Determination of depleted uranium in human hair by quadrupole inductively coupled plasma mass spectrometry: method development and validation

Abstract

The presence of depleted uranium (DU) in the environment and its possible transfer to humans has been a matter of concern for the potential risks to human health. Depleted uranium has been used for military purposes in the last few years of conflict due to its great availability and low cost. Uranium content has been mainly determined in biological fluids, such as urine and blood. Human hair can be considered as a good alternative indicator of exposure to DU thanks to its ability of accumulating chemical elements over a long period of time. Determining the isotopic composition of elements always requires using a sensitive and precise analytical technique. In this regard, isotope ratio quadrupole inductively coupled plasma mass spectrometry was considered as a qualified analytical technique and consequently employed in the present study in combination with a desolvating sample introduction system. The method was in-house validated at three different levels of concentration of depleted uranium (50, 500 and 1000 ng/l) according to common standards and guidelines. The limit of quantification of the method in hair was 7.21 μg/kg with a within-laboratory reproducibility, in terms of variation coefficient, of 16.8, 6.1 and 5.5% for the three levels, respectively. A novel analytical approach was applied to the specificity studies for hair contaminated by DU with different background levels of NU.

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Introduction

The process of enrichment of natural uranium (NU) produces depleted uranium (DU) as a by-product. Natural uranium contains 0.01% of ²³⁴U, 0.72% of ²³⁵U and 99.27% of ²³⁸U, whereas, DU, used to produce munitions, contains 0.2%–0.3% of ²³⁵U and 99.8%–99.7% of ²³⁸U. Because of its great availability, low cost (if compared with the cost of NU) and high density (19.07 g/cm³), DU has been also used in the production of tips of armour piercing bullets, cruise missile nose cones and as a part of protective armour for tanks.^1,2

The element uranium is a strong reducing agent particularly in aqueous systems,³ its metallic form presents high reactivity and its powder can spontaneously ignite at temperatures of 600–700 °C forming complex oxides, such as triuranium octaoxide (U₃O₈ predominately), uranium dioxide (UO₂) and uranium trioxide (UO₃). In an environment contaminated with DU particles, the mobilization of U from DU particles will be dependent on the oxidation state of U. As the weathering rate is higher for U₃O₈ than for UO₂, the mobilization of U from U₃O₈ particles should be increased compared to UO₂ particles, when in contact with body fluids or present in soil-water systems. Therefore, the presence of respiratory UO₂ and U₃O₈ particles and the mobilisation of U from oxidized DU particles should be taken into account in order to assess the health impact.⁴

Since the specific activity of DU is low (39.4 kBq/g), the radioactivity is considered weak. However, depending on the degree of exposure, DU may be dangerous for human health when ingested or inhaled because of its chemical toxicity.⁵

The presence of DU in the environment (soil and water) and its possible transfer to humans might be ascribed to its use for military purposes in the recent years of conflicts. In particular, when a contaminative aerosol of DU is produced and dispersed during a war conflict, the military personnel may inhale this dispersion thus potentially suffering adverse effects on their health.^3,5,6 In addition, this aerosol can be a potential hazard for population living in war areas; with regard to that, the World Health Organization published a monograph on possible health consequences for the inhabitants of the involved regions.⁷ A report on the occurrence of DU in Iraq after the Gulf War affirmed that the incidence of several cancers (including childhood leukaemia), congenital malformations and immune system diseases has increased for the residents.⁸

The content of uranium in human body has been mainly studied by analyzing urine^9–15 and blood,^16,17 since this kind of sample is relatively easy to obtain, if compared to other biological specimens, sampling of human hair can be a non-invasive alternative. Hair is considered to be a complementary tool as an indicator of human exposure to environmental pollution, even over a long period of time (from a few weeks to some months), for its ability to accumulate a certain number of substances, among which, chemical elements. The levels of these chemicals in hair may be even higher than in other biological matrices, like urine and blood. On the other hand, exogenous sources of these elements may contaminate hair surface thus affecting the results of the analysis, but this issue can be addressed by using a proper surface treating procedure. The main advantages offered by hair analysis are the simplicity of sampling, transport and handling in laboratory during sample preparation. Hair analysis was successfully applied in several sectors, namely, the assessment of environmental and occupational exposure, the forensic sciences, the evaluation of the nutritional status of patients and as a complementary tool of investigation in disease's diagnosis.^18–28

