INTRODUCTION:

The replication of the influenza virus predominantly occurs in the epithelial cells of both the upper and lower respiratory tract in humans.^1-3 This viral infection has the potential to spread rapidly within populations, leading to seasonal influenza epidemics worldwide each year.⁴ Particularly, in elderly or immunocompromised individuals, influenza virus infections can result in severe and even fatal outcomes.⁵

While influenza vaccination is considered a crucial preventive measure, its efficacy in immunocompromised individuals remains a subject of debate, despite efforts to enhance immunogenicity through various strategies such as exploring new vaccines, adjusting vaccination doses, timing, or incorporating adjuvants.^6-7

Influenza viruses A, B, C and D are the 4 types of seasonal viruses found. Influenza A viruses, depending on the combinations of the proteins on the surface of the virus, are classified into subtypes - A(H1N1), also known as the swine flu, and A(H3N2) influenza viruses. However, pandemics are known to be caused by only influenza type A viruses. Severity in terms of fever, leukopenia, and C-reactive protein is more in case of Influenza A H3N2 infection than A H1N1 or B. H3N2, a non-human influenza virus that normally circulates in pigs but can infect human beings, according to the US Centre for Disease Control and Prevention (CDC).⁸

Another recent development in India is the massive infection causing by Swine flu virus, a new strain of influenza virus- A/H1N1. Hospital in-patients admitted with novel influenza A (H1N1) infection are monitored and treated with antiviral therapy.⁹

Neuraminidase (NA) inhibitors currently stand as the most widely utilized class of anti-influenza drugs.¹⁰ Oseltamivir phosphate, an ethyl ester pro-drug administered orally is the best-known antiviral drug that slows the spread of influenza (flu) viruses (type A and B) between cells in the body.¹¹ However, the emergence of influenza viruses resistant to NA inhibitors poses a significant concern.^12-13 In addressing this issue, Baloxavir marboxil has been developed as an antiviral drug specifically for treating both influenza A and influenza B infections. Initially approved by the USFDA on October 24, 2018, for the treatment of acute uncomplicated influenza in patients aged 12 and older who have been symptomatic for no more than 48 hours, Baloxavir marboxil functions as an orally available small-molecule inhibitor of a cap-endonuclease, presenting a distinct mechanism of action compared to the neuraminidase inhibitor drug class commonly used for influenza infections.¹⁴ Baloxavir marboxil is a prodrug that is converted by hydrolysis to baloxavir, the active form that exerts anti-influenza virus activity

Numerous methods for estimating the concentration of baloxavir have been reported in the literature. Most of these methods employ oseltamivir or other antiviral drugs as internal standards, and the sensitivity of these methods tends to be lower. To align with regulatory requirements specifying that the internal standard should preferably be an isotope of the analyte, we present a novel, sensitive, and relatively straightforward LC–MS/MS method for estimating baloxavir in human plasma. This method utilizes baloxavir d5 as the internal standard and has undergone validation in accordance with FDA regulations, making it suitable for pharmacokinetic studies.¹⁵

MATERIALS AND METHODS:

Baloxavir and Baloxavir d5 were procured from BioOrganics, India.

Methanol (HPLC-grade), acetonitrile, ammonium acetate and formic acid of highest purity grade were purchased locally. In this study Milli Q purified water (Millipore, Milford, MA) was used.

In-house collected plasma were used for the experiments.

Preparation of analytes and internal standard solutions:

Stock solution of Baloxavir (80.0000µg/ml) was prepared in DMSO: Methanol: 50:50v/v. The stock solution of Baloxavir was then diluted to 25.068ng/ml to 15200.000ng/ml using DMSO: Methanol:50:50v/v as diluent.

Similarly, the internal standard, Baloxavir-d5, stock solution (80.0000 µg/ml) was prepared by dissolving in DMSO: Methanol:50:50v/v. It was then diluted to 100.000ng/ml using the same solution as diluent.

The concentration of each stock solution was corrected by considering its potency and actual amount weighed before dilution.

Preparation of calibration standards:

The calibration standards were prepared between the range of 0.505ng/ml to 302.724ng/ml in plasma by spiking 2% of the stock dilutions respectively. Stock dilutions were prepared in Methanol: DMSO:50:50v/v using respective stock solutions.All these bulk spiked samples were stored at about -70°C in aliquot of 200 µL.

