A critical analytical aspect on analytical protocols in the pharmaceutical analysis of H1N1 antiviral agent and its active metabolite

Background Oseltamivir (OSM) was the first active oral therapeutic inhibitor approved by the Food and Drug Administration in 1999 for the clinical management of the influenza virus. It is an ester-type prodrug of OSM carboxy-late in the market under the trade name Tamiflu ™ capsules, i.e., oseltamivir phosphate. Because of the ubiquitous application to alleviate influenza virus (flu virus) symptoms, it is imperative to develop systematic analytical protocols for quality control laboratories, bioequivalence, and pharmacokinetic analysis. Main body of the abstract This review provides complete state-of-the-art analytical protocols for quantifying OSM, as published in scientific journals and official compendia. Several studies use LC–MS/MS and HPLC/UV. Additionally, there are reports on UPLC, HPTLC, capillary electrophoresis, FTIR, voltammetry, potentiometry, spectrophotometric, and spectrofluorometry protocols for the drug. Many analytical protocols have also been documented to analyze OSM from environmental water, surface water, sewage discharge, the Neya River and treated sewage effluent and surface water. Conclusion The present review concludes with significant remarks on the methodology used to analyze OSM. Despite the therapeutic applicability of the drug, there are a limited number of comprehensive documents on analytical protocols for determining its concentration in various matrices. This lack of information is elusive, as the applicability and effectiveness of these protocols are crucial for ensuring the quality, efficacy, and safety of OSM


Background
Influenza is a highly acute infectious respiratory tract illness resulting from types A and B of influenza virus species.It spreads through droplets released when an infected person coughs or sneezes [1].The symptoms of the disease can range from mild to severe, affecting the respiratory system and other organs.Influenza can be particularly dangerous for young or elderly individuals with weakened immune systems or those with underlying health conditions, such as heart or lung diseases [2][3][4][5][6][7].
In 1969, P. Meindl and H. Tuppy developed the first inhibitor of influenza virus neuraminidase (IVN), which stopped virus replication; however, it had low potency and specificity.In 1993, scientist Von Itzstein and colleagues reported Zanamivir (ZMV) as a specific and potent IVN inhibitor that stopped the spread of the virus in both in vitro and in vivo studies.This development was demonstrated after the crystalline structure of IVN and its complex with neuraminic acid was reported by P. M. Colman and the research team in the 1980s [4,8].OSM was the first orally active inhibitor, documented in 1997 by Kim and research workers.In 1999, the Food and Drug Administration (FDA) approved the OSM for clinical use in various countries, such as the USA, Canada, Switzerland, and many other parts of the world [9].The compound OSM, previously known as GS4104 or RO-0796, is an ester-type prodrug of oseltamivir carboxylate (OSM-C), formerly known as GS4071 or RO64-08022.OSM is a modified antiviral generic version available in the market under the Tamiflu trade name, specifically oseltamivir phosphate (OSM-P).It has been proven to be an effective treatment for seasonal influenza, reducing the severity and duration of the illness.Studies have shown that OSM-C is 3-4 times more potent as an inhibitor than ZMV.In vitro, assessments have indicated that it is at least 10 6 times more selective for influenza virus (neuraminidases) than for parainfluenza virus, Newcastle disease virus, Vibrio cholerae, Clostridium perfringens, and human liver microsomes.
Despite its strong history, OSM has been the preferred neuraminidase inhibitor for the past two decades due to its prophylactic and therapeutic status.Scientists and academicians must comprehensively understand the latest updates for their professional and learning pursuits.Therefore, this review aims to briefly overview commonly used analytical protocols for detecting OSM in various samples and highlight the experimental conditions required for analysis.The second objective is to offer readers a wealth of analytical knowledge on the selected topic and emphasize essential background information such as physicochemical properties, chemistry, pharmacodynamics, and pharmacokinetic characteristics of OSM.

