DETERMINATION OF TOXIC HEAVY METALS IN CHOLIC ACID USING QUADRUPOLE INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY

Objective: The information on the presence of toxic heavy metals in pharmaceutical starting materials and finished product is very crucial from the viewpoint of human life and its hazardous impact on the worldwide environment. The present work deals with the detailed quantification of the toxic heavy metals, namely, V, Co, Ni, Cd, Hg, Pb, and As, present in colic acid using quadrupole inductively coupled plasma mass spectrometry (Q-ICPMS) with prior microwave-assisted digestion. Moreover, the preliminary characterization of commercially available cholic acid by FT-IR, NMR (1H and 13C), SEM-EDAX has also been carried out. Methods: Cholic acid of synthesis grade, Nitric acid (65%) AR. grade, ethylene diamine tetra acetic acid sodium salt AR grade, and certified reference metal stock standard solutions (1000 mg/L) of multiple elements prepared in 2–3% HNO3 of analytical grade were purchased from Merck (Darmstadt, Germany). All the samples were treated with nitric acid and microwave-assisted digestion. For the accurate determination of the elemental amount, various digested solutions and post-digestion diluents were tested. The linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) of the analytical technique were evaluated in accordance with the United States Pharmacopoeia 233 standard. Results and Discussion: The Q-ICPMS-based analytical method was validated for specificity, LOD, LOQ, linearity, accuracy, precision, and uncertainty. The estimated detection limits of the toxic heavy metals in cholic acid were in the range 2–180 μg/L. The quantification limits were in the range of 1.5–60 μg/L. Mean recoveries±standard deviations at different spiking levels were in the range 75.3±2.1–104.9±8.5%. The coefficients of variation were in the range of 0.5–8.1%. Conclusion: The precision of the analytical method, in terms of relative standard deviation, was below 1.95%. The uncertainty in the quantification of all the validated elements was found to be ≤1.70% for Sample 1.


INTRODUCTION
Cholic acid is a primary bile acid.Bile acids are biological compounds belonging to the steroidal family generated in humans (liver) and the majority of animals [1,2].Lipid-rich diet may be the source of the accumulation of toxic elements, such as Zn, Cu, Cd, Mn, and Ni in the liver, that produces bile acids.As a result of the accumulation of toxic heavy metals in the liver, the enzymatic activity is inhibited and the metabolic pathways are altered.Moreover, the presence of such toxic heavy metals increases the risk of tumor formation [3].
Monitoring and quantification of the presence of toxic heavy metals in the liver or the byproducts of the liver like those of bile acids is a necessity from a health perspective.Though a web of science search with the keywords, namely, cholic acid and toxic heavy metals shows four results, none of them match with either the objective or the outcome of the present study.The aim of this work is to have the complete information on the amount of toxic heavy metals present in cholic acid-containing drugs which are consumed by humans in everyday life and to ensure whether it is under the permissible limit set by the United States Pharmacopeia (USP) 233 standard.
Harmful effects of the presence of toxic heavy metals, such as vanadium (V), nickel (Ni), cadmium (Cd), mercury (Hg), lead (Pb), and arsenic (As) in water, food, drugs, and environment are well known and mankind is no stranger to bearing the heat of such contaminants and this needs no elaborate introduction [4][5][6][7][8][9][10][11][12].Pharmaceutical regulatory agencies have set the permitted levels of heavy metals in medication, which are consistently monitored using limit tests.These tests confirm that no inorganic impurities are introduced into the medications during any of the manufacturing phases.The USP, the British pharmacopeia, the European pharmacopeia, and the Japanese pharmacopeia are all jointly monitoring the total metal impurity contents in pharmaceutical products.However, the procedures adopted are non-specific, insensitive, and time-consuming, needing improvement in accuracy excepting the few new legislations namely USP 232 and 233.Thus, very sensitive and selective procedures are urgently needed for determining trace toxic heavy metals in pharmaceutical compounds, not only to meet the demanding regulatory criteria but also to ensure the safety and efficacy of medication intended for human consumption [13].
In Quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS), the energy source, namely, plasma is advantageous over other energy sources, such as flame ionization, because it allows ionization to occur in a chemically inert environment, preventing oxide formation and the ionization is more complete.Q-ICP-MS analysis of toxic heavy metals is superior to other methods such as atomic absorption spectrometry, X-ray fluorescence spectrometry, and ICP optical emission spectrometry owing to exceptionally low detection limits for a large range of elements.Some components can be measured to the billionth of a trillionth of a trillion [14].Many researchers have previously applied this sophisticated analytical technique of Q-ICP-MS for bioanalytic purposes successfully [15][16][17].In addition to Q-ICP-MS of the toxic heavy metals in cholic acid, for comparison, ICP-OES analysis was carried out.Moreover, cholic acid commercial sample was further systematically characterized using SEM-EDAX to know the purity of the sample Fig. 1.
The goal of this study is thus to develop a fast, effective, simple, and accurate method of cholic acid sample preparation in conjunction with Q-ICP-MS to accurately determination of above mentioned toxic heavy metal impurities in oral medicinal products in a single test.

