Introduction

Lead (Pb) is classified as a non-essential element and it has no known biological function, that even at very low concentrations is toxic to human health [1,2,3]. However, with advancements in health sciences, exposure to any level of Pb is recognized as detrimental to human health [4]. As a cumulative toxicant, it adversely affects multiple body systems including hematological, neurological, cardiovascular, gastrointestinal, and renal systems. Hence, various governing agencies like Occupational Safety and Health Administration (OSHA), USA have prescribed guidelines to protect and prevent the health consequences of Pb exposure [5]. Lead (Pb) smelting workers are occupationally exposed to high levels of Pb from airborne dust. A typical feed ore—Pb concentrate from captive mines consists of 63% Pb content in its composition. If low awareness of health hazards and poor hygiene practices exists at the workplace, such group of people may be potentially at high risk to develop Pb toxicity due to occupational exposure.

In medical surveillance, the blood-Pb level is considered as the gold standard marker to diagnose Pb-exposed individuals [6]. There is no identified threshold or safe Pb level below which there are no observable adverse health effects [7]. Hence, its precise assessment would play an essential role in the management of occupational Pb exposure.

Various analytical technics exist for estimating Pb levels in human blood samples such as atomic absorption spectrometry (AAS), anodic stripping voltammetry (ASV), Graphite Furnace Atomic Absorption Spectrometry (GF-AAS), inductively coupled plasma coupled with atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). These methods significantly differ in their analytical proficiencies [8, 9]. However, many forensic investigations reported ultra-trace level detection of Pb and other toxic elements in biological samples using AAS and AES techniques [10]. A comparative statement of detection limit and recovery performance of several analytical methods used for the analysis of Pb in blood samples is presented in Table 1 [11]

Table 1 Comparison of analytical method for estimation of Pb in blood samples [11]
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Besides, significantly reduced levels of Pb in blood are considered a clinical concern. Consequently, there is a growing interest in measuring very low blood-Pb levels. This warrants a need for analytical methods with a wide range, spanning across trace level detection to high-level estimation, applicable to population or sub-population from occupational exposure.

GF-AAS is one of the preferred analytical techniques for trace level detection of heavy metals in biological samples. It consumes as low as 10–50 µL sample volume. The entire sample is vaporized and atomized at 3000 °C within a small volume of graphite tube, leading to a dense atom population for better sensitivity.

Earlier, the development of the GF-AAS method reported a method detection limit (MDL) up to 1 µg/dL of Pb levels in human blood samples [12]. The right selection of sample preparation technic would determine the accuracy of elemental analysis in biological substances. However, sample preparation by microwave-assisted acid digestion method reported maximum recoveries for all experimental elements [13]. In the present study, we reported the development of the GF-AAS method for estimation of Pb levels in human blood samples with the lowest level detection up to 0.16 µg/dL and quantitative measurement up to 0.51 µg/dL.

Experimental

Reagents and chemicals

All chemicals and reagents used in the study were of analytical grade or certified reagent grade. Trace metal-free grade concentrated nitric acid (> 1 ppb concentration for 65 metals, ideal for analysis by GF or Flame AA, ICP-OES, and ICP-MS) was procured from fisher chemicals. 30% (w/v) hydrogen peroxide (H2O2) (ACS grade) solution was procured from Fisher chemical. NIST traceable standard reference material (1000 mg/L of Pb) was procured from Merck GmBH. Both ammonium dihydrogen phosphate (NH4H2PO4) and magnesium nitrate (Mg(NO3)2) analytical grade reagents for the preparation of matrix modifier solutions were procured from Merck GmBH. Deionized water (Millipore, Milli‐Q‐Elix3, ≤ 18 MΩ) was further double distilled for preparation of reagent standards and sample dilution.

Method development and optimization

The sample preparation was optimized for complete acid digestion by a two-step ramp temperature program as summarized in Table 2. The measurement of Pb concentration was optimized by instrumental parameters and the transversely heated graphite atomizer (THGA) furnace program as summarized in Table 3. The potential interference from light scattering and molecular absorption by matrix components was encountered using Zeeman background correction and chemical matrix modifiers (Mg(NO3)2 and NH4H2PO4) [8].

