Determination of trace cadmium in saliva samples using spray assisted droplet formation-liquid phase microextraction prior to the measurement by slotted quartz tube-flame atomic absorption spectrophotometry

BackgroundAn effective, green and rapid analytical strategy namely the simultaneous spray assisted droplet formation-liquid phase microextraction (S-SADF-LPME) method was developed for the determination of trace quantity of cadmium in saliva samples by using the slotted quartz tube-flame atomic absorption spectrophotometry (SQT-FAAS). By the developed method, external dispersive solvent usage for droplet formation was reduced to obtain a more environmental-friendly method.MethodsMethod consists of a simultaneous complexing and extraction step, which was based on spraying an extraction solvent containing a solid ligand into the aqueous sample solution, forming fine droplets without the use of dispersive solvent. The procedure was implemented using a customized, cost effective and portable spray apparatus to minimize the consumption of reagent, analysis time and operation steps. Thus, this methodology ensures better repeatability and accuracy while minimizing the relative errors caused by the experimental steps. Parameters including the buffer amount, extractant/ligand concentration, extraction solvent type, extraction/ligand solution volume, spraying number and vortex period were systemically optimized to lower the detection limit.ResultsUnder the optimal extraction conditions, 96.9-folds enhancement in the detection power of the traditional FAAS was achieved. The limit of detection and limit of quantification values of presented method were calculated to be 0.65 and 2.17 ng mL⁻¹, respectively. Accuracy and applicability of the optimized method was investigated by collecting saliva samples from smokers. Satisfactory percent recovery values wereachieved for cadmium with a low standard deviation in the acceptable range of 84.9–109.6 %.ConclusionThe developed dispersive solvent-free S-SADF-LPME technique presents a fast, simple, cost-effective and eco-friendly microextraction method based on the use of an easily accessible and functional spray apparatus.     

Determination of zinc fractions in human blood and seminal plasma by ultrafiltration and atomic absorption spectrophotometry

A simple and reliable method is described which combines ultrafiltration technique with atomic absorption spectrophotometry to determine the Zn fractions in human blood plasma and seminal plasma. Ultrafiltrable, loosely bound, and firmly bound Zn can be measured using this method in the presence or absence of ethylene-diaminetetraacetic acid (EDTA). The YMT membranes for the ultrafiltration must be rinsed thoroughly before use. In contrast to Zn in blood plasma, a large part of Zn in the seminal plasma was found to be ultrafiltrabe. This method can be applied to study the physiologically active part of Zn in body fluids related to various disease states.     

The improvement of sensitivity in lead and cadmium determinations using flame atomic absorption spectrometry

Various designs of quartz tubes of the slotted tube atom trap were examined to improve the sensitivity of flame atomic absorption spectrophotometry. The studied designs include the diameter length and the length of the upper slot of quartz tubes. Tubes having a diameter of 5 and 6 mm and an upper slot length of 2.3 cm produce the best sensitivity—as high as 6- and 10-fold enhancement for lead (Pb) and cadmium (Cd), respectively. The limits of quantitation were found to be 35 and 4 ng ml⁻¹ for Pb and Cd, respectively, by the optimized method. The achieved technique was applied to determine the concentrations of Pb and Cd in cancerous (malign) and noncancerous (adenoma) human thyroid tissues.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Measurement of zinc, copper, manganese, and iron concentrations in hair of pituitary dwarfism patients using flameless atomic absorption spectrophotometry

