High-Performance Liquid Chromatographic Determination of Quinolizidine Alkaloids in Radix Sophora Flavescens Using Tris(2,2′-Bipyridyl)Ruthenium(II) Electrochemiluminescence

Electrogenerated chemiluminescences (ECLs) of quinolizidine alkaloids including matrine (MT), sophocarpine (SC), and sophoridine (SRI) are studied. The light emission is caused by an electro-oxidation reaction between Ru(bpy)₃²⁺ and the tertiary amino group on the alkaloid compounds. A thin-layer flow cell equipped with a glassy carbon disk electrode (22.1mm²) at the potential of ⁺1.30V (vs. Ag/AgCl) was applied for ECL observation. MT, SC and SRI were separated and quantitatively determined within 25min by an ODS-80 Ts reversed-phase column with a mobile phase containing 80mmolL^–1 NaH₂PO₄–K₂HPO₄ buffer+acetonitrile (7:3)+40mmolL^–1 sodium dodecyl sulfate (pH 6.5). The determination limit at an S/N of 3 ranged from 3×10^–9gmL^–1 for MT, 6×10^–9gmL^–1 for SC and 1×10^–9gmL^–1 for SRI. The recoveries are from 92 to 108%, with repeatability ranging from 1.3 to 4.5% (relative standard deviation). The method was successfully applied to the determination of quinolizidine alkaloids in Sophora flavescens samples.     

Simple extraction of phencyclidine from human body fluids by headspace solid-phase microextraction (SPME)

Summary Phencyclidine (PCP) was found to be extractable by headspace solid-phase microextraction (SPME) from human whole blood and urine. Sample solutions were heated at 90°C in the presence of NaOH and K₂CO₃, and an SPME fiber was exposed in the headspace of a vial for 30 min. Immediately after withdrawal of the fiber, it was analyzed by gas chromatography with surface ionization detection (GC-SID). Recoveries of PCP were approximately 9.3–10.8% and 39.8–47.8% for whole blood and urine samples, respectively. The calibration curve for PCP showed good linearity in the range 2.5–100 ng mL^–1 whole blood and 0.5–100 ng mL^–1 urine. The detection limits were approximately 1.0 ng mL^–1 for whole blood and 0.25 ng mL^–1 for urine.     

On-line preconcentration and determination of cobalt by DPTH-gel chelating microcolumn and flow injection inductively coupled plasma atomic emission spectrometry

This paper reports the development of a new methodology for the determination of cobalt in biological samples by using a flow injection system with loaded DPTH-gel as solid phase to preconcentrate analytes. The procedure is based on the on-line preconcentration of cobalt on a microcolumn of 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide immobilized on silica gel (DPTH-gel). The trapped cobalt is then eluted with 1% tartaric acid and 1% citric acid (7.1 mL) and determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The analytical figures of merit for the determination of cobalt are as follows: detection limit (3S), 8.5 ng mL^–1; precision (RSD), 5.8% for 100 ng mL^–1 of cobalt; enrichment factor, 13 (using 7.3 mL of sample); sampling frequency, 40 h^–1 using a 60-s preconcentration time. For a 120-s preconcentration time (14.6 mL of sample volume) a detection limit of 5.7 ng mL^–1, an RSD under 5% at 50 ng mL^–1, an enrichment factor of 25, and a sampling frequency of 24 h^–1 were reported. The precision and accuracy of the method were checked by analysis of biological certified reference materials.     

Determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol by postcolumn derivatization witho-phthalaldehyde/2-mercapto-ethanol after ion-pair chromatography

