Determination of standard zinc values in the intact tissues of mice by ICP spectrometry

The most commonly encountered difficulties for the quantitative measurement of zinc in biological samples are the limited sample amount, total and effective digestion of connective and fatty residues, and low zinc concentrations. These problems often lead to the determination of lower zinc values than actually present, so that the sample preparation, digestion, and analytical procedure deserve careful attention. In this short communication, a new method for microwave tissue disintegration is described. The authors have obtained consistent and reproducible results with tissue samples of 0.5 g or less.     

Isotope ratio determination in boron analysis

Traditionally, boron (B) isotope ratios have been determined using thermal ionization mass spectrometry (TIMS) and, to some extent, secondary ion mass spectrometry (SIMS). Both TIMS and SIMS use a high-resolution mass analyzer, but differ in analyte ionization methods. TIMS uses electrons from a hot filament, whereas SIMS employs an energetic primary ion beam of Ga+, Cs+, or O- for analyte ionization. TIMS can be used in negative or positive ion modes with high sensitivity and precision of B isotope ratio determination. However, isobaric interferences may be a problem, if the sample is not well purified and/or memory of the previous sample is not removed. Time-consuming sample preparation, analyte (B) purification, and sample determination processes limit the applications of TIMS for routine analyses. SIMS can determine B and its isotope ratio in intact solid samples without destroying them, but has poorer resolution and sensitivity than TIMS, and is difficult to standardize for biological samples. Development of plasma-source mass spectrometry (MS) enabled the determination of B concentration and isotope ratio without requiring sample purification. Commonly used plasma-source MS uses an Ar inductively coupled plasma (ICP) as an ionization device interfaced to a low-resolution quadrupole mass analyzer. The quadrupole ICP-MS is less precise than TIMS and SIMS, but is a popular method for B isotope ratio determination because of its speed and convenience. B determination by ICP-MS suffers no spectroscopic interferences. However, sample matrices, memory effects, and some instrument parameters may affect the accuracy and precision of B isotope ratio determination if adequate precautions are not taken. New generations of plasma-source MS instruments using high-resolution mass analyzers provide better sensitivity and precision than the currently used quadrupole ICP-MS. Because of the convenience and high sample throughput, the high-resolution ICP-MS is expected to be the method of choice for B isotope ratio determination. The current state of instrumental capabilities is adequate for B isotope determination. However, precision and accuracy are primarily limited by sample preparation, introduction, and analytical methodology, including 1. Analyte loss and isotope fractionation during sample preparation. 2. The precision of B isotope determination in small samples, especially those containing low concentrations. 3. Difficult matrices. 4. Memory effects. Sample preparation by alkali fusion allows rapid and complete decomposition of hard-to-digest samples, but high-salt environments of the fused materials require extensive sample purification for B ratio determination. The alternative wet-ashing sample decomposition with HF also results in B loss and isotopic fractionation owing to the high volatility of BF3. Open-vessel dry- or wet-ashing methods usually do not work well for animal samples, and are also prone to B loss and contamination. Closed-vessel microwave digestion overcomes these problems, but the digests of biological materials have high C contents, which cause spectral interference on 11B and affect 11B/10B ratios. Exchange separation/preconcentration of B using exchange (cation or anion exchange, B-specific resin, e.g., Amberlite IRA-743) tend to cause B isotope fractionation, and C eluting from these resin columns may interfere with B isotope ratio determination. Memory effects of B that occur during sample determination may cause serious errors in B isotope ratio determination, especially when samples varying in B concentrations and/or isotope composition are analyzed together. Although the utilization of high-resolution plasma-source MS will undoubtedly improve analytical precision, it is the sample preparation, sample introduction, and analytical methodology that represent the primary limitation to accurate and precise B isotope ratio determination.     

