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A standard-free method of quantitative analysis, which is based on the fact that the total yield of continuous x-rays from the sample approximately corresponds to effective weight of the sample, was developed and has been applied to some typical bio-samples such as serum, whole blood, hair and untreated bone. In this work, the standard-free method was applied to untreated urine samples. This method allows us to perform sample preparation only by dropping 5 μl of urine sample onto a backing film. It requires neither a large amount of urine nor the internal standard. As the results, values of concentration of potassium for 4 samples agree well with the value obtained by the internal standard method within an error of 10%. The present method was also applied to 21 urine samples containing excess amount of urinary protein and / or sugar, and it is found that the present method is applicable to such abnormal urines. Owing to this method, target preparation can be performed at the place and time of sampling. It is quite convenient to environmental studies.

In order to address global environmental issues, a standard-free method developed by ourselves has been successfully applied to various kinds of bio-samples. Especially, a method for untreated hairs has been applied in many polluted areas to study human exposure to toxic elements. In addition to hair, nail is expected to give us valuable information about human exposure to toxic elements. However, the analysis requires relatively large amounts of samples and laborious sample preparation techniques which necessitate internal standards. In this work, we have developed a quantitative method for untreated human-nail analysis based on the standard-free method. It requires neither large amounts of nails nor complicated target preparation procedure. Furthermore, it is perfectly free from any ambiguity in target preparation such as volatilization of certain elements and contamination of the sample during chemical ashing. The optimum conditions of irradiating nail samples are established, and accuracy and reproducibility of the present method are confirmed. It is found that ultrasonic washing in distilled water is effective for many nail samples preventing the loss of elements from the sample. It is also found that elemental concentration in nails strongly depends on their sampling positions.

Nishina Memorial Cyclotron Center (NMCC) has been opened for nationwide-common utilization of positron nuclear medicine (PET) and PIXE since April 1993. At the present time, nearly 40 subjects of PIXE in various research fields are pursued here, and more than 50,000 samples have been analyzed up to the present. In order to perform quantitative analyses of diverse samples, technical developments in sample preparation, measurement and data analysis have been continuously carried out. Especially, a "standard-free method for quantitative analysis" made it possible to perform analysis of infinitesimal samples, powdered samples and untreated bio samples, which could not be well analyzed quantitatively in the past. The "standard-free method" and a "powdered internal standard method" made the process for target preparation quite easier. It has been confirmed that results obtained by these methods show satisfactory accuracy and reproducibility preventing any ambiguity coming from complicated target preparation processes.

We have developed standard-free methods for PIXE analysis of hair, nail, urine and serum, and they have widely been applied to studies on human exposure to some toxic elements caused by pollution in many countries. Although these samples are known to be quite suited for evaluating human exposure to toxic elements, there is a case where it is required to quantitatively analyze other samples taken from people. We have examined sweat, saliva, snivel and tear, which are expected to be useful for estimating concentration of various elements in a human body. However, sufficient quantities of them are difficult to be taken from a human body and it is required to establish standard-free methods for these samples. In the present work, standard-free methods for sweat and saliva were established and its accuracy and reproducibility are confirmed. It becomes possible to quantitatively analyze only a drop of sweat and saliva by means of this method. For snivel and tear, potassium concentration is obtained by the internal-standard method for a small-spot sample and the conversion coefficient required for the standard-free method was evaluated. It is found that analyses of these samples give us useful information about some specific elements. As the standard-free methods are free from dryness of samples, which becomes a serious problem in the case where the internal-standard method is applied, it is expected to give more essential information about elemental concentration in a human body.

We developed and reported standard-free methods for various samples such as hair, nail, urine, serum, sweat, snivel, tear and saliva, and they have been confirmed to be effective for addressing global environmental issues. It is expected that analyses of beard samples give us useful information about daily changes of elemental concentration in the body in comparison with the other samples. In this work, a quantitative method for untreated beard analysis based on the standard-free method has been developed. By means of the method, it becomes possible to quantitatively analyze extremely small quantity of beard samples, which are daily taken with ordinary electric shaver. The optimum conditions of washing samples have also been examined. It is found that some elements such as potassium, calcium and chlorine are lost by washing in distilled water and it is better to wash the sample in acetone. As a result, the standard-free method for beard samples has been successfully established and good accuracy and reproducibility were achieved.

