Validation of an Analytical Method for the Determination of Manganese and Lead in Human Hair and Nails Using Graphite Furnace Atomic Absorption Spectrometry

Abstract

This article describes the validation of analytical methods for the determination of Manganese (Mn) and lead (Pb) by graphite furnace atomic absorption spectrometry (GFAAS) in human hair and nail samples. Method validation parameters such as linearity, repeatability, reproducibility, and precision were determined. In addition, the limit of detection (LOD), the limit of quantification (LOQ), and measurement uncertainty were calculated. The developed method was linear in the concentration ranges of 0.001–0.015 and 0.002–0.020 µg·L⁻¹ of Mn and Pb, respectively. The determination coefficients obtained were greater than 0.995. The recoveries obtained after the addition of the standard concentration for the metals ranged from 84.80–107.98%, with a precision not exceeding 12.97% relative standard deviation. The calculated LOD and LOQ for Mn and Pb are within the ranges established by Commission Regulation (EU) No. 836/2011. The expanded uncertainty was estimated to be less than 9.93–6.59% for Mn and Pb. Matrix effects were also studied, finding a smooth effect in both matrices. The analysis of 30 samples of each type revealed the presence of Mn in 30 and Pb in 13 samples. Overall, the proposed validation method was considered optimal for the determination of Mn and Pb.

1. Introduction

The production of goods and services has currently caused concern on the part of regulatory and academic organizations dedicated to the identification, monitoring, and control of hazardous metal residues that can cause chronic and/or acute intoxication scenarios for humans and the environment [1,2]. Modern industries are characterized by the production and commercialization of products with a high presence of heavy metals that exceed the exposure limits for the receptor organisms. This situation is currently worsening due to the improper use of phytosanitary products (pesticides) and inorganic fertilizers in conventional extensive agriculture, the lack of integrated management of industrial waste deposited in ecosystems, and the use of conventional energy sources such as hydrocarbons contaminated with heavy metals such as lead, among others [3].

According to the study entitled “determination of heavy metals in urine of patients and tissue of corpses by atomic absorption spectroscopy”, the behavior of heavy metals in humans and the environment is cyclical: industry, atmosphere, soil, water, food, and humans interact in different environmental and biological matrices that can alter their degree of danger [3]. This cyclical behavior leads to scenarios of chronic and/or acute intoxication via ingestion, inhalation, and dermal contact mechanisms [3]. As a result, the accumulation in the body and the affectation of organs and glands such as the heart, brain, kidneys, bones, and liver, among others [3,4,5,6]. This situation has worsened in developing countries that have increased their industrial activities and, therefore, the availability and use of products with a high content of heavy metals, which is affecting the current population [3].

That said, in Colombia, heavy metal poisoning is recurrent in multiple areas and economic sectors, particularly the identification of heavy metals in the agricultural sector (food and non-food goods), due to the indiscriminate use of pesticides. Colombia is the country with the fifth highest use of pesticides in the South American continent after Argentina, Bolivia, Brazil, and Chile, resulting in annual consumption of 69,862 tons, which were used in the agricultural sector at the end of 2019 [7] these pesticides, organophosphates (OPPS) and carbamates are generally the most used due to the geographical location of the country [8]. This class of pesticides, within their chemical structures, possesses heavy metals, and the presence of Mn and Pb predominates. Therefore, several authors have conducted studies showing a positive association between metal levels in patients with diseases such as Alzheimer’s disease, breast cancer, hormonal imbalance, hypertension, rheumatoid arthritis, and pediatric neurocognitive impacts [9,10,11,12,13].

