Sample preparation

250mg of sample was taken in 50mL polyethylene tube and 0.2mL of 1000mg/L gold standard solution was added and then make up to 25mL with 2% nitric acid to make 10mg/mL sample solution.

Calibration standard preparation

For calibration standard stock solution preparation, according to ICH Q3D, Standard 1 containing 15ppm of As, 5ppm each of Pb and Cd, 30ppm of Hg, 50ppm of Co, 100ppm of V, 200ppm of Ni was diluted to 20mL with 2% HNO3. Then 0.2mL of 1000mg/L gold standard solution was added to each of them, and this stock solution was further diluted as per below table to make different level calibration standards (Table 2).

Table 2:Dilution for calibration standards. 100% level calibration standard solution was used as system check solution and the same was injected periodically to check performance of the system.

Spiked sample preparation

250mg of sample was taken in 50mL polyethylene tube, added amount of calibration standard stock solution as per below table, 0.2mL of gold standard solution and make up to 25mL with 2% nitric acid to make respective level spiked sample solution. In blank, calibration standards, sample blank, samples and spiked samples preparation 0.1mL of internal standard containing 5mg/L each of Yitrium and Bismuth were added to make final concentration as 20μg/L (Table 3).

Table 3:Dilution for spiked sample preparation.

Q-ICP-MS instruments optimization

The quantities of heavy metals were determined by quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS). Thermo Fisher Scientific, Model: iCAP RQ ICP-MS using Qtegra Intelligent Scientific Data Solution (ISDS) software equipped with Q Cell Collision Reaction Cell, RAPID Lens, with a quartz spray chamber, glass concentric nebulizer, online Internal Standard (ISTD) addition kit, exchangeable skimmer cones. The optimization of Q-ICP-MS is important because the nebulizer gas and make up gas flows should be adjusted to ensure plasma stability. The Q-ICP-MS was allowed to stabilize for 1h and the performance was optimized based on Radio Frequency (RF) power, autotune function in the control software and A tune B solution, the quadrupole ion deflector voltages were optimized to step wise find the settings that maximize signal intensity over the mass range, as well as mass calibration of Li, Co, In, Ba, Ce, Bi and U, sampling depth, argon flow rate, collision cell gas flow rate, lens voltage, sample uptake rate. The instrument was in helium Kinetic Energy Discrimination (He KED) technology mode which allow measurement of all analytes in a single mode. KED mode for analysis of V, Co, Ni, Cd, Hg, Pb and As.

Method validation criteria

Several parameters have been considered and evaluated for the method validation Like Linear Dynamic Range (LDR), method linearity, accuracy, precision, Limit of Detection (LOD), Limit of Quantification (LOQ), and Measurement of Uncertainty (MU). Analytical method validation of Q-ICP-MS for analysis of V, Co, Ni, Cd, Hg, Pb, and As was applied in accordance with ICH Q2(R1).

Q-ICP – MS analysis

The Q-ICP–MS-method used 6 replicate readings of 30 sweeps over the analyte mass-range with a dwell time of 100 μs for each mass per sweep. Sample aspiration followed by rinse program with 2% HNO3 kept for 60s. For running the instrument in KED-mode, 4.34mL min-1 of He gas was used.

Role and importance of internal standard in the detection of heavy toxic metals

The choice of an appropriate internal standard while using Q-ICP-MS for elemental analysis is very important since it shows very good impact on accuracy and precision of the result. The main role of internal standards is to stabilize the Q-ICP-MS instrument, rectification, or minimization of non-spectral reversible and irreversible matrix interferences. Non-spectral interferences can be divided in reversible, that is occurring while the sample is being measured, and irreversible matrix effects, that is clogging of the nebulizer and sampling orifices or deposition on the torch or in the ion lens stack. The errors associated with non-spectral interferences can be eliminated by appropriate calibration procedures, adapted sample preparation or limitation of the amount of sample delivered to nebulizer, plasma, and sampling devices, for example by the application of flow injection.

Optimization of Q-ICP-MS parameters

For accurate and correct results various instrumental parameters like torch alignment, the nebulizer gas and make up gas flows, Kinetic Energy Discrimination (KED), lens and mass calibration and resolution, detector voltage, dual detector should be optimized to achieve maximum instrumental performance. The plasma stability and position are most important parameter since this determine the sensitivity of instrument. To ensure this, the nebulizer gas and make up gas flows had to be adjusted. This instrumental requirement has been achieved by adjusting torch position and tuning for reduced oxide and doubly charged formation with a standard tuning solution containing 10ng/g of ⁷Li, ⁸⁹Y, ¹⁴⁰Ce and ²⁰⁵Tl in 2% HNO₃. The various optimized Q-ICP-MS parameters are reported in (Table 4).

