Ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride as a novel matrix for rapid quantitative and qualitative determination of serum free fatty acids by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

The blood free fatty acids (FFAs), which provide energy to the cell and act as substrates in the synthesis of fats, lipoproteins, liposaccharides, and eicosanoids, involve in a number of important physiological processes. In the present study, matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) with ammonia-treated N-(1-naphthyl) ethylenediamine dihydrochloride (ATNEDC) as a novel MALDI matrix in a negative ion mode was employed to directly quantify serum FFAs. Multiple point internal standard calibration curves between the concentration ratios of individual fatty acids to internal standard (IS, C_17:0) versus their corresponding intensity ratios were constructed for C_14:0, C_16:1, C_16:0, C_18:0, C_18:1, C_18:2, C_18:3, C_20:4, and C_22:6, respectively, in their mixture, with correlation coefficients between 0.991 and 0.999 and limits of detection (LODs) between 0.2 and 5.4 μM, along with the linear dynamic range of more than two orders of magnitude. The results indicate that the multiple point internal standard calibration could reduce the impact of ion suppression and improve quantification accuracy in the MALDI mode. The quantitative results of nine FFAs from 339 serum samples, including 161 healthy controls, 118 patients with hyperglycemia and 60 patients without hyperglycemia show that FFAs levels in hyperglycemic patient sera are significantly higher than those in healthy controls and patients without hyperglycemia, and elevated FFA levels are also associated with increased levels of fasting blood glucose (FBG) in hyperglycemic patient sera. Serum FFAs were identified on the basis of the observed accurate molecular masses and reliable isotope distributions obtained by MALDI-FTICR MS.     

Determination of priority pollutants in aqueous samples by dispersive liquid–liquid microextraction

A dispersive liquid–liquid microextraction (DLLME) method followed by high-performance liquid chromatography–triple quadrupole mass spectrometry has been developed for the simultaneous determination of linear alkylbenzene sulfonates (LAS C₁₀, C₁₁, C₁₂, and C₁₃), nonylphenol (NP), nonylphenol mono- and diethoxylates (NP₁EO and NP₂EO), and di-(2-ethylhexyl)phthalate (DEHP). The applicability of the method has been tested by the determination of the above mentioned organic pollutants in tap water and wastewater. Several parameters affecting DLLME, such as, the type and volume of the extraction and disperser solvents, sample pH, ionic strength and number of extractions, have been evaluated. Methanol (1.5 mL) was selected among the six disperser solvent tested. Dichlorobenzene (50 μL) was selected among the four extraction solvent tested. Enrichment factor achieved was 80. Linear ranges in samples were 0.01–3.42 μg L⁻¹ for LAS C₁₀–₁₃ and NP₂EO, 0.09–5.17 μg L⁻¹ for NP₁EO, 0.17–9.19 μg L⁻¹ for NP and 0.40–17.9 μg L⁻¹ for DEHP. Coefficients of correlation were higher than 0.997. Limits of quantitation in tap water and wastewater were in the ranges 0.009–0.019 μg L⁻¹ for LAS, 0.009–0.091 μg L⁻¹ for NP, NP₁EO and NP₂EO and 0.201–0.224 μg L⁻¹ for DEHP. Extraction recoveries were in the range from 57 to 80%, except for LAS C₁₀ (30–36%). The method was successfully applied to the determination of these pollutants in tap water and effluent wastewater from Seville (South of Spain). The DLLME method developed is fast, easy to perform, requires low solvent volumes and allows the determination of the priority hazardous substances NP and DEHP (Directive 2008/105/EC).     

