Determination of β-blockers in pharmaceutical preparations and human urine by high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection

A highly selective and sensitive detection method based on tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] electrogenerated chemiluminescence (ECL) has been developed for the quantitative determination of β-blockers in both pharmaceutical preparations and human urine samples. The ECL emission is based on the reaction between electro-oxidized Ru(bpy)₃³⁺ and the secondary amino groups on the β-blockers. The ECL intensities for the β-blockers were strongly dependent on the pH at which the ECL detections were conducted, with the maximum intensities being obtained at pH 9.0. Under the optimal condition, the detection limit for atenolol and metoprolol were almost 0.5 μM (50 pmol) and 0.08 μM (8 pmol), respectively, with S/N of 3 and a linear working range that extends four orders of magnitude with relative standard deviations below 5% for 10 replicate injected samples. The concentrations of atenolol and metoprolol were determined in pharmaceutical preparations using flow injection analysis with Ru(bpy)₃²⁺ ECL detection based on standard addition method. The determined values by the present method showed acceptable agreement with the stated values by manufacturers. The determination of the five β-blockers in human urine samples was performed using HPLC-Ru(bpy)₃²⁺ ECL detection. The resulting chromatogram was much simpler than that obtained with HPLC-UV absorbance detection.     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Determination of forsythiaside in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of forsythiaside in rat plasma after intravenous administration. Hesperidin was used as the internal standard. The drugs were separated on a reversed-phase C(18) column and detected at 332 nm. Good linearity was achieved in the range of 0.067-26.667 microg/mL. The intra- and inter-assay variation coefficients for this analysis were no more than 10.94 and 14.56%, respectively. The average recovery for forsythiaside was 87.42% from plasma. The analytical sensitivity and accuracy of this assay were adequate for characterization of the pharmacokinetics of intravenous administration of forsythiaside to rats and the assay has been successfully applied to provide pharmacokinetic data. The mean t(1/2Z) was 20.36, 19.40 and 23.62 min for 2, 5 and 20 mg/kg for forsythiaside after i.v. administration, respectively. The AUC(0-t) increased linearly from 40.64 to 624.14 microg min/mL after administration of the three doses.     

Sensitive determination of metal ions by liquid chromatography with tris(2,2'-bipyridine) ruthenium (II) complex electrogenerated chemiluminescence detection.

Emetine dithiocarbamate metal complex, which is prepared from emetine, carbon disulfide, and metal (II), was found to indicate a large chemiluminescence intensity on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II). Liquid chromatography equipped with the chemiluminescence detection was developed for analyzing trace metal ions by use of the metal complex formation. The mixture of the Cu(II) and Co(II) complexes as a model sample was injected into the LC system. The two metal complexes and an excess emetine were successfully separated. The Cu(II) and Co(II) complexes were determined over the range 1-300 nM (the detection limit of 650 fg) and 30-5000 nM (the detection limit of 17 pg), respectively.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

Determination of diphenhydramine by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection

Tris(2,2′-bipyridyl)ruthenium(II) electrochemiluminescence detection in a capillary electrophoresis separation system was used for the determination of diphenhydramine. In this study, platinum disk electrode (300 μm in diameter) was used as a working electrode and the influence of applied potential and buffer conditions were investigated. Under optimal conditions: 1.2 V applied potential, pH 8.50, 15 kV separation voltage and 10 mmol l⁻¹ running buffer, the calibration curve of diphenhydramine was linear over the range of 4×10⁻⁸ to 1×10⁻⁵ mol l⁻¹. This technique gave satisfactory precision, and relative standard deviations of migration times and chemiluminescence peak intensities were less than 1 and 6%, respectively. The technique was applied to animal studies for determination of diphenhydramine extracted from rabbit plasma and urine samples, and the extraction efficiency were between 92 and 98.5%.     

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (-)-epicatechin and (-)-epigallocatechin gallate solutions, were linear over the range of 0.1-50 microM. The detection limits (signal-to-noise ratio=3) were 0.8 pmol for (-)-epigallocatechin gallate and 1.2 pmol for (-)-epicatechin. The high-performance liquid chromatography-chemiluminescence (HPLC-CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.     