However, the use of biological matrices such as hair and nails for the analysis of levels of various toxic contaminants has several advantages over other biological matrices. Characteristics such as stability, low cost, easy collection, easy transport, and easy storage, among others, have demonstrated their potential application in various areas [14]. Different studies have carried out an analysis of metals in hair and nails as biomarkers of exposure and biomonitoring of concentrations of metals such as Mn and Pb, showing that the levels of heavy metals in the human body are associated with some factors such as geographical location, quality of life and work environment [15,16,17,18,19,20]. Hair and nails are bioindicators that allow the analysis of exposure levels and concentrations of different pollutants present in the human body [9]. These biological matrices allow digestion in an acid medium accompanied by an oxidizing agent to degrade the organic matter and then be analyzed by atomic absorption spectrometry with a graphite furnace. The levels of heavy metals in the human body are monitored to determine potential risks. Nearby sources of exposure or possible responses to ill health can be determined. This monitoring is usually performed with the analysis of hair samples. The environmental protection agency (EPA) considers it one of the most important biomarkers [21,22].

In this sense, this research seeks to characterize the population of interest and determine the concentration of Mn and Pb in samples of nails and hair of children between 7 and 10 years of age, since in Colombia there are no studies that develop methodologies for the determination of these metals by exposure to pesticides and other environmental contaminants. Additionally, it describes the method developed and validated using the atomic absorption technique in a graphite furnace and applied to a child population in a rural area located in the town of Sumapaz in the city of Bogota, Colombia.

2. Materials and Methods

2.1. Instrumentation

For this study, an atomic absorption spectrometer (model AA-7000, Shimadzu, Kyoto, Japan) equipped with Dual background correction system: high-speed self-reversal (SR-method) and deuterium lamp, graphite furnace (GFA-7000), and High-Performance Autosampler (ASC-7000) was used. For the analysis SR method, pyrolytic coating graphite tube and Single element hollow cathode lamps (HAMAMATSU PHOTONICS K.K., Hamamatsu, Japan) of Mn and Pb were used as light sources, and the operating conditions are shown in

Table 1. Instrumental conditions.

Parameter	Settings
	Mn	Pb
Lamp current (mA)	10 mA/600 mA	8 mA/300 mA
Lamp mode	BGC-D2	BGC-SR
Slit width (nm)	0.2 nm	0.5 nm
Wavelength (nm)	279.5 nm	283.3 nm

. To clean all human hair and nail samples, an Elmasonic model ultrasonic bath was used with a programmable temperature between 30–80 °C and a frequency of 37 kHz of ultrasound energy.

2.2. Reagents

All calibration curves were prepared using Manganese (Mn) and Lead (Pb) atomic absorption stock solutions (1.0 g·L⁻¹) purchased from PANREAC Applichem (Darmstadt, Germany) by making successive dilutions. For the preparation of all the solutions, ultrapure water obtained from an Elix Advantage 5 system (from Merck Millipore, Burlington, (Massachusetts, MA, USA) was used. All chemical reagents used were of standard analytical grade, including nitric acid (65%) hydrogen peroxide (30%), Triton X-100, acetone purchased from Merck (Darmstadt, Germany), and palladium nitrate matrix modifier Accustandard (Connecticut, CT, USA). Certified Reference Material CRM (ERM^®-DB001) of human hair trace elements was obtained from Institute for Reference Materials and Measurement (Geel, Belgium). European Commission.

2.3. Collection of Hair and Nail Samples

According to the location closest to the exposure of pesticides by crops, the locality of Sumapaz in the city of Bogotá, was selected for the collection of samples. A total of 30 hair samples of 5 cm in length from the tip, approximately 300 mg cut with sterilized stainless-steel scissors, and 30 nail samples of approximately 200 mg were taken from the hands and cut with sterilized nail clippers from boys and girls between the ages of 7 to 10 years old. The samples were sealed in paper envelopes and stored in a dry place at room temperature.

2.4. Cleaning Human Hair Procedure

All hair samples and certified reference material (CRM) were washed in a 1.0% m/v non-ionic detergent solution of (Triton X-100 Merck) for 15 min in an ultrasonic bath, then rinsed with Milli-Q water. Then 10 mL of 1.0 N HNO₃ was added, restarting the ultrasound bath for 10 min, and samples were rinsed again with Milli-Q water and finally transferred to filter paper disks (110 mm diameter, Whatman^®, Buckinghamshire, UK) and cleaned and dried at 70 °C for 4 h.