Evaluation of methods for total selenium determination in yeast

The selenium determination in biological materials by the classical fluorometric method (FM) is time-consuming and also hazardous, as it requires the destruction of the organic matrix samples with hot HNO₃/HClO₄ mixtures prior to analysis. Accordingly, commercial analytical laboratories are increasingly using faster instrumental methods; for sample digestion, avoid using HClO₄. Because of these procedural changes, the results obtained by commercial laboratories may be unreliable, especially for samples containing Se in organic forms. One such “difficult” substrate is Se yeast, which contains most of its Se as selenomethionine. To establish which methods for Se analysis and sample digestion are applicable, samples of Se yeast and of selenomethionine standards were sent to laboratories employing either flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), or hydride generation atomic absorption spectrometry (HGAAS). The result were compared with those obtained by FM and non-destructive instrumental neutron activation analysis (INAA). ICP-MS, after microwave digestion of sample with HNO₃/H₂O₂, produced results within 5% of the expected values, as did those obtained by FM and INAA. With FAAS, acceptable results were obtained after digestion with HNO₃/HCl. With HGAAS, sample digestion with HNO₃/H₂O₂ produced values that were systematically elevated by about 10% and exhibited standard deviations of ≥10%. Thus, current methods of sample digestion are applicable for Se yeast analysis by ICP-MS and FAAS, but not by HGAAS.     

电感耦合等离子质谱法(ICP-MS)法测定茵栀黄注射液中有害元素铅、砷、镉、汞、铜的含量

建立了电感耦合等离子体质谱法(ICP-MS)测定茵栀黄注射液中Pb、As、Cd、Hg、Cu 5种元素的方法。样品经微波消解后,直接用ICP-MS同时测定上述5种元素,结果5种元素的检出限分别在5～1250 ng/L之间;线性良好,线性相关系数均为r≥0.999;精密度RSD3.5%;回收率在95.7%～107.5%之间。方法操作简便、分析速度快、灵敏度高,各项分析性能指标均达到要求,适用于茵栀黄注射液中有害元素的测定。     

Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry

The analysis of trace elements in biological samples will extend our understanding of the impact that environmental exposure to these elements has on human health. Measuring arsenic content in nails has proven useful in studies evaluating the chronic body burden of arsenic. In this study, we developed methodology with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of total arsenic in nails. We assessed the utility of the washing procedures for removing surface contamination. Four types of preanalysis treatments (water bath, sonication, water bath plus sonication, and control) after sample decomposition by nitric acid were compared to evaluate the digestion efficiencies. In addition, we studied the stability of the solution over 1 wk and the effect of acidity on the arsenic signal. Arsenic content in the digested solution was analyzed by using Ar-N₂ plasma with Te as the internal standard. The results suggest that washing once with 1% Triton Χ-100 for 20 min for cleaning nail samples prior to ICP-MS analysis is satisfactory. Repeated measurement analysis of variance revealed that there was no significant difference among the various sample preparation techniques. Moreover, the measurements were reproducible within 1 wk, and acidity seemed to have no substantial influence on the arsenic signal. A limit of detection (on the basis of three times the standard deviation of the blank measurement) of 7 ng As/g toenail was achieved with this system, and arsenic recoveries from reference materials (human hair and nails) were in good agreement (95–106% recovery) with the certified/reference values of the standard reference materials. ICP-MS offers high accuracy and precision, as well as highthroughput capacity in the analysis of total arsenic in nail samples.     

Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF

BackgroundTrace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.Methods and resultsAfter centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.ConclusionTaken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.     

Applicability of microwave acid digestion to sample preparation of biological materials for analysis by particle-induced X-ray emission (PIXE)

A microwave acid digestion method for the preparation of biological samples for PIXE analysis is presented. The precision and accuracy of the entire PIXE analytical procedure, including the micro-wave digestion step, were evaluated by analyzing eight certified reference materials. For elements heavier than K, and for concentration levels from 2 μg/g upward, the total random error of a single analysis is in the range of 2–5%. The accuracy is better than 5%. The detection limits are down to 0.3 μg/g.

     

Isotope Ratio Measurements of Iron in Blood Samples by Multi-collector ICP-MS to Support Nutritional Investigations in Humans

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., ⁵⁴Cr⁺ on ⁵⁴Fe⁺ and ⁵⁸Ni⁺ on ⁵⁸Fe⁺) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008–0.05 (% RSD), 0.007–0.015 (% RSD), and 0.03–0.09 (% RSD) were obtained for ⁵⁴Fe/⁵⁶Fe, ⁵⁷Fe/⁵⁶Fe, and ⁵⁸Fe/⁵⁶Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.     

