HPLC Determination and Pharmacokinetic Study of Valdecoxib in Human Plasma

A sensitive, simple, and accurate high-performance liquid chromatographic method has been developed for determination of valdecoxib and the internal standard rofecoxib in human plasma. Protein was precipitated from plasma samples by addition of perchloric acid (HClO₄); the drug was then extracted with diethyl ether. Separation was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm i.d., 5 m particles) with ammonium acetate buffer-acetonitrile, 60:40 (v/v), containing 0.1% TEA, pH 6.5, as mobile phase. Detection and quantification were performed by UV-visible detection at 239 nm. Detection and quantification limits were 3 and 5 ng mL^–1, respectively. The linear concentration range for valdecoxib was 5–400 ng mL^–1. The validated RP HPLC method was used for determination of the pharmacokinetic data for the drug in humans.     

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     

Determination of Eupatilin in Human Plasma by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric method (LC-MS-MS) for the determination of eupatilin in human plasma has been developed. Eupatilin and an internal standard; (S)-N-(3-{3-fluoro-4-[6-(1-methyl-1H-tetrazol-5-yl)-pyridine-3-yl]-phenyl}-2-oxo-oxazolidin-5-ylmethyl)-acetamide (DA-7867) were extracted from human plasma by liquid-liquid extractionand analyzed on a phenyl-hexyl column using the mobile phase: acetonitrile-ammonium formate (10 mM, pH 3.0) (60:40, /). Analytes were detected using electrospray ionization-tandem mass spectrometry in multiple-reaction monitoring mode. The calibration curve was linear (r = 0.999) over the concentration range: 1.00–500 ng mL^–1 with a lower limit of quantification of 1.0 ng mL^–1 using a 100 L plasma sample. The precision (CV%) of this assay ranged: 2.4–7.0%, relative error: –7.0 to +2.0%. Recoveries of eupatilin ranged: 64.3–65.0%, with that of DA-7867 (internal standard) being 87.0 ± 5.3%.     

Liquid chromatographic–electrospray mass spectrometric determination of cyclosporin A in human plasma

A rapid, sensitive and selective liquid chromatography–mass spectrometry (LC–MS) assay has been developed for determination of cyclosporin A (CyA) in human plasma; cyclosporin B (CyB) was used as internal standard (IS). The method utilized a combination of a column-switching valve and a reversed-phase symmetry column. The mobile phase was a 25:75 (v/v) mixture of 10% aqueous glacial acetic acid and acetonitrile. Running time per single run was less than 10 min. Sample preparation included C₈ SPE of human plasma spiked with the analyte and internal standard, evaporation of the eluate to dryness at 50°C under N₂ gas, and finally reconstitution in the mobile phase. Detection of cyclosporin A and the IS was performed in selected ion-monitoring mode at m/z 601.3 and 594.4 Da for CyA and IS, respectively. Quantitation was achieved by use of the regression equation of relative peak area of cyclosporin to IS against concentration of cyclosporin. The method was validated according to FDA guideline requirements. The linearity of the assay in the range 5.0–400.0 ng mL^–1 was verified as characterized by the least-squares regression line Y=(0.00268±1.9×10^–4)X+(0.00078±1.8×10^–3), correlation coefficient, r=0.9986±1.1×10^–3 (n=48). Intra and inter-day quality-control measurements in the range 5.0–350.0 ng mL^–1 revealed almost 100% accuracy and 9% CV for precision. The mean absolute recovery of CyA was found to be 84.01±9.9% and the respective relative recovery was 100.3±9.19. The limit of quantitation (LOQ) achieved was 5 ng mL^–1. Eventually, stability testing of the analyte and IS in plasma or stock solution revealed that both chemicals were very stable when stored for long or short periods of time at room temperature or –20°C.     