Evaluation of C18 adsorbent cartridges for sampling and derivatization of primary amines in air

The sampling efficiency of C₁₈ solid-phase extraction cartridges was investigated for methylamine, ethylamine, propylamine, butylamine and pentylamine, in air. Determination of these analytes was based on derivatization with o-phthaldialdehyde-N-acetylcysteine (OPA-NAC) on the solid support and fluorescence detection at λ_excitation=330 nm and λ_emission=440 nm of the eluted derivatives. The calibration model derived from aqueous standards was statistically comparable with the calibration model for air standards. Aqueous amines can be used as standards. The method was useful for calculating short-term exposure limits (STEL). A sampling time of 15 min at 30 ml min⁻¹ was employed. Good recoveries for amines alone and their mixtures were obtained from air and water samples, 98±14 and 97±12% (mean ), respectively. Recovery values were independent of amine concentration. The detection limits varied between 0.16 and 0.52 μg per sample (0.35 and 1.18 mg m⁻³, respectively) and the calibration graphs were linear in the range 0.5-2 μg per sample except for pentylamine, which was 1.5-5 μg per sample. The utility of the method was demonstrated for the estimation of methylamine in two generated air samples.     

Rapid simultaneous determination of amines and organic acids in citrus using high-performance liquid chromatography

Rapid analytical method for the simultaneous separation and determination of amines and organic acids is a vital interest for quality control of citrus and their products. In the present study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid separation of three amines and two organic acids was developed. Chromatographic separation of compounds was achieved using Xbridge C₁₈ column at ambient temperature, with an isocratic mobile phase of 3 mM phosphoric acid at a flow rate of 1.0 mL min⁻¹. A photodiode array (PDA) detector was used to monitor the eluent at 223 nm and 254 nm with a total analysis time of 10 min. Extraction of amines and organic acids from citrus juice was optimized. The method was validated by tests of linearity, recovery, precision and ruggedness. The limit of detection (LOD) and limit of quantification (LOQ) for amines and ascorbic acid were determined to be 5 ng and 9.8 ng, respectively. All calibration curves showed good linearity (R² ≥ 0.9999) within the test ranges. The recoveries of the amines and organic acids ranged between 84% and 117%. The identity of each peak was confirmed by mass spectral (MS) analysis. The developed method was successfully applied to analyze the content of amines and organic acids in six different species and two varieties of citrus. Results indicate that mandarin and Marrs sweet orange contain high level of amines, while pummelo and Rio Red grapefruit had high content of ascorbic acid (137-251 μg mL⁻¹) and citric acid (5-22 mg mL⁻¹). Synephrine was the major amine present in Clementine (114 μg mL⁻¹) and Marrs sweet orange (85 μg mL⁻¹). To the best of our knowledge, this is the first report on simultaneous separation and quantification of amines and organic acids in Marrs sweet orange, Meyer lemon, Nova tangerine, Clementine, Ugli tangelo and Wekiwa tangelo.     

A chemiluminescence sensor for the determination of hydrogen peroxide

A chemiluminescence one-shot sensor for hydrogen peroxide is described. It is prepared by immobilization of cobalt chloride and sodium lauryl sulphate in hydroxyethyl cellulose matrix cast on a microscope cover glass. Luminol, sodium phosphate and the sample are mixed before use and applied on the membrane by a micropipette. The calibration graph is linear in the range 20-1600 μg/L, and the detection limit of the method (3σ) is 9 μg/L. A relative standard deviation of 4.5% was obtained for 100 μg/L H₂O₂ (n = 11). The sensor has been applied successfully to the determination of hydrogen peroxide in rainwater.     

Solid-phase microextraction—Gas chromatography to determine volatile organic sulfur compounds in the air at sewage treatment plants

Solid-phase microextraction (SPME) was applied to the determination of 7 volatile organic sulfur compounds (VOSCs), which were analysed by gas chromatography-mass spectrometry. The compounds studied were ethyl mercaptan (CH₃CH₂SH), dimethyl sulfide ((CH₃)₂S), carbon disulfide (CS₂), propyl mercaptan (C₃H₈S), butyl mercaptan (C₄H₁₀S), dimethyl disulfide ((CH₃)₂S₂) and 1-pentanethiol (C₅H₁₂S). Temperature and time conditions of SPME extraction were optimised and the method was validated, with good linearity in a calibration range between 0.1 and 1000 μg m⁻³. Method detection limits ranged between 0.01 and 0.08 μg m⁻³ and method quantification limits were between 0.10 and 0.25 μg m⁻³, allowing real samples taken from several different areas of a sewage treatment plant to be analysed. Repeatability of the method between samples went from 5.6% for pentanethiol up to 14.2% for carbon disulfide, and concentrations of total target compounds were found between 18 and 529 μg m⁻³, depending on the sampling site.     