Considerations in the determination of boron at low concentrations

Experimental evidence now supports the nutritional essentiality of boron (B) in some biological systems, and accordingly, the need for reliable analytical B data is increasing. However, the accurate determination of B in biological materials is a formidable challenge at low concentrations (<1 mg B/kg). Recent studies still show significant analytical discrepancies in the analysis of animal tissues and fluids, despite the development of instrumental techniques such as TIMS, ICP-MS, ICP-ES, ICAP, SIMS, NA-MS, PGAA, NRA, and so forth, which have demonstrated detection limits approaching or exceeding (μgB/kg concentrations. Since boric acid is both volatile and ubiquitous in nature, the chemical and physical pathways for B contamination and its loss are manifold, especially during sample preparation. An added obstacle is the inadequacy of biological reference materials certified for B below mg B/kg. With an emphasis toward sample preparation and ICP-MS analysis, examples are provided in this article to help the analyst avoid common problems associated with the analysis of B from biological sources. Topics that are discussed include contamination from Teflon vessels during microwave digestion, losses owing to freeze-drying, B isotopic variations, standards preparation, reagent backgrounds, and instrumental interferences.     

Determination of mercury in hair by square-wave anodic stripping voltammetry at a rotating gold disk electrode after microwave digestion

A simple and reliable method for the determination of mercury in hair on a rotating gold disk electrode using subtractive anodic stripping voltammetry without removal of oxygen is reported. Voltammetric and microwave parameters were optimized to obtain the best analytical results. Parameters such as supporting electrolyte concentration, influence of chloride in the Hg peak, deposition potential, scan rate, accumulation time, rotation rate, square-wave amplitude, and electrode conditioning were studied. Pressurized microwave-assisted digestion of hair, suitable for the accurate voltammetric determination of Hg, was evaluated using six acid mixtures and several time-power programs. Under the optimized conditions, no interference by copper, cadmium, lead, nickel, manganese, iron, or zinc was found at concentrations corresponding to their occurrence in normal hair. A calibration plot between 6,67 and 46,69 μg/L was linear, with r ² better than 0.999. The detection limit for a deposition time of 60 s at 25g, was calculated as 1.92 nM (3σ). Validation of the method was demonstrated with the use of a certified reference sample of hair. Eight real samples of hair (four unexposed children and four exposed persons) were also analyzed.     

Hair analysis for opiates: evaluation of washing and incubation procedures

Hair analysis of drugs of abuse has been a subject of interest from a clinical, social and forensic perspective for years because of the broad time detection window after intake in comparison to urine or blood. However, the correct and reliable interpretation of opiates findings in an authentic hair sample requires optimalisation and standardisation of decontamination and incubation procedures. Comparing various published methods, we have found some variability in them and no unequivocal recommended procedure for starting with a method directly. Therefore, various combinations of solvents, of various polarity, as washing solvents were tested for removing opiates from the external surface of real hair samples. The yields of opiates from these washings were compared with the yields from the interior of the hair matrix after digestion with various procedures. The opiates after digestion were cleaned up from resulting solution on extraction columns with mixed solid-phase and analysed by GC–MS in standard EI mode after silylation. The efficiencies of neutral (Söerensen buffer, pH 7.4), acid (0.1 M HCl) and basic (1 M NaOH) digestion of the hair matrix were evaluated and the relative recoveries for morphine, codeine, dihydrocodeine and hydrocodone were compared. As it is very problematic to imitate the reference hair sample with a specific amount of analytes incorporated inside, which can be used for calibration to get a close estimate of the quantities of analytes inside the solid authentic sample, the total digestion of a hair sample in basic medium was considered to be a very important reference basis for quantitative determinations. The ratios of hydrolysis of labile 6-acetylmorphine or acetylcodeine were tested and evaluated in practical routine conditions of acid or neutral digestion of hair. Comparing the three methods of incubation of authentic hair samples, the methods using 1 M NaOH or 0.1 M HCl yielded higher recoveries of total equivalents of morphine or codeine, whereas the incubation in Söerensen buffer allowed the reflection of real ratios of labile metabolites and/or parent compounds in an original sample. This method has been shown to be capable of detecting hydrocodone in hair with other opiates concomitantly and to indicate the drug abuse pattern of a person at various time intervals in the past.     

