INTRODUCTION:

Influenza infections pose a serious health threat, especially for vulnerable populations. While vaccination is a mainstay of prevention, limitations exist^1-5. Baloxavir marboxil, a prodrug that binds to particular molecular targets and strongly inhibits the influenza polymerase acidic (PA) protein, an enzyme solely linked with the virus, thereby prevents influenza infection^6-9. Analytical investigations of BXM's interaction with other drugs are less^10-12.

There are no reported methods for quantifying BXM using an internal isotopic standard. This study addresses the need for improved methods to quantify Baloxavir marboxil (BXM), an antiviral drug. We present a novel LC-API-MS/MS method for accurate BXM measurement in human plasma. This method offers advantages in simplicity, speed, and precision, making it valuable for pharmacokinetic studies and therapeutic drug monitoring.

Chemicals and Reagents:

Baloxavir marboxil (BXM) and Baloxavir marboxil d4 (IS) standard: Obtained from a commercial source.All chemicals are of analytical and HPLC grade. Human plasma with K2 EDTA wasobtained from a reputable blood bank. Stored at -20°C before use.

Liquid Chromatography (LC) and Mass Spectrometry (MS) Conditions:

LC System: Shimadzu HPLC API 2000 LC-MS/MS (or equivalent).

Ionization Mode: Positive atmospheric pressure ionization (ESI).

Chromatographic Separation:

· Column: Zorbax SB C18 (50mm x 4.6mm, 3.5μm particle size).

· Mobile Phase: 25:75 (v/v) mixture of HPLC-grade 5 mM ammonium acetate buffer and methanol

· Flow Rate: 1.0 mL/min

· Column Temperature: Ambient

Mass Spectrometry:

· Ion Spray Voltage: 5500 V

· Source Temperature: 550 °C

Gas Settings:

· Curtain Gas: Optimized at 40 V for both BXM and IS

· Collision Gas: Optimized at 6.00 V for BXM and adjusted for IS

· Dwell Time: 200 milli seconds

· Resolution: 1 unit (m/z)

· Detection Mode: Multiple Reaction Monitoring (MRM)

BXM Transition: 572.60 → 250.30 (m/z)

IS Transition: 576.60 → 254.30 (m/z)

Software: Analyst 1.4.2 (or equivalent) for LC and MS control.

Standard and Sample Preparation:

The internal standard (IS) and BXM stock solutions were 1.0mg/mL in 60:40 methanol-water. After preparation, these solutions were kept at 2-8°C. Diluting the stock solution with diluent provided BXM calibration standards. These working solutions were diluted with blank human plasma to create calibration standards. These standard concentrations were kept at 2-8°C. To evaluate technique performance during analysis, quality control (QC) samples were produced at varied BXM concentrations in human plasma. Stock solutions, working solutions, calibration standards, and QC samples were prepared and stored at -20°C.

Sample Preparation:

Plasma obtained from a cryogenic freezer. Added 25µL of 500.00ng/mL IS solution to each sample and mix well. TBME was the extraction solvent. After shaking for 20 minutes at 200RPM, the mixture was centrifuged at 4000RPM for 10 minutes at 4°C. The upper organic layer (about 4mL) was carefully transferred and evaporated at 45°C with liquid nitrogen. Reconstituted and vortexed residue in mobile phase. The reconstituted sample was analyzed using a 20µL aliquot in the LC-MS/MS Instrument. BXM concentrations in unknown samples were calculated using the calibration curve.

Method Validation:

The method's performance was rigorously evaluated following established guidelines for bioanalytical method validation^13-15.

To ensure the method's reliability, various tests were performed to assess its:

Selectivity: This evaluated potential interferences from other substances in human plasma samples.

System Suitability: This ensured consistent instrument performance throughout analysis.

Specificity: This confirmed the method's ability to measure BXM specifically, using plasma from different sources and treatments.

Calibration Curve: This established a relationship between the BXM concentration in a sample and the instrument's response.

Precision and Accuracy: This evaluated the method's consistency and correctness in measuring BXM at various concentrations.

Recovery and Matrix Effect: This assessed the efficiency of BXM extraction from plasma and potential interferences from plasma components on the measurement.