Preparation of Quality Control Samples:

Quality Control samples were similarly prepared at the concentrations of 0.526ng/ml for LOQQC, 1.422ng/ml for LQC, 32.320ng/ml for M1QC, 107.734ng/ml for MQC, 236.258ng/ml for HQC and 472.516ng/ml for DIQC in plasma by spiking 2% of the above stock dilutions respectively. The samples were then stored at about -70°C.

Sample preparation:

50 µL of internal standard mixture (Baloxavir-d5) was added to all RIA vials except blank. 100µL of sample was then added to each labeled RIA vial. 100µl of 1% Formic acid was added to respectively labeled RIA vials followed by 2.0 ml of extraction solvent (TBME:Ethyl Acetate ::70:30 v/v) to all samples. The samples were kept in vibramax at 2500rpm for 10 mins. The samples were centrifuged at 4000 rpm for 5 mins in refrigerated centrifuge at about 4°C. About 1.6ml of supernatant was transferred into respectively labeled RIA vials. The samples were dried at about 40°C in nitrogen evaporator and then reconstituted with 0.750ml of Acetonitrile: Milli-Q-Water:50:50 v/v solution. After vortexing, the samples were centrifuged at 11000rpm for 5mins at about 4°C. The samples were transferred to labeled HPLC vials and loaded into auto sampler.

Chromatography:

Baloxavir was estimated using LCMS/MS. The analyte was separated in the liquid chromatography followed by detection in the mass spectrometer. This is known as hyphenated technique which refers to a combination of two (or more) techniques to detect and separate chemicals from solutions. Here UPLC was used which has major advantages over HPLC in three areas: speed, resolution, and sensitivity.^16-17 The chromatographic method is described below.

10µL of sample was injected on a Acquity UPLC Peptide BEH C18 Column, 300Å, 1.7µm 2.1mm X 150 mm. Chromatographic separation was achieved using the following gradient (Table 1). Pump A contained 0.5% Formic acid and Pump B contained Methanol: Acetonitrile:80:20v/v. Flow rate was 0.3 ml/min without splitter in Waters UPLC attached to API 4000 Mass spectrometer (Applied Biosystems, USA). The column was maintained at 55⁰C in the column oven. The run time was 6.0 minutes.

Table 1: Gradient used for chromatographic separation

Time	Flow Rate	A%	B%
Initial	0.300	45.0	55.0
3.50	0.300	45.0	55.0
3.51	0.300	5.0	95.0
4.00	0.300	5.0	95.0
4.01	0.300	45.0	55.0
6.00	0.300	45.0	55.0

Mass Spectrometry:

Electrospray ionization (ESI) interface operated in positive atmospheric pressure ionization mode (API) was used for the multiple reaction monitoring (MRM). The protonated form of the analyte and the IS, [M+H] + ion was the precursor ion in the Q1 spectrum and was used as the precursor ion to obtain Q3 product ion spectra. Optimization of the collision energy and collision cell exit potential led to the most intense and consistent product ion Q3 MS spectra of the analyte and the IS. The source parameters like nebulizer gas (NED), collision gas (CAD), temperature and ion spray voltage were optimized to obtain adequate and reproducible response for the analyte.¹⁸

The operational conditions were optimized by infusing diluted stock solution of analyte and internal standard (Table 2).

Table 2: MS parameters for Baloxavir

Analyte	Q1	Q3	DP	EP	CE	CXP
Baloxavir	484.1	247.0	70	10	30	15
Baloxavir d5	489.1	252.1	70	10	30	15

Source temperature was set at 450°C. Nebulizer gas (GS1) and auxiliary gas (GS2) flows were 45 and 55 psi, respectively. Quadrupoles Q1 and Q3 were set on unit resolution.

MRM transitions were monitored as m/z 484.1→ 247.0 (Baloxavir), and m/z 489.1→ 252.1 (IS).

The MS/MS spectra for the parent ion 484.1 and the fragment giving the daughter ion 247.0 is shown in Figure 1.

Figure 1: MS/MS spectra for Baloxavir

Sample concentrations were calculated by linear regression analysis using the analyst software 1.7.2. Data was processed by peak area ratio. The concentration of unknown was calculated from the equation (Y= mX+ c) using regression analysis of spiked plasma calibration standards with reciprocal of the square of the drug concentration (1/X²).