The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis

Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples.     

Multianalysis of trace elements in mosses with inductively coupled plasma-mass spectrometry

As a part of an air-pollution biomonitoring survey, a procedure using inductively coupled plasma-mass spectrometry (ICP-MS) and microwave digestion was developed to achieve a high sample throughput and guarantee the accuracy of the results. This article presents an analytical method to measure 22 trace elements. As, Ba, Cd, Ce, Co, Cr, Cs, Cu, Fe, Hg, La, Mo, Ni, Pb, Rb, Sb, Sr, Th, Tl, U, V, W were analyzed in 563 mosses collected in France. The digestion was performed in polytetrafluoroethylene (PTFE) vessel using the mixture HNO₃-H₂O₂-HF. The data were reprocessed taking into account the drift curve calculated for each element. The detection limits (DL) calculation was based on the standard deviations of the reagent blanks concentrations. The DL varied from one batch to another, because of the heterogeneity of the mosses’ elemental contents. The DL ranged between 0.001 μg/g (Cs, Tl) and 70 μg/g (Fe) and were mainly around 0.01 μg/g (As, Cd, Ce, Co, Hg, La, Mo, Sb, Sr, U, V, W). The detection limits obtained were in agreement with the concentrations observed in the samples, except for Hg and Ni. The reproducibility between duplicates and the analytical precision were near 10%. The procedure was tested with the mosses’ reference materials.     

Screening and quantitation of multiclass drugs of abuse and pharmaceuticals in hair by fast liquid chromatography electrospray time-of-flight mass spectrometry

In this work, an automated screening method for the simultaneous identification and quantitation of 30 representative multiclass drugs (including opiates, cocaine and its main metabolite, cannabinoids, amphetamines and other stimulants in hair samples) has been developed using fast liquid-chromatography time-of-flight mass spectrometry (LC-TOFMS). The identification and quantitation of the drugs were carried out by liquid chromatography using a C(18) column (4.6×50 mm) with 1.8 μm particle size. Accurate mass measurements of ions of interest (typically [M+H](+)) by electrospray time-of-flight mass spectrometry in the positive ionization mode were used for unambiguous confirmation of the targeted species. Three sample preparation methodologies were evaluated: (a) direct methanolic extraction by sonication, (b) acidic extraction, and (c) alkaline digestion. Direct methanolic extraction showed better recoveries and cleaner extracts. The limits of detection obtained in hair matrix were as low as 5 pg mg(-1) for cocaine and cannabidiol, ranging from 5 to 75 pg mg(-1) for the studied species while the LOQ ranged from 15 to 250 pg mg(-1). The method has been applied to six hair samples from drug consumer volunteers, where the presence of at least one drug was confirmed by accurate mass measurements within 2 ppm (mass error) in most cases. The present study demonstrates the usefulness of LC-TOFMS for both screening and quantitation purposes in drug testing in hair. In addition, the possibility of non-target or a posteriori data analysis of samples or the extension of the procedure for testing for additional compounds offers interesting features for forensic analysis.     

Direct determination of selenium and other trace elements in serum samples by ICP-MS.