Different analytical techniques have been employed so far in hair analysis in order to quantify its content of chemical elements. Among them, inductively coupled plasma mass spectrometry (ICP-MS) is one of the most successful multielemental techniques thanks to its high performance and sensitivity.^22,29–34 Laser ablation inductively coupled plasma double focusing sector field mass spectrometry (LA-ICP-MS) was employed as an effective analytical tool for depth profiling of hair strands.³⁵

Inorganic mass spectrometry has demonstrated its ability to offer very accurate and precise measurements of isotope ratios in different samples.^36–39 The use of single-collector and multi-collector ICP-MS for isotope ratio determination has been fully investigated by different authors.⁴⁰ The conditions for a precise and accurate determination of ²³⁴U/²³⁸U ratios in geological materials were optimized by multiple collector (magnetic-sector) inductively coupled plasma mass spectrometry (MC-ICP-MS).⁴¹ Thermal ionization mass spectrometry (TIMS) is recognized as a very precise technique for the determination of the isotopic composition of different elements in the Periodic Table and it is appreciated for its capability of analyzing very small aliquots.^42,43

The presence of spectral interferences and matrix effects may influence the accuracy of the produced results, especially when biological samples are to be analysed. However, these interferences can be overcome by using suitable analytical approaches and/or appropriate instruments. An apparatus like sector field ICP-MS (SF-ICP-MS) is capable of discriminating the overlapping species by setting different resolutions, while different reaction gases can be used to reduce or eliminate chemically these interferences by dynamic reaction cell ICP-MS (DRC-ICP-MS). Sector field technology permits one to get lower detection limits with much higher sensitivity when compared with a quadrupole ICP-MS, nevertheless, a gain in sensitivity of one order of magnitude can be obtained by combining a high-efficiency sample introduction desolvating systems with this equipment.^44–47

Mostly, total uranium was determined in hair by means of ICP-MS,^48–51 only a few authors accomplished the determination of DU by isotope ratio measurements. In fact, the presence of NU in the environment makes this investigation extremely difficult.^52,53 A longitudinal distribution of uranium content in a single hair strand was determined by LA-ICP-MS.⁴⁸ In fact, this powerful analytical technique does not require any sample dissolution, this can be an advantage, especially in isotope ratio measurements on surfaces of biological samples, when the precision is a crucial factor.⁵⁴

In this article, a method for the quantification of DU in hair was developed by means of isotope ratio ICP-MS. A quadrupole mass spectrometer was chosen for determining the isotopic composition, because this instrument is cost-effective compared to a high-resolution apparatus and it is mostly used among routine analytical laboratories. Additionally, the method was in-house validated according to the standard ISO/IEC 17025 : 2005.⁵⁵ A novel analytical approach was applied to the specificity studies for hair with different background levels of NU, to the best of our knowledge there are no similar studies in this field.

Experimental

Sample treatment and analysis

Human hair specimens with different contents of uranium and a Certified Reference Material (CRM) on human hair were employed to develop and validate the method.

Human hair samples were collected from two groups of unexposed subjects living in urban areas of Italy. Sampling and washing of hair were carried out according to a previously established procedure.²² The washing procedure was necessary in order to remove the exogenous material on the hair surface. After washing, hair specimens were dried in an oven at 85 °C for approximately 16 h. The dried weights of the specimens were in the range of 0.2–0.3 g.

Dried samples were subsequently transferred and weighed (analytical balance Mod. BP 210 D, Sartorius Stedim Italy, Florence, Italy) into decontaminated high-pressure PTFE vessels, added with 2 ml of 67–69% Super Pure Nitric Acid (Romil, Cambridge, Great Britain) and left to stand overnight at room temperature in a chemical hood for the pre-digestion step. This step was carried out in order to avoid strong chemical reactions during the completion of digestion. The following day, the acid-assisted microwave (MW) digestion was performed (Ethos plus, Milestone, FKV, Sorisole, Bergamo, Italy) by adding 1 ml of 30% Super Pure Hydrogen Peroxide (Romil, Cambridge, Great Britain) and applying the following programme: 3 min at 250 W followed by 6 min cooling; 5 min at 250 W with 5 min cooling; a further 5 min at 450 W and finally 5 min at 500 W. After adequate cooling, the digested solutions were quantitatively transferred into Falcon^® tubes of 50 ml by adding high purity deionised water up to a volume of 20 ml. These solutions were stored in a refrigerator at +4 °C. A pool of hair samples of a volume of 500 ml was prepared by collecting the final digested solutions of 20 ml.

