytical ion-pair HPLC procedure used to determine phytic acid (IP6) and its degradation products (IP3–IP5) can be transformed to a preparative purification method. A commercial phytic acid (CPA) preparation was separated into its component fractions of IP3, IP4, IP5, and IP6 on two C 18 columns (1.8 and 4.2 ml) using 51% methanol containing 0.6–1% tetrabutylammonium hydroxide as ion-pair reagent and 0–0.025 M formic acid (pH 4.3) as eluent at 1.7 and 3.0 cm/min linear velocity, respectively, and 40°C. Elution was monitored at 40°C by a refractive index detector. Reproducible separation of CPA into four well-resolved peaks on these columns was possible after optimizing method variables, particularly the concentration of ion-pair reagent in the injected sample (>1.5%). The same separations were obtained after CPA loads were scaled up 25 times on a steel column (15 cm×19 mm I.D.), packed with Ethyl C 2 sorbent (10 μm) and on a 25 cm×21.2 mm I.D. C 18 column, but at a reduced linear velocity to increase the resolution. Therefore, this optimization of separation not only is useful for analysis of phytic acid and its degradation products but also it provides key parameters for scale up for further fractionation and characterization.

Early Onset of Lipid Peroxidation After Human Traumatic Brain Injury

BackgroundOn the basis of the contradiction between data on experimental head trauma showing oxidative stress-mediated cerebral tissue damage and failure of the majority of clinical trials using free radical scavenger...

Detection and separation of the S-adenosylmethionine-decarboxylase inhibitor SAM486A in human plasma and urine by reversed-phase ion-pairing high-performance liquid chromatography

A reversed-phase ion-pairing high-performance liquid chromatography method for the detection and separation of SAM486A in human plasma and urine is described. Precipitation of proteins was used for plasma sample preparation. Enrichment of SAM486A on a 5 μ C18 column using 0.05 M NaH 2PO 4 and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent was followed by isocratic elution onto a 5 μ C18 analytical column using 0.01 M NaH 2PO 4 and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent. Analysis time was 23.0 ± 0.1 min. The separation parameters were: capacacity factor = 6.21; plates/m = 15,002; peak tailing = 2.076. The method is linear between 5 ng/ml (detection limit) and 1000 ng/ml.