Pituitary dwarfism (hGHD) is known to be associated with trace element deficiency, which causes improper functioning of the involved endocrine system. Previously, we reported on the head hair concentrations of zinc, copper, manganese, and iron from a total of 418 normal subjects (154 male and 264 female). In this report, we analyzed the head hair concentrations of the same four trace metals of 103 hGHD children (60 male and 43 female) under treatment with human growth hormone (hGH). These subjects ranged in age from 5 to 18 yr. The results were compared with 338 agematched normal subjects (120 male and 218 female). Both male and female hGHD showed approx 1.7 times higher zinc concentrations than normal subjects. Cheruvanky et al. reported a similar trend but with a slightly lower difference between hGHD and normal subjects. The average copper content in the hair of both male and female subjects also showed higher values for the hGHD than for the normal subjects, a trend similar to the values reported by Teraoka et al. In the case of manganese, the concentrations in hair of the hGHD were approx 50% of the values in the normal subjects. Head hair concentrations of iron in the hGHD were commensurate with the normal subjects. Because the content of trace elements in hair varies with the age of subjects, as a control, we investigated the head hair concentration of zinc from 20 healthy girls ranging in age from 10 to 18 yr. The average zinc concentration decreased from 10 to 12 yr, but no clear relation to age was observed from 13 yr and older. These trends were similar to our previous report. The zinc concentration in hair and body weight gain over a year was negatively correlated. The age variation in the content of zinc, copper, manganese, and iron in hair was measured comparing hGHD with the normal subjects in various ages. Concerning the zinc-level variation of hGHD and normal subjects, there were conspicuous differences between hGHD and normal subjects. For copper, the variations in concentration with age were similar to zinc. Regarding the age variations for manganese, hGHD had lower concentrations in hair compared to the normal subjects throughout adolescence (11–18 yr). We have studied the effects between the hair and these trace element concentrations in hGHD before and after hGH administration. These results suggest that hGH affects the metabolism of these trace elements.     

Direct determination of selenium in rat blood plasma by Zeeman atomic absorption spectrometry

The method was developed to be applied for direct determination of selenium in rat plasma by graphite-furnace atomic absorption spectrometry with Zeeman background correction. Blood was obtained from CD rats of both sexes 2h after dosing in weeks 7 and 13 in order to acquire data on the levels of selenium in these animals during 13-week gavage administration of l-seleno-methylselenocysteine (SeMC), a new candidate chemopreventive agent under development. Application of the commonly used method of standard addition was found to be unsuitable to calculate the selenium content in rat plasma (within-run and between-run accuracy and precision parameters were less than 85%). Therefore, a new analytical method was developed. In this method, samples of rat plasma (50 microL) were diluted 10-fold with a reducing agent containing l-ascorbic acid, a modifier solution containing palladium chloride and Triton X-100. Samples were atomized in pyrolytically coated graphite tubes and peak height signals were measured. Selenium concentrations were determined by linear least squares regression analysis based on the standard curve generated in pooled rat blank plasma. Since selenium is normally present in plasma, a three-step approach was used to calculate selenium plasma levels. Initially selenium levels were determined based on the standard curve with selenium-spiked pool plasma. In the second step, background selenium levels in the pooled plasma were determined based on the same standard curve. In the third step, background level was added to the previously derived number. The relative errors were in the range from -4.6 to 11.4% (intra-day assay) and from -0.4 to 8.8% (inter-day assay) which proved good accuracy. The relative standard deviations were in the range from 1.88 to 4.70% (intra-day precision) and from 3.28 to 5.38% (inter-day precision). In rat plasma, the following dose-dependent selenium levels (mean+/-S.D.) in males and females, respectively, were observed at 13 weeks: 655.5+/-48.8 and 595.8+/-43.9 ng/mL (control group), 927.9+/-85.3 and 859.3+/-164.3 ng/mL (0.4 mg/kg per day dose group), 1238.9+/-182.4 and 1169.9+/-112.6 ng/mL (0.8 mg/kg per day dose group), and 1476.5+/-138.1 and 1320.1+/-228.6 ng/mL (2.0mg/kg per day dose group). No significant sex differences in selenium plasma levels were seen in the SeMC-treated groups. No significant differences in selenium plasma levels were seen between mean plasma levels at 7 and 13 weeks. The described method is simple, rapid, accurate, precise and can be easily applied in other laboratories for a large number of samples.     