Table 4:Typical ICP-MS instruments parameters for analysing samples. ^aRF: Radiofrequency; ^bQP: Quadrupole; ^cOctP: Octupole.

Method validation

Two decades ago, researchers were not completely aware of selectivity and specificity of an analytical method. On many occasions, they were intermixing these two very important terms, selectivity, and specificity. To create an awareness among analytical scientists for analytical methods, J. Vessman, differentiated these two important terms in detail in a report entitled, “Selectivity in Analytical Chemistry (IUPAC Recommendations 2001)”. One very clear definition of selectivity of a method refers to the extent to which it can determine analyte(s) in a complex mixture without interference from other components in the mixture. However, the same meaning has often been given to the term “specificity”. IUPAC clarified this overlap by expressing the view that “Specificity is the ultimate of Selectivity”. This is very important for all researchers who are working in analytical chemistry [10]. Primary isotopes of each element, ⁵¹V, ⁵⁹Co, ⁶⁰Ni, ¹¹¹Cd, ²⁰²Hg, ²⁰⁸Pb and ⁷⁵As were used in the present study to investigate the selectivity of the present method. Validation study was performed to determine and establish the reliability of the present method.

Estimated LOD

The term LOD is defined as the lowest concentration at which the instrument can detect but not quantify and the noise to signal ratio for LOD should be 1:3. LOD was estimated from the calibration function using Eq. (1) [11].

where, σ is standard deviation, S is slope derived from the calibration curve.

The estimated LODs were found to be 3.05, 1.66, 7.23, 0.25, 0.82, 0.16, 0.58μg/ L for V, Co, Ni, Cd, Hg, Pb and As, respectively. The minimum practical concentrations of tested elements in the analysed samples, which can be determined with acceptable accuracy (as per USP general chapter 233), were performed by analysing 3 replicates at 30μg/L for V, at 15μg/L for Co, at 60μg/L for Ni, at 1.5μg/L for Cd and Pb, at 9.0 μg/L for Hg and at 4.5μg/L for As, respectively. The results are reported in (Table 5).

Practical LOQ

The term LOQ is defined as the lowest concentration at which the instrument can detect and quantify. Control Threshold values (which is 30% of the PDE of the specific elemental impurity under consideration) as per ICH Q3D (R1) para 5.6 considered for verification as LOQ values and based on successful verification of precision, linearity (from LOQ) and recovery at LOQ level, below values considered as LOQ. The noise to signal ratio for LOQ should be 1:10. The estimated LOQs were found to be 30, 15, 60, 1.5, 9.0, 1.5 and 4.5 mg/ L for V, Co, Ni, Cd, Hg, Pb, and As, respectively. The results are reported in (Table 5).

Table 5:Results of estimated LODs, practical LOQs, and maximum permissible limits number of replicates=06.

Method linearity

According to Magnusson and Ornemark, if the value of calibration curve coefficient of determination (R2) is higher than 0.995, quantification result will be accurate as analytical response will be linear over certain concentration ranges. The method linearity was investigated over specific working range from different concentrations of reference standards [12,13].

Linearity of the calibration curves: The dynamic linear range found to be linear from 30 to 150μg/L for ⁵¹V, 15 to ^{75sup>μg/L for 59Co, 60 to 300μg/L for 60Ni, 1.5 to 7.5μg/L for 111Cd, 208Pb, 9 to 45μg/L for 202Hg and 4.5 to 22.5μg/L for 75As, respectively (Table 5) [14,15].}

Method linearity: The method linearity was checked using seven different levels of samples at 0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0mg/L for 51V, 0, 1.5, 2.5, 5.0, 7.5, 10.0, 12.5mg/L for ⁵⁹Co, 0, 6.0, 10.0, 20.0, 30.0, 40.0, 50.0mg/L for ⁶⁰Ni 0, 0.15, 0.25, 0.50, 0.75, 1.00, 1.25mg/L for¹¹¹Cd & ²⁰⁸Pb, 0, 0.9, 1.5, 3.0, 4.5, 6.0, 7.5mg/L for ²⁰²Hg and 0, 0.45, 0.75, 1.50, 2.25, 3.00, 3.75mg/L for 75As, respectively.

