(T1330-09-50) Quantitative Determination of Arsenic in Dog Blood and Human Blood by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for Arsenic Drug Development in Pre-Clinical and Clinical Studies

Purpose: Arsenic is the twentieth most abundant element in the earth's crust and is present in all living organisms. It is found in diet such as seafood, poultry, grains (especially rice), bread, cereal products, mushrooms, and dairy products. Specific forms of arsenic have been conventionally used as medicine. Arsenic is also contained in traditional Chinese medicine formulations and used for psoriasis, syphilis, asthma, joint pain (rheumatism), hemorrhoids, cough, itchiness, cancer, to reduce swelling (as an anti-inflammatory agent), and as a general tonic and pain-killer. In leukemia, arsenic trioxide increases death of cancer cells. To support arsenic drug development in pre-clinical and clinical phases, we hereby introduce validated methods for quantitative determination of arsenic in dog blood and human blood, respectively, by inductively coupled plasma-mass spectrometer (ICP-MS). The validated methods established linear range of arsenic at 0.5 – 1300 ng/mL in blood matrices, and the validation results met criteria set forth in regulated bioanalytical method validation guidelines.

Methods: Instruments: An Agilent 7850 ICP-MS equipped with SPS 4 Well Plate Kit, which allows for testing blood sample volume of 30 µL (before dilution). Arsenic standard solution (1000 µg/mL) and Germanium standard solution (100 µg/mL) were used to prepare standard and internal standard solutions, respectively. Biological Matrices: Dog blood, with K₂EDTA as anti-coagulant. Human blood, with K₂EDTA as anti-coagulant. Sample Preparation: Sample preparation were conducted with 10x dilution by alkaline diluent. The alkaline diluent was composed of 1% NH₄OH, 3% Butanol, 0.01% EDTA (H₄EDTA) and 0.01% Triton X 100. Calibration standards were prepared in dog blood or human blood in their respective methods at the concentrations of 0.5, 1.0, 5.0, 20.0 , 40.0, 200, 450, and 1300 ng/mL, respectively; Quality control (QC) sample concentrations were LLQC (0.5 ng/mL), LQC (1.5 ng/mL), GMQC (15 ng/mL), MQC (360 ng/mL), and HQC (1100 ng/mL) in respective blood matrices, and were prepared in 6 replicates at each QC concentration level. 30 µL of each calibrators and QCs were then added to 96-well plate, diluted (10x) collectively with the alkaline diluent and internal standard solution, vortexed, and centrifuged at 6200 rpm for 10 minutes at ambient temperature, before injected to Agilent 7850 ICP-MS.

Results: Linearity:  Three Accuracy/Precision runs were conducted for human blood and dog blood, respectively. The linearity results are summarized in below. Accuracy and Precision: Three Accuracy/Precision runs were conducted for human blood and dog blood, respectively. The accuracy/precision results are summarized in below. Matrix Effect: Matrix effect were assessed by spiking arsenic at GMQC (15.0 ng/mL) and HQC (1100 ng/mL) in 6 lots of human blood and dog blood, respectively. Neat solutions containing analyte and internal standard were prepared in diluent in singlet and the final concentrations were equivalent to respective GMQC and HQC samples. Matrix Factor (MF) of analyte or internal standard were calculated by dividing the mean signal intensity (in Counts Per Second, CPS) of the spiked blank samples of each lot by the corresponding mean CPS of neat solutions. The IS-normalized MF were calculated by dividing the analyte MF by internal standard MF. The results are summarized in below. Matrix Effect of Lipaemic Human Blood: Lipaemic matrix effect was assessed by spiking arsenic into lipaemic human blood (K₂EDTA) at GMQC (15.0 ng/mL) and HQC (1100 ng/mL), respectively.  Samples were prepared in triplicates at each concentration level. Lipaemic human blood (K₂EDTA) was prepared by adding lntralipid IV to human blood (K₂EDTA) to obtain a lipaemic human blood (K₂EDTA) with final lipid concentration at 12.3 mg/mL. Arsenic in the prepared lipaemic samples were then quantified, and the results were compared with respective nominal concentrations. Results are summarized in below.

Conclusion: Based on the results presented, bioanalytical methods were successfully validated to quantitatively determine arsenic in dog blood and human blood, respectively, in order to support arsenic drug development in pre-clinical and clinical phases. The methods need only 30 µL of blood sample for each testing, with high sensitivity (LLOQ at 0.5 ng/mL) and linearity in the range of 0.5 – 1300 ng/mL.

References: 1. US FDA Guidance for Industry, Bioanalytical Method Validation, May 2018.

Acknowledgments: No conflicts

Linearity Results of Accuracy/Precision Runs of the Method in Human Blood



Accuracy and Precision Results of the Method in Human Blood



Matrix Effect in Human Blood