Pharmacology of OSM
Neuraminidase inhibitors (NI) have been developed for prophylactic and therapeutic purposes against influenza in recent years.These inhibitors deliberately target the highly conserved viral enzyme surface area.The first kind of inhibitor to undergo a clinical trial is ZMV, which is not active when taken orally.Therefore, it is administrated through dry powder inhalation to the lungs.The hydrophobic side chain on OSM-C interacts with a hydrophobic pocket at the virus enzyme's active site.This binding prevents the enzyme from cleaving sialic acid residues on the surface of infected cells, inhibiting the release of new virus particles from the infected cells [18,19].In healthy volunteers, OSM-C reaches detectable levels in the bloodstream within 30 min after a single oral dose and is efficiently absorbed in the gastrointestinal tract.
The peak concentration of OSM-C in the bloodstream was reached 3-4 h after consumption.The oral bioavailability was high, at about 79% for a 150 mg dose compared to an intravenous dose.In a crossover and randomized study involving 18 volunteers, it was found that food did not significantly affect the absorption of OSM.However, the time taken to reach peak concentration (Cmax) or the total drug exposure (AUC) was delayed by 1 h [19,20].When OSM-C was administered intravenously, it was distributed into most body fluids.The volume of distribution at steady-state is 25.6L, equal to the total body water.OSM has a protein binding of 42% in human plasma specimens, while OSM-C has a protein binding of 3%.OSM is extensively metabolized through hepatic esterases, and OSM-C is primarily eliminated through renal excretion, involving both glomerular filtration and anionic renal tubular secretion.After administering a single oral dose of OSM ranging from 20 to 1000 mg, a mean renal clearance value of 21.7 L/h was recorded in healthy adult volunteers.
Approximately 63% of OSM-C was recovered from urine after intake of a 100 mg dose, and it was observed that about 20% of an oral dose of OSM was excreted in feces as either OSM or unchanged OSM.OSM-C clearance was found to be relatively slower in older individuals (around 65 years old) and much quicker in children (around 12 years old) compared to adults.In patients with severe kidney problems, OSM-C clearance was reduced [21,22].OSM is recommended at a dose of 75 mg (twice a day) for five consecutive days, with or without food, for adolescent and adult patients of all ages.Treatment should start within two days of the onset of symptoms.For infected patients with creatinine clearance less than 1.8 L/h (< 30 mL/min), a reduced dose of 75 mg once daily is recommended.Extra caution is needed for patients with creatinine clearance less than 0.6 L/h (< 10 mL/min).In the elderly, no dosage adjustment is required [18,19,23].A 12 mg/mL oral suspension is prescribed for children with influenza (older than 1 year) or adult patients who cannot swallow a capsule.In healthy adults, two doses of 75 mg of OSM for five consecutive days efficiently reduced the duration of naturally acquired influenza by 1.3-1.5 days and decreased the severity of illness by up to 38%.Common side effects include nausea, vomiting, dizziness, headache, diarrhea, nosebleeds, cough, redness of the eye, and insomnia [24].

A search of the state of the art for published reports on OSM
Numerous analytical reports on both qualitative and quantitative estimations of OSM from various samples are available.To review the research results and findings based on the gathered information, a literature analysis has been conducted using keywords such as IVN, bioanalytical protocols for OSM, and analytical protocols for OSM.This analysis involved selected methodologies, including hyphenated, spectroscopic, electrochemical, and chromatographic techniques, along with current issues of periodicals, citations of research and review articles and library database searches.Various academic search engines such as Google Scholar, Scopus, EBSCO, Analytical Sciences Digital Library, ProQuest Central, Web of Science, Semantic Scholar, and ScienceDirect were utilized.Examination of reports in the literature highlighted that a good percentage of analytical reports are available to determine OSM from diverse specimens.These investigations have their strengths and limitations.Over the past few years, many advancements and enhancements have been made to establish and validate analytical protocols for assessing the purity and quantification of a cited drug from various pharmaceutical or non-pharmaceutical specimens.
These protocols provide valuable data for analytical chemists to choose the most suitable protocol for their intended use.Regardless of the purpose for which the protocol was developed and validated, there is a constant need to improve these protocols.This includes enhancing the analytical response, implementing better sample preparation procedures, reducing the overall analysis time, minimizing toxic solvents and reagents, and adopting protocols that generate less hazardous waste materials.Developing an analytical protocol is challenging for analytical chemists or scientists/researchers because when the protocol is redeployed to other laboratories, it needs to be robust, precise, and accurate.Therefore, a proposed review would benefit researchers, analysts, and scientists by helping them develop new, rapid, robust analytical protocols for OSM.