Method accuracy

In the context of an analytical method, according to ICH guidelines, accuracy ‘is sometimes termed trueness’. The trueness of an analytical procedure reflects the closeness of agreement between the value that is either accepted as conventional true value or an accepted reference value with the observed value. Therefore, accuracy is an expression of both trueness and precision since both of these influences the result [16]. Accuracy can be measured by spiking the sample matrix with a known concentration of analyte standard and analyzing the sample using the “method to be validated.” The results of the accuracy are presented in (Table 6).

Table 6:Results of accuracy/recovery test of elements.

Precision study - repeatability and reproducibility

Precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings [17]. According to ICH [18], precision may be considered at three levels: repeatability, intermediate precision, and reproducibility (Figure 1). The precision was calculated in terms of relative standard deviation (RSD) by Eq. 2 and single estimation of precision uncertainty is calculated by Eq. 3, respectively [11].

Figure 1: Graphical abstract.

where,
xi=ith reading in the data set
μ=Mean of the data set
n=Number of readings in the data set
The results of repeatability test expressed as RSD were found to be 2.15%, 1.07%, 1.36%, 2.16%, 2.23%, 2.01% and 1.95% for ⁵¹V, ⁵⁹Co, ⁶⁰Ni, ¹¹¹Cd, ²⁰²Hg, ²⁰⁸Pb and ⁷⁵As, respectively. The results of the precision study are presented in (Table 7) and Linear regression analysis results are reported in (Table 8).

Table 7:Results of precision test of elements.

Table 8:Linear regression analysis.

Estimation of measurement uncertainty

According to S L R Ellison and A Williams (Eds). Eurachem/CITAC guide: Quantifying Uncertainty in Analytical Measurement, Third edition, (2012) ISBN 978-0-948926-30-3. available from www. eurachem.org., the term uncertainty (of measurement) is defined as “A parameter associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand”. There are various contributing factors like weighing of samples, sample and standard preparation, instrumental measurements, linearity measurement, laboratory repeatability and reproducibility. The expanded uncertainty was measured by multiplying the combined uncertainty, by using a coverage factor (k) of 2, at confidence level of 95% (Figure 2). The measurement uncertainties expressed as expanded uncertainties were estimated to be 10.18, 4.98, 19.69, 0.48, 3.03, 0.50 and 1.60mg/L for V, Co, Ni, Cd, Hg, Pb and As, respectively. The results of uncertainty of each element are presented in (Table 9).

Figure 2:Method linearity (a) V (3.0-25.0mg/L), (b) Co (1.5-12.5mg/L), (c) Ni (6.0-50.0mg/L), (d) Cd (0.15-1.25mg/ L), (e) Hg (0.9-7.5mg/L), (f) Pb (0.15-1.25mg/L) and (g) As (0.45-3.75 mg/L).

Table 9:Result of uncertainty tests of elements.

Bias study (recovery test)

The spiking levels used for recovery test were at 30, 100 and 150μg/L for 51V, 15, 50 and 75μg/L for 59Co, 60, 200 and 300μg/L for 60Ni, 1.5, 5 and 7.5μg/L for 111Cd, 208Pb, 9, 30 and 45μg/L for 202Hg and 4.5, 15 and 22.5μg/L for 75As, respectively. The mean recoveries±standard deviations at different levels varied between 83.1±0.5% and 106.7±2.1% with coefficient of variation expressed as relative standard deviation (RSD) were varied between 0.2% and 3.1% [19,20]. The results of recovery test are presented in (Table 6).

Table 10:Comparison of uncertainty statistics in three different commercial samples.

A Q-ICP-MS technique was used to validate the analytical method for simultaneous determination and quantification of seven toxic metals V, Co, Ni, Cd, Hg, Pb, As in theobromine (Table 10). The accuracy of the present method is (30-150% of target value) and precision value (n=6) were successfully achieved the criteria defined by WHO, AOAC, USFDA, ICH and USP 232/233. The validated method has been successfully evaluated in terms of LOD, LOQ, linearity, repeatability, reproducibility, accuracy, and precision. The proposed validated method is very simple, quick, easy, cost effective and reliable; therefore, it can be very useful for quantification of these toxic metals in routine laboratories analysis.

The help of Mr. Punam Shah, Bajaj Healthcare Private Ltd, Vadodara, Gujarat, India is gratefully acknowledged for providing three commercial samples of theobromine.

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