RESULTS AND DISCUSSION:

Method Development:

In the process of developing a straightforward and easily applicable method for determining Baloxavir in human plasma, UPLC-MS/MS was chosen as the preferred technique. Throughout the method development, various parameters such as mobile phase composition, column type, flow rate, injection volume, and sample volume, along with mass spectrometric, sample extraction, and internal standard parameters, were systematically optimized to achieve optimal results. Since we use reverse phase column, polar solvents such as water, acetonitrile, ethanol or methanol are generally used. The choice of mobile phase is governed by the physical properties of the solvent like polarity, miscibility with other solvents, chemical inertness, and toxicity. The polarity index gives an indication of the ability of a solvent to elute a compound from the column. ¹⁹ Despite using different branded RP-HPLC C8 and C18 columns for the separation of Baloxavir, peak distortion occurred due to tailing.

Efforts were made to address this issue by experimenting with different organic modifiers and buffers commonly used in reverse-phase chromatography. However, peak tailing persisted, likely attributed to nonspecific hydrophobic interactions with the acidic silanol groups present in the stationary phase. To counteract this, the mobile phase's pH was lowered to ensure complete protonation of residual silanol groups.

Formic acid was employed as the mobile phase buffer, initially at concentrations of 0.1% and 0.2%, which are commonly used strengths, but peak tailing persisted. Utilizing an end-capped peptide column (Acquity UPLC Peptide BEH C18 Column, 300Å, 1.7µm 2.1mm X 150 mm) and increasing the Formic acid concentration to 0.5% showed an improvement in peak shape, resolving the peak tailing issues.

Another challenge encountered during method development was variation in response due to potential multiple column long runner peaks in the sample. This was addressed by washing the column during the run, increasing the organic modifier concentration from 55% to 95% after the retention time of the analyte and internal standard.

Analyte (Baloxavir) and internal standard (Baloxavir d5) were eluted at 3.61 min and 3.56 min, respectively, within a total runtime of 6 minutes. Among various sample preparation methods, liquid-liquid extraction (LLE) was deemed suitable for its simplicity, high recovery, and minimal ion suppression effect. LLE, a cost-effective alternative to solid-phase extraction (SPE), demonstrated robustness, providing clean samples with good and reproducible recoveries for both analyte and internal standard.

Electrospray ionization (ESI) in positive ion mode was selected over atmospheric pressure chemical ionization (APCI) for its maximum response. The instrument was optimized for sensitivity and signal stability, and various source-dependent and compound-dependent parameters were fine-tuned for Baloxavir and the internal standard.

A gradient method using 0.5% Formic acid and Methanol:Acetonitrile::80:20v/v mobile phase was implemented for sharp peak resolution, achieving a total run time of 6 minutes—an ideal duration for high-throughput analysis. Retention times for Baloxavir and the internal standard were 3.61 min and 3.56 min, respectively.

Method Validation:

This method was validated as per the FDA Guidelines for specificity, linearity, intra- and inter-day precision and accuracy, and stability.¹²

Selectivity and specificity:

Selectivity indicates the ability of the bioanalytical method to quantify the analyte in the presence of other substances present in the matrix. The term specificity generally refers to a method that produces a response for a single analyte only. The terms selectivity and specificity have often been used interchangeably. For selectivity assessment, blank samples from at least 6 different sources are to be used.^20-21

To assess the selectivity of the method, eight individual human plasma lots, each including one lipemic and one hemolytic lot, were utilized. The examination involved comparing peak responses in blank lots with those of spiked LLOQ containing IS mixtures at the retention times of the analyte and internal standard. Figures 2 and 3 present representative chromatograms of blank plasma and blank plasma spiked with baloxavir at LLOQs, illustrating the method's selectivity. No interference was observed in the screened lots of the biological matrix for both the analyte and internal standard.

Figure 2: Representative chromatogram of blank plasma for Baloxavir

Figure 3: Representative chromatogram of lower limit of Quantification with IS for baloxavir

The signal-to-noise ratio for the analyte exceeded 60.053, while for the internal standard, it surpassed 1474.387 demonstrating acceptability (more than 5 is acceptable). The method's selectivity was confirmed at the LLOQ concentration of 0.505 ng/ml. Notably, even in the presence of the internal standard, no interference was noted in the screened lots of the biological matrix, further affirming the method's selectivity.

Linearity and Sensitivity:

Calibration curves comprising eight points were established, spanning concentrations from 0.505ng/ml to 302.724ng/ml for baloxavir. The analyte-to-internal standard peak-area ratio (y) was plotted against the nominal concentration ratio (x) of analyte to internal standard to evaluate the linearity of each calibration curve. Exceptional linearity was attained, with correlation coefficients exceeding 0.99 for all validation batches [Figure 4].