Analytical profile of OSM
Several analytical reports have been addressed in the literature since 1999 to estimate OSM individually.Here, we have categorized these published reports into two main classes: pharmacopoeial and non-pharmacopoeial reports.

Compendial analytical reports on OSM
Analytical protocols for OSM are officially addressed in the Indian Pharmacopoeia, British Pharmacopoeia, and United States Pharmacopeia.

Indian pharmacopoeia
The liquid-chromatographic (LC) protocol evaluated the OSM from capsule and oral suspension forms.The chromatographic conditions included a stainless steel column measuring 15 cm × 4.6 mm, packed with octylsilane bonded to porous silica (5 μm).The column temperature was set at 50 °C.The mobile phase consisted of a mixture of 66 volumes of a buffer solution prepared by dissolving 6.8 g of anhydrous monobasic potassium phosphate in 1000 mL of water and adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol (MeOH), and 23.5 volumes of acetonitrile (ACN).The flow rate was maintained at 1.0 mL/min, and detection was performed at 207.0 nm using a spectrophotometer.A sample volume of 15 μL was injected into the system [10].

British pharmacopoeia
The British Pharmacopoeia (BP) reported analytical protocols for analyzing OSM impurities.Table 1 depicts eight official impurities (A to H) documented in BP.
OSM-Impurity-A and C The LC was used to detect impurities A and C. The analysis was conducted using end-capped octylsilyl silica gel for chromatography R (5 μm), with a mixture of ACN: MeOH: potassium dihydrogen phosphate (135:245:620v/v), a flow rate of 1.2 mL/min, a column temperature of 50 °C, and detection at 207.0 nm using a spectrophotometer.The test solution and reference were injected at a volume of 15 μL.
OSM-Impurity-B The LC-MS was conducted to analyze impurity-B.The experiment utilized an end-capped octadecylsilyl silica gel for chromatography R (5 μm) column with a mobile phase comprised of ammonium acetate (AmAc): ACN: H 2 O (10:30:60%v/v).The flow rate was 1.5 mL/min, with the column temperature at 40 °C.A mass detector with an ESI-positive ionization source was employed, and the detection was performed at m/z 356.2.The injection volution used was 1.0 μL.
OSM-Impurity-H Gas chromatography (GC) estimates impurity-H under specific working conditions.These conditions include a poly(dimethyl)siloxane column, helium carrier gas with a flow rate of 1.2 mL/min and a split ratio 1:50.The analysis uses a flame ionization detector, and the injection volume is 1.0 μL [11].

United States pharmacopeia
The chromatographic conditions were as a column measuring 25 cm×4.6 mm packed with L7.The column temperature was maintained at 50 °C.The mobile phase consisted of a mixture of MeOH: ACN: sample solution A. Sample solution A was prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 980 mL of H 2 O, adjusting the pH to 6.0 using 1.0 M potassium hydroxide solution and then completing the volume to 1.0 L. The ratio of mobile phase components was 243:135:620v/v.The flow rate was set at 1.2 mL/min, and detection was performed at 207.0 nm using a UV detector.A sample volume of 15 μL was injected into the system [25].

Non-compendial analytical reports on OSM
Scientists, research scholars, and academicians have developed various non-pharmacopoeial protocols to accurately determine OSM in different matrices, in addition to pharmacopoeial protocols.The following section offers an overview of these non-pharmacopoeial protocols.