Dilution Integrity: This verified the accuracy of diluting BXM-spiked plasma samples without affecting the final concentration.

Ruggedness: This evaluated the method's performance across different analysts, columns, and instruments.

Stability Experiments: This assessed the stability of BXM in various conditions, such as injection solvent storage, incubation at room temperature, freeze-thaw cycles, and long-term freezing.

RESULTS:

Sample Preparation and Chromatography:

Sample preparation involved extracting BXM from human plasma using tert-butyl methyl ether (TBME) after evaluating various extraction solvents. Chromatographic separation utilized a Zorbax SB C18 column and a mobile phase consisting of 5 mM ammonium acetate buffer and methanol (25:75 ratio) at a flow rate of 0.5mL/min. The column temperature was maintained at 40°C.d4-Baloxavir Marboxil (d4 BXM) was chosen as the internal standard due to its structural similarity to BXM. The analysis runtime was approximately 150 seconds, with BXM and d4 BXM eluting at a retention time of around 1.2 minutes.

Both BXM and the internal standard were positively ionized using electrospray ionization (ESI), generating protonated precursor ions (M+H+). Instrument parameters like collision gas flow, dwell time, and temperature were optimized to maximize the detection response of the daughter ions.

Figure 1: Mass spectra of Baloxavir morboxil & Baloxavir-IS

Method Validation:

Selectivity and System Suitability:

Blank plasma samples from different sources were analyzed to confirm the method's ability to distinguish BXM from other components in human plasma.System performance was regularly monitored by evaluating the retention time and peak area consistency for BXM and the internal standard.

Sensitivity and Carryover Effect:

The lowest concentration of BXM that could be reliably measured (Limit of Quantification, LLOQ) was determined to be 10.160ng/mL. Measures were taken to minimize carryover effects, ensuring accurate measurements at low concentrations.

Specificity:

Analysis of blank plasma samples confirmed the absence of significant interferences from other biological molecules at the retention times of BXM and the internal standard.

Linearity:

The method demonstrated a linear relationship between the concentration of BXM in a sample and the instrument's response within a specific concentration range (10.160 to 1229.080ng/mL).

Table 1: Calibration Curve Standards Data of BolaxavirMarboxil

CC ID	Conc. (µg/mL)	N	Mean	SD	%CV	Recovery %
CC 1	10.16	6	10.06	0.075	0.76	99.01
CC 2	20.32	6	20.68	0.278	1.35	101.81
CC 3	60.84	6	62.49	1.551	2.48	102.73
CC 4	121.67	6	118.77	1.464	1.23	97.61
CC 5	243.58	6	230.05	11.315	4.92	94.53
CC 6	486.71	6	498.07	12.804	2.57	102.33
CC 7	737.45	6	729.98	4.834	0.66	98.99
CC 8	982.26	6	998.57	23.261	2.33	101.56
CC 9	1229.08	6	1246.73	16.674	1.34	101.44

Recovery:

The percentage of recovery was determined to be 75.80% for BXM and 52.46% for IS. (Table 2)

Table 2: Recovery Data of Bolaxavir marboxil

Analyte	ID	QC sample (ng/mL) n=6	% Recovery ±%CV
BXM	LQC	30.74	78.41±1.54
	MQC	153.74	72.94±1.55
	HQC	1024.95	76.05±0.65
BXM-IS	----	500	52.46±3.84

Precision and Accuracy:

The method's precision and accuracy were assessed through experiments involving multiple sample batches (inter-batch) and replicates within a single batch (intra-batch). Both precision (measured by %CV) and accuracy (% of actual concentration measured) were evaluated for different BXM concentration levels (LLOQ, LQC, MQC, HQC). The results demonstrated acceptable precision and accuracy across all quality control levels, ensuring reliable measurement of BXM in human plasma samples.