Downscaling a multicommuted flow injection analysis system for the photometric determination of iodate in table salt

In this work a downscaled multicommuted flow injection analysis setup for photometric determination is described. The setup consists of a flow system module and a LED based photometer, with a total internal volume of about 170 μL. The system was tested by developing an analytical procedure for the photometric determination of iodate in table salt using N,N-diethyl-henylenediamine (DPD) as the chromogenic reagent. Accuracy was accessed by applying the paired t-test between results obtained using the proposed procedure and a reference method, and no significant difference at the 95% confidence level was observed. Other profitable features, such as a low reagent consumption of 7.3 μg DPD per determination; a linear response ranging from 0.1 up to 3.0 m IO₃⁻, a relative standard deviation of 0.9% (n = 11) for samples containing 0.5 m IO₃⁻, a detection limit of 17 μg L⁻¹ IO₃⁻, a sampling throughput of 117 determination per hour, and a waste generation 600 μL per determination, were also achieved.     

Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M₁ (AFM₁) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC_α), detection capability (CC_β) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5, 1.0, and 1.5-fold the MRL) was 91% and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 μg kg⁻¹ and 0.015 μg kg⁻¹ while the CC_α and CC_β values were 0.058 μg kg⁻¹ and 0.065 μg kg⁻¹, respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.     

On-line high-performance liquid chromatography–ultraviolet–nuclear magnetic resonance method of the markers of nerve agents for verification of the Chemical Weapons Convention

This paper details an on-flow liquid chromatography–ultraviolet–nuclear magnetic resonance (LC–UV–NMR) method for the retrospective detection and identification of alkyl alkylphosphonic acids (AAPAs) and alkylphosphonic acids (APAs), the markers of the toxic nerve agents for verification of the Chemical Weapons Convention (CWC). Initially, the LC–UV–NMR parameters were optimized for benzyl derivatives of the APAs and AAPAs. The optimized parameters include stationary phase C₁₈, mobile phase methanol:water 78:22 (v/v), UV detection at 268 nm and ¹H NMR acquisition conditions. The protocol described herein allowed the detection of analytes through acquisition of high quality NMR spectra from the aqueous solution of the APAs and AAPAs with high concentrations of interfering background chemicals which have been removed by preceding sample preparation. The reported standard deviation for the quantification is related to the UV detector which showed relative standard deviations (RSDs) for quantification within ±1.1%, while lower limit of detection upto 16 μg (in μg absolute) for the NMR detector. Finally the developed LC–UV–NMR method was applied to identify the APAs and AAPAs in real water samples, consequent to solid phase extraction and derivatization. The method is fast (total experiment time ∼2 h), sensitive, rugged and efficient.     

Validation of a liquid chromatography–tandem mass spectrometry method for the identification and quantification of 5-nitroimidazole drugs and their corresponding hydroxy metabolites in lyophilised pork meat