Determination of phenolic compounds using high-performance liquid chromatography with Ce-Tween 20 chemiluminescence detection

A novel method for the simultaneous determination of phenolic compounds such as salicylic acid, resorcinol, phloroglucinol, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and m-nitrophenol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by phenolic compounds of the cerium(IV)-Tween 20 system in a sulfuric acid medium. The separation was carried out with an isocratic elution or with a gradient elution using a mixture of methanol and 1.5% acetic acid. For six phenolic compounds, the detection limits (3σ) were in the range 1.40-5.02 ng/ml and the relative standard deviations (n=11) for the determination of 0.1 μg/ml compounds were in the range 1.9-2.9%. The CL reaction was well compatible with the mobile phase of HPLC, no baseline drift often occurred in HPLC-CL detection was observed with a gradient elution. The method has been successfully applied to the determination of salicylic acid and resorcinol in Dermatitis Clear Tincture and p-hydroxybenzoic acid in apple juices.     

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).     

Tris(2,2'-bipyridine)ruthenium(II) electrogenerated chemiluminescence of alkaloid type drugs with solid phase extraction sample preparation

An electrogenerated chemiluminescence (ECL) method for the determination of pethidine, atropine, homatropine and cocaine is described. The optimum conditions were found to be similar for all of these compounds although the ECL emission intensity for cocaine was an order of magnitude lower than for pethidine due to their different chemical structures. Linear calibrations were obtained for all the compounds at pH 10 in borate buffer (0.05 mol l-1) at 1.3 V. Limits of detection of 6.8 x 10(-8), 2.2 x 10(-7), 3.2 x 10(-7) and 6.5 x 10(-7) mol l-1, respectively, were achieved for pethidine, atropine, homatropine and cocaine in standard solutions. Solid-phase extraction was used to separate the drugs from their matrix and the method was applied to the determination of spiked urine samples. The limits of quantitation for pethidine, atropine, homatropine and cocaine in urine were 1.0 x 10(-6), 2.0 x 10(-6), 2.0 x 10(-6) and 4.0 x 10(-6) mol l-1, respectively, with recoveries of between 90 and 110%.     

Quantification of zolpidem in human plasma by high-performance liquid chromatography with fluorescence detection

A simple, reliable HPLC method with fluorescence detection (excitation 320 and emission 388 nm) was developed and validated for quantitation of zolpidem in human plasma. Following a single-step liquid-liquid extraction, the analyte and internal standard (quinine) were separated using an isocratic mobile phase on a reversed-phase C(18) column. The lower limit of quantitation was 1.8 ng/mL, with a relative standard deviation of less than 5%. A linear dynamic range of 1.8-288 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.7-4.8 and 1.2-2.3%, respectively. The between-batch and within-batch accuracy was 95.3-100.4 and 95.5-102.7%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zolpidem in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Determination of carbamazepine by chemiluminescence detection using chemically prepared tris(2,2'-bipyridine)ruthenium(III) as oxidant.

A new chemiluminescence method for the determination of carbamazepine (CBZ) has been developed. The method is based on the chemiluminescence produced in the reaction of tris(2,2'-bipyridine)ruthenium(III) and CBZ in an acidic medium. The chemiluminescence intensity was enhanced by organic solvents in the reaction system. Under the optimum experimental conditions, the calibration curve was linear over the range 4.0 x 10(-3)-8.6 x 10(-7) mol/L for CBZ. The detection limit (S/N = 3) was 2.5 x 10(-7) mol/L and the relative standard deviation of six replicate measurements was 2.6% for 4.0 x 10(-4) mol/L of CBZ. The possible reaction mechanism were also discussed. The chemiluminescence method was successfully applied to assay the CBZ contents in pharmaceutical tablets.     

Determination of diclofenac in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitative determination of diclofenac potassium in plasma was developed. Naproxen was used as the internal standard. The drug and internal standard were isolated from plasma by extraction with dichloromethane and 2 M hydrochloric acid. Chromatographic separation was performed on a C18 column with methanol-water (68:32, v/v) adjusted to pH 3.2 with phosphoric acid as mobile phase. The oxidation potential for detection was established by constructing a voltammogram for diclofenac. The quantification limit for diclofenac in plasma was 5 ng mL(-1). Linearity of the method was confirmed in the range 5-2000 ng mL(-1), correlation coefficient 0.9998. Within-day relative standard deviations (RSDs) ranged from 0.66 to 14.00% and between-day RSDs from 0.59 to 15.78%. The method was successfully applied for the determination of pharmacokinetic parameters after ingestion of a 50 mg dose of diclofenac. Studies were performed on 18 healthy volunteers of both sexes.     