The Certified Reference Material BCR CRM no.397 “Human Hair” was used in the recovery and reproducibility studies as human hair in a suitable powder form. This material was not certified either for total uranium or isotope composition. A concentration of NU of 32 ng/g was quantified in this material. A fraction of this powder material weighing about 0.1 g was treated and digested as previously mentioned. The moisture content of the CRM was determined by strictly following the “instruction of use” reported in the certificate. In particular, a separate portion of CRM weighing about 0.1 g was dried in an oven at 90 °C for 16 h (successive weighings should not differ by more than 0.2 mg). A moisture content of 4.5% was obtained and the final results were all corrected for the dry mass.

Depleted uranium standard solution was prepared from ICP-MS calibration standard (Inorganic Ventures, Lakewood, NJ, USA) certified for the uranium content and the isotopic composition (²³⁵U, 0.204% ± 0.002 and ²³⁸U, 99.8% ± 0.1). A standard solution of 1 μg/l of DU was prepared from this certified calibration standard and used for the correction of the instrumental mass bias. Natural Uranium standard solution Standard Reference Material 3164 was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA).

Samples and calibrants were diluted and prepared for the analysis by high purity deionised water with a specific resistance > 18 MΩ cm (Easy Pure, PBI International, Milan, Italy). A dilution factor of 2 was applied. The quantification of uranium was carried out by using the addition calibration approach (matrix-matched calibration standards) and a logarithmic curve was adopted to fit the calibration points.

Two different mass spectrometers were employed in this investigation, namely, a Q-ICP-MS (Elan DRC II, Perkin Elmer, SCIEX, Norwalk, CT, USA) and a SF-ICP-MS (Element 2, Thermo-Finnigan, Bremen, Germany). A high-efficiency sample introduction desolvating system (Apex-IR, ESI, Omaha, NE, USA) was combined with the quadrupole mass spectrometer. This system was equipped with a MicroFlow PFA nebulizer operating at 200 μl/min, the heater was set at a temperature of 140 °C and the condenser at a temperature of 2 °C. Instrument settings and data acquisition parameters for the quadrupole spectrometer are listed in Table 1.

Table 1 Instrument settings and data acquisition parameters for Elan DRC II

RF power (W)	1300–1400
Gas flow rates (l/min)	Plasma, 15; Auxiliary, 1.0; Nebulizer, 0.8–1.0
Interface	Ni cones
Extraction lens voltage	Optimized for maximum I (^24Mg, ^115In, ^238U)
Analytical masses	^235U, ^238U
Dwell time (ms)	100
Sweeps/reading	400
Readings/replicate	1
Replicate	10
Integration time (s)	40
Settling time	200 μs

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DU percentage calculation

The quantitative analysis of the major isotope of uranium does not allow one to distinguish the exposure to DU from that one to NU (environmental). As reported in a previous study on the uranium isotopic composition in urine by ICP-MS, it is common practice to use a factor D in the DU determination. This factor is the percentage of ²³⁵U obtained from (R/1 + R) × 100, where R is the measured isotope ratio (²³⁵U/²³⁸U) corrected for the instrumental mass bias.¹³

The percentage of DU (P_DU) is linked to this factor D. The value of P_DU may vary from 0 to 100, being 0 when D = 0.72 (natural uranium) and 100 when D = 0.20 (depleted uranium). The P_DU is calculated by using the following mathematical equation:¹³

where A is the intensity of ²³⁵U in counts/s, 0.72 is the isotopic abundance of ²³⁵U in NU and −0.52 is the difference of the abundances of DU and NU (0.2–0.72).

Results and discussion

Interference study

Any possible spectral interference on masses ²³⁵U and ²³⁸U, originating from the mixture reagents and/or the matrix, was evaluated in a preliminary study. The formation of the argon-based polyatomic ions of Pt that potentially interfere on the isotopes ²³⁴U, ²³⁵U, ²³⁸U was prevented by using Ni sampler and skimmer cones.

