Determination of L-Dopa and L-Dopamine in Aqueous Solutions Using In-Loop SPME Coupled with LC

A new in-loop solid-phase microextraction (in-loop-SPME) technique, based on an aluminum capillary tube coupled to HPLC, is described for on-line isolation, concentration, and analysis of analytes from aqueous samples. L-Dopa and L-dopamine, in aqueous solutions, were selected as model compounds. The main conditions affecting extraction of the analytes from aqueous samples, desorption, injection, and chromatographic separation were investigated. The method is simple and reproducible. Using the proposed method, reliable determination of L-dopa and L-dopamine at parts-per-billion concentrations was achieved. The calibration plots were linear in the range of 2.5–1500 ng mL⁻¹ with correlation coefficients of 0.999 and 0.998 for L-dopa and L-dopamine, respectively. The detection limits were 0.5–1 ng mL⁻¹ with a relative standard deviation less than 4.1%. Concentration factors more than 100-fold were obtained for these compounds.     

Gas Chromatographic–Mass Spectrometric Method for Quantitation of Trimethylsilyl Derivatives of Nerve Agent Degradation Products in Human Plasma, Using Strong Anion–Exchange Solid-Phase Extraction

For unequivocal proof of the use of nerve agents such as sarin, soman, cyclohexylsarin, VX, and Russian VX, a simple and accurate method, gas chromatography–mass spectrometry (GC–MS) after trimethylsilyl derivatization, was explored for simultaneous determination of the corresponding alkyl methylphosphonic acids (AMPAs) and of methylphosphonic acid (MPA) in human plasma. GC–MS analysis was performed after solid-phase extraction, with a strong anion-exchange cartridge, from plasma samples previously deproteinized with mercuric acetate, and then derivatization with bis(trimethylsilyl)trifluoroacetamide containing 5% trimethylchlorosilane. All five AMPA derivatives and the MPA derivative were separated to baseline within 11 minutes without interference. Linear calibration plots were obtained over concentrations ranging from 50 ng mL⁻¹ to 5 µg mL⁻¹. The relative standard deviation of recoveries ranged from 1.9 to 9.7% and detection limits were 22 ng mL⁻¹ or below.Revised: 3 and 23 May 2005     

Kinetic Determination of Mercury(II) at Trace Level from Its Catalytic Effect on a Ligand Substitution Process

A catalytic kinetic method (CKM) is presented for the determination of mercury(II) based on its catalytic effect on the rate of substitution of N-methylpyrazinium ion (Mpz⁺) onto hexacyanoferrate(II). The progress of the reaction was monitored spectrophotometrically at 655 nm by registering the increase in absorbance of the product [Fe(CN)₅(Mpz]²⁻ under the reaction conditions: 5 × 10⁻³ mol L⁻¹ [Fe(CN)₆]⁴⁻), 5 × 10⁻⁵ mol L⁻¹ [Mpz⁺], T = 25.0 ± 0.1°C, pH 5.00 ± 0.02 and ionic strength, I = 0.1 mol L⁻¹ (KNO₃). Quantitative rate data at specified experimental conditions showed a linear dependence of the absorbance after fixed time A _t on the concentration of mercury(II) catalyst in the range 20.06–702.1 ng mL⁻¹. The maximum relative standard deviations and percentage errors for the determination of mercury(II) in the range of 20.06–200.6 ng mL⁻¹ were calculated to be 1.7 and 2.7% respectively. The detection limit was found to be 7.2 ng mL⁻¹ of mercury(II). Accuracy (expressed in terms of recoveries) was in the range of 98–103%. Figures of merit and interference due to many cations and anions was investigated and discussed. The applicability of the method was demonstrated by determining the mercury(II) in different synthetic samples and confirming the results using atomic absorption spectrophotometry. The proposed method allowed determination of mercury(II) in the range 20.06–702.1 ng mL⁻¹ with very good selectivity and an output of 30 samples h⁻¹.__________From Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 654–661.Original English Text Copyright © 2005 by Surendra Prasad.This article was submitted by the author in English.     