2.5. Cleaning Nails Procedure

There is no specific reference material available for nails. The validation parameters of this matrix are performed by recovering known standard additions to a set of unknown samples, according to (The Commission of The European Communities, 2002). Different studies carried out by various authors show acceptable results without the use of certified reference material from this matrix [2,4,16,17,18,19]. All nail samples were washed in a 1.0% m/v non-ionic detergent solution of (Triton X-100 Merck) for 15 min in an ultrasonic bath, then rinsed with Milli-Q water. Then, 10 mL of 1.0 N HNO₃ was added, restarting the ultrasound bath for 10 min, and the samples were rinsed again with acetone, rinsed again with Milli-Q water, and finally transferred to filter paper disks (110 mm diameter, Whatman^®) and cleaned and dried at 70 °C for 4 h.

2.6. Digestion Human Hair Samples Procedure

Reference material (CRM) and several samples of human hair (approximately ~80 mg of dry samples) were weighed in duplicate and transferred to glassware previously washed with 1.0 N HNO₃. Subsequently, the samples were mineralized by adding 1.5 mL of HNO₃ and heating at a temperature of 70 °C for 1 h. Then, the digestion products were treated with 3.0 mL of hydrogen peroxide H₂O₂ and heated to 70–80 °C for 2 h until the yellow color disappeared, then cooled and diluted to a volume of 10 mL.

2.7. Digestion Nails Samples Procedure

Several samples of human nails (approximately ~40 mg of dry samples) were weighed for duplicate and transferred to glassware previously washed with 1.0 N HNO₃. Subsequently, the samples were mineralized by adding 1.5 mL of HNO₃ and heating at a temperature of 70 °C for 1 h. Then, the digestion products were treated with 2.5 mL of hydrogen peroxide H₂O₂ and heated to 70–80 °C for 2 h until the yellow color disappeared, then cooled and diluted to a volume of 10 mL.

2.8. Analytical Procedures

Manganese and Lead analysis was performed using GFAAS following the oven schedule shown in

Table 2. Furnace graphite conditions.

Metal	Temperature (°C)-Ramp
	Ashing	Atomization	Cleaning Out
Mn	800/10-3	2200/0-3	2500
Pb	700/10-3	2000/0-3	2500

. All digested samples of Certified Reference Material (CRM), hair and nail were transferred to vials and taken to the autosampler, then analysis was scheduled using the “WizAard” software injection of a volume of 10 μL and repetition conditions of each sample in duplicate, establishing a relative standard deviation (%RSD) value less than <10% as data acceptance instrumental criteria.

2.9. Response Optimization with Palladium Matrix Modifier

The presence of complex matrix components can cause significant negative effects on the signals in the atomization stage, and the use of matrix modifiers to reduce or eliminate this effect has been reported by several authors [8,16,18,23,24,25,26,27]. The slope and intercept values of cluster calibration curves are compared in this study to see how different amounts of matrix modifier Pd (NO₃)₂ affect the instrumental response.

Cluster Analysis

In statistical data analysis, there are some exploratory data analysis tasks where the creation of groups (clusters) allows for identifying basic patterns in a dataset. Thus, cluster analysis provides tools to generate the batches depending on the type of information and variables. One of the most common algorithms for clustering is k-means, which is designed to create some groups where the elements on each of them show similarities. As said, “the k-means procedures consist of simply starting with k groups, each of which consists of a single random point, and thereafter adding each point to the group, the mean of that group is adjusted to take account of the new point” [28].

