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.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative determination of famotidine in human maternal plasma,umbilical cord plasma and urine using high‐performance liquid chromatography–mass spectrometry

Liquid chromatography with electrospray ionization mass spectrometry for the quantitative determination of famotidine in human urine, maternal and umbilical cord plasma was developed and validated. The plasma samples were alkalized with ammonium hydroxide and extracted twice with ethyl acetate. The extraction recovery of famotidine in maternal and umbilical cord plasma ranged from 53 to 64% and 72 to 79%, respectively. Urine samples were directly diluted with the initial mobile phase then injected into the HPLC system. Chromatographic separation of famotidine was achieved by using a Phenomenex Synergi? Hydro‐RP? column with a gradient elution of acetonitrile and 10 mm ammonium acetate aqueous solution (pH 8.3, adjusted with ammonium hydroxide). Mass spectrometric detection of famotidine was set in the positive mode and used a selected ion monitoring method. Carbon‐13‐labeled famotidine was used as internal standard. The calibration curves were linear (r² > 0.99) in the concentration ranges of 0.631–252 ng/mL for umbilical and maternal plasma samples and 0.075–30.0 µg/mL for urine samples. The relative deviation of method was <14% for intra‐ and inter‐day assays, and the accuracy ranged between 93 and 110%. The matrix effect of famotidine in human urine, maternal and umbilical cord plasma was less than 17%. Copyright © 2013 John Wiley & Sons, Ltd.     

Monitoring of the degradation kinetics of diatrizoate sodium to its cytotoxic degradant using a stability‐indicating high‐performance liquid chromatographic method

The X‐ray diagnostic agent sodium diatrizoate (DTA) was studied for chemical degradation. The 3,5‐diamino derivative was found to be the alkaline and acidic degradation product. The 3,5‐diamino degradate is also the synthetic precursor of DTA and it is proved to have cytotoxic and mutagenic effects. A sensitive, selective and precise high‐performance liquid chromatographic stability‐indicating method for the determination of DTA in the presence of its acidic degradation product and in pharmaceutical formulation was developed and validated. Owing to the high toxicity of the degradation product, the kinetics of the acidic degradation process was monitored by the developed RP‐HPLC method. The reaction was found to follow pseudo‐first order kinetics. The kinetic parameters such as rate constant (K ) and half‐life (t _½) were calculated under different temperatures and acid concentrations; activation energy was estimated from the Arrhenius plot. The developed RP‐HPLC method depends on isocratic elution of a mobile phase composed of methanol–water (25:75 v /v; pH adjusted with phosphoric acid), and UV detection at 238 nm. The method showed good linearity over a concentration range of 2–100 μg/mL with mean percentage recovery of 100.04 ± 1.07. The selectivity of the proposed method was tested using laboratory‐prepared mixtures. The proposed method has been successfully applied to the analysis of DTA in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with the official USP method. Validation of the proposed method was performed according to International Conference on Harmonization guidelines.     

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high‐performance liquid chromatography with 1‐phenyl‐3‐methyl‐5‐pyrazolone derivatization: application to gluconeogenesis studies

A new pre‐column derivative high‐performance liquid chromatography (HPLC) method for determination of d ‐glucose with 3‐O‐methyl‐d ‐glucose (3‐OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1‐phenyl‐3‐methy‐5‐pyrazolone at 70°C for 50 min. Glucose and 3‐OMG were extracted by liquid–liquid extraction and separated on a YMC‐Triart C₁₈ column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri‐ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39–25 μm (R² = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μm (0.070 mg/mL). Intra‐ and inter‐day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1–2.5 μm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous quantification of antofloxacin and its major metabolite in human urine by HPLC–MS/MS,and its application to a pharmacokinetic study

This study presents a high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous determination of antofloxacinin and its main metabolite – N ‐demethylated metabolite (N‐ DM) – in human urine. Ornidazole was used as the internal standard. This was a clinical urine recovery study, in which 10 healthy Chinese volunteers were intravenously administered a single 200 mg dose of antofloxacin hydrochloride. Compounds were extracted by albumen precipitation, after which samples were isocratically eluted using a Poroshell 120 SB‐C₁₈ column, and were analysed using HPLC–MS/MS under electronic spray ionization positive ion mode. The method was successfully applied in a urine pharmacokinetic study of antofloxacinin, with a detection range of 0.02/0.01 to 200/100 μg/mL (for antofioxacin/N‐ DM).The average percentages of antofioxacin/N‐ DM measured in urinary excretion frp, 10 volunteers were 54.9 ± 5.7/8.2 ± 2.5% in 120 h duration.     