High-Throughput Analysis of Sofalcone in Human Plasma by Use of Automated 96-Well Protein Precipitation and LC-MS-MS

A sensitive and selective method for the determination of sofalcone in human plasma was established by use of protein precipitation and liquid chromatography-tandem mass spectrometry. Plasma samples were transferred into 96-well plate using an automated sample handling system and spiked with 10 L of 2 g mL^–1 internal standard solution (d₃-sofalcone). 0.5 mL of acetonitrile was added to the 96-well plate and the plasma samples were then vortexed for 30 sec. After centrifugation, the supernatant was transferred into another 96-well plate and completely evaporated at 40 °C under a stream of nitrogen. The dry residue was reconstituted with mobile phase. All sample transfer and protein precipitation was automated through the application of both the PerkinElmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. The limit of quantitation of sofalcone was 2 ng mL^–1 using a sample volume of 0.2 mL for the analysis. The reproducibility of the method was evaluated by analyzing five samples at nine quality control (QC) levels over the nominal concentration range from 2 ng mL^–1 to 1000 ng mL^–1. Validation of the method showed that the assay has good precision and accuracy. Sofalcone and internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 451 and 454, and both gave a corresponding product ion at m/z 315. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of sofalcone in human plasma.     

Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-SiO₂) were synthesized by the sol–gel method. In the presence of Pb(Ac)₂ as a heavy atom perturber the particle can emit the intense and stable room-temperature phosphorescence (RTP) signal of R on a polyamide membrane, with _ex^max/_em^max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO₂ and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624–20.0 pg spot^–1 of human IgG (corresponding to a concentration range of 0.156–50.0 ng mL^–1, sample volume 0.40 L spot^–1). The regression equations of the working curves are I_p=71.27+7.208m_IgG (pg spot^–1) (r=0.9996). Detection limits calculated as 3S_b/k are 0.022 pg spot^–1. Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL^–1 IgG, respectively. This method is sensitive, accurate, and of high precision.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Spectrophotometric determination of 1-naphthylamine in urine with pentacyanonitrosylmanganate (II)

A new Spectrophotometric method is proposed for the determination of 1-naphthylamine (R), based on its reaction with Mn(CN)₅NO^2– to form Mn(CN)₅NH₂R^3– and measurement of the absorbance at 472 nm. In aqueous medium the molar absorptivity of the manganese complex is maximum ( = 8.0 × 10³1 · mole^–1 ·cm^–1) in the pH range 5.0–10.0, the colour develops more rapidly at pH 10.0. The absorptivity is increased by a factor of 3.5 if the complex is extracted as an ion-associate into chloroform. The extraction efficiency is 99.2% for a single-step extraction, and a concentration factor of 5 can also be achieved. Linearity of response extends over the range 0.04–1.4 gmg/ml 1-naphthylamine, the coefficient of variation being 1.4% at the 0.29 g/ml level (n = 6). The detection and determination limits are 0.005 and 0.018 g/ml, respectively. The method is selective enough to allow the determination of 1-naphthylamine in the presence of considerable amounts of other amines, such as aminophenols and phenylenediamines. Results obtained in the determination of 1-naphthylamine in human urine were very satisfactory.     

Determination of sulfur and selected trace elements in metallothionein-like proteins using capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell

The determination of sulfur in biologically relevant samples such as metalloproteins is described. The analytical methodology used is based on robust on-line coupling between capillary electrophoresis (CE) and octopole reaction cell inductively-coupled plasma mass spectrometry (ORC–ICP–MS). Polyatomic ions that form in the plasma and interfere with the determination of S at mass 32 are minimised by addition of xenon to the collision cell. The method has been applied to the separation and simultaneous element-specific detection of sulfur, cadmium, copper, and zinc in commercially available metallothionein preparations (MT) and metallothionein-like proteins (MLP) extracted from liver samples of bream (Abramis brama L.) caught in the river Elbe, Germany. Instrumental detection limits have been calculated according to the German standard procedure DIN 32645 for the determination of sulfur and some simultaneously measured trace elements in aqueous solution. For sulfur detection limits down to 1.3 g L^–1 (³⁴S) and 3.2 g L^–1 (³²S) were derived. For the other trace elements determined simultaneously detection limits ranging from 300 ng L^–1 (⁵⁸Ni) to 500 ng L^–1 (⁶⁶Zn, ⁵⁵Mn) were achieved. For quantification of sulfur and cadmium in a commercially available MT preparation under hyphenated conditions the use of external calibration is suggested. Finally, the need for proper sample-preparation technique will be discussed.     