2.10. Validation of Analytical Method

In the present study, validation parameters characteristic of analytical methods were evaluated following the recommendations of [29,30,31] and those reported by other authors [27,32]. Validation parameters such as linearity, accuracy, precision, the limit of detection (LOD) and limit of quantification (LOQ), and uncertainty were evaluated. The linearity of the method was determined by constructing calibration curves for Mn and Pb and analyzing the residuals generated by the linear regression of the response to the different concentrations, using the least-squares method and determining the correlation coefficient (R2), intercept at (y), slope and residual sum of squares. On the other hand, the method’s accuracy was determined by using samples spiked by adding known amounts of Mn and Pb standard solutions from certified reference material of human hair and nail samples. Accuracy was determined by calculating the repeatability and reproducibility of the analytical method by adding known amounts of standard solutions of Mn and Pb reference materials from human hair and nail samples at concentrations in the lower, middle, and upper regions of the calibration curve. The limit of detection (LOD) and quantification (LOQ) was determined by analyzing the response of the equipment to repetitions of blank samples of each matrix under study. Finally, finally, the expanded uncertainty was estimated considering several sources of uncertainty to establish a value for each of the contributions of each source separately to the uncertainty [29].

2.11. Study Area

Bogotá is the capital city of Colombia, located in the central part of the territory with an approximate area of 163,663 hectares, of which 23% are classified as urban areas, 75% are rural areas, and only 2% are areas for expansion purposes [33,34]. It has a population of 8,181,047 inhabitants, according to the census of Departamento Nacional de Estadistica (DANE) for 2018 [35]. The city is currently ranked as one of the most diverse and multicultural cities in the country due to its high presence of citizens from most of the geographical regions of Colombia and the high rate of foreign migrants that can be perceived in the city [34,35]. Administratively, Bogotá has a geographic division made up of 20 localities (18 urban and 2 rural) [34] (Figure 1).

The district of Sumapaz (Figure 2) is the largest in Bogotá, with an area of 78,097 ha, and is the only purely rural district. It is located between 3000 and 4100 m above sea level and has an average temperature ranging from 4.4 to 8.3 degrees Celsius. On the other hand, Usme (Figure 2) is the third-largest locality in Bogotá with an area of 21,506 hectares, composed of 10% urban land [34]. It is characterized by its rurality, which corresponds to 86% of its total area. It is located at an altitude between 2650 and 3750 m above sea level, characterized by a mountainous relief, which is related to the average temperature being in a range between 9 °C and 12 °C in the middle zone and in the highest parts or moorlands, between 6 °C to 9 °C [34].

3. Results and Discussion

3.1. Optimization from Response Using Matrix Modifier

The instrumental response of the analytical method was optimized by preparing several calibration curves in the linear ranges defined for Mn and Pb using CRM blanks and nail samples in triplicate by adding 10, 15, and 20 µL of Pd (NO₃)₂ palladium nitrate matrix modifier and then evaluating the correlation between the increase in sensitivity (slope) and the decrease of interferences (intersect) using a cluster analysis and ANOVA.

The analysis of the data was carried out by evaluating four groups to analyze Mn and Pb in nail and hair samples using a clustering method known as k-means; this technique was used to classify the results obtained in the several experiments with different amounts of matrix modifier. This analysis is proposed due to some differences between groups insomuch as the data depends exclusively on the slope and y-intersect; in addition, variance analysis was made to recognize differences between means from the variance in each group.

The comparison between groups and clusters observed shows that to quantify Pb in hair samples, the use of 15 µL is the most effective, increasing sensitivity, reducing matrix interference, and obtaining less variability; a similar behavior was observed in the analysis of Mn in the same matrix in the three groups: 10, 15, and 20 µL, as shown in Figure 3a,b. On the other hand, for the nail matrix, a decrease in matrix interferences is observed in groups 15 and 20 µL, and finally, the determination of Pb in this matrix does not show a negative or positive effect in any of the study groups, as shown in Figure 4a,b.

According to the ANOVA results obtained in

Table 3. Analysis of variance.