Quantitation of zopiclone and desmethylzopiclone in human plasma by high-performance liquid chromatography using fluorescence detection

A simple, reliable HPLC method using fluorescence detection (excitation 307 and emission 483 nm) was developed and validated for simultaneous quantitation of zopiclone and its metabolite desmethylzopiclone in human plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (zaleplon) were separated using an isocratic mobile phase on a reversed-phase C18 column. The lower limit of quantitation was 3 ng/mL for zopiclone and 6 ng/mL for desmethylzopiclone with a relative standard deviation of less than 5%. A linear dynamic range of 3-300 ng/mL for zopiclone and of 6-500 ng/mL for desmethylzopiclone was established. This HPLC method was validated with between-batch precision of 1.7-4.2% and 3.2-7.5% for zopiclone and desmethylzopiclone respectively. The between-batch accuracy was 99.4-111.5% and 101.6-104.8% for zopiclone and desmethylzopiclone, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zopiclone and desmethylzopiclone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days' storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Selective determination of acenaphthylene by flow injection analysis with tris(2,2'-bipyridine)ruthenium(II) chemiluminescence detection

A simple, rapid flow injection chemiluminescence (FI-CL) method has been developed for selective determination of acenaphthylene (ACY), based on the CL produced in the reaction of tris(2,2′-bipyridine)ruthenium(III) (Ru(bipy)₃³⁺) and ACY in an acidic buffer solution. Under the optimum experimental conditions, the calibration curve was linear over the range 5.0 × 10⁻³ to 4.0 × 10⁻⁷ mol L⁻¹ for ACY. The detection limit (S/N = 3) was 2.0 × 10⁻⁷ mol L⁻¹ and the relative standard deviation of 10 replicate measurements was 2.3% for 5.0 × 10⁻⁵ mol L⁻¹ of ACY. Selectivity of CL reaction of ACY from other 15 polycyclic aromatic hydrocarbons (PAHs) was investigated by flow injection method. The method was applied to determine the ACY content in soil.     

Pulse injection analysis with chemiluminescence detection: determination of citric acid using tris-(2,2'-bipyridine) ruthenium(II)

A chemiluminescence method for the determination of citric acid was developed. The method is based on the enhancement of citric acid on the chemiluminescence light emission of tris-(2,2'-bipyridine)ruthenium(II). In the presence of tris-(2,2'-bipyridine)ruthenium(II), upon the addition of Ce(IV), resulted in intense light emission. The emission intensity is greatly enhanced by the presence of citric acid. The linear range and detection limit of citric acid are 3.0x10(-8) approximately 6.0x10(-6) mol l(-1) and 3.0x10(-8) mol l(-1), respectively. The precision of the proposed method is determined by analyzing 11 samples containing 1.0x10(-7) mol l(-1) citric acid. The relative standard deviation is 3.0%. The enhanced mechanism of citric acid was studied. The method was evaluated by carrying out an interference study with common ions and compounds, by a recovery study and by analysis of human urine and orange juice. A satisfactory result was obtained.     

Simultaneous quantification of five anthraquinones in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive high-performance liquid chromatographic method with fluorescence detection (excitation 435 and emission 515 nm) was established and validated for quantification of five anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) in rat plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (1,8-dihydroxyanthraquinone) were separated on a reversed-phase C(18) column with water-phosphoric acid-methanol as mobile phase at a flow rate of 1 mL/min. The linear ranges of the calibration curves were 6.5-1300 ng/mL for aloe-emodin, 20-4000 ng/mL for rhein, 40-8000 ng/mL for emodin, 15-3000 ng/mL for chrysophanol and 13-2600 ng/mL for physcion. The lower limit of quantification was 6.5 ng/mL for aloe-emodin, 20 ng/mL for rhein, 40 ng/mL for emodin, 15 ng/mL for chrysophanol and 13 ng/mL for physcion. The mean accuracy was 94.3-105.1% for aloe-emodin, 90.3-108.8% for rhein, 92.6-106.7% for emodin, 95.8-103.8% for chrysophanol and 98.7-101.2% for physcion. The within-batch and between-batch precisions were < or = 5.5% and < or = 13.4%, respectively. This method is suitable for determining the five anthraquinones in plasma simultaneously and thus investigating the pharmacokinetics of anthraquinones from Xiexin decoction in rats.     

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

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of naproxen in human urine by high-performance liquid chromatography with direct electrogenerated chemiluminescence detection

A simple and sensitive liquid chromatographic method coupled with electrogenerated chemiluminescence (ECL) was described for the separation and quantification of naproxen in human urine. The method was based on the ECL of naproxen in basic NaNO₃ solution with a dual-electrode system. Factors affected the ECL emission were investigated. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of naproxen in the range of 4.0 × 10⁻⁸ g mL⁻¹ to 2.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 1.6 × 10⁻⁸ g mL⁻¹ (S/N = 3). Application of the method to the analyses of naproxen in human urine proved feasible.