We developed and reported a standard-free method for beard (including mustache and whiskers) samples that enables us to quantitatively analyze powdered beard samples of extremely small quantity. In order to investigate intake of essential elements and also exposure to toxic elements, daily changes of elemental concentration in the body give us essential information. Firstly, we have measured dependence of elemental concentration in beard, mustache and whiskers on the collecting regions in the face. Then, daily changes of elemental concentration in the body, which reflect daily ingestion of foods and waters, have been estimated by means of quantitative analyses of beard samples. These beard samples were taken from three persons everyday over successive 7, 14 and 33 days, respectively. As a result, some elements show long-term changes and a few toxic elements such as arsenic and lead exhibit changes in a period of a few days. We have also measured daily changes of elemental concentration in urine and beard taken from the same person at the same time every half day over 10 days, and clear correlation of daily changes of the arsenic concentration between urine and beard samples was observed. It is found that the standard-free method is quite effective for beard samples of very small quantities, and that beard analyses are quite suited to studies on daily changes of elemental concentration in the human body in comparison with the other samples depending on the elements.

Standard-free method for untreated hair samples in in-air PIXE has been developed. It is confirmed that the method gives us good sensitivity and accuracy within several minutes' measurement if more than twenty hairs are attached onto the target. Even in the case where the number of hairs is less than eight, which is regular for usual in-vacuum PIXE, 10-15 minutes measurement is found to be sufficient to achieve almost satisfactory sensitivity and accuracy for elements from Cl to Pb. As the present method allows us to carry out analyses without labor in target preparation, it is expected to be quite helpful in the studies on human exposure to toxic elements. Its availability will more and more increase when the method is combined with the method of simultaneous measurement of in-vacuum and in-air PIXE we have just developed.

A standard-free method for small lymph node and spleen samples taken from rats, and lymph node, esophagus, colon etc. taken from real patients has been developed in order to take measure to meet increasing demands for quantitative analyses of small amount of organs. It becomes possible to quantitatively analyze organ samples of less than 1 mg collected from small experimental animals and also from patients by biopsy, and accuracy and sensitivity of the method were examined by comparing the results with those obtained by a powdered-internal-standard method and a chemical-ashing method. It is found that the method is quite effective for estimating therapeutic effect of CDDP (Cisplatin) since it allows us to quantitatively evaluate uptake of CDDP into organs by analyzing small quantity of samples taken from actual patients by biopsy. It is expected that the method will become a powerful tool for studies not only in fundamental and clinical medicines but also on practical diagnosis and treatment.

We established and reported two years ago the original methods for evaluating daily changes of elemental concentration in a body by means of a standard-free method for powdered beard samples daily taken with electric shaver. It was found that the method is quite useful for investigating short- and long-term changes of elemental concentration in a body. However, the method for beard analysis is applicable only to men. In order to estimate daily changes of elemental concentration in a body for women and children, a new method which allows us to quantitatively analyze hair samples cut into 1 mm pieces has been developed and applied to three long hair samples taken from three persons. It is found that the method enables us to estimate both long- and short-term changes in elemental concentration in a body as well as beard analysis. It is found that sulfur keeps almost constant over a long period, and arsenic shows very rapid changes with a few days' period, while mercury shows only long-term changes with the period of a few months. These behaviors are almost the same as those observed in beard analyses. On the other hand, bromine shows a certain seasonal changes; its concentration shows a certain trend of increasing in summer and decreasing in winter. Lead and calcium show very long-term changes, and the behavior of strontium is quite similar to that of calcium. The method is expected to give us information about history of changes in elemental concentration in a human body over a few or more years. It is expected that the behavior of arsenic showing rapid elevation within a few days can be explained as a response to intakes of arsenic-rich foods. It is expected that the method gives us a clue to the identification of the main pathways of human exposure to certain toxic elements.

A standard-free method for hoof samples taken from cattle such as cow, calf, pony and sheep has been developed in order to estimate the state of health of these animals. The standard-free method developed for human nails was confirmed to be applicable to quantitative analysis of hoof samples since the shape of continuous X-rays is almost the same for nail and hoof taken from these ungulate animals. Accuracy and sensitivity of the present standard method were examined by comparing the results with those obtained by an internal-standard method combined with a chemical-ashing method, and it is confirmed that the method is applicable to hoof samples taken from domestic animals of many species. The method allows us to quantitatively analyze untreated hoof samples and to prepare the targets without complicated preparation technique which often brings ambiguous factors such as elemental loss from the sample and contamination of the sample during preparation procedure. It is also confirmed that halogens, which are important elements for estimating the state of health and are mostly lost during chemical-ashing, can be analyzed without problem by the present method. It is found that elemental concentration of more than twenty elements can be constantly analyzed and it is expected to be quite useful in order to estimate the state of health and to make diagnosis of domestic animals. It is also confirmed that elemental concentration of essential elements in hoof is not so changed depending on the positions in the sliced sample along both horizontal and vertical axis.