Determination of highly polar catecholamine with liquid chromatography–tandem mass spectrometry using weak cation-exchange stationary phase to increase retention time

This paper reports method development and validation work to determine highly polar bases, catecholamine compounds, using weak cation-exchange liquid chromatography of low ionic strength mobile phase with electrospray tandem mass spectrometry. Catecholamine compounds, such as epinephrine and norepinephrine, well-known biomarkers to diagnose hypertension disease, spiked in saline solutions are purified with solid phase extraction (SPE) using alumina powders. The extracts are loaded into a weak cation-exchange liquid chromatographic column via an injection loop and analyzed with electrospray-mass spectrometer. The de-salted extracts contain only small amounts of electrolytes to avoid saturating weak cation-exchange sites in the stationary phase with sodium ions. Using carefully selected mobile-phase solvents with optimized compositions (acetonitrile and water 10:90 v/v) and with dilute acid additives (acetic acid 0.1% v/v), we are able to elute catecholamine at sufficient retention times to avoid co-elution of saline matrix residues while maintaining adequate electrospray ionization efficiency of these compounds. Using epinephrine and norepinephrine standards, these methods are validated at the range of 5 to 500 ng mL^− 1. The measurement accuracy and precision of using epinephrine standards are within 12% and 5.3% respectively, whereas the accuracy and precision are within 6.0% and 4.2% respectively using epinephrine standards.The detection limits of epinephrine and norepinephrine are 0.10 ng mL^− 1 and 0.45 ng mL^− 1 respectively. The recovery percentages of our solid phase extraction methods using alumina powders are higher than 74%. When the validated calibration curves are used to determine epinephrine and norepinephrine in rat blood dialysates, the determination errors of accuracy and precision are both within 4%, while the determination errors are within 3% in rat blood plasma samples.     

HPLC Method for Determination of Rosiglitazone in Plasma

A fast and sensitive high performance liquid chromatography method for quantitative determination of rosiglitazone in human plasma has been developed. The extraction from plasma was performed using solid-phase extraction (SPE) on C4 silica (100 mg) disposable extraction cartridges (DEC). The separation of rosiglitazone and two metabolites was achieved on a Phenomenex® Synergi 4 µm MAX-RP (150 × 4.6 mm) column, protected by a guard column. The mobile phase was 0.01 M ammonium acetate, pH 7.0 - acetonitrile (65:35, v/v). (3S)-3-OH-quinidine was used as internal standard. The analytes were detected using fluorescence detection. The method was validated. The limit of quantitation was 1 ng mL⁻¹ and the detection limit was 0.25 ng mL⁻¹ for rosiglitazone in human plasma. The recovery was 90% for rosiglitazone. Linearity was observed over a range of 1-1000 ng mL⁻¹ (r²=0.9959). The intra- and inter-day precision (C.V.) did not exceed 8.7 %. Applicability of the method was demonstrated by a clinical pharmacokinetic study. A healthy volunteer received in two separate phases 4 mg and 8 mg rosiglitazone maleate as a single oral dose. Plasma concentrations were measured for 24 h in both phases.     

A sensitive and specific HPLC-MS method for the determination of sophoridine,sophocarpine and matrine in rabbit plasma

A sensitive and specific method was developed for the determination of sophoridine (SRI), sophocarpine (SC) and matrine (MT) in rabbit plasma by HPLC-MS. After an administration of Kuhuang by injection, blood samples were collected and extracted with methanol. The extract solutions were analysed by HPLC-MS method. The separation was performed on a ZORBAX Extend-C18 column using methanol/water/diethylamine (50:50:0.07, v/v/v) as mobile phase. The quinolizidine alkaloids were detected by using mass spectrometry in the SIM mode. There was a good linear relationship between peak area and concentration of analytes over the concentration range of 13.2–995.0 ng mL^–1 for SRI, 7.0–530.0 ng mL^–1 for SC and 8.8–655.0 ng mL^–1 for MT, respectively. The absolute recovery of this method was more than 57% for SRI, 87% for SC and 91% for MT. The accuracy of assay was more than 90%. The limits of detection (LODs) were 6.8 ng mL^–1 for SRI, 3.5 ng mL^–1 for SC and 4.2 ng mL^–1 for MT, respectively. The limits of quantitation (LOQs) were 13.2 ng mL^–1 for SRI, 7.0 ng mL^–1 for SC and 8.8 ng mL^–1 for MT, respectively. The intra-day and inter-day coefficients of variation (RSDs) were less than 10.1, 6.3 and 5.8% for SRI, SC and MT, respectively. The developed method was applied to determine the concentration–time profiles of SRI, SC and MT in rabbit plasma after injection of Kuhuang.     

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.     

Determination of photoirradiated high polar benzoylureas in tomato by HPLC with luminol chemiluminescence detection