Figure 4: Representative calibration curve for baloxavir

To assess accuracy, the concentrations of calibration standards were back calculated. The accuracy of each calibration point fell within the range of 0.47 to 2.62% for baloxavir.

To gauge the sensitivity of the method, six samples of the Lower Limit of Quantification (LLOQ) were processed and injected alongside three 'Precision and Accuracy' batches. The method demonstrated precision and accuracy within the ranges of 3.65% to 7.00% and 94.88% to 104.49% across the three batches, respectively. The quantification of Baloxavir at the LLOQ concentration of 0.505ng/ml was found to be both precise and accurate.

The analyte's limit of detection was identified as 0.127 ng/ml, and the signal-to-noise ratio exceeded 39.869, demonstrating the method's sensitivity for a pharmacokinetic study. With such a signal-to-noise ratio, it is evident that the Lower Limit of Quantification (LLOQ) for the analyte can be potentially reduced, or the plasma volume can be decreased. This widens the applicability of the method, especially in pediatric patients where sample volume constraints are often encountered.

Precision and accuracy:

Accuracy is a general measure of relative error (% RE). The mean value should not deviate by more than 20% in the case of LLOQ whereas it should be within 15% of the nominal value for other concentrations. The deviation of the mean from the nominal value serves as the measure of accuracy. Precision of an analytical method, on the other hand, describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. ²²

Precision and accuracy assessments for both intra- and inter-day batches of baloxavir involved six replicates of Quality Control (QC) samples (n=6) at four distinct concentrations: Lower Limit of Quantification (LLOQ), Low Quality Control (LQC), Middle Quality Control (MQC), and High-Quality Control (HQC). The concentrations for baloxavir corresponding to LLOQ, LQC, MQC, and HQC were 0.526 ng/mL, 1.422 ng/ml, 107.734 ng/mL, and 236.258 ng/mL, respectively. The results for precision and accuracy are detailed in Table 3. Intra-day and inter-day precisions for baloxavir were consistently within 3.95% for all analytes. The accuracy of the baloxavir assay ranged from 97.08% to 105.51% of the nominal values. Accuracy was expressed using the formula [(mean observed concentration)/(spiked concentration)] x 100%, while precision was evaluated using the relative standard deviation (RSD).

Table 3: Intra-day and inter-day accuracy and precision for the determination of baloxavir in human plasma

Parameter	QC ID	LOQQC		LQC			MQC			HQC
	Nominal Conc. (ng/ml)	0.526		1.422			107.734			236.258
	Mean calculated Conc (ng/ml)	Mean accuracy (%)	% CV	Mean calculated Conc (ng/ml)	Mean accuracy (%)	% CV	Mean calculated Conc (ng/ml)	Mean accuracy (%)	% CV	Calculated Conc (ng/ml)	Mean accuracy (%)	% CV
PA - 1	0.555	105.51	3.32	1.437	101.02	3.47	104.759	97.24	1.21	229.349	97.08	2.04
PA - 2	0.525	99.84	3.33	1.417	99.66	2.00	108.296	100.52	3.35	229.553	97.16	1.00
PA - 3	0.529	100.60	3.00	1.405	98.83	2.65	106.004	98.39	3.32	232.047	98.22	0.90
Inter-day	0.536	101.99	3.95	1.420	99.84	2.78	106.353	98.72	3.01	230.316	97.49	1.43

Table 4a: Matrix effects of baloxavir in human plasma

Matrix ID	LQC analyte area in absence of matrix	LQC analyte area in presence of matrix	LQC matrix factor for analyte	HQC analyte area in absence of matrix	HQC analyte area in presence of matrix	HQC matrix factor for analyte
PL_1770	11611	12851	107.85	1977865	1832561	94.39
PL_1771	11771	11597	97.32	1955505	1948431	100.35
PL_1772	11789	11722	98.37	1957426	1919047	98.84
PL_1794	12002	12360	103.72	1946332	1931307	99.47
PL_1795	11945	11305	94.87	1913594	1952115	100.54
PL_1797	12379	11122	93.34	1898529	1804220	92.93
LPL_1773		11238	94.31		1927070	99.25
HPL_1742		11352	95.27		1960924	101.00
Average	11916.167	11693.375	98.13	1941541.833	1909459.375	98.35
SD	265.626	608.957	5.11	29734.761	58376.804	3.01
%CV	2.23	5.21	5.21	1.53	3.06	3.06

