EFFLORESCENT, HYGROSCOPIC , DELIQUESCENCE , EFFLORESCENCE SUBSTANCE - DEFINITION

 EFFLORESCENT, HYGROSCOPIC , DELIQUESCENCE ,  EFFLORESCENCE SUBSTANCE -    DEFINITION

Some Important Characters of Compounds

EFFLORESCENT SUBSTANCE - DEFINITION

An efflorescent substance is a chemical which has water associated with its molecules, and which, when exposed to air, loses this water through evaporation. A common example of this phenomenon is the drying of cement. 

HYGROSCOPIC SUBSTANCE - DEFINITION

This is when substances absorb water from air, but not enough to form solutions. Examples of such substances include CaO,NaNO3​,NaCl, sucrose and CuO. Also, certain liquid substances absorb water from the air to get diluted, these are also regarded as being hygroscopic. Example, conc. H2​SO4​ and conc. HCl. lf a hydroscopic substance absorbs so much moisture that an aqueous solution is formed, the substance becomes deliquescent.

DEFINITION OF DELIQUESCENCE - DEFINITION

Deliquescence, the process by which a substance absorbs moisture from the atmosphere until it dissolves in the absorbed water and forms a solution.
Example are solid NaOH, CaCl2​, CaCl2​.6H2​O

DEFINITION OF EFFLORESCENCE - DEFINITION

Efflorescence, spontaneous loss of water by a hydrated salt, which occurs when the aqueous vapor pressure of the hydrate is greater than the partial pressure of the water vapor in the air. 
Examples are Na2​SO4​.10H2​O,  Na2​CO3​.10H2​O and FeSO4​.7H2​O 

 

Primary & Secondary Standard Definition, Use and Complete Guidelines

 

                       PRIMARY  AND SECONDRY STANDARD

Any chemical analysis can be considered valid only if the method of analysis is validated before adoption and results are reported against internationally recognized standard reference materials. Results of such analysis can be relied upon by consumers as well as other laboratories across the world.

Standards are primarily used for the following types of analytical studies:

• Assay
• Identification tests
• Limit tests for related substances
• Analytical method validation
• System suitability for analysis techniques and in particular spectroscopic and chromatographic analysis

It becomes necessary to understand the fine differences between different categories of standards used for chemical analysis. The standards are commonly grouped into two groups:

• Primary standards or certified reference materials or standard
• Secondary standards or working standards

Primary Standard OR Referance Standard

Primary standard is a reagent which is generally representative of the number of substance contains and easily weighed.A Primary standard is a reagent that’s stable, it’s not a hydrate /has no water of hydration, and has a high molecular weight.

A reference standard or reference material (RM) is a “material or substance one or more of whose property values are sufficiently homogeneous and well established to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials”, whereas a certified reference material (CRM) is a “reference material accompanied by a certificate issued by a certifying body, one or more of whose property values are certified by a technically valid procedure which establishes its traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence.” According to these definitions, CRMs form a subgroup of RMs, namely, that RMs which possesses additional characteristics - a certificate and traceable assigned values with an uncertainty statement.

A primary standard reference material is an ultra high purity grade compound used in analysis involving assay, identification or purity tests. It can be a single compound or a mixture having the analyte of interest in a specified and certified amount.

The impurities, if any, should be identified and controlled for use in assay studies. The material selected as a primary standard should be highly stable, free from water of hydration and bear traceability to a national or international standards body

In many cases it may not be possible to procure a reference material from such sources because of following reasons :

• The new molecule is encountered in R&D activity. In such case a reference may not be available from standard bodies. A laboratory can use a reference compound whose purity is established for its routine use.
• In- house primary reference material can be selected from a particular batch in manufacturing industries. After assigning its batch number its characteristic properties are documented for reference purpose and comparison with future production lots

Examples

Sodium carbonate Na2CO3
Sodium borate Na2B4O7
Potassium hydrogen iodate KH(IO3)2
Pure metals and their salts like Zn, Mg, Cu, Mn, Ag, AgNO3 , NaCl, KCl, KBr   – used in Acid base titrations
K2Cr2O7, KBrO3, KIO3, KI(IO3)2, NaC2O4, As2O3, pure iron – used in redox titrations

Eligibility criteria for a primary standard

A primary standard should satisfy the following conditions

primary standard

1. should be very pure
2. should neither be deliquescent (absorbing moisture) nor efflorescent (losing water)
3. should have high molecular weight so that weighing errors can be minimized
4. should be chemically stable
5. shall be readily soluble under given conditions
6. it should react stoichiometrically

When to use a primary standard?

