Inductively Coupled Plasma Mass Spectrometry Method for Plasma and Intracellular Antimony Quantification Applied to Pharmacokinetics of Meglumine Antimoniate

Figures & data

Figure 1. Proposed meglumine antimoniate chemical structure.

Proposed structural formula for meglumine antimoniate (364 Da) in aqueous solution.

Adapted with permission from [23].

Figure 2. Representative calibration curve for antimony in human plasma.

Calibration standards for antimony (25, 50, 250, 500, 2500, 5000, 7500 and 10000 ng/ml) were quantified using inductively coupled plasma mass spectrometry and calibration was fitted with a linear model based on weighting of 1/concentration². This calibration was obtained from the analytical run 1 and processed with the software Watson LIMS version 7.5 SP1.

Figure 3. Antimony plasma and intracellular concentration–time curves.

(A–E) Pharmacokinetic curves for antimony plasma and intracellular (peripheral blood mononuclear cell) levels. Samples were procured from five patients undergoing antileishmanial therapy with meglumine antimoniate. Samples were collected during the last day of treatment (day 20) and evaluated up to 24 h following drug administration.

Table 1. Clinical characteristics of cutaneous leishmaniasis patients.

Sociodemographic characteristics	n (%), n = 5
Age, mean (SD)	30.2 (12.61)
Sex, n (%):
– Male	4 (80)
– Female	1 (20%)
Race, n (%):
– Aboriginal	3 (60)
– Mestizo	2 (40)
Clinical outcome, n (%):
– Cure	5 (100)
Patients reporting ADR, n (%):
– At least one	5 (100)
Intensity of ADR, n (%):
– Mild	4 (80)
– Moderate	1 (20)
Most common type of reaction, n (%):
– Elevated amylases	3 (60)
– Decreased leukocyte count	3 (60)
– Headache	3 (60)
– Myalgia	3 (60)
Isolated species:
– Leishmania (Viannia) braziliensis	3/5
– Leishmania (Viannia) panamensis	1/5
– Not available	1/5

ADR: Adverse drug reaction; SD: Standard deviation.

Table 2. Agilent 7700x operating parameters.

Parameter	Setting
ICP–MS
RF power	1550 W
# of points per peak	3
Replicates	3
Integration time/mass	0.09 s (^115In); 0.09 s (^121Sb)
Total acquisition time	1.98 s
Nebulizer/carrier gas flow	1.05 l/min
Dilution mode	OFF
Integrated sample introduction system (ISIS-DS)
Load time	8 s
Load speed	0.45 rps
Probe rinse time	90 s
Post rinse time	30 s
Post rinse speed	0.5 rps
Loop tubing ID	2.29 mm

ICP–MS: Inductively coupled plasma mass spectrometry; ISIS–DS: Integrated sample introduction system with discrete sampling; RF: Radio frequency.

Table 3. Calibration curve regression parameters for antimony.

Run day	Analytical run number	Slope	Intercept	R-squared	LLOQ	LOQ
1	1	0.0039481	0.0089183	0.9966	25	10000
1	2	0.0038862	0.0082196	0.9979	25	10000
40	3	0.0039172	0.018297	0.9969	25	10000
68	4	0.0032206	0.0037602	0.9981	25	10000
68	5	0.0031721	0.0065024	0.9981	25	10000
75	6	0.0035459	0.0058869	0.9964	25	10000
132	7	0.00386	0.022976	0.9953	25	10000
Mean		0.0036500	0.0106515	0.9970
SD		0.0003376	0.0071493	0.0011
%CV		9.25	67.12	0.11

LLOQ: Lower limit of quantitation; LOQ: Limit of quantitation; SD: Standard deviation.

Table 4. Back-calculated calibration standards for antimony in human plasma.