Ion-pair adsorption film colorimetry — A new simple method for ppb level of aluminum ion in water sample

A new, highly sensitive and simple colorimetric method for trace aluminum(III) with 2,2-dihydroxyazobenzene, H₂L, is described, based on the ion-pair adsorption of the anionic Al chelate, [A1L₂ ^–, with crystal violet cation, CV⁺, on the surface of Polyvinylchloride film plasticized with dioctylphthalate. The blue violet species, CV⁺[A1L₂]^–, is enriched onto the transparent film, leading to a remarkable enhancement of the sensitivity, and the detection limits are 3 ng/ml by spectrophotometry and 5 ng/ml by visual colorimetry, respectively. Using spectrophotometer, a linear calibration curve is obtained over the concentration range of 0 to 50 ng/ml of Al. Further, the color system, consisting of red ([A1L₂]^–), yellow (H₂L), and blue violet (CV⁺), gave clear color changes suitable for visual determination of aluminum with an applicable range of 0 to above 3000 ng/ml. The four different color zones are khaki for 0–5 ng/ml, reddish-brown for 5–200 ng/ml, blue violet for 200 ng/ml-3g/ml, and colorless for more than 3g/ml. The proposed method has been successfully applied for the determination of aluminum in tap waters.     

Determination of Glimepiride in Human Plasma by LC-MS-MS and Comparison of Sample Preparation Methods for Glimepiride

A sensitive and selective method for quantitation of glimepiride in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry. Three different methods for the sample preparation of glimepiride and an internal standard were investigated (liquid-liquid extraction, solid-phase extraction and protein precipitation). Glipizide was used as an internal standard. Compounds were separated on a C₁₈ column with 80% acetonitrile and 20% deionized water (adjusted to pH 3.5 with acetic acid), as mobile phase at a flow rate of 200 L min^–1. By use of multiple reaction monitoring mode in MS-MS with liquid-liquid extraction and solid-phase extraction, glimepiride and glipizide were detected without severe interference from the human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]⁺) at m/z 491 and a corresponding product ion at m/z 352, and the internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 446 and a corresponding product ion at m/z 321. The limit of quantitation was 0.1 ng mL^–1, 0.5 ng mL^–1 and 1.0 ng mL^–1 when using liquid-liquid extraction, solid-phase extraction and protein precipitation, respectively. The validation, reproducibility, stability, and recovery of the different sample preparation methods were comparable and all the methods gave reliable results. The method has been successfully applied to pharmacokinetic study of glimepiride in human plasma.     

Simple plasma treatment for the quantitative determination of haloperidol by HPLC

Summary A method for the quantitative determination of haloperidol with benperidol as internal standard in human plasma is described. The method employs a silica cartridge SEP-PAK^TM for isolation of the drug containing fraction with buffer at pH 12. Final determination is performed by HPLC with UV-detector at 195 nm. The measuring range is between 2–200 ng haloperidol/ml plasma and corresponds to the therapeutic plasma concentration. At a therapeutic level of 25 ng/ml the coefficient of variation was determined to 7.4% (n=7). The lower limit of detection reached 2 ng/ml plasma.

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Einfache Probenaufarbeitung zur quantitativen HPLC-Bestimmung von Haloperidol in Plasma

Zusammenfassung Eine Methode zur quantitativen Bestimmung von Haloperidol, unter Verwendung von Benperidol als internem Standard, in Plasma wird beschrieben. Zur Aufarbeitung wird die Probe über SEP-PAK^TM-Silica-Säulen bei pH 12 extrahiert. Die Quantifizierung erfolgt mittels HPLC und UV-Detektion bei 195 nm. Der lineare Meßbereich liegt zwischen 2–200 ng Haloperidol/ml Plasma und stimmt somit mit dem erwarteten Blutspiegel überein. Bei einer therapeutischen Konzentration von 25 ng/ml betrug der Variationskoeffizient 7,4% (n=7). Das Detektionslimit war 2 ng/ml Plasma.