Summary A high-performance liquid chromatographic method is discribed for the determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol using Spherisorb ODS II stationary phase and mobile phase 30:70 (v/v) methanol: aqueous 1-octane sulfonic acid. Detection was fluorimetric following postcolumn derivatization with o-phthaladehyde/2-mercaptoethanol. The procedure was applied to the analysis of aqueous solutions and microcrystalline suspensions in liquid paraffin, prepared for investigation of the toxicological profile. The method was validated for selectivity, linearity of detector response, repeatability, limit of detection and quantitation. The HPLC method was selective. The instrumental limit of detection was 0.5 ng per injection (0.05 g mL^–1). The method detection limits were 0.5 g mL^–1 aqueous solution and 5 g mL^–1 liquid paraffin suspension, the quantitation limit 0.05 mg mL^–1 aqueous solution and 1.0 mg mL^–1 liquid paraffin. Linearity was within 0.94–47.1 g mL^–1. Intra-assay accuracy accounted for 99–100% in the range 0.05–226 mg mL^–1 aqueous solution, intra-assay precision for 2% (C.V.). For microcrystalline liquid paraffin suspensions with 1 and 250 mg mL^–1 99 and 109% was found for intra-assay accuracy. Intra-assay precision was 5% (C.V.). Reliable results over a wide concentration range can be obtained. The procedure is considered valid for determination of the analyte in aqueous solution or microcrystalline paraffin oil suspensions.     

2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol as a new analytical reagent for flow-injection spectrophotometric determination of trace vanadium(V)

A flow injection method using 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol-(Nitro-PAPS) as a new chromogenic reagent is presented for sensitive and rapid determination of vanadium. Nitro-PAPS reacts with vanadium(V) in weakly acidic medium to form a water soluble complex of molar absorptivity of 8.0 × 10⁴L mol^–1 cm^–1 at 592 nm (maximum absorption wavelength), which permits the straightforward application of a flow injection system to the sensitive determination of vanadium. Under the optimum conditions established, a linear calibration graph was obtained in the range 1–120 ng mL^–1. The relative standard deviation for 60 ng mL^–1 vanadium was 2.2% (n = 5) and the limit of detection was 1 ng mL^–1. The sample throughput is about 40 h^–1. Most inorganic and organic anions examined did not interfere even at concentrations of 3000–6000 times of vanadium. Interference from cobalt(II), copper(II) and nickel(II) at 200ng mL^–1 levels can be overcome by the addition of N-(dithio-carboxy)sarcosine. The recoveries for each 20 and 10 ng mL^–1 vanadium added to the river water were 98 and 97%, respectively.The authors express their thanks to Miss Miho Suzuki and Miss Hiroyo Yamada for their experimental assistance in part.     

Microdialysis sampling and high-performance liquid chromatography with chemiluminescence detection for in-vivo on-line determination and study of the pharmacokinetics of levodopa in blood

A simple, reliable, and reproducible method for in-vivo on-line separation and determination of levodopa has been based on microdialysis then high-performance liquid chromatography with chemiluminescence detection. The perfusate is perfused at a flow rate of 5 L min^–1. The concentration of levodopa in the dialysate is determined on line with a chemiluminescence system. The dialysate sample volume is approximately 20 L. The response of the system is linearly related to the concentration of levodopa in the range 1×10^–8 to 1×10^–6 g mL^–1 (r²=0.9995) with a detection limit (3) of 3×10^–9 g mL^–1 and sample throughput of 12 h^–1; RSD is 2.8% (n=11). The method has been successfully used to study the pharmacokinetics of levodopa in vivo; the values of the pharmacokinetics parameters C_max, AUC_0–t and T_max were 16.60, 20.92 ng mL^–1, and 90 min, respectively.     

A new and sensitive resonance-scattering method for determination of trace nitrite in water with rhodamine 6G

In the medium HCl–KI–rhodamine dye, NO₂^– reacts with excess I^– to form I₃^– and the I₃^– and rhodamine dye combine to form an association particle which gives three resonance-scattering (RS) peaks at 320 nm, 400 nm, and 595 nm. In systems containing rhodamine 6G (Rh6G), rhodamine B (RhB), rhodamine S (RhS), and butyl rhodamine B (BRhB) the resonance scattering intensity at 400 nm is proportional to nitrite concentrations in the range 2.3–276 ng mL^–1, 9.2–184 ng mL^–1, 9.2–184 ng mL^–1, and 9.2–92 ng mL^–1, respectively. Because of the high sensitivity, wide linear range, and good stability of the Rh6G system, it has been used for determination of nitrite in water samples, with satisfactory results. The spectral results have been used to verify that the formation of (Rh6G·I₃)_n association particles and their interface with the system are main factors that cause the RS enhancement.     