Selenium belongs to a group of trace elements of special interest in biological samples for clinical diagnosis. Selenium has antioxidizing functions and is essential for providing the organism with triiodothyronine produced from thyroxine. Among several analytical techniques used to determine the Se concentration in serum, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been used in the past because of its high sensitivity. Interference problems originating from different ions on the major Se isotopes have been described to be a limiting factor for the direct determination of Se in these matrices. Standard addition calibration or isotope dilution is often required to overcome carbon-enhanced ionisation effects in biological sample matrices. In most cases, the typical serum sample volume which is available for the analysis is limited to 0.5 ml or less, making multiple sample preparation for standard addition calibration impractical. Isotope dilution requires enriched isotopes and substantial sample preparation. Furthermore, the approximate Se concentration in every sample has to be known to adjust the appropriate amount of spike to each sample. Matrix matching with methanol has been described to overcome ionisation effects but we found limiting factors of this application when other trace elements are also determined within one sample run. This paper describes an effective sample preparation method which allows the direct determination of Se in serum without limiting the analytical capabilities for the additional determination of Al, Cu, Ni, Co, Cd, Mn and Zn in a single sample run by ICP-MS. Optimization procedures are presented and results of the analysis of reference samples are discussed, with a comparison of more than 150 serum data with those obtained by the GF-AAS method.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.     

Determination of urinary iodine by inductively coupled plasma mass spectrometry.

BACKGROUND: Mild iodine deficiency is endemic in many countries of Europe including Belgium. Fast, accurate and specific methods for quantification of urinary iodine are needed. We describe in this report a specific ICP-MS method for the quantification of urinary iodine. METHOD: Samples and iodate calibrators were diluted 20 times into aqueous solution containing triton X-100, 1.5% HCl and (103)Rh as an internal standard. Prior digestion or oxidation was not necessary. Results were compared with those obtained by Sandell-Kolthoff (S-K) spectrophotometric method. RESULTS: Comparison of both methods showed good agreement. The Passing-Bablok regression between both methods was ICP-MS=0.986 (S-K)-7.51. The Bland-Altman difference plot showed a small but significant mean difference of -13.3 microg/L for ICP-MS. The between-day coefficient of variation (CV) was 13% at 89 microg/L. Limit of detection was 4 microg/L and limit of quantification was 20 microg/L. No carryover effect has been observed on series containing up to 50 samples. CONCLUSION: The ICP-MS method described here is fast, accurate and specific for the quantification of urinary iodine. Compared to the S-K method the urinary iodine concentrations measured by the ICP-MS method were slightly, but significantly lower. Consequently, the results of studies using S-K method should be compared with caution with those using the ICP-MS method.     

Extraction of proteins and peptides from Coomassie Blue-stained sodium dodecyl sulfate--polyacrylamide gels.

A method is described for extracting proteins and peptides from stained sodecyl sulfate-polyacrylamide gels. Coomassie blue and sodium dodecyl sulfate present in stained gel sections are removed to allow subsequent analysis of the peptides (e.g., amino acid analysis or tryptic digestion and fingerprinting). The method is simple, requires no radioisotopes or special equipment, and can be carried out with a minimum of handling of the sample. The process can be used for samples at the nanomole level with recoveries of 90%.     

On‐target ultrasonic digestion of proteins

In the present work, we report a novel on‐target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on‐tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample‐handling steps and time are the features that make this method appealing to the many laboratories working with high‐throughput sample treatment.     

Evaluation of Status of Zinc, Copper, and Iron Levels in Biological Samples of Normal Children and Children with Night Blindness with Age Groups of 3–7 and 8–12 Years

The causes of night blindness in children are multifactorial, and particular consideration has been given to childhood nutritional deficiency, which is the most common problem found in underdeveloped countries. Such deficiency can result in physiological and pathological processes that in turn influence hair composition. This study was designed to compare the levels of zinc (Zn), copper (Cu), and iron (Fe) in scalp hair, blood, and urine of both genders of children with night blindness with age range of 3–7 and 8–12 years, comparing them to sex- and age-matched controls. A microwave-assisted wet acid digestion procedure was developed as a sample pretreatment, for the determination of zinc, copper, and iron in biological samples of children with night blindness. The proposed method was validated by using conventional wet digestion and certified reference samples of hair, blood, and urine. The digests of all biological samples were analyzed for Cu, Fe, and Zn by flame atomic absorption spectrometry using an air/acetylene flame. The results indicated significantly lower levels of Fe, Cu, and Zn in the biological samples (blood and scalp hair) of male and female children with night blindness, compared with control subjects of both genders. These data present guidance to clinicians and other professionals investigating the deficiency of essential trace metals in biological samples (scalp hair and blood) of children with night blindness.