Simultaneous UPLC‐MS/MS determination of antiepileptic agents for dose adjustment

Therapeutic drug monitoring (TDM) of anti‐epileptic drugs (AED) is a routine application. Carbamazepine (CRB) is monitored as the parent drug while oxcarbazepine (OXC) and eslicarbazepine acetate (ESL) are monitored as their active metabolite (eslicarbazepine; MHD). We have developed a UPLC‐MS/MS method for determining CRB, OXC, ESL and MHD in plasma or serum with a simplified extraction protocol. The developed method detects sildenafil (SLD), which clinically interferes with AED and is likely to be co‐administered in epileptic patients suffering from sexual insufficiency (60%). MHD was prepared in‐house. AED were simultaneously determined in presence of SLD using gatifloxacin as an internal standard (IS). Separation was achieved using acetonitrile, methanol and 100 mm ammonium acetate in water (32:3:65, v /v/v) on an Intersil^®RP‐HPLC column (250 × 4.6 mm, 5 μm). A one‐step extraction was performed by simultaneous protein and phospholipids precipitation. Detection was done by tandem mass spectrometry. No relative matrix effect was observed. The method was linear (0.5–40 μg/mL for CRB, ESL and MHD and 0.05–4 μg/mL for OXC), accurate and selective. Recoveries were 64.41 ± 5.07, 45.62 ± 1.73, 61.41 ± 4.77 and 60.33 ± 1.36 for CRB, OXC, ESL and MHD, respectively. The method was successfully applied for TDM of AED.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

On‐line flow‐through extraction–preconcentration‐large volume injection‐RP LC for trace determination of pyrethroids in Slovak soil

A rapid micro‐analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro‐sample (200 mg). It uses on‐line flow‐through extraction of soil micro‐samples (packed into a short glass column) with a methanol‐aqueous citric acid buffer mixture, successive on‐line SPE preconcentration of analytes from the extract and on‐line RP‐HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP‐18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on‐line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 μg/mL with correlation coefficients of linear regression (least‐squares method) in the range 0.998–0.999. Recovery studies were carried out at 0.25–1.00 μg/g dry soil fortification level and obtained recoveries were for K 81–84%, C 56–59% and for P 58–63%. Achieved LOD (confidence band) of studied pyrethroids were for large‐volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil “solid sampling HPLC”.     

Determination of carboplatin in canine plasma by high‐performance liquid chromatography

Carboplatin is an antineoplastic drug administered to treat different tumoral conditions in canine oncology. The objective of this study was to validate a high‐performance chromatographic (HPLC) method which could be applied in canine pharmacokinetic studies. Following ultrafiltration using a Centrifree device, standards, quality controls and plasma samples were separated by isocratic reversed‐phase HPLC on an Inertsil ODS‐2 (250 × 4.6 mm i.d.) analytical column and quantified using UV detection at 220 nm. The mobile phase was potassium phosphate (pH 4.5), with a flow‐rate of 1.0 mL/min. The procedure produced a linear curve (r² > 0.999) over the concentration range 1–200 μg/mL. The lower limit of quantification was 1 μg/mL. The intra‐assay and inter‐assay precision was ～90%. The overall recovery was ～90%. The method was illustrated with a preliminary pharmacokinetic analysis on nine dogs treated with carboplatin at our hospital. Carboplatin disposition followed a monocompartmental structure in dogs and was characterized by a short half‐life (50 min). Copyright © 2009 John Wiley & Sons, Ltd.     

Cetyl‐alcohol‐reinforced hollow fiber solid/liquid‐phase microextraction and HPLC–DAD analysis of ezetimibe and simvastatin in human plasma and urine

A new cetyl‐alcohol‐reinforced hollow fiber solid/liquid‐phase microextraction (CA–HF–SLPME) followed by high‐performance liquid chromatography–diode array detection (HPLC–DAD) method was developed for simultaneous determination of ezetimibe and simvastatin in human plasma and urine samples. To prepare the CA–HF–SLPME device, the cetyl‐alcohol was immobilized into the pores of a 2.5 cm hollow fiber micro‐tube and the lumen of the micro‐tube was filled with 1‐octanol with the two ends sealed. Afterwards, the prepared device was introduced into 10 mL of the sample solution containing the analytes with agitation. Under optimized conditions, calibration curves plotted in spiked plasma and urine samples were linear in the ranges of 0.363–25/0.49–25 μg L^?1 for ezetimibe/simvastatin and 0.193–25/0.312–25 μg L^?1 for ezetimibe/simvastatin in plasma and urine samples, respectively. The limit of detection was 0.109/0.174 μg L^?1 for ezetimibe/simvastatin in plasma and 0.058/0.093 μg L^?1 for ezetimibe/simvastatin in urine. As a potential application, the proposed method was applied to determine the concentration of selected analytes in patient plasma and urine samples after medication and satisfactory results were achieved. In comparison with reference methods, the CA–HF–SLPME–HPLC–DAD method demonstrates considerable potential in the biopharmaceutical analysis of selected drugs.     