Table 4b: Matrix effects of baloxavir d5 (IS) in human plasma

Matrix ID	LQC analyte area in absence of matrix	LQC analyte area in presence of matrix	LQC matrix factor for analyte	HQC analyte area in absence of matrix	HQC analyte area in presence of matrix	HQC matrix factor for analyte
PL_1770	376888	381176	99.81	377511	351571	92.99
PL_1771	380290	368839	96.58	384030	378370	100.08
PL_1772	382674	379554	99.39	376926	370393	97.97
PL_1794	381659	377895	98.95	375805	375265	99.26
PL_1795	381958	366562	95.98	379649	381996	101.04
PL_1797	387938	351708	92.09	374542	349046	92.32
LPL_1773		371727	97.34		375989	99.45
HPL_1742		379899	99.48		381599	100.93
Average	381901.167	372170.000	97.45	378077.167	370528.625	98.00
SD	3600.839	9925.289	2.60	3382.684	13031.298	3.45
%CV	0.94	2.67	2.67	0.89	3.52	3.52

Top of Form

Table 5a: Short and long –term stability of baloxavir (LLOQ) aqueous solution

Short-term stability of stock solution at 25⁰C for 26hrs			Short-term stability of working solution at 25⁰C for 24hrs			Long-term stability of stock solution at 2-8⁰C for 11days
Average area of stock solution	Average area of fresh stock solution	% Stability	Average area of working solution	Average area of fresh working solution	% Stability	Average area of stock solution	Average area of fresh stock solution	% Stability
14064.000	13574.667	103.76	13877.000	13574.667	102.38	14158.833	14109.000	100.72

Table 5b: Short and long –term stability of baloxavir (ULOQ) aqueous solution

Short-term stability of stock solution at 25⁰C for 26hrs			Short-term stability of working solution at 25⁰C for 24hrs			Long-term stability of stock solution at 2-8⁰C for 11days
Average area of stock solution	Average area of fresh stock solution	% Stability	Average area of working solution	Average area of fresh working solution	% Stability	Average area of stock solution	Average area of fresh stock solution	% Stability
2993173.167	3027082.333	99.02	3017757.000	3027082.333	99.83	2891363.167	3010329.667	96.40

iii) In-injector stability:

The stability for LQC and HQC samples kept in auto-sampler at 10⁰C for 88 h were: 101.64% and 96.92%, respectively. The stability of Baloxavir-d5 (IS) was found to be 98.98%.

iv) Wet extract stability:

The wet extract samples of Baloxavir, stored at room temperature, demonstrated stability for a period of 4 hours. When tested against freshly spikedcalibration standards as well as LQC and HQC samples, the stability was determined to be 103.54% and 98.57%, respectively.

v) Processed sample stability:

Six sets of both Low-Quality Control (LQC) and High-Quality Control (HQC) samples, which were bulk spiked, underwent processing according to the analytical test procedure. These samples were then stored at 2-8°C for a duration of 92 hours. Following the storage period, the samples were retrieved from the cooling cabinet and analyzed in comparison to a set of freshly prepared (freshly spiked, unfrozen) calibration standards. Additionally, six aliquots each of freshly prepared LQC and HQC samples were included in the analysis. The stability against freshly spiked Quality Control (QC) samples for LQC and HQC was determined to be 101.97% and 98.12%, respectively. The processed sample stability of Baloxavir, stored at 2-8°C, was confirmed for the entire 92-hour duration (results not shown).

According to FDA guidelines, the accepted range for all mentioned stability studies is that the mean concentration of stability samples should fall within 85-115% of the mean concentration of freshly prepared samples. Consequently, all analytes exhibited stability throughout the analysis process. Detailed results of the stability studies are presented in Table 6.

Lipemic and Hemolysis effect:

Six replicates of freshly spiked samples at Low-Quality Control (LQC), Middle-Quality Control (MQC), and High-Quality Control (HQC) levels in hemolyzed and lipemic plasma were processed and analyzed alongside a freshly spiked calibration curve standard.

Regarding the Lipemic effect, the precision values for LQC, MQC, and HQC were 4.43%, 0.92%, and 1.11%, respectively, with corresponding accuracies of 97.11%, 96.09%, and 97.79%. These values fell within the specified limits set by the FDA.