     

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.     

High-Performance Liquid Chromatographic Assay of Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in Human Plasma using a Solid-Phase Extraction Technique

A selective and sensitive liquid chromatographic method was developed for the determination of zonisamide in small volumes of plasma. Zonisamide and the internal standard methyl 4-hydroxybenzoate were extracted from 0.2 mL of plasma with solid-phase extraction columns and eluted with methanol. Analysis of the extracts was performed on a Symmetry C₁₈ column with ultra-violet spectrophotometric detection. The calibration curve was linear over the concentration range of 0.05–5 g mL^–1 in plasma. Recoveries were reasonable for routine analyses; the limit of quantification was 0.05 g mL^–1 with a signal-to-noise ratio of 5. This method could be useful for the pharmacokinetic study of zonisamide in a limited volume of human plasma and for therapeutic drug monitoring.     

A simple high-performance liquid chromatographic determination of mexiletine in human plasma at therapeutic levels

Summary A method for the quantitative determination of mexiletine in human plasma by high-performance liquid chromatography has been described. The plasma samples are buffered to pH 12 and extracted on Clin-Elut columns with diethylether-ethylacetate (1:1), after addition of the internal standard, the 2,4,6 methyl analogue of mexiletine. The minimum detectable amount of mexiletine is 50 ng in 0.5 ml plasma. Recovery is between 96–114% and the relative standard deviation at 1.5 ml^–1 level of mexiletine is 2.1% Accurate determinations of human plasma levels were performed after oral or intravenous treatment.Part of the work was presented at the 29. Kongreß der Deutschen Gesellschaft für Laboratoriumsmedizin, Hamburg 1985.     

Development of a stir-bar-sorptive extraction–liquid desorption–large-volume injection capillary gas chromatographic–mass spectrometric method for pyrethroid pesticides in water samples

Stir-bar-sorptive extraction followed by liquid desorption and large-volume injection capillary gas chromatography with mass spectrometric detection (SBSE–LD–LVI-GC–MS), had been applied for the determination of ultra-traces of eight pyrethroid pesticides (acrinathrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fenvalerate, and permethrin cis and trans isomers) in water samples. Instrumental calibration for selected-ion monitoring acquisition and conditions that could affect the SBSE–LD efficiency are fully discussed. By performing systematic assays on 30-mL water samples spiked at the 0.10 g L^–1 level it was established that stir-bars coated with 47 L polydimethylsiloxane, an equilibrium time of 60 min (750 rpm), 5% methanol as organic modifier, and acetonitrile as back-extraction solvent, provided the best analytical performance to monitor pyrethroid pesticides in water matrices. Good accuracy (81.8–105.0%) and remarkable reproducibility (<11.7%) were obtained, and the experimental recovery data were in good agreement with the theoretical equilibrium described by octanol–water partition coefficients (log K_O/W), with the exception of acrinathrin for which lower yields were measured. Excellent linear dynamic ranges between 25 and 400 ng L^–1 (r²>0.994), low quantification (3.0–7.5 ng L^–1) and detection (1.0–2.5 ng L^–1) limits were also achieved for the eight pyrethroid pesticides studied. The method was successfully used for analysis of tapwater and groundwater matrices spiked at the 0.10 g L^–1, revealing the suitability of the method for determination of pyrethroid pesticides in real samples. The method was shown be reliable and sensitive and a small volume of sample was required to monitor pyrethroids at ultra-trace levels, in compliance with international regulatory directives on water quality.     