Table 3: Intra and Inter-day Precision Data of Bolaxavir marboxil

Measured concentration (ng/mL)
Intra-day variation (Six replicates at each concentration)
Theoretical Concentration (nm/ml)	BXM
Theoretical Concentration (nm/ml)	Mean	SD	% CV	Accuracy (%)
LLOQ	9.98	0.82	8.26	97.20
LQC	30.16	3.04	10.09	98.09
MQC	135.98	2.91	2.15	88.45
HQC	935.36	14.84	1.59	91.26
Inter-day variation
LLOQ	10.34	1.06	10.31	100.74
LQC	29.541	2.619	8.87	96.08
MQC	138.076	3.923	2.84	89.81
HQC	939.235	14.008	1.49	91.64

Matrix Effect and Dilution Integrity:

The method was evaluated to account for potential interferences from plasma components on BXM measurements. Dilution experiments confirmed that diluting plasma samples did not affect the accuracy of BXM concentration measurements.

Table 4: Stability Data of BolaxavirMarboxil

Drug	Nominal conc. (ng/mL)	Stability	Mean ± SD^a (n=6) (ng/mL)	Accuracy (%)^b	Precision (%) % C.V.
BXM LQC	30.748	7.00 h (room temp.)	31.96±3.25	103.82	0.37
	30.748	7.00 h (room temp. IS)	31.81±2.48	103.57	1.62
	30.748	7 days (2-8 ˚C stock)	31.21±5.24	102.61	0.57
	30.748	7 days (2-8 ˚C IS)	31.82±6.24	103.59	1.13
	30.748	15.00 h (bench top)	29.32±8.94	98.73	1.20
	30.748	47.00 h (auto sampler)	29.36±2.15	99.75	1.54
	30.748	42 h (wet extract)	28.38±1.98	94.41	5.68
	30.748	23 h(reinjection)	27.63±2.51	89.90	0.49
	30.748	3 h (in whole blood)	27.97±3.21	90.18	2.16
	30.748	Freeze-thaw stability(4 cycles at-70±5 ˚C)	27.99±2.21	90.24	0.27
BXM HQC	1024.95	7.00 h (room temp.)	931.23±13.84	94.41	1.71
	1024.95	7.00 h (room temp. IS)	915.18±10.14	90.55	1.13
	1024.95	7 days (2-8 ˚C stock)	998.43±14.20	98.29	1.12
	1024.95	7 days (2-8 ˚C IS)	913.16±16.34	90.55	1.54
	1024.95	15.00 h (bench top)	889.61±11.78	88.83	5.68
	1024.95	47.00 h (auto sampler)	905.49±15.49	90.19	1.01
	1024.95	42 h (wet extract)	938.36±17.41	94.41	2.16
	1024.95	23 h(reinjection)	908.32±11.14	90.24	1.87
	1024.95	3 h (in whole blood)	1038.49±5.83	100.52	0.66
	1024.95	Freeze-thaw stability(4 cycles at-70±5 ˚C)	989.25±13.44	98.83	1.13

Ruggedness:

The method's performance remained consistent across different analysts, columns, and instruments, demonstrating its robustness.

Stability Studies:

BXM was found to be stable under various conditions, including storage in the injection solvent for extended periods, incubation at room temperature, freeze-thaw cycles, and long-term freezing.This ensures reliable BXM measurement in processed samples without significant degradation.Specific data points from Table 4 were omitted to maintain a general overview of the method's validation.

DISCUSSION:

· This study developed a rapid and sensitive method for measuring BXM in human plasma using liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

· Optimization of identified positive ion mode electrospray ionization (ESI) as the most suitable ionization method for BXM and the internal standard.

· The mobile phase composition was carefully chosen to improve sensitivity and minimize interferences from other substances in the plasma samples (matrix effect).

· Selecting an appropriate extraction solvent was crucial in reducing these interferences and ensuring accurate BXM measurement.

CONCLUSION:

The validated LC-MS/MS method offers a precise, accurate, and sensitive approach for quantifying BXM in human plasma. This method's lower limit of quantification makes it suitable for pharmacokinetic studies, and its rapid analysis time allows for efficient processing of multiple samples. The successful application of this method in human trials demonstrates its effectiveness for studying BXM pharmacokinetics.

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Received on 14.12.2022 Modified on 25.10.2023

Research J. Pharm. and Tech 2024; 17(6):2694-2698.

DOI: 10.52711/0974-360X.2024.00422