Whole blood and urine bioactive Hepcidin-25 determination using liquid chromatography mass spectrometry

Hepcidin is a small cysteine-rich signaling peptide that regulates blood serum iron concentrations [1–4]. Patients with chronic inflammation are known to have elevated levels of hepcidin in their blood and urine and often suffer from anemia as a result [5–10]. Measuring and quantifying the amount of active hepcidin in blood and urine can help to determine the cause and severity of the anemia thereby helping physicians determine the correct course of treatment [11–16]. We have developed a simple technique to isolate, chemically modify, and concentrate hepcidin from blood and urine coupled to high-pressure liquid chromatography mass spectrometry that can accurately and reproducibly measure and quantify the active hormone.     

A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.     

Determination of zinc in tissues of normal and dystrophic mice using electrothermal atomic absorption spectrometry and slurry sampling

An electrothermal atomic absorption spectrometric procedure for zinc determination in animal tissues is first optimized and then used to measure the metal content of different tissues from normal and dystrophic mice. The procedure involves minimal manipulation of the sample to overcome the severe contamination problems normally associated with the measurement of low zinc levels. The samples (recommended amount 10 mg) are slurried in 2 ml of a 10-mM tetramethylammonium hydroxide solution containing 0.1 g/100 ml silicone antifoam. After mild heating at 60 degrees C for 10 min and homogenization for 1 min, the suspensions are submitted to a 10-fold dilution and then injected into the electrothermal atomizer. Calibration is carried out using aqueous standard solutions of zinc prepared in the same suspension media. The reliability of the whole procedure is verified using three certified reference materials.     

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics

Background

The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow.

Result

Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification.

Conclusions

SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9087-4) contains supplementary material, which is available to authorized users.     

IgA肾病患者血液、尿液及咽拭子标本中穿通支原体的分离检出

目的探讨IgA肾病患者血液、尿液及咽拭子标本中穿通支原体（Mycoplasma penetrans,Mp）的分离检出率以及与病理型别相关性。方法采用分离培养法,共计从26例IgA肾病患者血液、尿液及咽拭子标本及38例正常对照相应标本中进行穿通支原体分离检测,对培养阳性标本用穿通支原体套式PCR进行证实。结果在11例（42．3％）患者血液与尿液或（和）咽拭子中同时分离到穿通支原体,单独尿液或咽拭子标本阳性分别为1例（3．8％）与7例（26．9％）。26例IgA肾病患者血液、尿液及咽拭子穿通支原体的分离检出率分别为42．3％、23．1％与57．7％;与38例正常对照组血液、尿液及咽拭子检出0例、2例（5．3％）与7例（18．4％）相比较,差异有非常显著性（P〈0．01）,在正常对照组中无2种以上标本同时检出穿通支原体。结论穿通支原体在ISA肾病患者的血液、尿液与咽拭子标本中均有较高的检出率且与病理型别有一定的相关性。     

Speciation of platinum in blood plasma and urine by micelle-mediated extraction and graphite furnace atomic absorption spectrometry

A highly sensitive and selective technique for the speciation of platinum by cloud point extraction prior to determination by graphite furnace atomic absorption spectrometry (GFAAS) was described. The separation of Pt(II) from Pt(IV) was performed in the presence of 4-(p-chlorophenyl)-1-(pyridin-2-yl)thiosemicarbazide (HCPTS) as chelating agent and Triton X-114 as a non-ionic surfactant. The extraction of Pt(II)–HCPTS complex needs temperature higher than the cloud point temperature of Triton X-114 and pH = 7, while Pt(IV) remains in the aqueous phase. The Pt(II) in the surfactant phase was analyzed by GFAAS, and the concentration of Pt(IV) was calculated by subtraction of Pt(II) from total platinum which was directly determined by GFAAS. The effect of pH, concentration of chelating agent, surfactant, and equilibration temperature were investigated. An enrichment factor of 42 was obtained for the preconcentration of Pt(II) with 50 mL solution. Under the optimum experimental conditions, the calibration curve was linear up to 30 μg L^?1 with detection limit of 0.08 μg L^?1 and the relative standard deviation was 1.8%. No considerable interference was observed due to the presence of coexisting anions and cations. The accuracy of the results was verified by analyzing different spiked samples (tap water, blood plasma and urine). The proposed method was applied to the speciation analysis of Pt in blood plasma and urine with satisfactory results.     