A simple RP‐HPLC method for quantification of columbianadin in rat plasma and its application to pharmacokinetic study

A rapid and sensitive reversed‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed to investigate pharmacokinetics of columbianadin, one of the main bioactive constituents in the roots of Angelica pubescens f. biserrata, in rat plasma after intravenous administration to rats at two doses of 10 and 20 mg/kg. The method involves a plasma clean‐up step using liquid–liquid extraction by diethyl ether, followed by RP‐HPLC separation and detection. Separation of columbianadin was performed on an analytical Diamonsil? ODS C₁₈ column, with a mobile phase of MeOH–H₂O (85 : 15, v/v) at a flow‐rate of 1.0 mL/min, and UV detection was set at 325 nm. The retention time of columbianadin and scoparone (internal standard) was 6.7 and 3.5 min, respectively. The calibration curve was linear over the range of 0.2–20.0 μg/mL (r² = 0.9986) in rat plasma. The lower limits of detection and quantification were 0.05 and 0.1 μg/mL, respectively. The extraction recovery from plasma was in the range of 81.61–89.93%. The intra‐ and inter‐day precisions (relative standard deviation) were between 1.01 and 9.33%, with accuracies ranging from 89.76 to 109.22%. The results indicated that the method established was suitable for the determination and pharmacokinetic study of columbianadin in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

RP‐HPLC‐DAD method for the estimation of embelin as marker in Embelia ribes and its polyherbal formulations

Evidence‐based herbal products with assured quality are assuming importance for complementary and alternative medicine. Traditional medicines by and large are not standardized and validated to meet the new requirements. In the present study, marker (embelin)‐based standardization of a major medicinal plant, Embelia ribes and its polyherbal formulations was attempted. Conditions for the quantitative extraction of the marker compound embelin from E. ribes fruits and herbal formulations were also optimized. Reversed‐phase high‐performance liquid chromatography, coupled with diode array detection (RP‐HPLC–DAD) for the quantification of embelin was developed and validated. Satisfactory results were obtained with respect to linearity (15–250 µg/mL), LOD (3.97 µg/mL), LOQ (13.2 µg/mL), recovery (99.4–103.8%) and precision (1.43–2.87%). The applicability of the method was demonstrated with selected phytopharmaceuticals. The present method was sensitive, accurate, simple and reproducible and therefore can be recommended for marker‐based standardization, and quality assurance of E. ribes herbal formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and sensitive LC–MS/MS method for quantification of quercetin‐3‐O‐β‐d‐glucopyranosyl‐7‐O‐β‐d‐gentiobioside in plasma and its application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method with good accuracy and precision was developed and validated for the pharmacokinetic study of quercetin‐3‐O‐β‐d ‐glucopyranosyl‐7‐O‐β‐d ‐gentiobioside (QGG) in Sprague–Dawley rats. Plasma samples were simply precipitated by methanol and then analyzed by LC–MS/MS. A Venusil® ASB C₁₈ column (2.1 × 50 mm, i.d. 5 μm) was used for separation, with methanol–water (50:50, v/v) as the mobile phase at a flow rate of 300 μL/min. The optimized mass transition ion‐pairs (m/z) for quantitation were 787.3/301.3 for QGG, and 725.3/293.3 for internal standard. The linear range was 7.32–1830 ng/mL with an average correlation coefficient of 0.9992, and the limit of quantification was 7.32 ng/mL. The intra‐ and inter‐day precision and accuracy were less than ±15%. At low, medium and high quality control concentrations, the recovery and matrix effect of the analyte and IS were in the range of 89.06–92.43 and 88.58–97.62%, respectively. The method was applied for the pharmacokinetic study of QGG in Sprague–Dawley rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Simple determination of betaine,l‐carnitine and choline in human urine using self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A sequential online extraction, clean‐up and separation system for the determination of betaine, l ‐carnitine and choline in human urine using column‐switching ion chromatography with nonsuppressed conductivity detection was developed in this work. A self‐packed pretreatment column (50 × 4.6 mm, i.d.) was used for the extraction and clean‐up of betaine, l ‐carnitine and choline. The separation was achieved using self‐packed cationic exchange column (150 × 4.6 mm, i.d.), followed by nonsuppressed conductivity detection. Under optimized experimental conditions, the developed method presented good analytical performance, with excellent linearity in the range of 0.60–100 μg mL⁻¹ for betaine, 0.75–100 μg mL⁻¹ for l ‐carnitine and 0.50–100 μg mL⁻¹ for choline, with all correlation coefficients (R²) >0.99 in urine. The limits of detection were 0.15 μg mL⁻¹ for betaine, 0.20 μg mL⁻¹ for l ‐carnitine and 0.09 μg mL⁻¹ for choline. The intra‐ and inter‐day accuracy and precision for all quality controls were within ±10.32 and ±9.05%, respectively. Satisfactory recovery was observed between 92.8 and 102.0%. The validated method was successfully applied to the detection of urinary samples from 10 healthy people. The values detected in human urine using the proposed method showed good agreement with the measurement reported previously.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.     

Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.