Hyphenated protocols
Hyphenated techniques are crucial for analyzing unknown organic compounds or medicinal candidates in natural extracts and biological specimens.The proposed roadmap addressed different hyphenated techniques, namely LC-MS/MS, GC-MS, and UHPLC-MS/MS, to evaluate OSM from different sources.Table 2 provides detailed information on the pharmaceutical/biological specimen, extraction technique, internal standard, operational conditions, interface, mass-to-charge ratio, and total analysis time, supporting our findings validity.This summary highlights several hyphenated investigations.Kanneti, R. et al. reported reliable and rugged solid-phase extraction (SPE) LC-MS/MS protocol.The authors have validated the protocols using 500 μL plasma samples with 0.6 mL/min isocratic flow rate and 1.0 min as total run time.The lower limit of quantitation (LLOQ) was recorded at 0.92, 5.22 ng/mL for the lowest concentration of standard curves, i.e., 0.92-745.95for OSM and 5.22-497.49ng/mL for OSM-C [26].Zhe-Yi Hu et al. improved the performance of the OSM and OSM-C assay in human plasma by using a pulsed gradient chromatographic protocol.They used sodium fluoride to inhibit esterase activity and prevent hydrolysis during sampling, ensuring the stability of the analyte [27].Gupta et al. studied high-throughput simultaneous bioanalysis of OSM and OSM-C with the aid of SPE using OSM-d5 and OSM-C13-d3 as internal standard (IS) in human plasma.The retention time (t R ) for both cited analytes was 1.56 and 1.11 min, respectively.Moreover, the extraction recovery average for targeting analytes was 94.4%, 92.4%, 93.1%, and 91.9%, correspondingly [28].Kromdijk et al. conducted ex vivo stability studies on OSM and OSM-C in human fluoride EDTA plasma and confirmed that OSM remains stable in whole blood in EDTA fluoride tubes for 8 h at 37 °C and for 24 h at 2-8 °C [29].Several reports have been highlighted, analyzing selected therapeutics in multiple biological specimens such as urine, saliva, cerebrospinal fluid, brain homogenate, and plasma from humans and rats [30][31][32].
Heinig and Bucheli developed a reliable method to estimate OSM and OSM-C levels in both rat and human specimens.The mean recoveries were close to 100% for rat plasma and human samples, ranging from 83.3 to 100.9% for cerebrospinal fluid and brain samples.OSM and OSM-C solutions remained stable for 24 h at room temperature and 4 weeks at − 20 °C for brain homogenate and plasma [30].UPLC-MS/MS protocol is documented to estimate 14 antiviral agents simultaneously along with OSM and OSM-C in chicken muscle tissues.The recovery rates of the proposed investigation are 56.2-113.4% with 1.7-10.3%RSD [33].Hoff, G. P., along with the research team, conducted analysis on the dried blood spots.The 5-1500 ng/mL linearity range and 20-1500 ng/mL were recorded for OSM and OSM-C, correspondingly with gradient elution followed by cited drugs eluted at 1.88 and 3.20 min [34].Wu et al. explored the application of UHPLC-MS/ MS to estimate the 11 antiviral agents (prohibited) from livestock and poultry feces.The study proposed a method that integrated extraction and cleanup into a single step.It involved extracting the same agents at a temperature of 90 °C using a mixture of MeOH and ACN in a 1:1 volume ratio, along with 0.5% glacial acetic acid.Researchers reported 71.5-112.5% extraction recoveries of 11 antiviral agents [35].Two LC-MS/MS protocols were reported in the literature to quantify cited drugs from poultry tissues and muscle.In both protocols, a gradient mode of analysis was performed using 50 mM FA in H 2 O (A) and 50 mM FA in ACN (B) [36].Lin, C.C., and the research team analyzed OSM and OSM-C in the placenta, amniotic fluid, fetus, and plasma of 13 day pregnant rats, finding the AUC of OSM-C in maternal plasma was 3.6-fold higher than OSM.They used modified SPE protocols with isocratic elution and studied pharmacokinetics with non-compartmental modeling [38].
From the survey for a roadmap of present work, it was noticed that there are also several hyphenated reports available on scholarly sources for analysis of OSM and its active metabolite from environmental water, surface water, sewage discharge, Neya River, and treated sewage effluent and surface water [40][41][42][43][44][45].In addition, another robust analytical technique, i.e., GC-MS application, was also documented in the literature for synergistic efficacy of a cited drug and Melissa officinalis essential oil.The authors reported that the estimation of volatile was executed on DB-IMS (30 m × 0.25 mm, 0.25 μm film thick- ness) using helium at 1.2 mL/min carrier gas rate of flow and 250 °C injection temperature [54].There is only one report available on GC-MS due to its limitations.It can only analyze volatile samples and is unsuitable for thermally stable active agents.Additionally, careful attention is required when introducing gas samples.