In pharmaceutical QC, the use of reference standards to calibrate the analytical procedure is mandatory when measurements are performed with relative methods such as HPLC in combination with a UV or MS detector. These measurements need to be traceable to a primary standard. This requirement is realized either by using the primary standard directly for the calibration purposes, or by using a secondary standard which is compared to the primary one.

ICH guideline Q7 states:

11.17 Primary reference standards should be obtained as appropriate for the manufacture of APIs. The source of each primary reference standard should be documented. Records should be maintained of each primary reference standard’s storage and use in accordance with the supplier’s recommendations. Primary reference standards obtained from an officially recognized source are normally used without testing if stored under conditions consistent with the supplier’s recommendations.

Officially recognized sources, however, are not specified in Q7, but the FDA does mention sources in their ‘Guidance for Industry on Analytical Procedures and Methods Validation for Drugs and Biologics’ but, interestingly, does not refer to these institutions as an official or definitive list:

Reference standards can often be obtained from the USP and may also be available through the European Pharmacopoeia, Japanese Pharmacopoeia, World Health Organization, or National Institute of Standards and Technology.

Instead, the FDA states that “reference materials from other sources should be characterized by procedures including routine and beyond routine release testing” and that producers “should consider orthogonal methods for reference material characterization”.

For primary RSs, both the ICH guideline and FDA guidance allow other sources than the “officially recognized sources”. Independent manufacturers can provide such primary standards, ideally characterized by processes like those outlined in the general text 5.12. of the European Pharmacopoeia (Ph.Eur).

Correct use of primary reference standards: what to keep in mind?

In essence, a primary RS needs to be fit for its intended purpose. A pharmacopoeial RS has been shown to be fit for its compendial purpose, but has not been demonstrated to be fit for any other purpose; this needs to be proven by the user. Consequently challenges of compendial standard use for non-compendial purposes have been reported in regulatory inspections. Other primary standards with fully documented CoAs can be used for most applications, providing they have been characterized appropriately. If a primary RS is used to establish a secondary standard then the secondary RS can only be used for the same purpose as the primary one.

Secondary or working standard

Secondary standard is a chemical that has been standardized against a primary standard for use in a specific analysis. Secondary standards are commonly used to calibrate analytical methods.

A secondary standard is a standard that is prepared in the laboratory for a specific analysis. It is usually standardized against a primary standard.

Primary standards come with a certificate of analysis and bear traceability to a globally recognized standards body. The cost is often too high for even milligram range quantities. Secondary standards or working standards are also high purity grade materials which are quantified in relation to primary standards and put to routine use in laboratories. Such working standards are assigned a limited validity depending on stability of the material and before expiry fresh working standards should be prepared for future use. It is important to realize that if an expired working standard is used in analysis then no credibility can be placed on the reported results.
These compounds are second-line materials. They consist in each instance of a material that is compared against the primary material, and used in its place.
It does not matter whether the secondary standard is compared against a pharmacopoeial primary standard, or against a primary standard obtained in-house or from a third source. A secondary standard can only be used for the same purposes as the primary standard. Thus if a primary standard was designed solely for a qualitative purpose (i.e. identification via IR, system suitability test or peak identification), then to use the corresponding secondary standard for quantitative purposes is not valid. For example, a large number of Ph.Eur. reference standards for APIs have been set up for IR comparisons only, and should not be used as a basis for quantitative secondary standards.

Handling and storage of standards

Standards play a crucial role in analysis so require to be preserved under specified conditions so that their authenticity is preserved over the prescribed storage periods.