Human biomonitoring of 73 elements in blood,serum, erythrocytes and urine

BackgroundHuman biomonitoring studies of trace elements in biological fluids are mostly limited to a certain number of elements or biological materials. In this study, we describe the significant extension of a biomonitoring to 73 elements being present in concentration ranges from ng/L to g/L in clinically relevant specimens such as blood, serum, erythrocytes and urine.MethodsThe samples were collected from 102 occupationally non-exposed inhabitants of northern Germany. The elements were determined either by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in the low concentration range or by inductively coupled plasma optical emission spectrometry (ICP-OES) for essential trace elements and electrolytes.ResultsMean values and selected percentiles of element concentrations are presented for all sample materials. From the results, we calculated the distribution of elements between plasma and blood cells. Application of ICP-MS/MS improves selectivity and accuracy in the determination of elements that are strongly spectrally interfered, such as Cr, Ge, Pd or Ti in blood samples.ConclusionsThis publication provides very valuable information for occupational or environmental hygienists, toxicologists and clinical chemists due to the particularly high number of determined elements and presented concentration ranges.     

Stability of (1R,2S)-(-)-Ephedrine hydrochloride in Candida albicans-inoculated urine and blood samples

The stability of drugs in biological evidence during collection and storage is of particular concern in forensic investigations. Microbes in the samples and other elements are an essential component of these investigations. In the current study, the HPLC method was used to examine the stability of (1R, 2S)-(-)-Ephedrine hydrochloride in plasma and urine samples inoculated with C. albicans after storage at 37 °C for 48 h and −20 °C for six months. In the stability experiment, MIC_50% of (1R, 2S)-(-)-Ephedrine hydrochloride was applied according to MIC and MFC that were determined in this work. This drug had MIC and MFC of 50 and 100 ppm, respectively. In HPLC analysis, the standard (1R, 2S)-(-)-Ephedrine hydrochloride had a retention time of 1.63 and was used to identify this drug in samples that had or had not been exposed to C. albicans. The findings demonstrated that within 48 h at 37 °C, C. albicans had an impact on the drug concentration (10% and more than 15%, respectively, were lost in plasma and urine samples inoculated with C. albicans). In plasma samples, the drug content remained stable at −20 °C for three months, although it degraded in urine samples after one month. In plasma and urine samples, the concentration reduction had surpassed 70% and 50% by the sixth month, respectively. The results of this investigation show that C. albicans can change the stability of (1R, 2S)-(-)-Ephedrine hydrochloride in plasma and urine samples that contain MIC_50% of Ephedrine hydrochloride.     

Iron in granulocytes of nephrotic patients determined by Zeeman electrothermal atomic absorption spectrophotometry

One of the major threats to patients undergoing maintenance hemodialysis is an increased susceptibility to infections caused by microorganisms, among whichYersinia orListeria are frequently recovered. In patients receiving hemodialysis, iron overload owing to multiple transfusions plays an important role in the mechanisms underlying the susceptibility to bacterial infections, partially mediated by impaired neutrophil function. In order to assess the true role of iron at the cellular level, an AAS method was developed to measure the iron content of granulocytes. Iron levels in the granulocytes were determined in an apparently healthy population and in a population of iron-overloaded renal hemodialysis patients. Granulocytes were isolated by a method modified from Böyum. The analyses were performed using pyrocoated graphite tubes, and in one of the char steps, oxygen was used to facilitate ashing. The mean iron level found in the granulocytes from apparently normal persons was 4.07 fg/cell (n=17) with a CV of 44%; the mean value for the dialysis patient group was 4.59 fg/cell (n=8) with a CV of 37%. There was no significant difference between the two groups,p=0.70.     