Table 2 Program for microwave acid digestion of blood samples and QC standards
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Table 3 Optimized experimental condition for GF-AAS instrument
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Method validation

The validation was based on standard test parameters viz. linearity, % recovery, precision [% relative standard deviation (RSD)], MDL, and method quantification limit (LOQm). The linearity of the calibration was determined by its correlation coefficient (R2). The linear curves with R2 value > 0.99 were selected for estimating blood-Pb levels. The recovery of Pb concentration was determined as per the standard spike method. The % recoveries range between 95 and 105% were considered acceptable for validation. The accuracy of the analysis was determined by the absorbance value reported by the GF-AAS manufacturer. The precision of analysis with < 10% RSD obtained from repeated measurement of the same sample) was accepted for validation. The MDL and LOQm were derived from the standard deviation (SD) of 7 replicates using the standard spike method [14].

Evaluation of blood lead level

Sample preparation

Sample preparation was performed by microwave-assisted acid digestion method using trace metal-free grade concentrated HNO3 and 30% H2O2 solution. 2.0 mL of whole blood samples, collected in Pb-free sodium heparin vial were digested with 2.0 mL of trace metal-free grade concentrated HNO3 and 0.5 mL of 30% H2O2 at 180 °C for 20 min along with blank in a microwave digestion system (START-D, Milestone, Italy) by two-step ramp program as summarized in Table 2. In the first step, the digestion mixture has gradually heated at 180 °C and 42 bar pressure for 10 min. In the second step, the same operating condition was maintained for another 10 min. The digested samples were cooled to room temperature and transferred to 5 mL Pb-free tubes. Finally, all samples were makeup to 5 mL volume with de-ionized double distilled water and preserved at 4 °C.

Instrumentation

All the measurements were performed using the GF-AAS instrument (model AAnalyst 800, manufactured by M/s. Perkin Elmer, USA). The instrument was equipped with a high-efficiency optical system, solid-state detector, graphite furnace sample introduction system, and THGA along with an integrated autosampler, and Zeeman background correction. All measurements were made using pyrolytically coated THGA tubes with end caps. The entire system was controlled by WinLab 32 software interface.

Preparation of reagents and calibration standards

The calibration standards of 1.0, 2.5, 5.0, 7.5, and 10.0 μg/dL of Pb were prepared from NIST traceable standard reference material (1000 mg/L of Pb, Merck GmBH) by serial dilution with 0.2% HNO3 solution in de-ionized double distilled water. The solutions of 10% NH4H2PO4 and 1% MgNO3 were diluted 5 times with 0.2% HNO3 and used as matrix modifier reagents.

Estimation of Pb levels in blood samples

The digested samples and blank along with a matrix modifier reagents were aspirated into the GF-AAS instrument. The instrumental parameters and the THGA furnace program were set as per optimized conditions as summarized in Table 3. The absorbance for samples was recorded at 283.3 nm wavelength and the unknown Pb levels (μg/dL) were determined from the calibration curve.

Study design and recruitment of subjects

In the present study, a total of 503 consenting adults (age > 18 years) from a Pb smelting plant were evaluated for their blood-Pb levels and associated health status. The essential ethical clearance was obtained from the Human Ethics Committee of the parent institute before initiating the study. The study followed all the methods and protocols for human experiments as recommended by the national ethical guidelines for biomedical and health research involving humans [15]. Informed written consent was obtained from each participant to utilize their details including blood-Pb levels for the present study. Sociodemographic and occupational details of the consenting participants were recorded using a semi-structured, pre-validated questionnaire. Clinical evaluation included general health examination and workplace-specific investigation. 5 mL venous blood was collected in heparin vials, from all consenting participants under aseptic precautions. All samples were transported under cold chain conditions and analyzed at the parent institute. Hemoglobin levels were estimated using Hemocue and categorized as ‘anemic’ and ‘normal’ based on WHO guidelines [16].

The clinical examination included the presence of respiratory, gastrointestinal, neurological, or musculoskeletal symptoms requiring medical supervision from work for over 24 h, in the last 1 year.

The blood pressure (BP) was measured on three occasions with about 5 min intervals between each reading, ensuring the participant was seated and relaxed [17] using a pre-calibrated digital sphygmomanometer (Omron Healthcare, Kyoto, Japan). The subsequent measurements of systolic BP (SBP) and diastolic BP (DBP) were recorded. The average of the second and third measurements was considered for the study. Individuals were categorized as pre-hypertensive, and hypertensive according to the guidelines described by the seventh report on prevention and control of high blood pressure recommended by the Joint National Committee [18].

Results and discussion

The present study was intended to develop an analytical method for detecting the lowest possible Pb levels in human blood samples, as the presence of Pb in biological samples is increasingly recognized as public health importance.