ANOVA
		Sum of Squares	df	Mean Square	F	Sig.
PB_C_PEND	Between Groups	0.000	3	0.000	26,909	0.000
	Within Groups	0.000	32	0.000
	Total	0.000	35
PB_C_Y	Between Groups	0.065	3	0.022	886,905	0.000
	Within Groups	0.001	32	0.000
	Total	0.066	35

from trials in hair, the groups conformed under the presence of blank, 10 µL, 15 µL, and 20 µL are equivalent to the cluster obtained under the k-means methodology. The ANOVA results validate the alternative hypotheses, which consider the differences between groups. In multiple comparisons, the results between clusters 2 and 4, which are groups of 10 µL and 20 µL, respectively, could belong to the same group.

3.2. Linearity

The instrumental response was evaluated by constructing different calibration curves with samples of hair (CRM) and nails enriched with stock solutions in a range of concentrations ranging from 0.001 to 0.015 mg/L and 0.002 to 0.020 mg/L of Mn and Pb, respectively, for the two matrices to evaluate the linear response of the method for the analysis of Mn and Pb in the selected matrices.

The least-squares analysis demonstrated the linearity of the analytical method, obtaining correlation coefficient values greater than or equal to 0.996 in

Table 4. Linearity data.

Matrix	Metal	Linear Range (µg·L−1)	Slope	Intercept	R2
Hair a	Mn	0.001–0.015	0.0578		0.998
	Pb	0.002–0.020	0.0114		0.996
Nails b	Mn	0.001–0.015	0.0756		0.999
	Pb	0.002–0.020	0.0071		0.998

^a CRM human hair (trace element); ^b Fortified Nail samples.

, demonstrating that there is a correlation that meets the acceptance criteria between the instrument response and the range of concentrations presented linearity in the range of concentrations under study, according to [36].

Precision, Repeatability, and Reproducibility

The accuracy of the analytical method was demonstrated by analyzing samples of (CRM) hair and nail samples enriched with stock solutions at the upper and lower levels of the calibration curve at 1, 5, and 15 µg·L⁻¹ and 2, 10, and 20 µg·L⁻¹ for Mn and Pb respectively. Due to the unavailability of certified reference material for the nail matrix, this study performed a recovery of unknown samples enriched according to what is reported in several studies related to this matrix [8,17,37]. On the other hand, the reproducibility of the method was evaluated by employing different analyst conditions, instruments, and different days during a period of 6 weeks. The results of %RSD obtained in the analysis of Mn and Pb in hair and nail samples are less than 15% (

Table 5. Precision and Uncertainty data.

Matrix	Metal	Analyte Concentration µg·g−1	Analyte Concentration µg·g−1	Precision (%RSD)		Uncertainty (%)
				Repeatability	Reproducibility
Hair a	Mn	0.625	0.657 ± 0.0127	2.65	9.88	9.93
	Pb	0.625	0.626 ± 0.1241	6.66	12.97	6.59
Nails b	Mn	1.25	1.288 ± 0.0626	6.65	6.39	8.63
	Pb	1.25	1.243 ± 0.0438	4.82	6.7	4.59

^a CRM human hair (trace element); ^b Fortified Nail samples.

), corroborating that the analytical method has a satisfactory precision that complies with the criteria described by the Commission of the European Communities [31].

3.3. Accuracy

The accuracy of the method was estimated by preparing (CRM) samples of hair and fortified nail samples by adding different known concentrations of Mn and Pb, then the samples were subjected to the same digestion process described in 2.6 and 2.7. The results of the accuracy evaluation of the analytical method are shown in

Table 6. Precision and Uncertainty data.