Following recommendations can prove useful:

• Store in amber coloured glass vials or bottles which are properly sealed or cappedand stored in controlled humidity conditions .
• Reseal vial or cap the bottle securely after use
• Temperature sensitive standards should be stored in cooling chambers or dedicated refrigerators between 2−80C
• Ensure that the standard is within its validity before use
• Do not return a new standard to original container to prevent contamination of the original stock.

The significant contribution of standards in any chemical analysis is emphasized in the article. It is indeed difficult to imagine a laboratory functioning without making use of standards and reference materials.

Preparation of Working Standard 

 Select any approved batch, the quality attributes of the selected batch will be reviewed critically with special emphasis on its assay and related substances. This batch shall have reviewed according to pharmaceutical standards and the selected batch shall have maximum assay/purity and the lowest related substance.  

Analyse the selected batch against the reference standard using logbook as per Annexure – VI and follow the stipulated control procedure which includes Description, Identication, Moisture Content, Assay, Perform Assay, and Moisture Content Analysis in triplicate.  The QC manager shall allocate the material abbreviation and assign a unique number to each working standard as below.

 

 

Noise and drift

In HPLC we deal with the time-dependent process. The appearance of the component from the column in the detector represented by the deflection of the recorder pen from the baseline. It is a problem to distinguish between the actual component and artifact caused by the pressure fluctuation, bubble, compositional fluctuation, etc. If the peaks are fairly large, one has no problem in distinguishing them. However, the smaller the peaks, the more important that the baseline be smooth, free of noise, and drift.

Baseline noise is the short time variation of the baseline from a straight line caused by electric signal fluctuations, lamp instability, temperature fluctuations and other factors. Noise usually has much higher frequency than actual chromatographic peak. Noise is normally measured "peak-to-peak": i.e., the distance from the top of one such small peak to the bottom of the next. Sometimes, noise is averaged over a specified period of time. Noise is the factor which limits detector sensitivity. In trace analysis, the operator must be able to distinguish between noise spikes and component peaks. A practical limit for this is a 3 x signal-to-noise ratio, but only for qualitative purposes. Practical quantitative detection limit better be chosen as 10x signal-to-noise ratio. This ensures correct quantification of the trace amounts with less than 2% variance. Figure below illustrates this, indicating the noise level of a baseline(measured at highest detector sensitivity) and the smallest peak which can be unequivocally detected.

Definition of noise, drift, and smallest detectable peak.

Another parameter related to the detector signal fluctuation is drift. Noise is a short-time characteristic of a detector, an additional requirement is that the baseline should deviate as little as possible from a horizontal line. It is usually measured for a specified time, e.g., 1/2 hour or one hour. Drift usually associated to the detector heat-up in the first hour after power-on. Figure also illustrates the meaning of drift.

COVID-19 DRUGS FAVIPIRAVIR HPLC -UV METHOD VALIDATION PROCESS

 COVID-19 DRUGS FAVIPIRAVIR HPLC -UV METHOD VALIDATION PROCESS

Favipiravir

   6-FLUORO-3-HYDROXYPYRAZINE-2-CARBOXAMIDE

   IUPAC NAME- 5-fluoro-2-oxo-1H-pyrazine-3-carboxamide

Molecular Weight -  157.1 g/mol                                     Favipiravir

DISCRIPTION -

Favipiravir (FVP), a pyrazine analog, has shown antiviral activity against a wide variety of viruses. It is considered to be worth further investigation as a potential candidate drug for COVID-19. It is not officially available in any pharmacopoeia. A rapid, simple, precise, accurate, and isocratic high performance liquid chromatography (HPLC) method has been developed for routine quality control of favipiravir in pharmaceutical formulations. Separation was carried out by C18 column. The mobile phase was a mixture of 50 mM potassium dihydrogen phosphate (pH 2.3) and acetonitrile (90:10, v/v) at a flow rate of 1 mL min−1. The ultraviolet (UV) detection and column temperature were 323 nm, and 30 °C, respectively. The run time was 15 min under these chromatographic conditions. Excellent linear relationship between peak area and favipiravir concentration in the range of 10–100 μg mL−1 has been observed (r2, 0.9999). Developed method has been found to be sensitive (limits of detection and quantification were 1.20 μg mL−1and 3.60 μg mL−1, respectively), precise (the interday and intraday relative standard deviation (RSD) values for peak area and retention time were less than 0.4 and 0.2%, respectively), accurate (recovery, 99.19–100.17%), specific and robust (% RSD were less than 1.00, for system suitability parameters). Proposed method has been successfully applied for quantification of favipiravir in pharmaceutical formulations.