LC Method for the Determination of Multiple Components in a Compound Cold Formulation

An isocratic liquid chromatographic method for determination of acetaminophen (AMP), caffeine (CAF), chlorphenamine maleate (CPM) and guaiacol glyceryl ether (GGE) in a compound cold formulation is described. Separation and quantitation were achieved on a Diamonsil C18 column using a binary mixture of methanol and 1.5% aqueous acetic acid (55: 45, v/v, pH 3.6) as mobile phase delivered at 0.4 mL min^–1. Single wavelength detection was at 220 nm for all four drugs and the run time was < 10 min. The linearity, accuracy and precision of the method were found to be acceptable over the concentration ranges: 16.0–127.8 g mL^–1 for AMP, 6.0–48.2 g mL^–1 for CAF, 5.0–40.0 g mL^–1 for CPM and 10.1–80.6 g mL^–1 for GGE.     

Use of High-Performance Liquid Chromatography–UV and Gas Chromatography–Mass Spectrometry for Determination of the Imidacloprid Content of Honeybees, Pollen, Paper Filters, Grass, and Flowers

Imidacloprid is a new insecticide with a wide range of action. Because honeybees are very sensitive to this substance, two techniques (HPLC–UV and GC–MS) which enable its detection in several matrices of both animal and vegetable origin were used to monitor its possible presence in cultivated land. In the first method quantification of imidacloprid in honeybees was achieved by use of the external standard method; the detection limit was 50 mg kg^–1, the linear range 0.05–1 mg mL^–1, recovery 60–83%, and the imprecision (coefficient of variation) 8.6% for repeatability and 11.8% for reproducibility. Recovery from pollen was 71–98% in the range 0.05–0.5 mg kg^–1. The repeatability was 9.2–13.9%. Imidacloprid can often be found in the environment, not as a simple molecule but as a group of degradation products. The GC–MS method could be used to quantify all these species as oxidation products and to determine the initial quantity of imidacloprid by use of a conversion factor. The liquid chromatographic analysis could be used to detect, in a standard solution, 10 ng mL^–1 derivatized 6-chloronicotinic acid. The linearity was good (R=0.999) over a wide concentration range (10 g mL^–1–10 ng mL^–1). Several samples with different matrices (filter paper placed on an pneumatic corn seed drill, grass, flowers, honeybees, etc.) obtained during the sowing period for imidacloprid-treated corn were analyzed. The quantification limit (LOQ) was 0.005 mg kg^–1 for grass and flowers, 0.002 mg kg^–1 for honeybees, and 0.024 mg kg^–1 for paper filters.     

Trace element determination in seawater by ICP-SFMS coupled with a microflow nebulization/desolvation system

Direct and simultaneous determination of Al, Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Pb, Sb, U, V and Zn in diluted (1:10 v:v) seawater from the Antarctic Ocean and the Venice Lagoon at the ng mL^–1 and pg mL^–1 level has been performed by using an inductively coupled plasma sector field mass spectrometer (ICP-SFMS). Samples were analysed by using a PFA microflow nebulizer coupled with a desolvation system or a PFA microflow nebulizer coupled with a Teflon spray chamber, respectively. Measurements were carried out at low (LR, m/m=300), medium (MR, m/m=3,000) and high (HR, m/m=7,500) resolutions depending on the studied isotope. To avoid contamination, sample pre-treatment was carried out in a clean laboratory equipped with a Class 100 vertical laminar flow hood. Concentration ranges (minimum–maximum in ng mL^–1) found in the Antarctic seawater samples (in depth profiles) were: Ag 0.0004–0.0018, As 0.69–1.32, Cd 0.031–0.096, Co 0.018–0.065, Cr 0.18–0.46, Cu 0.04–1.58, Fe 0.13–1.63, Mn 0.02–0.12, Mo 5.97–12.46, Pb 0.007–0.074, Sb 0.033–0.088, U 0.5–1.9, V 0.6–2.5 and Zn 0.16–0.80. Concentration ranges (min–max in ng mL^–1) found in the Venice Lagoon water samples (temporal profile from a benthic chamber experiment) were: Al 0.24–0.61, Ag 0.007–0.031, As 1.42–2.27, Cd 0.050–0.182, Co 0.440–1.461, Cr 0.15–0.34, Cu 0.81–2.46, Fe 0.25–1.66, Mn 11.6–31.7, Mo 6.50–10.6, Pb 0.047–0.225, Sb 0.240–0.492, U 1.7–3.3, V 1.3–2.8 and Zn 5.20–21.5. The detection limits range between 0.06 pg mL^–1 for Ag and U to 15 pg mL^–1 for Fe. In order to check the accuracy of the analytical procedure, measurements of the trace elements in a certified reference material (coastal Atlantic seawater, CASS-4-NRCC) were compared with the certified values. In addition, the results from the Antarctic and Venice Lagoon samples were compared with those obtained by using different analytical techniques.     