Matrix	Metal	Fortified Concentration (µg·g−1)	Concentration Recovery (µg·g−1)	Average Recovery (%)	RSD (%)
Hair a	Mn	0.125	0.118	94.21 ± 9.95	14.46
		0.625	0.630	99.24 ± 8.62	11.89
		1.875	1.590	84.8 ± 2.31	3.74
	Pb	0.250	0.257	102.86 ± 7.53	7.32
		1.250	1.276	102.12 ± 3.16	3.10
		2.500	2.429	97.14 ± 7.47	7.69
Nails b	Mn	0.250	0.270	107.98 ± 10.33	13.11
		1.250	1.213	97.04 ± 5.80	8.17
		3.750	3.788	101.02 ± 10.15	13.77
	Pb	0.500	0.434	86.72 ± 5.01	7.91
		2.500	2.394	94.97 ± 4.48	6.46
		5.000	4.880	97.61 ± 2.17	3.05

^a CRM human hair (trace element); ^b Samples were spiked with Mn and Pb by adding a standard solution.

. The average values obtained for the recoveries of each metal in hair and nail samples are in the ranges of 84.80–107.98% and 94.97–99.24% for Mn and Pb, respectively. On the other hand, all the calculated RSD values were lower than 15%, demonstrating an adequate accuracy according to the acceptance criteria established by the Commission of the European Communities [25].

3.4. LOD and LOQ

The efficiency of the instrument and the analytical method was determined by estimating the limits of detection (LOD) and quantification (LOQ), defined as the lowest concentration that generates a signal significantly different from that of the blank and the lowest analyte concentration that can be calculated under the described experimental conditions with adequate precision and accuracy, respectively. The LOD/LOQ values were determined by analyzing 10 blank samples of human hair (CRM) and nails and were calculated as 3SD/b/and 10SD/b, respectively. The results in

Table 7. Limit detection (LOD) and quantification (LOQ) data.

Matrix	Mn		Pb
	LOD µg·g−1	LOQ µg·g−1	LOD µg·g−1	LOQ µg·g−1
Human Hair	0.0486	0.0616	0.0318	0.0335
Nails	0.0844	0.1381	0.0420	0.0554

show that the obtained LOD/LOQ values for Mn and Pb in the hair matrix were 0.0486/0.0616 µg·g⁻¹ and 0.0318/0.0335 µg·g⁻¹, respectively, and 0.0884/0.1381 µg·g⁻¹ and 0.0420/0.0554 µg·g⁻¹ for the nail matrix, respectively, which reflects an increase in relation to hair; this increase is due to the absence of certified reference material for this matrix.

3.5. Measurement Uncertainty

According [38], uncertainty is the interval where the true value can be found with a higher probability, which is calculated by characterizing the dispersion of the values that could reasonably be attributed to the measurand. In this study, the uncertainty of the analytical method was estimated by estimating the expanded uncertainty by evaluating three groups: (1) instrumental uncertainty (calibration curve), (2) uncertainty of preparation of stock solutions (standard, volumetric material, and transfer pipette) and (3) uncertainty of sample preparation (transfer pipette). The sum of the contributions of each of the evaluated sources of uncertainty was expressed in RSD [27,39]. The results in Table 3 show the estimated uncertainty values for the analysis of Mn in hair and nails. The measurement expanded uncertainty was calculated with a coverage factor of 2, resulting in an approximate 95% level of confidence.

3.6. Analysis of Real Samples: Hair and Nails

Heavy metals are one of the most studied substances due to their toxicity and resulting effects on human health and the environment. These substances are present in the environment, but their concentration has increased considerably due to the intensification of anthropogenic activities [40]. Therefore, the validated analytical process for the determination of Mn and Pb was applied in the analysis of 30 hair samples and 30 nail samples randomly collected from children aged 7 to 10 years in the study area described in Section 2.11. All samples were analyzed in duplicate. The results show the presence of Mn levels in all the analyzed samples; the values are in the ranges of 0.838–3.331 μg·g⁻¹ and 2.033–8.931 μg·g⁻¹ for hair and nails, respectively. In Figure 5, on the other hand, the Mn levels found are in a range of 0.072–0.697 μg·g⁻¹ and 0.110–1.825 μg·g⁻¹ in 13 hair samples and 5 nail samples, respectively. In Figure 6, additionally, the RSD values obtained in the analyzed samples were less than <20%. The results also show that the concentration values of Mn and Pb are significantly higher in nail samples, which is consistent with what has been reported in other studies [14,41].