As no safe threshold was established for any levels of Pb in blood, the analytical methods with the lowest possible detections may be helpful to address populations or sub-population with trace levels or elevated blood-Pb concentrations either from environmental or occupational exposure [8]. The present study described the development and validation of a new GF-AAS method with a wide range of detection from trace levels (0.16 µg/dL) to elevated blood-Pb levels and its potential application in the clinical evaluation of Pb exposure among occupationally exposed Pb smelting plant workers.

Sample preparation by acid digestion renders complete decomposition of the organic matrix and transforms the elements into their free ionic forms. The microwave exposure facilitates the digestion process at lower temperature and pressure. The organic complexes are completely decomposed into CO2 and NO by the action of conc. HNO3 and H2O2 under microwave conditions, and mineralize the solution in aqueous form [19]. Remarkably, the sample preparation by microwave-assisted acid digestion was reported with maximum recoveries for all experimental elements [13]. In the present study, complete digestion of biological samples was ensured by a two-step ramp program, and optimum recoveries for all spike concentrations (over the entire calibration range) were obtained from optimized conditions as summarized in Table 2.

A GF-AAS instrument equipped with a high-resolution optical system and solid-state detector was used for the measurement of Pb levels. The graphite furnace sample introduction system was mounted to optimize short-term signal stability, whereas a solid-state detector for optimum signal to noise ratio. The accurate background correction for graphite furnace analysis was provided by longitudinal Zeeman-effect without the loss of light. The uniform temperature distribution was assured by a THGA across the entire length of the graphite tube. It can eliminate the memory effects and potential interferences that may occur with high-matrix sample analysis. The instrumental parameters for the optimized experimental condition are summarized in Table 3.

Method validation

The validation of the newly developed GF-AAS method for quantification of Pb levels in human blood samples was executed by evaluating standard performance parameters including linearity and selectivity, % recovery, precision, MDL, and LOQm as per standard guidelines [14].

Linearity and selectivity

The linearity of the method was determined from the 5-point calibration curve. As described in Fig. 1 the measured absorption revealed a linear correlation with the concentration of Pb over the entire range from 1 to 10 µg/dL and linear through zero.

Fig. 1
figure 1

Linearity was obtained from a 5-point calibration curve. The calibration curve was prepared from measured absorbance against each standard concentration of 1.0, 2.5, 5.0, 7.5, and 10.0 µg/dL Pb. The correlation coefficient (R2) value obtained from the slope of the calibration curve was 0.998

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The correlation coefficient (R2) obtained from linear regression was 0.998. However, the values at R2 > 0.99 may be acceptable for quantifying Pb levels [14], accordingly, the present method demonstrated acceptable linearity compliance. Selectivity refers to the competence of an analytical method to accurately measure an analyte in presence of interferences, under the stated analytical condition for the sample matrix being used [20]. Herein, to validate the selectivity, two calibration curves were developed by the addition of standards concentration within the linearity range to each aqueous and matrix (control blood) medium. Both the slopes were compared by student t test and there was no significant difference at a 95% confidence level. It confirmed that the developed method is selective to the analyte and linearity was not influenced by matrix interference. Hence, aqueous standards can be utilized for calibration while estimating Pb levels in blood samples. However, the matrix modifiers are used for the thermal stabilization of an analyte at optimum temperature conditions [21].

Estimation of recovery

Whether any loss of analyte, during sample preparation or due to matrix interferences during measurement, can be confirmed by the recovery studies [22]. In the present study, recovery assessment was carried out as per the standard addition method. Briefly, control blood samples were spiked at three different known concentrations (A) of Pb [23], followed by sample preparation by microwave acid digestion as described above. Finally, % recoveries were calculated from the measured concentrations (C) for all spike samples as summarized in Table 4.

Table 4 Recovery performance for blood-Pb level measurement
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The recovery values obtained for all spiked levels ranged between 95 and 105%. Conventionally, recovery values ranging between 94 and 106% were accepted for spiked blood specimens [24]. It suggests that the proposed method revealed acceptable recovery performance.

Estimation of MDL and LOQm

The proficiency of an analytical method is essentially evaluated by MDL and LOQm. MDL refers to the detection of the lowest concentration of an analyte that can reliably be distinguished from a noise signal, but not essentially is quantified. Whereas LOQm refers to the quantification of the lowest concentration of an analyte within acceptable levels of precision and accuracy [25]. In the present study, MDL and LOQm were calculated using the equation based on the literature method [14] and detailed in Table 5.

Table 5 Estimation of MDL and LOQm
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For the proposed method, the calculated values obtained for MDL and LOQm were 0.16 and 0.51 μg/dL of Pb, respectively. These figures suggested that the present method is able to detect approx. 21.8 times and quantify 6.80 times lower levels of Pb in blood than the reference blood-Pb value (3.5 μg/dL) suggested by Centres for Disease Control and Prevention (CDC) [26, 27].