Chromatographic protocols
Chromatographic techniques have been the primary method to assess pharmaceutical effectiveness in the past two decades.High-performance liquid-chromatography (HPLC) is acknowledged as a dominant analytical tool but is still the subject of ongoing research to enhance performance and accuracy.From 1999 to 2021, researchers commonly used HPLC with UV-visible, photodiode     array, fluorescence, and mass spectrometry detection for the pharmaceutical analysis of OSM and its metabolites.C18 is widely used in all HPLC reports for its longer carbon chain, hydrophobic nature, and interaction with analytes.ACN and MeOH are the most commonly used eluents and various buffer solutions.The most commonly reported HPLC-UV-visible detection wavelength is 207-254 nm, and 8 out of 13 quantifications were performed at 215 and 220 nm.Only one experiment was performed using fluorescent HPLC detection.The detailed chromatographic report specification for pharmaceutical analysis of OSM is presented in Table 3.
Charles and his colleagues have reported a reliable and rapid protocol for analyzing OSM in marketed capsules (Tamiflu and generic versions) using UV detection with a short elution time of less than 5 min.Sotalol hydrochloride was employed as the IS, and the average t R of the cited analytes were 3.34 ± 0.03 and 2.27 ± 0.02, respectively.The calibration range was 30-70 µg/ mL, and the mean recovery was 100.01-102.28%[55].Similarly, Green et al. performed calorimetric and HPLC protocols to test OSM alleged Tamiflu formulation [56].Analysis of cited drug was also performed by Chabai et al. using monolithic column [57].Four HPLC investigations have been addressed in the literature to quantify OSM in biological samples.Aydoğmuş et al. studied the reaction of 4-chloro-7-nitrobenzofurazan with OSM in a borate buffer solution at pH 8.50 and further quantified the same from the capsule and human plasma matrices with mexiletine hydrochloride as IS.OSM's LOD and LOQ were 5.70 and 17.38 ng/ mL from plasma.Further, 99.95% amount of the drug was estimated from capsules [60].Bahrami et al. use the procedure of SPE to extract OSM and OSM-C from human serum with a short run time < 10 min.The 98 ± 3% OSM-C and 85 ± 5% vanillin (IS) were extraction efficiencies from serum, and the proposed protocol is sensitive enough with 15 ng/mL of LOQ [61].On the same line, Fuke & co-researcher explored HPLC with UV detection to quantify OSM and OSM-C with floropipamide (IS) from biological materials (in case of death).All analytes were extracted through a cation exchange extraction cartridge [62].Five stability-indicating protocols were addressed for OSM using HPLC.Al-Bagary et al. reported alkaline degradation of OSM with the kinetic study.The degradation study was performed by exposing OSM to 0.1 M NaOH (10 mL) for 3 h [64].In a separate study, researchers reported that OSM is degraded in 0.05 N NaOH for 5 h (25% degradation), 1 N HCl at 85 °C for 25 min (10% degradation), 30% H 2 O 2 at 90 °C for 10 min (39% degradation).Whereas in thermal degradation, subjecting solid OSM at 120 °C for 1 h (22% degradation was observed) [65].
The chemical stability of OSM in an oral solution consisting of sodium benzoate (preservative) was studied using HPLC by Albert & Bockshorn [67].Rashed et al. document a stability-indicating UPLC protocol; a calibration curve was constructed in the 6-14 µg/mL range with 0.150 and 0.501 µg/mL LOD, and LOQ values were detected and quantified.The OSM was effective with 100.79 ± 1.23% from the pharmaceutical matrix [69].
Two HPTLC investigations were found for OSM in the literature.HPTLC is an often analytical tool with many benefits relative to other analytical tools, including the small volume of developmental phase needed and the rapid and low-cost analysis [70].A researcher reported the HPTLC protocol for assessment of OSM in pharmaceutical formulation counterfeited with ascorbic acid using (6.0:4.0:0.05v/v)MeOH: H 2 O: ammonia with retention factor (Rf) 0.70 and 0.83 for OSM and ascorbic acid.The OSM recovered from Taminil-N capsules was 102.51 ± 2.49% [71].