A liquid chromatography–electrospray ionisation tandem mass spectrometry method for the simultaneous detection and quantitation of 5-nitroimidazole veterinary drugs in lyophilised pork meat, the chosen format of a candidate certified reference material, has been developed and validated. Six analytes have been included in the scope of validation, i.e. dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), hydroxymetronidazole (MNZOH), hydroxyipronidazole (IPZOH), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). The analytes were extracted from the sample with ethyl acetate, chromatographically separated on a C₁₈ column, and finally identified and quantified by tandem mass spectrometry in the multiple reaction monitoring mode (MRM) using matrix-matched calibration and ²H₃-labelled analogues of the analytes (except for MNZOH, where [²H₃]MNZ was used). The method was validated in accordance with Commission Decision 2002/657/EC, by determining selectivity, linearity, matrix effect, apparent recovery, repeatability and intermediate precision, decision limits and detection capabilities, robustness of sample preparation method, and stability of extracts. Recovery at 1 μg/kg level was at 100% (estimates in the range of 101–107%) for all analytes, repeatabilities and intermediate precisions at this level were in the range of 4–12% and 2–9%, respectively. Linearity of calibration curves in the working range 0.5–10 μg/kg was confirmed, with r values typically >0.99. Decision limits (CCα) and detection capabilities (CCβ) according to ISO 11843-2 (calibration curve approach) were 0.29–0.44 and 0.36–0.54 μg/kg, respectively. The method reliably identifies and quantifies the selected nitroimidazoles in the reconstituted pork meat in the low and sub-μg/kg range and will be applied in an interlaboratory comparison for determining the mass fraction of the selected nitroimidazoles in the candidate reference material currently developed at IRMM.     

Determination of platinum, palladium and rhodium in biological and environmental samples by low temperature electrothermal vaporization inductively coupled plasma atomic emission spectrometry with diethyldithiocarbamate as chemical modifier

A novel method for inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of trace amounts of Pt(II), Pd(II) and Rh(III), based on gaseous compounds introduction into the plasma as their diethyldithiocarbamate complexes by electrothermal vaporization (ETV), was developed. At the temperature of 1100 °C, the trace amounts of Pt, Pd and Rh were vaporized into plasma. The factors affecting the formation of the chelates and their vaporization behaviors, such as ashing temperature and time, vaporization temperature and time, pH and the concentration of chelating reagents were studied in detail. Under the optimized conditions, the limits of detection (LODs) (3σ) of Pt, Pd and Rh for tested solutions were 5.4, 1.4 and 0.8 ng ml⁻¹, and for actual sample (auto-catalyst NIST SRM 2557) were 0.27, 0.07 and 0.04 μg g⁻¹, respectively. The relative standard deviations (RSDs) for Pt, Pd and Rh were 1.4, 2.6 and 2.4% (C_Pt=0.5 μg ml⁻¹, C_Pd,Rh=0.25 μg ml⁻¹, n=7), respectively. The linear ranges of calibration graphs for Pt, Pd and Rh cover three orders of magnitude. Compared with conventional electrothermal vaporization technique, using the reagent of diethyldithiocarbamate as chemical modifier could not only enhance the analytical sensitivities, but also reduce the vaporization temperature. By combination with a separation/preconcentration step, the proposed method had been successfully applied to the analysis of the artificial seawater, tap water and urine with recoveries ranging from 91 to 106%. The two certified reference material meager platinpalladium ore GBW 07293 and auto-catalyst NIST SRM 2557 was also analyzed for validation, and the determined values obtained were in good agreement with the certified values.     

Determination of selenium in human milk by electrothermal atomic absorption spectrometry and chemical modification

A method was developed for the determination of selenium in human milk using electrothermal atomic absorption spectrometry. The use of chemical modifiers as well as their implications during the pyrolysis step was examined. The chemical modifiers that were studied were Zr, Ir as well as the mixed modifier Zr-Ir. The Ir modifier stabilized selenium at 1000 °C, Zr at 800 °C, while the mixed modifier at 1200 °C. The effect of modifier mass was studied and was found that better results are achieved with addition of 2 μg Zr and 2 μg Ir. The characteristic masses of selenium in the presence of Zr, Ir and the mixed modifier were found to be 73.3, 18.0 and 14.7 pg, respectively, while the corresponding limits of detection were found 2.0, 0.50 and 0.41 μg l⁻¹. Consequently better results were obtained with the mixed modifier. The developed method was applied for the determination of selenium in human milk, which was digested with a HNO₃ + H₂O₂ mixture in a microwave oven. The limit of detection of the method was 1.37 μg l⁻¹, the characteristic mass, m₀, was 48.8 pg and the repeatability was less than 5% as R.S.D.(%). Matrix matched calibration was used. Recoveries were estimated to be 93-105%. The method was applied to breast milk of Greek women (n = 9) and the Se content was found to be in the range 16.7-42.6 μg l⁻¹ with mean value 27.4 ± 5.5 μg l⁻¹.     

Inorganic/organic mesoporous silica as a novel fiber coating of solid-phase microextraction

Mesoporous materials were employed as fast, sensitive and efficient fiber coatings of solid-phase microextraction (SPME) for the first time. Three micrometer as-synthesized C₁₆-MCM-41 particles were immobilized onto stainless steel wire with 100 μm coating thickness. In combination with high performance liquid chromatography (HPLC), extraction efficiency and selectivity of C₁₆-MCM-41 were investigated using aromatic hydrocarbons. Effect of extraction and desorption time, extraction temperature, stirring rate and ionic strength on extraction efficiency were examined. Aanalytical merits of SPME with C₁₆-MCM-41 coating were evaluated. The chromatographic peak area is proportional to the concentration of anthracene in the range 0.5-150 μg l⁻¹. The limit of detection was 0.05 μg l⁻¹ (S/N=3) and the relative standard deviation (R.S.D.) was 0.033%.     

Hollow fiber supported ionic liquid membrane microextraction for determination of sulfonamides in environmental water samples by high-performance liquid chromatography

By using ionic liquid as membrane liquid and tri-n-octylphosphine oxide (TOPO) as additive, hollow fiber supported liquid phase microextraction (HF-LPME) was developed for the determination of five sulfonamides in environmental water samples by high-performance liquid chromatography with ultraviolet detection The extraction solvent and the parameters affecting the extraction enrichment factor such as the type and amount of carrier, pH and volume ratio of donor phase and acceptor phase, extraction time, salt-out effect and matrix effect were optimized. Under the optimal extraction conditions (organic liquid membrane phase: [C₈MIM][PF₆] with 14% TOPO (w/v); donor phase: 4 mL, pH 4.5 KH₂PO₄ with 2 M Na₂SO₄; acceptor phase: 25 μL, pH 13 NaOH; extraction time: 8 h), low detection limits (0.1–0.4 μg/L, RSD ≤ 5%) and good linear range (1–2000 ng/mL, R² ≥ 0.999) were obtained for all the analytes. The presence of humic acid (0–25 mg/L dissolved organic carbon) and bovine serum albumin (0–100 μg/mL) had no significant effect on the extraction efficiency. Good spike recoveries over the range of 82.2–103.2% were obtained when applying the proposed method on five real environmental water samples. These results indicated that this present method was very sensitive and reliable with good repeatabilities and excellent clean-up in water samples. The proposed method confirmed hollow fiber supported ionic liquid membrane based LPME to be robust to monitoring trace levels of sulfadiazine, sulfamerazine, sulfamethazine, sulfadimethoxine and sulfamethoxazole in aqueous samples.     

Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography-mass spectrometry with precolumn derivatization

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the separation and analysis of d- and l-thyroxine was developed using R(−)/S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(−)/S(+)-DBD-PyNCS] as a chiral derivatization reagents. The T₄ derivatives with R(−)-DBD-PyNCS were efficiently separated on a reversed-phase column with water-acetonitrile containing 0.1% formic acid (41:59, v/v) as the eluent and analyzed using ESI-MS with negative selected ion monitoring (SIM) mode. The calibration curves of both the d-T₄ and l-T₄ were linear over the concentration range of 0.13-13 μg/ml. The detection limits (S/N = 3) were 28 ng/ml for d-T₄ and 40 ng/ml for l-T₄, respectively. The relative standard deviations (RSD, n = 5) were less than 3.6% at 1.3 μg/ml for both T₄ enantiomers. The proposed method was applied to the determination of l-T₄ enantiomer in a pharmaceutical formulation.     

FT-IR measurement of tagitinin C after solvent extraction from Tithonia diversifolia

Tagitinin C, an antiplasmodial compound, identified as one major compound of the subtropical medicinal plant, Tithonia diversifolia, was determined by FT-IR spectroscopy method. The crude ether extracts from aerial parts of the plant were evaporated to dryness and re-dissolved in tetrachloroethylene (C₂Cl₄) before analysis.The magnitude of the absorbance of the very specific CO stretching vibration (ν_CO) at 1664.8 cm⁻¹ was exploited in order to quantify tagitinin C. The determination coefficient (r²) of the calibration scale was 0.9994, the detection limit was lower than 3 μg ml⁻¹ and the quantification limit was lower than 10 μg ml⁻¹.Recovery values from 100.5 to 101.7% were found for spiked concentration levels from 19.91 to 89.95 μg ml⁻¹. The main characteristics of the curves obtained from the calibration standards and from the standard addition technique were not statistically different (Student t-test) suggesting that matrix effects were negligible.The results obtained for the determination of tagitinin C in the crude ether extract from aerial parts of T. diversifolia by LC and FT-IR spectroscopic method agreed well: 0.76±0.02 and 0.773±0.009, of tagitinin C in dried plant respectively.     

In-tube silylation in combination with thermal desorption gas chromatography-mass spectrometry for the determination of hydroxy polycyclic aromatic hydrocarbons in water

For the determination of hydroxy polycyclic aromatic hydrocarbons (OH-PAHs), a simple and sensitive method based on the silylation of OH-PAHs using N,O-bis(trimethylsilyl)trifluoro acetamide (BSTFA) in combination with thermal desorption gas chromatography-mass spectrometry (TD-GC-MS) is described. This method was performed by way of direct silylation in a TD unit (in-tube silylation) coupled to a GC inlet. Both a good detection limit (4.1-1200 pg L⁻¹, S/N = 3) and higher precision (relative standard deviation < 4% on average) were achieved for 21 OH-PAHs studied using the full scan mode (m/z = 40-550). These good results were due to the highly efficient derivatization of the OH-PAHs, which was attributed to not only the moisture-free environment and programmable heating in the TD tube for the in-tube silylation, but also to the constant vapor generation of BSTFA using a capillary introduction method. Although recoveries of 21 OH-PAHs from the spiked 3% NaCl solution ranged between 9 and 304%, those of 11 OH-PAHs fell between 70 and 130% (R.S.D. < 11%). Thus, the present method was applied to a seawater sample collected from an industrial port, and nine OH-PAHs including 1- and 2-OH-fluorenone and 1,8- and 2,6-OH-anthraquinone were determined at concentrations of 0.49-5.8 ng L⁻¹. Along with these OH-PAHs, significant amounts of several long chain fatty acids (C₁₂, C₁₆, C₁₈, C₂₀ and C₂₂) and bisphenol A were also identified in the seawater sample using reference data in a library of mass spectra (match factor: >80%).     

The use of experimental design in the development of an HPLC-ECD method for the analysis of captopril

An accurate, sensitive and specific high performance liquid chromatography-electrochemical detection (HPLC-ECD) method that was developed and validated for captopril (CPT) is presented. Separation was achieved using a Phenomenex^® Luna 5 μm (C₁₈) column and a mobile phase comprised of phosphate buffer (adjusted to pH 3.0): acetonitrile in a ratio of 70:30 (v/v). Detection was accomplished using a full scan multi channel ESA Coulometric detector in the “oxidative-screen” mode with the upstream electrode (E₁) set at +600 mV and the downstream (analytical) electrode (E₂) set at +950 mV, while the potential of the guard cell was maintained at +1050 mV. The detector gain was set at 300. Experimental design using central composite design (CCD) was used to facilitate method development. Mobile phase pH, molarity and concentration of acetonitrile (ACN) were considered the critical factors to be studied to establish the retention time of CPT and cyclizine (CYC) that was used as the internal standard. Twenty experiments including centre points were undertaken and a quadratic model was derived for the retention time for CPT using the experimental data. The method was validated for linearity, accuracy, precision, limits of quantitation and detection, as per the ICH guidelines. The system was found to produce sharp and well-resolved peaks for CPT and CYC with retention times of 3.08 and 7.56 min, respectively. Linear regression analysis for the calibration curve showed a good linear relationship with a regression coefficient of 0.978 in the concentration range of 2-70 μg/mL. The linear regression equation was y = 0.0131x + 0.0275. The limits of detection (LOQ) and quantitation (LOD) were found to be 2.27 and 0.6 μg/mL, respectively. The method was used to analyze CPT in tablets. The wide range for linearity, accuracy, sensitivity, short retention time and composition of the mobile phase indicated that this method is better for the quantification of CPT than the pharmacopoeial methods.     

A sequential injection fluorometric procedure for rapid determination of total protein in human serum

A sensitive procedure for the quantification of total protein bovine serum albumen (BSA) in human serum was presented with sequential injection sampling and fluorometric detection. A few microliters of sample and fluorescamine solutions were aspirated into the holding coil to facilitate the reaction of protein with fluorescamine by giving rise to a blue-green-fluorescent derivative. The derivative was afterwards excited by a 400 nm radiation from a UV radiator, and the emitted fluorescence was monitored at the wavelength of 470 nm. By loading 5.0 μl of sample and 4.0 μl of fluorescamine solution 0.075% (m/v), a linear calibration graph was obtained within 0.3-12.5 μg ml⁻¹, and a detection limit (3σ) of 0.1 μg ml⁻¹ was achieved, along with a sampling frequency of 40 h⁻¹ and a R.S.D. value of 2.1% at the 5.0 μg ml⁻¹ levels. Protein contents in human serums were analyzed by using the present procedure, and reasonable agreements were obtained with those obtained by a documented spectrophotometric (Biuret) method.     

Calibration free laser-induced breakdown spectroscopy of oxide materials

The quantitative determination of oxide concentration by laser-induced breakdown spectroscopy is relevant in various fields of applications (e.g.: analysis of ores, concrete, slag). Calibration free laser-induced breakdown spectroscopy and the multivariate calibration are among the methods employed for quantitative concentration analysis of complex materials. We measured the intensity of neutral and ionized atomic emission lines of oxide materials by laser-induced breakdown spectroscopy and we modified the calibration free laser-induced breakdown spectroscopy method to increase the accuracy. The concentration of oxides was obtained by using stoichiometric relations. Sample materials were prepared from oxide powder (Fe₂O₃, MgO, CaO) by mixing and pressing. The concentration was 9.8–33.3 wt.% Fe₂O₃, 7.6–33.3 wt.% MgO and 33.3–81.2 wt.% CaO for different samples. Nd:YAG laser (wavelength 1064 nm, pulse duration ≈ 6 ns) ablation was performed in air. The laser-induced plasma emission was measured by an Echelle spectrometer equipped with a sensitivity calibrated ICCD camera. The numerical calibration free laser-induced breakdown spectroscopy algorithm included the fast deconvolution of instrumental function, and the correction of self-absorption effects. The oxide concentration C_CF calculated from calibration free laser-induced breakdown spectroscopy results and the nominal concentration C_N were very close for all samples investigated. The relative error in concentration, |C_CF–C_N|/C_N, was < 10%, < 20%, and < 5% for Fe₂O₃, MgO, and CaO, respectively. The results indicate that this method can be employed for the analysis of major elements in multi-component technical materials.