Precision

The precision of an analytical method is defined as the proximity of individual test results for repeated measurements of the same sample under unique analytical conditions [28]. The precision of the proposed method was determined by repeated measurement of three replicates of three spiked samples of 0.25, 0.50, and 1.50 μg/dL of Pb concentrations for three successive days and expressed in terms of % RSD as summarized in Table 6. The values of % RSD for all three spiked levels were found within the acceptable limit (< 10% RSD) [23].

Table 6 Evaluation of precision of measurement
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Recently, an improved GF-AAS method was developed for the detection of low blood-Pb levels in compliance with the CDC blood-Pb reference value of 3.5 μg/dL [27]. The comparison of the detection limit between the present method and the established method [27] revealed that values of both MDL and (LOQ)m of the present method of 0.16 and 0.51 μg/dL were better than the values of existing method [27] of 0.2 and 1.0 μg/dL, respectively. This shows that the present method is capable of detecting even lower blood-Pb levels with improved accuracy and precision. A synopsis of performance parameters of the present analytical method is summarized in Table 7.

Table 7 Summary of performance parameters
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The assessment of all test parameters revealed successful validation of the proposed method. The test values for each parameter were observed within the acceptable limit as per standard guidelines. Consequently, the present method was implemented for the clinical evaluation of blood-Pb levels among occupationally exposed Pb smelting plant workers.

Evaluation of blood-Pb levels in occupationally exposed Pb smelting plant workers

Pb smelting workers are predominantly exposed to high Pb levels and prevalent with high blood-Pb levels. This occupational group is at potentially high risk to develop Pb toxicity if there exist low awareness and poor hygiene practices at the workplace. In the present study, occupationally exposed individuals engaged in various Pb smelting processes and activities were clinically evaluated as a part of their periodic medical investigation. The study cohort consisted of regular and contractual workers assigned administrative, supervisory, and labor tasks. The recruited participants were grossly engaged with Pb-smelting activities including handling the raw materials, furnace smelting, electrochemical refining, maintenance of the machinery, and miscellaneous activities.

Their blood-Pb levels were estimated using the newly developed GF-AAS method. The distribution of the study population and their blood-Pb levels concerning sociodemographic profile, employment factors, system-specific symptoms, and representative clinical parameters are summarized in Table 8.

Table 8 Distribution of study population and their blood-Pb levels
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It was observed that the study population had significantly higher levels of Pb in blood samples as per Occupational Standards and Guidelines for Lead suggested by OSHA [29]. Socio-economic status including tobacco usage and employment factors were found possible determinants for elevated blood-Pb levels among this group. The individuals with lower educational qualifications and backward economic status were found higher blood-Pb levels as compared to educationally and socially advanced groups. The participants belonging to contractual bases, worker class, or working near hazardous processes like raw material handling, furnace smelting, and electrochemical refining were shown elevated blood-Pb levels as compared to permanent or administrative employees. The study population demonstrated that system-specific symptoms were associated with their blood-Pb levels. It was observed that most of the system-specific symptoms (except abdominal discomfort) demonstrated by the study groups were found higher blood-Pb levels than the upper acceptable limit (40 µg/dL) in occupational exposure [29]. Evaluation of clinical parameters revealed that approx. 45.7% of individuals were found pre-hypertensive whereas, 25.8% were observed hypertensive as per the guidelines on prevention and control of high blood pressure prescribed by the Joint National Committee [18]. Similarly, a high prevalence of anemia was observed among the participants with blood-Pb levels > 40 µg/dL.

Conclusions

The present study revealed the development of an analytical method and its validation for precise measurement of Pb levels ranging from trace levels to higher concentrations in human blood samples using the GF-AAS technic. An overview of method performance is presented by validation results. The test results for each parameter including linearity, recovery, MDL, LOQm, and precision were observed within acceptable limits as per standard guidelines. The assessment of performance parameters revealed the competence of the proposed method for estimation of trace level detection of Pb in human blood samples. Finally, the method was implemented for the clinical evaluation of blood-Pb levels among occupationally exposed Pb smelting plant workers. The study population was found an average blood-Pb level of 42.80 ± 12.47 (mean ± SD). Sociodemographic status and employment factors were found possible determinants for the prevalence of high blood-Pb levels. The majority of system-specific symptoms were observed among study groups having mean blood-Pb levels higher than the upper acceptable limit (40 µg/dL) suggested by OSHA.