The standard-free method developed by ourselves 13 years ago has been widely applied to quantitative analyses of hairs such as head hair taken from human and body hair taken from companion and domestic animals. In the present work, the standard-free method for feather and down samples taken from wild birds such as swan, waxwing, osprey, heron and crow is developed. It is found that the standard-free method developed for human hairs can be successfully applied to feather samples without essential modification since the main constituents of feather are almost the same as those for human and animal hairs and, consequently, the shape of continuous X-rays is also the same. Accuracy and sensitivity of the present method were examined by comparing the results with those obtained by an internal-standard method combined with a chemical-ashing method. The method allows us to quantitatively analyze untreated feather samples of very small quantities and to prepare the target without complicated preparation technique. It is expected that the method will become a powerful tool for the studies not only on the mode of life of wild birds but also on environmental contamination by toxic elements.

The method of quantitative analysis of oil samples in in-air PIXE has been developed on the basis of a standard-free method. The components of the continuous X-rays originated from air and backing film can be exactly subtracted using a blank spectrum after normalization by the yields of Ar K-α X-rays. The method was developed using nine oil samples including standard oils and its accuracy was confirmed by comparing the results with those obtained by the internal-standard method. Validity of the method for practical oil samples was confirmed for various kinds of oils such as engine, machine and cooking oils. It was found that the method is effective for various kinds of oils whatever elements we designate as an index element.

A standard-free method for living plants in in-air PIXE has been developed in order to clarify the mechanism of elemental transportation and movement in farm products. The components of the continuous X-rays originated from air and a backing film can be exactly subtracted using a blank spectrum after normalization by the yields of Ar Kα X-rays. It is found by observing the yield of continuous X-rays with passage of time that water content is continuously decreasing during irradiation with a proton beam in a case of pinched leaves. In contrary, it is kept almost constant during irradiation for the living plants to which water is continuously provided through the roots. Stability of the yield of continuous X-rays is a required condition for a standard-free method, which makes use of the yield of continuous X-rays mainly emitted from water content. It is confirmed that potassium concentration shows no large position dependence on a leaf, and it keeps almost constant during irradiation, which also indicates that regular metabolism is going on. As potassium is always contained in all kinds of plants in large amount, it is designated as an index element. As a result, it is found that the potassium concentration obtained by the present standard-free method shows quite consistent values with those obtained by the internal-standard method. The present method is confirmed to be quite useful for investigating movement of not only toxic elements but also essential elements reflecting metabolism in plants.

As a part of research project for food risk assesment in Asian countries, we have collected a variety of samples including dairy products from Mongolia for PIXE analysis. However, some kinds of milk products or fats such as butter, margarine, mayonnaise and cream are often hard to be homogenized with internal standards because of their viscosity and oiliness, which makes their sample preparation a tiresome work. We have tried to develop efficient preparation and measurement methods for in-air PIXE analysis of those samples. For butter and margarine, liquid internal standard method was taken after melting them with water bath. Pasty samples like mayonnaise and cream were mixed with liquid standard in agate mortar. The applicability of these methods was examined by checking homogeneity of so prepared samples on the basis of Ca concentration. Along with the internal standard methods, a standard-free method has also been worked out for those fatty samples. The obtained results have been fairly satisfactory and we have applied the methods to the Mongolian dairy samples.

We developed and reported standard-free methods for various bio-samples in both invacuum and in-air PIXE and they have been applied to quantitative analyses of traces of bio-samples whose weights are less than 0.1 mg. In this study, we established a method of quantitative analysis for bio-samples of nearly $1\mu \text{g}$ in in-vacuum PIXE. In order to improve sensitivity and accuracy of analysis for smaller samples on the basis of the standard-free method, which makes use of continuous X-rays emitted from the sample, it is essential to design appropriate backing materials for supporting the sample. In the present study, we have examined various backing materials such as thinner and threadlike backings. As a result, it was found that a thread-like backing made of extended adhesive, which contains almost no impurity, is the most suitable for bio-samples of extremely small quantities, since it produces no large amount of continuous X-rays. The method has been applied to quantitative analyses of small insects and plants. Moreover, the method was applied to analyses of small marine organisms such as opossum shrimps and squilla shrimps which play important roles in a food chain in marine ecosystem. The results gave us valuable information about regeneration of marine ecosystem in the Sanriku district attacked by the huge tsunami that occurred on 11 March 2011.