Introduction

Chinese-borne coronavirus disease (COVID-19) spread rapidly and became an epidemic, affecting almost all countries and regions around the world. COVID-19 case death rate ranges from 1% to 7% according to the reports of World Health Organization (WHO). It caused all people in the world to change their lifestyle. It still threatens the entire World [1]. Since the outbreak of the COVID-19 began to affect the world, countries have implemented different treatment methods.

Active therapeutic alternatives are urgently needed as a rising COVID-19 pandemic and possible effects on global health [2]. Many medications such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical trials in several countries to assess their effectiveness and safety in treating coronavirus disease [3, 4]. So far, there is no gold standard for the treatment of COVID-19 since there is not enough evidence [5].

Favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide) is an analog of pyrazine (Fig. 1). Favipiravir (FVP) is an antiviral drug that was initially developed for influenza by Toyama Chemical. It selectively inhibits the RNA polymerase of RNA viruses, thus preventing viral reproduction. It displays antiviral activity against alpha-, filo-, bunya-, arena-, flavi-, and noroviruses [6, 7], as well as being active against the influenza virus.

Fig. 1.

After a pilot trial by Zhongnan Hospital of Wuhan University has found a better recovery rate in COVID-19 patients in the favipiravir group compared to the arbidol group [8], FVP is considered to be worth further investigation as a potential candidate drug for this disease.

According to the literature search, there are two published high performance liquid chromatography (HPLC) methods for determining FVP assay and impurities in active pharmaceutical ingredients [9, 10]. In both of these methods, a gradient HPLC mode was used for chromatographic separation and the run time was 60 min. FVP is not officially available in any pharmacopoeia and there is still a need for validated HPLC methods to determine FVP in pharmaceutical formulations.

REQUIRED CHEMICAL FOR TEST

Analytical grade chemicals were used without further purification in this study. Potassium dihydrogen phosphate (99.5–100.5%, Sigma–Aldrich), ortho-phosphoric acid (≥85%, Sigma–Aldrich), and HPLC-grade acetonitrile (≥99.9%, Sigma–Aldrich) were used. Deionized water was purified by a Milli-Q system (Millipore) with conductivity lower than 18.2 μS cm−1. FVP bulk powder and tablets (favicovir, 200 mg) were obtained from Atabay Pharmaceuticals and Fine Chemicals Inc (Istanbul, Turkey).

Stock standard solution

One hundred milligram pure drug was accurately weighed, dissolved in about 30 mL of deionized water and transferred to a 100 mL volumetric flask. Then the volume was completed to 100 mL with deionized water to obtain 1 mg mL−1 of stock solution. The resulting stock solution was sonicated and filtered through a 0.45 µm filter. The stock solution was further diluted with deionized water to obtain the required concentration of standard solutions (10–100 μg mL−1) before being injected into the system for analysis.

Sample solution

Ten FVP tablets were accurately weighed and transferred to a dry and clean mortar, then ground into a fine powder. Next, tablet powder equal to 250 mg FVP was transferred to a volumetric flask of 250 mL. About 100 mL deionized water was added and this flask was attached to a rotary shaker for 10 min. to completely disperse the ingredients. The mixture was sonicated for 30 min, diluted to volume with deionized water to give a solution containing 1,000 μg mL−1and then filtered through a 0.45 µm filter.

Determination of λmax

Standard solution (40 μg mL−1) was subjected to scanning between 200 and 800 nm on a ultraviolet (UV) spectrophotometer (Shimadzu UV-1800 spectrophotometer). λmax was obtained from the UV spectrum of standard solution.