This study reports the first analytical application of luminol chemiluminescence reaction for the sensitive detection of two benzoylurea insecticides (diflubenzuron and triflumuron). Off-line experiments demonstrated that previously irradiated traces of these benzoylurea insecticides largely enhanced the chemiluminescence emission yielded from the oxidation of luminol in methanol:water mixtures, by potassium permanganate in alkaline medium, the enhancement being proportional to the concentration of both pesticides. The two benzoylureas were determined in tomato samples by coupling liquid chromatography with post-column photoderivatization and detection based on this chemiluminescence reaction. Tomato samples were extracted using the QuEChERS method based on extraction with acetonitrile and dispersive solid-phase clean-up using primary and secondary amine (PSA). Interferences due to matrix effect were overcome by using matrix-matched standards. The optimised method was validated with respect to linearity, limits of detection and quantification, precision and accuracy. Under the optimised conditions, calibrations graphs were linear between 0.05 and 0.50 μg mL⁻¹ for diflubenzuron and between 0.10 and 1.00 μg mL⁻¹ for triflumuron. Method detection limits were 0.0025 and 0.0131 μg mL⁻¹ (equivalent to 0.0005 and 0.0026 mg kg⁻¹) and quantification limits were 0.05 and 0.10 μg mL⁻¹ (equivalent to 0.01 and 0.02 mg kg⁻¹) for diflubenzuron and triflumuron, respectively. In both cases, quantification limits were lower than the maximum residue levels (MRLs) established by the European legislation. The relative standard deviation of intra-day precision was below 10% and recoveries were between 79.7% and 94.2% for both pesticides.     

Determination of Diazepam in Human Plasma by Solid-Phase Microextraction and Capillary Gas Chromatography-Mass Spectrometry

A simple, sensitive, and reproducible solid-phase microextraction and capillary gas chromatography-mass spectrometry (SPME-GC-MS) method for determination of diazepam in human plasma is described. The optimum conditions for the SPME procedure were as following: direct extraction mode with a polydimethylsiloxane (PDMS) fiber (100 μm film thickness), 250 μL of sample plasma matrix modified with a solution containing sodium chloride (10% weight by volume) and 4.25 mL of a phosphate buffer solution (0.1mol L⁻¹, pH 6.9), extraction temperature 55°C under a magnetic stirring rate of 2500 rpm for 30 min, followed by the drug thermal desorption (250°C) in a GC injection port for 10 min. The limit of quantification of diazepam in plasma was 10.0 ng mL⁻¹, with a coefficient of variation lower than 14.0% and linearity from 10.0 to 1000.0 ng mL⁻¹, which allows diazepam analyses from sub to therapeutic levels.     

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.     

Comparative Evaluation of CE and HPLC for Determination of Cotinine in Human Urine

Two rapid and popular methods—capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) have been compared for analysis of cotinine in human urine. Cotinine was analyzed in less than 7 min, with detection limits of 5 and 3.2 ng mL⁻¹ for CE and HPLC, respectively. The performance of the methods was evaluated in terms of sensitivity, specificity, precision, accuracy, and limits of detection and quantification. Calibration plots were linear in the range 50–4,000 ng mL⁻¹, at least, and mean recoveries were satisfactory for both techniques. The methods were successfully used for quantification of cotinine in urine.     

Determination of ultra trace amounts of protein by 4-chlorosulfo-(2′-hyaroxylphenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] as the fluorescence spectral probe in AOT microemulsion

Experiments indicated that protein can enhance the fluorescence of the 4-chlorosulfo-(2′-hydroxylophenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] in the presence of bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) microemulsion. Based on this, a sensitive and reproducible fluorometric method for the determination of micro amount protein was developed. The calibration curves of four proteins were given. Under the optimum experimental conditions, the enhanced fluorescence intensity of the system was in proportional to the concentration of protein in the range of 0.1–11 μg mL⁻¹ for bovine serum albumin (BSA), 1.0–10 μg mL⁻¹ for human serum albumin (HSA), 1.0–50 μg mL⁻¹ for ovalbumin (Ova) and 2.5–18 μg mL⁻¹ for γ-globulins (γ-G). Their detection limits were 0.070, 0.071, 0.33 and 0.22 μg mL⁻¹, respectively. The ClSARP-Ti(IV) complex as a spectral probe can be used to the determination of protein in milk powder and oatmeal yielding with satisfactory results. Therefore, the proposed method is one of the most sensitive methods available. In addition, the interaction mechanism of this system is studied by multi-techniques.     

Analysis of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-methylsuccinimide in plasma

Summary A method for the determination of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in plasma was developed. 5-HNMP and 2-HMSI are metabolites to the widely used organic solvent N-methyl-2pyrrolidone (NMP). The 5-HNMP and 2-HMSI were purified from plasma by C8 solid phase extraction, derivatised by bistrimethylsilyl trifluoroacetamid, and analysed by gas chromatography with mass spectrometric detection. For 5-HNMP, the precision was 2–7 % (120 and 780 ng mL⁻¹) and the detection limit was 6 ng mL⁻¹ (m/z 98). For 2-HMSI, the precision was 2–9 % (160 and 1000 ng mL⁻¹) and the detection limit was 4 ng mL⁻¹ (m/z 144). The method is applicable for analysis of plasma samples from workers exposed to NMP.     

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.     