Spectrophotometric and spectrofluorometry
The spectrophotometric technique remains simple, costeffective, and comparable to other analytical techniques in terms of instrumentation, analysis time, less sample processing time, and reduced use of hazardous organic solvents.It is commonly used to measure various therapeutic agents in pure materials and pharmaceutical formulations [72].Twelve reports in the literature have estimated OSM using spectrophotometry and spectrofluorometry.These reports involve zero-order (D 0 ), first-order (D 1 ), ratio derivative, and difference spectrophotometry [58,74,75].Because the OSM is poorly absorbed in the UV-visible region, the authors were able to study its colorimetric reaction.
Several colorimetric reactions were used to detect OSM.These reactions involve congo red and bromochlorophenol blue salt [56] Aydoğmuş Z. has presented a highly accurate method, a fluorescent derivative of OSM, using fluorescamine in a borate buffer solution (pH 8.50).This method, which uses a less toxic organic compound and solvent than previously reported protocols, allows for the precise determination of OSM in an aqueous solution.As there is no need for extraction, OSM can be determined accurately in an aqueous solution and is stable for around 6 h [80].In another study, the Hantzsch

Sample treatment
Design a new selective and specific analytical protocol for quantifying the target analyte with excellent accuracy; choose the optimal IS significant step before the sample pretreatment begins.
The selection of an appropriate IS is of utmost importance.It should be chemically similar to the target assays being analyzed but not naturally present in a sample.The IS compensates for the loss of analyte at all stages of sample preparation and chromatographic analysis.Opting for compounds with the same physical and chemical properties as the target compounds is the most effective approach.In MS, a deuterated analog of the compound of interest is expected to be used.