At Nishina Memorial Cyclotron Center (NMCC), a large number of studies in various research fields have been pursued under nation-wide common usage since 1993. One of the features of NMCC’s PIXE is the performance of many problem-based studies in order to determine effective measures against a range of issues. Various samples must be quantitatively analyzed quickly and accurately to cope with environmental issues. We have developed several original methods for analyzing a variety of samples with good sensitivity. Specially designed absorbers were developed for sensitive analyses of samples composed of heavy elements. In addition, a powdered-internal-standard method was established for the accurate quantitative analyses of powdered samples whose matrixes are heavy elements. The standard-free method for bio-samples allowed us to quantitatively analyze untreated samples, micro samples of nearly 1 $\mu$g, and live bio-samples. We have been tackling a number of environmental problems in many countries in Southeast and East Asia using these original methods. We also wrestled with environmental problems caused by the massive tsunami that struck the Sanriku district of Japan in March 2011.

In this paper, we measured more than 4200 beard samples collected from a single person every day over a 12-year period and analyzed them based on a standard-free method. It was found that the sulfur concentration remained almost constant from a long-term perspective but showed small periodic elevations. Silicone, phosphorus and rubidium showed significant periodic changes with a cycle of several years. While manganese, strontium and molybdenum seemed to show long-term changes, there were no significant differences due to their large standard deviations. Concerning seasonal changes, silicone and phosphorus showed decreasing trends in summer, albeit without significant differences. The bromine concentration showed clear seasonal variations, rising in winter and falling in summer, suggesting the possible influence of seasonal changes in the elemental components in the airborne particles that pass over to Japan from the Asian Continent. These findings confirmed that beard analyses are useful not only for evaluating changes in trace element concentrations in the human body but also for clarifying the main pathways of the intake of these elements.

Internal Standard Method, which has been widely used for quantitative analysis of general bio-samples, requires certain skill in sample preparation since it sometimes accompanies a noticeable error due not only to inaccuracy of weight measurement both of a sample and of an internal standard but also to nonuniformity of the internal standard in the target. In this work, a standard-free method of quantitative analysis, based on the fact that the total yield of continuous x-rays from the sample approximately corresponds to effective weight of the sample, has been developed and applied to some typical bio-samples (NIST Bovine liver, a rat liver and a human serum). As a result, values of concentration obtained by the present method present good agreement with those obtained by the internal standard method. Further, in a case where target preparation is performed by unskilled person, the present method gives better results than those obtained by the internal standard method. It is also found that this method is applicable to almost all soft-tissue samples and, moreover, to targets prepared by a different method.

A standard-free method developed by ourselves has been applied to bio-medical samples of less than 1 mg or less than 1 μl, to which an ordinary internal standard method can not be accurately applied. As the result, correct values of potassium concentration in NIST-Bovine liver samples of nearly 0.5 mg were obtained. Furthermore, the standard-free method has been applied to small quantity of serum and whole-blood samples, and satisfactory results were obtained. For samples which easily peel off from a backing film, such as serum and whole blood, a carbon tape with an adhesive agent was also used as a backing film, and it is found that this method is also applicable. Moreover, the standard-free method was confirmed to be effective for a case where a specially designed x-ray absorber is used.

A Standard-free method, which has been developed by ourselves, was applied to powdered biomedical samples to which a conventional method of quantitative analysis, such as an internal standard method, can not be successfully applied. First, NIST-Bovine liver and NIES-rice targets, which are powdered as they are, were analyzed by the standard-free method and correct quantitative values of potassium and manganese concentration, which are consistent with those obtained by the conventional internal standard method, were obtained. Furthermore, for relative values of concentration of various elements, almost the best results we have ever had were obtained. Next, the present method has been successfully applied to a hard tissue sample (mouse bone), which is quite difficult to be accurately analyzed by the internal standard method. In order to solve the problem of falling off of powdered samples from a backing film, a carbon tape with an adhesive agent has been examined and found to be quite effective.