Considering the above, the mechanisms of intoxication that the children selected for the development of this analytical method could face are innumerable. Currently, the bioaccumulation of this type of substance in multiple environmental matrices and their chemical and physical transformation mechanisms facilitate the inhalation, ingestion, or dermal contact of these metals by humans, turning these chronic and/or acute intoxication scenarios into a public health problem. In the study entitled “Coexistence of diverse heavy metal pollution magnitudes: Health risk assessment of affected cattle and human population in some rural regions, Qena, Egypt”, the authors state that the presence of heavy metals considerably affects a rural population segment due to the bioaccumulation of these substances in environmental and biological matrices that subsequently come into contact with humans, such as the ingestion of contaminated milk produced on farms in the rural region of Qena, Egypt [42]. This phenomenon could be particularly evident in the rural area of Sumapaz, given the high use of pesticides, fertilizers, and Inorganic products with a high concentration of lead and manganese to produce previously contaminated agri-food and agricultural goods.

However, the effects of Pb and Mn exposure are an issue that sets off alarm bells for regulatory and research entities. Multiple studies have shown how these substances can affect human health in children and adults. These affectations attack the systems of the human body and each of the organs in general, showing cases of neuropsychiatric affectation among children from 0 to 4 years old and a decrease in the total weighted IQ [43]. In addition, deterioration is observed in the functions of the central nervous system with neuromotor impairment and the appearance of symptoms similar to Parkinson’s, the endocrine system with symptoms of renal and hepatic insufficiency and genetic alterations, mutagenic, teratogenic, and carcinogenic effects [40,44,45]. Finally, Ungureanu, E.L., and Mustatea, G., mention that contact with heavy metals can increase the incidence of antisocial behavior in children, making an alarming call for attention to organizations, given that the young population can absorb 4 to 5 times more lead and heavy metals than an adult, and thus increase the damage in the systems and organs exposed to the high concentration and bioaccumulation rates [40].

4. Conclusions

The use of a matrix modifier in the analysis of Mn and Pb in human hair significantly improves the instrumental response by decreasing the associated interferences. Additionally, it has been demonstrated that there is an increase in sensitivity in the analysis of Pb in this matrix. On the other hand, the analysis of Mn in nails shows a decrease in interferences with the addition of larger volumes of matrix modifiers. The results obtained from the validation parameters evaluated for the proposed analytical process comply with the criteria of linearity, repeatability, reproducibility, accuracy, LOD and LOQ, and uncertainty established in the Commission Regulation (EU) No. 836/2011. The correlation coefficient values obtained were greater than >0.995. The method’s accuracy ranged from 84.8% to 107.98%, and the precision measured as repeatability and reproducibility did not exceed 12.97% RSD. Furthermore, the values of LOD, LOQ, and uncertainty are satisfactory. In general, it can be concluded that the proposed method is suitable and reliable for the analysis of Mn and Pb in human hair and nails.

The application of the analytical process for the quantification of Mn and Pb in the samples collected in the study area showed normal levels of Mn, obtaining values ≤3331 and ≤8931 µg·g⁻¹ for hair and nails, respectively. Normal levels of Pb are also observed in some of the samples analyzed, with values ≤0.697 and ≤1825 µg·g⁻¹ for hair and nails, respectively. Although several authors have published studies on the application of metal analysis in various biological matrices, studies have not yet been carried out in Colombia to estimate the levels of these and other heavy metals of interest in matrices such as human hair and nails. the possibility of carrying out new studies in the country for the analysis of these metals as a biomarker to monitor the levels of these metals, their possible correlations with some diseases, and also as a bioindicator of the risk of exposure and its application in different social and occupational contexts.