Quantification of GS-441524 concentration in feline plasma using high performance liquid chromatography with fluorescence detection

Figures & data

Figure 1. Biosynthesis of remdesivir in human (individual image sourced from PubChem. CID 121304016 (GS-5734), CID 121313150 (GS-704277), CID 44468216 (GS-441524), and CID 56832906 (GS-443902).

Figure 2. Chromatograms depicting fluorescence excitation (left: max λ_ex: 241 nm) and emission spectrum (right: max λ_em 405 nm) of GS-441524 (100 ng/mL); y axis is absorbance; x axis is wavelength in nm.

Figure 3. Chromatograms of blank plasma monitored at λ_ex: 245 nm/λ_em: 395 nm (red trace) and at λ_ex: 250 nm/λ_em: 475 nm (black trace). Blank plasmas pre-spiked with mixtures of GS-441524 & remdesivir [78.1 (lower blue trace) and 313 ng/mL (higher blue trace), respectively] monitored at λ_ex: 250 nm/λ_em: 475 nm.

Figure 4. Typical chromatograms of blank plasma (black trace); blank plasma pre-spiked with a mixture of GS-441524, remdesivir, and GS-443902 (blue trace), a mixture of GS-441524, remdesivir, and alanine metabolite (red trace), and a mixture of medications often used in FIP patients for initial supportive care or sedation (see methods) (brown trace). No peaks are interfered with retention time (RT) of GS-441524 (RT: 4.78 min). Rt of remdesivir: 9.95 min; Rt: Alanine metabolite: 2.21 min; GS-443902 (1.98 min).

Table 1. Intra- and inter day precision and trueness of QCs (GS-441524).

Con(ng/mL)	Intraday (n = 3)			Interday (n = 3 × 3 days)
	Trueness	Precision		Trueness	Precision
	Bias (range)	Ave (± SD)	CV	Bias (range)	Ave (± SD)	CV
35.6	−1.6 to 6.1	35.1 (± 1.8)	5.2	−8.9 to 6.1	35.7 (± 1.4)	3.9
563	−3.4 to 7.5	554.7 (± 31.4)	5.7	−3.8 to 7.5	551.8 (± 21.7)	3.9
9000	−1.4 to 2.4	8977.7 (± 177.3)	2.0	0.2 to 1.4	8891.3 (± 80.7)	1.0

Table 2. Three cycle freeze and thaw stability (GS-441524) (n = 3).

	Initial (n = 3)	1st cycle (n = 3)	2nd cycle (n = 3)	3rd cycle (n = 3)
Ave ± SD	8938.8 ± 68.9	8787.2 ± 133.4	8690.6 ± 295.7	8842.7 ± 221.0
Bias		1.7	2.8	1.1
p-value		0.49	0.17	0.62
Ave ± SD	35.7 ± 0.7	36.6 ± 0.4	35.5 ± 1.0	36.5 ± 0.2
Bias		2.5	0.7	−2.1
p-value		0.29	0.65	0.16

Note: Bias & p-value of each cycle was determined from initial concentrations; p-value was determined from two tailed paired t-test.

Figure 5. Chromatograms of plasma GS-441524 after single IV injection of remdesivir (15 mg/kg BW); t = 0 hr (black trace), 1 hr (pink trace), 2 hr (blue trace), 8 hr (brown trace), 20 hr (green trace), and 24 hr (dark blue trace).

Figure 6. * Semi-logarithmic plasma GS-441524 concentrations vs. time curve in a cat with FIP following IV injection of remdesivir 15 mg/kg BW (red curve; n = 1); trough plasma GS-441524 concentrations after the administrations of remdesivir (9.87–15 mg/kg BW, IV or SC) in cats with FIP (n = 11); trough GS-441524 range from 23.7–190.1 ng/mL (84.9 ± 61.9 ng/mL, avg ± SD).

Table 3. Analytical methods to quantify GS-441524 in biological fluid (plasma).

Study	Compounds	Samples (Vol. uL)	Extraction	Injection vol (µL)	Standard	Instruments	Mobile phase (min)	Calibration range (LOQ)
Method in this study	GS-441524	Feline. plasma (50 uL)	Protein precipitation	10	External standard	UPLC-FLD (Ex 250 nm; Em 475 nm)	Gradient (15 min)	19.5–10,000 ng/mL (19.5 ng/mL)
Alvarez et al. 2020	GS-441524 Remdesivir	Human plasma (50 uL)	Protein precipitation	20	Internal standard	LC-MS/MS	Gradient (5 min)	5–2500 ng/mL (5 ng/mL)
Avataneo et al. 2020	GS-441524 Remdesivir	Human plasma	Protein precipitation	8	Internal standard	LC-MS/MS	Gradient (4 min)	0.98–1000 ng/mL (0.98 ng/mL)
Du et al. 2021	GS-441524 Remdesivir	Rat plasma (20 uL)	Protein precipitation	1	Internal standard	LC-MS/MS	Gradient (4.5 min)	2–1000 ng/mL
Habler et al. 2021	GS-441524 & Others	Human serum (50 uL)	Protein precipitation & Solid phase extraction	5	Internal standard	2D-UHPLC-MS/MS	Gradient (5 min)	22.2–186.8 ng/mL (22.2 ng/mL)

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Data availability statement

The data supporting the findings of this study are available from Dr. Benjamin Kimble ([email protected]) upon request.