Fluorometric Determination of Nitrogen Dioxide in Atmosphere Sampled by the Liquid Droplet Technique

A new, simple, sensitive and selective fluorometric method for the determination of nitrite has been developed. The reaction of nitrite with hydralazine in acidic medium, heated on a boiling water-bath for 15 min, produced a tetrazolo (5,1-a) phthalazine (Tetra-P). The product formed was measured at _ex = 274 nm and _em = 345 nm. The fluorescence intensity was valid over a nitrite concentration range 0.067–60.3 ng mL^–1, with a detection limit of 0.0091 ng mL^–1. The reproducibility of the proposed method was determined by running a different concentration of nitrite, 13.4, 33.5, and 46.9 ng mL^–1. The % recoveries and the relative standard deviations were found to be 100.6 ± 0.9, 99.9 ± 0.5, and 99.4 ± 1.1%, respectively. The proposed method was applied successfully to the determination of nitrogen dioxide sampled from the atmosphere using the liquid droplet method. The nitrogen dioxide our wind tunnel was controlled by an NO _x analyzer based on a chemiluminescence analyzer detector (CLAD 1000). A linear graph was obtained for the nitrogen dioxide in the wind tunnel vs. NO₂ sampled by the liquid droplet method. The effect of interference substances in the determination showed that cations and anions did not disturb the process. The results obtained were satisfactory when compared with the reference method.     

Selective Extraction and Inductively Coupled Plasma Atomic Emission Spectrophotometric Determination of Thorium Using a Chromogenic Crown Ether

A method for the preconcentration and trace determination of thorium with crown hydroxamic acid (NHDTAHA) is described. The thorium is extracted from the chloroform solution of NHDTAHA at pH 4 which gives a colourless extract, _max = 365 nm; = 9.4·10^–3 l·mol^–1·cm^–1. The extract is directly inserted in the plasma for ICP-AES measurements of thorium. A linear calibration graph was obtained between 15–150 ng·ml^–:1 of thorium. The proposed method has been applied for the determination of thorium in the presence of several diverse ions in monazite sand, rare earth sand and in sea water.     

Rapid spectrofluorimetric determination of lisinopril in pharmaceutical tablets using sequential injection analysis

The present work reports for the first time a simple and rapid method for the spectrofluorimetric determination of lisinopril (LSP) in pharmaceutical formulations using sequential injection analysis (SIA). The method is based on reaction of LSP with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol (borate buffer medium, pH=10.6). The emission of the derivative is monitored at 455 nm upon excitation at 346 nm. The various chemical and physical conditions that affected the reaction were studied. The calibration curve was linear in the range 0.3–10.0 mg L^–1 LSP, at a sampling rate of 60 injections h^–1. Consumption of OPA reagent was significantly reduced compared with conventional flow injection (FI) systems, because only 50 L of OPA was consumed per run. The method was found to be adequately precise (s_r=2% at 5 mg L^–1 LSP, n=10) and the 3 detection limit was 0.1 mg L^–1. The method was successfully applied to the analysis of two pharmaceutical formulations containing LSP. The results obtained were in good agreement with those obtained by use of high-performance liquid chromatography (HPLC), because the mean relative error, e_r, was <1.8%.     

Simultaneous measurements of formaldehyde and ozone in air by annular denuder-HPLC techniques

Summary A denuder sampling method combined with HPLC analysis for the simultaneous determination of formaldehyde and ozone in ambient air is described. It is based on the reactions of CH₂O and O₃ with 2,4-dinitrophenylhydrazine (DNPH) and 4-allyl-2-methoxyphenol (eugenol)_respectively, both acting as coatings of two annular denuders connected in series. Formaldehyde released from the ozonolysis of eugenol is quantitatively collected on a third downstream DNPH-coated denuder. The two DNPH denuders are then extracted and analyzed as hydrazone derivative by HPLC with UV absorbance detection.The stoichiometric factor of the eugenol-ozone reaction was found to be 2.0±0.1 moles of O₃ per mole of CH₂O. The limits of detection are 0.8gm^–3 CH₂O and 3gm^–3 O₃ for 100l air sampled, corresponding to 1-h sampling at 1.7l min^–1.