Analytical run number	25.0 ng/ml	50.0 ng/ml	250 ng/ml	500 ng/ml	2500 ng/ml	5000 ng/ml	7500 ng/ml	10000 ng/ml
1	21.9	48.5	246	494	2390	4980	7420	10300
	28.0	52.2	250	489	2440	5210	7630	10400
2	23.8	50.2	238	501	2470	4990	7370	10600
	27.1	47.2	243	485	2480	5180	7780	10200
3	23.8	45.3	247	512	2420	5260	7430	10600
	27.6	49.0	245	514	2400	5180	7620	9580
4	26.0	49.7	252	504	2510	5350	7580	10700
	25.1	46.3	240	483	2460	5050	7120	9970
5	24.3	47.2	246	486	2500	5150	7460	10400
	26.6	48.7	261	512	2430	4880	7130	10600
6	23.4	49.5	249	463	2260	5350	7630	9410
	25.7	54.0	258	508	2500	5200	7680	^† 13400
7	23.6	50.2	254	503	2390	4920	7180	10900
	27.8	44.2	241	501	2490	4830	7670	10900
Mean	25.3	48.7	248	497	2439	5109	7479	10351
SD	1.92	2.61	6.69	14.3	66.2	169	215	462
%CV	7.59	5.36	2.70	2.88	2.71	3.31	2.87	4.46
%Bias	1.20	-2.60	-0.80	-0.60	-2.44	2.18	-0.28	3.51
n	14	14	14	14	14	14	14	13

Reason for exclusion.

† >20% of acceptance criteria, not in statistics.

SD: Standard deviation.

Table 5. Accuracy and precision of antimony in human plasma.

Analytical run number	25.0 ng/ml	50.0 ng/ml	500 ng/ml	7500 ng/ml
1	24.8	55.1	486	7420
	26.1	48.6	486	7600
	26.5	47.9	491	7570
	23.2	46	510	7630
	24.2	50.6	495	7610
	26.1	50.6	498	7680
Mean	25.2	49.8	494.0	7585
SD	1.30	3.13	9.05	88.71
%CV	5.16	6.29	1.83	1.17
%Bias	0.80	-0.40	-1.20	1.13
n	6	6	6	6

SD: Standard deviation.

Table 6. Accuracy and precision of antimony in human peripheral blood mononuclear cells.

Analytical run number	25.0 ng/ml	50.0 ng/ml	500 ng/ml	7500 ng/ml
7	27.6	43.8	437	6390
	31.3	42.3	454	6440
	34.3	45	446	7050
	28.4	44.2	420	7310
	29.3	44	454	7450
Mean	30.2	43.9	442	6928
SD	2.68	3.13	9.05	88.70
%CV	8.88	7.14	2.05	1.28
%Bias	20.7	-12.3	-11.6	-7.63
n	5	5	5	5

SD: Standard deviation.

Table 7. Validated methods for antimony quantification in human samples.

	Anodic stripping voltammetry	Inductively coupled plasma mass spectrometry and on-line ion chromatography	Graphite furnace atomic absorption	Electrothermal atomic absorption Spectroscopy	Hydride generation atomic fluorescence spectrometry	Inductively coupled plasma mass spectrometry
Sample	Heparinized blood	Heparinized blood, plasma, urine, hair	Urine	Plasma, urine	Hair	Plasma, PBMCs
Amount of biological sample	5 ml	Blood, plasma and urine: ND^† Hair: 0.5–2.0 cm length (measured from the scalp)	2 g	ND	0.5 g	Plasma: 100 μl PBMCs: 2.27 × 10^6–25.7 × 10^6 cells
Extraction or digestion methods	ND^†	Blood, plasma and urine: ND^† Hair: nitric acid and hydrogen peroxide	Chelation with thenoyl-trifluoro-acetone in supercritical carbon dioxide	Nickelous nitrate hexahydrate in nitric acid and Triton X-100	Nitiric acid, hydrochloric acid and water in closed system	1% TMAH/1% EDTA
LLOQ	76 ng/ml	0.16 ng/ml for Sb^V 0.15 ng/ml for Sb^III	12.5 ng/ml	20 ng/ml	4.6 ng/g	25 ng/ml
CV at lowest quantification	1.3–3.4 %	0.9985 (R^2) for Sb^V 0.9989 (R^2) for Sb^III	2.8% (RSD)	11.1%	2.8% (RSD)	Plasma: 5.16% PBMCs: 8.88%
Quantification range of Sb concentrations	76–29,500 ng/ml	0–100 ng/ml for both Sb^V and Sb^III	2.5–200 ng/ml	0–200 ng/ml	ND^†	25–10000 ng/ml
Sb^V and Sb^III speciation	No	Yes	No	No	No	No
Reference	Chulay JD et al. (1988) [27]	Miekeley N et al. (2002) [12]	Hui-Ming L (2013) [28]	Cruz A et al. (2007) [11], de Aguilar MG et al. (2018) [29]	Cardozo MC et al. (2016) [30]	Current study

LLOQ: Lower limit of quantification; ND: Not described; PBMC: Peripheral blood mononuclear cell; TMAH: Tetramethylammonium hydroxide.

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