Chromatographic conditions

Chromatographic analysis was performed on a column of Inertsil ODS-3V C18 (4.6 mm × 250 mm, 5.0 μm). The mobile phase consisted of potassium dihydrogen phosphate 50 mM (pH 2.3) and acetonitrile (90:10, v/v). The mobile phase was filtered and degassed through a 0.45 μm membrane filter before use and then pumped at a flow rate of 1 mL min−1. The column has been thermostated at 30 °C. The run time was 15 min under these conditions.

Method validation

The analytical method validation has been performed as per ICH guidelines of Validation of Analytical Procedure: Q2 (R1) [11, 12]. The validation parameters such as system suitability, linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, specificity, precision, and robustness were addressed.

Linearity

Standard calibration has been prepared using six standard solutions within the concentration range of 10–100 μg mL−1. In optimized chromatographic conditions, each standard solution was chromatographed for 15 min three times. Least squares linear regression analysis of the average peak area versus concentration data were used to evaluate the linearity of the method.

Specificity/selectivity

Selectivity is the ability of the analytical method to produce a response for the analyte in the presence of other interference. The selectivity of the method was tested by comparing the chromatograms obtained for FVP standard, tablet, and blank solutions. The parameters retention time and tailing factor were calculated in order to prove that the method chosen was specific.

Limit of detection and limit of quantification

These values were determined using the standard error (s) and slope of the regression line (m) as shown in following equations:

$\begin{array}{c}\text{LOD}=3.3\ast \text{s}/\text{m}\\ \text{LOQ}=10\ast \text{s}/\text{m}\end{array}$

Precision

Precision was analyzed by calculating variations of the method in intraday (repeatability performed by analyzing standard solution on the same day) and inter-day (repeatability carried out by analyzing standard solution on three different days). Precision study was performed by injecting six times of standard solution at three different concentrations, 20, 40, and 60 μg mL−1 on the same day and three consecutive days.

Accuracy

Recovery studies were conducted by the standard addition technique to confirm the accuracy of the proposed method. In this method, 80, 100 and 120% of three different levels of pure drug were added to the previously analyzed sample solutions, and favipiravir recovery was calculated for each concentration.

Robustness

A robustness analysis was performed to determine the impact of minor yet systematic differences in chromatographic conditions. The modifications include different flow rates of the mobile phase (±0.1 mL min−1), acetonitrile ratio in the mobile phase (±1%) and column temperatures (±2 °C). After each change, System suitability parameters were checked by injecting the sample solution into the chromatographic system and the results were compared with those under the original chromatographic conditions.

Analysis of marketed formulations

Four milliliter of above prepared sample solution has been transferred into a volumetric flask of 100 mL and filled the mark with deionized water to prepare at the concentration of 40 μg mL−1 sample solution. This sample solution was filtered using 0.45 μm filter and then analyzed.

Solution stability

The stability of sample and standard solutions was monitored over a 24 h period. For this, standard and sample solutions were injected into the system at 8 h periods, and the peak area and retention time were evaluated. During the stability study, standard solutions have been stored at ambient temperature (25 °C) and protected from light.

Result and discussion

Determination of λmax

The wavelength corresponding to maximum absorbance (λmax) was determined as 323 nm from the UV spectrum of standard solution (Fig. 2).

Fig. 2.

UV spectrum (standard solution, 40 μg mL−1)