Table 4
Spectral parameters of spectrophotometry and spectrofluorometry reports LOD: limit of detection, LOQ: limit of quantitation, D 0 : zero-order spectrophotometry, D 1 : first-order spectrophotometry, TCNQ: 7,7,8,8-tetracyanoquinodimethane, Me 2 CO: acetone, H 2 O: water, RD: ratio difference spectrophotometry, A: difference spectrophotometry, MeOH: methanol, KMnO 4 : potassium permanganate, NaOH: sodium hydroxide, NBD-Cl: 4-chloro-7-nitrobenzo-2-oxo-1,3-diazole, FEM: fluorescence emission, FET: fluorescence excitation, FLA: fluorescamine, FMA: formaldehyde, λem: emission, λex: excitation, NQS: 1, 2-naphthoquinone-4-sulfonic acid, NR: not reported An excellent example of this is the use of OSM-d3, OSM-C-d3, [ 2 H 5 ]-OSM, [ 13 C, 2 H 3 ]-OSM-C, OSM-d2, OSM-H2, OSM-C-d2, and OSM-C-H2 [27-32, 34, 36, 40-44, 49, 53].However, apart from a deuterated analog or stable isotopes, in some cases, the compounds used as IS are drugs clinically explored to treat diseases are lidocaine hydrochloride, cephalexin, clopidogrel carboxylate [38,39,50].Sample treatment is a significant part of the analysis protocol.Developing a suitable sample preparation procedure involves several steps, such as extraction and purification, to obtain a final concentrate of the target analyte extract that is as free of matrix compounds as possible [92].The principle of green analytical chemistry emphasizes using direct analytical techniques to minimize the need for sample treatment [93].The goal is to reduce the environmental, health, and safety issues of traditional sample preparation methods.However, direct analysis may only sometimes be feasible, as the sample needs to be in the appropriate state, and higher sensitivity, selectivity, or throughput may be required.Therefore, proper sample preparation is crucial when developing an analytical protocol for the OSM and OSM-C.The protocols have yet to report direct analysis of biological specimens for OSM.Sample preparation is necessary to make biological samples suitable for the selected protocol for quantifying the same.To achieve greater sensitivity, selectivity, or performance, biological specimens should be void of proteins, salts, sugars, and other essential compounds to minimize interference and matrix effects of early and late elution components [94].
Most reports use SPE, PP, and LLE as standard extraction techniques.Applying SPE sample preparation is a slightly new technique for PP and LLE.It is more often used for OSM by scientists and researchers from biological and environmental specimens [26,28,31,32,[36][37][38][39][40][41][42][43][44]51].Off-line SPE with mixed phase cation (MPC)standard density (SD) 96-well plate using MeOH: 50 mM AmAc (90:10 v/v) revealed a high % recovery with adequate elution of the analyte [31].LC with strong cation exchangers separates and isolates acidified and basified sample extracts [36][37][38].Most reports use various ratios of ammonia in MeOH to elute analytes from SPE cartridges.Since SPE and LLE pretreatment techniques are unsuitable for animal tissues, a simple and rapid QuECh-ERS sample preparation method was used in chicken muscle [33] due to the large sample consumption and time-consuming sample treatment procedures of SPE, PP, and LLE.Online in-tube solid-phase microextraction was reported to be a more efficient method for extracting analytes in environmental waters, reducing analysis time and providing better sensitivity and precision than the abovementioned procedures.

Recent updates for current pharmaceutical research aspects of OSM
In order to wrap up our review of the OSM, we made an effort to incorporate the latest updates for 2021.During our investigation using various reputable databases, we were surprised to find that pharmaceutical researchers have been focusing on exploring OSM in different areas due to the impact of the pandemic and other viral infections.These include repurposing, clinical approaches, medicinal chemistry, development of pharmaceutical formulations, pharmaceutical analysis, and the evaluation of pharmacokinetics and pharmacodynamics [95][96][97][98][99][100][101][102][103][104][105][106][107][108][109].The details of the research scope, areas studied, and specific findings are outlined in Table 5.The exploration of the drug in clinical research has led to important discoveries regarding its potential use on its own or in combination with the effective treatment of COVID-19 and influenza patients.Medicinal chemistry research has been dedicated to developing new derivatives to combat resistant influenza.