Similarly, for the Hemolysis effect, the precision for LQC, MQC, and HQC were 2.50%, 2.57%, and 0.55%, while the accuracies were 99.59%, 97.81%, and 96.54%, respectively. These results also met the FDA specified limits (Table 7).

Table 6: Stability studies of baloxavir in plasma

Parameters	Bench-top stability for 5h		Freeze-thaw stability after 4 cycles		In-injector stability for 88h		Wet extract stability for 3h
Parameters	LQC	HQC	LQC	HQC	LQC	HQC	LQC	HQC
Nominal concentration (ng/ml)	1.422	236.258	1.422	236.258	1.422	236.258	1.422	236.258
Mean Calculated concentration (µg/ml)	1.407	233.865	1.488	235.237	1.445	228.975	1.472	232.877
SD	0.025	4.570	0.050	2.984	0.055	4.029	0.048	2.534
%CV	1.77	1.95	3.38	1.27	3.80	1.76	3.24	1.09
% Stability	98.95	98.99	104.46	99.57	101.64	96.92	103.54	98.57

Table 7: Lipemic and Hemolysis effect for Baloxavir

LQC

HQC

Nominal conc.

(ng/ml)

Mean

calculated

conc. (ng/ml)

Accuracy

(%)

% CV

Nominal conc.

(ng/ml)

Mean calculated conc. (ng/ml)

Accuracy

(%)

% CV

Lipe

mic

Hemo

lytic

Lipe

mic

Hemo

lytic

Lipe

mic

Hemo

lytic

Lipe

mic

Hemo

lytic

Lipe

mic

Hemo

lytic

Lipe

mic

Hemo

lytic

1.422

1.381

1.416

97.11

99.59

4.43

2.50

236.258

231.049

228.077

97.79

96.54

1.11

0.55

Table 8: Extended precision and accuracy of baloxavir

LQC

HQC

Nominal conc.

(ng/ml)

Mean calculated conc. (ng/ml)

Accuracy

(%)

% CV

Nominal conc.

(ng/ml)

Mean calculated conc. (ng/ml)

Accuracy

(%)

% CV

1.422

1.419

99.80

2.62

236.258

226.366

95.81

1.11

Extended precision and accuracy run:

A single set of Calibration Curve samples and 30 sets of Low Quality Control (LQC), Middle Quality Control (MQC), and High Quality Control (HQC) samples, forming a batch totaling 100 samples, were processed and subsequently analyzed. The precision and accuracy results are detailed in Table 8. For baloxavir, the precisions were 2.62% for LQC, 1.03% for MQC, and 1.11% for HQC, with corresponding accuracies of 99.80% for LQC, 97.50% for MQC, and 95.81% for HQC.

The LC–MS/MS method outlined in this study exhibited linearity across the concentration range of 0.505 to 302.724ng/ml in human plasma. The intra and inter-batch precision (%CV) remained below 4%, and % accuracy ranged from 97.08% to 105.51%. The overall % recovery for Baloxavir and baloxavir d5 exceeded 80%. Utilizing 100μL plasma samples enabled the attainment of lower limits of quantification. The method's selectivity, sensitivity, precision, and accuracy make it well-suited for pharmacokinetic analysis in a bioequivalence study. Additionally, the high sensitivity allows for its application in the analysis of pediatric samples, where sample volume constraints are commonly encountered.

In summary, the method employed in this study is both easy and expeditious to execute, characterized by commendable accuracy, precision, selectivity, and stability. The method's simplicity, coupled with a relatively short run time of 6.0 minutes per sample, renders it an appealing choice for high-throughput bioanalysis of Baloxavir.

REFERENCES:

1. Carrat F, Vergu E, Ferguson NM, Lemaitre M, Cauchemez S, Leach S, et al. Time lines of infection and disease in human influenza: a review of volunteer challenge studies. Am J Epidemiol. 2008; 167(7): 775-85.

2. Wright PF, Neumann G, Kawaoka Y. Orthomyxoviruses. In: Knipe DM, Howley PM, eds. Fields Virology. 5th edn. LippincottWilliams and Wilkins, 2007; 1691–740.

3. Taubenberger JK, Morens DM. The pathology of influenza virus infections. Annu Rev Pathol. 2008; 3: 499-522.

4. Fukao K, Noshi T, Yamamoto A, Kitano M, Ando Y, Noda T, et al. Combination treatment with the cap-dependent endonuclease inhibitor baloxavir marboxil and a neuraminidase inhibitor in a mouse model of influenza A virus infection. J Antimicrob Chemother. 2019; 74(3): 654-62.