Materials and solutions
Cholic acid of synthesis grade used in the study is procured from Suvidhinath laboratories.Nitric acid (65%), ethylene diamine tetra acetic acid sodium salt AR grade, and certified reference metal stock standard solutions (1000 mg/L) of V, Co, Ni, Cd, Hg, Pb, and As prepared in 2-3% HNO 3 of analytical grade were purchased from Merck (Darmstadt, Germany).Deionized water was prepared using a Milli-Q plus water purification system from Millipore (Bedford, MA, USA).Yttrium standard for ICP TraceCERT® (1000 mg/L Y in nitric acid), bismuth standard for ICP TraceCERT® (1000 mg/L Bi in nitric acid), nitric acid ≥69.0%,TraceSELECT™ for trace analysis from Honeywell were used for the study.All the autosampler vials, centrifuge tubes, and plastic bottles, were cleaned by soaking in 20% v/v HNO 3 analytical grade reagent for 4 h, followed by rinsing with deionized Milli-Q water thrice.Element impurities according to ICH Q3D, Standard 1 (containing 15 ppm of Arsenic (As), 5 ppm each of Lead (Pb) and Cadmium (Cd), 30 ppm of Mercury (Hg), 50 ppm of Cobalt (Co), 100 ppm of Vanadium (V), 200 ppm of Nickel (Ni) and three other elements i.e., 150 ppm each of Selenium (Se) and Silver (Ag) and 8 ppm of Thallium (Tl) multi-standard were procured from Sigma-Aldrich.

Sample preparation
Weighed accurately about 100 mg of cholic acid commercial sample into a 15 mL calibrated plastic tube.Transferred 90 mL of (65%) HNO 3 into 3000 mL volumetric flask containing 1000 mL of deionized water mixed well and diluted up to the mark with water, and shaken well.Added 3 mL of concentrated nitric acid to the sample in the sample tube and allowed the sample to digest with intermittent shaking.After sample digestion, when the sample became clear and no more fumes of nitric acid were evolved from the sample tube, the content is made up to 10 mL mark with water.

Microwave digestion
There are open and closed-vessel approaches to microwave-assisted digestion.A closed vessel method is appropriate for a majority of pharmaceutical applications.Digestion was performed using Mth 2018-001, STD 75 manufactured by PerkinElmer 16 position unit size microwave digestion system.Weighed accurately 0.2 g sample into 10 mL volumetric flask and mixed it with 7.0 mL conc.HNO 3 .Transferred into the digester vessel and selected the digestion method as above and digested the sample.Cooled to the room temperature and transferred into 10 mL volumetric flask and made up with purified water.Pipetted out 5.0 mL into 10 mL volumetric flask and dilute up to the mark with deionized water.Details of sample digestion were given in Table S1.

Standard stock solutions for calibration
Standard stock solutions for calibration were prepared by taking 1.0 mL of elemental impurities according to ICH Q3D standard, namely, 1 mL of a standard containing 100 ppm of Vanadium (V), 50 ppm of Cobalt (Co), 200 ppm of Nickel (Ni), 5 ppm of Cadmium (Cd), 30 ppm of Mercury (Hg), 5 ppm of Lead (Pb) and 15 ppm of Arsenic (As) (Table S2).The standard stock solutions were then diluted to 20 mL with 2% nitric acid.Then, these stock solutions were further diluted to make different levels of standards for calibration (Table 1).

Spiked sample solution
Weighed accurately about 100 mg of sample into 15 mL calibrated plastic tube.The amount of standard stock solution 2 to be added is specified in Table 2. Added 3 mL of conc.HNO 3 and allowed the sample to digest with intermittent shaking.After sample digestion, when the sample become clear and all the fumes of nitric acid ceased to evolve from the sample tube, the digestion was made up to the mark with deionized water.