Method development

Several preliminary studies were conducted to optimize the chromatographic conditions for the quantification of FVP. Mobile phases consisting of several buffer systems were tried at the beginning of the study; they could not meet the required system parameters. Then only potassium dihydrogen phosphate buffer system was tested without using organic modifiers, long analysis times were obtained. Different acetonitrile solution ratios were investigated to obtain optimum conditions. The acetonitrile ratio was determined as 10% against 50 mM potassium dihydrogen phosphate solution (pH = 2.3) due to the favipiravir peak being well shaped and symmetrical using this system. Eventually, it was found that the mobile phase consisting of 50 mM potassium dihydrogen phosphate (pH: 2.3 with ortho-phosphoric acid) and acetonitrile (90:10, v/v) provided stronger theoretical plates (>2,000) and peak tailing factor (<1.0). Mobile phase running at different flow rates (0.5–1.5 mL min−1) and containing mixtures of organic solvents and phosphate buffers, with ionic strengths and pH ranges were tested. Collectively, the best chromatographic conditions were achieved using an isocratic mobile phase comprising 50 mM potassium dihydrogen phosphate (pH = 2.3)-acetonitrile (90/10, v/v) at a flow rate of 1.0 mL min−1 on an Inertsil ODS-3V C18 column (4.6 mm × 250 mm, 5.0 μm) that was kept at 30 °C. The analysis was conducted at 30 °C, which offers a lot of advantages such as good chromatographic peak shape, enhanced column efficiency, and low-column pressure, in addition to being economic. The eluate was monitored using a UV detector set at 323 nm. Under the chromatographic conditions FVP were eluted at retention times 7.696. The tablet solution was analyzed for 60 min to ensure that there were no matrix components remaining in the column for much longer under the specified conditions. However, continuing the analysis after 15 min will increase both the analysis time and the cost. Overlapping peaks were not observed to overlap in samples from sample analyses injected into the system consecutively with 15 min of analysis time. Due to all these, the analysis time was determined as 15 min.

Method validation

Linearity

The stock standard solution of FVP was diluted appropriately with deionized water to obtain standard solutions within the concentration range of 10–100 μg mL−1. Each standard solution was injected three times into the HPLC system under the above-mentioned chromatographic working conditions. Linearity of the proposed method has been estimated at 6 concentration levels in the range of 10–100 μg mL−1 by regression analysis. The calibration curve was developed by plotting average peak area versus standard concentration (Fig. 3). The correlation coefficient, slope, and intercept of the regression line were determined using the least squares method. The relation between mean peak area Y (n = 3) and concentration, X expressed by equation Y = a + bX, was linear. Values of slope, intercept, and correlation coefficient (r) were 49.122, 82.598 and 0.9999, respectively as shown in Table 1. Lack of fit test was performed to evaluate linearity. In the Lack of fit test, P value (0.0516) greater than 0.05 indicates that the data satisfies the linearity condition. Overlay chromatogram of FVP standard solutions (10–100 μg mL−1) was demonstrated in Fig. 4A.

Fig. 3.

Parameter	Value
Linearity range (μg mL−1)	10–100
Slope	49.122
Intercept	82.598
Correlation coefficient	0.9999
Lack of fit
F	2.90
P	0.0516
SE of intercept	6.9024
SD of intercept	15.4340
LOD/LOQ (μg mL−1)	1.20/3.60

Fig. 4

A) Overlay chromatogram (standard solutions, 10–100 μg mL−1, λ: 323 nm). (B) Chromatogram (standard solution, 80 μg mL−1, λ: 323 nm). (C) Chromatogram (sample solution, 40 μg mL−1, λ: 323 nm). (D) Chromatogram (Blank solution, λ: 323 nm)

Specificity/selectivity

The chromatogram of FVP standard solution has been given in Fig. 4B. There is only one peak at the retention time of 7.696 min. The chromatogram of the tablet solution has been given in Fig. 4C. There is only one peak at the retention time of 7.696 min in this chromatograme. There are no other peaks caused by excipients and additives in this chromatograme. The chromatogram of the mobile phase has also given in Fig. 4D. There are no other peaks caused by contents of the mobile phase in this chromatograme. This indicates that the analytical method is specific. The parameters retention time and tailing factor were calculated in order to prove that the method chosen was specific. Retention time, theoretical plate number, and peak tailing factor values were 7.696, 13798, and 0.920, respectively. All of the values were within the accepted level.

Precision

Precision study was performed by injecting six times of standard solution at three different concentrations, 20, 40, and 60 μg mL−1 on the same day and three consecutive days. The precision data were given in Table 2. All RSD values for retention time and peak area for selected FVP concentrations were less than 0.5 and 2.0%, respectively. In this case, the method is precise and can be used for our intended purpose.