Discussion and conclusion
The present roadmap thoroughly and comprehensively outlines the cumulative protocol for assessing OSM, whether used alone or with numerous other conventional antiviral candidates.OSM has been a consistent choice as a neuraminidase inhibitor for prophylaxis and managing infections caused by type A (pandemic H1N1) and B influenza viruses over the past two decades.M2 and neuraminidase inhibitors, two distinct therapeutic agents, are employed to combat influenza infections.However, given the resistance of H1N1 isolates to M2 inhibitors, neuraminidase inhibitors, with OSM leading the way, have emerged as the ideal approach.Therefore, our work provides a comprehensive and detailed analytical profile of OSM from 1999 -2021, instilling confidence in its effectiveness.The author's research on OSM detection protocols highlights the effectiveness of hyphenated and chromatography techniques.These methods, including LC-MS/MS, UPLC-MS/MS, HPLC, HPTLC, UPLC, UV-Vis spectrophotometry, spectrofluorometry, FTIR, capillary electrophoresis, potentiometry, and voltammetry, have been extensively studied.Hyphenated techniques are the primary analytical technique reported in the literature review for OSM and OSM-C analysis in pharmaceutical and biological samples.These protocols demonstrate excellent specificity, reliable separation efficacy, a broad linearity range, and high automation.
Regarding detection sensitivity, the hyphenated protocols are more sensitive than the reported chromatographic protocols.Few documented reports highlighted OSM detection using LC-MS/MS in environmental water, surface water, sewage discharge, Neya River, treated sewage effluent, and surface water due to the emergence of OSM-resistant virus stains with the widespread use of TamifluTMcapsules.By surveying the reports 1999-2021, it is revealed that FA was the most common volatile additive used in solvent systems to improve the shape, resolution, and peak resolution of the OSM and, in the solvent system, dramatically decreased the interference by matrix effects.The most common ionization source is ESI; notwithstanding, ESI is the most widely employed ion source in the documented procedures.ACN is the major eluent in various reports in the solvent system, confirmed by LC-MS / MS and HPLC in 27 of 42 experiments, possibly due to its strong eluting power and favorable UV transmittance.To date, 13 to proceed into in vivo study [105] 12 Effectiveness of oseltamivir treatment on clinical failure in hospitalized patients with lower respiratory tract infection

Clinical
In hospitalized patients with influenza-associated lower respiratory tract infection, adding OSM to standard of care may reduce clinical failure compared to quality of practice alone [106] 13 Synthesis of OSM from m-anisic acid using a dissolving metal or electrochemical birch reduction

Medicinal chemistry
New biocatalysts-assisted approach for OSM using of birch reduction and use of enzymatic reduction to install absolute stereochemistry [107] 14 Design and synthesis of a series of C-5-NH 2 -acyl derivatives of OSM containing the pyrazole moiety Medicinal chemistry Many compounds were found to have a significant inhibitory effect on neuraminidase.In addition, an in silico ADME analysis revealed that the compounds were drug-like, with higher oral absorption rates and cell permeability than OSM [108] 15 OSM loaded PLGA nanoparticles targeted with spike protein binder peptide of SARS-CoV-2

Formulation and development
The OSM drug-loaded polymeric formulation targeted with a specific peptide may prove to be a viable option to more effective and controlled release medications in the treatment of COVID-19 [109] analytical reports have been explored in the literature to determine OSM using HPLC.
Several reports have been published on the analysis of OSM using spectral protocols, which are rapid and easy to use.However, these protocols are less effective in analyzing OSM samples of biological origin due to interference from components and additives commonly found in pharmaceutical products.Electrochemical protocols, on the other hand, are cost-effective, rapid, and robust and can accurately detect the sample of interest, even in complex and impure samples.Despite their advantages, only a few studies have reported the detection of OSM using electrochemical protocols in pharmaceutical and biological samples, and there are few documented protocols for pharmacokinetics and bioequivalence studies.Based on the literature, many official impurities of OSM are known, opening up the possibility of comprehensive toxicity assessments (in vivo and in vitro) of OSM.
Also, impurity profiling of OSM and its active metabolites (OSM-C) has yet to be achieved.Furthermore, the reports have yet to be established according to the principles of green analytical chemistry to minimize the side effects associated with analytical procedures on the environment and analysts/operators.Regulatory authorities recommend implementing a life cycle management approach to develop analytical protocols that reduce the costs of an analytical report during its life cycle.In conclusion, all analytical protocols have specific benefits and drawbacks; a suitable protocol is selected based on the protocol's requirements to achieve the intended outcomes.

Fig. 2
Fig.2The area chart displays the quantitative data relationship between the publication count and publication year [A] and sunburst chart revealed the percentage utility of analytical technique/methods for OSM[B]

Table 1
Impurities and related substances of OSM in Compendial Analytical Methodologies

Table 2
Overview on LC-MS/MS analysis conditions for OSM

Table 3
Chromatographic reports for pharmaceutical analysis of OSM