5. Lackenby A, Besselaar TG, Daniels RS, Fry A, Gregory V, Gubareva LV, et al. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-2017. Antiviral Res. 2018; 157: 38-46.

6. Bosaeed M, Kumar D. Seasonal influenza vaccine in immunocompromised persons. Hum Vaccin Immunother. 2018; 14(6): 1311-22.

7. Omoto S, Speranzini V, Hashimoto T, Noshi T, Yamaguchi H, Kawai M, et al. Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Scientific Reports. 2018; 8.

8. Jancy Rani E. H3N2 Influenza Virus: Is it Pandemic? A and V Pub International Journal of Nursing and Medical Research. 2023:56-9.

9. Kengar M, Jadhav A, Jadhav R, Jarag P, Shete S. Swine Flu and its Recent Medicines. Asian Journal of Pharmaceutical Analysis. 2019; 9: 30.

10. Hayden FG, Sugaya N, Hirotsu N, Lee N, de Jong MD, Hurt AC, et al. Baloxavir Marboxil for Uncomplicated Influenza in Adults and Adolescents. N Engl J Med. 2018; 379(10): 913-23.

11. Reddy S, Nayak N, Ahmed I, Thomas L, Mukhopadhyay A, Thangam S. Development and Validation of two LCMS/MS Methods for Simultaneous Estimation of Oseltamivir and its Metabolite in Human Plasma and Application in Bioequivalence Study. Asian Journal of Pharmaceutical Analysis. 2016; 6: 91.

12. Koshimichi H, Ishibashi T, Kawaguchi N, Sato C, Kawasaki A, Wajima T. Safety, Tolerability, and Pharmacokinetics of the Novel Anti-influenza Agent Baloxavir Marboxil in Healthy Adults: Phase I Study Findings. Clin Drug Investig. 2018; 38(12): 1189-96.

13. A New Stability Indicating RP-HPLC Method Development And Validation For Estimation Of Baloxavir Marboxil In Pharmaceutical Dosage Form. Journal of Pharmaceutical Negative Results. 2022: 970-83.

14. Gouda AS, Abdel-Megied AM, Rezk MR, Marzouk HM. LC-MS/MS-based metabolite quantitation of the antiviral prodrug baloxavir marboxil, a new therapy for acute uncomplicated influenza, in human plasma: Application to a human pharmacokinetic study. J Pharm Biomed Anal. 2023; 223: 115165.

15. Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM) May 2018, Available form: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf

16. Khan H, Ali J. UHPLC: Applications in Pharmaceutical Analysis. Asian Journal of Pharmaceutical Analysis. 2017; 7: 124.

17. Yousra Y, Fatima S, Rasheed N, Mohammad A. A Brief Review on Fundamentals of Analytical Chemistry. Asian Journal of Research in Pharmaceutical Science. 2017; 7: 13.

18. Shashikala P, Dodda S, Bakshi V. Development and Validation of Bioanalytical Method for the Estimation of Carisoprodol in Human Plasma using LC-MS/MS. Asian Journal of Pharmaceutical Analysis. 2015; 5: 181.

19. Khan H. Analytical Method Development in Pharmaceutical Research: Steps involved in HPLC Method Development. Asian Journal of Pharmaceutical Analysis. 2017; 7: 203.

20. Amruta S. Kadam, Nayana V. Pimpodkar, Puja S. Gaikwad, Sushila D. Chavan. Bioanalytical Method Validation. Asian Journal of Pharmaceutical Analysis. 2015; 5(4): 219-25.

21. Sahoo CK, Sudhakar M, Ramana DV, Satyanarayana K, Panda KC. Validation of Analytical Procedures- A Review. Asian Journal of Pharmaceutical Analysis. 2018; 8 (2): 109.

22. Saudagar R. B., Thete P. G. Bioanalytical Method Validation: A Concise Review. Asian Journal of Pharmaceutical Analysis, 2018; 8(2): 107-114

Received on 23.02.2024 Revised on 16.09.2024

Accepted on 08.01.2025 Published on 28.02.2025

Available online from March 03, 2025

Asian J. Pharm. Res. 2025; 15(1):13-21.

DOI: 10.52711/2231-5691.2025.00004

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

ii) Freeze thaw stability: