Determination of nonheme iron using inductively coupled plasma-atomic emission spectrometry

A technique for the rapid and accurate estimation of nonheme iron using inductively coupled plasma-atomic emission spectrometry is described. Yttrium was used as an internal standard. An external calibration method was used. The standards were prepared in a matrix composed of 2.5N HCl in 10% (w/v) trichloroacetic acid. The supernatant and coagulum fractions of liver nonheme iron were separated by the method of Drysdale and Ramsay with minor modification. The data determined by this procedure was compared and found to be agreement with data determined by the method of Hallgren. To evaluate the iron status of rats, hemoglobin and liver nonheme iron were determined. Hemoglobin and all of the nonheme iron fractions of the rats fed an iron-deficient diet were significantly lower than those of the rats fed an iron-sufficient diet. The blood content in the liver was estimated to be 80 microL/g from the blood iron concentration, and the difference between total and nonheme iron concentration in liver.     

Determination of major and trace elements in the liver of Wistar rats by inductively coupled plasma-atomic emission spectrometry and mass spectrometry

Inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) were used to determine age-related changes in the concentrations of constituent elements in the livers of Wistar rats of 1 week to 12 months old. At first, sample preparation and analytical conditions were investigated in order to set up a simple routine procedure for measuring multiple elements simultaneously. Seventeen elements in the standard reference samples of bovine and pork livers as well as rat liver samples could be determined with a reasonable precision and reproducibility. They were P, K, Na, Fe, Mg, Ca, Zn, Rb, Cu, Mn, Mo, Al, Co, Sr, Cs, Pb, and Cd in order of the levels of concentration in the adult rat livers. Of these elements, the five major elements (P, K, Na, Fe, Mg, Ca) were determined with ICP-AES and the others with ICP-MS. Although the number of animals was too small to draw a statistically definite conclusion, it seems that age-related changes in the concentrations of these elements could be categorized into three general patterns: (1) remaining essentially constant throughout the animal ages, as observed for P, K, Na, Mg, Ca, Rb, Sr, Cs, and Pb, (2) increasing with age, as observed for Fe, Mn, Mo, Co, and Cd, and (3) decreasing with age, especially in the early stages of growth, as observed for Cu and Zn.     

Determination of terbutaline enantiomers in biological samples using liquid chromatography with coupled columns

The purpose of this work was to develop and validate a method for the separation and determination of the enantiomers of terbutaline in plasma and intestinal juice. Terbutaline was extracted from plasma and intestinal juice by liquid-solid extraction on small C18 cartridges. The extract was then analyzed by coupled column liquid chromatography with amperometric detection. For chiral separation a beta-cyclodextrin phase was used. The within-day variation (Cv) on spiked plasma samples was in the range 0.8-6.4% at 3.8-33.8 nmol/liter for the (-)-enantiomer, and 2.6-23.0% at 1.3-11.3 nmol/liter for the (+)-enantiomer. The between-day variation on spiked plasma samples was 5.5% at 10.7 nmol/liter and 13.6% at 4.3 nmol/liter for the (-)-and (+)-enantiomers, respectively. The within-day variation for intestinal juice was in the range 0.7-1.5% at 5.6-30.0 mumol/liter for the (+)-enantiomer.     

The determination of phosphorus in Haemophilus influenzae type b conjugate vaccines by inductively coupled plasma-atomic emission spectrometry.

This study describes a method for the determination of phosphorus in lyophilized Haemophilus influenzae type b conjugate vaccines by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The concentration of polysaccharide is directly related to the concentration of phosphorus as measured in the laboratory. Phosphorus is present in the polyribosyl-ribitol phosphate (PRP) group of the Haemophilus influenzae type b conjugate vaccine. The repeating unit of PRP is 3-B-D ribose[1-1]ribitol-5-phosphate. Phosphorus in the final container is measured in microg per dose. The amount of PRP is calculated from this and reported in microg per dose. The Haemophilus influenzae type b conjugate vaccine was analyzed for phosphorus content within the range of 1.34 to 2.02 microg phosphorus per ml. The relative difference of phosphorus concentrations determined by the ICP-AES method from the phosphorus concentrations determined by the traditional colorimetric molybdate method ranged from 2.2 to 10.6%. Phosphorus spike recovery for the vaccine ranged from 93 to 99% (1.93+/-0.13 microg P/ml). The phosphorus determination of NIST SRM 3139 phosphorus spectrometric solution differed by 3.0% from the certified phosphorus value (10.00 mg P/ml).     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Determination of the concentration of complex boronated compounds in biological tissues by inductively coupled plasma atomic emission spectrometry