Instrumentation
Toxic heavy metal impurities in the cholic acid sample were determined by Agilent Technologies 5110 ICP-MS.The quantity of heavy metals, namely, V, Co, Ni, Cd, Hg, Pd, and As, were determined by Q-ICP-MS an iCAP RQ ICP-MS (Thermo Fisher Scientific) using QtegraTM software equipped with Q Cell Collision Reaction Cell, RAPID lens, with a quartz spray chamber, glass concentric nebulizer, online internal standard (ISTD) addition kit, and exchangeable skimmer cones.Optimization of Q-ICP-MS is important because the flow rates of the nebulizer gas and makeup gas should be adjusted to ensure the stability of the plasma.Q-ICP-MS was allowed to stabilize for 1 h and the performance was optimized based on radio frequency power, auto tune function in the control software, and A tune B solution, the quadrupole ion deflector voltages were optimized stepwise to find the settings that maximize signal intensity over the mass range, as well as for the 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 kept in KED mode for the analysis of V, Co, Ni, Cd, Hg, Pd, and As.

Criteria for validating the analytical method
For method validation, several criteria such as linear dynamic range, method linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and measurement of uncertainty were investigated and evaluated.In compliance with ICH Q2 (R1), Q-ICP-MS was used to validate the analytical method for the quantification of V, Co, Ni, Cd, Hg, Pd and As.

Q-ICP-MS analysis
Six replicate readings of 30 sweeps over the analyte mass range with a dwell time of 40 µs for each mass per sweep were used in a typical method of analysis.Sample aspiration followed by rinsing with 2% HNO 3 was done for 60 s.For running the instrument in KED mode, 4.34 mL min-1 of He gas was used.

Characterization of Cholic acid
Fourier transform infrared spectra of the samples were recorded at room temperature on a Perkin Elmer, U.S.A, spectrometer: (Model: Spectrum GX).The background due to air was measured, the adsorbent was added to KBr, and the sample was scanned 32 times over a frequency range of 400-4000 cm −1 . 1 H and 13 C NMR spectra were obtained using a Bruker, Model: 400 MHz FTNMR, Avance III Spectrophotometer, and [CDCl 3 ] acetone was used as a solvent.The chemical shifts are reported in ppm with respect to the TMS internal reference.SEM-EDAX analysis of the samples was carried out on Philips, Netherlands Model: ESEM EDAX XL-30 after depositing a gold coating.The voltage was 30 keV, and the field electron source was scanned at a resolution of 2 nm.

RESULTS AND DISCUSSION
Internal standard for the detection of toxic heavy metals While using Q-ICP-MS for elemental analysis, selecting an appropriate internal standard is critical.This would have a significant impact on the accuracy and precision of the results.

Optimization of operation parameters of Q-ICP-MS
Various optimized Q-ICP-MS parameters were reported (Table 3).

Method validation
In analytical chemistry, method validation is one of the technical aspects of the overall quality assurance scheme.Selectivity and specificity are determined by the element chosen and the corresponding possible potential interferences.It is always about "the extent to which the approach may be employed to determine the specific analytes in mixtures or matrices without interference from other components that behave similarly [18].The selectivity of the current approach was investigated using primary isotopes of each element, 51 V, 59 Co, 60 Ni, 111 Cd, 202 Hg, 208 Pb, and 75 As.A validation study was conducted to determine and prove the method's reliability.Some analytical characteristics were used to validate the approach.

Estimated LOD
The lowest concentration of an analyte in a sample that can be detected, but not necessarily quantified, is known as the LOD.It is a limit test that determines whether an analyte is above or below a certain threshold from the calibration function using Equation (1) [19].
where, σ is standard deviation S is slope derived from the calibration curve The estimated LODs were found to be 0.01, 0.01, 0.18, 0.002, 0.02, 0.02, and 0.10 µg/L for V, Co, Ni, Cd, Hg, Pd, and As, respectively.The minimum practical concentrations of tested elements in the analyzed samples, which can be determined with acceptable accuracy, were performed by analyzing 3 replicates at 30 µg/L for V, at 15 µg/L for Co.The estimated LODs were found to be 0.01, 0.01, 0.18, 0.002, 0.02, 0.02, and 0.10 µg/L for V, Co, Ni, Cd, Hg, Pd, and as, respectively.The minimum practical concentrations of tested elements in the analyzed samples, which can be determined with acceptable accuracy, were performed by analyzing 3 replicates at 30 µg/L for V, at 15 µg/L for Co, at 60 µg/L for Ni, at 1.5 µg/L for   4.

Estimated LOQ
The lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the method's stated operational circumstances is known as the LOQ.The noise-tosignal 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, µg/L for V, Co, Ni, Cd, Hg, Pb, and As, respectively.The results are reported in Table 4 [20].

Method linearity
The linearity of a test process is its ability (within a certain range) to deliver results that are directly proportional to the concentration of analyte in the sample, according to the CPMP guidelines [21].If the value of the calibration curve coefficient of determination (R2) is higher than 0.995, the quantification result will be accurate as analytical response will be linear over certain concentration ranges.The method linearity was investigated over a specific working range from different concentrations of reference standards.

Method accuracy
In the context of an analytical method, according to ICH guidelines, accuracy "is sometimes termed as trueness."The trueness of an analytical procedure reflects the closeness of agreement between the value that is either accepted as a conventional true value or an accepted Where, x i = i th reading in the data set µ = Mean of the data set n = Number of readings in the data set The results of the repeatability test expressed as RSD were found to be 2.74%, 2.46%, 1.95%, 5.02%, 3.45%, 2.56%, and 2.64%, for 111 Cd, 202 Hg, 208 Pb, 75 As, 51 V, 59 Co, and 60 Ni, respectively.Results of the linear regression analysis are reported in Table 5.

Estimation of measurement uncertainty
According to EURACHEM/CITAC GUIDE CG4, 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 such as 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%.The measurement uncertainties expressed as expanded uncertainties were estimated to be 13.3, 2.8, 16.7, 0.6, 4.1, 0.6, and 1.8, for V, Co, Ni, Cd, Hg, Pd, and As, reference value with the observed value.Therefore, accuracy is an expression of both trueness and precision since both of these influence the result [22].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 [23].

Precision study -repeatability and reproducibility
The precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple sampling.According to ICH, precision may be considered at three levels: repeatability, intermediate precision, and reproducibility.The precision was calculated in terms of relative standard deviation (RSD) using Equation 2and a single estimation of precision uncertainty was calculated using Equation 3, respectively.RSD = (S * 100)/x (2) respectively.The results on the uncertainty of each element in cholic acid are presented in Table 6.The study was extended to three more commercial samples of cholic acid and similar results were obtained as shown in Table 7.

Bias study (recovery test)
The spiking levels used for the recovery test were at 1.5, 5 and 7.5 µg/L for 111 Cd, 208 Pb, 4.5, 15, and 22.5 µg/L for 75 As, 9, 30, and 45 µg/L for 202 Hg, 15, 50 and 75 µg/L for 59 Co, 60, 200 and 300 µg/L for 60 Ni and 30, 100 and 150 µg/L for 51 V.The mean recoveries±standard deviations at different levels varied between 75.3±2.1% and 104.9±8.5% with coefficient of variation expressed as RSD ranged from 0.5% to 8.1%.Freshmen attending analytical chemistry courses are referred to the elegant review on ICP-MS technique by Wilschefski and Baxter.

CONCLUSION
The measurement of heavy metals in cholic acid using a microwaveaided acid digestion process and Q-ICP-MS analysis was satisfactorily confirmed in this study.For trace metal analysis of cholic acid, it is considered to be a good, reliable, and rapid approach.The accuracy of the present method is (30-150% of the target value) and precision value (n=6) successfully achieved the criteria defined by WHO, AOAC, USFDA, ICH, and USP 232/233.The proposed method was effectively applied for the routine analysis of heavy metals in cholic acid.LOD, LOQ, linearity, repeatability, reproducibility, accuracy, and precision have all been successfully assessed using the validated method.The suggested validated method is highly simple, quick, easy, cost-effective, and reliable, making it ideal for quantification of these hazardous metals in regular laboratory analysis.

Table 2 : Dilution for spiked sample preparation Level of spiked sample preparation (%) Amount of calibration standard stock solution to be added (mL)
LOQ: Limit of quantification

Table 1 : Dilution of standards for calibration Solution name (%) Volume of standard stock solution (mL)
LOQ: Limit of quantificationAsian J

Pharm Clin Res, Vol 16, Issue 12, 2023, 227-232 Kumar et al.
Cd, at 9.0 µg/L for Hg, at 1.5 µg/L for Pb, and at 4.5 µg/L for As.The results were reported in Table

Table 3 : Typical Q-ICP-MS instrument parameters for the analytical method
a RF: Radiofrequency; b QP: Quadrupole; c OctP: Octupole