Determination of V(V), Nb(V) and Ta(V) as their 2-(5-nitro-2-pyridylazo)-5-diethylaminophenol complexes by reversed-phase high performance liquid chromatography

A method for the reversed-phase liquid chromatographic separation and determination of V(V), Nb(V) and Ta(V) as 2-(5-nitro-2-pyridylazo)-5 diethylaminophenol (5-NO₂-PADAP) complexes is reported. The metal complexes were eluted in 9 min with a mobile phase of methanol-water (54 : 46, v/v) containing 10 mmol L^–1 acetate buffer (pH 3.0) on an ODS column. The detection limits for V, Nb and Ta were 0.09, 0.13 and 1.41 ng mL^–1, respectively, with S/N=3. The analysis of a reference sample of a mineral is discussed. The results corresponded to the certified values, and recoveries of 98.3–101.4% have been obtained.     

The Validation of an LC-MS Method for the Determination of Risperidone and its Active Metabolite 9-Hydroxyrisperidone in Human Plasma

A simple, specific and sensitive high performance liquid chromatography-mass spectrometry (LC-MS) method for the determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma has been developed and validated. The analytes were prepared through a single-step liquid-liquid extraction (LLE) procedure with the solvent methyl tert-butyl ether and quantitated by MS detection in the positive mode using selected ion monitoring (SIM). Each analytical run was completed within 9 min. Results showed that the LC-MS method enabled to detection of both compounds down to 0.1 ng.mL^–1 (S/N > 3) and the linear range was 0.2–24 ng.mL^–1, with the correlation coefficients above 0.99. At the concentration of 0.2, 0.5, 10 and 20 ng.mL^–1, the inter-day and intra-day RSD were both below 15%. The method has been successfully used to support the routine therapeutic drug monitoring (TDM) and the pharmacokinetics study of risperidone.     

Specific polyclonal-based immunoassays for sulfathiazole

A highly sensitive and specific enzyme-linked immunosorbent assay has been developed for detection of sulfathiazole (STZ, 4-amino-N-thiazol-2-yl-benzenesulfonamide). A set of haptens was synthesized in order to produce polyclonal antibodies against sulfonamides. Two ELISA formats (antibody-coated and conjugate-coated) were also investigated using all the serum/coating conjugate combinations that showed specific recognition. The developed ELISA succeeded in detection of STZ at concentrations as low as 0.03 ng mL^–1 over a measurable range of 0.12–6.71 ng mL^–1. Selectivity studies have demonstrated that other sulfonamides do not interfere significantly (<10%) with analysis of STZ by this immunochemical technique. Analysis of spiked bee honey samples by the developed ELISA method showed recoveries were good. The selectivity and sensitivity (IC₅₀=1.6 ng mL^–1) make it a suitable screening method for determination of low levels of STZ in food samples.     