The application of inductively coupled plasma atomic emission spectrometry (ICP-AES) to the determination of the concentration of complex boron-containing compounds in biological tissue samples is described. Tissue digestion is achieved with perchloric acid and hydrogen peroxide in 1 hr at 75 degrees C. The ICP-AES method gave a linear response for elemental boron concentration in the range 0.05 to 100 ppm and does not require the reduction of the boron to a simple species, such as boric acid. Complete recovery of boron in complex boron cluster compounds was obtained. The procedure has been applied to the determination of the boron content in compounds synthesised for neutron capture therapy and is suitable for use in biodistribution studies of such compounds.     

Multielement determination in small samples of human milk by inductively coupled plasma atomic emission spectrometry

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for routine analysis of small samples of human milk. The concentrations of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), and zinc (Zn) were determined in 203 milk samples from postpartum women at different stages of lactation after stepwise digestion in HNO₃, HCIO₄, and H₂O₂ under heat. Validation of the procedure was achieved using certified reference material of bovine liver (NBS 1577) with mean recoveries of 103.5%. The concentrations of the above elements in milk matrix were comparable with previously reported values. The analytical results from breast milk will provide reference information for mineral studies of Brazilian mothers and breast-fed infants.     

Determination of 235U and 238U in urine samples using sector field inductively coupled plasma mass spectrometry

The high sensitivity of SF-ICP-MS (sector field inductively coupled plasma mass spectrometry) using a torch with the "guard-electrode" (capacitive decoupled plasma) allows the determination of 238U (isotope abundance 99.2%) and 235U (0.8%) and their isotope ratio in human urine samples down to the physiological level of <10 ng/l total uranium. For sample preparation UV photolysis was used. Some quality criteria like for the detection limit, the reproducibility, recovery and the isotope ratio are given. The method can be applied in occupational as well as in environmental medicine because of its outstanding detection power.     

Determination of cobalt in water samples using solidified floating organic drop microextraction coupled with graphite furnace atomic absorption spectrometry

Abstract

A simple, rapid and inexpensive method of the solidified floating organic drop extraction (SFODME) technique coupled with graphite furnace atomic absorption spectrometry (GFAAS) has been developed for the determination of cobalt in water samples. 8-Hydroxyquinoline was used as a complex agent and 1-undecanol was used as the extraction solvent. The factors, including solvent types, solution pH, extractant volume and interfering ions, were investigated and optimised. Under the optimum conditions, the calibration graphs were linear in the range 0.05–10.0 ng mL^-1 cobalt, the limit of detection was 0.02 ng mL^-1, the limit of quantification was 0.05 ng mL^-1 and the relative standard deviation for 10 replicate measurements of 3 ng mL^-1 cobalt was 2.8%. The proposed method was successfully applied for the determination of cobalt in different water samples and the results were satisfactory.     

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP–OES). Application of ICP–OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP–HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS), and ³¹P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.     

Determination of carnitine and acylcarnitines in biological samples by capillary electrophoresis–mass spectrometry

We present fast LC–MS–MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid–liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC–MS–MS analysis.     

Determination of beta-phenylethylamine as its isothiocyanate derivative in biological samples by gas chromatography mass spectrometry

A detailed procedure of a gas chromatographic mass spectrometric assay for beta-phenylethylamine in biological samples, after its reaction with carbon disulphide to form the isothiocyanate derivative, is presented. Our method has advantages over the previous methods with the pentafluoropropionic derivative of beta-phenylethylamine in that the isothiocyanate derivative of beta-phenylethylamine is much more stable than the pentafluoropropionic derivative and that the background in selected ion monitoring is very low. Using the present method, the levels of beta-phenylethylamine in human urine, untreated and pargyline-treated rat brain were found to be 15.3 micrograms 24 h-1, 1.4 and 160 ng g-1 wet weight, respectively.     

Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry

We report an ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to quantify all-trans-retinal in biological samples of limited size (15–35 mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5-fmol lower limit of detection and a linear range from 5 fmol to 1 pmol. This assay revealed that extraocular concentrations of retinal range from approximately 2 to 40 pmol/g in multiple tissues—the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (k_d ? 0.4 nM) for its nuclear receptors (RARα, -β, and -γ), whereas retinal has low (if any) affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2- to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro and, therefore, provides an efficient method to study metabolism of retinol in vivo and in vitro.     

Determination of trichloroethylene in biological samples by headspace solid-phase microextraction gas chromatography/mass spectrometry

A simple, rapid and sensitive method for determination of trichloroethylene (TCE) in rat blood, liver, lung, kidney and brain, using headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS), is presented. A 100-microm polydimethylsiloxane (PDMS) fiber was selected for sampling. The major analytical parameters including extraction and desorption temperature, extraction and desorption time, salt addition, and sample preheating time were optimized for each of the biological matrices to enhance the extraction efficiency and sensitivity of the method. The lower limits of quantitation for TCE in blood and tissues were 0.25ng/ml and 0.75ng/g, respectively. The method showed good linearity over the range of 0.25-100ng TCE/ml in blood and 0.75-300ng TCE/g in tissues, with correlation coefficient (R(2)) values higher than 0.994. The precision and accuracy for intra-day and inter-day measurements were less than 10%. The relative recoveries of TCE respect to deionized water from all matrices were greater than 55%. Stability tests including autosampler temperature and freeze and thaw of specimens were also investigated. This validated method was successfully applied to study the toxicokinetics of TCE following administration of a low oral dose.     

Profiling biological samples using ultra performance liquid chromatography-inductively coupled plasma-mass spectrometry (UPLC-ICP-MS) for the determination of phosphorus and sulfur-containing metabolites

In this preliminary study UPLC-ICP-MS has been utilized to profile a range of different bio-fluids and tissue extracts for sulfur and phosphorus-containing metabolites. Particular attention has been given to the livers, plasma and urine from lean and obese Zucker rats, with a view to differentiating between them based solely on their respective sulfur or phosphorus profiles and/or their total sulfur and phosphorus content. In addition, bile and tumour extracts have been analysed to observe the nature of their profiles. To the best of our knowledge this is the first time ICP-MS has been used in a non-targeted metabonomic study. Results have shown lower limits of quantification for sulfur and phosphorus methods of 0.25 and 0.15 ng on column with CVs of 14.7% and 10.9% respectively. Total phosphorus analysis of the Zucker rat aqueous liver extracts, plasma and urine has shown the pattern of phosphorus concentrations to be statistically significantly different in the lean and obese Zucker rats. Chromatographic separation of the Zucker rat organic liver extracts and plasma allowed further differentiation between the lean and obese rats using their phosphorus profiles alone. In conclusion, this preliminary study has shown the potential of UPLC-ICP-MS to quantitatively discriminate between different species biofluids, fluids and tissues based solely on their phosphorus or sulfur concentrations and/or metabolomes.     

Detection of nanodiamonds in biological samples by EPR spectrometry

In model experiments in vitro, the applicability of the EPR spectrometry method for the detection of modified nanodiamonds (MNDs) in blood and homogenates of mouse organs has been established. A characteristic signal (g = 2.003, ΔH ≈ 10 G) is observed in the samples of biomaterials containing MNDs, the intensity of which increases linearly with the concentration of nanoparticles in the range of 1.6–200 μg MNDs per 1 mL of the sample. The EPR method in biomaterials reveals the presence of intrinsic paramagnetic centers, signals from which are superimposed on the signal from the MNDs. However, the intensity of these signals is small, which makes it possible to register the MNDs using EPR spectrometry with the necessary accuracy. The data obtained open up the prospects of using the EPR method for studies of the interorgan distribution, accumulation, and elimination of MNDs during their intravenous injection into experimental animals.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.