Simultaneous Determination of Cefepime and the Quinolones Garenoxacin, Moxifloxacin and Levofloxacin in Human Urine by HPLC-UV

A liquid chromatographic method with a C₁₈ column and acetonitrile/0.1 M phosphoric acid/ sodium hydroxide buffer (pH 3.0)/0.01 M n-octylamine (pH 3.0) as mobile phase in gradient mode has been developed and optimised for the simultaneous determination of the cephalosporin cefepime and the quinolones garenoxacin, levofloxacin and moxifloxacin. Identification and quantification was carried out with a diode-array UV detector, with working wavelengths of 256 nm for cefepime, 292 nm for levofloxacin, 294 nm for moxifloxacin and 282 nm for garenoxacin. The mobile flow-rate and sample volume injected were 1 mL min⁻¹ and 20 µL, respectively. The retention times and detection limits for each antibiotic were 4.9 min and 1.9 µg mL⁻¹ for cefepime, 7.5 min and 2.2 µg mL⁻¹ for levofloxacin, 8.9 min and 2.7 µg mL⁻¹ for moxifloxacin and 10.7 min and 1.8 µg mL⁻¹ for garenoxacin, respectively. The method was applied to the determination of the four molecules in spiked samples of human urine.     

Resonance Rayleigh scattering and resonance non-linear scattering method for the determination of aminoglycoside antibiotics with water solubility CdS quantum dots as probe

In pH 6.6 Britton–Robinson buffer medium, the CdS quantum dots capped by thioglycolic acid could react with aminoglycoside (AGs) antibiotics such as neomycin sulfate (NEO) and streptomycin sulfate (STP) to form the large aggregates by virtue of electrostatic attraction and the hydrophobic force, which resulted in a great enhancement of resonance Rayleigh scattering (RRS) and resonance non-linear scattering such as second-order scattering (SOS) and frequency doubling scattering (FDS). The maximum scattering peak was located at 310 nm for RRS, 568 nm for SOS and 390 nm for FDS, respectively. The enhancements of scattering intensity (ΔI) were directly proportional to the concentration of AGs in a certain ranges. A new method for the determination of trace NEO and STP using CdS quantum dots probe was developed. The detection limits (3σ) were 1.7 ng mL⁻¹ (NEO) and 4.4 ng mL⁻¹ (STP) by RRS method, were 5.2 ng mL⁻¹ (NEO) and 20.9 ng mL⁻¹ (STP) by SOS method and were 4.4 ng mL⁻¹ (NEO) and 25.7 ng mL⁻¹ (STP) by FDS method, respectively. The sensitivity of RRS method was the highest. The optimum conditions and influence factors were investigated. In addition, the reaction mechanism was discussed.     

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography–electrospray ionisation–mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25–1000 ng mL^–1 with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL^–1 for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.     

Micelle-Enhanced Spectrofluorimetric Method for the Determination of Antibacterial Trovafloxacin in Human Urine and Serum

A spectrofluorimetric method for the determination of trace amounts of the antibacterial trovafloxacin has been developed based on its native fluorescence in a micellar solution of sodium dodecyl sulfate (SDS). The wavelengths of excitation and emission were 270 nm and 410 nm, respectively. The optimised method allows the determination of 3.0–40.0 ng mL⁻¹ of trovafloxacin in 8 mM SDS solution and 0.1 M acetic acid-sodium acetate buffer solution (pH 5.5), with a relative standard deviation of 1.5% (for a level of 12.0 ng mL⁻¹) and a detection limit of 0.8 ng mL⁻¹. The method was applied to the determination of trovafloxacin in human urine and serum samples. It was validated using HPLC as a reference method. Recovery levels of the method reached 100% in all cases.     

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.     

UV–visible spectrum of the phenyl radical and kinetics of its reaction with NO in the gas phase

Pulse radiolysis transient UV–visible absorption spectroscopy was used to study the UV–visible absorption spectrum (225–575 nm) of the phenyl radical, C₆H₅(), and kinetics of its reaction with NO. Phenyl radicals have a strong broad featureless absorption in the region of 225–340 nm. In the presence of NO phenyl radicals are converted into nitrosobenzene. The phenyl radical spectrum was measured relative to that of nitrosobenzene. Based upon σ(C₆H₅NO)_{270 nm}=3.82×10⁻¹⁷ cm² molecule⁻¹ we derive an absorption cross-section for phenyl radicals at 250 nm, σ(C₆H₅())_{250 nm}=(2.75±0.58)×10⁻¹⁷ cm² molecule⁻¹. At 295 K in 200–1000 mbar of Ar diluent k(C₆H₅()+NO)=(2.09±0.15)×10⁻¹¹ cm³ molecule⁻¹ s⁻¹.