Simultaneous chiral separation and determination of carvedilol and 5′-hydroxyphenyl carvedilol enantiomers from human urine by high performance liquid chromatography coupled with fluorescent detection

A sensitive and specific high performance liquid chromatography coupled with fluorescent detection (HPLC-FL) and tandem mass spectrometry detection (HPLC-MS/MS) methods for separation and determination of carvedilol (CAR) enantiomers and 5′-hydroxyphenyl carvedilol (5′-HCAR) enantiomers has been developed and validated. The analysed compounds were extracted from human urine by solid phase extraction. Good enantioseparation of the studied enantiomers was achieved on CHIRALCEL® OD-RH column using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile in a gradient elution. The mass spectrometric data were acquired using the multiple reaction monitoring mode by positive electrospray ionisation. The method was validated over the concentration range from 25.0 ng mL^?1 to 200 ng mL^?1 for the analysed compounds. The limit of quantification varied from 14.2 ng mL^?1 to 24.2 ng mL^?1. Both the repeatability and inter-day precisions were below 10.0%, and the accuracy varied from ?13.2% to 3.77%. The extraction recoveries ranged from 79.2% to 108%. The present paper reports the method for the simultaneous determination of CAR enantiomers and their metabolite enantiomers (5′-HCAR) in human urine samples. This newly developed method was successfully used to analyse the aforementioned analytes in human urine samples obtained from patients suffering from cardiovascular disease.      

Determination of dextromethorphan and dextrorphan in urine by capillary zone electrophoresis: Application to the determination of debrisoquin-oxidation metabolic phenotype

Summary A capillary zone electrophoresis method has been developed for the determination of dextromethorphan and its metabolite, dextrorphan, in urine. A linear relationship was observed between the peak area and the concentration of both dextromethorphan and dextrorphan within the range of 490 ng mL^–1 to 500 g mL^–1 with a correlation coefficient of greater than 0.9999. The limit of detection was 80 ng mL^–1 for both compounds. The inter-day coefficients of variation for the concentrations of 2.5 g mL^–1 and 50 g mL^–1 were 6.2% and 4.1% for dextromethorphan, and 5.6% and 2.8% for dextrorphan (n=15). The method could be applied directly to the determination of dextromethorphan and dextrorphan in human urine without any sample pretreatment for the elimination of interfering compounds as is required in published highperformance liquid chromatography and gas chromatography methods. Using dextromethorphan as a probe of the debrisoquin-oxidation metabolic phenotype, the 44 healthy volunteers were phenotyped after oral administration of a 15 mg dose using both this capillary electrophoresis method and a high-performance liquid chromatography assay from the literature. Good agreement was found between the two methods.     

Simultaneous determination of gibberellic acid, indole-3-acetic acid and abscisic acid in wheat extracts by solid-phase extraction and liquid chromatography-electrospray tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of three representative phytohormones in plant samples: gibberellic acid (GA₃), indole-3-acetic acid (IAA) and abscisic acid (ABA). A solid-phase extraction (SPE) pretreatment method was used to concentrate and purify the three phytohormones of different groups from plant samples. The separation was carried out on a C₁₈ reversed-phase column, using methanol/water containing 0.2% formic acid (50:50, v/v) as the isocratic mobile phase at the flow-rate of 1.0 mL min⁻¹, and the three phytohormones were eluted within 7 min. A linear ion trap mass spectrometer equipped with electrospray ionization source was operated in negative ion mode. Selective reaction monitoring (SRM) was employed for quantitative measurement. The SRM transitions monitored were as 345 → 239, 301 for GA₃, 174 → 130 for IAA and 263 → 153, 219 for ABA. Good linearities were found within the ranges of 5–200 μg mL⁻¹ for IAA and 0.005–10 μg mL⁻¹ for ABA and GA₃. Their detection limits based on a signal-to-noise ratio of three were 0.005 μg mL⁻¹, 2.2 μg mL⁻¹ and 0.003 μg mL⁻¹ for GA₃, IAA and ABA, respectively. Good recoveries from 95.5% to 102.4% for the three phytohormones were obtained. The results demonstrated that the SPE-LC–MS/MS method developed is highly effective for analyzing trace amounts of the three phytohormones in plant samples.     