A previous publication on the subject of DU content determination in urine highlighted that the mass 235 can be interfered by the formation of a peak of undefined polyatomic ions at mass 234.81.¹⁴ The occurrence of this polyatomic species in digested hair samples was studied by SF-ICP-MS working in medium resolution (4000 m/Δm) in the mass range of 234.5–235.5 m/z. The spectra of two digested solutions of hair (one unspiked and the other spiked with a fixed amount of DU) were observed for this interference. Spectrum A in Fig. 1 represents the peak at mass 235.1 (isotope ²³⁵U) due to the NU in hair of the unspiked solution, while spectrum B shows the increase in signal intensity of the peak at mass 235 due to the spiked solution. Comparing the two spectra, it can be seen that there is no interfering peak at mass 234.81 as previously found by other authors analysing urine.¹³ Probably, the unknown molecular ion formed in the analysis of urine is not produced in hair.

Fig. 1 Typical SF-ICP-MS spectra in the mass range of 234.5–235.5 at mass resolution of 4.000 (m/∆m). Unspiked (A) and spiked (B) digested solution of hair with DU.

In a previous work, depleted uranium in fish was successfully determined by means of isotope ratio dynamic reaction cell ICP-MS using ammonia as a suitable gas to homogenise the distribution of the ion energy, typically deriving from the plasma (energy damping), and gain in precision.⁴⁷ The same gas was employed in the present study obtaining a different effect, a considerable and unexpected increase in the isotope 235 signal. In this matrix, ammonia probably forms unknown polyatomic species that hamper a reliable DU quantification. No other reaction gas was tested and the method was developed and validated in standard mode and the settling time was set at 200 μs as recommended by Perkin Elmer to improve measurement precision.

Instrumental detection limit and isotopic ratio measurement limit

For total uranium, the instrumental detection limit (IDL) was calculated by three times the standard deviation (sd) of measurements of 10 blank samples (a solution of 2 ml of HNO₃ + 1 ml of H₂O₂ diluted up to 20 ml with deionised water) and 10 spiked blank samples (5 ng/l).

The calculation of the isotopic ratio required the determination of the uranium minimum detectable quantity. Since the abundance of the ²³⁵U isotope is rather low (0.72% for NU and about 0.2% for DU) this parameter becomes critical. This minimum level, named isotopic ratio measurement limit (IRML), was calculated by dividing the IDL by the percentage of ²³⁵U present in uranium. The IDL and the IRML were found to be of 0.03 ng/l and 16 ng/l (DU), respectively.

Method validation

Method validation in analytical chemistry is considered one of the technical parts of the comprehensive scheme of quality assurance (QA). Single-laboratory method validation is commonly accepted in several circumstances, among these, when the feasibility of the method is to be ensured before approaching an expensive formal collaborative trial, when data from a collaborative trial is not available or conducting a collaborative study is impossible.⁵⁶

Method parameters to be evaluated were selected according to the current legislation and the available guidelines.^55,57,58 The following method characteristics were then evaluated: limit of detection and quantification of the method, working concentration range, specificity, trueness through recovery, within-laboratory reproducibility and stability of the analytical solutions.

Limits of the method

The limits of detection (LoD) and quantification (LoQ) of the method were calculated as 3 times and 10 times the deviation of factor D (fluctuating around the value 0.72) by running two series of 10 digested solutions of hair. These solutions were considered as a blank because of the absence of DU in those samples.

The final dilution and the mean weight of hair samples were taken into account for calculating those limits. Their values are shown in Table 2.