Std. conc. μg mL−1	Intraday precision			Interday precision
	Found conc. (6) (μg mL−1)	Peak area RSD (%)	Retention time RSD (%)	Found conc. (6) (μg mL−1)	Peak area RSD (%)	Retention time RSD (%)
20	20.02	0.178	0.015	19.92	0.195	0.034
40	40.35	0.066	0.004	40.08	0.082	0.012
60	60.24	0.041	0.002	60.10	0.064	0.025

 

Accuracy study

A known quantity of standard solution has been added to the sample solutions previously analyzed at three different levels (80%, 100% and 120%). The amount recovered for favipiravir has been calculated for three concentration. The recovery data were summarized in Table 3. Percent RSD values for all analyses were less than 2% indicating that excipients found in pharmaceutical formulations do not interfere and analytical method is very accurate.

Table 3.

Recovery data

---

Spiked level (%)	Amount added (μg mL−1)	Amount recovered (μg mL−1)	Recovery (%)	Average (%)	SD	RSD (%)
80	32	31.78	99.31	99.96	0.157	0.157
	32	31.74	99.19
	32	31.84	99.50
100	40	39.79	99.48	99.93	0.180	0.180
	40	39.75	99.38
	40	39.89	99.73
120	48	48.02	100.04	99.75	0.125	0.125
	48	48.08	100.17
	48	47.96	99.92

 

Robustness

The results showed that the change in flow rate and mobile phase concentration had little effect on the chromatographic behavior of FVP. The small change in the mobile phase flow rate and acetonitrile content have a small impact on the retention time of FVP. The change in the column temperature did not have a significant effect on the method. The results of this study, expressed as % RSD, were presented in Table 4.

Condition	Variation	Assay (%)	SD	RSD (%)
Mobile phase flow rate (1.00 mL min−1)	0.90 mL min−1	99.86	0.60	0.60
	1.10 mL min−1	99.94	0.62	0.62
Acetonitrile ratio in mobile phase (10%)	9%	100.12	0.67	0.67
	11%	99.96	0.71	0.71
Column temperature (30 °C)	28 °C	99.96	0.34	0.34
	32 °C	100.05	0.32	0.32

 

 

 

Solution stability

The stability of sample and standard solutions was monitored over a 24 h period. For this, standard and sample solutions were injected into the system at 8 h periods, and the peak area and retention time were evaluated. No changes in standard concentrations have been observed over a period of 24 h. The % RSD for peak area (n = 3) was 0.275% and the value for retention time (n = 3) was 0.12% for standard solution. The results have been demonstrated in Table 5. No major changes in active ingredient concentration have also been found in the tablet solution.

Table 5.

Standard solution stability (40 μg mL−1)

Time (h)	Peak area	Mean	SD	RSD (%)	Retention time (min)	Mean	SD	RSD (%)
8	2013.5	2019.7	5.5	0.275	7.594	7.596	0.009	0.115
	2021.3				7.606
	2024.2				7.589
16	2025.6	2020.0	5.2	0.257	7.597	7.599	0.015	0.192
	2019.2				7.614
	2015.3				7.585
24	2012.2	2018.1	6.5	0.323	7.610	7.597	0.011	0.150
	2025.1				7.588
	2017.1				7.594

 

 

Application of the method to the marketed tablets

The developed and validated method has been applied successfully for determination of FVP in pharmaceutical formulations. The result of assay of the marketed tablet of favipiravir is shown in Table 6. The results obtained are closely related to the amount indicated on the labels of the tablets. This shows that the method for content evaluation is useful.

Method application results

Formulation	Label claim (mg)	Amount of drug (mg)	% Assay ± SD
Favicovir tablet	200	200.35	100.18 ± 0.38

Conclusions

A very quick, cost-effective, precise and accurate HPLC method for the determination of FVP has been developed and validated in compliance with ICH guidance Q2. Besides the short run time (15 min), retention time (7.696) and flow rate of mobile phase (1 mL min−1) made the method attractive because these features save analysis time and cost. Potassium dihydrogen phosphate, used as a general purpose buffer, has many interesting properties. The most important of these features are good buffering capacity in the selected pH range, easy availability, low toxicity and cost, and greatly improved separation ability without colon degradation. In short, this method is sensitive, selective, reproducible and rapid for favipiravir in bulk and tablets. The accuracy and precision are within reasonable limits, the maximum of quantification is as small as 3.60 μg mL−1 and finally analytical method is reliable and robust.