Standardization and validation of a new atomic absorption spectroscopy technique for determination and quantitation of Aluminium adjuvant in immunobiologicals

In the present study, Aluminium quantification in immunobiologicals has been described using atomic absorption spectroscopy (AAS) technique. The assay was found to be linear in 25-125 microg/ml Aluminium range. The procedure was found to be accurate for different vaccines with recoveries of external additions ranging between 93.26 and 103.41%. The mean Limit of Variation (L.V.) for both intra- and inter-assay precision was calculated to be 1.62 and 2.22%, respectively. Further the procedure was found to be robust in relation to digestion temperature, alteration in acid (HNO(3) and H(2)SO(4)) ratio used for sample digestion and storage of digested vaccine samples up to a period of 15 days. After validation, AAS method was compared for its equivalency with routinely used complexometric titration method. On simultaneously applying on seven different groups of both bacterial and viral vaccines, viz., DPT, DT, TT, Hepatitis-A and B, Antirabies vaccine (cell culture) and tetravalent DPT-Hib, a high degree of positive correlation (+0.85-0.998) among AAS and titration methods was observed. Further AAS method was found to have an edge over complexometric titration method that a group of vaccines, viz., ARV (cell culture, adsorbed) and Hepatitis-A, in which Aluminium estimation is not feasible by pharmacopoeial approved complexometric titration method (possibly due to some interference in the sample matrix), this newly described and validated AAS assay procedure delivered accurate and reproducible results.     

Cobalt determination in serum and urine by electrothermal atomic absorption spectrometry

Cobalt determinations in biological fluids are of great interest in biological or toxicological research programs. Cobalturia is often chosen as an indicator for a biological monitoring program in occupational exposure to cobalt dusts. The method described here derives from the IUPAC reference method for nickel determination. It enables cobaltemia and cobalturia to be measured in small samples (1 mL). The mean usual values for cobalt in biological fluids are very low (2.7 nmol L⁻¹ for serum and 6.7 nmol L⁻¹ for urine), and therefore, thus require an analytical procedure with preconcentration and extraction. The sample is mineralized by wet acid digestion. After digestion, inorganic cobalt is extracted in form of ammonium pyrrolidine dithiocarbamate complex into isobutyl methyl ketone and measured in the organic layer by electrothermal atomic absorption spectrometry. The analytical parameters are described in detail. The extraction output is about 99%. The detection limits are 1.93 and 1.89 nmol L⁻¹ for serum and urine, respectively. Sensitivity (expressed as the concentration that gives a 0.044 absorbance) is 3.4 nmol L⁻¹ for serum and 3.3 nmol L⁻¹ for urine. Within-run precision ranged between 3.9 and 2.5% (coefficients of variation) for serum and 4.2 and 1.1% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. Between-run precision ranged between 4.3 and 3.3% (coefficients of variation) for serum and 4.2 and 2.3% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. At very low concentration, 5.7 nmol L⁻¹ for serum and 2.5 nmol L⁻¹ for urine, the between-run precision is, respectively, 19.5 and 28%. Linearity is effective between 0 and 272 nmol L⁻¹. Interferences and matrix effects are negligible for urine, serum, or plasma samples without hemoglobin. The method is easily applicable for routine determinations.     

Smoking does not influence cadmium concentrations in blood and urine in relatively high levels of environmental cadmium areas in Japan

We examined how the influence of smoking on blood and urinary cadmium (Cd) concentrations was modified by the level of environmental Cd. We measured blood and urinary Cd concentrations of 1134 men over 50 yr of age in three areas in Japan that were exposed to different levels of environmental Cd. Analysis of variance was used to compare Cd concentrations in blood and urine of smokers with those of nonsmokers living in the three areas. Correlation coefficients between the number of cigarets smoked per day or smoking indices (the number of cigarets smoked per day multiplied by the number of years smoked) and blood and urinary Cd concentrations were calculated. No significant difference in Cd concentrations between smokers and nonsmokers was observed in areas where the average Cd concentration in blood was over approx 2.4 ng/g, 2.0 μg/L in urine, and 2.3 μg/g creatinine in urine, respectively. Therefore, it was suggested that an influence of smoking on blood and urinary Cd concentrations was not observed in areas exposed to relatively high levels of environmental Cd.