LC-APCI-MS-MS methodology for determination of glybenclamide in human plasma

A liquid chromatographic/atmospheric pressure chemical ionization tandem mass spectrometric method (LC-APCI-MS-MS) for the determination of glybenclamide in human plasma is described. Glypizide, an analogue of glybenclamide, was used as internal standard. The analyte was extracted from plasma with diethyl ether/dichloromethane (70:30 v/v). The chromatography uses C₁₈ and 0.01 mol L^–1 acetic acid/acetonitrile (20:80 v/v) as stationary and mobile phase, respectively. Quantitation was preformed by using multiple reaction monitoring (MRM) of the precursor ion (m/z 494.2368.8) and the related product ion (m/z 446.0347.3) using the internal standard method. The analytical curve was linear in the range 1–300 ng mL^–1, and for a 400-L sample of human plasma, the limit of determination of the method was 1 ng mL^–1. The coefficients of variation of the method for intra-assay (within-run precision) and inter-assay (between-run precision) were less than 10%. The method was shown to be suitable for pharmacokinetic studies.An erratum to this article can be found at     

Flow injection chemiluminescence determination of naftopidil based on potassium permanganate oxidation in the presence of formaldehyde or formic acid

A flow injection method is proposed for the determination of naftopidil based upon the oxidation by potassium permanganate in a sulfuric acid medium and sensitized by formaldehyde and formic acid. The optimum chemical conditions for the chemiluminescence emission were 0.25 mM potassium permanganate and 4.0 M sulfuric acid. Two manifolds were tested and instrumental parameters such as the length of the reactor, injection volume and flow rate were compared. When using the selected manifold in the presence of 0.4 M formaldehyde, naftopidil gives a second-order calibration graph over the concentration range 0.1–40.0 mg L^–1 with a detection limit calculated (as proposed by IUPAC) of 92.5 ng mL^–1 and a standard deviation of 0.12 mg mL^–1 for ten samples of 10.0 mg L^–1 naftopidil. In the presence of 1.15 M formic acid, naftopidil gives a second-order calibration graph over the concentration range 0.05–40.0 mg L^–1 with a detection limit of 14.2 ng mL^–1 and a standard deviation of 0.37 mg mL^–1 for ten samples of 10.0 mg L^–1 naftopidil. In both cases, the determination is free from interferences from common excipients such as sucrose, glucose, lactose, starch and citric acid.     

Liquid Chromatographic Determination of Glutamine in Cerebrospinal Fluid Using 2-Hydroxynaphthaldehyde Derivatizing Reagent

Summary An analytical procedure has been developed for the selective determination of glutamine from cerebrospinal fluid (CSF) using 2-hydroxynaphthaldehyde derivatizing reagent. Arginine and tyramine could also be determined simultaneously. Separation was on a Phenomenex C-18, (150 × 4.6 mm i.d.) column with methanol: water (63:38 v/v) mobile phase at 1mL min^–1 and UV detection at 330nm. Detection limits for glutamine, arginine, and tyramine were 2.8 ng, 17.4 ng and 3.45 ng injection^–1 (5 L), respectively. A large number of amines and amino acids eluted did not affect the determination of glutamine. The analysis of CSF of four patients suffering from hydrocephalus for glutamine indicated concentrations within range 37.4–11.24 g mL^–1 with coefficient of variation 3.0–6.2%.     

Kinetic–Spectrophotometric Determination of Trace Silver(I) Using Its Catalytic Effect on the Oxidation Reaction of Fuchsin by Peroxodisulfate in the Presence of 1,10-Phenanthroline As an Activator

A kinetic spectrophotometric method for the determination of trace amounts of Ag(I) in the range of 2–20 ng mL^–1 is reported. The method is based on the catalytic effect of Ag(I) on the oxidation reaction of fuschin by potassium peroxodisulfate in the presence of 1,10-phenanthroline as an activator at pH = 5. The reaction is followed spectrophotometrically by measuring the change in absorbance (A) at 544 nm using a fixed time method (6 min). The reaction variables were optimized in order to achieve the highest sensitivity. The 3s criterion detection limit was 0.7 ng mL^–1, and the relative standard deviation for ten replicate measurements of 16 ng mL^–1 of Ag(I) was 0.95% (n = 10). The method was successfully applied to the determination of silver in expired black and white photographic films and results showed good agreements with those obtained by atomic absorption spectrometry.