Table 2 Method performance

Parameter	Value
IDL	0.03 ng/l
IRML	5 ng/l (NU); 16 ng/l (DU)
LoD, LoQ	12 ng/l, 36 ng/l
LoD, LoQ of the method	2.38 μg/kg; 7.21 μg/kg (DU)
Working concentration range	100–2000 ng/l (DU)
	Level 1 50 ng/l	Level 2 500 ng/l	Level 3 1000 ng/l
Recovery %	73	105	90
Within-laboratory reproducibility (CV %)	16.8	6.1	5.5

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Working concentration range

The working concentration range was investigated in order to verify the applicability of the method on the selected matrix over a certain concentration range. Six calibration standard solutions (0, 100, 250, 500, 1500, 2000 ng/l of DU) were prepared on a pool of digested hair containing 250 ng/l of NU. Subsequently a calibration curve was plotted by calculating the concentration of DU (C_DU) versus its concentration added using the formula: C_DU = (D_S − D_P)/(D_c − D_R); where D_S is the value of D for each spiked standard solution, D_P is the value of D of the pool of digested hair (250 ng/l of NU), D_c is the value of D obtained from the certified abundances of ²³⁵U and ²³⁸U (0.20358) and D_R is the value of D of NU. A working concentration range of 100 − 2000 ng/l was finally accepted as applicable on hair samples for DU.

Specificity

The term describes the extent to which a method uniquely reacts to a selected analyte and this characteristic is strictly dependent on the analytical technique of choice. The specificity study was performed in order to investigate the effect of potential interferent species on the quantification of the analyte in hair.

Since a natural level of NU might mask the real content of DU, two different studies, a qualitative and quantitative one, were carried out to determine the detectable and the quantifiable level of DU in the presence of NU.

For the qualitative test, a solution of digested hair containing 250 ng/l of NU was spiked with 50 ng/l of DU. The solution was divided into six aliquots, each of them was added with an increasing amount of NU (250, 500, 750, 1000, 1250, 1750 ng/l). All the aliquots were analysed for the isotopic ratio and the factor D was calculated as mentioned. A calibration plot was fitted with the logarithmic equation:

D = 0.035 ln (NU conc.) + 0.42 (b)

with a correlation coefficient of r² = 0.9031. Afterwards, ten samples of a digested solution of hair (containing only NU) were analysed and an average value of D = 0.7091 (R = 0.00714) was obtained with a standard deviation of 0.0015. By considering this standard deviation, the limit value of D that still allows one to determine DU is (0.7091 − 0.0015) = 0.7075. If this value of D is placed in the equation (b), a corresponding NU concentration of 2590 ng/l is achieved. The value of 2590 ng/l (digested solution), or 518 μg/kg (hair tissue), represents the limit concentration of NU that permits one to qualitatively detect DU at a concentration of 50 ng/l. At higher concentrations of NU or lower concentrations of DU, it becomes impossible to distinguish between them. In general, the method is capable to reveal DU up to a concentration of 1/50 of that of NU.

The quantitative study was done to verify the capability of the method to distinguish D values corresponding to two similar amounts of DU, i.e., 1.5 and 2 times the LoQ (50 and 75 ng/l). These two levels of DU were prepared in solutions containing three different concentrations of NU. A new digested solution of hair with a lower concentration of NU (50 ng/l) was used for this test. One unspiked and two spiked solutions were prepared (0, 500 and 1000 ng/l of NU). Subsequently, each of them was added with different concentrations of DU, namely, 0, 50 and 75 ng/l. The result of this quantitative study is shown in Fig. 2, it can be seen that there is a quite linear response between the concentration of DU and the factor D in function of three different levels of NU. The slope of the curve changes depending on the amount of NU, in particular, the sensitivity decreases at high concentration of NU. At high levels of NU, the values of D become very close to each other. It is reasonable to suppose that there is a limit value of NU above which the difference in D values could be too small to distinguish two close concentrations of DU. Starting from this assumption, this hypothesis was verified by evaluating the previous data at 50 and 75 ng/l of DU. To this aim, the values of the factor D and their differences obtained in 50, 550, 1050 ng/l of NU are reported in Table 3. This table shows that a NU concentration increase leads to a progressive decrease in the differences of D values. Moreover, the differences were plotted as a function of NU concentration in Fig. 3 and the following logarithmic equation was obtained:

D difference = −0.0144 ln (NU conc.) + 0.1067 (c)

with a correlation coefficient of r² = 0.9874.

Table 3 Differences in D values for two levels of DU versus different amounts of NU

NU (ng/l)	DU 50 ng/l	DU 75 ng/l	D difference
	Factor D ± sd
50	0.475 ± 0.0040	0.424 ± 0.0048	0.051
550	0.645 ± 0.0030	0.632 ± 0.0027	0.013
1050	0.667 ± 0.0021	0.658 ± 0.0017	0.009

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Fig. 2 Correlation plots between D values and DU concentrations. Effect of NU levels on the analytical response.

Fig. 3 Variation of D differences as a function of NU levels in analytical solutions containing 50 and 75 ng/l of DU.

If this limit difference is calculated using three times the standard deviation of 10 measurements of a solution containing 1050 ng/l of NU, a value of D difference of 0.0063 is finally achieved. This last value can be placed in the equation (c) obtaining a concentration of NU of 1066 ng/l (digested solution) or 212 μg/kg (hair tissue). This level of 1066 ng/l is supposed to be the limit of concentration of NU that allows one to quantify concentrations of DU close to the LoQ.

Recovery

The trueness of measurements was assessed through the recovery due to the lack of CRMs for depleted uranium in human hair. Three independent series of homogenous samples of about 0.1 g of the CRM powder were spiked with three different levels of concentration (50, 500 and 1000 ng/l) of DU.

The additions were made before digesting the samples and the whole recovery was calculated by running each series of independent samples in three different days. For each level of addition, three spiked and one unspiked sample were digested. A seven-point calibration curve was employed to quantify DU (50, 100, 200, 500, 1000 and 1500 ng/l).

An acceptance range of 90–110% was chosen for this research.⁵⁷ The results for the mean recovery are shown in Table 2. At a very low level (50 ng/l), close to the quantification limit, a satisfactory recovery was rather challenging to achieve, nevertheless, the recoveries for the other two levels of additions fell well within the established range.

Within-laboratory reproducibility

The within-laboratory reproducibility is the precision obtained in the same laboratory, under predetermined conditions over long time intervals, but some changes must be applied to the analytical conditions. The within-laboratory reproducibility can be calculated as the coefficient of variation expressed as percentage (CV %) and by analyzing a certain number of independent sets of samples in different days.

The analysis of three independent spiked CRM samples was repeated in three different days at the three levels of concentration (50, 500 and 1000 ng/l) in different operational conditions. In particular, the following changes have been applied: a) batches of reagents; b) pipettes; c) operators. The results of the within-laboratory reproducibility for the three levels of additions are summarized in Table 2.

A within-laboratory reproducibility (in terms of CV %) ≤ 20% was reported as acceptable for a mass fraction ≥ 10 μg/kg to 100 μg/kg.⁵⁷ In this study with a mass fraction below 10 μg/kg, the CV % found was good enough for the low levels of concentration.

Stability studies

The stability studies are required when the influence of storage conditions on the level of the analyte in the sample is unknown. In fact, the result of the analysis may be affected by the insufficient stability of the analyte in sample during the time of storage. It is important to check the stability of the analytical solutions in different storage conditions, after a precise and scheduled period of time. In this study, any possible loss of analyte was checked by storing a set of ten digested hair samples (containing 100 ng/l of DU) at three different temperatures, namely, +20 °C (daylight), +4 °C (refrigerator) and −20 °C (freezer). The DU content of each of the three sets of analytical solutions was analysed after a period of time of 2, 4, 6 and 9 weeks.

The recovery was taken into account to verify the stability of the solutions. At time zero, the mean recovery was 104%. The following minimum and maximum recoveries were obtained for the three storage conditions: 94% and 108% (+ 20 °C); 95% and 104% (+4 °C); 96% and 105% (−20 °C). The overall results confirmed the good stability of the solutions over a time of 9 weeks of storage.

Conclusions

The present article describes a precise and sensitive quantification method of depleted uranium in human hair. A background level of NU up to 20 times greater than that of DU can be accepted for the quantification of DU, this being the limit of applicability of this method. Whereas, with levels of NU ranging from 20 to 50 times higher the method can be used for screening purposes only.

The Q-ICP-MS exhibited high performances in spite of the very low level of the analyte measured and the combination with the desolvating sample introduction system turned out to be successful to gain adequate sensitivity.

The validation of the method was carried out at three different levels of concentration and the validation parameters were all characterized. The results obtained satisfied the requirements (when applicable) of the available guidelines.

The present method has never applied to human hair samples contaminated by DU because of the lack of samples, nevertheless, it is suitable to be applied to monitoring programmes.

Acknowledgements

The authors thank Gianluca Finocchiaro for editorial assistance on the manuscript.

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